KR102196711B1 - Pharmatheutical composition comprising Tenofovir disoproxil fumarate for preventing or treating of hepatic fibrosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis, containing tenofovir disoproxil fumarate (TDF) or a pharmaceutically acceptable salt thereof as an active ingredient, and more specifically, non -Provides a therapeutic use for viral liver fibrosis.

Description

Pharmaceutical composition comprising Tenofovir disoproxil fumarate for preventing or treating of hepatic fibrosis, containing tenofovir disoproxil fumaric acid as an active ingredient.

The present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis, containing tenofovir disoproxil fumarate (TDF) or a pharmaceutically acceptable salt thereof as an active ingredient, and more specifically, non -Provides a therapeutic use for viral liver fibrosis.

When the liver tissue is damaged and an inflammatory reaction occurs, hepatic stellate cells are activated to heal wounds in the liver tissue, and the process of combining a large amount of secreted collagen and extracellular matrix (ECM) proceeds. However, when the inflammatory reaction proceeds chronically and the wound healing process of the liver tissue is excessive, liver fibrogenesis occurs. If liver fibrosis continues to progress, a large amount of collagen in the liver tissue is deposited, and regeneration crystals are surrounded by the collagen, so that the liver tissue may develop into cirrhosis, indicating an abnormal structure.

Liver fibrosis is caused by a chronic inflammatory reaction in the liver tissue, and the causes of such inflammatory reactions are hepatitis due to viral infection, alcohol consumption, drug abuse, autoimmune disease, metabolic disorders, cholestasis and hepatitis due to other causes. It is known as. In particular, in addition to viral hepatitis caused by infection such as Hepatitis B virus, the incidence of non-viral hepatitis, which is mainly caused by non-alcoholic liver disease or metabolic disease, is increasing. The proportion of patients with viral liver fibrosis and mortality are also increasing.

Changes in the Prevalence of the Most Common Causes of Chronic Liver Diseases in the United States From 1988 to 2008 According to ZM Younossi et al Clinical Gastroenterology and Hepatology, June 2011 Volume 9, Issue 6, Pages　524-530., 1988-1994 Chronic liver diseases, including cirrhosis caused by CH-B (chronic hepatitis B) and HCV (chronic hepatitis C) viruses, occur at a similar rate as the times change from the period to the period from 2005 to 2008, but non-viral The cause of chronic liver disease 　 ALD (alcoholic liver disease) is slightly increased, and chronic liver disease caused by NAFLD (non-alcoholic fatty liver) is confirmed to increase continuously.

When hepatic fibrosis progresses to cirrhosis, it appears as an irreversible chronic disease in which normal restoration of liver tissue is difficult, and accordingly, it progresses to decompensated cirrhosis or liver cancer, resulting in a continuous increase in mortality.

According to Marcellin, Patrick, et al., The Lancet 381.9865 (2013): 468-475., it has been confirmed that patients with cirrhosis can also improve their condition. Therefore, it was previously thought that cirrhosis and hepatic fibrosis could not be improved, but recent studies have confirmed that hepatic fibrosis including cirrhosis can also improve after treatment. However, the cause has not yet been identified.

Specifically, tenofovir disoproxil fumarate (TDF) is known as an inhibitor against the nucleic acid reverse transcriptase of HBV virus or HIV virus, and is used as a therapeutic agent for chronic hepatitis B and AIDS. As a clinical study result, it has been reported that when TDF is continuously administered to chronic hepatitis B patients, the progression of hepatic fibrosis and hepatic lesions can also be suppressed as HBV is suppressed. Is known to be a clinical result of TDF inhibiting HBV, and thus no direct effect of TDF on the treatment of hepatic fibrosis has been reported.

Therefore, as a result of the present inventors trying to explore the use of TDF that can have other than the therapeutic effect of HBV virus, TDF inhibits the expression of hepatic fibrosis markers in activated hepatic stellate cells that induce the progression of hepatic fibrosis, and hepatic stellate cells The present invention was completed by confirming through cell experiments and animal experiments that TDF can be used as a direct hepatic fibrosis therapeutic agent since it induces apoptosis of the cells and induces cell death.

