KR20230120580A - Pharmaceutical Composition comprising Inhibitor of deubiquitinating enzyme for Preventing or Treating of Endoplasmic Reticulum Stress-related Diseases - Google Patents

Abstract

The deubiquitination enzyme inhibitor of the present invention inhibits the expression of USP42, a deubiquitination enzyme, and thus increases the degradation of endoplasmic reticulum stress proteins, thereby exhibiting preventive and therapeutic effects on liver diseases such as diabetes and fatty liver, including these It is expected that the pharmaceutical composition to be used as a therapeutic agent exhibiting excellent therapeutic effects on the endoplasmic reticulum stress-related diseases.

Description

Pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising a deubiquitinase inhibitor

The present invention relates to a pharmaceutical composition containing a deubiquitination enzyme inhibitor for preventing or treating endoplasmic reticulum stress-related diseases and a pharmaceutical preparation containing the composition.

The endoplasmic reticulum is a very important organelle that folds during protein biosynthesis. In the endoplasmic reticulum stress state, proteins in an unfolded state accumulate in the endoplasmic reticulum, resulting in various pathological conditions. Various pathological conditions or diseases known to be caused by endoplasmic reticulum stress include, for example, diabetes and liver disease.

Diabetes mellitus is a disease in which the concentration of glucose in the blood rises because the amount of insulin secretion that helps the food to be broken down into sugar and transported into the blood is insufficient or the normal function is not performed. According to the Korean Diabetes Association and the recent announcement by the Korea Centers for Disease Control and Prevention, the prevalence of diabetes in Korea is continuously increasing, and it is expected that the national health will be significantly threatened due to the increase in various chronic diseases and complications. If the homeostasis of blood sugar control due to diabetes is not maintained for a long time, hyperglycemia and insulin resistance (IR) can be induced. It is well known that it causes chronic complications such as vascular complications and increases the mortality rate therefrom. Therefore, early prevention and management of diabetes, which is an underlying disease of various chronic diseases, is an essential problem to improve public health. According to a recent study reporting the current status and characteristics of diabetes in Korea, a comparative analysis of the death rate by cause of death by country (as of 2013) by the Organization for Economic Cooperation and Development (OECD) showed that the mortality rate due to diabetes in Korea was It reached 28.9 per 100,000 population, which ranked 7th among all OECD countries.

The mortality rate of liver disease in Korea is very high at 23.5 per 100,000 people (37.8 males, 9.0 females), and is the 1st cause of death in the 40s (41.1/100,000) and the 2nd leading cause of death in the 50s (72.4/100,000). , it is currently the main cause of death for the middle-aged population in Korea, ranking third (10/100,000) as the cause of death in their 30s. Among the liver diseases, fatty liver is caused by accumulation of fat in the liver due to excessive fat or alcohol intake, increased fat synthesis in the liver, reduced neutral fat emission and combustion, and generally refers to cases in which the proportion of fat accumulated in the liver is 5% or more. On the other hand, fatty liver can be largely divided into alcoholic fat caused by excessive drinking and non-alcoholic fatty liver caused by liver and obesity, diabetes, hyperlipidemia or drugs. Alcoholic fatty liver occurs when excessive intake of alcohol promotes fat synthesis in the liver and prevents normal energy metabolism. On the other hand, non-alcoholic fatty liver often occurs in people suffering from obesity, insulin hypersensitivity, and diabetes. This phenomenon suggests that non-alcoholic fatty liver may be caused by an increase in the concentration of free fatty acids in the blood due to insulin resistance or excessive lipolysis.

Some people think that fatty liver is simply a phenomenon in which fat accumulates in the liver, but considering the fact that 50% of patients diagnosed with alcoholic fatty liver and 30% of patients diagnosed with non-alcoholic fatty liver develop cirrhosis, fatty liver is very serious. It should be regarded as one of the liver diseases. As such, fatty liver has emerged as a serious problem, but useful drugs for treating it are still lacking, and only exercise and diet therapy are recommended. Also, in fact, the treatment efficiency of fatty liver by this method is very low, and the development of an effective new therapeutic agent for fatty liver is required. However, an appropriate therapeutic agent for treating the disease has not been developed to date.

