Composition comprising 4'-thio-5-aza-2'- deoxycytidine for alleviating, preventing or treating liver diseases

Abstract

The present invention relates to a pharmaceutical composition comprising 4'-thio-5-aza-2'-deoxycytidine or a pharmaceutically acceptable salt thereof for preventing or treating liver diseases. The 4'-thio-5-aza-2'-deoxycytidine of the present invention exhibits excellent effects with respect to treatment and alleviation of liver diseases, particularly nonalcoholic fatty liver diseases and metabolic dysfunction steatohepatitis, and thus can be effectively used for alleviation and treatment of liver diseases.

Description

Composition for improving, preventing or treating liver disease comprising 4'-thio-5-aza-2'-deoxycytidine

[0001] The present invention relates to a composition for improving, preventing or treating liver disease, comprising 4'-thio-5-aza-2'-deoxycytidine.

[0002] The liver is a vital organ responsible for various metabolic processes, detoxification, breakdown, synthesis, and secretion in our bodies. A closer look at its functions reveals the following. First, the liver manages energy metabolism, metabolizing all nutrients absorbed from food into energy-producing substances and supplying or storing them throughout the body. Second, the liver synthesizes, stores, and distributes approximately 2,000 enzymes, albumin, serum proteins (clotting factors), bile acids, phospholipids, and fats such as cholesterol. Third, the liver excretes various metabolites through the bile duct into the duodenum, and its immune function plays a crucial role in sustaining our lives. Finally, the liver has detoxification and breakdown functions, eliminating drugs, toxic substances, and alcohol. However, this detoxification function can easily damage liver cells, leading to drug-induced, toxic, and alcohol-induced liver disease.

[0003] Among liver diseases, hepatic steatosis refers to a condition in which fat accumulates in liver cells. Medically, it refers to a pathological condition in which fat exceeds 5% of the total liver weight. Liver disease that includes this is recognized as the second most serious disease after cancer in terms of cause of death among adults in their 40s and 50s in developed countries.

[0004] Fatty liver disease can be broadly divided into alcoholic fatty liver disease (ALD) caused by excessive drinking and metabolic dysfunction fatty liver disease (MAFLD) caused by obesity, diabetes, hyperlipidemia, and medications. Metabolic dysfunction fatty liver disease refers to a broad range of diseases, including simple fatty liver disease without inflammation that occurs in patients who do not consume excessive amounts of alcohol, and diseases that progress due to this, including hepatocyte inflammation, liver fibrosis, and cirrhosis.

[0005] Metabolic fatty liver disease (MAFLD) describes a constellation of liver abnormalities that begins with hepatic steatosis (fatty liver). In some patients, MAFLD will progress to metabolic dysfunction steatohepatitis (MASH). MASH is histologically defined as hepatic steatosis accompanied by lobular inflammation and hepatocyte injury (ballooning), with or without fibrosis. MASH can progress to cirrhosis and hepatocellular carcinoma (HCC).

[0006] MAFLD is a global cause of chronic liver disease, steadily increasing alongside the rise in obesity and metabolic syndrome. In the United States, an estimated 24% of the population has MAFLD, with approximately one-third of these individuals also having MASH. MAFLD is associated with various comorbidities, including metabolic syndrome, diabetes, and cardiovascular disease (CVD). MAFLD is the fastest-growing cause of hepatic cirrhosis (HCC), a major cause of liver disease and liver transplantation. While significant progress has been made in elucidating the pathogenesis of MAFLD, identifying therapeutic targets, and discovering and developing novel agents, unresolved questions and unmet clinical needs remain in the field.

[0007] Currently, there is no established treatment for metabolically related fatty liver disease (NALFD), as it is associated with various factors, including diabetes, obesity, coronary artery disease, and lifestyle. Therefore, exercise and diet are recommended for fatty liver disease treatment. Furthermore, treatment options for NALFD are limited, and there are no FDA-approved medications to treat patients with this rapidly growing public health problem.

[0008] The present inventors have completed the present invention by confirming that 4'-thio-5-aza-2'-deoxycytidine (aza-T-dCyd) has a therapeutic advantage targeting the regulation of the epigenome in a situation where there is no practical treatment for existing metabolic abnormality steatohepatitis (MASH), and that aza-T-dCyd has a significant therapeutic effect in a MASH diet mouse model.

[0009] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating liver disease, comprising 4'-thio-5-aza-2'-deoxycytidine or a pharmaceutically acceptable salt thereof.

[0010] Another object is to provide a method for preventing or treating liver disease, comprising administering to a subject in need thereof the 4'-thio-5-aza-2'-deoxycytidine of the present invention, or a pharmaceutically acceptable salt thereof.

[0011] Another object is to provide a use of the 4'-thio-5-aza-2'-deoxycytidine of the present invention or a pharmaceutically acceptable salt thereof for preparing a medicament for preventing or treating liver disease.

[0012] Another object is to provide a composition comprising 4'-thio-5-aza-2'-deoxycytidine of the present invention or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of liver disease.

[0013] The present invention provides a pharmaceutical composition for preventing or treating liver disease, comprising 4'-thio-5-aza-2'-deoxycytidine represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0014] [Chemical Formula 1]

[0015]

[0016] According to the present invention, the 4'-thio-5-aza-2'deoxycytidine has a structure represented by the above chemical formula 1, and is also referred to as AZA or Aza-TdC in the present specification.

[0017] This 4'-thio-5-aza-2'deoxycytidine shows excellent improvement effects in the treatment of liver diseases.

