N-acylamino acid products and uses

Abstract

The present invention relates to N-acyl amino acid products and their use in the diagnosis and treatment of diseases.

Description

[0001] The introduction of sequence lists as a reference

[0002] As a separate part of the disclosure, this application contains a sequence list in computer-readable form (filename 55769A_Seqlisting.text; 2,597-byte - ASCII text file, created on August 17, 2021), which is incorporated herein by reference in its entirety. Invention Field

[0003] This invention relates to N-acyl amino acid products and their use in the diagnosis and treatment of diseases. Background Technology

[0004] Cardiovascular disease (CVD) caused by atherosclerosis is a leading cause of death worldwide. The most common chronic liver disease, non-alcoholic fatty liver disease (NAFLD), precedes and / or promotes the development of atherosclerosis. A portion of NAFLD patients develop more severe non-alcoholic steatohepatitis (NASH) and liver fibrosis, which can further accelerate the progression of atherosclerosis and CVD events. In fact, CVD is a leading cause of death in NAFLD patients, especially those with NASH.

[0005] There is still a need in the field for products and methods for the diagnosis and treatment of NASH, fibrosis, and CVD. Summary of the Invention

[0006] Abnormal lipid metabolism is a hallmark feature of both CVD and NAFLD. This paper anticipates that the regulation of abnormal metabolism by specific amino acids may also play a role in the pathogenesis of CVD and NAFLD. This application describes the use of N-acyl amino acids, fatty acid-conjugated amino acids, for the diagnosis and treatment of subjects suffering from one or more of the following conditions: steatohepatitis, fibrosis, or cardiovascular disease.

[0007] The first aspect of this article provides a method for treating cardiovascular disease conditions. The method involves administering a therapeutically effective amount of at least one N-acyl amino acid product to a subject suffering from a cardiovascular disease condition. Cardiovascular disease (CVD) conditions affect the heart and blood vessels, and include coronary artery disease, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease, aortic aneurysm, deep vein thrombosis, and pulmonary embolism.

[0008] The second aspect of this article provides a method for reducing fibrosis. The method comprises administering a therapeutically effective amount of at least one N-acyl amino acid product to a subject suffering from fibrosis.

[0009] The third aspect of this article provides a method for treating steatohepatitis. The method involves administering a therapeutically effective amount of at least one N-acyl amino acid product to a subject suffering from steatohepatitis.

[0010] The fourth aspect of this document provides N-acyl amino acid products and compositions, including pharmaceutical compositions. Exemplary N-acyl amino acid products include, but are not limited to, N-acylglycine, N-acylleucine, N-acyl-D-leucine, N-acylglycine-glycine-leucine, N-acylglycine-glycine-D-leucine, pharmaceutically acceptable salts thereof, or combinations of at least two of them.

[0011] The fifth aspect of this article provides methods for diagnosing disease conditions such as cardiovascular disease, fibrosis, or fatty liver disease. These methods include the detection of N-acyl amino acids, such as N-acylglycine, N-acylleucine, and / or N-acyl-D-leucine. Attached Figure Description

[0012] Figure 1 Metabolism and levels of inhibited N-acyl amino acids in NASH. pPCR analysis of hepatic expression of (A) Pm20d1 and (B) Glyat. LC-MS / MS analysis of hepatic concentrations of (C) N-oleoylglycine (C:18-Gly), (D) N-arachidonoylglycine (C20:4-Gly), and (E) N-oleoylleucine (C18:1-Leu). Data are mean ± SEM (n = 8). Compared with CD, *P<0.05; *P<0.01; ***P<0.001; compared with NASH+H2O, #P<0.05, ##P<0.01###P<0.001.

[0013] Figures 2A-2D The correlation between liver levels of N-acyl amino acids and NASH severity was investigated. Spearman correlations were calculated in mice (n=8–9) fed a CD (■) or NASH diet and treated with H2O (control, ▲), leucine (▼), glycine (◆), tripeptide-glycine-leucine (0.125 mg / g / d, ○), or tripeptide-glycine-leucine (0.5 mg / g / d, ●) for NAFLD activity (NAS) and the levels of N-oleoylglycine (C181-Gly), N-arachidonicylglycine (C20:4-Gly), and N-oleoylleucine (C18:1-Leu) in the liver and the following parameters: (A) fatty liver as assessed by lipid extraction and TG quantification, (B) inflammatory infiltration as assessed by F4 / 80 positive area, (C) fibrosis score as assessed by Sirius Red staining, and (D) NAFLD activity score (NAS).

[0014] Figures 3A-3C The association between hepatic levels of N-acyl amino acids and circulating cardiometabolic risk factors. Spearman correlations were calculated between hepatic levels of N-oleoylglycine (C18:1-Gly), N-arachidonicylglycine (C20:4-Gly), and N-oleoylleucine (C18:1-Leu) and plasma levels of (A) ALT, (B) MCP-1, and (C) TC in mice (n=8–9) fed a CD (■) or NASH diet and treated with H2O (control, ▲), leucine (▼), glycine (◆), tripeptide-glycine-glycine-leucine (0.125 mg / g / d, ○), or tripeptide-glycine-glycine-leucine (0.5 mg / g / d, ●).

[0015] Figure 4 N-acyl amino acids directly activate PPARα. (A, B) COS-1 cells were co-transfected with PPREx3-TK-luciferase, PPARα, and Renilla. Twenty-four hours post-transfection, cells were treated for 24 hours with 10 μM PPARα agonist WY-14643, 1 mM glycine or tripeptide glycine-glycine-leucine or 10 μM N-oleoylglycine (C18:1-Gly), N-arachidonicylglycine (C20:4-Gly), or N-oleoylleucine (C18:1-Leu). Luciferase activity was normalized by Renilla. Compared with CTL, ***P<0.001.

[0016] Figures 5A-5C The correlation between hepatic levels of N-acyl amino acids and the expression of PPARα target genes was investigated. Spearman correlations were calculated between hepatic levels of N-oleoylglycine (C18:1-Gly), N-arachidonicylglycine (C20:4-Gly), and N-oleoylleucine (C18:1-Leu) and the expression of (A) Ppargc1a, (B) Acot3, and (C) Acadl in mice (n=8–9) fed a CD (■) or NASH diet and treated with H2O (control, ▲), leucine (▼), glycine (◆), tripeptide-glycine-glycine-leucine (0.125 mg / g / d, ○), or tripeptide-glycine-glycine-leucine (0.5 mg / g / d, ●).

[0017] Figure 6A-6D. N-acyl amino acids stimulate lipid utilization via FAO. (A, B) Oxygen consumption rate (OCR) and dependence on FAO assessed using a Seahorse XFe96 analyzer. HepG2 cells were stimulated with 10 μM N-arachidonicylglycine (C20:4-Gly), N-oleoylleucine (C18:1-Leu), or a mediator (ethanol, EtOH), and then treated with 6 μM of the CPT1 inhibitor etomoxir (n=8). (C, D) Lipid biosynthesis and hydrolysis assessed by monitoring the incorporation of [3H]-acetate (3.3 μCi / ml) into TG in HepG2 cells treated with 10 μM N-arachidonicylglycine (C20:4-Gly), N-oleoylleucine (C18:1-Leu), or a mediator (ethanol, EtOH) (n=6).