Proteins can be degraded and synthesized through ubiquitin/deubiquitinase, and ubiquitin binds to the target protein by E1, E2, and E3 during the ubiquitin/deubiquitinization process and is degraded through proteasome activity, whereas deubiquitinase It is known that protein degradation can be prevented by separating ubiquitin from a target protein. However, studies on methods for preventing and treating liver diseases such as diabetes and acute/chronic fatty liver by regulating the expression of endoplasmic reticulum stress-related proteins by inhibiting deubiquitinase have not been reported to date.

Korea Patent No. 1949309

One aspect is to provide a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases, including a deubiquitination enzyme inhibitor.

Another aspect is to provide a health functional food composition for preventing or improving endoplasmic reticulum stress-related diseases, including a deubiquitination enzyme inhibitor.

Another aspect is to provide a pharmaceutical formulation for preventing or treating endoplasmic reticulum stress-related diseases, including the pharmaceutical composition.

1. A pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising a deubiquitination enzyme inhibitor.

2. The pharmaceutical composition according to 1 above, wherein the deubiquitination enzyme is USP42.

3. The method of 1 above, wherein the deubiquitination enzyme inhibitor is an antisense nucleotide, aptamer, small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA) that binds complementary to deubiquitination enzyme mRNA And at least one selected from the group consisting of RNA interference (RNAi), a pharmaceutical composition.

4. The pharmaceutical composition according to 1 above, wherein the deubiquitination enzyme inhibitor is siRNA consisting of the nucleotide represented by SEQ ID NO: 2.

5. The pharmaceutical composition according to 1 above, wherein the deubiquitination enzyme inhibitor inhibits degradation of endoplasmic reticulum stress proteins.

6. The pharmaceutical composition according to 1 above, wherein the deubiquitination enzyme inhibitor inhibits activation of at least one selected from PERK and IRE1.

7. The pharmaceutical composition according to 1 above, wherein the deubiquitination enzyme inhibitor inhibits the expression of at least one selected from ATF4 and CHOP.

8. The pharmaceutical composition according to 1 above, wherein the endoplasmic reticulum stress-related disease is at least one selected from the group consisting of diabetes, fatty liver, liver fibrosis, hepatitis, and liver cancer.

9. A health functional food composition for preventing or improving endoplasmic reticulum stress-related diseases, including a deubiquitination enzyme inhibitor.

10. A pharmaceutical formulation for preventing or treating endoplasmic reticulum stress-related diseases comprising the composition of 1 above.

Pharmaceutical compositions and preparations for preventing or treating endoplasmic reticulum stress-related diseases containing the deubiquitination enzyme inhibitor of the present invention inhibit the expression of USP42, a deubiquitination enzyme, thereby increasing the degradation of endoplasmic reticulum stress proteins, such as diabetes and fatty liver, etc. It can show preventive and therapeutic effects of liver diseases.

