WO2018159900A1 - Srebp-1 decoy oligodeoxynucleotide and pharmaceutical composition for preventing or treating fatty liver disease containing same as active ingredient - Google Patents

Abstract

The present invention relates to SREBP-1 decoy oligodeoxynucleotide and a pharmaceutical composition for preventing or treating fatty liver disease containing the same as an active ingredient. More particularly, the SREBP-1 decoy oligonucleotide according to the present invention controls the expression of genes related to lipid biosynthesis by inhibiting the transcription factor activity of SREBP-1, which is a transcription factor, thereby suppressing the accumulation of fat in the liver when a fatty liver-induced animal model is treated with the SREBP-1 decoy oligodeoxynucleotide. Moreover, the SREBP-1 decoy oligonucleotide exhibits the effects of reducing blood alanine reducing, aspartate transaminase, total cholesterol, and neutral fat indices, as well as transaminase liver and blood inflammation indices. Due to such effects, the SREBP-1 decoy oligonucleotide can be used as a pharmaceutical composition for preventing or treating fatty liver disease.

Description

SRPEP-1 decoy oligodioxynucleotide and pharmaceutical composition for the prevention or treatment of fatty liver disease containing the same as an active ingredient

The present invention relates to a pharmaceutical composition for the prevention or treatment of SREBP-1 decoy oligodioxynucleotide and fatty liver disease containing the same as an active ingredient, and more particularly, to inhibit the transcription factor action of the transcription factor SREBP-1 Prevention of fatty liver disease containing SREBP-1 decoy oligodioxynucleotide and its active ingredient, which suppresses the accumulation of fat in liver tissue and reduces the inflammatory response by controlling the expression of genes related to biosynthesis Or to a pharmaceutical composition for treatment.

Gene therapy is a very effective method for treating and preventing various diseases by specifically inhibiting expression of specific genes by regulating the gene expression system and delivering it to a disease site. In particular, the method of administering decoy oligodeoxynucleotide (decoy ODN) to transcription factors in gene therapy has recently been attempted as a tool for reducing the expression of specific genes by inhibiting the action of transcription factors. Theoretically, a strategy to inhibit the activity of these transcription factors would be to inject decoy ODN into the cell, which binds to the transcription factor of interest in the cell. That is, since the DNA binding site of the transcription factor is bound by the decoy, the transcription factor cannot bind to the promoter of the target gene, and the activity of the transcription factor is interrupted, thereby reducing the expression of a specific gene.

Fatty liver refers to a state in which fat is accumulated in liver cells, and in the normal liver, fat accounts for about 5%, and more fat is accumulated in this state. Fatty liver deteriorates and the mass of fat in the hepatocytes is pushed to important components of the cells, including the nucleus, to one side, reducing the function of the hepatocytes. This leads to impaired circulation of blood and lymph in the liver. When this happens, hepatocytes cannot receive oxygen and nutrients properly, and liver function decreases.

Fatty liver can be divided into alcoholic fatty liver and non-alcoholic fatty liver. Alcoholic fatty liver is caused by alcohol. The more alcohol you drink, the more likely it is, and if you continue to consume it, your liver's ability to metabolize alcohol will be worse. It also occurs well in poor nutrition. Some of the alcoholic fatty livers can die of alcoholic hepatitis and cirrhosis.

Non-alcoholic fatty liver occurs when a person consumes a lot of fat regardless of alcohol, because the liver does not synthesize much fat or release it. Non-alcoholic fatty liver occurs when there are obesity, hyperlipidemia, and diabetes. It can also be caused by medications such as steroids. Nonalcoholic fatty liver can also progress to nonalcoholic fatty hepatitis.

Alcoholic fatty liver should be treated for alcohol. If you have non-alcoholic fatty liver, maintaining a healthy lifestyle, such as losing weight with dietary control and proper exercise, can help with treatment.