[0018] Metabolic dysfunction steatohepatitis (MASH), mentioned above, is a liver disease that can progress to severe diseases such as cirrhosis or liver cancer. The expression of DNA methyltransferases (DNMTs) increases in liver tissue from MASH patients. Specifically, gene expression levels of DNMT1 and DNMT3A increase as MASH progresses in liver tissue from patients with metabolic dysfunction steatohepatitis (Figure 6).

[0019] Based on these characteristics, the present invention confirmed through experiments on animal models that 4'-thio-5-aza-2'deoxycytidine of the present invention can be usefully used as a DNMT inhibitor to improve and treat liver diseases including metabolic steatohepatitis.

[0020] In particular, the 4'-thio-5-aza-2'-deoxycytidine of the present invention exhibits excellent efficacy in the treatment of liver diseases, particularly metabolic steatohepatitis and metabolic-related fatty liver disease, by inhibiting fat accumulation in the liver, inhibiting the expression of DNMT, and reducing the expression of FASN, a key gene for free fatty acid synthesis, and SCD-1, which is related to fatty acid metabolism.

[0021] Additionally, it may restore metabolic homeostasis by lowering lipid accumulation and reducing inflammation, and may exhibit excellent therapeutic efficacy by reversing hypermethylation induced by MASLD and restoring transcriptional activity of key regulatory genes mediated by HNF4a and PPARa.

[0022] In the present invention, the liver disease may be at least one disease selected from the group consisting of simple fatty liver, metabolically abnormal fatty liver disease (MAFLD), liver inflammation, metabolic steatohepatitis (MASH), metabolic liver disease, cholestatic liver disease, liver fibrosis, cirrhosis, and liver failure.

[0023] The above cholestatic liver disease may be at least one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and a combination thereof.

[0024] The above liver disease may be metabolic steatohepatitis accompanied by fatty liver, hepatic fibrosis, or cirrhosis.

[0025] Additionally, in the present invention, the liver disease may include liver cancer caused by metabolic abnormality steatohepatitis.

[0026] The term "prevention" as used herein refers to any action that suppresses or delays the onset of a liver disease-related condition through the administration of a composition. In the present invention, "treatment" refers to any action that improves or beneficially alters the symptoms of the condition through the administration of a composition.

[0027] In the present invention, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, and include, for example, inorganic ion salts manufactured with calcium, potassium, sodium, and magnesium; inorganic acid salts manufactured with hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, iodic acid, perchloric acid, and sulfuric acid; organic acid salts manufactured with acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid; sulfonic acid salts manufactured with methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and naphthalenesulfonic acid; Amino acid salts made from glycine, arginine, lysine, etc.; and amine salts made from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc.; however, the types of salts meant in the present invention are not limited by these listed salts.

[0028] In addition, the 4'-thio-5-aza-2'-deoxycytidine represented by the chemical formula 1 of the present invention includes not only pharmaceutically acceptable salts but also all salts, hydrates and solvates that can be prepared by conventional methods.

[0029] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, and may be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.

[0030] The pharmaceutically acceptable carriers include, but are not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the pharmaceutical composition of the present invention may include, but is not limited to, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, and other pharmaceutically acceptable additives.

[0031] When the pharmaceutical composition of the present invention is formulated as an oral solid preparation, it includes tablets, pills, powders, granules, capsules, etc., and such solid preparations may include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., and include, but are not limited to, lubricants such as magnesium stearate and talc.

[0032] When the pharmaceutical composition of the present invention is formulated as an oral liquid, it includes a suspension, a solution, an emulsion, a syrup, etc., and includes, but is not limited to, a diluent such as water or liquid paraffin, a wetting agent, a sweetener, a fragrance, a preservative, etc.

[0033] When the pharmaceutical composition of the present invention is formulated for parenteral use, it includes a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, and a suppository. Non-aqueous solvents and suspensions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Bases for suppositories include, but are not limited to, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0034] The composition may be administered in single or multiple doses in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to prevent or treat a disease at a reasonable benefit / risk ratio applicable to medical prevention or treatment, and the effective dosage level may be determined according to factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, drugs used in combination or concurrently with the composition of the present invention used, and other factors well known in the medical field. For example, 4'-thio-5-aza-2'-deoxycytidine represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof may be administered at 0.0001 to 100 mg / kg per day, and the administration may be administered once a day or in several divided doses.

[0035] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes, for example, oral administration, intrathecal, intra-auricular, intraperitoneal or intravenous, intramuscular, subcutaneous, intrauterine epidural, sublingual, or intracerebrovascular injection, but is not limited thereto.

[0036] The pharmaceutical composition of the present invention may contain 4'-thio-5-aza-2'-deoxycytidine represented by chemical formula 1 or a pharmaceutically acceptable salt thereof in an amount of 0.01 to 95 wt%, preferably 1 to 80 wt%, based on the total weight of the composition.

[0037] The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Furthermore, the pharmaceutical composition of the present invention can be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer the amount that achieves maximum efficacy with the minimum amount possible without causing side effects, a determination readily made by those skilled in the art.

[0038] The term "subject" of the present invention encompasses an animal or human whose symptoms can be improved by administration of a pharmaceutical composition according to the present invention. By administering a therapeutic composition according to the present invention to a subject, liver disease can be effectively prevented and treated.

[0039] The term "administration" in the present invention refers to introducing a given substance into a human or animal by any suitable method. The therapeutic composition according to the present invention may be administered orally or parenterally via any common route, as long as it can reach the target tissue. Furthermore, the therapeutic composition according to the present invention may be administered by any device capable of transporting the active ingredient to target cells.

[0040] Another aspect of the present invention provides a method for preventing or treating liver disease, comprising administering 4'-thio-5-aza-2'-deoxycytidine represented by the above chemical formula 1, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

[0041] In the present invention, the term “4'-thio-5-aza-2'-deoxycytidine represented by chemical formula 1” or “liver disease” is as described above.

