阅读说明：本技术 用于减少或治疗纤维化的组合物和方法 (Compositions and methods for reducing or treating fibrosis ) 是由 迈克尔·哈米尔 瑞弗·亚非恩 李中伟 哈利·路易特哈特 卢克·S·哈姆 斯维特拉娜·马鲁基 于 2019-06-19 设计创作，主要内容包括：本公开提供了用于减少或治疗例如患有纤维化病状或病症的对象的纤维化的组合物和方法。(The present disclosure provides compositions and methods for reducing or treating fibrosis in a subject, e.g., having a fibrotic condition or disorder.)

1.A method for reducing fibrosis in a subject comprising administering to a subject in need thereof an effective amount of a composition comprising:

a) (ii) a leucine amino acid entity, wherein,

b) an arginine amino acid entity, wherein the arginine amino acid entity,

c) a glutamine amino acid entity; and

d) an N-acetylcysteine (NAC) -entity;

with the following conditions:

the fibrosis is not liver fibrosis and is not,

thereby reducing fibrosis of the subject.

2. A method of treating a fibrotic condition or disorder in a subject in need thereof comprising administering to the subject an effective amount of a composition comprising:

a) a leucine-amino acid entity, wherein the amino acid entity is a leucine-amino acid entity,

b) an arginine-amino acid entity, wherein,

c) a glutamine-amino acid entity; and

d) NAC-entity;

with the following conditions:

the fibrotic condition or disorder is not a liver fibrosis condition or disorder,

thereby treating fibrotic conditions or disorders.

3.A composition for reducing fibrosis in a subject comprising an effective amount of a composition comprising:

a) (ii) a leucine amino acid entity, wherein,

b) an arginine amino acid entity, wherein the arginine amino acid entity,

c) a glutamine amino acid entity; and

d) an N-acetylcysteine (NAC) -entity;

with the following conditions:

the fibrosis is not liver fibrosis.

4.A composition for treating a fibrotic condition or disorder in a subject in need thereof comprising an effective amount of a composition comprising:

a) a leucine-amino acid entity, wherein the amino acid entity is a leucine-amino acid entity,

b) an arginine-amino acid entity, wherein,

c) a glutamine-amino acid entity; and

d) NAC-entity;

with the following conditions:

the fibrotic condition or disorder is not a liver fibrosis condition or disorder.

5. The method of claim 1 or 2, or composition for use of claim 3 or 4, wherein the fibrotic condition or disorder is selected from a pulmonary fibrotic condition or disorder, a cardiac or vasculature fibrotic condition or disorder, a renal fibrotic condition or disorder, a pancreatic fibrotic condition or disorder, a skin fibrotic condition or disorder, a gastrointestinal fibrotic condition or disorder, a bone marrow or hematopoietic fibrotic condition or disorder, a nervous system fibrotic condition or disorder, an ocular fibrotic condition or disorder, or a combination thereof.

6. The method of any one of claims 1,2 or5, or the composition for use of any one of claims 3-5, wherein administration of the composition results in reduction or inhibition of one, two, three, four, five, six or more (e.g., all) of:

(a) formation or deposition of tissue fibrosis;

(b) the size, cellularity, composition, or cellular content of fibrotic lesions;

(c) fibrosis damaged collagen;

(d) fibrosis-damaged collagen or hydroxyproline content;

(e) expression or activity of a fibrogenic protein;

(f) fibrosis associated with inflammatory responses; or

(g) Weight loss associated with fibrosis.

7. The method of any one of claims 1,2, 5 or 6, or the composition for use of any one of claims 3-6, wherein the method further comprises determining one, two, three, four, five, six, seven, eight, nine, ten, 11 or more (e.g., all) of the following levels: (a) col1a 1; (b) FGF-21; (c) hydroxyproline content; (d) IL-1 β; (e) matrix Metalloproteinases (MMPs), e.g., MMP-13, MMP-2, MMP-9, MT1-MMP, MMP-3, or MMP-10; (f) the N-terminal fragment of type III collagen (proC 3); (g) PIIINP (N-terminal propeptide of type III collagen); (h) alpha-smooth muscle actin (alpha SMA); (i) TGF-beta; (j) tissue Inhibitors of Metalloproteinases (TIMP); for example, TIMP1 or TIMP 2; (k) acta 2; or (l) Hsp 47.

8. The method of any one of claims 1,2 or 5-7, or the composition for use of any one of claims 3-7, wherein the total wt% of (a) - (d) is greater than the total wt% of one, two or three of the other amino acid entity components, non-amino acid entity protein components (e.g., whey protein) or non-protein components in the composition (e.g., in dry form).

9. The method of any one of claims 1,2 or 5-8, or the composition for use of any one of claims 3-8, wherein said composition comprises a combination of 18 or fewer amino acid entities.

10. The method of any one of claims 1,2 or 5-9, or the composition for use of any one of claims 3-9, wherein the composition does not comprise peptides of more than 20 amino acid residues in length (e.g. whey proteins), or if peptides of more than 20 amino acid residues in length are present, the peptides are present in less than 10 weight (wt)% of the total weight of the protein components or all components of the composition (e.g. in dry form). .

11. The method of any one of claims 1,2 or5 to 10, or the composition for use of any one of claims 3 to 10, wherein at least one, two, three or four (e.g., all) of methionine (M), tryptophan (W), valine (V) or cysteine (C) is absent or, if present, is present at less than 10 wt% of the composition (e.g., in dry form).

12. The method of any one of claims 1,2 or 5-11, or the composition for use of any one of claims 3-11, wherein at least one, two, three or more (e.g., all) of (a) - (d) are selected from table 1.

13. The method of any one of claims 1,2, or 5-12, or the composition for use of any one of claims 3-12, wherein the composition further comprises one or both of (e) an isoleucine amino acid entity or (f) a valine amino acid entity.

14. The method of any one of claims 1,2 or 5-13, or the composition for use of any one of claims 3-13, wherein the weight ratio of said leucine amino acid entity, said arginine amino acid entity, said glutamine amino acid entity and said NAC-amino acid entity is 1 +/-20%: 1.5 +/-20%: 2 +/-20%: 0.15+/-20 percent.

15. The method of claim 13 or 14, or the composition for use of claim 13 or 14, wherein the weight ratio of said leucine entity, said isoleucine amino acid entity, said valine amino acid entity, said arginine amino acid entity, said glutamine amino acid entity and said NAC-amino acid entity is 1 +/-20%: 0.5 +/-20%: 0.5 +/-20%: 1.5 +/-20%: 2 +/-20%: 0.15+/-20 percent.

16. The method of any one of claims 1,2 or 5-15, or the composition for use of any one of claims 3-15, wherein the composition comprises:

a) a leucine amino acid entity selected from the group consisting of:

i) l-leucine or a salt thereof,

ii) a dipeptide or salt thereof, or a tripeptide or salt thereof, comprising L-leucine, or

iii) beta-hydroxy-beta-methylbutyric acid (HMB) or a salt thereof;

b) an arginine amino acid entity selected from:

i) l-arginine or a salt thereof,

ii) a dipeptide or salt thereof or a tripeptide or salt thereof comprising L-arginine,

iii) creatine or a salt thereof, or

iv) a dipeptide or salt thereof, or a tripeptide or salt thereof, comprising creatine;

c) the glutamine amino acid entity is L-glutamine or a salt thereof, or a dipeptide or a salt thereof comprising L-glutamine, or a tripeptide or a salt thereof; and

d) the NAC entity is NAC or a salt thereof, or a dipeptide comprising NAC or a salt thereof.

17. The method of any one of claims 1,2 or5 to 16, or the composition for use of any one of claims 3 to 16, wherein the composition further comprises one or both of:

e) l-isoleucine or a salt thereof, or a dipeptide or a salt thereof, or a tripeptide or a salt thereof, comprising L-isoleucine; or

f) L-valine or a salt thereof, or a dipeptide or a salt thereof or a tripeptide comprising L-valine or a salt thereof.

18. The method of any one of claims 1,2 or 5-17, or the composition for use of any one of claims 3-17, wherein the composition comprises:

a) the leucine amino acid entity is L-leucine or a salt thereof;

b) the arginine amino acid entity is L-arginine or a salt thereof;

c) the glutamine amino acid entity is L-glutamine or a salt thereof; and

d) the NAC entity is NAC or a salt thereof.

19. The method of any one of claims 1,2 or 5-18, or the composition for use of any one of claims 3-18, wherein the composition comprises:

a) the leucine amino acid entity is L-leucine or a salt thereof;

b) the arginine amino acid entity is L-arginine or a salt thereof;

c) the glutamine amino acid entity is L-glutamine or a salt thereof;

d) the NAC entity is NAC or a salt thereof;

e) the isoleucine amino acid entity is L-isoleucine or a salt thereof; and

f) the valine amino acid entity is L-valine or a salt thereof.

20. The method of any one of claims 1,2 or 5-19, or the composition for use of any one of claims 3-19, wherein the composition is formulated with a pharmaceutically acceptable carrier.

21. The method of any one of claims 1,2 or 5-20, or the composition for use of any one of claims 3-20, wherein the composition is formulated as a dietary composition.

22. The method of claim 21, or the composition for use of claim 21, wherein the dietary composition is selected from a medical food, a functional food or a supplement.

Background

Fibrosis (fibrosis) is a serious health problem characterized by the formation of excess fibrous connective tissue at least partially as a result of repair or reactive processes, such as in response to injury. In fibrosis, abnormal accumulation of extracellular matrix proteins can lead to scarring and thickening of the affected tissue. Fibrosis can occur in various organs including the lung, liver, heart, kidney, pancreas, skin and brain. Multiple conditions (conditions) and disorders (disorders) are associated with fibrosis, such as cardiomyopathy, hypertension, arterial stiffness, chronic hepatitis c infection, crohn's disease, adult respiratory distress syndrome, and sarcoidosis. Currently available treatments for fibrotic conditions have limited efficacy.

In view of the limitations of available treatments, there remains a need for anti-fibrotic agents, e.g., dietary compositions and therapeutics, that reduce fibrosis in subjects.

Disclosure of Invention

Provided herein are compositions comprising amino acid entities for use in improving or reducing fibrosis in a subject, e.g., a subject having a fibrotic condition or disorder. The compositions are useful in methods of reducing and/or treating (e.g., reversing, reducing, ameliorating, or preventing) fibrosis in a subject (e.g., a human) in need thereof. The method may further comprise monitoring the subject for an improvement in one or more symptoms of fibrosis following administration of the composition comprising the amino acid entity.

In one aspect, the invention features a method for reducing fibrosis in a subject, comprising administering to a subject in need thereof an effective amount of a composition (e.g., an active moiety) comprising:

a) (ii) a leucine amino acid entity, wherein,

b) an arginine amino acid entity, wherein the arginine amino acid entity,

c) a glutamine amino acid entity; and

d) an N-acetylcysteine (NAC) entity;

thereby reducing fibrosis of the subject.

In some embodiments, the fibrosis is not liver fibrosis.

In another aspect, the invention features a method of treating a fibrotic condition or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a composition (e.g., an active moiety) comprising:

a) (ii) a leucine amino acid entity, wherein,

b) an arginine amino acid entity, wherein the arginine amino acid entity,

c) a glutamine-amino acid entity; and

d) NAC-entity;

thereby treating the condition or disorder of fibrosis.

In some embodiments, the fibrotic condition or disorder is not a liver fibrosis condition or disorder.

In another aspect, the invention features a composition for reducing fibrosis in a subject, comprising an effective amount of a composition comprising:

a) (ii) a leucine amino acid entity, wherein,

b) an arginine amino acid entity, wherein the arginine amino acid entity,

c) a glutamine amino acid entity; and

d) an N-acetylcysteine (NAC) -entity;

with the following conditions:

the fibrosis is not liver fibrosis.

In another aspect, the invention features a composition for treating a fibrotic condition or disorder in a subject in need thereof, comprising an effective amount of a composition comprising:

a) a leucine-amino acid entity, wherein the amino acid entity is a leucine-amino acid entity,

b) an arginine-amino acid entity, wherein,

c) a glutamine amino acid entity; and

d) NAC-entity;

with the following conditions:

the fibrotic condition or disorder is not a liver fibrosis condition or disorder.

In some embodiments, the fibrotic condition or disorder is selected from a pulmonary fibrotic condition or disorder, a cardiac or vasculature fibrotic condition or disorder, a renal fibrotic condition or disorder, a pancreatic fibrotic condition or disorder, a skin fibrotic condition or disorder, a gastrointestinal fibrotic condition or disorder, a bone marrow or hematopoietic fibrotic condition or disorder, a nervous system fibrotic condition or disorder, an ocular fibrotic condition or disorder, or a combination thereof.

In some embodiments, administration of the composition (e.g., active moiety) results in a reduction or inhibition of one, two, three, four, five, six, or more (e.g., all) of: (a) formation or deposition of tissue fibrosis; (b) the size, cellularity, composition, or cellular content of fibrotic lesions; (c) fibrosis damaged collagen; (d) fibrosis-damaged collagen or hydroxyproline content; (e) expression or activity of a fibrogenic protein; (f) fibrosis associated with inflammatory responses; or (g) weight loss associated with fibrosis.

In some embodiments, the method further comprises determining one, two, three, four, five, six, seven, eight, nine, ten or more (e.g., all) of the following levels: (a) col1a 1; (b) FGF-21; (c) hydroxyproline content; (d) IL-1 β; (e) matrix Metalloproteinases (MMPs), such as MMP-13, MMP-2, MMP-9, MT1-MMP, MMP-3, or MMP-10; (f) the N-terminal fragment of type III collagen (proC 3); (g) PIIINP (N-terminal propeptide of type III collagen); (h) alpha-smooth muscle actin (alpha SMA); (i) TGF-beta; (j) tissue Inhibitors of Metalloproteinases (TIMP); for example, TIMP1 or TIMP 2; or (k) Hsp 47.

In some embodiments, the composition (e.g., active portion) further comprises one or both of (e) an isoleucine-amino acid entity or (f) a valine amino acid entity.

In some embodiments, the total weight% (wt%) of (a) - (d) or (a) - (f) is greater than the total weight% of one, two or three of the other amino acid entity components, non-amino acid entity protein components (e.g., whey protein) or non-protein components of the composition (e.g., in dry form), e.g., (a) - (d) or (a) - (f) is at least 50 wt%, 75 wt% or 90 wt% of the total weight of one or two or all of the amino acid entity components (e.g., in dry form) in the composition.

In some embodiments, the composition comprises a combination of 18 or less, 15 or less, or 10 or less amino acid entities, e.g., the combination comprises at least 42, 75, or 90 weight percent of the total weight of the amino acid entity components or all components in the composition (e.g., in dry form).

In some embodiments, the composition does not comprise a peptide that is greater than 20 amino acid residues in length (e.g., whey protein), or if a peptide that is greater than 20 amino acid residues in length is present, the peptide is present at less than 10%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or less by weight of the total weight of the non-amino acid solid protein component or all components of the composition (e.g., in dry form).

In some embodiments, at least one, two, three, or more (e.g., all), or if present, less than, for example, 10 weight percent, 1 weight percent, 0.5 weight percent, 0.1 weight percent, 0.05 weight percent, 0.01 weight percent, 0.001 weight percent, or less of the total weight of all components in the composition (e.g., in dry form) is absent from the composition. In some embodiments, one, two, three, or more (e.g., all) of methionine, tryptophan, valine, or cysteine, if present, is present in free form. In some embodiments, one, two, three, or more (e.g., all) of methionine, tryptophan, valine, or cysteine, if present, is present in salt form.

In some embodiments, methionine, tryptophan, valine, or cysteine, if present, may be present in the oligopeptide, polypeptide, or protein, provided that the protein is not whey, casein, whey protein, or any other protein useful as a nutritional supplement, medical food, or similar product, whether present as an intact protein or a protein hydrolysate (hydrosate).

In some embodiments, at least one, two, three, four, five, or more (e.g., all) of (a) - (f) are selected from table 1.

In some embodiments, the weight ratio of leucine amino acid entities, arginine amino acid entities, glutamine amino acid entities, and NAC-amino acid entities is 1 +/-20%: 1.5 +/-20%: 2 +/-20%: 0.15+/-20 percent. In some embodiments, the weight ratio of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities, and NAC-amino acid entities is 1 +/-20%: 0.5 +/-20%: 0.5 +/-20%: 1.5 +/-20%: 2 +/-20%: 0.15+/-20 percent.

In some embodiments, the composition (e.g., active portion) comprises:

a) a leucine amino acid entity selected from the group consisting of: i) l-leucine or a salt thereof, ii) a dipeptide or a salt thereof comprising L-leucine, or a tripeptide or a salt thereof, or iii) beta-hydroxy-beta-methylbutyrate (HMB) or a salt thereof;

b) an arginine amino acid entity selected from: i) l-arginine or a salt thereof, ii) a dipeptide or a salt thereof, or a tripeptide or a salt thereof comprising L-arginine, iii) creatine or a salt thereof, or iv) a dipeptide or a salt thereof, or a tripeptide or a salt thereof comprising creatine;

c) the glutamine amino acid entity is L-glutamine or a salt thereof, or a dipeptide or a salt thereof comprising L-glutamine, or a tripeptide or a salt thereof; and

d) the NAC entity is NAC or a salt thereof, or a dipeptide comprising NAC or a salt thereof.

In some embodiments, the composition (e.g., active portion) further comprises one or both of: e) l-isoleucine or a salt thereof or a dipeptide or a salt thereof comprising L-isoleucine, or a tripeptide or a salt thereof; or f) L-valine or a salt thereof or a dipeptide or a salt thereof or a tripeptide comprising L-valine or a salt thereof.

In some embodiments, the composition (e.g., active portion) comprises: a) the leucine amino acid entity is L-leucine or a salt thereof; b) the arginine amino acid entity is L-arginine or a salt thereof; c) the glutamine amino acid entity is L-glutamine or a salt thereof; and d) the NAC entity is NAC or a salt thereof.

In some embodiments, the composition (e.g., active portion) comprises: a) the leucine amino acid entity is L-leucine or a salt thereof; b) the arginine amino acid entity is L-arginine or a salt thereof; c) the glutamine amino acid entity is L-glutamine or a salt thereof; d) the NAC entity is NAC or a salt thereof; e) the isoleucine amino acid entity is L-isoleucine or a salt thereof; and f) the valine amino acid entity is L-valine or a salt thereof.

In some embodiments, the compositions (e.g., active portion) are formulated with a pharmaceutically acceptable carrier.

In some embodiments, the composition (e.g., active portion) is formulated as a dietary composition.

Drawings

Fig. 1A-1B are graphs showing the effect of treatment with amino acid composition (amino acid composition a-1) on NAFLD activity score, balloon enlargement (balloon) and fibrosis in the STAM mouse model (fig. 1A) and the FATZO mouse model (fig. 1B).

FIG. 2 is a schematic representation of a treatment regimen for administering amino acid compositions to STAM and FATZO mice.

Figures 3A-3E are a series of graphs and images showing the effect of treating STAM and FATZO mice with amino acid compositions on NAFLD Activity Score (NAS), steatosis, inflammation, and liver fibrosis (as determined using histology).

FIG. 4 is a genetic map of the pattern of hepatic gene expression after treatment with the amino acid composition in STAM mice, showing inhibition of the fibrogenic TGF-b signaling pathway (fibrotic signaling pathway).

FIG. 5 is a series of graphs showing the protein levels of MCP-1 and MIP-1 as ligands for the C-C chemokine receptors type 2 (CCR2) and type 5 (CCR5) after treatment with amino acid compositions.

Figure 6 is a series of microscopic images showing the histology of the liver of FATZO mice after administration of the indicated amino acid composition (H & E staining or Sirius Red staining for collagen deposition (Sirius Red stain).