An object of the present invention is to provide a novel use of Tenofovir disoproxil fumarate (TDF).

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, containing as an active ingredient tenofovir disoproxil fumarate (TDF) or a pharmaceutically acceptable salt thereof do.

In addition, the present invention provides a food composition for preventing or improving liver fibrosis, containing TDF or a pharmaceutically acceptable salt thereof as an active ingredient.

In a preferred embodiment of the present invention, the liver fibrosis may be non-viral liver fibrosis or AKT signaling system related liver fibrosis, and the non-viral liver fibrosis is liver fibrosis, alcoholic cirrhosis, nonalcoholic cirrhosis, Any one or more diseases selected from the group consisting of autoimmune cirrhosis, primary biliary cholangitis-related cirrhosis, Wilson's disease-related cirrhosis, hemoglobin-related cirrhosis, portal cirrhosis, postnecrotic cirrhosis, malnutrition-related cirrhosis, and cardiac cirrhosis. I can.

In addition, the daily dose of Tenofovir disoproxil fumarate (TDF) or a pharmaceutically acceptable salt thereof may be 0.1 mg/kg to 300 mg/kg.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, containing tenofovir disoproxil fumarate (TDF) or a pharmaceutically acceptable salt thereof as an active ingredient.

The TDF provided by the present invention inhibits the progression of hepatic fibrosis by inhibiting the expression of a marker protein of the progression of hepatic fibrosis in an activated hepatic stellate cell model, and increases apoptosis by inducing the apoptosis of hepatic stellate cells. , Can significantly inhibit the progress of liver fibrosis. At this time, self-killing can be induced by regulating the activation of the AKT (especially PI3K-AKT) signaling system.

The TDF of the present invention can exhibit an effect of inhibiting the progression of hepatic fibrosis even in a non-HBV-infected hepatic fibrosis cell model when compared with a drug used as a therapeutic agent for the conventional hepatitis B virus. That is, TDF can have a direct therapeutic effect on liver fibrosis caused by a non-viral cause. Along with this, TDF does not cause liver and kidney toxicity, but rather can have an anti-inflammatory action in the liver.

1 is a result of confirming the degree of apoptosis by TDF in hepatic stellate cells.
2 is a result of confirming the degree of self-killing by TDF in hepatic stellate cells.
3(a) and (b) are results of confirming the protein expression level of self-killing signal transduction by TDF in hepatic stellate cells.
4 is a result of confirming which signaling system is self-killing by TDF in hepatic stellate cells.
5(a) to (c) are results of animal experiments confirming the antifibrotic effect by taking TDF.
6 is a result of an animal experiment confirming the degree of self-killing by taking TDF in hepatic stellate cells.
7 is a result of an animal experiment confirming liver and kidney toxicity after taking TDF.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, containing as an active ingredient tenofovir disoproxil fumarate (TDF) or a pharmaceutically acceptable salt thereof.

In the pharmaceutical composition of the present invention, the TDF is used as an antiviral agent against HIV-1 and HBV. The TDF is preferably a pro-drug present in the form of fumarate of tenofovir, as shown in the following [Formula 1]:

[Formula 1]

.

.

The TDF of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, butin-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, me Toxibenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol Rate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is a conventional method, e.g., by dissolving the TDF in an excess acid aqueous solution, and precipitating this salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be manufactured by making it. It can also be prepared by heating the same amount of TDF and an acid or alcohol in water, and then evaporating and drying the mixture, or by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt can be made using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).

In addition, the TDF of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method, for example, TDF is dissolved in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and an excessive amount of organic acid is added or an aqueous acid solution of an inorganic acid It can be prepared by precipitation or crystallization after adding Subsequently, the solvent or excess acid is evaporated from the mixture and dried to obtain an addition salt, or the precipitated salt can be prepared by suction filtration.

