METHODS OF PREDICTING LIVER FAT CONTENT AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/212,003, filed 17 June 2021 and U.S. Provisional Application No. 63/270,444, filed 21 October 2021, the contents of each of which are hereby incorporated by reference in their entirety.

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 prevalent 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).

Proton density fat fraction (PDFF), calculated based on magnetic resonance imaging (MRI) readings, is one approach to assessing liver fat content and grading steatohepatitis. However, MRI requires specialized equipment and technical support. There is a need for alternative methods for assessing liver fat content and grading steatohepatitis.

SUMMARY

Disclosed herein, at least in part, is a method for evaluating a subject by acquiring a value of liver fat content.

In one aspect, the disclosure provides a method of evaluating a subject (e.g., evaluating the effectiveness of an amino acid composition in a subject), comprising acquiring a value of liver fat content for the subject.

In some embodiments, the value of liver fat content comprises a measure of: i) a level of threonine (e.g., wherein the level of threonine comprises a percent change from a baseline level of threonine); ii) a level of lysine (e.g., wherein the level of lysine comprises a percent change from a baseline level of lysine); iii) a level of ornithine (e.g., wherein the level of ornithine comprises a percent change from a baseline level of ornithine); iv) a level of asparagine (e.g., wherein the level of asparagine comprises a percent change from a baseline level of asparagine); and v) a level of serine (e.g., wherein the level of serine comprises a percent change from a baseline level of serine).

The present disclosure also provides methods of evaluating the effectiveness of an amino acid composition in a subject. In some embodiments, the method of evaluating the effectivness of an amino acid composition includes acquiring a value of liver fat content for the subject. In some embodiments, the value of liver fat content comprises a measure of: i) a level of threonine (e.g., wherein the level of threonine comprises a percent change from a baseline level of threonine); ii) a level of lysine (e.g., wherein the level of lysine comprises a percent change from a baseline level of lysine); iii) a level of ornithine (e.g., wherein the level of ornithine comprises a percent change from a baseline level of ornithine); iv) a level of asparagine (e.g., wherein the level of asparagine comprises a percent change from a baseline level of asparagine); and v) a level of serine (e.g., wherein the level of serine comprises a percent change from a baseline level of serine).

In another aspect, the disclosure provides a method of treating a subject having a liver disease (e.g., NASH). In some embodiments, the method comprises: a) administering to the subject a effective amount of an amino acid composition comprising a L-amino acid entity, optionally an I-amino acid entity, optionally a V- amino acid entity, an R-amino acid entity, a Q-amino acid entity, and aNAC entity; and b) acquiring a value of liver fat content for the subject. In some embodiments, acquiring the value of liver fat content comprises a measure of one or more of (e.g., 2, 3, 4, or all of): i) a level of threonine (e.g., wherein the level of threonine comprises a percent change from a baseline level of threonine); ii) a level of lysine (e.g., wherein the level of lysine comprises a percent change from a baseline level of lysine); iii) a level of ornithine (e.g., wherein the level of ornithine comprises a percent change from a baseline level of ornithine); iv) a level of asparagine (e.g., wherein the level of asparagine comprises a percent change from a baseline level of asparagine); or v) a level of serine (e.g., wherein the level of serine comprises a percent change from a baseline level of serine); thereby treating the subject.

In some embodiments of any of the methods disclosed herein, wherein acquiring a value of liver fat content further comprises a measure of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or all of): vi) a level of creatine (e.g., wherein the level of creatine comprises a percent change from a baseline level of creatine); vii) a level of phenylalanine (e.g., wherein the level of phenylalanine comprises a percent change from a baseline level of phenylalanine); viii) a level of acetyl-l-camitine (e.g., wherein the level of acetyl-l-camitine comprises a percent change from a baseline level of acetyl-l-carnitine); ix) a level of glutamate (e.g., wherein the level of glutamate comprises a percent change from a baseline level of glutamate); x) a level of glutamine (e.g., wherein the level of glutamine comprises a percent change from a baseline level of glutamine); xi) a level of methionine (e.g., wherein the level of methionine comprises a percent change from a baseline level of methionine); xii) a level of proline (e.g., wherein the level of proline comprises a percent change from a baseline level of proline); xiii) a level of histidine (e.g., wherein the level of histidine comprises a percent change from a baseline level of histidine); xiv) a level of carnitine (e.g., wherein the level of carnitine comprises a percent change from a baseline level of carnitine); or xv) a level of tyrosine (e.g., wherein the level of tyrosine comprises a percent change from a baseline level of tyrosine).

In some embodiments, acquiring a value of liver fat content for the comprises a measure of one or more (e.g., 2, 3, 4, 5, or all) of: i) a level of threonine (e.g., wherein the level of threonine comprises a percent change from a baseline level of threonine); ii) a level of ornithine (e.g., wherein the level of ornithine comprises a percent change from a baseline level of ornithine); iii) a level of lysine (e.g., wherein the level of lysine comprises a percent change from a baseline level of lysine); iv) a level of glutamate (e.g., wherein the level of glutamate comprises a percent change from a baseline level of glutamate); v) a level of serine (e.g., wherein the level of serine comprises a percent change from a baseline level of serine); and vi) a level of phenylalanine (e.g., wherein the level of phenylalanine comprises a percent change from a baseline level of phenylalanine).

In another aspect, the disclosure provides methods of evaluating the effectivenss of an amino acid composition in a subject. In some embodiments, the method of evaluating the effectivness of an amino acid composition includes acquiring a value of liver fat content for the subject. In some embodiments, the value of liver fat content comprises the measure of one or more (e.g., 2, 3, 4, 5, or all) of: i) a level of threonine (e.g., wherein the level of threonine comprises a percent change from a baseline level of threonine); ii) a level of ornithine (e.g., wherein the level of ornithine comprises a percent change from a baseline level of ornithine); iii) a level of lysine (e.g., wherein the level of lysine comprises a percent change from a baseline level of lysine); iv) a level of glutamate (e.g., wherein the level of glutamate comprises a percent change from a baseline level of glutamate); v) a level of serine (e.g., wherein the level of serine comprises a percent change from a baseline level of serine); and vi) a level of phenylalanine (e.g., wherein the level of phenylalanine comprises a percent change from a baseline level of phenylalanine).

In another aspect, the disclosure provides a method of treating a subject having a liver disease (e.g., NASH). In some embodiments, the method comprises: a) administering to the subject a effective amount of an amino acid composition comprising a L-amino acid entity, optionally an I-amino acid entity, optionally a V- amino acid entity, an R-amino acid entity, a Q-amino acid entity, and aNAC entity; and b) acquiring a value of liver fat content for the subject. In some embodiments, acquiring the value of liver fat content comprises a measure of one or more of (e.g., 2, 3, 4, or all of): i) a level of threonine (e.g., wherein the level of threonine comprises a percent change from a baseline level of threonine); ii) a level of ornithine (e.g., wherein the level of ornithine comprises a percent change from a baseline level of ornithine); iii) a level of lysine (e.g., wherein the level of lysine comprises a percent change from a baseline level of lysine); iv) a level of glutamate (e.g., wherein the level of glutamate comprises a percent change from a baseline level of glutamate); v) a level of serine (e.g., wherein the level of serine comprises a percent change from a baseline level of serine); and vi) a level of phenylalanine (e.g., wherein the level of phenylalanine comprises a percent change from a baseline level of phenylalanine).

In some embodiments, of any of the methods disclosed herein, acquiring a value of liver fat content futher comprises the measure of one or more (e.g., 2, 3, or all) of: vii) a level of creatine (e.g., wherein the level of creatine comprises a percent change from a baseline level of creatine); viii) a level of leucine (e.g., wherein the level of leucine comprises a percent change from a baseline level of leucine); ix) a level of glycine (e.g., wherein the level of glycine comprises a percent change from a baseline level of glycine); x) a level of arginine (e.g., wherein the level of arginine comprises a percent change from a baseline level of arginine).

In some embodiments of any of the methods disclosed herein, acquiring a value of liver fat content further comprises a measure of a level of 2-hydroxybutyric acid (e.g., wherein the level of 2-hydroxybutyric acid comprises a percent change from a baseline level of 2- hydroxybutyric acid).

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content comprises a measure of the level of threonine, the level of ornithine, and the level of lysine.

In some embodiments, acquiring the value of liver fat content comprises measuring the level of no more than 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, or 15 amino acids or metabolites thereof.

In some embodiments, acquiring the value of liver fat content comprises measuring the level of no more than 5, 6, 7, 8, 9, 10, 11, 2, 13, 14, or 15 analytes.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content comprises measuring the level of threonine, the level of glutamine, the level of lysine, the level of ornithine, and the level of asparagine.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content comprises measuring the level of threonine and ornithine.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content comprises measuring the level of threonine, ornithine, and lysine.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content comprises measuring the level of threonine, ornithine, and glutamate.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content comprises measuring the level of threonine, ornithine, serine, and phenylalanine.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content comprises measuring the level of threonine, ornithine, glutamate, and lysine.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content measuring the level of threonine, ornithine, lysine, serine, and phenylalanine.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content measuring the level of threonine, ornithine, glutamate, serine, and phenylalanine. In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content measuring the level of threonine, ornithine, lysine, glutamate, serine, and phenylalanine.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content further comprises measuring one or more of: a) the level of arginine and glycine; b) the level of creatine; or c) the level of leucine.

In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, and creatine. In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, and phenylalanine. In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, and acetyl-L-carnitine.

In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, and glutamate. In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, glutamate, and glutamine. In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, glutamate, glutamine, and methionine. In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-camitine, glutamate, glutamine, methionine, and proline. In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-camitine, glutamate, glutamine, methionine, proline, and histidine. In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, glutamate, glutamine, methionine, proline, histidine, and carnitine. In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-camitine, glutamate, glutamine, methionine, proline, and histidine. In some embodiments, the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-camitine, glutamate, glutamine, methionine, proline, histidine, carnitine, and tyrosine.

In some embodiments, the measuring comprises using a separation step followed by a detection step. In some embodiments, the separation step is selected from HPLC, UPLC, ion exchange chromatography (IEC), gas chromatography, and capillary electrophoresis (CE). In some embodiments, the detection step is selected from mass spectrometry (MS) (e.g., tandem mass spectrometry (MS/MS)).

In some embodiments of any of the methods disclosed herein, the subject was treated with an amino acid composition prior to measurement of the level of threonine, the level of glutamine, the level of lysine, the level of ornithine, and the level of asparagine, e.g., about 1, 2, 3, 4, 6, 8, 10, 12, 14, or 16 weeks prior.

In some embodiments of any of the methods disclosed herein, the subject was treated with an amino acid composition prior to measurement of the level of threonine, the level of serine, the level of lysine, the level of ornithine, the level of phenylalanine, and the level of glutamate, e.g., about 1, 2, 3, 4, 6, 8, 10, 12, 14, or 16 weeks prior.

In some embodiments, the subject has, or is identified as having, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), or alcoholic fatty liver disease (AFLD).

In some embodiments, the subject has, or is identified as having, diabetes, e.g., type II diabetes.

In some embodiments, the subject is not diabetic.

In some embodiments, after the subject received the amino acid composition, the subject experienced a reduction in liver fat content of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

In some embodiments, an MRI is contraindicated for the subject.