[0042] The term "subject" of the present invention refers to any animal that has developed or may develop a liver disease, and may typically be an animal that can exhibit a beneficial effect by treatment with 4'-thio-5-aza-2'-deoxycytidine represented by Chemical Formula 1 of the present invention, or a pharmaceutically acceptable salt thereof, but includes without limitation any subject that has symptoms of liver disease or is likely to have such symptoms. As described above, by administering the pharmaceutical composition of the present invention to a subject, the liver disease can be effectively prevented or treated.

[0043] Another aspect of the present invention provides a use of 4'-thio-5-aza-2'-deoxycytidine represented by the chemical formula 1 of the present invention or a pharmaceutically acceptable salt thereof for preparing a medicament for preventing or treating liver disease.

[0044] Another aspect of the present invention provides a composition comprising 4'-thio-5-aza-2'-deoxycytidine represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of liver disease.

[0045] Another aspect of the present invention provides a use of 4'-thio-5-aza-2'-deoxycytidine represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of liver disease.

[0046] Another aspect of the present invention provides a food composition for preventing or improving liver disease, comprising 4'-thio-5-aza-2'-deoxycytidine represented by the following chemical formula 1 or a food-wise acceptable salt thereof.

[0047] [Chemical Formula 1]

[0048]

[0049] The term "improvement" of the present invention means any act in which liver disease is improved or beneficially changed by administration of the composition of the present invention.

[0050] In the food composition of the present invention, the 4'-thio-5-aza-2'-deoxycytidine and its salt are preferably contained in an amount of 0.00001 to 0.01 wt% relative to the food composition. If the amount is less than 0.00001 wt%, the effect is minimal, and if the amount exceeds 0.01 wt%, the increase in effect relative to the amount used is minimal, making it uneconomical.

[0051] Food-wise acceptable salt may be applied in the same manner as pharmaceutically acceptable salt.

[0052] The food composition of the present invention may be, for example, any one selected from among noodles, gum, dairy products, ice cream, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, alcoholic beverages, alcohol, vitamin complexes, and other health supplements, but is not necessarily limited thereto.

[0053] When the food composition of the present invention is used as a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method.

[0054] In the present invention, the food composition includes a health functional food.

[0055] The above “health functional food” refers to a food manufactured and processed using raw materials or ingredients with functionality useful to the human body as defined in Act No. 6727 on Health Functional Foods, and “functionality” refers to consumption for the purpose of obtaining a useful effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects.

[0056] The food composition and health functional food of the present invention may include additional ingredients. For example, they may include biotin, folate, pantothenic acid, vitamins A, C, D, E, B1, B2, B6, B12, niacin, etc. In addition, they may include minerals such as chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), and calcium (Ca). In addition, they may include amino acids such as cysteine, valine, lysine, and tryptophan. In addition, food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), bactericides (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (pasting agents), film-forming agents, antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), gum bases, antifoaming agents, solvents, and improvers can be added. The above additives can be selected depending on the type of food and used in an appropriate amount.

[0057] The 4'-thio-5-aza-2'-deoxycytidine of the present invention exhibits excellent effects in the treatment and improvement of liver diseases, particularly fatty liver disease related to metabolic disorders and metabolic steatohepatitis.

[0058] Figure 1 shows the results of methylation profile analysis performed in a cohort of MASLD patients.

[0059] Figure 2 shows the results of investigating the expression patterns involved in methylation regulation in human MASLD subjects by performing transcriptome analysis to identify methylation regulators in MASLD.

[0060] Figure 3 shows the results confirming the increase in DNMT1 expression in the European MASLD cohort (GSE1352521).

[0061] Figures 4 and 5 show the results of Pearson correlation analysis performed on the MASLD sample cohort.

[0062] Figure 6 shows a box plot of changes in the DNMT (DNA methyltransferase) gene in a cohort of patients with metabolic abnormality steatohepatitis (Korean cohort (A, B), European cohort GSE135251 (C, D)).

[0063] Figure 7 shows the results of analyzing the DNMT inhibitory effect of 4'-thio-5-aza-2'-deoxycytidine of the present invention in a MASH-induced mouse model ((A) liver tissue, (B) histological analysis using H&E, Oil red O, and immunohistochemical analysis results for macrophage index (F4 / 80+); (C) liver, body weight, and liver-body weight ratio).

[0064] Figure 8 shows the results of gene expression analysis related to lipid accumulation regulation in a MASH-induced mouse model.

[0065] Figure 9 is a schematic diagram for confirming the pharmacological inhibitory efficacy of DNMT1 in a mouse model of diet-induced MASLD to confirm the involvement of DNMT1 in MASLD.

[0066] Figure 10 shows the results of phenotypic and histological analysis following administration of 5-Aza-4′-thio-2′-deoxycytidine (Aza-TdC), a DNMT1 inhibitor.

[0067] Figure 11 shows the results of histological score analysis according to administration of 5-Aza-4′-thio-2′-deoxycytidine (Aza-TdC), a DNMT1 inhibitor.

[0068] Figures 12 and 13 are diagrams showing changes in gene expression involved in lipid absorption (Cd36), transport (FABPs), droplet formation, and storage following administration of 5-Aza-4′-thio-2′-deoxycytidine (Aza-TdC), a DNMT1 inhibitor.

[0069] Figures 14 and 15 are diagrams showing changes in gene expression involved in inflammation and fibrosis following administration of 5-Aza-4′-thio-2′-deoxycytidine (Aza-TdC), a DNMT1 inhibitor.

