OA19827A

OA19827A - Amino acid compositions and methods for the treatment of liver diseases.

Info

Publication number: OA19827A
Application number: OA1201900243
Authority: OA
Inventors: Michael Hamill; Raffi AFEYAN; Chung-Wei Lee; Harry LUITHARDT; David Berry
Original assignee: Axcella Health Inc.
Priority date: 2016-12-19
Filing date: 2017-12-19
Publication date: 2021-05-26

Abstract

This disclosure provides pharmaceutical compositions comprising amino acid entities and uses thereof. Methods for improving liver function and for treating liver diseases comprising administering an effective amount of the compositions to a subject in need thereof are also disclosed.

Description

AMINO ACID COMPOSITIONS AND METHODS FOR THE TREATMENT OF LIVER DISEASES

RELATED APPLICATIONS

This application claims priority to U.S. Serial No. 62/436,073 filed December 19, 2016, U.S. Serial No. 62/443,205 filed January 6, 2017, U.S. Serial No. 62/491,773 filed April 28, 2017, U.S. Serial No. 62/545,322 filed August 14, 2017, and U.S. Serial No. 62/576,267 filed October 24,2017, the contents of which are each incorporated herein by reference in their entireties.

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BACKGROUND

Non-alcoholic fatty liver disease (NAFLD) is a disease characterized by fatty deposits in the liver due to causes other than alcohol. NAFLD is the most prévalent liver disease in developed countries and affects close to 25% of the people in the United States. Non-alcoholic steatohepatitis (NASH) is the most severe form of NAFLD, which can lead to inflammation of the liver, fibrosis, cirrhosis, chronic liver failure, and hepatocellular carcinoma (HCC).

Currently, there are no approved thérapies for treating NASH or NAFLD. Accordingly, there is an unmet need for new treatments in NAFLD and NASH.

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SUMMARY

Disclosed herein, at least in part, is a composition including at least four different amino acid entities.

In some embodiments, the composition is capable of one, two, three, four, five, or six or allof:

a) decreasing or preventing liver fibrosis;

b) decreasing or preventing liver injury;

c) decreasing or preventing hépatocyte inflammation;

d) improving, e.g., increasing, glucose tolérance;

e) decreasing or preventing steatosis;

f) decreasing or preventing hépatocyte ballooning; or

g) improving gut function.

In some embodiments, the composition comprises a leucine (L)-amino acid entity, an arginine (R)-amino acid entity, a glutamine (Q)-amino acid entity; and an antioxidant or reactive oxygen species (ROS) scavenger (e.g., a N-acetylcysteine (NAC) entity, e.g., NAC). In some embodiments, at least one amino acid entity is not provided as a peptide of more than 20 amino acid residues in length.

In some embodiments:

(i) an amino acid entity (e.g., at least one, two, or three of the amino acid entities) of (a) is selected from Table 2; and/or (ii) (A) one or both of the R-amino acid entity and the Q-amino acid entity are présent at a higher amount (wt. %) than the L-amino acid entity, or (B) the composition further comprises a serine (S)-amino acid entity. .

In any of the aspects and embodiments disclosed herein, the wt. ratio of the L-amino acid entity, the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or sait thereof is about 0.5 to 3 : 0.5 to 4 : 1 to 4 : 0.1 to 2.5, e.g., the wt. ratio of the L-amino acid entity, the Ramino acid entity, the L-glutamine or a sait thereof, and the NAC or sait thereof is about 1 : 1.5 : 2 : 0.15 or about 1 : 1.5 : 2 : 0.3. In certain embodiments, the wt. ratio of the L-amino acid entity, the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or sait thereof is about 1 +/-15% : 1.5 +/- 15% : 2 +/- 15% : 0.15 +/-15% or about 1 +/-15% : 1.5 +/- 15% : 2 +/15% : 0.3 +/- 15%. In any of the aforesaid embodiments in this paragraph, the wt. ratio of the Lamino acid entity, the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or sait thereof is about 1 : 0.75 : 2 : 0.15 or about 1 : 0.75 : 2 : 0.3. In any of the aforesaid embodiments in this paragraph, the wt. ratio of the L-amino acid entity, the R-amino acid entity, the Lglutamine or a sait thereof, and the NAC or sait thereof is about 1 +/- 15% : 0.75 +/- 15%: 2 +/15%: 0.15 +/-15% or about 1 +/-15%: 0.75 +/- 15% : 2 +/- 15% : 0.3 +/- 15%.

In any of the aspects and embodiments disclosed herein, the wt. ratio of the L-amino acid entity, the I-amino acid entity, the V-amino acid entity, the R-amino acid entity, the L-glutamine or sait thereof, and the NAC or sait thereof is about 1 : 0.5: 0.5: 1.5 : 2 : 0.15 or about 1 : 0.5: 0.5: 1.5:2:0.3.

In any of the aspects and embodiments disclosed herein, the composition further comprises one or both of L-glycine and L-serine. In some embodiments, the composition comprises an L-amino acid entity, an I-amino acid entity, an V-amino acid entity, an R-amino

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I I I acid entity, an L-glutamine or a sait thereof, an NAC or a sait thereof, and an L-glycine. In certain embodiments, the composition comprises an L-amino acid entity, an l-amino acid entity, a V-amino acid entity, an R-amino acid entity, an L-glutamine or a sait thereof, an NAC or a sait thereof, and an L-serine. In certain embodiments, the composition comprises an L-amino acid 5 entity, an l-amino acid entity, an V-amino acid entity, an R-amino acid entity, an L-glutamine or a sait thereof, an NAC or a sait thereof, an L-glycine, and an L-serine. In any of the aforesaid embodiments in this paragraph, the wt. ratio of the L-amino acid entity, the l-amino acid entity, the V-amino acid entity, the R-amino acid entity, the L-glutamine or sait thereof, and the NAC or sait thereof is about 1 : 0.5: 0.5: 1.5 : 2 : 0.15 or about 1 : 0.5: 0.5: 1.5 :2 : 0.3. In any of the aforesaid embodiments in this paragraph, the wt. ratio of the L-amino acid entity, the l-amino acid entity, the V-amino acid entity, the R-amino acid entity, the L-glutamine or sait thereof, and the NAC or sait thereof is about 1 +/-15% : 0.5 +/- 15% : 0.5 +/- 15% : 1.5 +/-15% : 2 +/-15% : 0.15 +/- 15% or about 1 +/- 15% : 0.5 +/- 15%: 0.5 +/- 15%: 1.5+/- 15% : 2 +/- 15%: 0.3+/- 15%.

In any of the aspects and embodiments disclosed herein, the composition comprises about 15 0.5 g to about 10 g of the L-amino acid entity, about 0.25 g to about 5 g of the l-amino acid entity, about 0.25 g to about 5 g of the V-amino acid entity, about 0.5 g to about 20 g of the Ramino acid entity, about 1 g to about 20 g of the L-glutamine or a sait thereof, and about 0.1 g to about 5 g of the NAC or a sait thereof, e.g., the composition comprises about 1 g of the L-amino acid entity, about 0.5 g of the l-amino acid entity, about 0.5 g of V-amino acid entity, about 1.5 g of R-amino acid entity, about 2 g of L-glutamine or a sait thereof, and about 0.15 g or about 0.3 g of NAC or a sait thereof. In certain embodiments, the composition comprises about 0.15 g of NAC. In certain embodiments, the composition comprises about 0.3 g of NAC. In embodiments, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the II I

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I amino acid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acid entity, about 4 g 25 of L-glutamine or a sait thereof, and about 0.9 g of NAC or a sait thereof.

In any of the aspects and embodiments disclosed herein,, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the l-amino acid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or a sait thereof, about 0.9 g of NAC or a sait thereof, and about 6 g of L-serine or a sait thereof. In embodiments, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the l-amino acid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or a sait thereof, about 0.9 g of NAC or a sait thereof, and about 6.67 g of L-serine or a sait thereof.

In embodiments, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the l-amino acid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or a sait thereof, about 0.9 g of NAC or a sait thereof, about 9 g of L-serine or 5 a sait thereof, and about 9 g of L-glycine or a sait thereof. In embodiments, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the l-amino acid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or a sait thereof, about 0.9 g of NAC or a sait thereof, about 3.33 g of L-serine or a sait thereof, and about 3.33 g of L-glycine or a sait thereof.

In one aspect, the invention features a composition including free amino acids, wherein the amino acids include arginine, glutamine, N-acetylcysteine, and a branched-chain amino acid chosen from one, two, or ail of leucine, isoleucine, and valine.

In any of the aspects and embodiments disclosed herein, the branched-chain amino acid is leucine, isoleucine, and valine.

In any of the aspects and embodiments disclosed herein, the wt ratio of leucine, isoleucine, valine, arginine, glutamine, N-acetylcysteine is 1 : 0.5 : 0.5 : 1.5 : 2 : 0.15. In certain embodiments, the wt ratio of leucine, isoleucine, valine, arginine, glutamine, N-acetylcysteine is 1 +/-15% : 0.5 +/- 15%: 0.5 +/-15%: 1.5 +/-15%: 2 +/- 15%: 0.15+/- 15%.

In any of the aspects and embodiments disclosed herein, a total weight (wt) of the amino acids is about 2 g to about 60 g. In some embodiments, the total wt of the amino acids is about 6 g, about 12 g, about 18 g, about 24 g, or about 48 g.

In any of the aspects and embodiments disclosed herein, the composition includes about 0.5 g to about 10 g of leucine, about 0.25 g to about 5 g of isoleucine, about 0.25 g to about 5 g of valine, about 1 g to about 20 g of arginine, about 1 g to about 20 g of glutamine, and about 0.1 g to about 5 g of N-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the composition includes about 1 g of leucine, about 0.5 g of isoleucine, about 0.5 g of valine, about 1.5 g of arginine, about 2 g of glutamine, and about 0.15g of N-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the composition includes about 2 g of leucine, about 1 g of isoleucine, about 1 g of valine, about 3.0 g of arginine, about 4 g of glutamine, and about 0.3 g of N-acetylcysteine.

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In any of the aspects and embodiments disclosed herein, the composition includes about 4 g of leucine, about 2 g of isoleucine, about 2 g of valine, about 6.0 g of arginine, about 8 g of glutamine, and about 0.6 g of N-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the amino acids include about 10 wt % to about 30 wt % leucine, about 5 wt % to about 15 wt % isoleucine, about 5 wt % to about 15 wt % valine, about 15 wt % to about 40 wt % arginine, about 20 wt % to about 50 wt % glutamine, and about 1 wt % to about 8 wt % n-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the amino acids include about 16 wt % to about 18 wt % leucine, about 7 wt % to about 9 wt % isoleucine, about 7 wt % to about 9 wt % valine, about 28 wt % to about 32 wt % arginine, about 31 wt % to about 34 wt % glutamine, and about 1 wt % to about 5 wt % n-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the amino acids include about 16.8 wt % leucine, about 8.4 wt % isoleucine, about 8.4 wt % valine, about 30.4 wt % arginine, about 33.6 wt % glutamine, and about 2.5 wt % n-acetylcysteine.

In some embodiments of any of the compositions or methods disclosed herein (wherein the ratios discussed in (1)-(26) below are weight ratios):

1) the ratio of the L-amino acid entity to the l-amino acid entity is at least 1.5:1, or at least 1.75:1, and not more than 2.5:1 or not more than 3:1, e.g., the ratio of the L-amino acid entity to the l-amino acid entity is about 2:1;

2) the ratio of L-amino acid entity to V-amino acid entity is at least 2:1, at least 3:1, at least 3.5:1, at least 4:1, or at least 5:1, and not more than 6: 1, e.g., the ratio of L-amino acid entity to V-amino acid entity is about 4:1;

3) the ratio of the L-amino acid entity to the R-amino acid entity is at least 1:1, at least 3.5:3, at least 4:3, or at least 2:1, and not more than 5:2, e.g., the ratio of the L-amino acid entity to the R-amino acid entity is about 4:3;

4) the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is at least 0.5:1, or at least 0.75:1, and not more than 1.5 to 1 or not more than 2:1, e.g., the ratio ofthe L-amino acid entity to the L-glutamine or sait thereof is about 1:1;

5) the ratio of the L-amino acid entity to the NAC entity or a sait thereof is at least 2:1, at least 3:1, at least 3.5:1, or at least 4:1, and not more than 5 to 1 or not more than 6:1, e.g., the ratio of the L-amino acid entity to the NAC entity or sait thereof is about 4:1 (e.g., 4:0.9);

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6) optionally wherein the ratio of the L-amino acid entity to the S-amino acid entity or a sait thereof is greater than 1:3, greater than 1.5:3, about 2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g., the ratio of the L-amino acid entity to the S-amino acid entity is about 2:3, or the ratio of the L-amino acid entity to the S-amino acid entity is about 3:5; or

7) a combination of two, three, four, five, or six of (1)-(6).

In some embodiments of any of the compositions or methods disclosed herein:

8) the ratio of l-amino acid entity to V-amino acid entity is at least 1:1, at least 1.5:1, or about 2:1, and not more than 2.5:1 or not more than 3:1, e.g., the ratio of l-amino acid entity to V-amino acid entity is about 2:1;

9) the ratio ofthe l-amino acid entity to the R-amino acid entity is greater than 1:3, greater than 1.5:3, or about 2:3, and not more than 2.5:3 or not more than 1:1, e.g., the ratio of the l-amino acid entity to the R-amino acid entity is about 2:3;

10) the ratio of the l-amino acid entity to the L-glutamine or a sait thereof is at least 1:4, at least 1:3, or about 1:2, and not more than 1:1 or not more than 2:1, e.g., the ratio of the Iamino acid entity to the L-glutamine or sait thereof is about 1:2;

11) the ratio of the l-amino acid entity to the NAC entity or a sait thereof is at least 1:1, at least 1.5:1, or about 2:1, and not more than 2.5:1 or not more than 3:1, e.g., the ratio of the Iamino acid entity to the NAC entity or sait thereof is about 2:1 (e.g., 2:0.9);

12) optionally wherein the ratio of the l-amino acid entity to the S-amino acid entity or a sait thereof is greater than 1:4, greater than 1.5:4, about 1:3, or about 3:10, and not more than 1.5:3 or 2:3, e.g., the ratio of the l-amino acid entity to the S-amino acid entity is about 1:3, or the ratio of the l-amino acid entity to the S-amino acid entity is about 3:10; or

13) a combination of two, three, four, or five of (8)-(12).

In some embodiments of any of the compositions or methods disclosed herein:

14) the ratio of the V-amino acid entity to the R-amino acid entity is greater than 1:4, greater than 1.5:4, or about 1:3, and not more than 1:2 or not more than 1:1, e.g., the ratio ofthe V-amino acid entity to the R-amino acid entity is about 1:3;

15) the ratio of the V-amino acid entity to the L-glutamine or a sait thereof is greater than 1:5, or greater than 1.5:5, about 1:4, and not more than 1.5:4 or not more than 1:3, e.g., the ratio of the V-amino acid entity to the L-glutamine or sait thereof is about 1:4;

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16) the ratio of the V-amino acid entity to the NAC entity or a sait thereof is at least 1:2, at least 1.5:2, or about 1:1, and not more than 1.5:1 or not more than 2:1, e.g., the ratio of the Vamino acid entity to the NAC entity or sait thereof is about 1:1 (e.g., 1:0.9);

17) optionally wherein the ratio of the V-amino acid entity to the S-amino acid entity or a 5 sait thereof is greater than 1:8, greater than 1:7, about 1:6, or about 3:20, and not more than 1.5:6 or 1:3, e.g., the ratio of the V-amino acid entity to the S-amino acid entity is about 1:6, or the ratio of the V-amino acid entity to the S-amino acid entity is about 3:20; or

18) a combination of two, three, or four of (14)-(17).

In some embodiments of any of the compositions or methods disclosed herein:

19) the ratio of the R-amino acid entity to the L-glutamine or a sait thereof is greater than

1:2, greater than 1.25:2, or about 3:4, and not more than 3.5:4 or not more than 1:1, e.g., the ratio of the R-amino acid entity to the L-glutamine or sait thereof is about 3:4;

20) the ratio of the R-amino acid entity to the NAC entity or a sait thereof is at least 4:1, at least 4:1.5, or about 3:1, and not more than 3:1.5 or not more than 3:2, e.g., the ratio of the R- amino acid entity to the NAC entity or sait thereof is about 3:1 (e.g., 3:0.9);

21) optionally wherein the ratio of the R-amino acid entity to the S-amino acid entity or a sait thereof is greater than 1:4, greater than 1:3, about 1:2„ or about 9:20, and not more than 1.5:2 or 1:1, e.g., the ratio of the R-amino acid entity to the S-amino acid entity is about 1:2, or the ratio of the R-amino acid entity to the S-amino acid entity is about 9:20; or

22) a combination of two or three of (19)-(21).

In some embodiments of any of the compositions or methods disclosed herein:

23) the ratio of the L-glutamine to the NAC entity or a sait thereof is at least 5:1, at least 5:1.5, or about 4:1, and not more than 4:1.5 or not more than 3:1, e.g., the ratio of the Lglutamine to the NAC entity or sait thereof is about 4:1 (e.g., 4:0.9);

24) optionally wherein the ratio of the L-glutamine to the S-amino acid entity or a sait thereof is greater than 1:3, greater than 1.25:3, about 2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g., the ratio of the L-glutamine to the S-amino acid entity is about 2:3, or the ratio of the Lglutamine to the S-amino acid entity is about 3:5; or

25) a combination of (23) and (24).

In some embodiments of any of the compositions or methods disclosed herein:

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26) the ratio of the NAC entity to the S-amino acid entity or a sait thereof is greater than | 1:8, greater than 1:7, or about 1:6, and not more than 1:5 or not more than 1.5:5, e.g., the ratio of the NAC entity to the S-amino acid entity is about 1:6 (e.g., 0.9:6 or 2.7:20).

| In an embodiment, the composition satisfies the properties of (1)-(7) defined above.

In certain embodiments, the composition satisfies the properties of at least 2, 3, 4, 5, 6, or | 7 of any of properties (1)-(26) defined above.

In an embodiment, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the | V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, and

NAC or a sait thereof is 12 : 6 : 3 : 9 : 12 : 2.7.

| 10 In an embodiment, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the

V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, | NAC or a sait thereof, and the S-amino acid entity is 12 : 6 : 3 : 9 : 12 : 2.7 : 18.

In an embodiment, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the | V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof,

NAC or a sait thereof, and the S-amino acid entity is 12 : 6 : 3 : 9 : 12 : 2.7 : 20.

| In an embodiment, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the

V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, and

NAC or a sait thereof is 12 +/-15% : 6 +/-15% : 3 +/- 15% : 9 +/-15% : 12 +/- 15% : 2.7 +/15%.

J 20 In an embodiment, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the

V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof,

NAC or a sait thereof, and the S-amino acid entity is 12 +/- 15% : 6 +/- 15% : 3 +/-15% : 9 +/* 15% : 12 +/- 15% : 2.7 +/- 15% : 18 +/-15%.

| In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, | NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 : 6 : 3 : 9 : 12 : 2.7 : 9: 9.

In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V- amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 : 6 : 3 : 9 : 12 : 2.7 : 10: 10.

In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof,

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NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 +/-15% : 6 +/-15% : 3 +/- 15% : 9 +/- 15% : 12 +/- 15% : 2.7 +/- 15% : 9 +/- 15% : 9 +/- 15%. In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 +/- 15% : 6 +/- 15% : 3 +/- 15% : 9 +/- 15% : 12 +/15% : 2.7 +/- 15% : 10 +/-15% : 10 +/-15%.

In any of the aspects and embodiments disclosed herein, the composition further includes one or more pharmaceutically acceptable excipients.

In some embodiments, the excipients are selected from the group consisting of citric acid, lecithin, a sweetener, a dispersion enhancer, a flavoring, a bittemess masking agent, and a natural or artificial coloring.

In some embodiments, the composition is in the form of a solid, powder, solution, or gel. In some embodiments, the amino acids consist of leucine, isoleucine, valine, arginine, glutamine and N-acetylcysteine.

Another aspect of the invention features a dietary composition including the composition of any one of the foregoing aspects or embodiments, e.g., wherein the dietary composition is chosen from a medical food, a functional food, or a supplément.

In some embodiments, the dietary composition is chosen from a medical food, a functional food, or a supplément.

In some embodiments, the subject has type 2 diabètes and/or a relatively high BMI.

In some embodiments, the subject has non-alcoholic fatty liver disease (NAFLD).

In some embodiments, the subject has non-alcoholic fatty liver (NAFL).

In some embodiments, the subject has pédiatrie NAFLD.

In some embodiments, the patient has steatosis.

In some embodiments, the subject has non-alcoholic steatohepatitis (NASH).

In some embodiments, the subject has fibrosis.

In some embodiments, the subject has cirrhosis.

In some embodiments, the subject has AFLD.

In some embodiments, the subject has ASH.

In some embodiments, the subject has hepatocarcinoma, an increased risk of liver failure, or an increased risk of death.

In some embodiments, the subject has type 2 diabètes.

In some embodiments, the composition promûtes weight loss in the subject.

In some embodiments of the method or the dietary composition for use, the composition is administered at a dose of about 15 g/d to about 90 g/d.

In some embodiments of the method or the dietary composition for use, the composition is administered at a dose of about 18 g/d, about 24 g/d, about 36/d, about 54 g/d, or about 72 g/d.

In some embodiments of the method or the dietary composition for use, the composition is administered one, two, to three times per day.

In some embodiments of the method or the dietary composition for use, the composition is administered at a dose of about 6 g, about 8 g, about 12 g, about 16 g, about 18 g, or about 24 g three times per day.

One embodiment provides a nutritional supplément, dietary formulation, fimctional food, medical food, food, or beverage comprising a composition described herein. Another embodiment provides a nutritional supplément, dietary formulation, fimctional food, medical food, food, or beverage comprising a composition described herein for use in the management of any of the diseases or disorders described herein. The composition disclosed herein can be used to improve liver function in a subject with fatty liver disease, such as non-alcoholic fatty liver disease (NAFLD; e.g. NAFL or non-alcoholic steatohepatitis (NASH)) or alcoholic fatty liver disease (AFLD; e.g., alcoholic steatohepatitis (ASH)). Thus, a method, including a dosage regimen, for treating (e.g., inhibiting, reducing, ameliorating, or preventing) various liver disorders, diseases, or symptoms thereof using the amino acid entity compositions is disclosed herein. The composition can also be used as a dietary composition, e.g., a medical food, a fimctional food, or a supplément.

Another aspect of the invention features a method for treating one or more symptoms selected from the group consisting of decreased fat metabolism, hépatocyte apoptosis, hépatocyte ballooning, inflammation of adipose tissue, inflammation of hepatic tissue, fibrosis, and oxidative stress, wherein the method includes administering to a subject in need thereof an effective amount of the composition of any one of aspects or embodiments disclosed herein.

In some embodiments, the subject has non-alcoholic fatty liver disease (NAFLD).

In some embodiments, the subject has non-alcoholic fatty liver (NAFL).

In some embodiments, the subject has pédiatrie NAFLD.

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In some embodiments, the patient has steatosis.

In some embodiments, the subject has non-alcoholic steatohepatitis (NASH).

In some embodiments, the subject has alcoholic fatty liver disease (AFLD).

In some embodiments, the subject has alcoholic steatohepatitis (ASH)).

In some embodiments, the subject has fibrosis.

In some embodiments, the subject has cirrhosis.

In some embodiments, the subject has one, two, or more (e.g., ail) of hepatocarcinoma, an increased risk of liver failure, or an increased risk of death.

In some embodiments, the subject has type 2 diabètes.

Another aspect of the invention features a method for treating non-alcoholic fatty liver disease (NAFLD) including administering to a subject in need thereof an effective amount of the composition of any one of the aspects or embodiments disclosed herein.

In some embodiments, the subject has NAFL.

In some embodiments, the subject has pédiatrie NAFLD.

In some embodiments, the patient has steatosis.

Another aspect of the invention features a method for treating non-alcoholic steatohepatitis (NASH) including administering to a subject in need thereof an effective amount of the composition of any one of the aspects or embodiments disclosed herein.

In some embodiments, the subject has fibrosis.

Another aspect of the invention features a method for treating AFLD including administering to a subject in need thereof an effective amount of the composition of any one of the aspects or embodiments disclosed herein.

In some embodiments, the subject has ASH.

Another aspect of the invention features a method for treating cirrhosis including administering to a subject in need thereof an effective amount of the composition of any one of the aspects or embodiments disclosed herein.

In some embodiments, administering the composition results in an improvement in one or more metabolic symptoms in the subject. In some embodiments, the improvement in one or more metabolic symptoms is selected from the following: increased free fatty acid and lipid

I metabolism, improved mitochondrial function, white adipose tissue (WAT) browning, decreased reactive oxygen species (ROS), increased levels of glutathione (GSH), decreased hepatic ^e inflammation, decreased hépatocyte ballooning, improved gut barrier function, increased insulin sécrétion, or improved glucose tolérance.

^— In some embodiments, the increased free fatty acid and lipid metabolism occurs in the liver.

^e In some embodiments, administration of the composition results in an improvement in one or more metabolic symptoms after a treatment period of 24 hours.

In some embodiments, the method further includes determining the level of one, two,

I three, four, five, six, seven, eight, nine, ten, or more (e.g., ail) of the following:

a) alanine aminotransferase (ALT);

Ib) aspartate aminotransferase (AST);

c) adiponectin;

Id) N-terminal fragment of type ΙΠ collagen (proC3);

e) caspase-cleaved keratin 18 fragments (M30 and M65);

If) IL-1 beta;

g) C-reactive protein;

Ih)PIHNP;

i) a tissue inhibitor of metalloproteinase (TIMP); e.g., ΊΊΜΡ1 or TIMP2;

110 j) MCP-1;

k)FGF-21;

- 1) Collai;

B m) Acta2;

n n) a matrix metalloproteinase (MMP), e.g., MMP-13, MMP-2, MMP-9, MT1-MMP,

I 15 MMP-3, or MMP-10;

- o)ACOXl;

I p) IL-10; or _ q) NF-kB.

In some embodiments, administration of the composition results in an improvement in — one or more of a)-q) after a treatment period of 24 hours.

H In some embodiments, the composition is administered prior to a meal.

I ¹²

I

In some embodiments, the composition is administered concurrent with a meal.

In some embodiments, the composition is administered following a meal.

In some embodiments, the composition is administered with a second agent.

In some embodiments, the second agent is selected from the group consisting of a famesoid X receptor (FXR) agonist, a stearoyl CoA desaturase inhibitor, a CCR2 and CCR5 chemokine antagonist, a PPAR alpha and delta agonist, a caspase inhibitor, a galectin-3 inhibitor, an acetyl CoA carboxylase inhibitor, or an ileal sodium bile acid co-transporter inhibitor.Another aspect of the invention provides a method of maintaining or improving liver health comprising administering to a subject an effective amount of any of the compositions described herein. Another embodiment provides a method of providing nutritional support or supplémentation to a subject suffering from NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH) comprising administering to the subject an effective amount of a composition described herein. Yet another embodiment provides a method of providing nutritional supplémentation that aids in the management of NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH) to a subject comprising administering to the subject in need thereof an effective amount of a composition described herein.

Additional features and embodiments of the présent invention include one or more of the following.

Another aspect of the invention features a composition comprising:

a) a L-amino acid entity chosen from L-leucine or a sait thereof, or P-hydroxy-βmethybutyrate (HMB) or a sait thereof, or a combination of L-leucine or a sait thereof and HMB or a sait thereof;

b) an R-amino acid entity chosen from:L-arginine or a sait thereof, ornithine or a sait 5 thereof, or creatine or a sait thereof, or a combination of two or three of L-argininc or a sait thereof, ornithine or a sait thereof, or creatine or a sait thereof;

c) L-glutamine or a sait thereof; and

d) N-acetylcysteine (NAC) or a sait thereof.

In an embodiment, L-leucine is provided as part of a dipeptide comprising L-lcucine, or a 10 sait thereof, or a tripeptide comprising L-leucine, or a sait thereof.

In an embodiment, L-arginine is provided as part of a dipeptide comprising L-arginine, or a sait thereof, or a tripeptide comprising L-arginine, or a sait thereof.

In an embodiment L-glutamine is provided as part of a dipeptide comprising Lglutamine, or a sait thereof, or a tripeptide comprising L- glutamine, or a sait thereof.

In an embodiment NAC is provided as a part of a dipeptide comprising NAC, or a sait thereof, or a tripeptide comprising NAC, or a sait thereof.

In some embodiments of any ofthe compositions or methods disclosed herein, one, two, three, or four of méthionine (M), tryptophan (W), valine (V), or cysteine (C) is absent, or if ’ présent, is présent at a percentage of the composition by weight (wt. %) of less than 10%. In some embodiments, the total wt. % of (a)-(d) is greater than the total wt. % of any other amino acid entity in the composition.

In some embodiments of any of the compositions or methods disclosed herein, one, two, three, or four ofthe amino acids in (a)-(d) is provided as part of a dipeptide or tripeptide, e.g., m an amount of at least 10 wt. % of the composition. In certain embodiments, the dipeptide is a homodipeptide or heterodipeptide ofany ofthe amino acids in (a)-(d), e.g., one, two, three, or four ofthe amino acids m (a)-(d) is a homodipeptide or heterodipeptide. In certain embodiments, the tripeptide is a homotripeptide or heterotripeptide of any of (a)-(d), e.g., one, two, three, or four of (a)-(d) is a homotripeptide or heterotripeptide.

In some embodiments of any ofthe compositions or methods disclosed herein, (a) is a Lamino acid entity dipeptide or a sait thereof (e.g., a L-leucine dipeptide or a sait thereof). In some embodiments, (a) is a homodipeptide. In some embodiments, (a) is a heterodipeptide, e.g., 20 Ala-Leu.

In some embodiments of any ofthe compositions or methods disclosed herein, (b) is a Larginme dipeptide or a sait thereof. In some embodiments, (b) is a homodipeptide. In some embodiments, (b) is a heterodipeptide, e.g., Ala-Arg.

In some embodiments of any of the compositions or methods disclosed herein, (c) is a L25 glutamine dipeptide or a sait thereof. In some embodiments, (c) is a homodipeptide, e.g., GinGin. In some embodiments, (c) is a heterodipeptide, e.g., Ala-Gln.

In some embodiments of any ofthe compositions or methods disclosed herein:

f) a wt. % ofthe L-glutamine or a sait thereof in the composition is greater than the wt. % of the R-amino acid entity;

g) the wt. % ofthe L-glutamine or a sait thereof in the composition is greater than the wt.

% of the L-amino acid entity;

h) the wt. % ofthe R-amino acid entity in the composition is greater than the wt. % ofthe L-amino acid entity; or

i) a combination of two or three of (f)-(h).

In some embodiments of any of the compositions or methods disclosed herein,the wt. % of the L-glutamine or a sait thereof in the composition is at least 5% greater than the wt. % ofthe R-amino acid entity, e.g., the wt. % of the L-glutamine or a sait thereof is at least 10%, 15%, 20%, or 25% greater than the wt. % of the R-amino acid entity.

In some embodiments of any ofthe compositions or methods disclosed herein,the wt. % of the L-glutamine or a sait thereof in the composition is at least 20% greater than the wt. % of the L-amino acid entity, e.g., the wt. % of the L-glutamine or a sait thereof in the composition is at least 25%, 30%, 35%, 40%, 45%, or 50% greater than the wt. % of the L-amino acid entity.

In some embodiments of any ofthe compositions or methods disclosed herein,the wt. % ofthe R-amino acid entity in the composition is at least 10% greater than the wt. % ofthe Lamino acid entity, e.g., the wt. % ofthe R-amino acid entity in the composition is at least 15%, 20%, 25%, or 30% greater than the wt. % of the L-amino acid entity.

In some embodiments of any of the compositions or methods disclosed herein:

j) the ratio of the L-amino acid entity to the R-amino acid entity is at least 1:4, or at least 2:5, and not more than 3:4, e.g., the ratio of L-amino acid entity to R-amino acid entity is about 2:3;

k) the ratio ofthe L-amino acid entity to the L-glutamine or a sait thereof is at least 1:4, or at least 1:3, and not more than 3:4, e.g., the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is about 1:2;

1) the ratio ofthe R-amino acid entity to the L-glutamine or a sait thereof is at least 1:4, or at least 1:2, and not more than 6:7, e.g., the ratio of the R-amino acid entity to the L-glutamine or a sait thereof is about 3:4; or

m) a combination of two or three of (j)-(l).

In an embodiment, the composition satisfies the properties of (j)-(l) defined above.

In certain embodiments, the composition satisfies the properties of at least 2,3, or 4 of any of properties (j)-(m) defined above.

I

In some embodiments of any of the compositions or methods disclosed herein, the composition further comprises one or both of an isoleucine (I)-amino acid-entity and a vahne (V)-amino acid-entity, e.g., both the l-amino acid-entity and the V-amino acid-entity are présent. In some embodiments of any of the compositions or methods disclosed herein:

n) the wt. % of the L-amino acid-entity in the composition is greater than or equal to the

I wt. % of the l-amino acid-entity and the V-amino acid-entity in combination;

o) the wt. % of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid- | entity in combination in the composition is greater than or equal to the wt. % of the L-glutamine or a sait thereof;

p) the wt. % of the L-amino acid-entity, the l-amino acid-entity, and the V-ammo acid- entity in combination in the composition is less than the wt. % of the R-amino acid entity;

Iq) the wt. % of the R-amino acid entity and the L-glutamine or a sait thereof m the composition is greater than the wt. % of the L-amino acid-entity, the l-amino acid-entity, and the

V-amino acid-entity in combination; or

r) _a combination of two, three, or four of (n)-(q).

I In some embodiments of any of the compositions or methods disclosed herem:

s) the wt. % of the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or | a sait thereof is at least 50% of the composition, or at least 70% of the composition, but not more than 90% of the composition;

t) the wt. % of the NAC or a sait thereof is at least 1%, or at least 2%, but not more than

10% of the composition;

Iu) the wt. % of the L-amino acid-entity, the l-amino acid-entity, and the V-ammo acidentity in combination is at least 15%, or at least 20%, but not more than 50% of the composition;

I’ v) the wt. % of the R-amino acid entity, the L-giutamine or a sait thereof, and the NAC or a sait thereof is at least 40%, or at least 50%, but not more than 80% of the composition; or

Iw) a combination of two, three, or four of (s)-(v).

In some embodiments of any of the compositions or methods disclosed herem:

Ix) the ratio of the L-amino acid entity to the l-amino acid entity is at least 1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or not more than 3:1, e.g., the ratio of the L-ammo acid | 30 entity to the l-amino acid entity is about 2:1 ;

I

J i..

y) the ratio of L-amino acid entity to V-amino acid entity is at least 1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or not more than 3:1, e.g., the ratio of L to V is about 2:1;

z) the ratio of the L-amino acid entity to the R-amino acid entity is greater than 1:3, greater than 1.5 to 3, and less than 3:3, e.g., the ratio of the L-amino acid entity to the R-amino acid entity is about 2:3;

aa) the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is greater than 1:4, greater than 1.5 to 4 and less than 4:4, or less than 3:4, e.g., the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is about 1:2; or bb) a combination of two, three, or four of (x)-(aa).

In an embodiment, the composition satisfies the properties of (x)-(aa) defined above.

In certain embodiments, the composition satisfies the properties of at least 2, 3, 4, or 5 of any of properties (x)-(bb) defined above.

In some embodiments of any of the compositions or methods disclosed herein:

cc) the ratio of the l-amino acid entity to the V-amino acid entity is at least 0.5:1, or at least 0.75:1, and not more than 1.5 to 1 or not more than 2:1, e.g., the ratio of the L-amino acid entity to the l-amino acid entity is about 1:1;

dd) the ratio of the l-amino acid entity to the R-amino acid entity is at least 0.5:3, or at least 0.75:3, and not more than 2:3, or not more than 1.5:3, e.g., the ratio of the L-amino acid entity to the l-amino acid entity is about 1:3;

ee) the ratio of the l-amino acid entity to the L-glutamine or a sait thereof is at least 0.5:4, or at least 0.75:4, and not more than 3:4, or not more than 2:4, e.g., the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is about 1:4; or ff) or a combination of two or three of (cc)-(ee).

In an embodiment, the composition satisfies the properties of (cc)-(ec) defined above.

In certain embodiments, the composition satisfies the properties of at least 2,3, or 4 of any of properties (cc)-(ff) defined above.

In some embodiments of any of the compositions or methods disclosed herein:

gg) the ratio ofthe L-amino acid entity to the V-amino acid entity is at least 1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or not more than 3:1, e.g., the ratio of the L-amino acid entity to the V-amino acid entity is about 2:1 ;

I I , I I

I ¹⁰I

I 15 I

I । 20

I

I 25 I

I

I 19827

I hh) the ratio of the L-amino acid entity to the R-amino acid entity is greater than 1:3 or greater than 1.5 to 3, and less than 3:3, e.g., the ratio of the L-amino acid entity to the R-amino acid entity is about 2:3;

ii) the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is greater than 1:4 or greater than 1.5 to 4, and less than 4:4 or less than 3:4, e.g., the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is about 1:2; or jj) a combination of two or three of (gg)-(ii).

In an embodiment, the composition satisfies the properties of (gg)-(ii) defined above.

In certain embodiments, the composition satisfies the properties of at least 2,3, or 4 of any of properties (gg)-(jj) defined above.

In some embodiments of any of the compositions or methods disclosed herein:

kk) the ratio of the V-amino acid entity to the L-glutamine or a sait thereof is at least 0.5:4, or at least 0.75:4, and not more than 3:4, or not more than 2:4, e.g., the ratio of the Lamino acid entity to the L-glutamine or a sait thereof is about 1:4;

11) the ratio of the V-amino acid entity to the R-amino acid entity is at least 0.5:3, or at least 0.75:3, and not more than 2:3, or not more than 1.5:3, e.g., the ratio of the V-amino acid entity to the R-amino acid entity is about 1:3;

mm) the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acidentity in combination to the R-amino acid entity, L-glutamine or a sait thereof, and NAC or a sait thereof is at least 1:4, or at least 2:3, or not more than 5:7, or not more than 6:7, e.g., the ratio is about 6:11; or nn) a combination of two or three of (kk)-(mm).

In an embodiment, the composition satisfies the properties of (kk)-(mm) defined above.

In certain embodiments, the composition satisfies the properties of at least 2, 3, or 4 of any of properties (kk)-(nn) defined above.

In some embodiments of any of the compositions or methods disclosed herein, the composition further comprises an S-amino acid entity.

In some embodiments of any of the compositions or methods disclosed herein:

| 18

I

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I I I I

I I I I I I I

2) the ratio ofL-amino acid entity to V-amino acid entity is at least 2:1, at least 3-1 at least 3.5:1, at least 4:1, or at least 5:1, and not more than 6: 1, e.g., the ratio of L-amino acid entity to V-ammo acid entity is about 4:1;

3) the ratio ofthe L-amino acid entity to the R-amino acid entity is at least 1:1 at least .5:3, at least 4:3, or at least 2:1, and not more than 5:2, e.g„ the ratio ofthe L-amino acid entity to the R-amino acid entity is about 4:3;

4) the ratio ofthe L-amino acid entity to the L-glutamine or a sait thereof is at least 0 5-1 or at least 0.75:1, and not more than 1.5 to 1 or not more than 2:1, e.g., the ratio ofthe L-amino ' acid entity to the L-glutamine or sait thereof is about 1:1;

5) the ratio of the L-amino acid entity to the NAC entity or a sait thereof isat least 21 at east 3:1, at least 3.5:1, or at least 4:1, and not more than 5 to 1 or not more than 6:1, e g the ’ ratio ofthe L-amino acid entity to the NAC entity or sait thereof is about 4:1 (e.g 4-0 9)·

6) optionally wherein the ratio ofthe L-amino acid entity to the S-amino acid enti’ty or a sa t thereof is greater than 1:3, greater than 1.5:3, about 2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g., the ratio ofthe L-amino acid entity to the S-amino acid entity is about 2-3 or the ratio ofthe L-amino acid entity to the S-amino acid entity is about 3:5; or ’

7) a combination of two, three, four, five, or six of (1)-(6).

In some embodiments ofany ofthe compositions or methods disclosed herein:

8) the ratio of l-amino acid entity to V-amino acid entity is at least 1:1, at least 1 51 or 20 about 2:1, and not more than 2.5:1 or notmore than 3:1, e.g., the ratio of l-amino acid entity’to V-amino acid entity is about 2:1;

9) the ratio ofthe l-amino acid entity to the R-amino acid entity is greater than 1:3, greater than 1.5:3, or about 2:3, and not more than 2.5:3 or not more than 1:1, e.g., Jhe ratio of the l-amino acid entity to the R-amino acid entity is about 2:3;

10) the ratio ofthe l-amino acid entity to the L-glutamine or a sait thereof is at least 1 -4 at least 1:3, or about 1:2, and not more than 1:1 or not more than 2:1, e.g., the ratio ofthe I- ’ amino acid entity to the L-glutamine or sait thereof is about 1:2;

11) the ratio ofthe l-amino acid entity to the NAC entity or a sait thereof is at least 11 atleast 1.5:1, or about 2:1, and not more than 2.5:1 ornotmorethan3:l,e.g.,theratioofthel- ^moactd entity to the NAC entity or sait thereof is about 2:1 (e g 2-0 9)·

I

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I

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I I i ²⁰

I

I 25

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12) optionally wherein the ratio of the l-amino acid entity to the S-amino acid entity or a sait thereof is greater than 1:4, greater than 1.5:4, about 1:3, or about 3:10, and not more than 1.5:3 or 2:3, e.g., the ratio of the l-amino acid entity to the S-amino acid entity is about 1:3, or the ratio ofthe l-amino acid entity to the S-amino acid entity is about 3:10; or

13) a combination of two, three, four, or five of (8)-(12).

In some embodiments of any of the compositions or methods disclosed herein:

14) the ratio of the V-amino acid entity to the R-amino acid entity is greater than 1:4, greater than 1.5:4, or about 1:3, and not more than 1:2 or not more than 1:1, e.g., the ratio of the V-amino acid entity to the R-amino acid entity is about 1:3;

17) optionally wherein the ratio of the V-amino acid entity to the S-amino acid entity or a sait thereof is greater than 1:8, greater than 1:7, about 1:6, orabout3:20, and not more than 1.5:6 or 1:3, e.g., the ratio of the V-amino acid entity to the S-amino acid entity is about 1:6, or the ratio of the V-amino acid entity to the S-amino acid entity is about 3:20; or

18) a combination of two, three, or four of (14)-(17).

In some embodiments of any of the compositions or methods disclosed herein:

19) the ratio of the R-amino acid entity to the L-glutamine or a sait thereof is greater than 1:2, greater than 1.25:2, or about 3:4, and not more than 3.5:4 or not more than 1:1, e.g., the ratio of the R-amino acid entity to the L-glutamine or sait thereof is about 3:4;

20) the ratio of the R-amino acid entity to the NAC entity or a sait thereof is at least 4:1, at least 4:1.5, or about 3:1, and not more than 3:1.5 or not more than 3:2, e.g., the ratio of the Ramino acid entity to the NAC entity or sait thereof is about 3:1 (e.g., 3:0.9);

21) optionally wherein the ratio of the R-amino acid entity to the S-amino acid entity or a sait thereof is greater than 1:4, greater than 1:3, about 1:2, or about 9:20, and not more than 1.5:2 or 1:1, e.g., the ratio of the R-amino acid entity to the S-amino acid entity is about 1:2, or the ratio of the R-amino acid entity to the S-amino acid entity is about 9:20; or | 20

I

22) a combination of two or three of (19)-(21).

In some embodiments of any of the compositions or methods disclosed herein:

25) a combination of (23) and (24).

In some embodiments of any of the compositions or methods disclosed herein:

26) the ratio of the NAC entity to the S-amino acid entity or a sait thereof is greater than 1:8, greater than 1:7, or about 1:6, and not more than 1:5 or not more than 1.5:5, e.g., the ratio of the NAC entity to the S-amino acid entity is about 1:6 (e.g., 0.9:6 or 2.7:20).

In an embodiment, the composition satisfies the properties of (1)-(7) defined above.

In certain embodiments, the composition satisfies the properties of at least 2, 3, 4, 5, 6, or 7 of any of properties (1)-(26) defined above.

In an embodiment, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, and NAC or a sait thereof is 12 : 6 : 3 : 9 : 12 : 2.7.

In an embodiment, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, and the S-amino acid entity is 12 : 6 : 3 : 9 : 12 : 2.7 : 18.

In an embodiment,'the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, and the S-amino acid entity is 12 : 6 : 3 : 9 : 12 : 2.7 : 20.

In an embodiment, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, and NAC or a sait thereof is 12 +/-15% : 6 +/- 15% : 3 +/-15% : 9 +/-15% : 12 +/-15% : 2.7 +/15%.

In an embodiment, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, and the S-amino acid entity is 12 +/- 15% : 6 +/- 15% : 3 +/- 15% : 9 +/15% : 12 +/- 15% : 2.7 +/- 15% : 18 +/- 15%.

In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 : 6 : 3 : 9 : 12 : 2.7 : 9: 9. In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the Vamino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 : 6 : 3 : 9 : 12 : 2.7 : 10: 10.

In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 +/- 15% : 6 +/-15% : 3 +/-15% : 9 +/-15% : 12 +/-15% : 2.7 +/-15% : 9 +/-15% : 9 +/- 15%. In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 +/-15% : 6 +/-15% : 3 +/- 15% : 9 +/-15% : 12 +/15% : 2.7 +/- 15% : 10 +/- 15% : 10 +/- 15%.

In some embodiments of any of the compositions or methods disclosed herein:

oo) a wt. % ofthe L-amino acid entity in the composition is greater than the wt. % ofthe NAC or a sait thereof;

pp) awt. % ofthe R-amino acid entity in the composition is greater than the wt. % ofthe NAC or a sait thereof;

qq) a wt. % of the L-glutamine or a sait thereof in the composition is greater than the wt. % of the NAC or a sait thereof; or rr) a combination of two or three of (oo)-(qq).

In some embodiments of any of the compositions or methods disclosed herein, at least one of (a)-(d) is a free amino acid, e.g., two, three, or four of (a)-(d) are a free amino acid, e.g., at least 50 wt. % of the total wt. of the composition is one or more amino acid entities in free form.

In some embodiments of any of the compositions or methods disclosed herein, at least one of (a)-(d) is in a sait form, e.g., one, two, three, or four of (a)-(d) is in a sait form, e.g., at least 10 wt. % of the total wt. of the composition is one or more amino acid entities in sait form.

In some embodiments of any of the compositions or methods disclosed herein, the composition is capable of one, two, three, four, five, or ail of:

a) b) c) d) e) f)

g) decreasing or preventing liver fibrosis;

decreasing or preventing liver injury;

decreasing or preventing hépatocyte inflammation;

improving, e.g., increasing, glucose tolérance;

decreasing or preventing steatosis;

decreasing or preventing hépatocyte ballooning; or improving gut function.

In some embodiments of any of the compositions or methods disclosed herein, the composition further comprises one or both of L-glycine and L-serine. In certain embodiments, the composition comprises an L-amino acid entity, an I-amino acid entity, an V-amino acid entity, an R-amino acid entity, an L-glutamine or a sait thereof, an NAC or a sait thereof, and an L-glycine. In certain embodiments, the composition comprises an L-amino acid entity, an Iamino acid entity, an V-amino acid entity, an R-amino acid entity, an L-glutamine or a sait thereof, an NAC or a sait thereof, and an L-serine. In certain embodiments, the composition comprises an L-amino acid entity, an I-amino acid entity, an V-amino acid entity, an R-amino acid entity, an L-glutamine or a sait thereof, an NAC or a sait thereof, an L-glycine, and an Lserine. In any of the aforesaid embodiments in this paragraph, the wt. ratio of the L-amino acid entity, the I-amino acid entity, the V-amino acid entity, the R-amino acid entity, the L-glutamine or sait thereof, and the NAC or sait thereof is about 1 : 0.5: 0.5: 1.5 : 2 : 0.15 or about 1 : 0.5: 0.5: 1.5 : 2 : 0.3.

In some embodiments of any of the compositions or methods disclosed herein, the wt. ratio of the L-amino acid entity, the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or sait thereof is about 0.5 to 3 : 0.5 to 4 : 1 to 4 : 0.1 to 2.5, e.g., the wt. ratio of the Lamino acid entity, the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or sait thereof is about 1 : 1.5 : 2 : 0.15, about 1 : 1.5 : 2 : 0.225, about 1 : 1.5 : 2 : 0.3, or about 1 : 1.5 : 2 : 0.5. In any of the aforesaid embodiments in this paragraph, the wt. ratio of the L-amino acid ι 19827

I entity, the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or sait thereof is

I about 1 : 0.75 : 2 : 0.15, about 1 : 0.75 : 2 : 0.225, about 1 : 0.75 : 2 : 0.3, or about 1 : 0.75 : 2 :

0.5.

ITn some embodiments, the wt. ratio of the L-amino acid entity, the l-amino acid entity, 5 the V-amino acid entity, the R-amino acid entity, the L-glutamine or sait thereof, and the NAC or — sait thereof is about 1 : 0.5: 0.5: 1.5 :2 : 0.15, about 1 : 0.5: 0.5: 1.5 :2 : 0.225, about 1 : 0.5: 0.5: 1.5 : 2 : 0.3, or about 1 : 0.5: 0.5: 1.5 : 2 : 0.5.

_ In some embodiments of any of the compositions or methods disclosed herein, the composition comprises about 0.5 g to about 10 g of the L-amino acid entity, about 0.25 g to — 10 about 5 g of the l-amino acid entity, about 0.25 g to about 5 g of the V-amino acid entity, about

0.5 g to about 20 g of the R-amino acid entity, about 1 g to about 20 g of the L-glutamine or a — sait thereof, and about 0.1 g to about 5 g of the NAC or a sait thereof, e.g., the composition comprises about 1 g of the L-amino acid entity, about 0.5 g of the l-amino acid entity, about 0.5 — g of V-amino acid entity, about 1.5 g of R-amino acid entity, about 2 g of L-glutamine or a sait

I 15 thereof, and about 0.15 g, about 0.225 g, about 0.3 g, or about 0.5 g of NAC or a sait thereof. In _ certain embodiments, the composition comprises about 0.15 g of NAC. In certain embodiments,

H the composition comprises about 0.3 g of NAC. In embodiments, the composition comprises _ about 4 g of the L-amino acid entity, about 2 g of the l-amino acid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or a sait thereof, and _ 20 about 0.9 g of NAC or a sait thereof. In embodiments, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the l-amino acid entity, about 1 g of V-amino acid entity, _ about 3 g of R-amino acid entity, about 4 g of L-glutamine or a sait thereof, about 0.9 g of NAC | or a sait thereof, and about 6 g of L-serine or a sait thereof. In embodiments, the composition _ comprises about 4 g of the L-amiho acid entity, about 2 g of the l-amino acid entity, about 1 g of | 25 V-amino acid entity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or a sait thereof, about 0.9 g of NAC or a sait thereof, and about 6.67 g of L-serine or a sait thereof. In

H embodiments, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the Iamino acid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acid entity, about 4 g

I of L-glutamine or a sait thereof, about 0.9 g of NAC or a sait thereof, about 3 g of L-serine or a sait thereof, and about 3 g of L-glycine or a sait thereof. In embodiments, the composition

I comprises about 4 g of the L-amino acid entity, about 2 g of the l-amino acid entity, about 1 g of

V-amino acid entity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or a sait thereof, about 0.9 g of NAC or a sait thereof, about 3.33 g of L-serine or a sait thereof, and about 3.33 g of L-glycine or a sait thereof.

In some embodiments of any of the compositions or methods disclosed herein, the composition comprises:

a) L-Leucine or a sait thereof;

b) L-Isoleucine or a sait thereof;

c) L-Valine or a sait thereof;

d) L-Arginine or a sait thereof;

e) L-Glutamine or a sait thereof; and

f) NAC or a sait thereof.

In an embodiment, L-Leucine is provided as part of a dipeptide comprising L-Leucine, or a sait thereof, or a tripeptide comprising L-Leucine, or a sait thereof.

In an embodiment, L-Isoleucine is provided as part of a dipeptide comprising LIsolcucine, or a sait thereof, or a tripeptide comprising L- Isoleucine, or a sait thereof.

In an embodiment, L-Valine is provided as part of a dipeptide comprising L- Valine, or a sait thereof, or a tripeptide comprising L- Valine, or a sait thereof.

In some embodiments of any of the compositions or methods disclosed herein, the composition comprises a combination of 4 to 20 different amino acid entities, e.g., a combination of 5 to 15 different amino acid entities.

In some embodiments of any of the compositions or methods disclosed herein, at least two, three, four, or more amino acid entities arc not comprised in a peptide of more than 4,5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15,16, 17,18, 19, or 20 amino acid residues in length.

Another aspect of the invention features a method for improving liver function, wherein the method comprises administering to a subject in need thereof an effective amount of a composition comprising:

a) a L-amino acid entity chosen from L-leucine or a sait thereof, or β-hydroxy-pmethybutyrate (HMB) or a sait thereof, or a combination of L-leucine or a sait thereof and HMB or a sait thereof;

b) an R-amino acid entity chosen from:L-arginine or a sait thereof, omithine or a sait thereof, or creatine or a sait thereof, or a combination of two or three of L-arginine or a sait thereof, omithine or a sait thereof, or creatine or a sait thereof;

c) L-glutamine or a sait thereof; and

d) N-acetylcysteine (NAC) or a sait thereof.

Another aspect of the invention features a method for treating one or more symptoms selected from the group consisting of decreased fat metabolism, hépatocyte apoptosis, hépatocyte ballooning, inflammation of adipose tissue, inflammation of hepatic tissue, fibrosis, liver injury, steatosis, glucose tolérance, and oxidative stress, wherein the method comprises administering to a subject in need thereof an effective amount of a composition comprising:

a) a L-amino acid entity chosen from L-leucine or a sait thereof, or β-hydroxy-βmethybutyrate (HMB) or a sait thereof, or a combination of L-leucine or a sait thereof and HMB or a sait thereof;

c) L-glutamine or a sait thereof; and

d) N-acetylcysteine (NAC) or a sait thereof.

Another aspect of the invention features a method for treating fatty liver disease, wherein the method comprises administering to a subject in need thereof an effective amount of a composition comprising:

a) a L-amino acid entity chosen from L-leucine or a sait thereof, or p-hydroxy-pmethybutyrate (HMB) or a sait thereof, or a combination of L-leucine or a sait thereof and HMB or a sait thereof;

c) L-glutamine or a sait thereof; and

d) N-acetylcysteine (NAC) or a sait thereof.

In some embodiments of any of the compositions or methods disclosed herein, e.g., of any of the methods described herein, the subject has a disease or disorder selected from the group consisting of non-alcoholic fatty liver (NAFL), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic fatty liver disease (AFLD), and alcoholic steatohepatitis (ASH). In certain embodiments, the subject has pédiatrie NAFLD.

In some embodiments of any of the compositions or methods disclosed herein, e.g., of any of the methods described herein, the subject has a high BMI, obesity, gut leakiness, gut dysbiosis, or gut microbiome disturbance.

In some embodiments of any of the compositions or methods disclosed herein, e.g., of any ofthe methods described herein, the subject has cirrhosis, hepatocarcinoma, an increased risk of liver failure, an increased risk of death, metabolic syndrome, or type 2 diabètes.

In some embodiments of any of the compositions or methods disclosed herein, e.g., of any ofthe methods described herein, the subject has increased levels ofinflammatory cytokines relative to a normal subject, e.g., the subject has increased levels of TNFa relative to a normal subject e.g., without the one or more symptoms or without the fatty liver disease.

In some embodiments, e.g., of any ofthe methods described herein, the subject exhibits muscle atrophy or has a decreased ratio of muscle tissue to adipose tissue relative to a normal subject, e.g., without the one or more symptoms or without a fatty liver disease, e.g., the subject exhibits muscle atrophy without one or both of fibrosis or cirrhosis.

In some embodiments, e.g., of any of the methods described herein, the subject exhibits reverse lipid transport from adipose tissue to liver tissue.

