Therapeutic use of exosomes containing super-repressor-IκB (srIκB) for liver diseases

Abstract

The present invention relates to the therapeutic use of exosomes containing super-repressor-IκB (srIκB) for liver diseases.

Description

Technical Field

[0001] The present invention relates to the therapeutic use of exosomes containing super-repressor-IκB (srIκB) for liver diseases.

Background Art

[0002] Liver diseases vary in type and severity from fatty liver to cirrhosis, and since there are no initial symptoms, they are often discovered only after they have progressed significantly. The causes of liver diseases are diverse, including infections by viruses and bacteria, alcohol and toxic substances, excessive accumulation of fat and heavy metals, and abnormal immune reactions. Although various therapeutic agents are used for such liver diseases depending on the cause and type of the disease, many cases cannot be treated with conventionally known therapeutic agents, and liver diseases still rank first as a cause of death worldwide. Therefore, the development of drugs for treating liver diseases is still desired. On the other hand, in recent years, exosomes have attracted much attention as a novel biocarrier for gene / drug delivery. Exosomes are extracellular vesicles (EVs) that play an important role in cell-to-cell communication by delivering bioactive substances to recipient cells or by affecting the signal transduction pathways of target cells.

[0003] The inventors of the present invention have developed exosomes containing a bioactive substance that exhibits a liver disease preventive or therapeutic effect, and have confirmed that the exosomes exhibit an excellent liver disease therapeutic effect by stably and effectively delivering the bioactive substance to liver cells and tissues, and provide the exosomes of the present invention as a liver disease preventive or therapeutic agent.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

[0005] [Non-Patent Document 1] https: / / tabula-muris-senis.ds.czbiohub.org / [Non-Patent Document 2] https: / / david.ncifcrf.gov / [Non-Patent Document 3] Remmerie, A. et al., Immunity 2020, 53, 641-657 [Non-Patent Document 4] Bonnardel, J.; T’Jonck,W. et al., Immunity 2019, 51, 638-654 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] An object of the present invention is to provide a pharmaceutical composition for preventing or treating liver diseases, comprising exosomes containing super-repressor-IκB (srIκB) as an active ingredient. [Means for Solving the Problems]

[0007] One aspect for realizing the present invention relates to a pharmaceutical composition for preventing or treating liver diseases, comprising exosomes containing super-repressor-IκB (srIκB) as an active ingredient.

[0008] Another aspect for realizing the present invention relates to a method for preventing or treating liver diseases, comprising the step of treating an individual at risk of liver diseases with exosomes containing super-repressor-IκB (srIκB) protein. [Effects of the Invention]

[0009] Exosomes containing super-repressor-IκB (srIκB) according to the present invention are useful for the prevention or treatment of liver diseases.

Brief Description of Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] The mode for carrying out the present invention will be described. Note that each description and embodiment disclosed in the present invention is applicable to other descriptions and embodiments as well. That is, any combination of various elements disclosed in the present invention is included in the present invention. Also, the present invention is not limited to the following specific descriptions.

[0012] Moreover, those having ordinary knowledge in the relevant technical field will be able to recognize and confirm many equivalents of the specific aspects of the present invention described in this application using only ordinary experiments. Furthermore, it is intended that such equivalents are also included in the present invention.

[0013] One aspect of realizing the present invention is to provide a pharmaceutical composition for preventing or treating liver diseases containing exosomes. Specifically, the exosomes mean exosomes containing super-repressor-IκB (srIκB). Super-repressor-IκB (srIκB) is an active ingredient contained in the exosomes of the present invention that brings about a preventive or therapeutic effect on liver diseases and is mixed with cargo proteins.

[0014] The exosomes may contain super-repressor-IκB (srIκB).

[0015] The "super-repressor-IκB (srIκB)" in the present invention refers to a protein that is not phosphorylated by IκB kinase (IKK) and is not degraded by a proteolytic enzyme complex. Since the srIκB is an S32A, S36A mutant form of the IκB protein (IκBα), in the present invention, the IκB protein or a fragment thereof is also included. The super-repressor-IκB (srIκB) is an amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 95% sequence homology with them, or a fragment thereof, but is not limited to the above examples.

[0016] Hereinafter, srIκB is also referred to as a cargo protein.

[0017] The "Exosome" in the present invention refers to a vehicle capable of carrying a cargo protein, and the cargo protein can be loaded by various known methods. As an example of a known method for loading a cargo protein, a method of overexpressing a cargo protein in exosome-producing cells and loading it into exosomes, a method of overexpressing a fusion protein in exosome-producing cells using a vector in which an exosome-specific marker and a cargo protein are fused to enhance the loading efficiency and loading it into exosomes, a method of loading a cargo protein into exosomes by a photodynamically reversible protein-protein interaction, etc. can be mentioned, but it is not limited thereto.

[0018] Specifically, regarding the method for producing exosomes containing the super-repressor-IkB (srIκB) protein of the present invention, in order to provide a composition and method for producing the exosomes of the present invention, the entire contents of Patent Documents 2 and 3 are incorporated herein by reference. The "exosomes containing a super-repressor" in the present invention are used interchangeably with "Exo-SrIκB".

