Peptides for treating muscle atrophy

Abstract

To provide peptides that may be used to promote muscle growth and muscle health, reduce muscle loss, and support immune and / or inflammatory responses in subjects, particularly those exhibiting muscle atrophy.SOLUTION: The present invention relates to a composition comprising a peptide having up to 50 amino acids and comprising a specific sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a number of peptides and compositions comprising one or more peptides. Use of the peptides or compositions to treat or prevent muscle wasting in a subject is also contemplated. [Background technology]

[0002] Muscle atrophy is a human condition characterized by muscle wasting, increased protein breakdown, increased systemic inflammation, and downregulation of newly synthesized proteins in affected muscle types. Symptoms of this condition include significant weakness in one or more limbs, with one arm or leg becoming significantly smaller than the other. It is commonly associated with aging (often as part of sarcopenia), subjects who have been physically inactive for extended periods, such as bedridden patients, astronauts, and injured athletes, subjects with eating disorders, subjects with metabolic diseases, and subjects with diseases characterized by nerve, muscle, or joint degeneration, such as ALS, MS, muscular dystrophy, Guillain-Barré syndrome, sarcopenia, osteoarthritis, polio, rheumatoid arthritis, spinal muscular atrophy, and polymyositis.

[0003] Ribosomal protein S6 is one of 33 proteins that, along with one molecule of 18S rRNA, constitute the small 40S ribosomal subunit (4). rpS6 directly interacts with the m7GpppG 5'-cap-binding complex required for translation initiation and represents a regulatory convergence point for signaling pathways that control translation initiation in response to cues for cell growth and proliferation. rpS6 undergoes inducible phosphorylation in response to mitogens and cell proliferation stimuli, and this phosphorylation is conserved among vertebrates, invertebrates, plants, and fungi (5). In higher eukaryotes, phosphorylation occurs at a cluster of five serine residues at the carboxyl terminus of rpS6: Ser-235, Ser-236, Ser-240, Ser-244, and Ser-247 (6). Drosophila rpS6 contains a similar configuration of five phosphorylation sites, but its homolog in Saccharomyces cerevisiae contains two Ser residues corresponding to mammalian Ser-235 and Ser-236 (4). Phosphorylation of rpS6 begins at Ser-236, followed by phosphorylation of Ser-235, Ser-240, Ser-244, and Ser-247 (7, 8). Phosphorylation of rpS6 at the C-terminal residue increases its affinity for the m7GpppG cap, strongly suggesting that rpS6 phosphorylation promotes mRNA translation initiation.

[0004] Carboxyl-terminal phosphorylation of rpS6 is regulated by at least two signaling pathways. The p70 ribosomal S6 kinases, S6K1 and S6K2, play a major role in rpS6 C-terminal phosphorylation in response to insulin, serum, and amino acid stimulation (4). S6K1 and S6K2 phosphorylate Ser-240 and Ser-244 but are dispensable for phosphorylation of Ser-235 and Ser-236 in intact cells (13). The activity of S6K1 and S6K2 is directly regulated by mTOR, the mammalian target of rapamycin, in response to growth and mitogenic cues. Inhibition of mTOR by rapamycin causes a dramatic decrease in rpS6 phosphorylation in mammalian cells (14). mTOR also phosphorylates the translational repressor 4E-BP1, causing its dissociation from the m7GpppG 5'-cap-binding complex. By combining S6K and 4E-BP1 phosphorylation, mTOR positively regulates protein translation in response to favorable growth conditions. The RAS / ERK pathway also regulates rpS6 phosphorylation independently of mTOR through activation of the p90 ribosomal S6K kinases RSK1 and RSK2 (12). RSK1 and RSK2 phosphorylate rpS6 at Ser-235 and Ser-236 in response to phorbol esters, serum, and oncogenic RAS, and phosphorylation of both residues is required for cap binding (13). Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to overcome at least one of the above problems. [Means for solving the problem]

[0006] Applicant discovered a number of peptides derived from Vicia faba (Faba bean) proteins that can phosphorylate ribosomal protein S6 (rpS6) in a dose-dependent manner across a range of concentrations in in vitro cell assays (Figures 1-10 and 12). RPS6 is an important substrate for protein kinases and is phosphorylated by growth factors and mitogens during cell proliferation and division. This is a key step in the synthesis of new proteins in skeletal muscle tissue. The described peptides also have the ability to reduce the expression of mRNA transcripts (TRIM63 and FBXO32) that are directly linked to increased protein degradation leading to progressive skeletal muscle atrophy (Figures 14-15). Furthermore, increased muscle atrophy is associated with a systemic increase in circulating TNFα. Some of the peptides described herein also result in a reduction in TNFα expression in circulating immune cells. The peptides can be used to promote muscle growth and muscle health, reduce muscle loss, and support immune and / or inflammatory responses in subjects, particularly those exhibiting muscle atrophy, such as the elderly, physically inactive people, and subjects with indications characterized by muscle atrophy (i.e., MS and polio).

[0007] According to a first aspect of the present invention, TIKLPAGT SEQ ID NO: 1 IEDPGQFPT SEQ ID NO:2 HLPSYSPSPQ SEQ ID NO: 3 KGDIIAIPSGIPY SEQ ID NO: 4 LDWYKGPT SEQ ID NO:5 SRGPIYSN SEQ ID NO: 6 LERGDTIKIPAGT SEQ ID NO: 7 TIKIPAGT SEQ ID NO:8 SYSPSPQ SEQ ID NO: 9 IGSSSSPDIYNPQAGRIKT SEQ ID NO: 10 IDPNGLHLPSYSPSPQL SEQ ID NO: 11 LVNRDDEEDLRVLDLVIP SEQ ID NO: 12 ITGQVLHPNGGTVVNA SEQ ID NO: 13 ALEPDNR SEQ ID NO: 14 LREQSQQNECQLER SEQ ID NO: 15 VAGKGIPWDKQDPGEEAIES SEQ ID NO: 16 VGRRGGQHQQEEESEEQKD SEQ ID NO: 17 YDEEKEQGEEEIRK SEQ ID NO: 18 HLPSYSPSP SEQ ID NO: 19 SYSPSP SEQ ID NO: 20 The present invention provides a peptide comprising or consisting essentially of an amino acid sequence selected from the group consisting of: or a functional variant thereof (hereinafter referred to as "the peptide of the present invention").

[0008] In one embodiment, the peptide comprises SEQ ID NO: 20. Examples of such peptides include SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 19. In another aspect, the present invention provides modified peptides of the present invention. The peptides can be modified by any method described herein, for example, N-terminal, C-terminal, or amino acid side chain modification, PEGylation, cyclization, or lipidation.

[0009] In another aspect, the invention provides a conjugate comprising a peptide of the invention conjugated (typically covalently conjugated) to a binding partner.

[0010] The term "peptides of the invention" includes modified peptides and conjugates.

[0011] In another aspect, the invention provides a composition comprising one, more or all of the peptides of the invention, for one, two, three, four or five of the peptides of the invention.

[0012] In one embodiment, the composition comprises a peptide comprising SEQ ID NO:20, eg, one, two, three of SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:19.

[0013] In one embodiment, the composition comprises or consists essentially of SEQ ID NO:1 or SEQ ID NO:8, for example SEQ ID NO:7 and SEQ ID NO:8.

[0014] In one embodiment, the composition is a powder optionally containing additional peptides. The powder may be a protein hydrolysate that may be supplemented with peptides and / or may be provided as an edible powder. In one embodiment, the powder comprises about 0.0001 to about 1.0%, about 0.0001 to about 0.2%, about 0.001 to about 0.1%, or about 0.001 to about 0.1% (w / w) of one or more peptides of the present invention. In one embodiment, the powder is substantially free of intact proteins. In one embodiment, the composition is a food, beverage, or nutritional supplement. In another embodiment, the composition is a topical composition.

[0015] In one embodiment, the powder comprises the peptides of SEQ ID NOs: 1 and 3, typically in an amount of about 0.01 to 0.2% (w / w). In one embodiment, the powder comprises the peptides of SEQ ID NOs: 1, 2, 3 and 5, typically in an amount of about 0.001 to 0.2% (w / w). In one embodiment, the powder comprises the peptides of SEQ ID NOs: 1, 2, 3, 4 and 5, typically in an amount of about 0.001 to 0.2% (w / w).

[0016] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a peptide of the present invention in combination with a pharmaceutically acceptable excipient.

[0017] In another aspect, the present invention provides a method for treating or preventing muscle atrophy in a subject, comprising administering a therapeutically effective amount of a peptide or composition of the present invention to the subject. The treatment can be administered by a pharmaceutical composition, a nutritional supplement, or a food or beverage containing the peptide or composition of the present invention. In one embodiment, the subject is an elderly subject or a physically inactive subject, for example, a subject with a physical injury.

[0018] In another aspect, the present invention provides a method for treating or preventing muscle atrophy in a subject having a disease or condition characterized by muscle atrophy, comprising administering to the subject a therapeutically effective amount of a peptide or composition of the present invention. Examples of diseases or conditions characterized by muscle atrophy include physical injuries, eating disorders, metabolic diseases (including type I and type II diabetes), and diseases characterized by nerve, muscle, or joint degeneration, such as ALS, MS, muscular dystrophy, Guillain-Barré syndrome, osteoarthritis, polio, rheumatoid arthritis, spinal muscular atrophy, cachexia, sarcopenia, malnutrition, and polymyositis.

[0019] In another aspect, the present invention provides a method of promoting muscle synthesis in a subject, comprising administering to the subject a therapeutically effective amount of a peptide or composition of the present invention.

[0020] In another aspect, the invention provides a method for reducing muscle loss in a subject, comprising administering to the subject a therapeutically effective amount of a peptide or composition of the invention.

[0021] In another aspect, the invention provides a method of supporting or enhancing an immune or inflammatory response in a subject, comprising administering to the subject a therapeutically effective amount of a peptide or composition of the invention.

[0022] In another aspect, the present invention provides a method for protecting muscle during periods of muscle breakdown in a subject (e.g., during weight exercise), comprising administering to the subject a therapeutically effective amount of a peptide or composition of the present invention.

[0023] In another aspect, the present invention provides a method for increasing the abundance of muscle fibers (particularly type I or type II muscle fibers) in a subject, comprising the step of administering to the subject a therapeutically effective amount of a peptide or composition of the invention.

[0024] In any embodiment, the subject can be healthy, young, or elderly.

[0025] In any embodiment, the peptide or composition may be administered orally (eg, in a drink, food, or pharmaceutical composition).

[0026] In one embodiment, the peptide comprises SEQ ID NO: 20. Examples of such peptides include SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 19.

[0027] In any embodiment, the peptide comprises or consists essentially of SEQ ID NO:1 or SEQ ID NO:8, for example, SEQ ID NO:7 and SEQ ID NO:8.

[0028] In another aspect, the present invention provides a nucleic acid encoding a peptide of the present invention.

[0029] In another aspect, the present invention provides an expression vector comprising DNA encoding a peptide of the present invention, which vector is configured for heterologous expression of the peptide of the present invention in a host cell (hereinafter referred to as the "expression vector of the present invention").

[0030] In another aspect, the present invention provides host cells, particularly bacterial or mammalian producer cells, engineered to heterologously express a peptide of the invention (hereinafter referred to as "transformed cells of the invention"). In one embodiment, the transformed host cell comprises an expression vector of the invention.