In some embodiments, e.g., of any of the methods described herein, the subject is treated with a composition, e.g., any composition as described herein.In some embodiments of any of the aspects described herein:

(i) an amino acid entity (e.g., at least one, two, or three of the amino acid entities) of (a) is selected from Table 2; and/or '(ii) one or both of the R-amino acid entity and the Q-amino acid entity are présent at a higher amount (wt. %) than the L-amino acid entity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are graphs showing the effect of treatment with an amino acid composition (Amino Acid Composition A-l) on the NAFLD activity score, ballooning, and fibrosis in the STAM mouse model (FIG. IA) and in the FATZO mouse model (FIG. IB).

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FIG. 2 is a schematic showing the metabolic symptoms of patients with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and cirrhosis prior to administration of a composition comprising amino acid entities described herein (top) and the improvement in patients with NAFLD, NASH, and cirrhosis after administration of the 5 composition (bottom).

FIG. 3 is a schematic showing treatment regimens for administration of an amino acid composition to STAM and FATZO mice.

FIGS. 4A-4E are a sériés of graphs and images showing the effect of treating STAM and FATZO mice with an amino acid composition on the NAFLD activity score (NAS), steatosis, 10 inflammation, and liver fibrosis as determined with histology.

FIGS. 5A-5B are images showing the levels of liver unsaturated fatty acids and acylcamitines of STAM mice treated with the amino acid composition.

FIG. 6 is an image of a gene map of the liver gene expression pattern following treatment with the amino acid composition in STAM mice showing activation of ACOX1.

FIGS. 7A-7D are images of gene maps of the liver gene expression pattern following treatment with the amino acid composition in STAM mice showing upstream regulator activation of anti-inflammatory IL-10 (FIG. 7A); inhibition of pro-inflammatory NF-kB (FIG. 7B), interferons, IL-lb, and IL-2 (FIG. 7C); and suppression of the fibrogenic TGF-b signaling pathway.

FIG. 8 is a sériés of graphs showing MCP-1 and MIP-1 protein levels, which are the ligands of C-C chemokine receptor types 2 (CCR2) and 5 (CCR5), following treatment with the amino acid composition.

FIGS. 9A-9L are a sériés of microscopy images shown lipid accumulation in primary human hepatôcytes following treatment with vehicle control (FIGS. 9A-9Î)), a LIVRQNAC 25 amino acid composition (FIGS. 9E-9H), or free fatty acids and TNFa (FF+TNF; FIGS. 9I-9L).

FIG. 10 is a sériés of microscopy images showing liver histology (H&E stain or Sirius Red stain for collagen déposition) from FATZO mice after administration of the indicated amino acid compositions.

FIG. 11 is a sériés of microscopy images showing liver histology from FATZO mice after 30 administration of the indicated amino acid compositions.

FIG. 12 is a sériés of graphs showing NAFLD activity scores (top left panel), Sirius Red staining (top right panel), steatosis levels (bottom left panel), inflammation levels (bottom middle panel), and ballooning (bottom right panel) observed in fixed liver tissues from FATZO mice after administration of the indicated amino acid compositions.

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DETAILED DESCRIPTION

The présent invention provides, at least in part, methods and compositions comprising at least four different amino acid entities. In some embodiments, the composition is capable of one, two, three, four, five, or six or ail of:

a) decreasing or preventing liver fibrosis;

b) decreasing or preventing liver injury;

c) decreasing or preventing hépatocyte inflammation;

d) improving, e.g., increasing, glucose tolérance;

e) decreasing or preventing steatosis;

f) decreasing or preventing hépatocyte ballooning; or

g) improving gut function.

In some embodiments, at least one amino acid entity in the compositions is not a peptide of more than 20 amino acid residues in length.

In some embodiments, the composition comprises a leucine (L)-amino acid entity, an 20 arginine (R)-amino acid entity, a glutamine (Q)-amino acid entity; and an antioxidant or reactive oxygen species (ROS) scavenger (e.g., a N-acetylcysteine (NAC) entity, e.g., NAC). In some embodiments, at least one amino acid entity is not a peptide of more than 20 amino acid residues in length. In some embodiments, the composition is capable of improving gut barrier function.

The composition described herein can be administered to a subject to provide a bénéficiai 25 effect in one or both of improving liver function or treating (e.g., revering, reducing, ameliorating, or preventing) a liver disease (e.g., a fatty liver disease). A subject that may be treated with the composition include a subject having non-alcoholic fatty liver disease (NAFLD; e.g., pédiatrie NAFLD), such as a subject with non-alcoholic steatohepatitis (NASH) or NAFL, or subjects with alcoholic fatty liver disease (AFLD), such as alcoholic steatohepatitis (ASH). In 30 particular, the subject may hâve one, two, or more (e.g., ail) of a high BMI, obesity, fibrosis, or cirrhosis. The subject may also hâve one, two, or more (e.g., ail) of gut leakiness, gut dysbiosis, or gut microbiome disturbance.

The subject may exhibit an improvement in liver function or liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)) after administration of a composition comprising a L-amino acid entity, a R-amino acid entity, a Q-amino acid entity; and an antioxidant or ROS scavenger, e.g., a NAC entity, e.g., NAC. For example, the amino acid entity composition may be administered to the subject 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 at a dose of about 15 total grams per day to about 90 total grams per day (e.g., a total of about 48 g or a total of about 72 g per day).

Treatment with the amino acid entity composition can resuit in improved liver function in a subject, e.g., by one, two, three, four, five or more (e.g., ail) of increasing free fatty acid and lipid metabolism, improving mitochondrial function, browning of white adipose tissue (WAT), decreasing reactive oxygen species (ROS), increasing levels of glutathione (GSH), decreasing hepatic inflammation, improving gut barrier function, increasing insulin sécrétion, or improving glucose tolérance.

In some embodiments, the composition is for use as a médicament in improving liver function in a subject (e.g., a subject with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)). In some embodiments, the composition including amino acid entities is for use as a médicament in treating (e.g., reversing, reducing, ameliorating, or preventing) a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)) in a subject.

In some embodiments, the composition is for use in the manufacture of a médicament for improving liver function in a subject (e.g., a subject with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)). In some embodiments, the composition including amino acid entities is for use in the manufacture of a médicament for treating (e.g., reversing, reducing, ameliorating, or preventing) a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)) in a subject.

Additionally, the compositions can be used in methods of dietary management of a subject (e.g., a subject without a liver disease or with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)).

One embodiment provides a nutritional supplément, dietary formulation, functional food, medical food, food, or beverage comprising a composition described herein. Another embodiment provides a nutritional supplément, dietary formulation, functional food, medical food, food, or beverage comprising a composition described herein for use in the management of any of the diseases or disorders described herein.

One embodiment provides a method of maintaining or improving liver health comprising administering to a subject an effective amount of a composition described herein. Another embodiment provides a method of providing nutritional support or supplémentation to a subject with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)) comprising administering to the subject an effective amount of a composition described herein. Yet another embodiment provides a method of providing nutritional supplémentation that aids in the management of liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)) comprising administering to the subject in need thereof an effective amount of a composition described herein.

Définitions

Ternis used in the claims and spécification are defined as set forth below unless otherwise specified.

It must be noted that, as used in the spécification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictâtes otherwise.

As used herein, the term “amino acid entity” refers to an amino acid in one or both of free form or sait form, an amino acid residue of a peptide (e.g., of a dipeptide, oligopeptide, or .

polypeptide), a dérivative of an amino acid, a precursor of an amino acid, or a métabolite of an amino acid.

As used herein the term “XXX amino acid entity” refers to an amino acid entity that if a free amino acid, comprises free XXX or XXX in sait form; if a peptide, refers to a peptide comprising an XXX residue; if a dérivative, refers to a dérivative of XXX; if a precursor, refers to a precursor of XXX; and if a métabolite, refers to a XXX métabolite. For example, where XXX is leucine (L), then L-amino acid entity refers to free L or L in sait form, a peptide comprising a L residue, a L dérivative, a L precursor, or a métabolite of L; where XXX is arginine (R), then R-amino acid entity refers to free R or R in sait form, a peptide comprising a R residue, a R dérivative, a R precursor, or a métabolite of R; where XXX is glutamine (Q), then Q-amino acid entity refers to free Q or Q in sait form, a peptide comprising a Q residue, a Q dérivative, a Q precursor, or a métabolite of Q; and where XXX is N-acetylcysteine (NAC), then NAC-amino acid entity refers to free NAC or NAC in sait form, a peptide comprising a NAC residue, a NAC dérivative, a NAC precursor, or a métabolite of NAC.

“About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or précision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

An “amino acid” refers to an organic compound having an amino group (-NH2), a carboxylic acid group (-C(=O)OH), and a side chain bonded through a central carbon atom, and includes essential and non-essential amino acids, as well as natural and unnatural amino acids.

The proteogenic amino acids, shown below, are known by three- and one-letter abbreviations in addition to their full names. For a given amino acid, these abbreviations are used interchangeably herein. For example, Leu, L or leucine ail refer to the amino acid leucine; Ile, I or isoleucine ail refer to the amino acid isoleucine; Val, V or valine ail refer to the amino acid valine; Arg, R or arginine ail refer to the amino acid arginine; and Gin, Q or glutamine ail refer to the amino acid glutamine.

Likewise, the non-natural amino acid dérivative N-acetylcysteine may be referred to interchangeably by “NAC” or “N-acetylcysteine.”

Amino acids may be présent as D- or L- isomers. Unless otherwise indicated, amino acids referred to herein are L-isomers of amino acids.

Table 1. Amino acid names and abbreviations.

Amino acid	Three-letter	One-letter
Alanine	Ala	A
Arginine	Arg	R
Asparagine	Asn	N
Aspartic acid	Asp	D
Cysteine	Cys	C
Glutamic acid	Glu	E

Glutamine	Gin	Q
Glycine	Gly	G
Histidine	His	H
Isoleucine	Ile	I
Leucine	Leu	L
Lysine	Lys	K
Méthionine	Met	M
Phenylalanine	Phe	F
Proline	Pro	P
Serine	Ser	S
Threonine	Thr	T
Tryptophan	Trp	W
Tyrosine	Tyr	Y
Valine	Val	V

The term “effective amount” as used herein means an amount of an amino acid, or pharmaceutical composition which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., provide a positive clinical response). The effective amount of an active ingrédient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable excipient(s) and/or carrier(s) utilized, and like factors with the knowledge and expertise of the attending physician.

A “pharmaceutical composition” described herein comprises at least one amino acid and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is used as a therapeutic, a nutraceutical, a medical food, or as a supplément.

The term “pharmaceutically acceptable” as used herein, 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 animais without excessive toxicity, irritation, allergie response, or other problem or complication, commensurate with a reasonable benefît/risk ratio.

A composition, formulation or product is “therapeutic” if it provides a bénéficiai clinical effect. A bénéficiai clinical effect can be shown by lessening the progression of a disease and/or alleviating one or more symptoms of the disease.

A “unit dose” or “unit dosage” as used herein means an amount or dose of medicine prepared in an individual packet or container for convenience, safety, or monitoring. A “unit dose” or “unit dosage” comprises the drug product or drug products in the form in which they are marketed for use, with a spécifie mixture of active ingrédients and inactive components (excipients), in a particular configuration (such as a capsule shell, for example), and apportioned into a particular dose.

As used herein, the terms “treat,” “treating,” or “treatment” of a liver disease refer in one embodiment, to ameliorating, e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH), (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discemible by the patient. In yet another embodiment, “treat,” “treating,” or “treatment” refers to modulating a symptom of a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)), either physically, (e.g., stabilization of a discemible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treat,” “treating,” or “treatment” refers to preventing or delaying the onset or development or progression of a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)).

Détermination of amino acid weight percent and amino acid ratios in a composition

The weight ratio of a particular amino acid or particular amino acids in a composition or mixture of amino acids is the ratio of the weight of the particular amino acid or amino acids in the composition or mixture compared to the total weight of amino acids présent in the composition or mixture. This value is calculated by dividing the weight of the particular amino acid or of the particular amino acids in the composition or mixture by the weight of ail amino acids présent in the composition or mixture.

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Compositions comprising Amino Acid Entities

The présent disclosure provides compositions, e.g., pharmaceutical compositions, comprising amino acid entities. These pharmaceutical compositions are made up of amino acid entities including amino acids in one or both of free form or sait form, amino acid residues of a peptide (e.g., of a dipeptide, oligopeptide, or polypeptide), dérivatives of an amino acid, precursors of an amino acid, or métabolites of an amino acid. For example, the compositions can include a leucine (L)-amino acid entity, a arginine (R)-amino acid entity, a glutamine (Q)-amino acid entity; and an antioxidant or reactive oxygen species (ROS) scavenger, e.g., a Nacetylcysteine (NAC) entity, e.g., NAC (Table 2). In particular, at least one amino acid entity is not a peptide of more than 20 amino acid residues in length.

Table 2. Amino acid entities include amino acids, precursors, métabolites, and dérivatives of the compositions described herein.

	Exemplary Amino Acid	Precursors	Métabolites	Dérivatives
L	L-Leucine	Oxo-leucine	HMB (betahydroxy-betamethybutyrate); Oxo-leucine; Isovaleryl-CoA	D-Leucine; N- Acetyl-Leucine
I	L-Isoleucine	2-Oxo-3-methylvalerate; Threonine	2-Oxo-3 -methylvalerate; Methylbutyrl-CoA	D-Isoleucine; NAcetylIsoleucine
V	L-Valine	2-Oxo-valerate	Isobutryl-CoA; 3- HIB-CoA; 3-HIB	D-Valine; N- Acetyl-Valine
R	L-Arginine	Argininosuccinate; Citrulline; Aspartate; Glutamate	Omithine; Citrulline; Agmatine; Creatine	D-Arginine; NAcetyl-Arginine;
Q	L-Glutamine	Glutamate	Carbamoyl-P; Glutamate	D-Glutamine; NAcetylGlutamine;

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NAC	N- Acetylcysteine	Serine; Acetylserine; Cystathionine;	Glutathione; Cystathionine; Homocystéine; Méthionine	D-Cysteine; LCysteine; Cystine; Cysteamine
S	L-Serine	Phosphoserine, Phydroxypyruvate, LGlycine	Glycine, Tryptophan, Acetylserine, Cystathionine, Phosphatidylserine 1

It is contemplated that alternatives to serine that can be an S-amino acid entity include, for example, glycine, threonine, or a combination of serine and glycine (e.g., a 1:1 ratio of serine and glycine).

In some embodiments, the total weight of the L-amino acid entity, R-amino acid entity, Q-amino acid entity; and ROS scavenger, e.g., a NAC entity, e.g., NAC, is greater than the total wt. of other amino acid entities in the composition. In certain embodiments, two, three, or more (e.g., ail) of méthionine (M), trytophan (W), or valine (V) may be absent from the amino acid entity composition, or if présent, are présent at less than 2 weight (wt.) %.

In some embodiments, one or both of the R-amino acid entity and the Q-amino acid entity are présent at a higher amount (wt. %) than the L-amino acid entity. The R-amino acid entity can be présent, e.g., at an amount of at least 2 wt. %, at least 3 wt. %, at least 4 wt. %, at least 5 wt. %, at least 6 wt. %, at least 7 wt. %, or at least 8 wt. % greater than the L-amino acid entity. The Q-amino acid entity can be présent, e.g., at an amount of at least 2 wt. %, at least 3 wt. %, at least 4 wt. %, or at least 5 wt. % greater than the L-amino acid entity.

In some embodiments, the L-amino acid entity is selected from the group consisting of a precursor, a métabolite, and a dérivative. In certain embodiments, the L-amino acid entity is selected from the group consisting of L-leucine, P-hydroxy-p-methybutyrate (HMB), oxoleucine, isovaleryl-CoA, D-leucine, and n-acetylleucine. In'one embodiment, the L-amino acid entity is L-leucine. In another embodiment, the L-amino acid entity is HMB.

In some embodiments, the R-amino acid entity is selected from the group consisting of a precursor, a métabolite, and a dérivative. In certain embodiments, the R-amino acid entity is

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I I I 1 selected from the group consisting of L-arginine, D-arginine, omithine, argininosuccinate, citrulline, aspartate, glutamate, agmatine, and N-acetyl-arginine. In one embodiment, the Ramino acid entity is L-arginine. In one embodiment, the R-amino acid entity is creatine. In another embodiment, the R-amino acid entity is omithine.

In some embodiments, the Q-amino acid entity is selected from the group consisting of a precursor, a métabolite, and a dérivative. In certain embodiments, the Q-amino acid entity is selected from the group consisting of L-glutamine, glutamate, carbamoyl-P, glutamate, Dglutamine, and n-acetylglutamine. In one embodiment, the Q-amino acid entity is L-glutamine.

In some embodiments, the NAC-amino acid entity is selected from the group consisting of a precursor, a métabolite, and a dérivative. In certain embodiments, the NACamino acid entity is selected from the group consisting NAC, serine, acetylserine, cystathionine, cystathionine, homocystéine, méthionine, glutathione, D-cysteine, and L-cysteine. In one embodiment, the NAC entity is NAC. In one embodiment, the NAC entity is glutathione.

In various embodiments, the composition further comprises one or two additional branched-chain amino acid (BCAA)-entities, e.g., one or both of an isoleucine (I)-amino acidentity and a valine (V)-amino acid-entity. In some embodiments, both the l-amino acid-entity and the V-amino acid-entity are présent. In certain embodiments, the L-entity is présent at a higher amount (% by weight) than one or both of the l-amino acid-entity and the V-amino acidentity (e.g., the L-entity is présent at an amount of at least 10 wt. %, at least 15 wt. %, at least 20 wt. %, at least 25 wt. %, at least 30 wt. %, at least 35 wt. %, at least 40 wt. %, at least 45 wt. %, or at least 50 wt. % greater than one or both of the l-amino acid-entity and the V-amino acidentity).

In some embodiments, the l-amino acid entity is selected from the group consisting of a sait, a precursor, a métabolite, and a dérivative. In certain embodiments, the l-amino acid entity is selected from the group consisting of L-isoleucine, 2-oxo-3-methyl-valerate, threonine, 2-oxo-3-methyl-valerate, methylbutyrl-CoA, D-isoleucine, and N-acetyl-isoleucine. In one embodiment, the l-amino acid entity is L-isoleucine.

In some embodiments, the V-amino acid entity is selected from the group consisting of a precursor, a métabolite, and a dérivative. In certain embodiments, the V-amino acid entity is selected from the group consisting of L-valine, 2-oxo-valerate, isobutryl-CoA, 3-HIB-CoA, 3HIB, D-valine, and N-acetyl-valine. In one embodiment, the l-amino acid entity is L-valine.

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In some embodiments, the composition comprises L-leucine or a leucine métabolite (e.g., HMB), L-arginine or an L-arginine métabolite (e.g., creatine or omithine), L-glutamine, and NAC or a NAC métabolite, e.g., glutathione. In one embodiment, the composition comprises L-leucine, L-arginine, L-glutamine, and NAC. In one embodiment, the composition comprises HMB, creatine, L-glutamine, and glutathione. In one embodiment, the composition comprises HMB, omithine, L-glutamine, and glutathione. In one embodiment, the composition comprises HMB, L-arginine, L-glutamine, and NAC. In one embodiment, the composition comprises L-leucine, creatine, L-glutamine, and NAC. In one embodiment, the composition comprises L-leucine, omithine, L-glutamine, and NAC. In one embodiment, the composition comprises L-leucine, L-arginine, L-glutamine, and glutathione.

In some embodiments, the weight (wt.) ratio of the L-amino acid entity, the R-amino acid entity, the Q-amino acid entity, and the NAC-amino acid entity is about 0.5 to 3 : 0.5 to 4 :1 to 4 : 0.1 to 2.5. In one embodiment, the wt. ratio of the L-amino acid entity, the R-amino acid entity, the Q-amino acid entity, and the NAC-amino acid entity is about 1 : 1.5 : 2 : 0.15.

In some embodiments, the wt. ratio of the L-amino acid entity, the l-amino acid entity, the V-amino acid entity, the R-amino acid entity, the Q-amino acid entity, and the NACamino acid entity is about 0.5 to 2 : 0.1 to 1 : 0.1 to 1 : 0.5 to 3 : 0.5 to 4 : 0.1 to 0.5. In an embodiment, the wt. ratio of the L-amino acid entity, the l-amino acid entity, the V-amino acid entity, the R-amino acid entity, the Q-amino acid entity, and the NAC-amino acid entity is about

1:0.5:0.5:1.5:2:0.15.

In various embodiments, the total wt. of amino acids présent is about 2 g to about 60 g. In certain embodiments, the total wt. of amino acids présent is about 6 g, about 12 g, about 18 g, about 24 g, or about 48 g. In one embodiment, the total wt. of amino acids présent is about 6 g. In one embodiment, the total wt. of amino acids présent is about 12 g. In one embodiment, the total wt. of amino acids présent is about 18 g. In an embodiment, the total wt. of amino acids présent is about 24 g. In one embodiment, the total wt. of amino acids présent is about 48 g.

In some embodiments, the composition comprises about 0.5 g to about 10 g of the Lamino acid entity, about 0.25 g to about 5 g of the l-amino acid entity, about 0.25 g to about 5 g of the V-amino acid entity, about 1 g to about 20 g of the R-amino acid entity, about 1 g to about

20 g of the Q-amino acid entity, and about 0.1 g to about 5 g of the NAC-amino acid entity. In an embodiment, the composition comprises about 1 g of the L-amino acid entity, about 0.5 g of

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I I I the l-amino acid entity, about 0.5 g of V-amino acid entity, about 1.5 g of R-amino acid entity, about 2 g of Q-amino acid entity, and about 0.15 g of NAC-amino acid entity. In an embodiment, the composition comprises about 2 g of the L-amino acid entity, about 1 g of the Iamino acid entity, about 1 g of the V-amino acid entity, about 3 g of the R-amino acid entity, 5 about 4 g of the Q-amino acid entity, and about 0.3 g of the NAC-amino acid entity. In an embodiment, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the Iamino acid entity, about 2 g of the V-amino acid entity, about 6 g of the R-amino acid entity, about 8 g of the Q-amino acid entity, and about 0.6 g of the NAC-amino acid entity.

In some embodiments, the the amino acids comprise about 10 wt % to about 30 wt % 10 leucine, about 5 wt % to about 15 wt % isoleucine, about 5 wt % to about 15 wt % valine, about 15 wt % to about 40 wt % arginine, about 20 wt % to about 50 wt % glutamine, and about 1 wt % to about 8 wt % n-acetylcysteine. In certain embodiments, the amino acids comprise about 16 wt % to about 18 wt % leucine, about 7 wt % to about 9 wt % isoleucine, about 7 wt % to about 9 wt % valine, about 28 wt % to about 32 wt % arginine, about 31 wt % to about 34 wt % glutamine, and about 1 wt % to about 5 wt % n-acetylcysteine. In an embodiment, the amino acids comprise about 16.8 wt % leucine, about 8.4 wt % isoleucine, about 8.4 wt % valine, about 30.4 wt % arginine, about 33.6 wt % glutamine, and about 2.5 wt % n-acetylcysteine.

In any of the foregoing embodiments, at least one amino acid entity is a free amino acid, e.g., one, two, three, or more (e.g., ail) amino acid entities are a free amino acid. In some embodiments, the L-amino acid entity, the R-amino acid entity, the Q-amino acid entity, and the NAC-amino acid entity is a free amino acid entity. In certain embodiment, the L-amino acid entity, the l-amino acid entity, the V-amino acid entity, the R-amino acid entity, the Q-amino acid entity, and the NAC-amino acid entity a free amino acid.

In any of the foregoing embodiments, at least one amino acid entity is in a sait form, e.g., 25 one, two, three, or more (e.g., ail) of the amino acid entities is in a sait form. In some embodiments, wherein the L-amino acid entity, the R-amino acid entity, the Q-amino acid entity, and the NAC-amino acid entity is in a sait form. In certain embodiments, the L-amino acid entity, the l-amino acid entity, the V-amino acid entity, the R-amino acid entity, the Q-amino acid entity, and the NAC-amino acid entity is in a sait form.

In any of the foregoing embodiments, the composition comprises a combination of 2 to different amino acid entities, e.g., 5 to 15 different amino acid entities.

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In some embodiments, the NAC entity is more stable than cysteine. In certain embodiments, the NAC entity does not comprise cysteine.

In some embodiments, the composition further comprises one, two, three, four, five, six, seven, eight, nine, ten, or more (e.g., ail) or more of serine, glycine, glutamine, HMB, arginine, L-leucine, citrulline, glutamine, omithine, L-cysteine, cystine, or glutathione.

In some embodiments, the composition further comprises serine.

In some embodiments, the composition further comprises glycine.

In some embodiments, the composition further comprises camitine.

In some embodiments, the composition includes arginine, glutamine, N-acetylcysteine, and a branched-chain amino acid (BCAA) chosen from one, two, or ail of leucine, isoleucine, and valine.

In some embodiments, the BCAA is leucine.

In some embodiments, the BCAA is isoleucine.

In some embodiments, the BCAA is valine.

In some embodiments, the BCAA is leucine and isoleucine.

In some embodiments, the BCAA is leucine and valine.

In some embodiments, the BCAA is isoleucine and valine.

In some embodiments, the BCAA is leucine, isoleucine, and valine.

In particular, the composition may consist of leucine, isoleucine, valine, arginine, glutamine, and N-acetylcysteine.

In some embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.5: 2 : 0.1-0.3. In some embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine and Nacetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.5: 2 : 0.15. In some embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine and Nacetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.5: 2 : 0.25.

In some embodiments, the amino acids leucine, isoleucine, valine, arginine (e.g., arginine HCl), glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.51.81 : 2 : 0.1-0.3. In some embodiments, the amino acids leucine, isoleucine, valine, arginine (e.g., arginine HCl), glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.5-1.81 : 2 : 0.15. In some embodiments, the amino acids leucine, isoleucine, valine,

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I arginine (e.g., arginine HCl), glutamine, and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.5-1.81 : 2 : 0.25.

In some embodiments, the amino acids leucine, isoleucine, valine, arginine (e.g., arginine HCl), glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.81 : 2 5 : 0.1-0.3. In some embodiments, the amino acids leucine, isoleucine, valine, arginine (e.g., arginine HCl), glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.81 : 2 : 0.15. In some embodiments, the amino acids leucine, isoleucine, valine, arginine (e.g., arginine HCl), glutamine, and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.81 :2:0.25.

In some embodiments, the amino acids leucine, isoleucine, valine, arginine HCl, glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.5 : 2 : 0.1 to 0.3. In some embodiments, the amino acids leucine, isoleucine, valine, arginine HCl, glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.5 :2 : 0.25. In some embodiments, the amino acids leucine, isoleucine, valine, arginine HCl, glutamine and N- acetylcysteine are présent in a weight ratio of 1 : 0.5 : 0.5 : 1.5 : 2 : 0.15.

In some embodiments, the amino acids leucine, isoleucine, valine, arginine HCl, glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.81 : 2 : 0.1 to 0.3. In some embodiments, the amino acids leucine, isoleucine, valine, arginine HCl, glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.81 : 2 : 0.25.

In some embodiments, the amino acids leucine, isoleucine, valine, arginine HCl, glutamine and N-acetylcysteine are présent in a weight ratio of 1 : 0.5 : 0.5 : 1.81 : 2 : 0.15.

In some embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.81 : 2 : 0.1-0.3. In some embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine and N- acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.81 : 2 : 0.15. In some embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine, and Nacetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.81 : 2 : 0.25.

In some embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine and N-acetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.5 : 2 : 0.1 to 0.3. In some embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine and Nacetylcysteine are présent in a weight ratio of about 1 : 0.5 : 0.5 : 1.5 : 2 : 0.25. In some

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I embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine and Nacetylcysteine are présent in a weight ratio of 1 : 0.5 : 0.5 : 1.5 : 2 : 0.15.

In some embodiments, a total weight (wt) of the amino acids is about 2 g to about 60 g.

In some embodiments, the total weight of amino acids présent is about 5 g, about 6 g, about 7 g, about 11g, about 12g, about 13 g, about 14 g, about 15 g, about 16 g, about 17 g, about 18 g, about 19 g, about 20 g, about 21 g, about 22 g, about 23 g, about 24 g, about 25 g, about 26 g, about 27 g, about 28 g, about 29 g, about 30 g, about 31g, about 32 g, about 33 g, about 34 g, about 35 g, about 36 g, about 37 g, about 38 g, about 39 g, about 40 g, about 41 g, about 42 g, about 43 g, about 44 g, about 45 g, about 46 g, about 47 g, about 48 g, about 49 g, or about 50 g.

In certain embodiments, the total wt of the amino acids is about 6 g.

In certain embodiments, the total wt of the amino acids is about 12 g.

In certain embodiments, the total wt of the amino acids is about 18 g.

In certain embodiments, the total wt of the amino acids is about 24 g.

In certain embodiments, the total wt of the amino acids is about 48 g.

In some embodiments, the composition includes about 0.5 g to about 10 g of leucine, about 0.25 g to about 5 g of isoleucine, about 0.25 g to about 5 g of valine, about 1 g to about 20 g of arginine, about 1 g to about 20 g of glutamine, and about 0.1 g to about 5 g of Nacetylcysteine.

In some embodiments, the composition includes at least 1 g of leucine, at least 0.5 g of isoleucine, at least 0.5 g of valine, at least 1.5 g of arginine (or 1.81 g of arginine HCl), at least 2 g of glutamine, and at least 0.15 g of N-acetylcysteine.

In some embodiments, the composition includes about 1 g of leucine, about 0.5 g of isoleucine, about 0.5 g of valine, about 1.5 g of arginine (or 1.81 g of arginine HCl), about 2 g of glutamine, and about 0.15 g of N-acetylcysteine.

In some embodiments, the composition includes at least 2 g of leucine, at least 1 g of isoleucine, at least 1 g of valine, at least 3.0 g of arginine (or 3.62 g of arginine HCl), at least 4 g of glutamine, and at least 0.3 g of N-acetylcysteine.

In some embodiments, the composition includes about 2 g of leucine, about 1 g of isoleucine, about 1 g of valine, about 3.0 g of arginine (or 3.62 g of arginine HCl), about 4 g of glutamine, and about 0.3 g of N-acetylcysteine.

In some embodiments, the composition includes at least 4 g of leucine, at least 2 g of isoleucine, at least 2 g of valine, at least 6.0 g or arginine (or 7.24 g of arginine HCl), at least 8 g of glutamine, and at least 0.6 g of N-acetylcysteine.

In some embodiments, the composition includes about 4 g of leucine, about 2 g of isoleucine, about 2 g of valine, about 6.0 g or arginine (or 7.24 g of arginine HCl), about 8 g of glutamine, and about 0.6 g of N-acetylcysteine.

In some embodiments, the composition includes at least 1.0 g of leucine, at least 0.5 g of isoleucine, at least 0.5 g of valine, at least 1.5 g of arginine, at least 2.0 g of glutamine, or at least 0.15 g of N-acetylcysteine. In some embodiments, the composition includes about 1.0 g of leucine, about 0.5 g of isoleucine, about 0.5 g of valine, about 1.5 g of arginine, about 2.0 g of glutamine, or about 0.15 g of N-acetylcysteine.

In some embodiments, the composition includes at least 1.0 g of leucine, at least 0.5 g of isoleucine, at least 0.5 g of valine, at least 1.5 g of arginine, at least 2.0 g of glutamine, and at least 0.25 g of N-acetylcysteine. In some embodiments, the composition includes about 1.0 g of leucine, about 0.5 g of isoleucine, about 0.5 g of valine, about 1.5 g of arginine, about 2.0 g of glutamine, and about 0.25 g of N-acetylcysteine.

In some embodiments, the amino acids of the composition include about 10 wt % to about 30 wt % leucine, about 5 wt % to about 15 wt % isoleucine, about 5 wt % to about 15 wt % valine, about 15 wt % to about 40 wt % arginine, about 20 wt % to about 50 wt % glutamine, and about 1 wt % to about 8 wt % n-acetylcysteine.

In some embodiments, the amino acids of the composition include about 16 wt % to about 18 wt % leucine, about 7 wt % to about 9 wt % isoleucine, about 7 wt % to about 9 wt % valine, about 28 wt % to about 32 wt % arginine, about 31 wt % to about 34 wt % glutamine, and about 1 wt % to about 5 wt % n-acetylcysteine.

In some embodiments, the amino acids of the composition include about 16.8 wt % leucine, about 8.4 wt % isoleucine, about 8.4 wt % valine, about 30.4 wt % arginine, about 33.6 wt % glutamine, and about 2.5 wt % n-acetylcysteine.

In some embodiments, the composition comprises one or more excipients selected from the group consisting of: citric acid, lecithin, a sweetener, a dispersion enhancer, a flavoring, a bittemess masking agent, and a natural or artificial coloring.

In some embodiments, the composition comprises citric acid.

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In some embodiments, the composition is in the form of a solid, powder, solution, or gel.

In certain embodiments, the composition is in the form of a powder (e.g. in a packet)

In some embodiments, the composition includes one or more pharmaceutically acceptable excipients, wherein the amino acids comprise leucine, arginine, glutamine, and N- acetylcysteine. An aspect of the présent disclosure provides a composition comprising free amino acids and one or more pharmaceutically acceptable excipients, wherein the amino acids consist of leucine, arginine, glutamine, and N-acetylcysteine. In some embodiments, the amino acids leucine, arginine, glutamine, N-acetylcysteine and glycine are présent in a weight ratio of 1 : 1.5 : 2 : 0.15. In some embodiments, the composition comprises at least 1.0 g of leucine, at least 1.5 g of arginine, at least 2.0 g of glutamine, or at least 0.15 g of N-acetylcysteine. In some embodiments, the composition comprises at least 1.5 g of arginine and at least 2.0 g of glutamine. In some embodiments, the amino acids leucine, arginine, glutamine, and Nacetylcysteine are présent in weight % of each compared to total amino acid weight of 20.4 to 22.6%, 30.6 to 33.9%, 40.9 to 45.2%, and 3.1 to 3.4%, respectively. In some embodiments, the amino acids leucine, arginine, glutamine, and N-acetylcysteine, are présent in weight % of each compared to total amino acid weight of 21.5%, 32.3%, 43.0%, and 3.2%, respectively.

In some embodiments, the composition further includes a famesoid X receptor (FXR) agonist, a stearoyl CoA desaturase inhibitor, a CCR2 and CCR5 chemokine antagonist, a PPAR alpha and delta agonist, a caspase inhibitor, a galectin-3 inhibitor, an acetyl CoA carboxylase inhibitor, or an ileal sodium bile acid co-transporter inhibitor. In some embodiments, the composition further comprises an FXR agonist. In certain embodiments, the FXR agonist is obeticholic acid. In some embodiments, the composition further includes one or more of: LMB763, LJN-452, emricasan, and cenicriviroc.

An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine

HCl, glutamine, and N-acetylcysteine as its amino acid entities in a wt. ratio of 1 : 0.5 : 0.5 : 1.81 : 2 : 0.15 (Table 3). An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine, glutamine, and N-acetylcysteine as its amino acid entities in a wt. ratio of 1 : 0.5 : 0.5 : 1.5 : 2 : 0.15 (Table 4).

Table 3. Exemplary amino acid components of the composition including Arginine HCl.

Amino acid

wt. ratio

wt. %

g / packet

g dose #1

g dose #2

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Leucine	1	16.78	LOOg	2g	4g
Isoleucine	0.5	8.39	0.50 g	1 g	2g
Valine	0.5	8.39	0.50 g	1 g	2g
Arginine HCl	1.81	30.37	1.81g	3.62 g	7.24 g
Glutamine	2	33.56	2.00 g	4g	8g
N-acetylcysteine	0.15	2.52	0.15 g	0.3 g	0.6 g
Total amino acids			5.96 g	^g	~24 g

Table 4. Exemplary amino acid components of the composition including Arginine.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	17.70	1-00 g	2	4
Isoleucine	0.5	8.85	0.50 g	1	2
Valine	0.5	8.85	0.50 g	1	2
Arginine	1.5	26.55	1.5 g	3	6
Glutamine	2	35.4	2.00 g	4	8
N-acetylcysteine	0.15	2.65	0.15 g	0.3	0.6
Total amino acids			5.65g	11.3 g	22.6 g

HCl, glutamine, and N-acetylcysteine as its amino acid entities in a wt. ratio of 1 : 0.5 : 0.5 : 0.905 : 2 : 0.15 (Table 5). An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine, glutamine, and N-acetylcysteine as its amino acid entities in a wt. ratio of 1 : 0.5 : 0.5 : 0.75 : 2 : 0.15 (Table 6).

Table 5. Exemplary amino acid components of the composition including Arginine HCl.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	19.78	1.00 g	2g	4g
Isoleucine	0.5	9.89	0.50 g	1 g	2g
Valine	0.5	9.89	0.50 g	1 g	2g
Arginine HCl	0.905	17.90	0.905 g	1.81g	3.62 g

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Glutamine	2	39.56	2.00 g	4g	8g
N-acetylcysteine	0.15	2.97	0.15 g	0.3 g	0.6 g
Total amino acids			5-06 g	~10g	~20 g

Table 6. Exemplary amino acid components of the composition including Arginine.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	20.41	1.00 g	2	4
Isoleucine	0.5	10.20	0.50 g	1	2
Valine	0.5	10.20	0.50 g	1	2
Arginine	0.75	15.31	0.75 g	1.5	3
Glutamine	2	40.82	2.00 g	4	8
N-acetylcysteine	0.15	3.06	0.15 g	0.3	0.6
Total amino acids			4.9 g	9.8 g	19.6 g

An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine 5 HCl, glutamine, and N-acetylcysteine as its amino acid entities in a wt. ratio of 1 : 0.5 : 0.25 : 0.905 : 1 : 0.225 (Table 7). An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine, glutamine, and N-acetylcysteine as its amino acid entities in a wt. ratio of 1 : 0.5 : 0.25 : 0.75 : 1 : 0.225 (Table 8).

Table 7. Exemplary amino acid components of the composition including Arginine HCl.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	25.77	1.00 g	2g	4g
Isoleucine	0.5	12.89	0.50 g	1 g	2g
Valine	0.25	6.44	0.25 g	0.50 g	1 g
Arginine HCl	0.905	23.32	0.905 g	1.81g	3.62 g
Glutamine	1	25.77	1.00 g	2g	4g
N-acetylcysteine	0.225	5.80	0.225 g	0.45 g	0.9 g
Total amino acids			3.88 g	7-76 g	15.52 g

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Table 8. Exemplary amino acid components of the composition including Arginine.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	26.85	1.00 g	2	4
Isoleucine	0.5	13.42	0.50 g	1	2
Valine	0.25	6.71	0.25 g	0.5	1
Arginine	0.75	20.13	0.75 g	1.5	3
Glutamine	1	26.85	1.00 g	2	4
N-acetylcysteine	0.225	6.04	0.225 g	0.45	0.9
Total amino acids			3.725 g	7.45 g	14.9 g

An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine HCl, glutamine, N-acetylcysteine, and serine as its amino acid entities in a wt. ratio of 1 : 0.5 :

0.25 : 0.905 : 1 : 0.225 : 0.667 (Table 9). An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine, glutamine, N-acetylcysteine, and serine as its amino acid entities in a wt. ratio of 1 : 0.5 : 0.25 : 0.75 : 1 : 0.225 : 1.5 (Table 10).

Table 9. Exemplary amino acid components of the composition including Arginine HCl.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	18.59	1.00 g	2g	4g
Isoleucine	0.5	9.29	0.50 g	1 g	2g
Valine	0.25	4.65	0.25 g	0.50 g	1 g
Arginine HCl	0.905	16.82	0.905 g	1.81 g	3.62 g
Glutamine	1	18.59	1.00 g	2g	4g
N-acetylcysteine	0.225	4.18	0.225 g	0.45 g	0.9 g
Serine	1.5	27.88	1.5	3	6
Total amino acids			531g	10.76 g	21.52 g

Table 10. Exemplary amino acid components of the composition including Arginine.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	19.14	1.00 g	2	4

Isoleucine	0.5	9.57	0.50 g	1	2
Valine	0.25	4.78	0.25 g	0.5	1
Arginine	0.75	14.35	0.75 g	1.5	3
Glutamine	1	19.14	1.00 g	2	4
N-acetylcysteine	0.225	4.31	0.225 g	0.45	0.9
Serine	1.5	28.71	1.5	3	6
Total amino acids			5.225	10.45	20.9

An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine HCl, glutamine, N-acetylcysteine, and serine as its amino acid entities in a wt. ratio of 1 : 0.5 : 0.25 : 0.905 : 1 : 0.225 : 0.667 (Table 11). An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine, glutamine, N-acetylcysteine, and serine as its amino acid entities in a wt. ratio of 1 : 0.5 : 0.25 : 0.75 : 1 : 0.225 : 1.667 (Table 12).

Table 11. Exemplary amino acid components of the composition including Arginine HCl.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	18.02	1.00 g	2g	4g
Isoleucine	0.5	9.01	0.50 g	1 g	2g
Valine	0.25	4.50	0.25 g	0.50 g	1 g
Arginine HCl	0.905	16.31	0.905 g	1.81g	3.62 g
Glutamine	1	18.02	1.00 g	2g	4g
N-acetylcysteine	0.225	4.05	0.225 g	0.45 g	0.9 g
Serine	1.667	30.09	1.67 g	3.33 g	6.67 g
Total amino acids			5.55 g	11.09 g	22.19 g

Table 12. Exemplary amino acid components of the composition including Arginine.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	18.54	1.00 g	2	4
Isoleucine	0.5	9.27	0.50 g	1	2
Valine	0.25	4.64	0.25 g	0.5	1

Arginine	0.75	13.91	0.75 g	1.5	3
Glutamine	1	18.54	1.00 g	2	4
N-acetylcysteine	0.225	4.17	0.225 g	0.45	0.9
Serine	1.667	30.92	1.67 g	3.33 g	6.67 g
Total amino acids			5.395 g	10.78 g	21.57 g

The disclosure also provides a composition including at least four different amino acid entities (e.g., four, five, six, or more different amino acid entitites), in which the composition is capable of one, two, three, four, five, or ail of:

a) one or both of decreasing or preventing one or both of liver fibrosis or liver injury;

b) one or both of decreasing or preventing hépatocyte inflammation;

c) improving, e.g., increasing, glucose tolérance;

d) one or both of decreasing or preventing steatosis; or

e) one or both of decreasing or preventing hépatocyte ballooning, provided that at least one amino acid entity is not a peptide of more than 20 amino acid residues in length.

In some embodiments, the composition includes at least four different amino acid entities (e.g., four, five, six, or more different amino acid entities) that decreases or prevents one or both of liver fibrosis or liver injury. For instance, the reducing and/or inhibiting liver fibrosis and/or liver injury comprises can include reducing a level of one or both of collagen, e.g., type I and III collagen or α-smooth muscle actin (aSMA).

In some embodiments, the composition includes at least four different amino acid entities (e.g., four, five, six, or more different amino acid entities) that decreases or prevents hépatocyte inflammation. In some embodiments, the reducing and/or inhibiting liver fibrosis and/or liver injury includes reducing a level or activity of one, two, three, four, or more (e.g., ail) of a matrix metalloproteinase (MMP) (e.g., MMP-13, MMP-2, MMP-9, MT1-MMP, MMP-3, or MMP-10), a tissue inhibitor of metalloproteinase (TIMP) (e.g., TIMP1), aspartate transaminase (AST), alanine transaminase (ALT), or N-terminal fragment of type ΙΠ collagen (proC3).

In some embodiments, the decreasing or preventing hépatocyte inflammation comprises reducing a level or activity of one, two, three, four, five, six, seven or more (e.g., ail) of NF-kB, interférons, IL-lb, IL-2, MCP-1, MIP-1, a caspase-cleaved keratin 18 fragments (e.g., one or both of M30 or M65), or C-reactive protein. In an embodiment, the decreasing or preventing hépatocyte inflammation comprises increasing a level or activity of IL-10.

In an embodiment, the improving, e.g., increasing, glucose tolérance, comprises increasing a level or activity of adiponectin. In an embodiment, the improving, e.g., increasing, glucose tolérance, comprises decreasing a level or activity of FGF-21.

In certain embodiments, the hépatocyte inflammation comprises LPS induced hépatocyte inflammation.

In some embodiments, the composition is capable of enhancing fatty acid oxidation, e.g., one or both of reducing levels of unsaturated fatty acids or increasing levels of acylcamitine (e.g., in a STAM mouse model or a FATZO mouse model). In certain embodiments, the réduction in levels of unsaturated fatty acids is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the level of change shown in Table 53, e.g., measured as described in Example 9. In certain embodiments, the increase in levels of acylcamitine is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the level of change shown in Table 53, e.g., measured as described in Example 9.

In certain embodiments, the composition is capable of reducing, or reduces, liver fibrosis or liver injuiy by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of alanine transaminase (ALT), e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, or reduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of aspartate transaminase (AST), e.g., an antibodybased détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, or reduces, alanine transaminase (ALT) by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of ALT, e.g., an antibody-based détection assay,

e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, or reduces, aspartate transaminase (AST) by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of AST, e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, or reduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of hydroxyproline, e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, or reduces, hydroxyproline levels by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of hydroxyproline, e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, or reduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using LX-2 cells, e.g., levels of Collai, Acta2, and/or TIMP2 in 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 7, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; NAC; or an amino acid composition comprising L-arginine, L-glutamine, and NAC).

In certain embodiments, the composition is capable of reducing, or reduces, expression of one or more collagen biomarkers (e.g., Collai, Acta2, and/or TIMP2) by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using LX-2 cells, e.g., levels of Collai, Acta2, and/or TIMP2 in 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 7, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition

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In certain embodiments, the composition is capable of reducing, or reduces, expression of one or more collagen biomarkers (e.g., Collai) by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using primary hepatic stellate cells, e.g., levels of Collai in primary hepatic stellate cells, e.g., as assessed using an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 12, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, Lvaline, L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

In certain embodiments, the composition is capable of increasing, or increases, expression of one or more collagen biomarkers (e.g., procollagen lal) by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using primary hepatic stellate cells, e.g., levels of procollagen lalin primary hepatic stellate cells, e.g., as assessed using an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 12, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising Lleucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

In certain embodiments, the composition is capable of reducing, or reduces, hépatocyte inflammation by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using HepG2 cells, e.g., decreased activity, e.g., decreased TNFa-induced activity of NF-kB in a reporter assay in HepG2 cells, as described in Example 8, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising Lleucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; or NAC).

In certain embodiments, the composition is capable of reducing, or reduces, TNFainduced activity of NF-kB in HepG2 cells by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%,

45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using HepG2 cells, e.g., decreased activity, e.g., decreased TNFa-induced activity of NF-kB in a reporter assay in HepG2 cells, as described in Example 8, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; or NAC).

In certain embodiments, the composition is capable of increasing, or increases, glucose tolérance, e.g., in a STAM mouse model or in a FATZO mouse model, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of glucose levels, e.g., using glucose oxidase, e.g., using a glucometer, e.g., as described in Example 5, e.g., relative to a reference composition (e.g., a vehicle control or a positive control, e.g., metformin).

In certain embodiments, the composition is capable of increasing, or increases, blood glucose metabolism, e.g., in a STAM mouse model or in a FATZO mouse model, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of glucose levels, e.g., using glucose oxidase, e.g., using a glucometer, e.g., as described in Example 5, e.g., relative to a reference composition (e.g., a vehicle control or a positive control, e.g., metformin).

In certain embodiments, the composition is capable of decreasing, or decreases, steatosis and/or inflammation by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2, e.g., in primary hépatocytes, e.g., using an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 10, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, Lisoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, and Lglutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

In certain embodiments, the composition is capable of decreasing, or decreases, MCP1/CCL2 levels by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2, e.g., in primary hépatocytes, e.g., using an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 10, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, Lisoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, and L54

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glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).In certain embodiments, the | composition is capable of decreasing, or decreases, TNFa infiammatory response by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2 or an | assay of IL-6, e.g., in primary hepatic stellate cells, e.g., using an antibody-based détection assay,

e.g., an ELISA, e.g., as described in Example 11, e.g., relative to a reference composition (e.g., a | vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid | composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

| 10 In certain embodiments, the composition is capable of decreasing, or decreases,

MCP1/CCL2 levels and/or IL-6 levels by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or | 10%, as detected using an assay of MCP1/CCL2 or an assay of IL-6, e.g., in primary hepatic stellate cells, e.g., using an antibody-based détection assay, e.g., an ELISA, e.g., as described in | Example 11, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition | comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising Lleucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine;

| isoleucine; leucine; or NAC).

In any of the foregoing embodiments, the reference composition comprises a single | 20 amino acid entity, e.g., a L-amino acid entity, an I-amino acid entity, a V-amino acid entity, a Ramino acid entity, a Q-amino acid entity, or a NAC-amino acid entity, each assayed separately as | a free amino acid, or a combination of amino acid entities (e.g., a L-amino acid entity, an Iamino acid entity, and a V-amino acid entity; a R-amino acid entity, a Q-amino acid entity, and a | NAC-amino acid entity; a L-amino acid entity, an I-amino acid entity, V-amino acid entity, a R25 amino acid entity, and a Q-amino acid entity). In certain embodiments, the reference | composition comprises vehicle (e.g., PBS or saline).

In some embodiments, the composition that decreases and/or prevents liver fibrosis | and/or liver injury comprises one or more branched-chain amino acid (BCAAs), one or more conditionally essential amino acid (CEAAs), and an antioxidant or reactive oxygen species j| 30 (ROS) scavenger.

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In some embodiments, the composition that decreases and/or prevents hépatocyte inflammation comprises one or more BCAAs, one or more CEAAs, and an antioxidant or ROS scavenger.

In some embodiments, the composition that increases glucose tolérance comprises one or more BCAAs, one or more CEAAs, and an antioxidant or ROS scavenger.

In some embodiments, the composition that decreases and/or prevents steatosis comprises one or more BCAAs, one or more CEAAs, and an antioxidant or ROS scavenger.

In some embodiments, the composition that decreases and/or prevents hépatocyte ballooning comprises one or more BCAAs, one or more CEAAs, and an antioxidant or ROS scavenger.

In an embodiment, the BCAA comprises a L-amino acid entity. In an embodiment, the BCAAs comprise a L-amino acid entity and an l-amino acid entity. In an embodiment, the BCAAs comprise a L-amino acid entity and a V-amino acid entity. In an embodiment, the BCAAs comprise a L-amino acid entity, a V-amino acid entity, and an l-amino acid entity. In an embodiment, the CEAA comprises a R-amino acid entity. In an embodiment, the CEAA comprises a Q-amino acid entity. In an embodiment, the CEAA comprises a R-amino acid entity and a Q-amino acid entity. In an embodiment, the antioxidant or ROS scavenger comprises a NAC entity, e.g., NAC.

In some embodiments, the composition comprises a) a L-amino acid entity, an R-amino acid entity, and a Q-amino acid entity; and b) an antioxidant or ROS scavenger, e.g., a NAC entity, e.g., NAC.

In some embodiments, the composition further comprises an l-amino acid-entity or a Vamino acid-entity. In other embodiments, the composition further comprises an l-amino acidentity and a V-amino acid-entity.

Production of the Amino Acid Compositions

Amino acids used to make the compositions may be agglomerated, and/or instantized to aid in dispersai and/or solubilization.

The amino acid compositions of the présent disclosure may be made using amino acids and amino acid dérivatives from the following sources, or other sources may used: FUSIBCAA™ Instantized Blend (L-Leucine, L-Isoleucine and L-Valine in 2:1:1 weight ratio),

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FUSIL™ Instantized L-Leucine, L-Arginine HCl, and L-Glutamine may be obtained from | Ajinomoto Co., Inc; N-acetyl-cysteine may be obtained from Spectrum Chemical.

To produce the amino acid compositions of the instant disclosure, the following general | steps may be used: the starting materials (individual amino acids and excipients) may be blended 5 in a blending unit, followed by vérification of blend uniformity and amino acid content, and | filling of the blended powder into stick packs or other unit dosage form. The content of stick packs or other unit dosage forms may be dispersed in water at time of use for oral administration.

I

Formulations | 10 The pharmaceutical compositions of the présent disclosure may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, | émulsions, dispersible powders or granules, syrups or élixirs, medical food products, nutraceuticals), for topical use (for example as creams, ointments, gels, or aqueous or oily | solutions or suspensions), for administration by inhalation (for example as finely divided 15 powder) or for parental administration (for example as a stérile aqueous or oily solution for | intravenous, subcutaneous, intramuscular dosing or as a suppository for rectal dosing).

| Excipients

The amino acid compositions of the présent disclosure may be compounded or

H 20 formulated with one or more excipients. Non-limiting examples of suitable excipients include a tastant, a flavorant, a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a | lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.

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

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

J 30 In some embodiments, the composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starches, pregelatinized starches, gelatin,

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In some embodiments, the composition comprises a lubricant as an excipient. Nonlimiting examples of suitable lubricants include magnésium stéarate, calcium stéarate, zinc stéarate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnésium lauryl sulfate, and light minerai oil.

In some embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, xanthan gum, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, 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 and modified starches thereof, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, 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. Flavoring agents can be chosen from synthetic flavor oils and flavoring aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; and combinations thereof. In some embodiments, the flavoring agent is selected from cinnamon oils; oil of wintergreen; peppermint oils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oil such as lemon oil, orange oil, grape and grapefruit oil; and fruit essences including apple, peach, pear, strawberry, raspbeny, 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 sait;

dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro dérivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like. Also contemplated are hydrogenated starch hydrolysates and the synthetic sweetener 3,6-dihydro-6-methyl-l,2,3-oxathiazin-4-one-2,2dioxide, particularly the potassium sait (acesulfame-K), and sodium and calcium salts thereof.

In some embodiments, the composition comprises a coloring agent. Non-limiting examples of suitable color agents include food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), and extemal drug and cosmetic colors (Ext. D&C). The coloring agents can be used as dyes or their corresponding lakes.

Particular excipients may include one or more of: citric acid, lecithin, (e.g. Alcolec F100), sweeteners (e.g. sucralose, sucralose micronized NF, acesulfame potassium (e.g. Ace-K)), a dispersion enhancer (e.g. xanthan gum (e.g. Ticaxan Rapid-3)), flavorings (e.g. vanilla custard #4306, Nat Orange WONF #1326, lime 865.0032U, and lemon 862.2169U), a bittemess masking agent (e.g. 936.2160U), and natural or artificial colorings (e.g. FD&C Yellow 6).

Methods of Treatment

The composition as described herein can be administered to improve liver function, e.g., in a patient with a liver disease. The composition as described herein can also be administered to treat (e.g., reverse, reduce, ameliorate, or prevent) a disorder, e.g., a liver disease in a subject. The présent disclosure provides methods of treating a liver disease selected from fatty liver disease (steatohepatitis), alcoholic steatohepatitis (ASH), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. In particular, an effective amount of the composition can be administered (e.g., according to a dosage regimen described herein) to treat a subject with non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), or cirrhosis.

Patients with Liver Disease

In some embodiments, a subject has fatty liver disease selected from NAFLD and AFLD.

In some embodiments, the subject has pédiatrie NAFLD. In some embodiments, the subject with NAFLD has NASH or NAFL. In some embodiments, the subject with AFLD has ASH.

In certain embodiments, the subject exhibits symptoms of gut leakiness. In certain embodiments, the subject has gut dysbiosis. In certain embodiments, the subject has gut microbiome disturbance. The subject may hâve increased levels of inflammatory cytokines, e.g., increased TNFa, relative to a normal subject without a fatty liver disease.

In certain embodiments, the subject exhibits muscle atrophy, e.g., has a decreased ratio of muscle tissue to adipose tissue, e.g., relative to a normal subject without a fatty liver disease. For example, the subject exhibits muscle atrophy without fibrosis and/or cirrhosis.

In certain embodiments, the subject exhibits reverse lipid transport from adipose tissue to liver tissue.

In some embodiments, the subject has fibrosis. The subject may hâve cirrhosis. The subject may also hâve a metabolic syndrome.

In certain embodiments, the subject has one, two, or more (e.g., ail) of hepatocarcinoma, an increased risk of liver failure, or an increased risk of death.