In the pharmaceutical composition of the present invention, in that TDF directly induces death of activated hepatic stellate cells, which is the main cause of liver fibrosis progression, the liver fibrosis is viral hepatic fibrosis (HBV infection) and non-viral It is preferable to include all of hepatic fibrosis, specifically, more preferably non-viral hepatic fibrosis, but is not limited thereto. More specifically, the non-viral liver fibrosis is liver fibrosis, alcoholic cirrhosis, non-alcoholic cirrhosis, autoimmune cirrhosis, primary biliary cholangitis-related cirrhosis, Wilson's disease-related cirrhosis, hemoglobin-related cirrhosis, portal cirrhosis, postnecrotic cirrhosis, It is more preferable that it is one or more diseases selected from the group consisting of cirrhosis and cardiac cirrhosis related to malnutrition, but is not limited thereto.

On the other hand, in that TDF induces self-killing by inactivating the AKT (especially PI3K-AKT) signaling system, the liver fibrosis may be AKT (especially, PI3K-AKT) signaling system-related liver fibrosis. On the other hand, TDF has no significant effect on ERK, P38 and JNK signaling systems.

The composition of the present invention may have an anti-inflammatory effect in the liver in addition to the use of preventing or treating liver fibrosis.

When the composition of the present invention is used as a pharmaceutical product, a pharmaceutical composition containing TDF or a pharmaceutically acceptable salt thereof as an active ingredient may be formulated and administered in various oral or parenteral dosage forms as follows at the time of clinical administration. , But is not limited thereto.

Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and the like.These formulations include diluents (e.g., lactose, dexte) in addition to the active ingredients. Rose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, optionally such as starch, agar, alginic acid or sodium salts thereof. Disintegrants or boiling mixtures and/or absorbents, colorants, flavors, and sweeteners.

The pharmaceutical composition containing TDF or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient can be administered parenterally, and parenteral administration is a method of injecting subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. By. At this time, in order to formulate a formulation for parenteral administration, the TDF or a pharmaceutically acceptable salt thereof is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which can be prepared in ampoules or vials unit dosage form. . The composition may be sterilized and/or contain adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts and/or buffers for controlling osmotic pressure, and other therapeutically useful substances, which are conventional methods of mixing, granulation. It can be formulated according to the method of painting or coating.

In addition, the dosage of TDF of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease. Specifically, the daily dose of TDF of the present invention may be 0.1 mg/kg to 300 mg/kg, and more specifically, the daily dose of TDF of the present invention based on a mouse is a body surface area commonly used in animal experiments. Conversion factor (BSA-CF) is a value multiplied by 12.33, which is 1.2 mg/kg to 246.6 mg/kg, and the adult human standard daily dose of TDF of the present invention may be 0.1 mg/kg to 20 mg/kg. According to the judgment of the pharmacist, it may be dividedly administered once or several times a day at regular time intervals.

In addition, the present invention provides a food composition for preventing or improving liver fibrosis, containing as an active ingredient tenofovir disoproxil fumarate (TDF) or a pharmaceutically acceptable salt thereof.

In the food composition of the present invention, since the TDF is the same as described above for the pharmaceutical composition, a duplicate description will be omitted.

In the food composition of the present invention, since the liver fibrosis is the same as described above with respect to the pharmaceutical composition, a duplicate description will be omitted.

The food composition according to the present invention can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruit, canned food, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g. ham, sausage) Corn beef), bread and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, sweets, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , Vegetable protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.), etc., can be prepared by adding the TDF of the present invention. In addition, nutritional supplements are not limited thereto, but may be prepared by adding the TDF of the present invention to capsules, tablets, pills, and the like. In addition, the health functional food is not limited thereto, but for example, the TDF of the present invention itself is prepared in the form of tea, juice, and drinks, and liquefied, granulated, encapsulated, and powdered so that it can be consumed (healthy beverage). can do. In addition, in order to use the TDF of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate. In addition, it can be prepared in the form of a composition by mixing the TDF of the present invention with a known active ingredient known to have an effect of inhibiting or treating liver fibrosis.