[0018] Figure 7 Experimental design for NASH studies in mice.

[0019] Figure 8 A-8C.N-oleoylleucine (C18:1-Leu) reduced body weight without affecting obesity. Body composition analysis based on NMR at weeks 21–22 (n=8): (A) body weight, (B) fat percentage, and (C) lean body mass percentage. Data are mean ± SEM. Statistical differences were compared by one-way ANOVA followed by Tukey post-hoc test or by Kruskal-Wallis test followed by Dunn post-hoc test. ***P < 0.001 compared to SD; ###P < 0.001 compared to NASH; AAAP < 0.001 compared to NASH+C18:1.

[0020] Figure 9 A-9D N-oleoylleucine (C18:1-Leu) had no significant effect on systemic energy balance in NASH. Metabolic parameters (n=8) were assessed using the Integrated Laboratory Animal Monitoring System (CLAMS) at weeks 21–22: (A) respiratory exchange rate (RER), (B) energy expenditure (EE), (C) food intake, and (D) total activity. Data are mean ± SEM. Statistical differences were compared by one-way ANOVA followed by Tukey's post-hoc test or by Kruskal-Wallis test followed by Dunn's post-hoc test. *P<0.05, **P<0.01, ***P<0.001 compared with SD.

[0021] Figure 10A-10B. N-oleoylleucine (C18:1-Leu) significantly reduced hepatomegaly. (A) Gross morphology of the liver and (B) liver weight to body weight ratio at the endpoint (n = 8–10). Data are mean ± SEM. Statistical differences were compared by Kruskal-Wallis test followed by Dunn's post-hoc test. ***P < 0.001 compared to SD; ##P < 0.01 compared to NASH; ^P < 0.05 compared to NASH+C18:1.

[0022] Figure 11 A-11C,N-oleoylleucine (C18:1-Leu) reduced circulating liver enzymes. Circulating liver enzymes at the endpoint: (A) alanine aminotransferase (ALT), (B) aspartate aminotransferase (ASP), and (C) alkaline phosphatase (ALP) (n = 8–10). Data are mean ± SEM. Statistical differences were compared by one-way ANOVA followed by Tukey's post-hoc test or by Kruskal-Wallis test followed by Dunn's post-hoc test. Compared with SD, **P < 0.01, ***P < 0.001; compared with NASH, #P < 0.05, ##P < 0.01.

[0023] Figure 12 A-12D.N-oleoylleucine (C18:1-Leu) significantly reduced diet-induced NASH. (A) Hematoxylin and eosin (H&E) histology of the liver (scale bar = 50 μm). (BE) H&E histology was used to score (B) fatty liver (0-3), (C) lobular inflammation (0-3), and (D) hepatocyte ballooning (0-2). NAFLD activity score (NAS) was calculated as the sum of the above scores (n = 8-10). Data are mean ± SEM. Statistical differences were compared by Kruskal-Wallis test followed by Dunn's post-hoc test. ***P < 0.001 compared with SD, #P < 0.05 compared with NASH, and ^P < 0.05 compared with NASH+C18:1.

[0024] Figure 13 A-13B. N-oleoylleucine (C18:1-Leu) significantly reduced fatty liver. (A) Oil Red O (ORO) histology of the liver (scale bar = 100 μm). (B) Plasma total cholesterol (TC) (n = 8-10). Data are mean ± SEM. Statistical differences were compared by Kruskal-Wallis test followed by Dunn's post-hoc test. **P < 0.01, ***P < 0.001 compared with SD.

[0025] Figure 14A-14C,N-oleoylleucine (C18:1-Leu) significantly reduced NASH diet-induced liver and systemic inflammation. (A) Immunohistochemistry of liver F4 / 80 (scale bar = 50 μm). (B) Plasma CCl2 motif chemokine ligand 2 (CCL2), and (C) CCL5 (n = 8–10). Data are mean ± SEM. Statistical differences were compared by one-way ANOVA followed by Tukey post-hoc analysis or by Kruskal-Wallis test followed by Dunn post-hoc analysis. **P < 0.01, ***P < 0.001 compared with SD, #P < 0.05 compared with NASH.

[0026] Figure 15 A-15B. N-oleoylleucine (C18:1-Leu) significantly reduced NASH diet-induced liver fibrosis. (A) Sirius histology of the liver (scale bar = 50 μm). (B) Fibrosis score based on Sirius histology (n = 8–10). Data are mean ± SEM. Statistical differences were compared by Kruskal-Wallis test followed by Dunn's post-hoc test. **P < 0.01, ***P < 0.001 compared with SD, #P < 0.05 compared with NASH.

[0027] Figure 16 Experimental design for a study of atherosclerosis in mice.

[0028] Figure 17 A-17B.N-oleoylleucine (C18:1-Leu) treatment had no significant effect on body weight and plasma cholesterol in atherosclerotic mice. (A) Body weight and (B) Total plasma cholesterol (TC) at the endpoint. Data are mean ± SEM. Statistical differences were compared using unpaired t-tests.

[0029] Figure 18 N-oleoylleucine (C18:1-Leu) significantly reduced atherosclerosis. Hematologic and epithelial eosinophil (H&E) histology of the aortic sinus was used to quantify plaque area. Data are mean ± SEM. Statistical differences were compared using the Mann-Whitney test. *P < 0.05.

[0030] Figure 19 N-oleoylleucine (C18:1-Leu) significantly reduced the number of diseased macrophages. Mac-2 immunohistochemistry of the aortic sinus was used to quantify the content of diseased macrophages. Data are mean ± SEM. Statistical differences were compared using the Mann-Whitney test. **P < 0.01. Detailed Implementation

[0031] N-acyl amino acid products are products in which the acyl group of a long-chain fatty acid is covalently linked to an amino acid.

[0032] The amino acid component of the N-acyl amino acid products described herein may be glycine or leucine, or a peptide containing both glycine and leucine. The peptide may be, for example, a dipeptide or a tripeptide. Except for glycine, common amino acids contain at least one chiral carbon atom. Leucine exists in two stereoisomers, called the L-isomer and the D-isomer. Most naturally occurring proteins and peptides consist only of the L-isomer. Unless D-leucine is specified, the N-acyl amino acid products containing leucine described herein contain L-leucine.

[0033] Exemplary dipeptide amino acid components are glycine-glycine, glycine-leucine, glycine-D-leucine, leucine-leucine, D-leucine-leucine, D-leucine-D-leucine, and leucine-D-leucine.

[0034] An exemplary tripeptide amino acid component is glycine-glycine-leucine and glycine-glycine-D-leucine.

[0035] In this article, the long-chain fatty acid component of the N-acyl amino acid product can be a polyunsaturated fatty acid or a nitro fatty acid.

[0036] An exemplary N-acyl amino acid product is N-palmitoylglycine.

[0037] An exemplary N-acyl amino acid product is N-stearoylglycine.

[0038] An exemplary N-acyl amino acid product is N-oleoylglycine.