In some embodiments, the subject has type 2 diabètes.

In some embodiments, the subject with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)) is a mammal (e.g., a human). In some embodiments, the subject has been diagnosed with NAFLD, NASH or cirrhosis. In some embodiments, the subject has not received prior treatment with a composition as described herein (e.g., the subject is a naïve subject). In some embodiments, the subject with NAFLD, NASH or cirrhosis has diabètes (e.g., type 2 diabètes).

In some embodiments, the subject has NAFLD. In some embodiments, the subject has NAFL. In certain embodiments, the subject (e.g., a child or an adolescent) has pédiatrie NAFLD. In some embodiments, the subject has hepatic steatosis. In some embodiments, a subject with pédiatrie NAFLD has steatosis.

In some embodiments, the subject has non-alcoholic steatohepatitis (NASH). In some embodiments, the subject with NASH has fibrosis.

In some embodiments, the subject has cirrhosis. In some embodiments, the subject with cirrhosis has fibrosis. In some embodiments, the subject with cirrhosis has hepatocarcinoma. In some embodiments, the subject with cirrhosis has an increased risk of liver failure. In some embodiments, the subject with cirrhosis has hepatocarcinoma, an increased risk of liver failure, and an increased risk of death.

In some embodiments, a subject exhibits a symptom of liver disease (e.g. NAFLD, NASH, or cirrhosis), e.g., a metabolic symptom, prior to administration of the composition. In some embodiments, a subject exhibits a metabolic symptom of liver disease (e.g. NAFLD, NASH, or cirrhosis) selected from one, two, three, four, five, six, or more (e.g., ail) of decreased fat metabolism, hépatocyte apoptosis, hépatocyte ballooning, inflammation of adipose tissue, inflammation of hepatic tissue, hépatocyte ballooning, oxidative stress (e.g., reactive oxygen species (ROS), decreased gut barrier function, decreased insulin sécrétion, or decreased glucose tolérance (e.g., relative to a healthy subject without a liver disease).

In some embodiments, a subject exhibits modulated (e.g., increased) levels of a biomarker prior to administration of the composition. In some embodiments, a subject exhibits modulated levels of a biomarker selected from one, two, three, four, five, six, seven, eight, nine, or more (e.g., ail) of ACOX1; IL-10; NF-kB, an interferon, IL-2; glutathione (GSH); alanine aminotransferase (ALT); aspartate aminotransferase (AST); adiponectin; N-terminal fragment of type III collagen (proC3); caspase-cleaved keratin 18 fragments (M30 and M65); IL-Ιβ; Creactive protein; ΡΠΙΝΡ; ΊΊΜΡ1; MCP-1; or FGF-21 (e.g., relative to a healthy subject without a liver disease).

In some embodiments, the subject exhibits increased levels of ALT, e.g., relative to a healthy subject without a liver disease.

In some embodiments, the subject exhibits increased levels of AST, e.g., relative to a healthy subject without a liver disease.

Improvement in Symptoms of Liver Disease

The composition as described herein can be administered to treat (e.g., reverse, reduce, ameliorate, or prevent) a subject (e.g., a human) with a liver disease, thereby improving a symptom of a liver disease in the patient. In some embodiments, the composition is administered to a subject with NAFLD. In some embodiments, the composition is administered to a subject with NAFL. In some embodiments, the composition is administered to a subject with NASH. In some embodiments, the composition is administered to a subject with cirrhosis of the liver.

In some embodiments, administration of a composition (e.g., at a dosage regimen described herein) results in an improvement in one or more symptoms of NAFLD, e.g., a metabolic symptom of NAFLD, in a subject.

In some embodiments, administration of the composition results in increased free fatty acid and lipid metabolism in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD). In some embodiments, administration of the composition results in improved mitochondrial function in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD). In some embodiments, administration of the composition results in white adipose tissue (WAT) browning in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD).

In some embodiments, administration of the composition results in decreased reactive oxygen species (ROS) in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD). In some embodiments, administration of the composition results in increased levels of glutathione (GSH) in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD).

In some embodiments, administration of the composition results in decreased hepatic inflammation in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD). In some embodiments, administration of the composition results in decreased hépatocyte ballooning in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD).

In some embodiments, administration of the composition results in improved gut barrier function in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD).

In some embodiments, administration of the composition results in increased insulin sécrétion in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD). In some embodiments, administration of the composition results in improved glucose tolérance in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD).

In some embodiments, the composition reduces or inhibits liver fibrosis in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD). In some embodiments, the composition reduces or inhibits liver fibrosis in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD).

In some embodiments, the composition reduces liver fat in a subject with NAFLD (e.g., a subject with pédiatrie NAFLD). In some embodiments, the composition reduces liver enzyme levels (e.g., ALT or AST) in blood or plasma from a subject with NAFLD (e.g., a subject with pédiatrie NAFLD).

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In some embodiments, administration of a composition (e.g., at a dosage regimen described herein) including amino acid entities results in an improvement in one or more symptoms of NASH, e.g., a metabolic symptom of NASH, in a subject.

In some embodiments, administration of the composition results in increased free fatty acid and lipid metabolism in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes). In some embodiments, administration of the composition results in improved mitochondrial function in a subject with NASH. In some embodiments, administration of the composition results in white adipose tissue (WAT) browning in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes).

In some embodiments, administration of the composition results in decreased reactive oxygen species (ROS) in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes). In some embodiments, administration of the composition results in increased levels of glutathione (GSH) in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes).

In some embodiments, administration of the composition results in decreased hepatic inflammation in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes). In some embodiments, administration of the composition results in decreased hépatocyte ballooning in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes).

In some embodiments, administration of the composition results in improved gut barrier function in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes).

In some embodiments, administration of the composition results in increased insulin sécrétion in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes). In some embodiments, administration of the composition results in improved glucose tolérance in a 25 subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes).

In some embodiments, the composition reduces or inhibits liver fibrosis in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes). In some embodiments, the composition reduces or inhibits liver fibrosis in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes).

In some embodiments, the composition reduces liver fat in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabètes). In some embodiments, the composition

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In some embodiments, administration of a composition (e.g., at a dosage regimen described herein) including amino acid entities results in an improvement in one or more 5 symptoms of cirrhosis, e.g., a metabolic symptom of cirrhosis, in a subject.

In some embodiments, administration of the composition results in decreased reactive oxygen species (ROS) in a subject with cirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death). In some embodiments, administration of the composition results in increased levels of glutathione (GSH) in a subject with cirrhosis (e.g., a 10 subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death).

In some embodiments, administration of the composition results in decreased hepatic inflammation in a subject with cirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death). In some embodiments, administration of the composition results in decreased hépatocyte ballooning in a subject with cirrhosis (e.g., a subject 15 with hepatocarcinoma, increased risk of liver failure, and increased risk of death).

In some embodiments, administration of the composition results in improved gut barrier function in a subject with cirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death).

In some embodiments, administration of the composition results in increased insulin 20 sécrétion in a subject with cirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death). In some embodiments, administration of the composition results in improved glucose tolérance in a subject with cirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death).

In some embodiments, the composition reduces or inhibits liver fibrosis in a subject with 25 cirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death). In some embodiments, the composition reduces or inhibits liver fibrosis in a subject with cirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death).

In some embodiments, the composition reduces liver fat in a subject with cirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death). In some embodiments, the composition reduces liver enzyme levels (e.g., ALT or AST) in blood or plasma from a subject with cirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death).

Dosage Regimens

The composition can be administered according to a dosage regimen described herein to treat (e.g., inhibit, reduce, ameliorate, or prevent) a disorder, e.g., a liver disease in a subject (e.g., a human). In some embodiments, the subject has NAFLD. In some embodiments, the subject has NAFL. In some embodiments, the subject has NASH. In some embodiments, the subject has cirrhosis.

The composition can be provided to a patient with a liver disease (e.g., NAFL, NASH, or cirrhosis) in either a single or multiple dosage regimens. In some embodiments, doses are administered, e.g., twice daily, three times daily, four times daily, five times daily, six times daily, seven times daily, or more. 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 for at least 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or longer. In some embodiments, the composition is administered chronically, e.g., 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 at a dose of about 2 g to about 60 20 g total amino acids, e.g., 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 at a dose of about 5 g to about 15 g, about 10 g to about 20 g, about 20 g to about 40 g, or about 30 g to about 50 g total amino acids, e.g., 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 25 times per day).

In some embodiments, the composition is administered at a dose of about 5 g to about 10 g total amino acids, e.g., 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 at a dose of about 6 g total amino acids, e.g., once per day, twice per 30 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 an embodiment, the composition is administered at a dose of about 6 g total amino acids three times per day.

In some embodiments, the composition is administered at a dose of about 10 g to about 20 g total amino acids, e.g., 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 at a dose of about 12 g total amino acids, e.g., 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 an embodiment, the composition is administered at a dose of about 12 g total amino acids three times per day.

In some embodiments, the composition is administered at a dose of about 20 g to about 40 g total amino acids, e.g., 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 at a dose of about 18 g total amino acids, e.g., 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 an embodiment, the composition is administered at a dose of about 18 g total amino acids three times per day.

In some embodiments, the composition is administered at a dose of about 20 g to about 40 g total amino acids, e.g., 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 at a dose of about 24 g total amino acids, e.g., 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 an embodiment, the composition is administered at a dose of about 24 g total amino acids three times per day.

In some embodiments, the composition is administered at a dose of about 30 g to about 50 g total amino acids, e.g., 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 at a dose of about 48 g total amino acids, e.g., 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 an embodiment, the composition is administered at a dose of about 48 g total amino acids three times per day.

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In some embodiments, the composition is administered at a dose of about 5 grams, about 8 grams, about 9 grams, about 10 grams, about 11 grams, about 12 grams, about 13 grams, about 14 grams, about 15 grams, about 16 grams, about 17 grams, about 18 grams, about 19 about grams, about 20 grams, about 21 grams, about 22 grams, about 24 grams, about 25 grams, about 5 26 grams, about 27 grams, about 28 grams, about 29 grams, or about 30 grams total amino acids (e.g., about 12 g or about 24 g) , e.g., 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 every 2 hours, every 3 hours, every 4 hours, every 5 hours, eveiy 6 hours, every 7 hours, every 8 hours, every 9 hours, or every 10 10 hours to a subject with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g.,

ASH)).

In an embodiment, the composition is administered to a subject with NAFLD prior to a meal. In an embodiment, the composition is administered to a subject with NAFLD concurrent with a meal. In an embodiment, the composition is administered to a subject with NAFLD 15 following ameal.

In an embodiment, the composition is administered to a subject with NAFL prior to a meal. In an embodiment, the composition is administered to a subject with NAFL concurrent with a meal. In an embodiment, the composition is administered to a subject with NAFL following a meal.

In an embodiment, the composition is administered to a subject with NASH prior to a meal. In an embodiment, the composition is administered to a subject with NASH concurrent with a meal. In an embodiment, the composition is administered to a subject with NASH following a meal.

In an embodiment, the composition is administered to the subject with cirrhosis prior to a 25 meal. In an embodiment, the composition is administered to a subject with cirrhosis concurrent with a meal. In an embodiment, the composition is administered to a subject with cirrhosis following a meal.

In an embodiment, the composition includes at least 1 g of leucine, at least 0.5 g of isoleucine, at least 0.5 g of valine, at least 1.5 g of arginine (or 1.81 g of arginine HCl), at least 2 30 g of glutamine, and at least 0.15 g of N-acetylcysteine for administration three times per day (e.g., for a total of at least 18 g per day).

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In an embodiment, the composition includes about 1 g of leucine, about 0.5 g of isoleucine, about 0.5 g of valine, about 1.5 g of arginine (or 1.81 g of arginine HCl), about 2 g of glutamine, and about 0.15 g of N-acetylcysteine for administration three times per day (e.g., for a total of about 18 g per day).

In an embodiment, the composition includes at least 2 g of leucine, at least 1 g of isoleucine, at least 1 g of valine, at least 3.0 g of arginine (or 3.62 g of arginine HCl), at least 4 g of glutamine, and at least 0.3 g of N-acetylcysteine for administration three times per day (e.g., a total of at least 36 g per day).

In an embodiment, the composition includes about 2 g of leucine, about 1 g of isoleucine, about 1 g of valine, about 3.0 g or arginine (or 3.62 g of arginine HCl), about 4 g of glutamine, and about 0.3 g of N-acetylcysteine for administration three times per day (e.g., a total of about 36 g per day).

In an embodiment, the composition includes at least 4 g of leucine, at least 2 g of isoleucine, at least 2 g of valine, at least 6.0 g of arginine (or 7.24 g of arginine HCl), at least 8 g of glutamine, and at least 0.6 g of N-acetylcysteine for administration three times per day (e.g., a total of at least 72 g per day).

In an embodiment, the composition includes about 4 g of leucine, about 2 g of isoleucine, about 2 g of valine, about 6.0 g of arginine (or 7.24 g of arginine HCl), about 8 g of glutamine, and about 0.6 g of N-acetylcysteine for administration three times per day (e.g., a total of about 72 g per day).

In an embodiment, the composition includes at least 1 g of leucine, at least 0.5 g of isoleucine, at least 0.5 g of valine, at least 0.75 g of arginine (or 0.905 g of arginine HCl), at least 2 g of glutamine, and at least 0.15 g of N-acetylcysteine for administration three times per day (e.g., for a total of at least 18 g per day).

In an embodiment, the composition includes about 1 g of leucine, about 0.5 g of isoleucine, about 0.5 g of valine, about 0.75 g of arginine (or 0.905 g of arginine HCl), about 2 g of glutamine, and about 0.15 g of N-acetylcysteine for administration three times per day (e.g., for a total of about 18 g per day).

In an embodiment, the composition includes at least 2 g of leucine, at least 1 g of isoleucine, at least 1 g of valine, at least 1.5 g of arginine (or 1.81 g of arginine HCl), at least 4 g

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I of glutamine, and at least 0.3 g of N-acetylcysteine for administration three times per day (e.g., a total of at least 36 g per day).

In an embodiment, the composition includes about 2 g of leucine, about 1 g of isoleucine, about 1 g of valine, about 1.5 g or arginine (or 1.81 g of arginine HCl), about 4 g of glutamine, 5 and about 0.3 g of N-acetylcysteine for administration three times per day (e.g., a total of about 36 g per day).

In an embodiment, the composition includes at least 4 g of leucine, at least 2 g of isoleucine, at least 2 g of valine, at least 3.0 g of arginine (or 3.62 g of arginine HCl), at least 8 g of glutamine, and at least 0.6 g of N-acetylcysteine for administration three times per day (e.g., a 10 total of at least 72 g per day).

In an embodiment, the composition includes about 4 g of leucine, about 2 g of isoleucine, about 2 g of valine, about 3.0 g of arginine (or 3.62 g of arginine HCl), about 8 g of glutamine, and about 0.6 g of N-acetylcysteine for administration three times per day (e.g., a total of about 72 g per day).

In an embodiment, the composition includes at least 1 g of leucine, at least 0.5 g of isoleucine, at least 0.25 g of valine, at least 0.75 g of arginine (or 0.905 g of arginine HCl), at least 1 g of glutamine, and at least 0.225 g of N-acetylcysteine for administration three times per day (e.g., for a total of at least 18 g per day).

In an embodiment, the composition includes about 1 g of leucine, about 0.5 g of 20 isoleucine, about 0.25 g of valine, about 0.75 g of arginine (or 0.905 g of arginine HCl), about 1 g of glutamine, and about 0.225 g of N-acetylcysteine for administration three times per day (e.g., for a total of about 18 g per day).

In an embodiment, the composition includes at least 2 g of leucine, at least 1 g of isoleucine, at least 0.5 g of valine, at least 1.5 g of arginine (or 1.81 g of arginine HCl), at least 2 25 g of glutamine, and at least 0.45 g of N-acetylcysteine for administration three times per day (e.g., a total of at least 36 g per day).

In an embodiment, the composition includes about 2 g of leucine, about 1 g of isoleucine, about 0.5 g of valine, about 1.5 g or arginine (or 1.81 g of arginine HCl), about 2 g of glutamine, and about 0.45 g of N-acetylcysteine for administration three times per day (e.g., a total of about 30 36 g per day).

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In an embodiment, the composition includes at least 4 g of leucine, at least 2 g of isoleucine, at least 1 g of valine, at least 3 g of arginine (or 3.62 g of arginine HCl), at least 4 g of glutamine, and at least 0.9 g of N-acetylcysteine for administration three times per day (e.g., a total of at least 72 g per day).

In an embodiment, the composition includes about 4 g of leucine, about 2 g of isoleucine, about 1 g of valine, about 3 g of arginine (or 3.62 g of arginine HCl), about 4 g of glutamine, and about 0.9 g of N-acetylcysteine for administration three times per day (e.g., a total of about 72 g per day).

In an embodiment, the composition includes at least 1 g of leucine, at least 0.5 g of 10 isoleucine, at least 0.25 g of valine, at least 0.75 g of arginine (or 0.905 g of arginine HCl), at least 1 g of glutamine, at least 0.225 g of N-acetylcysteine, and at least 1.5g or about 1.67 g of serine for administration three times per day (e.g., for a total of at least 18 g per day or for a total of at least 20 g per day).

In an embodiment, the composition includes about 1 g of leucine, about 0.5 g of 15 isoleucine, about 0.25 g of valine, about 0.75 g of arginine (or 0.905 g of arginine HCl), about 1 g of glutamine, about 0.225 g of N-acetylcysteine, and about 1.5 g or about 1.67 g of serine for administration three times per day (e.g., for a total of about 18 g per day or for a total of at least 20 g per day).

In an embodiment, the composition includes at least 2 g of leucine, at least 1 g of 20 isoleucine, at least 0.5 g of valine, at least 1.5 g of arginine (or 1.81 g of arginine HCl), at least 2 g of glutamine, at least 0.45 g of N-acetylcysteine, and at least 3 g or about 3.33 g of serine for administration three times per day (e.g., a total of at least 36 g per day or for a total of at least 40 g per day).

In an embodiment, the composition includes about 2 g of leucine, about 1 g of isoleucine, 25 about 0.5 g of valine, about 1.5 g or arginine (or 1.81 g of arginine HCl), about 2 g of glutamine, about 0.45 g of N-acetylcysteine, and about 3 g or about 3.33 g of serine for administration three times per day (e.g., a total of about 36 g per day or for a total of at least 40 g per day).

In an embodiment, the composition includes at least 4 g of leucine, at least 2 g of isoleucine, at least 1 g of valine, at least 3 g of arginine (or 3.62 g of arginine HCl), at least 4 g of 30 glutamine, at least 0.9 g of N-acetylcysteine, and at least 6 g or about 6.67 g of serine for administration three times per day (e.g., a total of at least 90 g per day).

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In an embodiment, the composition includes about 4 g of leucine, about 2 g of isoleucine, | about 1 g of valine, about 3 g of arginine (or 3.62 g of arginine HCl), about 4 g of glutamine, about 0.9 g of N-acetylcysteine, and about 6 g or about 6.67 g of serine for administration three | times per day (e.g., a total of about 90 g per day).In some embodiments, the composition comprises four stick packs, each stick pack comprising 25% of the quantity of each amino acid | included in the composition (e.g., as described herein).

| Secondary Agents

In some embodiments, the method further comprises administering a famesoid X receptor | 10 (FXR) agonist, a stearoyl CoA desaturase inhibitor, a CCR2 and CCR5 chemokine antagonist, a

PPAR alpha and delta agonist, a caspase inhibitor, a galectin-3 inhibitor, an acetyl CoA | carboxylase inhibitor, or an ileal sodium bile acid co-transporter inhibitor prior to, concurrently with, or after administration of the amino acid composition.

| In some embodiments, the method further includes administering an FXR agonist. In some embodiments, the FXR agonist is obeticholic acid. In some embodiments, the method | further includes administering one or more of: LMB-763, LJN-452, emricasan, and cenicriviroc.

। Dietary Compositions

IThe composition including amino acid entities can be dietary compositions, e.g., chosen 20 from a medical food, a functional food, or a supplément.

IThe composition including amino acid entities can be for use as a dietary composition, e.g., chosen from a medical food, a functional food, or a supplément. In some embodiments, the

I dietary composition is for use in a method comprising adminstering the composition to a subject.

In some embodiments, the subject has one or both of type 2 diabètes or a relatively high 125 BMI.

In some embodiments, the subject has fatty liver disease.

Iln some embodiments, the subject has NAFLD (e.g., pédiatrie NAFLD). In an embodiment, the subject has NASH. In an embodiment, the subject has NAFL.

। In some embodiments, the subject has AFLD. In an embodiment, the subject has ASH.

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In some embodiments, the subject has one, two, three, four, or more (e.g., ail) of fibrosis, | cirrhosis, hepatocarcinoma, an increased risk of liver failure, or an increased risk of death.

In some embodiments, the composition promûtes weight loss in the subject.

| In some embodiments, administration of the dietary composition results in an improvement in one or more metabolic symptoms in the subject, e.g., one or more metabolic | symptoms is selected from the following: increased free fatty acid and lipid metabolism (e.g., in the liver), improved mitochondrial function, white adipose tissue (WAT) browning, decreased | reactive oxygen species (ROS), increased levels of glutathione (GSH), decreased hepatic inflammation, decreased hépatocyte ballooning, improved gut barrier function, increased insulin | 10 sécrétion, or glucose tolérance. In certain embodiments, administration of the composition results in an improvement in one or more metabolic symptoms after a treatment period of 24 | hours.

The method can further include determining the level of one, two, three, four, five, six, | seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more (e.g., ail) of the following:

a) alanine aminotransferase (ALT);

| b) aspartate aminotransferase (AST);

c) adiponectin;

| d) N-terminal fragment of type ΠΙ collagen (proC3);

e) caspase-cleaved keratin 18 fragments (M30 and M65);

| 20 f) IL-1 beta;

g) C-reactive protein;

| h) ΡΠΙΝΡ;

i) a tissue inhibitor of metalloproteinase (TIMP); e.g., ΊΊΜΡ1 or TIMP2;

| j) MCP-1;

* 25 k)FGF-21;

| 1) Collai;

m) Acta2;

n) a matrix metalloproteinase (MMP), e.g., MMP-13, MMP-2, MMP-9, MT1-MMP, * MMP-3, or MMP-10;

o) ACOX1;

p) IL-10; or

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q)NF-kB.

In certain embodiments, administration of the composition results in an improvement in one or more of a)-q) after a treatment period of 24 hours.

In some embodiments, the subject exhibits increased levels of one or both of ALT or

AST prior to administraton of the composition, e.g., relative to a healthy subject without a liver disease. In some embodiments, administration of the composition results in a decrease in levels of one or both of ALT or AST.

Methods of Providing an Amino Acid to a Subject

The présent disclosure features a method of providing amino acid entities to a subject comprising administering to the subject an effective amount of a composition described herein, e.g., a composition comprising a leucine (L)-amino acid entity, a arginine (R)-amino acid entity, a glutamine (Q)-amino acid entity; and an antioxidant or reactive oxygen species (ROS) scavenger, e.g., a N-acetylcysteine (NAC) entity, e.g., NAC. In some embodiments, at least one amino acid entity is not a peptide of more than 20 amino acid residues in length.

The présent disclosure also features a method of increasing one, two, three, or more (e.g., ail) amino acid entities in a subject comprising administering to the subject an effective amount of the composition described herein. In some embodiments, administration of the composition 20 results in an increase in the amino acid entities in one, two, or more (e.g., ail) of blood, plasma, or sérum of the subject, e.g., in a blood, plasma, or sérum sample from the subject.

Biomarkers

Any of the methods disclosed herein can include evaluating or monitoring the effectiveness of administering a composition including amino acid entities to a subject with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)).

In embodiments, the value of effectiveness to the composition in treating a subject with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)) comprises a measure of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or more (e.g., ail) of the following:

a) alanine aminotransferase (ALT);

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b) aspartate aminotransferase (AST);

c) adiponectin;

d) N-terminal fragment of type m collagen (proC3);

e) caspase-cleaved keratin 18 fragments (M30 and M65);

f) IL-1 beta;

g) C-reactive protein;

h) ΡΠΙΝΡ;

i) a tissue inhibitor of metalloproteinase (TIMP); e.g., ΊΊΜΡ1 or TIMP2;

j) MCP-1;

k)FGF-21;

1) Collai;

m) Acta2;

n) a matrix metalloproteinase (MMP), e.g., MMP-13, MMP-2, MMP-9, MT1-MMP, MMP-3, or MMP-10;

o)ACOXl;

p) IL-10; or q)NF-kB.

In some embodiments of any of the methods disclosed herein, the measure of one or more of a)-q) is obtained from a sample acquired from the subject with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)). In some embodiments, the sample is chosen from a blood sample (e.g., a plasma sample) or a liver sample.

In some embodiments, the subject is evaluated prior to receiving, during, or after receiving, a composition including amino acid entities.

In some embodiments, administration of the composition including amino acid entities (e.g., at a dose of about 2 g to about 60 g total amino acids, e.g., about 6 g, about 12 g, about 18 g, about 24 g, or about 48 g), results in an improvement in one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or more (e.g., ail) of the following:

a) alanine aminotransferase (ALT);

b) aspartate aminotransferase (AST);

c) adiponectin;

d) N-terminal fragment of type III collagen (proC3);

e) caspase-cleaved keratin 18 fragments (M30 and M65);

f) IL-1 beta;

g) C-reactive protein;

h) PIIINP;

i) a tissue inhibitor of metalloproteinase (TIMP); e.g., TIMP1 or TIMP2;

j) MCP-1;

k)FGF-21;

1) Collai;

m) Acta2;

o) ACOX1;

p) IL-10; or q) NF-kB.

In some embodiments, administration of the composition including amino acid entities (e.g., at a dose of about 2 g to about 60 g total amino acids, e.g., about 6 g, about 12 g, about 18 g, about 24 g, or about 48 g three times daily), results in an improvement in one, two, three, four, five, six, seven, eight, nine, ten, or more (e.g., ail) of a)-k) after a treatment period of, about 24 hours, about 72 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or 12 weeks. In certain embodiments, administration of the composition results in an improvement in one, two, three, four, five, or more (e.g., ail) of a)-k) after a treatment period of about 2 weeks.

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NUMBERED EMBODIMENTS | The invention is further described with reference to the following numbered embodiments.

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1. A composition comprising:

| a) a leucine (L)-amino acid entity, an arginine (R)-amino acid entity, and a glutamine (Q)-amino acid entity; and | b) an antioxidant or reactive oxygen species (ROS) scavenger, e.g., a N-acetylcysteine (NAC) entity, e.g., NAC;

| 10 provided that:

c) at least one amino acid entity is not provided as a peptide of more than 20 amino acid | residues in length, and optionally wherein:

| (i) an amino acid entity (e.g., at least one, two, or three of the amino acid entities) of (a) is selected from Table 2; or | (ii) (A) one or both of the R-amino acid entity and the Q-amino acid entity are présent at a higher amount (wt. %) than the L-amino acid entity, or (B) the composition further comprises a | serine (S)-amino acid entity.

1 A. The composition of embodiment 1, wherein the composition satisfies the property of (i)·

IB. The composition of any of the preceding embodiments, wherein the composition satisfies the property of (ii)(A).

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IC. The composition of any of the preceding embodiments, wherein the composition ^e satisfies the property of (ii)(B).

ID. The composition of any of the preceding embodiments, wherein the composition 30 further comprises an S-amino acid entity, and wherein the S-amino acid entity is présent at a * higher amount than any other amino acid entity.

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2. The composition of any of embodiments 1-1D, wherein the composition comprises an | amino acid and three amino acid entities.

| 3. The composition of any of embodiments 1-1D, wherein the composition comprises an amino acid precursor and three amino acid entities. ·

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4. The composition of any of embodiments 1-1D, wherein the composition comprises an | amino acid métabolite and three amino acid entities.

| 10 5· The composition of any of embodiments 1-1D, wherein the composition comprises an amino acid dérivative and three amino acid entities.

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6. The composition of any of embodiments 1-1D, wherein the composition comprises two | amino acids and two amino acid entities.

| 7. The composition of any of embodiments 1-1D, wherein the composition comprises two amino acid precursors and two amino acid entities.

8. The composition of any of embodiments 1-1D, wherein the composition comprises two | 20 amino acid métabolites and two amino acid entities.

| 9. The composition of any of embodiments 1-1D, wherein the composition comprises two amino acid dérivatives and two amino acid entities.

10. The composition of any of embodiments 1-1D, wherein the composition comprises | three amino acids and one amino acid entity.

| 11. The composition of any of embodiments 1-1D, wherein the composition comprises three amino acid precursors and one amino acid entity.

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12. The composition of any of embodiments 1-1D, wherein the composition comprises | three amino acid métabolites and one amino acid entity.

| 13. The composition of any of embodiments 1-1D, wherein the composition comprises three amino acid dérivatives and one amino acid entity.

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14. The composition of any of embodiments 1-2, wherein the composition comprises ΤΙ leucine, a R-amino acid entity, and a Q-amino acid entity, and an antioxidant or ROS scavenger,

e.g., a NAC entity.

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15. The composition of any of embodiments 1-2, 2, 14, or 380, wherein the composition | comprises L-leucine, R-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger,

e.g., a NAC entity.

16. The composition of any of embodiments 1-2, 14, or 381, wherein the composition | comprises L-leucine, argininosuccinate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

17. The composition of any of embodiments 1-2, 14, or 382, wherein the composition | 20 comprises L-leucine, citrulline, a Q-amino acid entity, and an antioxidant or ROS scavenger,

e.g., a NAC entity.

18. The composition of any of embodiments 1-2, 14, or 383, wherein the composition H comprises L-leucine, aspartate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

19. The composition of any of embodiments 1-2, 14, or 384, wherein the composition | comprises L-leucine, L-glutamate, a Q-amino acid entity, and an antioxidant or ROS scavenger,

e.g., a NAC entity.

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20. The composition of any of embodiments 1-2, 14, or 385, wherein the composition comprises L-leucine, omithine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

21. The composition of any of embodiments 1-2, 14, or 386, wherein the composition comprises a L-leucine, agmatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

22. The composition of any of embodiments 1-2, 14, or 387, wherein the composition 10 comprises a L-leucine, creatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

23. The composition of any of embodiments 1-2, 14, or 388, wherein the composition comprises L-leucine, D-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, 15 e.g., a NAC entity.

24. The composition of any of embodiments 1-2, 14, or 389, wherein the composition comprises L-leucine, N-acetyl-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

25. The composition of any of embodiments 1-2,14, or 428, wherein the composition comprises L-leucine, a R-amino acid entity, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

26. The composition of any of embodiments 1-2, 14, or 429, wherein the composition comprises L-leucine, a R-amino acid entity, glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

27. The composition of any of embodiments 1-2, 14, or 430, wherein the composition 30 comprises L-leucine, a R-amino acid entity, carbamoyl-P, and an antioxidant or ROS scavenger, e.g., a NAC entity.

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28. The composition of any of embodiments 1-2, 14, or 431, wherein the composition comprises L-leucine, a R-amino acid entity, D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

29. The composition of any of embodiments 1-2, 14, or 432, wherein the composition comprises L-leucine, a R-amino acid entity, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

30. The composition of any of embodiments 1-2, 14, or 445, wherein the composition comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and NAC.

31. The composition of any of embodiments 1-2, 14, or 446, wherein the composition comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and serine.

32. The composition of any of embodiments 1-2, 14, or 447, wherein the composition comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and acetylserine.

33. The composition of any of embodiments 1-2,14, or 448, wherein the composition 20 comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and cystathionine.

34. The composition of any of embodiments 1-2, 14, or 449, wherein the composition comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and glutathione.

35. The composition of any of embodiments 1-2, 14, or 450, wherein the composition comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and homocystéine.

36. The composition of any of embodiments 1-2, 14, or 451, wherein the composition comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and méthionine.

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37. The composition of any of embodiments 1-2, 14, or 452, wherein the composition | comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and D-cysteine.

| 38. The composition of any of embodiments 1-2, 14, or 453, wherein the composition comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and L-cysteine.

39. The composition of any of embodiments 1-2, 14, or 454, wherein the composition | comprises L-leucine, a R-amino acid entity, a Q-amino acid entity, and cystine.

| 10 40. The composition of any of embodiments 1-2, 14, 380, or 428, wherein the composition comprises L-leucine, L-arginine, L-glutamine, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

| 41. The composition of any of embodiments 1-2, 14, 381, or 429, wherein the composition comprises L-leucine, argininosuccinate, glutamate, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

| 42. The composition of any of embodiments 1-2, 14, 382, or 431, wherein the composition comprises L-leucine, citrulline, D-glutamine, and an antioxidant or ROS scavenger, £ 20 e.g., a NAC entity.

| 43. The composition of any of embodiments 1-2, 14, or 383, wherein the composition comprises L-leucine, aspartate, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a fl NAC entity.

^B 25 | 44. The composition of any of embodiments 1-2,14,380, or 445, wherein the composition comprises L-leucine, L-arginine, a Q-amino acid entity, and NAC.

45. The composition of any of embodiments 1-2, 14, 381, or 446, wherein the | 30 composition comprises L-leucine, argininosuccinate, a Q-amino acid entity, and serine.

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46. The composition of any of embodiments 1-2, 14, 382, or 447, wherein the composition comprises L-leucine, citrulline, a Q-amino acid entity, and acetylserine.

47. The composition of any of embodiments 1-2, 14, 383, or 448, wherein the composition comprises L-leucine, aspartate, a Q-amino acid entity, and cystathionine.

48. The composition of any of embodiments 1-2, 14, 384, or 449, wherein the composition comprises L-leucine, glutamate, a Q-amino acid entity, and glutathione.

49. The composition of any of embodiments 1-2,14, 385, or 450, wherein the composition comprises L-leucine, omithine, a Q-amino acid entity, and homocystéine.

50. The composition of any of embodiments 1-2, 14, 386, or 451, wherein the composition comprises L-leucine, agmatine, a Q-amino acid entity, and méthionine.

51. The composition of any of embodiments 1-2, 14, 387, or 452, wherein the composition comprises L-leucine, creatine, a Q-amino acid entity, and D-cysteine.

52. The composition of any of embodiments 1-2, 14, 388, or 453, wherein the composition comprises L-leucine, D-arginine, a Q-amino acid entity, and L-cysteine.

53. The composition of any of embodiments 1-2,14, 389, or 454, wherein the composition comprises L-leucine, N-acetyl-arginine, a Q-amino acid entity, and cystine.

54. The composition of any of embodiments 1-2, 14,428, or 445, wherein the composition comprises L-leucine, a R-amino acid entity, L-glutamine, and NAC.

55. The composition of any of embodiments 1-2, 14,429, or 446, wherein the composition comprises L-leucine, a R-amino acid entity, glutamate, and serine.

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56. The composition of any of embodiments 1-2,14,430, or 447, wherein the composition comprises L-leucine, a R-amino acid entity, carbamoyl-P, and acetylserine.

57. The composition of any of embodiments 1-2, 14, 432, or 448, wherein the composition comprises L-leucine, a R-amino acid entity, N-acetyl-glutamine, and cystathionine.

58. The composition of any of embodiments 1-2, 14, 433, or 449, wherein the composition comprises L-leucine, a R-amino acid entity, L-glutamine, and glutathione.

59. The composition of any of embodiments 1-2, 14, or 450, wherein the composition comprises L-leucine, a R-amino acid entity, glutamate, and homocystéine.

60. The composition of any of embodiments 1-2, 14, or 451, wherein the composition comprises L-leucine, a R-amino acid entity, carbamoyl-P, and méthionine.

61. The composition of any of embodiments 1-2, 14, or 452, wherein the composition comprises L-leucine, a R-amino acid entity, N-acetyl-glutamine, and D-cysteine.

62. The composition of any of embodiments 1-2,14, or 453, wherein the composition 20 comprises L-leucine, a R-amino acid entity, L-glutamine, and L-cysteine.

63. The composition of any of embodiments 1-2, 14, or 454, wherein the composition comprises L-leucine, a R-amino acid entity, a glutamate, and cystine.

64. The composition of any of embodiments 1-2, 14, 380, or 445, wherein the composition comprises L-leucine, L-arginine, L-glutamine, and NAC.

65. The composition of any of embodiments 1-2, 14, 381, or 446, wherein the composition comprises L-leucine, argininosuccinate, glutamate, and serine.

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66. The composition of any of embodiments 1-2, 14, 382, or 447, wherein the | composition comprises L-leucine, citrulline, carbamoyl-P, and acetylserine.

| 67. The composition of any of embodiments 1-2, 14, 383, or 448, wherein the composition comprises L-leucine, aspartate, D-glutamine, and cystathionine.

68. The composition of any of embodiments 1-2, 14,384, or 449, wherein the | composition comprises L-leucine, glutamate, L-glutamine, and glutathione.

J 10 69. The composition of any of embodiments 1-2, 14, 385, or 450, wherein the composition comprises L-leucine, omithine, glutamate, and homocystéine.

70. The composition of any of embodiments 1-2, 14, 386, or 451, wherein the composition comprises L-leucine, agmatine, carbamoyl-P, and méthionine.

| 71. The composition of any of embodiments 1-2, 14, 387, or 452, wherein the composition comprises L-leucine, creatine, D-glutamine and D-cysteine.

72. The composition of any of embodiments 1-2, 14, 388, or 453, wherein the | 20 composition comprises L-leucine, D-arginine, a Q-amino acid entity, and L-cysteine.

| 73. The composition of any of embodiments 1-2, 14, 389, or 454, wherein the composition comprises L-leucine, N-acetyl-arginine, argininosuccinate, and cystine.

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74. The composition of embodiment 1 or 3, wherein the composition comprises oxo leucine, a R-amino acid entity, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

75. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises 30 oxo-leucine, a R-amino acid entity, and a Q-amino acid entity, and an antioxidant or ROS ^— scavenger, e.g., a NAC entity.

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76. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, L-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

77. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, argininosuccinate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

78. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, citrulline, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

79. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, aspartate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

80. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, glutamate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

81. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, omithine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

82. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, agmatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

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83. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, creatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

84. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, D-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenge, e.g., a NAC entity.

85. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, N-acetyl-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

86. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

87. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

88. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, carbamoyl-P, and an antioxidant or ROS scavenger, e.g., a NAC entity.

89. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

90. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

I 91. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and NAC.

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92. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises | oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and serine.

| 93. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and acetylserine.

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94. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises | oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and cystathionine.

| 95. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and glutathione.

96. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises | oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and homocystéine.

| 20 97. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and méthionine.

98. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises

H oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and D-cysteine.

* 25 | 99. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and L-cysteine.

100. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and a NAC entity.

101. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, a Q-amino acid entity, and cystine.

102. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, L-arginine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

103. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, argininosuccinate, glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

104. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, citrulline, D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

105. The composition of embodiment 1-1D, 3, or 74,wherein the composition comprises oxo-leucine, aspartate, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

106. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, L-arginine, a Q-amino acid entity, and NAC.

107. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, argininosuccinate, a Q-amino acid entity, and serine.

108. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, citrulline, a Q-amino acid entity, and acetylserine.

109. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, aspartate, a Q-amino acid entity, and cystathionine.

110. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, glutamate, a Q-amino acid entity, and glutathione.

111. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, omithine, a Q-amino acid entity, and homocystéine.

112. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, agmatine, a Q-amino acid entity, and méthionine.

113. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, creatine, a Q-amino acid entity, and D-cysteine.

114. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, D-arginine, a Q-amino acid entity, and L-cysteine.

115. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, N-acetyl-arginine, a Q-amino acid entity, and cystine.

116. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, L-glutamine, and NAC.

117. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, glutamate, and serine.

118. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, carbamoyl-P, and acetylserine.

119. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, N-acetyl-glutamine, and cystathionine.

120. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, L-glutamine, and glutathione.

121. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, glutamate, and homocystéine.

122. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises 5 oxo-leucine, a R-amino acid entity, carbamoyl-P, and méthionine.

123. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, N-acetyl-glutamine, and D-cysteine.

124. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, L-glutamine, and L-cysteine.

125. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, a R-amino acid entity, a glutamate, and cystine.

126. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, L-arginine, L-glutamine, and NAC.

127. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises 20 oxo-leucine, argininosuccinate, glutamate, and serine.

128. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, citrulline, carbamoyl-P, and acetylserine.

129. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, aspartate, D-glutamine, and cystathionine.

130. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, N-acetyl-glutamine, L-glutamine, and glutathione.

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131. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises | oxo-leucine, ornithine, glutamate, and homocystéine.

| 132. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, agmatine, carbamoyl-P, and méthionine.

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133. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises | oxo-leucine, creatine, D-glutamine and D-cysteine.

| 10 134. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises oxo-leucine, D-arginine, a Q-amino acid entity, and L-cysteine.

135. The composition of embodiment 1-1D, 3, or 74, wherein the composition comprises B oxo-leucine, N-acetyl-arginine, argininosuccinate, and cystine.

| 136. The composition of embodiment 1-1D or 4, wherein the composition comprises

HMB, a R-amino acid entity, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., | a NAC entity.

| 20 137. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, L-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a | NAC entity.

138. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, argininosuccinate, a Q-amino acid entity, and an antioxidant or ROS scavenger, | e.g., a NAC entity.

139. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, citrulline, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a | 30 NAC entity.

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140. The composition of embodiment 1-1D, 4, or 136, wherein the composition | comprises HMB, aspartate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a

NAC entity.

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141. The composition of embodiment 1-1D, 4, or 136, wherein the composition £ comprises HMB, glutamate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a

NAC entity.

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142. The composition of embodiment 1-1D, 4, or 136, wherein the composition | 10 comprises HMB, omithine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a

NAC entity.

143. The composition of embodiment 1-1D, 4, or 136, wherein the composition | comprises HMB, agmatine, a Q-amino acid entity, and an antioxidant or ROS scavenger e.g., a

NAC entity.

144. The composition of embodiment 1-1D, 4, or 136, wherein the composition

I comprises HMB, creatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a

NAC entity.

| 20

145. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, D-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenge e.g., a

NAC entity.

146. The composition of embodiment 1-1D, 4, or 136, wherein the composition | comprises HMB, N-acetyl-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger,

e.g., a NAC entity.

147. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, L-glutamine, and an antioxidant or ROS scavenger, e.g., an antioxidant, e.g., a NAC entity.

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I 148. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, glutamate, and an antioxidant or ROS scavenger, e.g., an | antioxidant, e.g., a NAC entity.

| 149. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, carbamoyl-P, and an antioxidant or ROS scavenger, e.g., | an antioxidant, e.g., a NAC entity.

| 10 150. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, D-glutamine, and an antioxidant or ROS scavenger, e.g., | an antioxidant, e.g., a NAC entity.

| 151. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, N-acetyl-glutamine, and an antioxidant or ROS | scavenger, e.g., an antioxidant, e.g., a NAC entity.

| 152. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, a Q-amino acid entity, and NAC.

| 20

153. The composition of embodiment 1-1D, 4, or 136, wherein the composition | comprises HMB, a R-amino acid entity, a Q-amino acid entity, and serine.

154. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, a Q-amino acid entity, and acetylserine.

155. The composition of embodiment 1-1D, 4, or 136, wherein the composition | comprises HMB, a R-amino acid entity, a Q-amino acid entity, and cystathionine.

156. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, a Q-amino acid entity, and glutathione.

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157. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, a Q-amino acid entity, and homocystéine.

158. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, a Q-amino acid entity, and méthionine.

159. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, a Q-amino acid entity, and D-cysteine.

160. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, a Q-amino acid entity, and L-cysteine.

161. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, a Q-amino acid entity, and cysteine.

162. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, a Q-amino acid entity, and cystine.

163. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, L-arginine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

164. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, argininosuccinate, glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

165. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, citrulline, D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC 30 entity.

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166. The composition of embodiment 1-1D, 4, or 136, wherein the composition | comprises HMB, aspartate, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a

NAC entity.

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167. The composition of embodiment 1-1D, 4, or 136, wherein the composition

S comprises HMB, L-arginine, a Q-amino acid entity, and NAC.

168. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, argininosuccinate, a Q-amino acid entity, and serine.

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169. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, citrulline, a Q-amino acid entity, and acetylserine.

170. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, aspartate, a Q-amino acid entity, and cystathionine.

171. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, glutamate, a Q-amino acid entity, and glutathione.

172. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, omithine, a Q-amino acid entity, and homocystéine.

173. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, agmatine, a Q-amino acid entity, and méthionine.

174. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, creatine, a Q-amino acid entity, and D-cysteine.

175. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, D-arginine, a Q-amino acid entity, and L-cysteine.

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176. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, N-acetyl-arginine, a Q-amino acid entity, and cystine.

177. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, L-glutamine, and NAC.

178. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, glutamate, and serine.

179. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, carbamoyl-P, and acetylserine.

180. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, N-acetyl-glutamine, and cystathionine.

181. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, L-glutamine, and glutathione.

182. The composition of embodiment 1-1D, 4, or 136, wherein the composition 20 comprises HMB, a R-amino acid entity, glutamate, and homocystéine.

183. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, carbamoyl-P, and méthionine.

184. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, N-acetyl-glutamine, and D-cysteine.

185. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, a R-amino acid entity, L-glutamine, and L-cysteine.

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186. The composition of embodiment 1-1D, 4, or 136, wherein the composition | comprises HMB, a R-amino acid entity, a glutamate, and cystine.

| 187. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, L-arginine, L-glutamine, and NAC.

188. The composition of embodiment 1-1D, 4, or 136, wherein the composition | comprises HMB, argininosuccinate, glutamate, and serine.

| 10 189. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, citrulline, carbamoyl-P, and acetylserine.

I

190. The composition of embodiment 1-1D, 4, or 136, wherein the composition

B comprises HMB, aspartate, D-glutamine, and cystathionine.

* 15 | 191. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, N-acetyl-glutamine, L-glutamine, and glutathione.

192. The composition of embodiment 1-1D, 4, or 136, wherein the composition | 20 comprises HMB, omithine, glutamate, and homocystéine.

193. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, agmatine, carbamoyl-P, and méthionine.

194. The composition of embodiment 1-1D, 4, or 136, wherein the composition | comprises HMB, creatine, D-glutamine and D-cysteine.

195. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, D-arginine, a Q-amino acid entity, and L-cysteine.

| 30 | 97

196. The composition of embodiment 1-1D, 4, or 136, wherein the composition comprises HMB, N-acetyl-arginine, argininosuccinate, and cystine.

197. The composition of embodiment 1-1D, or 4, wherein the composition comprises 5 isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

198. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, L-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

199. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, argininosuccinate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

200. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, citrulline, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

201. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, aspartate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

202. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, glutamate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

203. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, omithine, a Q-amino acid entity, and an antioxidant or ROS 30 scavenger, e.g., a NAC entity.

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204. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, agmatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

205. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, creatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

206. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, D-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

207. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, N-acetyl-arginine, a Q-amino acid entity, and an antioxidant or ROS 15 scavenger, e.g., a NAC entity.

208. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

209. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

210. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, carbamoyl-P, and an antioxidant or ROS . scavenger, e.g., a NAC entity.

211. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

212. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

£ 213. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and NAC.

I

214. The composition of embodiment 1-1D, 4, or 197, wherein the composition

I 10 comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and serine.

215. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleiyl-CoA, a R-amino acid entity, a Q-amino acid entity, and acetylserine.

216. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and cystathionine.

217. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and glutathione.

218. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and homocystéine.

| 219. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and méthionine.

220. The composition of embodiment 1-1D, 4, or 197, wherein the composition | comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and D-cysteine.

| 30 221. The composition of embodiment 1-1 D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and L-cysteine.

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I 222. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and cysteine.

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223. The composition of embodiment 1-1D, 4, or 197, wherein the composition | comprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity, and cystine.

| 224. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, L-arginine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a | 10 NAC entity.

| 225. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, argininosuccinate, glutamate, and an antioxidant or ROS scavenger,

H e.g., a NAC entity.

| 226. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, citrulline, D-glutamine, and an antioxidant or ROS scavenger, e.g., a | NAC entity.

B 20 227. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, aspartate, N-acetyl-glutamine, and an antioxidant or ROS scavenger,

J e.g., a NAC entity.

228. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, L-arginine, a Q-amino acid entity, and NAC.

229. The composition of embodiment 1-1D, 4, or 197, wherein the composition | comprises isovaleryl-CoA, argininosuccinate, a Q-amino acid entity, and serine.

230. The composition of embodiment 1-1D, 4, or 197, wherein the composition * comprises isovaleryl-CoA, citrulline, a Q-amino acid entity, and acetylserine.

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231. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, aspartate, a Q-amino acid entity, and cystathionine.

232. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, glutamate, a Q-amino acid entity, and glutathione.

233. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, ornithine, a Q-amino acid entity, and homocystéine.

234. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, agmatine, a Q-amino acid entity, and méthionine.

235. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, creatine, a Q-amino acid entity, and D-cysteine.

236. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, D-arginine, a Q-amino acid entity, and L-cysteine.

237. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, N-acetyl-arginine, a Q-amino acid entity, and cystine.

238. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, L-glutamine, and NAC.

239. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, glutamate, and serine.

240. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, carbamoyl-P, and acetylserine.

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241. The composition of embodiment 1-1D, 4, or 197, wherein the composition | comprises isovaleryl-CoA, a R-amino acid entity, N-acetyl-glutamine, and cystathionine.

| 242. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, L-glutamine, and glutathione.

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243. The composition of embodiment 1-1D, 4, or 197, wherein the composition | comprises isovaleryl-CoA, a R-amino acid entity, glutamate, and homocystéine.

| 10 244. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, carbamoyl-P, and méthionine.

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245. The composition of embodiment 1-1D, 4, or 197, wherein the composition | comprises isovaleryl-CoA, a R-amino acid entity, N-acetyl-glutamine, and D-cysteine.

| 246. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, a R-amino acid entity, L-glutamine, and L-cysteine.

247. The composition of embodiment 1-1D, 4, or 197, wherein the composition

I 20 comprises isovaleryl-CoA, a R-amino acid entity, a glutamate, and cystine.

248. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, L-arginine, L-glutamine, and NAC.

249. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, argininosuccinate, glutamate, and serine.

250. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, citrulline, carbamoyl-P, and acetylserine.

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251. The composition of embodiment 1-1D, 4, or 197, wherein the composition | comprises isovaleryl-CoA, aspartate, D-glutamine, and cystathionine.

| 252. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, N-acetyl-glutamine, L-glutamine, and glutathione.

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253. The composition of embodiment 1-1D, 4, or 197, wherein the composition | comprises isovaleryl-CoA, omithine, glutamate, and homocystéine.

| 10 254. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, agmatine, carbamoyl-P, and méthionine.

255. The composition of embodiment 1-1D, 4, or 197, wherein the composition | comprises isovaleryl-CoA, creatine, D-glutamine and D-cysteine.

| 256. The composition of embodiment 1-1D, 4, or 197, wherein the composition comprises isovaleryl-CoA, D-arginine, a Q-amino acid entity, and L-cysteine.

257. The composition of embodiment 1-1D, 4, or 197, wherein the composition | 20 comprises isovaleryl-CoA, N-acetyl-arginine, argininosuccinate, and cystine.

| 258. The composition of embodiment 1-1D or 5, wherein the composition comprises Dleucine, a R-amino acid entity, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g.,

H a NAC entity.

| 259. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, L-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, | e.g., a NAC entity.

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260. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, argininosuccinate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

261. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, citrulline, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

262. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, aspartate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

263. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, glutamate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

264. The composition of embodiment 1-1D, 5, or 258, wherein composition comprises Dleucine, omithine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

265. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, agmatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

266. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, creatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

267. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, D-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

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I 268. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, N-acetyl-arginine, a Q-amino acid entity, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

| 269. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, L-glutamine, and an antioxidant or ROS scavenger, | e.g., a NAC entity.

| 10 270. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, glutamate, and an antioxidant or ROS scavenger, | e.g., a NAC entity.

| 271. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, carbamoyl-P, and an antioxidant or ROS scavenger, | e.g., a NAC entity.

| 272. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, D-glutamine, and an antioxidant or ROS scavenger, | 20 e.g., a NAC entity.

273. The composition of embodiment 1-1D, 5, or 258, wherein the composition | comprises D-leucine, a R-amino acid entity, N-acetyl-glutamine, and an antioxidant or ROS scavenger, ee.g., a NAC entity.

274. The composition of embodiment 1-1D, 5, or 258, wherein the composition | comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and NAC.

275. The composition of embodiment 1-1D, 5, or 258, wherein the composition * comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and serine.

g 30

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276. The composition of embodiment 1-1D, 5, or 258, wherein the composition | comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and acetylserine.

| 277. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and cystathionine.

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278. The composition of embodiment 1-1D, 5, or 258, wherein the composition | comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and glutathione.

| 10 279. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and homocystéine.

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280. The composition of embodiment 1-1D, 5, or 258, wherein the composition | comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and méthionine.

| 281. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and D-cysteine.

282. The composition of embodiment 1-1D, 5, or 258, wherein the composition 20 comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and L-cysteine.

283. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and cysteine.

284. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, a Q-amino acid entity, and cystine.

285. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, L-arginine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

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286. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, argininosuccinate, glutamate, and an antioxidant or ROS scavengere.g., a NAC entity.

287. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, citrulline, D-glutamine, and an antioxidant or ROS scavengere.g., a NAC entity.

288. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, aspartate, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

289. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, L-arginine, a Q-amino acid entity, and NAC.

290. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, argininosuccinate, a Q-amino acid entity, and serine.

291. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, citrulline, a Q-amino acid entity, and acetylserine.

292. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, aspartate, a Q-amino acid entity, and cystathionine.

293. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, glutamate, a Q-amino acid entity, and glutathione.

294. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, omithine, a Q-amino acid entity, and homocystéine.

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295. The composition of embodiment 1-1D, 5, or 258, wherein the composition | comprises D-leucine, agmatine, a Q-amino acid entity, and méthionine.

| 296. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, creatine, a Q-amino acid entity, and D-cysteine.

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297. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, D-arginine, a Q-amino acid entity, and L-cysteine.

| 10 298. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, N-acetyl-arginine, a Q-amino acid entity, and cystine.

299. The composition of embodiment 1-1 D, 5, or 258, wherein the composition | comprises D-leucine, a R-amino acid entity, L-glutamine, and NAC.

300. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, glutamate, and serine.

301. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, carbamoyl-P, and acetylserine.

302. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, N-acetyl-glutamine, and cystathionine.

303. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, L-glutamine, and glutathione.

304. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, glutamate, and homocystéine.

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305. The composition of embodiment 1-1D, 5, or 258, wherein the composition | comprises D-leucine, a R-amino acid entity, carbamoyl-P, and méthionine.

| 306. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, a R-amino acid entity, N-acetyl-glutamine, and D-cysteine.

307. The composition of embodiment 1-1D, 5, or 258, wherein the composition | comprises D-leucine, a R-amino acid entity, L-glutamine, and L-cysteine.

| 10 308. The composition of embodiment 1-1D, 5, or 258, wherein the composition ' comprises D-leucine, a R-amino acid entity, a glutamate, and cystine.

309. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, L-arginine, L-glutamine, and NAC.

310. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, argininosuccinate, glutamate, and serine.

|

311. The composition of embodiment 1-1D, 5, or 258, wherein the composition | 20 comprises D-leucine, citrulline, carbamoyl-P, and acetylserine.

312. The composition of embodiment 1-1D, 5, or 258, wherein the composition * comprises D-leucine, aspartate, D-glutamine, and cystathionine.

313. The composition of embodiment 1-1D, 5, or 258, wherein the composition | comprises D-leucine, N-acetyl-glutamine, L-glutamine, and glutathione.

314. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, omithine, glutamate, and homocystéine.

g 30

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315. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, agmatine, carbamoyl-P, and méthionine.

316. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, creatine, D-glutamine and D-cysteine.

317. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, D-arginine, a Q-amino acid entity, and L-cysteine.

318. The composition of embodiment 1-1D, 5, or 258, wherein the composition comprises D-leucine, N-acetyl-arginine, argininosuccinate, and cystine.