[0019] The exosome has a diameter of about 50 nm to about 200 nm, specifically, a diameter of about 50 nm to about 150 nm, but is not limited thereto.

[0020] The "liver disease" in the present invention means a disease occurring in the liver, specifically, liver fibrosis, liver cirrhosis, fatty liver, alcoholic liver disease, cholestasis or a combination thereof, but is not limited to the above diseases as long as there are abnormalities in the liver tissue and function.

[0021] The "liver fibrosis" in the present invention is the result of the injury recovery process in response to repeated liver injuries. Different from liver cirrhosis, it is reversible, consists of thin fibrils, and is known to have no nodule formation. It recovers normally when the cause of liver injury disappears, but if such a liver fibrosis process continues repeatedly, it progresses to liver cirrhosis.

[0022] The "liver cirrhosis" in the present invention is a chronic disease that develops by repeating the regeneration of hepatocytes and the increase of fibrous tissue, and is pathologically accompanied by necrosis and fibrosis. Ultimately, it progresses to liver cirrhosis complications such as liver failure and diseases such as liver cancer, leading to death. In particular, since there are no subjective symptoms in the early stage and it is discovered after progression, treatment is very difficult.

[0023] The "fatty liver" in the present invention is a disease in a state where fat accumulates in hepatocytes, specifically, alcoholic fatty liver caused by excessive drinking, and non-alcoholic fatty liver caused by obesity, diabetes, dyslipidemia, and metabolic syndrome.

[0024] The "alcoholic liver disease" in the present invention is a liver disease caused by excessive alcohol consumption. Specifically, it includes various disease groups such as alcoholic fatty liver, alcoholic steatohepatitis, alcoholic cirrhosis, and alcoholic liver fibrosis, but is not limited thereto.

[0025] Specifically, the "alcoholic liver disease" means various liver disease groups caused by chronic alcohol intake. As the first stage of alcoholic liver disease, alcoholic fatty liver appears in which fat accumulates in hepatocytes due to continuous alcohol intake. As the disease progresses, excessive fat accumulation, necrosis of hepatocytes, and acute inflammatory reactions appear, and this stage is called alcoholic hepatitis. Subsequently, as the final stage, it progresses to alcoholic cirrhosis in which collagen accumulates in liver tissue and liver fibrosis appears. Therefore, regarding such alcoholic liver diseases, it is rare for the above stages to progress in sequence or to appear in the form of a single disease. Most often, the progression stages overlap and appear in the form of a complex disease group.

[0026] The "cholestasis" in the present invention means a disease in which bile stagnates in the liver due to autoimmune diseases or biliary atresia. Cholestasis is broadly classified into extrahepatic cholestasis (such as biliary compression, biliary atresia, biliary stricture, Caroli's disease, etc.) and intrahepatic cholestasis. Cholestasis is often used interchangeably with cholestasis liver disease. The cholestasis liver disease includes, in descending order of frequency, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), Alagille syndrome (AS), etc.

[0027] The composition of the present invention shows (a) a decrease in the relative weight of the liver, (b) a decrease in jaundice, (c) a decrease in the number of liver abscesses, (d) a decrease in liver fibrosis, (e) a decrease in the T-BIL value, (f) an increase in the HDL value, (g) a decrease in the quantitative change of a-SMA in hepatic stellate cells, (h) a decrease in the serum ALT / AST value, and / or (i) a decrease in alcoholic fatty liver, and can prevent or treat liver diseases, but is not limited thereto.

[0028] The exosome of the present invention or a pharmaceutical composition containing the same may further contain a pharmaceutically acceptable excipient. In the case of oral administration, binders, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. can be used as the excipient. In the case of injections, buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used. In the case of topical administration, bases, lubricants, preservatives, etc. can be used, but are not particularly limited thereto.

[0029] The dosage form of the composition of the present invention can be manufactured in various forms by mixing with pharmaceutically acceptable excipients as described above. For example, it may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories. The composition of the present invention may be an aqueous solution or suspension of physiologically acceptable exosomes. In the case of injections, it can be manufactured in disposable ampoules or multiple-dose forms.

[0030] In addition, the composition is formulated into a single-dose preparation suitable for administration into a patient's body by a conventional method in the pharmaceutical field, and can be administered orally or by a parenteral administration route including skin, intravenous (IV), intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intraluminal of the digestive tract, topical, sublingual, intravaginal, or rectal routes using the administration methods commonly used in the art, but is not limited thereto.

[0031] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of drug that is the active ingredient, along with various relevant factors such as the disease to be treated, the route of administration, the age, sex, and weight of the patient, and the severity of the disease. Specifically, the composition of the present invention contains the exosomes in a pharmaceutically effective amount, but is not limited thereto.

[0032] Containing the exosomes in a pharmaceutically effective amount means containing them to such an extent that the intended pharmacological activity (for example, prevention, improvement, or treatment of liver disease) can be obtained by the exosomes of the present invention, and also means containing them at a level where there is no toxicity or side effect to the administered individual, or at a slight level that is pharmaceutically acceptable, but is not limited thereto. Such a pharmaceutically effective amount is determined by comprehensively considering the number of administrations, the patient, the dosage form, etc.