1 is a diagram showing the mechanism of protein degradation and synthesis by ubiquitin and deubiquitin enzymes.
Figure 2 is a diagram confirming whether the siRNA action of the deubiquitination enzyme (USP42) in mouse adipocytes.
Figure 3 is a diagram confirming the effect of deubiquitination enzyme (USP42) deficiency in an acute fatty liver model. 3A is a diagram illustrating a method of inducing fatty liver formation in mice by intraperitoneal injection of tunicamycin (1 mg/kg) twice after constructing an animal model deficient in the USP42 gene. 3B and 3C are diagrams confirming that fatty liver induced by tunicamycin is suppressed in USP42-deficient animal models, and the weight of the reduced liver also increases again. Figure 3D is a view confirming that triglyceride, which is increased in fatty liver models induced by tunicamyin, is suppressed in USP42 deficient mouse models. Figure 3E is a diagram confirming the morphological changes of liver tissue in a fatty liver induced mouse model by hematoxylin and eosin staining (H&E staining). Figure 3F is a diagram confirming the concentrations of aspartate aminotransferase (GOT), alanine aminotransferase (GPT), and alkaline phosphatase (ALP) among enzymes contained in hepatocytes through blood analysis.
Figure 4 is a diagram confirming the expression of endoplasmic reticulum stress-related proteins according to deubiquitination enzyme (USP42) inhibition.
5 is a diagram confirming the effect of deubiquitination enzyme (USP42) overexpression in an acute fatty liver model. 5A is a diagram showing changes in tunicamycin-induced endoplasmic reticulum stress after USP42 DNA was transfected into cancer cells to determine whether the deubiquitination enzyme overexpression system using DNA works. 5B is a diagram confirming that fatty liver was induced only by USP42 gene overexpression as a result of inducing fatty liver formation in mice by intraperitoneally injecting tunicamycin twice after preparing an animal model with USP42 gene overexpression. Figure 5C is a diagram confirming that the weight of liver tissue is reduced by the USP42 gene overexpression animal model and tunicamycin administration in which fatty liver is induced, and is restored again in the USP42 cysteine 120 mutation type overexpression animal model. 5D is a diagram confirming that triglyceride is increased in USP42 overexpression fatty liver-induced animal models and suppressed in USP42 cysteine 120 mutant type overexpression animal models. Figure 5E is a diagram confirming the concentrations of aspartate aminotransferase (GOT), alanine aminotransferase (GPT), and alkaline phosphatase (ALP) among enzymes contained in hepatocytes through blood analysis.
Figure 6 is a diagram confirming the expression of endoplasmic reticulum stress-related proteins according to overexpression of deubiquitination enzyme (USP42).
7 is a diagram confirming changes in endoplasmic reticulum stress-related protein expression according to the presence or absence of USP42 expression in hepatocytes obtained from mouse liver tissue.
8 is a diagram confirming insulin resistance according to inhibition of deubiquitination enzyme (USP42) in pancreatic beta cells.
Figure 9 is a diagram confirming the endoplasmic reticulum stress-induced apoptosis changes according to deubiquitination enzyme (USP42) inhibition in cancer cells.
10 is a diagram confirming the relationship of BMI1 to the induction of endoplasmic reticulum stress by regulation of deubiquitination enzyme (USP42). Figure 9A is a result of confirming the expression of Polycomb protein (Polycomb protein) to identify a specific mechanism according to the regulation of deubiquitination enzyme (USP42) expression in cancer cells. 9B and 9C are results confirming the relevance of BMI1 to USP42-mediated endoplasmic reticulum stress induction.
11 is a diagram confirming the steatohepatitis inhibitory effect according to the inhibition of BMI1 expression.

The present inventors have confirmed that the deubiquitination enzyme inhibitor suppresses the expression of USP42, a deubiquitination enzyme, and thus increases the degradation of endoplasmic reticulum stress proteins, thereby exhibiting preventive and therapeutic effects on liver diseases such as diabetes and fatty liver.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising a deubiquitination enzyme inhibitor.

In the present specification, deubiquitination enzyme refers to a proteolytic enzyme that serves to recognize and remove ubiquitin molecules bound to target proteins.

In one embodiment of the present invention, the deubiquitination enzyme may be USP42.

In this specification, "USP42" is one of the deubiquitination enzymes that remove conjugated ubiquitin from specific proteins to regulate various cellular processes. In one embodiment of the invention, USP42 is capable of inhibiting the degradation of endoplasmic reticulum stress proteins.

In the present specification, "deubiquitination enzyme inhibitor" inhibits the role of recognizing and removing ubiquitin molecules bound to the target protein of the deubiquitination enzyme, thereby achieving the purpose of inhibiting fat synthesis and accumulation Materials may be included without limitation.

More specifically, the deubiquitination enzyme inhibitor of the present invention may be a USP42 inhibitor, and preferably the deubiquitination enzyme inhibitor is an antisense nucleotide, aptamer, small interfering RNA (siRNA), short hairpin RNA ( shRNA), micro RNA (miRNA) and RNA interference (RNAi), or an antibody specific to the USP42 enzyme.

In one embodiment of the present invention, the deubiquitination enzyme inhibitor may be a siRNA composed of the nucleotide represented by SEQ ID NO: 2, and the siRNA composed of the nucleotide represented by SEQ ID NO: 2 may inhibit the expression of USP42 .

In the present invention, the siRNA may include all that are chemically modified by a general method known in the art to prevent rapid degradation by in vivo nucleases. For example, a hydrogen atom, a halogen atom, an -O-alkyl group, an -O-acyl group, or an amino group, specifically, H, OR , R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br, I, etc. (R is an alkyl or aryl, preferably an alkyl group having 1 to 6 carbon atoms , R' may be an alkylene, preferably an alkylene having 1 to 6 carbon atoms), phosphorothioate, phosphorodithioate, alkylphosphonate, phosphoroamidate, boranophosphate, etc. can be formulated as

In addition, the chemical modification is such that at least one nucleotide included in the siRNA of the present invention is a nucleic acid of any one of LNA (locked nucleic acid), UNA (unlocked nucleic acid), morpholino, and PNA (peptide nucleic acid) It may be characterized as being substituted to have an analogue form. Preparation of these LNA, UNA, PNA, morpholino, etc. can be prepared by knowledge known in the art based on the siRNA sequence information provided in the present invention, and existing literature can be referred to (Veedu RN, Wengel J. Chem Biodivers. .