319. The composition of embodiment 1-1D or 5, wherein the composition comprises Nacetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

320. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, L-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

321. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, argininosuccinate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

322. The composition of embodiment 1-1D, 5, or 319„ wherein the composition comprises N-acetyl-leucine, citrulline, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

323. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, aspartate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

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I 324. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, glutamate, a Q-amino acid entity, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

| 325. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, omithine, a Q-amino acid entity, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

| 10 326. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, agmatine, a Q-amino acid entity, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

| 327. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, creatine, a Q-amino acid entity, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

| 328. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, D-arginine, a Q-amino acid entity, and an antioxidant or ROS | 20 scavenger, e.g., a NAC entity.

329. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, N-acetyl-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

330. The composition of embodiment 1-1D, 5, or 319, wherein the composition ^— comprises N-acetyl-leucine, a R-amino acid entity, L-glutamine, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

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331. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

332. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, carbamoyl-P, and an antioxidant or ROS scavenger, e.g., a NAC entity.

333. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

334. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

335. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and NAC.

336. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and serine.

337. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and acetylserine.

338. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and cystathionine.

339. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and glutathione.

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340. The composition of embodiment 1-1D, 5, or 319, wherein the composition | comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and homocystéine.

| 341. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and méthionine.

I

342. The composition of embodiment 1-1D, 5, or 319, wherein the composition | comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and D-cysteine.

| 10 343. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and L-cysteine.

I

344. The composition of embodiment 1-1D, 5, or 319, wherein the composition

H comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and cysteine.

| 345. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acid entity, and cystine.

346. The composition of embodiment 1-1D, 5, or 319, wherein the composition | 20 comprises N-acetyl-leucine, L-arginine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

347. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, argininosuccinate, glutamate, and an antioxidant or ROS scavenger, 25 e.g., a NAC entity.

348. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, citrulline, D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

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349. The composition of embodiment 1-1D, 5, or 319, wherein the composition | comprises N-acetyl-leucine, aspartate, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

I

350. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, L-arginine, a Q-amino acid entity, and NAC.

351. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, argininosuccinate, a Q-amino acid entity, and serine.

352. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, citrulline, a Q-amino acid entity, and acetylserine.

353. The composition of embodiment 1-1D, 5, or 319, wherein the composition 15 comprises N-acetyl-leucine, aspartate, a Q-amino acid entity, and cystathionine.

354. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, glutamate, a Q-amino acid entity, and glutathione.

355. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, omithine, a Q-amino acid entity, and homocystéine.

356. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, agmatine, a Q-amino acid entity, and méthionine.

357. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, creatine, a Q-amino acid entity, and D-cysteine.

358. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, D-arginine, a Q-amino acid entity, and L-cysteine.

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359. The composition of embodiment 1-1D, 5, or 319, wherein the composition | comprises N-acetyl-leucine, N-acetyl-arginine, a Q-amino acid entity, and cystine.

| 360. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, L-glutamine, and NAC.

I

361. The composition of embodiment 1-1D, 5, or 319, wherein the composition | comprises N-acetyl-leucine, a R-amino acid entity, glutamate, and serine.

| 10 362. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, carbamoyl-P, and acetylserine.

363. The composition of embodiment 1-1D, 5, or 319, wherein the composition

B comprises N-acetyl-leucine, a R-amino acid entity, N-acetyl-glutamine, and cystathionine.

* 15 | 364. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, L-glutamine, and glutathione.

365. The composition of embodiment 1-1D, 5, or 319, wherein the composition | 20 comprises N-acetyl-leucine, a R-amino acid entity, glutamate, and homocystéine.

| 366. The composition of embodiment 1-ID, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, carbamoyl-P, and méthionine.

367. The composition of embodiment 1-1D, 5, or 319, wherein the composition | comprises N-acetyl-leucine, a R-amino acid entity, N-acetyl-glutamine, and D-cysteine.

368. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, a R-amino acid entity, L-glutamine, and L-cysteine.

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369. The composition of embodiment 1-1D, 5, or 319, wherein the composition | comprises N-acetyl-leucine, a R-amino acid entity, a glutamate, and cystine.

| 370. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, L-arginine, L-glutamine, and NAC.

371. The composition of embodiment 1-1D, 5, or 319, wherein the composition | comprises N-acetyl-leucine, argininosuccinate, glutamate, and serine.

| 10 372. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, citrulline, carbamoyl-P, and acetylserine.

373. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, aspartate, D-glutamine, and cystathionine.

^B 15

374. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, N-acetyl-glutamine, L-glutamine, and glutathione.

375. The composition of embodiment 1-1D, 5, or 319, wherein the composition | 20 comprises N-acetyl-leucine, omithine, glutamate, and homocystéine.

376. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, agmatine, carbamoyl-P, and méthionine.

377. The composition of embodiment 1-1D, 5, or 319, wherein the composition

I comprises N-acetyl-leucine, creatine, D-glutamine and D-cysteine.

378. The composition of embodiment 1-1D, 5, or 319, wherein the composition comprises N-acetyl-leucine, D-arginine, a Q-amino acid entity, and L-cysteine.

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379. The composition of embodiment 1-1D, 5, or 319, wherein the composition | comprises N-acetyl-leucine, N-acetyl-arginine, argininosuccinate, and cystine.

380. The composition of embodiment 1-1D or 2, wherein the composition comprises a Lamino acid entity, L-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

381. The composition of embodiment 1-1D or 2, wherein the composition comprises a Lamino acid entity, argininosuccinate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

382. The composition of embodiment 1-1D, 3, or 4, wherein the composition comprises a L-amino acid entity, citrulline, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

383. The composition of embodiment 1-1D or 3, wherein the composition comprises a Lamino acid entity, aspartate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

384. The composition of embodiment 1-1D or 3, wherein the composition comprises a Lamino acid entity, glutamate, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

385. The composition of embodiment 1-1D or 4, wherein the composition comprises a Lamino acid entity, omithine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

386. The composition of embodiment 1-1D or 4, wherein the composition comprises a Lamino acid entity, agmatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

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387. The composition of embodiment 1-1D or 4, wherein the composition comprises a Lamino acid entity, creatine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

388. The composition of embodiment 1-1D or 5, wherein the composition comprises a Lamino acid entity, D-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

389. The composition of embodiment 1-1D or 5, wherein the composition comprises a Lamino acid entity, N-acetyl-arginine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

390. The composition of embodiment 1-1D, 3, or 384, wherein the composition comprises L-leucine, glutamate, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

391. The composition of embodiment 1-1D, 4, or 385, wherein the composition comprises L-leucine, omithine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

392. The composition of embodiment 1-1D, 4, or 386, wherein the composition comprises a L-amino acid entity, agmatine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

393. The composition of embodiment 1-1D, 4, or 387, wherein the composition comprises a L-amino acid entity, creatine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

394. The composition of embodiment 1-1D, 4, or 388, wherein the composition comprises a L-amino acid entity, D-arginine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

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I 395. The composition of embodiment 1-1D, 4, or 389, wherein the composition comprises a L-amino acid entity, D-arginine, N-acetyl-arginine, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

| 396. The composition of embodiment 1-1D or 380, wherein the composition comprises a

L-amino acid entity, L-arginine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC | entity.

| 10 397. The composition of any of embodiments 1-2, or 381, wherein the composition comprises a L-amino acid entity, argininosuccinate, glutamate, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

| 398. The composition of embodiment 1-1D, 3,4, or 382, wherein the composition comprises a L-amino acid entity, citrulline, carbamoyl-P, and an antioxidant or ROS scavenger, | e.g., a NAC entity.

J 399. The composition of embodiment 1-1D, 3, or 383, wherein the composition comprises a L-amino acid entity, aspartate, glutamate, and an antioxidant or ROS scavenger, e.g., j 20 a NAC entity.

400. The composition of embodiment 1-1D, 3, or 384, wherein the composition comprises a L-amino acid entity, glutamate, D-glutamine, and an antioxidant or ROS scavenger,

e.g., a NAC entity.

401. The composition of embodiment 1-1D, 4, or 385, wherein the composition comprises a L-amino acid entity, omithine, N-acetyl-glutamine, and an antioxidant or ROS | scavenger, e.g., a NAC entity.

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402. The composition of embodiment 1-1D, 4, or 386, wherein the composition comprises a L-amino acid entity, agmatine, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

403. The composition of embodiment 1-1D, 4, or 387, wherein the composition comprises a L-amino acid entity, creatine, glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

404. The composition of embodiment 1-1D, 5, or 388, wherein the composition comprises a L-amino acid entity, D-arginine, carbamoyl-P, and an antioxidant or ROS scavenger, e.g., a NAC entity.

405. The composition of embodiment 1-1D, 5, or 389, wherein the composition comprises a L-amino acid entity, N-acetyl-arginine, glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

406. The composition of embodiment 1-1D, 3, 80, or 445, wherein the composition comprises a L-amino acid entity, L-arginine, L-glutamine, and NAC.

407. The composition of any of embodiments 1-2, 381, or 446, wherein the composition comprises a L-amino acid entity, argininosuccinate, glutamate, and serine.

408. The composition of embodiment 1-1D, 3,4, 382, or 447, wherein the composition comprises a L-amino acid entity, citrulline, carbamoyl-P, and acetylserine.

409. The composition of embodiment 1-1D, 3,383, or 448, wherein the composition comprises a L-amino acid entity, aspartate, glutamate, and cystathionine.

410. The composition of embodiment 1-1D, 3, 384, or 449, wherein the composition comprises a L-amino acid entity, glutamate, D-glutamine, and glutathione.

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411. The composition of embodiment 1-1D, 4,385, or 448, wherein the composition comprises a L-amino acid entity, omithine, N-acetyl-glutamine, and cystathionine.

412. The composition of embodiment 1-1D, 4,386, or 450, wherein the composition comprises a L-amino acid entity, agmatine, L-glutamine, and homocystéine.

413. The composition of embodiment 1-1D, 4,387, or 451, wherein the composition comprises a L-amino acid entity, creatine, glutamate, and méthionine.

414. The composition of embodiment 1-1D, 5, 388, or 454, wherein the composition comprises a L-amino acid entity, D-arginine, carbamoyl-P, and D-cysteine.

415. The composition of embodiment 1-1D, 5, 389, or 453, wherein the composition comprises a L-amino acid entity, N-acetyl-arginine, glutamate, and L-cysteine.

416. The composition of embodiment 1-1D, 380, or 454, wherein the composition comprises a L-amino acid entity, L-arginine, L-glutamine, and cystine.

417. The composition of embodiment 1-1D, 6, or 445, wherein the composition comprises a L-amino acid entity, L-arginine, a Q-amino acid, and NAC.

418. The composition of any of embodiments 1-2, or 446, wherein the composition comprises a L-amino acid entity, argininosuccinate, a Q-amino acid, and serine.

419. The composition of embodiment 1-1D, 3, or 447, wherein the composition comprises a L-amino acid entity, citrulline, a Q-amino acid, and acetylserine.

420. The composition of embodiment 1-1D, 4, or 448, wherein the composition comprises a L-amino acid entity, aspartate, a Q-amino acid, and cystathionine.

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421. The composition of embodiment 1-1D, 3, or 449, wherein the composition g comprises a L-amino acid entity, glutamate, a Q-amino acid, and glutathione.

g 422. The composition of embodiment 1-1D, 4, or 448, wherein the composition comprises a L-amino acid entity, omithine, a Q-amino acid, and cystathionine.

423. The composition of embodiment 1-1D, 4, or 450, wherein the composition g comprises a L-amino acid entity, agmatine, a Q-amino acid, and homocystéine.

g 10 424. The composition of embodiment 1-1D, 4, or 451, wherein the composition comprises a L-amino acid entity, creatine, a Q-amino acid, and méthionine.

425. The composition of embodiment 1-1D, 5, or 452, wherein the composition

I comprises a L-amino acid entity, D-arginine, a Q-amino acid, and D-cysteine.

* 15 g 426. The composition of embodiment 1-1D, 5, or 453, wherein the composition comprises a L-amino acid entity, N-acetyl-arginine, a Q-amino acid, and L-cysteine.

427. The composition of embodiment 1-1D, 5, or 454, wherein the composition g 20 comprises a L-amino acid entity, L-arginine, a Q-amino acid, and cystine.

428. The composition of embodiment 1-1D or 2, wherein the composition comprises a L- * amino acid entity, a R-amino acid entity, L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

429. The composition of embodiment 1-1D, 3, or 4, wherein the composition comprises a * L-amino acid entity, a R-amino acid entity, glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

I I

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430. The composition of embodiment 1-1D or 4, wherein the composition comprises a L| amino acid entity, a R-amino acid entity, carbamoyl-P, and an antioxidant or ROS scavenger,

e.g., a NAC entity.

I

431. The composition of embodiment 1-1D or 5, wherein the composition comprises a L| amino acid entity, a R-amino acid entity, D-glutamine, and an antioxidant or ROS scavenger,

e.g., a NAC entity.

432. The composition of embodiment 1-1D or 5, wherein the composition comprises a L| 10 amino acid entity, a R-amino acid entity, N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

433. The composition of embodiment 1-1D, 5, or 431, wherein the composition | comprises a L-leucine, a R-amino acid entity, D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

434. The composition of embodiment 1-1D, 4 or 430, wherein the composition comprises | a L-leucine, L-arginine, carbamoyl-P, and an antioxidant or ROS scavenger, e.g., a NAC entity.

435. The composition of any of embodiments 1-2,428, or 445, wherein the composition comprises a L-amino acid entity, a R-amino acid entity, L-glutamine, and NAC.

436. The composition of embodiment 1-1D, 4,429, or 446, wherein the composition comprises a L-amino acid entity, a R-amino acid entity, glutamate, and serine.

437. The composition of embodiment 1-1D, 4,430, or 447, wherein the composition * comprises a L-amino acid entity, a R-amino acid entity, carbamoyl-P, and acetylserine.

438. The composition of embodiment 1-1D, 5,431, or 448, wherein the composition | 30 comprises a L-amino acid entity, a R-amino acid entity, D-glutamine, and cystathionine.

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439. The composition of embodiment 1-1D, 5, 432, or 449, wherein the composition | comprises a L-amino acid entity, a R-amino acid entity, N-acetyl-glutamine, and glutathione.

| 440. The composition of any of embodiments 1-2, 428, or 450, wherein the composition comprises a L-amino acid entity, a R-amino acid entity, L-glutamine, and homocystéine.

441. The composition of embodiment 1-1D, 3,4,429, or 451, wherein the composition | comprises a L-amino acid entity, a R-amino acid entity, glutamate, and méthionine.

| 10 442. The composition of embodiment 1-1D, 4,430, or 452, wherein the composition comprises a L-amino acid entity, a R-amino acid entity, carbamoyl-P, and D-cysteine

I

443. The composition of embodiment 1-1D, 5, 431, or 453, wherein the composition

H comprises a L-amino acid entity, a R-amino acid entity, D-glutamine, and L-cysteine.

444. The composition of embodiment 1-1D, 5, 432, or 454, wherein the composition comprises a L-amino acid entity, a R-amino acid entity, N-acetyl-glutamine, and cystine.

445. The composition of embodiment 1-1D or 5, wherein the composition comprises a ΤΙ 20 amino acid entity, a R-amino acid entity, a Q-amino acid entity, and NAC.

446. The composition of embodiment 1-1D or 3, wherein the composition comprises a Lamino acid entity, a R-amino acid entity, a Q-amino acid entity, and serine.

447. The composition of embodiment 1- ID or 3, wherein the composition comprises a ΤΙ amino acid entity, a R-amino acid entity, a Q-amino acid entity, and acetylserine.

fl 448. The composition of embodiment 1-1D or 3, wherein the composition comprises a L* amino acid entity, a R-amino acid entity, a Q-amino acid entity, and cystathionine.

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449. The composition of embodiment 1-1D or 4, wherein the composition comprises a L| amino acid entity, a R-amino acid entity, a Q-amino acid entity, and glutathione.

| 450. The composition of embodiment 1-1D or 4, wherein the composition comprises a L- amino acid entity, a R-amino acid entity, a Q-amino acid entity, and homocystéine.

I

451. The composition of embodiment 1 -1D or 4, wherein the composition comprises a ΤΙ amino acid entity, a R-amino acid entity, a Q-amino acid entity, and méthionine.

| 10 452. The composition of embodiment 1-1D or 5, wherein the composition comprises a Lamino acid entity, a R-amino acid entity, a Q-amino acid entity, and D-cysteine.

I

453. The composition of embodiment 1-1D or 5, wherein the composition comprises a ΤΙ amino acid entity, a R-amino acid entity, a Q-amino acid entity, and L-cysteine.

| 454. The composition of embodiment 1-1D or 5, wherein the composition comprises a Lamino acid entity, a R-amino acid entity, a Q-amino acid entity, and cystine.

455. The composition of embodiment 1-1D or 2, wherein the composition comprises a ΤΙ | 20 amino acid, omithine, a Q-amino acid entity, and an antioxidant or ROS scavenger, e.g., a NAC ~ entity.

' 456. The composition of embodiment 1-1D or 455, wherein the composition comprises

H L-leucine, omithine, l-glutamine, and NAC.

| 457. The composition of embodiment 1-1D or 455, wherein the composition comprises

HMB, omithine, l-glutamine, and NAC.

। 458. The composition of any of the foregoing embodiments, wherein the composition | 30 comprises L-leucine or a leucine métabolite (e.g., HMB), L-arginine or an L-arginine métabolite (e.g., creatine), l-glutamine, and NAC or a NAC métabolite, e.g., glutathione.

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459. The composition of any of the foregoing embodiments, wherein the composition comprises L-leucine or a leucine métabolite (e.g., HMB), L-arginine or an L-arginine métabolite (e.g., creatine), L-glutamine, and NAC or a NAC métabolite, e.g., glutathione.

460. The composition of any of the previous embodiments, further comprising an isoleucine (I)-amino acid entity.

461. The composition of embodiment 460, wherein the l-amino acid entity is an amino acid.

462. The composition of embodiment 460 or 461, wherein the amino acid entity is Lisoleucine.

463. The composition of embodiment 460, wherein the l-amino acid entity is an amino acid precursor.

464. The composition of embodiment 460 or 463, wherein the l-amino acid entity is 2oxo-3-methyl-valerate.

465. The composition of embodiment 460 or 463, wherein the l-amino acid entity is threonine.

466. The composition of embodiment 460, wherein the l-amino acid entity is an amino acid métabolite.

467. The composition of embodiment 460 or 466, wherein the l-amino acid entity is 2oxo-3 -methy 1-valerate

468. The composition of embodiment 460 or 466, wherein the l-amino acid entity is methylbutyrl-CoA.

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I 469. The composition of embodiment 460, wherein the l-amino acid entity is an amino acid dérivative.

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470. The composition of embodiment 460 or 469, wherein the l-amino acid entity is D| isoleucine.

| 471. The composition of embodiment 460 or 469, wherein the l-amino acid entity is Nacetyl-isoleucine.

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472. The composition of any of the previous embodiments, further comprising a valine | (V)-amino acid entity.

| 473. The composition of embodiment 472, wherein the V-amino acid entity is an amino acid.

474. The composition of embodiment 472 or 473, wherein the V-amino acid entity is ΤΙ valine.

| 20 475. The composition of embodiment 472, wherein the V-amino acid entity is an amino acid precursor.

476. The composition of embodiment 472 or 475, wherein the V-amino acid entity is 2B oxo-valerate.

^B 25 | 477. The composition of embodiment 472, wherein the V-amino acid entity is an amino acid métabolite.

478. The composition of embodiment 472 or 477, wherein the V-amino acid entity is | 30 isobutryl-CoA.

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479. The composition of embodiment 472 or 477, wherein the V-amino acid entity is 3- | HIB-CoA.

£ 480. The composition of embodiment 472 or 477, wherein the V-amino acid entity is 3-

HIB.

481. The composition of embodiment 472, wherein the V-amino acid entity is an amino | acid dérivative.

| 10 482. The composition of embodiment 472 or 481, wherein the V-amino acid entity is Dvaline.

483. The composition of embodiment 472 or 481, wherein the V-amino acid entity is ΝΑ acetyl-valine.

| 484. The composition of any of the preceding embodiments, further comprising Lglycine.

485. The composition of any of the preceding embodiments, further comprising an S| 20 amino acid entity (e.g., L-serine, phosphoserine, P-hydroxypyruvate, L-glycine, tryptophan, acetylserine, cystathionine, phosphatidylserine, or any combination thereof, e.g., L-serine and L| glycine).

486. The composition of any of the preceding embodiments, further comprising camitine.

_ 487. The composition of any of the preceding embodiments, comprising:

H a) a L-amino acid entity chosen from L-leucine or a sait thereof, or β-hydroxy-β- _ methybutyrate (HMB) or a sait thereof or a combination of L-leucine or a sait thereof and HMB ora sait thereof;

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b) an R-amino acid entity chosen from L-arginine or a sait thereof, omithine or a sait thereof, or creatine or a sait thereof or a combination of two or three of L-arginine or a sait thereof, omithine or a sait thereof, or creatine or a sait thereof;

c) L-glutamine or a sait thereof; and

d) N-acetylcysteine (NAC) or a sait thereof.

488. The composition of any of the preceding embodiments, wherein the L-Leucine is provided as part of a dipeptide comprising L-Leucine, or a sait thereof, or a tripeptide comprising L-Leucine, or a sait thereof.

489. The composition of any of the preceding embodiments, wherein the L-Arginine is provided as part of a dipeptide comprising L-Arginine, or a sait thereof, or a tripeptide comprising L-Arginine, or a sait thereof.

490. The composition of any of the preceding embodiments, wherein the L-Glutamine is provided as part of a dipeptide comprising L-Glutamine, or a sait thereof, or a tripeptide comprising L-Glutamine, or a sait thereof

491. The composition of any of the preceding embodiments, wherein the NAC is provided as a part of a dipeptide comprising NAC, or a sait thereof, or a tripeptide comprising NAC, or a sait thereof.

492. The composition of any of the preceding embodiments, wherein one, two, three, or four of méthionine (M), tryptophan (W), valine (V), or cysteine (C) is absent, or if présent, is présent at less than 10 weight (wt.) % of the composition.

493. The composition of any of the preceding embodiments, wherein the total wt. % of (a)-(d) is greater than the total wt. % of any other amino acid entity in the composition.

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494. The composition of any of the preceding embodiments, wherein one, two, three, or | four of the amino acids in (a)-(d) is provided as part of a dipeptide or tripeptide, e.g., in an amount of at least 10 wt. % of the composition.

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495. The composition of embodiment 494, wherein the dipeptide is a homodipeptide or | heterodipeptide of any of the amino acids in (a)-(d), e.g., one, two, three, or four of the amino acids in (a)-(d) is a homodipeptide or heterodipeptide.

496. The composition of embodiment 494, wherein the tripeptide is a homotripeptide or | 10 heterotripeptide of any of the amino acids in (a)-(d), e.g., one, two, three, or four of the amino acids in (a)-(d) is a homotripeptide or heterotripeptide.

497. The composition of any of the preceding embodiments, wherein (a) is a L-amino

B acid entity dipeptide or a sait thereof (e.g., a L-leucine dipeptide or a sait thereof) * 15 | 498. The composition of embodiment 497, wherein (a) is a homodipeptide or a heterodipeptide, e.g., Ala-Leu.

499. The composition of any of the preceding embodiments, wherein (b) is a L-arginine £ 20 dipeptide or a sait thereof.

500. The composition of embodiment 499, wherein (b) is a homodipeptide or a heterodipeptide, e.g., Ala-Arg.

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501. The composition of any of the preceding embodiments, wherein (c) is a L-glutamine | dipeptide or a sait thereof.

502. The composition of embodiment 501, wherein (c) is a homodipeptide, e.g., Gln* Gin, or wherein (c) is a heterodipeptide, e.g., Ala-Gln.

503. The composition of any of the preceding embodiments, wherein:

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f) a wt. % of the L-glutamine or a sait thereof in the composition is greater than the wt. % | of the R-amino acid entity;

g) the wt. % of the L-glutamine or a sait thereof in the composition is greater than the wt.

| % of the L-amino acid entity;

h) the wt. % of the R-amino acid entity in the composition is greater than the wt. % of the | L-amino acid entity; or

i) a combination of two or three of (f)-(h).

504. The composition of any of the preceding embodiments, wherein the wt. % of the L| 10 glutamine or a sait thereof in the composition is at least 5% greater than the wt. % of the R- amino acid entity, e.g., the wt. % of the L-glutamine or a sait thereof is at least 10%, 15%, 20%, g or 25% greater than the wt. % of the R-amino acid entity g 505. The composition of any of the preceding embodiments, wherein the wt. % of the ΤΙ 5 glutamine or a sait thereof in the composition is at least 20% greater than the wt. % of the L amino acid entity, e.g., the wt. % of the L-glutamine or a sait thereof in the composition is at least 25%, 30%, 35%, 40%, 45%, or 50% greater than the wt. % of the L-amino acid entity.

506. The composition of any of the preceding embodiments, wherein the wt. % of the Rg 20 amino acid entity in the composition is at least 10% greater than the wt. % of the L-amino acid entity, e.g., the wt. % of the R-amino acid entity in the composition is at least 15%, 20%, 25%, g or 30% greater than the wt. % of the L-amino acid entity.

507. The composition of any of the preceding embodiments, wherein:

j) the ratio of the L-amino acid entity to the R-amino acid entity is at least 1:4, or at least

2:5, and not more than 3:4, e.g., the ratio of L-amino acid entity to R-amino acid entity is about

2:3;

k) the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is at least 1:4, * or least 1:3, and not more than 3:4, e.g., the ratio of the L-amino acid entity to the L-glutamine or g 30 a sait thereof is about 1:2;

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1) the ratio of the R-amino acid entity to the L-glutamine or a sait thereof is at least 1:4, or least 1:2, and not more than 6:7, e.g., the ratio of the R-amino acid entity to the L-glutamine or a sait thereof is about 3:4; or

m) a combination of two or three of (j)-(l).

508. The composition of any of the preceding embodiments, further comprising one or both of an isoleucine (I)-amino acid-entity and a valine (V)-amino acid-entity, e.g., both the Iamino acid-entity and the V-amino acid-entity are présent.

509. The composition of embodiment 508, wherein:

n) the wt. % of the L-amino acid-entity in the composition is greater than or equal to the wt. % of the l-amino acid-entity and the V-amino acid-entity in combination;

o) the wt. % of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acidentity in combination in the composition is greater than or equal to the wt. % of the L-glutamine or a sait thereof;

p) the wt. % of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acidentity in combination in the composition is less than the wt. % of the R-amino acid entity;

q) the wt. % of the R-amino acid entity and the L-glutamine or a sait thereof in the composition is greater than the wt. % of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination; or

r) a combination of two, three, or four of (n)-(q).

510. The composition of embodiment 508 or 509, wherein:

s) the wt. % of the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or a sait thereof is at least 50% of the composition, or at least 70% of the composition, but not more than 90% of the composition;

t) the wt. % of the NAC or a sait thereof is at least 1%, or at least 2%, but not more than 10% of the composition;

u) the wt. % of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acidentity in combination is at least 15%, or at least 20%, but not more than 50% of the composition;

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v) the wt. % of the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC | or a sait thereof is at least 40%, or at least 50%, but not more than 80% of the composition; or w) a combination of two, three, or four of (s)-(v).

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511. The composition of any of embodiments 508-510, wherein:

| x) the ratio of the L-amino acid entity to the l-amino acid entity is at least 1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or not more than 3:1, e.g., the ratio of the L-amino acid | entity to the l-amino acid entity is about 2:1;

y) the ratio of L-amino acid entity to V-amino acid entity is at least 1.5:1, or at least | 10 1.75:1, and not more than 2.5 to 1 or not more than 3:1, e.g., the ratio of L to V is about 2:1;

z) the ratio of the L-amino acid entity to the R-amino acid entity is greater than 1:3, | greater than 1.5 to 3, and less than 3:3, e.g., the ratio of the L-amino acid entity to the R-amino acid entity is about 2:3;

| aa) the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is greater than

1:4, greater than 1.5 to 4 and less than 4:4, or less than 3:4, e.g., the ratio of the L-amino acid | entity to the L-glutamine or a sait thereof is about 1:2; or bb) a combination of two, three, or four of (x)-(aa).

512. The composition of any of embodiments 508-511, wherein:

| 20 ce) the ratio ofthe l-amino acid entity to the V-amino acid entity is at least .5:1, or at least .75:1, and not more than 1.5 to 1 or not more than 2:1, e.g., the ratio of the L-amino acid | entity to the l-amino acid entity is about 1:1 ;

dd) the ratio of the l-amino acid entity to the R-amino acid entity is at least .5:3, or at | least .75:3, and not more than 2:3, or not more than 1.5:3, e.g., the ratio ofthe L-amino acid entity to the l-amino acid entity is about 1:3;

| ee) the ratio of the l-amino acid entity to the L-glutamine or a sait thereof is at least .5:4, or at least .75:4, and not more than 3:4, or not more than 2:4, e.g., the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is about 1:4; or fï) or a combination of two or three of (cc)-(ee).

513. The composition of any of embodiments 508-512, wherein:

134 gg) the ratio of the L-amino acid entity to the V-amino acid entity is at least 1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or not more than 3:1, e.g., is the ratio of the L-amino acid entity to the V-amino acid entity is about 2:1;

hh) the ratio ofthe L-amino acid entity to the R-amino acid entity is greater than 1:3, greater than 1.5 to 3, and less than 3:3, e.g., the ratio ofthe L-amino acid entity to the R-amino acid entity is about 2:3;

ii) the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is greater than 1:4, greater than 1.5 to 4 and less than 4:4, or less than 3:4, e.g., the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is about 1:2; or jj) a combination of two or three of (gg)-(ii).

514. The composition of any of embodiments 508-513, wherein:

kk) the ratio of the V-amino acid entity to the L-glutamine or a sait thereof is at least .5:4, or at least .75:4, and not more than 3:4, or not more than 2:4, e.g., the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is about 1:4;

11) the ratio of the V-amino acid entity to the R-amino acid entity is at least .5:3, or at least .75:3, and not more than 2:3, or not more than 1.5:3, e.g., the ratio ofthe V-amino acid entity to the R-amino acid entity is about 1:3;

515. The composition of any of the preceding embodiments, wherein:

oo) a wt. % of the L-amino acid entity in the composition is greater than the wt. % of the NAC or a sait thereof;

pp) a wt. % of the R-amino acid entity in the composition is greater than the wt. % of the NAC or a sait thereof;

qq) a wt. % of the L-glutamine or a sait thereof in the composition is greater than the wt. % of the NAC or a sait thereof; or

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I rr) a combination of two or three of (oo)-(qq).

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1516. The composition of any of the preceding embodiments, wherein at least one of the 5

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I amino acids of (a)-(d) is a free amino acid, e.g., two, three, or four of the amino acids of (a)-(d) are a free amino acid, e.g., at least 50 wt. % of the total wt. of the composition is one or more amino acid entities in free form.

517. The composition of any of the preceding embodiments, wherein at least one of the amino acids of (a)-(d) is in a sait form, e.g., one, two, three, or four of the amino acids of (a)-(d) is in a sait form, e.g., at least 10 wt. % of the total wt. of the composition is one or more amino acid entities in sait form.

518. The composition of embodiment 517, wherein at least 10 wt. % of the total wt. of the composition is one or more amino acid entities in sait form.

519. The composition of any of the preceding embodiments, wherein the composition is capable of one, two, three, four, five, or ail of:

a) decreasing or preventing liver fibrosis;

b) decreasing or preventing liver injury;

c) decreasing or preventing hépatocyte inflammation;

d) improving, e.g., increasing, glucose tolérance;

e) decreasing or preventing steatosis;

f) decreasing or preventing hépatocyte ballooning; or

g) improving gut function.

520. The composition of any of the preceding embodiments, wherein the composition further comprises a serine (S)-amino acid entity, e.g., a S-amino acid entity chosen from Lserine, phosphoserine, P-hydroxypyruvate, L-glycine, tryptophan, acetylserine, cystathionine, cysteine, phosphatidylserine, and D-serine or a combination thereof, e.g., a combination of Lserine and L-glycine.

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I 521. The composition of embodiment 520, wherein the composition comprises an Lamino acid entity, an l-amino acid entity, an V-amino acid entity, an R-amino acid entity, an L| glutamine or a sait thereof, an NAC or a sait thereof, and an L-glycine.

| 522. The composition of embodiment 520, wherein the composition comprises an Lamino acid entity, an l-amino acid entity, an V-amino acid entity, an R-amino acid entity, an L| glutamine or a sait thereof, an NAC or a sait thereof, and an L-serine.

523. The composition of embodiment 520, wherein the composition comprises an Lamino acid entity, an l-amino acid entity, an V-amino acid entity, an R-amino acid entity, an ΤΙ glutamine or a sait thereof, an NAC or a sait thereof, an L-glycine, and an L-serine.

524. The composition of any of embodiments 520-523, wherein the wt. ratio of the L15 amino acid entity, the l-amino acid entity, the V-amino acid entity, the R-amino acid entity, the L-glutamine or sait thereof, and the NAC or sait thereof is about 1 : 0.5: 0.5: 1.5 : 2 : 0.15 or about 1 : 0.5: 0.5: 1.5 : 2 : 0.3.

525. The composition of any of the preceding embodiments, wherein the wt. ratio of the K 20 L-amino acid entity, the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or * sait thereof is about 0.5 to 3 : 0.5 to 4 : 1 to 4 : 0.1 to 2.5, e.g., the wt. ratio of the L-amino acid entity, the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or sait thereof is about 1 : 1.5 : 2 : 0.15 or about 1 : 1.5 : 2 : 0.3.

526. The composition of embodiment 525, wherein the wt. ratio of the L-amino acid entity, the R-amino acid entity, the L-glutamine or a sait thereof, and the NAC or sait thereof is ® about 1 : 0.75 : 2 : 0.15 or about 1 : 0.75 : 2 : 0.3.

527. The composition of any of the preceding embodiments, wherein the wt. ratio of the | 30 L-amino acid entity, the l-amino acid entity, the V-amino acid entity, the R-amino acid entity,

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528. The composition of any of embodiments 13-33, wherein the composition comprises about 0.5 g to about 10 g of the L-amino acid entity, about 0.25 g to about 5 g of the l-amino acid entity, about 0.25 g to about 5 g of the V-amino acid entity, about 0.5 g to about 20 g of the R-amino acid entity, about 1 g to about 20 g of the L-glutamine or a sait thereof, and about 0.1 g to about 5 g of the NAC or sait thereof, e.g., the composition comprises about 1 g of the L-amino acid entity, about 0.5 g of the l-amino acid entity, about 0.5 g of V-amino acid entity, about 1.5 g of R-amino acid entity, about 2 g of L-glutamine or a sait thereof, and about 0.15 g or about 0.3 g of NAC or sait thereof.

529. The composition of embodiment 528, wherein the composition comprises about 4 g ofthe L-amino acid entity, about 2 g ofthe l-amino acid entity, about 1 g ofV-amino acid entity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or a sait thereof, and about 0.9 g of NAC or a sait thereof.

530. The composition of any ofthe preceding embodiments, wherein the composition comprises:

a) L-leucine or a sait thereof;

b) L-isoleucine or a sait thereof;

c) L-valine or a sait thereof;

b) L-arginine or a sait thereof;

e) L-glutamine or a sait thereof; and

f) NAC or a sait thereof.

531. The composition of embodiment 530, wherein the L-Leucine is provided as part of a dipeptide comprising L-Leucine, or a sait thereof, or a tripeptide comprising L-Leucine, or a sait thereof.

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532. The composition of embodiment 530 or 531, wherein the L-Isoleucine is provided | as part of a dipeptide comprising L-Isoleucine, or a sait thereof, or a tripeptide comprising LIsoleucine, or a sait thereof.

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533. The composition of any of embodiments 530-532, wherein the L-Valine is provided | as part of a dipeptide comprising L-Valine, or a sait thereof, or a tripeptide comprising L-Valine, or a sait thereof.

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534. The composition of any of embodiments 530-533, wherein the L-Arginine is | 10 provided as part of a dipeptide comprising L-Arginine, or a sait thereof, or a tripeptide comprising L-Arginine, or a sait thereof.

535. The composition of any of embodiments 530-534, wherein the L-Glutamine is | provided as part of a dipeptide comprising L-Glutamine, or a sait thereof, or a tripeptide comprising L-Glutamine, or a sait thereof.

536. The composition of any of embodiments 530-535, wherein the NAC is provided as | a part of a dipeptide comprising NAC, or a sait thereof, or a tripeptide comprising NAC, or a sait thereof.

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537. The composition of any of the preceding embodiments, wherein the composition | comprises a combination of 4 to 20 different amino acid entities, e.g., a combination of 5 to 15 different amino acid entities.

538. The composition of any of the preceding embodiments, wherein at least two, three, | four, or more amino acid entities is not a peptide of more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15,16, 17, 18, 19, or 20 amino acidresidues in length.

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539. A method for improving liver function, wherein the method comprises | 30 administering to a subject in need thereof an effective amount of a composition of any of the preceding embodiments.

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540. The method of embodiment 539, wherein the L-Leucine is provided as part of a dipeptide comprising L-Leucine, or a sait thereof, or a tripeptide comprising L-Leucine, or a sait thereof.

541. The method of embodiment 539 or 540, wherein the L-Arginine is provided as part of a dipeptide comprising L-Arginine, or a sait thereof, or a tripeptide comprising L-Arginine, or a sait thereof.

542. The method of any of embodiments 539-541, wherein the L-Glutamine is provided as part of a dipeptide comprising L- Glutamine, or a sait thereof, or a tripeptide comprising LGlutamine, or a sait thereof.

543. The method of any of embodiments 539-542, wherein the NAC is provided as a part of a dipeptide comprising NAC, or a sait thereof, or a tripeptide comprising NAC, or a sait thereof.

544. A method for treating one or more symptoms selected from the group consisting of decreased fat metabolism, hépatocyte apoptosis, hépatocyte ballooning, inflammation of adipose tissue, inflammation of hepatic tissue, fibrosis, liver injury, steatosis, glucose tolérance, and oxidative stress, wherein the method comprises administering to a subject in need thereof an effective amount of a composition of any of the preceding embodiments.

545. The method of embodiment 544, wherein the L-Leucine is provided as part of a dipeptide comprising L-Leucine, or a sait thereof, or a tripeptide comprising L-Leucine, or a sait thereof.

546. The method of embodiment 544 or 545, wherein the L-Arginine is provided as part of a dipeptide comprising L-Arginine, or a sait thereof, or a tripeptide comprising L-Arginine, or a sait thereof.

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547. The method of any of embodiments 544-546, wherein the L-Glutamine is provided as part of a dipeptide comprising L- Glutamine, or a sait thereof, or a tripeptide comprising LGlutamine, or a sait thereof.

548. The method of any of embodiments 544-547, wherein the NAC is provided as a part of a dipeptide comprising NAC, or a sait thereof, or a tripeptide comprising NAC, or a sait thereof.

549. A method for treating fatty liver disease, wherein the method comprises administering to a subject in need thereof an effective amount of a compositionof any of the preceding embodiments.

550. The method of embodiment 549, wherein the L-Leucine is provided as part of a dipeptide comprising L-Leucine, or a sait thereof, or a tripeptide comprising L-Leucine, or a sait thereof.

551. The method of embodiment 549 or 550, wherein the L-Arginine is provided as part of a dipeptide comprising L-Arginine, or a sait thereof, or a tripeptide comprising L-Arginine, or a sait thereof.

552. The method of any of embodiments 549-551, wherein the L-Glutamine is provided as part of a dipeptide comprising L- Glutamine, or a sait thereof, or a tripeptide comprising LGlutamine, or a sait thereof.

553. The method of any of embodiments 549-552, wherein the NAC is provided as a part of a dipeptide comprising NAC, or a sait thereof, or a tripeptide comprising NAC, or a sait thereof.

554. The method of any of the preceding embodiments, wherein the subject has a disease or disorder selected from the group consisting of non-alcoholic fatty liver (NAFL), non-alcoholic ¹⁴¹

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| 555. The method of embodiment 554, wherein the subject has pédiatrie NAFLD.

| 556. The method of any of the preceding embodiments, wherein the subject has a high

BMI, obesity, gut leakiness, gut dysbiosis or gut microbiome disturbance.

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557. The method of any of the preceding embodiments, wherein the subject has | 10 cirrhosis, hepatocarcinoma, an increased risk of liver failure, an increased risk of death, metabolic syndrome, or type 2 diabètes.

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558. The method of any ofthe preceding embodiments, wherein the subject has | increased levels of inflammatory cytokines relative to a normal subject, e.g., the subject has increased levels of TNFa relative to a normal subject e.g., without the one or more symptoms or | without the fatty liver disease.

| 559. The method of any ofthe preceding embodiments, wherein the patient exhibits muscle atrophy or has a decreased ratio of muscle tissue to adipose tissue relative to a normal | 20 subject, e.g., without the one or more symptoms or without a fatty liver disease, e.g., the patient exhibits muscle atrophy without one or both of fibrosis or cirrhosis.

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560. The method of any of the preceding embodiments, wherein the subject exhibits

B reverse lipid transport from adipose tissue to liver tissue.

* 25 | 561. The composition of any of the preceding embodiments, wherein the composition comprises free amino acids, wherein the amino acids comprise arginine, glutamine, N- | acetylcysteine, and a branched-chain amino acid chosen from one, two, or ail of leucine, isoleucine, and valine.

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562. The composition of embodiment 561, wherein the branched-chain amino acid is leucine, isoleucine, and valine.

563. The composition of embodiment 561 or 562, wherein the wt ratio of leucine, isoleucine, valine, arginine, glutamine, N-acetylcysteine is 1 : 0.5 : 0.5 : 1.5 : 2 : 0.15.

564. The composition of any of embodiments 561-563, wherein a total weight (wt) of the amino acids is about 2 g to about 60 g.

565. The composition of embodiment 564, wherein the total wt of the amino acids is about 6 g, about 12 g, about 18 g, about 24 g, or about 48 g.

566. The composition of any of embodiments 561-565, wherein the composition comprises about 0.5 g to about 10 g of leucine, about 0.25 g to about 5 g of isoleucine, about 0.25 g to about 5 g of valine, about 1 g to about 20 g of arginine, about 1 g to about 20 g of glutamine, and about 0.1 g to about 5 g of N-acetylcysteine.

567. The composition of embodiment 566, wherein the composition comprises about 1 g of leucine, about 0.5 g of isoleucine, about 0.5 g of valine, about 1.5 g of arginine, about 2 g of glutamine, and about 0.15 g of N-acetylcysteine.

568. The composition of embodiment 566, wherein the composition comprises about 2 g of leucine, about 1 g of isoleucine, about 1 g of valine, about 3.0 g of arginine, about 4 g of glutamine, and about 0.3 g of N-acetylcysteine.

569. The composition of embodiment 566, wherein the composition comprises about 4 g of leucine, about 2 g of isoleucine, about 2 g of valine, about 6.0 g of arginine, about 8 g of glutamine, and about 0.6 g of N-acetylcysteine.

570. The composition of any of embodiments 561-566, wherein the amino acids comprise about 10 wt % to about 30 wt % leucine, about 5 wt % to about 15 wt % isoleucine,

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I I about 5 wt % to about 15 wt % valine, about 15 wt % to about 40 wt % arginine, about 20 wt % to about 50 wt % glutamine, and about 1 wt % to about 8 wt % n-acetylcysteine.

571. The composition of embodiment 570, wherein the amino acids comprise about 16 wt % to about 18 wt % leucine, about 7 wt % to about 9 wt % isoleucine, about 7 wt % to about 9 wt % valine, about 28 wt % to about 32 wt % arginine, about 31 wt % to about 34 wt % glutamine, and about 1 wt % to about 5 wt % n-acetylcysteine.

572. The composition of embodiment 571, wherein the amino acids comprise about 16.8 wt % leucine, about 8.4 wt % isoleucine, about 8.4 wt % valine, about 30.4 wt % arginine, about 33.6 wt % glutamine, and about 2.5 wt % n-acetylcysteine.

573. The composition of any of the preceding embodiments, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

574. The composition of embodiment 573, wherein the excipients are selected from the group consisting of citric acid, lecithin, a sweetener, a dispersion enhancer, a flavoring, a bittemess masking agent, and a natural or artificial coloring.

575. The composition of any of the preceding embodiments, wherein the composition is in the form of a solid, powder, solution, or gel.

576. The composition of any of the preceding embodiments, wherein the amino acids consist of leucine, isoleucine, valine, arginine, glutamine and N-acetylcysteine.

577. A method for treating one or more symptoms selected from the group consisting of decreased fat metabolism, hépatocyte apoptosis, hépatocyte ballooning, inflammation of adipose tissue, inflammation of hepatic tissue, fibrosis, and oxidative stress, wherein the method comprises administering to a subject in need thereof an effective amount of the composition of any one of embodiments 561-576.

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578. The method of embodiment 577, wherein the subject has non-alcoholic fatty liver disease (NAFLD).

579. The method of embodiment 577 or 578, wherein the subject has pédiatrie NAFLD.

580. The method of embodiment 578 or 579, wherein the patient has steatosis.

581. The method of embodiment 577, wherein the subject has non-alcoholic steatohepatitis (NASH).

582. The method of embodiment 581, wherein the subject has fibrosis.

583. The method of embodiment 577, wherein the subject has cirrhosis.

584. The method of embodiment 583, wherein the subject has hepatocarcinoma, an increased risk of liver failure, or an increased risk of death.

585. The method of any of embodiments 577-584, wherein the subject has type 2 diabètes.

586. A method for treating non-alcoholic fatty liver disease (NAFLD) comprising administering to a subject in need thereof an effective amount of the composition of any of embodiments 561-576.

587. The method of embodiment 586, wherein the subject has pédiatrie NAFLD.

588. The method of embodiment 586 or 587, wherein the patient has steatosis.

589. A method for treating non-alcoholic steatohepatitis (NASH) comprising administering to a subject in need thereof an effective amount of the composition of any of embodiments 561-576.

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590. The method of embodiment 589, wherein the subject has fibrosis.

591. A method for treating cirrhosis comprising administering to a subject in need thereof an effective amount of the composition of any of embodiments 561-576.

592. The method of embodiment 591, wherein the subject has hepatocarcinoma, an increased risk of liver failure, or an increased risk of death.

593. The method of any of embodiments 577-592, wherein administering the composition results in an improvement in one or more metabolic symptoms in the subject.

594. The method of embodiment 593, wherein the improvement in one or more metabolic symptoms is selected from the following: increased free fatty acid and lipid metabolism, improved mitochondrial function, white adipose tissue (WAT) browning, decreased reactive oxygen species (ROS), increased levels of glutathione (GSH), decreased hepatic inflammation, decreased hépatocyte ballooning, improved gut barrier function, increased insulin sécrétion, or glucose tolérance.

595. The method of embodiment 594, wherein the increased free fatty acid and lipid metabolism occurs in the liver.

596. The method of embodiment 594 or 595, wherein administration of the composition results in an improvement in one or more metabolic symptoms after a treatment period of 24 hours.

597. The method of any of embodiments 577-596, wherein the method further comprises determining the level of one, two, three, four, five, six, seven, eight, nine, ten, or more (e.g., ail) of the following:

a) alanine aminotransferase (ALT);

b) aspartate aminotransferase (AST);

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c) adiponectin;

d) N-terminal fragment of type m collagen (proC3);

e) caspase-cleaved keratin 18 fragments (M30 and M65);

f) IL-1 beta;

g) C-reactive protein;

h) ΡΠΙΝΡ;

i)TIMPl;

j)MCP-l;or

k)FGF-21.

598. The method of embodiment 597, wherein administration of the composition results in an improvement in one or more of a)-k) after a treatment period of 24 hours.

599. The method of any of embodiments 577-598, wherein the composition is administered prior to a meal.

600. The method of any of embodiments 577-598, wherein the composition is administered concurrent with a meal.

601. The method of any of embodiments 577-598, wherein the composition is administered following a meal.

602. The method of any of embodiments 577-601, wherein the composition is administered with a second agent.

603. The method of embodiment 602, wherein the second agent is selected from the group consisting of a famesoid X receptor (FXR) agonist, a stearoyl CoA desaturase inhibitor, a CCR2 and CCR5 chemokine antagonist, a PPAR alpha and delta agonist, a caspase inhibitor, a galectin-3 inhibitor, an acetyl CoA carboxylase inhibitor, or an ileal sodium bile acid cotransporter inhibitor.

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604. A dietary composition comprising the composition of any of embodiments 561-576, | e.g., wherein the dietary composition is chosen from a medical food, a functional food, or a supplément.

605. The composition of any of embodiments 561-576 for use as a dietary composition, | e.g., wherein the dietary composition is chosen from a medical food, a functional food, or a supplément.

606. The dietary composition of embodiment 605, wherein the subject has type 2 | diabètes and/or a relatively high BMI.

| 607. The dietary composition of any of embodiments 605 or 606, wherein the subject has non-alcoholic fatty liver disease (NAFLD).

608. The dietary composition of any of embodiments 605-607, wherein the subject has | pédiatrie NAFLD.

| 609. The dietary composition of any of embodiments 605-608, wherein the patient has steatosis.

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610. The dietary composition of any of embodiments 605-609, wherein the subject has | non-alcoholic steatohepatitis (NASH).

| 611. The dietary composition of embodiment 610, wherein the subject has fibrosis.

| 612. The dietary composition of any of embodiments 604-606, wherein the subject has cirrhosis.

613. The dietary composition of embodiment 612, wherein the subject has | hepatocarcinoma, an increased risk of liver failure, or an increased risk of death.

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614. The dietary composition of any of embodiments 604-613, wherein the subject has type 2 diabètes.

615. The dietary composition of any of embodiments 604-614, wherein the composition promotes weight loss in the subject.

616. The method or dietary composition of any of the preceding embodiments, wherein the composition is administered at a dose of about 15 g/d to about 90 g/d.

617. The method or dietary composition of embodiment 616, wherein the composition is administered at a dose of about 18 g/d, about 24 g/d, about 36/d, about 54 g/d, or about 72 g/d.

618. The method or dietary composition of any of the preceding embodiments, wherein the composition is administered one, two, to three times per day.

619. The method or dietary composition of any of the preceding embodiments, wherein the composition is administered at a dose of about 6 g, about 8 g, about 12 g, about 16 g, about 18 g, or about 24 g three times per day.

620. The composition of any of the preceding embodiments, wherein:

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4) the ratio of the L-amino acid entity to the L-glutamine or a sait thereof is at least 0.5:1, or at least 0.75:1, and not more than 1.5 to 1 or not more than 2:1, e.g., the ratio of the L-amino acid entity to the L-glutamine or sait thereof is about 1:1 ;

7) a combination of two, three, four, five, or six of (1)-(6).

621. The composition of embodiment 620, wherein:

9) the ratio of the l-amino acid entity to the R-amino acid entity is greater than 1:3, greater than 1.5:3, or about 2:3, and not more than 2.5:3 or not more than 1:1, e.g., the ratio of the l-amino acid entity to the R-amino acid entity is about 2:3;

13) a combination of two, three, four, or five of (8)-(12).

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622. The composition of embodiment 620 or 621, wherein:

16) the ratio of the V-amino acid entity to the NAC entity or a sait thereof is at least 1:2, at least 1.5:2, or about 1:1, and not more than 1.5:1 or not more than 2:1, e.g., the ratio oftheVamino acid entity to the NAC entity or sait thereof is about 1:1 (e.g., 1:0.9);

17) optionally wherein the ratio of the V-amino acid entity to the S-amino acid entity or a sait thereof is greater than 1:8, greater than 1:7, about 1:6, or about 3:20, and not more than 1.5:6 or 1:3, e.g., the ratio of the V-amino acid entity to the S-amino acid entity is about 1:6, or the ratio of the V-amino acid entity to the S-amino acid entity is about 3:20; or

18) a combination of two, three, or four of (14)-(17).

623. The composition of any of embodiments 620-622, wherein:

22) a combination of two or three of (19)-(21).

624. The composition of any of embodiments 620-623, wherein:

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23) the ratio of the L-glutamine to the NAC entity or a sait thereof is at least 5:1, at least I I 5 I I I ¹⁰I « ₅I I | 20 l ₅I I

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5:1.5, or about 4:1, and not more than 4:1.5 or not more than 3:1, e.g., the ratio of the Lglutamine to the NAC entity or sait thereof is about 4:1 (e.g., 4:0.9);

25) a combination of (23) and (24).

625. The composition of any of embodiments 620-624, wherein:

626. The composition of any of embodiments 620-625, wherein the composition satisfies the properties of (1)-(7) defined above.

627. The composition of any of embodiments 620-626, wherein the composition satisfies the properties of at least 2, 3,4, 5, 6, or 7 of any of properties (1)-(26) defined above.

628. The composition of any of embodiments 620-627, wherein the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, and NAC or a sait thereof is 12 : 6 : 3 : 9 : 12 : 2.7.

629. The composition of any of embodiments 620-628, wherein the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, and the S-amino acid entity is 12 :6:3:9:12:2.7:18.

630. The composition of any of embodiments 620-629, wherein the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino

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I acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, and the S-amino acid entity is 12 : 6 : 3 : 9 : 12 :2.7 : 20.

631. The composition of any of embodiments 620-630, wherein the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, and NAC or a sait thereof is 12 +/-15% : 6 +/-15% : 3 +/- 15% : 9 +/-15% : 12 +/- 15% : 2.7 +/- 15%.

632. The composition of any of embodiments 620-631, wherein the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, and the S-amino acid entity is 12 +/- 15% : 6 +/- 15% : 3 +/- 15% : 9 +/- 15% : 12 +/- 15% : 2.7 +/- 15% : 18 +/- 15%.

633. The composition of any of embodiments 620-632, wherein the ratio of the L-amino acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 : 6 : 3 : 9 : 12 : 2.7 : 9: 9. In certain embodiments, the ratio of the L-amino acidentity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L- glycine is 12 : 6 : 3 : 9 : 12 : 2.7 : 10: 10.

634. The composition of any of embodiments 620-633, wherein the ratio of the L-amino * acid-entity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the 25 L-glycine is 12 +/-15% : 6 +/-15% : 3 +/-15% : 9 +/-15% : 12 +/- 15% : 2.7 +/- 15% : 9 +/-

15% : 9 +/- 15%. In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acid- ^e entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 +/- 15% : 6 +/- 15% : 3 +/- 15% : 9 +/- 15% : 12 +/- 15% : 2.7 +/- 15% : 10 +/- 15% : 10 +/- 15%.

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635. A pharmaceutical composition comprising the composition of any of the preceding embodiments.

636. The composition of any of the preceding embodiments, wherein the L-amino acid entity is chosen from the group consisting of L-leucine, P-hydroxy-P-methybutyrate (HMB), oxo-leucine, isovaleryl-CoA, D-leucine, and n-acetyl-leucine, or a combination thereof.

637. The composition of any of the preceding embodiments, wherein the R-amino acid entity is chosen from the group consisting of L-arginine, omithine, argininosuccinate, citrulline, aspartate, glutamate, agmatine, creatine, D-arginine, and N-acetyl-arginine, or a combination thereof.

638. The composition of any of the preceding embodiments, wherein the Q-amino acid entity is chosen from the group consisting of L-glutamine, glutamate, carbamoyl-P, glutamate, D-glutamine, and n-acetylglutamine, or a combination thereof.

639. The composition of any of the preceding embodiments, wherein the NAC-amino acid entity is chosen from the group consisting of NAC, serine, acetylserine, cystathionine, glutathione, homocystéine, méthionine, D-cysteine, L-cysteine, cysteamine, and cystine, or a combination thereof.

640. The composition of any of the preceding embodiments, wherein the S-amino acid entity is chosen from the group consisting of L-serine, phosphoserine, P-hydroxypyruvate, Lglycine, tryptophan, acetylserine, cystathionine, and phosphatidylserine.

641. A dietary composition comprising the composition of any of the preceding embodiments, wherein the dietary compositions is chosen from a medical food, a functional food, or a supplément.

642. A method of providing amino acid entities to a subject comprising administering to the subject an effective amount of the composition of any of the preceding embodiments.