[0033] The pharmaceutical composition of the present invention contains the above components (active ingredients) in an amount of 0.01 to 99% by weight per volume, but is not particularly limited thereto.

[0034] The total effective amount of the composition of the present invention may be administered to the patient as a single dose or as multiple doses. The content of the active ingredient in the pharmaceutical composition of the present invention may be changed according to the degree of the disease. Specifically, the dosage of the exosomes of the present invention may be about 1×10 6 ~1×10 15 pn per kg of body weight per day. However, the dosage of the exosomes is determined by considering various factors such as the route of administration and the number of treatment sessions of the pharmaceutical composition, as well as the age, weight, health status, sex, severity of the disease, diet, excretion rate, etc. of the patient. Considering these, a person with ordinary knowledge in the art will be able to determine an appropriate effective dosage according to the specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its dosage form, route of administration, and method of administration as long as it exhibits the effects of the present invention.

[0035] Another aspect for implementing the present invention provides a method for preventing or treating liver diseases, which includes exosomes containing a super-repressor (SR)-IκB (srIκB) protein.

[0036] The following production examples are for more specifically explaining the present invention, and the present invention is not limited to these production examples.

[0037] Production Example 1. Preparation of exosomes containing a target protein (Exo-SrIκB) Exosomes containing super-repressor (SR)-IκB (srIκB) as a target protein were prepared by the method disclosed in Patent Document 4.

[0038] Production Example 2. Preparation of control group exosomes (Exo-naive) Expi293F cells (Thermofisher) were cultured under light irradiation conditions for 4 days in the same manner as in Production Example 1, and then the culture medium was harvested and purified to obtain Exo-naive.

[0039] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for more specifically explaining the present invention, and the present invention is not limited to these examples.

[0040] [Example 1] Evaluation of efficacy in a biliary stricture-induced liver cirrhosis mini-pig model (Exo-SrIκB loaded with SrIκB) 1-1. Preparation of a biliary stricture-induced animal model In order to confirm that the exosomes containing the super-repressor-IκB (srIκB) of the present invention can prevent or treat liver diseases, animal models of liver diseases induced by various causes were prepared and used for confirmation.

[0041] First, a biliary stricture-induced animal model was established, and the efficacy of exosomes containing super-repressor-IκB (Exo-SrIκB) was confirmed.

[0042] Specifically, a biliary stricture mini-pig model was established by performing Endobiliary radiofrequency ablation (EB-RFA) guided Endoscopic etrograde cholangiogram (ERC) on mini-pigs using an RFA catheter with a temperature sensor function (ELRA RF catheter; STARmed). The site where the inserted RFA electrode abutted the bile duct wall was designated as EB-RFA, and it was performed using the device at Radiofrequency energy: 80°C, 7W, for 90 seconds (Table 1).

[0043]

Table 1

[0044] Three weeks after RFA induction, a stent (Nexent biliary stent, total length: 4 cm) was inserted into EB-RFA to open the occluded bile duct, and the animals were dissected five weeks after RFA induction.

[0045] A test substance (vehicle-buffer, Exo-SrIκB) was intravenously administered (Intravenous, i.v.) for 1 hour using an infusion pump four times at 1, 2, 3, and 4 weeks after RFA induction. The individual dose was calculated based on the body weight immediately before administration and converted to a concentration of 1.6e+10 pn / kg / 1 mL.

[0046] Six times in total, blood was collected before administration of the test substance (vehicle-buffer, Exo-SrIκB) at 0 hours (before induction), 1, 2, 3, 4, and 5 weeks after RFA induction.

[0047] The method for establishing the above-described specific animal model is shown in Figure 1.

[0048] 1-2. Confirmation of the relative weight of the liver and liver tissue findings after autopsy and histopathological examination After the test, the animals were anesthetized, blood was collected from the vein, then exsanguinated, and then the liver tissue was excised individually. The weight of the excised liver tissue was measured and recorded in a photograph (Appendix 9.8). Then, the excised liver tissue was fixed with 10% buffered neutral formalin solution. The fixed tissue was trimmed to a certain thickness, and then through a general tissue processing process, it was paraffin-embedded to prepare tissue sections of 4-5 μm, and then H&E (Hematoxylin & Eosin) staining, a staining method, was performed.

[0049] Next, the relative weight (%) of the liver was measured and the average was calculated. The results are shown in Figure 2.

[0050] As a result, as shown in Figure 2, G1 (vehicle control group) was 3.5, G2 (Exo-SrIκB administration group) was 2.9, and G3 (normal control group) was 1.8. It was confirmed that G1 (vehicle control group) increased to about twice that of G3 (normal control group), and it was confirmed that G2 (Exo-SrIκB administration group) decreased by 17% compared to G1 (vehicle control group) (b in Figure 2).

[0051] In addition, as a result of confirming liver abscess as a clinical symptom, as shown in Figure 3, liver abscess was observed in the vehicle control group, but not in the Exo-SrIκB administration group of the present invention.