When using the TDF of the present invention as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as an additional ingredient, like a normal drink. The natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, It may contain glycerin, alcohol or a carbonating agent. In addition, the health food of the present invention may contain flesh for the manufacture of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients may be used independently or in combination. The ratio of these additives is not very important, but it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1: Drug treatment of LX2 cells

LX2 cells were cultured in DMEM medium at 5 x 10 ⁵ cells/ml in 100 mm plate. After 24 hours of incubation, ETV, LAM, and TDF were treated with 10, 50, 100, and 200 uM, respectively, followed by additional culture for 24 hours.

Example 2: Western blot analysis

After culturing according to Example 1, cells were collected using trypsin, and then proteins were extracted using RIPA protein extraction buffer. The same amount of each sample was electrophoresed on a 10% SDS-PAGE gel. The protein of the gel after electrophoresis was transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) through an electric flow and blocked with a blocking buffer (5% skim milk in PBS) at room temperature for 30 minutes. After blocking was completed, the mixture was washed 3 times with TBS-T for 10 minutes each, and then reacted with the primary antibody. Each primary antibody was diluted 1:500 in 1% BSA-containing TBS buffer and reacted overnight in a low temperature room (4° C.), and then washed three times with TBS-T for 10 minutes to remove non-specific reactions. As a secondary antibody, horseradish peroxidase (HRP) is linked rabbit IgG antibody (anti-rabbit IgG, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) or mouse IgG antibody (anti-mouse IgG, Santa Cruz Biotechnology Inc. .) was reacted at room temperature for 2 hours. After the completion of the secondary antibody reaction, it was washed three times for 10 minutes using TBS-T, and the expression of each protein was visualized using an ECL (enhanced chemiluminesecent system) solution (Amersham Biosciences) and Las-4000 (Fujifilm Corporation, Japan). Made it.

In the present invention, the products shown in Table 1 below were used as the primary antibody.

name manufacturer Bell PARP Sigma Rabbit Cleaved-caspase3 Cell Signaling Rabbit Bcl-xl Sigma Rabbit Bcl-2 Abcam Mouse PI3K Cell Signaling Rabbit p-PI3K Cell Signaling Rabbit AKT Cell Signaling Rabbit p-AKT Cell Signaling Rabbit ERK Santa Cruz Rabbit p-ERK Cell Signaling Rabbit p38/MAPK Cell Signaling Rabbit p-p38/MAPK Cell Signaling Rabbit JNK Santa Cruz Mouse p-JNK Cell Signaling Rabbit α-SMA Sigma-Aldrich Mouse α-tubulin Sigma Mouse β-actin Sigma-Aldrich Mouse

Example 3: Evaluation of apoptosis (MTT assay)

To evaluate apoptosis, 7000 cells were cultured in a 96 well plate with LX2 cells and HSC-T6 (Rat hepatic stellate cells) in 10% DMEM medium for 24 hours, and then ETV, LAM, and TDF were respectively 10, 50, 100, After 200 uM treatment, it was further cultured for 24 hours. Cells cultured in DMEM medium containing 10% FBS without drug treatment were used as controls. After 24 hours of drug treatment, 5 ug/ml of thiazolyl blue tetrazolium blue (m5655, Sigma) was mixed in DMEM medium with 10% FBS, and 200 ul was added to each well for 1 hour at 37°C. After incubation and removal of the medium, 100 ul of DMSO was added and mixed evenly, and the absorbance was measured at 540 nm.

Example 4: Self-killing evaluation (Annexin V assay)

LX2 cells were cultured in a 6 well plate at 1 x 10 ⁵ cells/ml in 10% DMEM medium for 24 hours, and each drug was treated with 10, 50, 100, 200 uM, and then cultured for 24 hours. After collecting the cells by treatment with trypsin, the cells were fluorescently stained using the FITC-Annexin V apoptosis detection kit (556547, BD Biosciences) and measured using the FACS machine BD Canto II (BD Biosciences), and the result was FlowJo software (FlowJo, LLC, USA).