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643. A method of manufacturing or making a composition comprising forming a composition comprising the following:

a) a L-amino acid entity,

b) an R-amino acid entity,

c) a Q-amino acid entity;

d) a NAC entity, e.g., NAC; and optionally, e) an S-amino acid entity;

provided that:

f) at least one amino acid entity is not provided as a peptide of more than 20 amino acid residues in length, wherein:

(i ) the amino acid entity of (a) is selected from Table 2; and (ii ) one or both of the R-amino acid entity and the Q-amino acid entity are présent at a higher amount (wt. %) than the L-amino acid entity.

644. The method of any of the preceding embodiments, wherein:

3) the ratio of the L-amino acid entity to the R-amino acid entity is at least 1:1, at least 3.5:3, at least 4:3, or at least 2:1, and not more than 5:2, e.g., the ratio ofthe L-amino acid entity to the R-amino acid entity is about 4:3;

6) optionally wherein the ratio of the L-amino acid entity to the S-amino acid entity or a sait thereof is greater than 1:3, greater than 1.5:3, about 2:3, or about 3:5, and not more than

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2.5: 3 or 1:1, e.g., the ratio of the L-amino acid entity to the S-amino acid entity is about 2:3, or the ratio of the L-amino acid entity to the S-amino acid entity is about 3:5; or

7) a combination of two, three, four, five, or six of (1)-(6).

645. The method of embodiment 644, wherein:

11) the ratio of the l-amino acid entity to the NAC entity or a sait thereof is at least 1:1, at least 1.5:1, or about 2:1, and not more than 2.5:1 or not more than 3:1, e.g., the ratio ofthelamino acid entity to the NAC entity or sait thereof is about 2:1 (e.g., 2:0.9);

12) optionally wherein the ratio of the l-amino acid entity to the S-amino acid entity or a saitthereofis greaterthan 1:4, greaterthan 1.5:4, about 1:3, orabout3:10, andnotmorethan 1.5:3 or 2:3, e.g., the ratio of the l-amino acid entity to the S-amino acid entity is about 1:3, or the ratio of the l-amino acid entity to the S-amino acid entity is about 3:10; or

13) a combination of two, three, four, or five of (8)-(12).

646. The method of embodiment 644 or 645, wherein:

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18) a combination of two, three, or four of (14)-(17).

647. The method of any of embodiments 644-646, wherein:

20) the ratio of the R-amino acid entity to the NAC entity or a sait thereof is at least 4:1, at least 4:1.5, or about 3:1, and not more than 3:1.5 or not more than 3:2, e.g., the ratio oftheRamino acid entity to the NAC entity or sait thereof is about 3:1 (e.g., 3:0.9);

22) a combination of two or three of (19)-(21).

648. The method of any of embodiments 644-647, wherein:

25) a combination of (23) and (24).

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649. The method of any of embodiments 644-648, wherein:

650. The method of any of embodiments 644-649, wherein the composition satisfies the properties of (1)-(7) defined above.

651. The method of any of embodiments 644-650, wherein the composition satisfies the properties of at least 2, 3, 4, 5, 6, or 7 of any of properties (1)-(26) defined above.

652. The method of any of embodiments 644-651, wherein the ratio of the L-amino acidentity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, and NAC or a sait thereof is 12 : 6 : 3 : 9 : 12 : 2.7.

653. The method of any of embodiments 644-652, wherein the ratio of the L-amino acidentity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, and the S-amino acid entity is 12 : 6 : 3:9:12:2.7:18.

654. The method of any of embodiments 644-653, wherein the ratio of the L-amino acidentity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, and the S-amino acid entity is 12 : 6 : 3 : 9 : 12 : 2.7 : 20.

655. The method ofany of embodiments 644-654, wherein the ratio ofthe L-amino acidentity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, and NAC or a sait thereof is 12 +/-15% :6+/-15% : 3 +/15% : 9 +/- 15% : 12 +/- 15% : 2.7 +/- 15%.

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656. The method of any of embodiments 644-655, wherein the ratio of the L-amino acidentity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, and the S-amino acid entity is 12 +/15% : 6 +/-15% : 3 +/- 15% : 9 +/- 15% : 12 +/- 15% : 2.7 +/- 15% : 18 +/- 15%.

657. The method of any of embodiments 644-656, wherein the ratio of the L-amino acidentity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the Lglycine is 12 : 6 : 3 : 9 : 12 : 2.7 : 9: 9. In certain embodiments, the ratio of the L-amino acidentity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the Lglycine is 12 : 6 : 3 : 9 : 12 : 2.7 : 10: 10.

658. The method of any of embodiments 644-657, wherein the ratio of the L-amino acidentity, the l-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the Lglycine is 12 +/- 15% : 6 +/-15% : 3 +/- 15% : 9 +/- 15% : 12 +/-15% : 2.7 +/- 15% : 9 +/-15% : 9 +/- 15%. In certain embodiments, the ratio of the L-amino acid-entity, the l-amino acidentity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a sait thereof, NAC or a sait thereof, the S-amino acid entity, and the L-glycine is 12 +/-15% : 6 +/- 15% : 3 +/- 15% : 9 +/- 15% : 12 +/- 15% : 2.7 +/- 15% : 10 +/-15% : 10 +/-15%.

659. The composition or method of any of the preceding embodiments, wherein the composition is capable of enhancing fatty acid oxidation, e.g., one or both of reducing levels of unsaturated fatty acids or increasing levels of acylcamitine (e.g., in a STAM mouse model or a FATZO mouse model). In certain embodiments, the réduction in levels of unsaturated fatty acids is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the level of change shown in Table 53, e.g., measured as described in Example 9. In certain embodiments, the increase in levels of acylcamitine is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the level of change shown in Table 53, e.g., measured as described in Example 9.

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660. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of alanine transaminase (ALT), e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

661. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of aspartate transaminase (AST), e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

662. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, alanine transaminase (ALT) by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of ALT, e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

663. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, aspartate transaminase (AST) by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of AST, e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

664. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of hydroxyproline, e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

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665. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, hydroxyproline levels by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of hydroxyproline, e.g., an antibody-based détection assay, e.g., an ELISA, e.g., as described in

Example 4, e.g., relative to a reference composition (e.g., a vehicle control).

666. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using LX-2 cells, e.g., levels of Collai, Acta2, and/or TIMP2 in 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 7, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; NAC; or an amino acid composition comprising L-arginine, L-glutamine, and NAC).

667. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, expression of one or more collagen biomarkers (e.g., Collai, Acta2, and/or TIMP2) by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using LX-2 cells, e.g., levels of Collai, Acta2, and/or ΊΊΜΡ2 in 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 7, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; or NAC).

668. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, expression of one or more collagen biomarkers (e.g., Collai) by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using primary hepatic stellate cells, e.g., levels of

Collai in primary hepatic stellate cells, e.g., as assessed using an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 12, e.g., relative to a reference composition

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I I (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

669. The composition or method of any of the preceding embodiments, wherein the composition is capable of increasing, or increases, expression of one or more collagen biomarkers (e.g., procollagen lal) by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using primary hepatic stellate cells, 10 e.g., levels of procollagen lalin primary hepatic stellate cells, e.g., as assessed using an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 12, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising Lleucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, 15 and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

670. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, hépatocyte inflammation by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using HepG2 cells, e.g., decreased activity, e.g., decreased TNFa-induced activity of NF-kB in a reporter assay in HepG2 cells, as described in Example 8, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, Lisoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, and L25 glutamine; or NAC).

671. The composition or method of any of the preceding embodiments, wherein the composition is capable of reducing, or reduces, TNFa-induced activity of NF-kB in HepG2 cells by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 30 85%, or 90% as detected using HepG2 cells, e.g., decreased activity, e.g., decreased TNFa- induced activity of NF-kB in a reporter assay in HepG2 cells, as described in Example 8, e.g.,

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I relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, Lvaline, L-arginine, and L-glutamine; or NAC).

672. The composition or method of any of the preceding embodiments, wherein the composition is capable of increasing, or increases, glucose tolérance, e.g., in a STAM mouse model or in a FATZO mouse model, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of glucose levels, e.g., using glucose oxidase, e.g., using a glucometer, 10 e.g., as described in Example 5, e.g., relative to a reference composition (e.g., a vehicle control or a positive control, e.g., metformin).

673. The composition or method of any of the preceding embodiments, wherein the composition is capable of increasing, or increases, blood glucose metabolism, e.g., in a STAM 15 mouse model or in a FATZO mouse model, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of glucose levels, e.g., using glucose oxidase, e.g., using a · glucometer, e.g., as described in Example 5, e.g., relative to a reference composition (e.g., a vehicle control or a positive control, e.g., metformin).

674. The composition or method of any of the preceding embodiments, wherein the composition is capable of decreasing, or decreases, steatosis and/or inflammation by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2, e.g., in primary hépatocytes, e.g., using an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 10, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

675. The composition or method of any of the preceding embodiments, wherein the composition is capable of decreasing, or decreases, MCP1/CCL2 levels by at least 1%, 2%, 3%,

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4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2, e.g., in primary hépatocytes, e.g., using an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 10, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising Lleucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

676. The composition or method of any of the preceding embodiments, wherein the composition is capable of decreasing, or decreases, TNFa inflammatory response by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2 or an assay of IL-6, e.g., in primary hepatic stellate cells, e.g., using an antibody-based détection assay, e.g., an ELISA, e.g., as described in Example 11, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and NAC; an amino acid composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

677. The composition or method of any of the preceding embodiments, wherein the composition is capable of decreasing, or decreases, MCP 1/CCL2 levels and/or IL-6 levels by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of

MCP1/CCL2 or an assay of IL-6, e.g., in primary hepatic stellate cells, e.g., using an antibodybased détection assay, e.g., an ELISA, e.g., as described in Example 11, e.g., relative to a reference composition (e.g., a vehicle control; an amino acid composition comprising L-leucine, 25 L-isoleucine, L-valine; an amino acid composition comprising L-arginine, L-glutamine, and

NAC; an amino acid composition comprising L-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

678. The composition of any of the preceding embodiments for use as a médicament.

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679. The composition of any of the preceding embodiments for use in a method as £ disclosed herein.

680. The use of a composition of any of the preceding embodiments in the manufacture * 5 of a médicament.

681. The use of a composition of any of the preceding embodiments in the manufacture g of a médicament for treating any of the disorders or conditions disclosed herein.

Although many of the above embodiments are shown in dépendent form, it is | contemplated that any of the embodiments or combinations thereof may be in independent form.

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EXAMPLES

The Example below is set forth to aid in the understanding of the inventions, but is not intended to, and should not be construed to, limit its scope in any way.

Example 1: Method of producing the amino acid compositions

The amino acid compositions of the instant disclosure and formulations thereof may be made according to methods known in the art. They may also be made by the methods described below.

The starting materials (individual amino acids and excipients) are blended and sieved to generate a powder blend, which is filled into stick packs. The contents of the stick packs are dispersed in water at time of use for oral administration. An example of the mixing and reconstitution protocols, and stick pack formulations made thereby, are provided below.

Mixing protocol

1. Ingrédients were weighed into a container.

2. The container was sealed and placed in a Turbula mixer and contents mixed on low setting for 2 minutes.

3. The blended powder was sieved using a No. 14 screen and any clumps not passing through the sieve were broken apart.

4. The blended and sieved powder was transferred back to the container and mixed in a Turbula mixer on low for 10 minutes.

Table 13. Stick pack formulations

	Placebo	Formulation of Amino Acid Composition A-l	Formulation of Amino Acid Composition A-2
Ingrédient	Amount per stick pack (g)
FUSI-BCAA™ Instantized Blend (2:1:1 L:I:V)	0.00	2.00	2.00
(contains L-Leucine)	N/A	(1.00)	(1.00)
(contains L-Isoleucine)	N/A	(0.50)	(0.50)
(contains L-Valine)	N/A	(0.50)	(0.50)
L-Arginine HCl	0.00	1.50	1.81
L-Glutamine	0.00	2.00	2.00

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N-Acetylcysteine	0.00	0.25	0.15
Citric Acid	0.98	0.67	0.67
Lecithin (Alcolec F100)	0.59	0.83	0.83
Acesulfame Potassium	0.04	0.05	0.05
Sucralose micronized NF	0.02	0.03	0.03
Xanthan Gum (Ticaxan Rapid-3)	0.24	0.24	0.24
Vanilla Custard #4306	0.06	0.06	0.06
Maltrin QD M500 maltodextrin NF	5.75	0.00	0.00
Nat Orange WONF #1326	0.36	0.36	0.36
Lime 865.0032U	0.05	0.05	0.05
Lemon 862.2169U	0.05	0.05	0.05
Bittemess Masking 936.2160U	0.12	0.12	0.12
FD&C Yellow 6	0.01	0.01	0.01
FD&C Red 40(1:100 in M500)	0.0667	0.00	0.0000
Total (g)	8.33	8.22	8.42

Reconstitution protocol

Stick pack formulations were reconstituted according to the following protocol:

1. A total (g) “amount per stick pack” of powder blend was weighed.

2. About 118.3 g (4 oz) of cold filtered water was weighed into a sealable container.

3. The “amount per stick pack” of the powder blend was transferred to the sealable container and the container was sealed.

4. The container was shaken vigorously for 20 to 30 seconds.

Example 2: Analytical characterization of the amino acid compositions

Described below are methods used to characterize some of the physicochemical properties in formulations of the amino acid compositions prepared according to Example 1.

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Identification and Assay. The identification and assay of % label claim of each amino acid présent in Formulations of Amino Acid Composition A-l and Amino Acid Composition A-2 was evaluated by reversed phase HPLC.

Amino acid analysis

Briefly, an amino acid analysis method using reversed-phase high pressure liquid chromatography (HPLC) was developed to measure free amino acid content (except for Nacetylcysteine) in formulations of amino acid compositions described herein following resuspension. Column and chromatographie conditions were modified from Agilent Technical Note: “Automated Amino Acid Analysis Using an Agilent Poroshell HPH-C18 Column (Agilent Application Note 5991-5571EN)”. Primary amino acids in the sample are derivatized online using the Agilent 1260 or 1290 UPLC well-plate autosampler using o-phthaldialdehyde (OPA). Séparation is achieved using an Agilent ZORBAX Eclipse Plus column (4.6 mm ID x 100 mm, 3.5 pm). The OPA-derivatives of primary amino acids are detected using fluorescence (FLD) at 340 nm emission/450 nm excitation wavelengths and UV détection at 338 nm. Individual amino acids are expected to elute according to known représentative chromatograms of amino acid standards. Concentrations of amino acids in samples are determined by fitting a sample peak area to a standard curve. Alternatively, amino acid analysis may be performed using derivatization with AccQ-Tag chemistry and standards (Waters).

Amino acid analysis: N-acetylcysteine

For N-acetylcysteine (NAC), an HPLC test method was developed based on the United States Pharmacopeia Monograph Chapter 39 (USP <39>) for “Acetylcysteine” to détermine the content of N-acetylcysteine of reconstituted powder of formulations described herein. This HPLC method involves the use of a reversed-phase column without any derivatization step. The séparation was done using a column of C-18 backbone as the stationaiy phase, and 0.05 M ΚΉ2ΡΟ4 as the mobile phase. UV détection was performed at 214 nm. The column was then flushed with 5% acetonitrile to remove any residual sample components at the end of each injection. At the end of the sequence, a low flow “system flush” procedure involving stronger organic solvents is used to preserve the column for storage. N-Acetylcysteine is expected to

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I elute according to known représentative chromatograms of standards. Concentrations of N| acetylcysteine in samples are determined by fitting a sample peak area to a standard curve.

Results for Formulation of Amino Acid Composition A-l (compared to theoretical g | per serving) are shown in Table 14. For glutamine, a mean mass per serving of 1.84 g was observed; compared to 2.00 g theoretical per serving, this gives a % agreement (or % label claim | value) of 92%. For arginine, a mean mass per serving of 1.69 g was observed; compared to 1.50 g theoretical per serving, this gives a % agreement (or % label claim value) of 113%. For valine, | a mean mass per serving of 0.51 g was observed; compared to 0.50 g theoretical per serving, this gives a % agreement (or % label claim value) of 101%. For isoleucine, a mean mass per serving | 10 of 0.52 g was observed; compared to 0.50 g theoretical per serving, this gives a % agreement (or % label claim value) of 104%. For leucine, a mean mass per serving of 1.04 g was observed;

| compared to 1.00 g theoretical per serving, this gives a % agreement (or % label claim value) of

104%. For N-acetylcysteine (NAC), a mean mass per serving of 0.28 g was observed; compared | to 0.25 g theoretical per serving, this gives a % agreement (or % label claim value) of 111 %.

Overall, the amino acids and amino acid dérivatives in the Formulation of Amino Acid

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Composition A-l had a range of % label claims of 92-113%.

Table 14. % Label Claim Results: Formulation of Amino Acid Composition A-l

Sample Name	g of Individual Amino Acid/ per serving of Amino Acid Composition A-l
GLN	ARG	VAL	ILE	LEU	NAC
AA Comp A-l	1.84	1.70	0.51	0.52	1.05	0.28
AA Comp A-l	1.82	1.68	0.50	0.52	1.04	0.28
AA Comp A-l	1.84	1.70	0.51	0.52	1.05	0.28
Mean	1.84	1.69	0.51	0.52	1.04	0.28
Theoretical g/serving for each AA	2.00	1.50	0.50	0.50	1.00	0.25
% Agreement Observed/ Theoreticai*100	92	113	101	104	104	111

Results for Formulation of Amino Acid Composition A-2 (compared to theoretical g per serving) are shown in Table 15. For glutamine, a mean mass per serving of 2.102 g was observed; compared to 2.00 g theoretical per serving, this gives a % agreement (or % label claim

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Table 15. % Label Claim Results: Formulation of Amino Acid Composition A-2

Sample Name	g of Individual Amino Acid/ per serving of Amino Acid Composition A-2
GLN	ARG	VAL	ILE	LEU	NAC
AA Comp A-2	2.09	1.92	0.54	0.53	1.05	0.16
AA Comp A-2	2.10	1.90	0.53	0.53	1.06	0.15
AA Comp A-2	2.11	1.96	0.54	0.53	1.06	0.15
AA Comp A-2	2.11	1.90	0.54	0.53	1.06	0.15
Mean	2.102	1.922	0.536	0.531	1.058	0.153
Theoretical g/serving for each AA	2.00	1.50	0.50	0.50	1.00	0.15
% Agreement Observed/ Theoretical*100	105.1	106.6	107.5	106.2	105.8	101.7

Example 3: Pharmacokinetic characterization of the amino acid compositions

The amino acid compositions of the présent disclosure were characterized in rodent and human subjects for their pharmacokinetic effects on amino acid concentrations in response to ingestion of the compositions.

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Rat Pharmacokinetics

The pharmacokinetic effects of a formulation of Amino Acid Composition A-l were tested in rats. After an overnight fast, rats were given the formulation by oral gavage. Portai vein and jugular vein blood was collected just before the dose, and at 5, 15, 30, 60, 120,240 and 360 minutes thereafter. Plasma concentration of amino acid levels were measured, and maximum concentration (Cmax), time of maximum concentration (T_max), and half-life (T1/2) were determined. Maximum concentration values are corrected for baseline endogenous amino acid levels. Results for rat PK studies are shown below in Tables 16-21.

Table 16. Leucine Rat PK - Formulation of Amino Acid Composition A-l

Dose (mg/kg)	C (μΜ) max ^v* ^z	^max (h)	Τ-Λ (h)
266	421	1.0	1.2

Table 17. Isoleucine Rat PK - Formulation of Amino Acid Composition A-l

Dose (mg/kg)	c_max (μΜ)	T_m„ (»)	T (h)
133	176	0.6	1.0

Table 18. Valine Rat PK - Formulation of Amino Acid Composition A-l

Dose (mg/kg)	C (μΜ) max ^z	T_m„ (h)	T* (h)
133	323	0.9	1.5

Table 19. Arginine Rat PK - Formulation of Amino Acid Composition A-l

Dose (mg/kg)	C (μΜ) max ^z	T_max (h)	(h)
399	896	1.0	1.1

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Table 20. Glutamine Rat PK - Formulation of Amino Acid Composition A-l

Dose (mg/kg)	C_max (PM)	Tmax (h)	T (h)
531	300	1.8	4.1

Table 21. N-acetylcysteine Rat PK - Formulation of Amino Acid Composition A-l

Dose (mg/kg)	C (μΜ) max ^z	T_m„ (h)	τ_κ (h)
66	34	0.9	0.8

Allometric scaling was assumed to couvert rat mg/kg doses into human équivalent mg/kg doses. A comparison of these équivalent mg/kg doses and human gram doses gram (assuming bodyweight of 70 kg) is shown in Table 22.

Table 22. Amino Acid Doses: Comparison of Rat and Human

Dose	Leu	Ile	Val	Arg	Gin	NAC
Rat (mg/kg)	266	133	133	399	531	66
Human (mg/kg)	43	21	21	64	86	11
Human (g)	3	1.5	1.5	4.5	6	0.75

Human Pharmacokinetics

The impacts of orally administered Formulation of Amino Acid Composition A-l prepared according to Example 1 on amino acid pharmacokinetics was evaluated in six apparently healthy human subjects between the âges of 18 and 40. Changes in plasma concentrations of amino acids in response to ingestion of the Formulation of Amino Acid Composition A-l at two doses (High: 3 stick packs, ~18 g of amino acids; vs. Low: 1 stick pack, ~6 g of amino acids) were determined. Blood samples (3 mL) were collected after an initial baseline and in spécifie intervals thereafter [i.e., 0 (pre-administration), 15, 30, 60, 90, 120, 150, 180,210, and 240 minutes]. Plasma concentration of amino acid levels were measured, and

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Table 23. Leucine Human PK - Formulation of Amino Acid Composition A-l

Dose	Dose (g)	^max (μΜ)	(h)	T -¹½ (h)	AUC_Iast (μΜ-h)
HIGH	3.0	294	0.8	1.1	471
LOW	1.0	117	0.8	1.3	153

Table 24. Isoleucine Human PK - Formulation of Amino Acid Composition A-l

Dose	Dose (g)	^max (μΜ)	^Tm„ (h)	T (h)	AUC_)ast (μΜ-h)
HIGH	1.5	141	0.7	0.8	194
LOW	0.5	52	0.8	0.6	54

Table 25. Valine Human PK - Formulation of Amino Acid Composition A-l

Dose	Dose (g)	^max (μΜ)	T„„ (I>)	T,, (h)	AUC_Iast (μΜ-h)
HIGH	1.5	238	0.8	1.3	400
LOW	0.5	89	0.8	1.7	101

Table 26. Arginine Human PK - Formulation of Amino Acid Composition A-l

Dose

Dose (g)

(μΜ)

T„ (h)

T (h)

AUC_|as( (μΜ-h)

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HIGH	4.5	177	0.8	1.6	311
LOW	1.5	69	0.8	1.3	111

Table 27. Glutamine Human PK - Formulation of Amino Acid Composition A-l

Dose	Dose (g)	C _v (μΜ) max ^v* ⁷	(h)	τ_κ (h)	AUC_last (μΜ-h)
HIGH	6.0	190	0.9	2.9	332
LOW	2.0	103	1.1	3.0	186

Example 4: Therapeutic Amino Acid Composition A-l treatment improves liver fibrosis in an animal model of chemically induced fibrosis

Amino Acid Composition A-l was tested for its ability to affect liver fibrosis in a model of chemically induced liver fibrosis. A commonly used model of experimental hepatic fibrosis is induced chemically in mice using carbon tetrachloride; CCI4 (Gideon Smith, Animal Models of Cutaneous and Hepatic Fibrosis; Progress in Molecular Biology and Translational Science, Vol. 105, pp. 371- 408). CCI4 causes inflammation, hépatocyte damage, necrosis and fibrosis after 4 weeks of treatment and cirrhosis after 8 weeks. Liver fibrosis induced in mice by carbon tetrachloride (CCI4) resembles important properties of human liver fibrosis including inflammation, régénération and fiber formation.

Animais

Male BALB/c mice 7 to 8 weeks of âge were used for this study. Animais were housed four per cage, kept on a standard 12 hr light cycle and given free access to water and standard mouse chow. Food and water were available ad libitum.

Procedure

Animais were dosed with 5% CCU or vehicle intraperitoneally (IP) typically 3 days a week for 4 weeks. CC14 was formulated weekly. 10 ml/kg of Amino Acid Composition A-l at 23 mg/ml, 76 mg/ml or 153 mg/ml was dosed by oral gavage twice daily. Animais were

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I weighed twice weekly and blood was collected via retro-orbital sinus once per week for sérum. After four weeks, blood was collected for sérum isolation and mice were euthanized via cervical dislocation. Two lobes of liver were removed — the left lobe was placed in a tube containing 10% formalin for histopathology, while the right lobe was weighed and placed in a beadbeater tube containing 2.3 mm zirconia beads and 2x volume of 1:100 protease inhibitor (Sigma Aldrich, #P8340). Tissue samples were homogenized for 2 minutes in a beadbeater machine and immediately spun down at 3,000 rpm for 15 minutes at 4°C. Sérum was analyzed for ALT/AST levels at weeks 2 and 4. Homogenized liver samples were further evaluated for Hydroxyproline (Hyp) content to identify formation of liver fibrosis.

Hydroxyproline (week 4)

Hydroxyproline (4-hydroxyproline, Hyp) is a common nonproteinogenic amino acid and is used as an indirect measure of the amount of collagen présent, indicative of fibrosis. Hepatic Hyp content levels in CCL-treated animais were significantly higher than vehicle treated animais. Data are mean ± standard déviation (stdev); “Comp A-l”: Amino Acid Composition A1; *p<0.05 compared to vehicle control by unpaired T test. Raw data are shown in Table 28.

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Table 28. Hepatic Hyp content level results

	Hydroxyproline
Vehicle/Sham	Vehicle/CCL4	Comp A-l, 23mg/ml	Comp A-l, 76mg/ml	Comp A-l, 153mg/ml
mean	0.160	0.263*	0.280	0.228	0.201
stdev	0.067	0.107	0.104	0.124	0.057

AST levels and ALT levels

Aspartate transaminase (AST) and alanine transaminase (ALT) are commonly measured clinical biomarkers of liver health. Both AST and ALT levels were significantly elevated in CCL administered animais for the entire duration of the study, suggesting that liver damage has occurred. Data are mean ± standard déviation (stdev); “Comp A-l”: Amino Acid Composition A-l; p values are compared to vehicle/CC14 control; by one-tailed T test; n.s. not signifîcant. Raw data are shown in Tables 29 and 30.

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Table 29. ALT level results

	Liver ALT
Vehicle/Sham	Vehicle/CCL4	Comp A-l, 23mg/ml	Comp A-l, 76mg/ml	Comp A-l, 153mg/ml
mean	1608.4	4153.4	3694.9	3023.4	2992.7
stdev	1099.5	1427.4	2106.4	1343.8	1674.2
			n.s.	p<0.05	p=0.0371

Table 30. AST level results

	Liver AST
Vehicle/Sham	Vehicle/CCL4	Comp A-l, 23mg/ml	Comp A-l, 76mg/ml	Comp A-l, 153mg/ml
mean	155.8	933.6	879.2	554.7	680.4
stdev	69.7	237.0	527.3	336.6	431.2
			n.s.	p<0.01	p=0.0394

SUMMARY

Treatment with Amino Acid Composition A-l resulted in réduction of chemicallyinduced fibrosis as indicated by reduced levels of hydroxyproline, a marker for collagen production, and in improvement of clinical biomarkers of liver damage as indicated by réduction in levels of liver enzymes ALT and AST (Tables 31-33).

Table 31. Hepatic Hyp content level results: raw data

Hydroxyproline
Vehicle/Sham	Vehicle/CCL4	Comp A-l, 23mg/ml	Comp A-l, 76mg/mï	Comp A-l, 153mg/ml
0.122	0.241	0.246154	0.190323	0.248649
0.277	0.318	0.529578	0.174684	0.24
0.152	0.298	0.234783	0.226549	0.18
0.108	0.493	0.216393	0.169128	0.174233
0.123	0.2	0.294737	0.175887	0.133333
0.108	0.196	0.22439	0.107692	0.135758
0.232	0.183	0.305512	0.212389	0.210219
	0.177	0.393064	0.316191	0.150265
		0.272897	0.612174	0.231293
		0.192683	0.18018	0.308824

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		0.164341	0.218803
			0.203279
			0.17971

Table 32. ALT level results: raw data

	Jver ALT
Vehicle/Sham	Vehicle/CCL4	Comp A-l, 23mg/ml	Comp A-l, 76mg/ml	Comp A-l, 153mg/ml
685.0737	4963.448	1299.647	4325.237	2611.524
2623.343	578.7053	5069.816	4325.237	2150.594
1606.933	5235.278	5566.202	2304.237	1866.945
3805.214	2115.138	5188.003	1051.454	696.8924
779.6234	4384.331	3828.851	1488.746	1725.121
637.7988	4207.05	330.5123	4313.419	3722.483
1417.834	5471.652	649.6176	4112.501	5211.641
1311.466	5105.273	1441.471	2859.717	4797.986
	3462.471	5495.29	2564.249	1216.916
	4147.957	4892.536	5318.009	1796.033
	5436.196	5329.828	2836.079	5069.816
	3852.489	5247.097	2457.881	5046.179
	5034.36		1346.922

Table 33. AST level results: raw data

	_<iver AST
Vehicle/Sham	Vehicle/CCL4	Comp A-l, 23mg/ml	Comp A-l, 76mg/ml	Comp A-l, 153mg/ml
95.37346	908.3081	315.7015	703.1751	508.1721
57.38585	1050.129	928.5682	720.9027	335.9616
239.7263	877.918	1389.484	371.4167	379.0142
194.1412	660.1224	1047.596	262.5189	211.8688
123.231	599.3423	589.2123	267.5839	510.7046
102.971	675.3175	181.4787	819.6704	885.5156
237.1938	1470.525	285.3115	629.7324	1214.742
196.6737	1070.389	305.5715	414.4693	941.2307
	733.5651	1690.853	505.6396	252.3889
	976.6858	1100.779	1485.72	297.974
	1088.116	1232.469	356.2217	1437.602
	918.4382	1483.187	406.8718	1189.416
	1108.376		267.5839

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Example 5: Therapeutic treatment with Amino Acid Composition A-l improves oral glucose tolérance in a pre-clinical animal model

Amino Acid Composition A-l and metformin were tested for their ability to affect glucose tolérance in a genetically obese B6.Cg-Lep^ob/J (ob/ob) mouse model (Maida A, et al., 2010, PMID: 20972533).

Model description

B6.Cg-Lep^ob/J (ob/ob) mice harbor a spontaneous mutation of leptin (Lep) gene. ob/ob mice exhibit hyperphagia, obesity, and metabolic syndrome/T2DM-like symptoms, e.g. hyperglycemia, hyperinsulinemia, and insulin résistance, ob/ob mice hâve impaired intestinal barrier function, gut microbial translocation, and an inflammatory, fîbrogenic phenotype of hepatic stellate cells (Brun P et al., 2004, PMID: 17023554). ob/ob mice develop skeletal muscle hypoplasia in quadriceps femoris, similar to the effect of aging in humans (Hamrick MW et al., 2004, PMID: 15003785). ob/ob mice exhibit intolérance to glucose and insulin. Metformin lowers plasma glucose (Cool B, et al., Cell Metab 2006, PMID: 16753576), liver triglycéride, and reverses NAFLD in ob/ob mice (Lin HZ et al., 2000, PMID: 10973319; Cool B, et al., Cell Metab 2006, PMID: 16753576). A single dose of metformin treatment reduces blood glucose and improves glucose tolérance (OGTT) in C57/BL6.

Experimental design

Eight-week-old male ob/ob mice were subjected to treatment of test articles (Amino Acid Composition A-l and metformin) followed by oral glucose tolérance test (OGTT) on Day 3. Mice were randomized by body weight and unfasted blood glucose on Day -1. Body weight was recorded daily in the moming before AM dosing on Day 1, Day 2, and Day 3. Test articles were dosed by oral gavage at 10 ml/kg. Dosage of a test article was calculated based on daily body weight. Treatment schedule and dose are listed in the following section (Table 34). AM doses were administered at 0700, and PM doses were administered at 1800. Oral glucose tolérance test (OGTT) was performed after 6-hour fasting on Day 3.

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Table 34. Treatment schedule

Group	Test article	#	Dosing Schedule
Group 1	Vehicle	N=5	Vehicle dosed on Day 1 and Day 2 at 0700 and 1800, and Day 3 at 0700 and 30 min before OGTT for a total of 6 doses.
Group 2	Metformin	N=5	Metformin hydrochloride (450 mg/kg, QD PO at the beginning of dark cycle) dosed on Day -1, Day 1, and Day 2 at 1800, and at 30 min before OGTT on Day 3 for a total of 4 doses.
Group 3	Amino Acid Composition A-l	N=5	Amino Acid Composition A-l (1500 mg/kg, BID PO at 0700 and 1800) dosed on Day 1 and Day 2 at 0700 and 1800, and Day 3 at 0700 and 30 min before OGTT for a total of 6 doses.
Group 4	Amino Acid Composition A-l	N=5	Amino Acid Composition A-l (3000 mg/kg, BID PO at 0700 and 1800) dosed on Day 1 and Day 2 at 0700 and 1800, and Day 3 at 0700 and 30 min before OGTT for a total of 6 doses.

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Baseline glucose and biochemistry (insulin, triglycéride and cholestérol)

Mice were fasted for 6 hours prior to OGTT test. Food was removed at 0700 hours on

Day 3; water was provided during fasting. Blood samples were collected from tail snip or facial puncture at -30 min (relative to OGTT) into K2EDTA tubes for baseline glucose and blood biochemistry (insulin, triglycéride, and cholestérol). Blood glucose was measured by a glucometer (SDI StatStrip Xpress or équivalent). Plasma was collected in K2EDTA and saved at -80°C.

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Oral glucose tolérance test (OGTT)

Mice were bled for baseline glucose and plasma at -30 min. Test articles were then dosed by oral gavage at -30 min. Glucose was administered per os (P.O.) at a dosage of 2.0 g/kg body weight. Blood glucose levels were measured at 0 min immediately prior to glucose injection and 15 then at 15, 30, 60,120 and 240 minutes thereafter (shown as 0.25, 0.5, 1,2, and 4 hours in Table 35 below).

Results are shown in Table 35. Data are mean ± standard déviation (stdev). (p values by Dunnett’s multiple comparisons: **p<0.005 compared to vehicle control; ***p<0.001 compared to vehicle control; ****p<0.0005 compared to vehicle control.) 20

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Results

Table 35. OGTT results: Mean Blood glucose levels (mg/dl) and standard déviations (stdev)

	Vehicle	Amino Acid Comp A-l, 1500 mg/kg	Amino Acid Comp A-l, 3000 mg/kg	Metformin
Timepoint (hours)	mean blood glucose level (mg/dl)	stdev	mean blood glucose level (mg/dl)	stdev	mean blood glucose level (mg/dl)	stdev	mean blood glucose level (mg/dl)	stdev
-1	241.8	108.3	245.6	89.4	229.6	78.2	196.4	59.8
0	282.6	47.0	374.6	97.6	303.0	77.1	199.4	62.6
0.25	655.0	107.2	575.6	73.8	456.2**	36.6	353.6****	73.6
0.5	640.6	92.6	555.2	84.0	513.0	47.9	390.2***	99.5
1	378.0	111.1	386.6	27.5	316.4	86.1	317.6	116.9
2	236.6	54.8	243.5	18.4	230.0	101.1	158.2	44.0
4	197.8	53.3	214.8	56.8	179.8	81.3	109.4	29.0

SUMMARY

Treatment with Amino Acid Composition A-l resulted in improvement of oral glucose tolérance, as indicated by improved blood glucose clearance upon oral glucose loading. In addition, 3-day treatment with Amino Acid Composition A-l did not alter baseline blood glucose in ob/ob mice (Table 36).

Table 36. OGTT results: Blood glucose levels (mg/dl) raw data

Timepoint (hours)	Vehicle
-1	211	141	211	219	427
0	239	256	273	284	361
0.25	741	676	514	578	766
0.5	551	621	604	630	797
1	305	317	327	369	572
2	182	243	230	203	325
4	146	203	167	188	285

Timepoint

Amino Acid Comp A-l, 1500 mg/kg

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(hours)
-1	224	220	190	191	403
0	331	548	347	316	331
0.25	526	702	580	531	539
0.5	532	419	621	587	617
1	431	365	367	395	375
2	246		243	220	265
4	192		193	175	299

Timepoint (hours)	Amino Acid	Zomp A-l	, 3000 mg/kg
-1	137	294	179	214	324
0	242	359	203	329	382
0.25	412	490	438	443	498
0.5	513	482	467	512	591
1	240	351	220	342	429
2	161	334	148	160	347
4	120	235	110	139	295

Timepoint (hours)	Metformin
-1	265	248	183	120	166
0	274	234	220	129	140
0.25	387	397	427	310	247
0.5	462	439	482	307	261
1	365	399	431	239	154
2	154	183	219	124	111
4	118	93	155	101	80

Example 6: Therapeutic t reatment of NAFLD, NASH, and HCC with Amino Acid Composition A-l in a pre-clinical animal model

Amino Acid Composition A-l and Obeticholic acid (6a-ethyl-chenodeoxycholic acid; “OCA”) were tested for their ability to treat NASH in the ST AM™ model (Stelic Institute & Co., Tokyo, Japan; Saito K. et al., 2015 Sci Rep 5: 12466). Two additional groups of normal C57BL/6 mice fed standard chow and vehicle treated STAM™ mice were included as Controls.

Ail animais receiving treatment or vehicle were treated starting at 6 weeks until 9 weeks of âge. Compounds were administered via oral gavage, with a dose volume of 10 ml/kg. Amino Acid

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Composition A-l was administered twice daily at a dose of 1500 mg/kg, and OC A was | administered once daily at a dose of 30 mg/kg.

| STAM™ mouse model description

STAM™ is a model for non-alcoholic steatohepatitis (NASH) and hepatocellular | carcinoma (HCC), developed by SMC Laboratories, Inc. and created by the combination of

Chemical and dietary interventions using C57BL/6 mice (Saito K. étal., 2015 Sci Rep 5: 12466).

| Mice are treated with a low dose of streptozotocin at birth and fed a high fat diet starting at 4 weeks. Evidence of fatty liver is présent by 5 weeks, followed by NASH by 7 weeks and fibrosis | 10 by 9 weeks.

| Induction of NASH

NASH was induced in 53 male mice by a single subcutaneous injection of 200 pg streptozotocin (STZ, Sigma-Aldrich, USA) solution 2 days after birth and feeding with high fat 15 diet (HFD, 57 kcal% fat, Cat# HFD32, CLEA Japan, Japan) after 4 weeks of âge.

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Route of drug administration, préparation of dosing solutions and treatment doses | Amino Acid Composition A-l, OCA and Vehicle (described below) were administered by oral route in a volume of 10 mL/kg. Amino Acid Composition A-l was solubilized in | 20 deionized water to 150 mg'ml(lOX). OCA (Advanced ChemBlocks Inc.) was resuspended in

0.5% methycellulose in water to 3 mg/ml (10X). Amino Acid Composition A-l was | administered at a dose of 1500 mg/kg twice daily (9 am and 7 pm). OCA was administered at a dose of 30 mg/kg once daily (9 am).

Histological analyses | Liver samples from mice in Group 2 (Vehicle), 3 (Amino Acid Composition A-l) and 4 (OCA) were used for the following assays. For HE staining, sections were eut from paraffin | blocks of liver tissue prefixed in Bouin’s solution and stained with Lillie-Mayer’s Hematoxylin (Muto Pure Chemicals Co., Ltd., Japan) and eosin solution (Wako Pure Chemical Industries).

NAFLD Activity score (NAS) was calculated according to the criteria of Kleiner (Kleiner D.E. et * al., Hepatology, 2005;41:1313).

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EXPERIMENTAL DESIGN

Study groups

Group 1: STZ: Ten néonatal STZ-primed mice were fed with a normal diet ad libitum without any treatment until 9 weeks of âge.

Group 2: Vehicle: Ten NASH mice were orally administered vehicle (10% phosphate buffered saline, pH 7.2) in a volume of 10 mL/kg twice daily (9 am and 7 pm) from 6 to 9 weeks of âge.

Group 3: Amino Acid Composition A-l: Ten NASH mice were orally administered water for irrigation supplemented with Amino Acid Composition A-l at a dose of 1500 mg/kg twice daily (9 am and 7 pm) from 6 to 9 weeks of âge.

Group 4: OCA: Ten NASH mice were orally administered 0.5% methylcellulose supplemented with OCA at a dose of 30 mg/kg once daily (9 am) from 6 to 9 weeks of âge.

Group 5: Normal: Ten normal mice were fed with a normal diet ad libitum without any treatment until 9 weeks of âge.

Group 6: HFD: Ten normal mice were fed with a high fat diet ad libitum without any treatment until 9 weeks of âge.

Histological analysis results: HE staining, NAFLD activity score and q-smooth muscle actin staining

Non-alcoholic fatty liver disease activity score results

The non-alcoholic fatty liver disease (NAFLD) activity score was assessed via histological analysis and grading of H&E stained liver sections from each animal. This score is the sum of three individual scores that grade the degree of steatosis (0-3), inflammation (0-2), and hépatocyte ballooning (0-2). Ail tissues were graded using the scoring criteria of Kleiner et al. (Kleiner et al. Hepatology. 2005; 41(6): 1313-21). Results are shown in Table 37. Data are mean ± standard déviation (stdev). Normal C57BL/6 mice fed standard chow had a mean score of 0 +/- 0. Vehicle treated STAM™ mice had a mean score of 4.7 +/- 0.67. Amino Acid Composition A-l treated mice had a mean score of 3.1 +/- 0.74. OCA treated mice had a mean score of 2.9 +/- 0.74. Both Amino Acid Composition A-l and OCA were statistically different

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Similarly, Amino Acid Composition A-l treated mice showed a mean ballooning score | of 0.4 +/- 0.52, compared to a mean ballooning score for vehicle treated STAM™ mice of 1.6 +/5 0.52, and a mean ballooning score for OCA treated mice of 0.3 +/- 0.48. Both Amino Acid | Composition A-l and OCA were statistically different from vehicle for ballooning score when compared using Dunnett’s multiple comparisons test (Amino Acid Composition A-l p=0.0001, I OCAp=0.0001). Raw data are shown in Tables 37-40.

Table 37. NAFLD Activity Score

NAFLD Activity Score (NAS)
Condition	Normal C57BL/6 mice	Vehicle-treated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
Mean	0	4.7	3.1	2.9
stdev	0	0.67	0.74	0.74

Table 38. NAFLD Activity: Steatosis Score

	Steatosis
Normal C57BL/6 mice	Vehicletreated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
Mean	0	1	0.9	0.8
stdev	0	0.00	0.32	0.42

Table 39. NAFLD Activity: Inflammation Score

	Inflammation
Normal C57BL/6 mice	Vehicletreated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
Mean	0	2.1	1.8	1.8
stdev	0	0.32	0.63	0.79

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Table 40. NAFLD Activity: Ballooning Score

Ballooning

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	Normal C57BL/6 mice	Vehicletreated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
Mean	0	1.6	0.4	0.3
stdev	0	0.52	0.52	0.48

Fibrosis: Sirius Red staining results

Fibrosis was assessed by analysis of Sirius red positively stained cell area from stained liver sections from each animal. Images were quantified using the percent of positively stained area was used as a measure of fibrosis. Results of this analysis are shown in Table 39. Data are mean ± standard déviation (stdev). Normal C57BL/6 mice fed standard chow had a mean positive area of 0.286 +/- 0.09. Vehicle treated STAM™ mice had a mean positive area of 1.1 +/- 0.26. Amino Acid Composition A-l treated mice had a mean positive area of 0.828 +/- 0.33. OCA treated mice had a mean score of 0.776 +/- 0.25. Amino Acid Composition A-l and OCA were statistically different from vehicle when compared using Dunnett’s multiple comparisons test (Amino Acid Composition A-l p=0.00494, OCA p<0.016). Raw data are shown in Table 41.

Table 41. Fibrosis (mean positively stained area, Sirius red)

Condition	Normal C57BL/6 mice	Vehicle-treated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
Mean	0.286	1.1	0.828	0.776
stdev	0.09	0.26	0.33	0.25

Similarly to the statistically significant improvement in the NAFLD activity score, ballooning, and fibrosis in the STAM mouse model after treatment with Amino Acid Composition A-l (FIG. 1 A), a statistically significant improvement in the NAFLD activity score, ballooning, and fibrosis was determined in the high-fat, high fructose and cholestérol diet (HFFC) mouse model after treatment with Amino Acid Composition A-l (FIG. IB).

α-Smooth muscle actin (α-SMA) staining results

Liver sections of ail mice were stained for the marker α-smooth muscle actin (aSMA) to identity activated hepatic stellate cells. Images were quantified using the percent of positively

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| Data are mean ± standard déviation (stdev); p values are compared to vehicle-treated STAM mice control; by one-tailed T test.

| Normal C57BL/6 mice fed standard chow had a mean positive area of 0.682 +/- 0.26.

Vehicle treated STAM™ mice had a mean positive area of2.128 +/- 0.50. Amino Acid | Composition A-l treated mice had a mean positive area of 1.657 +/- 0.84. OCA treated mice had a mean score of 1.562 +/- 0.31.

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Table 42. Activated hepatic stellate cells (mean positively stained area, α-smooth muscle actin)

Condition	Normal C57BL/6 mice	Vehicle-treated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
Mean	0.682	2.128	1.657	1.562
stdev	0.26	0.50	0.84	0.31
			p=0.073	p<0.05

SUMMARY

Treatment with Amino Acid Composition A-l significantly reduced NASH severity to levels équivalent to Famesoid X Receptor (FXR) inhibition by OCA (which is currently under clinical investigation by Intercept Pharmaceuticals, Inc. for treatment of NASH), as indicated by significant réduction in NAFLD Activity Score (NAS) (mean NAS: 3.1 +/- 0.74 for Amino Acid Composition A-l vs. vehicle treated STAM™ mice mean score of 4.7 +/- 0.67, compared to OCA treated mice mean score of 2.9 +/- 0.74), and development of fibrosis as indicated by the downregulation of hepatic stellate cell activation (mean aSMA positively stained area: 1.657 +/20 0.84 for Amino Acid Composition A-l vs. vehicle treated STAM™ mice mean area of 2.128 +/- 0.50, compared to OCA treated mice mean area of 1.562 +/- 0.31).

Table 43. NAFLD Activity Score: raw data

Normal C57BL/6 mice	Vehicle-treated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
0	6	3	4
0	5	4	2

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5	4	2
4	3	4
5	2	3
5	2	3
4	3	2
4	3	3
4	3	3
5	4	3

Table 44. NAFLD Activity: Steatosis Score: raw data

Steatosis
Normal C57BL/6 mice	Vehicletreated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
0	1	1	1
0	1	1	1
0	1	1	1
0	1	1	1
0	1	0	1
0	1	1	0
0	1	1	1
0	1	1	0
0	1	1	1
0	1	1	1

Table 45. NAFLD Activity: Inflammation Score: raw data

Inflammation
Normal C57BL/6 mice	Vehicletreated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
0	3	1	2
0	2	2	1
0	2	2	1
0	2	2	2
0	2	1	2
0	2	1	3
0	2	2	1
0	2	2	3
0	2	2	2
0	2	3	1

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Table 46. NAFLD Activity: Ballooning Score: raw data

	lallooning
Normal C57BL/6 mice	Vehicletreated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
0	2	1	1
0	2	1	0
0	2	1	0
0	1	0	1
0	2	1	0
0	2	0	0
0	1	0	0
0	1	0	0
0	1	0	0
0	2	0	1

Table 47. Fibrosis (mean positively stained area, Sirius red): raw data

Normal C57BL/6 mice	Vehicle-treated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
0.26	0.79	1.07	0.36
0.35	1.43	0.58	0.56
0.19	1.44	0.48	1.1
0.31	1.36	0.58	1.19
0.19	1.04	1.07	0.89
0.36	0.75	0.34	0.91
0.24	1.07	0.86	0.66
0.37	1.13	1.43	0.72
0.18	0.83	0.96	0.68
0.41	1.16	0.91	0.69

Table 48. Activated hepatic stellate cells (mean positively stained area, α-smooth muscle actin): raw data

Normal C57BL/6 mice	Vehicle-treated STAM mice	Amino Acid Composition A-l treated STAM mice	OCA treated STAM mice
0.47	2.16	0.81	1.46
0.59	2.77	1.35	1.51
1.13	2.21	1.3	1.49

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0.52	1.5	3.03	1.17
0.75	2.87	2.04	1.49
0.46	1.93	0.97	1.5
0.37	1.6	3.08	1.13
0.85	1.46	1.91	2.03
0.62	2.36	1.15	1.87
1.06	2.42	0.93	1.97

Example 7: Réduction of fibrogenic gene expression in hepatic stellate cells treated with an amino acid composition

Hepatic stellate cells in a healthy liver are in the space of Disse, between the hépatocytes and liver sinusoïdal endothélial cells. In response to liver injury hepatic stellate cells become activated, proliférative and contractile, increase production of aSMA, sécrétion of type I and ΠΙ collagens and spécifie MMP and ΊΊΜΡ proteins. LX-2 cells were selected as a model of activated hepatic stellate cells and used to test whether spécifie amino acid compositions would reduce fibrogenic gene expression induced with TGFpi.

LX-2 hepatic stellate cells (Millipore) were seeded on day 0 at 1.67E4 cells per well in collagen I coated 96-well microplates (ThermoFisher) in Dulbecco’s Modified Eagle Medium (DMEM, Corning) supplemented with 2% heat inactivated fêtai bovine sérum (HI-FBS, HyClone) and 0.2% Primocin (InVivoGen) and incubated overnight at 37°C, 5% CO2. On day 1, cells were washed twice with 150 pL per well DPBS (Gibco) and replaced with amino acid free DMEM (US Biologicals) containing a defined custom amino acid concentration based on the mean physiological concentrations in blood based on values published in the Human Metabolome DataBase (1,2,3), with 25 mM Glucose, 1 mM Sodium Pyruvate and a dose curve of defined amino acid compositions LIVRQ+N-Acetylcysteine, LIVRQ, RQ+N-Acetylcysteine, Nacetylcysteine, LIV at 40X the concentration présent in the basal HMDB (Human Metabolome Database (Wishart DS, Tzur D, Knox C, et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan;35(Database issue):D521-6. 17202168)) derived amino acid concentrations or individually with leucine, isoleucine, valine, arginine, glutamine or cysteine at 50X the HMDB derived concentrations. Combinations containing N-acetylcysteine were dosed with 10 mM. Cells were pretreated for 6 hours at 37°C, 5% CO2. After pretreatment, TGFpi (R&D Systems) or vehicle was spiked into each well for a final

189 concentration of 5 ng/mL and cells were incubated under this stimulus for a further 12 hours at 37°C, 5%CO2.

After 12 hour incubation, RNA was prepared as described using the FastLane Cell Multiplex Kit (Qiagen) as described in the manufacturera protocol. Two microlitera of cell lysate was utilized in subséquent qRT-PCR experiments using the FastLane Cell Multiplex Kit at a reduced final qPCR reaction volume of 20 pL. Quantitative PCR was conducted on lysâtes to détermine collagen-lal expression normalized to β-actin housekeeping expression using the AACt method using TaqMan primer probes (Integrated DNA Technologies: Coll Al, Hs.PT.58.15517795; Actb, Hs.PT.39a.22214847; Acta2, Hs.PT.56a.24853961; Timp2, Hs.PT.58.14780594).

Results:

Table 49 shows the Collai, Acta2, and Timp2 gene expression in LX-2 cells treated with amino acid combinations compared to vehicle with or without TGFpi stimulus. LIVRQ+NAcetylcysteine, LIVRQ, RQ+N-Acetylcysteine, and N-acetylcysteine reduced Collai expression and Timp2 expression. LIVRQ+N-acetylcysteinc shows the largest réduction of Collai, Acta2, and Timp2 gene expression. LIVRQ-N-acetylcysteine reduces Acta2 expression signifïcantly greater than N-Acetylcysteine alone, RQ+N-acetylcysteine, and LIV. LIVRQ+N-acetylcysteine reduces Timp2 expression signifïcantly greater than any of the other combinations (Table 49).

Table 49.

TGFP 1	Amino Acid Suppleme nt	Collai	Acta2	Timp2
Mean	Std. Deviati on	Numb erof value s	Mean	Std. Deviati on	Numb erof values	Mean	Std. Deviatio n	Number of values
Yes	Vehicle	2.861	0.3151	4	0.801	0.1149	4	1.658	0.2791	4
No	Vehicle	1.042	0.3102	4	1.006	0.1190	4	1.022	0.2400	4
Yes	LIVRQNA C	1.267	0.4106	4	0.292	0.0969	4	0.535	0.0306	4
Yes	LIVRQ	1.787	0.2926	4	0.267	0.0637	4	0.975	0.2006	4
Yes	RQNAC	1.664	0.3320	4	0.487	0.1042	4	0.897	0.1932	4
Yes	NAC	1.659	0.4695	4	0.647	0.1097	4	1.076	0.0681	4
Yes	LIV	2.831	0.3404	3	0.793	0.0812	4	1.927	0.0944	4

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Table 50 shows the Collai expression of individual amino acids with or without TGFpi | stimulus at IX or 50X the HMDB derived amino acid concentration. Individually, only cysteine showed a significant decrease in Collai expression at 50X.

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Table 50.

	Collai
TGFpi	Amino Acid Supplément	Mean	Std. Déviation	Number of values
No	Vehicle	1.015	0.1832	8
Yes	IX CYS	2.491	0.1588	4
Yes	50X CYS	1.695	0.3310	4
Yes	IX ILE	2.020	0.1451	4
Yes	50XILE	2.028	0.3667	4
Yes	IX LEU	1.901	0.3360	4
Yes	50X LEU	2.372	0.4153	4
Yes	IX VAL	2.093	0.2157	4
Yes	50X VAL	2.203	0.5762	4

	Collai
TGFpl	Amino Acid Supplément	Mean	Std. Déviation	Number of values
No	Vehicle	1.010	0.1510	8
Yes	IX ARG	1.620	0.6691	4
Yes	50X ARG	1.970	0.7740	4

	Collai
TGFP1	Amino Acid Supplément	Mean	Std. Déviation	Number of values
No	Vehicle	1.012	0.1681	8
Yes	1XGLN	2.340	0.7069	4
Yes	50XGLN	2.194	0.3359	4

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Example 8: Réduction in hépatocyte inflammation after treatment with an amino acid composition

The ability of amino acids to influence hépatocyte inflammation was assessed using HepG2 Hepatocellular Carcinoma cells stably expressing NF-kB luciferase reporter System (Signosis, Inc.). HepG2 cells were seeded on day 0 in 4.5e4 in a 96-well microplates (ThermoFisher) in Dulbecco’s Modified Eagle Medium (DMEM, Corning) supplemented with 0.1% heat inactivated fêtai bovine sérum (HI-FBS, HyClone) and 0.2% Primocin (InVivoGen) and incubated overnight at 37°C, 5% CO2. On day 1, cells were washed once with 150 pL per well DPBS (Gibco) and replaced with amino acid free DMEM (US Biologicals) containing a 10 defined custom amino acid concentration based on the mean physiological concentrations in blood based on values published in the Human Metabolome Database (Wishart DS, Tzur D, Knox C, et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007

Jan;35(Database issue):D521-6. 17202168). with 25 mM Glucose, 1 mM Sodium Pyruvate and a dose curve of defined amino acid compositions (i.e. vehicle, LIVRQ+N-acetylcysteine,

LIVRQ, RQ+N-acetylcysteine, N-acetylcysteine alone, LIV or individually with Leucine, Isoleucine, Valine, Arginine, Glutamine, and Cysteine) at 50X (Table 51). Cells were pretreated in the defined media for 12 hours at 37°C, 5% CO2. After pretreatment, TNFa (R&DSystems) or vehicle was spiked into each well for a final concentration of 100 pM and cells were incubated under this stimulus for an additional 6 hours at 37°C, 5%CÜ2. After 12-hour incubation, cells were washed lx in 150ul cold PBS and lysed using Passive Lysis Buffer and luciferase assay was performed according to manufacturer’s protocol (Signosis). Firefly luciferase activity was assessed using a Bio-Tek SynergyH4 plater reader and luminometer (Sitcheran R*, Comb WC, Cogswell PC, Baldwin AS*. Essential rôle for epidermal growth factor receptor in glutamate receptor signaling to NF-kappaB. Mol Cell Biol. (2008) Aug;28(16):5061-70. Epub 2008 Jun 9).