[0039] An exemplary N-acyl amino acid product is N-docosahexaenoylglycine.

[0040] An exemplary N-acyl amino acid product is N-arachidonicylglycine.

[0041] An exemplary N-acyl amino acid product is N-palmitoylleucine.

[0042] An exemplary N-acyl amino acid product is N-stearoylleucine.

[0043] An exemplary N-acyl amino acid product is N-oleoylleucine.

[0044] An exemplary N-acyl amino acid product is N-docosahexaenoylleucine.

[0045] An exemplary N-acyl amino acid product is N-arachidonicylleucine.

[0046] An exemplary N-acyl amino acid product is N-palmitoyl D-leucine.

[0047] An exemplary N-acyl amino acid product is N-stearoyl D-leucine.

[0048] An exemplary N-acyl amino acid product is N-oleoyl D-leucine.

[0049] An exemplary N-acyl amino acid product is N-docosahexaenoyl D-leucine.

[0050] An exemplary N-acyl amino acid product is N-arachidonicoyl D-leucine.

[0051] An exemplary N-acyl amino acid product is N-palmitoylglycine-glycine-leucine.

[0052] An exemplary N-acyl amino acid product is N-stearoylglycine-glycine-leucine.

[0053] An exemplary N-acyl amino acid product is N-oleoylglycine-glycine-leucine.

[0054] An exemplary N-acyl amino acid product is N-docosahexaenoylglycine-glycine-leucine.

[0055] An exemplary N-acyl amino acid product is N-arachidonicylglycine-glycine-leucine.

[0056] An exemplary N-acyl amino acid product is N-palmitoylglycine-glycine-D-leucine.

[0057] An exemplary N-acyl amino acid product is N-stearoylglycine-glycine-D-leucine.

[0058] An exemplary N-acyl amino acid product is N-oleoylglycine-glycine-D-leucine.

[0059] An exemplary N-acyl amino acid product is N-docosahexaenoylglycine-glycine-D-leucine.

[0060] An exemplary N-acyl amino acid product is N-arachidonicylglycine-glycine-D-leucine.

[0061] The fatty acid component of the N-acyl amino acid product described herein can be a polyunsaturated fatty acid (PUFA), such as linoleic acid, conjugated linoleic acid, or omega-3 fatty acid. Exemplary omega-3 fatty acids include, but are not limited to, docosahexaenoic acid, alpha-linolenic acid, or eicosapentanoic acid. The fatty acid component of the N-acyl amino acid product described herein can be a metabolite of an omega-3 fatty acid, such as a furan fatty acid or resolvin. An exemplary furan fatty acid is 3-carboxy-4-methyl-5-propyl-2-furanopropionic acid. An exemplary resolvin is resolvin D.

[0062] In this article, the fatty acid component of the N-acyl amino acid product can be a nitro fatty acid, such as 10-nitro-octadec-9-enoic acid, 9-nitro-octadec-9-enoic acid, nitrated ω-3 fatty acids (including, but not limited to, linolenic acid, α-linolenic acid, eicosapentaenoic acid, docosahexanoic acid, and stearidonic acid), nitrated ω-5 fatty acids (including, but not limited to, myristone acid), nitrated ω-6 fatty acids (including, but not limited to, linoleic acid, γ-linoleic acid, dihomo-gamma-linoleic acid, and arachidonic acid), nitrated ω-7 fatty acids (including, but not limited to, conjugated linoleic acid and palmitoleic acid), or nitrated ω-9 fatty acids (including, but not limited to, oleic acid and erucic acid).

[0063] Combinations of different N-acyl amino acid products are also provided. For example, combinations of two or more of N-arachidonic glycine, N-oleoylleucine, and N-oleoyl D-leucine are provided. As yet another example, a combination of N-arachidonic glycine and N-oleoylleucine is provided. As yet another example, combinations of two or more of N-arachidonic glycine-glycine-leucine, N-oleoylglycine-glycine-leucine, N-arachidonic glycine-glycine-D-leucine, and N-oleoylglycine-glycine-D-leucine are provided. As a further example, combinations of two or more of N-arachidonic glycine, N-oleoylleucine, N-arachidonic glycine-glycine-leucine, N-oleoylglycine-glycine-leucine, N-arachidonic glycine-glycine-D-leucine, and N-oleoylglycine-glycine-D-leucine are provided.

[0064] The N-acyl amino acid products described herein also include pharmaceutically acceptable salts. Pharmaceutically acceptable salts are well-known in the art. For example, Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). Examples of such salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids. Examples of metal salts include alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium, magnesium, and barium salts; and aluminum salts. Examples of salts with organic bases include salts formed with trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylenediamine. Examples of salts with inorganic acids include salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Examples of salts with organic acids include salts formed with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.

[0065] The N-acyl amino acid products or their pharmaceutically acceptable salts described herein can be synthesized and / or administered as prodrugs in their original synthetic form. A prodrug is a compound that, under physiological conditions in vivo, is converted to the product described herein through reactions such as enzymes or gastric acid; specifically, a compound that, according to enzymes, is converted to a glycine tripeptide molecule or its pharmaceutically acceptable salt through oxidation, reduction, hydrolysis, etc.; or a compound that, according to gastric acid, is converted to a glycine tripeptide molecule through hydrolysis, etc. See, for example, IYAKUHIN no KAIHATSU (Development of Pharmaceuticals), Vol. 7, Design of Molecules, pp. 163-198, published by HIROKAWA SHOTEN (1990).

[0066] The peptide components of the N-acyl amino acid products described herein can be generated using peptide synthesis methods known in the art. Peptide synthesis can employ condensation reactions, for example, in solid-phase or liquid-phase synthesis methods. If the resulting product has a protecting group, the protecting group is removed. Examples of known peptide synthesis methods include those described in the following: M. Bodanszky and MA Ondetti: Peptide Synthesis, Interscience Publishers, New York (1966); Schroeder and Luebke: The Peptide, Academic Press, New York (1965); Nobuo Izumiya et al.: Peptide Gosei-no-Kiso to Jikken (Basics and experiments of peptide synthesis), published by Maruzen Co. (1975); Haruaki Yajima and Shunpei Sakakibara: Seikagaku Jikken Koza (Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry of Proteins) IV, 205 (1977); and Haruaki Yajima ed.: Zoku Iyakuhin no Kaihatsu (A sequel to Development of Pharmaceuticals), Vol. 14, Peptide Synthesis, published by Hirokawa. Shoten Publishing.

[0067] The compositions provided herein comprise at least one N-acyl amino acid product, or a combination of N-acyl amino acid products.

[0068] The pharmaceutical compositions provided herein comprise pharmaceutically acceptable excipients and at least one N-acyl amino acid product or a combination of two or more N-acyl amino acid products.

[0069] Pharmaceutical compositions suitable for delivering the N-acyl amino acid products described herein and methods for their preparation will be apparent to those skilled in the art. Exemplary standard considerations and methods are provided in Remington's Pharmaceutical Sciences, The Science and Practice of Pharmacy, 22nd Edition, Lippincott Williams & White, Baltimore, MD (2013).