In addition, the siRNA of the present invention may include variants having one or more substitutions, insertions, deletions, and combinations thereof, which are functional equivalents having changes that do not reduce its activity. For example, the siRNA of the present invention may exhibit 80% or more homology with the siRNA of each corresponding SEQ ID NO, preferably 90% or more, and more preferably 95% or more. Such homology can be readily determined by comparing the sequence of nucleotides to corresponding portions of the target gene using computer algorithms well known in the art, such as the Align or BLAST algorithms.

In one embodiment of the present invention, the deubiquitination enzyme inhibitor may promote degradation of endoplasmic reticulum stress proteins as a mechanism for preventing and treating diabetes.

In one embodiment of the present invention, the deubiquitination enzyme inhibitor may promote degradation of endoplasmic reticulum stress proteins as a mechanism for inhibiting fat synthesis and accumulation in the liver.

In one embodiment of the present invention, the deubiquitination enzyme inhibitor may effectively prevent, treat, or improve diabetes and fatty liver by inhibiting activation of one or more selected from PERK and IRE1.

In one embodiment of the present invention, the deubiquitination enzyme inhibitor may effectively prevent, treat, or improve diabetes and fatty liver by inhibiting the expression of one or more selected from ATF4 and CHOP.

In the present specification, the endoplasmic reticulum refers to a very important organelle that folds during protein biosynthesis. In the endoplasmic reticulum stress state, proteins in an unfolded state accumulate in the endoplasmic reticulum, which can cause various pathological conditions.

In one embodiment of the present invention, the "endoplasmic reticulum stress-related disease" may include liver disease, degenerative brain disease, arteriosclerosis, diabetes, diabetic complications such as diabetic eye disease and diabetic kidney disease, Alzheimer's disease and cystic fibrosis. can

In one embodiment of the present invention, the pharmaceutical composition may exhibit effects of improving, preventing, and treating diabetes symptoms. In one embodiment of the present invention, “liver disease” refers to a group consisting of fatty liver, liver fibrosis, hepatitis, and liver cancer. It may be one or more selected from.

As used herein, "fatty liver" refers to simple fatty liver, alcoholic fatty liver, nutritional fatty liver, starvation fatty liver, obese fatty liver, diabetic fatty liver, steatohepatitis, non-alcoholic fatty liver disease (non-alcoholic fatty liver) It may be at least one selected from the group consisting of -alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and fibrosis.

In the present specification, "non-alcoholic fatty liver" refers to a disease caused by accumulation of excess energy in the form of neutral fat in the liver due to bad lifestyles such as lack of exercise and high-calorie meals. If left untreated, it can progress from hepatitis, liver cirrhosis to liver cancer, but until now there is no suitable treatment, so treatment through exercise and improvement in dietary life is mainly performed. In the present invention, the non-alcoholic fatty liver includes various types of liver diseases ranging from simple non-alcoholic fatty liver with only fat and almost no liver cell damage, chronic non-alcoholic steatohepatitis with severe and persistent liver cell damage, and cirrhosis.

As used herein, "liver fibrosis" refers to liver cirrhosis, hepatorenal syndrome, hepatitis hepatis, metabolic liver disease, chronic liver disease, hepatitis B virus infection, hepatitis C virus infection. Hepatitis D virus infection, Schistosomiasis, alcoholic liver disease, nonalcoholic steatohepatitis, diabetes, protein deficiency, coronary artery disease, autoimmune hepatitis, α-1-Antitrypsin deficiency And it may be one selected from the group consisting of primary biliary cirrhosis, but is not limited thereto.

In one embodiment of the present invention, the liver fibrosis or liver cirrhosis may be caused by non-alcoholic steatohepatitis.

As used herein, the term "prevention" refers to all activities of suppressing or delaying the onset of bone diseases by the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to all activities in which symptoms of bone disease are improved or beneficially changed by the pharmaceutical composition according to the present invention.