TNFa -stimulated NF-kB activity was unaffected by treating cells in 50x Leucine,

Isoleucine, Valine, Arginine, and Glutamine, relative to the lx Plasma amino acid baseline media. Pretreating cells in 50x Cysteine did resuit in a significant blunting of TNFa-induced NFkB activity. Combinatorial treatments with the single amino acids did hâve varying effects on the NF-kB reporter activity, but importantly, the combination of ail 6 amino acids together (LIVRQNAC) resulted in the most significant inhibition of TNFa induced NF-kB activity in liver cells (Table 51).

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Table 51

TNFa	Amino Acid Supplément	NF-kB Reporter Activity
Mean	Std. Déviation	Number of values
lOOpM	Vehicle (Ix AA)	8865.50	333.05	2
lOOpM	LIVRQNAC	3960.50	678.12	2
lOOpM	LIVRQ	5685.00	1453.81	2
lOOpM	RQNAC	5618.00	926.31	2
lOOpM	NAC	6852.00	1023.89	2
lOOpM	LIV	5911.00	422.85	2
lOOpM	IxL	5811.00	134.35	2
lOOpM	50xL	6070.50	58.69	2
lOOpM	1x1	8129.50	713.47	2
lOOpM	50x1	8937.50	17.68	2
lOOpM	IxV	7255.50	557.91	2
lOOpM	50xV	5992.00	644.88	2
lOOpM	IxR	10170.50	140.71	2
lOOpM	50xR	9760.00	1083.29	2
lOOpM	lxGlu	8201.00	2091.62	2
lOOpM	50x Glu	7313.50	1054.30	2
lOOpM	Ix Cys	9968.50	1614.33	2
lOOpM	50x Cys	6820.50	23.34	2

Example 9: Treatment with an Amino Acid Composition améliorâtes NASH progression in two rodent models by impacting lipid metabolism, inflammation, and fibrosis

The amino acid composition is formulated to simultaneously target multiple mechanisms of disease pathology to safely and effectively treat NASH (Table 52). As described herein, the effïcacy of the amino acid composition was studied in two established mouse models of NASH to détermine the effect of the amino acid composition on signs and symptoms associated with NASH and related disorders (FIG. 2).

Table 52. Exemplary amino acid components of the amino acid composition.

Amino acid	wt. ratio	wt. %	g / packet	g dose #1	g dose #2
Leucine	1	16.78	1.00 g	2g	4g
Isoleucine	0.5	8.39	0.50 g	1 g	2g

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Valine	0.5	8.39	0.50 g	1 g	2g
Arginine HCl	1.81	30.37	L81g	3.62 g	7.24 g
Glutamine	2	33.56	2.00 g	4g	8g
N-acetylcysteine	0.15	2.52	0.15 g	0.3 g	0.6 g
Total amino acids				cd2_g	—24 g

Animal studies

The STAM™ mouse is a model for non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC), developed by SMC Laboratories, Inc. Evidence of fatty liver is 5 présent by 5 weeks of âge, followed by NASH by 7 weeks of âge, and fibrosis by 9 weeks of âge. Male STAM mice were generated in C57BL/6 mice, which received a low dose streptozotocin 2 days after birth and were fed a high fat diet (57% kcal fat, HFD32, CLEA Japan, Inc.) starting at 4 weeks old (Saito K. et al., 2015 Sci Rep 5: 12466; hereby incorporated by reference in its entirety). The amino acid composition was administered to STAM mice at a dose of 1.6 m/kg twice daily for 3 weeks starting at 6 weeks of âge. One group of vehicle treated STAM mice was included as a control. Unfasted mice were euthanized at 9 weeks old. Plasma and liver samples were harvested for further analysis (FIG. 3).

The FATZO™ mouse is an inbred, polygénie model of obesity, metabolic syndrome, and NASH, developed by Crown Bioscience, Inc (Peterson RG. Et al., 2017 PLoS One; hereby incorporated by reference in its entirety). Male FATZO mice were fed a high fat, fructose, and cholestérol (HFFC) diet (40% kcal fat, D12079B, Research Diets, Inc. and 5% fructose in drinking water) starting at 6 weeks old to induce NAFLD and NASH. Evidence of fatty liver is présent by 4 weeks post induction, followed by NASH by 16 weeks post induction and fibrosis by 20 weeks of induction. The designed amino acid composition was administered at a dose of

3.0 g/kg twice daily for 4 weeks starting at 16 weeks post induction (FIG. 3). One group of vehicle treated FATZO mice was included as control. Unfasted mice were euthanized at 20 weeks post-induction. Plasma and liver samples were harvested for further analysis.

Histological Analysis

The 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 whole slides.

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The livers were evaluated by veterinary pathologists blind to sample ID using the NASH | Clinical Research Network (CRN) liver histological scoring system (Kleiner DE, et al., 2015, hereby incorporated by reference in its entirety). The NASH CRN Scoring System assesses | progression of steatosis, lobular inflammation, hépatocyte ballooning, degeneration, and fibrosis.

One cross section of liver for each case was analyzed with the NASH score system. Steatosis, | lobular inflammation, and fibrosis progression was assessed on a 0-3 scale. Ballooning degeneration was assessed on a 0-2 scale.

| The Positive Pixel Count algorithm of the Aperio Automatic Image Quantitation was used to quantify the percentage of a spécifie stain présent in a scanned slide image. A range of | 10 color (range of hues and saturation) and three intensity ranges (weak, positive, and strong) were masked and evaluated. The algorithm counted the number and intensity-sum in each intensity | range, along with three additional quantifies: average intensity, ratio of strong/total number, and average intensity of weak positive pixels.

| A spécifie positive pixel algorithm was used for imaging the Sirius Red and Oil Red O liver sections. The positive pixel algorithm was modified to distinguish between the orange and | blue colors. Alterations from the normal “hue value” (0.1 to 0.96) and “color saturation” (0.04 to .29), were made for the Sirius Red évaluation. Vasculature and artifacts were excluded from | analysis.

| 20 Liver Triglycéride and Cholestérol Measurement

Liver total lipid-extracts were obtained by Folch’s method (Folch J. et al., J. Biol.

| Chem.l957;226:497; hereby incorporated by reference in its entirety). Liver samples were homogenized in chloroform-methanol (2:1, v/v) and incubated overnight at room température.

| After washing with chloroform-methanol-water (8:4:3, v/v/v), the extracts were evaporated to dryness, and dissolved in isopropanol. Liver triglycéride and cholestérol contents were measured | by the Triglycéride E-test and Cholestérol E-test, respectively.

Gene Expression Analysis

Liver RNA samples were converted into cDNA libraries using the Illumina TruSeq

StrandedmRNA samplepréparation kit (Illumina# RS-122-2103). Transcriptome were * analyzed at Q2 Solutions (Morrisville, NC). RNA Seq data were normalized and analyzed using

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Ingenuity Pathway Analysis (QIAGEN Bioinformatics). Mouse liver gene expression at the | pathway level was focused on because it is translatable to human NAFLD (Teufel A, et al.,

Gastroenterology, 2016, hereby incorporated by reference in its entirety).

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Métabolite Analysis | Metabolic profiling based on both capillaiy electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and LC-TOFMS platforms was performed at Human Metabolome | Technologies (Yamagata, Japan). Métabolites in the samples were identified by comparing the migration time and m/z ratio with authentic standards and quantified by comparing their peak | 10 areas with those of authentic standards.

| Liver Cytokine/Chemokine Measurement

The levels of IL-lb, MCP-1, and MIP-1 protein in liver were quantified using the

B multiplex ELISA Assay (Meso Scale Discovery, Rockville, Maryland).

* 15

B The Amino Acid Composition Improves Ballooning and Fibrosis in Both STAM and

FATZO mice | Treatment with the amino acid composition significantly reduced NAFLD activity scores (NAS) in both STAM and FATZO mice (FIG. 4A). Treatment with the amino acid composition | 20 also significantly decreased hépatocyte ballooning in STAM mice (FIG. 4B). Scores of steatosis and inflammation were not changed according to histological measures by treatment of STAM | mice with the amino acid composition. The Sirius Red-positive, fibrosis area was significantly lowered by treating the STAM mice with the amino acid composition, while the Oil Red O area | was not changed by treating the STAM mice with the amino acid composition (FIG. 4C). Liver triglycéride and cholestérol levels were not changed.

Treatment with the amino acid composition also significantly decreased hépatocyte ballooning in FATZO mice (FIG. 4D). Scores of steatosis and inflammation as well as liver triglycéride and cholestérol levels were not changed in the FATZO mice treated with the amino acid composition treatment. The Sirius Red-positive, fibrosis area was significantly lowered by | 30 treatment of the FATZO mice with the amino acid composition, while the Oil Red O area was

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I not changed by treatment of the FATZO mice with the amino acid composition treatment (FIG.

| 4E).

| The Amino Acid Composition Enhances Fatty Acid Oxidation

NAFLD is characterized by hepatic lipid accumulation. Liver triglycéride is attributable | to a précisé balance between acquisition by de novo lipogenesis and uptake of non-esterified fatty acids from the plasma, versus disposai by fatty acid oxidation and by the sécrétion of | triglyceride-rich lipoproteins (Kawano Y, Cohen DE, J Gastroenterol. 2013, hereby incorporated by reference in its entirety). Compared to control mice, STAM mice had higher liver unsaturated | 10 fatty acids, which were reduced by treatment with the amino acid composition (FIG. 5 A and

Table 53). Liver acylcamitines in STAM mice were increased by treatment with the amino acid | composition, suggesting enhanced fatty acid beta-oxidation (FIG. 5B and Table 53).

| Table 53. P-values and fold changes for liver acylcarnitine and unsaturated fatty acids following treatment of STAM mice with the amino acid composition (treated) compared to H control.

Lipid	KEGG ID	HMDBID	Control p-val	Control fold change	Treated p-val	Treated fold change
AC(13:1)	NoID	NoID		-1.61	7.94E-02	1.32
FA(14:3)	NoID	NoID	5.17E-03	1.69	4.83E-01	-1.24
FA(20:3)	NoID	NoID	6.97E-06	18.29	1.35E-01	-2.17
FA(22:4)	NoID	NoID	7.12E-07	34.79	3.15E-01	-1.79
FA(22:5)-1	NoID	NoID	2.39E-02	3.31	1.19E-01	-1.80
FA(22:5)-2	NoID	NoID	2.11E-04	3.03	1.69E-02	-1.92
Linoleic acid	C01595	HMDB00673	9.90E-04	3.18	1.68E-02	-1.77
Linolenic acid	C06427	HMDB01388	3.57E-05	35.88	4.08E-02	-1.99
Oleic acid	C00712	HMDB00207	1.95E-04	18.05	3.67E-02	-1.88
Palmitoleic acid	C08362	HMDB03229	9.84E-05	2.69	2.70E-02	-1.48

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Différentiel gene expression patterns in the liver impacted by treatment with the amino | acid composition were interpreted in the context of the upstream regulator Systems biology knowledgebase framework developed by Ingenuity Pathway Analysis. Computed z-scores

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I indicated that the gene expression patterns are consistent with activation of ACOX1, which encodes peroxisomal fatty acid oxidation, as an upstream regulator (FIG. 6 and Table 54).

Table 54. P-values and fold changes for gene expression associated with the ACOX1 pathway following treatment of STAM mice with the amino acid composition (treated) compared to control.

gene	IPAupstream regulator	ACOX1 path	IPA_gene name	Control fold change	Control p-val	Treated fold change	Treated p-val
Akrlc6	ACOX1	ACOX1	AKR1C4	-1.68	1.88E-06	1.207	8.606E-02
C9	ACOX1	ACOX1		-3.10	7.81E-07	1.370	1.678E-02
Ces3a	ACOX1	ACOX1		-2.10	2.69E-06	1.379	3.900E-02
Ces3b	ACOX1	ACOX1		-3.16	1.05E-07	1.476	8.274E-02
Cyp2c50	ACOX1	ACOX1	Cyp2c54	-1.72	1.24E-04	1.243	6.999E-02
Cyp4al2 a	ACOX1	ACOX1		-1.59	4.60E-03	1.293	8.589E-02
Cyp7bl	ACOX1	ACOX1		-4.45	4.29E-04	1.408	7.877E-02
Egfr	NFKB;ACOX1	ACOX1		-1.98	1.31E-04	1.348	1.187E-02
Gstpl	ACOX1	ACOX1		-2.31	2.56E-06	1.281	2.924E-02
Mupl	ACOX1	ACOX1		-7.69	1.47E-03	1.781	7.683E-02
Mupll	ACOX1	ACOX1		-2.47	9.01E-03	1.703	5.779E-02
Mupl4	ACOX1	ACOX1		-2.05	1.27E-02	1.395	4.890E-02
Mupl 6	ACOX1	ACOX1		-6.27	4.38E-03	1.465	7.558E-02
Mup6	ACOX1	ACOX1		-1.73	2.27E-02	1.330	5.784E-02
Selenbp2	TGFB;IL10; ACOX1	ACOX1	Selenbpl	-15.77	3.73E-05	3.015	2.916E-02
Serpinal c	TGFB;ACOX1	ACOX1		-2.25	7.22E-09	1.290	5.612E-02
Serpinal e	TGFB;ACOX1	ACOX1		-43.20	3.93E-08	2.361	1.852E-02
Slc4a4	ACOX1	ACOX1		1.55	7.00E-06	-1.209	4.682E-03
Trib3	IL2;NFKB;AC 0X1	ACOX1		2.40	3.14E-04	-1.472	1.987E-02

The Amino Acid Composition Tempers Inflammation Pathways

Inflammation is a “second-hit” of NASH. The differential gene expression patterns in the liver as a resuit of treatment with the amino acid composition yielded z-scores within IPA analysis associated with upstream regulator activation of anti-inflammatory IL-10 (FIG. 7A) and

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I inhibition of pro-inflammatory NF-kB (FIG. 7B and Table 55), interferons, IL-lb, and IL-2 | (FIG. 7C and Table 55). At the protein level, treatment with the amino acid composition significantly down-regulated hepatic MCP-1 and MIP-1, which are the ligands of C-C | chemokine receptor types 2 (CCR2) and 5 (CCR5), respectively (FIG. 8). Thus, treatment with the amino acid composition tempered the immune system toward an anti-inflammatory state, | which may dampen NASH progression.

| Table 55. P-values and fold changes for gene expression associated with the ACOX1 pathway following treatment of STAM mice with the amino acid composition (treated)

H 10 compared to control.

Rene	IPA upstream regulator	IPA gene name	IL10_ path	ILlb_ path	IL2 path	NFKB path	TGFB path	Control fold change	Control pval	Treated fold change	Treated p-val
Abcbla	NFKB; IL10		IL10			NFKB		3.02	1.55E-06	-1.239	1.047E-01
Abcblb	NFKB; ILIO		IL10			NFKB		-2.24	4.90E-O3	1.272	9.745E-02
Acta!	TGFB						TGFB	7.96	5.87E-03	-1.849	1.044E-0I
Adorai	TGFB						TGFB	1.94	1.99E-05	-1.222	3.262E-02
AK0074 36	NFKB	ADAMTS9				NFKB		2.32	6.38E-03	-1.788	6.451E-02
AK0436 76	ILlb	PFKP		ILlb				1.68	3.12E-O3	-1.462	1.586E-02
AK1541 84	ILlb; TGFB	CYBA		ILlb			TGFB	1.99	1.O2E-O3	-1.222	9.247E-02
AK1580 38	IL2	ΝΑ VI			IL2			1.62	2.63E-02	-1.659	1.587E-02
Atf5	ILlb;IL2			ILlb	IL2			1.71	3.00E-04	-1.223	9.622E-02
Bcl2ald	ILlb; IL2; NFKB			ILlb	IL2	NFKB		4.00	1.74E-04	-1.733	3.064E-02
Capn5	IL2				IL2			1.51	2.05E-05	-1.235	5.908E-03
Ccrl	ILlb;IL2;TGFB			ILlb	1L2		TGFB	2.82	1.28E-O3	-1.352	7.496E-02
Cd274	ILlb;IL2;NFKB			ILlb	IL2	NFKB		2.37	1.7IE-07	-1.282	1.5O6E-O2
Cd83	ILlb;IL2;NFKB;T GFB			ILlb	IL2	NFKB	TGFB	2.41	5.36E-07	-1.434	6.661E-03
Chstll	TGFB						TGFB	2.91	7.87E-06	-1.308	1.881E-02
CIec2i	TGFB						TGFB	1.80	1.01E-03	-1.226	5.963 E-02
Egfr	NFKB					NFKB		-1.98	1.31E-O4	1.348	1.187E-O2
Entpdl	IL2				1L2			1.87	1.84E-04	-1.218	8.413E-02
FrC!	TGFB						TGFB	49.56	1.03E-03	-1.478	4.472E-02
Gabrd	TGFB						TGFB	5.82	1.48Ε-Ό4	-1.511	5.978E-02
Gbp4	ILlb;IL10	Gbp6	IL10	ILlb				1.60	1.01E-03	-1.234	5.234E-02
Gbp5	IL10		IL10					1.81	7.82E-05	-1.217	6.120E-02
Gm8909	ILlb;NFKB;IL10	HLA-Λ	IL10	ILlb		NFKB		4.03	8.19E-O4	-1.943	2.569E-02

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Gpr85	TGFB						TGFB	2.46	1.77E-02	-1.685	6.628E-02
Gucy2c	TGFB						TGFB	2.35	8.97E-O3	-1.395	1.080E-O1
Hk2	ILlb;lL2			ILlb	IL2			2.00	2.01E-04	-1.301	9.834E-02
Hsdl7b6	TGFB						TGFB	2.89	4.20E-05	-1.244	1.036E-02
Illm	ILlb;NFKB;TGFB ;IL10		IL10	ILlb		NFKB	TGFB	4.43	5.94E-O9	-1.273	1.951 E-02
Lama3	ILlb			ILlb				-3.09	1.90E-05	1.650	5.542E-02
Lck	IL2				IL2			1.92	8.87E-O4	-1.246	3.O27E-O2
Lifr	ILlb;IL2;TGFB			ILlb	IL2		TGFB	-4.43	2.43E-05	1.406	2.099E-02
Msrl	TGFB						TGFB	1.58	1.I3E-O3	-1.226	5.836E-02
Mstlr	TGFB						TGFB	2.08	4.17E-03	-1.523	1.147E-02
Nlrp3	TGFB						TGFB	1.93	1.30E-03	-1.506	2.456E-02
P2ryl4	TGFB						TGFB	3.29	4.67E-05	-1.268	4.039E-02
Pcskl	ILlb			ILlb				2.07	9.00E-03	-2.070	8.15OE-O2
Pla2g4a	ILlb;TGFB			ILlb			TGFB	2.10	7.94E-05	-1.265	1.064E-01
Plbl	ILlb			ILlb				2.01	2.09 E-02	-1.696	2.324E-02
Rgsl6	ILlb;ÏL2;NFKB			ILlb	IL2	NFKB		7.68	2.47E-05	-I.6O4	4.319E-02
Saa4	ILlb			ILlb				-1.72	4.76E-02	1.283	9.771 E-02
Selenbp2	TGFB;IL10	Selenbpl	IL10				TGFB	-15.77	3.73E-O5	3.015	2.916E-02
Sema3b	TGFB						TGFB	4.12	4.78E-05	-1.285	9.461 E-02
Serpinal c	TGFB						TGFB	-2.25	7.22E-09	1.290	5.612E-02
Serpinal e	TGFB						TGFB	-43.20	3.93E-O8	2.361	1.852ΕΌ2
Serpina3 k	ILlb;NFKB;TGFB			ILlb		NFKB	TGFB	-2.95	1.85E-O8	1.500	1.125E-02
Serpinb2	ILlb;NFKB			ILlb		NFKB		1.94	4.98E-O2	-1.935	1.865E-02
Slc23a2	TGFB						TGFB	2.00	1.02E-05	-1.258	2.229E-02
Slc2a6	NFKB;IL10		IL10			NFKB		1.79	2.68E-02	-1.281	9.775E-02
Slc7al	NFKB;TGFB					NFKB	TGFB	1.64	3.96E-O3	-1.324	8.657E-02
Slc7al 1	ILlb;ILI0		IL10	ILlb				65.45	1.35E-03	-1.869	1.068E-01
Tkl	ILlb			ILlb				-2.40	4.99E-06	1.295	4.036E-02
TIrll	IL10		IL10					1.66	6.11E-03	-1.368	6.OO5E-O2
TIr2	ILlb;IL2;NFKB;T GFB;IL10		IL10	ILlb	IL2	NFKB	TGFB	2.12	5.32E-O5	-1.300	5.430E-02
Trib3	IL2;NFKB				IL2	NFKB		2.40	3.14E-04	-1.472	1.987E-O2
Xcll	IL2				IL2			2.52	1.22E-03	-1.796	6.279E-02

The Amino Acid Composition Prevents Fibrogenesis Pathways

Fibrosis is at the nexus of several biologie processes, such as metabolic dysrégulation, inflammation, and cell death. Lipid accumulation in hépatocytes and chronic inflammation induce fibrogenic activation of hepatic stellate cells (Wobser H, et al., Cell Res. 2009, which is

200 hereby incorporated by reference in its entirety). The liver gene expression pattern resulting from treatment with the amino acid composition was consistent with the suppression of the fibrogenic TGF-b signaling pathway (Fig. 7D).

Increasing evidence implicates that CCR2/CCR5 and their ligands, including MCP1/MIP-l, promote macrophage recruitment and hepatic stellate cell activation which contribute to fibrosis following liver tissue damage (Lefebvre E, et al., PLoS One 2016, which is hereby incorporated by reference in its entirety). The amino acid composition displayed a potent antifibrotic activity in the STAM model of NASH via reducing hepatic TGF-b signaling and MCP-1 and MIP-1 proteins (FIG. 8).

Conclusion

The amino acid composition demonstrated consistent disease modifying activity in both ST AM and FATZO mouse models of NASH including improvement in NAS and amelioration of ballooning and fibrosis. The activity of the amino acid composition appears to be driven, at least in part, via increase in fatty acid oxidation, réduction in levels of key cytokines and transcription pathways associated with liver inflammation and fibrosis.

Example 10: Hepatocvte rnodel for steatosis and inflammation

Hépatocyte lipotoxicity appears to be a central driver of hepatic cellular injury via oxidative stress and endoplasmic réticulum (ER) stress. The ability of amino acids to influence steatosis (lipid accumulation) and inflammation in hépatocytes was assessed using human primary hépatocytes (Lonza, TRL).

Cell seeding and maintenance

Primary hépatocytes lot nos. from two healthy human donors were seeded on day 0 at density of 6e04 cells in 96 well optical microplates (Thermofîsher) in hépatocyte plating media (William’s E medium (Gibco) supplemented with 10% heat-inactivated FBS (Atlanta Bio), 2mM Glutamax (Gibco), Ix ITS plus (R&D Systems), and 0.2% Primocin (InVivoGen) and incubated for 6 hours at 37°C, 5% CO2. After 6 hours, cells were washed twice with 150 ul William’s E medium and incubated overnight at 37°C, 5% CO2 with serum-free hépatocytes culture media (Hépatocytes defined medium (Corning)) supplemented with 5 ug human recombinant EGF

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I (Corning), 2mM Glutamax (Gibco), and lx Penicillin/Streptomycin. On day 1, cells were washed twice with 150 pL per well William’s E medium (Gibco) and incubated for 24h in the hépatocyte culture media in the same conditions described above.

Amino acids pre-treatment

On day 2, cells were washed twice with 150 ul DPBS IX (Gibco) and maintained in amino acid-free WEM (US Biologicals) containing a defined custom amino acid concentration based on the mean physiological concentrations in blood. The values are published in the Human Metabolome Database (Wishart DS, Tzur D, Knox C, et al., HMDB: the Human

Metabolome Database. Nucleic Acids Res. 2007 Jan; 35(Database issue):D521-6. 17202168; which is hereby incorporated by reference in its entirety). This custom media is supplemented with 11 mM Glucose, 0.272 mM Sodium Pyruvate, and a dose curve of defined amino acid compositions (i.e., vehicle, LIVRQ+N-acetylcysteine, LIVRQ, RQ+N-acetylcysteine, Nacetylcysteine alone, LIV, or individually with L-Leucine, L-Isoleucine, L-Valine, L-Arginine,

L-Glutamine, and L-Cysteine) at various ranges of concentrations. Cells were maintained in this defined media for 24 hours at 37°C, 5% CO2.

Co-treatment with free fatty acids and different amino acids combination

After pre-treatment, cells were exposed to free fatty acids (FFA) at 250 uM with a ratio 20 of 2:1 (Oleate:Palmitate) supplemented with TNF-α (Thermofisher) at 1 ng/ml or vehicle. Cells were incubated with the FF As mixture and the different amino acids combinations for 24 hours at 37°C, 5%CÜ2. After 24 hours incubation, media was removed for cytokine analysis and replaced by fresh media containing the same stimulus conditions and amino acid concentrations. Cells were incubated for an additional 48 hours for a total of 72 hours of FFA and

TNFa stimulation.

Cytokine Analysis after 24h by ELISA

Human CCL2 (MCP-1) was measured by ELISA (Human CCK2/MCP-1 DuoSet ELISA, R&D Systems) at 1/5 or 1/10 dilution in IX Reagent Diluent (Reagent Ancillary Kit 2, R&D

Systems). Data were normalized to the spécifie per well cell density determined by nuclei count stained by Hoechst 3342 (Life technologies) in the fluorescence microscopy described below.

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Intracellular lipid accumulation analysis after 72h by fluorescence microscopy

After 72 hours, cells were washed twice in lOOul PBS Ix (Gibco), fixed with 4% Paraformaldéhyde, and washed twice with PBS Ix (100 ul). After fixation, lipids were stained 5 with HCS LipidTOX Red Neutral (Thermofîsher Scientific) diluted lOOOx and nuclei were stained with Hoechst 3342 (Life Technologies) diluted to 4ug/ml. The LipidTOX™ neutral lipid stain has an extremely high affinity for neutral lipid droplets that was detected by fluorescence microscopy using a high content imager (Molecular Devices).

Results

Lipid accumulation and steatosis phenotypes

Primary human hépatocytes from healthy donors were found to hâve low levels of lipid accumulation (FIG. 9A-9D). Treatment of the cells with free fatty acids (FF) + TNFa induced lipid accumulation (FIG. 9I-9L) with a macro-steatosis phenotype. Treatment with LIVRQNAC 15 changed the hépatocyte phenotypes from macro-steatosis to micro-steatosis (FIG. 9E-9H).

MCP1/CCL2 sécrétion

Tables 56-59 show the baseline subtracted sécrétion of MCP1/CCL2 in primary human hépatocytes cells from two healthy donors (donor 1 for Tables 56 and 57, and donor 2 for Tables 20 58 and 59). LIVRQNAC, LIVRQNAC+G, LIVRQNAC+S, LIVRQ and RQNAC signifïcantly decreased MCP1/CCL2 sécrétion in both donors. The combination LIV, however, signifïcantly increased MCP1/CCL2 sécrétion only in one of the donors. The addition of arginine (R) and glutamine (Q) to a combination of LIV decreased the sécrétion of MCP1/CCL2 in both donors compared to LIV alone. Individually, N-acetyl cysteine and glutamine are shown to signifïcantly 25 decrease MCP1/CCL2 sécrétion, while arginine increased MCP1 sécrétion. Isoleucine, Leucine and Valine did not hâve an effect on MCP1/CCL2 sécrétion.

Table 56. Changes in MCP1 expression for donor 1 upon administration of amino acid compositions

MCP1 expression relative to Control - Donor 1

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Amino Acid Supplément	Conc. (X)	Mean	Std. Déviation	Number of values	Pvalue*	Significance
LIVRQNAC	40	-24.1616	0.032252	3	0.0001	****
LIVRQNAC	30	-22.2916	2.119583	3	0.0001	♦♦♦*
LIVRQNAC	20	-18.4363	0.850597	3	0.0005	***
LIVRQNAC	10	-14.3383	1.854977	3	0.0074	**
LIVRQNAC	1	0	1.048045	3
LIVRQNAC+G	40	-22.0824	0.873105	3	0.0001	*♦
LIVRQNAC+G	30	-19.2605	1.611788	3	0.0003	***
LIVRQNAC+G	20	-17.5807	2.893835	3	0.0009	***
LIVRQNAC+G	10	-13.7521	3.068991	3	0.0106	*
LIVRQNAC+G	1	0	1.682719	3
LIVRQNAC+S	40	-32.4703	0.340537	3	0.0001	*♦
LIVRQNAC+S	30	-30.768	1.339048	3	0.0001	****
LIVRQNAC+S	20	-25.5964	1.854519	3	0.0001	♦ ♦♦*
LIVRQNAC+S	10	-17.8326	1.974033	3	0.0008	***
LIVRQNAC+S	1	2.37E-15	18.41384	3
LIV	40	15.52052	6.323205	3	0.0094	**
LIV	30	12.3111	10.02706	3	0.0475	*
LIV	20	12.6686	4.109608	3	0.0401	♦
LIV	10	-5.18869	1.579468	3	0.6477	ns
LIV	1	-1.2E-15	8.178943	3
LIVRQ	40	-25.9576	0.484283	3	0.0028	**
LIVRQ	30	-23.6562	2.599721	3	0.0099
LIVRQ	20	-13.4723	3.427666	3	0.6401	ns
LIVRQ	10	-9.22141	7.599407	3	0.9986	ns
LIVRQ	1	-8.23198	5.80889	3
RQNAC	40	-21.4681	2.903892	3	0.0003	***
RQNAC	30	-17.1873	5.202568	3	0.0038	**
RQNAC	20	-12.1782	2.907484	3	0.0506	ns
RQNAC	10	-8.89378	4.748653	3	0.206	ns
RQNAC	1	1.18E-15	10.02527	3
N-Acetyl Cysteine	40	-17.6065	1.211739	3	0.0009	***
N-Acetyl Cysteine	20	-10.8919	2.27818	3	0.0545	ns
N-Acetyl Cysteine	10	-2.49755	8.795693	3	0.9424	ns
N-Acetyl Cysteine	5	-0.76286	7.457085	3	0.9991	ns
N-Acetyl Cysteine	0	0	6.716428	3

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Table 57. Changes in MCP1 expression for donor 1 upon administration of single amino acid compositions

Amino Acid Supplément	Conc. (μΜ)	MCP1 expression relative to Control- Donor 1
Mean	Std. Déviation	Number of values	Pvalue*	Significance
Valine	23420	14.16805	19.23365	3	0.6777	ns
Valine	11710	77.73396	137.82	3	0.9998	ns
Valine	4684	23.6867	46.48697	3	0.2502	ns
Valine	234	-2.4E-15	13.86902	3
Arginine	5440	10.9386	4.79774	3	0.0057	**
Arginine	2720	6.526801	4.266971	3	0.1517	ns
Arginine	1088	5.114414	4.685563	3	0.3321	ns
Arginine	109	2.37E-15	0.666016	3
Glutamine	22484	-21.8392	1.113443	3	0.0004	***
Glutamine	11242	-9.00139	1.68951	3	0.2459	ns
Glutamine	3747	-0.89805	6.374471	3	0.9991	ns
Glutamine	749	0	9.549143	3
Isoleucine	6639	-0.205	2.292188	3	0.9998	ns
Isoleucine	3320	-2.41722	2.382379	3	0.4907	ns
Isoleucine	1328	-0.30729	2.409691	3	0.9992	ns
Isoleucine	66	-1.2E-15	3.163838	3
Leucine	15270	-1.36762	3.37035	3	0.8675	ns
Leucine	7635	1.895506	3.757642	3	0.6872	ns
Leucine	3054	3.340489	3.016641	3	0.2201	ns
Leucine	153	5.92E-16	3.132507	3
N-Acetyl Cysteine	10000	-17.6065	1.211739	3	0.0009
N-Acetyl Cysteine	5000	-10.8919	2.27818	3	0.0545	ns
N-Acetyl Cysteine	2500	-2.49755	8.795693	3	0.9424	ns
N-Acetyl Cysteine	1000	-0.76286	7.457085	3	0.9991	ns
N-Acetyl Cysteine	0	0	6.716428	3

Table 58. Changes in MCP1 expression for donor 2 upon administration of amino acid compositions

Amino Acid Supplément	Conc. (X)	MCP1 expression relative to Control - Donor 2
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	-24.5376	1.632923	3	0.0001	***♦
LIVRQNAC	30	-13.6824	2.562571	3	0.0001	****

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LIVRQNAC	20	-8.42053	1.545343	3	0.0001	****
LIVRQNAC	10	2.126223	0.453924	3	0.0007	***
LIVRQNAC	1	-4.7E-15	0.412226	3
LIVRQNAC+G	40	-35.3651	2.08381	3	0.0007	***
LIVRQNAC+G	30	-30.3247	5.225183	3	0.001	***
LIVRQNAC+G	20	-17.0719	4.522244	3	0.0119	*
LIVRQNAC+G	10	-14.2586	2.767898	3	0.049	*
LIVRQNAC+G	1	-7.1E-15	7.613666	3
LIVRQNAC+S	40	-35.8381	1.404782	3	0.0001	****
LIVRQNAC+S	30	-30.9946	2.372062	3	0.0001	****
LIVRQNAC+S	20	-16.8831	3.223007	3	0.0004	***
LIVRQNAC+S	10	-5.60595	10.2119	3	0.1887
LIVRQNAC+S	1	2.37E-15	4.4168	3
LIV	40	-46.7898	8.664441	3	0.3692	ns
LIV	30	-34.5953	16.84743	3	0.6246	ns
LIV	20	-28.0851	31.84348	3	0.7684	ns
LIV	10	-11.0006	72.74556	3	0.9889	ns
LIV	1	9.47E-15	60.93638	3
LIVRQ	40	-129.802	7.067989	3	0.0008	***
LIVRQ	30	-110.034	4.53852	3	0.0042	**
LIVRQ	20	-33.3611	31.87706	3	0.6524
LIVRQ	10	-3.30904	71.03267	3	0.9999
LIVRQ	1	-4.7E-15	46.12987	3
RQNAC	40	-133.48	1.908424	3	0.0006	***
RQNAC	30	-123.712	1.043889	3	0.0013	**
RQNAC	20	-109.575	5.533323	3	0.0044	*♦
RQNAC	10	-55.8583	22.72309	3	0.2273
RQNAC	1	1.42E-14	43.79031	3
N-Acetyl Cysteine	10000	-28.4419	1.694	3	0.0001	***
N-Acetyl Cysteine	5000	-10.5725	4.362178	3	0.0012	**
N-Acetyl Cysteine	2500	-4.0591	5.600773	3	0.0572	ns
N-Acetyl Cysteine	1000	1.602474	3.423109	3	0.0001	♦ ***
N-Acetyl Cysteine	0	0	2.068861	3

Table 59. Changes in MCP1 expression for donor 2 upon administration of single amino acid compositions__________________________________________________________________

MCP1 expression relative to Control- Donor 2

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Amino Acid Supplément	Conc. (μΜ)	Mean	Std. Déviation	Number of values	P-value*	Signifîcance
Valine	23420	-30.7921	22.55378	3	0.6118	ns
Valine	11710	38.24762	28.44112	3	0.4268	ns
Valine	4684	10.79011	51.87642	3	0.9835	ns
Valine	234	-1.4E-14	30.91388	3
Arginine	5440	8.493664	22.98385	3	0.9913	ns
Arginine	2720	24.06261	63.49489	3	0.7429	ns
Arginine	1088	24.95224	52.94171	3	0.7192	ns
Arginine	109	-4.7E-15	11.27976	3
Glutamine	22484	-138.873	10.74317	3	0.0001	****
Glutamine	11242	-90.6558	15.43989	3	0.0037	**
Glutamine	3747	-45.0574	41.63249	3	0.2474	ns
Glutamine	749	2.84E-14	59.86955	3	0.7631
Isoleucine	6639	18.62132	26.01824	3	0.5663	ns
Isoleucine	3320	-5.64461	7.719105	3	0.9882	ns
Isoleucine	1328	26.62309	5.65413	3	0.2613	ns
Isoleucine	66	0	4.245462	3
Leucine	15270	-26.6436	10.08177	3	0.2607	ns
Leucine	7635	-2.98815	21.00205	3	0.9989	ns
Leucine	3054	16.11014	8.662188	3	0.68	ns
Leucine	153	-4.7E-15	7.63396	3
N-Acetyl Cysteine	10000	-28.4419	1.694	3	0.0001	***
N-Acetyl Cysteine	5000	-10.5725	4.362178	3	0.0012	**
N-Acetyl Cysteine	2500	-4.0591	5.600773	3	0.0572	ns
N-Acetyl Cysteine	1000	1.602474	3.423109	3	0.0001	****
N-Acetyl Cysteine	0	0	2.068861	3

Example 11: Hepatic Stellate Cell - TNFa Inflammatory Response

Methods

Primary human hepatic stellate cells were obtained from Samsara Sciences based on the following criteria for selecting donors: adult âge (between 18 and 50 years), normal BMI (>18.5 and <25), and absence of confounding liver disease. Primary human hepatic stellate cells grown in Complété HSC Medium to ~80% confluence in T75 or Tl50 flasks below passage 10 were

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I I seeded into stérile, collagen I coated, 96-well optical plastic microplates (ThermoScientific, 152036) at 4000 cells per well (~1250 cells per cm²) and incubated for 6 hours at 37°C, 5% CO2 in a humidified incubator.

After 6 hours, plates were removed from the incubator and the medium gently pipetted off and 5 washed once with 150 pL per well DPBS. The DPBS was removed and the pretreatment medium (± single amino acid dropout, 1XHMDB DMEM + 3% dialyzed FBS + 0.2% Primocin, ± supplémentai amino acid dose; see experiment for medium composition) was applied to the cells at 150 pL per well. Plates were retumed to the incubator overnight, ~14-15 hours.

After overnight pretreatment, the medium was removed from the cells, and the same 10 pretreatment medium, now supplemented with 3 ng/mL TNFa is applied. Each plate contained 3 ng/mL TNFa in IX human plasma amino acid (HMDB or P AA) concentration medium, 0 ng/mL in 1XHMDB, and 3 ng/mL TNFa + 50 nM Bengamide in 1XHMDB to serve as Controls. Plates were incubated for 12 hours at 37°C, 5% CO2.

After 12 hour stimulus with TNFa, supematant was removed and frozen at -80°C in two 15 separate aliquots. Plates were washed gently once with DPBS and 100 pL per well of 1XHMDB DMEM + 3% dialyzed FBS + 0.2% Primocin +10% CCK-8 viability reagent (Dojindo). Plates were incubated for 1 hour at 37°C, 5% CO2.

After 1 hour of incubation, viability was measured on the Synergy plate reader (Absorbance at 977 (test), 900 (reference), and 450 (CCK8) nm). Immediately, the medium was 20 removed and the plates were fixed with 70 pL per well 4% paraformaldéhyde in PBS at room température for 20 minutes, followed by two 150 pL PBS washes, and stored with 100 pL per well PBS at 4°C until immunofluorescence staining.

Human CCL2/MCP1 and Human IL-6 were measured by ELISA (Human CCK2/MCP-1 DuoSet ELISA, R&D Systems; Human IL-6 DuoSet ELISA, R&D Systems) at 1/5 and 1/20 dilution in IX Reagent Diluent (Reagent Ancillaiy Kit 2, R&D Systems). Data were normalized to the spécifie per well cell density determined by Hoechst stained nuclei count.

Results

Pro-inflammatory MCP-1 Chemokine Sécrétion

Tables 60-63 show per-cell normalized MCP-1 chemokine sécrétion in primary human hepatic stellate cells from two donors as a fold change from the plasma amino acid background.

208 .

Statistical signifîcance calculated by one-way ANOVA with Dunnett’s multiple comparison test within each treatment group. LIVRQNAC+G and RQNAC singifïcantly decrease MCP-1 sécrétion in both donors. LIVRQNAC, LIVRQNAC+S reduced MCP 1 sécrétion and was statistically significant in one of two donors. Individually, each of valine, arginine, and leucine had no significant impact on MCP-1 sécrétion. Glutamine reduced MCP1 sécrétion in both donors but was only statistically significant in one of two donors. N-acetyl cysteine significantly reduced MCP-1 sécrétion in both donors.

Table 60. Changes in MCP1 sécrétion for donor 3 upon administration of amino acid compositions

Amino Acid Supplément	Conc. (X)	Fold Change MCP1 Sécrétion Normalized Per Cell
Mean	Std. Déviation	Number of values	Signifîcance	P-value
LIVRQNAC	40	0.6237	0.2500	3	ns	0.2763
LIVRQNAC	30	0.6180	0.2436	3	ns	0.2657
LIVRQNAC	20	0.5679	0.1728	3	ns	0.1863
LIVRQNAC	10	0.5548	0.2139	3	ns	0.1694
LIVRQNAC	1	1.0000	0.3619	3
LIVRQNAC+G	40	0.6216	0.0903	3	*♦	0.0036
LIVRQNAC+G	30	0.6742	0.0549	3	♦ *	0.0095
LIVRQNAC+G	20	0.6373	0.0888	3	**	0.0047
LIVRQNAC+G	10	0.7075	0.0610	3	*	0.0179
LIVRQNAC+G	1	1.0000	0.1704	3
LIVRQNAC+S	40	0.5911	0.1451	3	ns	0.2045
LIVRQNAC+S	30	0.5932	0.1943	3	ns	0.2077
LIVRQNAC+S	20	0.5760	0.1681	3	ns	0.1828
LIVRQNAC+S	10	0.6820	0.2396	3	ns	0.3845
LIVRQNAC+S	1	1.0000	0.4098	3
LIV	40	1.2677	0.5786	3	ns	0.7802
LIV	30	1.3632	0.5837	3	ns	0.8368
LIV	20	1.3336	0.4754	3	ns	0.7964
LIV	10	1.3745	0.5427	3	ns	0.9132
LIV	1	1.0000	0.3186	3
LIVRQ	40	1.3042	0.4140	3	ns	0.7695
LIVRQ	30	1.2208	0.4403	3	ns	0.9036
LIVRQ	20	0.9915	0.3521	3	ns	0.9999
LIVRQ	10	0.9968	0.3907	3	ns	0.9999

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LIVRQ	1	1.0000	0.4257	3
RQNAC	40	0.3220	0.0282	3	***♦	0.0001
RQNAC	30	0.4353	0.0941	3	****	0.0001
RQNAC	20	0.4629	0.0998	3	♦**	0.0001
RQNAC	10	0.6513	0.0925	3	**	0.0028
RQNAC	1	1.0000	0.1132	3
N-Acetyl Cysteine	40	0.4485	0.0587	3	***	0.0002
N-Acetyl Cysteine	20	0.5413	0.1018	3	♦	0.0009
N-Acetyl Cysteine	10	0.6565	0.0502	3	**	0.007
N-Acetyl Cysteine	5	0.8492	0.1515	3	ns	0.2738
N-Acetyl Cysteine	0	1.0000	0.1142	3

Table 61. Changes in MCP1 sécrétion for donor 3 upon administration of single amino acid compositions

Amino Acid Supplément	Conc. (μΜ)	Fold Change MCP1 Sécrétion Normalized Per Cell
Mean	Std. Déviation	Number of values	Significance	P-value
Valine	23420	1.2651	0.1295	3	ns	0.1126
Valine	11710	1.0204	0.1126	3	ns	0.9956
Valine	4684	1.0630	0.0878	3	ns	0.8999
Valine	234	1.0000	0.2008	3
Arginine	5440	0.7840	0.2753	3	ns	0.7069
Arginine	2720	0.8821	0.2249	3	ns	0.9264
Arginine	1088	0.9435	0.3221	3	ns	0.9903
Arginine	109	1.0000	0.3404	3
Glutamine	22484	0.6212	0.1952	3	ns	0.2465
Glutamine	11242	0.6106	0.2085	3	ns	0.226
Glutamine	3747	0.6036	0.2596	3	ns	0.2135
Glutamine	749	0.7048	0.2473	3	ns	0.4593
Glutamine	562	1.0000	0.2185	3
Isoleucine	6639	1.2084	0.1334	3	ns	0.284
Isoleucine	3320	1.2169	0.0589	3	ns	0.2565
Isoleucine	1328	1.5550	0.2070	3	♦*	0.0038
Isoleucine	66	1.0000	0.1188	3
Leucine	15270	1.1808	0.2601	3	ns	0.5156
Leucine	7635	1.3054	0.1748	3	ns	0.1491
Leucine	3054	1.1479	0.0605	3	ns	0.6605
Leucine	153	1.0000	0.0784	3
N-Acetyl Cysteine	10000	0.4485	0.0587	3		0.0002
N-Acetyl Cysteine	5000	0.5413	0.1018	3	***	0.0009
N-Acetyl Cysteine	2500	0.6565	0.0502	3	♦*	0.007

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N-Acetyl Cysteine	1000	0.8492	0.1515	3	ns	0.2738
N-Acetyl Cysteine	0	1.0000	0.1142	3

Table 62. Changes in MCP1 sécrétion for donor 4 upon administration of amino acid compositions

Amino Acid Supplément	Conc. (X)	Fold Change MCP1 Sécrétion Normalized Per Cell
Mean	Std. Déviation	Number of values	Significance	P-value
LIVRQNAC	40	0.7791	0.0740	3	ns	0.1328
LIVRQNAC	30	0.6333	0.1114	3	*	0.0116
LIVRQNAC	20	0.6997	0.1013	3	*	0.0352
LIVRQNAC	10	0.8114	0.1271	3	ns	0.2216
LIVRQNAC	1	1.0000	0.1607	3
LIVRQNAC+G	40	0.6738	0.0979	3	♦	0.0454
LIVRQNAC+G	30	0.7117	0.0783	3	ns	0.0794
LIVRQNAC+G	20	0.6735	0.1127	3	*	0.0452
LIVRQNAC+G	10	0.7682	0.0563	3	ns	0.1778
LIVRQNAC+G	1	1.0000	0.2452	3
LIVRQNAC+S	40	0.5780	0.0781	3	♦*	0.0025
LIVRQNAC+S	30	0.5393	0.1185	3	*♦	0.0013
LIVRQNAC+S	20	0.6487	0.0732	3	**	0.0085
LIVRQNAC+S	10	0.6872	0.0118	3	*	0.017
LIVRQNAC+S	1	1.0000	0.1803	3
LIV	40	0.7010	0.1399	3	**	0.0059
LIV	30	0.8883	0.0530	3	ns	0.3745
LIV	20	0.9284	0.0579	3	ns	0.7114
LIV	10	0.8663	0.0569	3	ns	0.2428
LIV	1	1.0000	0.0928	3
LIVRQ	40	1.2235	0.0592	3	ns	0.4365
LIVRQ	30	1.1653	0.0558	3	ns	0.6679
LIVRQ	20	0.8845	0.2698	3	ns	0.862
LIVRQ	10	1.0110	0.0738	3	ns	0.9999
LIVRQ	1	1.0000	0.3016	3
RQNAC	40	0.4312	0.0994	3	**♦	0.0006
RQNAC	30	0.3910	0.0649	3	***	0.0003
RQNAC	20	0.5579	0.2079	3	**	0.0037
RQNAC	10	0.5545	0.0663	3	**	0.0035
RQNAC	1	1.0000	0.0987	3
N-Acetyl Cysteine	40	0.5011	0.0756	3	**♦	0.0001
N-Acetyl Cysteine	20	0.6728	0.1024	3	**	0.003

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N-Acetyl Cysteine	10	0.8033	0.1101	3	ns	0.058
N-Acetyl Cysteine	5	0.6437	0.0648	3	♦*	0.0017
N-Acetyl Cysteine	0	1.0000	0.0673	3

Table 63. Changes in MCP1 sécrétion for donor 4 upon administration of single amino acid compositions

Amino Acid Supplément	Conc. (μΜ)	FoldC	lange MCP1 Sécrétion Normalized Per Cell
Mean	Std. Déviation	Number of values	Signifïcance	P-value
Valine	23420	1.1525	0.0406	3	ns	0.9999
Valine	11710	1.1544	0.1743	3	ns	0.8877
Valine	4684	1.0942	0.0846	3	ns	0.3545
Valine	234	1.0000	0.1464	3
Arginine	5440	0.9456	0.0639	3	ns	0.9076
Arginine	2720	1.0446	0.0741	3	ns	0.9449
Arginine	1088	1.0453	0.1733	3	ns	0.9423
Arginine	109	1.0000	0.1486	3
Glutamine	22484	0.7039	0.0544	3	*♦	0.0065
Glutamine	11242	0.7129	0.2237	3	♦ ♦	0.0077
Glutamine	3747	0.6639	0.0467	3	♦ ♦	0.0027
Glutamine	749	0.7782	0.0860	3	*	0.0452
Glutamine	562	1.0000	0.0709	6
Isoleucine	6639	0.9103	0.0536	3	ns	0.5597
Isoleucine	3320	0.8830	0.0872	3	ns	0.3538
Isoleucine	1328	1.3338	0.1099	3	♦ ♦	0.0044
Isoleucine	66	1.0000	0.0853	3
Leucine	15270	1.5745	0.0844	3	ns	0.1886
Leucine	7635	1.7129	0.6026	3	ns	0.0885
Leucine	3054	1.5342	0.1746	3	ns	0.2332
Leucine	153	1.0000	0.2040	3
N-Acetyl Cysteine	10000	0.5011	0.0756	3	**♦	0.0001
N-Acetyl Cysteine	5000	0.6728	0.1024	3	**	0.003
N-Acetyl Cysteine	2500	0.8033	0.1101	3	ns	0.058
N-Acetyl Cysteine	1000	0.6437	0.0648	3	**	0.0017
N-Acetyl Cysteine	0	1.0000	0.0673	3

IL-6 Cytokine Sécrétion

Tables 64-67 show per-cell normalized IL-6 cytokine sécrétion in primary human hepatic stellate cells from two donors as a fold change from the plasma amino acid background.

Statistical signifïcance calculated by one-way ANOVA with Dunnett’s multiple comparison test

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I I within each treatment group. LIVRQNAC, LIVRQNAC+S and RQNAC significantly reduced IL-6 sécrétion in one of two donors. LTVRQNAC+G, LIVRQNAC+S and RQNAC decreased IL-6 sécrétion in both donors. LIV and LIVRQ did not hâve a signifîcant impact on IL-6 sécrétion in either donor. Individually, valine, arginine, isoleucine, and leucine had no signifîcant effect on IL-6 sécrétion. N-acetyl cysteine reduced IL-6 sécrétion in both donors but was only statistically signifîcant in one of two donors. Glutamine significantly reduced IL-6 sécrétion in both donors.

Table 64. Changes in IL-6 cytokine sécrétion for donor 1 upon administration of amino acid compositions

Amino Acid Supplément	Conc. (X)	Fold Change IL-6 Sécrétion Normalized Per Cell
Mean	Std. Déviation	Number of values	Significance	P-value
LIVRQNAC	40	0.4857	0.0915	3	·	0.0004
LIVRQNAC	30	0.5667	0.0941	3	**	0.0014
LIVRQNAC	20	0.6671	0.0431	3	*·	0.0088
LIVRQNAC	10	0.6579	0.1231	3	**	0.0074
LIVRQNAC	1	1.0000	0.1361	3
LIVRQNAC+G	40	0.4995	0.1427	3	ns	0.0949
LIVRQNAC+G	30	0.5722	0.2185	3	ns	0.1679
LIVRQNAC+G	20	0.6185	0.1769	3	ns	0.2376
LIVRQNAC+G	10	0.7040	0.2809	3	ns	0.4276
LIVRQNAC+G	1	1.0000	0.3513	3
LIVRQNAC+S	40	0.5397	0.1569	3	*	0.0105
LIVRQNAC+S	30	0.5513	0.1190	3	•	0.0122
LIVRQNAC+S	20	0.6264	0.1593	3	«	0.0338
LIVRQNAC+S	10	0.6799	0.1218	3	ns	0.0703
LIVRQNAC+S	1	1.0000	0.1671	3
LIV	40	1.3536	0.4767	3	ns	0.6216
LIV	30	1.2423	0.3135	3	ns	0.8437
LIV	20	1.2321	0.4818	3	ns	0.8611
LIV	10	1.1421	0.3489	3	ns	0.9704
LIV	1	1.0000	0.1647	3
LIVRQ	40	0.8274	0.2003	3	ns	0.7863
LIVRQ	30	0.8880	0.2175	3	ns	0.938
LIVRQ	20	0.8468	0.1100	3	ns	0.8431
LIVRQ	10	0.9247	0.2696	3	ns	0.984
LIVRQ	1	1.0000	0.3311	3
RQNAC	40	0.3958	0.0947	3	•	0.0109

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RQNAC	30	0.4433	0.1317	3	*	0.0177
RQNAC	20	0.4936	0.1079	3	*	0.0297
RQNAC	10	0.5729	0.1741	3	ns	0.0674
RQNAC	1	1.0000	0.3440	3
N-Acetyl Cysteine	40	0.5716	0.2306	3	ns	0.2067
N-Acetyl Cysteine	20	0.6121	0.1718	3	ns	0.2729
N-Acetyl Cysteine	10	0.7354	0.2816	3	ns	0.5703
N-Acetyl Cysteine	5	0.7141	0.2509	3	ns	0.5098
N-Acetyl Cysteine	0	1.0000	0.3472	3

Table 65. Changes in IL-6 cytokine sécrétion for donor 1 upon administration of single amino acid compositions

Amino Acid Supplément	Conc. (μΜ)	Fold Change IL-6 Sécrétion Normalized Per Cell
Mean	Std. Déviation	Number of values	Significance	P-value
Valine	23420	1.0404	0.2175	3	ns	0.9949
Valine	11710	0.9562	0.3332	3	ns	0.9935
Valine	4684	0.9790	0.1777	3	ns	0.9993
Valine	234	1.0000	0.2868	3
Arginine	5440	0.7776	0.1994	3	ns	0.6927
Arginine	2720	1.0231	0.4381	3	ns	0.9993
Arginine	1088	0.9828	0.2957	3	ns	0.9997
Arginine	109	1.0000	0.1728	3
Glutamine	22484	0.5138	0.0818	3	·*	0.0046
Glutamine	11242	0.5136	0.1189	3	**	0.0046
Glutamine	3747	0.5460	0.0891	3	··	0.0072
Glutamine	749	0.6320	0.1181	3	*	0.0249
Glutamine	562	1.0000	0.2226	3
Isoleucine	6639	1.0859	0.1489	3	ns	0.764
Isoleucine	3320	1.1156	0.0776	3	ns	0.5903
Isoleucine	1328	1.0233	0.1536	3	ns	0.9922
Isoleucine	66	1.0000	0.1276	3
Leucine	15270	1.0767	0.0246	3	ns	0.853
Leucine	7635	1.1215	0.0872	3	ns	0.6249
Leucine	3054	1.1762	0.2273	3	ns	0.3655
Leucine	153	1.0000	0.1535	3
N-Acetyl Cysteine	10000	0.5716	0.2306	3	ns	0.2067
N-Acetyl Cysteine	5000	0.6121	0.1718	3	ns	0.2729
N-Acetyl Cysteine	2500	0.7354	0.2816	3	ns	0.5703
N-Acetyl Cysteine	1000	0.7141	0.2509	3	ns	0.5098
N-Acetyl Cysteine	0	1.0000	0.3472	3

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Table 66. Changes in IL-6 cytokine sécrétion for donor 2 upon administration of amino acid compositions

Amino Acid Supplément	Conc. (X)	Fold Change IL-6 Sécrétion Normalized Per Cell
Mean	Std. Déviation	Number of values	Significance	P-value
LIVRQNAC	40	0.9911	0.1150	3	ns	0.9998
LIVRQNAC	30	0.9560	0.0473	3	ns	0.9404
LIVRQNAC	20	1.0008	0.1450	3	ns	0.9999
LIVRQNAC	10	1.0845	0.0707	3	ns	0.6567
LIVRQNAC	1	1.0000	0.0553	3
LIVRQNAC+G	40	0.8055	0.1705	3	ns	0.4153
LIVRQNAC+G	30	0.8218	0.1567	3	ns	0.4855
LIVRQNAC+G	20	0.9236	0.1642	3	ns	0.9342
LIVRQNAC+G	10	1.1076	0.2097	3	ns	0.8216
LIVRQNAC+G	1	1.0000	0.0416	3
LIVRQNAC+S	40	0.9508	0.0933	3	ns	0.967
LIVRQNAC+S	30	0.8581	0.0364	3	ns	0.4836
LIVRQNAC+S	20	0.8289	0.0765	3	ns	0.3356
LIVRQNAC+S	10	0.8487	0.1018	3	ns	0.432
LIVRQNAC+S	1	1.0000	0.2312	3
LIV	40	0.9122	0.0773	3	ns	0.8233
LIV	30	1.0994	0.0987	3	ns	0.7586
LIV	20	1.0400	0.2330	3	ns	0.9857
LIV	10	0.9579	0.1077	3	ns	0.9828
LIV	1	1.0000	0.0540	3
LIVRQ	40	0.9327	0.0639	3	ns	0.8313
LIVRQ	30	0.8421	0.1125	3	ns	0.2361
LIVRQ	20	0.7871	0.0932	3	ns	0.0841
LIVRQ	10	0.8693	0.0750	3	ns	0.3744
LIVRQ	1	1.0000	0.1428	3
RQNAC	40	0.8711	0.0816	3	ns	0.5267
RQNAC	30	0.7460	0.1133	3	ns	0.0843
RQNAC	20	0.7838	0.0708	3	ns	0.1544
RQNAC	10	0.8781	0.1566	3	ns	0.5705
RQNAC	1	1.0000	0.1557	3
N-Acetyl Cysteine	40	0.7064	0.0418	3	ns	0.0508
N-Acetyl Cysteine	20	0.8111	0.1049	3	ns	0.2549
N-Acetyl Cysteine	10	0.9180	0.2230	3	ns	0.8353
N-Acetyl Cysteine	5	0.9161	0.1067	3	ns	0.8252
N-Acetyl Cysteine	0	1.0000	0.0632	3

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Table 67. Changes in IL-6 cytokine sécrétion for donor 2 upon administration of single amino acid compositions

Amino Acid Supplément	Conc. (μΜ)	Fold Change IL-6 Sécrétion Normalized Per Cell
Mean	Std. Déviation	Number of values	Significance	P-value
Valine	23420	0.9015	0.0930	3	ns	0.4967
Valine	11710	0.9218	0.1179	3	ns	0.6516
Valine	4684	1.0383	0.1014	3	ns	0.9291
Valine	234	1.0000	0.0696	3
Arginine	5440	0.8895	0.0897	3	ns	0.547
Arginine	2720	0.9401	0.1611	3	ns	0.8654
Arginine	1088	0.9924	0.0692	3	ns	0.9996
Arginine	109	1.0000	0.1263	3
Glutamine	22484	0.5993	0.0611	3	****	0.0001
Glutamine	11242	0.6478	0.0371	3		0.0001
Glutamine	3747	0.7100	0.0356	3	***	0.0003
Glutamine	749	0.7673	0.0222	3		0.0017
Glutamine	562	1.0000	0.1027	6
Isoleucine	6639	1.1648	0.1125	3	ns	0.1448
Isoleucine	3320	0.9096	0.0916	3	ns	0.5304
Isoleucine	1328	1.1020	0.0987	3	ns	0.4446
Isoleucine	66	1.0000	0.0641	3
Leucine	15270	1.0183	0.1155	3	ns	0.9795
Leucine	7635	0.9574	0.0590	3	ns	0.8187
Leucine	3054	1.0011	0.0618	3	ns	0.9999
Leucine	153	1.0000	0.0277	3
N-Acetyl Cysteine	10000	0.7064	0.0418	3	ns	0.0508
N-Acetyl Cysteine	5000	0.8111	0.1049	3	ns	0.2549
N-Acetyl Cysteine	2500	0.9180	0.2230	3	ns	0.8353
N-Acetyl Cysteine	1000	0.9161	0.1067	3	ns	0.8252
N-Acetyl Cysteine	0	1.0000	0.0632	3

Example 12: TGFBl Fibrogenic Gene Expression of Hepatic Stellate Cell

Primary human hepatic stellate cells were obtained from Samsara Sciences based on the following criteria for selecting donors: adult âge (between 18 and 50 years), normal BMI (>18.5 and <25), and absence of confounding liver disease. Cells grown in Complété HSC Medium to ~80% confluence in T75 or T150 flasks below passage 10 were seeded into stérile, collagen I coated, 96-well optical plastic microplates (ThermoScientific, 152036) at 6000 cells per well

216 (~1250 cells per cm2) and incubated overnight at 37°C, 5% CO2 in a humidifïed incubator in DMEM with 2% Fêtai Bovine Sérum and 1% Antibiotic-Antimycotic.

