Pharmaceutical composition for prevention and treatment of fibrosis and liver diseases

Abstract

The present invention relates to a pharmaceutical composition for prevention and treatment of fibrosis and liver diseases, the pharmaceutical composition comprising, as an active ingredient, a benzothiazole aniline-based compound having the structure of chemical formula 1, wherein the pharmaceutical composition can effectively inhibit the expression of factors such as free fatty acid (FFA) or TGF-β1 and the expression of downstream proteins of related signals.

Description

Pharmaceutical composition for the prevention and treatment of fibrosis and liver disease

[0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of fibrosis and liver disease, and more particularly, to a pharmaceutical composition for the treatment or prevention of fibrosis or metabolic disease, comprising a benzothiazole aniline compound as an active ingredient.

[0002] Fibrosis is defined as the abnormal increase or accumulation of fibrous tissue within tissues, and is considered a key pathological feature in the development and progression of various diseases. Fibrosis is a connective tissue that normally forms to heal or replace damaged tissue, playing a positive role in promoting structural stability and functional recovery. However, when this fibrous tissue formation becomes abnormally excessive, it can develop into fibrosis, which severely impairs the normal function of the tissue or organ. This can occur due to chronic irritation, a persistent inflammatory response, or an abnormal regulation of the wound healing process.

[0003] Fibrosis manifests in various forms depending on the tissue and organ. For example, pulmonary fibrosis is caused by excessive accumulation of fibrous tissue within the lungs, leading to decreased lung function, symptoms such as shortness of breath and coughing, and in severe cases, can be life-threatening. Liver fibrosis, due to the abnormal accumulation of fibrous tissue within the liver, impairs liver function and carries a risk of developing cirrhosis and liver failure in the long term. Fibrosis can also occur in various organs, including cardiac fibrosis, renal fibrosis, and dermal fibrosis, resulting in functional impairment and structural stiffening of the affected organ.

[0004] Most fibrosis treatments developed to date focus on alleviating symptoms or delaying disease progression. For example, anti-inflammatory or antifibrotic drugs reduce inflammation and inhibit fibrosis. However, these treatments offer limited efficacy and fall short of eliminating the underlying cause of fibrosis or completely curing the disease. In particular, existing medications can have reduced efficacy or cause side effects with long-term use, leading to a persistent need for safer and more effective treatments.

[0005] Therefore, it is crucial to more precisely understand the pathogenesis of fibrosis and develop novel treatment strategies based on this understanding. In particular, if fundamental treatments can be developed that can suppress the early stages of fibrosis or halt its progression, it would be possible to prevent organ dysfunction caused by fibrosis and dramatically improve patient prognosis. Against this backdrop, the development of innovative pharmaceutical compositions and treatments that can delay or treat the progression of fibrosis is currently a key research topic in the fields of medicine and pharmacy.

[0006] The purpose of the present invention is to provide a pharmaceutical composition for the prevention and treatment of fibrosis or metabolic diseases, which has excellent efficacy and few side effects by inhibiting target substances that cause fibrosis or metabolic diseases.

[0007] In one aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of fibrosis and liver disease, comprising a compound having a structure represented by the following chemical formula 1.

[0008] [Chemical Formula 1]

[0009]

[0010] In one embodiment, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating fibrosis, including pulmonary fibrosis, renal fibrosis, liver fibrosis, skin fibrosis, and sclerosis.

[0011] In one embodiment, the pharmaceutical composition may be characterized by inhibiting the expression of free fatty acid (FFA) or TGF-β1 and the expression of downstream proteins of related signals.

[0012] In one embodiment, the pharmaceutical composition may comprise metabolic fatty liver disease (MAFLD), metabolic steatohepatitis (MASH), liver fibrosis, cardiac fibrosis, pulmonary fibrosis, kidney fibrosis, inflammatory bowel disease, rheumatic disease, steatosis, inflammation, hepatitis, pulmonary hypertension, obesity, diabetes, or hyperlipidemia.

[0013] The pharmaceutical composition for the prevention and treatment of fibrosis and liver disease, which comprises a benzothiazole aniline compound according to the present invention as an active ingredient, can effectively suppress the expression of factors such as free fatty acid (FFA) or TGF-β1 and the expression of downstream proteins of related signals.

[0014] The present invention can effectively improve, prevent and treat fibrosis including pulmonary fibrosis, renal fibrosis, liver fibrosis, skin fibrosis and cirrhosis, and metabolic diseases including metabolic fatty liver disease (MAFLD), metabolic steatohepatitis (MASH), liver fibrosis, obesity, diabetes, hyperlipidemia, hepatic steatosis, inflammation and hepatitis by regulating the expression of the above factors.

[0015] Figure 1 is a drawing showing the structure of a benzothiazole aniline compound, which is an effective ingredient of a compound according to the present invention.

[0016] Figure 2 is a drawing showing data confirming cytotoxicity through the results of cell viability analysis of a compound according to the present invention.

[0017] FIG. 3 is a drawing including results visually showing that a compound according to the present invention can suppress hepatic steatosis and fibrosis in a concentration-dependent manner in a cell model induced by FFA and TGF-β1.

[0018] Figure 4 is a drawing visually showing data verifying the inhibitory effect of the compound according to the present invention on fibrosis and hepatic steatosis at the molecular level.

