WO2022266480A1 - Procédés de prédiction de la teneur en graisse hépatique et leurs utilisations - Google Patents

Procédés de prédiction de la teneur en graisse hépatique et leurs utilisations Download PDF

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WO2022266480A1
WO2022266480A1 PCT/US2022/034053 US2022034053W WO2022266480A1 WO 2022266480 A1 WO2022266480 A1 WO 2022266480A1 US 2022034053 W US2022034053 W US 2022034053W WO 2022266480 A1 WO2022266480 A1 WO 2022266480A1
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level
amino acid
subject
baseline
acid entity
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PCT/US2022/034053
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English (en)
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Michael HAMILL
Szilvia KIRIAKOV
Matthew Russell
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Axcella Health Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine

Definitions

  • Non-alcoholic fatty liver disease 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 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 calculated based on magnetic resonance imaging (MRI) readings, is one approach to assessing liver fat content and grading steatohepatitis.
  • MRI magnetic resonance imaging
  • MRI requires specialized equipment and technical support.
  • Disclosed herein, at least in part, is a method for evaluating a subject by acquiring a value of liver fat content.
  • 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.
  • 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).
  • a level of threonine e.g., wherein the level of threonine
  • the present disclosure also provides methods of evaluating the effectiveness of an amino acid composition in a subject.
  • the method of evaluating the effectivness of an amino acid composition includes acquiring a value of liver fat content for the subject.
  • 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)
  • the disclosure provides a method of treating a subject having a liver disease (e.g., NASH).
  • 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.
  • 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
  • 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
  • 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 measure of one or
  • the disclosure provides methods of evaluating the effectivenss of an amino acid composition in a subject.
  • the method of evaluating the effectivness of an amino acid composition includes acquiring a value of liver fat content for the subject.
  • 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
  • the disclosure provides a method of treating a subject having a liver disease (e.g., NASH).
  • 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.
  • 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 one or more of (
  • 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).
  • 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
  • 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).
  • acquiring the value of liver fat content comprises a measure of the level of threonine, the level of ornithine, and the level of lysine.
  • 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.
  • 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.
  • 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.
  • acquiring the value of liver fat content comprises measuring the level of threonine and ornithine.
  • acquiring the value of liver fat content comprises measuring the level of threonine, ornithine, and lysine.
  • acquiring the value of liver fat content comprises measuring the level of threonine, ornithine, and glutamate.
  • acquiring the value of liver fat content comprises measuring the level of threonine, ornithine, serine, and phenylalanine.
  • acquiring the value of liver fat content comprises measuring the level of threonine, ornithine, glutamate, and lysine.
  • liver fat content measuring the level of threonine, ornithine, lysine, serine, and phenylalanine.
  • 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.
  • 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.
  • 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.
  • 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.
  • the value of liver fat content comprises the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, glutamate, glutamine, and methionine.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the measuring comprises using a separation step followed by a detection step.
  • the separation step is selected from HPLC, UPLC, ion exchange chromatography (IEC), gas chromatography, and capillary electrophoresis (CE).
  • the detection step is selected from mass spectrometry (MS) (e.g., tandem mass spectrometry (MS/MS)).
  • 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.
  • 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.
  • 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).
  • NASH non-alcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • AFLD alcoholic fatty liver disease
  • the subject has, or is identified as having, diabetes, e.g., type II diabetes.
  • the subject is not diabetic.
  • the subject 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%.
  • an MRI is contraindicated for the subject.
  • 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.
  • 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.
  • PDFF proton density fat fraction
  • MRI multiparametric magnetic resonance imaging
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the liver fat content is predictive of the PDFF level measured 8 weeks later (e.g., at week 16 of the amino acid composition).
  • a decrease in the level of threonine compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of lysine compared to the baseline is indicative of decreased liver fat content.
  • an increase in the level of ornithine compared to the baseline is indicative of decreased liver fat content.
  • an increase in the level of asparagine compared to the baseline is indicative of decreased liver fat content.
  • increase in the level of serine compared to the baseline is indicative of decreased liver fat content.
  • an increase in the level of creatine compared to the baseline is indicative of decreased liver fat content.
  • an increase in the level of phenylalanine compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of acetyl-l-carnitine compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of glutamate compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of glutamine compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of methionine compared to the baseline is indicative of decreased liver fat content.
  • an increase in the level of proline compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of histidine compared to the baseline is indicative of decreased liver fat content.
  • an increase in the level of carnitine compared to the baseline is indicative of decreased liver fat content.