After the overnight incubation, plates were removed from the incubator and the medium was gently pipetted off and washed twice with 150 pL per well DPBS. The DPBS was removed and the pretreatment medium (± single amino acid dropout, 1XHMDB DMEM + 1% AntibioticAntimycotic, 10 mM HEPES, ± supplémentai amino acid dose; see experiment for medium composition) was applied to the cells at 150 pL per well. Plates were returned to the incubator for 10.5 hours.

After 10.5 hour pretreatment, the medium was removed from the cells, and the same pretreatment medium, now supplemented with 3 ng/mL TGFpi, was applied. Each plate contained 3 ng/mL TGFpi in IX human plasma amino acid (HMDB or PAA) concentration medium, 0 ng/mL in 1XHMDB, and 3 ng/mL TGFpl + 20 pM Silybin in 1XHMDB to serve as Controls. Plates were then incubated for 24 hours at 37°C, 5% CO2.

After 24 hour stimulus, supematant was removed and frozen at -80°C in two separate aliquots. The cells were then washed with 125 pL per well Buffer FCW (FastLane Cell Multiplex NR Kit, Qiagen, 216713). The wash buffer was immediateiy removed and 50 pL of Cell Processing Mix (containing genomic DNA Wipeout buffer) was applied to lyse cells, incubating for 10 minutes at room température. RNA lysate was then transferred to 96-well qPCR plates, sealed, and gDNA was digested on thermal cycler at 75°C for 5 minutes. RNA lysate was frozen at -80°C.

Each 20 pL one-step RT-qPCR reaction contained 4 pL of RNA lysate. Gene expression of Collai, Timp2, and Gapdh were multiplexed using the HEX, Cy5, and FAM fluorescent channels, respectively, with commercially available primer-probe mixes (the Human Collai Primer-Probe Set, HEX; the Human Timp2 Primer-Probe Set, Cy5; and the Human Gapdh Primer-Probe Set, FAM from IDT). Gene expression was evaluated using the AACq method within each single amino acid dropout and supplémentation by normalizing to its own IX HMDB concentration.

Human Procollagen lal was measured from the supematant by ELISA (Human ProCollagen I alpha 1 DuoSet ELISA, R&D Systems) at 1/100 dilution in IX Reagent Diluent (Reagent Ancillary Kit 2, R&D Systems).

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Collai Gene Expression

Tables 68, 69, 69-1, 69-2, 69-3, and 69-4 show the mean fold change in Collai gene expression in primary human hepatic stellate cells from three different healthy donors. LIVRQNAC and LIVRQNAC+S showed significantly decreased Collai gene expression in two of three donors. LIVRQNAC+G and RQNAC showed significantly decreased Collai expression in ail three donors. LIVRQ showed a significant change in Collai gene expression in only one donor. LIV alone did not significantly change Collai gene expression.

Each of leucine, isoleucine, valine, and arginine did not significantly change Collai gene expression in any donor when the amino acid was administered alone. Glutamine decreased Collai gene expression in two of three donors. N-acetyl cysteine significantly reduced Collai gene expression in ail three donors.

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Table 68. Fold change of Collai gene expression after administration of an amino acid composition, normalized to Gapdh expression in a first donor

Amino Acid Supplément	Conc. (X)	Collai Fold Expression Relative to Control
Mean	Std. Déviation	Number of values	P-value*	Signifîcance
LIVRQNAC	40	0.91	0.08	4	ns	0.401
LIVRQNAC	30	0.87	0.10	4	ns	0.1073
LIVRQNAC	20	0.88	0.04	4	ns	0.1483
LIVRQNAC	10	0.90	0.08	4	ns	0.3035
LIVRQNAC	1	1.00	0.10	4
LIVRQNAC+G	40	0.73	0.15	4	♦♦	0.0053
LIVRQNAC+G	30	0.79	0.08	4	*	0.0252
LIVRQNAC+G	20	0.84	0.08	4	ns	0.1181
LIVRQNAC+G	10	0.79	0.11	4	*	0.0286
LIVRQNAC+G	1	1.00	0.03	4
LIVRQNAC+S	40	0.79	0.05	4	«	0.0325
LIVRQNAC+S	30	0.86	0.13	4	ns	0.1848
LIVRQNAC+S	20	0.96	0.10	4	ns	0.9287
LIVRQNAC+S	10	0.85	0.12	4	ns	0.1566
LIVRQNAC+S	1	1.00	0.10	4
LIV	40	0.93	0.03	4	ns	0.5561
LIV	30	1.04	0.07	4	ns	0.8872
LIV	20	1.04	0.09	4	ns	0.9069

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LIV	10	1.05	0.10	4	ns	0.8156
LIV	1	1.00	0.07	4
LIVRQ	40	0.75	0.03	4	***	0.001
LIVRQ	30	0.73	0.05	4		0.0004
LIVRQ	20	0.80	0.03	4	♦♦	0.0054
LIVRQ	10	0.84	0.08	4	• *	0.0208
LIVRQ	1	1.01	0.13	4
RQNAC	40	0.51	0.07	4	♦*	0.0001
RQNAC	30	0.49	0.02	4		0.0001
RQNAC	20	0.59	0.04	4	♦♦♦♦	0.0001
RQNAC	10	0.68	0.07	4	♦♦♦♦	0.0001
RQNAC	1	1.00	0.11	4
N-Acetyl Cysteine	40	0.76	0.06	4	♦♦	0.0011
N-Acetyl Cysteine	20	1.02	0.08	4	ns	0.9921
N-Acetyl Cysteine	10	1.07	0.08	4	ns	0.5517
N-Acetyl Cysteine	5	1.00	0.08	4	ns	0.9999
N-Acetyl Cysteine	0	1.00	0.06	4

Table 69. Fold change of Collai gene expression after administration of a single amino acid composition, normalized to Gapdh expression in the first donor

Amino Acid Supplément	Conc. (μΜ)	Collai Fold Expression Relative to Control
Mean	Std. Déviation	Number of values	P-value*	Signifïcance
Valine	23420	1.00	0.05	4	ns	0.9996
Valine	11710	1.09	0.17	4	ns	0.5528
Valine	4684	1.05	0.11	4	ns	0.8851
Valine	234	1.00	0.08	4
Arginine	5440	1.12	0.18	4	ns	0.2151
Arginine	2720	1.03	0.03	4	ns	0.9625
Arginine	1088	0.99	0.06	4	ns	0.9989
Arginine	109	1.00	0.03	4
Glutamine	22484	0.53	0.01	4		0.0001
Glutamine	11242	0.62	0.05	4	**««	0.0001
Glutamine	3747	0.70	0.03	3	****	0.0001
Glutamine	749	1.00	0.07	4	ns	0.9999
Glutamine	562	1.00	0.07	3
Isoleucine	6639	1.11	0.07	4	ns	0.7553
Isoleucine	3320	1.10	0.14	4	ns	0.7944
Isoleucine	1328	1.05	0.22	4	ns	0.9831
Isoleucine	66	1.01	0.21	4

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Leucine	15270	0.99	0.10	4	ns	0.994
Leucine	7635	1.12	0.16	4	ns	0.5049
Leucine	3054	1.11	0.15	4	ns	0.5499
Leucine	153	1.00	0.11	4
N-Acetyl Cysteine	10000	0.76	0.06	4	**	0.0011
N-Acetyl Cysteine	5000	1.02	0.08	4	ns	0.9921
N-Acetyl Cysteine	2500	1.07	0.08	4	ns	0.5517
N-Acetyl Cysteine	1000	1.00	0.08	4	ns	0.9999
N-Acetyl Cysteine	0	1.00	0.06	4

Table 69-1. Fold change of Collai gene expression after administration of an amino acid composition, normalized to Gapdh expression in second donor.

Amino Acid Supplément	Conc. (X)	Collai Fold Expression Relative to Control
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	0.72	0.05	4	****	0.0001
LIVRQNAC	30	0.72	0.02	4	♦ ♦♦♦	0.0001
LIVRQNAC	20	0.70	0.03	4	♦ ♦♦♦	0.0001
LIVRQNAC	10	0.71	0.08	4	****	0.0001
LIVRQNAC	1	1.00	0.02	4
LIVRQNAC+G	40	0.60	0.09	4	♦ »**	0.0001
LIVRQNAC+G	30	0.68	0.07	4	««*	0.0001
LIVRQNAC+G	20	0.71	0.09	4		0.0003
LIVRQNAC+G	10	0.69	0.06	4	♦	0.0002
LIVRQNAC+G	1	1.00	0.07	4
LIVRQNAC+S	40	0.66	0.02	4	*♦♦♦	0.0001
LIVRQNAC+S	30	0.69	0.06	4	♦ ***	0.0001
LIVRQNAC+S	20	0.76	0.05	4	***	0.0002
LIVRQNAC+S	10	0.77	0.04	4	♦ **	0.0003
LIVRQNAC+S	1	1.00	0.11	4
LIV	40	1.20	0.21	4	ns	0.1032
LIV	30	1.10	0.09	4	ns	0.6074
LIV	20	1.10	0.04	4	ns	0.6031
LIV	10	1.02	0.08	4	ns	0.9981
LIV	1	1.00	0.11	4
LIVRQ	40	1.23	0.13	4	ns	0.1945
LIVRQ	30	1.12	0.13	4	ns	0.7176
LIVRQ	20	1.08	0.24	4	ns	0.8874
LIVRQ	10	1.14	0.16	4	ns	0.5632
LIVRQ	1	1.00	0.11	4

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RQNAC	40	0.54	0.03	4	****	0.0001
RQNAC	30	0.55	0.06	4	*♦♦♦	0.0001
RQNAC	20	0.58	0.04	4	****	0.0001
RQNAC	10	0.73	0.04	4		0.0007
RQNAC	1	1.01	0.16	4
N-Acetyl Cysteine	40	0.57	0.06	4	♦ ♦♦♦	0.0001
N-Acetyl Cysteine	20	0.69	0.06	4		0.0001
N-Acetyl Cysteine	10	0.69	0.09	4	♦ ♦♦	0.0001
N-Acetyl Cysteine	5	0.69	0.05	4	***	0.0001
N-Acetyl Cysteine	0	1.00	0.10	4

Table 69-2. Fold change of Collai gene expression after administration of a single amino acid composition, normalized to Gapdh expression in second donor.

Amino Acid Supplément	Conc. (μΜ)	Collai Fold Expression Relative to Control
Mean	Std. Déviation	Number of values	P-value*	Signifies nce
Valine	23420	1.05	0.03	4	ns	0.9194
Valine	11710	0.98	0.11	4	ns	0.9827
Valine	4684	1.05	0.18	4	ns	0.8893
Valine	234	1.00	0.11	4
Arginine	5440	1.15	0.10	4	ns	0.2773
Arginine	2720	1.15	0.14	4	ns	0.2759
Arginine	1088	0.99	0.15	4	ns	0.9938
Arginine	109	1.00	0.12	4
Glutamine	22484	0.86	0.07	4	ns	0.1411
Glutamine	11242	0.91	0.09	4	ns	0.4365
Glutamine	3747	1.04	0.14	4	ns	0.9811
Glutamine	749	1.02	0.13	4	ns	0.9988
Glutamine	562	1.01	0.12	8
Isoleucine	6639	1.03	0.07	4	ns	0.8931
Isoleucine	3320	0.99	0.08	4	ns	0.9841
Isoleucine	1328	0.97	0.10	4	ns	0.9157
Isoleucine	66	1.00	0.02	4
Leucine	15270	1.13	0.14	4	ns	0.0811
Leucine	7635	1.05	0.05	4	ns	0.7277
Leucine	3054	1.06	0.03	4	ns	0.5342
Leucine	153	1.00	0.03	4
N-Acetyl Cysteine	10000	0.57	0.06	4	***♦	0.0001
N-Acetyl Cysteine	5000	0.69	0.06	4	****	0.0001
N-Acetyl Cysteine	2500	0.69	0.09	4	***	0.0001

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N-Acetyl Cysteine	1000	0.69	0.05	4		0.0001
N-Acetyl Cysteine	0	1.00	0.10	4

Table 69-3. Fold change of Collai gene expression after administration of an amino acid composition, normalized to Gapdh expression in third donor.

Amino Acid Supplément	Conc. (X)	Collai Fold Expression Relative to Control
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	0.81	0.09	4	««	0.008
LIVRQNAC	30	0.70	0.06	4	**♦	0.0001
LIVRQNAC	20	0.79	0.08	4	*♦	0.0035
LIVRQNAC	10	0.79	0.07	4	♦ ♦	0.0039
LIVRQNAC	1	1.00	0.06	4
LIVRQNAC+G	40	0.63	0.10	4		0.0002
LIVRQNAC+G	30	0.64	0.02	4	♦ ♦♦	0.0003
LIVRQNAC+G	20	0.75	0.14	4	♦ ♦	0.005
LIVRQNAC+G	10	0.71	0.11	4	♦ ♦	0.0017
LIVRQNAC+G	1	1.00	0.03	4
LIVRQNAC+S	40	0.79	0.11	4	«	0.0316
LIVRQNAC+S	30	0.79	0.04	4	*	0.0309
LIVRQNAC+S	20	0.77	0.09	4	*	0.0208
LIVRQNAC+S	10	0.85	0.09	4	ns	0.1434
LIVRQNAC+S	1	1.01	0.16	4
LIV	40	1.00	0.16	4	ns	0.9999
LIV	30	0.94	0.16	4	ns	0.8685
LIV	20	1.08	0.08	4	ns	0.6767
LIV	10	0.93	0.04	4	ns	0.7713
LIV	1	1.00	0.05	4
LIVRQ	40	1.00	0.05	4	ns	0.9999
LIVRQ	30	1.07	0.13	4	ns	0.8753
LIVRQ	20	1.10	0.13	4	ns	0.6983
LIVRQ	10	1.05	0.21	4	ns	0.9641
LIVRQ	1	1.00	0.07	4
RQNAC	40	0.64	0.05	4	·♦*	0.0003
RQNAC	30	0.70	0.13	4	♦♦	0.0018
RQNAC	20	0.66	0.05	4		0.0005
RQNAC	10	0.87	0.15	4	ns	0.2175
RQNAC	1	1.00	0.04	4
N-Acetyl Cysteine	40	0.62	0.01	4	♦	0.0005
N-Acetyl Cysteine	20	0.73	0.10	4	*♦	0.0083

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N-Acetyl Cysteine	10	0.82	0.09	4	ns	0.0909
N-Acetyl Cysteine	5	0.91	0.12	4	ns	0.4954
N-Acetyl Cysteine	0	1.01	0.16	4

Table 69-4. Fold change of Collai gene expression after administration of a single amino acid composition, normalized to Gapdh expression in second donor.

Amino Acid Supplément	Conc. (μΜ)	Collai Fold Expression Relative to Control
Mean	Std. Déviation	Number of values	P-value*	Significance
Valine	23420	1.13	0.12	4	ns	0.7199
Valine	11710	1.27	0.31	4	ns	0.1735
Valine	4684	1.22	0.16	4	ns	0.3247
Valine	234	1.01	0.13	4
Arginine	5440	1.02	0.09	4	ns	0.9702
Arginine	2720	0.99	0.09	4	ns	0.9973
Arginine	1088	0.95	0.02	4	ns	0.5384
Arginine	109	1.00	0.05	4
Glutamine	22484	0.81	0.11	4	«	0.0113
Glutamine	11242	0.81	0.11	4	**	0.0087
Glutamine	3747	1.00	0.03	4	ns	0.9999
Glutamine	749	0.96	0.07	4	ns	0.8697
Glutamine	562	1.00	0.10	8
Isoleucine	6639	1.03	0.04	4	ns	0.9974
Isoleucine	3320	0.94	0.13	4	ns	0.8329
Isoleucine	1328	0.94	0.17	4	ns	0.7947
Isoleucine	66	1.02	0.20	4
Leucine	15270	1.07	0.12	4	ns	0.9535
Leucine	7635	1.00	0.16	4	ns	0.998
Leucine	3054	1.08	0.23	4	ns	0.9185
Leucine	153	1.01	0.19	4
N-Acetyl Cysteine	10000	0.62	0.01	4	***	0.0005
N-Acetyl Cysteine	5000	0.73	0.10	4	♦*	0.0083
N-Acetyl Cysteine	2500	0.82	0.09	4	ns	0.0909
N-Acetyl Cysteine	1000	0.91	0.12	4	ns	0.4954
N-Acetyl Cysteine	0	1.01	0.16	4

Procollagen lal Sécrétion

223

Tables 70, 71, 71-1, 71-2,71-3, and 71-4 show the fold change in procollagen lal in primary human hepatic stellate cells from three different healthy donors normalized to their respective baseline amino acid conditions. Statistical significance calculated by one-way ANOVA with Dunnett’s multiple comparison test within each treatment group. The combination LIV signifïcantly increased procollagen lal sécrétion in ail three donors. The addition of arginine (R) and glutamine (Q) to a combination of LIV counteracted the profibrogenic effect of LIV alone. LIVRQNAC, LIVRQNAC+G, LIVRQNAC+S and RQNAC signifïcantly decreased procollagen lal sécrétion in ail three donors. Individually, N-acetyl cysteine was shown to signifïcantly decrease procollagen lal sécrétion in two of the three donors. Valine signifïcantly increased procollagen lal sécrétion in only one of two donors, while isoleucine and arginine signifïcantly increased procollagen lal sécrétion in two of three donors. In other words, glutamine administered individually did not hâve a significant impact on procollagen lal sécrétion. As such, the réduction of the profibrogenic effect of LIV with arginine and glutamine relative to that of LIV alone would not hâve been expected based on the effect of individual amino acid treatments.

Table 70. Fold change of procollagen lal sécrétion after administration of an amino acid composition in a first donor

Amino Acid Supplément	Conc. (X)	Procollagen lal Sécrétion (Fold Change of IX)
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	0.6283	0.0585	3	***	0.0001
LIVRQNAC	30	0.5975	0.0709	3		0.0001
LIVRQNAC	20	0.6504	0.0622	4	***	0.0001
LIVRQNAC	10	0.8287	0.0936	4	*	0.0277
LIVRQNAC	1	1.0000	0.0908	4
LIVRQNAC+G	40	0.5288	0.0402	3	***	0.0006
LIVRQNAC+G	30	0.6297	0.0200	3	♦*	0.0042
LIVRQNAC+G	20	0.5926	0.0634	4	**	0.001
LIVRQNAC+G	10	0.7404	0.0920	4	*	0.0267
LIVRQNAC+G	1	1.0000	0.2151	4
LIVRQNAC+S	40	0.5900	0.0450	3	***	0.0003
LIVRQNAC+S	30	0.5562	0.1242	3	***	0.0002
LIVRQNAC+S	20	0.6844	0.0638	3	**	0.0022
LIVRQNAC+S	10	0.7003	0.0946	3	**	0.0032

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LIVRQNAC+S	1	1.0000	0.0311	3
LIV	40	1.3017	0.1474	3	ns	0.0518
LIV	30	1.3358	0.1922	3	*	0.0305
LIV	20	1.2592	0.0747	3	ns	0.0997
LIV	10	1.0149	0.1089	3	ns	0.9997
LIV	1	1.0000	0.0828	3
LIVRQ	40	1.0070	0.1716	3	ns	0.9999
LIVRQ	30	1.0190	0.1103	3	ns	0.9983
LIVRQ	20	1.1403	0.0516	3	ns	0.3875
LIVRQ	10	1.0454	0.0908	3	ns	0.9609
LIVRQ	1	1.0000	0.0935	3
RQNAC	40	0.3622	0.0166	3	****	0.0001
RQNAC	30	0.4232	0.0819	3	****	0.0001
RQNAC	20	0.5819	0.0574	3	***	0.0001
RQNAC	10	0.8181	0.0703	3	*	0.0313
RQNAC	1	1.0000	0.0967	3
N-Acetyl Cysteine	40	0.5076	0.0154	3	··	0.0001
N-Acetyl Cysteine	20	0.6593	0.0914	3	***	0.0003
N-Acetyl Cysteine	10	0.7939	0.0715	3	**	0.01
N-Acetyl Cysteine	5	0.9175	0.0519	3	ns	0.3855
N-Acetyl Cysteine	0	1.0000	0.0686	3

Table 71. Fold change of procollagen lal sécrétion after administration of a single amino acid composition in the first donor

Amino Acid Supplément	Conc. (μΜ)	Procollagen lal Sécrétion (Fold Change of IX)
Mean	Std. Déviation	Number of values	P-value*	Significance
Valine	23420	1.2139	0.0544	3	ns	0.1392
Valine	11710	1.2069	0.0881	3	ns	0.155
Valine	4684	1.1203	0.1908	3	ns	0.5111
Valine	234	1.0000	0.1389	4
Arginine	5440	1.0646	0.0939	3	ns	0.4155
Arginine	2720	1.1757	0.0466	3	*	0.01
Arginine	1088	1.0291	0.0615	4	ns	0.8428
Arginine	109	1.0000	0.0389	4
Glutamine	22484	1.0564	0.1293	3	ns	0.8468
Glutamine	11242	1.0888	0.0261	3	ns	0.5648
Glutamine	3747	1.0757	0.1003	4	ns	0.6356
Glutamine	749	0.9790	0.0836	4	ns	0.993
Glutamine	562	1.0000	0.0596	3

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Isoleucine	6639	1.2144	0.1129	3	ns	0.0537
Isoleucine	3320	1.1366	0.0938	3	ns	0.2411
Isoleucine	1328	0.9229	0.0614	3	ns	0.6321
Isoleucine	66	1.0000	0.0953	3
Leucine	15270	1.1710	0.1043	3	ns	0.094
Leucine	7635	1.0915	0.0832	3	ns	0.4736
Leucine	3054	1.1410	0.1245	4	ns	0.1424
Leucine	153	1.0000	0.0481	4

Table 71-1. Fold change of procollagen lal sécrétion after administration of an amino acid composition in the second donor

Amino Acid Supplément	Conc. (X)	Procollagen lal Sécrétion (Fold Orange of IX)
Mean	Std. Déviation	Number of values	P-value*	Signifîcance
LIVRQNAC	40	0.7465	0.0551	3	**	0.0041
LIVRQNAC	30	0.6829	0.0991	3	***	0.0007
LIVRQNAC	20	0.6922	0.0281	4		0.0004
LIVRQNAC	10	0.7879	0.0748	4	**	0.0085
LIVRQNAC	1	1.0000	0.1141	4
LIVRQNAC+G	40	0.6372	0.0267	3	****	0.0001
LIVRQNAC+G	30	0.7347	0.0324	3	****	0.0001
LIVRQNAC+G	20	0.6716	0.0552	4	****	0.0001
LIVRQNAC+G	10	0.7823	0.0579	4	***	0.0001
LIVRQNAC+G	1	1.0000	0.0580	4
LIVRQNAC+S	40	0.8756	0.0372	3	ns	0.1229
LIVRQNAC+S	30	0.7340	0.0432	3	*♦	0.0019
LIVRQNAC+S	20	0.7405	0.0491	3	**	0.0022
LIVRQNAC+S	10	0.7472	0.0710	3	**	0.0027
LIVRQNAC+S	1	1.0000	0.1031	3
LIV	40	1.4409	0.0697	3	****	0.0001
LIV	30	1.3679	0.0156	3		0.0001
LIV	20	1.3418	0.1090	3	***	0.0002
LIV	10	1.2176	0.0343	3	**	0.0057
LIV	1	1.0000	0.0396	3
LIVRQ	40	0.9851	0.0534	3	ns	0.9965
LIVRQ	30	1.0185	0.0735	3	ns	0.9921
LIVRQ	20	0.9212	0.0215	3	ns	0.4893
LIVRQ	10	0.9558	0.0580	3	ns	0.8556
LIVRQ	1	1.0000	0.1134	3
RQNAC	40	0.6363	0.0432	3	***	0.0002

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RQNAC	30	0.6154	0.0196	3		0.0001
RQNAC	20	0.7060	0.0851	3	***	0.0009
RQNAC	10	0.8385	0.0248	3	*	0.041
RQNAC	1	1.0000	0.1071	3
N-Acetyl Cysteine	40	0.8383	0.0378	3	ns	0.4053
N-Acetyl Cysteine	20	0.7378	0.1347	3	ns	0.1002
N-Acetyl Cysteine	10	0.8877	0.2282	3	ns	0.6842
N-Acetyl Cysteine	5	0.8387	0.0832	3	ns	0.407
N-Acetyl Cysteine	0	1.0000	0.0808	3

Table 71-2. Fold change of procollagen lal sécrétion after administration of a single amino acid composition in the second donor

Amino Acid Supplément	Conc. (μΜ)	Procollagen lal Sécrétion (Fold Change of IX)
Mean	Std. Déviation	Number of values	P-value*	Signifïcance
Valine	23420	1.3068	0.0963	3	**	0.0019
Valine	11710	1.2877	0.1122	3	**	0.0029
Valine	4684	1.2865	0.0717	4	**	0.0018
Valine	234	1.0000	0.0589	4
Arginine	5440	1.1304	0.0187	3	ns	0.0937
Arginine	2720	1.0722	0.0791	3	ns	0.4483
Arginine	1088	1.0126	0.0822	4	ns	0.989
Arginine	109	1.0000	0.0778	4
Glutamine	22484	0.7143	0.0566	3	»·	0.0058
Glutamine	11242	0.7080	0.0246	3	**	0.005
Glutamine	3747	0.7541	0.0860	4	*	0.0102
Glutamine	749	0.9191	0.1171	4	ns	0.5776
Glutamine	562	1.0000	0.1003	3
Isoleucine	6639	1.5423	0.1489	3	**	0.006
Isoleucine	3320	1.4940	0.0238	3	*	0.0102
Isoleucine	1328	1.4811	0.2307	3	*	0.0117
Isoleucine	66	1.0000	0.1264	3
Leucine	15270	0.9518	0.0406	3	ns	0.9292
Leucine	7635	1.2628	0.1763	3	ns	0.0607
Leucine	3054	1.0781	0.1735	4	ns	0.7374
Leucine	153	1.0000	0.0681	4

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Table 71-3. Fold change of procollagen lal sécrétion after administration of an amino acid composition in the third donor

Amino Acid Supplément	Conc. (X)	Procollagen loi Sécrétion (Fold Change of IX)
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	0.9052	0.0344	3	ns	0.5685
LIVRQNAC	30	0.7456	0.0895	3	«	0.0192
LIVRQNAC	20	0.7817	0.0680	4	*	0.03
LIVRQNAC	10	0.9774	0.1451	4	ns	0.9927
LIVRQNAC	1	1.0000	0.1116	4
LIVRQNAC+G	40	0.7040	0.0080	3	**	0.002
LIVRQNAC+G	30	0.6249	0.0819	3	***	0.0003
LIVRQNAC+G	20	0.6863	0.1334	4	***	0.0006
LIVRQNAC+G	10	1.0068	0.0642	4	ns	0.9998
LIVRQNAC+G	1	1.0000	0.0724	4
LIVRQNAC+S	40	0.9190	0.0772	3	ns	0.3351
LIVRQNAC+S	30	0.8107	0.0596	3	*	0.0101
LIVRQNAC+S	20	0.8878	0.0129	3	ns	0.1296
LIVRQNAC+S	10	0.9814	0.0458	3	ns	0.9852
LIVRQNAC+S	1	1.0000	0.0780	3
LIV	40	1.3233	0.0667	3	**	0.0024
LIV	30	1.2510	0.1070	3	*	0.0125
LIV	20	1.2702	0.0639	3	**	0.0079
LIV	10	1.1912	0.1049	3	ns	0.0532
LIV	1	1.0000	0.0521	3
LIVRQ	40	1.2020	0.1119	3	ns	0.1081
LIVRQ	30	1.1380	0.0955	3	ns	0.3407
LIVRQ	20	0.9489	0.1179	3	ns	0.9263
LIVRQ	10	1.0786	0.0764	3	ns	0.7564
LIVRQ	1	1.0000	0.1056	3
RQNAC	40	0.6590	0.0860	3	**	0.0012
RQNAC	30	0.6708	0.0407	3	**	0.0016
RQNAC	20	0.9135	0.1192	3	ns	0.5063
RQNAC	10	0.8783	0.0515	3	ns	0.245
RQNAC	1	1.0000	0.0740	3
N-Acetyl Cysteine	40	0.6962	0.0189	3	*	0.0125
N-Acetyl Cysteine	20	0.8521	0.0709	3	ns	0.2666
N-Acetyl Cysteine	10	0.9391	0.1250	3	ns	0.8641
N-Acetyl Cysteine	5	1.0897	0.1245	3	ns	0.6511
N-Acetyl Cysteine	0	1.0000	0.1133	3

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Table 71-4. Fold change of procollagen lal sécrétion after administration of a single amino acid composition in the third donor

Amino Acid Supplément	Conc. (μΜ)	Procollagen lal Sécrétion (Fold Change of IX)
Mean	Std. Déviation	Number of values	P-value*	Significance
Valine	23420	1.1139	0.1077	3	ns	0.5315
Valine	11710	1.0498	0.1773	3	ns	0.918
Valine	4684	1.0428	0.1036	4	ns	0.9323
Valine	234	1.0000	0.1203	4
Arginine	5440	1.2125	0.0862	3	*	0.0112
Arginine	2720	1.1314	0.0820	3	ns	0.1114
Arginine	1088	1.0623	0.0629	4	ns	0.5378
Arginine	109	1.0000	0.0760	4
Glutamine	22484	1.0121	0.0730	3	ns	0.9989
Glutamine	11242	1.1204	0.1056	3	ns	0.2356
Glutamine	3747	0.9734	0.0900	4	ns	0.9747
Glutamine	749	1.0317	0.0644	4	ns	0.9538
Glutamine	562	1.0000	0.0447	3
Isoleucine	6639	1.4465	0.0958	3	**	0.0014
Isoleucine	3320	1.2703	0.0352	3	*	0.024
Isoleucine	1328	1.2687	0.0374	3	*	0.0247
Isoleucine	66	1.0000	0.1629	3
Leucine	15270	0.9892	0.0260	3	ns	0.9979
Leucine	7635	1.2027	0.0693	3	ns	0.0638
Leucine	3054	1.1399	0.1385	4	ns	0.1844
Leucine	153	1.0000	0.1077	4

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Example 13: Cytokine Sécrétion in Primary Human Macrophages । Isolation of Peripheral Blood Mononuclear Cell (PBMC)

Unpurified bufïy coats (Research Blood Components) were carefully poured into 50mL | centrifuge tubes and diluted with room température Dulbecco’s Phosphate Buffered Saline (dPBS) with Calcium and Magnésium (Gibco). Diluted bufïy coats were further divided into | 10 four total 5OmL centrifuge tubes at 20mL per tube. Lymphocyte Séparation Medium (Corning) was carefully pipetted to the bottom of each centrifuge tube. Mixtures were centrifuged at 850 X g for 32 minutes at 20°C with 0 décélération and accélération.

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The PBMC layer was separated from other components after centrifugation and added to | new 50mL centrifuge tube containing 25mL dPBS. Total volume was brought up to 50mL with dPBS and centrifuged at 600 X g for 10 minutes at 20°C with accélération of 9, décélération of | 5. Supematant was carefully removed from cell pellets. The cell pellets were resuspended using lOmL dPBS. Total volume was then brought up to 50mL using dPBS and centrifuged at 450 X g φ for 5 min at 20°C with accélération of 9, décélération of 9. The supematant removal and cell pellet resuspension was repeated again.

| The supematant was then carefully removed from cell pellets. Cell pellets were resuspended in lOmL dPBS without calcium or magnésium and filtered through a 70 uM cell j|| 10 strainer. The total PBMC number was determined using a Cellometer K2 automated cell counter. A total of 5E6 cells were saved for flow cytométrie analysis. Remaining cells were | centrifuged at 490 X g for 5 minutes at 20°C with accélération of 9, décélération of 9.

CD 14+ Cell Sélection

CD14+ cells were selected using EasySep™ Human CD14 Positive Sélection Kit H * (STEMCELL Technologies). Cells were resuspended in cold EasySep™ Buffer (STEMCELL Technologies) at 1X10⁸ cells/mL. A total of 100 uL/mL EasySep™ Human CD14 Positive Sélection Cocktail Π was added to the cell suspension, mixed, and incubated at room température for 10 minutes. A total of 100 uL/mL RapidSpheres were added to the mixture and incubated at W 20 room température for 3 minutes after mixing, then RoboSep buffer was added to bring up the total volume to 10mL. The mixture in a 15 mL tube was placed in magnet and incubated at room température for 3 minutes. Supematant was discarded and 10 mL fresh EasySep™ buffer was * added to 15mL tube. The addition of RoboSep buffer, mixing, and discarding of supematant was was repeated two more times.

^— 25 Négative and positive fractions were centrifuged at 490 X g for 5 minutes at 20°C with accélération of 9, décélération of 9, and resuspended in DMEM (Gibco) and 10% Heat ^e Inactivated Fêtai Bovine Sérum (Atlanta Bio) and Penicillin/Streptomycin. Cells were counted and centrifuged again at 490 X g for 5 minutes at 20°C with accélération of 9, décélération of 9.

* After centrifugation, cell were resuspended in DMEM (Gibco) and 10% Heat Inactivated Fêtai | 30 Bovine Sérum (Atlanta Bio) and Penicillin/Streptomycin containing 500 U/mL GM- and plated

I

230 at 1-2 X 10⁶ cells/mL on 10cm tissue culture plates. Cells were kept in 37°C, 5%CO2 in between feedings/harvest.

CD 14+ Cell Feedîng

Cells were fed every 3-4 days by removing media and unattached cells, centrifuging at 490 X g for 5 minutes at 20C with accélération of 9, décélération of 9, and resuspending in fresh DMEM (Gibco) and 10% Heat Inactivated Fêtai Bovine Sérum (Atlanta Bio) and Penicillin/Streptomycincontaining 500 U/mL GM-CSF. Resuspended cells were seeded back onto 10cm tissue culture plates and incubated at 37°C, 5%CO2.

Macrophage Harvest

After complété cell attachment, culture supematant was removed and cultures were washed IX with 5mL PB S. A total of 3mL room température Cellstripper was added and cultures were incubated at 37°C, 5% CO2 for approximately 10 minutes until cells were rounded and beginning to detach. Cell scraper was used to completely detach cells from plate. Collected cell were spun down at 490g for 5 min at room température and resuspended in 10% DMSO in Heat Inactivated Fêtai Bovine Sérum and immediately frozen in -80C.

Screen

Primary human PMBC derived macrophages were seeded on day 0 at 3.0E4 cells per well in 96-well microplates (ThermoFisher) in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with penicillin-streptomycin (Hyclone) and 10% heat inactivated fêtai bovine sérum (HI-FBS) (Atlanta Bio) and incubated overnight at 37°C, 5% CO2. On day 1, cells were washed once with 150uL per well DPBS (Gibco) and treated with 75uL of:

a.

b.

Amino acid free DMEM (US Biologicals) containing a defined custom amino acid concentration based on the mean physiological concentrations in blood based on values published in the Human Metabolome Database (HMDB), with 6mM glucose, ImM sodium pyruvate, 10 mM HEPES, 0.2% primocin (InVivoGen); or The same medium described above with one amino acid at various concentrations including complété dropout.

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On day 2, cells were treated with 75uL of the same médiums described above supplemented with 0.30ng/mL lipopolysaccharide (LPS) (Sigma) for a final concentration of 0.15ng/mL LPS. Control wells were treated with luM BX-795 (Tocis), luM TAK242 (Sigma), 0.15ng/mL LPS, or phosphate buffered saline (PBS).

On day 3, the supematant was collected and immediately frozen in -80°C freezer. Cells were washed once with 150uL DPBS and viability was assessed using the WST-8 Cell Prolifération Cytotoxicity Assay (Dojindo). Following the assay, cells were washed twice with 150uL PBS and fixed with 4% paraformaldéhyde for 5 min followed by two additional washes with 150uL PBS. Protein levels in supematant samples were analyzed by ELISA for IL-6 and TNFa using commercially available kits (R&D Systems) according to manufacturer-supplied protocols. Results are shown in Tables 71-5 through 71-10 below.

Table 71-5. IL-6 Measurements: Donor 1

Amino Acid Supplément	Conc. (X)	Donor 1 IL-6 Measurements
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	-619.787	114.1592	3	0.0001
LIVRQNAC	30	-525.849	63.87122	3	0.0001	***♦
LIVRQNAC	20	-290.199	33.5584	3	0.0092	♦♦
LIVRQNAC	10	51.81434	183.3933	3	0.9479	ns
LIVRQNAC	1	0	148.7761	3	na	na
- LIVRQNAC+G	40	-1099.11	44.1139	3	0.0001	****
LIVRQNAC+G	30	-903.836	107.7113	3	0.0001	****
LIVRQNAC+G	20	-616.626	114.7826	3	0.0001	****
LIVRQNAC+G	10	-367.918	98.44611	3	0.0007	***
LIVRQNAC+G	1	0	172.9553	3	na	na
LIVRQNAC+S	40	-968.997	90.53282	3	0.0001	****
LIVRQNAC+S	30	-798.326	52.89122	3	0.0001	♦*
LIVRQNAC+S	20	-506.804	63.85224	3	0.0001	«***
LIVRQNAC+S	10	-243.259	114.742	3	0.0365	*
LIVRQNAC+S	1	0	259.8506	3	na	na
LIV	40	4.918642	62.7077	3	0.9999	ns
LIV	30	86.01907	128.1151	3	0.7604	ns
LIV	20	112.1501	83.62436	3	0.564	ns
LIV	10	54.22668	63.10515	3	0.9392	ns
. LIV	1	0	75.98804	3	na	na
LIVRQ	40	322.0706	73.87715	3	0.0033	**

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LIVRQ	30	297.8004	34.60168	3	0.0072	**
LIVRQ	20	604.021	203.8836	3	0.0001	****
LIVRQ	10	289.1798	57.78952	3	0.0095	♦*
LIVRQ	1	0	93.58494	3	na	na
RQNAC	40	-911.011	12.65475	3	0.0001	****
RQNAC	30	-766.912	26.23659	3	0.0001	****
RQNAC	20	-511.403	32.15983	3	0.0001	****
RQNAC	10	-201.63	6.477522	3	0.1054	ns
RQNAC	1	0	174.9658	3	na	na
N-Acetyl Cysteine	40	-914.194	56.77271	3	0.0001	****
N-Acetyl Cysteine	20	-553.802	85.27013	3	0.0001	****
N-Acetyl Cysteine	10	-121.142	53.05191	3	0.4973	ns
N-Acetyl Cysteine	5	308.1772	263.4651	3	0.0052	**
N-Acetyl Cysteine	0	0	45.08485	3	na	na

Amino Acid Supplément	Conc. (μΜ)	Donor 1IL-6 Measurements
Mean	Std. Déviation	Number of values	P-value*	Significance
Valine	23420	-106.268	155.3559	3	0.7885	ns
Valine	11710	-97.25	77.26313	3	0.8339	ns
Valine	4684	-85.9843	74.99317	3	0.8841	ns
Valine	234	0	124.8497	3	na	na
Arginine	5440	357.4394	154.8508	3	0.0159	*
Arginine	2720	-186.57	85.86105	3	0.3477	ns
Arginine	1088	-181.36	131.6475	3	0.3722	ns
Arginine	109	0	282.0306	3	na	na
Glutamine	22484	440.1437	114.443	3	0.0022	**
Glutamine	11242	397.1745	23.36272	3	0.0064	**
Glutamine	3747	291.5443	81.30853	3	0.0623	ns
Glutamine	749	0	73.06692	3	na	na
Isoleucine	6639	-218.332	146.5098	3	0.221	ns
Isoleucine	3320	-15.8843	89.88616	3	0.9998	ns
Isoleucine	1328	25.98372	323.6109	3	0.9984	ns
Isoleucine	66	0	48.21125	3	na	na
Leucine	15270	84.46122	68.15253	3	0.8902	ns
Leucine	7635	-69.9873	99.00843	3	0.9398	ns
Leucine	3054	244.9743	355.6551	3	0.1442	ns
Leucine	153	0	61.85589	3	na	na

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Treatment with LIVRQNAC, LIVRQNAC + G, LIVRQNAC + S, RQNAC, and NAC | signifïcantly reduced LPS-induced IL-6 sécrétion in primary human monocyte-derived macrophages. Treatment with LIVRQ signifïcantly increased IL-6 sécrétion, while LIV had no | effect. Arginine and glutamine administered alone increased IL-6 sécrétion while other amino acids alone did not effect IL-6 sécrétion. Two Way ANOVA Dunnett Multiple Comparisons was performed for statistical analysis. Mean values represented as baseline subtracted values.

Table 71-6. IL-6 Measurements: Donor 2

Amino Acid Supplément	Conc. (X)	Donor 2 IL-6 Measurements
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	-27.1916	1.853569	3	0.0003
LIVRQNAC	30	-21.5766	1.709414	3	0.0045	»♦
LIVRQNAC	20	-8.20655	8.458638	3	0.5143	ns
LIVRQNAC	10	-1.71581	6.104437	3	0.9965	ns
LIVRQNAC	1	-2.4E-15	11.85079	3
LIVRQNAC+G	40	-33.2001	3.55425	3	0.0001	****
LIVRQNAC+G	30	-30.8468	0.854995	3	0.0001	****
LIVRQNAC+G	20	-18.4318	4.870421	3	0.0187	*
LIVRQNAC+G	10	14.63551	21.82024	3	0.0824	ns
LIVRQNAC+G	1	2.37E-15	8.607557	3
LIVRQNAC+S	40	-26.5993	2.963677	3	0.0004	***
LIVRQNAC+S	30	-14.2166	1.460268	3	0.0954	ns
LIVRQNAC+S	20	-8.2522	2.917345	3	0.5095	ns
LIVRQNAC+S	10	8.127841	1.783214	3	0.5227	ns
LIVRQNAC+S	1	0	6.232673	3
LIV	40	34.10306	1.950493	3	0.0001	****
LIV	30	31.10835	9.757211	3	0.0001	****
LIV	20	20.32684	3.17293	3	0.0081	**
LIV	10	15.10204	9.179111	3	0.0697	ns
LIV	1	-7.1E-15	4.738966	3
LIVRQ	40	49.62156	17.37012	3	0.0001	****
LIVRQ	30	42.9625	7.798872	3	0.0001	****
LIVRQ	20	48.38603	13.08566	3	0.0001	«*
LIVRQ	10	45.99191	15.19687	3	0.0001	****
LIVRQ	1	1.18E-15	6.324379	3
RQNAC	40	-36.5521	1.877658	3	0.0001	****
RQNAC	30	-26.3768	0.744676	3	0.0004	***
RQNAC	20	-18.7428	1.353649	3	0.0164	*

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RQNAC	10	-3.74427	4.74578	3	0.9393	ns
RQNAC	1	2.37E-15	12.26314	3
N-Acetyl Cysteine	40	-33.7585	0.895842	3	0.0001	****
N-Acetyl Cysteine	20	-24.9999	1.083467	3	0.0008	***
N-Acetyl Cysteine	10	-9.75111	2.381012	3	0.3617	ns
N-Acetyl Cysteine	5	-0.79458	5.988677	3	0.9998	ns
N-Acetyl Cysteine	0	-2.4E-15	1.900091	3

Amino Acid Supplément	Conc. (μΜ)	Donor 2 IL-6 Measurements
Mean	Std. Déviation	Number of values	P-value*	Significance
Valine	23420	4.395899	10.35903	3	0.973	ns
Valine	11710	-1.19605	7.303571	3	0.9998	ns
Valine	4684	-4.52846	4.069907	3	0.97	ns
Valine	234	-4.7E-15	9.361734	3
Arginine	5440	-12.4164	0.292618	3	0.5017	ns
Arginine	2720	-13.6102	2.1177	3	0.4207	ns
Arginine	1088	-9.70116	9.286942	3	0.6995	ns
Arginine	109	2.37E-15	14.30728	3
Glutamine	22484	34.38845	7.467725	3	0.0026	**
Glutamine	11242	63.31441	35.02748	3	0.0001	****
Glutamine	3747	22.51543	9.686139	3	0.0721	ns
Glutamine	749	2.37E-15	2.203881	3
Isoleucine	6639	-1.77438	10.22772	3	0.999	ns
Isoleucine	3320	2.305485	1.328015	3	0.9975	ns
Isoleucine	1328	-2.31776	9.121049	3	0.9974	ns
Isoleucine	66	0	12.3413	3
Leucine	15270	47.59735	16.64049	3	0.0001	****
Leucine	7635	30.46065	7.144005	3	0.0087	**
Leucine	3054	29.60609	13.39676	3	0.0111	*
Leucine	153	7.11E-15	6.308577	3

Treatment with LIVRQNAC, LIVRQNAC + G, LIVRQNAC + S, RQNAC, and NAC significantly reduced LPS-induced IL-6 sécrétion in primary human monocyte-derived macrophages. Treatment with LIVRQ and LIV significantly increased IL-6 sécrétion. Glutamine 5 and leucine administered alone increased IL-6 sécrétion, while the other amino acids alone had no effect. Two Way ANOVA Dunnett Multiple Comparisons was performed for statistical analysis. Mean values represented as baseline subtracted values.

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Table 71-7. IL-6 Measurements: Donor 3

Amino Acid Supplément	Conc. (X)	Donor 3 IL-6 Measurements
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	-18.2445	4.129349	3	0.7529	ns
LIVRQNAC	30	-16.8219	1.366045	3	0.8001	ns
LIVRQNAC	20	-13.4826	12.48206	3	0.8948	ns
LIVRQNAC	10	-34.4539	37.38053	3	0.2356	ns
LIVRQNAC	1	-1.4E-14	14.03982	3
LIVRQNAC+G	40	-54.4799	5.467815	3	0.0228	*
LIVRQNAC+G	30	-48.3118	1.960574	3	0.0513	ns
LIVRQNAC+G	20	-55.792	7.763897	3	0.019	*
LIVRQNAC+G	10	-44.8309	14.34972	3	0.0783	ns
LIVRQNAC+G	1	0	26.01471	3
LIVRQNAC+S	40	-14.5337	15.82418	3	0.868	ns
LIVRQNAC+S	30	-25.9127	10.00119	3	0.479	ns
LIVRQNAC+S	20	-25.8862	21.61536	3	0.48	ns
LIVRQNAC+S	10	-11.9742	10.3333	3	0.9277	ns
LIVRQNAC+S	1	-4.3E-14	15.34164	3
LIV	40	10.21257	37.58938	3	0.9576	ns
LIV	30	-32.6891	24.862	3	0.2771	ns
LIV	20	27.66715	39.40901	3	0.4207	ns
LIV	10	9.44789	71.20002	3	0.9677	ns
LIV	1	-4.7E-14	27.50075	3
LIVRQ	40	74.9145	12.55033	3	0.001	***
LIVRQ	30	120.1764	20.21514	3	0.0001	****
LIVRQ	20	77.12007	11.45452	3	0.0007	**♦
LIVRQ	10	67.95483	43.58345	3	0.003	**
LIVRQ	1	-2.4E-14	27.62048	3
RQNAC	40	-45.9765	5.740028	3	0.0683	ns
RQNAC	30	-53.3845	16.45009	3	0.0265	*
RQNAC	20	-65.6761	3.400465	3	0.0044	**
RQNAC	10	-32.8776	33.99103	3	0.2724	ns
RQNAC	1	-2.8E-14	23.14404	3
N-Acetyl Cysteine	40	-140.851	4.662272	3	0.0001	****
N-Acetyl Cysteine	20	-122.656	8.219985	3	0.0001	****
N-Acetyl Cysteine	10	-103.586	28.4385	3	0.0001	****
N-Acetyl Cysteine	5	-70.3269	8.563896	3	0.0021	**
N-Acetyl Cysteine	0	-9.5E-15	11.75797	3

Amino Acid Supplément

Donor 3 IL-6 Measurements

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	Conc. (μΜ)	Mean	Std. Déviation	Number of values	P-value*	Signifîcance
Valine	23420	-29.2004	25.98066	3	0.4329	ns
Valine	11710	-43.8022	8.331697	3	0.1239	ns
Valine	4684	-30.0609	8.478329	3	0.4072	ns
Valine	234	4.26E-14	17.2027	3
Arginine	5440	-6.80983	0.643932	3	0.9922	ns
Arginine	2720	-7.50318	22.06663	3	0.9888	ns
Arginine	1088	31.5786	70.48311	3	0.3642	ns
Arginine	109	0	17.26952	3
Glutamine	22484	108.5158	55.59202	3	0.0001	****
Glutamine	11242	98.4903	58.37	3	0.0001	★*
Glutamine	3747	25.35457	16.40416	3	0.556	ns
Glutamine	749	3.79E-14	16.54987	3
Isoleucine	6639	-16.3663	8.09174	3	0.9718	ns
Isoleucine	3320	0	19.80362	3	0.9928	ns
Isoleucine	1328	-28.9897	13.10903	3	0.6593	ns
Isoleucine	66	-6.69039	13.72995	3
Leucine	15270	#	#	3	#	#
Leucine	7635	#	#	3	U	#
Leucine	3054	#	#	3	#	#
Leucine	153	#	#	3	#	#

Leucine was not measured in Exp3 due to technical error #

Treatment with LIVRQNAC + G, LIVRQNAC + S, RQNAC, and NAC significantly reduced LPS-induced IL-6 sécrétion in primary human monocyte-derived macrophages. Treatment with LIVRQ increased IL-6 sécrétion, while LIV and LIVRQNAC had no statistically significant effects on IL-6 sécrétion. Glutamine administered alone significantly increased IL-6 sécrétion, while other amino acids alone had no effect. Two Way ANOVA Dunnett Multiple Comparisons was performed for statistical analysis. Mean values represented as baseline subtracted values.