[0031] The present invention also provides a method for producing a peptide of the present invention, comprising the steps of providing a transformed cell of the present invention, culturing the transformed host cell to result in heterologous expression of a recombinant peptide of the present invention by the host cell, and recovering the recombinant peptide of the present invention.

[0032] The present invention also provides a method of engineering a cell for heterologous expression of a peptide of the present invention, which comprises the step of transforming a cell with an expression vector of the present invention, whereby the transformed cell is capable of heterologous expression of a peptide of the present invention.

[0033] Other aspects and preferred embodiments of the present invention are defined and described in the other claims set forth below. [Brief explanation of the drawings]

[0034] [Figure 1] FIG. 1 shows the rPS6 phosphorylation activity of SEQ ID NO: 1 at various peptide concentrations. [Figure 2] FIG. 1 shows the rPS6 phosphorylation activity of SEQ ID NO: 2 at various peptide concentrations. [Figure 3] FIG. 1 shows the rPS6 phosphorylation activity of SEQ ID NO: 3 at various peptide concentrations. [Figure 4] FIG. 1 shows the rPS6 phosphorylation activity of SEQ ID NO: 4 at various peptide concentrations. [Figure 5] FIG. 1 shows the rPS6 phosphorylation activity of SEQ ID NO: 5 at various peptide concentrations. [Figure 6] FIG. 1 shows the rpS6 phosphorylation activity of peptides of SEQ ID NOs: 1 to 5 at 0.5 μg / ml. [Figure 7] Figure 1 shows the rPS6 phosphorylation activity of peptides SEQ ID NO:8; SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:10; SEQ ID NO:16; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:17; SEQ ID NO:18 at 0.05 μg / ml for 20 minutes following a starvation protocol (one-way ANOVA; *p<0.05 **p<0.01 ***p<0.001; mean±SEM; N=4): [Figure 8] Figure 1 shows the effect of SEQ ID NO: 6 on S6 phosphorylation. C2C12 cells were treated with peptide (0.005-0.5 μg / ml) for 30 minutes following a starvation protocol (Student's t-test or one-way ANOVA; *p<0.05**p<0.01***p<0.001; mean ± SEM; N=4). [Figure 9]Figure 1 shows the effect of SEQ ID NO: 9 containing the motif SYSPSP (SEQ ID NO: 20) on S6 phosphorylation. C2C12 cells were treated with peptide (5 μg / ml) for 30 minutes following a starvation protocol (Student's t-test or one-way ANOVA; *p < 0.05 **p < 0.01 ***p < 0.001; mean ± SEM; N = 4). [Figure 10] Figure 1 shows the effect of SEQ ID NO: 11, which contains the motif SYSPSP (SEQ ID NO: 20), on S6 phosphorylation. C2C12 cells were treated with peptide (0.005-0.5 μg / ml) for 30 minutes following a starvation protocol. (Student's t-test or one-way ANOVA; *p<0.05, **p<0.01, ***p<0.001; mean ± SEM; N=4). [Figure 11] Figure 1 shows the effect of peptide treatment on TNF-α secretion. THP-1 macrophages were treated with Neuritas peptide (0.5 μg / ml) for 24 hours, followed by stimulation with LPS (100 ng / ml) for 24 hours. SEQ ID NO: 14; SEQ ID NO: 8; SEQ ID NO: 13; SEQ ID NO: 15. (One-way ANOVA; *p<0.05, **p<0.01, ***p<0.001; mean±SEM; N=4). [Figure 12] Figure 1 shows the effect of peptide treatment on S6 phosphorylation. C2C12 cells were treated with SEQ ID NO: 2 (0.5-5 μg / ml) for 30 minutes according to a starvation protocol. (One-way ANOVA; * p<0.05, ** p<0.01, *** p<0.001; mean ± SEM; N = 3). [Figure 13] Figure 1 shows the effect of peptide treatment on TNF-α secretion. THP-1 macrophages were treated with SEQ ID NO:2 (0.05-5 μg / ml) for 24 hours, followed by stimulation with LPS (100 ng / ml) for 24 hours. (One-way ANOVA; *p<0.05, **p<0.01, ***p<0.001; mean±SEM; N=3). [Figure 14]Figure 1 shows the effect of Neuritas peptides on atrophy-related gene expression. PCR analysis was performed on atrophy-induced C2C12 cells showing the effect of SEQ ID NO: 19 (containing the SYSPSP motif) on TRIM63 (N = 6) gene expression. Cells were treated with dexamethasone (0.3 μg / ml) for 24 hours, and pep_MU12PE (0.5-5 μg / ml) was added 30 minutes before the end of dexamethasone treatment. (One-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; mean ± SEM). [Figure 15] Figure 1 shows the effect of Neuritas peptides on atrophy-related gene expression. PCR analysis was performed on atrophy-induced C2C12 cells showing the effect of SEQ ID NO: 19 (containing the SYSPSP motif) on FBXO (N=5) gene expression. Cells were treated with dexamethasone (0.3 μg / ml) for 24 hours, and pep_MU12PE (0.5-5 μg / ml) was added 30 minutes before the end of dexamethasone treatment. (One-way ANOVA; *p<0.05, **p<0.01, ***p<0.001; mean ± SEM). [Figure 16] This figure shows that NPN_1 (a powder composition containing SEQ ID NOs: 3 and 8) significantly prevented muscle loss in the soleus muscle after unloading. C57BL / 6 mice were treated with BBI (113.3 mg / kg / day), casein (650 mg / kg / day), or NPN_1 (650 mg / kg / day) for 18 days. (One-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; N = 10). [Figure 17] NPN_1 reduces connective tissue and intermuscular fat mass and increases muscle fiber density. (A) Skeletal muscle immunostaining and hematoxylin and eosin (H&E) staining showing the effect of NPN_1 on type I (red) and type IIa (green) muscle fiber density. Quantification of the effect of treatment on type I (B) and type IIa (C) fiber density is shown (* p < 0.05, ** p < 0.01, *** p < 0.001; N = 5). DETAILED DESCRIPTION OF THE INVENTION

[0035] All publications, patents, patent applications, and other references mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference and the contents of which were set forth in full.

[0036] Definitions and General Settings

[0037] As used herein, unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meaning that such terms may enjoy in the art:

[0038] Unless the context otherwise requires, the use of the singular herein shall be read to include the plural, and vice versa. The terms "a" or "an," when used in reference to an object, shall be construed as referring to one or more of that object. That is, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0039] As used herein, the term "comprise," or variations thereof, such as "comprises" or "comprising," should be read to indicate the inclusion of an enumerated integer (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., feature, element, characteristic, property, method / process step, or limitation) but not the exclusion of other integers or groups of integers. Thus, as used herein, the term "comprise" is inclusive or open-ended and does not exclude additional, unenumerated integers or method / process steps.

[0040] As used herein, the term "disease" is used to define an abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, disorder, condition, illness, state, or syndrome in which physiological function is impaired, regardless of the nature of the etiology (or whether an etiological basis for the disease has actually been established). Thus, conditions resulting from infection, trauma, injury, surgery, radiation ablation, poisoning, or nutritional deficiency are included.

[0041] As used herein, the term "treatment" or "treating" refers to an intervention (e.g., administration of a drug to a subject) that cures, ameliorates, or alleviates the symptoms of a disease or eliminates its cause (or reduces the effects of) it. In this context, the term is used interchangeably with the term "therapy."

[0042] Furthermore, the terms "treatment" or "treating" refer to an intervention (e.g., administration of a drug to a subject) that prevents or delays the onset or progression of a disease, or reduces its incidence (or eradicates) within the treated population. In this context, the term treatment is used synonymously with the term "prevention."

[0043] As used herein, an effective or therapeutically effective amount of a peptide of the present invention is defined as an amount that can be administered to a subject without undue toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit / risk ratio, but sufficient to provide the desired effect, e.g., treatment or prevention manifested by a permanent or temporary improvement in the subject's condition. This amount will vary from subject to subject, depending on the individual's age and general condition, the method of administration, and other factors. Therefore, it is not possible to specify an exact effective amount, but one of ordinary skill in the art can determine an appropriate "effective" amount in any individual case using routine experimentation and general background knowledge. Therapeutic results in this context include eradication or alleviation of symptoms, relief of pain or discomfort, prolonged survival, improved mobility, and other markers of clinical improvement. A therapeutic result need not be a complete cure.

[0044] In the context of the above-defined treatment and effective amount, the term subject (which should be read as including "individual," "animal," "patient," or "mammal," as the context allows) is defined as any subject in need of treatment, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, livestock, farm animals, zoo animals, sport animals, and pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and cows; primates such as apes, monkeys, orangutans, and chimpanzees; canines such as dogs and wolves; felines such as cats, lions, and tigers; equines such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters, and guinea pigs. In a preferred embodiment, the subject is a human.

[0045] As used herein, the term "peptide" refers to a polymer typically composed of 5 to 50 amino acid monomers linked together via peptide bonds. Peptides of and for use in the present invention (including fragments and variants thereof) can be produced in whole or in part by chemical synthesis or by expression from nucleic acids. For example, peptides of and for use in the present invention can be readily prepared according to well-established standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, e.g., J.M. Stewart and J.D. Young, Solid-Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, IL (1984); M. Bodantzky and A. Bodantzky, The Practice of Peptide Synthesis, Springer-Berg, New York (1984)). If necessary, any of the peptides used in the present invention can be chemically modified to enhance their stability. Chemically modified peptides or peptide analogs include any functional chemical equivalent of a peptide characterized by its increased stability and / or efficacy in vivo or in vitro for practicing the present invention. The term peptide analog also refers to any amino acid derivative of a peptide as described herein. Peptide analogs can be generated by procedures including, but not limited to, side chain modification, incorporation of unnatural amino acids and / or their derivatives during peptide synthesis, the use of cross-linking agents, and other methods that impose conformational constraints on peptides or their analogs. Examples of side chain modifications include reductive alkylation by reaction with aldehydes followed by reduction with NaBH4; amidation with methylacetimidate; acetylation with acetic anhydride; carbamylation of amino groups with cyanates; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS); alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; pyridoxylation of lysines with pyridoxa-5'-phosphate followed by reduction with NaBH4, etc.The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal. Carboxyl groups can be modified by carbodiimide activation via o-acylisourea formation followed by subsequent derivatization, for example, to the corresponding amide. Sulfhydryl groups can be modified by carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; mixed disulfide formation with other thiol compounds; reaction with maleimide, maleic anhydride, or other substituted maleimides; formation of mercury derivatives using 4-chloromercuribenzoic acid, 4-chloromercuriphenylsulfonic acid, phenylmercuric chloride, 2-chloromercuri-4-nitrophenol, and other mercuric compounds; and carbamylation with cyanate at alkaline pH. Tryptophan residues can be modified by methods such as oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halides. Tyrosine residues can be converted to form 3-nitrotyrosine derivatives by nitration with tetranitromethane. Modification of the imidazole ring of histidine residues can be achieved by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids. Modifications of peptide structures include the generation of retro-inverso peptides containing reverse sequences encoded by D-amino acids.

[0046] The term "peptides of the invention" collectively refers to peptides comprising or consisting essentially of an amino acid sequence selected from SEQ ID NOs: 1 to 20, and therapeutically effective variants thereof. The peptides of the invention may be recombinant peptides.