Fig. 7 is a series of microscopic images showing liver histology from FATZO mice after administration of the indicated amino acid composition.

Fig. 8 is a series of graphs showing the NAFLD activity score (upper left panel), sirius red staining (upper right panel), level of steatosis (lower left panel), inflammation (lower middle panel), and ballooning observed in fixed liver tissue from FATZO mice after administration of the indicated amino acid compositions.

FIGS. 9A-9B are a series of graphs showing the effect of treatment of human subjects with amino acid compositions on the level of proC3 (FIG. 9A) in addition to PIIINP and TIMP-1 (FIG. 9B).

Detailed Description

Described in part herein are compositions (e.g., active fractions) comprising amino acid entities and methods of reducing fibrosis by administering an effective amount of the compositions. The compositions can be administered to treat or prevent a fibrotic condition or disorder in a subject in need thereof. The amino acid entities and the relative amounts of the amino acid entities in the composition have been carefully selected, for example, to reduce fibrosis in subjects (e.g., subjects with fibrotic conditions or disorders) that require coordination of a number of biological, cellular, and molecular processes. The compositions allow for multi-path beneficial effects on tissue physiology to optimize modulation of signaling pathways involved in the fibrotic response and reduce deposition (and improve absorption) of extracellular matrix in fibrosis. In particular, the compositions have been specifically tailored to reduce fibrogenic gene/protein expression, reduce inflammation associated with fibrosis, and inhibit pathways associated with fibrosis.

In the examples described in detail below, the compositions of the present invention improve fibrosis and reduce fibrogenic gene and protein expression.

Definition of

Terms used in the claims and specification are as defined below, unless otherwise indicated.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "amino acid entity" refers to a L-amino acid in free form or in salt form (or both), with less than 20 amino acid residues in the peptide (e.g., oligopeptides, such as dipeptides or tripeptides), derivatives of amino acids, precursors of amino acids, or metabolites of amino acids (see, e.g., table 1). Amino acid entities include amino acid derivatives, amino acid precursors, amino acid metabolites or salt forms of amino acids that are capable of affecting the biological function of the free L-amino acid. An amino acid entity does not include native polypeptides or proteins of greater than 20 amino acid residues in either intact or modified form (e.g., hydrolyzed form).

Amino acid salts include any ingestible salts. For pharmaceutical compositions, the salt form of the amino acid (e.g., active moiety) present in the composition should be a pharmaceutically acceptable salt. In a particular example, the salt form is the hydrochloride (HCl) salt form of an amino acid.

In some embodiments, the derivative of the amino acid entity comprises an amino acid ester (e.g., an alkyl ester, such as an ethyl or methyl ester of the amino acid entity) or a keto acid.

Table 1. amino acid entities include amino acids, precursors, metabolites and derivatives of the compositions described herein.

"about" and "approximately" generally refer to an acceptable degree of error in the measured quantity given the nature or accuracy of the measurement. Exemplary degrees of error are within 15 percent (%) of a given value or range of values, typically within 10%, and more typically within 5%.

"amino acid" refers to a compound having an amino group (-NH)₂) Carboxylic acid groups (-C (═ O) OH) and side chains bonded through a central carbon atom, including essential and non-essential amino acids as well as natural, non-protein and non-natural amino acids.

The term "active moiety" as used herein refers to a combination of four or more amino acid entities having the ability to have a physiological effect, e.g., anti-fibrotic effect, as described herein in general. For example, the active moiety may rebalance metabolic dysfunction in a subject having a disease or disorder. The active moiety of the invention may comprise other biologically active ingredients. In some examples, the active moiety comprises a defined combination of four or more amino acid entities, as described in detail below. In other embodiments, the active moiety consists of a defined combination of amino acid entities, as described in detail below.

The individual amino acid entities are present in the composition (e.g., active moiety) in various amounts or ratios, which can be expressed as a weight (e.g., in grams), a weight ratio of the amino acid entities to each other, a molar amount, a weight percent of the composition, a molar percent of the composition, a caloric content, a percent caloric contribution to the composition, and the like. Generally, the invention will provide the grams of amino acid entities, the weight percentage of amino acid entities relative to the weight of the composition (i.e., the weight of all amino acid entities and any other biologically active ingredients present in the composition), or the ratio, in dosage form. In some embodiments, the composition, e.g., the active moiety, is provided as a pharmaceutically acceptable formulation (e.g., a pharmaceutical product).

As used herein, the term "effective amount" refers to an amount of an active of the invention in a composition of the invention, particularly a pharmaceutical composition of the invention, that is sufficient to alleviate symptoms and/or ameliorate the condition to be treated (e.g., provide a desired clinical response). The effective amount of active for use in the compositions will vary depending upon the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy (concurrentthery), the particular active employed, the particular pharmaceutically acceptable excipient and/or carrier employed, and like factors known to the attending physician.

A "pharmaceutical composition" as described herein comprises at least one "active moiety" and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is used as a therapeutic agent. Other compositions (GMP; pharmaceutical grade components) that do not need to meet pharmaceutical standards may be used as nutraceuticals, medical foods or supplements, which are referred to as "consumer health compositions".

As used herein, the term "pharmaceutically acceptable" refers to amino acids, materials, excipients, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In a particular embodiment, "pharmaceutically acceptable" means free of detectable endotoxin or that the endotoxin level is below a level acceptable in a pharmaceutical product.

In a particular embodiment, "pharmaceutically acceptable" refers to a standard used by the pharmaceutical industry or an agency or entity supervising the pharmaceutical industry (e.g., a governmental or trade agency or entity) to ensure that one or more product quality parameters are within an acceptable range for a drug, pharmaceutical composition, treatment, or other therapeutic agent. The product quality parameter may be any parameter prescribed by a pharmaceutical industry or institution or entity (e.g., a governmental or trade institution or entity), including but not limited to composition; composition homogeneity; the dosage; dose uniformity; presence, absence and/or amount of contaminants or impurities; and sterility grade (e.g., presence, absence, and/or content of microorganisms). Examples of government regulatory agencies include: the Federal Drug Administration (FDA), the European drug administration (EMA), the Swiss drug administration, the Chinese Food and Drug Administration (CFDA) or the Japanese drug and medical device administration (PMDA).

The term "pharmaceutically acceptable excipient" refers to a physiologically compatible ingredient in a pharmaceutical formulation other than an active agent. Pharmaceutically acceptable excipients may include, but are not limited to, buffers, sweeteners, dispersion enhancers, flavoring agents, bitterness masking agents, natural colors, artificial colors, stabilizers, solvents, or preservatives. In a particular embodiment, the pharmaceutically acceptable excipient comprises one or both of citric acid or lecithin.

As used herein, the term "non-amino acid entity protein component" refers to a peptide (e.g., a polypeptide or oligopeptide), a fragment thereof, or a degraded peptide. Exemplary non-amino acid solid protein components include, but are not limited to, one or more of whey protein, egg albumin, soy protein, casein, hemp protein, pea protein, brown rice protein, or fragments or degraded peptides thereof.

As used herein, the term "non-protein component" refers to any component of the composition other than a protein component. Exemplary non-protein components may include, but are not limited to, carbohydrates (e.g., monosaccharides (e.g., dextrose, glucose, or fructose), disaccharides, oligosaccharides, or polysaccharides). Lipids (e.g., sulfur-containing lipids (e.g., alpha-lipoic acid), long chain triglycerides, omega 3 fatty acids (e.g., EPA, DHA, STA, DPA, or ALA), omega 6 fatty acids (GLA, DGLA, or LA), medium chain triglycerides, or medium chain fatty acids); vitamins (e.g., vitamin a, vitamin E, vitamin C, vitamin D, vitamin B6, vitamin B12, biotin, or pantothenic acid); minerals (zinc, selenium, iron, copper, folate, phosphorus, potassium, manganese, chromium, calcium or magnesium); or sterols (e.g., cholesterol).

A composition, formulation or product is "therapeutic" if it provides the desired clinical effect. The desired clinical effect may be manifested by reducing the progression of the disease and/or alleviating one or more symptoms of the disease.

A "unit dose" includes one or more pharmaceutical products in a form marketed for use, having a specific mixture of active and inactive ingredients (excipients), having a specific configuration (e.g. capsule shell), and being dispensed as a specific dose (e.g. multiple stick packs).

As used herein, the term "treating or treatment" of fibrosis (e.g., a fibrotic condition or disorder) refers to ameliorating fibrosis (e.g., slowing, arresting or reducing the development of fibrosis or at least one clinical symptom thereof); alleviating or improving at least one physical parameter, including those parameters that may not be discernible by the patient; and/or preventing or delaying the onset, development or progression of fibrosis.

Compositions comprising amino acid entities (e.g., active moieties)

The compositions (e.g., active portions) of the invention described herein comprise amino acid entities, such as the amino acid entities shown in table 1.

In some embodiments, the leucine amino acid entity is selected from L-leucine, β -hydroxy- β -methylbutyrate (HMB), oxo-leucine (α -ketoisocaproic acid (KIC)), isovaleryl-CoA, N-acetyl-leucine, or a combination thereof.

In some embodiments, the arginine amino acid entity is selected from L-arginine, creatine, argininosuccinate, aspartic acid, glutamic acid, agmatine, N-acetyl-arginine, or a combination thereof.

In some embodiments, the glutamine amino acid entity is selected from the group consisting of L-glutamine, glutamate, carbamoyl-P, glutamate, N-acetylglutamine, or a combination thereof.

In some embodiments, the NAC-amino acid entity is selected from NAC, acetylserine (acetylserine), cystathionine, homocysteine (homocystereine), glutathione, or a combination thereof.

In some embodiments, the isoleucine amino acid entity is selected from L-isoleucine, 2-oxo-3-methyl-pentanoic acid (α -keto- β -methylpentanoic acid (KMV)), methylbutanoyl-CoA, N-acetyl-isoleucine, or a combination thereof.

In some embodiments, the valine amino acid entity is selected from L-valine, 2-oxo-pentanoic acid (α -Ketoisovalerate (KIV)), isobutyryl-CoA, N-acetyl-valine, or a combination thereof.

In certain embodiments, the serine amino acid entity is selected from the group consisting of L-serine, phosphoserine, P-hydroxypyruvate, glycine, acetylserine, cystathionine, phosphatidylserine, or a combination thereof. In some embodiments, the serine amino acid entity is selected from L-serine or L-glycine. In one embodiment, the serine amino acid entity is L-serine. In another embodiment, the serine amino acid entity is L-glycine. In another embodiment, the serine amino acid entity is L-glycine and L-serine (e.g., in a 1:1 weight ratio of L-glycine to L-serine).

The compositions described herein can further comprise one, two, three, four, five or more (e.g., all) or more of L-serine, L-glycine, creatine, or glutathione.

In some embodiments, the composition comprises a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity (e.g., L-glutamine or a salt thereof), a NAC entity, and L-serine.

In some embodiments, the composition comprises a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity (e.g., L-glutamine or a salt thereof), a NAC entity, and L-glycine.

In some embodiments, the composition comprises a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity (e.g., L-glutamine or a salt thereof), a NAC entity, L-glycine, and L-serine.

In some embodiments, the composition comprises a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity (e.g., L-glutamine or a salt thereof), and a NAC entity. In some embodiments, one, two, three, four, five, or more (e.g., all) of (a) - (f) are in the form of free amino acids in the composition, e.g., at least: 42 wt%, 75 wt%, 90 wt% or more of the total weight of the amino acid entity component or total component is one, two, three, four, five or more (e.g., all) of (a) - (f) in the composition in free amino acid form (e.g., in dry form).

In some embodiments, one, two, three, four, five or more (e.g., all) of (a) - (f) are in salt form in the composition, e.g., at least: 0.01 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 5 wt%, or 10 wt% or more of the total weight of the amino acid entity component or total component is one, two, three, four, five or more (e.g., all) of (a) - (f) in salt form in the composition.

In some embodiments, one, two, three, four, five or more (e.g., all) of (a) - (f) are provided as part of a dipeptide or tripeptide, e.g., in at least the following amounts: 0.01 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 5 wt%, or 10 wt% or more of the amino acid entity component or the total component of the composition.

In some embodiments, the composition comprises, consists essentially of, or consists of:

a) (ii) a leucine amino acid entity, wherein,

b) an arginine amino acid entity, wherein the arginine amino acid entity,

c) a glutamine amino acid entity; and

d) an N-acetylcysteine (NAC) entity.

In some embodiments, the composition (e.g., active portion) comprises, consists essentially of, or consists of:

a) a leucine amino acid entity selected from the group consisting of: i) l-leucine or a salt thereof, ii) a dipeptide or a salt thereof, or a tripeptide or a salt thereof comprising L-leucine, or iii) beta-hydroxy-beta-methylbutyrate (HMB) or a salt thereof;

b) an arginine amino acid entity selected from: i) l-arginine or a salt thereof, ii) a dipeptide or a salt thereof, or a tripeptide or a salt thereof comprising L-arginine, iii) creatine or a salt thereof, or v) a dipeptide or a salt thereof, or a tripeptide or a salt thereof comprising creatine;

c) the glutamine amino acid entity is L-glutamine or a salt thereof, or a dipeptide including L-glutamine or a salt thereof, or a tripeptide or a salt thereof; and

d) the NAC entity is NAC or a salt thereof or a dipeptide comprising NAC or a salt thereof.

In some embodiments, the composition (e.g., active portion) further comprises, consists essentially of, or consists of one or both of: e) l-isoleucine or a salt thereof, or a dipeptide or a salt thereof, or a tripeptide or a salt thereof, comprising L-isoleucine; or f) L-valine or a salt thereof, or a dipeptide or a salt thereof, or a tripeptide or a salt thereof, comprising L-valine.

In some embodiments, the composition (e.g., active portion) comprises, consists essentially of, or consists of: a) l-leucine or a salt thereof; b) l-arginine or a salt thereof; c) l-glutamine or a salt thereof; and d) NAC or a salt thereof.

In some embodiments, the composition (e.g., active moiety) is capable of reducing or preventing fibrosis. For example, reducing or inhibiting one or both of fibrosis includes reducing the level of one or both of collagen, e.g., type I and type III collagen, or alpha-smooth muscle actin (alpha SMA).

In certain embodiments, the composition (e.g., active moiety) is capable of reducing or decreasing fibrosis by at least 5%, 10%, or 15%, as measured using a hydroxyproline assay, e.g., an antibody-based detection assay, e.g., an ELISA, e.g., as described in example 1, e.g., relative to a reference composition (e.g., a vector control).

In certain embodiments, the composition (e.g., active moiety) is capable of reducing or decreasing liver fibrosis or liver damage by at least 20%, 50%, or 65%, as detected using LX-2 cells, e.g., as assessed using a nucleic acid amplification method, e.g., PCR or qRT-PCR, e.g., as described in example 3, e.g., the level of Col1a1 and/or TIMP2 in LX-2 cells relative to a reference composition (e.g., a vector control, a single amino acid entity, or a combination of amino acid entities).

In some embodiments, the composition (e.g., active moiety) is capable of reducing or decreasing fibrosis of one or more hepatocyte types (e.g., one, two or three of hepatocytes, stellate cells, or macrophages, e.g., in a three-culture of hepatocytes, stellate cells, and macrophages), e.g., as detected by a change (e.g., a decrease) in the level of a fibrotic marker, e.g., one, two, three or more (e.g., all) of procollagen I α 1, MCP-1, YKL40, or GRO α (CXCL1), e.g., by at least 20%, 30%, 40%, or 50%, e.g., as assessed using an antibody-based detection assay (e.g., ELISA), e.g., as described in example 9, e.g., relative to a reference composition (e.g., a lower concentration of composition, a carrier control, a protein, a, A single amino acid entity or a combination of amino acid entities). In certain embodiments, the composition results in a reduction of one, two, three, or more (e.g., all) of:

(i) a level of procollagen I α 1 (e.g., a reduction in procollagen I α 1 level of at least 20%, 30%, 40%, or 50%);

(ii) MCP-1 level (e.g., at least 50%, 60%, 70%, 80%, or 90% reduction in MCP-1 level);

(iii) a YKL40 level (e.g., a reduction in YKL40 level of at least 70%, 80%, 90%, or 95%); or

(iv) The level of GRO α (CXCL1) (e.g., a decrease in GRO α (CXCL1) level of at least 15%, 20%, 25%, or 30%).

In some embodiments, the activity of the composition (e.g., the active moiety) is assessed by contacting one or more hepatocyte types (e.g., one, two, or three of hepatocytes, stellate cells, or macrophages) in culture, e.g., hepatocyte types isolated from a membrane (e.g., a permeable membrane, e.g., Transwell), with the composition under the conditions described in example 9.

In certain embodiments, the composition (e.g., active moiety) is capable of reducing or reducing liver fibrosis or liver damage, as detected by proliferation of stellate cells, e.g., the level of DNA synthesis in stellate cells, e.g., by at least 50%, 60%, 70%, or 80%, e.g., as assessed using nuclear staining, e.g., EdU (5-ethynyl-2' -deoxyuridine), e.g., as described in example 10, e.g., relative to a reference composition (e.g., vector control (PBS), a single amino acid entity, or a combination of amino acid entities).

i. Measurement of

The composition (e.g., active portion) can include 0.5g +/-20% to 10g +/-20% leucine amino acid entity, 1g +/-20% to 15g +/-20% arginine amino acid entity, 0.5g +/-20% to 20g +/-20% glutamine amino acid entity, and 0.1g +/-20% to 5g +/-20% NAC entity.

Exemplary compositions can include 1g of a leucine amino acid entity, 0.5g of an isoleucine amino acid entity, 0.5g of a valine amino acid entity, 1.5g or 1.81g of an arginine amino acid entity, 2g of a glutamine amino acid entity, and 0.15g of a NAC entity (e.g., g/package (packet) as shown in table 2).

TABLE 2 exemplary compositions including L-arginine (R) or L-arginine hydrochloride (R HCl) forms

In some embodiments, a composition (e.g., an active portion) includes 1g +/-20% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.5g +/-20% valine amino acid entity, 1.5g +/-20% arginine amino acid entity, 2g +/-20% glutamine amino acid entity, and 0.15g +/-20% NAC entity. In some embodiments, the composition includes 1g +/-15% leucine amino acid entity, 0.5g +/-15% isoleucine amino acid entity, 0.5g +/-15% valine amino acid entity, 1.5g +/-15% arginine amino acid entity, 2g +/-15% glutamine amino acid entity, and 0.15g +/-15% NAC entity. In some embodiments, the composition includes 1g +/-10% leucine amino acid entity, 0.5g +/-10% isoleucine amino acid entity, 0.5g +/-10% valine amino acid entity, 1.5g +/-10% arginine amino acid entity, 2g +/-10% glutamine amino acid entity, and 0.15g +/-10% NAC entity. In some embodiments, the composition includes 1g +/-5% leucine amino acid entity, 0.5g +/-5% isoleucine amino acid entity, 0.5g +/-5% valine amino acid entity, 1.5g +/-5% arginine amino acid entity, 2g +/-5% glutamine amino acid entity, and 0.15g +/-5% NAC entity. In some embodiments, the composition comprises 1g of a leucine amino acid entity, 0.5g of an isoleucine amino acid entity, 0.5g of a valine amino acid entity, 1.5g or 1.81g of an arginine amino acid entity, 2g of a glutamine amino acid entity, and 0.15g of a NAC entity.