Example 5: Animal experiment

The 8-week-old mice were divided into 5 groups, the group without treatment (Normal), the group without treatment (TAA) after 10 weeks of injection of the chemical substance thioacetamide (TAA) inducing fibrosis, and 10 TAA. Group fed ETV 1 mg/kg daily (TAA+ETV) via oral gavage while infusion for weeks, group fed LAM 100 mg/kg daily (TAA+LAM) with TAA infusion for 10 weeks, TAA While injecting for 10 weeks, the experiment was performed in a group fed with TDF 200 mg/kg daily (TAA+TDF). The amount of each drug was tested in a non-toxic dose based on the amount calculated through the body surface area conversion factor (BSA-CF) of the amount actually taken by humans. After the mice were sacrificed 10 weeks later, additional experiments were performed.

Example 6: Collagen Quantification Experiment

To quantify collagen in mouse liver tissue, collagen accumulation was visualized through Sirius red staining using Picro-Sirius Red Staining Kit (Abcam, Cambridge, UK), and each 5 sites of the stained tissue were captured and ImageJ software (NIH , USA). In addition, the amount of hydroxyproline in the liver was quantified using a Hydroxyproline assay kit (BioVision, Milpitas, CA).

Example 7: Real-time polymerase chain reaction (Quantitative real-time PCR)

RNA was extracted from mouse liver tissue and cDNA was synthesized. The expression of Col1a1 and Timp1 was confirmed and analyzed using a Light Cycler 480 instrument (Roche Applied Science, Indianapolis, IN) using a TaqMan probe.

Example 8: Fluorescence staining (Immunofluorescence staining)

Mouse liver tissue fixed on OCT-embedding compound was cut to a thickness of 7-μm, fixed in 4% paraformaldehyde, and then conjugated with α-SMA antibody and Anti-mouse IgG conjugated with Alexa Fluor 546 (Invitrogen). Staining was performed using an antibody, and nuclei were stained using DAPI for control staining. In addition, for TUNEL staining, cells that were apoptotic were stained using an in situ cell death detection kit (Roche), and this was visualized through Confocal Microscopy (Zeiss, Jena, Germany).

1 is a result of confirming the degree of apoptosis by TDF in hepatic stellate cells. Specifically, when confirming the apoptosis after 10, 50, 100, 200 uM treatment of ETV, LAM, and TDF in LX2, a human-derived hepatic stellate cell, and HSC-T6, a rat-derived hepatocyte, at each concentration, ETV and LAM are While it did not affect the apoptosis of hepatic astrocytes, it was confirmed that the apoptosis effect of hepatic astrocytes appeared when TDF was treated. Through these results, it was confirmed that TDF inhibits fibrosis by inducing apoptosis of hepatic stellate cells.

2(a) and (b) are results of confirming the degree of self-killing by TDF in hepatic stellate cells. Specifically, it was confirmed that self-killing occurs after TDF treatment when ETV, LAM, and TDF were treated at different concentrations in LX2, a human-derived hepatic stellate cell, and HSC-T6, a rat-derived hepatic stellate cell. Through this result, it was confirmed that apoptosis caused by TDF is caused by apotosis.

In order to reconfirm this, the change in the expression level of the protein in the signaling pathway inducing self-killing was confirmed. After the ETV, LAM or TDF was treated and further cultured, the cell morphology was first observed under a microscope. Then, the cells were collected and Western blot was performed in the method of Example 2.

FIG. 3 is a result of confirming the protein expression level of self-killing signal transduction by TDF in hepatic stellate cells, showing that the expression of Bcl-xl, which inhibits apoptosis, was reduced when TDF 200 μM was treated compared to other groups. It was confirmed that caspase-3 and PARP proteins were cleaved and activated. Through this result, it was confirmed that autocell death through TDF is through the self-kill signal.