[0047] The term "therapeutically effective variant" as applied to a reference peptide refers to a peptide having an amino acid sequence substantially identical to the reference peptide and that is therapeutically effective as defined below. Thus, for example, this term should be interpreted to include variants altered with respect to one or more amino acid residues. Preferably, such alterations include insertions, additions, deletions, and / or substitutions of no more than five, more preferably no more than four, even more preferably no more than three, and most preferably only one or two amino acids. Insertions, additions, and substitutions of both natural and modified amino acids are contemplated. Variants may have conservative amino acid changes, where the introduced amino acid is structurally, chemically, or functionally similar to that being replaced. Generally, variants will have at least 70% amino acid sequence identity, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and ideally at least 95%, 96%, 97%, 98%, or 99% sequence identity with the reference antimicrobial fragment. As used herein, the term "sequence identity" should be understood to include both sequence identity and similarity. That is, a variant (or homolog) that shares 70% sequence identity with a reference sequence is a variant (or homolog) in which any 70% of the aligned residues of the variant (or homolog) are identical or conservative substitutions for corresponding residues in the reference sequence, over the entire length of the sequence. Sequence identity is the amount of exact match between two different sequences, where gaps are not counted and the measurement pertains to the shorter of the two sequences. With respect to the term "sequence homology," this term should be understood to mean that a variant (or homolog) shares a defined percent similarity or identity with a reference sequence when a percentage of aligned residues of the variant (or homolog) are identical to or conservative substitutions for corresponding residues in the reference sequence, and the variant (or homolog) shares the same function as the reference sequence.

[0048] This alignment and percent homology or sequence identity can be determined using software programs known in the art, for example, one alignment program is BLAST using default parameters. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi.

[0049] "Therapeutically effective" as applied to the peptides of the invention in the context of muscle synthesis means a peptide that is capable of producing a significant increase in rpS6 phosphorylation compared to a control in the phospho-S6 cellular assay described below, and this applies particularly to peptides of SEQ ID NOS: 1-3, 8-12, and 16-20.

[0050] "Therapeutically effective" as applied to the peptides of the invention in the context of supporting an immune or inflammatory response means a peptide that is capable of reducing TNF-α secretion in THP-1 macrophages compared to a control in the TNF-α secretion cell assay described below, and this applies particularly to peptides of SEQ ID NOS: 2, 8, and 13-15.

[0051] "Composition": The present invention also relates to compositions comprising one or more of the peptides of the present invention. The peptides, or part or all of the peptides, may be modified or provided as conjugates. The composition may be a food ingredient powder, a food beverage, a nutritional supplement, a pharmaceutical composition, or a topical composition. In one embodiment, the composition is a sports nutrition product, e.g., a beverage, a snack, or a supplement. In one embodiment, the composition is a beverage. In one embodiment, the composition is a bakery product. In one embodiment, the composition is a dairy product. In one embodiment, the composition is a snack product. In one embodiment, the composition is a baked extruded food product. In one embodiment, the composition is a milk powder. In one embodiment, the composition is an infant formula product. In one embodiment, the composition is a confectionery product. In one embodiment, the composition is yogurt. In one embodiment, the composition is a yogurt drink. In one embodiment, the composition is an ice cream product. In one embodiment, the composition is a frozen food product. In one embodiment, the composition is a breakfast cereal. In one embodiment, the composition is bread. In one embodiment, the composition is a flavored milk drink. In one embodiment, the composition is a confectionery bar. In one embodiment, the composition is tea or a tea product. In one embodiment, the composition is the base of an extruded snack product. In one embodiment, the composition is a fried snack product. In one embodiment, the composition is a nutritional supplement. In one embodiment, the composition is a sports nutrition product. In one embodiment, the composition is a baby food product. In one embodiment, the composition is a specialty food for immunocompromised individuals. In one embodiment, the composition is a food for geriatric patients. In one embodiment, the composition is an animal feed. In one embodiment, the composition is an animal feed supplement. In one embodiment, the composition is a medical food.

[0052] The composition may be a topical or pharmaceutical composition. The peptides of the present invention are used in the topical or pharmaceutical compositions of the present invention at a therapeutically effective concentration to achieve the desired effect; preferably between 0.00000001% (wt) and 20% (wt), based on the total weight of the composition; preferably 0.000001% (wt) to 15% (wt), more preferably 0.0001% (wt) to 10% (wt), and even more preferably 0.0001% (wt) to 5% (wt). Ideally, the peptides of the present invention are used in a concentration of about 0.00001% w / w to about 0.5% w / w [0.1 to 5000 ppm], more preferably 0.00005 w / w to about 0.05 w / w [0.5 to 500 ppm], and most preferably about 0.0001 w / w to about 0.01 w / w [1 to 100 ppm]. Ideally, the peptides of the present invention are preferably used at about 0.0001% w / w to about 0.004% w / w of the composition.

[0053] The dosage of the compositions of the present invention for use in foods and food or nutritional supplements (i.e., edible compositions) ranges broadly from 0.2 to 100 g / day. In one embodiment, the daily dosage is 1 to 10 g / day, ideally about 3 to 8 g / day. In one embodiment, the daily dosage is 10 to 20 g / day. In one embodiment, the daily dosage is 20 to 30 g / day. In one embodiment, the daily dosage is 30 to 40 g / day. In one embodiment, the daily dosage is 10 to 20 g / day. In one embodiment, the daily dosage is about 5 g / day, ideally about 3 to 8 g / day. In one embodiment, the dosage is 2 to 1000 mg / day / kg body weight. In one embodiment, the dosage is 10 to 500 mg / day / kg body weight. In one embodiment, the dosage is 10 to 100 mg / day / kg body weight. In one embodiment, the dosage is 30 to 70 mg / day / kg body weight. The dosage of the peptides of the present invention for food supplements can be 0.00001 mg to 0.01 mg or dose per day.

[0054] Foods may be foods for specific medicinal purposes (FSMPs), which are defined as foods specially formulated, processed, and intended for the dietary management of a disease, disorder, or medical condition in individuals being treated under medical supervision. These foods are intended for the exclusive or partial nutritional supplementation of people whose nutritional requirements cannot be met through conventional foods. Dosages can range from 50 to 500 grams per day, depending on the patient's age and condition. When administered as a food for special medical purposes or as a medical food, the daily dosage can be 50 to 500 grams.

[0055] "Topical Composition": The present invention also relates to a topical composition comprising a peptide or composition of the present invention. It is understood that a topical composition may comprise multiple peptides. In one embodiment, the topical composition comprises substantially all peptides. The topical composition of the present invention may be presented in a formulation selected from the group consisting of a cream, multiple emulsions, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, lotions, gels, cream gels, hydroalcoholic solutions, hydroglycolic solutions, cosmetics, personal care products, hydrogels, liniments, serums, soaps, dusting powders, pastes, semisolid formulations, liniments, serums, shampoos, conditioners, ointments, rinse-off formulations, talc, mousses, powders, sprays, aerosols, solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, patches, gel patches, bandages, adhesive systems, water-in-oil emulsions, oil-in-water emulsions, and silicone emulsions.

[0056] Pharmaceutical Composition: A further aspect of the present invention relates to pharmaceutical compositions comprising a peptide of the present invention or a composition of a peptide of the present invention mixed with one or more pharmaceutically acceptable diluents, excipients, or carriers. While the peptides and compositions of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient, or diluent, particularly for human treatment. Pharmaceutical compositions can be for human or animal use in human and veterinary medicine. Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein can be found in "Handbook of Pharmaceutical Excipients, 2nd Edition" (1994), edited by A. Wade and P. J. Weller. In particular, formulations for topical delivery are described in "Topical Drug Delivery Formulations," edited by David Osborne and Antonio Aman, Taylor & Francis, the entire contents of which are incorporated herein by reference. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 1985). Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol, and water. The choice of pharmaceutical carrier, excipient, or diluent can be determined taking into account the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain any suitable binder, lubricant, suspending agent, coating agent, or solubilizer as, or in addition to, the carrier, excipient, or diluent. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, dyes, and even flavorings can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

[0057] The peptides or compositions of the present invention can be adapted for topical, oral, rectal, parenteral, intramuscular, intraperitoneal, intraarterial, intrabronchial, subcutaneous, intradermal, intravenous, nasal, vaginal, buccal, or sublingual administration. For oral administration, compressed tablets, pills, tablets, gellules, drops, and capsules are particularly used. Preferably, these compositions contain 1 to 250 mg, more preferably 10-100 mg, of active ingredient per dose. Other dosage forms include solutions or emulsions that can be injected intravenously, intraarterially, subcutaneously, intradermally, intraperitoneally, or intramuscularly and are prepared from sterile or sterilizable solutions. Pharmaceutical compositions of the present invention can also be in the form of suppositories, vaginal rings, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions, or dusting powders. The compositions of the present invention can be formulated for topical delivery. Topical delivery generally refers to delivery to the skin, but can also refer to delivery to epithelial cells, e.g., to body cavities lined with the lungs or respiratory tract, the gastrointestinal tract, or the buccal cavity. In particular, formulations for local delivery are described in "Topical drug delivery formulations," Taylor & Francis, edited by David Osborne and Antonio Aman, the entire contents of which are incorporated herein by reference. Compositions or formulations for delivery to the respiratory tract are described in O'Riordan et al. (Respir Care, 2002, November 47), EP2050437, WO2005023290, US2010098660, and US20070053845. Compositions and formulations for delivering active ingredients to the ileum, particularly the proximal ileum, include microparticles and microencapsulates in which the active agent is encapsulated within a protective matrix formed of a polymer or milk protein that is acid-resistant but dissolves easily in the more alkaline ileal environment. Examples of such delivery systems are described in EP1072600.2 and EP13171757.1. An alternative to transdermal administration is through the use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin.The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.

[0058] The injectable form may contain 10 to 1000 mg, preferably 10 to 250 mg, of the active ingredient per dose. Compositions may be formulated in unit dosage form, ie, in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.

[0059] Those skilled in the art can easily determine the appropriate dose of one of the present compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage most appropriate for an individual patient, which will depend on a variety of factors, including the activity of the particular compound used, the metabolic stability and duration of action of that compound, age, body weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, severity of the particular condition, and the individual being treated. The dosages disclosed herein are exemplary of the average case. Of course, there may be individual instances in which higher or lower dosage ranges are merited, and such are within the scope of the present invention. Where appropriate, the agent may be administered at a dose of 0.01 to 30 mg / kg body weight, e.g., 0.1 to 10 mg / kg, more preferably 0.1 to 1 mg / kg body weight. In an exemplary embodiment, one or more doses of 10 to 300 mg / day, or more preferably 10 to 150 mg / day, are administered to a patient for treatment of an inflammatory disorder.

[0060] In a particularly preferred embodiment, the methods and uses of the present invention include the administration of the peptides or compositions of the present invention in combination with one or more other active agents, such as existing antimicrobial agents or pharmacological enhancers available on the market. In such cases, the compounds of the present invention can be administered sequentially, simultaneously or sequentially with one or more other active agents.

[0061] In one embodiment of the present invention, the peptides of the present invention may be administered in the form of a conjugate comprising a peptide, a linker, and an antibody molecule, with the aim of increasing the half-life of the conjugate in vivo.