In some embodiments, a composition (e.g., an active portion) includes 1g +/-20% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.5g +/-20% valine amino acid entity, 1.5g +/-20% arginine amino acid entity, 2g +/-20% glutamine amino acid entity, and 0.3g +/-20% NAC entity. In some embodiments, the composition includes 1g +/-15% leucine amino acid entity, 0.5g +/-15% isoleucine amino acid entity, 0.5g +/-15% valine amino acid entity, 1.5g +/-15% arginine amino acid entity, 2g +/-15% glutamine amino acid entity, and 0.3g +/-15% NAC entity. In some embodiments, the composition includes 1g +/-10% leucine amino acid entity, 0.5g +/-10% isoleucine amino acid entity, 0.5g +/-10% valine amino acid entity, 1.5g +/-10% arginine amino acid entity, 2g +/-10% glutamine amino acid entity, and 0.3g +/-10% NAC entity. In some embodiments, the composition includes 1g +/-5% leucine amino acid entity, 0.5g +/-5% isoleucine amino acid entity, 0.5g +/-5% valine amino acid entity, 1.5g +/-5% arginine amino acid entity, 2g +/-5% glutamine amino acid entity, and 0.3g +/-5% NAC entity. In some embodiments, the composition comprises 1g of a leucine amino acid entity, 0.5g of an isoleucine amino acid entity, 0.5g of a valine amino acid entity, 1.5g or 1.81g of an arginine amino acid entity, 2g of a glutamine amino acid entity, and 0.3g of a NAC entity.

Exemplary compositions can include 1g of a leucine amino acid entity, 0.5g of an isoleucine amino acid entity, 0.5g of a valine amino acid entity, 0.75g or 0.905g of an arginine amino acid entity, 2g of a glutamine amino acid entity, and 0.15g of a NAC entity (e.g., as shown in the g/package of table 3).

TABLE 3 exemplary compositions including L-arginine (R) or L-arginine hydrochloride (R HCl) forms

In some embodiments, a composition (e.g., an active portion) includes 1g +/-20% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.5g +/-20% valine amino acid entity, 0.75g +/-20% arginine amino acid entity, 2g +/-20% glutamine amino acid entity, and 0.15g +/-20% NAC entity. In some embodiments, the composition includes 1g +/-15% leucine amino acid entity, 0.5g +/-15% isoleucine amino acid entity, 0.5g +/-15% valine amino acid entity, 0.75g +/-15% arginine amino acid entity, 2g +/-15% glutamine amino acid entity, and 0.15g +/-15% NAC entity. In some embodiments, the composition includes 1g +/-10% leucine amino acid entity, 0.5g +/-10% isoleucine amino acid entity, 0.5g +/-10% valine amino acid entity, 0.75g +/-10% arginine amino acid entity, 2g +/-10% glutamine amino acid entity, and 0.15g +/-10% NAC entity. In some embodiments, the composition includes 1g +/-5% leucine amino acid entity, 0.5g +/-5% isoleucine amino acid entity, 0.5g +/-5% valine amino acid entity, 0.75g +/-5% arginine amino acid entity, 2g +/-5% glutamine amino acid entity, and 0.15g +/-5% NAC entity. In some embodiments, the composition comprises 1g of a leucine amino acid entity, 0.5g of an isoleucine amino acid entity, 0.5g of a valine amino acid entity, 0.75g or 0.905g of an arginine amino acid entity, 2g of a glutamine amino acid entity, and 0.15g of a NAC entity.

In some embodiments, a composition (e.g., an active portion) includes 1g +/-20% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.5g +/-20% valine amino acid entity, 0.75g +/-20% arginine amino acid entity, 2g +/-20% glutamine amino acid entity, and 0.3g +/-20% NAC entity. In some embodiments, the composition includes 1g +/-15% leucine amino acid entity, 0.5g +/-15% isoleucine amino acid entity, 0.5 +/-15% valine amino acid entity, 0.75g +/-15% arginine amino acid entity, 2g +/-15% glutamine amino acid entity, and 0.3g +/-15% NAC entity. In some embodiments, the composition includes 1g +/-10% leucine amino acid entity, 0.5g +/-10% isoleucine amino acid entity, 0.5 +/-10% valine amino acid entity, 0.75g +/-10% arginine amino acid entity, 2g +/-10% glutamine amino acid entity, and 0.3g +/-10% NAC entity. In some embodiments, the composition includes 1g +/-5% leucine amino acid entity, 0.5g +/-5% isoleucine amino acid entity, 0.5 +/-5% valine amino acid entity, 0.75g +/-5% arginine amino acid entity, 2g +/-5% glutamine amino acid entity, and 0.3g +/-5% NAC entity. In some embodiments, the composition comprises 1g of a leucine amino acid entity, 0.5g of an isoleucine amino acid entity, 0.5g of a valine amino acid entity, 0.75g or 0.905g of an arginine amino acid entity, 2g of a glutamine amino acid entity, and 0.3g of a NAC entity.

Exemplary compositions can include 1g of a leucine amino acid entity, 0.5g of an isoleucine amino acid entity, 0.25g of a valine amino acid entity, 0.75g or 0.905g of an arginine amino acid entity, 1g of a glutamine amino acid entity, and 0.225g of a NAC entity (e.g., as shown in the g/package of table 4).

TABLE 4 exemplary compositions including L-arginine (R) or L-arginine hydrochloride (R HCl) forms

In some embodiments, a composition (e.g., an active portion) includes 1g +/-20% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.25g +/-20% valine amino acid entity, 0.75g +/-20% arginine amino acid entity, 1g +/-20% glutamine amino acid entity, and 0.225g +/-20% NAC entity. In some embodiments, the composition includes 1g +/-15% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.25g +/-20% valine amino acid entity, 0.75g +/-15% arginine amino acid entity, 1g +/-15% glutamine amino acid entity, and 0.225g +/-15% NAC entity. In some embodiments, the composition includes 1g +/-10% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.25g +/-20% valine amino acid entity, 0.75g +/-10% arginine amino acid entity, 1g +/-10% glutamine amino acid entity, and 0.225g +/-10% NAC entity. In some embodiments, the composition includes 1g +/-5% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.25g +/-20% valine amino acid entity, 0.75g +/-5% arginine amino acid entity, 1g +/-5% glutamine amino acid entity, and 0.225g +/-5% NAC entity. Exemplary compositions can include 1g of a leucine amino acid entity, 0.5g of an isoleucine amino acid entity, 0.25g of a valine amino acid entity, 0.75g or 0.905g of an arginine amino acid entity, 1g of a glutamine amino acid entity, 0.225g of a NAC entity, and 1.5g of a serine amino acid entity (e.g., g/package as shown in Table 5).

TABLE 5 exemplary compositions including L-arginine (R) or L-arginine hydrochloride (R HCl) forms

In some embodiments, the composition comprises 1g +/-20% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.25g +/-20% valine amino acid entity, 0.75g +/-20% arginine amino acid entity, 1g +/-20% glutamine amino acid entity, 0.225g +/-20% NAC-amino acid entity, and 1.5g +/-20% serine amino acid entity. In some embodiments, the composition comprises 1g +/-15% leucine amino acid entity, 0.5g +/-15% isoleucine amino acid entity, 0.25g +/-15% valine amino acid entity, 0.75g +/-15% arginine amino acid entity, 1g +/-15% glutamine amino acid entity, 0.225g +/-15% NAC-amino acid entity, and 1.5g +/-15% serine amino acid entity. In some embodiments, the composition comprises 1g +/-10% leucine amino acid entity, 0.5g +/-10% isoleucine amino acid entity, 0.25g +/-10% valine amino acid entity, 0.75g +/-10% arginine amino acid entity, 1g +/-10% glutamine amino acid entity, 0.225g +/-10% NAC-amino acid entity, and 1.5g +/-10% serine amino acid entity. In some embodiments, the composition comprises 1g +/-5% leucine amino acid entity, 0.5g +/-5% isoleucine amino acid entity, 0.25g +/-5% valine amino acid entity, 0.75g +/-5% arginine amino acid entity, 1g +/-5% glutamine amino acid entity, 0.225g +/-5% NAC-amino acid entity, and 1.5g +/-5% serine amino acid entity.

Exemplary compositions can comprise 1g of a leucine amino acid entity, 0.5g of an isoleucine amino acid entity, 0.25g of a valine amino acid entity, 0.75g or 0.905g of an arginine amino acid entity, 1g of a glutamine amino acid entity, 0.225g of a NAC entity, and 1.667g of a serine amino acid entity (e.g., g/package as shown in table 6).

TABLE 6 exemplary compositions including L-arginine (R) or L-arginine hydrochloride (R HCl) forms

In some embodiments, the composition comprises 1g +/-20% leucine amino acid entity, 0.5g +/-20% isoleucine amino acid entity, 0.25g +/-20% valine amino acid entity, 0.75g +/-20% arginine amino acid entity, 1g +/-20% glutamine amino acid entity, 0.225g +/-20% NAC-amino acid entity, and 1.667g +/-20% serine amino acid entity. In some embodiments, the composition comprises 1g +/-15% leucine amino acid entity, 0.5g +/-15% isoleucine amino acid entity, 0.25g +/-15% valine amino acid entity, 0.75g +/-15% arginine amino acid entity, 1g +/-15% glutamine amino acid entity, 0.225g +/-15% NAC-amino acid entity, and 1.667g +/-15% serine amino acid entity. In some embodiments, the composition comprises 1g +/-10% leucine amino acid entity, 0.5g +/-10% isoleucine amino acid entity, 0.25g +/-10% valine amino acid entity, 0.75g +/-10% arginine amino acid entity, 1g +/-10% glutamine amino acid entity, 0.225g +/-10% NAC-amino acid entity, and 1.667g +/-10% serine amino acid entity. In some embodiments, the composition comprises 1g +/-5% leucine amino acid entity, 0.5g +/-5% isoleucine amino acid entity, 0.25g +/-5% valine amino acid entity, 0.75g +/-5% arginine amino acid entity, 1g +/-5% glutamine amino acid entity, 0.225g +/-5% NAC-amino acid entity, and 1.667g +/-5% serine amino acid entity.

ii, ratio of

Exemplary compositions may include a weight (wt.) ratio of 1 +/-15%: 0.5 +/-15%: 0.5 +/-15%: 1.5 +/-15%: 2 +/-15%: 0.15 +/-15% or 1 +/-15%: 0.5 +/-15%: 0.5 +/-15%: 1.81 +/-15%: 2 +/-15%: 0.15 ± 15% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-10% by weight of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities, and NAC-amino acid entities: 0.5 +/-10%: 0.5 +/-10%: 1.5 +/-10%: 2 +/-10%: 0.15 +/-10% or 1 +/-10%: 0.5 +/-10%: 0.5 +/-10%: 1.81 +/-10%: 2 +/-10%: 0.15 +/-10%. In some embodiments, the composition comprises 1 +/-5% by weight of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities, and NAC-amino acid entities: 0.5 +/-5%: 0.5 +/-5%: 1.5 +/-5%: 2 +/-5%: 0.15 +/-5% or 1 +/-5%: 0.5 +/-5%: 0.5 +/-5%: 1.81 +/-5%: 2 +/-5%: 0.15 +/-5%. In some embodiments, the composition comprises a weight ratio of 1: 0.5: 0.5: 1.5: 2: 0.15 or 1: 0.5: 0.5: 1.81: 2: 0.15 of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, and a NAC-amino acid entity.

Exemplary compositions may include a weight (wt.) ratio of 1 +/-20%: 0.5 +/-20%: 0.5 +/-20%: 1.5 +/-20%: 2 +/-20%: 0.3 +/-20% or 1 +/-20%: 0.5 +/-20%: 0.5 +/-20%: 1.81 +/-20%: 2 +/-20%: 0.3 ± 20% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-15% by weight: 0.5 +/-15%: 0.5 +/-15%: 1.5 +/-15%: 2 +/-15%: 0.3 +/-15% or 1 +/-15%: 0.5 +/-15%: 0.5 +/-15%: 1.81 +/-15%: 2 +/-15%: 0.3 ± 15% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-10% by weight: 0.5 +/-10%: 0.5 +/-10%: 1.5 +/-10%: 2 +/-10%: 0.3 +/-10% or 1 +/-10%: 0.5 +/-10%: 0.5 +/-10%: 1.81 +/-10%: 2 +/-10%: 0.3 ± 10% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-5% by weight: 0.5 +/-5%: 0.5 +/-5%: 1.5 +/-5%: 2 +/-5%: 0.3 +/-5% or 1 +/-5%: 0.5 +/-5%: 0.5 +/-5%: 1.81 +/-5%: 2 +/-5%: 0.3+ -5% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, and NAC-amino acid entity. In some embodiments, the composition comprises a weight ratio of 1: 0.5: 0.5: 1.5: 2: 0.3 or 1: 0.5: 0.5: 1.81: 2: 0.3 of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, and a NAC-amino acid entity.

Exemplary compositions may include a weight (wt.) ratio of 1 +/-20%: 0.5 +/-20%: 0.5 +/-20%: 0.75 +/-20%: 2 +/-20%: 0.15 +/-20% or 1 +/-20%: 0.5 +/-20%: 0.5 +/-20%: 0.905 +/-20%: 2 +/-20%: 0.15 ± 20% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-15% by weight: 0.5 +/-15%: 0.5 +/-15%: 0.75 +/-15%: 2 +/-15%: 0.15 +/-15% or 1 +/-15%: 0.5 +/-15%: 0.5 +/-15%: 0.905 +/-15%: 2 +/-15%: 0.15 ± 15% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-10% by weight: 0.5 +/-10%: 0.5 +/-10%: 0.75 +/-10%: 2 +/-10%: 0.15 +/-10% or 1 +/-10%: 0.5 +/-10%: 0.5 +/-10%: 0.905 +/-10%: 2 +/-10%: 0.15 ± 10% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-5% by weight: 0.5 +/-5%: 0.5 +/-5%: 0.75 +/-5%: 2 +/-5%: 0.15 +/-5% or 1 +/-5%: 0.5 +/-5%: 0.5 +/-5%: 0.905 +/-5%: 2 +/-5%: 0.15+ -5% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, and NAC-amino acid entity. In some embodiments, the composition comprises a weight ratio of 1: 0.5: 0.5: 0.75: 2: 0.15 or 1: 0.5: 0.5: 0.905: 2: 0.15 of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, and a NAC-amino acid entity.

Exemplary compositions may include a weight (wt.) ratio of 1 +/-20%: 0.5 +/-20%: 0.5 +/-20%: 0.75 +/-20%: 2 +/-20%: 0.3 +/-20% or 1 +/-20%: 0.5 +/-20%: 0.5 +/-20%: 0.905 +/-20%: 2 +/-20%: 0.3 ± 20% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-15% by weight: 0.5 +/-15%: 0.5 +/-15%: 0.75 +/-15%: 2 +/-15%: 0.3 +/-15% or 1 +/-15%: 0.5 +/-15%: 0.5 +/-15%: 0.905 +/-15%: 2 +/-15%: 0.3 ± 15% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-10% by weight: 0.5 +/-10%: 0.5 +/-10%: 0.75 +/-10%: 2 +/-10%: 0.3 +/-10% or 1 +/-10%: 0.5 +/-10%: 0.5 +/-10%: 0.905 +/-10%: 2 +/-10%: 0.3 ± 10% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities and NAC-amino acid entities. In some embodiments, the composition comprises 1 +/-5% by weight: 0.5 +/-5%: 0.5 +/-5%: 0.75 +/-5%: 2 +/-5%: 0.3 +/-5% or 1 +/-5%: 0.5 +/-5%: 0.5 +/-5%: 0.905 +/-5%: 2 +/-5%: 0.3+ -5% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, and NAC-amino acid entity. In some embodiments, the composition comprises a weight ratio of 1: 0.5: 0.5: 0.75: 2: 0.3 or 1: 0.5: 0.5: 0.905: 2: 0.3 of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, and a NAC-amino acid entity.

Exemplary compositions may include a weight (wt.) ratio of 1 +/-20%: 0.5 +/-20%: 0.25 +/-20%: 0.75 +/-20%: 1 +/-20%: 0.225 +/-20% or 1 +/-20%: 0.5 +/-20%: 0.25 +/-20%: 0.905 +/-20%: 1 +/-20%: 0.225 +/-20% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, and NAC-amino acid entity. In some embodiments, the composition comprises 1 +/-15% by weight: 0.5 +/-15%: 0.25 +/-15%: 0.75 +/-15%: 1 +/-15%: 0.225 +/-15% or 1 +/-15%: 0.5 +/-15%: 0.25 +/-15%: 0.905 +/-15%: 1 +/-15%: 0.225 +/-15% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, and NAC-amino acid entity. In some embodiments, the composition comprises 1 +/-10% by weight: 0.5 +/-10%: 0.25 +/-10%: 0.75 +/-10%: 1 +/-10%: 0.225 +/-10% or 1 +/-10%: 0.5 +/-10%: 0.25 +/-10%: 0.905 +/-10%: 1 +/-10%: 0.225 +/-10% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, and NAC-amino acid entity. In some embodiments, the composition comprises 1 +/-5% by weight: 0.5 +/-5%: 0.25 +/-5%: 0.75 +/-5%: 1 +/-5%: 0.225 +/-5% or 1 +/-5%: 0.5 +/-5%: 0.25 +/-5%: 0.905 +/-5%: 1 +/-5%: 0.225 +/-5% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, and NAC-amino acid entity. In some embodiments, the composition comprises a weight ratio of 1: 0.5: 0.25: 0.75: 1: 0.225 or 1: 0.5: 0.25: 0.905: 1: 0.225 of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, and a NAC-amino acid entity.

Exemplary compositions comprising amino acid entities may comprise 1 +/-20% by weight: 0.5 +/-20%: 0.25 +/-20%: 0.75 +/-20%: 1 +/-20%: 0.225 +/-20%: 1.5 +/-20% or 1 +/-20%: 0.5 +/-20%: 0.25 +/-20%: 0.905 +/-20%: 1 +/-20%: 0.225 +/-20%: 1.5 +/-20% of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, a NAC-amino acid entity, and a serine amino acid entity. In some embodiments, the composition comprises 1 +/-15% by weight: 0.5 +/-15%: 0.25 +/-15%: 0.75 +/-15%: 1 +/-15%: 0.225 +/-15%: 1.5 +/-15% or 1 +/-15%: 0.5 +/-15%: 0.25 +/-15%: 0.905 +/-15%: 1 +/-15%: 0.225 +/-15%: 1.5 +/-15% of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, a NAC-amino acid entity, and a serine amino acid entity. In some embodiments, the composition comprises 1 +/-10% by weight: 0.5 +/-10%: 0.25 +/-10%: 0.75 +/-10%: 1 +/-10%: 0.225 +/-10%: 1.5 +/-10% or 1 +/-10%: 0.5 +/-10%: 0.25 +/-10%: 0.905 +/-10%: 1 +/-10%: 0.225 +/-10%: 1.5 +/-10% of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, a NAC-amino acid entity, and a serine amino acid entity. In some embodiments, the composition comprises 1 +/-5% by weight: 0.5 +/-5%: 0.25 +/-5%: 0.75 +/-5%: 1 +/-5%: 0.225 +/-5%: 1.5 +/-5% or 1 +/-5%: 0.5 +/-5%: 0.25 +/-5%: 0.905 +/-5%: 1 +/-5%: 0.225 +/-5%: 1.5 +/-5% of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, a NAC-amino acid entity, and a serine amino acid entity. In some embodiments, the composition comprises a weight ratio of 1: 0.5: 0.25: 0.75: 1: 0.225: 1.5 or 1: 0.5: 0.25: 0.905: 1: 0.225: 1.5 of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, a NAC-amino acid entity, and a serine amino acid entity.