[0019] FIG. 5 is a drawing visually illustrating the mechanism of the pharmaceutical composition for inhibiting fibrosis, demonstrating by immunofluorescence analysis that the compound according to the present invention effectively inhibits α-SMA and Col-1 expression in TGF-β1-induced fibrosis.

[0020] FIG. 6 is a drawing including results visually and quantitatively demonstrating that a compound according to the present invention can effectively reduce intracellular fat accumulation and triglyceride content in a FFA-induced hepatic steatosis model.

[0021] FIG. 7 is a diagram showing an animal experiment plan for creating a MASH in vivo model that provides a basis for evaluating the efficacy of a compound according to the present invention at various doses.

[0022] Figure 8 is a diagram showing the results of analyzing body weight changes over time in a MASH mouse model, showing the efficacy of the compound according to the present invention in suppressing body weight gain during the MASH induction process.

[0023] FIG. 9 is a diagram showing data evaluating the effect of a compound according to the present invention on improving liver toxicity and histopathological changes in a MASH mouse model, showing that the compound according to the present invention is effective in reducing liver toxicity and improving pathological changes.

[0024] FIG. 10a is a drawing showing the results of evaluating pathological changes in liver tissue in a MASH mouse model using Sirius Red, F4 / 80, and H&E staining, showing that the compound according to the present invention can effectively suppress liver fibrosis, inflammation, and fat accumulation, thereby improving pathological changes in liver tissue.

[0025] Figure 10b shows analysis data quantifying the results of Figure 10a.

[0026] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention is susceptible to various modifications and variations, and thus specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. Throughout the description of each drawing, similar reference numerals have been used to designate similar components.

[0027] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" indicate the presence of a feature, step, operation, component, part, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, steps, operations, components, parts, or combinations thereof.

[0028] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0029]

[0030] The present invention relates to a pharmaceutical composition for the prevention and treatment of fibrosis and liver diseases, particularly metabolic steatohepatitis (MASH) and metabolic fatty liver disease (MAFLD). The pharmaceutical composition of the present invention comprises a compound that can inhibit the progression of fibrosis, improve liver function, and effectively reduce fat accumulation and inflammatory responses. Through this, the present invention overcomes the limitations of existing therapeutic agents and can contribute to the fundamental treatment of the target disease and improved prognosis for patients.

[0031] The present invention has demonstrated therapeutic efficacy in various nonclinical models, and is based on experimental evidence supporting the drug's efficacy and safety. Furthermore, the present invention has the potential to fundamentally block the progression of the disease by targeting key pathological mechanisms of liver fibrosis and inflammation.

[0032] The pharmaceutical composition according to the present invention can be used for the prevention or treatment of metabolic diseases such as metabolic fatty liver disease (MAFLD), metabolic steatohepatitis (MASH), steatosis, obesity, diabetes, hyperlipidemia, and fibrotic diseases such as liver fibrosis, cardiac fibrosis, pulmonary fibrosis, renal fibrosis, pulmonary hypertension, and rheumatic diseases. In addition, the present invention can be used for inflammatory diseases such as inflammatory bowel disease, inflammation, and hepatitis, which are diseases closely related to metabolism or fibrosis, and other diseases in which metabolic disorders and fibrosis play a core role in the pathological mechanism may fall within the scope of the rights according to the present invention.

[0033] Below, the structure of the compound according to the present invention is described through drawings, and the therapeutic effect and safety of the drug confirmed through various non-clinical models are described.

[0034] Figure 1 shows the structure of a benzothiazole aniline compound, which is an effective ingredient of a compound according to the present invention. The compound used in the present invention is a benzothiazole aniline compound, and the benzothiazole aniline compound means a compound having a benzothiazole structure containing an aniline functional group. Here, the aniline functional group means a structure in which an amino group (-NH2) is bonded to a benzene ring (phenyl group) (Ph-NH2) or a modification thereof (Ph-NHR, Ph-NR2), and the benzothiazole structure means an aromatic compound in which a benzene ring (hexagonal aromatic ring) and a thiazole ring (pentagonal aromatic ring containing nitrogen and sulfur) are condensed, as follows.

[0035] <Benzothiazole structure>

[0036]

[0037] These structural features play a crucial role in inhibiting key mechanisms of fibrosis and inflammation. Benzothiazoles act as inhibitors of enzymes that regulate oxidative stress and inflammatory responses, and their aniline functional groups can interact with specific proteins to modulate the expression of genes associated with fibrosis.

[0038] This compound blocks the progression of fibrosis by inhibiting the TGF-β1 / SMAD pathway, and reduces the inflammatory response and fibrosis in liver tissue by reducing the expression of PPAR-γ. In addition, it exhibits anti-inflammatory effects by reducing the expression of fibrosis marker proteins such as α-SMA and Col-1, and suppressing macrophage infiltration and the expression of inflammatory cytokines. In addition, it was confirmed to suppress fat accumulation and reduce the levels of triglycerides (TG) and total cholesterol (TC) in liver disease models. In conclusion, the compound of the present invention is a compound that can have pharmacological activity that can fundamentally suppress the progression of diseases by targeting the main pathological mechanisms of fibrosis and liver disease.

[0039]

[0040] Below, experimental results that can confirm the stability and therapeutic effect of a benzothiazole aniline compound according to one embodiment of the present invention are described.