[0062] Modified Peptides "Modified Peptide": In one embodiment, the peptides of the present invention (including peptide variants) can be modified peptides. The term "modified peptide" is used interchangeably with the term peptide derivative. In one embodiment, the term "modified peptide" refers to a peptide that has been modified to exhibit one or more of the following properties compared to the unmodified peptide: increased plasma half-life; increased lipophilicity of the peptide; increased renal clearance of the modified peptide; or improved resistance of the modified peptide to proteolysis, typically while retaining rpS6 phosphorylation activity. Various methods for modifying the peptides of the present invention to exhibit these properties are disclosed herein, including conjugation of the peptide to a binding partner (e.g., an albumin-binding small molecule, a large macromolecule, a long-lived plasma protein, or an antibody or antibody fragment), cyclization, addition of an N- or C-terminus or side chain, protecting groups, substitution of L-amino acids with D-isomers, amino acid modification, increased plasma protein binding, and increased albumin binding. Modified peptides include, but are not limited to, peptides substituted with one or more groups defined herein, conjugated to a binding partner, or cyclized. Generally, peptides are modified to increase their half-life in vivo in animals. Various methods of modification are described below.

[0063] In one embodiment, the modification may be any modification that provides the peptides and / or compositions of the present invention with increased cell penetration ability. In one embodiment, the modification may be any modification that increases the half-life of the compositions or peptides of the present invention. In one embodiment, the modification may be any modification that increases the activity of the compositions or peptides of the present invention. In one embodiment, the modification may be any modification that increases the selectivity of the compositions or peptides of the present invention. In one embodiment, the group is a protecting group. The protecting group can be an N-terminal protecting group, a C-terminal protecting group, or a side chain protecting group. The peptide can have one or more of these protecting groups.

[0064] Those skilled in the art are aware of suitable techniques for reacting amino acids with these protecting groups. These can be added by preparative methods known in the art, such as those outlined in paragraphs

[0104] to

[0107] of US2014120141. The groups can remain on the peptide or can be removed. Protecting groups can be added during synthesis.

[0065] In an embodiment of the present invention, the peptide may be substituted with one or more groups selected from those having 1 to 29 carbon atoms, straight or branched, long or short, saturated or unsaturated, hydroxyl, amino, aminoacyl, sulfate or sulfide groups, or unsubstituted. N-acyl derivatives include acyl groups derived from acetic acid, capric acid, lauric acid, myristic acid, octanoic acid, palmitic acid, stearic acid, behenic acid, linoleic acid, linolenic acid, lipoic acid, oleic acid, isosteric acid, elide acid, 2-ethylhexanoic acid, coconut oil fatty acid, tallow fatty acid, hardened tallow fatty acid, palm kernel fatty acid, lanolin fatty acid, or similar acids. These may be substituted or unsubstituted. If substituted, they are preferably substituted with hydroxyl, SO3H, SH, or SS, among others, but are not limited thereto.

[0066] In one embodiment of the present invention, the peptide is R1-X-R2. The R1 and / or R2 groups are attached to the amino terminus (N-terminus) and carboxyl terminus (C-terminus) of the peptide sequence, respectively. In one embodiment, the peptide is R1-X. Alternatively, the peptide is X-R2. Preferably, R1 is H, C1-4 alkyl, acetyl, benzoyl or trifluoroacetyl. X is a peptide of the invention. R2 is OH or NH2.

[0067] In one embodiment, R1 is selected from the group formed by H, an acyclic substituted or unsubstituted aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and R5-CO-, wherein R5 is selected from the group formed by H, an acyclic substituted or unsubstituted aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclyl, and substituted or unsubstituted heteroarylalkyl; R2 is selected from the group formed by -NR3R4, -OR3, and -SR3, where R3 and R4 are independently selected from the group formed by H, acyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl; and R1 and R2 are not an α-amino acid.

[0068] According to another preferred embodiment, R2 is -NR3R4, -OR3 or -SR3, and R3 and R4 are independently selected from the group formed by H, substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C2-C24 alkenyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), substituted or unsubstituted C2-C24 alkynyl, substituted or unsubstituted C3-C24 cycloalkyl, substituted or unsubstituted C5-C24 cycloalkenyl, substituted or unsubstituted C8-C24 cycloalkynyl, substituted or unsubstituted C6-C30 aryl, substituted or unsubstituted C7-C24 aralkyl, substituted or unsubstituted 3-10 membered heterocyclyl ring, and substituted or unsubstituted heteroarylalkyl having 2 to 24 carbon atoms and 1 to 3 atoms other than carbon, wherein the alkyl chain is 1 to 6 carbon atoms. Optionally, R3 and R4 may be joined by a saturated or unsaturated carbon-carbon bond and, together with the nitrogen atom, form a ring. More preferably, R2 is -NR3R4 or -OR3, and R3 and R4 are independently selected from the group formed by H, substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C2-C24 alkenyl, substituted or unsubstituted C2-C24 alkynyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C6-C15 aryl, and 3-10 membered substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl having a 3-10 membered ring and an alkyl chain of 1-6 carbon atoms. More preferably, R3 and R4 are selected from the group formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. Even more preferably, R3 is H, and R4 is selected from the group formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. According to an even more preferred embodiment, R2 is selected from -OH and -NH2.

[0069] According to another embodiment of the present invention, R1 is selected from the group formed by H, acetyl, lauroyl, myristoyl or palmitoyl, R2 is -NR3R4 or -OR3, R3 and R4 are independently selected from H, methyl, ethyl, hexyl, dodecyl and hexadecyl, preferably R2 is -OH or -NH2. More preferably, R1 is acetyl or palmitoyl, and R2 is -NH2. In a preferred embodiment, the acyl group is attached to the N-terminus of at least one amino acid of the peptide.

[0070] In one embodiment of the present invention, the peptide is modified to include a side chain protecting group. The side chain protecting group can be one or more of a benzyl or benzyl-based group, a t-butyl-based group, a benzyloxy-carbonyl (Z) group, and an allyloxycarbonyl (alloc) protecting group. The side chain protecting group can be derived from an achiral amino acid, such as achiral glycine. The use of an achiral amino acid helps stabilize the resulting peptide and also facilitates the convenient synthetic route of the present invention. Preferably, the peptide further includes a modified C-terminus, preferably an amidated C-terminus. The achiral residue can be α-aminoisobutyric acid (methylalanine). It will be understood that the specific side chain protecting group used will depend on the peptide sequence and the type of N-terminal protecting group used.

[0071] In one embodiment of the present invention, the peptide is conjugated, linked, or fused to one or more polyethylene glycol polymers or other compounds, such as molecular weight increasing compounds. The molecular weight increasing compound is any compound that increases the molecular weight of the resulting conjugate, typically by 10% to 90%, or 20% to 50%, and may have a molecular weight between 200 and 20,000, preferably between 500 and 10,000. The molecular weight increasing compound may be PEG, any water-soluble (amphiphilic or hydrophilic) polymer moiety, PEG homo- or copolymers, monomethyl-substituted polymers of PEG (mPEG), and polyoxyethyleneglycerol (POG), polyamino acids such as polylysine, polyglutamic acid, and polyaspartic acid, particularly those in their L-conformation, pharmacologically inactive proteins such as albumin and gelatin, fatty acids, oligosaccharides, lipid amino acids, and dextran. The polymer moiety may be linear or branched and may have a molecular weight of 500 to 40,000 Da, 5,000 to 10,000 Da, or 10,000 to 5,000 Da. The compound may be any suitable cell-permeable compound, such as tat peptide, penetratin, pep-1, etc. The compound may be an antibody molecule. The compound may be a lipophilic moiety or a polymer moiety.

[0072] Lipophilic substituents and polymeric substituents are known in the art. Lipophilic substituents include N, O, or S atoms that form part of acyl groups, sulfonyl groups, esters, sulfonyl esters, thioesters, amides, or sulfonamides. The lipophilic moiety can comprise a hydrocarbon chain having 4 to 30 carbon atoms, preferably 8 to 12 carbon atoms. It can be linear or branched, saturated or unsaturated. The hydrocarbon chain can be further substituted. It can be a cycloalkane or a heterocycloalkane.

[0073] The peptide may be modified at the N-terminus, C-terminus, or both. The polymer or compound is preferably linked to an amino, carboxyl, or thio group, and may be linked via the N-terminus or C-terminus of the side chain of any amino acid residue. The polymer or compound may be conjugated to the side chain of any suitable residue.

[0074] The polymer or compound can be conjugated via a spacer, which can be a natural or unnatural amino acid, succinic acid, lysyl, glutamyl, asparagyl, glycyl, beta-alanyl, gamma-aminobutanoyl.

[0075] The polymer or compound can be conjugated via an ester, sulfonyl ester, thioester, amide, carbamate, urea, sulfonamide. Those skilled in the art will be aware of suitable means for preparing the described conjugates.

[0076] Peptides can be chemically modified, for example, by covalent attachment to a polymer, to extend their circulatory half-life.Exemplary polymers and methods for attaching such polymers to peptides are shown, for example, in U.S. Patent Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.Additional exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) moieties.

[0077] The peptides of the present invention can be modified in one or more ways to manipulate storage stability, pharmacokinetics, and / or any aspect of the peptide's biological activity, such as potency, selectivity, and drug interactions. Possible chemical modifications include, but are not limited to, conjugation to the peptide of one or more of polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polypropylene glycol, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, colominic acid or other carbohydrate-based polymers, polymers of amino acids, and biotin derivatives. PEG conjugation of proteins at Cys residues is disclosed, for example, in Goodson, RJ & Katre, NV (1990) Bio / Technology 8, 343 and Kogan, TP (1992) Synthetic Comm. 22, 2417.

[0078] Modified peptides can also include sequences in which one or more residues have been modified (i.e., by phosphorylation, sulfation, acylation, PEGylation, etc.), as well as variants containing one or more modified residues relative to the parent sequence. Amino acid sequences can also be modified with labels capable of providing a directly or indirectly detectable signal, including, but not limited to, radioisotope, fluorescent, and enzyme labels. Fluorescent labels include, for example, Cy3, Cy5, Alexa, BODIPY, fluorescein (e.g., FluorX, DTAF, and FITC), rhodamine (e.g., TRITC), auramine, Texas Red, AMCA Blue, and Lucifer Yellow. Preferred isotopic labels include H, C, P, S, Cl, Cr, Co, Co, Fe, Y, I, I, and Re. Preferred enzyme labels include peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase and peroxidase, and alkaline phosphatase (see, e.g., U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043). Enzymes can be conjugated by reaction with cross-linking molecules such as carbodiimides, diisocyanates, glutaraldehyde, and the like. Enzyme labels can be detected visually or measured by calorimetry, spectrophotometry, fluorescence spectrophotometry, amperometry, or gasometry. Other labeling systems, such as avidin / biotin, tyramide signal amplification (TSA®), etc., are known in the art and are commercially available (see, e.g., ABC kit, Vector Laboratories, Inc., Burlingame, Calif.; NEN®, Life Science Products, Inc., Boston, Mass.).

[0079] In one embodiment, the peptide, variant, and / or composition is modified to enhance drug performance. In one embodiment, the peptide, variant, and / or composition is modified to increase stability, permeability, maintain efficacy, avoid toxicity, and / or increase half-life. Modifications may be as described above. For example, modifications may be N- and C-terminal protection, which may involve modified amino acids, cyclization, amino acid substitution, and / or conjugation to macromolecules or large polymers or long-lived plasma proteins. Strategies for extending half-life may be those disclosed by Strohl et al. (BioDrugs, 2015), Schlapschy et al. (Protein Eng Des Sel. 2013), Podust, VN et al. (Protein Eng Des Sel. 2013), Zhang, L et al. (Curr Med Chem. 2012), Gaberc-Porekar, V et al. (Curr Opin Drug Discov Devel. 2008). Examples include PEGylation, lipidation (covalent attachment of fatty acids to peptide side chains), fusion to the Fc domain and human serum albumin, fusion with hydrophilic amino acid polymers (such as XTEN or PAS), and / or fusion with half-life extending proteins.