[0070] Figure 16 is a diagram showing the results of protein analysis for Plin2, CD36, and SCD1 following administration of 5-Aza-4′-thio-2′-deoxycytidine (Aza-TdC), a DNMT1 inhibitor.

[0071] Figure 17 is a diagram showing the results of single nucleotide RNA sequencing (snRNA-seq) performed on liver tissue of each diet group.

[0072] Figure 18 shows the results of epigenome analysis using a MASH-induced mouse model ((A) DNA methylation heatmap for changes in differentially methylated sites (DMPs) in a MASH-induced mouse model; (B) Gene ontology (GO) analysis results of hypermethylated and hypomethylated DMPs using the GREAT tool).

[0073] Figure 19 is a diagram showing the decrease in methylation in promoter regions including specific CpG sites such as cg47230786 of Slc38a3, cg43959116 of C1ra, cg40013516 of Il6ra, and cg33066766 of Prlr after DNMT1 inhibition.

[0074] Figure 20 is a diagram showing the correlation between hypermethylation and decreased gene expression under MASLD conditions.

[0075] Figure 21 shows strong binding of HNF4α and PPARα near the promoter regions of genes including Prlr, C1ra, Il6ra, Pck1, Gls2, Klf15, and Slc38a3.

[0076] Figure 22 shows that CpG sites near the promoter-TSS region of Pck1 show restored methylation after pharmacological inhibition of DNMT1, as verified by pyrosequencing.

[0077] Figures 23 and 24 show the results of transcriptome analysis using a MASH-induced mouse model. Figure 23 shows Cnetplot, which is used to show the potential biological complexity of the same genes that can belong to multiple annotation categories of GO terms of AZA differentially expressed genes (DEGs) using a cluster profiler; Figure 24 shows a heatmap for specific metabolic-related pathways of upregulated and downregulated AZA differentially expressed genes (DEGs).

[0078] Figure 25 is a diagram illustrating, but not limited to, a treatment mechanism for MASLD due to inhibition of DNMT1.

[0079] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention.

[0080] <Experimental Method>

[0081] DNA methylation microarray experiments and data analysis

[0082] The EPIC BeadChip (Illumina) was used for methylation array analysis. 1 μg of genomic DNA extracted from human liver tissue was treated with 20 μL of sodium bisulfite solution provided in the EZ DNA Methylation-Gold kit (Zymo Research, Orange, CA). The bisulfite-converted DNA (4 μL) was then amplified using the Infinium Methylation Assay kit (Illumina). The amplified DNA was then hybridized to the EPIC BeadChip and scanned using the Illumina iSCAN system.

[0083] To assess the degree of CpG methylation, the SeSAMe package (v1.20.0) was utilized within R software (v4.3.1). Preprocessing included several steps: 'qualityMask', 'infniumIChannel', 'dyeBiasNL', 'pOOBAH', and 'noob', which aimed to filter out low-quality probe data, correct for dye bias and channel switching, and eliminate background subtraction. In particular, CpG probes that failed in more than 10% of samples, non-CpG probes, probes targeting chromosome X or Y, and flagged probes were excluded from the analysis. Additionally, measurements with a detection P-value <0.05 were considered to represent a significant signal above background.

[0084] Pyrosequencing

[0085] CpG sites of the gene of interest were selected for quantification of methylation levels. Genomic DNA from mouse liver tissue was treated with sodium bisulfite using the EZ DNA Methylation Gold Kit (Zymo Research, Orange, CA). Bisulfite-modified DNA (100 ng) was amplified using specific primers in a 15-µl reaction volume of 2x DreamTaq Hot Start Green PCR Master Mix (ThermoFisher, MA, USA). PCR was performed with an initial denaturation at 95°C for 10 min, followed by 45 cycles of 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, and a final incubation at 72°C for 7 min. Pyrosequencing was performed according to the manufacturer's specifications using sequencing primers and run on a PyroMark Q48 Autoprep using PyroMark Q48 Advanced CpG reagents (QIAGEN, Valencia, CA).

[0086] RNA-seq data analysis

[0087] RNA sequencing (RNA-seq) data included 92 liver samples from 28 controls, 16 MASL, and 48 MASH subjects. Total RNA extracted from each liver sample was subjected to RNA sequencing using the HiSeq2500 platform. Reads were then mapped and quantified using the human genome (hg19 / GRCh37) reference based on GENCODE v19. Publicly available data were also obtained from GEO (GSE135251). The counts matrix was normalized to the trimmed mean of the M-values ​​(i.e., TMM) using the edgeR package (version 4.0.11), then converted to counts per million and subsequently log-transformed to generate normalized values. Expression values ​​of complement genes were extracted from this normalized matrix. Clinical data related to NAS and fibrosis stages were then used to explore the expression patterns of complement genes across the MASLD spectrum using boxplots for visualization.

[0088] single nuclear RNA sequencing (snRNA-seq)

[0089] For single-nucleotide RNA-seq (snRNA-seq) data, the Seurat package (v4.4.0) was used for a systematic analysis workflow. Initially, Seurat objects were independently generated for each sample group (Control, MASLD, and AZA-TdC). These Seurat objects were filtered to exclude cells with fewer than 5,000 genes, more than 20,000 reads, and mitochondrial gene expression exceeding 15%. After merging the datasets, the combined Seurat objects were normalized and scaled using the NormalizeData and ScaleData functions. Variable genes were identified using the Find Variable Features function, and the top 2,000 genes were selected for further analysis. Principal component analysis (PCA) was performed using the RunPCA function, followed by batch correction using the RunHarmony function in the Harmony1 package. Clustering was performed using the FindNeighbors and FindClusters functions, with a resolution adjusted to generate 10-15 clusters based on 20 principal components. Uniform Manifold Approximation and Projection (UMAP) was used to visualize the data. Cell clusters were annotated using the singleR2 computational tool. Manual annotation was also performed based on established biological knowledge, and similar preprocessing steps were performed after generating separate Seurat objects for hepatocyte subset analysis. Differential expression analysis was performed using the Wilcox test to compare hepatocyte subsets under MASLD and MASLD+Aza-TdC conditions.