[0070] Depending on the specific route of administration and dosage form, the pharmaceutical compositions described herein are formulated with pharmaceutically acceptable excipients, such as carriers, solvents, stabilizers, adjuvants, diluents, etc. Components of the pharmaceutical composition may be included to modify, maintain, or preserve, for example, the composition's pH, molar osmolarity, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. Compositions are typically formulated to achieve physiologically compatible pH, ranging from about 3 to about 11, from about pH 3 to about pH 7, or from about pH 5.0 to about pH 8, depending on the formulation and route of administration.

[0071] Suitable excipients include, for example, sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a typical excipient when the pharmaceutical composition is administered intravenously. Saline solutions (including, but not limited to, sodium chloride solutions) and aqueous solutions of glucose and glycerol can be used as liquid excipients, particularly for injectable solutions. Additional suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rich, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. A larger list of anticipated excipients includes, but is not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, triglycerides, etc.). HCl, citrates, phosphates, other organic acids); fillers (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); colorants; flavorings and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight peptides; salt-forming counterions (such as sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); Solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, dehydrated sorbitol esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethorphan, lecithin, cholesterol, tyloxapal); stability enhancers (sucrose or sorbitol); strain enhancers (e.g., alkali metal halides (in one respect, sodium chloride or potassium chloride, mannitol or sorbitol); delivery media; diluents; and / or carrier molecules, including large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive viral particles.

[0072] The pharmaceutical compositions described herein can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The pharmaceutical compositions described herein can be formulated for the immediate release and / or modified release of N-acyl amino acid products.

[0073] The pharmaceutical compositions described herein may be administered via any suitable route, such as intravenous, oral, ocular, intradermal, subcutaneous, intraperitoneal, or intramuscular routes. Oral administration is contemplated using delivery media known in the art that minimize the degradation of N-acyl amino acids in the gastrointestinal tract, including, but not limited to, microspheres, liposomes, enteric-coated dry emulsions, tablets, or nanoparticles.

[0074] A kit for administering an N-acyl amino acid product or combination of products to a subject in need includes the N-acyl amino acid product composition described herein, instructions for using the N-acyl amino acid product composition, and optional additional second therapeutic agent or therapy.

[0075] The exemplary stock solution composition of this article comprises N-arachidonicylglycine and / or N-oleoylleucine diluted to a final concentration of 50 mg / ml in 100% ethanol. N-arachidonicylglycine is stored at -80°C, and N-oleoylleucine is stored at -20°C. The exemplary pharmaceutical composition of this article is then freshly prepared from the stock solution composition by dilution in a mixture of 100% ethanol and sterile saline solution (0.9% NaCl) at a ratio of 4:15:81 (v:v:v) to produce a composition of N-acyl amino acid product at a concentration of 2 mg / ml.

[0076] This document describes an exemplary stock solution composition comprising N-acylglycine-glycine-leucine and / or N-acylglycine-glycine-D-leucine, comprising a lyophilized cake prepared in a formulation buffer consisting of 10 mM glutamic acid, 2% glycine, 1% sucrose, and 0.01% polysorbate 20 to pH 4.25. The exemplary pharmaceutical composition is then prepared by reconstitution with a volume of sterile diluent, such as sterile isotonic saline or water, for example 0.5 mL to about 10 mL, or for example 2.2 mL of sterile water, to produce a composition of N-acyl amino acid product at a concentration of 1 g / mL to about 100 g / mL.

[0077] A method is provided for administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of the N-acyl amino acid product or combination thereof described herein.

[0078] The subject can be a mammal, and a mammal can be, for example, a laboratory animal or a human, and the human subject includes adult, adolescent and pediatric subjects.

[0079] As used herein, a “therapeutic effective amount” refers to an amount of N-acyl amino acid product sufficient to demonstrate a detectable therapeutic effect. The effect is detected by improvement in the clinical condition and / or reduction, elimination, or inhibition of the development of specific symptoms or events associated with the condition. The precise effective amount for a subject will depend on the subject’s weight, size, and health status; the nature and severity of the condition; and the product or combination of products selected for administration. Therapeutic effective amounts are determined through routine testing within the skill and judgment of a clinician.

[0080] The pharmaceutical compositions described herein may be administered to the subject via any suitable route as indicated above. For example, the compositions of the present invention may be administered via intravenous, oral, ocular, intradermal, intraperitoneal, subcutaneous, or intramuscular routes.

[0081] Those skilled in the art will understand that the effective dose varies in part depending on the delivered molecule, the indication for use of the composition, the route of administration, and the size (weight, body surface or organ size) and condition (age and general health status) of the subject. Therefore, clinicians may titrate the dose and modify the route of administration to obtain the best therapeutic effect. Therapeutic effective doses may include, but are not limited to, doses from about 1 mg / kg to about 10,000 mg / kg, about 1 mg / kg to about 1,000 mg / kg, about 0.1 mg / kg to about 1,000 mg / kg, about 1 mg / kg to about 1,000 mg / kg, about 1,000 mg / kg to about 10,000 mg / kg, or about 1 mg / kg to about 500 mg / kg, calculated based on the subject's body weight. An exemplary therapeutic effective dose is about 100 mg to about 200 g. An exemplary therapeutic effective dose is about 100 mg / kg to about 200 mg / kg. Another exemplary therapeutically effective dose is about 0.01 mg / kg to about 200 mg / kg. Another exemplary therapeutically effective dose is about 10 mg / kg to about 200 mg / kg. Another exemplary therapeutically effective dose is about 1 mg to about 10 mg. Dosage can be given once daily, twice or three times daily, every other day, twice weekly, once weekly, once monthly, or once every six months. Delivery can also be by continuous infusion. The methods described herein can be used to treat, for example, cardiovascular conditions, steatohepatitis, and fibrosis.

[0082] The term "treating" (or other forms of the term, such as "treatment" or "treat") is used herein to mean applying the compositions of the present invention to alleviate a subject's condition and / or reduce, suppress, or eliminate specific symptoms or events associated with the condition. Therefore, the term "treating" includes preventing a subject from developing a condition, particularly when a subject is prone to acquiring a condition; reducing or suppressing a condition; and / or improving or reversing a condition. Within the scope of the methods of the present invention relating to condition prevention, it should be understood that the term "prevention" does not require the complete avoidance of a condition.

[0083] Cardiovascular conditions are diseases of the heart and blood vessels, including, but not limited to: coronary artery disease – a disease of the blood vessels that supply the heart muscle; cerebrovascular disease – a disease of the blood vessels that supply the brain; peripheral artery disease – a disease of the blood vessels that supply the arms and legs; rheumatic heart disease – damage to the heart muscle and heart valves caused by rheumatic fever from streptococcal bacteria; congenital heart disease – a structural malformation of the heart present at birth; aortic aneurysm – an abnormal protrusion of the aortic wall; and deep vein thrombosis and pulmonary embolism – blood clots in the leg veins that can shift and move to the heart and lungs. Treatment of cardiovascular conditions results in one or more of the following: mitigation detectable by standard techniques, including but not limited to: reduction of atherosclerotic plaques (e.g., demonstrated by ultrasound imaging), increase in cardiac function, reduction of myocardial hypertrophy [e.g., demonstrated by ultrasound imaging, computed tomography, magnetic resonance imaging, or analysis of biomarkers such as troponin and / or BMP (or other biomarkers, such as those listed on page e101 of Tang et al., Circulation, 116:e99-e109 (2007))], decrease in blood pressure, reduction of inflammatory states (e.g., demonstrated by analysis of circulating inflammatory markers such as MCP-1, C-reactive protein, serum amyloid A protein, heat shock protein 65, interleukin-6, and leukocyte adhesion molecules), and reduction in aortic diameter.