In some embodiments, the subject is evaluated after a determination that the subject is claustrophobic, is too large to fit in a standard MRI scanner (e.g., is greater than about 300, 350, 400, 450, or 500 lbs), or has a metallic implant. In some embodiments, the subject, e.g., prior to treatment, has one or more of (e.g., two or all of) a proton density fat fraction (PDFF) >10%, a corrected T1 [cTl] >830 msec by multiparametric magnetic resonance imaging (MRI), and fasting aspartate aminotransferase >20 IU/L. In some embodiments, the baseline level of threonine is the level of threonine in a biological sample from the subject, wherein the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of i). In some embodiments, the baseline level of lysine is the level of lysine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of ii). In some embodiments, the baseline level of ornithine is the level of ornithine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of iii). In some embodiments, the baseline level of asparagine is the level of asparagine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of iv). In some embodiments, the baseline level of serine is the level of serine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of v). In some embodiments, the baseline level of creatine is the level of creatine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of vi). In some embodiments, the baseline level of phenylalanine is the level of phenylalanine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of vii). In some embodiments, the baseline level of acetyl-l-camitine is the level of acetyl-l-carnitine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of viii). In some embodiments, the baseline level of glutamate is the level of glutamate in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of ix). In some embodiments, the baseline level of glutamine is the level of glutamine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of x). In some embodiments, the baseline level of methionine is the level of methionine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of xi). In some embodiments, the baseline level of proline is the level of proline in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of xii). In some embodiments, the baseline level of histidine is the level of histidine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of xiii). In some embodiments, the baseline level of carnitine is the level of carnitine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of xiv). In some embodiments, the baseline level of tyrosine is the level of tyrosine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of xv). In some embodiments, the baseline level of 2-hydroxybutyric acid is the level of 2-hydroxybutyric acid in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of 2-hydroxybutyric acid.

In some embodiments, the baseline level of threonine is the level of threonine in a biological sample from the subject, wherein the biological sample was collected before administration of the amino acid composition to the subject.

In some embodiments, the baseline level of lysine is the level of lysine in a biological sample from the subject, wherein the biological sample was collected before administration of the amino acid composition to the subject.

In some embodiments, the baseline level of ornithine is the level of ornithine in a biological sample from the subject, wherein the biological sample was collected before administration of the amino acid composition to the subject.

In some embodiments, the baseline level of asparagine is the level of asparagine in a biological sample from the subject, wherein the biological sample was collected before administration of the amino acid composition to the subject.

In some embodiments, the baseline level of serine is the level of serine in a biological sample from the subject, wherein the biological sample was collected before administration of the amino acid composition to the subject.

In some embodiments, the baseline level of creatine is the level of creatine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of phenylalanine is the level of phenylalanine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of acetyl-l-camitine is the level of acetyl-1- carnitine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject. In some embodiments, the baseline level of glutamate is the level of glutamate in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of glutamine is the level of glutamine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of methionine is the level of methionine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of proline is the level of proline in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of histidine is the level of histidine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of carnitine is the level of carnitine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of tyrosine is the level of tyrosine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of 2-hydroxybutyric acid is the level of 2- hydroxybutyric acid in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of threonine is the level of threonine in a biological sample from the subject, wherein the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of i). In some embodiments, the baseline level of ornithine is the level of ornithine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of ii). In some embodiments, the baseline level of lysine is the level of lysine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of iii). In some embodiments, the baseline level of glutamate is the level of glutamate in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of iv). In some embodiments, the baseline level of serine is the level of serine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of v). In some embodiments, the baseline level of phenylalanine is the level of phenylalanine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of vi). In some embodiments, the baseline level of creatine is the level of creatine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of vii). In some embodiments, the baseline level of leucine is the level of leucine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of viii). In some embodiments, the baseline level of glycine is the level of glycine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of ix). In some embodiments, the baseline level of arginine is the level of arginine in a biological sample from the subject, in which the biological sample was collected 2, 3, 4, 6, or 8 weeks prior to the measure of x).

In some embodiments, the baseline level of threonine is the level of threonine in a biological sample from the subject, wherein the biological sample was collected before administration of the amino acid composition to the subject.

In some embodiments, the baseline level of ornithine is the level of ornithine in a biological sample from the subject, wherein the biological sample was collected before administration of the amino acid composition to the subject.

In some embodiments, the baseline level of lysine is the level of lysine in a biological sample from the subject, wherein the biological sample was collected before administration of the amino acid composition to the subject.

In some embodiments, the baseline level of glutamate is the level of glutamate in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of serine is the level of serine in a biological sample from the subject, wherein the biological sample was collected before administration of the amino acid composition to the subject. In some embodiments, the baseline level of phenylalanine is the level of phenylalanine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of creatine is the level of creatine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of leucine is the level of leucine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of glycine is the level of glycine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of arginine is the level of arginine in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of 2-hydroxybutyric acid is the level of 2- hydroxybutyric acid in a biological sample from the subject, in which the biological sample was collected before the administration of the amino acid composition to the subject.

In some embodiments, the biological sample is a blood serum sample or a blood plasma sample. In some embodiments, the biological sample is a fasting blood plasma sample.

In some embodiments, the liver fat content is indicative of the PDFF level measured 6 weeks later or 8 weeks later (e.g., at week 12 of administration of the amino acid composition or at week 16 of the administration of the amino acid composition).

In some embodiments, the liver fat content is predictive of the PDFF level measured 8 weeks later (e.g., at week 16 of the amino acid composition). In some embodiments, a decrease in the level of threonine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of lysine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of ornithine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of asparagine compared to the baseline is indicative of decreased liver fat content. In some embodiments, increase in the level of serine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of creatine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of phenylalanine compared to the baseline is indicative of decreased liver fat content.

In some embodiments, a decrease in the level of acetyl-l-carnitine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of glutamate compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of glutamine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of methionine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of proline compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of histidine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of carnitine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an decrease in the level of tyrosine compared to the baseline is indicative of decreased liver fat content.

In some embodiments, a change (e.g., an increase or a decrease, e.g., a decrease) in the level of glycine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of leucine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a change (e.g., an increase or a decrease, e.g., an increase) in the level of arginine compared to the baseline is indicative of decreased liver fat content.

In some embodiments, responsive to said value of liver fat content, performing one or more of (e.g., 2, 3, 4, 5, 6, or all of):

(i) identifying the subject as responsive or non-responsive to the amino acid composition;

(ii) identifying the subject as in need of a diagnostic, e.g., an assay for PDFF, e.g., an MRI assay for PDFF;

(iii) performing a diagnostic assay on the subject, e.g., an assay for PDFF, e.g., an MRI assay for PDFF;

(iv) ending administration of the amino acid composition;

(v) continuing administration of the amino acid composition; (vi) changing (e.g., increasing or decreasing) the dosage of the amino acid composition;

(vii) changing (e.g., increasing or decreasing) the frequency of administration of the amino acid composition;

(viii) beginning administration of a second composition, e.g., a second amino acid composition.

In some embodiments, responsive to said value of liver fat content, performing i). In some embodiments, responsive to said value of liver fat content, performing ii). In some embodiments, responsive to said value of liver fat content, performing iii). In some embodiments, responsive to said value of liver fat content, performing iv). In some embodiments, responsive to said value of liver fat content, performing v). In some embodiments, responsive to said value of liver fat content, performing vi). In some embodiments, responsive to said value of liver fat content, performing vii). In some embodiments, responsive to said value of liver fat content, performing viii).

In some embodiments, responsive to said value of liver fat content, performing i) and ii). In some embodiments, responsive to said value of liver fat content, performing i), ii), and iii). In some embodiments, responsive to said value of liver fat content, performing i), ii), iii), and iv).

In some embodiments, responsive to said value of liver fat content, performing i), ii), iii), iv), and v). In some embodiments, responsive to said value of liver fat content, performing i), ii), iii), iv), v), and vi). In some embodiments, responsive to said value of liver fat content, performing i), ii), iii), iv), v), vi), vii), and viii).

In some embodiments, responsive to said value of liver fat content, performing ii) and iii). In some embodiments, responsive to said value of liver fat content, performing ii), iii), and iv). In some embodiments, responsive to said value of liver fat content, performing ii), iii), iv), and v). In some embodiments, responsive to said value of liver fat content, performing ii), iii), iv), v), and vi). In some embodiments, responsive to said value of liver fat content, performing ii), iii), iv), v), vi), vii), and viii).

In some embodiments, responsive to said value of liver fat content, performing iii) and iv). In some embodiments, responsive to said value of liver fat content, performing iii), iv), and v). In some embodiments, responsive to said value of liver fat content, performing iii), iv), v), and vi). In some embodiments, responsive to said value of liver fat content, performing iii), iv), v), vi), vii), and viii).

In some embodiments, responsive to said value of liver fat content, performing iv) and v). In some embodiments, responsive to said value of liver fat content, performing iv), v), and vi).

In some embodiments, responsive to said value of liver fat content, performing iv), v), vi), vii), and viii).

In some embodiments, responsive to said value of liver fat content, performing v) and vi). In some embodiments, responsive to said value of liver fat content, performing v), vi), vii), and viii).

In some embodiments, responsive to said value of liver fat content, performing vi), vii), and viii).

In some embodiments, responsive to said value of liver fat content, performing vii) and viii).

In some embodiments, identifying the subject as responsive or non-responsive to the amino acid composition comprises:

(i) identifying the subject as a responder;

(ii) identifying the subject as a non-responder; or

(iii)identifying the subjects degree of responsiveness.

In some embodiments, a responder has a reduction in liver fat content. In some embodiments, a responder has at least 30% reduction in liver fat content. In some emdobiments, the reduction in liver fat content is measure after at least 6 weeks (e.g., after 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 17, or 18 weeks).

In some embodiments, the amino acid composition comprises: a) a leucine (L)-amino acid entity, a arginine (R)-amino acid entity, and a glutamine (Q)-amino acid entity; and b) a N-acetylcysteine (NAC) entity, e.g., NAC. In some embodiments, the total wt. % of (a)-(b) is greater than the total wt. % of any other amino acid entity in the composition. In some embodiments, the amino acid composition comprises c) 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; d) the ratio of the L-amino acid entity to the Q amino acid entity 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 Q-amino acid entity is about 1 :2; e) the ratio of the R-amino acid entity to the Q amino acid entity 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 Q-amino acid entity is about 3:4; or f) a combination of two or three of c)-e).

In some embodiments, the amino acid composition further comprises one or two additional branched-chain amino acid (BCAA)-entities, e.g., one or both of an isoleucine (I)- amino acid-entity and a valine (V)-amino acid-entity, optionally wherein the two additional BCAA-entities are chosen from Table 2.

In some embodiments, the amino acid composition comprises a 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 certain embodiments, 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 Q-amino acid entity, and the NAC-amino 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 some embodiments, a 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 (e.g., 1 ± 20%) : about 1.5 (e.g., 1.5 ± 20%): about 2 (e.g., 2 ± 20%): about 0.15 (e.g., 0.15 ± 20%). In some embodiments, a 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 (e.g., 1 ± 20%) : about 1.81 (e.g., 1.81 ± 20%): about 2 (e.g., 2 ± 20%): about 0.15 (e.g., 0.15 ± 20%). In some embodiments, a 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 (e.g., 1 ± 15%) : about 1.81 (e.g., 1.81 ± 15%): about 2 (e.g., 2 ± 215%): about 0.15 (e.g., 0.15 ± 15%).

In certain embodiments, a 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 (e.g., 1 ± 20%) : about 1.5 (e.g., 1.5 ± 20%) : about 0.6 (e.g., 0.6 ± 20%): about 0.15 (e.g., 0.15 ± 20%).

In some embodiments, the amino acid composition comprises: a) a leucine amino acid entity, b) a arginine amino acid entity, c) glutamine amino acid entity, d) a N-acetylcysteine (NAC) entity, and e) one or both of serine amino acid entity or a carnitine entity.

In certain embodiments, the total wt. % of (a)-(e) is greater than the total wt. % of other amino acid entities in the composition (e.g., in dry form). In some embodiments, the amino acid composition comprises one or both of: the wt. % of the serine amino acid entity is at least 32 wt. % of the amino acid entity components or total components in the composition; or the wt. % of the carnitine entity is at least 2 wt. % of the amino acid entity components or total components in the composition. In some embodiments, the amino acid composition further comprises: (f) an isoleucine amino acid entity.

In some embodiments, the amino acid entities of the amino acid compositions described herein are selected from Table 2.