[0080] Modifications of peptides to increase their in vivo half-life are described, for example, in the following documents:

[0081] Strategies to improve plasma half life time of peptide and protein drugs. Werle M, Bernkop-Schnurch A. Amino Acids. 2006 Jun; 30(4): 351-67. Due to the clear advantages of long-acting peptide and protein drugs, strategies to extend the plasma half-life of such compounds are highly sought after. Short plasma half-lives are generally due to rapid renal clearance and enzymatic degradation during systemic circulation. Peptide / protein modifications can potentially extend plasma half-life. By shortening the overall amino acid content of somatostatin and replacing L-analog amino acids with D-amino acids, the plasma half-life of the derivative octreotide was 1.5 hours, compared with only minutes for somatostatin. A PEG (2,40K) conjugate of INF-alpha-2b demonstrated a 330-fold increased plasma half-life compared to the native protein. The purpose of this review was to provide an overview of possible strategies for extending plasma half-life, such as N- and C-terminal modifications and PEGylation, as well as methods for evaluating the efficacy of drug modifications. Furthermore, basic data on the most important proteolytic enzymes in human blood, liver, and kidney, as well as their cleavage specificities and inhibitors, are provided to predict the enzymatic cleavage of peptide and protein drugs during systemic circulation.

[0082] Strategic Approaches to Optimizing Peptide ADME Properties. Li Di AAPS J. 2015 Jan; 17(1):134-143. Strategies for stabilizing peptides against proteolysis Many approaches are available to enhance peptide stability through structural modifications. Some approaches not only improve stability but also enhance other ADME properties. For example, cyclization can increase stability and permeability, and conjugation to polymers can improve stability and reduce renal clearance. It is important to maintain efficacy and avoid toxicity while improving peptide stability and ADME properties. N- and C-terminal protection Many proteolytic enzymes in blood / plasma, liver, or kidney are exopeptidases, aminopeptidases, and carboxypeptidases, which degrade peptide sequences from the N- and C-termini. N- and / or C-terminal modifications often improve peptide stability. In many instances, N-acetylation and C-amidation have been reported to increase resistance to proteolysis. Substitution of L-amino acids with D-amino acids Substitution of natural L-amino acids with unnatural D-amino acids reduces substrate recognition and binding affinity of proteolytic enzymes, improving their stability. One example is vasopressin, which contains L-arginine and has a half-life of 10–35 minutes in humans. The half-life of its D-Arg analog, desmopressin, is 3.7 hours in healthy human volunteers. In a study of bicyclic peptide inhibitors of the cancer-associated protease urokinase-type plasminogen activator (uPA), replacement of a specific glycine with D-serine not only increased potency by 1.8-fold but also increased stability in mouse plasma by 4-fold. Amino acid modification Modifications of natural amino acids can improve peptide stability by introducing steric hindrance or interfering with enzyme recognition. For example, the half-life of gonadotropin-releasing hormone is very short (minutes), whereas the half-life of buserelin in humans, in which one Gly is replaced by t-butyl-D-Ser and another by an ethylamide, is much longer. ·Cyclization Cyclization imposes conformational constraints, reducing peptide flexibility and increasing stability and permeability. Depending on the functional groups, peptides can be cyclized head-to-tail, head / tail-to-side chain, or side chain-to-side chain. Cyclization is typically performed via lactamization, lactonization, and sulfide-based bridging. Disulfide bridges can create folding and conformational constraints and improve potency, selectivity, and stability. Many disulfide-rich peptides, such as linaclotide, lepirudin, and ziconotide, are on the market or in preclinical or clinical development. Many methods for peptide cyclization, including on-resin cyclization, side-chain cyclization (lactam bridging), cyclization by orthogonal coupling, enzyme-catalyzed cyclization, peptide size-specific cyclization, and thiazole / oxazole ring-containing cyclic peptides, have been described by Davies et al. (J. Peptide Sci, 9: 471-501 (2003)). Conjugation to polymers Conjugation to macromolecules (e.g., polyethylene glycol (PEG), albumin) is an effective strategy to improve peptide stability and reduce renal clearance.

[0083] Renal clearance Many peptides demonstrate promising pharmacological activity in vitro but fail to demonstrate in vivo efficacy due to their very short in vivo half-lives (minutes). The rapid clearance and short half-life of peptides hinder successful drug development. The primary causes of rapid peptide clearance from the systemic circulation are enzymatic proteolysis and / or renal clearance. Glomerular pore diameters are approximately 8 nm, making hydrophilic peptides with MW <2-25 kDa susceptible to rapid filtration through the renal glomerulus. Because peptides are not readily reabsorbed from the renal tubules, they often have high renal clearance and short half-lives. Other minor pathways of peptide clearance are endocytosis and proteasomal and hepatic degradation. Comparison of systemic and renal clearance in animal models provides useful information regarding whether renal clearance is likely to be the primary excretion pathway. In patients with impaired renal function, it may be necessary to adjust the dose of peptide drugs to avoid drug accumulation and exposure to high drug concentrations, as inappropriate dosing in patients with impaired renal function can lead to toxicity or ineffective treatment. Several strategies have been developed to reduce peptide renal clearance and extend half-life; these are outlined below. Improved plasma protein binding Binding of peptides to membrane or serum proteins reduces renal clearance. An example is the cyclic peptide drug octreotide, a treatment for endocrine tumors. It binds to lipoproteins and has a half-life of approximately 100 minutes in humans (unbound fraction 0.65). Covalent binding to albumin-binding small molecules Covalent attachment of albumin-binding small molecules to peptides can reduce glomerular filtration, improve proteolytic stability, and extend half-life by indirectly interacting with albumin via the highly conjugated small molecule. Conjugation to large polymers Conjugating peptides to large synthetic or natural polymers or carbohydrates increases molecular weight and hydrodynamic volume, potentially reducing renal clearance. Common polymers used for peptide conjugation are PEG, polysialic acid (PSA), and hydroxyethyl starch (HES). Fusion to long-lived plasma proteins Plasma proteins such as albumin and immunoglobulin (IgG) fragments have long half-lives of 19–21 days in humans. Due to their high molecular weight (67–150 kDa), these proteins have low renal clearance, and binding to the neonatal Fc receptor (FcRn) reduces their excretion by pinocytosis through the vascular epithelium. Covalent conjugation of peptides to albumin or IgG fragments can decrease renal clearance and extend their half-lives.

[0084] Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. William R. Strohl BioDrugs. 2015; 29(4):215-239.

[0085] Schlapschy, M, Binder, U, Borger, C et al. PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins. Protein Eng Des Sel. 2013; 26(8):489-501.

[0086] Podust, VN, Sim, BC, Kothari, D et al. Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer. Protein Eng Des Sel. 2013; 26(11):743-53.

[0087] Zhang, L, Bulaj, G. Converting Peptides into Drug Leads by Lipidation. Curr Med Chem. 2012; 19(11):1602-18.

[0088] Gaberc-Porekar, V, Zore, I, Podobnik, B et al. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug Discov Devel. 2008; 11(2):242-50.

[0089] Dr. Ronald V. Swanson - Long-Lived Peptide Evolution and Novel Hybrid Approaches for Peptide Half-Life Extension Technology. From Drug Discovery World online, Spring 2014.

[0090] PEGylation PEGylation, which involves attaching long chains of the hydrophilic polymer polyethylene glycol to molecules of interest, was originally conceived as a modification to prevent the immune system from recognizing foreign proteins, thereby enabling their usefulness as therapeutics. Once antibodies against the unmodified drug form, protein drugs are rapidly neutralized and eliminated. Unexpectedly, PEGylation improved the pharmacokinetics of proteins even in the absence of anti-drug antibodies. 1 By simply increasing the size of the drug molecule, PEGylation slowed the filtration of the drug by the kidney. The empirical observation that increasing size or hydrodynamic radius leads to reduced renal clearance and increased half-life subsequently became the primary rationale for PEGylation of protein and peptide drugs. PEGylation can have various effects on molecules, such as making proteins and peptides more water-soluble or protecting them from degradation by proteolytic enzymes. PEGylation can also affect the binding of therapeutic proteins to their cognate cellular receptors, usually reducing affinity. Changes in the size, structure, and linkage mode of the PEG polymer can affect the biological activity of the conjugated drug. First-generation PEGylation methods were fraught with challenges. However, the chemistry of PEGylation is remarkably simple. The process involves the covalent attachment of polyethylene glycol chains to reactive side chains on proteins or peptides. For example, PEG easily binds to the amino groups of lysines on the surface of proteins or peptides. 2 The reaction is pH-dependent. At high pH (above 8.0), the amino groups on lysine side chains covalently attach to PEG via N-hydroxysuccinimide. This method typically yields a family of products with varying numbers of PEG chains attached to different sites on the protein, rather than a single, discrete product. 3 The first approved PEGylated drugs were Pegademase bovine (PEGylated bovine adenosine deamidase) as an enzyme replacement therapy for severe combined immunodeficiency and Pegaspargase (PEGylated asparaginase) for the treatment of acute lymphoblastic leukemia. 1 Although these drugs were complex mixtures of various PEGylated species, they offered improved therapeutic properties over native enzymes, including extended serum half-lives and reduced protein immunogenicity. Due to the inherent polydispersity of PEG, quality and batch-to-batch reproducibility were challenging. Despite this limitation, two PEGylated interferons (pegylated interferon alfa-2b and peggylated interferon alfa-2a), which are heterogeneous populations of multiple monoPEGylated positional isomers, have been FDA-approved for the treatment of hepatitis C. These agents were launched in 2001 and 2002, respectively. The basic PEGylation technology has undergone numerous refinements and variations. Second-generation PEGylation processes have introduced the use of branched structures and alternative chemistries for PEG attachment. In particular, PEGs bearing cysteine-reactive groups, such as maleimide and iodoacetamide, allow for targeting of PEGylation to a single residue within a peptide or protein, reducing heterogeneity in the final product but not eliminating the heterogeneity due to the polydispersity of PEG itself. Although the original rationale for PEGylation was to reduce immunogenicity, there are nonetheless several examples of immunogenic PEGylated proteins. One example is PEGylated urate oxidase, an enzyme that reduces plasma uric acid levels in patients with gout. In clinical trials, a relatively high percentage of gout patients failed to respond to treatment and developed antibodies specific to PEG but not to the uricase protein. 2 PEGylated liposomes are generally considered non-immunogenic, but several studies have found them to be immunogenic. PEGylated liposomes induce a strong anti-PEG immunoglobulin M (IgM) response. Furthermore, multiple injections of PEG-glucuronidase have been shown to induce the production of specific anti-PEG IgM antibodies, facilitating the clearance of PEG-modified proteins from the body. The main potential drawback of using PEG as a modifying agent is its non-biodegradability. The U.S. Food and Drug Administration (FDA) has approved PEG for use as a vehicle in pharmaceuticals, including injectable, topical, rectal, and nasal formulations. PEG exhibits little toxicity and is excreted intact from the body either through the kidneys (for PEG <30 kDa) or feces (for PEG >20 kDa). Repeated administration of some PEGylated proteins to animals has resulted in the observation of renal tubular cell vacuolation. Recently, choroid plexus epithelial cell vacuolation has also been observed in toxicity studies using proteins conjugated with large (≥40 kDa) PEG. Choroid plexus epithelial cells produce cerebrospinal fluid and form the blood-CSF barrier. While the long-term adverse effects of cell vacuolation are unknown, it is certainly an undesirable outcome for some potential therapeutics. One possible alternative is to use biodegradable polymers instead of PEG. Polymers such as hydroxyethyl starch (HES) are considered alternatives. HES is nontoxic, biodegradable, and used as a plasma substitute. The HES process functions similarly to PEGylation in that it reduces renal clearance by increasing the hydrodynamic radius of the peptide, but its biodegradability may reduce its tendency for accumulation. However, HES and other proposed biodegradable polymeric PEG alternatives are polydisperse, similar to PEG, making characterization of the end products and metabolites difficult. One novel solution that alleviates both concerns is to use defined polypeptides as polymer components. This approach is described below.