In one embodiment of the present invention, the pharmaceutical composition can improve hepatic inflammation or systemic inflammation, inhibit lipid accumulation in the liver, and hepatic sinusoid vascular endothelial cells ( It may also reduce the capillarization of liver sinusoidal endothelial cells (LSECs).

In addition, the pharmaceutical composition can reduce the expression of alanine aminotransferase (ALT) or aspartate aminotransferase (AST), triglycerides (TG) or total cholesterol (total Cholesterol, TC) content may be reduced.

The pharmaceutical composition may reduce the expression of a lipogenesis-related factor, the lipogenesis-related factor refers to a factor known to be involved in the synthesis of conventional lipids, for example, SREBP1c (sterol regulatory element-binding transcription factor-1c), ACC (acetyl-CoA carboxylase), PPARγ (peroxisome proliferator-activated receptors γ), or FAS (fatty acid synthase).

The pharmaceutical composition can reduce the expression of pro-inflammatory factors, which refer to those conventionally known as inflammatory factors, for example, F4/80, MCP-1 (monocyte chemoattractant protein-1) , IL-1β (interleukin 1β), IL-1α (interleukin 1α), TNF-α (tumor necrosis factor-α), IL-6 or IL-8.

The pharmaceutical composition can reduce the expression of a pro-fibrotic (fibrosis-related) factor, the fibrosis-related factor refers to a factor conventionally known as a factor involved in fibrosis. For example, it may be α-smooth muscle actin (α-SMA), collagen 1α (Col1α), collagen 4α (Col4α), or transforming growth factor-β (TGF-β).

The pharmaceutical composition can reduce the expression of endothelial adhesion molecules, such as E-selectin, intercellular adhesion molecule-1 (ICAM-1), It may be vascular cell adhesion molecule-1 (VCAM-1) or cluster of differentiation 31 (CD31).

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, intranasally, inhaled or topically applied) depending on the desired method, and the dosage is determined according to the condition of the patient. and body weight, disease severity, drug form, route of administration and time, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dose level is the type of disease, severity, drug activity, drug sensitivity, administration time, route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, body weight, absorption rate, inactivity rate and excretion rate of the active ingredient in the body, disease type, and concomitant drugs. In addition, in the cycle, it can be administered daily or every other day, or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

In one embodiment of the present invention, the deubiquitination enzyme is 0.1 μg to 3.0 μg, 0.1 μg to 2.5 μg, 0.1 μg to 2.0 μg, 0.15 μg to 2.0 μg, 0.2 μg to 2.0 μg, 0.25 μg to 0.25 μg to the pharmaceutical composition. μg to 2.0 μg, 0.25 μg to 1.5 μg or 0.25 μg to 1.0 μg.

When the compounds are administered as a composition, they may be formulated with a pharmaceutically acceptable vehicle or carrier in suitable amounts to provide a suitable dosage form.

Meanwhile, the composition may further include a carrier, an excipient, and a diluent used in preparing a pharmaceutical composition.

The carrier is commonly used and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc., and, if necessary, an antioxidant , and other conventional additives such as a buffer may be further included.

In addition, excipients, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to formulate formulations for injections such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, but is not limited thereto.

Regarding a suitable pharmaceutically acceptable carrier and formulation, it can be preferably formulated according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in dosage form, but may be formulated into an injection form, an injection form, a spray form, an inhalant form, or an external skin preparation.

In addition, the composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions.

Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and the solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, in addition to the tectorgenin and its fractions. , lactose, gelatin, etc. can be mixed and prepared. In addition to the above excipients, lubricants such as magnesium styrate and talc may be used.

Liquid preparations for oral administration may include suspensions, internal solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to simple diluents such as water and liquid paraffin.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspending agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, and glycerogeratin may be used.

In addition, the present invention provides a health functional food composition for preventing or improving endoplasmic reticulum stress-related diseases comprising a deubiquitination enzyme inhibitor.

As used herein, the term "improvement" refers to any activity that at least reduces the parameters related to the condition being treated, for example, the severity of symptoms.

In this case, the health functional food composition may be used simultaneously with or separately from a drug for treatment before or after the onset of the disease in order to prevent or improve the disease.

In the health functional food composition of the present invention, the active ingredient may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined depending on its purpose of use (for prevention or improvement). In general, the composition of the present invention can be added in an amount of preferably 15% by weight or less, preferably 10% by weight or less, based on the raw material during production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range.