  • an decrease in the level of tyrosine compared to the baseline is indicative of decreased liver fat content.
  • a change in the level of glycine compared to the baseline is indicative of decreased liver fat content.
  • an increase in the level of leucine compared to the baseline is indicative of decreased liver fat content.
  • 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.
  • liver fat content responsive to said value of liver fat content, performing one or more of (e.g., 2, 3, 4, 5, 6, or all of):
  • identifying the subject as in need of a diagnostic e.g., an assay for PDFF, e.g., an MRI assay for PDFF;
  • performing i), ii), iii), iv), and v responsive to said value of liver fat content, performing i), ii), iii), iv), 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).
  • performing iii) and iv 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).
  • performing iv) and v responsive to said value of liver fat content, performing iv), v), and vi).
  • performing v) and vi responsive to said value of liver fat content, performing v), vi), vii), and viii).
  • identifying the subject as responsive or non-responsive to the amino acid composition comprises:
  • 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,
  • 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.
  • NAC N-acetylcysteine
  • the total wt. % of (a)-(b) is greater than the total wt. % of any other amino acid entity in the composition.
  • 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).
  • 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.
  • BCAA branched-chain amino acid
  • 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.
  • 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.
  • 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%).
  • a wt 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%).
  • 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%).
  • 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%).
  • 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.
  • 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).
  • 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.
  • the amino acid composition further comprises: (f) an isoleucine amino acid entity.
  • amino acid entities of the amino acid compositions described herein are selected from Table 2.
  • 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.
  • the 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%.
  • 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.
  • 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%.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the branched-chain amino acid is leucine, isoleucine, and valine.
  • 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%.
  • 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).
  • 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).
  • 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.
  • 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.
  • the composition comprises about
  • the composition comprises about
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • composition further comprises one or more pharmaceutically acceptable excipients.
  • 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.
  • the composition is in the form of a solid, powder, solution, or gel.
  • the amino acids consist of leucine, isoleucine, valine, arginine, glutamine and N-acetylcysteine.
  • the subject has type 2 diabetes and/or a relatively high BMI.
  • the subject has non-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • the subject has non-alcoholic fatty liver (NAFL).
  • NAFL non-alcoholic fatty liver
  • the subject has pediatric NAFLD.
  • the patient has steatosis.
  • the subject has non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • the subject has fibrosis.
  • the subject has cirrhosis.
  • the subject has AFLD.
  • the subject has ASH. In some embodiments, the subject has hepatocarcinoma, an increased risk of liver failure, or an increased risk of death.
  • the subject has type 2 diabetes.
  • the amino acid composition is administered at a dose of about 15 g/d to about 90 g/d.
  • 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.
  • the amino acid composition is administered one, two, to three times per day.
  • 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.
  • administering the composition results in an improvement in one or more metabolic symptoms in the subject.
  • 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.
  • the increased free fatty acid and lipid metabolism occurs in the liver.
  • 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).
  • 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.
  • the composition is administered concurrent with a meal.
  • the composition is administered with a second agent.
  • 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.
  • FXR farnesoid X receptor
  • 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.
  • 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;
  • 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, or a dipeptide
  • NAC NAC
  • 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
  • carnitine entity is L-carnitine or a salt thereof or a dipeptide or salt thereof, or tripeptide or salt thereof, comprising L-camitine
  • 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
  • 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.
  • 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.
  • HMB P-hydroxy-P- methylbutyrate
  • oxo-leucine isovaleryl-CoA
  • D-leucine D-leucine
  • n-acetyl-leucine or a combination thereof.
  • the R-amino acid entity is chosen from L-arginine, ornithine, argininosuccinate, agmatine, creatine, and N-acetyl-arginine, or a combination thereof.
  • the Q-amino acid entity is chosen from L-glutamine, carbamoyl-P, glutamate, and n-acetylglutamine, or a combination thereof.
  • the NAC-amino acid entity is chosen from NAC, cystathionine, glutathione, homocysteine, and cysteamine, or a combination thereof.
  • 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.
  • 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.
  • the S-amino acid entity is chosen from L-serine, Phosphoserine, P-hydroxypyruvate, Acetylserine, Phosphatidylserine, or a combination thereof.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • the measuring includes using a separation step followed by a detection step.
  • the separation step is selected from HPLC, UPLC, ion exchange chromatography (IEC), gas chromatography, and capillary electrophoresis (CE).
  • the detection step is selected from mass spectrometry (MS) (e.g., tandem mass spectrometry (MS/MS)).