[0041] (cell culture)

[0042] In one embodiment of the present invention, the cell culture conditions used utilized culture media and environmental conditions optimized for the characteristics of each cell line. Human hepatic stellate cell line LX-2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium supplemented with 10% Fetal Bovine Serum (FBS), 1% Antibiotic-Antimycotic (AA), and 2 mM L-Glutamine. Human skin fibroblast cell line HS27 cells, which use the same DMEM medium, were cultured in addition to 10% FBS and 1% AA. Human lung fibroblast cell line MRC-5 cells were cultured in Minimum Essential Medium (MEM) with Earle's Balanced Salts (MEM / EBSS) supplemented with 10% FBS and 1% AA, and human renal proximal tubule cell line HK-2 cells were cultured in RPMI 1640 Medium supplemented with 10% FBS and 1% AA.

[0043] All cells were cultured in a CO₂ incubator (Thermo Scientific) at 37°C and 5% CO₂ to maintain physiological conditions and provide an optimal growth environment. These culture conditions were established to meet the physiological needs of each cell line and ensure experimental reproducibility and reliability.

[0044] (Cytotoxicity test)

[0045] In one embodiment of the present invention, an experiment was performed using CCK-8 (Cell Counting Kit-8) to evaluate the cytotoxicity of the compound according to the present invention and confirm the viability. CCK-8 is an experimental method based on water-soluble tetrazolium salt (WST-8), and uses the principle that the amount of formazan dye produced by intracellular dehydrogenase is directly proportional to the number of living cells and inversely proportional to cytotoxicity. This allows for indirect measurement of cytotoxicity. The experiment was conducted with four types of cells: LX-2, MRC-5, HK-2, and HS27. Each cell was seeded at a density of 2 × 10⁴ / well in a 96-well plate, and then stabilized for more than 24 hours under conditions of 37°C and 5% CO₂. Afterwards, the compound according to the present invention was treated to each well at concentrations of 0, 1, 5, 10, 25, 50, 75, 100, and 200 μM, and cultured for 24 hours under the same conditions. After culture, 10 μL of CCK-8 solution was added to each well, and the well was further cultured for 2 hours, and the absorbance was measured at 450 nm using a microplate reader. The collected data were analyzed and graphed using the GraphPad Prism application, and statistical significance was confirmed through one-way ANOVA and Tukey's multiple comparison test.

[0046] Figure 2 shows a statistical analysis to confirm cytotoxicity through the results of cell viability analysis of the compound according to the present invention, and *, **, and *** in Figure 2 represent significance probability (p), respectively, and are marks showing that the difference between data can occur with a probability of less than 0.05, 0.01, and 0.001, respectively, and thus are significant statistics. As a result of the experiment, it can be confirmed that the cell viability decreased in a concentration-dependent manner as the concentration of the compound according to the present invention increased in all LX-2, MRC-5, HK-2, and HS27 cells. In particular, LX-2 and HK-2 cells showed a significant decrease in viability at a concentration of 10 μM or higher, and MRC-5 and HS27 cells showed a rapid decrease at a concentration of 25 μM or higher.

[0047] Table 1 below quantitatively represents the above results. Cytotoxicity (%) was calculated as [(experimental value - blank value) / control value] x 100.

[0048] Cell line [IC 50 ] (μM)LX-220.29 ± 1.77MRC-522.50 ± 1.23HK-29.72 ± 0.45HS2713.39 ± 0.45

[0049]

[0050] These results suggest that the compound of the present invention can exhibit cytotoxicity within a specific concentration range, and that sensitivity may vary depending on the cell line. These data can serve as valuable baseline data for establishing the effective concentration range of the compound of the present invention and assessing its pharmacological applicability.

[0051] (Induction of hepatic steatosis and fibrosis in vitro using FFA and TGF-β1)

[0052] Hepatic steatosis was induced by treating hepatic stellate cells (HSCs) with free fatty acid (FFA), and fibrosis was induced by treating them with transforming growth factor-beta (TGF-β1).

[0053] For this purpose, 10 mM FFA stock solutions were prepared by mixing oleic acid (C18:1) and palmitic acid (C16:0) in ethanol, respectively. The oleic acid solution was prepared by mixing 28.246 mg of oleic acid (MW 282.46, Sigma-Aldrich) in 10 ml of ethanol (Duksan), and the palmitic acid solution was prepared by mixing 25.642 mg of palmitic acid (MW 256.42, Sigma-Aldrich) in 10 ml of ethanol. Before treating with FFA to induce hepatic steatosis, hepatic stellate cells (LX-2) were cultured in a CO2 incubator at 37°C and 5% carbon dioxide. After 24 hours, a medium containing 200 μM FFA (oleic acid: palmitic acid = 2:1, molar ratio) and a compound according to the present invention (concentrations of 0, 1, 5, and 10 μM) was added to the cells cultured in a cell culture dish. The cells treated with FFA and the compound according to the present invention were further cultured in a CO2 incubator under the same conditions (37°C, 5% CO2) for 24 hours. The morphology of the cells after treatment was imaged using a Nikon inverted microscope.