In addition to containing the active ingredient, the health functional food composition of the present invention may contain other ingredients as essential ingredients without particular limitation. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients like a normal beverage. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. can The ratio of the natural carbohydrates can be appropriately determined by a person skilled in the art.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. These components can be used independently or in combination, and the ratio of these additives can also be appropriately selected by those skilled in the art.

“Deubiquitination enzyme”, “USP42”, “endoplasmic reticulum stress-related diseases”, “prevention” and the like may be within the aforementioned range.

In addition, the present invention provides a pharmaceutical formulation for preventing or treating endoplasmic reticulum stress-related diseases, including the pharmaceutical composition.

In addition, the present invention provides a method for preventing or treating endoplasmic reticulum stress-related diseases comprising administering the pharmaceutical composition to a subject.

In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. means mammals.

"Deubiquitination enzyme", "USP42", "administration", "prevention", "treatment" and the like may be within the foregoing range.

In addition, the present invention provides a use of the pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases.

In addition, the present invention provides a use of a deubiquitination enzyme inhibitor for preparing a drug for treating endoplasmic reticulum stress-related diseases.

In addition, the present invention provides the use of a composition comprising a deubiquitination enzyme inhibitor for preparing a drug for treating endoplasmic reticulum stress-related diseases.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples and experimental examples.

Example

Example 1. Production of deubiquitination enzyme deficient animal model

Nanoparticles were attached to USP42 siRNA (50 μg/1 mice), a deubiquitination enzyme, and intravenously injected three times through the tail of mice (C57BL/6, black mice) to construct an animal model deficient in the deubiquitination enzyme gene ( see Table 1).

base sequence Control siRNAs UUCUCCGAACGUGUCACGU (SEQ ID NO: 1) USP42 siRNA AUACAGUCUACCUCGAACG (SEQ ID NO: 2)

Example 2. Production of deubiquitination enzyme gene overexpression animal model

A deubiquitination enzyme (USP42) DNA (30 μg/1 mouse) was reacted with in vivo-jetPEI and then intravenously injected three times through the tail of the mouse to construct an animal model overexpressing the deubiquitination enzyme gene.

Experimental example

Experimental Example 1. siRNA action of deubiquitination enzyme (USP42) in mouse adipocytes

To confirm the action of the USP42 siRNA prepared in Example 1, mouse adipocytes, 3T3-L1, were treated with USP42 siRNA. As a result, it was confirmed that the induction of endoplasmic reticulum stress by tunicamycin was suppressed when USP42 siRNA was treated (see FIG. 2).

Experimental Example 2. Effects of Deubiquitination Enzyme (USP42) Deficiency in Acute Fatty Liver Model

After constructing an animal model deficient in the USP42 gene, tunicamycin (1 mg/kg) was intraperitoneally injected twice to induce fatty liver formation in mice (see FIG. 3A ). As a result, fatty liver induced by tunicamycin was suppressed in the USP42-deficient animal model, and the weight of the reduced liver increased again (see Figs. 3B and 3C), and the increased triglyceride in the fatty liver model induced by tunicamycin was It was confirmed that it was inhibited in the USP42 deficient mouse model (see FIG. 3D).

In addition, hematoxylin and eosin staining (H&E staining) was used to confirm morphological changes in liver tissue in a mouse model of fatty liver induction. As a result, it was confirmed that vacuoles produced in liver tissue by tunicamycin did not appear in the USP42-deficient mouse model (see FIG. 3E).

In addition, the concentrations of aspartate aminotransferase (GOT), alanine aminotransferase (GPT), and alkaline phosphatase (ALP) among enzymes contained in hepatocytes were confirmed through blood analysis. As a result, it was confirmed that corresponding enzymes increased with liver damage were suppressed in the USP42 deficient mouse model (see FIG. 3F).

Experimental Example 3. Investigation of endoplasmic reticulum stress-related protein expression according to inhibition of deubiquitination enzyme (USP42)

In the case of the fatty liver induced mouse model, it is difficult to maintain endoplasmic reticulum homeostasis due to the induction of endoplasmic reticulum stress. Induction of endoplasmic reticulum stress through unconjugated protein response causes various metabolic diseases. Since fatty liver formation is suppressed in the USP42 deficiency model, changes in the expression of endoplasmic reticulum stress-related proteins were investigated.