  • 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.
  • 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.
  • one or more e.g., 1, 2, 3, 4, 5, or all
  • 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).
  • NASH non-alcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • AFLD alcoholic fatty liver disease
  • the subject has, or is identified as having, diabetes, e.g., type II diabetes.
  • the subject is not diabetic.
  • the subject 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%.
  • the amino acid composition comprises at least four different amino acid entities.
  • 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.
  • 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).
  • ROS reactive oxygen species
  • 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).
  • NAFLD non-alcoholic fatty liver disease
  • NAFLD non-alcoholic steatohepatitis
  • AFLD alcoholic fatty liver disease
  • ASH alcoholic steatohepatitis
  • 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.
  • NAFLD e.g., NASH or NAFL
  • AFLD e.g., ASH
  • ASH liver function or liver disease
  • 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.
  • 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.
  • WAT white adipose tissue
  • ROS reactive oxygen species
  • GSH glutathione
  • 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)).
  • a liver disease e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)
  • 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.
  • NAFLD e.g., NASH or NAFL
  • AFLD e.g., ASH
  • 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)).
  • a liver disease e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)
  • 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.
  • NAFLD e.g., NASH or NAFL
  • AFLD e.g., ASH
  • 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.
  • 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.
  • 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;
  • XXX is arginine (R)
  • 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;
  • XXX is glutamine (Q)
  • 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-
  • “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.
  • 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.
  • 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
  • Gin, Q or glutamine all refer to the amino acid glutamine.
  • 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.
  • 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.
  • 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.
  • 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.
  • the amino acid composition is provided as a pharmaceutically acceptable preparation (e.g., a pharmaceutical product).
  • an effective amount 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.
  • the pharmaceutical composition is used as a therapeutic, a nutraceutical, a medical food, or as a supplement.
  • pharmaceutically acceptable refers to amino acids, materials, excipients, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. 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).
  • 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.
  • 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).
  • the liver disease is NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH).
  • “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • “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.
  • “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)).
  • the present disclosure provides methods of evaluating a subject (e.g., evaluating the effectiveness of an amino acid composition in a subject).
  • evaluating a subject includes acquiring a value of liver fat content for the subject.
  • evaluating the effectiveness of an amino acid composition in a subject includes acquiring a value of liver fat content for the subject.
  • 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.
  • 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.
  • the value of liver fat content includes the measure of threonine, lysine, ornithine, asparagine, serine, creatine, phenylalanine, acetyl-L-carnitine, glutamate, glutamine, and methionine.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the measuring includes a separation step followed by a detection step.
  • the separation step substantially reduces the number of undesired compounds (e.g., compounds not used in determining the value of liver fat content).
  • the separation step substantially separates the analytes for analysis.
  • the separation step is selected from HPLC, UPLC, ion exchange chromatography (IEC), gas chromatography, and capillary electrophoresis (CE).
  • the detection step comprises mass spectrometry (e.g., tandem mass spectrometry (MS/MS).
  • the mass spectrometry analysis can comprise Fourier transform (FT), time-of- flight (TOF), matrix assisted laser desorption ionization (MALDI), ion trap, and quadrupole.
  • FT Fourier transform
  • TOF time-of- flight
  • MALDI matrix assisted laser desorption ionization
  • ion trap ion trap
  • quadrupole ion trap
  • 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).
  • the separation step is immediately followed by the detection step (e.g., HPLC-MS/MS, UPLC-MS/MS, CE-MS (e.g, FTMS).
  • internal standards e.g., stable isotope analogs for each amino acid
  • internal standards 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.
  • the measuring includes a deproteinization step of the sample prior to the separation step.
  • 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.
  • methods of precipitating proteins from a biological sample include the use of sulfosalicylic acid (SSA) or trichloracetic acid (TCA).
  • SSA sulfosalicylic acid
  • TCA trichloracetic acid
  • protein precipitation is followed by centrifugation, and/or filtration.
  • 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.
  • the subject was treated with an amino acid composition about 1, 2, 3, 4, 6, 8, 10, 12, 14, or 16 weeks prior.
  • the subject was treated with an amino acid composition about 8 weeks prior.
  • 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%.
  • MRI is contraindicated for the subject.
  • the reduction in liver fat content is measured by MRI-PDFF.
  • 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).
  • NASH non-alcoholic steatohepatitis
  • ASH alcoholic steatohepatitis
  • AFLD alcoholic fatty liver disease
  • the subject has, or is identified as having diabetes, e.g., type II diabetes. In some embodiments, the subject is not diabetic.