[0090]

[0091] <Example>

[0092] Example 1. Alterations in MASLD Methylation and Increased DNMT1 Expression in Patients with Metabolic Dysfunction-Associated Fatty Liver Disease (MASLD)

[0093] To investigate the association between MASLD and DNA methylation regulation, we conducted analyses aimed at clarifying the relationship between DNA methylation status and MASLD progression.

[0094] We performed methylation profiling in a cohort of 106 MASLD patients using the genome-wide Illumina 850K array. Regarding the progression of MASLD, differentially hypermethylated and hypomethylated CpG sites were represented as heatmaps, respectively, and are presented in Figure 1 . Specifically, comparing controls and MASLD patients, we identified 607 hypermethylated and 423 hypomethylated differentially methylated sites (DMPs) (FDR <0.001 and delta β value <0.1).

[0095] To identify methylation regulators in MASLD, transcriptome analysis was performed to investigate the expression patterns involved in methylation regulation in human MASLD subjects, and the results are shown in Figure 2 (control, n=30; MASLD, n=100). As can be seen in Figure 2, a positive correlation was observed between the DNMT (DNA methyltransferase) family and the progression of MASLD, and in particular, the expression of DNMT1 and DNMT3a was confirmed to be differentially elevated in MASLD subjects compared to the control group.

[0096] Furthermore, consistent with the results in the Korean MASLD cohort, an increase in DNMT1 expression was also observed in the European MASLD cohort (GSE1352521) compared to the control group, as shown in Figure 3. As can be seen in Figure 3, the meta-analysis of the MASLD cohort further demonstrates a significant correlation between DNMT1 expression and MASLD.

[0097] Additionally, as shown in Figures 4 and 5, Pearson correlation analysis in a cohort of 130 Korean MASLD samples showed that DNMT1 expression was positively correlated with NAS, ballooning, lobular and portal inflammation scores, and DNMT1 showed a positive correlation with clinical parameters related to insulin resistance, including insulin, HOMA-IR, Hb1Ac and adipose insulin resistance.

[0098] Example 2. Confirmation of the efficacy of liver disease treatment using an animal model.

[0099] Example 2-1. MASH-induced mouse model

[0100] To establish the MASH model, C58BL / 6J mice were fed the MASH diet (protein, 9.8%, fat, 52.9%, carbohydrate, 37.3%, #TD.190142, Teklad Custom Diet) and sucrose solution (D-glucose = 18.9 g / L, fructose = 23.1 g / L) starting at 8 weeks of age. After 14 weeks, the mice were divided into three groups, and each group was administered the drug once a day by oral gavage.

[0101] Example 2-2. Confirmation of DNMT inhibition effect in MASH-induced mouse model

[0102] In order to confirm the effect of 4'-thio-5-aza-2'-deoxycytidine of the present invention on improving the MASH phenomenon, C58BL / 6J mice were fed a normal diet (NC) or a MASH diet for 12 weeks, and then 4'-thio-5-aza-2'-deoxycytidine, a DNMT inhibitor of the present invention, was administered intraperitoneally for 4 weeks, and then the experimental mice were sacrificed. Afterwards, the size of the liver was visually confirmed through an autopsy, and the liver tissue of each mouse was collected and the gross characteristics and histological analysis of the liver tissue were performed with H&E (Haematoxylin and Eosin) and Oil Red O, and immunohistochemical analysis for the macrophage index (F4 / 80+) was performed to confirm the degree of MASH progression. In addition, the liver, body weight, and liver-body weight ratio were measured, and the results are shown in Fig. 7.

[0103] As can be seen in Figure 7, hepatomegaly induced by the MASH diet was significantly attenuated by 4'-thio-5-aza-2'-deoxycytidine administration (MASH+AZA) (Figure 7A).

[0104] As can be seen from the Oil Red O staining results in Figure 7B, excessive lipid accumulation in the livers of mice with MASH was significantly reduced by administration of 5-aza-2'-deoxycytidine (Figure 7B).

[0105] In addition, when the results of immunohistochemical analysis using F4 / 80 staining were compared with the livers of the normal diet-fed mouse group (NC) to confirm macrophage expression as a pathological indicator of inflammation, F4 / 80 expression induced by MASH was significantly suppressed in the MASH diet mice (NHSH+AZA) administered 5-aza-2'-deoxycytidine due to the inhibition of DNMT by 5-aza-2'-deoxycytidine administration (Fig. 7B).

[0106] In addition, in the case of hepatomegaly, a representative symptom of MASH, the group administered 5-aza-2'-deoxycytidine (NHSH+AZA) showed a significant improvement in body weight / body mass ratio compared to the group fed a normal diet (NC) (Fig. 7C).

[0107] Therefore, it was shown that the 5-aza-2'-deoxycytidine of the present invention has a remarkable effect in improving the MASH phenomenon due to inhibition of DNMT.

[0108] Example 2-3. Analysis of gene expression related to lipid accumulation regulation in a mouse model induced by MASH.

[0109] To confirm the effect of 4'-thio-5-aza-2'-deoxycytidine of the present invention on the expression of genes related to lipid accumulation regulation, C58BL / 6J mice were fed a normal diet (NC) or a MASH diet for 18 weeks, and then 4'-thio-5-aza-2'-deoxycytidine, a DNMT inhibitor of the present invention, was administered daily for 4 weeks. The expression of genes regulating lipid accumulation was analyzed by Western blot. The results are shown in Fig. 8.