As a result, it was confirmed that the activation of sensors (PERK, IRE1) that promote unspliced protein responses in the fatty liver induced mouse model was suppressed in the USP42-deficient model, and the protein expression of ATF4 and CHOP was also suppressed (see FIG. 4). This indicates that the USP42 gene deficiency can inhibit fatty liver formation caused by endoplasmic reticulum stress.

Experimental Example 4. Effect of overexpression of deubiquitination enzyme (USP42) in acute fatty liver model

To confirm the function of the DNA-based deubiquitination enzyme overexpression system, cancer cells were transfected with USP42 DNA and changes in tunicamycin-induced endoplasmic reticulum stress were examined. As a result, when the USP42 gene was overexpressed, the expression of ATF4 and CHOP increased. In addition, when the mutant type in which cysteine 120, the catalytic domain of USP42, was substituted with alanine was overexpressed, it was confirmed that the expression of ATF4 and CHOP decreased (see FIG. 5A). Therefore, it was found that the presence or absence of USP42 expression is important when endoplasmic reticulum stress is induced.

After constructing an animal model overexpressing the USP42 gene, tunicamycin was injected intraperitoneally twice to induce fatty liver formation in mice. As a result, overexpression of the USP42 gene alone induced fatty liver. On the other hand, in an animal model in which the cysteine 120 mutation type of USP42 was overexpressed, fatty liver was not induced even when tunicamycin was administered (see FIG. 5B).

The weight of liver tissue was reduced by the overexpression of the USP42 gene and the administration of tunicamycin, and was restored in the overexpression animal model of the cysteine 120 mutation type of USP42 (see FIG. 5C).

Triglyceride was increased in USP42 overexpression fatty liver-induced animal model and suppressed in USP42 cysteine 120 mutant type overexpression animal model (see Fig. 5D).

In addition, the concentrations of aspartate aminotransferase (GOT), alanine aminotransferase (GPT), and alkaline phosphatase (ALP) among enzymes contained in hepatocytes were confirmed through blood analysis. As a result, it was confirmed that the enzymes increased according to liver damage caused by overexpression of USP42 were suppressed in an animal model in which USP42 cysteine 120 mutant type was overexpressed (see FIG. 5E ).

From these results, it was confirmed that fatty liver was formed when USP42 was overexpressed.

Experimental Example 5. Investigation of endoplasmic reticulum stress-related protein expression according to overexpression of deubiquitination enzyme (USP42)

Expression of endoplasmic reticulum stress-related proteins was investigated in the liver tissues of USP42 gene overexpressing animal models. Increased protein expression of ATF4 and CHOP upon USP42 overexpression did not appear in liver tissues of animal models overexpressing the USP42 cysteine 120 mutation. In addition, a-SMA and vimentin, which are important marker proteins for liver fibrosis, were increased in liver tissues overexpressing the USP42 gene (see FIG. 6).

These results indicate that USP42 can induce fatty liver as well as hepatic fibrosis.

Experimental Example 6. Investigation of endoplasmic reticulum stress-related protein expression according to the presence or absence of USP42 expression in mouse hepatocytes

Expression of endoplasmic reticulum stress-related proteins was investigated according to the presence or absence of USP42 expression in hepatocytes obtained from mouse liver tissue.

As a result, it was confirmed that ATF4 and CHOP, which were increased by Tunicamycin treatment, were suppressed in the absence of USP42 expression using siRNA. Conversely, when the USP42 gene was overexpressed, the expression of ATF4 and CHOP increased, and when the mutant type was overexpressed, the expression of ATF4 and CHOP decreased (see FIG. 7).

These results indicate that USP42 can induce endoplasmic reticulum stress.

Experimental Example 7. Insulin resistance investigation according to inhibition of deubiquitination enzyme (USP42) in pancreatic beta cells

It is known that thapsigargin is an intracellular calcium pump inhibitor that can promote insulin secretion from pancreatic beta cells. Accordingly, the relevance of USP42 to insulin resistance in pancreatic beta cells as well as hepatocytes was confirmed.

As a result, it was confirmed that since insulin secretion is promoted by thapsigargin, the amount of insulin in pancreatic beta cells was reduced, which was restored when USP42 expression was suppressed (see FIG. 8 ). This indicates that USP42 is related to diabetic disease.