One protein that stimulates the synthesis of fatty acids in the liver is the sterol regulatory element-binding protein (SREBP). This gene is a lipid homeostatic regulatory transcription factor that plays a role in increasing gene transcriptional activity of enzymes important for fatty acid biosynthetic pathways and has three forms: SREBP-1a, SREBP-1c and SREBP-2. SREBP-1c mainly induces fatty acid synthesis and glucose metabolism of insulin, while SREBP-2 is relatively involved in cholesterol synthesis. In addition, SREBP-1a is involved in both pathways. SREBP-1 involved in the synthesis of fatty acids contains the SRE-1 and E-box motif moieties, DNA binding sites that bind for transcription in the nucleus.

 Fatty liver has been identified as the cause and severity of the disease, but it is often not practiced by patients with fatty liver who need to improve symptoms through diet control and exercise. Therefore, there is a need for developing an effective fatty liver treatment drug.

Korean Patent Publication No. 2016-0089000 discloses a novel synthetic oligodioxynucleotide that simultaneously inhibits DNA and RNA, and a pharmaceutical composition for the prevention and treatment of fibrotic diseases containing the same as an active ingredient, Korean Patent No. 0874798 The present invention discloses a circular dumbbell-shaped decoy oligonucleotide comprising a DNA binding site, and Korean Patent No. 1161118 discloses a novel Chi decoy oligodioxynucleotide and a pharmaceutical composition for preventing and treating renal failure containing the same as an active ingredient. Although disclosed, SREBP-1 decoy oligodioxynucleotide of the present invention and a pharmaceutical composition for preventing or treating fatty liver disease containing the same as an active ingredient is not disclosed.

The present invention is derived from the above requirements, the present invention provides a pharmaceutical composition for the prevention or treatment of fatty liver disease containing SREBP-1 decoy oligodioxynucleotide and the same as an active ingredient, SREBP-1 as an active ingredient The present invention was completed by confirming that decoy oligodioxynucleotides reduce liver fat content and decrease hepatic function indicators, blood total cholesterol, triglycerides and inflammatory indices in fatty liver-derived animal models.

In order to solve the above problems, the present invention is covalently linked to the SRE-1 oligodioxynucleotide of SEQ ID NO: 1 and the E-box motif oligodeoxynucleotide of SEQ ID NO: 2, DNA of two loop structure and transcription factor Provided is a circular dumbbell-shaped SREBP-1 decoy oligodioxynucleotide consisting of a stem structure capable of binding to a binding site.

The present invention also provides a pharmaceutical composition for the prevention or treatment of fatty liver disease comprising a dumbbell-type SREBP-1 decoy oligodioxynucleotide as an active ingredient.

The present invention relates to a pharmaceutical composition for the prevention or treatment of SREBP-1 decoy oligodioxynucleotide and fatty liver disease containing the same as an active ingredient, the SREBP-1 decoy oligodioxynucleotide of the present invention in a fatty liver-derived animal model It has the effect of inhibiting the fat content of the liver, and has the effect of reducing the liver function indicator, blood total cholesterol, triglyceride and inflammatory index. Therefore, the composition of the present invention using SREBP-1 decoy oligodioxynucleotide as an active ingredient may be used as a functional material for the prevention and treatment of fatty liver disease.

1 is a diagram showing the structure of SREBP-1 decoy oligodioxynucleotide. It shows the structure of the SREBP-1 decoy oligodioxynucleotide including the SRE-1 and E-box motif moieties that bind to DNA.

Figure 2 is a result of confirming the degree of damage recovery and fat accumulation of liver tissue according to administration of SREBP-1 decoy oligodioxynucleotide using an animal model. NC is a negative control group of the general diet, SREBP-1 decoy ODN is a group treated with SREBP-1 decoy oligodioxynucleotide in the general diet group, HFD + ODN is a group treated with the oligodioxynucleotide negative control group in the high-fat diet group, and HFD + SREBP-1 decoy ODN is a group treated with SREBP-1 decoy oligodioxynucleotide in the high fat diet group.