Exemplary compositions may include a weight (wt.) ratio of 1 +/-20%: 0.5 +/-20%: 0.25 +/-20%: 0.75 +/-20%: 1 +/-20%: 0.225 +/-20%: 1.667 +/-20% or 1 +/-20%: 0.5 +/-20%: 0.25 +/-20%: 0.905 +/-20%: 1 +/-20%: 0.225 +/-20%: 1.667 +/-20% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, NAC-amino acid entity, and serine amino acid entity. In some embodiments, the composition comprises 1 +/-15% by weight: 0.5 +/-15%: 0.25 +/-15%: 0.75 +/-15%: 1 +/-15%: 0.225 +/-15%: 1.667 +/-15% or 1 +/-15%: 0.5 +/-15%: 0.25 +/-15%: 0.905 +/-15%: 1 +/-15%: 0.225 +/-15%: 1.667 +/-15% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, NAC-amino acid entity, and serine amino acid entity. In some embodiments, the composition comprises 1 +/-10% by weight: 0.5 +/-10%: 0.25 +/-10%: 0.75 +/-10%: 1 +/-10%: 0.225 +/-10%: 1.667 +/-10% or 1 +/-10%: 0.5 +/-10%: 0.25 +/-10%: 0.905 +/-10%: 1 +/-10%: 0.225 +/-10%: 1.667 +/-10% leucine amino acid entity, isoleucine amino acid entity, valine amino acid entity, arginine amino acid entity, glutamine amino acid entity, NAC-amino acid entity, and serine amino acid entity. In some embodiments, the composition comprises 1 +/-5% by weight: 0.5 +/-5%: 0.25 +/-5%: 0.75 +/-5%: 1 +/-5%: 0.225 +/-5%: 1.667 +/-5% or 1 +/-5%: 0.5 +/-5%: 0.25 +/-5%: 0.905 +/-5%: 1 +/-5%: 0.225 +/-5%: 1.667+ -5% of leucine amino acid entities, isoleucine amino acid entities, valine amino acid entities, arginine amino acid entities, glutamine amino acid entities, NAC-amino acid entities and serine amino acid entities. In some embodiments, the composition comprises a weight ratio of 1: 0.5: 0.25: 0.75: 1: 0.225: 1.667 or 1: 0.5: 0.25: 0.905: 1: 0.225: 1.667 of a leucine amino acid entity, an isoleucine amino acid entity, a valine amino acid entity, an arginine amino acid entity, a glutamine amino acid entity, an NAC-amino acid entity, and a serine amino acid entity.

In some embodiments, the composition comprises 10 to 30 weight% +/-15% leucine amino acid entities, 5 to 15 weight% +/-15% isoleucine amino acid entities, 5 to 15 weight% +/-15% valine amino acid entities, 15 to 40 weight% +/-15% arginine amino acid entities, 20 to 50 weight% +/-15% glutamine amino acid entities, and 1 to 8 weight% +/-15% NAC entities.

In some embodiments, the composition comprises 10% +/-15% to 30% +/-15% by weight leucine amino acid entities. In some embodiments, the composition comprises 5% +/-15% to 15% +/-15% isoleucine amino acid entities by weight. In some embodiments, the composition comprises 5% +/-15% to 15% +/-15% valine amino acid entities by weight. In some embodiments, the composition comprises 15% +/-15% to 40% +/-15% arginine amino acid entities by weight. In some embodiments, the composition comprises 20% +/-15% to 50% +/-15% glutamine amino acid entities by weight. In some embodiments, the composition comprises 1% wt. +/-15% to 8% wt. +/-15% NAC entities.

In some embodiments, the composition comprises 16 to 18 weight% +/-15% leucine amino acid entities, 7 to 9 weight% +/-15% isoleucine amino acid entities, 7 to 9 weight% +/-15% valine amino acid entities, 28 to 32 weight% +/-15% arginine amino acid entities, 31 to 34 weight% +/-15% glutamine amino acid entities, and 1 to 5 weight% +/-15% NAC entities.

In some embodiments, the composition comprises 16% +/-15% to 18% +/-15% by weight leucine amino acid entities. In some embodiments, the composition comprises 7% +/-15% to 9% +/-15% isoleucine amino acid entities by weight. In some embodiments, the composition comprises from 7% +/-15% to 9% +/-15% valine amino acid entities by weight. In some embodiments, the composition comprises 28% +/-15% to 32% +/-15% arginine amino acid entities by weight. In some embodiments, the composition comprises 31% +/-15% to 34% +/-15% glutamine amino acid entities by weight. In some embodiments, the composition comprises 1% +/-15% to 5% +/-15% NAC entities by weight.

In some embodiments, the composition comprises 16.8% +/-15% leucine amino acid entities, 8.4% +/-15% isoleucine amino acid entities, 8.4% +/-15% valine amino acid entities, 30.4% +/-15% arginine amino acid entities, 33.6% +/-15% glutamine amino acid entities, and 2.5% +/-15% NAC entities by weight.

Relationships of amino acid entities

In some embodiments, the composition (e.g., active moiety) has one or more of the following properties:

a) the weight% of Q-amino acid entities in the composition is greater than the weight% of the R-amino acid entities;

b) the weight% of Q-amino acid entities in the composition is greater than the weight% of L-amino acid entities;

c) the weight% of the R-amino acid entities in the composition is greater than the weight% of the L-amino acid entities; or

d) A combination of two or three of (a) - (c).

In some embodiments, the wt.% of glutamine amino acid entities in the composition is greater than the wt.% of arginine amino acid entities, e.g., the wt.% of glutamine amino acid entities in the composition is at least 5% greater than the wt.% of arginine amino acid entities, e.g., the wt.% of glutamine amino acid entities is at least 10% or 25% greater than the wt.% of arginine amino acid entities.

In some embodiments, the wt.% of glutamine amino acid entities in the composition is greater than the wt.% of leucine amino acid entities, e.g., the wt.% of glutamine amino acid entities in the composition is at least 20% greater than the wt.% of leucine amino acid entities, e.g., the wt.% of glutamine amino acid entities in the composition is at least 25% or 50% greater than the wt.% of leucine amino acid entities.

In some embodiments, the weight% of arginine amino acid entities in the composition is greater than the weight% of leucine amino acid entities, e.g., the weight% of arginine amino acid entities in the composition is at least 10% greater than the weight% of leucine amino acid entities, e.g., the weight% of arginine amino acid entities in the composition is at least 15% or 30% greater than the weight% of leucine amino acid entities.

In some embodiments, the weight% of leucine amino acid entities in the composition is greater than the weight% of isoleucine amino acid entities in the composition, e.g., the weight% of leucine amino acid entities in the composition is at least 25 weight% greater than the weight% of isoleucine amino acid entities in the composition.

In some embodiments, the wt.% of leucine amino acid entities in the composition is greater than the wt.% of valine amino acid entities in the composition, e.g., the wt.% of leucine amino acid entities in the composition is at least 25 wt.% greater than the wt.% of valine amino acid entities in the composition.

In some embodiments, the wt.% of arginine amino acid entities, glutamine amino acid entities, and NAC entities is at least 50 wt.% or 70 wt.% of the amino acid entities in the composition, but no more than 90 wt.% of the amino acid entities in the composition.

In some embodiments, the wt% of NAC entities is at least 1 wt% or 2 wt% of the amino acid entity component or all components in the composition, but not more than 10 wt% or more of the amino acid entity component or all components in the composition.

In some embodiments, the combination of isoleucine and valine amino acid entities is at least 15% or 20% by weight of the amino acid entity component or all components in the composition, but not more than 50% by weight of the amino acid entity component or all components in the composition;

in some embodiments, the glutamine amino acid entity and NAC entity are at least 40% or 50% by weight of the amino acid entity component or all components in the composition, but not more than 90% by weight of the amino acid entity component or all components in the composition.

In some embodiments, the composition (e.g., active portion) further comprises a serine amino acid entity, e.g., the serine amino acid entity is present in a higher amount than any other amino acid entity component in the composition. In some embodiments, the wt.% of serine amino acid entities is at least 20 wt.% or more of the amino acid entities or all components in the composition.

Excluded or restricted amino acid molecules from the composition

In some embodiments, the composition does not comprise peptides (e.g., protein supplements) greater than 20 amino acid residues in length selected from or derived from one, two, three, four, five or more (e.g., all) of egg white protein, soy protein, casein protein, hemp protein, pea protein, or brown rice protein, or if present, in less than the following amounts: the total weight (e.g., in dry form) of the non-amino acid entity protein component or all components in the composition is 10 weight (wt)%, 5 wt.%, 1 wt.%, 0.1 wt.%, 0.05 wt.%, 0.01 wt.%.

In some embodiments, the composition comprises a combination of 3 to 19, 3 to 15, or 3 to 10 different amino acid entities; for example, the combination comprises: at least 42 wt.%, 75 wt.%, or 90 wt.% of the total weight% (e.g., in dry form) of the amino acid entity components or all components in the composition.

In some embodiments, the dipeptide or salt thereof or tripeptide or salt thereof is present at less than: 10 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.001 wt% or less of the total weight (e.g., in dry form) of the amino acid entity components or all components in the composition.

In some embodiments, at least 50%, 60%, 70% or more of the total grams of amino acid entity components in the composition (e.g., in dry form) are from one, two, three, four, five, seven, eight, nine or more (e.g., all) of (a) - (j).

In some embodiments, at least 50%, 60%, 70% or more of the heat from the amino acid entity component or all of the components in the composition (e.g., in dry form) is from one, two, three, four, five, seven, eight, nine or more (e.g., all) of (a) - (j).

In some embodiments, carbohydrates (e.g., one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of dextrose, maltodextrose, sucrose, dextrin, fructose, galactose, glucose, glycogen, high fructose corn syrup, honey, inositol, invert sugar, lactose, levulose, maltose, molasses, sugar cane, or xylose) are not present in the composition, or if present, are present in less than the following amounts: for example, 10 wt%, 5 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.001 wt% or less of the total weight of the composition (in dry form).

In some embodiments, vitamins (e.g., one, two, three, four, five, six, or seven of vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, vitamin C, or vitamin D) are not present in the composition, or if present, are present in less than the following amounts: for example, 10 wt%, 5 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.001 wt% or less of the total weight of the composition (in dry form).

In some embodiments, one or both of nitrate or nitrite is not present in the composition, or if present, is present in less than the following amounts: for example, 10 wt%, 5 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.001 wt% or less of the total weight of the composition (in dry form).

In some embodiments, the 4-hydroxyisoleucine is not present in the composition, or if present, is present in an amount less than: for example, 10 wt%, 5 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.001 wt% or less of the total weight of the composition (in dry form).

In some embodiments, the probiotic (e.g., bacillus probiotic) is not present in the composition, or if present, is present in less than the following amounts: for example, 10 wt%, 5 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.001 wt% or less of the total weight of the composition (in dry form).

In some embodiments, phenyl acetate (phenyl acetate) is not present in the composition, or if present, is present in an amount less than: for example, 10 wt%, 5 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.001 wt% or less of the total weight of the composition (in dry form).

In some embodiments, gelatin is not present in the composition (e.g., a gelatin capsule), or if present, is present in an amount less than: for example, 10 wt%, 5 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.001 wt% or less of the total weight of the composition (in dry form).

In some embodiments, one, two or three of S-allylcysteine, S-allylmercaptocysteine, or fructosyl-arginine are not present in the composition, or if present, are present in less than the following amounts: for example, 10 wt%, 5 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.001 wt% or less of the total weight of the composition (in dry form).

Uses, e.g. methods of treatment

The compositions (e.g., active fractions) of the invention as described herein can be administered to ameliorate or reduce fibrosis, e.g., to treat or prevent a fibrotic condition or disorder in a subject. The method comprises administering to a subject in need thereof a composition described herein in an amount sufficient to reduce or inhibit fibrosis in the subject. The compositions can be administered to improve tissue repair, for example in patients with fibrotic conditions or disorders.

In some embodiments, the subject has fibrosis or has been diagnosed with a fibrotic condition or disorder. In some embodiments, the subject having a fibrotic condition or disorder is a human. In some embodiments, the subject has not received prior treatment with the composition (e.g., a subject on initial treatment: (a) subject))。

The disclosure features methods for improving or reducing fibrosis, comprising administering to a subject in need thereof an effective amount of a composition disclosed herein (e.g., an active moiety). The compositions may be administered according to a dosage regimen described herein to treat a subject having a fibrotic condition or disorder.

In some embodiments, the compositions (e.g., active moieties) described herein are used as a medicament to treat (e.g., reverse, reduce, ameliorate, or prevent) fibrosis in a subject (e.g., a subject having a fibrotic condition or disorder). In some embodiments, the compositions (e.g., active moieties) described herein are used for the manufacture of a medicament for treating (e.g., reversing, reducing, ameliorating, or preventing) fibrosis in a subject (e.g., a subject having a fibrotic condition or disorder).

In certain embodiments, reducing or treating fibrosis comprises reducing one, two, three, four, five or more (e.g., all) of: formation or deposition of tissue fibrosis; the size, cellularity (e.g., fibroblast or immune cell number), composition, or cellular content of the fibrotic lesion; fibrosis-damaged collagen or hydroxyproline content; expression or activity of a fibrogenic protein; fibrosis associated with inflammatory responses; or weight loss associated with fibrosis. In some embodiments, reducing fibrosis increases survival of the subject.

Exemplary fibrotic diseases include, but are not limited to, multisystemic (e.g., systemic sclerosis, multifocal fibrosis, scleroderma-like graft-versus-host disease in bone marrow transplant recipients, nephrogenic systemic fibrosis, or scleroderma) and organ-specific disorders (e.g., fibrosis of the lung, heart, kidney, pancreas, skin, brain, and other organs). In certain embodiments, the fibrotic condition is a fibrotic condition of the lung, a fibrotic condition of the heart or vasculature, a fibrotic condition of the kidney, a fibrotic condition of the skin, a fibrotic condition of the gastrointestinal tract, a fibrotic condition of the bone marrow or hematopoietic tissue, a fibrotic condition of the nervous system, a fibrotic condition of the eye, or a combination thereof.

In certain embodiments, the fibrotic condition is primary fibrosis. In one embodiment, the fibrotic disease is idiopathic. In other embodiments, the fibrotic condition is associated with (e.g., secondary to) the following: diseases (e.g., infectious diseases, inflammatory diseases, autoimmune diseases, and/or connective diseases); a toxin; damage (e.g., environmental hazards (e.g., asbestos, coal dust, and/or polycyclic aromatic hydrocarbons), smoking, or wounds); medical treatment (e.g., surgical incision, chemotherapy, or radiation); or a combination thereof.

In certain embodiments, the fibrotic disorder is a fibrotic disorder of the lung. In certain embodiments, the fibrotic condition of the lung is selected from one or more of: pulmonary fibrosis, Idiopathic Pulmonary Fibrosis (IPF), common interstitial pneumonia (UIP), interstitial lung disease, Cryptogenic Fibrotic Alveolitis (CFA), bronchiectasis, and scleroderma lung disease. In one embodiment, the fibrosis of the lung is secondary to a disease, toxin, injury, medical treatment, or a combination thereof.

For example, fibrosis of the lung may be associated with (e.g., secondary to) one or more of: disease processes such as asbestosis and silicosis; occupational hazards; an environmental contaminant; smoking; autoimmune connective tissue diseases (e.g., rheumatoid arthritis, scleroderma, and Systemic Lupus Erythematosus (SLE)); connective tissue diseases (e.g., sarcoidosis); or an infectious disease (e.g., infection, particularly chronic infection). In one embodiment, the fibrotic condition of the lung is associated with an autoimmune connective tissue disease (e.g., scleroderma or lupus, e.g., SLE).

In other embodiments, pulmonary fibrosis includes, but is not limited to, pulmonary fibrosis associated with Chronic Obstructive Pulmonary Disease (COPD), acute respiratory distress syndrome, scleroderma, pleural fibrosis, chronic asthma, acute lung syndrome, amyloidosis, bronchopulmonary dysplasia, kaplan syndrome, Dressler syndrome, histiocytosis X, idiopathic pulmonary hemosiderosis, lymphangiosarcoidosis, mitral stenosis, polymyositis, pulmonary edema, pulmonary hypertension (e.g., idiopathic pulmonary arterial hypertension (IPH)), pneumoconiosis, radiation therapy (e.g., radiation-induced fibrosis), rheumatoid disease, scherfsier's disease, systemic lupus erythematosus, systemic sclerosis, tropical pulmonary eosinophilia, sarcoidosis, wiry-kefir disease (Weber-Christian disease), Wegener's granulomatosis, Whipple's disease, or exposure to toxins or irritants (e.g., drugs such as amiodarone, bleomycin, busulfan, carmustine, chloramphenicol, hexamethonium, methotrexate (methotrexate), dimethylargoline, mitomycin C, nitrofurantoin, penicillamine, pellomycin or pralol, or inhalation talc or dust, e.g., coal dust, silica). In certain embodiments, the pulmonary fibrosis is associated with an inflammatory disorder of the lung, such as one or both of asthma or COPD.

In certain embodiments, the fibrotic disorder is a fibrotic disorder of the kidney. In certain embodiments, the fibrotic condition of the kidney is selected from one or more of: kidney fibrosis (e.g., chronic kidney fibrosis), kidney disease associated with one or both of injury or fibrosis (e.g., chronic kidney disease associated with diabetes (e.g., diabetic nephropathy)), lupus, scleroderma of the kidney, glomerulonephritis, focal segmental glomerulosclerosis, IgA nephropathy fibrosis associated with human Chronic Kidney Disease (CKD), chronic progressive kidney disease (CPN), tubulointerstitial fibrosis, ureteral obstruction, chronic uremia, chronic interstitial nephritis, radiation nephropathy, glomerulosclerosis, Progressive Glomerulonephropathy (PGN), endothelial/thrombotic microangiopathy injury, HIV-associated kidney disease, or fibrosis associated with exposure to toxins, irritants or chemotherapeutic agents. In one embodiment, the fibrotic condition of the kidney is renal scleroderma. In some embodiments, the fibrotic condition of the kidney is transplant nephropathy, diabetic nephropathy, lupus nephritis, Focal Segmental Glomerulosclerosis (FSGS), endothelial/thrombotic microangiopathy injury, or HIV-associated nephropathy (HIVVAN).

In other embodiments, the fibrotic condition is associated with leprosy or tuberculosis.

In other embodiments, the compositions described herein are used to treat hyperproliferative fibrotic diseases, e.g., non-cancerous fibrotic diseases. In one embodiment, the hyperproliferative fibrotic disease is multi-system or organ specific. Exemplary hyperproliferative fibrotic diseases include, but are not limited to, multisystem diseases (e.g., systemic sclerosis, multifocal fibrosis, scleroderma-like graft-versus-host disease in bone marrow transplant recipients, nephrogenic systemic fibrosis, or scleroderma) and organ-specific disorders (e.g., fibrosis of the eye, lung, heart, kidney, pancreas, skin, and other organs).

In certain embodiments, the fibrotic disorder is a fibrotic disorder of the heart. In certain embodiments, the fibrotic condition of the heart is myocardial fibrosis (e.g., myocardial fibrosis associated with radiation myocarditis, complications of surgical procedures (e.g., myocardial post-operative fibrosis); infectious diseases (e.g., chagas disease, bacterial, trichinosis, or fungal myocarditis)); granuloma; metabolic storage disorders (e.g., cardiomyopathy, hemochromatosis); developmental disorders (e.g., endocardial fibroelastosis); arteriosclerosis, or exposure to toxins or irritants (e.g., drug-induced cardiomyopathy, drug-induced cardiotoxicity, alcoholic cardiomyopathy, cobalt poisoning, or exposure). In certain embodiments, the myocardial fibrosis is associated with an inflammatory disorder of the cardiac tissue (e.g., myocardial sarcoidosis). In some embodiments, the fibrotic condition is a fibrotic condition associated with myocardial infarction. In some embodiments, the fibrotic condition is a fibrotic condition associated with congestive heart failure.