[0084] Events related to cardiovascular conditions that can be alleviated through treatment as described in this article include, but are not limited to, heart failure, decompensation (e.g., cardiac or hepatic), myocardial infarction, and aneurysm.

[0085] Steatohepatitis is a type of fatty liver disease characterized by inflammation of the liver along with fat accumulation. Nonalcoholic steatohepatitis (NASH) causes liver damage similar to that seen in steatohepatitis caused by heavy alcohol consumption. Macroscopically and microscopically, NASH is characterized by lobular and / or portal vein inflammation, varying degrees of fibrosis, hepatocellular death, and pathological angiogenesis. In the most severe cases, NASH can progress to cirrhosis, hepatocellular carcinoma, and liver failure. The treatment of steatohepatitis described in this article can be monitored using the subject's NAFLD activity score. The NAFLD activity score (NAS) can be calculated according to the criteria of Kleiner et al., Hepatology, 41:1313-1321 (2005). NAS scores of 0–2 are not considered diagnostic for NASH, NAS scores of 3–4 are considered non-diagnostic, ambiguous, or positive for NASH, while NAS scores of 5–8 are primarily considered diagnostic for NASH. Serial liver biopsies from subjects who may have NASH can be used to assess changes in the NAS score and serve as an indicator of changes in disease status. An increased score indicates progression, a unchanged score indicates stabilization, and a decreased score indicates regression of NASH. Treatment of steatohepatitis in this study resulted in one or more mitigating effects detectable by standard techniques, including, but not limited to: a reduction in hepatic fat (e.g., demonstrated by lipid staining with Oil Red O, biochemical analysis of triglycerides, or ultrasound imaging), a reduction in inflammatory states (e.g., demonstrated by histology as described in Table 1 above by Kleiner, or by analysis of circulating inflammatory markers such as MCP-1, C-reactive protein, serum amyloid A protein, heat shock protein 65, interleukin-6, and leukocyte adhesion molecules), a reduction in damaged hepatocytes (e.g., demonstrated by histology), and a reduction in atherosclerotic plaques (e.g., demonstrated by ultrasound imaging).

[0086] Fibrosis is a pathological wound healing process in which connective tissue replaces normal parenchymal tissue to the extent that it leads to extensive tissue remodeling and the formation of permanent scar tissue. Excessive accumulation of extracellular matrix components, such as collagen produced by fibroblasts, results in the formation of permanent fibrotic scars. Fibrosis is scored from 0 to 4 (0: no fibrosis; 1: perisinusoidal or portal venous fibrosis; 2: perisinusoidal and portal venous fibrosis; 3: bridging fibrosis; 4: cirrhosis). See, for example, Kleiner, Table 1 above. An increasing score indicates progression, a unchanged score indicates stabilization, and a decreasing score indicates regression of fibrosis. Treatment of fibrosis described in this article results in a reduction of fibrosis detectable by standard techniques in one or more of the liver, heart, lungs, kidneys, skin, and adipose tissue. Treatment of fibrosis described in this article may also result in a reduction of fibrosis detectable by standard techniques in one or more of the bile ducts, gallbladder, or other structures involved in bile production and transport. For example, collagen accumulation is routinely detected by methods such as Picrosirius Red or Masson trichrome staining, or by the detection of hydroxyproline.

[0087] The treatment described herein may include treatment with one or more N-acyl amino acid products in combination with a second therapeutic agent, such as other lipid-lowering agents and / or glucose-lowering agents. Other lipid-lowering agents and / or glucose-lowering agents include, but are not limited to, statins, fibrates, SGLT2 inhibitors, metformin, and enterokinins.

[0088] The diagnostic methods described in this article involve detecting N-acyl amino acids in the body, such as N-acylglycine, N-acylleucine, and / or N-acyl-D-leucine. The diagnostic expectations presented include preliminary diagnosis and / or monitoring of disease progression / regression. Liver levels of these N-acyl amino acids are negatively correlated with the severity of fatty liver, fibrosis, inflammation, and hypercholesterolemia.

[0089] Example

[0090] This invention is illustrated by the following examples, which include a long-term dietary model of NASH characterized by the coexistence of steatohepatitis and fibrosis in mice. Unbiased analysis of liver gene expression, confirmed by RNA sequencing followed by qPCR, revealed that genes encoding enzymes that catalyze the condensation of fatty acids and various amino acids (containing the peptidase M20 domain, Pm20d1), particularly glycine (glycine-N-acyltransferase, Glyat), are repressed in NASH. Targeted metabolomics showed that the levels of N-oleoylglycine (C18:1-Gly), N-arachidonicylglycine (C20:4-Gly), and N-oleoylleucine (C18:1-Leu) were significantly reduced in the livers of mice with NASH. This reduction was alleviated by long-term treatment with free glycine or tripeptide glycine-glycine-leucine. The liver levels of the aforementioned N-acyl amino acids were significantly and negatively correlated with the severity of fatty liver, fibrosis, inflammation, and hypercholesterolemia, while positively correlated with the expression of target genes of peroxisome proliferator-activated receptor-α (PPARα), a major regulator of fatty acid β-oxidation (FAO). Using Seahorse and luciferase assays, it was found that N-acyl amino acids directly activate PPARα, stimulating mitochondrial respiration and FAO. In conclusion, N-acyl amino acids mediate hepatic lipid utilization and improve energy metabolism, thus constituting an effective treatment for CVD, steatohepatitis, and fibrosis.

[0091] Example 1

[0092] Tripeptide glycine-glycine-leucine protects against [the virus] by regulating liver metabolism and N-acyl amino acid levels. NASH

[0093] To explore the therapeutic potential of tripeptide glycine-glycine-leucine for NASH, an experimental approach mimicking advanced NAFLD was used. As described, C57BL / 6J mice were fed a high-fat, high-fructose, and high-cholesterol diet (NASH diet) for 12 weeks. After NASH was confirmed, the mice were randomized to receive oral administration of 0.125 or 0.5 mg / g / day of tripeptide glycine-glycine-leucine, an equivalent amount of leucine, glycine, or H2O for an additional 12 weeks on the NASH diet. Mice fed a low-fat control diet (CD) and administered H2O served as controls.