[0052] Furthermore, as a result of confirming jaundice symptoms as a clinical symptom, as shown in Figure 4, jaundice symptoms were observed in the gums and sclera of the eyes in the vehicle control group, but not in the Exo-SrIκB administration group of the present invention.

[0053] As a result of observing the subcutaneous cross-section after laparotomy on the day of autopsy, jaundice symptoms that were not observed during the observation period were observed in the excipient control group, and were additionally confirmed in the Exo-SrIκB administration group. It was confirmed that they were slightly observed in the Exo-SrIκB administration group compared to the excipient control group.

[0054] Next, based on the findings from observing the liver tissue, scoring and evaluation were performed by dividing into four items: Piecemeal necrosis, Lobular necrosis, Histological activity score, and Fibrosis. The results are shown in Figure 5.

[0055] As shown in Figure 5, as a result of calculating the scores observed in the liver tissue by the average, in G1 (excipient control group), Piecemeal necrosis was 2.8, Lobular necrosis was 2.0, Histological activity score was 3.0, Fibrosis was 3.8, and the total score was 11.5.

[0056] In contrast, in G2 (Exo-SrIκB administration group) of the present invention, Piecemeal necrosis was 1.5, Lobular necrosis was 1.5, Histological activity score was 2.0, Fibrosis was 2.5, and the total score was 7.5.

[0057] Therefore, in the G2 (Exo-SrIκB administration group) of the present invention, compared with the G1 (vehicle control group), the reduction was 46% in piecemeal necrosis, 25% in lobular necrosis, 33% in histological activity score, and 34% in fibrosis, respectively, and a 35% reduction was confirmed in the total score.

[0058] 1-3. Confirmation of collagen fiber distribution To confirm the distribution of collagen fibers, MT (Masson's Trichrome) staining that stains only collagen fibers was performed. All tissue slides of each individual were photographed, and the stained area with respect to the total area was measured and the average value was calculated.

[0059] The fibrotic part of the liver tissue was stained and then measured (%), and calculated by the mean and standard deviation. The results are shown in Fig. 6.

[0060] As a result, as shown in Fig. 6, G1 (vehicle control group) was 8.1, G2 (Exo-SrIκB administration group) was 5.0, and G3 (normal control group - Sham) was 3.5. It was confirmed that the G2 (Exo-SrIκB administration group) of the present invention was reduced by 38.3% compared with the G1 (vehicle control group).

[0061] 1-4. Confirmation of blood biochemical examination The blood obtained in Example 1-1 was centrifuged at 3000 rpm, 4 °C for 10 minutes, and the upper layer of serum was collected. Then, 11 items including T-BIL (Total bilirubin) and HDL-C (High density lipoprotein cholesterol) were measured using an automatic blood biochemical analyzer (7180, Hitachi, Japan).

[0062] Blood biochemical examinations were performed separately before the RFA surgery, before the administration of the test substance, and on the autopsy day. The results calculated by the mean and standard deviation of the blood analysis on the autopsy day are shown in Fig. 7.

[0063] As shown in Fig. 7, in G1 (vehicle control group), it was confirmed that T-BIL was 2.09 mg / dL and HDL-C was 27.7 mg / dL. In G2 (Exo-SrIκB administration group), it was confirmed that T-BIL was 0.87 mg / dL and HDL-C was 37.4 mg / dL.

[0064] In most of the measured items, there was no difference between G1 (vehicle control group) and G2 (Exo-SrIκB administration group) of the present invention. However, in terms of T-BIL concentration, G2 (Exo-SrIκB administration group) decreased to 1 / 2.4 of G1 (vehicle control group), and in terms of HDL value, it was confirmed that it increased 1.4 times.

[0065] In short, in the animal model of biliary stricture-induced cirrhosis, the composition containing exosomes (Exo-SrIκB) containing the super-repressor (SR)-IκB (srIκB) of the present invention reduces the relative weight (%) of the liver compared to the vehicle control group, reduces liver abscesses, and also effectively reduces liver fibrosis in MT staining for confirming liver tissue fibrosis. As a result of blood biochemical tests, it was confirmed that while significantly reducing the T-BIL value, it exerts the effect of increasing HDL. From these results, it is confirmed that Exo-SrIκB of the present invention effectively treats fatty liver, cholestasis, liver fibrosis, and cirrhosis, and further exerts excellent liver protection and liver function improvement effects.

[0066] [Example 2] Evaluation of the efficacy of Exo-SrIκB using a TAA-induced liver fibrosis animal model 2-1. Preparation of animal model Next, an animal model of liver fibrosis induced by TAA (Thioacetamide (172502-500G, SIGMA)) was prepared to evaluate the efficacy of Exo-SrIκB of the present invention.

[0067] Specifically, a mouse model of liver fibrosis was established by intraperitoneally injecting C57BL / 6 male mice (9 weeks old) with 200 mg / kg of TAA three times a week for 8 weeks.