FIG. 3 shows the results of immunochemical staining of the expression level of cholesterol synthase (HMG-CoA reductase, HMGCR) in liver tissue following administration of SREBP-1 decoy oligodioxynucleotide in an animal model. NC is a negative control group of the general diet, SREBP-1 decoy ODN is a group treated with SREBP-1 decoy oligodioxynucleotide in the general diet group, HFD + ODN is a group treated with the oligodioxynucleotide negative control group in the high-fat diet group, and HFD + SREBP-1 decoy ODN is a group treated with SREBP-1 decoy oligodioxynucleotide in the high fat diet group.

Figure 4 is a result of confirming the degree of protein expression of the biosynthesis-related genes of lipids in liver tissue following administration of SREBP-1 decoy oligodioxynucleotide using an animal model. NC is a negative control without any treatment, ODN is an oligodioxynucleotide negative control.

5 is a result of confirming the expression level of the inflammatory factor IL-6 in the serum according to the administration of SREBP-1 decoy oligodioxynucleotide in the animal model. NC is a negative control group of the general diet, SREBP-1 decoy ODN is a group treated with SREBP-1 decoy oligodioxynucleotide in the general diet group, HFD + ODN is a group treated with the oligodioxynucleotide negative control group in the high-fat diet group, and HFD + SREBP-1 decoy ODN is a group treated with SREBP-1 decoy oligodioxynucleotide in the high fat diet group. * Indicates that IL-6 expression was statistically significantly increased in the HFD group that was fed a high-fat diet compared to NC, which is a p <0.005. † indicates that IL-6 expression was statistically significantly reduced when SREBP-1 decoy ODN was administered compared to ODN, and † means p <0.005.

Figure 6 is a result of confirming the mRNA expression level of inflammatory factors TNF-α, IL-1β, IL-6 in the liver tissue according to administration of SREBP-1 decoy oligodioxynucleotide in the animal model. NC is a negative control group of the general diet, SREBP-1 decoy ODN is a group treated with SREBP-1 decoy oligodioxynucleotide in the general diet group, HFD + Con ODN is a high-fat diet group treated with an oligodioxynucleotide negative control group together Group and HFD + SREBP-1 decoy ODN is a group treated with SREBP-1 decoy oligodioxynucleotide in the high-fat diet group.

In order to achieve the object of the present invention, the present invention is covalently linked to the SRE-1 oligodioxynucleotide of SEQ ID NO: 1 and the E-box motif oligodioxynucleotide of SEQ ID NO: 2, two loop structures and transcription factors It provides a circular dumbbell-shaped SREBP-1 decoy oligodioxynucleotide consisting of a stem structure capable of binding to the DNA binding site.

The dumbbell-type SREBP-1 decoy oligodioxynucleotide is a SRE-1 oligodioxynucleotide (SRE-1 ODN) consisting of the nucleotide sequence of SEQ ID NO: 1 and an E-box motif oligodioxynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 (E-box motif ODN) is covalently linked and consists of two loop and stem structures. The stem structure is a site capable of binding to the DNA binding site of the transcription factor.

SRE-1 ODN: 5'-GAATTCGTGTGGGGTGATTGAAAACAATCACCCCACAC-3 '(SEQ ID NO: 1)

E-box motif ODN: 5'-GAATTCGTATCACGTGATGAAAACATCACGTGATAC-3 '(SEQ ID NO: 2)

The present invention also provides a pharmaceutical composition for the prevention or treatment of fatty liver disease comprising the dumbbell-type SREBP-1 decoy oligodioxynucleotide as an active ingredient.

The fatty liver disease includes alcoholic fatty liver disease and non-alcoholic fatty liver disease, preferably non-alcoholic fatty liver disease. The non-alcoholic fatty liver disease is characterized by any one selected from the group consisting of simple fatty liver, nutrient fatty liver, hunger fatty liver, obese fatty liver, diabetic fatty liver, fatty hepatitis, liver fibrosis and cirrhosis.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent in addition to the dumbbell-shaped SREBP-1 decoy oligodioxynucleotide. In addition, it may be used in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods, but is not limited thereto. . Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose. Or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin, and the like may be used as the base of the suppository. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

Suitable dosages of the pharmaceutical compositions according to the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be.