  • the baseline level of the amino acid is the level of the amino acid in a biological sample from the subject.
  • the biological sample was collected before the administration of the amino acid composition to the subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an increase in the level of phenylalanine compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of acetyl-l-carnitine compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of glutamate compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of glutamine compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of methionine compared to the baseline is indicative of decreased liver fat content.
  • an increase in the level of proline compared to the baseline is indicative of decreased liver fat content.
  • a decrease in the level of histidine compared to the baseline is indicative of decreased liver fat content.
  • an increase in the level of carnitine compared to the baseline is indicative of decreased liver fat content.
  • an decrease in the level of tyrosine compared to the baseline is indicative of decreased liver fat content.
  • 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.
  • an increase in the level of leucine compared to the baseline is indicative of decreased liver fat content.
  • 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.
  • the method includes performing one or more of (e.g., 2, 3, 4, 5, 6, or all of):
  • identifying the subject as in need of a diagnostic e.g., an assay for PDFF, e.g., an MRI assay for PDFF;
  • identifying the subject as responsive or non-responsive to the amino acid composition includes: i) identifying the subject as a responder;
  • 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
  • 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.
  • the compositions can include five amino acid entities and an antioxidant or reactive oxygen species (ROS) scavenger.
  • ROS reactive oxygen species
  • 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).
  • L leucine
  • R arginine
  • Q glutamine
  • ROS reactive oxygen species
  • 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).
  • NAC N-acetylcysteine
  • 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.
  • 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.
  • 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.
  • Table 2 Amino acid entities include amino acids, precursors, metabolites, and derivatives of the compositions described herein.
  • 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).
  • 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.
  • HMB P-hydroxy-P-methylbutyrate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • BCAA branched-chain amino acid
  • I isoleucine
  • V valine
  • both the I-amino acid-entity and the V-amino acid-entity are present.
  • 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.
  • the I-amino acid entity is selected from the group consisting of a salt, a precursor, a metabolite, and a derivative.
  • 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.
  • the I-amino acid entity is L-isoleucine.
  • 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.
  • 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.
  • the composition comprises L- leucine, L-arginine, L-glutamine, and NAC.
  • the composition comprises HMB, creatine, L-glutamine, and glutathione.
  • the composition comprises HMB, ornithine, L-glutamine, and glutathione.
  • 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.
  • 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.
  • the 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.
  • 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%).
  • 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.
  • 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.
  • 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.
  • the wt the 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%.
  • the wt the 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%.
  • the wt the ratio of the L-amino acid entity, the I-amino acid entity
  • the wt the 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%.
  • the wt the 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%.
  • the wt the ratio of the L-amino acid entity, the I-amino
  • the wt the 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%.
  • the wt the ratio of the L-amino acid entity, the I-amino acid entity, the R
  • the total wt. of amino acids present is about 2 g to about 60 g.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition comprises a combination of 2 to 20 different amino acid entities, e.g., 5 to 15 different amino acid entities.
  • the NAC entity is more stable than cysteine. In certain embodiments, the NAC entity does not comprise cysteine.
  • 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.
  • the composition further comprises serine.
  • the composition further comprises glycine.
  • the composition further comprises carnitine.
  • 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.
  • BCAA branched-chain amino acid
  • the BCAA is leucine.
  • the BCAA is isoleucine.
  • the BCAA is valine.
  • the BCAA is leucine and isoleucine.
  • the BCAA is leucine and valine.
  • the BCAA is isoleucine and valine. In some embodiments, the BCAA is leucine, isoleucine, and valine.
  • composition may consist of leucine, isoleucine, valine, arginine, glutamine, and N-acetyl cysteine.
  • the composition may consist of leucine, isoleucine, arginine, glutamine, N-acetylcysteine, and one or both of serine or carnitine.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a total weight (wt) of the amino acids is about 2 g to about 60 g.
  • 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.
  • the total wt of the amino acids is about 6 g.
  • the total wt of the amino acids is about 12 g.
  • the total wt of the amino acids is about 18 g.
  • the total wt of the amino acids is about 24 g.
  • the total wt of the amino acids is about 48 g.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition comprises citric acid.
  • 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)
  • 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.
  • the amino acids leucine, arginine, glutamine, N-acetylcysteine and glycine are present in a weight ratio of 1 : 1.5 : 2 : 0.15.
  • 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.
  • 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.
  • 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.