[0054] Figure 3a shows the concentration-dependent inhibitory effect of the compound (ENT101) of the present invention on morphological changes in hepatic stellate cells (LX-2) in a FFA-treated hepatic steatosis model. In particular, at concentrations of 5 μM and 10 μM, pathological morphological changes in cells were significantly reduced, suggesting that the compound of the present invention is effective in alleviating fat accumulation and cell damage. These results suggest the potential of the compound of the present invention as a therapeutic agent for hepatic steatosis and related diseases.

[0055] TGF-β1 (R&D Systems) was dissolved in 4 mM hydrochloric acid to prepare a stock solution at a concentration of 10 μg / ml. The stock solution was aliquoted and stored in an ultra-low temperature freezer (Deep Freezer Ultra-Low Freezers, Eppendorf) for use as needed.

[0056] Hepatic stellate cells (LX-2), human lung fibroblasts (MRC-5), renal fibroblasts (HK-2), and skin fibroblasts (HS27) were seeded at 1 × 10 in each 60 mm culture dish. 6 The cells were seeded at a density of 100 cells / dish and stabilized for 24 hours at 37°C and 5% CO2. Afterwards, each cell was treated with a compound according to the present invention (0, 0.1, 0.5, 1, 5, 10 μM) and TGF-β1 (5, 10 ng / ml) and cultured for 24 hours under the same conditions. The morphological changes of the treated cells were imaged using a Nikon inverted microscope.

[0057] Figures 3b-e visually demonstrate the dose-dependent inhibition effect of compounds according to the present invention on TGF-β1-induced fibroblast activation in various fibrosis models of liver (LX-2), lung (MRC-5), kidney (HK-2), and skin (HS27). This highlights the potential of compounds according to the present invention to act as signaling inhibitors in tissue-specific fibrotic pathogenesis, supporting their potential as therapeutic agents for fibrosis.

[0058] To confirm the signaling mechanism of TGF-β1, LX-2 cells were cultured by treating them with the compound according to the present invention (10 μM) and TGF-β1 (5 ng / ml) for various periods of time (0.5, 1, 3, 6, 16, and 24 hours). To observe the morphological changes of the cells according to the treatment time during the experiment, images were acquired using a Nikon inverted microscope. After treatment with TGF-β1, a change to a spindle-shaped morphology was observed in all cells, which showed a typical characteristic of fibroblast activation.

[0059] The results are presented in the images in Figure 3f, which visually depict the effects of the compounds of the present invention on TGF-β1-induced signaling pathways and cell morphology changes over time. This demonstrates the time-dependent antifibrotic effects of the compounds of the present invention.

[0060] (Western blot experiment)

[0061] Proteins were extracted from cells treated with compounds according to the present invention (concentrations: 0, 1, 5, 10 μM) and FFA (200 μM) or TGF-β1 (5, 10 ng / ml), and Western blot analysis was performed. For protein extraction, a homogenization solution was prepared by adding a protease inhibitor cocktail and a phosphatase inhibitor (PhosSTOP) to 1x RIPA lysis buffer, and the cultured cells were washed with 1x DPBS and collected using a cell scraper. The collected cells were centrifuged in a microcentrifuge (1,500 rpm, 5 min, 4°C), the supernatant was removed, and the cells were homogenized with 1x RIPA lysis buffer and incubated on ice for 30 min. After centrifugation at 13,000 rpm for 10 minutes, the supernatant was separated, and the protein concentration was quantified by measuring the absorbance at 595 nm using a BCA protein assay kit. The quantified protein was mixed with 5x sample buffer at a ratio of 4:1, denatured at 95°C for 10 minutes, cooled on ice, and used as a Western blot analysis sample.

[0062] SDS-PAGE was performed using a 10% polyacrylamide gel. After loading the protein size marker and the prepared sample, electrophoresis was performed at 100 V to separate the proteins according to their size. The proteins after electrophoresis were transferred at 30 mA for 100 minutes using a system consisting of Mini Trans-Blot filter paper and PVDF membrane. After transfer, the membrane was blocked with a 3% BSA solution for 60 minutes to prevent nonspecific binding. Afterwards, each protein was treated with a specific primary antibody for Western blot analysis, and the treatment conditions were set according to the protein to be tested. Through this process, the effect of the compound according to the present invention on changes in protein expression induced by FFA and TGF-β1 treatment could be confirmed, and the mechanism of action of the compound according to the present invention was visually analyzed.

[0063] The primary antibodies and processing conditions used for Western blot analysis are listed in Table 2 below.

[0064] Primary antibodyDilution ratioCatalog No ManufacturerFibronectin1:1000ab2413abcamCol-11:1000MA1-26771invitrogenPPAR-γ1:1000ab209350abcamTGF-β1:10003709CSTN-Ca dherin1:100013116CSTα-SMA1:100019245CSTSMAD2 / 31:10008685CSTp-SMAD2 / 31:10008828CSTAceCS11:10003658CSTAcetyl-CoA Carboxylase1:10003676CSTATP-Citrate Lyase1:10004332CSTFatty Acid Synthase1:10003180CSTLipin 11:100014906CSTACSL11:10009189CSTβ-actin1:1000sc-477778santa cruzSecondary antibodyDilution ratioCatalog NoManufacturerGoat Anti-rabbit IgG, HRP conjugated1:20007074SCSTGoat Anti-mouse IgG, HRP conjugated1:20007076SCST

[0065] Figures 4a-d show the results of Western blot analysis to evaluate changes in the expression of fibrosis-related proteins induced by TGF-β1 (Transforming Growth Factor-beta 1). Each figure shows the effect of the compound (ETN101) according to the present invention on the expression of fibrosis-related proteins (Col-1, Fibronectin, PPAR-γ, N-cadherin, α-SMA) in LX-2, MRC-5, HK-2, and HS27 cells.