Table 71-8. TNFalpha Measurements: Donor 1

Amino Acid Supplément	Conc. (X)	Donor 1 TNFa Measurements
Mean	Std. Déviation	Number of values	P-value*	Signifîcance
LIVRQNAC	40	-422.74	4.347575	3	0.0001	****
LIVRQNAC	30	-389.74	1.004633	3	0.0001	****
LIVRQNAC	20	-336.69	3.007435	3	0.0001	****
LIVRQNAC	•10	-246.04	27.61929	3	0.0001	****

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LIVRQNAC	1	0	36.31082	3
LIVRQNAC+G	40	-490.92	4.427614	3	0.0001	***★
LIVRQNAC+G	30	-447.73	9.819865	3	0.0001	****
LIVRQNAC+G	20	-377.32	5.837159	3	0.0001	****
LIVRQNAC+G	10	-268.29	9.642365	3	0.0001	****
LIVRQNAC+G	1	0	37.44353	3
LIVRQNAC+S	40	-415.03	4.800449	3	0.0001	****
LIVRQNAC+S	30	-379.44	4.694868	3	0.0001	****
LIVRQNAC+S	20	-323.77	7.971135	3	0.0001	****
LIVRQNAC+S	10	-209.59	21.15676	3	0.0001	****
LIVRQNAC+S	1	0	30.0492	3
LIV	40	60.37	20.26331	3	0.0065	**
LIV	30	42.09	22.95664	3	0.0865	ns
LIV	20	63.37	37.24144	3	0.004	♦*
LIV	10	45.61	44.71078	3	0.0556	ns
LIV	1	0	10.49958	3
LIVRQ	40	6.38	17.1283	3	0.9909	ns
LIVRQ	30	-6.72	18.9622	3	0.989	ns
LIVRQ	20	38.38	39.85515	3	0.1333	ns
LIVRQ	10	-18.95	10.84371	3	0.6982	ns
LIVRQ	1	0	36.96184	3
RQNAC	40	-408.44	1.179877	3	0.0001	·*
RQNAC	30	-390.41	1.341282	3	0.0001	·*
RQNAC	20	-338.2	3.284307	3	0.0001	****
RQNAC	10	-251.35	4.121085	3	0.0001	****
RQNAC	1	0	51.06933	3
N-Acetyl Cysteine	40	-644.49	2.42197	3	0.0001	****
N-Acetyl Cysteine	20	-561.33	8.435064	3	0.0001	****
N-Acetyl Cysteine	10	-446.88	12.22132	3	0.0001	****
N-Acetyl Cysteine	5	-326.24	11.10173	3	0.0001	****
N-Acetyl Cysteine	0	0	42.00516	3

Amino Acid Supplément	Conc. (μΜ)	Donor 1 TNFa Measurements
Mean	Std. Déviation	Number of values	P-vafue*	Signifïcance
Valine	23420	-14.98	20.86784	3	0.9928	ns
Valine	11710	-41.77	36.61662	3	0.7784	ns
Valine	4684	-40.37	32.31016	3	0.7974	ns
Valine	234	0	24.8661	3
Arginine	5440	62.06	48.80326	3	0.4786	ns
Arginine	2720	5.12	15.47951	3	0.9998	ns

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Arginine	1088	-24.33	17.74317	3	0.9577	ns
Arginine	109	0	18.5366	3
Glutamine	22484	-103.07	27.02483	3	0.0985	ns
Glutamine	11242	-65.24	23.02631	3	0.4346	ns
Glutamine	3747	-45.7	28.56445	3	0.7222	ns
Glutamine	749	0	30.75138	3
Isoleucine	6639	-40.95	78.56369	3	0.7896	ns
Isoleucine	3320	-96.3	45.66981	3	0.1339	ns
Isoleucine	1328	-42.68	21.07739	3	0.7657	ns
Isoleucine	66	0	115.9559	3
Leucine	15270	-46.21	29.00402	3	0.7148	ns
Leucine	7635	-23.04	40.08864	3	0.965	ns
Leucine	3054	42.04	77.19161	3	0.7746	ns
Leucine	153	0	157.6578	3

Treatment with LIVRQNAC, LIVRQNAC + G, LIVRQNAC + S, RQNAC, and NAC significantly reduced LPS-induced TNFa sécrétion in primary human monocyte-derived macrophages. Treatment with LIV increased TNFa sécrétion, while LIVRQ had no signfîciant effects on TNFa sécrétion. None of the individually administered amino acids had an effect on TNFa sécrétion. Two Way ANOVA Dunnett Multiple Comparisons was performed for statistical analysis. Mean values represented as baseline subtracted values.

Table 71-9. TNFalpha Measurements: Donor 2

Amino Acid Supplément	Conc. (X)	Donor 2 TNFa Measurements
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	-98.1341	2.118962	3	0.0001	·***
LIVRQNAC	30	-85.1019	1.385677	3	0.0001	****
LIVRQNAC	20	-64.3364	10.07525	3	0.0001	****
LIVRQNAC	10	-38.3512	5.120689	3	0.0001	***·
LIVRQNAC	1	0	5.45587	3
LIVRQNAC+G	40	-91.3454	5.994009	3	0.0001	****
LIVRQNAC+G	30	-82.4397	4.200763	3	0.0001	****
LIVRQNAC+G	20	-61.247	8.702492	3	0.0001	****
LIVRQNAC+G	10	-23.9913	7.471422	3	0.008	**
LIVRQNAC+G	1	-4.7E-15	4.578295	3
LIVRQNAC+S	40	-74.1572	4.163823	3	0.0001	****
LIVRQNAC+S	30	-64.0016	5.549308	3	0.0001	****

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LIVRQNAC+S	20	-47.5673	3.970363	3	0.0001	****
LIVRQNAC+S	10	-28.635	7.390447	3	0.0012	♦*
LIVRQNAC+S	1	-4.7E-15	7.564883	3
LIV	40	49.84155	4.092799	3	****	0.0001
LIV	30	29.1118	14.72509	3	***	0.001
LIV	20	30.17595	5.797518	3	***	0.0006
LIV	10	16.68974	10.85983	3	ns	0.0974
LIV	1	0	10.41523	3
LIVRQ	40	64.1705	27.82953	3	****	0.0001
LIVRQ	30	50.92104	6.955429	3	****	0.0001
LIVRQ	20	45.65882	19.0128	3	****	0.0001
LIVRQ	10	32.37038	19.44425	3	***	0.0002
LIVRQ	1	-4.7E-15	5.942707	3
RQNAC	40	-84.147	5.821583	3	****	0.0001
RQNAC	30	-77.9626	1.626776	3	****	0.0001
RQNAC	20	-63.3754	3.494595	3	****	0.0001
RQNAC	10	-37.6072	1.88043	3	****	0.0001
RQNAC	1	-9.5E-15	4.727924	3
N-Acetyl Cysteine	40	-103.984	0.720962	3	0.0001	****
N-Acetyl Cysteine	20	-88.6528	0.668195	3	0.0001	****
N-Acetyl Cysteine	10	-70.8382	12.08717	3	0.0001	****
N-Acetyl Cysteine	5	-54.1596	11.06287	3	0.0001
N-Acetyl Cysteine	0	9.47E-15	2.926881	3

Amino Acid Supplément	Conc. (μΜ)	Donor 2 TNFa Measûrements
Mean	Std. Déviation	Number of values	P-value*	Significance
Valine	23420	-1.25079	12.85688	3	0.9991	ns
Valine	11710	-0.83505	8.524018	3	0.9998	ns
Valine	4684	-0.00221	5.127759	3	0.9999	ns
Valine	234	-4.7E-15	8.717375	3
Arginine	5440	-0.57378	8.672536	3	0.9999	ns
Arginine	2720	-3.76334	2.467885	3	0.9594	ns
Arginine	1088	-12.7222	4.764842	3	0.2488	ns
Arginine	109	1.42E-14	3.511446	3
Glutamine	22484	11.50181	6.216029	3	0.3311	ns
Glutamine	11242	20.03996	11.90208	3	0.0279	*
Glutamine	3747	9.338214	9.748253	3	0.5134	ns
Glutamine	749	-9.5E-15	7.275868	3
Isoleucine	6639	19.25756	5.097831	3	0.0365	*
Isoleucine	3320	10.26061	7.861148	3	0.4307	ns

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Isoleucine	1328	2.918887	1.921961	3	0.9836	ns
Isoleucine	66	4.74E-15	6.264135	3
Leucine	15270	46.68507	11.63209	3	0.0001	****
Leucine	7635	41.97528	6.512087	3	0.0001	****
Leucine	3054	31.74019	11.56537	3	0.0002	***
Leucine	153	0	0.482598	3

Treatment with LIVRQNAC, LIVRQNAC + G, LIVRQNAC + S, RQNAC, and NAC significantly reduced LPS-induced TNFa sécrétion in primary human monocyte-derived macrophages. Treatment with LIV and LIVRQ increased TNFa sécrétion. Leucine, isoleucine, and glutamine administered individually increased TNFa sécrétion, while the other amino acids had no effect. Two Way ANOVA Dunnett Multiple Comparisons was performed for statistical analysis. Mean values represented as baseline subtracted values.

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Table 71-10. TNFalpha Measurements: Donor 3

Amino Acid Supplément	Conc. (X)	Donor 3 TNFa Measurements
Mean	Std. Déviation	Number of values	P-value*	Significance
LIVRQNAC	40	-18.7507	2.487301	3	0.0001	****
LIVRQNAC	30	-15.5979	0.932399	3	0.0006	***
LIVRQNAC	20	-10.7042	3.013527	3	0.026	•
LIVRQNAC	10	-8.49034	2.434812	3	0.1029	ns
LIVRQNAC	1	0	4.067982	3
LIVRQNAC+G	40	-14.6552	3.149813	3	0.0013	**
LIVRQNAC+G	30	-11.6973	2.026588	3	0.0129	*
LIVRQNAC+G	20	-8.0218	0.671662	3	0.1331	ns
LIVRQNAC+G	10	-4.8035	1.658348	3	0.5453	ns
LIVRQNAC+G	1	-2.4E-15	5.625453	3
LIVRQNAC+S	40	-14.247	1.800575	3	0.0018	**
LIVRQNAC+S	30	-15.1388	1.568817	3	0.0009	***
LIVRQNAC+S	20	-12.4722	3.334857	3	0.0073
LIVRQNAC+S	10	-6.72057	1.833554	3	0.2549	ns
LIVRQNAC+S	1	0	4.171555	3
LIV	40	14.07984	11.14252	3	0.002	**
LIV	30	1.759786	1.102706	3	0.9748	ns
LIV	20	14.51396	10.41503	3	0.0014	**
LIV	10	8.560957	12.86074	3	0.0989	ns
LIV	1	2.37E-15	3.660423	3

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LIVRQ	40	25.84453	0.659584	3	0.0001	****
LIVRQ	30	33.74883	5.974096	3	0.0001	****
LIVRQ	20	20.94481	2.163828	3	0.0001	****
LIVRQ	10	15.45187	3.942596	3	0.0007	»»»
LIVRQ	1	0	4.575346	3
RQNAC	40	-21.5102	1.191926	3	0.0001	·*
RQNAC	30	-20.8898	2.622446	3	0.0001	··**
RQNAC	20	-19.9558	3.302225	3	0.0001	****
RQNAC	10	-9.09425	5.483242	3	0.0725	ns
RQNAC	1	0	6.189505	3
N-Acetyl Cysteine	40	-55.3093	0.809363	3	0.0001	««
N-Acetyl Cysteine	20	-48.4373	1.563179	3	0.0001	****
N-Acetyl Cysteine	10	-41.7266	3.533914	3	0.0001	★***
N-Acetyl Cysteine	5	-33.6246	0.253484	3	0.0001	****
N-Acetyl Cysteine	0	4.74E-15	8.55997	3

Amino Acid Supplément	Conc. (μΜ)	Donor 3 TNFa Measurements
Mean	Std. Déviation	Number of values	P-value*	Significance
Valine	23420	3.688279	7.532913	3	0.8962	ns
Valine	11710	-2.59866	2.586099	3	0.9674	ns
Valine	4684	0.126	0.903014	3	0.9999	ns
Valine	234	-2.4E-15	2.731283	3
Arginine	5440	-1.76662	4.067694	3	0.992	ns
Arginine	2720	-0.96691	4.86075	3	0.9991	ns
Arginine	1088	3.131153	10.346	3	0.9384	ns
Arginine	109	3.55E-15	4.325877	. 3
Glutamine	22484	29.14034	17.71417	3	0.0001	****
Glutamine	11242	18.00238	14.58602	3	0.0061	>>
Glutamine	3747	1.935546	2.127977	3	0.9887	ns
Glutamine	749	0	5.196592	3
Isoleucine	6639	-1.66019	4.262718	3	0.9938	ns
Isoleucine	3320	3.308901	3.745411	3	0.9262	ns
Isoleucine	1328	-6.22991	0.48195	3	0.5976	ns
Isoleucine	66	-2.4E-15	3.844593	3
Leucine	15270	#	#	3	#	#
Leucine	7635	#	#	3	#	#
Leucine	3054	#	#	3	#	#
Leucine	153	#	#	3	#	#

# Leucine was not measured in Exp3 due to technical error

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Treatment with LIVRQNAC, LIVRQNAC + G, LIVRQNAC + S, RQNAC, and NAC | significantly reduced LPS-induced TNFa sécrétion in primary human monocyte-derived macrophages. Treatment with LIV and LIVRQ increased TNFa sécrétion. Individually | administered amino acids had no significant effect on TNFa sécrétion, except for glutamine which increased TNFa sécrétion. Two Way ANOVA Dunnett Multiple Comparisons was | performed for statistical analysis. Mean values represented as baseline subtracted values.

| Example 14: Treatment of NAFLD Patients with an Amino Acid Composition

The study described herein features the administration of a composition including amino | 10 acids to treat patients with NAFLD. The composition can include about 2 g of leucine, about 1 g of isoleucine, about 1 g of valine, about 3.62 g of arginine, about 4 g of glutamine, and about | 0.3 g of N-acetylcysteine for administration three times per day (e.g., a total of about 36 g per day). The composition can also include about 2 g of leucine, about 1 g of isoleucine, about 1 g | of valine, about 3.62 g of arginine, about 4 g of glutamine, and about 0.6 g of N-acetylcysteine 15 for administration three times per day (e.g., a total of about 37 g per day).

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Alternatively, the composition can include about 4 g of leucine, about 2 g of isoleucine, | about 2 g of valine, about 7.24 g of arginine, about 8 g of glutamine, and about 0.6 g of Nacetylcysteine for administration two or three times per day (e.g., a total of about 48 g or a total | 20 of about 72 g per day). The composition can also include about 4 g of leucine, about 2 g of isoleucine, about 2 g of valine, about 7.24 g of arginine, about 8 g of glutamine, and about 1.2 g | of N-acetylcysteine for administration two or three times per day (e.g., a total of about 49 g or a total of about 73 g per day).

| For each composition, the dose can be administered prior to, concurrently with, or following a meal. Alternatively, the composition is not administered immediately before, with, | or after a meal. The amino acid composition can be administered for a period of at least 12 weeks, e.g., for 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 16 weeks. In particular, the amino | acid composition is administered for a period of at least 16 weeks, e.g., for 16 weeks. The composition can be administered orally.

| 30 Key criteria for selecting NAFLD patients for enrollment in a clinical study of the amino acid composition include: 1) a diagnosis of NAFLD; 2) type 2 diabètes; 3) a relatively high BMI;

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4) a NAFLD Fibrosis Score of less than 0.6; 5) a liver biopsy; and 7) a MRI and/or CT | assessment of the liver. The patients can hâve type 2 diabètes in addition to NAFLD.

Fatty liver disease can be document by a prior history of steatosis confirmed within 3 | months of screening by at least one of the following methods: liver fat by MRI with a PDFF > 5 8%; fibroscan with Control Atténuation Parameter > 300 dB/m; or liver biopsy indicating non- | NASH NAFLD steatosis > Grade I. If the patient does not hâve this documented prior history of steatosis within 3 months of screening, then a liver fat score of >10% must be documented at the time of screening using the following formula:

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Predicted percent liver fat = 10^Λ(-0.805 + (0.282 * metabolic syndrome [yes =l/no= 0]) + (0.078 *type 2 diabètes [yes =2/no= 0]) + (0.525 * logl0(insulin mU/L)) + (0.521 * logl0(AST U/L)) - (0.454 *loglO(AST/ALT))³⁴

Patients can be on stable exercise, diet and lifestyle routine within 3 months prior to screening, with no major body weight fluctuations, e.g., subjects should be within ±3% of their body weight over the last 3 months at the time of screening. Patients can hâve a body mass index (BMI) > 32 kg/m2 at screening. For sites whose MRI equipment cannot accommodate a patient with a BMI of > 45 kg/m2, an upper limit between 40 to 45 kg/m2 may be applied.

Patients must be on a stable dose of glucose-lowering médication (which can include 20 metformin, sulfonylureas, dipeptidyl peptidase-4 (DPP-4) inhibitors, sodium-glucose cotransporter 2 (SGLT2) inhibitors, or long-acting basal insulin) for at least 3 months before Screening and plan to remain on the same médication without anticipated dose adjustments of their médications for the duration of the study.· Patients may be included in the study if they are concurrently treated with anti-hypertensive médications (e.g., beta blockers, hydrochlorothiazide, 25 ACE inhibitors, angiotensin receptor blockers), médications for dyslipidemia (e.g., statins, fibrates), and médication for hypothyroidism (e.g., lévothyroxine), so long as they hâve been on stable doses and regimen of these médications for at least 3 months before Screening and plan to remain on the same médication without anticipated dose adjustments of their médications for the duration of the study. Patients may be on vitamin suppléments (e.g. multivitamins; vitamin E <400 lU/day). However, they must be on stable doses and regimen of these vitamin suppléments for at least 3 months before screening without anticipated dose adjustments for the duration of the study.

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Primary endpoints in the study include the safety and tolerability of administering the | composition including amino acids to patients with NAFLD. Secondary endpoints indicative of an improvement in symptoms of NAFLD include the following: 1) intrahepatic fat réduction by | MRI; and 2) assessment of biomarkers involved in liver biochemistry, fibrosis/apoptosis, and meteabolism. The following biomarkers can be assessed in a sample (e.g., a plasma or liver | sample) from a NAFLD patient: a) alanine aminotransferase (ALT); b) aspartate aminotransferase (AST); c) adiponectin; d) N-terminal fragment of type ΙΠ collagen (proC3); e) | caspase-cleaved keratin 18 fragments (M30 and M65); f) IL-1 beta; g) C-reactive protein; h) PTTTNP; i) TIMP1; j) MCP-1; k) FGF-21; or 1) gamma glutamyl transferase (GGT). For

J 10 example, a subject with NAFLD can exhibit a decrease in levels of one, two, or more (e.g., ail) of ALT, AST, or GGT after treatment with the composition.

| The patient may exhibit a mean change in plasma glucose, insulin, homeostatic model assessment insulin résistance (HOMA IR), lipid profile, hemoglobin Aie (HbAlc) and other | metabolic parameters from, e.g., baseline to Weeks 6 and 12, including changes in plasma glucose and insulin levels in the setting of an oral glucose tolérance test (oGTT) from, e.g., | baseline to Weeks 6 and 12. The patient may exhibit a mean change in body weight from, e.g., baseline to Weeks 6 and 12.

B Administration of the amino acid composition can resuit in an improvement in the

NAFLD activity score, glucose tolérance, hépatocyte inflammation, liver fibrosis or liver injury, | 20 steatosis, or hépatocyte ballooning in the patient.

Example 15. Treatment of NASH in a Mouse Model with an Amino Acid Composition Induction of NASH in Mice

In one example, the effects of LIVRQNAC and related amino acid compositions in the obesity, metabolism-driven non-alcoholic steatohepatitis (NASH) in FATZO mouse model was | examined.

MATERIALS AND METHODS

Induction of NASH in Mice

NASH was induced in 60 male FATZO mice by a western diet (Research Diet # ® D12079B; fat 40 %kcal, protein 17 %kcal, carbohydrate 43 %kcal) supplemented with 5%

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I I fructose in the drinking water (WDF) during a 16 week induction phase. Diets and water were available ad libitum. Littermate control male FATZO mice fed with a control diet (n=6, Purina # 5008; fat 17 %kcal, protein 27 %kcal, carbohydrate 56 %kcal) and stérile water were set up for control purpose. Mice were housed in plastic cages with microisolator. Sterilized bedding was replaced once a week. Mice were housed three per cage and maintained on a twelve hour light cycle throughout study duration. Room température was monitored daily and maintained at 2225°C. Body weight was recorded every week during the induction phase.

Study Design

Following 16 weeks diet induction, 6 mice remained on control diet (group 1, Control) while 60 induced mice were randomized on body weight and plasma glucose (fed) for assignment to the following treatments. FATZO mice were administered with test articles starting at 16 weeks post western diet NASH induction for 4 weeks. Test articles were administered by oral gavage. Animais were euthanized at 20 weeks post western diet NASH induction, and tissues were harvested for analysis.

Group	(n)	Treatment (oral)	Diet
1	6	Vehicle	5008 WDF
2	10	Vehicle	D12079B + 5% Fructose
3	10	LIVRQNAC (1500 mg/kg)	D12079B + 5% Fructose
4	10	LIVRQNAC (3000 mg/kg)	D12079B + 5% Fructose
5	10	LIVRQNAC+G (3885 mg/kg)	D12079B + 5% Fructose
6	10	LRQNAC (2469 mg/kg)	D12079B + 5% Fructose
7	10	Obeticholic acid (OCA) 30 mg/kg/day	D12079B + 5% Fructose

Test Articles

LIVRQNAC, LIVRQNAC+G, LRQNAC, and OCA (Advanced ChemBlocks, Inc.), incipient, and water for irrigation were provided by Axcella Health, Inc. 0.5%

Methylcellulosewas provided by CrownBio, Inc. Dosing solutions were prepared according to Appendix 1. TA compounds (amino acid compositions) were amino acid blends formuiated fresh daily in water for irrigation (Baxter # 27F7114) and the excipients 0.125% Xanthan Gum, 1.5

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I I I mM Sodium Lauryl Sulfate and 0.28% Lecithin. Obeticholic acid (OCA) was suspended in 0.5% methylcellulose in water for irrigation. Ail test articles were stored refrigerated. TA compounds were provided in frozen powder form by the sponsor. Dosing was continued for 4 weeks.

Leucine dosages of LIVRQNAC+G and LRQNAC were matched to that of LIVRQNAC.

Amino Acid Compositions

Ingrédient	Grade	Supplier	Supplier Part Number	Lot Number
Fusi-BCAA Unflavored (2:1:1 LLeu:L-Ile:L-Val)	Instantized (0.30.9% Lecithin)	Ajinomoto (AjiPure)	33555	OH704
L-Arginine HCl	USP	Sigma (Ajinomoto)	A4599	CDB0352V
L-Arginine HCl	USP	Sigma (Ajinomoto)	A4599	CDB0352V
L-Glutamine	USP	Ajinomoto	32824	R014A003
Glycine	USP	Ajinomoto	30359	R015T008
Acetylcysteine (NAC)	USP	Spectrum Chemical	AC126	1FI0576

	LIVRQNAC	LIVRQNAC+G	LRQNAC
Ingrédient	Daily Dose (g)	Daily Dose (g)	Daily Dose (g)
Fusi-BCAA Unflavored (2:1:1 L-Leu:L- Ile:L-Val)	24.0	24.0
Fusil (L-Leucine)			12.0
L-Arginine HCl	18.0	18.0	18.0
L-Glutamine	24.0	24.0	24.0
Glycine		20.0
Acetylcysteine (NAC)	1.8	1.8	1.8
AMINO ACIDS =	67.8	87.8	55.8

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Test Articles Administration

LIVRQNAC, LIVRQNAC+G, LRQNAC, OCA and Vehicle were administered by oral gavage at a volume of 10 mL/kg throughout the study. Dosages were calculated by daily body weight. LIVRQNAC, LIVRQNAC+G, LRQNAC, and Vehicle were administered twice per day (BID), while OCA was administered once a day (QD) in the moming. Mice receiving OCA once per day (QD), and one vehicle QD. Doses were administered by oral gavage at 0700 and 1800 by oral gavage for 4 weeks.

Body Weight and Blood Glucose

The viability, clinical signs and behavior were monitored daily. Body weight was recorded daily during the dosing period. Blood samples were collected weekly in the AM (0700) via tail clip for glucose measurement (StatStrip glucometer).

Necropsv and Sample Harvest

Animais were anesthetized with CO2 inhalation and exsanguinated via cardiac puncture for euthanasia. Terminal blood samples (K2EDTA) were obtained by cardiac puncture in anesthetized animais at termination. Samples were provided frozen to Axcella Health. Organ weights (total liver, gonadal fat pads) were recorded. Pancréas, and small intestine and gonadal fat pads were fixed in 10% Buffered Formalin and prepared as directed in protocol. A section of small intestine, gonadal fat pad and liver were also snap frozen in liquid nitrogen and shipped to the sponsor.

Histological Analyses

The liver tissues were fixed in Bouin’s solution at 4°C for 24 hours followed by baths of standard concentrations of alcohol then xylene to préparé the tissues for paraffin embedding. After being embedded in paraffin and cooled, five-micron sections were eut and stained for routine H&E and Picric Sirius Red. A section of both right and left lobes of the livers were frozen in OCT for analysis of lipid content with Oil-Red-) staining. The Aperio whole slide digital imaging System (Scan Scope CS, Vista, CA) was used for imaging. Ail slides were imaged at 20x. The scan time ranged from 1.5 minutes to a maximum time of 2.25 minutes. The

248 whole images were housed and stored in their Spectrum software system and images were shot from the whole slides.

The livers were evaluated using the NASH liver criteria for scoring. In this mouse study, one cross section of liver for each case was analyzed with the NASH score System. According to the published NASH CRN Scoring System, this scoring system comprises of NAFLD Activity Score (NAS), fibrosis stage and identification of NASH by pattern récognition. The NAS can range from 0 to 8 and is calculated by the sum of scores of steatosis (0-3), lobular inflammation (0-3) and hépatocyte ballooning (0-2) from H&E stained sections. Fibrosis was scored (0-4) from picrosirius red stained slides. The NASH system is used for human liver 18 gauge biopsies. Steatosis, lobular inflammation, hépatocyte, balloon degeneration, fibrosis, NAS and the presence of NASH by pattern récognition were systematically assessed. In this study we evaluated one total cross section of liver per mouse in this study. This is about 15 times the size of an 18 gauge human liver biopsy. The pathology score was determined as 0, +1, +2, or +3. The lésions were scored on location (periportal, centrilobular, and mid zonal) and fat accumulation (focal, periportal, and/ or centrilobular). The other part of the score was distribution of the lésions: focal, multifocal and/or diffuse. Also, mild, moderate and severity of the lésions. These parameters made up the total NASH score.

Ail immunohistochemical staining steps were performed using the Dako FLEX SYSTEM on an automated immunostainer; incubations were done at room température and Tris buffered saline plus 0.05% Tween 20, pH 7.4 (TBS - Dako Corp.) was used for ail washes and diluents. Thorough washing was performed after each incubation. Primary antibodies included anti-mouse SMA, F4/80, Mac-2, and Picric Sirius Red. Control sections were treated with an isotype control using the same concentration as primary antibodies to verify the staining specificity.

White adipose tissue (WAT) adipocyte size was analyzed from the H&E stained sections. Using the Aperio Image Scope application, 3 localized régions (edge of tissue, tissue not surrounding vascular area, tissue surrounding vascular area) of each tissue specimen were assessed by measuring the area of 10 largest adipocytes of the région. Within each tissue, 10 hot spots of each régions were quantified (um²) and averaged.

Pancreatic beta-islet cells were identified by immunohistochemical staining.

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Image Analysis

Aperio Automatic Image Quantitation was employed to quantify positive pixels of immunohistochemical staining, Oil-Red O, and Sirius Red staining. The Positive Pixel Count algorithm was used to quantify the percentage of a spécifie stain présent in a scanned slide image. A range of color (range of hues and saturation) and three intensity ranges (weak, positive, and strong) were masked and evaluated. The algorithm counted the number and intensity-sum in each intensity range, along with three additional quantities: average intensity, ratio of strong/total number, and average intensity of weak positive pixels. The positive pixel algorithm was modified to distinguish between the orange and blue colors. Alterations from the normal “hue value” (0.1 to 0.96) and “color saturation” (0.04 to 0.29), were made for the Sirius Red évaluation. Vasculature and artifacts were excluded from analysis.

Liver Gene Expression Analysis

Liver gene expression of MCP-1 and ΜΙΡ-la was measured by quantitative PCR.

Liver Cytokine and Chemokine Measurement

Liver IL-lb, MCP-1, and MIP-1 protein levels were quantified using the multiplex ELISA Assay (Meso Scale Discovery, Rockville, Maryland).

Statistical Analysis

Statistical analyses of liver histological scores were performed using Bonferroni Multiple Comparison Test on GraphPad Prism 6 (GraphPad Software Inc., USA). P values < 0.05 were considered statistically significant. Results were expressed as mean ± SEM. Comparisons were made between Group 2 (Vehicle) and the following groups; Group 3 (LIVRQNAC 1,500 mg/kg), Group 4 (LIVRQNAC 3,000 mg/kg), Group 5 (LIVRQNAC+G, 3,885 mg/kg), and (LRQNAC, 2,469 mg/kg).

RESULTS

Body and Liver Weight

Feeding the western diet supplemented with fructose (WDF) for 16 weeks elicited significant effects on body weight compared to control fed animais. Prior to administration of

250 test agent, animais fed the WDF were significantly heavier (47.6 ± 0.45 vs. 43.9 ± 1.03g; p< 0.01) compared to animais fed the control diet.

Body weight decreased compared to baseline values in ail treatment groups; there were no signifîcant différences 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 (1500 mg/kg), LIVRQNAC (3000 mg/kg), LIVRQNAC+G, LRQNAC, and OCA, respectively; p< 0.4992).

Liver weight (% body weight) was significantly higher in vehicle treated animais fed WDF compared to control diet (7.22 ± 0.3 vs. 5.05 ± 0.24%; p< 0.0001); however, in animais fed WDF, no signifîcant effects compared to vehicle were noted in any treatment group (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 (1500 mg/kg), LIVRQNAC (3000 mg/kg), LIVRQNAC+G, LRQNAC, and OCA, respectively; p< 0.7450).

Blood Glucose

Feeding the western diet supplemented with fructose (WDF) for 16 weeks elicited signifîcant effects on glycemia compared to control fed animais. Prior to administration of test agent, animais fed the WDF had significantly lower glucose (160.0 ± 3.01 vs. 218.3 ± 28.6 mg/dL; p< 0.0001) compared to animais fed the control diet.

Blood glucose, although higher in control animais at baseline, remained relatively stable during 4 weeks of compound administration. When averaged over the dosing period, there were no signifîcant différences in average blood glucose compared to vehicle for any treatment group (166.0 ± 9.7, 157.1 ± 4.6, 154.6 ± 2.3, 159.4 ± 3.8, 155.5 ± 3.8, 153.6 ± 3.0 and 169.7 ± 6.3 mg/dL for control, vehicle, LIVRQNAC (1500 mg/kg), LIVRQNAC (3000 mg/kg), LIVRQNAC+G, LRQNAC, and OCA, respectively; p< 0.1587).

Liver Triglycéride and Cholestérol

Liver triglycéride and cholestérol content were similarly elevated after WDF feeding compared to vehicle treated animais fed control diet (liver triglycéride p<0.0040 ; liver cholestérol: p< 0.0001). Among animais fed WDF, there were no signifîcant différences in liver triglycéride (p<0.1206) when compared to vehicle for any treatment group. While OCA reduced

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I liver cholestérol content compared to vehicle by 32% (p<0.05), no amino acid composition treatment group affected liver cholestérol as compared to WDF feeding vehicle group.

Liver	Vehicle	LIVRQNA C 1.5 g/kg	LIVRQNA C 3.0 g/kg	LIVRQNAC+ G	LRQNAC	OCA
Triglycerid	31.49+5.8	47.63+1.19	47.94+1.37	50.57+1.58	49.47+1.4	49.81+1.6
e	5					3
Cholestérol	8.37+0.06	7.74+0.318	7.48+0.697	6.42+0.648	7.84+0.10	5.63+0.49
	5				4	5

Liver Histology

FATZO mice fed with the control diet developed mild steatosis and no inflammation, ballooning, or fibrosis (FIG. 10). FATZO mice fed with the WDF and treated with vehicle developed significant steatosis, mild inflammation, ballooning, and fibrosis. In contrast to predominantly macrovesicular steatosis in the vehicle groups, a mixture of predominantly microvesicular and diminished macrovesicular steatosis was observed in LIVRQNAC, LIVRQNAC+G and LRQNAC groups, as shown in FIG. 11.

The NAFLD activity score is calculated from histological scoring of steatosis (0-3), inflammation (0-3), and ballooning (0-2) in fixed liver tissues. In WDF fed animais, ail amino acid composition treatments produced a significant réduction in the NAS compared to the vehicle treatment group (FIG. 12). LIVRQNAC and amino acid composition treatments reduced liver steatosis as compared to vehicle, although only LIVRQNAC+G and LRQNAC reached statistical significance (p<0.05), while LIVRQNAC did not (LIVRQNAC 3.0 g/kg, p=0.12). Ail amino acid composition treatments significantly attenuated hépatocyte ballooning, the biomarker of lipotoxicity and cell death. Amino acid composition treatments did not significantly alter liver inflammation. In conclusion, amino acid composition-associated improvement of liver pathology is mainly attributed to atténuation of hépatocyte ballooning.

There was no significant effect of OCA on the NAS score and NAS components compared to vehicle.

Liver Vehicle LIVRQNAC LIVRQNAC LIVRQNAC+G LRQNAC OCA

Pathology 1.5 g/kg 3.0 g/kg

NAS 3.65+0.183 2.70+0.213 2.89+0.111 2.83+0.186 2.72+0.147 3.72+0.147

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Steatosis	1.8+0.133	1.6+0.163	1.44+0.176	1.33+0.167	1.33+0.167	1.78+0.147
Inflammation	0.9+0.1	1.0+0.0	1.0+0.0	1.0+0.0	1.0+0.0	1.0+0.0
Ballooning	0.95+0.05	0.1+0.1	0.44+0.176	0.50+0.144	0.39+0.111	0.94+0.056

Livers from vehicle treated animais demonstrated a mild fibrosis; score of 0.8+0.1. Only livers from animais treated with LIVRQNAC (1500 mg/kg) demonstrated a significant réduction in fibrosis when compared to the vehicle treated group, (0.2±0.1 versus 0.8±0.1, p<0.01), but not with LIVRQNAC (3000 mg/kg), LIVRQNAC+G or LRQNAC. Sirius Red collagen staining demonstrated that ail amino acid composition treatments had signifïcantly lower collagen déposition compared to vehicle (LIVRQNAC 1500 mg/kg, p<0.01; LIVRQNAC 3000 mg/kg, p<0.01; LIVRQNAC+G, p=0.09; LRQNAC, p<0.05). OCA did not affect liver fibrosis score or Sirius Red collagen staining area.

Fibrosis	Vehicle	LIVRQNAC 1.5g/kg	LIVRQNAC 3.0g/kg	LIVRQNAC+G	LRQNAC	OCA
Fibrosis	0.8+0.133	0.2+0.133	0.44+0.176	0.44+0.176	0.33+0.167	0.67+0.167
Sirius	1.82+0.279	0.77+0.116	0.72+0.092	0.107+0.218	0.79+0.183	1.59+0.36
Red

Consistent with liver triglycéride levels, amino acid composition treatments did not alter liver Oil Red O staining area compared to vehicle group. OCA reduced Oil Red O staining area (p<0.05).

Oil Red O	Vehicle	LIVRQNA C 1.5g/kg	LIVRQNA C 3.0g/kg	LIVRQNAC+ G	LRQNAC	OCA
OU Red O	0.32+0.01	0.28+0.022	0.30+0.022	0.26+0.023	0.29+0.01	0.24+0.02
	9				8	1
Triglycerid	31.49+5.8	47.63+1.19	47.94+1.37	50.57+1.58	49.47+1.4	49.81+1.6
e	5					3

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Liver Gene Expression

MCP-1 (CCL2) and ΜΙΡ-la (CCL3) are proinflammatory chemokines that médiate liver inflammation via macrophage and neutrophil recruitment. MCP-1 and ΜΙΡ-la are the ligands of CCR2 and CCR5, respectively, which serve the promising therapeutic targets to treat liver fibrosis in NASH. MCP-1 and ΜΙΡ-la RNA expression levels in the liver were significantly upregulated in the WDF fed mice as compared to control diet-fed mice, as shown in Tables 74 and 75.

Table 74. Fold change in MCP-1 mRNA levels after administration of amino acid compositions

MCP-1	Ctrl diet	Vehicle	LIVRQNAC (3000 mg/kg)	LIVRQNAC+G (3885 mg/kg)	LRQNAC (2469 mg/kg)
Mean	0.1457	1.079	1.396	0.6102	0.8777
SEM	0.0291	0.1956	0.3414	0.09597	0.2315

Table 75. Fold change in ΜΙΡ-la mRNA levels after administration of amino acid compositions

MlP-la	Ctrl diet	Vehicle	LIVRQNAC (3000 mg/kg)	LIVRQNAC+G (3885 mg/kg)	LRQNAC (2469 mg/kg)
Mean	0.08328	1.194	1.67	0.814	1.514
SEM	0.02141	0.25	0.3366	0.1029	0.525

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LIVRQNAC and LRQNAC treatments did not significantly alter liver MCP-1 and MIPla RNA expression as compared to vehicle group. LIVRQNAC+G treatment resulted in slightly lower liver MCP-1 RNA expression as compared to vehicle group (p=0.054) and LIVRQNAC group (p<0.05). Similarly, LIVRQNAC+G treatment resulted in slightly lower liver MCP-1 . RNA expression as compared to vehicle group although the différence was not significant (p=0.19) and LIVRQNAC group (p<0.05).

Liver Chemokines and Cytokines

Consistent with RNA data (FIG. 25), liver MCP-1 and ΜΙΡ-la protein levels were elevated in the WDF fed mice as compared to control diet-fed mice, as shown in Tables 76 and 77.

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Table 76. Mean liver MCP-1 protein levels after administration of amino acid compositions

MCP-1	Ctrl diet	Vehicle	LIVRQNAC (3000 mg/kg)	LIVRQNAC+G (3885 mg/kg)	LRQNAC (2469 mg/kg)
Mean	41.47	278.3	392	221.1	247.1
SEM	7.463	61.41	83.97	36.6	75.16

Table 77. Mean liver ΜΙΡ-la protein levels after administration of amino acid compositions

MlP-la	Ctrl diet	Vehicle	LIVRQNAC (3000 mg/kg)	LIVRQNAC+G (3885 mg/kg)	LRQNAC (2469 mg/kg)
Mean	23.16	191.8	282.9	142.8	141.1
SEM	5.429	30.03	58.88	17.04	24.36

Liver MCP-1 and MIP-la protein levels were also positively correlated with RNA expression levels, as shown in Tables 78 and 79.

Table 78. Corrélations between MCP-1 protein and RNA levels after administration of amino acid compositions

Ctrl diet	y=0.0022x+0.0542	R2=0.3202
Vehicle	y=0.0029x+0.3316	R2=0.7986
LIVRQNAC (3000 mg/kg)	y=0.0036x+0.0144	R2=0.7831
LIVRQNAC+G (3885 mg/kg)	y=0.0018x+0.2542	R2=0.3988
LRQNAC (2469 mg/kg)	y=0.0027x+0.2969	R2=0.6857

Table 79. Corrélations between ΜΙΡ-la protein and RNA levels after administration of amino acid compositions

Ctrl diet	y=0.001x+0.0593	R2=.O69
Vehicle	y=0.0057x+0.191	R2=0.4202
LIVRQNAC (3000 mg/kg)	y=0.0051x+0.2334	R2=0.7887
LIVRQNAC+G (3885 mg/kg)	y=0.0045x+0.1817	R2=0.4403
LRQNAC (2469 mg/kg)	y=0.0064x+0.1814	R2=0.4875

LIVRQNAC and LRQNAC treatments did not significantly alter liver MCP-1 and MIPla protein levels as compared to vehicle group. LTVRQNAC+G treatment slightly lowered liver

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MCP-1 (p=0.095) and ΜΙΡ-la (p<0.05) protein levels as compared to LIVRQNAC group.

Additionally, liver MCP-1 and ΜΙΡ-la protein levels positively correlated, as shown in Table 80.

Table 80. Corrélations between MCP-1 and ΜΙΡ-la protein levels after administration of 5 amino acid compositions
	Ctrl diet	y=0.6803x-5.0537	R2=0.8744
Vehicle	y=0.389x+83.574	R2=0.6325
LIVRQNAC (3000 mg/kg)	y=0.6615x+23.609	R2=0.8903
LIVRQNAC+G (3885 mg/kg)	y=0.4437x+44.728	R2=0.9082
LRQNAC (2469 mg/kg)	y=0.3108x+75.901	R2=0.9241
Proinflammatory cytokines IL-lb, IL-6, TNFa, and CXCL1 protein levels in liver were elevated in the WDF fed mice as compared to control diet-fed mice, as shown in Tables 81-84. 10 Table 81. Mean liver IL-lb protein levels after administration of amino acid compositions
	IL-lb	Ctrl diet	Vehicle	LIVRQNAC (3000 mg/kg)	LIVRQNAC+G (3885 mg/kg)	LRQNAC (2469 mg/kg)
Mean	10.53	22.31	22.11	14.42	28.85
SEM	1.248	6.063	5.739	3.299	10.41
Table 82. Mean liver IL-6 protein levels after administration of amino acid compositions
	IL-6	Ctrl diet	Vehicle	LIVRQNAC (3000 mg/kg)	LIVRQNAC+G (3885 mg/kg)	LRQNAC (2469 mg/kg)
Mean	5.838	8.452	7.298	5.77	6.71
SEM	0.3536	2.723	2.043	1.06	1.625
Table 83. Mean liver CXCL1 protein levels after administration of amino acid 15 compositions
	CXCL1	Ctrl diet	Vehicle	LIVRQNAC (3000 mg/kg)	LIVRQNAC+G (3885 mg/kg)	LRQNAC (2469 mg/kg)
Mean	41.42	105.9	119.6	77.77	84.9
SEM	2.487	13.26	26.15	8.023	10.49
Table 84. Mean liver TNFa protein levels after administration of amino acid compositions
	TNFa	Ctrl diet	Vehicle	LIVRQNAC (3000 mg/kg)	LIVRQNAC+G (3885 mg/kg)	LRQNAC (2469 mg/kg)
Mean	1.703	3.71	4.574	2.974	4.119
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I SEM I 0,5641 | 0.4647 | 0.5654 | 0.1513 | 0.8341 |

LIVRQNAC, LIVRQNAC+G, and LRQNAC treatments did not signifïcantly alter IL-lb, IL-6, TNFa, and CXCL1 protein levels as compared to vehicle. Liver TNFa levels were lower by LIVRQNAC+G treatment as compared to LIVRQNAC.

SUMMARY

Based on clinical observations, WDF-fed FATZO mice gained more body weight that those fed with a control diet. Fed blood glucose levels were comparable between WDF-fed and control diet-fed mice despite of the différence in body weight change. Ail treatments were well 10 tolerated in FATZO mice. Both WDF-fed and control diet-fed mice lose body weight during the treatment period, which may be due to the stress associated with administration of test articles or vehicle via oral gavage twice a day.

NAS was signifïcantly attenuated in ail amino acid composition treatment groups as compared to vehicle, predominantly attributing to ballooning score. Hépatocyte ballooning was 15 signifïcantly reduced in ail the amino acid composition treatment groups. Steatosis was signifïcantly reduced in LIVRQNAC+G and LRQNAC treatment groups. LIVRQNAC also lowered steatosis, although the différence was not significant. Inflammation was not affected by amino acid composition treatments. Despite the histological improvement in steatosis score in LIVRQNAC+G and LRQNAC treatment groups, liver triglycéride, cholestérol, and Oil-Red O 20 staining remained unchanged by amino acid composition treatments. Consistent with the histological and biochemical data, de novo lipogenesis enzymes FASN and ACACA RNA levels were not affected by amino acid composition treatment.

Although liver triglycéride levels were not affected by amino acid composition treatments, the characteristics of hépatocyte steatosis were differed by amino acid composition 25 treatments. Liver of the WDF-fed mice (vehicle group) demonstrated predominantly macrovesicular steatosis. In contrast, macrovesicular steatosis was diminished, and a mixture of microvesicular and macrovesicular steatosis in ail amino acid composition treatment groups. The biological meaning and mechanism of amino acid compositions on macro- to microvesicular steatosis phenotypes merit further investigation.

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Liver fibrosis score in FATZO model of NAFLD was significantly attenuated by LIVRQNAC treatment at low dose but not at high dose. LIVRQNAC+G and LRQNAC had no effect on fibrosis. Nonetheless, Sirius Red collagen staining demonstrated that LIVRQNAC, LIVRQNAC+G and LRQNAC significantly reduced collagen déposition in the liver.

Consistent with liver inflammation scores, liver RNA and protein levels of the proinflammatory chemokine MCP-1 and ΜΙΡ-la and cytokines IL-lb, IL-6, TNFa, and CXCL1 were not significantly affected by amino acid composition treatment. It is of interest to note that LIVRQNAC+G (équivalent to LIVRQNAC plus Glycine) treatment had lower liver MCP-1, ΜΙΡ-la, and TNFa as compared to LIVRQNAC.

Increased liver oxidative stress associated with inflammation is observed during NAFLD and NASH. Glutathione (GSH) is a pivotai endogenous anti-oxidant which can counteract reactive oxygen species. Glycine and its direct metabolic precursor, serine, are substrates for GSH biosynthesis. Thus, serine and/or glycine supplémentation helps replenish GSH and améliorâtes NAFLD and NASH. LIVRQNACG treatment had lower inflammation chemokines and cytokines in the liver, supporting that supplémentation of glycine or serine is bénéficiai in NAFLD and NASH.

In conclusion, ail three amino acid compositions (LIVRQNAC, LIVRQNAC+G and LRQNAC) tested in FATZO mice attenuate NAFLD activity scores, hépatocyte ballooning, and fibrosis. These amino acid compositions can be used to treat NASH. Glycine-containing amino 20 acid compositions can further reduce liver inflammation which results in reduced liver fibrosis.

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

Ail references, issued patents and patent applications cited within the body of the instant spécification are hereby incorporated by reference in their entirety, for ail purposes.

Claims

CLAIMS | 1. A composition comprising: (a) L-leucine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, | 5 comprising L-leucine; (b) an arginine amino acid entity chosen from: | (i) L-arginine or a sait thereof, (ii) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-arginine, | (iii) omithine or a sait thereof,

| (1) a wt. % of the L-glutamine or a sait thereof or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-glutamine of (c) in the composition is greater than the wt. % of the | arginine amino acid entity of (b);

15 (2) the wt. % ofthe L-glutamine or a sait thereof or a dipeptide or sait thereof, or | tripeptide or sait thereof, comprising L-glutamine of (c) in the composition is greater than or equal to the wt. % of the leucine amino acid entity of (a);

(1) the ratio of the leucine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-leucine to the arginine amino acid entity is at least 1:4, and not more than 3:4;

(1) the wt. % of the L-glutamine or a sait thereof, or the dipeptide or sait thereof, or | tripeptide or sait thereof, comprising L-glutamine in the composition is:

(i) greater than the wt. % of the arginine amino acid entity; or | (ii) equal to or greater than the wt. % of the leucine or a sait thereof, or a dipeptide

30 or sait thereof, or tripeptide or sait thereof, comprising L-leucine;

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I (2) the wt. % of the arginine amino acid entity in the composition is greater than the wt. % of the L-leucine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-leucine; or (3) the wt. % of the arginine amino acid entity in (b), the L-glutamine or a sait thereof or the dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-glutamine in (c), and the NAC or a sait thereof or the dipeptide or sait thereof, or tripeptide or sait thereof, comprising NAC in (d) in combination is at least 50% of the composition, when the composition is in powder form, but not more than 90% of the composition, when the composition is in powder form.
(2) the ratio of the leucine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-leucine to the L-glutamine or a sait thereof, or the dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-glutamine is at least 1:4, and not more than 3:4;

| 2. The composition of claim 1, wherein méthionine, tryptophan, and cysteine are présent at less than 2 wt. % of the composition when the composition is in powder form; or | wherein méthionine, tryptophan, or cysteine are absent in the composition.

| 25 3. The composition of claim 1 or 2, wherein:
| (3) the wt. % ofthe arginine amino acid entity of (b) in the composition is greater than the wt. % of the leucine amino acid entity of (a); or | 20 (4) a combination oftwo or three of (1)-(3).

| 20. The composition for use of any one of claims 13-19, wherein:

(i) (a)-(d) are each a free amino acid or a sait thereof, and wherein the composition | further comprises L-isoleucine or a sait thereof and L-valine or a sait thereof;

25 (ii) (a)-(d) are each a free amino acid or a sait thereof, and wherein the composition fl further comprises L-isoleucine or a sait thereof and L-valine or a sait thereof, and wherein a ratio of the L-leucine or a sait thereof of (a), the L-isoleucine or a sait thereof, the L-valine or a sait I thereof, the L-arginine or a sait thereof of (b), the L-glutamine or a sait thereof of (c), and the

NAC or a sait thereof of (d) is, respectively, 1 +/- 15% : 0.5 +/-15%: 0.5 +/- 15%: 1.5 +/- 15%: 2 | 30 +/-15%: 0.15+/- 15%; or

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I (iii) (a)-(d) are each a free amino acid or a sait thereof, and wherein the composition | further comprises L-isoleucine or a sait thereof, L-valine or a sait thereof, and L-serine, and wherein a ratio of the L-leucine or a sait thereof of (a), the L-isoleucine or a sait thereof, the L| valine or a sait thereof, the L-arginine or a sait thereof of (b), the L-glutamine or a sait thereof of 5 (c), the NAC or a sait thereof of (d), and the L-serine is, respectively, 12 +/- 15% : 6 +/- 15% : 3 | +/- 15% : 9 +/-15% : 12 +/-15% : 2.7 +/-15% : 18 +/-15%.

| 21. A pharmaceutical composition for use in treating liver dysfunction or liver disease in a human, wherein the pharmaceutical composition consists essentially of:

(3) the ratio of the arginine amino acid entity to the L-glutamine or a sait thereof, or the dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-glutamine is at least 1:4, and not more than 6:7; or (4) the wt. ratio of: (a) the leucine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-leucine, (b) the arginine amino acid entity, (c) the Lglutamine or a sait thereof, or the dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-glutamine, and (d) the NAC or sait thereof, or the dipeptide or sait thereof, or tripeptide or sait thereof, comprising NAC is, respectively, 1 +/- 15% : 1.5 +/- 15%: 2 +/-15%: 0.15 +/- 15%; 1 +/- 15%: 1.5 +/-15% : 2 +/-15% : 0.3 +/- 15%; or 1 +/- 15%: 0.75 +/-15% : 1 +/- 15% : 0.225 +/- 15%.
4. The composition of any one of claims 1-3, wherein:
5 thereof, comprising L-valine; or | (b) one or both of the arginine amino acid entity or the L-glutamine or a sait thereof, or the dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-glutamine are | présent at a higher amount (wt. %) than the leucine amino acid entity.

5 (i) L-leucine or a sait thereof, (ii) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-leucine, or (iii) P-hydroxy-P-methylbutyrate (HMB) or a sait thereof;

(b) an arginine amino acid entity chosen from:

5. The composition of any one of claims 1-4, further comprising one or both of:

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I (1) L-isoleucine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait | thereof, comprising L-isoleucine; or (2) L-valine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, | comprising L-valine.
| 6. The composition of claim 5, wherein the composition comprises:

(a) L-leucine or a sait thereof;

| (b) L-arginine or a sait thereof;

(c) L-glutamine or a sait thereof;
7. The composition of any one of claims 1-6, wherein:

| 20 (i) at least one of (a)-(d) is a free amino acid;

(ii) at least one of (a)-(d) is in a sait form;

| (iii) the composition comprises a combination of not more than 15 different amino acid entities; or | (iv) the composition further comprises a serine amino acid entity chosen from L-serine or

25 a sait thereof, L-glycine, or a combination thereof.
8. The composition of any one of claims 1-7, wherein:

| the arginine amino acid entity is chosen from:

i) L-arginine or a sait thereof, | 30 ii) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-arginine, iii) omithine or a sait thereof, or

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I iv) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising omithine.
9. The composition of any one of claims 1-8, wherein the wt. % of the NAC or a sait thereof, or the dipeptide or sait thereof, or tripeptide or sait thereof, comprising NAC is at least 5 1%, but not more than 10% of the composition, when the composition is in powder form.
| 10 (a) L-leucine or a sait thereof;

(b) L-arginine or a sait thereof;

| (c) L-glutamine or a sait thereof;

(d) N-acetylcysteine (NAC) or a sait thereof;

| (e) L-isoleucine or a sait thereof;

| 10 19. The composition for use of any one of claims 13-18, wherein the composition comprises:

10 (i) L-arginine or a sait thereof, (ii) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-arginine, (iii) omithine or a sait thereof, (iv) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising omithine, (v) creatine or a sait thereof, or

15 (vi) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising creatine;

(c) L-glutamine or a sait thereof or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-glutamine; and (d) N-acetylcysteine (NAC) or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising NAC, .

wherein when the composition is in powder form at least 50 wt. % ofthe total wt. ofthe composition is one or more amino acid entities in free form, and wherein the total wt. % of (a)20 (d) is greater than the total wt. % of other amino acid entities in the composition.

17. The composition for use of claim 16, wherein the fatty liver disease is:

(i) non-alcoholic fatty liver (NAFL) or non-alcoholic fatty liver disease (NAFLD);

25 (ii) non-alcoholic steatohepatitis (NASH); or (iii) alcoholic fatty liver disease (AFLD) or alcoholic steatohepatitis (ASH).

18. The composition for use of any one of claims 13-17, wherein: (a) the composition further comprises:

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I ( i) a serine amino acid entity chosen from L-serine or glycine;

| (ii) L-isoleucine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-isoleucine; or | (iii) L-valine or a sait thereof or a dipeptide or sait thereof, or tripeptide or sait

10. A dietary composition comprising the composition of any one of claims 1-9.

| 10 (d) NAC or a sait thereof;

(e) L-isoleucine or a sait thereof; and | (f) L-valine or a sait thereof;

and/or wherein the wt. ratio of the L-leucine or a sait thereof, the L-isoleucine or a sait | thereof, the L-valine or a sait thereof, the L-arginine or a sait thereof, the L-glutamine or a sait

15 thereof, and the NAC or sait thereof is, respectively, 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%: 1.5+/- 15% : 2 +/15%: 0.3+/- 15%.

10 (iv) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising omithine, | (v) creatine or a sait thereof, or (vi) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising creatine;

| ' (c) L-glutamine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-glutamine; and j 15 (d) N-acetylcysteine (NAC) or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising NAC;

| wherein when the composition is in powder form, at least 50 wt. % of the total wt. of the composition is one or more amino acid entities in free form, and wherein the total wt. % of | (a)-(d) is greater than the total wt. % of other amino acid entities in the composition.
11. A pharmaceutical composition comprising the composition of any one of claims 1 -9.
12. A pharmaceutical composition consisting essentially of:

(a) L-leucine or a sait thereof;

(b) L-arginine or a sait thereof;

(c) L-glutamine or a sait thereof;

15 (d) NAC or a sait thereof;

(e) L-isoleucine or a sait thereof;

(f) L-valine or a sait thereof; and (g) one or more pharmaceutically acceptable excipients.

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13. A composition for use in treating liver dysfunction or liver disease in a human subject, wherein the composition comprises:

(a) a leucine amino acid entity chosen from:

(i) L-leucine or a sait thereof, (ii) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-leucine, 25 or (iii) P-hydroxy-P-methylbutyrate (HMB) or a sait thereof;

(b) an arginine amino acid entity chosen from:

(i) L-arginine or a sait thereof, (ii) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-arginine, 30 (iii) omithine or a sait thereof,

262 (iv) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising ornithine, (v) creatine or a sait thereof, or (vi) a dipeptide or sait thereof, or tripeptide or sait thereof, comprising creatine;

(c) L-glutamine or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising L-glutamine; and (d) N-acetylcysteine (NAC) or a sait thereof, or a dipeptide or sait thereof, or tripeptide or sait thereof, comprising NAC, wherein when the composition is in powder form at least 50 wt. % of the total wt. of the composition is one or more amino acid entities in free form, and wherein the total wt. % of (a)(d) is greater than the total wt. % of other amino acid entities in the composition.
14. The composition for use of claim 13, wherein the subject has:

(i) a disease or disorder selected from non-alcoholic fatty liver (NAFL), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic fatty liver disease (AFLD), or alcoholic steatohepatitis (ASH); or (ii) cirrhosis, hepatocarcinoma, metabolic syndrome, type 2 diabètes, obesity, or a combination thereof.

15. The composition for use of claim 13 or 14, wherein administration of the composition results in two or more of:

decreasing or preventing liver fibrosis;

decreasing or preventing liver injury;

decreasing or preventing hépatocyte inflammation;

improving glucose tolérance;

decreasing or preventing steatosis;

decreasing or preventing hépatocyte ballooning;

improving gut function;

decreasing or preventing fîbrogenic gene expression;

decreasing or preventing liver or macrophage expression or sécrétion of inflammatory cytokines or chemokines; or decreasing or preventing hépatocyte lipid accumulation.

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16. A composition for use in treating a fatty liver disease, wherein the composition comprises:

(a) a leucine amino acid entity chosen from:
15 (f) L-valine or a sait thereof; and | (g) one or more pharmaceutically acceptable excipients.