[0110] As can be seen in Fig. 8, in the MASH+ZAZ group, the levels of fatty acid synthase (FASN) and stearoyl-CoA deoxidase-1 (SCD1), which are important enzymes in lipid synthesis and key enzymes in monounsaturated fatty acid synthesis, were found to decrease at the translational level by 4'-thio-5-aza-2'-deoxycytidine treatment.

[0111] Through this, we confirmed that inhibiting DNMT through 4'-thio-5-aza-2'-deoxycytidine treatment in MASH improved impaired lipid metabolism.

[0112] Example 2-4. Confirmation of reduced hepatomegaly and lipid accumulation following DNMT1 inhibition in a MASLD mouse model.

[0113] Similar to the experiments in Examples 2-1 to 2-3 above, we additionally confirmed the reduction in hepatomegaly and lipid accumulation following DNMT1 inhibition in a MASLD mouse model. Specifically, as shown in Figure 9, to confirm the involvement of DNMT1 in MASLD, the pharmacological inhibition efficacy of DNMT1 was confirmed in a mouse model of diet-induced MASLD.

[0114] For this purpose, 6-week-old male C57BL / 6N(J) mice were purchased from ORIENTBIO (Korea) and housed at the Korea Research Institute of Bioscience and Biotechnology. The mice were housed in a facility with controlled conditions of 23°C, 40-50% humidity, and a 12-h light-dark cycle, and had free access to water during the study period. All procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. At 8 weeks of age, the mice were randomly divided into three groups. The control group was maintained on a standard diet (SD) (#2018S, Teklad Global 18% protein rodent diet, ENVIGO), and the MASLD group was provided with a MASLD diet (#TD.190142, Teklad Custom Diet, ENVIGO, USA) supplemented with a sucrose solution (18.9 g / L D-glucose, 23.1 g / L fructose) for 18 weeks. A third group followed the same MASLD diet for 18 weeks but received additional oral administration of the DNMT1-specific inhibitor 5-Aza-4′-thio-2′-deoxycytidine (Aza-TdC) for the final 4 weeks before sacrifice.

[0115] As shown in Fig. 10, administration of 5-Aza-4′-thio-2′-deoxycytidine (Aza-TdC), a DNMT1 inhibitor, significantly attenuated the progression of MASLD in the liver by reducing hepatomegaly, a common phenotype in diet-induced MASLD. Histological analysis using BODIPY staining confirmed that excessive lipid accumulation in the liver induced by the MASLD diet was significantly suppressed after DNMT1 antagonism. In addition, immunohistochemical analysis confirmed a decrease in the presence of macrophages and lymphocytes, which showed a decrease in anti-F4 / 80 Ab reactivity in the livers of mice treated with 5-Aza-TdC compared to MASLD alone. These results confirmed that reducing immune cell infiltration through DNMT1 inhibition can also reduce inflammation associated with MASLD.

[0116] In addition, as can be confirmed in Fig. 11, the results of the analysis through histological scores showed that hepatic steatosis, lobular inflammation, and hepatocellular ballooning were reduced, and the NAS (MAFLD Activity Score) score was lowered through DNMT1 regulation.

[0117] Example 2-5. Confirmation of improved expression of genes related to lipid metabolism and inflammation through DNMT1 inhibition.

[0118] To elucidate the molecular mechanisms and therapeutic potential of DNMT1 inhibition in MASLD, we used RNA-seq analysis. Specifically, DNMT1 inhibition significantly reduced the expression of genes involved in lipid uptake (Cd36), transport (FABPs), droplet formation, and storage, demonstrating its potential to improve MASLD.

[0119] Specifically, as shown in Figures 12 and 13, the SCD1 gene, an enzyme essential for fatty acid desaturation, was downregulated, and the expression of genes involved in lipid absorption, including Cd36, was restored, as well as the expression of genes involved in lipid transport (Fabp1-4) and droplet dynamics (Plin2, Plin3, and Cideac). This demonstrates that DNMT1 plays a crucial role in regulating lipid metabolism under MASLD conditions.

[0120] Furthermore, as shown in Figures 14 and 15, bulk RNA-seq analysis revealed a significant decrease in the expression of markers such as Adgre1, F4 / 80, TNF-α, IL-6, and TGF-β1, indicating attenuation of inflammation. Furthermore, the expression of collagen genes involved in fibrosis, such as Col1a2, Col3a1, Col4a2, and Col5a2, was partially reduced, suggesting that DNMT1 inhibition can alleviate the fibrotic process through its anti-inflammatory effects.

[0121] Moreover, as confirmed through Figure 16, protein analysis also confirmed that the elevated levels of Plin2, CD36, and SCD1 in MALSD were restored after Aza-TdC administration.

[0122] Additionally, to further investigate hepatocyte-specific changes, single-nucleotide RNA sequencing (snRNA-seq) was performed on liver tissues from each diet group. As shown in Fig. 17, DNMT1 inhibition resulted in a significant downregulation of lipid metabolism genes in hepatocytes, which was consistent with the RNA-seq results.

[0123] Example 3. Epigenomic analysis using a MASH-induced mouse model

[0124] To identify methylation changes induced by 4'-thio-5-aza-2'-deoxycytidine in a mouse model of MASH, a methylation array experiment was performed using the EPIC BeadChip (Illumina). Specifically, 1 μg of genomic DNA collected from mouse liver tissue was treated with 20 μL of sodium bisulfite solution included in the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA). The bisulfite-converted DNA (4 μL) was amplified using the InfiniumMethylation Assay kit (Illumina). The amplified DNA was hybridized to the EPIC BeadChip and scanned using the Illumina iSCAN system. CpG methylation values ​​were calculated as mean-β values ​​using the minfipackage (version 1.26.2) in R software, and functional normalization was used to remove technical variation. Measurements with a detection p-value less than 0.05 were considered to have a signal significant above background.