[0066] The results in Figures 4a-d suggest that ETN101 exhibits antifibrotic effects by inhibiting the expression of TGF-β1-induced fibrosis-related proteins. This inhibitory effect gradually increased with ETN101 concentration, supporting a concentration-dependent antifibrotic mechanism of action. Consequently, the compound of the present invention can be evaluated as a potential antifibrotic treatment.

[0067] Figure 4e shows the results of Western blot analysis performed to evaluate the change in the expression of fibrosis-related proteins induced by TGF-β1 (5 ng / ml) and the compound ETN101 (10 μM) according to the present invention in LX-2 cells. Figures 4a-d show the results of Western blot analysis performed to evaluate the change in the expression of fibrosis-related proteins induced by TGF-β1 (Transforming Growth Factor-beta 1). Each figure shows the effect of the compound (ETN101) according to the present invention on the expression of fibrosis-related proteins (Col-1, Fibronectin, PPAR-γ, N-cadherin, α-SMA) in LX-2, MRC-5, HK-2, and HS27 cells.

[0068] The results in Figures 4a-d suggest that ETN101 exhibits antifibrotic effects by inhibiting the expression of TGF-β1-induced fibrosis-related proteins. This inhibitory effect gradually increased with ETN101 concentration, supporting a concentration-dependent antifibrotic mechanism of action. Consequently, the compound of the present invention can be evaluated as a potential antifibrotic treatment.

[0069] Figure 4e shows the results of analyzing the changes in protein expression of SMAD2 / 3 and p-SMAD2 / 3 after simultaneous treatment of LX-2 cells with TGF-β1 (5 ng / ml) and ETN101 (10 μM), a compound according to the present invention. The total expression of SMAD2 / 3 protein increased by TGF-β1 treatment, and there was no significant change in the expression level over time in the group simultaneously treated with TGF-β1 and ETN101. On the other hand, the expression of p-SMAD2 / 3, which was initially increased by TGF-β1 treatment, was suppressed in the group simultaneously treated with TGF-β1 and ETN101, showing a significant decrease especially in the early stage (0.5 to 3 hours). This suggests that ETN101 can effectively block the TGF-β1 signaling pathway.

[0070] Figure 4f shows the results of analyzing the changes in the expression of proteins related to lipogenesis and metabolism according to the ETN101 concentration (0, 1, 5, 10 μM) in LX-2 cells induced by FFA (200 μM). The expression of acetyl-CoA carboxylase (ACC), ATP-citrate lyase (ATP), and fatty acid synthase (FAS) increased by FFA treatment, but was suppressed by ETN101 treatment in a concentration-dependent manner. The expression of AceCS1 and Lipin 1 also increased by FFA treatment, but ETN101 decreased their expression in a concentration-dependent manner. In the case of ACSL1, the expression suppression effect was not evident even with ETN101 treatment, but the expression of TGF-β1 decreased in a concentration-dependent manner, indicating that ETN101 is effective in suppressing TGF-β1 expression.

[0071] In summary, Figures 4e and 4f demonstrate that ETN101 has the potential to alleviate the pathological processes of fibrosis and hepatic steatosis by inhibiting the TGF-β1 signaling pathway and reducing adipogenesis. These results suggest that ETN101 has potential as an antifibrotic and antisteatosis drug.

[0072] (immunocytochemistry)

[0073] In this experiment, LX-2, MRC-5, HK-2, and HS27 cells were seeded at a density of 5 × 10⁴ / well in 4-well chamber slides, respectively, and stabilized for 24 hours under 37°C, 5% CO2 conditions. The stabilized cells were treated with compounds according to the present invention (concentrations: 0, 0.1, 0.5, 1, 5, 10 μM) and TGF-β1 (5, 10 ng / ml), and cultured in a CO2 incubator for 24 hours.

[0074] After incubation, cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes and washed three times with TBS for 5 minutes each. Afterwards, cells were treated with 0.3% Triton X-100 for 15 minutes and washed three times with TBS for 10 minutes each. Cells were incubated in blocking solution (5% NGS and 5% BSA) for 1 hour at room temperature, and then treated with primary antibodies anti-α-SMA and anti-Col-1 (1:500) at 4°C.

[0075] After primary antibody incubation, cells were washed three times with 1x TBS for 10 minutes each. Subsequently, secondary antibodies Alexa Fluor™ 555 and Alexa Fluor™ 488 (1:1000) were incubated for 1 hour at room temperature, and nuclei were visualized with Antifade Mounting Medium containing DAPI.

[0076] Fluorescence expression images were captured using a Nikon fluorescence microscope (Ti-2U), and fluorescence expression was analyzed using Image J software. Three images were randomly selected for analysis of all samples, and the results were expressed graphically using GraphPad Prism software. Statistical significance was confirmed using one-way ANOVA and Tukey's test.

[0077] Figure 5 shows the results of an experiment that can confirm the effect of the compound (ETN101) according to the present invention on inhibiting α-SMA and Col-1 expression in LX-2, MRC-5, HK-2, and HS27 cells induced by TGF-β1.