  • the composition further comprises an FXR agonist.
  • the FXR agonist is obeticholic acid.
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • 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).
  • MMP matrix metalloproteinase
  • TIMP1 tissue inhibitor of metalloproteinase
  • AST aspartate transaminase
  • ALT alanine transamin
  • 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.
  • the decreasing or preventing hepatocyte inflammation comprises increasing a level or activity of IL-10.
  • the improving, e.g., increasing, glucose tolerance comprises increasing a level or activity of adiponectin.
  • the improving, e.g., increasing, glucose tolerance comprises decreasing a level or activity of FGF-21.
  • the hepatocyte inflammation comprises LPS induced hepatocyte inflammation.
  • 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).
  • the reference composition comprises vehicle (e.g., PBS or saline).
  • 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.
  • an antioxidant or ROS scavenger e.g., a NAC entity, e.g., NAC.
  • 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.
  • the amino acid composition comprises LIRQNacCarS.
  • compositions e.g., an Active Moiety
  • 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- BCAATM Instantized Blend (L-Leucine, L-Isoleucine and L-Valine in 2: 1 : 1 weight ratio), FUSILTM 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.
  • 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.
  • 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).
  • 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
  • topical use for example as creams,
  • the amino acid compositions of the present disclosure may be compounded or formulated with one or more excipients.
  • 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.
  • the excipient comprises a buffering agent.
  • suitable buffering agents include citric acid, sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • the excipient comprises a preservative.
  • suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • the composition comprises a binder as an excipient.
  • 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.
  • the composition comprises a lubricant as an excipient.
  • 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.
  • the composition comprises a dispersion enhancer as an excipient.
  • 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.
  • the composition comprises a disintegrant as an excipient.
  • the disintegrant is a non-effervescent disintegrant.
  • 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.
  • the disintegrant is an effervescent disintegrant.
  • suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
  • 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.
  • 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.
  • the excipient comprises a sweetener.
  • 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.
  • 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.
  • the composition comprises a coloring agent.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • NASH non-alcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • NAFL non-alcoholic fatty liver
  • 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):
  • identifying the subject as responsive or non-responsive to the amino acid composition comprises:
  • 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.
  • 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.
  • the subject exhibits symptoms of gut leakiness.
  • the subject has gut dysbiosis.
  • 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.
  • 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.
  • the subject exhibits muscle atrophy without fibrosis and/or cirrhosis.
  • the subject exhibits reverse lipid transport from adipose tissue to liver tissue.
  • the subject has fibrosis.
  • the subject may have cirrhosis.
  • the subject may also have a metabolic syndrome.
  • the subject has one, two, or more (e.g., all) of hepatocarcinoma, an increased risk of liver failure, or an increased risk of death.
  • the subject has type 2 diabetes.
  • the subject with a liver disease is a mammal (e.g., a human).
  • the subject has been diagnosed with NAFLD, NASH or cirrhosis.
  • the subject has not received prior treatment with a composition as described herein (e.g., the subject is a naive subject).
  • the subject with NAFLD, NASH or cirrhosis has diabetes (e.g., type 2 diabetes).
  • 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.
  • the subject has non-alcoholic steatohepatitis (NASH). In some embodiments, the subject with NASH has fibrosis.
  • NASH non-alcoholic steatohepatitis
  • 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.
  • 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.
  • a subject exhibits a metabolic symptom of liver disease (e.g.
  • NAFLD NAFLD, NASH, or cirrhosis
  • 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
  • ROS reactive oxygen species
  • 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.
  • a subject e.g., a human
  • the amino acid composition is administered to a subject with NAFLD.
  • the amino acid composition is administered to a subject with NAFL.
  • the amino acid composition is administered to a subject with NASH.
  • the amino acid composition is administered to a subject with cirrhosis of the liver.
  • administering results in an improvement in one or more symptoms of NAFLD, e.g., a metabolic symptom of NAFLD, in a subject.
  • administration of the amino acid composition results in a decares in the liver fat content of the subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a change e.g., an increase or a decrease, e.g., a decrease
  • 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.
  • 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.
  • a change e.g., an increase or a decrease, e.g., an increase
  • 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).
  • WAT white adipose tissue
  • 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).
  • ROS reactive oxygen species
  • 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).
  • 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).
  • administration of the amino acid composition results in improved gut barrier function in a subject with NAFLD (e.g., a subject with pediatric NAFLD).
  • 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).
  • 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).
  • 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).