In some embodiments, the fibrotic condition is associated with an autoimmune disease selected from scleroderma or lupus, e.g., systemic lupus erythematosus.

In some embodiments, the fibrotic condition is systemic. In some embodiments, the fibrotic condition is systemic sclerosis (e.g., restrictive systemic sclerosis, diffuse systemic sclerosis, or systemic sclerosis scleroderma), nephrogenic systemic fibrosis, cystic fibrosis, chronic graft versus host disease, or atherosclerosis.

In some embodiments, the fibrotic condition is scleroderma. In some embodiments, scleroderma is localized, e.g., macular or linear scleroderma. In some embodiments, the condition is systemic sclerosis, e.g., restrictive systemic sclerosis, diffuse systemic sclerosis, or systemic sclerosis-like scleroderma.

In other embodiments, the fibrotic condition affects one or more tissues selected from the group consisting of: tendon, cartilage, skin (e.g., skin epidermis or endothelium), cardiac tissue, vascular tissue (e.g., arteries, veins), pancreatic tissue, lung tissue, kidney tissue, uterine tissue, ovarian tissue, neural tissue, testicular tissue, peritoneal tissue, colon, small intestine, biliary tract, intestine, bone marrow, hematopoietic tissue, or ocular (e.g., retina) tissue.

In some embodiments, the fibrotic disorder is a fibrotic disorder of the eye. In some embodiments, the fibrotic condition is glaucoma, macular degeneration (e.g., age-related macular degeneration), macular edema (e.g., diabetic macular edema), retinopathy (e.g., diabetic retinopathy), or dry eye.

In certain embodiments, the fibrotic disorder is a fibrotic disorder of the skin. In certain embodiments, the fibrotic condition of the skin is selected from one or more of the following: skin fibrosis (e.g., hypertrophic scars, keloids), scleroderma, nephrogenic systemic fibrosis (e.g., resulting from exposure to gadolinium, which is often used as a contrast agent for MRI in patients with severe renal failure), and keloids.

In certain embodiments, the fibrotic disorder is a fibrotic disorder of the gastrointestinal tract. In certain embodiments, the fibrotic condition is selected from one or more of: fibrosis associated with scleroderma; radiation-induced intestinal fibrosis; fibrosis associated with a foregut inflammatory disorder (e.g., barrett's esophagus or chronic gastritis), and/or fibrosis associated with a post-inflammatory bowel disease (e.g., Inflammatory Bowel Disease (IBD), ulcerative colitis, or crohn's disease). In some embodiments, the fibrotic condition of the gastrointestinal tract is fibrosis associated with scleroderma.

In one embodiment, the fibrotic condition is a chronic fibrotic condition or disorder. In certain embodiments, the fibrotic condition is associated with an inflammatory condition or disorder.

In some embodiments, the fibrosis and/or inflammatory condition is osteomyelitis, e.g., chronic osteomyelitis.

In some embodiments, the fibrotic condition is amyloidosis. In certain embodiments, the amyloidosis is associated with chronic osteomyelitis.

In some embodiments, the fibrotic condition or disorder is a fibrotic condition or disorder of the liver. In certain embodiments, the fibrotic condition of the liver is selected from: non-alcoholic fatty liver disease (NAFL), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Alcoholic Fatty Liver Disease (AFLD) or Alcoholic Steatohepatitis (ASH). In some embodiments, the fibrotic condition of the liver is selected from: cirrhosis, cholestatic liver disease (e.g., Primary Biliary Cirrhosis (PBC)), bile duct injury, bile fibrosis, or cholangiopathy.

In some embodiments, the fibrotic condition or disorder is not a liver fibrosis condition or disorder. In some embodiments, the fibrotic condition or disorder is not a muscle fibrotic condition or disorder.

Dosage regimen

The compositions (e.g., active fractions) can be administered according to the dosage regimen described herein to reduce or treat fibrosis. For example, the composition can be administered to a subject at a dose of 2g +/-20% per day to 90g +/-20% per day (e.g., 72g +/-20% total amino acid entities per day) for a treatment period of, e.g., two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, or longer.

In some embodiments, the composition may be provided to a subject having a fibrotic condition or disorder in a single or multiple dose regimen. In some embodiments, the dose is administered two times per day, three times per day, four times per day, five times per day, six times per day, seven times per day, or more. In some embodiments, the composition is administered once, twice, or three times per day. In some embodiments, the composition is administered for at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 2 weeks. In some embodiments, the composition is administered chronically (e.g., for more than 30 days, e.g., 31 days, 40 days, 50 days, 60 days, 3 months, 6 months, 9 months, one year, two years, or three years).

In some embodiments, the composition is administered prior to a meal. In other embodiments, the composition is administered concurrently with the meal. In other embodiments, the composition is administered after a meal.

The composition may be administered every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, or every 10 hours to ameliorate or reduce fibrosis in a subject (e.g., a subject with a fibrotic condition or disorder).

In some embodiments, the composition comprises four stick packs, e.g., each stick pack comprises 25% +/-15% of the amount of each amino acid entity comprised in the composition described herein. In some embodiments, four stick packs are administered three times per day. In some embodiments, the composition comprises three stick packs, e.g., each stick pack comprises 33.3% +/-15% of the amount of each amino acid entity comprised in the composition described herein. In some embodiments, three stick packs are administered three times per day.

In some embodiments, the composition is administered at a dose of about 2g +/-20% to 50g +/-20% of total amino acid entities, such as once per day, twice per day, three times per day, four times per day, five times per day, or six times per day (e.g., three times per day). In some embodiments, the composition is administered three times daily at a dose of 2g +/-20% to 10g +/-20% total amino acid entities, e.g., 8g +/-20% or 10g +/-20% total amino acid entities is administered three times daily. In some embodiments, the composition is administered three times daily at a dose of 10g +/-20% to 20g +/-20% total amino acid entities, e.g., 11g +/-20%, 12g +/-20%, 15g +/-20%, 16g +/-20%, or 20g +/-20% total amino acid entities three times daily. In some embodiments, the composition is administered three times daily at a dose of 20g +/-20% to 30g +/-20% total amino acid entities, e.g., 21g +/-20%, 22g +/-20%, 23g +/-20%, or 24g +/-20% total amino acid entities three times daily.

Preparation of active fractions and pharmaceutical compositions

The disclosure features methods of making or preparing the compositions (e.g., active moieties) of the foregoing invention. The amino acid entities used to prepare the composition may be agglomerated and/or rapidly dissolved to aid dispersion and/or dissolution.

The compositions may be prepared using amino acid entities from the following sources, or other sources may be used: for example, FUSI-BCAA instant blends (L-leucine, L-isoleucine and L-valine in a weight ratio of 2:1: 1), instant L-leucine and other acids are available from Ajinomoto co. The pharmaceutical amino acid solid raw material can be used for preparing medicinal amino acid solid products. Food (or supplement) grade amino acid entity raw materials can be used for producing dietary amino acid entity products.

To prepare the compositions of the present disclosure, the following general procedure may be used: the starting materials (the individual amino acid entities and excipients) can be blended in a blending unit, then the homogeneity and content of the amino acid entities of the blend verified, and the blended powder filled into a stick pack or other unit dosage form. The contents of the stick pack or other unit dosage form may be dispersed in water for oral administration.

The food supplement and medical nutritional composition of the invention will be in a form suitable for oral administration.

When raw materials, such as pharmaceutical grade amino acid entities and/or excipients, are combined into a composition, contaminants may be present in the composition. The composition meets the criteria for a contamination level when the composition comprises substantially no (e.g., comprises less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, or 0.001% (w/w)) contaminants. In some embodiments, the compositions described in the methods herein do not comprise a contaminant. Contaminants include any substance that is not intentionally present in the composition (e.g., pharmaceutical grade amino acid entities and excipients, e.g., orally administered components, may be intentionally present) or has a negative impact on product quality parameters of the composition (e.g., side effects in a subject, reduced efficacy, reduced stability/shelf life, discoloration, odor, off-taste, off-texture/mouthfeel, or increased segregation of components of the composition). In some embodiments, the contaminants comprise microorganisms, endotoxins, metals, or combinations thereof. In some embodiments, the level of contamination, for example, by metals, lecithin, choline, endotoxins, microorganisms, or other contaminants of each part of the composition (e.g., contaminants from the raw material) is below the level allowed in the food.

Excipient

The amino acid compositions disclosed herein can be compounded or formulated with one or more excipients. Non-limiting examples of suitable excipients include tastants, flavors, buffers, preservatives, stabilizers, binders, compactants, lubricants, dispersion enhancers, disintegrants, flavors, sweeteners, and colorants.

In some embodiments, the excipient comprises a buffer. Non-limiting examples of suitable buffering agents include citric acid, sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.

In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobial agents, such as parabens, chlorobutanol, and phenol.

In some embodiments, the composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyl oxazolidinone, polyvinyl alcohol, C12-C18 fatty acid alcohols, polyethylene glycol, polyols, sugars, oligosaccharides, and combinations thereof.

In some embodiments, the composition comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, Sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate and light mineral oil.

In some embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersing agents include starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, xanthan gum, bentonite, purified lignocellulose, sodium starch glycolate, isomorphous silicates, and microcrystalline cellulose as high HLB emulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as an excipient. In some embodiments, the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized starch and modified starches, sweeteners, clays such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar gum, locust bean gum, karaya gum, pectin and tragacanth. In some embodiments, the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments, the excipient comprises a flavoring agent. The flavoring agent may be selected from synthetic flavoring oils and flavoring aromatics; a natural oil; extracts from plants, leaves, flowers and fruits; and combinations thereof. In some embodiments, the flavoring agent is selected from cinnamon oil; wintergreen oil; peppermint oil; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oils such as lemon oil, orange oil, grape and grapefruit oil; and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot.

In some embodiments, the excipient comprises a sweetener. Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as the sodium salt; dipeptide sweeteners, such as aspartame; dihydrochalcone (dihydrochalcone) compounds, glycyrrhizin; stevia (stevioside); chlorinated derivatives of sucrose, such as sucralose (sucralose); and sugar alcohols such as sorbitol, mannitol, xylitol, and the like. Hydrogenated starch hydrolysates and synthetic sweeteners 3, 6-dihydro-6-methyl-1, 2, 3-oxathiazin-4-one-2, 2-dioxide, in particular the potassium (acesulfame-K) and sodium and calcium salts thereof, are also contemplated.

In some embodiments, the composition comprises a colorant. Non-limiting examples of suitable colorants include food, drug and cosmetic colorants (FD & C), drug and cosmetic colorants (D & C), and topical drug and cosmetic colorants (ext.d & C). Colorants can be used as dyes or their corresponding lakes.

Particular excipients may include one or more of the following: citric acid, lecithin (e.g., Alcolec F100), sweeteners (e.g., sucralose micronized NF, acesulfame potassium (e.g., Ace-K)), dispersion enhancers (e.g., xanthan gum (e.g., Ticaxan Rapid-3)), flavorants (e.g., vanilla custard) #4306, Nat Orange WONF #1326, lime 865.0032U, and lemon 862.2169U), bitterness blockers (e.g., 936.2160U), and natural or artificial colors (e.g., FD & C Yellow No. 6 (Yellow 6)). Exemplary ingredient contents for each stick pack are shown in table 7.

TABLE 7 ingredient content in each stick pack

In another embodiment, the excipients are limited to citric acid, sweeteners (e.g., sucralose), xanthan gum, flavoring agents (e.g., vanilla custard #4036), flavoring agents (e.g., Nat Orange WONF #1362), and coloring agents (e.g., FD & C Yellow 6). Excipients specifically do not include lecithin (table 8).

TABLE 8 exemplary Contents in each stick pack

Dietary compositions

The composition (e.g., active fraction) comprising the amino acid entity may be formulated and used as a dietary composition, e.g., selected from a medical food, a functional food or a supplement. In such embodiments, the raw materials and the final product should meet food standards.

The composition of any aspect and embodiment disclosed herein may be used as a dietary composition, for example selected from a medical food, a functional food or a supplement. In some embodiments, the dietary composition is used in a method comprising administering the composition to a subject. The composition can be used in dietary compositions for the purpose of improving or reducing fibrosis.

In some embodiments, the dietary composition is selected from a medical food, a functional food, or a supplement. In some embodiments, the composition is in the form of a nutritional supplement, dietary formulation, functional food, medical food, or beverage comprising the composition described herein. In some embodiments, a nutritional supplement, dietary formulation, functional food, medical food, or beverage comprising a composition described herein is used to manage fibrosis (e.g., in a subject having a fibrotic condition or disorder).

The disclosure features methods of improving fibrosis comprising administering to a subject an effective amount of a dietary composition described herein.

The disclosure features methods of providing nutritional support or supplementation to a subject having fibrosis (e.g., a subject having a fibrotic condition or disorder), including administering to the subject an effective amount of a composition described herein.

The present disclosure features methods of providing nutritional support or supplementation that aids in managing fibrosis (e.g., a fibrotic condition or disorder), including administering to a subject in need thereof an effective amount of a composition described herein.

In some embodiments, the subject has or has been diagnosed with a fibrotic condition or disorder. In other embodiments, the subject does not have a fibrotic condition or disorder.

In addition, the compositions may be used in methods of dietary management of a subject (e.g., a subject without fibrosis).

In some embodiments, the subject has a pulmonary fibrosis condition or disorder. In some embodiments, the subject has a fibrotic condition or disorder of the heart or vasculature. In some embodiments, the subject has a renal fibrotic condition or disorder. In some embodiments, the subject has a pancreatic fibrotic condition or disorder. In some embodiments, the subject has a fibrotic condition or disorder of the skin. In some embodiments, the subject has a gastrointestinal fibrotic condition or disorder. In some embodiments, the subject has a myelogenous or hematopoietic fibrotic condition or disorder. In some embodiments, the subject has a fibrotic condition or disorder of the nervous system. In some embodiments, the subject has an ocular fibrotic condition or disorder.

Biomarkers

Any of the methods disclosed herein can include assessing or monitoring the effectiveness of administering a composition (e.g., an active moiety) of the invention as described herein to a subject having fibrosis (e.g., a subject having a fibrotic condition or disorder). The method includes obtaining a value for the effectiveness of the composition such that the value is indicative of the effectiveness of the therapy.

In some embodiments, the subject exhibits an elevated level of proC3, e.g., relative to a healthy subject without fibrosis. In some embodiments, the subject exhibits elevated ALT levels, e.g., relative to a healthy subject without fibrosis. In some embodiments, the subject exhibits elevated AST levels, e.g., relative to a healthy subject without fibrosis. In some embodiments, the subject exhibits an elevated level of TIMP (e.g., TIMP1 or TIMP2), e.g., relative to a healthy subject without fibrosis. In some embodiments, the subject exhibits an elevated level of Col1a1, e.g., relative to a healthy subject without fibrosis. In some embodiments, the subject exhibits an elevated Acta2 level, e.g., relative to a healthy subject without fibrosis. In some embodiments, the subject exhibits an elevated level of Hsp47, e.g., relative to a healthy subject without fibrosis. In some embodiments, the subject exhibits an elevated hydroxyproline level, e.g., relative to a healthy subject without fibrosis.

In some embodiments, administration of a composition (e.g., an active moiety) to a subject in a dosage regimen described herein reduces the level or activity of one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16 or more (e.g., all) of: (a) the N-terminal fragment of type III collagen (proC 3); (b) tissue Inhibitor of Metalloproteinases (TIMP) protein; for example, TIMP1 or TIMP 2; (c) col1a 1; (d) acta 2; (e) ALT; (f) AST; (g) hydroxyproline; (h) TGF-beta; (i) MCP-1; (j) MIP-1; (k) collagen, such as type I and type III collagen; (l) Alpha-smooth muscle actin (alpha SMA); (m) PIIINP; (n) Hsp 47; (o) procollagen I α 1; (p) YKL 40; or (q) GRO α (CXCL 1).

Methods of assessment (e.g., screening)

In another aspect, disclosed herein is a method or assay for evaluating a composition as described herein. The method comprises the following steps: (a) contacting one or more hepatocyte types (e.g., one, two or three of hepatocytes, stellate cells, or macrophages, e.g., in a triple culture of hepatocytes, stellate cells, and macrophages), e.g., hepatocyte types isolated by a membrane (e.g., a permeable membrane, e.g., Transwell) with the composition under the conditions described in example 9; and (b) detecting the level of one, two, three or more (e.g., all) of a fibrosis marker, e.g., procollagen I α 1, MCP-1, YKL40, or GRO α (CXCL 1)). In some embodiments, a change (e.g., a decrease) in the level of a marker of fibrosis (e.g., one, two, three, or more (e.g., all) of procollagen I α 1, MCP-1, YKL40, or GRO α (CXCL1)) indicates that the composition is suitable for reducing or treating fibrosis. In some embodiments, the composition results in a reduction, e.g., at least 10%, 20%, 30%, 40%, 50% or more, e.g., a reduction, e.g., indicating that the composition is suitable for reducing or treating a reduction in fibrosis, in the level of a marker of fibrosis (e.g., one, two, three, or more (e.g., all) of procollagen I α 1, MCP-1, YKL40, or GRO α (CXCL 1)). In certain embodiments, the composition results in a reduction of one, two, three, or more (e.g., all) of:

(i) a level of procollagen I α 1 (e.g., a reduction in procollagen I α 1 level of at least 20%, 30%, 40%, or 50%);

(ii) MCP1 levels (e.g., at least a 50%, 60%, 70%, 80%, or 90% reduction in MCP1 levels);

(iii) a YKL40 level (e.g., a reduction in YKL40 level of at least 70%, 80%, 90%, or 95%); or

(iv) The level of GRO α (CXCL1) (e.g., a decrease in GRO α (CXCL1) level of at least 15%, 20%, 25%, or 30%).

In some embodiments, one or more hepatocyte types (e.g., hepatocytes, stellate cells, and macrophages) are present in the co-culture, e.g., hepatocyte types separated by a membrane (e.g., a permeable membrane, e.g., Transwell) (e.g., hepatocytes separated by a membrane from one or both of stellate cells or macrophages) are present in the culture, e.g., the ratio of hepatocytes to macrophages to stellate cells is about 10:2:1 (e.g., the ratio of hepatocytes to astrocytes to macrophages separated by a membrane (e.g., a permeable membrane, e.g., Transwell) is about 10:2: 1).

In some embodiments, the detecting step comprises obtaining a sample, e.g., a culture sample from a Transwell plate as described in example 9, and measuring the level of a fibrotic marker (e.g., one, two, three, or more (e.g., all) of procollagen I α 1, MCP-1, YKL40, or GRO α (CXCL 1)).

Examples

The following examples are intended to aid in the understanding of the present invention, but are not intended to, and should not be construed to, limit its scope in any way.

Example 1 therapeutic amino acid composition A-1 treatment ameliorates hepatic fibrosis in an animal model of chemically-induced fibrosis

The ability of amino acid composition a-1 to affect liver fibrosis was tested in a chemically induced liver fibrosis model. Use of carbon tetrachloride (CCl) in mice₄) The common model of chemical induction of experimental liver Fibrosis (Gideon Smith, Animal Models of Cutaneous and Hepatic Fibrosis; progress in Molecular Biology and Molecular Science, Vol.105, pp.371-408). CCl₄Inflammation, hepatocyte injury, necrosis and fibrosis were induced after 4 weeks of treatment, and cirrhosis was induced after 8 weeks. By tetrachloroCarbon conversion (CCl)₄) Induced liver fibrosis in mice resembles important characteristics of human liver fibrosis, including inflammation, regeneration, and fibrogenesis.

Male BALB/c mice 7-8 weeks old were used for this study. Four animals were housed per cage, maintained in a standard 12 hour light cycle, and had free access to water and standard mouse chow. Food and water are available ad libitum.