[0078] In Fig. 5a, α-SMA expression in LX-2 cells increased with TGF-β1 treatment, but α-SMA expression gradually decreased when treated with ETN101 at various concentrations, and was particularly suppressed to the control level or lower at a concentration of 10 μM. In Fig. 5b, Col-1 expression in LX-2 cells also increased with TGF-β1 treatment, but decreased with ETN101 treatment, and was most effectively suppressed at a concentration of 10 μM.

[0079] In Figure 5c, α-SMA expression in MRC-5 cells increased with TGF-β1 treatment, but decreased with increasing ETN101 concentration, and was suppressed to the control level or lower at a concentration of 1 μM. In Figure 5d, Col-1 expression in MRC-5 cells increased with TGF-β1 treatment, but decreased with increasing ETN101 concentration, and the inhibitory effect was evident at a concentration of 1 μM.

[0080] In Figure 5e, α-SMA expression in HK-2 cells was increased by TGF-β1 treatment, but decreased by ETN101 treatment, and was suppressed to the control level or lower at a concentration of 1 μM. In Figure 5f, Col-1 expression in HK-2 cells was increased by TGF-β1 treatment, but decreased as the ETN101 treatment concentration increased, and the effect was prominent at a concentration of 1 μM.

[0081] In Fig. 5g, α-SMA expression in HS27 cells was increased by TGF-β1 treatment, but was decreased by ETN101 treatment in a concentration-dependent manner, and was suppressed to the control level or higher at a concentration of 1 μM. In Fig. 5h, Col-1 expression in HS27 cells was increased by TGF-β1 treatment, but was decreased as the ETN101 treatment concentration increased, and the effect was evident at a concentration of 1 μM.

[0082] The results of the eight drawings presented in Figure 5 above suggest that ETN101 exhibits an antifibrotic effect by inhibiting TGF-β1-induced fibrotic signals.

[0083] (Oil Red O (ORO) dyeing)

[0084] To evaluate the change in intracellular lipid accumulation content according to FFA treatment, ORO staining was used. The ORO staining solution was prepared by mixing it with distilled water in a ratio of 6:4 and filtering it through a 0.2 μm filter. The experiment was conducted on LX-2 cells, and the culture medium containing FFA and the compound according to the present invention was removed and washed twice with 1X DPBS. Thereafter, the cells were fixed with 4% PFA for 2 hours, and the fixed cells were washed three times with 1X DPBS and then pretreated with 60% isopropanol for 5 minutes.

[0085] After pretreatment, cells were stained with ORO solution at room temperature for 2 hours, and nuclear staining was performed with hematoxylin for 1 minute to visually confirm cell structure. After staining, cells were washed three times with 1X DPBS, the supernatant was removed, completely dried, and observed under a microscope.

[0086] Quantitative analysis of lipid accumulation content was performed by culturing cells at a density of 2 × 10⁴ cells / well in 96-well plates and performing the same staining process. After staining, the dye was eluted with 100% isopropanol, and the absorbance was measured at 450 nm using a microplate reader. The final measured values ​​were graphed using GraphPad Prism software, and statistical significance was confirmed using one-way ANOVA and Tukey's test.

[0087] Figure 6a shows the results of analyzing the changes in lipid accumulation induced by FFA (free fatty acid) treatment in LX-2 cells using Oil Red O (ORO) staining. The upper microscopic images show that lipid accumulation significantly increased in FFA-treated LX-2 cells, while in the ETN101-treated group, lipid accumulation tended to gradually decrease in a concentration-dependent manner. In the control group, lipid accumulation was barely visible, but in the group treated with 200 μM FFA alone, the number and size of intracellular lipid droplets increased significantly. When ETN101 was treated at concentrations of 1, 5, and 10 μM, lipid accumulation was inhibited in a concentration-dependent manner, and in particular, 10 μM showed an inhibitory effect close to the control group.

[0088] The quantitative analysis graph below shows that the lipid accumulation content significantly increased in the FFA-only treatment group compared to the control group, while in the ETN101-treated group, lipid accumulation significantly decreased as the concentration increased. Statistical analysis results showed that the lipid accumulation inhibition effect was significantly higher as the ETN101 concentration increased, and the inhibitory effect was strongest at a concentration of 10 μM. These results suggest that ETN101 is effective in inhibiting FFA-induced lipid accumulation and may have an anti-lipid accumulation effect through a concentration-dependent action.

[0089] (Triglyceride, Triglyceride (TG) analysis)

[0090] The neutral fat content in LX-2 cells was measured using a TG assay kit (Asanpharm). The experiment was conducted with LX-2 cells seeded in 6-well plates at a density of 5 × 10 5 The cells were seeded at a density of 10 / well and stabilized at 37°C and 5% CO2 for 24 hours. Afterwards, a medium containing the compound according to the present invention (0, 1, 5, 10 μM) and 200 μM FFA (oleic acid: palmitic acid = 2:1, molar ratio) was added, and the cells were cultured for an additional 24 hours.