[0094] method

[0095] animal

[0096] Animal procedures were approved by the Institutional Animal Care & Use Committee of the University of Michigan (UM) (PRO00008239) and conducted in accordance with institutional guidelines. Seven-week-old male C57BL mice were used, sourced from Jackson Laboratories. After one week of acclimatization, mice were fed an unlimited diet of either a low-fat control diet (CD, Research Diets D17072805, 10% fat) or a high-fat, high-fructose, and high-cholesterol diet (NASH diet, Research Diets D17010103). After confirming NASH, mice were randomized to receive oral administration of 0.125 or 0.5 mg / g / day of tripeptide glycine-glycine-leucine (Beijing Shuanglu Pharmaceutical Co., Ltd.), an equivalent amount of leucine (0.17 mg / g / day, Sigma-Aldrich L8912), glycine (0.33 mg / g / day, Sigma-Aldrich G5417), or H2O for an additional 12 weeks on the NASH diet.

[0097] Histology and Immunohistochemistry

[0098] All histological procedures were performed by the In Vivo Animal Core (IVAC) Histology Laboratory at UM. Technicians were unaware of the experimental group's procedures. Formalin-fixed tissues were processed with fractional alcohol and cleared with xylene using an automated VIP5 or VIP6 tissue processor (TissueTek, Sakura-Americas), followed by infiltration with molten paraffin. The tissues were sectioned at a thickness of 4 μm using a Histostar Embedding Station (ThermoFisher Scientific) and mounted on slides using an M355S rotary microtome (ThermoFisher Scientific). The slides were stained with hematoxylin and eosin (H&E, ThermoFisher Scientific). For Sirius red staining, slides were treated with 0.2% phosphomolybdic acid for 3 minutes and then transferred to 0.1% Sirius red (Rowley Biochemical Inc.) saturated in picric acid for 90 minutes, followed by transfer to 0.01N hydrochloric acid for 3 minutes.

[0099] Frozen sections were prepared for Oil Red O staining. Formalin-fixed liver samples were cryoprotected overnight at 4°C in 20% sucrose, blotted dry, then blast-frozen in liquid nitrogen with an OCT compound (Tissue-Tek, Cat#4583) and stored at -80°C until ready for cryosectioning. Before sectioning, the frozen blocks were acclimated to approximately -20°C and sectioned at 5 μm using a cryostat (Cryotome) SME (Thermo-Shandon, Cat#77200227). Slides were stored at -80°C until staining. Before staining, liver slides were thawed to room temperature for 30 minutes. Slides were post-fixed in 10% neutral buffered formalin for 20 minutes, rinsed in DDW, and then rinsed in 60% isopropanol for 5 minutes before being placed in working oil red O-isopropanol stain (Rowley Biochemical Inc., H-503-1B). The slides were then rinsed in 60% isopropanol followed by three DDW changes. Finally, the slides were counterstained with Harris hematoxylin and mounted in Aqua-Mount (Lerner Laboratories, Cat#13800) aqueous mounting medium.

[0100] Immunohistochemical staining was performed on an IntelliPATH FLX automated immunohistochemical stainer (Biocare Medical) after blocking endogenous peroxidase and nonspecific binding. Detection was then performed using a commercial detection system based on horseradish peroxidase-based biotin-free polymers, with visualization using diaminobenzidine chromogen and nuclear counterstaining using hematoxylin. Specific to F4 / 80 (Bio-Rad ABD Serotec, Cat#MCA497), rat monoclonal primary antibody (clone CI:A3-1) was diluted to 1:400 in DaVinci Diluent (Biocare Medical, Cat#PD900) and incubated for 60 minutes, followed by detection using a Rat-on-Mouse HRP-Polymer (Biocare Medical, Cat#RT517) two-step probe-polymer incubation for 10 minutes and 30 minutes, respectively.

[0101] NAFLD activity and fibrosis score

[0102] H&E staining was used to score NAFLD activity (NAS). Steroidosis was scored from 0 to 3 (0: <5% steatosis; 1: 5-33%; 2: 34-66%; 3: >67%). Hepatocellular ballooning was scored from 0 to 2 (0: normal hepatocytes; 1: normal-sized hepatocytes with pale cytoplasm; 2: pale and enlarged hepatocytes, at least 2-fold). Lobular inflammation was scored from 0 to 2 based on the number of inflammatory lesions counted at 20X (0: none; 1: <2 lesions; 2: ≥2 lesions). NAS was calculated as the sum of the steatosis, hepatocellular ballooning, and lobular inflammation scores. Liver fibrosis was scored from 0 to 4 using Sirius red staining (0: no fibrosis; 1: perisinusoidal or portal fibrosis; 2: perisinusoidal and portal fibrosis; 3: bridging fibrosis; 4: cirrhosis).

[0103] plasma analysis

[0104] Clinical chemistry assays of ALT and AST were performed by UM IVAC on a Liasys 330 Chemical Analyzer (AMSDiagnostics) using manufacturer-supplied reagents and procedures. Plasma total cholesterol was measured using the Wako Diagnostics kit (999-02601). Plasma MCP-1 was measured using the Mouse CCL2 / JE / MCP-1 Quantikine ELISA kit (R&D Systems).

[0105] RNA sequencing and data analysis

[0106] Total RNA was extracted from mouse liver samples using the QIAGEN RNeasy kit. Library preparation and sequencing were performed by the UM DNA Sequencing Core. RNA quality was assessed using TapeStation (Agilent, Santa Clara, CA). All samples had an RNA integrity number (RINs) >8.5. Samples were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760L) with a Poly(A) mRNA Magnetic Isolation Module (NEB, E7490L) and NEBNext Multiple Oligos for Illumina Unique Dual (NEB, E6440L), where 10 ng–1 μg of total RNA was subjected to poly(A)mRNA purification. The mRNA was then fragmented and replicated into first-strand cDNA using a mixture of reverse transcriptase and dUTP. Samples underwent end repair and dA-tailing, followed by ligation with the NEBNext adaptor. The products were purified and enriched by PCR to produce the final cDNA library. The quality and quantity of the final library were checked using TapeStation (Agilent) and qPCR with the Kapa Library Quantification Kit (Kapa Biosystems, KK4835) for the Illumina sequencing platform. The library was sequenced with paired ends on a NovaSeq 6000 sequencing system (Illumina).

[0107] The quality of the raw FASTQ files was checked using FastQC v0.11.8 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Trimmomatic v.0.35 was used to cleanse low-quality reads with the following parameters: SLIDINGWINDOW:4:20MINLEN:25. The resulting high-quality reads were then plotted onto the mouse reference genome (GRCm38.90) using HISAT2 v.2.1.0.13. Gene expression quantification was performed using HTSeq-counts v0.6.0 based on GRCm38.90 genome annotation. The R package DESeq2 was then used to identify differentially expressed genes (DEGs). Genes with an adjusted p-value less than 0.05 and an absolute fold change greater than 2 were considered significant DEGs. The clusterProfiler package was then used to analyze upregulated and downregulated DEGs of the significantly enriched KEGG pathway. The significance of enrichment was determined by a right-tailed Fisher exact test followed by a Benjamini-Hochberg multiple test.