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 salt thereof, and the NAC or salt 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 salt thereof, and the NAC or salt 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 salt thereof, and the NAC or salt 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 L- amino acid entity, the R-amino acid entity, the L-glutamine or a salt thereof, and the NAC or salt 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 L-glutamine or a salt thereof, and the NAC or salt 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 salt thereof, and the NAC or salt 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 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 I-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 salt thereof, and about 0.1 g to about 5 g of the NAC or a salt thereof, e.g., the composition comprises about 1 g of the L-amino acid entity, about 0.5 g of the I-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 salt thereof, and about 0.15 g or about 0.3 g of NAC or a salt 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 I- 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 salt thereof, and about 0.9 g of NAC or a salt 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 I-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 salt thereof, about 0.9 g of NAC or a salt thereof, and about 6 g of L-serine or a salt thereof. In embodiments, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the I-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 salt thereof, about 0.9 g of NAC or a salt thereof, and about 6.67 g of L-serine or a salt thereof.

In embodiments, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the I-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 salt thereof, about 0.9 g of NAC or a salt thereof, about 9 g of L-serine or a salt thereof, and about 9 g of L-glycine or a salt thereof. In embodiments, the composition comprises about 4 g of the L-amino acid entity, about 2 g of the I-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 salt thereof, about 0.9 g of NAC or a salt thereof, about 3.33 g of L-serine or a salt thereof, and about 3.33 g of L-glycine or a salt thereof.

In one aspect, the invention features a composition including free amino acids, wherein the amino acids comprise arginine, glutamine, N-acetylcysteine, and a branched-chain amino acid chosen from one, two, or all 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 some embodiments, the total wt. % of LIVRQNac is greater than the total wt. % of one, two, or three of other amino acid entity components, non-amino acid entity protein components (e.g., whey protein), or non-protein components in the composition (e.g., in dry form), e.g., LIVRQNac is at least: 50 wt. %, 75 wt. %, or 90 wt. % of the total wt. of one or both of amino acid entity components or total components in the composition (e.g., in dry form).

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

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 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.

In any of the aspects and embodiments disclosed herein, 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.

In any of the aspects and embodiments disclosed herein, 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.

In any of the aspects and embodiments disclosed herein, 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.

In any of the aspects and embodiments disclosed herein, the amino acids comprise 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 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 any of the aspects and embodiments disclosed herein, 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 an embodiment, the ratio of the L-amino acid-entity, the I-amino acid-entity, and the V-amino acid-entity in combination to the R-amino acid entity, L-glutamine or a salt thereof, and NAC or a salt thereof is 12 +/- 15% : 6 +/- 15% : 3 +/- 15% : 9 +/- 15% : 12 +/- 15% : 2.7 +/- 15%.

In any of the aspects and embodiments disclosed herein, the composition further comprises 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 bitterness 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.

In some embodiments, the subject has type 2 diabetes 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 pediatric 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 diabetes.

In some embodiments of the method, the amino acid composition is administered at a dose of about 15 g/d to about 90 g/d.

In some embodiments of the method, the amino acid 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, the amino acid composition is administered one, two, to three times per day.

In some embodiments of the method, the amino acid 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.

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 metabolism, improved mitochondrial function, white adipose tissue (WAT) browning, decreased reactive oxygen species (ROS), increased levels of glutathione (GSH), decreased hepatic inflammation, decreased hepatocyte ballooning, improved gut barrier function, increased insulin secretion, or improved glucose tolerance.

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

In some embodiments, the composition is administered prior to a meal (e.g., about 10 minutes prior, about 15 minutes prior, about 20 minutes prior, about 25 minutes prior, about 30 minutes prior, about 35 minutes prior, about 40 minutes prior, about 45 minutes prior, about 50 minutes prior, about 55 minutes prior, or about 60 minutes prior).

In some embodiments, the composition is administered concurrent with 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 farnesoid 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 supplementation 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 supplementation 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 present invention comprise one or more of the following.

In some embodiments, when present: a) the leucine amino acid entity is chosen from: i) L-leucine or a salt thereof, ii) a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-leucine, or iii) P-hydroxy-P-methylbutyrate (HMB) or a salt thereof; b) the arginine amino acid entity is chosen from: i) L-arginine or a salt thereof, ii) a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-arginine, iii) a dipeptide or salt thereof, or tripeptide or salt thereof, comprising creatine; or iv) creatine or a salt thereof, c) the glutamine amino acid entity is L-glutamine or a salt thereof or a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-glutamine; d) the NAC entity is NAC or a salt thereof or a dipeptide or salt thereof, comprising

NAC; e) one or both of: i) the serine amino acid entity is L-serine or a salt thereof or a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-serine; or ii) the carnitine entity is L-carnitine or a salt thereof or a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-camitine; f) the isoleucine amino acid entity is chosen from: i) L-isoleucine or a salt thereof, ii) a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L- isoleucine, or

(iii) 2-Oxo-3 -methyl -valerate or a salt thereof or Methylbutyrl-CoA or a salt thereof, and g) the valine amino acid entity is chosen from: i) L-valine or a salt thereof, ii) a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-valine, or iii) Isobutyryl-CoA or a salt thereof, 3-HIB-CoA or a salt thereof, or 3-HIB or a salt thereof.

In some embodiments, the L-amino acid entity is chosen from L-leucine, P-hydroxy-P- methylbutyrate (HMB), oxo-leucine, isovaleryl-CoA, D-leucine, and n-acetyl-leucine, or a combination thereof.

In some embodiments, the R-amino acid entity is chosen from L-arginine, ornithine, argininosuccinate, agmatine, creatine, and N-acetyl-arginine, or a combination thereof.

In some embodiments, the Q-amino acid entity is chosen from L-glutamine, carbamoyl-P, glutamate, and n-acetylglutamine, or a combination thereof.

In some embodiments, the NAC-amino acid entity is chosen from NAC, cystathionine, glutathione, homocysteine, and cysteamine, or a combination thereof.

In some embodiments, the I-amino acid entity is chosen from L-isoleucine, 2-oxo-3- methyl-valerate, 2-oxo-3 -methyl-valerate, methylbutyryl-CoA, and N-acetyl-isoleucine, or a combination thereof.

In some embodiments, the V-amino acid entity is chosen from L-valine, 2-oxo-valerate, isobutyryl-CoA, 3-HIB-CoA, and N-acetyl-valine, or a combination thereof.

In some embodiments, the S-amino acid entity is chosen from L-serine, Phosphoserine, P-hydroxypyruvate, Acetylserine, Phosphatidylserine, or a combination 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 are 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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows bar graphs showing changes from baseline (day 1, “Dl”) at week 8 (“W8”) and week 16 (“W16”) of the study as performed in Example 2. The order of the bars matches the order of the groups shown in the legend.

FIG. 2A is a volcano plot showing significance of amino acid change versus amino acid concentration % change from baseline.

FIG. 2B is a graph showing the strong correlation with predictions of PDFF changes at week 16 based on metabolic data at week 8 of amino acid composition administration. The prediction was based on a statistical model trained on metabolomic data.

FIGs. 3A-3C are images showing the feature selection identified by Lasso analysis and random forest regression. FIG. 3 A is a schematic showing model of polar metabolomics and PDFF liver fat; Random Forest model of PDFF liver fat and amino acid profile; and Lasso model of PDFF liver fat and amino acid profile. FIG. 3B is a Venn Diagram identifying the top change in PDFF predictor amino acids identifies by one or more of Random Forest model (top left), polar metabolomics and PDFF liver fat Lasso model (top left), and PDFF liver fat and amino acid profile Lasso model (bottom). FIG. 3C are bar graphs identifying the top change in PDFF predictor amino acids identifies by polar metabolomics and PDFF liver fat Lasso model (top), Random Forest model (middle), and PDFF liver fat and amino acid profile Lasso model (bottom).

DETAILED DESCRIPTION

The present invention provides, at least in part, methods of evaluating a subject comprising acquiring a value of liver fat content for the subject. The value of liver fat content for the subject can be indicative of a subject’s response to administration of an amino acid composition (e.g., an amino acid composition described herein). Furthermore, the liver fat content for a subject can be predictive of a subject’s response to the administration of an amino acid composition (e.g., an amino acid composition described herein). The value of liver fat content can be used in a non-invasive, alternative method for grading steatohepatitis (e.g., an alternative to MRI-PDFF). Acquiring a value of liver fat content can be used for subjects that, including but not limited to, are claustrophobic, are too large to fit in a standard MRI scanner (e.g., is greater than about 300, 350, 400, 450, or 500 lbs), or have a metallic implant.

In some embodiments of any of the methods disclosed herein, acquiring the value of liver fat content comprises measuring the level of threonine, the level of glutamine, the level of lysine, the level of ornithine, and the level of asparagine. In some embodiments, the measuring includes using a separation step followed by a detection step. In some embodiments, the separation step is selected from HPLC, UPLC, ion exchange chromatography (IEC), gas chromatography, and capillary electrophoresis (CE). In some embodiments, the detection step is selected from mass spectrometry (MS) (e.g., tandem mass spectrometry (MS/MS)).

In some embodiments of any of the methods disclosed herein, the subject was treated with an amino acid composition prior to measurement of the level of threonine, the level of glutamine, the level of lysine, the level of ornithine, and the level of asparagine, e.g., about 1, 2, 3, 4, 6, 8, 10, 12, 14, or 16 weeks prior.

In some embodiments of any of the methods disclosed herein, the subject was treated with an amino acid composition prior to measurement of one or more (e.g., 1, 2, 3, 4, 5, or all) of the level of threonine, the level of ornithine, the level of lysine, the level of glutamate, the level ofserine, and the level of phenyalalnine, e.g., about 1, 2, 3, 4, 6, 8, 10, 12, 14, or 16 weeks prior.

In some embodiments, the subject has, or is identified as having, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), or alcoholic fatty liver disease (AFLD).

In some embodiments, the subject has, or is identified as having, diabetes, e.g., type II diabetes.

In some embodiments, the subject is not diabetic.

In some embodiments, after the subject received the amino acid composition, the subject experienced a reduction in liver fat content of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the amino acid composition comprises at least four different amino acid entities. In some embodiments, the composition is capable of one, two, three, four, five, or six or all of: a) decreasing or preventing liver fibrosis; b) decreasing or preventing liver injury; c) decreasing or preventing hepatocyte inflammation; d) improving, e.g., increasing, glucose tolerance; e) decreasing or preventing steatosis; f) decreasing or preventing hepatocyte ballooning; or g) improving gut function.

In some embodiments, the amino acid 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, the composition is capable of improving gut barrier function.

The amino acid compositions described herein can be administered to a subject to provide a beneficial 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 amino acid compositions include a subject having non-alcoholic fatty liver disease (NAFLD; e.g., pediatric 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 particular, the subject may have one, two, or more (e.g., all) of a high BMI, obesity, fibrosis, or cirrhosis. The subject may also have one, two, or more (e.g., all) 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 composition can result in improved liver function in a subject, e.g., by one, two, three, four, five or more (e.g., all) 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 secretion, or improving glucose tolerance.

In some embodiments, the amino acid composition is for use as a medicament 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 amino acid composition is for use as a medicament 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 amino acid composition is for use in the manufacture of a medicament 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 amino acid composition is for use in the manufacture of a medicament 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.

Definitions

Terms used in the claims and specification are defined as set forth below unless otherwise specified.

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

As used herein, the term “amino acid entity” refers to a (L)-amino acid in free form or salt form (or both), the L-amino acid residue in a peptide smaller than 20 amino acid residues (e.g., oligopeptide, e.g., a dipeptide or a tripeptide), a derivative of the amino acid, a precursor of the amino acid, or a metabolite of the amino acid (see, e.g., Table 2). An amino acid entity includes a derivative of the amino acid, a precursor of the amino acid, a metabolite of the amino acid, or a salt form of the amino acid that is capable of effecting biological functionality of the free L-amino acid. An amino acid entity does not include a naturally occurring polypeptide or protein of greater than 20 amino acid residues, either in whole or modified form, e.g., hydrolyzed form.