[0091] After treatment, the culture medium was removed, the cells were washed three times with PBS, and the cells were collected using a scraper and centrifuged (10,000 rpm, 5 min, 4°C) to separate the supernatant. Then, lysis buffer was added to the pellet and centrifuged again (12,000 rpm, 5 min, 4°C). 120 μl of enzyme buffer solution was added to 80 μl of the collected supernatant, mixed, and incubated for 10 minutes in a CO2 incubator at 37°C, and the absorbance was measured at 550 nm.

[0092] As a positive control, the standard reagent in the TG assay kit was used, and protein quantification was performed using the BCA method (total 25 μg). The absorbance measurement results were graphed using the GraphPad Prism program, and statistical significance was verified using one-way ANOVA (Tukey's test). The triglyceride content was calculated using the following formula. The triglyceride content of the standard solution is 300 mg / dL.

[0093] TG levels (%) = [absorbance of sample / absorbance of standard] × 300

[0094] Figure 6b shows the results of analyzing changes in neutral fat content according to FFA (free fatty acid) treatment in LX-2 cells according to the concentration of the compound (ETN101) according to the present invention. The graph of Figure 6b shows neutral fat content as a relative ratio (%), and shows a tendency for neutral fat content to significantly decrease as the concentration of the compound according to the present invention increases compared to cells treated only with FFA.

[0095] Specifically, the group treated with only FFA showed a significant increase in neutral fat content compared to the control group, and when treated with the compound according to the present invention at 1, 5, and 10 μM, the neutral fat content showed a gradual decrease. As a result of statistical analysis, the group treated with the compound according to the present invention showed a significant decrease compared to the FFA treated group, indicating that the compound according to the present invention is effective in inhibiting neutral fat accumulation induced by FFA.

[0096] (Efficacy evaluation in an animal model of metabolic dysfunction-associated steatohepatitis (MASH))

[0097] A metabolic steatohepatitis (MASH) animal model was created using C57BL / 6 mice fed a choline-deficient, high-fat diet (CDAHFD) for 10 weeks. The compound of the present invention was then administered orally daily for 7 weeks, and the improvement in liver fibrosis and related liver disease markers was evaluated.

[0098] Figure 7 shows the animal experiment plan for constructing a MASH in vivo model that provides the basis for evaluating the efficacy of the compound according to the present invention at various doses. The body weights of the mice were measured and recorded 19 times in total: once per week during the disease induction period (3 times in total for 3 weeks), twice per week during the drug administration period (15 times in total for 7 weeks), and once on the day of necropsy. The experiment was conducted on 46 mice, and the mice were maintained under standard experimental conditions maintaining a 12:12 h light-dark cycle. The experimental groups were divided into a vehicle control group (n=4) maintained on a normal diet and 5 groups that received oral administration of vehicle or various concentrations of the compound according to the present invention (20 mg / kg, 40 mg / kg, 60 mg / kg, 80 mg / kg) in the CDAHFD model. Drug administration was performed once daily for a total of 7 weeks, and blood and liver tissue samples were collected and analyzed before the start of drug administration, 3 weeks after administration, and 7 weeks after administration.

[0099] In one embodiment, the oral administration dose may be 20 to 80 mg / kg, preferably 40 to 80 mg / kg.

[0100] Figure 8, which records the body weight change of mice, shows the effect of administration of choline-deficient and high-fat diet (CDAHFD) and the compound according to the present invention (ETN101) on body weight change in a metabolic steatohepatitis (MASH) model. The group that consumed only CDAHFD during the disease induction period showed a continuous decrease in body weight, and symptoms related to metabolic disorders and liver damage were observed. On the other hand, the group administered 20 mg / kg of ENT101 showed a similar tendency to the CDAHFD model group, and the groups administered 40 mg / kg and 60 mg / kg showed a slight body weight stabilization effect, showing a slight improvement compared to the CDAHFD group, and the group administered 80 mg / kg showed a tendency for body weight to decrease.

[0101] Figure 9a shows the results of evaluating the effects of ETN101 on liver damage and lipid metabolism in a CDAHFD-induced metabolic steatohepatitis (MASH) mouse model. Alanine transaminase (ALT) and aspartate transaminase (AST) levels, key indicators of liver damage, were significantly increased in the CDAHFD group compared to the control group. However, in groups administered ETN101 at various concentrations (20 mg / kg to 80 mg / kg), ALT and AST levels decreased in a dose-dependent manner.

[0102] Total cholesterol (TC) and triglyceride (TG) levels were used as indicators to evaluate lipid metabolism. TC levels slightly decreased in the CDAHFD group, but TG levels remained similar across all groups, indicating no significant changes following ETN101 administration.

[0103] These results suggest that ETN101 is effective in alleviating CDAHFD-induced liver damage and may contribute to improved liver function by reducing ALT and AST levels. Although the effects on TC and TG were limited, the overall hepatoprotective effect of ETN101 was demonstrated.

[0104] After blood collection, mice were euthanized, and the abdominal cavity was exposed to harvest spleen, kidney, and liver tissues. The total tissue weight of each individual was measured. Care was taken to avoid rupturing the gallbladder during liver extraction, and if bile contaminated the liver, it was washed with PBS.