The concentration of the active ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time, etc., and is not limited to a specific range of concentration.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

Example 1.Decoy Oligodioxynucleotide Construction

The base sequence of the decoy oligodioxynucleotide (hereinafter decoy ODN) used in Example 1 is as follows.

SREBP-1 Decoy ODN:

SRE-1 ODN: 5'-GAATTCGTGTGGGGTGATTGAAAACAATCACCCCACAC-3 '(SEQ ID NO: 1)

E-box motif ODN: 5'-GAATTCGTATCACGTGATGAAAACATCACGTGATAC-3 '(SEQ ID NO: 2)

Con ODN:

5'- GAATTCCAGGTACGGCAAAAAATTGCCGTACCTG-3 '(SEQ ID NO: 3)

The produced oligodioxynucleotide (ODN) was denatured at 95 ° C. for 3 minutes and then annealed while lowering the temperature to 80-25 ° C. The annealed ODN was ligated with T4 ligase for 16-18 hours to produce spherical Con ODN and SREBP-1 decoy ODN. The structure of the ligated spherical dumbbell-shaped SREBP-1 decoy ODN is shown in FIG. 1. Ligation decoy ODN was identified on a 15% non-denaturating gel.

Example 2 Production of Fatty Liver Induced Animal Model

In order to produce a fatty liver-induced animal model, a high fat diet (HFD: 21% fat and 1% cholesterol) was consumed for 12 weeks, and 10 µg decoy at 2 weeks intervals during high fat diet. ODN was injected through the tail vein of the rat. The NC diet fed commercial rodent feed and infused with decoy ODN under the same conditions. After 12 weeks, liver tissues were extracted or blood was used for the experiment.

Example 3 Confirmation of Liver Tissue Injury and Fat Accumulation in Animal Models

Liver extracted from the animal model of Example 2 was prepared with paraffin sections, and H & E staining was performed to confirm the degree of damage and fat accumulation of liver tissue. As shown in FIG. 2, the liver of the subject fed the Con ODN diet was shown to have a higher degree of damage and fat accumulation than the general diet group, but the SREBP-1 decoy ODN was injected while the diet was fed a high fat diet. Hepatic tissues significantly reduced liver tissue damage and fat accumulation compared to Con ODN injection group. Therefore, it was confirmed that SREBP-1 decoy ODN has an effect of suppressing liver damage and fat accumulation induced by high fat diet.

Example 4 Blood Analysis of Animal Models

Animal models of Example 2 were measured for blood alanine aminotransferase (ALT), aspartate aminotransferase (AST, aspartate aminotransferase), total cholesterol and triglyceride index using a commercial assay kit. As a result, as shown in Table 1, the activity of plasma alanine aminotransferase and aspartic acid aminotransferase, which are indicators of liver function, was significantly increased in the high-fat diet group compared to the normal diet group, and SREBP-1 decoy When ODN was injected, the activity of alanine aminotransferase and aspartic acid aminotransferase was significantly decreased compared to the Con ODN injection group. In addition, the total cholesterol and triglyceride index was significantly increased in the high-fat diet group compared to the normal diet group, and the total cholesterol and triglyceride index was significantly higher in the SREBP-1 decoy ODN-injected group than the Con ODN-injected group. Decreased. Therefore, SREBP-1 decoy ODN may play a role in preventing hepatic deterioration induced by high fat diet.