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 salt form; if a peptide, refers to a peptide (e.g., a dipeptide or a tripeptide) comprising an XXX residue; if a derivative, refers to a derivative of XXX; if a precursor, refers to a precursor of XXX; and if a metabolite, refers to a XXX metabolite. For example, where XXX is leucine (L), then L-amino acid entity refers to free L or L in salt form, a peptide (e.g., a dipeptide or a tripeptide) comprising a L residue, a L derivative, a L precursor, or a metabolite of L; where XXX is arginine (R), then R-amino acid entity refers to free R or R in salt form, a peptide (e.g., a dipeptide or a tripeptide) comprising a R residue, a R derivative, a R precursor, or a metabolite of R; where XXX is glutamine (Q), then Q-amino acid entity refers to free Q or Q in salt form, a peptide (e.g., a dipeptide or a tripeptide) comprising a Q residue, a Q derivative, a Q precursor, or a metabolite of Q; and where XXX is N- acetylcysteine (NAC), then NAC-amino acid entity refers to free NAC or NAC in salt form, a peptide (e.g., a dipeptide or a tripeptide) comprising a NAC residue, a NAC derivative, a NAC precursor, or a metabolite of NAC.

“About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 15%, more typically within 10%, and even more typically, within 5% of a given value or range of values.

An “amino acid” refers to an organic compound having an amino group (-NFL), a carboxylic acid group (-C(=0)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 all refer to the amino acid leucine; lie, I or isoleucine all refer to the amino acid isoleucine; Val, V or valine all refer to the amino acid valine; Arg, R or arginine all refer to the amino acid arginine; and Gin, Q or glutamine all refer to the amino acid glutamine. Likewise, the non-natural amino acid derivative N-acetylcysteine may be referred to interchangeably by “NAC” or “N-acetylcysteine.”

Amino acids may be present 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.

As used herein, the term “amino acid composition” means a combination of three or more amino acid entities that, in aggregate, has a physiological effect as described herein. An amino acid composition described herein can contain other biologically active ingredients or inactive ingredients. For instance, an amino acid composition may comprise ingredients that are other than amino acids.

The individual amino acid entities are present in the composition, e.g., the amino acid composition, in various amounts or ratios, which can be presented as amount by weight (e.g., in grams), ratio by weight of amino acid entities to each other, amount by mole, amount by weight percent of the composition, amount by mole percent of the composition, caloric content, percent caloric contribution to the composition, etc. Generally, this disclosure will provide grams of an amino acid entity in a dosage form, i.e., the weight of an amino acid entity relative to the weight of the total composition to define the weight percent of the amino acid entity. In some embodiments, the amino acid composition, is provided as a pharmaceutically acceptable preparation (e.g., a pharmaceutical product).

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., to positively modify one, two, or more of a subject’s symptoms, e.g., provide a positive clinical response). The effective amount of an active ingredient 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 supplement.

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 animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. This may be a standard used by the pharmaceutical industry or by agencies or entities (e.g., government or trade agencies or entities) regulating the pharmaceutical industry to ensure one or more product quality parameters are within acceptable ranges for a medicine, pharmaceutical composition, treatment, or other therapeutic. A product quality parameter can be any parameter regulated by the pharmaceutical industry or by agencies or entities, e.g., government or trade agencies or entities, including but not limited to composition; composition uniformity; dosage; dosage uniformity; presence, absence, and/or level of contaminants or impurities; and level of sterility (e.g., the presence, absence and/or level of microbes). Exemplary government regulatory agencies include: Federal Drug Administration (FDA), European Medicines Agency (EMA), SwissMedic, China Food and Drug Administration (CFDA), or Japanese Pharmaceuticals and Medical Devices Agency (PMDA).

A composition, formulation or product is “therapeutic” if it provides a beneficial clinical effect. A beneficial 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 specific mixture of active ingredients 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 the liver disease (e.g.,, slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In some embodiments, the liver disease is NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH). In another embodiment, “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible 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 discernible 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)). Methods of evaluating subjects

The present disclosure provides methods of evaluating a subject (e.g., evaluating the effectiveness of an amino acid composition in a subject). In some embodiments, evaluating a subject includes acquiring a value of liver fat content for the subject.

Also provided are methods of evaluating the effectiveness of an amino acid composition in a subject. In some embodiments, evaluating the effectivness of an amino acid composition in a subject includes acquiring a value of liver fat content for the subject.

In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, and creatine. In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, and phenylalanine. In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, and acetyl-L-carnitine.

In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, and glutamate. In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, glutamate, and glutamine. In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, glutamate, glutamine, and methionine. In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-camitine, glutamate, glutamine, methionine, and proline. In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-camitine, glutamate, glutamine, methionine, proline, and histidine. In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, glutamate, glutamine, methionine, proline, histidine, and carnitine. In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-camitine, glutamate, glutamine, methionine, proline, and histidine. In some embodiments, the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-camitine, glutamate, glutamine, methionine, proline, histidine, carnitine, and tyrosine. In some embodiments, the value of liver fat content includes the measure of threonine and ornithine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, and lysine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, and glutamate. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, and serine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, and phenylalanine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, and glutamate. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, glutamate, and serine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, glutamate, serine, and phenylalanine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, and serine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, serine, and phenylalanine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, and phenylalanine.

In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, and creatine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, creatine, and leucine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, creatine, leucine, and glycine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, creatine, leucine, glycine, and arginine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, and leucine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, leucine, and glycine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, leucine, glycine, and arginine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, creatine, and glycine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, creatine, glycine, and arginine. In some embodiments, the value of liver fat content includes the measure of threonine, ornithine, lysine, glutamate, serine, phenylalanine, creatine, leucine, and arginine.

In some embodiments, the measuring includes a separation step followed by a detection step. In some embodiments, the separation step substantially reduces the number of undesired compounds (e.g., compounds not used in determining the value of liver fat content). In some embodiments, the separation step substantially separates the analytes for analysis. In some embodiments, the separation step is selected from HPLC, UPLC, ion exchange chromatography (IEC), gas chromatography, and capillary electrophoresis (CE). In some embodiments, the detection step comprises mass spectrometry (e.g., tandem mass spectrometry (MS/MS). In some embodiments, the mass spectrometry analysis can comprise Fourier transform (FT), time-of- flight (TOF), matrix assisted laser desorption ionization (MALDI), ion trap, and quadrupole. In some embodiments, the analytes are collected after elution during the separation step and later analyzed during the detection step (e.g., liquid chromatography followed by MALDI-MS). In some embodiments, the separation step is immediately followed by the detection step (e.g., HPLC-MS/MS, UPLC-MS/MS, CE-MS (e.g, FTMS).

In some embodiments, for methods of detection using mass spectrometry, internal standards (e.g., stable isotope analogs for each amino acid) can be used during the detection step. One of skill in the art will understand what internal standards to employ when performing the methods described herein.

In some embodiments, the measuring includes a deproteinization step of the sample prior to the separation step. In some embodiments, the deproteinization step includes the precipitation of proteins from the sample. Methods for precipitation of proteins from a biological sample are well known to one of skill in the art. By way of example, and without any limitation, methods of precipitating proteins from a biological sample include the use of sulfosalicylic acid (SSA) or trichloracetic acid (TCA). In some embodiments, protein precipitation is followed by centrifugation, and/or filtration.

In some embodiments, the subject was treated with an amino acid composition prior to measurment of the level of threonine, the level of glutamine, the level of lysine, the level of ornithine, and the level of asparagine. In some embodiments, the subject was treated with an amino acid composition about 1, 2, 3, 4, 6, 8, 10, 12, 14, or 16 weeks prior. In some embodiments, the subject was treated with an amino acid composition about 8 weeks prior. In some embodiments, after the subject received the amino acid composition, the subject experienced a reduction in liver fat content of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, MRI is contraindicated for the subject. In some embodiments, the reduction in liver fat content is measured by MRI-PDFF.

In some embodiments, the subject has, or is identified as having, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), or alcoholic fatty liver disease (AFLD).

In some embodiments, the subject has, or is identified as having diabetes, e.g., type II diabetes. In some embodiments, the subject is not diabetic.

In some embodiments, the baseline level of the amino acid is the level of the amino acid in a biological sample from the subject. In some embodiments, the biological sample was collected before the administration of the amino acid composition to the subject. In some embodiments, the baseline level of threonine is the level of threonine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of lysine is the level of lysine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of ornithine is the level of ornithine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of asparagine is the level of asparagine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of serine is the level of serine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of creatine is the level of creatine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of phenylalanine is the level of phenylalanine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of acetyl-L-carnitine is the level of acetyl-L-carnitine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of glutamate is the level of glutamate in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of glutamine is the level of glutamine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of methionine is the level of methionine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of proline is the level of proline in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of histidine is the level of histidine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of carnitine is the level of carnitine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of tyrosine is the level of tyrosine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject.

In some embodiments, the baseline level of threonine is the level of threonine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of ornithine is the level of ornithine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of lysine is the level of lysine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of glutamate is the level of glutamate in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of serine is the level of serine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of phenylalanine is the level of phenylalanine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of creatine is the level of creatine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of leucine is the level of leucine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of glycine is the level of glycine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject. In some embodiments, the baseline level of arginine is the level of arginine in a biological sample (e.g., a blood serum sample or a blood plasma sample) from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the administration of the amino acid composition to the subject.

In some embodiments, a decrease in the level of threonine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of lysine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of ornithine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of asparagine compared to the baseline is indicative of decreased liver fat content. In some embodiments, increase in the level of serine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of creatine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of phenylalanine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of acetyl-l-carnitine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of glutamate compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of glutamine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of methionine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of proline compared to the baseline is indicative of decreased liver fat content. In some embodiments, a decrease in the level of histidine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of carnitine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an decrease in the level of tyrosine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a change (e.g., an increase or a decrease, e.g., a decrease) in the level of glycine compared to the baseline is indicative of decreased liver fat content. In some embodiments, an increase in the level of leucine compared to the baseline is indicative of decreased liver fat content. In some embodiments, a change (e.g., an increase or a decrease, e.g., an increase) in the level of arginine compared to the baseline is indicative of decreased liver fat content.

In some embodiments, responsive to said value of liver fat content, the method includes performing one or more of (e.g., 2, 3, 4, 5, 6, or all of):

(i) identifying the subject as responsive or non-responsive to the amino acid composition;

(ii) identifying the subject as in need of a diagnostic, e.g., an assay for PDFF, e.g., an MRI assay for PDFF;

(iii) ending administration of the amino acid composition;

(iv) continuing administration of the amino acid composition;

(v) changing (e.g., increasing or decreasing) the dosage of the amino acid composition;

(vi) changing (e.g., increasing or decreasing) the frequency of administration of the amino acid composition; beginning administration of a second therapy, e.g., a second amino acid composition.

In some embodiments, identifying the subject as responsive or non-responsive to the amino acid composition includes: i) identifying the subject as a responder;

(ii) identifying the subject as a non-responder; or

(iii)identifying the subjects degree of responsiveness.

Determination 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 present 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 all amino acids present in the composition or mixture. It is understood that Nac is considered to be an amino acid for the purpose of this calculation.

Compositions comprising Amino Acid Entities

The present 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 salt form, amino acid residues of a peptide (e.g., of a dipeptide, oligopeptide, or polypeptide), derivatives of an amino acid, precursors of an amino acid, or metabolites of an amino acid. For example, the compositions can include five amino acid entities and an antioxidant or reactive oxygen species (ROS) scavenger. In some embodiments, the five amino acid entities can be a leucine (L)-amino acid entity, an arginine (R)-amino acid entity, a glutamine (Q)-amino acid entity, and the antioxidant or reactive oxygen species (ROS) scavenger can be a N-acetylcysteine (NAC) entity, e.g., NAC (Table 2).

In some embodiments, the five amino acid entities can be a leucine (L)-amino acid entity, an isoleucine (I)-amino acid entity, a valine (V)-amino acid entity, an arginine (R)-amino acid entity, a glutamine (Q)-amino acid entity, and the antioxidant or reactive oxygen species (ROS) scavenger can be a N-acetylcysteine (NAC) entity, e.g., NAC (Table 2).