[0108] Quantitative real-time PCR analysis

[0109] Total RNA was extracted from mouse liver samples using the QIAGEN RNeasy kit (QIAGEN). RNA was reverse transcribed into cDNA using SuperScript III and random primers (Invitrogen). Specific transcripts were evaluated using a real-time PCR system (Bio-Rad) with iQ SYBR Green Supermix (Bio-Rad) and a normalized ΔΔCt threshold cycling method. Gene expression was normalized relative to Gapdh. Primer pairs used for qPCR were obtained from Integrated DNA Technologies and are listed below:

[0110]

[0111] Liver analysis

[0112] Liver samples were rapidly removed from mice subjected to euthanasia, flash-frozen in liquid nitrogen, and maintained at -80°C. The LC-MS / MS method used for the detection and quantification of N-acyl amino acids was developed by the U-M Pharmacokinetics and Mass Spectrometry Core. Technicians were unaware of the experimental group's activity. N-acyl amino acid levels in the liver were normalized relative to tissue weight and expressed as ng / g liver tissue. For TG quantification, frozen liver samples (100 mg) were homogenized in PBS and centrifuged (14,000 RPM, 20 min). The supernatant was collected, and the protein concentrations were analyzed using Bio-Rad Bradford assays. To assess liver lipid composition, lipids were extracted from the supernatant using a 3:2 (v:v) ratio of hexane (≥99%, Sigma-Aldrich 32293) and isopropanol (≥99.5%, Fisher Scientific A426-4), with the hexane phase evaporated for 48 hours. The amount of liver TG was determined by spectrophotometry using the commercially available Wako Diagnostics kit (994-02891).

[0113] result

[0114] At the endpoint, the elevated plasma levels of NAFLD markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were attenuated by glycine or tripeptide-glycine-leucine. Therefore, glycine or tripeptide-glycine-leucine significantly reduced NASH diet-induced hepatomegaly, and histological analysis revealed lower levels of fatty liver, inflammation (F4 / 80 macrophage staining), and fibrosis (Sirius red staining) with NAFLD activity scores significantly reduced by tripeptide-glycine-leucine at 0.5 mg / g / day.

[0115] Unbiased analysis of liver gene expression via RNA sequencing revealed that the gene encoding an enzyme that catalyzes fatty acids and various amino acids (including Pm20d1 containing the peptidase M20 domain) and specifically condenses with glycine (glycine-N-acyltransferase, Glyat) was downregulated in mice with NASH, and this downregulation was reversed by treatment with the tripeptide glycine-glycine-leucine. These results were confirmed by qPCR analysis. Figure 1 A, B). Therefore, targeted metabolomics revealed that levels of N-oleoylglycine (C18:1-Gly), N-arachidonicylglycine (C20:4-Gly), and N-oleoylleucine (C18:1-Leu) were significantly reduced in the livers of mice with NASH, which was relieved by long-term treatment with glycine or tripeptide glycine-glycine-leucine. Figure 1 CE).

[0116] Example 2

[0117] Liver levels of N-acyl amino acids are associated with markers of fatty liver disease, fibrosis, and cardiovascular disease. To examine the relationship between N-acyl amino acids and NASH, we analyzed the correlation between liver levels of N-acyl amino acids from mice in Example 1 and indicators of NASH severity, namely fatty liver (quantitative liver triglycerides, TG), inflammation (F4 / 80 immunohistochemistry), fibrosis (Sirius red staining), and total NAS. We found that liver levels of N-acyl amino acids were correlated with fatty liver (… Figure 2A ), inflammation Figure 2B ), fibrosis (2C) and NAS ( Figure 2D Significant and negative correlations were found between the levels of N-arachidonicoglycine (C20:4-Gly) and the above indicators (P<0.0001).

[0118] Then, to examine the relationship between N-acyl amino acids and other cardiometabolic risk factors, we analyzed the correlation between liver levels of N-acyl amino acids in the mice of Example 1 and plasma levels of ALT (a marker of liver injury), monocyte chemoattractant protein 1 (MCP-1, an inflammatory marker), and total cholesterol (TC, one of the strongest risk factors for CVD). Similar to NASH indicators ( Figure 2A -D), liver levels of N-acyl amino acids were significantly and negatively correlated with ALT, MCP-1, and TC, with the most significant correlation found for N-arachidonicylglycine (C20:4-Gly) (P<0.0001). Figure 3A -C).

[0119] Example 3

[0120] N-acyl amino acids directly activate PPARα

[0121] Unbiased RNA sequencing analysis described in Example 1 revealed that major pro-inflammatory and pro-fibrotic pathways were enriched in the livers of mice with NASH. In contrast, in the livers of mice fed a NASH diet and treated with tripeptide-glycine-glycine-leucine, the most significantly upregulated pathways were associated with energy metabolism and FAO. Specifically, hepatic expression of PPARα, the major regulator of FAO, and its target genes was suppressed in NASH. This suppression was reversed in the livers of mice treated with glycine or tripeptide-glycine-leucine.

[0122] Next, we used an in vitro luciferase-based system to test whether glycine or tripeptide glycine-glycine-leucine directly activates PPARα.

[0123] method

[0124] COS-1 and HepG2 cells were obtained from the American Type Culture Collection (ATCC) and cultured at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin-streptomycin (Pen-Strep, Gibco). For luciferase assays, COS-1 cells were seeded in 96-well plates. At 60-70% confluence, cells were transfected using Lipofectamine 3000 (Invitrogen) with PPREx3-TK-luciferase, PPARα, and Renilla constructs at doses of 80 ng, 10 ng, and 10 ng, respectively. Twenty-four hours post-transfection, cells were serum-starved and treated for 24 hours with 10 μM PPARα activator WY-14643 (Cayman Chemicals, 70730), 1 mM glycine, or tripeptide glycine-glycine-leucine, or 10 μM N-acyl amino acid (Cayman Chemicals, C20:4-Gly 90051, C18:1-Leu 20064). Luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega) and normalized by Renilla assay.

[0125] result

[0126] Although the known PPARα agonist WY-14643 significantly increased luciferase activity at 10 μM, neither glycine nor tripeptide glycine-glycine-leucine showed significant effects at concentrations up to 1 mM. Figure 4 A). We then tested the PPARα response to activation of N-acyl amino acids, which were found to be elevated in the livers of mice fed a CD diet or those fed a NASH diet and treated with glycine or tripeptide-glycine-leucine. Figure 1 Similar to WY-14643, 10 μM of N-oleoylglycine (C18:1-Gly), N-arachidonicylglycine (C20:4-Gly), or N-oleoylleucine (C18:1-Leu) significantly increased luciferase activity. Figure 4 B).

[0127] In vivo, hepatic levels of N-acyl amino acids were significantly and positively correlated with the expression of key PPARα target genes, which play major roles in regulating mitochondrial biogenesis and FAO, including peroxisome proliferation activation receptor, γ, and coactivator 1α (Ppargc1a). Figure 5A Acyl-CoA thioesterase 3 (Acot3, Figure 5B ) and acyl-CoA dehydrogenase, long chain (Acadl, Figure 5C Therefore, N-acyl amino acids directly activate PPARα in vitro and are associated with the expression of its key target genes in vivo.

[0128] Example 4

[0129] N-acyl amino acids stimulate lipid utilization via fatty acid β-oxidation.

[0130] To assess the direct effect of N-acyl amino acids on lipid utilization via FAO, we used the Seahorse assay in HepG2 cells.