[0091] Lipidization The second major chemical modification method for extending peptide half-life is lipidation, which involves the covalent attachment of fatty acids to the peptide side chain. 4 Originally conceived and developed as a method for extending the half-life of insulin, lipidation shares the same basic mechanism of half-life extension as PEGylation: increasing the hydrodynamic radius and reducing renal filtration. However, the lipid moiety itself is relatively small, and its effect is indirectly mediated through noncovalent attachment of the lipid moiety to circulating albumin. Albumin is a large (67 kDa) and highly abundant protein in human serum (35–50 g / L), naturally functioning to transport lipid-containing molecules throughout the body. Binding to plasma proteins can also protect peptides from attack by peptidases through steric hindrance, similar to that seen with PEGylation. Lipidation results in decreased peptide water solubility, but this can be tuned by manipulating the linker between the peptide and the fatty acid, for example, by using glutamic acid or mini-PEGs within the linker. Linker manipulation and alteration of the lipid moiety may affect self-aggregation and slow biodistribution independent of albumin, potentially contributing to increased half-life. Following the pioneering work on insulin, lipidation of various peptides has been investigated, particularly those in the diabetes field, such as human glucagon-like peptide-1 (GLP-1) analogs, glucose-dependent insulinotropic polypeptides, and especially GLP-1R / glucagon receptor co-agonists. Two lipidated peptide drugs are currently FDA-approved for human use: the GLP-1 analog liraglutide and insulin detemir, both of which are long-acting antidiabetic agents. A pharmacologically relevant difference between PEGylation and lipidation is that therapeutically active peptides are covalently attached to much larger PEGs, whereas small fatty acyl peptide conjugates are non-covalently attached to larger albumin molecules, with the conjugated and unconjugated forms thought to exist in equilibrium. This can lead to differences in biodistribution, potentially resulting in different pharmacology due to the potential for access to receptors localized in different tissues to elicit different effects. In some cases, a more restricted biodistribution is desirable, while in other cases, greater tissue penetration is important. An interesting variation of the PEG approach that addresses this issue was developed by Santi et al., utilizing releasable PEG conjugates with predictable cleavage rates. PEGylation and lipidation confer protection against proteases and peptidases by shielding through steric hindrance, and directly or indirectly extend circulating half-life through increased hydrodynamic radius. Both methods utilize chemical conjugation and are flexible in that they are independent of the means used to produce the modified peptide, whether biologically or synthetically produced. The advantage of using synthetic peptides is that they can incorporate unnatural amino acids designed to address certain issues, including instability due to known proteolytic cleavage loads. They also allow for more flexibility in selecting conjugation sites, which is important when activity or potency is highly dependent on modified residues, such as free or C-terminal amides.

[0092] Classical gene fusions: Fc and HSA Classical genetic fusion to long-lived serum proteins offers an alternative method for half-life extension that differs from chemical conjugation to PEG or lipids. Two major proteins have traditionally been used as fusion partners: antibody Fc domains and human serum albumin (HAS). Fc fusion involves the fusion of peptides, proteins, or receptor exodomains to the Fc portion of an antibody. Both Fc and albumin fusions achieve half-life extension not only by increasing the size of peptide drugs but also by utilizing the body's natural recycling mechanism, the neonatal Fc receptor, FcRn. The pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein within endosomes. Fusions based on these proteins can have half-lives ranging from 3 to 16 days, which is much longer than typical PEGylated or lipidated peptides. Fusion to antibody Fc can improve the solubility and stability of peptide or protein drugs. An example of a peptide-Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin, the same protein utilized by fatty acylated peptides, is another common fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. The key difference between Fc and albumin is the dimeric nature of Fc, whereas HAS is a monomeric structure. Depending on the choice of fusion partner, the fusion peptide can be presented as either a dimer or a monomer. The dimeric nature of peptide-Fc fusions can result in avidity effects if the target receptors are in sufficiently close proximity or are themselves dimeric. This may or may not be desirable depending on the target.

[0093] Engineered Polypeptide Fusions: XTEN and PAS An interesting variation on the recombinant fusion concept is the development of low-complexity sequences designed as fusion partners for essentially unstructured hydrophilic amino acid polymers, functional analogs of PEG. The inherent biodegradability of the polypeptide platform makes it attractive as a potentially more benign alternative to PEG. Another advantage is the precise molecular structure of the recombinant molecule, as opposed to the polydispersity of PEG. Unlike HSA and Fc peptide fusions, which require maintaining the three-dimensional folding of the fusion partner, recombinant fusions to unstructured partners can often be subjected to harsh conditions, such as higher temperatures or HPLC purification. The most advanced polypeptide in this class, called XTEN (Amunix), is 864 amino acids long and consists of six amino acids (A, E, G, P, S, and T). The polymer's biodegradability makes it significantly larger than the commonly used 40 kDa PEG, resulting in a greater half-life extension. The fusion of XTEN with peptide drugs extends half-lives by 60–130 times compared to the native molecule. Two fully recombinantly engineered XTEN products, VRS-859 (Exenatide-XTEN) and VRS-317 (Human Growth Hormone-XTEN), have entered clinical trials. In a Phase Ia trial, VRS-859 was found to be well tolerated and effective in patients with type 2 diabetes. VRS-317 reported superior pharmacokinetic and pharmacodynamic properties compared to previously studied rhGH products and offers the potential for once-monthly administration. A second polymer based on a similar conceptual consideration is PAS (XL-Protein GmbH), a random coil polymer composed of a more restricted set of only three small, uncharged amino acids: proline, alanine, and serine. Whether the differences in the biophysical properties of PAS and the more negatively charged XTEN may contribute to differences in biodistribution and / or in vivo activity is unknown, but this will become clear as these polypeptides are incorporated into more therapeutics and the behavior of the fusions is characterized. Regardless of whether the partner is Fc, HSA, XTEN, or PAS, all peptide-protein fusions are genetically encoded and consequently suffer from similar constraints. One limitation is that only naturally occurring amino acids can be incorporated, unlike methods using chemical conjugation, which allow the use of synthetic peptides incorporating unnatural amino acids. Methods that overcome this by expanding the genetic code have been developed by companies such as Ambrx and Sutro, but are not yet widely used. A second limitation is that either the N- or C-terminus of the peptide must be fused to the partner. Often, the peptide termini are involved in receptor interactions, and genetic fusion to one or both termini can significantly impair activity. The site of PEG or lipid conjugation can be anywhere on the peptide, allowing it to be optimized to maximize the biological activity of the resulting therapeutic.

[0094] Hybrid method for fusing synthetic peptides with half-life-extending proteins Gene fusion has previously offered the potential for longer half-life extension, but it lacks the advantages offered by methods utilizing chemical conjugation, PEGylation, and lipidation, in terms of conjugation site flexibility and the incorporation of unnatural amino acids or peptide backbone modifications. One of the first efforts to combine the benefits of gene fusion and chemical conjugation for half-life extension was undertaken by researchers at the Scripps Research Institute in La Jolla, whose technology later became the basis for the biotechnology company CovX. 10,11 These researchers developed a platform using catalytic aldolase antibodies to form a reversible covalent enamine bond between the active site lysine of the antibody and a β-diketone incorporated into a peptide or small molecule. The resulting conjugate is called CovXBody®. This approach combines the functional properties of peptide drugs or small molecules with the long serum half-life of antibodies via chemical conjugation rather than gene fusion. Following the initial demonstration of this technology, researchers expanded the use of the CovX-Body® prototype, which is based on an integrin-targeting peptidomimetic pharmacophore. At least three molecules based on this construct are in clinical development: CVX-096, a GLp-1R agonist; CVX-060, an angiopoietin-2 binding peptide; and CVX-045, a thrombospondin mimetic. Recently, XTEN polypeptides have also been used in a chemical conjugation mode,12 more directly analogous to PEG. The first example of an XTENized peptide generated using this method is GLP2-2G-XTEN, which is chemically conjugated to an XTEN protein polymer using maleimide-thiol chemistry. The chemically conjugated GLP2-2G-XTEN molecule demonstrated comparable in vitro activity, in vitro plasma stability, and pharmacokinetics in rats to recombinant fusion GLP2-2G-XTEN. The number and spacing of reactive groups, such as lysine or cysteine ​​side chains, in the fully designed sequence of an XTEN or PAS polypeptide can be precisely controlled through site-specific variation due to the limited amino acid set from which they are composed. This provides a greater degree of flexibility for methods that can utilize Fc or albumin, whose sequences naturally contain many reactive groups, in contrast to Covx technology, which relies on reactive residues in highly specialized active sites. In addition, the lack of tertiary structure in XTEN or PAS should provide more flexibility in the conditions and chemicals used for coupling and purification of the conjugate. In summary, hybrid peptide half-life extension methods are emerging that combine the advantages of chemical conjugation and gene fusion methods while overcoming their respective limitations. These methods enable the creation of molecules based on recombinant polypeptide-based partners that confer longer half-lives but free the therapeutic peptide moiety from the constraints of being composed exclusively of natural L-amino acids or as linear, unidirectional polypeptides fused at either the N- or C-terminus, thus opening the door to a broad range of long-acting peptide-based drugs.

[0095] "Muscle atrophy" is defined as a loss of muscle mass and is characterized by complete or partial muscle wasting and resulting weakness. It typically occurs in individuals who do not exercise adequately, do not eat properly, or both. It is also present in subjects with muscle diseases such as myopathies (i.e., muscular dystrophies) and is a comorbidity of several diseases / conditions, including eating disorders, cancer, AIDS, COPD, ALS, physical injuries that limit movement, and degenerative conditions. It is also commonly seen in older adults as part of the normal aging process.

[0096] "Elderly subject" refers to a subject who is at least 65 years of age.

[0097] "Diseases or conditions characterized by muscle atrophy" refer to diseases or conditions that include muscle atrophy as a symptom. Examples include physical injuries (i.e., injuries that render a subject physically inactive, such as injuries to muscles, nerves, bones, cartilage, ligaments, intervertebral discs, head, or joints), diseases that confine a subject to bed or home (i.e., cancer, infectious diseases, etc.), eating disorders, cachexia, sarcopenia, malnutrition, metabolic diseases (including type I and type II diabetes), and nerve, muscle, or joint degenerative diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson's disease, Huntington's disease, muscular dystrophy, Guillain-Barré syndrome, osteoarthritis, polio, rheumatoid arthritis, spinal atrophy, and polymyositis. Neurodegenerative diseases include ALS, MS, Parkinson's disease, and Huntington's disease. Muscle degenerative diseases include myopathies (including polymyositis), muscular dystrophies, Guillain-Barré syndrome, and spinal muscular atrophy. Joint degenerative diseases include arthritis, rheumatoid arthritis, and osteoarthritis.