In some embodiments, the amino acid composition comprises: a) a leucine amino acid entity, b) a arginine amino acid entity, c) glutamine amino acid entity; d) a N-acetylcysteine (NAC) entity; and e) a serine amino acid entity. In some embodiments, the composition comprises: a) a leucine amino acid entity, b) a arginine amino acid entity, c) glutamine amino acid entity; d) a NAC entity; and e) a carnitine entity. In some embodiments, the composition comprises: a) a leucine amino acid entity, b) a arginine amino acid entity, c) glutamine amino acid entity; d) a NAC entity; and e) a serine amino acid entity and a carnitine entity.

Amino acid compositions comprising LRQNac and LIVRQNac, as well as methods of making and using the compositions, are described in WO2018/118941, incorporated herein by reference in its entirety. Amino acid compositions comprising LRQNac and LIVRQNac, as well as methods of making and using the compositions, are described in WO2019/246221, incorporated herein by reference in its entirety. Table 2. Amino acid entities include amino acids, precursors, metabolites, and derivatives of the compositions described herein. It is contemplated that a composition described herein may comprise, e.g., as an alternatives to serine,, glycine, threonine, or a combination of serine and glycine (e.g., a 1:1 ratio of serine and glycine).

In some embodiments, the L-amino acid entity is selected from the group consisting of a precursor, a metabolite, and a derivative. In certain embodiments, the L-amino acid entity is selected from the group consisting of L-leucine, P-hydroxy-P-methylbutyrate (HMB), oxo- leucine, isovaleryl-CoA, and n-acetyl-leucine. 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 I-amino acid entity is selected from the group consisting of a precursor, a metabolite, and a derivative. In certain embodiments, the I-amino acid entity is selected from the group consisting of L-isoleucine, 2-Oxo-3 -methyl-valerate, methylbutyl-CoA, and N-Acetyl-isoleucine. In one embodiment, the I-amino acid entity is L-isoleucine.

In some embodiments, the V-amino acid entity is selected from the group consisting of a precursor, a metabolite, and a derivative. In certain embodiments, the V-amino acid entity is selected from the group consisting of L-Valine, 2-Oxo-valerate, isobutyl-COA, 3-HIB-COA, 3- HIB, and N-acetyl-valine. In one embodiment, the V-amino acid is L-valine.

In some embodiments, the R-amino acid entity is selected from the group consisting of a precursor, a metabolite, and a derivative. In certain embodiments, the R-amino acid entity is selected from the group consisting of L-arginine, ornithine, argininosuccinate, citrulline, agmatine, creatine, and N-acetyl-arginine. In one embodiment, the R-amino acid entity is L- arginine. In one embodiment, the R-amino acid entity is creatine. In another embodiment, the R-amino acid entity is ornithine.

In some embodiments, the Q-amino acid entity is selected from the group consisting of a precursor, a metabolite, and a derivative. In certain embodiments, the Q-amino acid entity is selected from the group consisting of L-glutamine, carbamoyl-P, 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 metabolite, and a derivative. In certain embodiments, the NAC-amino acid entity is selected from the group consisting NAC, serine, acetylserine, cystathionine, cystathionine, homocysteine, methionine, glutathione, D-cysteine, and L-cysteine. In one embodiment, the NAC entity is NAC. In one embodiment, the NAC entity is glutathione. In some embodiments, the Carnitine (Car) amino acid entity is selected from the group consisting of a precursor, a metabolite, and a derivative. In certain embodiments, the Car amino acid entity is selected from the group consisting of L-Carnitine, 6-N-trimethyllysine; N6- Trimethyl-3-OH-lysine, Acetyl-L-Carnitine (ALCAR); Proprionyl-L-Camitine (PLCAR); L- Camitine L-Tartrate.

In some embodiments, the S-amino acid entity is selected form the group consisting of a precursor, a metabolite, and a derivative. In certain embodiments, the S-amino acid entity is selected from the group consisting of L-serine; Phosphoserine, P-hydroxypyruvate; and phosphatidyl serine .

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 acid- entity and a valine (V)-amino acid-entity. In some embodiments, both the I-amino acid-entity and the V-amino acid-entity are present. In certain embodiments, the L-entity is present at a higher amount (% by weight) than one or both of the I-amino acid-entity and the V-amino acid- entity (e.g., the L-entity is present 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 I-amino acid-entity and the V-amino acid- entity).

In some embodiments, the I-amino acid entity is selected from the group consisting of a salt, a precursor, a metabolite, and a derivative. In certain embodiments, the I-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 I-amino acid entity is L-isoleucine.

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

In some embodiments, the composition comprises L-leucine or a leucine metabolite (e.g., HMB), L-arginine or an L-arginine metabolite (e.g., creatine or ornithine), L-glutamine, and NAC or a NAC metabolite, 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, ornithine, 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, ornithine, 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 : 0.225. 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 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 : 1.81 : 2 : 0.15. In an 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 : 1.61 : 2 : 0.15.

In some embodiments, a 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 (e.g., 1 ± 20%) : about 1.81 (e.g., 1.8 ± 20%): about 2 (e.g., 2 ± 20%): about 0.15 (e.g., 0.15 ± 20%). In some embodiments, a 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 (e.g., 1 ± 15%) : about 1.81 (e.g., 1.81 ± 15%): about 2 (e.g., 2 ± 215%): about 0.15 (e.g., 0.15 ± 15%).

In some embodiments, 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 Q-amino acid entity, and the NAC-amino 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 I-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 an embodiment, 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 Q-amino acid entity, and the NAC-amino acid entity is about 1 : 0.5 : 0.5 : 1.81 : 2 : 0.15. In some embodiments, the wt. ratio of the L-amino acid entity, the I-amino acid entity, the R-amino acid entity, the Q-amino acid entity, the NAC entity, the CAR-entity, and the S- amino acid entity of 3 +/-20% : 1.5 +/-20% : 4 +/-20% : 2 +/-20% : 1.3 +/-20% : 0.9 +/-20% : 7.5 +1-20%. In some embodiments, the wt. ratio of the L-amino acid entity, the I-amino acid entity, the R-amino acid entity, the Q-amino acid entity, the NAC entity, the CAR-entity, and the S- amino acid entity of 3 +/-15% : 1.5 +/-15% : 4 +/-15% : 2 +/-15% : 1.3 +/-15% : 0.9 +/-15% : 7.5 +/-15%. In some embodiments, the wt. ratio of the L-amino acid entity, the I-amino acid entity, the R-amino acid entity, the Q-amino acid entity, the NAC entity, the CAR-entity, and the S- amino acid entity of 3 +/-10% : 1.5 +/-10% : 4 +/-10% : 2 +/- 10% : 1.3 +/-10% : 0.9 +/-10% : 7.5 +/-10%. In some embodiments, the wt. ratio of the L-amino acid entity, the I-amino acid entity, the R-amino acid entity, the Q-amino acid entity, the NAC entity, the CAR-entity, and the S- amino acid entity of 3 +1-5% : 1.5 +1-5% : 4 +1-5% : 2 +1-5% : 1.3 +1-5% : 0.9 +1-5% : 7.5 +1-5%.

In various embodiments, the total wt. of amino acids present is about 2 g to about 60 g.

In certain embodiments, the total wt. of amino acids present 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 present is about 6 g. In one embodiment, the total wt. of amino acids present is about 12 g. In one embodiment, the total wt. of amino acids present is about 18 g. In an embodiment, the total wt. of amino acids present is about 24 g. In one embodiment, the total wt. of amino acids present is about 48 g.

In some embodiments, 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 I-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 the I-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 I- amino acid entity, about 1 g of the V-amino acid entity, about 3 g of the R-amino acid entity, 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 I- amino 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 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., all) 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 I-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 salt form, e.g., one, two, three, or more (e.g., all) of the amino acid entities is in a salt 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 salt form. In certain embodiments, the L-amino acid entity, the I-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 salt form.

In any of the foregoing embodiments, the composition comprises a combination of 2 to 20 different amino acid entities, e.g., 5 to 15 different amino acid entities.

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., all) or more of serine, glycine, glutamine, HMB, arginine, L-leucine, citrulline, glutamine, ornithine, 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 carnitine.

In some embodiments, the composition includes arginine, glutamine, N-acetylcysteine, and a branched-chain amino acid (BCAA) chosen from one, two, or all 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-acetyl cysteine.

In certain embodiments, the composition may consist of leucine, isoleucine, arginine, glutamine, N-acetylcysteine, and one or both of serine or carnitine.

In some embodiments, the amino acids leucine, isoleucine, valine, arginine, glutamine and N-acetylcysteine are present 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 N- acetylcysteine are present 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 N- acetylcysteine are present 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 HC1), glutamine and N-acetylcysteine are present in a weight ratio of about 1 : 0.5 : 0.5 : 1.5- 1.81 : 2 : 0.1-0.3. In some embodiments, the amino acids leucine, isoleucine, valine, arginine (e.g., arginine HC1), glutamine and N-acetylcysteine are present 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, arginine (e.g., arginine HC1), glutamine, and N-acetylcysteine are present in a weight ratio of about 1 : 0.5 : 0.5 : 1.5-1.81 : 2 : 0.25.

In some embodiments, the amino acid composition comprises, consists essentially of, or consists of: a) a leucine amino acid entity, b) a arginine amino acid entity, c) glutamine amino acid entity; d) a NAC entity; and e) one or both of a serine amino acid entity and a carnitine entity.

In some embodiments, the composition (e.g., the Active Moiety) comprises, consists essentially of, or consists of: a) an leucine amino acid entity chosen from: i) L-leucine or a salt thereof, ii) a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-leucine, or iii) P-hydroxy-P-methylbutyrate (HMB) or a salt thereof; b) an arginine amino acid entity chosen from: i) L-arginine or a salt thereof, ii) a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-arginine, iii) ornithine or a salt thereof, iv) a dipeptide or salt thereof, or tripeptide or salt thereof, comprising ornithine, v) creatine or a salt thereof, or vi) a dipeptide or salt thereof, or tripeptide or salt thereof, comprising creatine; c) the glutamine amino acid entity is L-glutamine or a salt thereof or a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-glutamine; d) the NAC entity is NAC or a salt thereof or a dipeptide or salt thereof, comprising NAC; and e) one or both of: i) the serine amino acid entity is L-serine or a salt thereof or a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-serine; or ii) the carnitine entity is L-carnitine or a salt thereof or a dipeptide or salt thereof, comprising L-carnitine.

In some embodiments, the composition (e.g., an Active Moiety) comprises, consists essentially of, or consists of: a) the leucine amino acid entity is L-leucine or a salt thereof; b) the arginine amino acid entity is L-arginine or a salt thereof; c) the glutamine amino acid entity is L-glutamine or a salt thereof; d) the NAC entity is NAC or a salt thereof; e) one or both of: i) the serine amino acid entity is L-serine or a salt thereof, or ii) the carnitine entity is L-camitine or a salt thereof; and f) the isoleucine amino acid entity is L-isoleucine or a salt thereof.

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 present is about 5 g, about 6 g, about 7 g, about 11 g, 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 31 g, 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 N- acetylcysteine.