[0131] method

[0132] HepG2 cells were obtained from the American Type Culture Collection (ATCC) and cultured at 37°C and 5% CO2 in Dulbecco's modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin-strepmycin (Pen-Strep, Gibco). Oxygen consumption rate (OCR) and dependence on FAO were assessed using a Seahorse XFe96 analyzer (Agilent). HepG2 cells were seeded at 2.5 x 10⁴ cells / well in XF96 cell culture microplates (Agilent). The next day, the XFe96 sensor tubes were hydrated according to the manufacturer's instructions. Cells (port 1) were treated with N-acyl amino acids (10 μM) or a mediator (EtOH), ethoxysulfuron (Agilent, 6 μM, port 2), and finally with rotenone + antimycin A (R / A, Agilent, port 3).

[0133] For TG biosynthesis and hydrolysis assays, HepG2 cells were seeded in 12-well plates. At 60–70% confluence, cells were treated with N-acyl amino acids (10 μM) or a mediator (EtOH) in serum-free medium supplemented with 0.1% BSA and stimulated at 37°C for 3 h with [3H]-acetate (3.3 μCi / ml, ART 0202, American Radiolabeled Chemicals) to assess the rate of TG biosynthesis. In some wells, cells were washed twice with PBS ([3H]-acetate withdrawal) and incubated again for 3 h in serum-free medium supplemented with 0.1% BSA with N-acyl amino acids (10 μM) or a mediator (EtOH) to assess the rate of TG hydrolysis. At the end of the incubation period, cells were washed twice with PBS. Cellular lipids were extracted using a 3:2 (v:v) ratio of hexane (≥99%, Sigma-Aldrich 32293) and isopropanol (≥99.5%, Fisher Scientific A426-4), with the hexane phase evaporated for 48 hours. Lipids were then separated by thin-layer chromatography (TLC) on silica gel plates (60F254, M1057150001, Fisher Scientific) and visualized in a 130:30:1.5 (v:v:v) ratio of hexane / diethyl ether (≥99.9%, 309966, Sigma-Alrich) / acetic acid (≥99.7%, A38-212, Fisher Scientific). TG spots were visualized by iodine vapor (identified using appropriate standards), and [3H]-labeling was counted using a Tri-Carb 2810TR liquid scintillation analyzer (PerkinElmer). The data were normalized relative to protein levels and expressed as counts per minute (CPM) / mg of cell protein.

[0134] result

[0135] Acute stimulation with N-arachidonicylglycine (C20:4-Gly) or N-oleoylleucine (C18:1-Leu) significantly increased the cellular oxygen consumption rate (OCR), which was attenuated by blocking FAO with ethosylcarnitine palmitoyltransferase-1 (CPT-1) inhibitor ethosyl, a key player in regulating mitochondrial fatty acid uptake and subsequent β-oxidation. Figure 6A, B). We then assessed the rates of lipid biosynthesis and its hydrolysis by monitoring the incorporation of [3H]-labeled acetate into TG with and without N-acyl amino acids. Both N-arachidonicylglycine (C20:4-Gly) and N-oleoylleucine (C18:1-Leu) attenuated the rate of TG biosynthesis; however, only N-arachidonicylglycine (C20:4-Gly) significantly accelerated the rate of TG hydrolysis. Figure 6 (C, D). These results demonstrate that N-acyl amino acids, particularly N-arachidonicylglycine (C20:4-Gly), directly stimulate lipid utilization via FAO, highlighting their therapeutic potential for steatohepatitis, fibrosis, and CVD.

[0136] Example 5

[0137] Treatment of NASH in mice

[0138] Figure 7 The experimental design for NASH studies in mice is shown.

[0139] method

[0140] C57BL / 6J mice were fed a standard diet (SD) or a non-alcoholic steatohepatitis (NASH) diet for 16 weeks. After NASH confirmation, the mice were randomized to receive 10 mg / kg / day (IP) N-oleoylleucine (C18:1-Leu) or an equivalent amount of oleic acid (C18:1) or a mediator (EtOH) for an additional 6 weeks on the NASH diet. Control mice were fed the SD diet and administered the mediator.

[0141] result

[0142] Figure 8 It was confirmed that C18:1-Leu reduces body weight without affecting obesity.

[0143] Figure 9 It was confirmed that C18:1-Leu had no significant effect on the whole-body energy balance in NASH.

[0144] Figure 10 C18:1-Leu was confirmed to significantly reduce hepatomegaly.

[0145] Figure 11 C18:1-Leu was confirmed to reduce circulating liver enzymes.

[0146] Figure 12 C18:1-Leu was confirmed to significantly reduce diet-induced NASH.

[0147] Figure 13C18:1-Leu was confirmed to significantly reduce fatty liver.

[0148] Figure 14 C18:1-Leu was confirmed to significantly reduce NASH diet-induced liver and systemic inflammation.

[0149] Figure 15 C18:1-Leu was confirmed to significantly reduce NASH diet-induced liver fibrosis.

[0150] Example 6

[0151] Treatment of atherosclerosis in mice

[0152] Figure 16 The experimental design for a study on atherosclerosis in mice is shown.

[0153] method

[0154] Apolipoprotein E-deficient (Apoe-'-) mice were fed a Western diet (WD) for 8 weeks. Mice were then randomized to receive 7.5 mg / kg / d (IP) N-oleoylleucine (C18:1-Leu) or an equivalent amount of oleic acid (C18:1) for an additional 4 weeks on WD (n=10).

[0155] result

[0156] Figure 17 It was confirmed that C18:1-Leu treatment had no significant effect on body weight and plasma cholesterol in atherosclerotic mice.

[0157] Figure 18 C18:1-Leu was confirmed to significantly reduce the area of ​​atherosclerotic plaques.

[0158] Figure 19 C18:1-Leu was confirmed to significantly reduce the number of diseased macrophages.

[0159] While the invention has been described with reference to various embodiments and examples, it should be understood that variations and modifications will occur to those skilled in the art. Therefore, the invention should only be subject to the limitations set forth in the claims.

[0160] All documents cited in this application are incorporated herein by reference in their entirety, with particular attention paid to their references. sequence list <110> University of Michigan Board of Trustees <120> N-Acylamino Acid Products and Their Uses <130> 30275 / 55769A / PC <150> 63 / 067,175 <151> 2020-08-18 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 1 ctgcgacttc aacagcaact 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 2 gagttgggat agggcctctc 20 <210> 3 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 3 ctgcatcttg gactctaatg gac 23 <210> 4 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 4 gcgatattac ttttctccgt gtg 23 <210> 5 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 5 caaagtatag ccacctgttc acc 23 <210> 6 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 6 gatcttttgg ggctgtagtt tct 23 <210> 7 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 7 atcacgttca aggtcaccct ac 22 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 8 ttctgcttct gcctctctct ct 22 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 9 gctcagtcac cctcaggtaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 10 aagtttccgc cgatgttgga 20 <210> 11 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 11 ctatattgcg aattacggca ca 22 <210> 12 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 12 acaccttgct tccattgaga at 22

Claims

1. Use of N-oleoylleucine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of non-alcoholic steatohepatitis in the body.

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