Figure 4 is a result of confirming which signaling system is self-killing by TDF in hepatic stellate cells. Specifically, PI3K-AKT, ERK, P38 and JNK signaling systems known to induce self-killing were identified. There was no effect on the expression and phosphorylation of ERK, P38 and JNK, but after TDF treatment. It was confirmed that the PI3K-AKT signaling system was deactivated. From these results, it was predicted that TDF induced self-killing by regulating the activation of the PI3K-AKT signaling system.

5(a) to (c) are results of animal experiments confirming the antifibrotic effect by taking TDF. As a result, it was confirmed that collagen was significantly reduced in mice taking TDF through Sirius red staining, and hydroxyproline was significantly reduced in mice taking TDF compared to mice taking other drugs. Confirmed. The expression of α-SMA was also close to the normal level after TDF administration, and the expressions of Col1a1 and Timp1 were also significantly reduced in TDF-treated mice. Through this, it was confirmed that a significant anti-fibrotic effect was observed after TDF administration in a non-viral hepatic fibrosis mouse model.

6 is a result of an animal experiment confirming the degree of self-killing by taking TDF in hepatic stellate cells. Specifically, as a result of observing cells undergoing apoptosis after performing α-SMA and TUNEL staining, which is an activated hepatic stellate cell marker protein in the liver tissue of each mouse, mice not taking drugs or treated with ETV and LAM In contrast, a wide range of apoptosis could be observed, not in a specific region, whereas in mice taking TDF, apoptosis occurred depending on the region where activated hepatic stellate cells were located. Through these results, it was verified that TDF can exert an antifibrotic effect by inducing self-killing of activated hepatic stellate cells not only in cell experiments but also in animal experiments.

7 is a result of an animal experiment confirming liver and kidney toxicity after taking TDF. Specifically, in order to confirm liver and kidney toxicity by TDF, ALT and Creatinine levels were measured. In both results, toxicity to liver and kidney by TDF was not observed, and in particular, it was confirmed that ALT levels were significantly reduced in the group of mice taking TDF. Through this, it can be expected that not only the stability of TDF, which induces apoptosis specifically only in hepatic stellate cells, but also TDF inactivates inflammatory cells in the liver and alleviates the inflammatory response.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

Claims (8)

Tenofovir disoproxil fumarate (Tenofovir disoproxil fumarate, TDF) or a pharmaceutically acceptable salt thereof containing as an active ingredient, non-viral hepatic fibrosis prevention or treatment pharmaceutical composition.
delete The method of claim 1, wherein the non-viral hepatic fibrosis is liver fibrosis, alcoholic cirrhosis, nonalcoholic cirrhosis, Wilson's disease-related cirrhosis, hemoglobin-related cirrhosis, portal cirrhosis, post-necrotic cirrhosis, cirrhosis related to nutritional deficiency A pharmaceutical composition for preventing or treating non-viral hepatic fibrosis, characterized in that at least one selected from the group consisting of.
According to claim 1, wherein the non-viral hepatic fibrosis is AKT signaling system related non-viral hepatic fibrosis, characterized in that, non-viral hepatic fibrosis prevention or treatment pharmaceutical composition.
The pharmaceutical composition for preventing or treating non-viral hepatic fibrosis according to claim 1, wherein the composition does not cause side effects due to liver and kidney toxicity.
The method of claim 1, wherein the daily dosage of Tenofovir disoproxil fumarate (TDF) or a pharmaceutically acceptable salt thereof is 0.1 mg/kg to 300 mg/kg, non- A pharmaceutical composition for preventing or treating viral liver fibrosis.
Tenofovir disoproxil fumarate (Tenofovir disoproxil fumarate, TDF) or a pharmaceutically acceptable salt thereof as an active ingredient, non-viral liver fibrosis prevention or improvement food composition containing as an active ingredient.
delete

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