  • liver enzyme levels e.g., ALT or AST
  • administering results in an improvement in one or more symptoms of NASH, e.g., a metabolic symptom of NASH, in a subject.
  • 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).
  • WAT white adipose tissue
  • 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).
  • ROS reactive oxygen species
  • 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).
  • GSH glutathione
  • 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).
  • 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).
  • a subject with NASH e.g., a subject with NAFLD, fibrosis, and type 2 diabetes.
  • 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).
  • 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).
  • 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.
  • liver enzyme levels e.g., ALT or AST
  • 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).
  • ROS reactive oxygen species
  • 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).
  • GSH glutathione
  • 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).
  • 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).
  • a subject with cirrhosis e.g., a subject with hepatocarcinoma, increased risk of liver failure, and increased risk of death.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • a disorder e.g. , a liver disease in a subject (e.g., a human).
  • the subject has NAFLD.
  • the subject has NAFL.
  • the subject has NASH.
  • the subject has cirrhosis.
  • the amino acid composition can be provided to a patient with a liver disease (e.g., liver cancer).
  • a liver disease e.g., liver cancer
  • doses are administered, e.g., twice daily, three times daily, four times daily, five times daily, six times daily, seven times daily, or more.
  • the composition is administered for at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 2 weeks.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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).
  • the composition is administered at a dose of about 12 g total amino acids three times per day.
  • 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).
  • 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).
  • the composition is administered at a dose of about 18 g total amino acids three times per day.
  • 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).
  • 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).
  • the composition is administered at a dose of about 24 g total amino acids three times per day.
  • 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).
  • 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).
  • the composition is administered at a dose of about 48 g total amino acids three times per day.
  • 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).
  • 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)).
  • a liver disease e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • FXR farnesoid X receptor
  • the method further includes administering an FXR agonist.
  • the FXR agonist is obeticholic acid.
  • the method further includes administering one or more of: LMB-763, LJN-452, emricasan, and cenicriviroc.
  • 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.
  • the dietary composition is for use in a method comprising adminstering the composition to a subject.
  • the subject has one or both of type 2 diabetes or a relatively high
  • the subject has fatty liver disease.
  • the subject has NAFLD (e.g., pediatric NAFLD). In an embodiment, the subject has NASH. In an embodiment, the subject has NAFL.
  • NAFLD e.g., pediatric NAFLD.
  • the subject has NASH.
  • NAFL e.g., NAFL.
  • 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.
  • the composition promotes weight loss in the subject.
  • 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.
  • administration of the composition results in an improvement in one or more metabolic symptoms after a treatment period of 24 hours.
  • 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.
  • 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
  • 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.
  • the container was sealed and placed in a Turbula mixer and contents mixed on low setting for 2 minutes.
  • the blended powder was sieved using a No. 14 screen and any clumps not passing through the sieve were broken apart.
  • 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.
  • Example 2 Safety, Tolerability, and Biologic Activity of LIVRQNac and LIRQNacCarS in Subjects With Nonalcoholic Fatty Liver Disease
  • 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.
  • 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.
  • 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.
  • 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.
  • MRI-PDFF liver fat content
  • 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.
  • ANCOVA covariance
  • 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,.
  • PEAEs Product-emergent AEs
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CE-FTMS Capillary Electrophoresis Fourier Transform Mass Spectrometry
  • Samples were prepared according to manufacturer’s protocols (Human Metabolome Technologies, Yamagata, Japan), filtered, concentrated and resuspended in ultrapure water immediately before measurement.
  • 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.
  • 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.
  • 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.
  • Baseline corrected metabolite concentration mean per group and timepoint was used for building clusters using correlation metric.
  • 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.
  • branched chain amino acids valine, isoleucine, leucine
  • glutamine shows decreases in LIVRQNac treatment group despite dosing.
  • 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.
  • 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.
  • 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
  • ornithine and arginine concentration changes from baseline at week 6 showed significant slope for PDFF change at week 6.
  • 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.

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Abstract

Cette divulgation concerne, par exemple, un procédé d'évaluation d'un sujet par l'acquisition d'une valeur de la teneur en graisse hépatique. La teneur en graisse hépatique du sujet peut indiquer la réponse d'un sujet à l'administration d'une composition d'acides aminés (par exemple, une composition d'acides aminés comprenant LRQNac). La teneur en graisse hépatique peut être utilisée dans un procédé alternatif non invasif permettant de déterminer la teneur en graisse hépatique, ou pour classer la stéatohépatite (par exemple, une alternative à la MRI-PDFF).
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