[0098] As used herein, the term "expression vector of the invention" refers to any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements) suitable for expressing the peptides of the invention in cells. Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid molecule encoding the peptide comprises, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 12, 355-59 (1997)), a compacted nucleic acid vector (e.g., U.S. Pat. No. 6,077,835 and / or WO 00 / 70087), or a plasmid vector such as pBR322, pUC19 / 18, or pUC118 / 119. Such nucleic acid vectors and their uses are well known in the art (see, eg, US Pat. Nos. 5,589,466 and 5,973,972). In one embodiment, the DNA comprises an expression control sequence.

[0099] In one embodiment, the vector is suitable for expressing the peptides of the present invention in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, 1989, J. Biol. Chem. 264, 5503-5509), and pET vectors (Novagen, Madison, Wis.). In one embodiment, the expression vector may also, or alternatively, be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as yeast alpha factor, alcohol oxidase, and PGH (reviewed in F. Ausubel et al., ed., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York; and Grant et al., 1987, Methods in Enzymol. 153, 516-544). In other embodiments, the expression vector is suitable for expression in baculovirus-infected insect cells (Kost, T; and Condreay, JP, 1999, Current Opinion in Biotechnology 10(5): 428-33).

[0100] Expression control sequences are engineered to control and drive the transcription of a gene of interest and the subsequent expression of the protein in various cell systems. Plasmids combine an expressible gene of interest with expression control sequences (i.e., expression cassettes) containing desired elements, such as, for example, a promoter, enhancer, selectable marker, operator, etc. In the expression vectors of the invention, the nucleic acid molecule encoding the peptide may contain or be associated with any suitable promoter, enhancer, selectable marker, operator, repressor protein, polyA termination sequence, and other expression-enhancing elements.

[0101] As used herein, a "promoter" refers to a DNA sequence sufficient to direct the transcription of a DNA sequence to which it is operably linked, i.e., to which it is linked, in such a manner as to permit transcription of the nucleotide sequence encoding a peptide of the present invention when the appropriate signals are present. Expression of the nucleotide sequence encoding the peptide can be placed under the control of any promoter or enhancer element known in the art. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter / enhancer or CMV major IE (CMV-MIE) promoter, as well as RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC), and HIV LTR promoters). In some embodiments, the vector comprises a promoter selected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC, and HIV LTR.

[0102] The nucleic acid molecules of the invention may also be operably linked to an efficient poly(A) termination sequence, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also include a regulatable, inducible promoter (inducible, repressible, developmentally regulatable) as opposed to a constitutive promoter such as CMV IE (those skilled in the art will recognize that such terms are indeed descriptors of the degree of gene expression under certain conditions).

[0103] Selectable markers are well-known elements in the art. Under selective conditions, only cells that express appropriate selectable markers can survive. Generally, selectable marker genes express proteins, usually enzymes, that confer resistance to various antibiotics in cell culture. Under other selective conditions, cells that express fluorescent protein markers can be visualized and therefore selected. Embodiments include β-lactamase (bla) (β-lactam antibiotic resistance or ampicillin resistance gene or ampR), bls (blastidine resistance acetyltransferase gene), bsd (blastidine-S deaminase resistance gene), bsr (blastidine-S resistance gene), Sh ble (Zeocin® resistance gene), hygromycin phosphotransferase (hpt) (hygromycin resistance gene), tetM (tetracycline resistance gene or tetR), neomycin phosphotransferase II (npt) (neomycin resistance gene or neoR), kanR (kanamycin resistance gene), and pac (puromycin resistance gene).

[0104] In certain embodiments, the vector comprises one or more selectable marker genes selected from the group consisting of bla, bls, BSD, bsr, Shble, hpt, tetR, tetM, npt, kanR, and pac. In other embodiments, the vector comprises one or more selectable marker genes encoding green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyanofluorescent protein (CFP), enhanced cyanofluorescent protein (eCFP), or yellow fluorescent protein (YFP).

[0105] For purposes of the present invention, gene expression in eukaryotic cells may be tightly regulated using a strong promoter controlled by an operator that is in turn regulated by a regulatory protein, which may be a recombinant "regulatory fusion protein" (RFP). RFPs essentially consist of a transcription blocking domain and a ligand binding domain that regulates its activity. An example of such an expression system is described in US20090162901 A1, which is incorporated herein by reference in its entirety.

[0106] As used herein, "operator" refers to a DNA sequence introduced within or near a gene, such that the gene can be regulated by RFP binding to the operator, thereby preventing or allowing transcription of the gene of interest, i.e., the nucleotide encoding the peptide of the present invention. Numerous operators in prokaryotic cells and bacteriophages have been well characterized (Neidhardt, ed. Escherichia, coli, and Salmonella; Cellular and Molecular Biology 2d. Vol. 2 ASM Press, Washington, DC 1996). These include, but are not limited to, the operator region of the LexA gene of E. coli, which binds to the LexA peptide, and the lactose and tryptophan operators, which bind to the repressor proteins encoded by the Lad and trpR genes of E. coli. These also include bacteriophage operators from the lambda PR and phage P22 ant / mnt genes, which bind to the repressor proteins encoded by lambda cI and P22 arc. In some embodiments, when the transcription blocking domain of RFP is a restriction enzyme such as NotI, the operator is the recognition sequence for that enzyme. Those skilled in the art will recognize that the operator must be located adjacent to or 3' to the promoter so that it can control transcription by the promoter. For example, U.S. Patent No. 5,972,650, incorporated herein by reference, specifies that the tetO sequence is within a specific distance from the TATA box. In certain embodiments, the operator is preferably located immediately downstream of the promoter. In other embodiments, the operator is located within 10 base pairs of the promoter.

[0107] In an exemplary cell expression system, cells are engineered to express the tetracycline repressor protein (TetR), and a protein of interest is placed under the transcriptional control of a promoter whose activity is regulated by TetR. Two tandem TetR operators (tetO) are placed immediately downstream of the CMV-MIE promoter / enhancer within the vector. Transcription of the gene encoding the protein of interest, directed by the CMV-MIE promoter within such a vector, can be blocked by TetR in the absence of tetracycline or some other suitable inducer (e.g., doxycycline). In the presence of the inducer, the TetR protein cannot bind to tetO, resulting in transcription and subsequent translation (expression) of the protein of interest. (See, e.g., U.S. Patent No. 7,435,553, the entire contents of which are incorporated herein by reference.)

[0108] The vectors of the present invention may also use the Cre-lox recombination tool to facilitate integration of a gene of interest into the host genome. The Cre-lox strategy requires at least two components: 1) Cre recombinase, an enzyme that catalyzes recombination between two loxP sites; and 2) a loxP site (e.g., a specific 34-base pair sequence consisting of an 8-bp core sequence where recombination occurs and two flanking 13-bp inverted repeats) or a mutant loxP site. (See, e.g., Araki et al., 1995, PNAS 92: 160-4; Nagy, A. et al., 2000, Genesis 26: 99-109; Araki et al., 2002, Nuc Acids Res 30(19): e103; and US20100291626A1, all of which are incorporated herein by reference.) In another recombination strategy, the yeast-derived FLP recombinase can be utilized with the consensus sequence FRT (see also, for example, Dymecki, SM, 1996, PNAS 93 (12): 6191-6196).

[0109] As used herein, the term "host cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic organisms (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pertis, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Tricoplusia ni, etc.), non-human animal cells, mammalian cells, human cells, or cell fusions (e.g., hybridomas or quadromas, etc.). In certain embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In other embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV 1, kidney (e.g., HEK 293, 293 EBNA, MSR 293, MDCK, HaK, BHK 21), HeLa, HepG2, WI 38, MRC 5, Colo 25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the foregoing. In some embodiments, the cells comprise one or more viral genes, e.g., retinal cells (e.g., PER.C6® cells) expressing viral genes. In some embodiments, the cells are CHO cells. In other embodiments, the cells are CHO K1 cells.

[0110] As used herein, the term "transformed cell of the invention" refers to a host cell that contains a nucleic acid stably integrated into the cellular genome that comprises a nucleotide sequence that encodes expression of a peptide of the invention. In another embodiment, the invention provides a cell that contains a non-integrated (i.e., episomal) nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, that comprises a sequence that encodes expression of a peptide of the invention. In other embodiments, the invention provides a cell line produced by stably transfecting a host cell with a plasmid that comprises an expression vector of the invention.

[0111] As used herein, the term "engineered" as applied to a cell means genetically manipulated using recombinant DNA techniques, which generally involves the synthesis of a suitable expression vector (see above), followed by transfection of the expression vector into a host cell (generally stable transfection).

[0112] As used herein, the term "heterologous expression" refers to the expression of a nucleic acid in a host cell that does not naturally harbor the nucleic acid. The insertion of a nucleic acid into a heterologous host is accomplished by recombinant DNA techniques. [Example]

[0113] Example The present invention will now be described with reference to specific examples, which are merely exemplary and for purposes of illustration only. They are not intended in any way to limit the scope of the claimed exclusive rights or the invention described. These examples constitute the best mode presently contemplated for carrying out the invention.

[0114] material and method Cell culture assay preparation: Preparation of C2C12 Preparation of growth medium: To 500 mL of 4.5 g / L glucose DMEM, add 5 mL of L-glutamine solution (final concentration: 1%), 5 mL of penicillin-streptomycin (final concentration: 1%), and 50 mL of sterile-filtered fetal bovine serum (final concentration: 10%) that has been preheated at 55 °C for 30 min. Preparation of differentiation medium: To 500 mL of 4.5 g / L glucose DMDM, add 5 mL of L-glutamine solution (final concentration: 1%), 5 mL of penicillin-streptomycin (final concentration: 1%), and 10 mL of heat-inactivated horse serum (final concentration: 2%). Preparation of starvation medium: To 500 mL of 4.5 g / L glucose DMEM, add 5 mL of L-glutamine solution (final concentration: 1%) and 5 mL of penicillin-streptomycin (final concentration: 1%).

[0115] S6 phosphorylation assay workflow Day 1: In a 96-well plate, 2400 cells / well (8,000 cells / cm) were plated in 100 μl / well of growth medium. 2 ) and allow them to attach and grow for 48 hours at 37°C, 5% CO2. (See Thawing and Subculturing C2C12 SOP 58 for details.) Day 3: Remove growth medium and add 100 μl / well of differentiation medium. If possible, add fresh differentiation medium daily and differentiate for 7 days at 37°C, 5% CO2. (See C2C12 SOP 60 Differentiation for details.) Day 8: Remove differentiation medium and add 100 μl / well of starvation medium to starve the cells for 3 hours at 37°C, 5% CO2. Then, remove starvation medium and add 100 μl / well of HBSS and incubate at 37°C, 5% CO2 for 1 hour to remove amino acids from the cells. Place the peptide and hydrolysate in a 2 ml test tube and dilute the peptide / hydrolysate with HBSS to the desired concentration (typically 0.5 μg / ml and 5 μg / ml). A minimum volume of 300 μl is required to treat three wells (100 μl / well). A 0.1 μM insulin treatment serves as a positive control. All treatments should be completed in triplicate. A treatment time of 30 minutes is recommended.

[0116] Cell culture assay preparation: THP-1 cell culture Human monocytic leukemia (THP-1) cells (ECACC collection; Sigma-Aldrich, St. Louis, MO, USA) were maintained in Roswell Park Memorial Institute medium (RPMI 1640, Lonza, Basel, Switzerland) supplemented with 1% L-glutamine, 10% heat-inactivated FBS, 1% penicillin-streptomycin, and 10% sterile-filtered fetal bovine serum preheated to 55°C for 30 min.