Blood analysis of animal models NC SREBP-1 Decoy ODN HFD + Con ODN HFD + SREBP-1 Decoy ODN AST (U / ℓ) 109.33 ± 4.2 111.5 ± 6.7 261.25 ± 35.2 245.13 ± 27.2 ALT (U / ℓ) 81.77 ± 2.9 97.31 ± 5.2 245.73 ± 23.8 238.16 ± 19.6 Total Cholesterol (mg / dℓ) 108.5 ± 5.2 125.41 ± 3.5 382.32 ± 45.1 267.34 ± 33.4 Triglycerides (mg / dℓ) 112.75 ± 4.2 111.2 ± 2.9 419.2 ± 24.3 287.11 ± 22.5

Example 5 Expression Changes of Cholesterol-related Synthesis Genes in Animal Models

In the animal model of Example 2, expression of cholesterol synthase (HMG-CoA reductase, HMGCR), a gene involved in biosynthesis of cholesterol, was observed. Immunohistochemical staining was performed to confirm expression of HMGCR protein in liver tissue, and the results are shown in FIG. 3. The expression of HMGCR protein was significantly increased when Con ODN was injected into the high-fat diet group compared to the normal diet group, and the expression of HMGCR protein was decreased when SREBP-1 decoy ODN was injected into the high-fat diet group compared to the Con ODN injection group. The expression of HMGCR protein in the SREBP-1 decoy ODN-injected group was similar to that of the normal diet group. Thus, treatment of SREBP-1 decoy ODN can inhibit cholesterol biosynthesis by reducing the expression of HMGCR protein.

Example 6 Expression Changes of Lipid Biosynthesis Related Factors in Animal Models

Liver tissue was extracted from the animal model of Example 2 and confirmed by Western blot protein expression of genes related to lipid biosynthesis. Lipid biosynthesis-related proteins were selected from SREBP-1C, Fatty acid syntase (FAS), Acetyl-CoA carboxylase (ACC), and Steroyl-CoA desaturase (SCD-1). As a result, it was confirmed that the expression of lipid biosynthetic proteins was induced by a high fat diet as shown in FIG. 4, and it was confirmed that expression of SREBP-1 decoy ODN was significantly reduced. Thus, the biosynthesis of lipids induced by high fat diet can be reduced by inhibiting the expression of related genes by SREBP-1 decoy ODN treatment, which in turn can improve fat accumulation in liver tissue by SREBP-1 decoy ODN. There will be.

Example 7 Analysis of Inflammation-Related Factors in Animal Models

In the animal model of Example 2, it was confirmed that the expression of IL-6 increased by high-fat diet decreased in the group injected with SREBP-1 decoy ODN as shown in FIG. 5. In addition, as shown in Figure 6, the group injected with SREBP-1 decoy ODN with a high-fat diet reduced the mRNA expression of inflammatory factors TNF-α, IL-1β, IL-6 in liver tissue. Therefore, SREBP-1 decoy ODN may reduce the inflammatory response of the liver induced by high fat diet and may help prevent hepatitis.

Claims (5)

SRE-1 oligodioxynucleotide of SEQ ID NO: 1 and E-box motif oligodeoxynucleotide of SEQ ID NO: 2 are covalently linked to form a stem structure capable of binding to two loop structures and DNA binding sites of transcription factors A circular dumbbell shaped SREBP-1 decoy oligodioxynucleotide. A pharmaceutical composition for preventing or treating fatty liver disease, comprising the dumbbell-type SREBP-1 decoy oligodioxynucleotide of claim 1 as an active ingredient. The pharmaceutical composition for preventing or treating fatty liver disease according to claim 2, wherein the fatty liver disease is non-alcoholic fatty liver disease or alcoholic fatty liver. The fatty liver according to claim 3, wherein the non-alcoholic fatty liver disease is any one selected from the group consisting of simple fatty liver, nutrient fatty liver, hunger fatty liver, obese fatty liver, diabetic fatty liver, fatty hepatitis, liver fibrosis and liver cirrhosis. Pharmaceutical compositions for the prevention or treatment of diseases. The pharmaceutical composition for preventing or treating fatty liver disease according to claim 2, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent in addition to the dumbbell-type SREBP-1 decoy oligodioxynucleotide.

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