[0068] In a preliminary experiment, it was confirmed that the increase in blood biochemistry biomarkers and histopathological fibrosis began 2 weeks after the administration of 200 mg / kg of TAA. Therefore, the administration time of Exo-SrIκB was determined to be 2 weeks after TAA administration. The administration of Exo-SrIκB was performed by intravenous bolus injection three times a week for 6 weeks. On the same administration day as TAA, TAA was administered in the morning and Exo-SrIκB was administered in the afternoon. Subsequently, the body weight of the animals was measured for 8 weeks, and the dosage was administered according to the body weight. Exo-SrIκB was administered at a dosage of 4E+11 pn / kg.

[0069] When administering 200 mg / kg of TAA, the mortality rate of animals within 2 weeks reaches 20-30%. Therefore, the groups were formed around 1.5 weeks after TAA administration, which was before the administration of Exo-SrIκB. The groups were divided into a sham control group, a negative control group administered only 200 mg / kg of TAA, and a group administered Exo-SrIκB, with 9 animals selected for each group. Among these, 4 animals from each group were used for the ICG (indocyanine green) test.

[0070] Finally, after 8 weeks, autopsy was performed, whole blood was collected, and serum was separated. Subsequently, the livers of the mice were excised to separate proteins, and a part was fixed for histopathological experiments. Four animals from each group for the ICG test were tested the next day. 5 mg / kg of ICG was intravenously administered, and blood was collected 15 minutes later to measure the ICG concentration present in the blood.

[0071] The preparation of the animal model is shown in Figure 8.

[0072] 2-2. Analysis results of the ICG test More than 90% of ICG is taken up by hepatocytes and excreted only from the liver. The retention rate after ICG administration is used as an index for liver function evaluation.

[0073] Specifically, 5 mg / kg of ICG was administered to animals, and blood was collected 15 minutes later to evaluate the ICG concentration. The results are shown in Figure 9.

[0074] As a result, as shown in Figure 9, an increase in the ICG concentration was confirmed in the blood of the negative control group compared to the mock control group. In contrast, in the Exo-SrIκB administration group of the present invention, it was confirmed that the ICG concentration significantly decreased by about 52.9% compared to the negative control group.

[0075] 2-3. Analysis Results of Blood Biochemistry Biomarkers The blood concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as markers of liver injury. The results are shown in Figure 9.

[0076] Note that AST and ALT are aminotransferases present in the liver, are involved in gluconeogenesis, and are released into the blood when the liver is damaged, so they are enzymes used for the diagnosis of liver diseases.

[0077] As a result, as shown in Figure 10, AST and ALT increased in the negative control group compared to the mock control group, while it was confirmed that they were suppressed in the Exo-SrIκB administration group of the present invention.

[0078] 2-4. Analysis Results of Fibrosis Markers Proteins were isolated from the livers of mice, and changes in a-SMA (a-smooth muscle actin), a fibrosis marker, were measured by Western blotting. The amount of protein loaded in the experiment was normalized by the amount of a-tubulin and analyzed. The results are shown in Figure 11.

[0079] As shown in Figure 11, compared with the sham control group, the amount of a-SMA in the liver tissue of the negative control group increased, while the amount of a-SMA in the Exo-SrIκB administration group of the present invention decreased.

[0080] 2-5. Analysis results of hepatocyte necrosis Fixed liver tissue was stained with hematoxylin and eosin (H&E staining) to evaluate the degree of hepatocyte necrosis, and scoring was performed by a histopathology expert. The results are shown in Figure 12.

[0081] As shown in Figure 12, compared with the sham control group, hepatocyte necrosis in the negative control group increased significantly, while it decreased in the Exo-SrIκB administration group of the present invention.

[0082] In summary, in a liver fibrosis animal model prepared by intraperitoneal administration of TAA, which causes liver injury, the blood concentrations of AST and ALT related to liver injury were significantly decreased in the group administered with Exo-SrIκB of the present invention. In addition, ICG, which is excreted only from the liver, was intravenously administered, and then the blood concentration was measured. As a result, in the Exo-SrIκB administration group of the present invention, it was confirmed that the ICG concentration in the blood significantly decreased compared with the negative control group.

[0083] Furthermore, as a result of analyzing the amount of a-SMA, it was confirmed that compared with the sham control group, a-SMA increased in the negative control group, while it decreased in the group administered with Exo-SrIκB. As a result of scoring the quantitative change of necrotic cells, it was confirmed that the group administered with Exo-SrIκB exerted an effect of decreasing compared with the negative control group.

[0084] From these results, it can be seen that Exo-SrIκB can be effectively used for the treatment of hepatic fibrosis, liver cirrhosis, and fatty liver by suppressing hepatocyte necrosis and liver fibrosis.

[0085] [Example 3] Evaluation of the efficacy of Exo-SrIκB against ALD (Alcoholic liver disease) 3-1. Preparation of animal model In order to confirm the therapeutic efficacy of Exo-SrIκB of the present invention in an acute / chronic alcohol-induced liver injury mouse model (Ethanol-induced Steatohepatitis, NIAAA model), an animal model was prepared as follows.