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 HC1), 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 HC1), 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 HC1), 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 HC1), 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 HC1), 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 HC1), 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 amino acid composition includes 1 g +/- 20% of a leucine amino acid entity, 0.5 g +/- 20% of an isoleucine amino acid entity, 1.33 g +/- 20% of an arginine amino acid entity, 0.67 g +/- 20% of a glutamine amino acid entity, 0.43 g +/- 20% of a NAC entity, 0.30 g +/- 20% of a carnitine entity, and 2.5 g +/- 20% of a serine amino acid entity. In some embodiments, the amino acid composition includes 1 g +/- 15% of a leucine amino acid entity, 0.5 g +/- 15% of an isoleucine amino acid entity, 1.33 g +/- 15% of an arginine amino acid entity, 0.67 g +/- 15% of a glutamine amino acid entity, 0.43 g +/- 15% of a NAC entity, 0.30 g +/- 15% of a carnitine entity, and 2.5 g +/- 15% of a serine amino acid entity. In some embodiments, the amino acid composition includes 1 g +/- 10% of a leucine amino acid entity, 0.5 g +/- 10% of an isoleucine amino acid entity, 1.33 g +/- 10% of an arginine amino acid entity, 0.67 g +/- 10% of a glutamine amino acid entity, 0.43 g +/- 10% of a NAC entity, 0.30 g +/- 10% of a carnitine entity, and 2.5 g +/- 10% of a serine amino acid entity. In some embodiments, the amino acid composition includes 1 g +/- 5% of a leucine amino acid entity, 0.5 g +/- 5% of an isoleucine amino acid entity, 1.33 g +/- 5% of an arginine amino acid entity, 0.67 g +/- 5% of a glutamine amino acid entity, 0.43 g +/- 5% of a NAC entity, 0.30 g +/- 5% of a carnitine entity, and 2.5 g +/- 5% of a serine amino acid entity.

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 bitterness masking agent, and a natural or artificial coloring.

In some embodiments, the composition comprises citric acid.

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 present 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 present 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 N- acetylcysteine are present 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 present 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 farnesoid 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: LMB- 763, LJN-452, emricasan, and cenicriviroc. An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine HC1, 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 HC1.

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

An exemplary Amino Acid Composition includes leucine, isoleucine, valine, arginine HC1, 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 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 : 0.25 : 0.75 : 1 : 0.225 (Table 6). Table 5. Exemplary amino acid components of the composition including Arginine HC1.

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

An exemplary Amino Acid Composition includes leucine, isoleucine, arginine HC1, glutamine, serine, carnitine, and N-acetylcysteine as its amino acid entities in a wt. ratio of 1.0 : 0.5 : 1.61 : 0.67 : 2.5 : 0.33 : 0.15 (Table 7). Table 7. Exemplary amino acid components of the composition including Arginine HC1.

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 all 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 hepatocyte inflammation; c) improving, e.g., increasing, glucose tolerance; d) one or both of decreasing or preventing steatosis; or e) one or both of decreasing or preventing hepatocyte ballooning.

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 a-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 hepatocyte 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., all) 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 III collagen (proC3).

In some embodiments, the decreasing or preventing hepatocyte inflammation comprises reducing a level or activity of one, two, three, four, five, six, seven or more (e.g., all) of NF-kB, interferons, 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 hepatocyte inflammation comprises increasing a level or activity of IL-10. In an embodiment, the improving, e.g., increasing, glucose tolerance, comprises increasing a level or activity of adiponectin. In an embodiment, the improving, e.g., increasing, glucose tolerance, comprises decreasing a level or activity of FGF-21.

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

In any of the foregoing embodiments, the reference composition comprises a single amino acid entity, e.g., a L-amino acid entity, an I-amino acid entity, a V-amino acid entity, a R- amino 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 I- amino 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 R- 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 amino acid 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 I-amino acid-entity or a V- amino acid-entity. In other embodiments, the composition further comprises an I-amino acid- entity and a V-amino acid-entity.

In some embodiments, the amino acid composition comprises LIRQNacCarS.

Production of the Amino Acid Compositions

The present disclosure features a method of manufacturing or making a composition (e.g., an Active Moiety) of the foregoing invention. Amino acid entities used to make the compositions may be agglomerated, and/or instantized to aid in dispersal and/or solubilization.

The amino acid compositions of the present disclosure may be made using amino acids and amino acid derivatives from the following sources, or other sources may used: e.g., FUSI- BCAA™ Instantized Blend (L-Leucine, L-Isoleucine and L-Valine in 2: 1 : 1 weight ratio), FUSIL™ Instantized L-Leucine, L- Arginine HC1, and L-Glutamine may be obtained from Ajinomoto Co., Inc; N-acetyl-cysteine may be obtained from Spectrum Chemical. Pharma grade amino acid entity raw materials may be used in the manufacture of pharmaceutical amino acid entity products. Food (or supplement) grade amino acid entity raw materials may be used in the manufacture of dietary amino acid entity products.

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 in a blending unit, followed by verification 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.

Formulations

The pharmaceutical compositions of the present disclosure may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs, 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 powder) or for parental administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular dosing or as a suppository for rectal dosing).

Excipients

The amino acid compositions of the present disclosure may be compounded or 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.

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

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

In some embodiments, the composition comprises a binder as an excipient. Non-limiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.

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

In some embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable 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 com starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro crystalline 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, raspberry, cherry, plum, pineapple, and apricot.

In some embodiments, the excipient comprises a sweetener. Non-limiting examples of suitable sweeteners include glucose (com syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as the sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives 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-2,2- dioxide, particularly the potassium salt (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 external 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 bitterness masking agent (e.g. 936.2160U), and natural or artificial colorings (e.g. FD&C Yellow 6).

Methods

The present disclosure provides methods of treating a subject having a liver disease (e.g., a liver disease selected from non-alcoholic steatohepatitis (NASH), fatty liver disease (steatohepatitis), alcoholic steatohepatitis (ASH), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), liver fibrosis, and cirrhosis. In particular, the method includes administering to the subject an effective amount (e.g., according to a dosage regimen described herein) of an amino acid composition comprising a L-amino acid entity, optionally an I-amino acid entity, optionally a V-amino acid entity, an R-amino acid entity, a Q-amino acid entity, and a NAC entity; and acquiring a value of liver fat content (e.g., as described herein) for the subject to treat a subject with non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or cirrhosis.

In some embodiments, the method includes, responsive to said value of liver fat content, performing one or more of (e.g., 2, 3, 4, 5, 6, or all of):

(i) identifying the subject as responsive or non-responsive to the amino acid composition; (ii) identifying the subject as in need of a diagnostic, e.g., an assay for PDFF, e.g., an MRI assay for PDFF;

(iii) ending administration of the amino acid composition;

(iv) continuing administration of the amino acid composition;

(v) changing (e.g., increasing or decreasing) the dosage of the amino acid composition;

(vi) changing (e.g., increasing or decreasing) the frequency of administration of the amino acid composition;

(vii) beginning administration of a second therapy, e.g., a second amino acid composition.

In certain embodiments, identifying the subject as responsive or non-responsive to the amino acid composition comprises:

(i) identifying the subject as a responder;

(ii) identifying the subject as a non-responder; or

(iii)identifying the subjects degree of responsiveness.

Patients with Liver Disease

In some embodiments, the subject was treated with an amino acid composition prior to acquiring a value of liver fat content (e.g., as described herein) for the subject.

In some embodiments, a subject has fatty liver disease selected from NAFLD and AFLD. In some embodiments, the subject has pediatric 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 have 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 have cirrhosis. The subject may also have a metabolic syndrome.

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

In some embodiments, the subject has type 2 diabetes.

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 naive subject). In some embodiments, the subject with NAFLD, NASH or cirrhosis has diabetes (e.g., type 2 diabetes).

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 pediatric NAFLD. In some embodiments, the subject has hepatic steatosis. In some embodiments, a subject with pediatric 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., all) of decreased fat metabolism, hepatocyte apoptosis, hepatocyte ballooning, inflammation of adipose tissue, inflammation of hepatic tissue, hepatocyte ballooning, oxidative stress (e.g., reactive oxygen species (ROS), decreased gut barrier function, decreased insulin secretion, or decreased glucose tolerance (e.g., relative to a healthy subject without a liver disease). Improvement in Symptoms of Liver Disease

The amino acid 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 amino acid composition is administered to a subject with NAFLD. In some embodiments, the amino acid composition is administered to a subject with NAFL. In some embodiments, the amino acid composition is administered to a subject with NASH. In some embodiments, the amino acid composition is administered to a subject with cirrhosis of the liver.

In some embodiments, administration of an amino acid 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 amino acid composition results in a decares in the liver fat content of the subject.

In some embodiments, administration of the amino acid composition results in an increase in the level of threonine compared to a level of threonine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in an increase in the level of lysine compared to a level of lysine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in a decrease in the level of ornithine compared to a level of ornithine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in a decrease in the level of asparagine compared to a level of asparagine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in a decrease in the level of serine compared to a level of serine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in a decrease in the level of creatine compared to a level of creatine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in a decrease in the level of phenylalanine compared to a level of phenylalanine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in an increase in the level of acetyl-L-camitine compared to a level of acetyl-L-carnitine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in an increase in the level of glutamate compared to a level of glutamate in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in an increase in the level of glutamine compared to a level of glutamine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in an increase in the level of methionine compared to a level of methionine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in a decrease in the level of proline compared to a level of proline in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject. In some embodiments, the amino acid composition results in an increase in the level of histidine compared to a level of histidine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in a decrease in the level of carnitine compared to a level of carnitine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in an decrease in the level of tyrosine compared to a level of tyrosine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in a change (e.g., an increase or a decrease, e.g., a decrease) in the level of glycine compared to a level of glycine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in an increase in the level of leucine compared to a level of leucine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

In some embodiments, the amino acid composition results in a change (e.g., an increase or a decrease, e.g., an increase) in the level of arginine compared to a level of arginine in a biological sample from the subject, where the biological sample was collected prior (e.g., within 0, 1, 2, 3, 4, 5, or more days prior) to the initial administration of the amino acid composition to the subject.

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

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

In some embodiments, administration of the amino acid composition results in decreased hepatic inflammation in a subject with NAFLD (e.g., a subject with pediatric NAFLD). In some embodiments, administration of the amino acid composition results in decreased hepatocyte ballooning in a subject with NAFLD (e.g., a subject with pediatric NAFLD).

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

In some embodiments, administration of the amino acid composition results in increased insulin secretion in a subject with NAFLD (e.g., a subject with pediatric NAFLD). In some embodiments, administration of the amino acid composition results in improved glucose tolerance in a subject with NAFLD (e.g., a subject with pediatric NAFLD).

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

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

In some embodiments, administration of an amino acid 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 amino acid 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 diabetes). In some embodiments, administration of the amino acid composition results in improved mitochondrial function in a subject with NASH. In some embodiments, administration of the amino acid composition results in white adipose tissue (WAT) browning in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabetes).

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

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

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

In some embodiments, administration of the amino acid composition results in increased insulin secretion in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabetes). In some embodiments, administration of the amino acid composition results in improved glucose tolerance in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabetes).

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

In some embodiments, the amino acid composition reduces liver fat in a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabetes). In some embodiments, the amino acid composition reduces liver enzyme levels (e.g., ALT or AST) in blood or plasma from a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabetes). In some embodiments, administration of an amino acid composition (e.g., at a dosage regimen described herein) including amino acid entities results in an improvement in one or more symptoms of cirrhosis, e.g., a metabolic symptom of cirrhosis, in a subject.

In some embodiments, administration of the amino acid 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 amino acid composition results in increased levels of glutathione (GSH) 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 amino acid 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 amino acid composition results in decreased hepatocyte ballooning 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 amino acid 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 amino acid composition results in increased insulin secretion 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 amino acid composition results in improved glucose tolerance 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid composition is administered at a dose of about 2 g to about 60 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 amino acid 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 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 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. 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 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, every 6 hours, every 7 hours, every 8 hours, every 9 hours, or every 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 following a meal.

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 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 HC1), 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 1.5 g of arginine (or 1.81 g of arginine HC1), 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 HC1), 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 HC1), 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 HC1), 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 HC1), 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 HC1), 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 HC1), 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 HC1), 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 1.5 g or arginine (or 1.81 g of arginine HC1), 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 3.0 g of arginine (or 3.62 g of arginine HC1), 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 3.0 g of arginine (or 3.62 g of arginine HC1), 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 HC1), 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 isoleucine, about 0.25 g of valine, about 0.75 g of arginine (or 0.905 g of arginine HC1), 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 HC1), at least 2 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 HC1), 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 36 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 HC1), 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 HC1), 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 isoleucine, at least 0.25 g of valine, at least 0.75 g of arginine (or 0.905 g of arginine HC1), 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 isoleucine, about 0.25 g of valine, about 0.75 g of arginine (or 0.905 g of arginine HC1), 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 isoleucine, at least 0.5 g of valine, at least 1.5 g of arginine (or 1.81 g of arginine HC1), 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, about 0.5 g of valine, about 1.5 g or arginine (or 1.81 g of arginine HC1), 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 HC1), at least 4 g of 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). 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 HC1), 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 farnesoid X receptor (FXR) agonist, a stearoyl Co A 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

The amino acid composition can be dietary compositions, e.g., chosen from a medical food, a functional food, or a supplement.