[0117] TNF-A Secretion Assay Workflow THP-1-derived TNF released into the supernatant was assessed using a TNF-α ELISA kit (BioLegend, San Diego, CA, USA) according to the manufacturer's instructions. To differentiate into macrophages, THP-1 cells were seeded into 6-well plates (2 × 10 6 Well -1), and then treated with 100 nM phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) for 72 hours at 37°C and 5% CO2. After incubation, nonadherent cells were aspirated, and adherent cells were treated with NPN_1 (0.5–5 μg / mL) in duplicate or triplicate. After 24 hours of incubation, lipopolysaccharide (LPS) from Escherichia coli O127:B8 (Sigma-Aldrich, St. Louis, MO, USA) was added to 100 ng / mL for 24 hours at 37°C and 5% CO2. The supernatant was collected, and THP-1-derived TNF released into the supernatant was assessed using a TNF-α ELISA kit (BioLegend, San Diego, California, USA) according to the manufacturer's instructions.

[0118] RNA isolation from C2C12 cells and real-time QPCR C2C12 cells were seeded into 6-well plates and allowed to grow and differentiate at 37°C and 5% CO2. Subsequently, cells were starved for 24 hours in starvation medium at 37°C and 5% CO2. To induce atrophy, cells were treated with 100 μM dexamethasone solubilized in DMEM-LM (30030, BIOSCIENCES) supplemented with 1% penicillin-streptomycin for 24 hours at 37°C and 5% CO2, according to Menconi et al. (2008)

[33] . Thirty minutes before the end of atrophy induction, cells were treated with NPN, added on top of the dexamethasone treatment, and incubated for 30 minutes at 37°C and 5% CO2. Dilutions were calculated to achieve the desired concentrations in a final volume of 2 mL per well. An equal volume of the treatment solution added to each well was first removed from the dexamethasone treatment solution without disturbing the cells. C2C12 cells were lysed with TRIzol (Invitrogen, Carlsbad, USA), and total RNA was extracted using the Purelink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions. Total RNA (1 μg) was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA, USA). Quantitative PCR was performed using a TagMan probe-based method. For experiments using C2C12, mRNA expression was detected using TagMan fluorescent gene expression probes (ABI Biosystems, CA, USA) for Trim63 (Mm 01185221_m1) and Fbxo32 (Mm 00499518_m1). A master mix containing primers / probes and TagMan® Gene Expression Master Mix (ABI Biosystems, CA, USA) was added to 1 μL of cDNA template. A final volume of 9 μL was pipetted in duplicate onto a Roche optical 96-well reaction plate, and real-time PCR was performed on a Roche LightCycler 480 real-time PCR instrument. The threshold cycle (Ct) for each well was calculated using the instrument software.Data analysis was performed using the ΔΔCt method, with raw data normalized by the B2M housekeeping gene (Mm 00437762_m1) included in the plate. Because dexamethasone was used to induce atrophy in C2C12 cells, all gene expression was compared to dexamethasone. Subsequently, NPN_1 was added to examine whether it could attenuate the atrophic effect caused by dexamethasone. Results were expressed as fold changes relative to the control.

[0119] Disuse atrophy test protocol in mice This study was conducted in collaboration with Melior Discovery (USA). Twelve-week-old male C57b1 / 6J mice (N = 10 / group) were randomly assigned to treatment groups based on body weight (10 days after ring implantation). Ethical approval was granted by the International Association for Religious Freedom (IARF #: MLR-I15) and therefore was conducted in accordance with the ethical standards set forth by the Institutional Animal Care and Use Committee (IACUC). This study consisted of five treatment groups: (1) healthy control (weight-bearing control), (2) hindlimb unloading (HU) control vehicle (atrophy), (3) Bowman-Birk inhibitor (BBI; 113.3 mg / kg positive control), (4) casein (650 mg / kg; positive control), and (5) NPN_1 (650 mg / kg). Briefly, before lowering the hindlimbs, a caudal ring was formed using 2-0 sterile surgical steel wire, which was passed through the 5th, 6th, or 7th intervertebral space, from which the mouse was suspended. The vertebral position of the caudal ring was selected to adequately balance the animal's weight without interfering with defecation. The animal was suspended by a swivel harness attached to the top of the cage. The animal's body was maintained at a 30° elevation angle, so that only the forelimbs maintained contact with the cage floor. During this procedure, the animal was allowed to move around within the cage and had free access to food and water. Animal height was checked daily and adjusted as necessary

[34] . Based on IACUC guidelines, mice were given 7 days for acclimation and conditioning, followed by 10–13 days of recovery after tail ring implantation. The primary endpoint of this study was to assess the wet weight of the soleus muscle contained within the hindlimb of control and test mice directly after 19 days of hindlimb suspension. Additionally, fixed muscle samples (5 / group) were sent to CaresBio Laboratory LLC for immunofluorescence (IF) staining of type I and type IIa muscle fiber markers and image analysis. Soleus muscle tissue samples (10 / group) were also sent to Cellomatics Biosciences LTD for gene expression analysis.

[0120] Medication and muscle wet mass All mice received either NPN_1, BBI, or casein from days 1 to 18. On day 19, animals were sacrificed by cervical dislocation, blood / plasma samples were collected, and soleus muscles were isolated and weighed using a digital platform balance. Wet muscle weights were normalized to body weight (mg / g). One soleus muscle was snap-frozen, and the other was fixed in 4% fresh PBS-buffered formaldehyde.

[0121] Immunofluorescence analysis of soleus muscle Collected soleus muscles were post-fixed in 4% fresh PBS-buffered formaldehyde. Five samples were randomly selected from each treatment group for immunofluorescence analysis. The muscles were paraffin-embedded and sectioned. Immunofluorescent labeling was used to stain type I and type IIa muscle fibers. Image analysis was performed on representative areas of each sample for both staining labels. Upon sample processing, sections (8 μM thick) were cut, and immunofluorescent staining was performed as previously described

[35] . Briefly, slides were subjected to heat-induced antigen retrieval in citrate buffer (10 mM, pH 6.0), blocked with a nonspecific antigenic blocker, and then incubated overnight with primary antibodies ab11083 (dilution 1:100) and ab91506 (dilution 1:200). Both primary antibody concentrations were determined after optimization on test slides. Corresponding fluorescently conjugated secondary antibodies (Alexa 594 and Alexa 488) were applied for 1 hour at room temperature. To visualize nuclei, 4,6-diamidino-2-phenylindole (DAPI) was added to the secondary antibody.

[0122] Image acquisition and analysis Slides were scanned using a customized computer-controlled microscope (xy stage and z controller, Zeiss Microscope, Carl Zeiss GmbH, Jena, Germany) with X4 and X10 objectives. Images were analyzed using image analysis software based on MATLAB (R2011b, MathWorks). A baseline scan was performed using the HU control group. Image analysis algorithms were applied to images generated from tissue microscope slides stained with secondary antibody controls to generate background scores. The control / baseline was used to generate an algorithm for distinguishing between signals and signal-to-noise ratios, which was applied to all images. Each marker was quantified using single-channel analysis. Automatic background subtraction was performed. Intensity scores for all markers were then calculated, corresponding to the mean signal intensity divided by the local area. Significant differences in the relative staining area and mean relative intensity of staining for different groups in mouse muscle tissue were calculated. Raw data are presented; no normalization was performed.

[0123] RNA isolation and gene array from mouse flounder tissues Gene expression analysis was performed using soleus muscle tissue samples. 300 μL of homogenization solution with 3 μL of proteinase K (Quantigene Plex Assay, Invitrogen) was added to 10 mg of frozen tissue to prepare a concentrated lysate. One 5 mm stainless steel bead was added to the tube and placed in a Bullet Blender® homogenizer. The tissue was then homogenized at speed 10 for 2 minutes. The tube was cooled at room temperature, and this process was repeated until no visible particles remained. The tissue lysate was then incubated at 65°C for 30 minutes, followed by centrifugation at 16,000 × g for 15 minutes, and the supernatant was immediately used in the assay kit (according to the manufacturer's guidelines). Net mean fluorescence intensity (MFI) was obtained from Luminex for all genes.

[0124] Table 1. Fold-regulated gene expression after NPN_1 treatment. RNA was extracted from soleus muscle tissue samples taken from control, vehicle-, and NPN_1-treated animals (N=10 / group). Genes related to myogenesis and mitochondrial biogenesis were upregulated.

[0125] [Table 1]

[0126] composition

[0127] [Table 2]

[0128] [Table 3]

[0129] [Table 4]

[0130] [Table 5]

[0131] [Table 6]

[0132] [Table 7]

[0133] [Table 8]

[0134] [Table 9]

[0135] The topical composition may be applied topically to a subject suffering from muscle wasting or to a healthy individual after strenuous physical activity.

[0136] It will be understood that the percentages are examples only and any suitable percentages can be used depending on the application.

[0137] The emulsion is prepared in the following manner: Phase A: Ultres 10 (carbomer) is dispersed in water and allowed to swell for 20 minutes, then phase B is added and heated to 75°C. Phase C is heated separately to 75°C. The two phases are mixed under stirring, homogenized, phase D is added, neutralized with phase E, cooled to reach 30°C, then phases F and G are added and the pH is adjusted to 6 with NaOH. It will be understood that this is only an example and that any suitable method known in the art may be used.

[0138] equivalent The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice are expected to occur to those skilled in the art in light of these descriptions. These modifications and variations are intended to be included within the scope of the claims appended hereto.

Claims

1. A composition comprising a peptide having a maximum of 50 amino acids and comprising the sequence of SEQ ID NO:

20.

2. 2. The composition of claim 1, wherein the peptide has 6 to 20 amino acids.

3. The composition of claim 2, wherein the peptide comprising the sequence of SEQ ID NO: 20 is selected from SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO:

19.

4. 4. The composition according to any one of claims 1 to 3, comprising a peptide having a maximum of 50 amino acids and comprising the sequence of SEQ ID NO:

8.

5. The composition of claim 4, wherein the peptide having up to 50 amino acids and comprising the sequence of SEQ ID NO: 8 is selected from SEQ ID NO: 8 and SEQ ID NO:

7.

6. The composition of claim 1, comprising peptides of SEQ ID NO: 3 and SEQ ID NO:

8.

7. The composition of claim 1 , wherein the composition is an edible powder.

8. A method for promoting muscle synthesis in a healthy subject, comprising orally administering to said subject a therapeutically effective amount of the composition of any one of claims 1 to 7.

9. A method for inhibiting muscle loss in a healthy subject, comprising orally administering to said subject a therapeutically effective amount of the composition according to any one of claims 1 to 7.

10. A method for ameliorating inflammatory responses in a healthy subject, comprising orally administering to said subject a therapeutically effective amount of a composition according to any one of claims 1 to 7.

11. 8. The composition of any one of claims 1 to 7 for use in a method for preventing or treating muscle wasting in a subject.

12. An isolated peptide having a maximum of 50 amino acids and comprising SEQ ID NO:

20.

13. 13. The isolated peptide of claim 12, having 6 to 20 amino acids.

14. 14. The isolated peptide of claim 13, selected from SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:

19.

15. 15. Use of an isolated peptide according to any one of claims 12 to 14 or a composition according to any one of claims 1 to 7 to promote muscle synthesis in a healthy subject.

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