[0125] Additionally, Gene Ontology Analysis (GO) was performed to determine the functions of genes whose DNA methylation was altered by 4'-thio-5-aza-2'-deoxycytidine.

[0126] Specifically, gene ontology (GO) analysis was performed using the Genomic Regions Enrichment of Annotations Tool (GREAT, version 4.0.4) (https: / great.stanford.edu / great / public / html / ), which assigns biological meaning to a set of non-coding genomic regions by examining the annotations of surrounding genes.

[0127] To identify genes associated with these regions, we defined a window encompassing the basal plus extension settings (5 kb upstream, 1 kb downstream) of differentially methylated CpG sites (DMPs). This window was used to examine CpGs located in regulatory regions distal to the transcription start site (TSS).

[0128] The analysis results are as shown in Figure 18.

[0129] Epigenomic analysis of liver tissue from a MASH-induced mouse model revealed that the methylation changes caused by MASH were reversed by DNMT inhibition by 4'-thio-5-aza-2'-deoxycytidine treatment. That is, the 4'-thio-5-aza-2'-deoxycytidine-treated (AZA) group was confirmed to show a DNA methylation profile similar to that of the liver tissue of normal mice (Fig. 18A). Many genes related to lipid metabolism and lipid transport were found in the MASH-induced mouse model, in which DNA methylation increased and then decreased by 4'-thio-5-aza-2'-deoxycytidine treatment, and many genes related to inflammatory responses were found in the MASH-induced mouse model, in which DNA methylation decreased and then increased by 4'-thio-5-aza-2'-deoxycytidine treatment (Fig. 18B).

[0130] Additionally, methylation at specific CpG sites within the promoter region of the target gene showed differential methylation changes.

[0131] Specifically, as shown in Figure 19, methylome array data showed a significant decrease in methylation in promoter regions containing specific CpG sites, such as cg47230786 of Slc38a3, cg43959116 of C1ra, cg40013516 of Il6ra, and cg33066766 of Prlr, after DNMT1 inhibition, which was verified by pyrosequencing of genomic DNA. These genes, known to be involved in the regulation of lipid metabolism or inflammation, were generally regulated by HNFa and PPARa. That is, as confirmed in Figure 20, they exhibited hypermethylation under MASLD conditions, which correlated with decreased expression. After DNMT1 inhibition, these promoters switched to a hypomethylated state, which was accompanied by a significant induction of expression, as this promoted the recruitment of transcription factors and restoration of transcriptional activity.

[0132] Additionally, to further evaluate the roles of HNF4a and PPARa in transcriptional regulation, publicly available chromatin immunoprecipitation sequencing (ChIP-seq) datasets (GSE90533 and GSE61817) were analyzed. The results are shown in Figure 21 . As can be seen in Figure 21 , strong binding of HNF4α and PPARα was observed near the promoter regions of genes including Prlr, C1ra, Il6ra, Pck1, Gls2, Klf15, and Slc38a3, which overlapped with binding sites showing altered methylation. That is, deletion of HNF4a and PPARa significantly reduced binding in the promoter regions of Slc38a3, C1ra, Il6ra, and Prlr, thereby confirming that these genes are direct targets of HNF4a and PPARa.

[0133] Furthermore, in the MASLD mouse model, DNMT1 inhibition via Aza-TdC treatment restored the recruitment of HNF4a and PPARa to the promoter region, which was reduced in MASLD compared to controls. This suggests that changes in DNA methylation induced by DNMT1 inhibition may promote the binding affinity of HNF4a and PPARa to the promoter region, thereby contributing to the restoration of gene expression and the attenuation of lipid accumulation or inflammation in MALD.

[0134] Additionally, as shown in Figure 22, CpG sites near the promoter-TSS region of Pck1 showed restored methylation after pharmacological inhibition of DNMT1, as verified by pyrosequencing.

[0135] Example 4. Transcriptome analysis using a MASH-induced mouse model

[0136] To identify changes in the set of genes related to lipid metabolism following inhibition of DNMT by 4'-thio-5-aza-2'-deoxycytidine in a mouse model of MASH, single nuclear RNA sequencing (snRNA-seq) analysis was performed, and the specific methods are as follows:

[0137] (1) General analysis of single-cell transcriptome data:

[0138] Seurat objects of single-cell transcriptome data from three different sample groups (control (NC), MASH, and AZA) were merged into a single, combined object. Integration of these datasets was performed using the 'Harmony' package, followed by data normalization, data scaling, and variable feature finding functions to identify the top 2,000 feature sets, respectively. The overall analysis was performed using the 'Seurat' v4.4.0 package in R. In the initial preprocessing step, genes expressed in less than 10 cells were filtered out. Furthermore, cells were excluded based on predefined criteria: mitochondrial gene expression exceeded 15%, the number of feature genes was less than 5,000, and the number of reads was less than 20,000.

[0139] (2) Cell cluster annotation:

[0140] The 'singleR' computational tool was used to annotate cell clusters. This approach involved comparing annotated reference data with marker genes selected to optimally distinguish cell phenotypes. Manual annotation was also performed, leveraging prior biological knowledge.

[0141] (3) Differential expression analysis:

[0142] To identify differences between clusters, differential gene expression analysis was performed using the Wilcox test implemented in Seurat. Each gene considered for analysis had to be expressed in at least 50% of cells in both groups.