The amino acid composition can be for use as a dietary composition, e.g., chosen from a medical food, a functional food, or a supplement. In some embodiments, the 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 diabetes or a relatively high

BMI.

In some embodiments, the subject has fatty liver disease.

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

In some embodiments, the composition promotes 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 hepatocyte ballooning, improved gut barrier function, increased insulin secretion, or glucose tolerance. In certain embodiments, administration of the composition results in an improvement in one or more metabolic symptoms after a treatment period of 24 hours.

In certain embodiments, administration of the composition positively modifies the symptoms and/or conditions to be treated (e.g., to positively modify one, two, or more of a subject’s symptoms, e.g., provide positive clinical response) after a treatment period of 24 hours.

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. Exemplary stick pack formulations are shown in Table 6, below. Mixing protocol

1. Ingredients 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 6. Stick pack formulations

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. An amino acid composition comprising LIRQNacCarS, e.g., as described herein, can be made according to a similar protocol. Example 2: Safety, Tolerability, and Biologic Activity of LIVRQNac and LIRQNacCarS in Subjects With Nonalcoholic Fatty Liver Disease

Objectives: LIVRQNac and LIRQNacCarS are novel, orally administered amino acid compositions, specifically designed to simultaneously support multiple metabolic and fibroinflammatory pathways associated with nonalcoholic fatty liver disease (NAFLD). This study assessed safety, tolerability, and biologic activity of LIVRQNac and LIRQNacCarS in subjects with NAFLD.

Methods: In this multi center, 16-week, placebo-controlled, single-blind, randomized clinical study in NAFLD subjects stratified by type 2 diabetes (T2D), LIVRQNac 24 g, LIRQNacCarS 13.5 g or 20.3 g, or placebo were administered twice daily. Key metabolism measurement magnetic resonance imaging-proton density fat fraction [MRI-PDFF] was evaluated. Safety outcomes included adverse events and standard laboratory assessments.

Results: Baseline characteristics of the 102 enrolled subjects, including 40 with T2D, were consistent with presumed nonalcoholic steatohepatitis (NASH). LIVRQNac showed consistently greater biologic activity than LIRQNacCarS or placebo. Week- 16 changes from baseline with LIVRQNac versus placebo include: MRI-PDFF -22.9% versus -5.7%, . Week-16 changes from baseline with LIRQNacCarS 20.3 g include: MRI-PDFF -8.1%. A greater proportion of subjects treated with LIVRQNac achieved clinically a relevant threshold of: >30% MRI-PDFFat Week-16. Study products were safe and well tolerated with stable lipid and weight profiles.

Conclusions: Both compositions showed multitargeted activity on biological pathways relevant to NAFLD. LIVRQNac demonstrated the greatest activity over 16 weeks, warranting continued clinical investigation in NASH subjects.

Methods

Study design

This 16-week, multicenter, randomized, single-blind, placebo-controlled study was performed under US Food and Drug Administration regulations and guidance in support of research with food, and in accordance with the tenets of the Declaration of Helsinki, and complied with International Council for Harmonisation Harmonized Tripartite Guideline for Good Clinical Practice and all applicable local regulations. The protocol was approved by a central institutional review board (IntegReview) before study initiation. All subjects provided written informed consent. Study population

Male and female subjects aged >18 years with NAFLD were recruited from outpatient clinics in the Summit Clinical Research network if they had proton density fat fraction (PDFF) >10%, corrected T1 [cTl] >830 msec by multiparametric magnetic resonance imaging (MRI), and fasting aspartate aminotransferase >20 IU/L. Body weight (BW) was required to be stable (±5% within 3 months preceding the screening visit with the expectation that subjects would not engage in lifestyle interventions/changes during the study). Subjects with T2D were required to have stable glycemic control on their existing medications (thiazolidinediones, glucagon-like peptide-1 analogs/GLP-1 receptor agonists, and prandial/short-acting insulins were exclusionary), with a screening hemoglobin Ale (HbAlc) <9.5%. Key exclusion criteria were current or history of significant alcohol consumption, liver disease (other than NAFLD or NASH) and/or hepatic decompensation, current or planned use of dietary supplements containing proteins or amino acids, ketones, or fish oils, and instability in chronic conditions.

Intervention

Subjects were randomized 2:2:2: 1 to receive twice-daily LIVRQNac 24 g (22.6 g free AA); LIRQNacCarS (13.5 g or 20.3 g, the latter isocaloric and isonitrogenous to LIVRQNac); or a calorie-, excipient-, and color-matched placebo 24 g orally. Randomization occurred via an interactive web response system using a stratified design in blocks of 7 to ensure even allocation of T2D subjects across all arms. Subjects were blinded to treatment assignment; personnel dispensing study product were unblinded to package LIRQNacCarS into high or low doses and to instruct subjects on the number of packages to consume daily based on dose. Subjects maintained their usual dietary and physical activity pattems/regimens for the study duration.

The study products were packaged in dry powder stick packs. Each LIVRQNac stick pack was composed of leucine 1.00 g, isoleucine 0.50 g, valine 0.50 g, arginine HC1 1.81 g, glutamine 2.00 g, NAC 0.15 g (5.65 g free AA/stick pack), and each LIRQNacCarS stick pack was composed of leucine 1.00 g, isoleucine 0.50 g, arginine HC1 1.61 g, glutamine 0.67 g, serine 2.50 g, carnitine 0.33 g, NAC 0.43 g (6.76 g free AA/stick pack). Each dose (2-4 stick packs) was to be reconstituted as an orange-flavored suspension in 8 oz (-240 mL) of water and administered 30 minutes before a meal. The initial dose was administered at the Day 1 (baseline) visit.

Assessments

Demographics and clinical characteristics were collected on Day 1. Subsequent study visits were scheduled at Weeks 1, 2, 4, 8, 12, and 16 and a safety follow-up at Week 18. Multiparametric MRI examinations and oral glucose tolerance tests were performed at baseline (Day 1), Week 8, and Week 16 to characterize liver fat content (MRI-PDFF). Adverse events (AEs), vital signs, electrocardiograms, safety laboratory tests (including fasting lipid profiles), and physical examinations (including BW) were collected.

Statistical analysis

All analyses were performed at the significance level of 2-sided 0.05 and considered exploratory. The safety population included all subjects receiving >1 dose of study product and subjects were analyzed according to the product/dose received on Day 1. The per-protocol population included all randomized subjects who had >1 postbaseline MRI, received >80% and <120% of study product, and had no major protocol deviations.

Analysis of covariance (ANCOVA) for continuous endpoints and the Cochran-Mantel- Haenszel test for binary endpoints were applied (both adjusted for baseline T2D status); summary statistics were reported based on observed data collected at each visit. Absolute or relative change from baseline at Weeks 8 and 16 of various biomarkers, lipid profiles, and other clinical parameters were summarized and pair-wise comparisons with placebo were performed. Least squares means were estimated through ANCOVA models adjusted by baseline value and T2D status. Responder analyses of clinically relevant thresholds of biologic activity correlated to histologic improvements in NAFLD activity score was also performed (eg, percentage of subjects at Week 16 achieving reductions of >30% in MRI-PDFF,.

RESULTS

Subject disposition and baseline characteristics

Four hundred and eighty-eight (488) subjects signed consent and were screened at 17 study centers. Of these, 102 subjects were randomized and received >1 dose of study product (placebo, n = 15; LIVRQNac 24 g BID, n = 29; LIRQNacCarS 13.5 g BID, n = 26; LIRQNacCarS 20.3 g BID, n = 32) (Table 7). Mean cohort age was 50.2 years (Table 7). Mean (standard error; SE) BW in the LIVRQNac and LIRQNacCarS groups ranged between 102.3 (4.6) and 106.2 (4.6) kg and mean (SE) body mass index ranged between 36.8 (1.36) and 38.5 (1.50) kg/m ² . Baseline demographics were generally well balanced across groups with the exception of higher BW in the placebo group (Table 7). Baseline characteristics were phenotypically consistent with presumed NASH (mean ALT 54.4 U/L, FibroScan 13.0 kPa, and Pro-C3 16.80 ng/mL) and insulin resistance (mean HOMA-IR 10.9) and were similar among groups except for lower HOMA-IR in the placebo group. Forty subjects (39.2%) had comorbid T2D. Mean baseline HbAlc was 7.43% in T2D subjects.

Table 7. Subject demographics and baseline characteristics in the overall safety population Biologic activity of LIVRQNac and LIRQNacCarS

On a background of stable lifestyle regimen and despite the provision of -210 kcal/day with the study products, BW remained stable over the 16-week duration across all treatment arms (mean % BW [SE] change: -0.6% [0.28]) at Week 16 versus baseline (Table 8).

Table 8. Summary of body weight and serum lipid profile changes in the overall safety population at Week 16

Compared with placebo, while both LIVRQNac and LIRQNacCarS reduced liver fat content at Weeks 8 and 16, reductions with LIVRQNac were more pronounced. Relative reduction in MRI-PDFF with LIVRQNac was significantly greater than the corresponding isonitrogenous composition (LIRQNacCarS 20.3g); at Week 8: -20.7% versus -8.8%, Week 16: -22.9% versus -8.1%, P < 0.05 (FIG. 1).

Per-protocol population findings showed similar trends to the safety population, with more notable and consistent improvement in metabolic and fibroinflammatory biomarkers after LIVRQNac treatment versus placebo or LIRQNacCarS (Table 9). Biologic activity with LIVRQNac tended to be more pronounced in those with comorbid T2D than in the overall population; LIVRQNac showed greater reductions in MRI-PDFF (-31.2 versus -8.3%), ALT (-34.6 versus -13.9%), and cTl (-105.1 versus -42.7 msec) than placebo. Detailed analysis including the positive changes on glucose homeostasis induced by LIVRQNac in the T2D subgroup will be reported in a subsequent publication.

Table 9. Change from baseline in MRI-PDFF in the per-protocol population

Safety and tolerability

Product-emergent AEs (PEAEs) reported by subjects receiving LIVRQNac and LIRQNacCarS were mild or moderate (Table 10). The only PEAEs reported by >10% of subjects in any arm were gastrointestinal (diarrhea, nausea, reduced appetite), upper respiratory tract infection, and headache. Gastrointestinal AEs were generally mild and transient, and resolved without intervention (eg, no anti diarrheal, antiperistaltic, or antiemetic agents required) within 2 to 3 weeks. Two serious AEs were reported (1 with LIVRQNac, 1 with LIRQNacCarS 20.3 g); both were determined unrelated to study product. No clinically significant changes in safety laboratory tests including serum lipid levels (Table 2), vital signs, physical exams, or electrocardiograms were reported. Discontinuations due to AEs were noted in 1 subject each in placebo and LIVRQNac arms, and 2 in the LIRQNacCarS 20.3-g arm (Table 10).

Table 10. Summary safety findings in the overall safety population at Week 16

DISCUSSION

LIVRQNac and LIRQNacCarS were safe and well tolerated over 16 weeks and demonstrated clinically relevant multitargeted activity on liver structure and function assessed by biomarkers related to metabolic and fibroinflammatory pathways in a population of presumed NASH subjects. Notably, these positive findings were observed without confounding from BW or serum lipid changes across treatment arms.