[0086] Specifically, 8- to 10-week-old C57BL / 6JWT male mice approved by the Animal Experiment Performance Management Committee of the Korea Advanced Institute of Science and Technology (KAIST) were used. The mice were fed a liquid diet with 4.5% EtOH for 10 days.

[0087] For the 3-day continuous injection experiment, the mice were randomly divided into Exo-Naive (10 9 particles / day / mouse), low-dose Exo-srIκB (10 8 particles / day / mouse), or high-dose Exo-srIκB (10 9 particles / day / mouse) groups. The exosomes at the indicated doses were intravenously injected every 24 hours for 3 consecutive days. Six hours after the last injection, acute EtOH (4 g kg -1 of 40% EtOH gavage) was administered to the mice, and the mice were sacrificed 6 hours later.

[0088] Hepatocytes (HEP), hepatic stellate cells (HSC), Kupffer cells (KC), and liver mononuclear cells (MNC) were isolated from C57BL / 6J WT male mice.

[0089] 3-2. Identification of the major target cell types of Exo-srIκB To identify the major target cell types of Exo-srIκB, the transcriptional profiles of various hepatocytes were examined using scRNA-seq analysis.

[0090] Specifically, single-cell RNA sequencing (scRNA-seq) analysis of liver tissues from normal mice was performed by searching the Tabula Muris Senis website (Non-Patent Document 1) or by searching the NCBI Gene Expression Omnibus (accession number: GSE132042).

[0091] KEGG (Kyoto encyclopedia of genes and genomes) pathway and Gene Ontology analyses were performed using DAVID (Database for Annotation, Visualization and Integrated Discovery) (Non-Patent Document 2). For the scRNA-seq analysis of human liver specimens, accession number: GSE136103 was used in the NCBI Gene Expression Omnibus.

[0092] As a result of analyzing the scRNA-seq of normal mouse liver, a total of nine clusters of Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC), ductal epithelial cells, hepatic stellate cells (HSC), hepatocytes (HEPs), myeloid cells, NK cells, B cells, and dendritic cells (DCs) were identified (a in Figure 14).

[0093] In addition, it was confirmed that the expression levels of genes related to the exosome uptake process, such as clathrin-dependent endocytosis (Ap2a2 and Picalm), caveolin-mediated endocytosis (Cav2 and Pascin2), and vesicle fusion (Stx3 and Snap23), were high in LSECs or KCs (b in Figure 14). Since KCs are anatomically located on LSECs, it is judged appropriate that KCExo-srIκB is delivered to KCs after intravenous injection.

[0094] 3-3.Confirmation of increased expression levels of genes related to the Exosome Uptake Process Next, to confirm whether the inflammatory activation of KCs affects the expression levels of genes related to the exosome uptake process, bulk RNA-seq analysis of KCs was performed. Bulk RNA-seq of primary KCs from mice treated with vehicle or LPS used accession number: GSE86397 in the NCBI Gene Expression Omnibus.

[0095] As a result, an increase in the expression levels of genes related to the clathrin-dependent or caveolin-mediated endocytosis pathway was confirmed. In addition, an increase in the gene expression levels of the inflammatory response and NF-kB signaling pathway was confirmed in KCs treated with LPS (lipopolysaccharide) compared to KCs treated with vehicle (c-e in Figure 14). In addition, in the scRNA-seq analysis of MARCO+ human KCs isolated from cirrhotic patients, it was confirmed that the expression levels of genes related to clathrin-dependent endocytosis, caveolin-mediated endocytosis, and positive regulation of NF-κB transcription factor activity were upregulated compared to the control group (f, g in Figure 14).

[0096] From these results, it can be seen that Exo-srIκB is effectively delivered to activated KCs and suppresses NF-κB-mediated inflammation in ALD.

[0097] 3-4.Confirmation of suppression of inflammatory gene expression in mice treated with LPS in vitro The efficient delivery of Exo-srIκB to KCs and its inhibitory effect on LPS-induced inflammatory gene expression were confirmed. Delivery into KCs was confirmed by DiI labeling of Exo-srIκB. Specifically, isolated and purified Exo-srIκB (1.0×10 12 particles) was cultured with 10 μL of DiI for 30 minutes at 37°C in the dark. Then, up to 4 mL of 1× filtered phosphate-buffered saline was added and transferred to an Amicon® Ultra-4 centrifugal filter device (Merck, Rahway, NJ, USA). The solution was eluted by centrifugation at 3200×g for 15 minutes at 4°C until the residual volume was approximately 200 μL. First, to examine the delivery of Exo-SrIκB into KCs, mouse KCs were treated with DiI-labeled Exo-SrIκB at each time (1 hour or 3 hours) and each dose (KC:Exo-SrIκB = 1:1000 or 1:10,000). After 1 hour, it was not confirmed that Exo-SrIκB was loaded onto KCs in vivo, but after 3-hour treatment, it was confirmed that Exo-SrIκB was delivered to approximately 8% of KCs at the low dose (KC:Exo-SrIκB = 1:1000) and approximately 70% of KCs at the high dose (KC:Exo-SrIκB = 1:10,000) (a-c in Figure 15).