[0143] (4) Hepatocyte subset analysis:

[0144] We generated a Seurat object using hepatocytes. We then calculated highly variable genes within this hepatocyte subset. After scaling and normalizing the expression matrix, we constructed an SNN graph using 20 principal components. The resolution parameter was fine-tuned to obtain 10-15 clusters for each hepatocyte.

[0145] (5) Differential expression analysis of hepatocyte subsets:

[0146] To gain a deeper understanding of the AZA hepatocyte phenotype, gene expression levels were assessed through differential gene expression analysis between MASH hepatocytes and 4'-thio-5-aza-2'-deoxycytidine (AZA) hepatocytes.

[0147] (6) Functional Analysis:

[0148] To explore the potential molecular mechanisms of AZA and identify key pathways and associated genes, we performed gene set enrichment analysis (GSEA) of ranked 4'-thio-5-aza-2'-deoxycytidine (AZA) differentially expressed genes (DEGs) using ClusterProfiler v4.8.3.

[0149] The analysis results are as shown in Figures 23 and 24.

[0150] As shown in Figures 23 and 24, the disruption of the lipid metabolism-related gene set caused by MASH was reversed by inhibition of DNMT, resulting in a gene expression profile similar to that of normal liver tissue. Therefore, transcriptomic analysis of mouse liver tissue confirmed that 4'-thio-5-aza-2'-deoxycytidine (AZA) has excellent therapeutic efficacy in liver disease.

[0151] The results of the analysis according to the present invention are summarized in Figure 25. Inhibition of DNMT1 restored metabolic homeostasis by reducing TAG accumulation and inflammation, thereby attenuating the progression of MASLD. DNMT1 inhibition reversed MASLD-induced hypermethylation and restored the transcriptional activity of key regulatory genes mediated by HNF4a and PPARa. Pharmacological inhibition of DNMT1 attenuated hypermethylation around the promoter regions of target genes such as Slc38a3, C1ra, Il6ra, and Prlr. These changes in DNA methylation due to DNMT1 inhibition enhanced the binding affinity of HNF4a and PPARa to the promoter regions, restoring gene expression and attenuating lipid accumulation and inflammation in MASLD livers. Furthermore, increased DNMT1 levels interfere with HNF4a binding through hypermethylation, thereby inhibiting PCK1 transcription and activating downstream signaling pathways.

[0152] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A pharmaceutical composition for preventing or treating liver disease comprising 4'-thio-5-aza-2'-deoxycytidine represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] 2. A pharmaceutical composition according to claim 1, wherein the liver disease is at least one selected from the group consisting of simple fatty liver, metabolically abnormal fatty liver disease (MAFLD), liver inflammation, metabolic steatohepatitis (MASH), metabolic liver disease, cholestatic liver disease, liver fibrosis, cirrhosis, and liver failure.

3. A pharmaceutical composition according to claim 2, wherein the cholestatic liver disease is at least one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

4. A pharmaceutical composition according to claim 1, wherein the liver disease is fatty liver, hepatic fibrosis, or cirrhosis accompanied by metabolic steatohepatitis.

5. A pharmaceutical composition according to claim 1, wherein the liver disease is liver cancer caused by metabolic abnormality steatohepatitis.

6. A pharmaceutical composition according to claim 1, wherein the liver disease is at least one selected from the group consisting of metabolically abnormal fatty liver disease (MAFLD) and metabolically abnormal steatohepatitis (MASH).

7. A method for preventing or treating liver disease, comprising administering to a subject in need thereof 4'-thio-5-aza-2'-deoxycytidine represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] 8. A method according to claim 7, wherein the liver disease is at least one selected from the group consisting of simple fatty liver, metabolically abnormal fatty liver disease (MAFLD), liver inflammation, metabolic steatohepatitis (MASH), metabolic liver disease, cholestatic liver disease, liver fibrosis, cirrhosis, and liver failure.

9. A method according to claim 8, wherein the cholestatic liver disease is at least one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

10. A method according to claim 7, wherein the liver disease is fatty liver disease, hepatic fibrosis, or metabolic steatohepatitis accompanied by cirrhosis.

11. A method according to claim 7, wherein the liver disease is liver cancer caused by metabolic abnormality fatty liver disease.

12. A method according to claim 7, wherein the liver disease is at least one selected from the group consisting of metabolically abnormal fatty liver disease (MAFLD) and metabolically abnormal steatohepatitis (MASH).

13. Use of 4'-thio-5-aza-2'-deoxycytidine or a pharmaceutically acceptable salt thereof represented by the following chemical formula 1 for manufacturing a medicine for preventing or treating liver disease: [Chemical Formula 1] 14. The use according to claim 13, wherein the liver disease is at least one selected from the group consisting of simple fatty liver, metabolically abnormal fatty liver disease (MAFLD), liver inflammation, metabolic steatohepatitis (MASH), metabolic liver disease, cholestatic liver disease, liver fibrosis, cirrhosis, and liver failure.

15. The use according to claim 14, wherein the cholestatic liver disease is at least one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

16. In the 13th paragraph, the liver disease is fatty liver, hepatic fibrosis or cirrhosis accompanied by metabolic steatohepatitis.

17. The use according to claim 13, wherein the liver disease is liver cancer caused by metabolic abnormality fatty liver disease.

18. The use according to claim 13, wherein the liver disease is at least one selected from the group consisting of metabolic fatty liver disease (MAFLD) and metabolic steatohepatitis (MASH).

19. Use of 4'-thio-5-aza-2'-deoxycytidine represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of liver disease: [Chemical Formula 1] 20. A composition comprising 4'-thio-5-aza-2'-deoxycytidine represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of liver disease: [Chemical Formula 1]

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