Reductions from baseline in measures of liver fat content (MRI-PDFF) tended to be greater and were more consistently demonstrated with LIVRQNac versus isocaloric placebo. At Week 16, a greater proportion of subjects who received LIVRQNac (~35%— 40%) versus placebo (~8%— 25%) achieved >30% jMRI-PDFF, each of these thresholds has been correlated to improved histopathologic outcomes.

Lipotoxicity and insulin resistance are considered hallmarks in the pathogenesis of NASH, with an imbalance in fatty acid biosynthesis and inability of mitochondria to adequately metabolize free fatty acids. Reduced liver fat content and improved insulin sensitivity following LIVRQNac treatment were observed without BW changes despite ingestion of additional -210 calories/day and maintenance of pre-study diet/exercise regimens. These findings imply that LIVRQNac may promote fatty acid oxidation to induce these key physiological changes. Biochemical data from primary human hepatocytes treated with LIVRQNac support this hypothesis and are consistent with reported activities of BCAAs to promote fat oxidation and ketogenesis. Both amino acid compositions were safe and generally well tolerated, consistent with published short- and long-term studies that have long established the safety of AA administration. However, consistently greater biologic activity was observed with LIVRQNac versus LIRQNacCarS, although these amino acid compositions were isocaloric and isonitrogenous. Thus, our findings of differential biologic activity in this rigorously controlled study highlight a critical insight: while administration of AA mixtures may result in comparable safety profiles, biologic activity is highly dependent on the specific amino acid composition.

A key strength of this study is the standardization of calorie- and nitrogen-matched study products in the randomized groups including the control (placebo) arm. Other strengths include patient blinding to product allocation and the 16-week administration providing sufficient exposure to adequately assess safety, tolerability, and biologic activity. Although this was an early-stage study, baseline characteristics (Table 7) indicated that subjects enrolled had presumed NASH. Baseline body mass index was also indicative of a comorbidly obese, insulin-resistant population, factors associated with NASH onset and progression. Body weight was required to be stable for at least 3 months prior to enrollment and subjects were not allowed to engage in any new lifestyle interventions during the study. Other strengths included oral administration with high compliance and ease of administration. There are also important limitations to consider. Because this was an early-phase study, it was not powered for statistical significance, although clinically relevant conclusions can be drawn between study groups based on the strong consistency of changes seen across several metabolic and fibroinflammatory markers, many of which correlate with histologic outcomes. Given the relatively small sample size in this study, potential bias-factor(s) might not be balanced at baseline among study groups, which may require further investigation in future studies.

In conclusion, the systematic characterization of specific amino acid compositions in subjects with NAFLD adds to the depth of knowledge on the safety and physiologic activity of amino acid compositions and highlights that specificity of amino acid composition matters for optimal effects. Our findings support the potential of a novel amino acid composition, LIVRQNac, to simultaneously address the multifactorial pathogenesis of NAFLD/NASH, their key comorbidities, representing a unique modality with a coordinated multitargeted mechanism of action without major safety or tolerability issues. Example 3: Metabolomics data collection

Data were collected on fasting plasma blood draw from patients using amino acid measurements at baseline, weeks 2, 4, 8, 12 and 16 and polar metabolite measurements at baseline, weeks 8 and 16.

Amino acid analysis

Plasma concentration of amino acids (AAs) L, I, V, R and Q in LIVRQNac, other non- dosed amino acids and N-acetylcysteine were determined by a UPLC - MS/MS (ultra performance liquid chromatography - tandem mass spectrometry) method with stable isotope- labeled internal standards used to normalize the response for each analyte. Dipotassium ethylenediaminetetraacetic acid was used as an anticoagulant. The standards were spiked into a commercially available stripped human plasma matrix. Verification of assay selectivity, precision and accuracy were confirmed using fortified authentic human plasma. The linearity for the analytes ranged up to 80 mM. The lower limit of quantification was determined to be 0.25 to 2.5 mM, depending on the analyte.

Polar metabolite analysis

Metabolome analysis was performed in 252 samples of human plasma using Capillary Electrophoresis Fourier Transform Mass Spectrometry (CE-FTMS) in two modes for cationic and anionic metabolites. 518 metabolites (297 metabolites in Cation mode and 221 metabolites in Anion mode) were detected.

Samples were prepared according to manufacturer’s protocols (Human Metabolome Technologies, Yamagata, Japan), filtered, concentrated and resuspended in ultrapure water immediately before measurement.

Compounds were measured in the Cation and Anion modes of CE-FTMS based metabolome analysis. Peaks detected in CE-FTMS analysis were extracted using automatic integration software (MasterHands ver. 2.18.0.1 developed at Keio University) to obtain peak information including m/z, migration time (MT), and peak area. Peak areas were converted to relative peak areas. Absolute quantification was performed in target metabolites. All metabolite concentrations were calculated by normalizing peak area for each metabolite to the area of internal standard and by using standard curves obtained by single-point calibrations. Among target metabolites, 97 metabolites (54 in Cation and 43 in Anion Mode, respectively) were detected and quantified.

Data processing

Amino acid measurements were baseline-corrected to yield percent change from baseline at each timepoint. Polar metabolite measurements were baseline corrected to yield log2 fold change measurements at each timepoint.

Statistical analysis

In order to investigate if there are significant changes in fasting metabolite levels upon LIVRQNac or placebo treatment over time, a two-sided t-test was conducted with the null hypothesis that there is no change from baseline. Using mean baseline corrected metabolite concentration measurements per timepoint and treatment group. The resulting p-values were corrected by Benjamini-Hochberg procedure and visualized on Volcano plots.

Among dosed amino acids, a significant increase in arginine was found at weeks 2, 4 in LIVRQNac group while the rest of the amino acids and timepoints remained non-significant. However, multiple non-dosed amino acids showed significant decrease from baseline at multiple timepoints such as threonine, proline, tyrosine, lysine, tryptophan, methionine. Nondosed amino acids significantly decreased at a single timepoint were tryptophan, serine, glycine. Ornithine, a direct metabolite of arginine, was the only significantly increased non-dosed amino acid.

Unsupervised machine learning

Hierarchical clustering

Baseline corrected metabolite concentration mean per group and timepoint was used for building clusters using correlation metric.

Hierarchical clustering separated LIVRQNac and placebo groups, further supporting the notion of a distinct LIVRQNac metabolic profile. Clusters within the profile separate most dosed amino acids that either increase (arginine and ornithine) or do not change (branched chain amino acids) from nondosed that decrease from baseline upon LIVRQNac treatment. The notion that branched chain amino acids (valine, isoleucine, leucine) do not change in fasting plasma despite dosing suggests that they are actively metabolized.

Note that glutamine shows decreases in LIVRQNac treatment group despite dosing.

Supervised machine learning

Data preparation

Features were built from baseline corrected metabolite concentration changes at week 8. Labels were baseline corrected MRI PDFF measurements at week 16. Each patient represented a data point including all patients in the safety cohort of the study.

Model building

Lasso regression, a multivariate linear regression model with shrinkage, was performed on both datasets.

Random Forest regression, an ensemble method, was performed on the amino acid dataset. Feature importances across these models were visualized on boxplots and top features identified.

Validation

1. Shared features across datasets

If the most important amino acid features of Lasso regression that predict PDFF change are compared across the two datasets, they largely overlap.

2. Most important features are shared across models

If the most important features of the amino acid dataset are compared according to Lasso and Random Forest regressions, they largely overlap, with the exception of arginine being selected by Random Forest regression but dropped by Lasso regression. This exception is understandable as arginine is collinear with ornithine, and Lasso selects the most important one from all collinear features.

3. Significant linear relationship at a different timepoint

Machine learning models were trained on week 8 metabolite changes from baseline and week 16 PDFF change from baseline. This validation is checking for significant linear relationship between metabolite changes at week 16 and PDFF change at week 16. Lysine and ornithine have significant slopes.

4. Significant linear relationship in a different study

The metabolite changes explaining PDFF change were tested for linear relationship to PDFF change in a different patient population, from an earlier study. Here, ornithine and arginine concentration changes from baseline at week 6 showed significant slope for PDFF change at week 6.

Example Summary: Metabolic profile changes predictive of observed reduction in liver fat content

The relationship between plasma metabolomic changes and MRI-PDFF changes observed clinically with LIVRQNac and LIRQSNacCar were assessed. The impact of changes on fasting plasma profile of dosed and non-dosed AAs on MRI-PDFF was also evaluated.

As described above, in a multicenter, randomized, placebo (PBO)-controlled study of LIVRQNac in subjects with NAFLD, fasting plasma samples were analyzed for change from baseline in AAs and polar metabolites over 16 weeks. Hierarchical clustering was conducted to identify metabolite profile differences between LIVRQNac and PBO. Subsequently, machine learning models (Random Forest and Lasso, using leave-one-out cross-validation, as described herein) were fitted to quantify the degree that the distinct metabolic profile of LIVRQNac predicts the observed reduction in MRI-PDFF.

Baseline characteristics were consistent with presumed NASH in 102 subjects who received >1 study product dose. Improvements with LIVRQNac vs PBO in MRI-PDFF were seen at week 16 (-22.9% vs -5.7% PBO; p=.0583). Commensurate changes in LIVRQNac metabolic profile relative to PBO were found, including changes in dosed and non-dosed AAs (FIG. 2A). Statistically significant increases were observed in dosed AAs arginine (and ornithine, its direct metabolite); non-dosed AAs, including threonine, proline, tyrosine, were significantly decreased from baseline at multiple timepoints with LIVRQNac. The branched chain AAs included in LIVRQNac remained unchanged. The machine learning model identified a metabolic signature at week 8 that best explains PDFF changes at week 16 (FIG. 2B; Table 11). Table 11. Amino acid changes that predict the observed changes in MRI-PDFF

These results were robust to different model classes and metabolomic datasets.

This example demonstrates that LIVRQNac treatment has a distinct metabolic signature in fasting plasma predictive of reductions in MRI-PDFF in NAFLD that includes significant changes across dosed and non-dosed AAs, as well as polar metabolites. These findings add to a growing body of clinical data suggesting an association between the metabolic profile of LIVRQNac and improvements in MRI-PDFF, consistent with its proposed multifactorial metabolic impact. Example 4. Validation, significant linear relationship in a different study, and identification of top amino acid predictors of PDFF response across studies and models.

The metabolite changes explaining PDFF change were tested for linear relationship to PDFF change in a different patient population from an earlier study of LIVRQNac. This earlier study was a multicenter, open-label, 12-week, safety and tolerability study in 32 subjects with NAFLD and type 2 diabetes treated with LIVRQNac 24 g TID. In patients with liver fat >10% at baseline (n=23), mean PDFF decreased from 17.8% at baseline to 13.9% at Week 12, a mean change of -4.1% Top predictive features shared across models were validated on the earlier study data.

Here, ornithine and arginine concentration changes from baseline at week 6 showed significant slope for PDFF change at week 6.

Linear regression analyses demonstrated significant correlation between increases in amino acids and decreases in PDFF in LIVRQNac-treated subjects in both early clinical studies. Feature selection across model classes and metabolomic datasets arrived at a shared set of

AAs as most predictive of PDFF changes in study subjects (FIG. 3, , and Table 12).

Table 12. regression coefficients from Random Forest and Lasso models The top “change in PDFF” predictor amino acids were identified in Lasso analysis of polar metabolomics and PDFF liver fat data sets [identified as: ornithine, threonine, lysine, serine, creatine, phenylalanine], in Lasso analysis of amino acid profile and PDFF liver fat data sets [identified as: threonine, glutamate, ornithine, serine, phenylalanine, leucine], and in Random Forest analysis of amino acid profile and PDFF liver fat data sets [identified as: ornithine, lysine, glycine, serine, threonine, leucine] (FIGs. 3A-3C ).

Predictor amino acids common to all three sets are ornithine and threonine. Predictor amino acids common to two or three sets are ornithine, threonine, lysine, serine, phenylalanine and glutamate. The overall set of amino acids identified as top predictors of PDFF response is ornithine, threonine, lysine, serine, phenylalanine, glutamate, arginine, glycine, leucine, and creatine.