Intraperitoneal (IP) administration of 5% CCl to animals₄Or vehicle, typically 3 days per week for 4 weeks. CCl4 was formulated once a week. 10ml/kg of amino acid composition A-1 was administered twice daily by oral gavage (oral gavage) at 23mg/ml, 76mg/ml or 153 mg/ml. Animals were weighed twice weekly and blood was collected via the retro-orbital sinus once weekly for serum. After four weeks, blood was collected for serum separation and mice were sacrificed by cervical dislocation. Two leaves of the liver were removed-the left leaf was placed in a tube containing 10% formalin for histopathology while the right leaf was weighed and placed in a bead mill tube containing 2.3mm zirconia beads and 2 volumes of 1:100 protease inhibitor (Sigma Aldrich, # P8340). The tissue samples were homogenized in a bead mill for 2 minutes and immediately centrifuged at 3,000rpm for 15 minutes at 4 ℃. Serum was analyzed for ALT/AST levels at weeks 2 and 4. The hydroxyproline (Hyp) content of homogenized liver samples was further evaluated to identify the formation of liver fibrosis.

Hydroxyproline (week 4)

Hydroxyproline (4-hydroxyproline, Hyp) is a common non-proteinogenic amino acid and is used as an indirect measure of the amount of collagen present, indicating fibrosis. CCl₄Liver Hyp levels were significantly higher in the treated animals than in the vehicle treated animals. Data are mean ± standard deviation (STDEV); "composition A-1": amino acid composition A-1; comparison with vehicle control<0.05. The raw data are shown in table 9.

TABLE 9 results of hepatic hydroxyproline content levels

AST levels and ALT levels

Aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) are common clinical biomarkers for liver health. CCl was administered throughout the study period₄The levels of both AST and ALT in the animals of (a) were significantly elevated, indicating the development of liver damage. Data are mean ± standard deviation (STDEV); "composition A-1": amino acid composition A-1; p-value was compared to vehicle/CCl 4 control; passing single tail T test; n.s. was not significant. The raw data are shown in tables 29 and 30.

TABLE 10 ALT level results

TABLE 11 AST level results

Brief description of the drawings

Treatment with amino acid composition a-1 resulted in a reduction in chemically induced fibrosis, as indicated by reduced hydroxyproline (a marker of collagen production) levels, and in an improvement in clinical biomarkers of liver injury, as indicated by reduced levels of the liver enzymes ALT and AST (tables 12-14).

Table 12 results for hepatic hydroxyproline content levels: raw data

Table 13 ALT level results: raw data

Table 14 AST level results: raw data

Example 2 therapeutic treatment (therapeutic treatment) of NAFLD, NASH and HCC with amino acid composition A-1 in preclinical animal models.

In STAM^TMModel (Stelic Institute)&Co, Tokyo, Japan; amino acid composition a-1 and obeticholic acid (6 α -ethyl-chenodeoxycholic acid; "OCA") for treating NASH. Two additional groups of normal C57BL/6 mice fed standard diet and vehicle-treated STAM were included^TMMice served as controls. All animals receiving treatment or vehicle were treated from 6 weeks of age until 9 weeks of age. The compounds were administered by oral gavage in a dose volume of 10 ml/kg. Amino acid composition A-1 was administered twice daily at a dose of 1500mg/kg and OCA was administered once daily at a dose of 30 mg/kg.

STAM^TMIs a model of non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC), developed by SMC laboratories, and generated by a combination of chemical and dietary interventions using C57BL/6 mice (Saito K et al, 2015Sci Rep5: 12466). Mice were treated at birth with a low dose of streptozotocin (streptozotocin) and fed a high fat diet starting at 4 weeks. Evidence of fatty liver occurs 5 weeks ago, followed by NASH 7 weeks ago and fibrosis 9 weeks ago.

NASH was induced in 53 male mice by a single subcutaneous injection of 200 μ g streptozotocin (STZ, Sigma-Aldrich, USA) solution 2 days after birth and feeding a high fat diet (HFD, 57 kcal% fat, Cat # HFD32, CLEA Japan, Japan) after 4 weeks of age.

Amino acid composition A-1, OCA and vehicle (described below) were administered by oral route in a volume of 10 mL/kg. Amino acid composition A-1 was dissolved in deionized water to 150mg/ml (10X). The OCA (Advanced ChemBlocks Inc.) was resuspended in 0.5% aqueous methylcellulose to 3mg/ml (10X). Amino acid composition A-1 was administered twice daily (9am and 7pm) at a dose of 1500 mg/kg. OCA was administered once daily at a dose of 30mg/kg (9 am).

Liver samples from mice from group 2 (vehicle), group 3 (amino acid composition a-1) and group 4 (OCA) were used for the following assays. For HE staining, sections were sectioned from paraffin blocks of liver tissue previously fixed in Bouin's solution and stained with Lillie-Mayer's hematoxylin (Muto Pure Chemicals co., ltd., japan) and eosin solution (Wako Pure Chemical Industries). The NAFLD Activity Score (NAS) was calculated according to the Kleiner standard (Kleiner D.E. et al, Hepatology, 2005; 41: 1313).

Research group

Group 1: STZ: ten neonatal STZ-primed mice were fed a normal diet ad libitum without any treatment until 9 weeks of age.

And 2, group: carrier: from 6 to 9 weeks of age, ten NASH mice were orally administered vehicle (10% phosphate buffered saline, ph7.2) twice daily (9am and 7pm) in a volume of 10 mL/kg.

And 3, group: amino acid composition a-1: from 6 to 9 weeks of age, ten NASH mice were orally administered with infusion water supplemented with amino acid composition a-1 twice daily (9am and 7pm) at a dose of 1500 mg/kg.

4 groups are as follows: OCA: from 6 to 9 weeks of age, ten NASH mice were orally administered with 0.5% methylcellulose supplemented with OCA once daily (9am) at a dose of 30 mg/kg.

And 5, group: and (3) normal: ten normal mice were fed a normal diet ad libitum without any treatment until 9 weeks of age.

6 groups are as follows: HFD: ten normal mice were fed the high fat diet ad libitum without any treatment until 9 weeks of age.

Histological results: HE staining, NAFLD activity scoring and alpha-smooth muscle actin staining

By histological analysis and H from each animal&E grading evaluation of stained liver sections Non Alcoholic Fatty Liver Disease (NAFLD) activity scores. The score is the sum of three separate scores that rank the degree of steatosis (0-3), inflammation (0-2), and hepatocyte ballooning (0-2). All tissues were graded using the scoring criteria of Kleiner et al (Kleiner et al, Hepatology, 2005; 41 (6): 1313-21). The results are shown in Table 15. Data are mean ± standard deviation (Stdev). Normal C57BL/6 mice were fed standard feed with an average score of 0 +/-0. Vector-treated STAM^TMThe average score of the mice was 4.7 +/-0.67. Amino acid composition A-1 treated mice had an average score of 3.1 +/-0.74. The average score of OCA-treated mice was 2.9 +/-0.74. Both amino acid composition a-1 and OCA were statistically different from the vehicle in NAFLD activity scores when compared using the Dunnett's multiple comparison test (amino acid composition a-1p ═ 0.0001, OCA p ═ 0.0001).

Similarly, amino acid composition A-1 treated mice showed a mean balloonlike expansion score of 0.4+/-0.52, in contrast to vector-treated STAM^TMThe mean balloonlike expansion score for mice was 1.6+/-0.52 and for OCA-treated mice was 0.3 +/-0.48. Both amino acid composition a-1 and OCA were statistically different from the vehicle in balloon-like expansion score when compared using Dunnett's multiple comparison test (amino acid composition a-1p ═ 0.0001, OCA p ═ 0.0001). The raw data are shown in tables 15-18.

TABLE 15 NAFLD Activity scores

Table 16.NAFLD activity: steatosis scoring

Table 17.NAFLD activity: inflammation score

Table 18.NAFLD activity: balloon-like dilation score

Fibrosis: results of Sirius Red (Sirius Red) staining

Fibrosis was assessed by analyzing the sirius red positive stained cell area of stained liver sections of each animal. Images were quantified using the percentage of positively stained area as a measure of fibrosis. The results of this analysis are shown in table 19. Data are mean ± standard deviation (Stdev). Normal C57BL/6 mice had an average positive area of 0.286+/-0.09 after feeding standard feed. The mean positive area of the vector-treated STAM mice was 1.1 +/-0.26. The average positive area of the mice treated with amino acid composition A-1 was 0.828 +/-0.33. The average score of OCA-treated mice was 0.776 +/-0.25. Amino acid composition a-1 and OCA were statistically different from the vehicle when compared using Dunnett's multiple comparison test (amino acid composition a-1 p-0.00494, OCA p < 0.016). The raw data are shown in table 19.

TABLE 19 fibrosis (average positive staining area, sirius red)

Similar to the statistically significant improvement in NAFLD activity score, balloon-like enlargement and fibrosis in the STAM mouse model after treatment with amino acid composition a-1 (fig. 1A), the statistically significant improvement in NAFLD activity score, balloon-like enlargement and fibrosis was determined in the high fat, high fructose and cholesterol diet (HFFC) mouse model after treatment with amino acid composition a-1 (fig. 1B).

Staining results for alpha-smooth muscle actin (alpha-SMA)

Liver sections of all mice were stained for the marker α -smooth muscle actin (α SMA) to identify activated hepatic stellate cells. Images were quantified using the percentage of positively stained area as a measure of stellate cell activation. The results are shown in Table 20. Data are mean ± standard deviation (STDEV); p-value is compared to vehicle-treated STAM mouse controls; by single tail T test. The average positive area after normal C57BL/6 mice were fed standard feed was 0.682 +/-0.26. Vector-treated STAM^TMThe average positive area of the mice was 2.128 +/-0.50. The average positive area of the mice treated with amino acid composition A-1 was 1.657 +/-0.84. The average score of OCA-treated mice was 1.562 +/-0.31.

TABLE 20 activated hepatic stellate cells (mean positively stained area, alpha-smooth muscle actin)

Treatment with amino acid composition A-1 significantly reduced the severity of NASH to a level comparable to inhibition of Farnesoid X Receptor (FXR) by OCA (currently under clinical study by Intercept Pharmaceuticals, Inc. for treatment of NASH), as shown by a significant reduction in NAFLD Activity Score (NAS) (3.1 +/-0.74 for the average NAS of amino acid composition A-1 compared to 2.9+/-0.74 for the average score of OCA-treated mice, 4.7+/-0.67 for the average score of vehicle-treated STAM mice), and reduced the development of fibrosis as shown by downregulation of hepatic stellate cell activation (1.657 +/-0.84 for the average alpha positive SMA staining area of amino acid composition A-1 compared to 1.562+/-0.31 for the average area of OCA-treated mice, relative to vehicle-treated STAM)^TMAverage area 2.128+/-0.50 for mice. .

Table 21 NAFLD activity score: raw data

Table 22.NAFLD activity: steatosis score: raw data

Table 23 NAFLD activity: inflammation scoring: raw data

Table 24.NAFLD activity: balloon-like expansion score: raw data

Table 25 fibrosis (mean positive staining area, sirius red): raw data

Table 26. activated hepatic stellate cells (mean positive staining area, α -smooth muscle actin): raw data

Example 3 treatment with amino acid composition reduces expression of fibrogenic genes in hepatic stellate cells

Hepatic stellate cells in healthy liver are located in the space of dise between hepatocytes and hepatic sinusoidal endothelial cells. In response to liver injury, hepatic stellate cells become activated, proliferate and contract, increasing production of α SMA, secretion of type I and type III collagen, and specific MMP and TIMP proteins. LX-2 cells were selected as a model of activated hepatic stellate cells and used to test whether a particular amino acid composition would reduce TGF β 1-induced fibrogenic gene expression.

On day 0, LX-2 hepatic stellate cells (Millipore) were seeded in collagen I-coated 96-well microplates (ThermoFisher) with 1.67E4 cells per well in Dulbecco's Modified Eagle Medium (DMEM, Corning) supplemented with 2% heat-inactivated fetal bovine serum (HI-FBS, HyClone) and 0.2% primocin (invivogen) and cultured overnight at 37 ℃, 5% CO 2. The cells were washed and the medium was replaced with amino acid free DMEM (us biologicals) containing a specific tailored concentration of amino Acids based on the mean physiological concentration in blood based on the values published in the Human Metabolome Database (1,2,3), and a dose profile of the specific amino acid composition LIVRQ + N-acetylcysteine, LIVRQ, RQ + N-acetylcysteine, LIV, which is the base HMDB (HMDB: the Human Metabolome database.nucleic Acids res.2007 jan; 35 (databasesu): D521-6.17202168) 40 times the concentration of the derived amino acids, or with leucine, isoleucine, valine, arginine, glutamine or cysteine, respectively, 50 times the concentration of HMDB derived. The combination containing N-acetylcysteine was administered at 10 mM. Cells were pretreated for 6 hours at 37 ℃ with 5% CO 2. After pretreatment, TGF beta 1 (R)&D Systems) or vehicle were incorporated into each well to a final concentration of 5ng/mL, and the cells were cultured under this stimulus at 37 ℃ under 5% CO2 for an additional 12 hours.

After 12 hours of culture, RNA extraction and quantitative PCR were performed on the lysates to determine the expression of collagen-1A 1 normalized to β -actin housekeeping expression using the Δ Δ Ct method using TaqMan primer probes (Integrated DNA Technologies: Col1A1, Hs.PT.58.15517795; Actb, Hs.PT.39a.22214847; Acta2, Hs.PT.56a.24853961; Timp2, Hs.PT.58.80594).

Table 27 shows the gene expression of Col1a1, Acta2 and Timp2 in LX-2 cells treated with amino acid combinations compared to vectors with or without TGF β 1 stimulation. LIVRQ + N-acetylcysteine, LIVRQ, RQ + N-acetylcysteine and N-acetylcysteine reduced Col1a1 expression and Timp2 expression. LIVRQ + N-acetylcysteine showed the greatest reduction in gene expression for Col1a1, Acta2, and Timp 2. LIVRQ-N-acetylcysteine significantly reduced the expression of Acta2 more than N-acetylcysteine, RQ + N-acetylcysteine, and LIV alone. LIVRQ + N-acetylcysteine significantly reduced the expression of Timp2 more than any other combination (table 27).

Table 27.

Table 28 shows Col1a1 expression for each amino acid, with or without TGF β 1 stimulus, at 1 × or 50 × HMDB derived amino acid concentrations. Alone, at 50X, only cysteine showed a significant reduction in Col1a1 expression.

Table 28.

Example 4 amino acid composition treatment improves progression of NASH in two rodent models by affecting lipid metabolism and fibrosis

The amino acid composition is formulated to simultaneously target multiple disease pathology mechanisms to safely and efficiently treat NASH (table 29). As described herein, the efficacy of amino acid compositions was studied in two established mouse models of NASH to determine the effect of amino acid compositions on signs and symptoms associated with NASH and related disorders.

TABLE 29 exemplary amino acid composition of amino acid composition.

STAM^TMMice are models of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) developed by SMC Laboratories, Inc. Evidence of fatty liver exists at 5 weeks of age, followed by NASH at 7 weeks of age and fibrosis at 9 weeks of age. Male STAM mice are generated in C57BL/6 mice, which receive a low dose of streptozotocin 2 days after birth and are fed a high fat diet (57% kcal fat, HFD32, CLEA Japan, Inc.) beginning at 4 weeks of age (Saito K et al, 2015Sci Rep5: 12466; incorporated herein by reference in its entirety). The amino acid composition was administered to STAM mice twice daily for 3 weeks at a dose of 1.6m/kg, starting at 6 weeks of age. A panel of vector-treated STAM mice was included as a control. Non-fasted mice were euthanized at 9 weeks of age. Plasma and liver samples were collected for further analysis (figure 2).

FATZO^TMMice are polygenic models of inbred obesity, metabolic syndrome and NASHType, developed by Crown Bioscience, Inc (Peterson rg.et, 2017PLoS one; incorporated herein by reference in its entirety). Male FATZO mice were fed a high fat, fructose and cholesterol (HFFC) diet (40% kcal fat, D12079B, Research Diets, inc., and 5% fructose drinking water solution) starting at 6 weeks of age to induce NAFLD and NASH. Evidence of fatty liver development 4 weeks post-induction followed by NASH development 16 weeks post-induction and fibrosis development 20 weeks post-induction. The designed amino acid composition was administered twice daily for 4 weeks at a dose of 3.0g/kg starting 16 weeks after induction (fig. 2). One group of vector treated FATZO mice served as controls. Non-fasted mice were euthanized at 20 weeks post induction. Plasma and liver samples were collected for further analysis.

An Aperio ScanScope CS Whole slide digital imaging System (Vista, CA) was used for imaging in H & E, Picric Sirius Red, SMA, F4/80. Images were captured from the entire slide.

The liver was evaluated by a veterinary pathologist blinded to sample ID using the NASH Clinical Research Network (CRN) liver histology scoring system (Kleiner DE et al, 2015, incorporated herein by reference in its entirety). The NASH CRN scoring system assesses the progression of steatosis, lobular inflammation, hepatocellular ballooning, degeneration, and fibrosis. One cross section of the liver was analyzed for each case using the NASH scoring system. Steatosis, lobular inflammation and fibrosis progression were assessed on a scale of 0-3. Balloon-like expansion degeneration was evaluated on a 0-2 scale.

The Positive Pixel Count algorithm of adaptive Automatic Image quantification is used to quantify the percentage of specific staining present in scanned slide images. The color range (hue and saturation range) and three intensity ranges (weak, positive and strong) were masked and evaluated. The algorithm counts the number and intensity sums in each intensity range, and three additional quantities: average intensity, ratio of strong/total, and average intensity of weak positive pixels.

Sirius Red and oil Red o (oil Red o) liver slices were imaged using a specific positive pixel algorithm. The positive pixel algorithm was modified to distinguish between orange and blue. For the evaluation of sirius red, changes from the normal "hue value" (0.1-0.96) and "color saturation" (0.04-0.29) were made. The vasculature and artifacts were excluded from the analysis.

Total lipid extracts from the liver were obtained by the Folch's method (Folch J et al, J.biol.chem.1957; 226: 497; incorporated herein by reference in its entirety). Liver samples were homogenized in chloroform-methanol (2:1, v/v) and incubated overnight at room temperature. After washing with chloroform-methanol-water (8:4:3, v/v/v), the extract was evaporated to dryness and dissolved in isopropanol. Liver triglyceride and cholesterol levels were measured by the triglyceride E test and the cholesterol E test, respectively.

The liver RNA samples were converted into cDNA libraries using the Illumina TruSeq Stranded mRNA sample preparation kit (Illumina # RS-122-2103). Transcriptomes were analyzed under Q2 solution (Morrisville, NC). RNA seq data was normalized and analyzed using an Ingeneity Pathway Analysis (QIAGEN Bioinformatics). Expression of mouse liver gene at the pathway level was focused on as it could be converted to human NAFLD (Teufel a et al, Gastroenterology, 2016, which is incorporated herein by reference in its entirety).

Metabolic profiling based on capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and LC-TOFMS platforms was performed in Human metamolome Technologies (Yamagata, Japan). Metabolites in the sample are identified by comparing migration time and m/z ratio to the true standard and quantified by comparing their peak areas to the peak areas of the true standard.

The level of IL-1b protein in the liver was quantified using a multiplex ELISA assay (Meso Scale Discovery, Rockville, Maryland).