[0098] To confirm the role of Exo-SrIκB in suppressing the LPS-induced inflammatory response in KCs, mouse KCs were pretreated with Exo-Naive or Exo-SrIκB and then treated with LPS. qRT-PCR was performed by the following method.

[0099] As a result of qRT-PCR analysis, it was confirmed that the expression levels of inflammatory genes (Il1b, Tnf, Il6, and Ccl2) increased by LPS were normalized by Exo-SrIκB treatment (d in Figure 15). In addition, it was confirmed that LPS treatment significantly increased the uptake of Exo-srIκB by KC (a - c in Figure 15).

[0100] From these results, it was confirmed that Exo-SrIκB was effectively taken up by KC cells activated by inflammation.

[0101] 3 - 5.Confirmation of the therapeutic effects of Exo-SrIκB on alcoholic fatty liver (AFL) and alcoholic liver injury (ALI) Based on the results confirmed in the above-mentioned experiments, the purpose was to confirm the therapeutic effect of Exo-SrIκB in acute / chronic alcoholic liver disease experimental models. EtOH was supplied for 10 days, and then the mice were randomly divided into two groups and injected intravenously with Exo-Naive or Exo-SrIκB (a in Figure 16). For histological analysis, similar regions of the left and middle lobes of the mouse liver were used for histological analysis. The liver tissue was fixed overnight at room temperature with 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MO, USA). After removing paraffin and rehydrating, the paraffin-embedded tissue was sliced to a thickness of 4 μm and stained with H&E (Hematoxylin & Eosin), a staining method.

[0102] As a result, by confirming the measurement of intrahepatic TG, a significant decrease in alcoholic fatty liver (AFL) was confirmed in Exo-srIκB-treated mice compared to Exo-Naive-treated mice (b, c in Figure 16).

[0103] For biochemical analysis, liver TG (triglyceride) was measured. Specifically, liver lipids were extracted from approximately 20 - 30 mg of frozen liver tissue using a chloroform / methanol (2:1 ratio) solution. Subsequently, the freeze-dried liver lipids were resuspended in 5% bovine serum albumin (BSA) saline. Using a VetTest chemical analyzer (IDEXX Laboratories, Westbrook, ME, USA), the serum levels of liver TG, alanine aminotransferase (ALT), aspartate aminotransferase (AST), TG, and total cholesterol (TC) were measured.

[0104] As a result, a decrease in the blood concentrations of ALT / AST was observed with Exo-SrIκB, and there were no differences in blood TG and TC concentrations and body weight (d, i in Figure 16).

[0105] Next, to confirm the delivery of Exo-srIκB into the liver, the NF-kB level in the nuclear fraction of the whole liver tissue was measured by Western blot. The nuclear protein level was analyzed by normalizing it to the expression level of lamin B1 for each sample. Densitometry analysis was performed using ImageJ (National Institute of Health, Bethesda, MD, USA).

[0106] As a result, a significant decrease in NF-kB nuclear translocation was confirmed in the mice treated with Exo-SrIκB (e in Figure 16). Also, in the mice treated with Exo-SrIκB, compared to the control group mice, the expression levels of CXCL1 and CCL2 in the liver were suppressed, and the number of liver mononuclear cells (MNC) per liver tissue decreased (f, g in Figure 16).

[0107] Furthermore, flow cytometry was performed to confirm whether the number of infiltrating macrophages decreased. As a result, a decrease in F4 / 80+CD11b+ infiltrating macrophages was observed in the Exo-SrIκB treatment group, and in particular, a significant decrease in F4 / 80+Ly6Chi pro-inflammatory macrophages was confirmed. In contrast, the frequency of F4 / 80+Ly6Clow anti-inflammatory macrophages was confirmed to increase in the Exo-SrIκB treatment group. However, in Ly6G+CD11b+ neutrophils or lymphocytes, it was confirmed that the two groups showed similar frequencies (h in Fig. 16).

[0108] 3-6. Mitigating effects of continuous administration of Exo-srIκB on alcoholic liver injury (ALI), alcoholic fatty liver (AFL), and alcoholic fibrosis (ALF) in mice The purpose was to confirm the therapeutic and preventive effects of various Exo-srIκB on alcohol-associated liver injury (ALI).

[0109] Specifically, WT mice were supplied with EtOH for 9 days (4 g kg -1 of 40% EtOH gavage), and before alcohol administration, they were randomly divided into three groups and administered Exo-Naive (10 9 particles / day / mouse), low-dose Exo-srIκB (10 8 particles / day / mouse), or high-dose Exo-srIκB (10 9 particles / day / mouse) continuously for 3 days (a in Fig. 17).

[0110] As a result, compared with control group mice having similar body weights and serum TG and TC levels, it was confirmed that all serum ALT / AST values significantly decreased in low-dose and high-dose Exo-srIκB injection mice (b in Fig. 17 and g - i in Fig. 17).

[0111] In addition, to confirm the total fat content, as a result of performing H&E staining and Oil Red O staining, it was confirmed that lipid accumulation in the intermediate region HEP (apoptotic hepatocytes) was significantly suppressed by Exo-srIκB treatment (c, d in Figure 17).