Experimental Example 8. Investigation of endoplasmic reticulum stress-induced apoptosis changes according to inhibition of deubiquitination enzyme (USP42) in cancer cells

In cancer cells, changes in endoplasmic reticulum stress-induced apoptosis according to inhibition of deubiquitination enzyme (USP42) were confirmed.

As a result, it was confirmed that the induction of endoplasmic reticulum stress by tunicamycin treatment in liver cancer cells was reduced when USP42 expression was deficient using siRNA (see FIG. 9A). In addition, it was confirmed that apoptosis induced by tunicamycin in renal cancer cells was inhibited when USP42 expression was deficient using siRNA (see FIG. 9B ).

This indicates that USP42 is involved in liver cancer formation.

Experimental Example 9. Investigation of BMI1 relevance in induction of endoplasmic reticulum stress by regulation of deubiquitination enzyme (USP42)

In order to identify a specific mechanism according to the regulation of deubiquitination enzyme (USP42) expression in cancer cells, the expression of Polycomb protein was confirmed. As a result, BMI1 mRNA expression was specifically increased by overexpression of USP42, and suppressed when USP42 cysteine 120 mutation was overexpressed (see FIG. 10A).

In addition, the relevance of BMI1 to USP42-mediated endoplasmic reticulum stress induction was confirmed. The protein expressions of ATF4 and CHOP, which were increased by USP42 overexpression, were decreased when BMI1 was deficient (see Fig. 10B). Conversely, ATF4 and CHOP expressions, which were decreased upon USP42 deficiency, were increased upon BMI1 overexpression (see FIG. 10C ).

This indicates that USP42 is involved in the induction of endoplasmic reticulum stress through the regulation of BMI1, a Polycomb protein.

Experimental Example 10. Investigation of steatohepatitis inhibitory effect according to BMI1 expression inhibition

The Bmi-1 gene was deficient in a steatohepatitis model through a high-fat diet using High Fat High Sucrose (HFHS) (see FIG. 11A). As a result, it was confirmed that steatohepatitis induced by the HFHS diet was inhibited when Bmi-1 was deficient, and increased neutral fat was also reduced (see FIGS. 11B and 11C).

Thereafter, morphological changes in liver tissue were confirmed in the steatohepatitis mouse model by hematoxylin and eosin staining (H&E staining) and Oil red O staining. As a result, vacuoles produced by the HFHS high-fat diet did not appear in the BMI1 deficient mouse model (see Fig. 11D).

In addition, BMI1 protein expression was analyzed in the liver tissue of the BMI1 deficiency model (see FIG. 11E).

In addition, as a result of confirming the concentration of aspartate aminotransferase (GOT) among the enzymes contained in hepatocytes through blood analysis, it was confirmed that GOT, which increased according to liver damage, was suppressed in the BMI1 deficient mouse model (see FIG. 11F ).

Claims (10)

A pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases, comprising a deubiquitination enzyme inhibitor.
The pharmaceutical composition according to claim 1, wherein the deubiquitination enzyme is USP42.
The method according to claim 1, wherein the deubiquitination enzyme inhibitor is an antisense nucleotide, aptamer, small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA) and RNA that complementarily bind to deubiquitination enzyme mRNA At least one selected from the group consisting of interference (RNAi), a pharmaceutical composition.
The pharmaceutical composition according to claim 1, wherein the deubiquitination enzyme inhibitor is siRNA consisting of the nucleotide represented by SEQ ID NO: 2.
The pharmaceutical composition of claim 1, wherein the deubiquitination enzyme inhibitor inhibits degradation of endoplasmic reticulum stress proteins.
The pharmaceutical composition of claim 1, wherein the deubiquitination enzyme inhibitor inhibits activation of one or more selected from PERK and IRE1.
The pharmaceutical composition of claim 1, wherein the deubiquitination enzyme inhibitor inhibits the expression of one or more selected from ATF4 and CHOP.
The pharmaceutical composition according to claim 1, wherein the endoplasmic reticulum stress-related disease is at least one selected from the group consisting of diabetes, fatty liver, liver fibrosis, hepatitis, and liver cancer.
A health functional food composition for preventing or improving endoplasmic reticulum stress-related diseases, comprising a deubiquitination enzyme inhibitor.
A pharmaceutical formulation for preventing or treating endoplasmic reticulum stress-related diseases, comprising the composition of claim 1.