The amino acid composition improves balloon-like expansion and fibrosis of both STAM and FATZO mice

Treatment with amino acid compositions significantly reduced NAFLD Activity Score (NAS) in both STAM and FATZO mice (fig. 3A). Treatment with the amino acid composition also significantly reduced balloon-like expansion of hepatocytes in the STAM mice (fig. 3B). The scores for steatosis and inflammation were not changed by treating the stamp mice with the amino acid composition according to histological determination. Sirius red-positive fibrosis area was significantly reduced after treatment of the STAM mice with the amino acid composition, whereas oil red O area was unchanged after treatment of the STAM mice with the amino acid composition (fig. 3C). Liver triglyceride and cholesterol levels were not altered.

Treatment with the amino acid composition also significantly reduced balloon-like expansion of hepatocytes in FATZO mice (fig. 3D). The scores for steatosis and inflammation and liver triglyceride and cholesterol levels were not changed in FATZO mice treated with the amino acid composition. The area of sirius red-positive fibrosis was significantly reduced when the amino acid composition was used to treat the FATZO mice, whereas the oil red O area was unchanged when the amino acid composition was used to treat the FATZO mice (fig. 3E).

The amino acid composition prevents fiber generation pathway

Fibrosis is a link of several biological processes, such as metabolic disorders, inflammation and cell death. Lipid accumulation and chronic inflammation in hepatocytes induce fibrotic activation of hepatic stellate cells (Wober H et al, Cell res.2009, which is incorporated herein by reference in its entirety). Treatment with amino acid compositions resulted in liver gene expression patterns consistent with inhibition of the fibrogenic TGF-b signaling pathway (figure 4).

There is increasing evidence that CCR2/CCR5 and its ligands, including MCP-1/MIP-1, promote macrophage recruitment and hepatic stellate cell activation leading to fibrosis following liver tissue injury (Lefebvre E et al, PLoS One2016, incorporated herein by reference in its entirety). The amino acid composition showed potent anti-fibrotic activity in the STAM model of NASH by reducing hepatic TGF-b signaling and MCP-1 and MIP-1 proteins (FIG. 5).

In the STAM and FATZO mouse models of NASH, the amino acid composition showed consistent disease-altering activity, including improvement of NAS and improvement of balloon-like expansion and fibrosis. The activity of the amino acid composition appears to be driven at least in part by increased fatty acid oxidation, decreasing the level of transcriptional pathways associated with fibrosis.

Example 5 TGF-beta 1 fibrogenic Gene expression by hepatic stellate cells

Primary human hepatic stellate cells were obtained from Samsara Sciences. Cells were grown to-80% confluence in complete HSC medium, seeded below passage 10 in T75 or T150 flasks into sterile, collagen I-coated 96-well optical plastic microplates (ThermoScientific, 152036) and cultured overnight in DMEM containing 2% fetal bovine serum and 1% antibiotic-antifungal agent in a humidified incubator at 37 ℃ in 5% CO 2. After overnight incubation, plates were washed and pretreated with medium + -single amino acid deletion (amino acid drop), 1xhMDB DMEM + -supplemented amino acid dose for 10.5 hours. After 10.5 hours of pretreatment, the same pretreatment medium supplemented with 3ng/mL of TGF β 1 was used and incubated at 37 ℃ under 5% CO2 for 24 hours. After 24 hours of stimulation, the supernatant was removed, RNA was extracted, and gene expression was assessed in each single amino acid deletion and supplementation by normalization to its own 1 × HMDB concentration using the Δ Δ Cq method.

Human procollagen I α 1 was measured by ELISA (human procollagen I α 1DuoSet ELISA, R & D Systems) from supernatants diluted at 1/100 in 1X reagent dilutions (reagent kit 2, R & D Systems).

Col1a1 Gene expression

Tables 30, 31-1, 31-2, 31-3, and 31-4 show the mean fold change in Col1a1 gene expression in primary human hepatic stellate cells from three different healthy donors. LIVRQNAC and LIVRQNAC + S showed a significant reduction in Col1a1 gene expression in two of the three donors. LIVRQNAC + G and RQNAC showed significant reduction in Col1a1 expression in all three donors. LIVRQ showed significant changes in Col1a1 gene expression in only one donor. LIV alone did not significantly alter Col1a1 gene expression.

Each of leucine, isoleucine, valine, and arginine did not significantly alter Col1a1 gene expression in any donor when the amino acids were administered alone. Glutamine reduced Col1a1 gene expression in two of the three donors. N-acetylcysteine significantly reduced the expression of the Col1a1 gene in all three donors.

TABLE 30 fold change in Col1a1 gene expression following administration of amino acid composition, normalized to Gapdh expression in the first donor

TABLE 31 fold change in Col1a1 gene expression following administration of a single amino acid composition, normalized to Gapdh expression in the first donor

Table 31-1 fold change in Col1a1 gene expression after administration of the amino acid composition, normalized to Gapdh expression in the second donor.

Table 31-2 fold change in Col1a1 gene expression after administration of the amino acid composition, normalized to Gapdh expression in the second donor.

Table 31-3 fold change in Col1a1 gene expression after administration of the amino acid composition, normalized to Gapdh expression in the third donor.

Tables 31-4 fold change in Col1a1 gene expression after administration of a single amino acid composition, normalized to Gapdh expression in the second donor.

Procollagen I alpha 1 secretion

Tables 32, 33-1, 33-2, 33-3, and 33-4 show fold changes in procollagen I.alpha.1 in procollagen hepatic stellate cells from three different healthy donors, normalized to their respective baseline amino acid conditions. Statistical significance was calculated by one-way ANOVA and Dunnett's multiple comparison test in each treatment group. In all three donors, LIV combinations significantly increased procollagen I.alpha.1 secretion. The addition of arginine (R) and glutamine (Q) to the combination of LIV counteracted the profibrotic effect of LIV alone. LIVRQNAC, LIVRQNAC + G, LIVRQNAC + S and RQNAC significantly reduced procollagen I α 1 secretion in all three donors. Separately, N-acetylcysteine was shown to significantly reduce procollagen I α 1 secretion in two of the three donors. Valine significantly increased procollagen I α 1 secretion from only one of the two donors, while isoleucine and arginine significantly increased procollagen I α 1 secretion from two of the three donors. In other words, glutamine alone had no significant effect on procollagen I α 1 secretion. Therefore, based on the effect of amino acid treatment alone, the effect of LIV with arginine and glutamine to reduce profibrosis compared to LIV alone was unexpected.

TABLE 32 fold change in procollagen 1. alpha.1 secretion following administration of amino acid composition in first donor

TABLE 33 fold change in procollagen 1. alpha.1 secretion following administration of a Single amino acid composition in a first donor

TABLE 33-1 fold Change in procollagen 1. alpha.1 secretion after administration of the amino acid composition in the second donor

TABLE 33-2 fold Change in procollagen 1. alpha.1 secretion after administration of Single amino acid composition in second Donor

TABLE 33-3 fold Change in procollagen 1. alpha.1 secretion after administration of amino acid composition in third Donor

TABLE 33-4 fold Change in procollagen 1. alpha.1 secretion after administration of Single amino acid composition in third Donor

Example 6 treatment of NASH in a mouse model with amino acid compositions

NASH induction in mice

In one embodiment, the role of LIVRQNAC and related amino acid compositions in obesity, metabolically driven non-alcoholic steatohepatitis (NASH) in a mouse model of FATZO is examined.

NASH was induced in 60 male FATZO mice during the 16-week induction period by adding 5% fructose to the drinking Water (WDF) on a western diet (study diet # D12079B; fat 40% kcal, protein 17% kcal, carbohydrate 43% kcal). Diet and water were available ad libitum. Littermates control male FATZO mice fed with a control diet (n 6, Purina # 5008; fat 17% kcal, protein 27% kcal, carbohydrate 56% kcal) and sterile water were established as controls. Mice were housed in plastic cages with micro-isolators. The sterile pad was replaced once a week. Mice received three per cage and maintained a twelve hour photoperiod throughout the study. The room temperature was monitored daily and maintained at 22-25 ℃ and body weight was recorded weekly during the induction period.

After 16 weeks of diet induction, 6 mice were kept on the control diet (group 1, control), while 60 induced mice were randomly assigned to the following treatments according to body weight and plasma glucose (feeding). Test items were administered to the FATZO mice for 4 weeks, starting 16 weeks after western dietary NASH induction. The test article was administered by oral gavage. Animals were euthanized 20 weeks after western dietary NASH induction and tissues were harvested for analysis.

LIVRQNAC, LIVRQNAC + G, LRQNAC and OCA (Advanced ChemBlocks, Inc.), excipients and irrigation water were supplied by Axcella Health, Inc. 0.5% methylcellulose was supplied by CrownBio, Inc. Dosing solutions were prepared as in appendix 1. The TA compound (amino acid composition) was an amino acid-doped compound freshly prepared daily in perfusion water and vehicle 0.125% xanthan gum, 1.5mM sodium lauryl sulfate, and 0.28% lecithin (Baxter #27F 7114). Obeticholic acid (OCA) was suspended in 0.5% methylcellulose in perfusion water. All test items were kept refrigerated. The sponsor provided the TA compound as a frozen powder. Dosing continued for 4 weeks.

The leucine dose of LIVRQNAC + G and LRQNAC matches the dose of LIVRQNAC.

LIVRQNAC, LIVRQNAC + G, LRQNAC, OCA and vehicle were administered by oral gavage in a volume of 10mL/kg throughout the study. The dose was calculated by daily body weight. LIVRQNAC, LIVRQNAC + G, LRQNAC and vehicle were administered twice daily (BID), while OCA was administered once daily in the morning (QD). Mice received oca (QD), and a carrier QD once a day. Doses were administered by oral gavage for 4 weeks at 0700 and 1800.

Vitality, clinical signs and behavior were monitored daily. Body weights were recorded daily during dosing. Blood samples were collected weekly in AM (0700) by tail clip for glucose measurement (StatStrip glucometer).

Animals were anesthetized with CO2 inhalation and exsanguinated by cardiac puncture for euthanasia. Terminal blood samples (K2EDTA) were obtained by cardiac puncture at the end of anesthetized animals. Samples were provided to Axcella Health in frozen form. Organ weights (total liver, gonadal fat pad) were recorded. Pancreatic, small intestinal and gonadal fat pads were fixed in 10% buffered formalin and prepared as directed in the protocol. Sections of small intestine, gonadal fat pad and liver were also snap frozen in liquid nitrogen and transported to the sponsor.

Liver tissue was fixed in Bouin solution at 4 ℃ for 24 hours and then immersed in standard alcohol and then xylene to prepare the tissue for paraffin embedding. After paraffin embedding and cooling, five micron sections were cut and stained for regular H & E and Picric acid (Picric) sirius red. Sections of the right and left lobes of the liver were frozen in OCT for analysis of lipid content with oil red staining. Aperio full-slide digital imaging System (Scan Scope CS, Vista, CA) was used for imaging. All slides were imaged at 20 x. The scan time ranged from 1.5 minutes to a maximum time of 2.25 minutes. The entire image was housed and stored in its Spectrum software system, and images were taken from the entire slide.

Liver was assessed using NASH liver scoring criteria. In this mouse study, one liver cross-section per case was analyzed using the NASH scoring system. The scoring system includes NAFLD Activity Scoring (NAS), fibrosis stage and identification of NASH by pattern recognition according to the disclosed NASH CRN scoring system. NAS can range from 0 to 8 and is calculated by the sum of scores from H & E stained sections for steatosis (0-3), lobular inflammation (0-3) and hepatocyte ballooning (0-2). Fibrosis was scored from picric acid sirius red stained slides (0-4). The NASH system is used for human liver biopsy No. 18. The presence of steatosis, lobular inflammation, hepatocellular ballooning degeneration, fibrosis, NAS and NASH by pattern recognition was assessed systematically. In this study, we evaluated one total cross-section of the liver per mouse in this study. This is about 15 times the size of a 18 th human liver biopsy. The pathology score was determined to be 0, +1, +2, or + 3. The location of the lesion (loss) (periportal, leaflet center (centromeric), and median (midzonal)) and fat accumulation (focal), periportal, and/or leaflet center) were scored. Another part of the score is the distribution of foci, multifocal and/or diffuse lesions. In addition, mild, moderate and severe lesions. These parameters constitute the total NASH score.

All immunohistochemical staining steps were performed on an automated immunostaining instrument using the Dako FLEX system; incubations were performed at room temperature and Tris buffered saline plus 0.05% Tween20, pH7.4(TBS-Dako Co.) was used for all washes and diluents. Thorough washing was performed after each incubation. Primary antibodies include anti-mouse SMA, F4/80, Mac-2, and sirius red picric acid. Control sections were treated with isotype control using the same concentration as the primary antibody to verify staining specificity.

From H&E stained sections were analyzed for White Adipose Tissue (WAT) adipocyte size. Using Aperio Image Scope Application, 3 local regions (tissue margin, tissue not surrounding blood vessels, tissue surrounding blood vessels) of the region were evaluated by measuring the area of 10 largest adipocytes per tissue sample. Within each tissue, 10 hot spots per region were quantified (μm)²) And averaged.

Pancreatic β -islet cells were identified by immunohistochemical staining.

Aperio automated Image quantification (automated Image quantification) was used to quantify the positive pixels of immunohistochemical staining, oil red O and sirius red staining. A positive pixel count algorithm is used to quantify the percentage of a particular stain present in the scanned slide image. The color range (hue and saturation range) and three intensity ranges (weak, positive and strong) were masked and evaluated. The algorithm counts the number and intensity sums in each intensity range, and three additional quantities: average intensity, ratio of strong/total, and average intensity of weak positive pixels. The positive pixel algorithm was modified to distinguish between orange and blue. For sirius red evaluation, changes were made from the normal "hue value" (0.1-0.96) and "color saturation" (0.04-0.29). The vasculature and artifacts were excluded from the analysis.

Liver IL-1b protein levels were quantified using a multiplex ELISA assay (Meso Scale Discovery, Rockville, Maryland).

Statistical analysis of hepatic histology scores were performed on GraphPad Prism 6(GraphPad software, USA) using a Bonferroni multiple comparison test. P values < 0.05 were considered statistically significant. Results are expressed as mean ± SEM. A comparison was made between group 2 (vector) and the following groups: group 3 (LIVRQNAC1,500mg/kg), group 4 (LIVRQNAC3,000mg/kg), group 5 (LIVRQNAC + G3,885 mg/kg), and (LRQNAC2, 469mg/kg).

Body weight and liver weight

Feeding fructose supplemented Western Diet (WDF) for 16 weeks resulted in a significant effect on body weight compared to control-fed animals. Animals fed WDF were significantly heavier (47.6 + -0.45 vs. 43.9 + -1.03 g; p < 0.01) than animals fed the control diet prior to administration of the test agent.

Weight loss compared to baseline values in all treatment groups; there was no significant difference in weight loss compared to vehicle (7.6. + -. 0.9, -6.9. + -. 1.3, -6.8. + -. 1.4, -5.7. + -. 1.2, -6.4. + -. 1.0, -4.7. + -. 1.6 and-3.9. + -. 1.5% for control, vehicle, LIVRQNAC (1500mg/kg), LIVRQNAC (3000mg/kg), LIVRQNAC + G, LRQNAC and OCA, respectively; p < 0.4992).

Vehicle-treated animals fed WDF had significantly higher liver weights (% by weight) compared to the control diet (7.22 ± 0.3 vs 5.05 ± 0.24%; p < 0.0001); however, in the animals fed WDF, no significant effect was found in any of the treated groups compared with the vehicle (7.22. + -. 03, 7.14. + -. 0.3, 7.19. + -. 0.26, 6.69. + -. 0.18, 7.02. + -. 0.5 and 6.81. + -. 0.2 for vehicle, LIVRQNAC (1500mg/kg), LIVRQNAC (3000mg/kg), LIVRQNAC + G, LILRQNAC and OCA, respectively; p < 0.7450).

Histology of liver

FATZO mice fed with the control diet developed mild steatosis, ballooning or fibrosis (fig. 6). FATZO mice fed WDF and treated with vehicle experienced significant steatosis, ballooning and fibrosis. In contrast to the major macrovesicular steatosis in the vehicle group, a mixture of predominantly vesicular and reduced macrovesicular steatosis was observed in the LIVRQNAC, LIVRQNAC + G and LRQNAC groups, as shown in fig. 7.

NAFLD activity scores were calculated from histological scores for steatosis (0-3) and ballooning (0-2) in fixed liver tissue. In WDF fed animals, all amino acid composition treatments resulted in a significant NAS reduction compared to vehicle treated group (figure 8). Treatment with LIVRQNAC and amino acid composition reduced hepatic steatosis compared to vehicle, although only LIVRQNAC + G and LRQNAC achieved statistical significance (p < 0.05), but not LIVRQNAC (LIVRQNAC3.0G/kg, p ═ 0.12). Treatment with all amino acid compositions significantly reduced biomarkers of hepatocyte ballooning, lipotoxicity and cell death. In summary, the improvement in liver pathology associated with amino acid composition is mainly due to the attenuation of hepatocyte ballooning. Compared to vehicle, OCA had no significant effect on NAS score and NAS components.

Liver from vehicle treated animals showed mild fibrosis; score 0.8 ± 0.1. When compared to the vehicle-treated group, only the livers from animals treated with LIVRQNAC (1500mg/kg) showed a significant reduction in fibrosis (0.2 ± 0.1 versus 0.8 ± 0.1, p < 0.01), whereas treatment with LIVRQNAC (3000mg/kg), LIVRQNAC + G or LRQNAC did not. Sirius red collagen staining demonstrated significantly lower collagen deposition with all amino acid composition treatments compared to vehicle (LIVRQNAC1500mg/kg, p < 0.01; LIVRQNAC3000 mg/kg, p < 0.01; LIVRQNAC + G, p ═ 0.09; LRQNAC, p < 0.05). OCA did not affect hepatic fibrosis scores or sirius red collagen staining areas.

Liver chemokines and cytokines

As shown in table 34, the proinflammatory cytokine IL-1b protein level was elevated in the liver of WDF-fed mice compared to control diet-fed mice.

TABLE 34 mean hepatic IL-1b protein levels following administration of amino acid compositions

Brief description of the drawings

Based on clinical observations, WDF-fed FATZO mice gained more weight than those fed with the control diet. All treatments were well tolerated in the FATZO mice. Both WDF-fed mice and control diet-fed mice lost weight during treatment, which may be due to stress associated with the twice-daily administration of the test article or vehicle by oral gavage.

NAS was significantly attenuated in all amino acid composition treatment groups compared to vehicle, mainly due to balloon-like expansion score. The hepatocyte ballooning was significantly reduced in all amino acid composition treated groups. In LIVRQNAC + G and LRQNAC treated groups, steatosis was significantly reduced. LIVRQNAC also reduced steatosis, although the difference was not significant. Consistent with histological and biochemical data, amino acid composition treatment did not affect the RNA levels of the fat de novo lipogenesis (de novo lipogenesis) enzymes FASN and ACACA.

The characteristics of hepatic cell steatosis differ from amino acid composition treatment. The liver of WDF-fed mice (vehicle group) showed predominantly bullous steatosis. In contrast, in all amino acid composition-treated groups, macrovesicular steatosis was reduced and was a mixture of vesicular and macrovesicular steatosis. The biological significance and mechanism of the amino acid composition on the large-vesicular to small-vesicular steatosis phenotype deserves further study.

Low dose LIVRQNAC treatment, but not high dose LIVRQNAC treatment, significantly attenuated liver fibrosis scores in the FATZO model of NAFLD. LIVRQNAC + G and LRQNAC had no effect on fibrosis. Nevertheless, sirius red collagen staining demonstrated that LIVRQNAC, LIVRQNAC + G and LRQNAC significantly reduced collagen deposition in the liver.