[0112] To confirm whether cell death of HEP and non-parenchymal cells in the liver tissue decreased, the Terminal deoxynucleotidyl TUNEL (transferase dUTP nick end labeling, Abcam, Cambridge, UK) assay was performed according to the manufacturer's instructions, images were captured using an Olympus BX51 microscope equipped with a CCD camera (Olympus, Tokyo, Japan), and analyzed with DP2-BSW.

[0113] As a result, it was confirmed that treatment with Exo-srIκB decreased cell death of HEP and non-parenchymal cells in the intermediate region of Exo-Naive-treated mice (c, d in Figure 17).

[0114] Finally, the expression levels of genes (ACTA2, COL1A1, and TAGLN) related to fibrotic activation in isolated hepatic stellate cells (HSC) were significantly decreased in the low-dose Exo-srIκB group compared to the control group, and it was also confirmed that they decreased in the high-dose Exo-srIκB group (f in Figure 17).

[0115] 3-7. Mitigating effect of alcoholic hepatitis by suppressing KC activity To explore the mechanism of the protective effect of Exo-SrIκB in ARLD, KC, the main target cell of Exo-SrIκB, was analyzed from the Exo-SrIκB continuous administration animal models of Examples 3-6. Based on known studies confirming that in the progression of non-alcoholic steatohepatitis, germ-derived KC (emKC) gradually disappears and is ultimately replaced by bone marrow-derived macrophages (bmKC), the aim was to analyze KC (Non-Patent Documents 3, 4). In particular, CLEC2 and TIM4 were used as markers for emKC (CLEC2+TIM4+), and they were distinguished from bmKC (CLEC2+TIM4-), and flow cytometry was performed.

[0116] As a result, there was no difference in frequency among the three groups of F4 / 80hiCD11b + KC, emKC (CLEC2+TIM4+), and bmKC (CLEC2+TIM4-) (a, b in Figure 18).

[0117] Next, for histological analysis, similar regions of the left and middle lobes of the mouse liver were used for histological analysis. As a result, in the immunostaining of liver tissue, a similar number of CLEC4F (a marker for emKC and bmKC) expressing cells were also confirmed (c in Figure 18). However, in the liver tissue of Exo-Naive-treated mice, there were morphological changes in central CLEC4F+KC, and it was confirmed that these changes disappeared with Exo-SrIκB treatment, so functional changes in KC by Exo-SrIκB were confirmed (c in Figure 18).

[0118] Also, a decrease in nuclear translocation of NF-κB and a decrease in the expression levels of pro-inflammatory genes (TNF, IL1b, and Il6) were confirmed in KC or the whole liver tissue isolated from Exo-srIκB-treated mice compared to control group mice (d, e in Figure 18), and a decrease in IL-6 level was also confirmed in the serum of Exo-srIκB-treated mice (f in Figure 18). Furthermore, in isolated KC and HEP from Exo-srIκB-treated mice, the expression levels of CXCL1 and CCL2 decreased compared to control group mice (e in Figure 18 and a in Figure 19).

[0119] Indeed, in Exo-SrIκB-treated mice, the number of MPO+ neutrophils was significantly decreased in non-damaged liver tissues, particularly in the central part (g in Figure 18). Also, FSChi granulocytes such as Ly6G+CD11b+ neutrophils were at the same frequency in all groups, but compared with control group mice, in Exo-srIκB-treated mice, it was confirmed that the frequency of F4 / 80+CD11b+ macrophages, particularly F4 / 80+Ly6Chi pro-inflammatory macrophages, decreased in the liver, and the frequency of F4 / 80+Ly6Clow anti-inflammatory macrophages increased (h in Figure 18 and d in Figure 19).

[0120] From these results, it was confirmed that continuous administration of Exo-srIκB suppresses inflammatory KCs in ALD and exerts a hepatoprotective effect.

[0121] In summary, when the Exo-SrIκB of the present invention is administered, it effectively suppresses fat accumulation, cell death, acute inflammation, and liver fibrosis in liver tissues, so it was confirmed that it can be provided as an effective therapeutic agent for alcoholic liver disease in which these symptoms appear complexly.

[0122] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be construed as including all modifications or variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

**Claim 1** A pharmaceutical composition for preventing or treating liver diseases, comprising exosomes containing super-repressor-IκB (srIκB) as an active ingredient. **Claim 2** The pharmaceutical composition according to claim 1, wherein the liver disease is liver fibrosis, liver cirrhosis, fatty liver, alcoholic liver disease, cholestasis or a combination thereof. **Claim 3** The pharmaceutical composition according to claim 2, wherein the alcoholic liver disease is alcoholic fatty liver, alcoholic steatohepatitis, alcoholic liver cirrhosis, alcoholic liver fibrosis or a combination thereof. **Claim 4** The composition according to claim 1, wherein the composition is administered orally, transdermally, intraperitoneally, intravenously (IV), intramuscularly, subcutaneously or in combination thereof. **Claim 5** The dosage of the exosome is 1×10 ６ pn / kg to 1×10 １５ pn / kg, and the composition according to claim 1.

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