[0105] Figure 9b shows the results of analyzing the weights of the liver, spleen, and kidneys collected from mice. The left graph shows liver weight, which significantly increased due to CDAHFD, but decreased in a dose-dependent manner in the group administered ETN101. The middle graph shows spleen weight, and no significant change in spleen weight was observed in any group. The right graph shows kidney weight, and the weight of both the left and right kidneys was included in the kidney weight, and no significant difference was observed in any group. The photos below show the macroscopic findings of the livers extracted from each group. In the CDAHFD group, the liver size was larger and the color was lighter, whereas in the ETN101-administered group, the size and color of the livers were similar to those of the control group. ETN101 showed a dose-dependent effect of improving the increased liver weight due to CDAHFD, suggesting the possibility of improving liver fibrosis and steatohepatitis.

[0106] Figure 9c is an image comparing the macroscopic findings of liver tissues extracted from each group. While the liver in the vehicle group exhibited normal size and color, the CDAHFD group exhibited increased liver size, paler color, and traces of fat accumulation. In the ETN101-administered group, liver size decreased and color tended to approach normal with increasing dose. This suggests that ETN101 is effective in alleviating CDAHFD-induced liver damage.

[0107] After weighing, the anterior and posterior surfaces of the livers were photographed and cut into sections according to the intended purpose. Sections were fixed in 10% neutral buffered formalin (NBF) and processed into paraffin blocks for each animal. The following endpoints were evaluated by qualitative histological evaluation and quantitative histomorphometry by a certified veterinary pathologist.

[0108] Figure 10a is a photograph of each tissue slide after hematoxylin & eosin (H&E) staining was completed, observed and photographed using an optical microscope, and Figure 10b is a graph showing a quantitative evaluation of the histopathological quantitative evaluation of liver tissue by scoring the degree of steatosis, inflammation, and ballooning degeneration according to the metabolic abnormality fatty liver disease (MAFLD) activity score criteria.

[0109] The degree of fibrosis was assessed by measuring the area of ​​red-stained collagen fibers using Picro-Sirius Red-stained slides. Analysis of inflammatory cells (macrophages) was performed using F4 / 80 immunohistochemical staining (IHC). In this process, rabbit anti-F4 / 80 antibody (1:500) was used as the primary antibody, and detection was performed using the Vectastain Elite ABC kit and 3,3'-diaminobenzidine (DAB). After counterstaining with hematoxylin, the slides were observed and photographed under a light microscope.

[0110] The captured tissue images were analyzed using Image-Pro software, and tissue sections were segmented using a software application with uniform thresholding parameters. The target area in each IHC- or histochemically stained slide was quantified as a percentage of the total area, and data were calculated based on this.

[0111] In Sirius Red staining, the staining area significantly increased in the CDAHFD group due to the accumulation of collagen fibers, and in the groups administered ETN101 at different concentrations, the collagen fiber staining area tended to decrease in a concentration-dependent manner.

[0112] F4 / 80 staining results showed that macrophage accumulation was evident in the CDAHFD group, but in the ETN101 administration group, the macrophage staining area decreased in a concentration-dependent manner and the inflammatory response was alleviated.

[0113] In H&E staining analysis, the CDAHFD group showed severe fat accumulation and inflammation in hepatocytes, whereas in the ETN101 administration group, steatosis and inflammation symptoms gradually improved depending on the concentration.

[0114] Quantitative analysis results showed that, across all three graphs—Sirius Red, F4 / 80, and H&E—the efficacy of ETN101 in alleviating fibrosis, inflammation, and steatosis significantly improved with increasing ETN101 administration concentration. These results strongly suggest that ETN101 can effectively suppress liver fibrosis, inflammation, and fat accumulation at high concentrations.

[0115]

[0116] In the above-described examples of the present invention, the efficacy of ETN101 for the prevention and treatment of metabolic steatohepatitis (MASH) and liver disease was demonstrated. In the CDAHFD model, ETN101 exhibited therapeutic effects such as inhibition of liver fibrosis, alleviation of inflammation, and reduction of fat accumulation, which were confirmed through histological analysis (Picro-Sirius Red, F4 / 80, H&E) and biochemical marker analysis (ALT, AST).

[0117] ETN101 showed excellent efficacy in a concentration-dependent manner and has great potential as a new treatment option compared to existing treatments, so it is expected to be a significant breakthrough in the development of liver disease treatments.

[0118]

[0119] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

Claims

1. A pharmaceutical composition for the prevention and treatment of fibrosis and liver disease, comprising a compound having a structure of the following chemical formula 1: [Chemical Formula 1] 2. In paragraph 1, The above pharmaceutical composition is a pharmaceutical composition for preventing or treating fibrosis including lung fibrosis, kidney fibrosis, liver fibrosis, skin fibrosis and sclerosis. Pharmaceutical composition for the prevention and treatment of fibrosis and liver disease.

3. In paragraph 2, The pharmaceutical composition is characterized in that it inhibits the expression of free fatty acid (FFA) or TGF-β1 and the expression of downstream signal regulatory proteins. Pharmaceutical composition for the prevention and treatment of fibrosis and liver disease.

4. In paragraph 1, The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of diseases including metabolic fatty liver disease (MAFLD), metabolic steatohepatitis (MASH), liver fibrosis, cardiac fibrosis, pulmonary fibrosis, kidney fibrosis, inflammatory bowel disease, rheumatic disease, steatosis, inflammation, hepatitis, pulmonary hypertension, obesity, diabetes or hyperlipidemia. Pharmaceutical composition for the prevention and treatment of fibrosis and liver disease.

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