In summary, all three amino acid compositions tested in FATZO mice (LIVRQNAC, LIVRQNAC + G, and LRQNAC) attenuated NAFLD activity scores, hepatocyte ballooning and fibrosis. These amino acid compositions are useful for treating NASH. Glycine-containing amino acid compositions can further reduce pathways, leading to reduced liver fibrosis.

Example 7 treatment of a subject with an amino acid composition.

The studies described herein are characterized by administering to a subject having type 2 diabetes (T2DM) and non-alcoholic fatty liver disease (NAFLD) a composition comprising an amino acid. The purpose of this PRE-IND and IRB approved study was to determine the safety and tolerability of amino acid compositions and their impact on the structure and function of human physiology by observing various markers of fibrosis, inflammation, insulin sensitivity, glucose and lipid metabolism, and apoptosis after 6 and 12 weeks of administration. The composition includes about 1g L-leucine, about 0.5g L-isoleucine, about 0.5g L-valine, about 1.5g L-arginine (or 1.81g L-arginine HCl), about 2.0g L-glutamine, and about 0.15g N-acetylcysteine per stick pack for administration three times per day (e.g., a total of about 72g per day, or about 24g per day) in four stick packs.

In this study, subjects received the amino acid composition three times per day for 12 weeks. The amino acid was provided in powder form to dissolve in 12 ounces of water. The participant amino acid compositions were provided over a 12 week study period.

The main efficacy indicators (outome measures) of this study are safety and tolerability. Secondary efficacy indicators are the examination of the effects on human physiology by biomarkers related to metabolism, inflammation and fibrosis. Assessments were made at baseline (day 1), at study weeks 6 and 12.

Key criteria for selecting an object include the following: male or female aged 18 to 70 years, inclusive; willing and able to provide written informed consent; the medical history of T2DM or hemoglobin A1c (HbA1c) is more than or equal to 6.5% and less than 10% during screening; fatty liver disease was documented by one of the following criteria: a. the prior history of steatosis was confirmed within 3 months of screening by at least one of the following methods: liver fat measured by MRI, PDFF is more than or equal to 8%; the Control Attenuation Parameter (Control Attenuation Parameter) of the Fibroscan is more than or equal to 300 dB/m; liver biopsy showed non-NASH NAFLD steatosis > grade I. If the patient had no prior history of such documented steatosis disease within 3 months of the screening (as noted in 4 a), a liver fat score of ≧ 10% must be recorded at the time of screening using the formula:

predicted liver fat percentage is 10 (-0.805+ (0.282 metabolic syndrome [ is ═ 1/no ═ 0]) + (0.078 diabetes type 2 [ is ═ 2/no ═ 0]) + (0.525 log10 (insulin mU/L)) + (0.521 log10(AST U/L)) - (0.454 log10(AST/ALT))34

Note that: insulin, ALT and AST should be measured in fasting serum samples. Subjects must undergo stable exercise, diet and lifestyle routines within 3 months prior to screening without major weight fluctuations, i.e., subjects should be within ± 3% of their body weight within the last 3 months at the time of screening. The Body Mass Index (BMI) during screening is more than or equal to 32kg/m 2. For locations where the MRI apparatus cannot accommodate patients with a BMI ≧ 45kg/m2, an upper limit of 40-45kg/m2 may be applied. Patients must receive a stable dose of glucose-lowering drug (which may include metformin, sulfonylureas, dipeptidyl peptidase-4 [ DPP-4] inhibitors, sodium-glucose co-transporter 2(sodium-glucose co-transporter2) [ SGLT2] inhibitors or long-acting basal insulin) for at least 3 months prior to screening and are scheduled to retain the same drug during the study without expecting drug dose adjustments thereof. See section 8 below for a complete list of excluded diabetes-related drugs. Subjects may be included in the study if they receive simultaneous treatment with antihypertensive drugs (e.g., beta blockers, hydrochlorothiazides, ACE inhibitors, angiotensin receptor blockers), drugs for dyslipidemia (e.g., statins, fibrates) and drugs for hypothyroidism (e.g., levothyroxine) as long as they take stable doses and courses of these drugs for at least 3 months prior to screening and are scheduled to maintain the same drugs during the study without expecting drug dose adjustments thereof. The subject may take a vitamin supplement (e.g., multivitamins; vitamin E < 400 IU/day). However, they must take stable doses and courses of these vitamin supplements for at least 3 months prior to screening without the expected dose adjustments. Female subjects with childbearing potential (childbearing potential) must have a negative serum pregnancy test at screening and must agree to use an efficient contraceptive method during the course of the study and during the course of sexual intercourse 30 days after the last dose of study treatment. Fertility potential refers to those female subjects with ovarian failure who have not undergone hysterectomy, bilateral ovariectomy, or medical record, or women <50 years of age with any duration of amenorrhea.

LIVRQNAC reduces plasma pro-C3 and other key fibrotic biomarkers at week 12, supporting inhibition of fibrogenesis. Mean levels of plasma proC3, PIIINP and TIMP-1 were determined at baseline (day 1) and at weeks 6 and 12. FIG. 9A shows the mean value of Pro-C3 over time (ng/ml, +/-SEM) in a specified number of subjects. LIVRQNAC significantly (p < 0.05) reduced pro-C3 levels at week 12 compared to day 1. FIG. 9B shows that LIVRQNAC tended to decrease PIIINP and TIMP-1 levels (ng/ml, +/-SEM) at weeks 6 and 12 relative to day 1.

The findings of this study indicate that amino acid compositions have good safety and tolerability characteristics and affect biomarkers of human structure and function associated with fibrosis.

Example 8 TGF-beta 1 fibrogenic Gene expression by hepatic stellate cells

Primary human hepatic stellate cells were obtained from Samsara Sciences according to the following criteria for donor selection: adult age (between 18 and 50 years), normal BMI (> 18.5 and < 25), and no confounding liver disease. Cells grown to-80% confluence in complete HSC medium were seeded below generation 10 in T75 or T150 flasks into sterile, type I collagen-coated 96-well optical plastic microplates (ThermoScientific, 152036) at 6000 cells per well (-1250 cells/cm 2) and cultured overnight in DMEM containing 2% fetal bovine serum and 1% antibiotic-antifungal agent in a humidified incubator at 37 ℃ and 5% CO 2.

After overnight incubation, the plates were removed from the incubator, the medium was gently pipetted out and washed twice with 150 μ L DPBS per well. DPBS was removed and pre-treatment medium (. + -. single amino acid deletion,. + -. 1X HMDB DMEM + 1% antibiotic-antimycotic, 10mM HEPES,. + -. supplemented with amino acid dose; see experiment) was applied to the cells at 150. mu.L/well. The plate was returned to the incubator for 10.5 hours.

After 10.5 hours of pretreatment, the medium was removed from the cells and the same pretreatment medium now supplemented with 3ng/mL TGF β 1 was used. Each plate contained 1X human plasma amino acid (HMDB or PAA) concentration medium containing 3ng/mL of TGF β 1, 1X HMDB containing 0ng/mL of TGF β 1, and 1X HMDB containing 3ng/mL of TGF β 1+20 μ M Silibinin (Silybin) for use as controls. The plates were then incubated at 37 ℃ under 5% CO2 for 24 hours.

After 24 hours stimulation, the supernatant was removed and frozen in two separate aliquots at-80 ℃. Cells were then washed with buffer FCW (FastLane Cell Multiplex NR Kit, Qiagen, 216713) at 125 μ L per well. Immediately remove the wash buffer, apply 50 μ L of cell treatment mixture (containing genomic DNA clearing buffer) to lyse the cells, incubate for 10 min at room temperature. The RNA lysates were then transferred to 96-well qPCR plates, sealed, and the gDNA5 was digested on a thermal cycler at 75 ℃ for minutes. RNA lysates were frozen at-80 ℃.

Each 20. mu.L one-step RT-qPCR reaction contained 4. mu.L of RNA lysate. Gene expression of Hsp47 and Gapdh was multiplexed using HEX and FAM fluorescence channels, respectively, with a commercially available primer-probe mix (human Hsp47 primer-probe set, HEX; and human Gapdh primer-probe set, FAM from IDT). In each single amino acid deletion and supplementation, gene expression was assessed using the Δ Δ Cq method by normalization against its own 1 × HMDB concentration.

Results

Hsp47 gene expression

Tables 35, 36, 37, 38, 39 and 40 show the mean fold-change in Hsp47 gene expression in primary human hepatic stellate cells from three different healthy donors. LIVRQNac, LIVRQNacG, LIVRQNacS, RQNac, and N-acetylcysteine reduced Hsp47 gene expression in all three donors. LIVRQ reduced Hsp47 in only one of the three donors, and LIV had no significant effect on Hsp47 gene expression.

Leucine, isoleucine and valine did not significantly alter Hsp47 gene expression in any donor when the amino acids were administered alone. Arginine significantly increased Hsp47 gene expression in two of the three donors when the amino acids were administered alone. When administered alone, glutamine significantly increased Hsp47 gene expression in one of the three donors. N-acetylcysteine significantly reduced the expression of the Hsp47 gene in all three donors.

TABLE 35 fold change in Hsp47 gene expression following administration of amino acid composition, normalized to Gapdh expression in first donors

TABLE 36 fold change in Hsp47 gene expression following administration of a single amino acid composition, normalized to Gapdh expression in the first donor

Table 37 fold change in Hsp47 gene expression after administration of the amino acid composition, normalized to Gapdh expression in the second donor.

Table 38 fold change in Hsp47 gene expression after administration of a single amino acid composition, normalized to Gapdh expression in the second donor.

TABLE 39 fold change in Hsp47 gene expression following administration of amino acid composition, normalized to Gapdh expression in third donors

Table 40 fold change in Hsp47 gene expression after administration of a single amino acid composition, normalized to Gapdh expression in the second donor.

Example 9 reconstruction of the liver microenvironment to interrogate the triple culture model for fibrosis

Cell seeding and maintenance

A three-culture model of the three major cell types including liver (hepatocytes, kupffer cells, and stellate cells) was developed to evaluate the effect of the amino acid combination L-leucine, L-isoleucine, L-valine, L-arginine, L-glutamine, and N-acetylcysteine (LIVRQNAC) on fibrosis.

Hepatocytes, macrophages and astrocytes isolated from healthy donors were co-cultured using 96-well or 12-well transwell (corning).

Primary human hepatic stellate cells obtained from Samsara Sciences and grown to-80% confluence in complete HSC medium in T150 flasks were seeded onto the lower surface of a transwell membrane previously coated with collagen (Corning).

Primary human PBMC-derived macrophages were also added to the lower surface of the membrane once seeded with astrocytes. In Transwell, both cells were plated on hepatocyte plating medium (William's E medium (Gibco) supplemented with 10% heat-inactivated FBS (Atlanta Bio), 2mM Glutamax (Gibco) and 0.2% Primocin (InVivoGen)) at 37 ℃ with 5% CO₂The cells were incubated for 6 hours.

After 6 hours of culture, primary hepatocytes from healthy human donors were seeded on collagen gels on the upper surface of transwell. The three cultures were plated in hepatocyte plating medium as described above at 37 ℃ with 5% CO₂And (5) culturing. After 6 hours, the cells were washed once and 5% CO at 37 deg.C₂The liver cells were plated in the medium overnight. On day 1, cells were washed once and 5% CO in hepatocyte defined medium (Corning) supplemented with 2mM Glutamax (Gibco) and 1 XPicillin/streptomycin (P/S) at 37 ℃₂Incubate overnight.

Pretreatment with amino acids

On day 2, cells were washed twice with DPBS 1x (gibco) and kept at:

a. amino acid free WEM (american biologics) supplemented with 11mM glucose (Sigma), 0.272mM sodium pyruvate (Sigma), 1x P/s (gibco), and containing defined custom amino acid concentrations based on average physiological concentrations in blood; or

b. The same medium as above, wherein one concentration of the defined amino acid composition LIVRQNAC is 30X or 40X.

Cells were grown in defined media (a and b) at 37 ℃ with 5% CO₂The lower part is maintained for 24 hours.

Co-treatment with free fatty acids and different amino acid compositions

After 24 hours of pretreatment, cells were maintained in the same medium as above and exposed to 250 μ M Free Fatty Acids (FFAs) with a 2:1 (oleate: palmitate) ratio supplemented with 1ng/ml ± LIVRQNAC TNF- α (thermoliser). At 37 deg.C, 5% CO₂After 24 hours of subculture, the medium was removed from each side of the Transwell, and the cells were cultured under the same conditions as above for another 48 hours.

24 hours later cytokines/chemokines and procollagen I.alpha.1 were analyzed by ELISA

The multiple recombinant analytes were analyzed with supernatants from both sides of a 96-well transwell plate: IL6, IL8, MCP1, IP10, Gro α and procollagen I α 1(Fireplex kit, Abcam). YKL40 was measured in supernatants collected from 12-well transwell plates by ELISA (human chitinase 3-like protein 1(YKL40) Quantikine ELISA, R & D Systems).

Procollagen I alpha 1 secretion

Table 41 shows the fold change in procollagen I α 1 secreted by astrocytes treated with (FFAs TNF α) + LIVRQNAC at 30x, normalized to FFAs + TNF α baseline. Statistical significance calculated by T-test showed that LIVRQNAC significantly reduced the secretion of procollagen I.alpha.1. The levels of procollagen I α 1 on the hepatocyte side were measured, showing no difference between the two treatments (table 42).

TABLE 41 fold change in procollagen I.alpha.1 secretion by astrocytes in triplicate cultures following administration of 30 XLIVRQNAC compared to 1 XLIVRQNAC

TABLE 42 fold change in procollagen I.alpha.1 levels measured from the hepatocyte side in three cultures following 30 XLIVRQNAC administration compared to 1 XLIVRQNAC

Tables 43 and 44 show fold-change in cytokines and chemokines secreted from the macrophage and stellate or hepatocyte sides treated with 30x FFA + TNF α + LIVRQNAC, respectively, normalized to FFA + TNF α baseline (1x LIVRQNAC). Several pro-inflammatory cytokines (IL-6, IL-8, IP-10, aad GROalpha (CXCL1)) and chemokines (MCP1) were measured, which have been assigned chemoattractant properties and have been shown to be upregulated in NASH patients. Statistical significance calculated by T-test showed that treatment with liVRQNAC at 30X significantly reduced IL-6, IP-10, GRO α (CXCL1) and MCP1 levels compared to control liVRQNAC at 1X. IL-8 levels were also reduced when treated with 30x LIVRQNAC, however, did not show statistical significance compared to 1x LIVRQNAC.

TABLE 43 fold-change in cytokines and chemokines secreted by macrophages and astrocytes following administration of 30 XLIVRQNAC compared to 1 XLIVRQNAC

TABLE 44 fold change in cytokines and chemokines secreted by hepatocytes after 30 XLIVRQNAC administration compared to 1 XLIVRQNAC

Tables 45 and 46 show the fold change in YKL-40 secretion by macrophages and astrocytes or hepatocytes treated with FFA TNF α + LIVRQNAC at 40x, normalized to LIVRQNAC1 x.

Plasma levels of YKL40 (also known as chitinase 3-like protein 1[ CHI3L1]) are increased in several inflammatory diseases, including NASH. It has been shown that YKL40 plasma levels increase with the progression of fibrosis in NAFLD patients. Statistical significance calculated by T-test showed that 40x LIVRQNAC significantly reduced hepatocyte YKL40 levels. YKL-40 levels measured from the macrophage and astrocyte side also decreased when treated with LIVRQNAC40x, but did not show statistical significance compared to LIVRQNAC 1x treatment.

TABLE 45 fold change in YKL40 secretion by astrocytes and macrophages following 40 XLIVRQNAC administration compared to 1 XLIVRQNAC

TABLE 46 fold change in hepatocyte YKL40 secretion following 40 XLIVRQNAC administration compared to 1 XLIVRQNAC

Example 10 TGF-beta 1-induced proliferation of hepatic stellate cells

Proliferation of hepatic stellate cells is a key phenotypic feature of activated hepatic stellate cells. Primary human hepatic stellate cells were obtained from Samsara Sciences according to the following criteria for donor selection: adult age (between 18 and 50 years), normal BMI (> 18.5 and < 25), and no confounding liver disease. Cells from three different donors were grown to-80% confluence in complete HSC medium in T75 or T150 flasks at 6000 cells/well (-1250 cells/cm) under conditions of less than 10 passages²) Inoculated into sterile, type I collagen-coated 96-well optical plastic microplates (Thermoscientific, 152036) and incubated at 37 ℃ with 5% CO₂In a humidified incubator of (1), overnight in DMEM containing 2% fetal bovine serum and 1% antibiotic-antifungal agent.

After overnight incubation, the plates were removed from the incubator, the medium was gently pipetted out and washed twice with 150 μ L DPBS per well. DPBS was removed and pre-treatment medium (1 × HMDB amino acid DMEM + 1% antibiotic-antifungal agent, 10mM HEPES, ± supplementary treatment dose of multiple of HMDB amino acid concentration (X)) was administered to the cells at 150 μ L per well. Each treatment and dose was tested in three replicate wells per plate. Vehicle controls were tested in 6 replicate wells per plate. Plates were incubated overnight. After overnight pretreatment, the medium was removed from the cells and the same pretreatment medium supplemented with 3ng/mL TGF β 1 was used. To determine proliferation, cells were labeled with 10 μ M EdU (5-ethynyl-2' -deoxyuridine), which was incorporated into DNA during active DNA synthesis. The plates were then incubated at 37 ℃ with 5% CO₂And culturing for 24 hours.

After 24 hours stimulation, the supernatant was removed and frozen in two separate aliquots at-80 ℃. Then the cells were washed with DPBS and lysed with 4% paraformaldehydeThe solution was fixed for 20 minutes. Cells were permeabilized with 0.1% Triton X-100 and click-iT was used^TMEdU Alexa Fluor^TMThe 555HCS assay (Invitrogen) labeled EdU according to the manufacturer's instructions. The nucleus is labeled with the cell-permeable DNA binding dye Hoechst 33342.

Cells were imaged using an ImageXpress microscopy confocal high content imager (Molecular Devices) using a 10x Plan Apo objective. Twelve frames are imaged per well. EdU labeled with Alexa Fluor555 was detected in the Texas Red channel. Nuclei labeled with Hoechst33342 were detected in the DAPI channel. Image analysis was performed using MetaXpress6.2.3.733 edition (Molecular Devices). The number of proliferating cells (defined as those nuclei positive for the EdU marker (EdU +)) and total nuclear counts were determined for each condition. The percent EdU positive cells (% EdU +) was determined as the number of EdU positive nuclei divided by the total number of nuclei per well. Nuclear counts and fold change in% EdU + cells were calculated relative to baseline amino acid (1 × HMDB) vector (PBS) conditions stimulated with 3ng/mL TGF β 1. The mean of each phenotype measurement in wells of 3ng/mL TGF β 1 treated PBS vector was defined as baseline. The phenotype measurements in each well were divided by the baseline. A score equal to 1 indicates no change from baseline. A score less than or greater than 1 means a decrease or an increase, respectively. Statistical analysis (mean, standard deviation calculation and two-tailed t-test) was performed on the log2 transformation scores.

Results

Table 47 shows log2 transformation of fold change in percentage of actively proliferating EdU positive cells relative to PBS vector conditions in primary human hepatic stellate cells from three different donors. LIVRQNAC reduced the percentage of actively proliferating EdU positive cells in all three donors relative to 3ng/mL of TGF β 1 vector. Table 48 shows log2 transformation of fold change in nuclear counts relative to PBS vector conditions in primary human hepatic stellate cells from three different donors. LIVRQNAC reduced nuclear counts relative to 3ng/mL TGF β 1 vector under the highest two dose conditions in two of the three donors tested.

Watch 47.

Watch 48.

While the present invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

All references, issued patents and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.