WO2023149768A1 - Composition pharmaceutique comprenant un inhibiteur de la désubiquitinase pour la prévention ou le traitement de maladies associées au stress du réticulum endoplasmique - Google Patents

Composition pharmaceutique comprenant un inhibiteur de la désubiquitinase pour la prévention ou le traitement de maladies associées au stress du réticulum endoplasmique Download PDF

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WO2023149768A1
WO2023149768A1 PCT/KR2023/001666 KR2023001666W WO2023149768A1 WO 2023149768 A1 WO2023149768 A1 WO 2023149768A1 KR 2023001666 W KR2023001666 W KR 2023001666W WO 2023149768 A1 WO2023149768 A1 WO 2023149768A1
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endoplasmic reticulum
usp42
reticulum stress
pharmaceutical composition
liver
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PCT/KR2023/001666
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English (en)
Korean (ko)
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권택규
우선민
서승언
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주식회사 내쉬원
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Priority claimed from KR1020230014865A external-priority patent/KR20230120583A/ko
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Publication of WO2023149768A1 publication Critical patent/WO2023149768A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/13—Nucleic acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • proteins in an unfolded state accumulate in the endoplasmic reticulum, resulting in various pathological conditions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising a deubiquitination enzyme inhibitor.
  • 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.
  • RNAi RNA interference
  • deubiquitination enzyme inhibitor is at least one siRNA selected from the group consisting of nucleotides represented by SEQ ID NO: 2 and SEQ ID NO: 3.
  • 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 including a deubiquitination enzyme inhibitor.
  • a pharmaceutical formulation for preventing or treating endoplasmic reticulum stress-related diseases comprising the composition of 1 above.
  • 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 deubiquitination enzymes USP42 and TRABID, thereby increasing the degradation of endoplasmic reticulum stress proteins, thereby preventing diabetes and It can show preventive and therapeutic effects of liver diseases such as fatty liver.
  • 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.
  • GAT aspartate aminotransferase
  • GPT alanine aminotransferase
  • ALP alkaline phosphatase
  • 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.
  • GAT aspartate aminotransferase
  • GPT alanine aminotransferase
  • ALP alkaline phosphatase
  • Figure 6 is a diagram confirming the expression of endoplasmic reticulum stress-related proteins according to overexpression of deubiquitination enzyme (USP42).
  • FIG. 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.
  • FIG. 8 is a diagram confirming insulin resistance according to inhibition of deubiquitination enzyme (USP42) in pancreatic beta cells.
  • FIG. 9 is a diagram confirming changes in endoplasmic reticulum stress-induced apoptosis according to inhibition of deubiquitination enzyme (USP42) in cancer cells.
  • FIG. 10 is a diagram confirming the relationship of BMI1 to the induction of endoplasmic reticulum stress by regulating deubiquitination enzyme (USP42).
  • Figure 10A 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.
  • 10B and 10C are results confirming the relevance of BMI1 to USP42-mediated endoplasmic reticulum stress induction.
  • FIG. 11 is a diagram confirming the steatohepatitis inhibitory effect according to the inhibition of BMI1 expression.
  • FIG. 12 is a diagram confirming the siRNA action of deubiquitination enzyme (TRABID) in mouse adipocytes.
  • TRABID deubiquitination enzyme
  • 13 is a diagram confirming the effect of deubiquitination enzyme (TRABID) deficiency in an acute fatty liver model.
  • 13A 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 TRABID gene.
  • 13B and 13C are diagrams confirming that fatty liver induced by tunicamycin is suppressed in TRABID-deficient animal models, and the reduced weight of the liver also increases again.
  • Figure 13D is a diagram confirming that the increase in triglyceride in the fatty liver model induced by tunicamyin is suppressed in the TRABID deficient mouse model.
  • Figure 13E 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).
  • 13F 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.
  • GAT aspartate aminotransferase
  • GPT alanine aminotransferase
  • ALP alkaline phosphatase
  • Figure 14 is a diagram confirming the expression of endoplasmic reticulum stress-related proteins according to deubiquitination enzyme (TRABID) inhibition.
  • TRABID deubiquitination enzyme
  • 16 is a diagram confirming the effect of deubiquitination enzyme (TRABID) overexpression in an acute fatty liver model.
  • TRABID deubiquitination enzyme
  • FIG. 17 is a diagram confirming insulin resistance according to deubiquitination enzyme (TRABID) inhibition in pancreatic beta cells.
  • TRABID deubiquitination enzyme
  • TRABID deubiquitination enzyme
  • 19 is a diagram confirming the effect of a TRABID inhibitor in an acute fatty liver model.
  • 20 is a diagram confirming the expression of endoplasmic reticulum-related proteins according to TRABID expression and activity regulation in mouse hepatocytes.
  • the present inventors confirmed that the deubiquitination enzyme inhibitor inhibits the expression of deubiquitination enzymes USP42 and TRABID, thereby increasing the degradation of endoplasmic reticulum stress proteins, thereby exhibiting preventive and therapeutic effects on liver diseases such as diabetes and fatty liver.
  • the present invention provides a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising a deubiquitination enzyme inhibitor.
  • deubiquitination enzyme refers to a proteolytic enzyme that serves to recognize and remove ubiquitin molecules bound to target proteins.
  • the deubiquitination enzyme may be one or more selected from the group consisting of USP42 and TRABID. In one embodiment, the deubiquitination enzyme may be USP42. In one embodiment, the deubiquitination enzyme may be TRABID.
  • 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.
  • TRABID is one of deubiquitination enzymes that remove ubiquitin conjugated in specific proteins to regulate various cellular processes.
  • TRABID can inhibit the degradation of endoplasmic reticulum stress proteins.
  • 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.
  • the deubiquitination enzyme inhibitor of the present invention may be a USP42 inhibitor or a TRABID inhibitor, and preferably the deubiquitination enzyme inhibitor is an antisense nucleotide, aptamer, or small interfering RNA (siRNA) against the USP42 or TRABID gene. , short hairpin RNA (shRNA), micro RNA (miRNA) and RNA interference (RNAi), or an antibody specific to USP42 or TRABID enzymes.
  • shRNA short hairpin RNA
  • miRNA micro RNA
  • RNAi RNA interference
  • the deubiquitination enzyme inhibitor may be one or more siRNAs selected from the group consisting of nucleotides represented by SEQ ID NO: 2 and SEQ ID NO: 3.
  • 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 .
  • the deubiquitination enzyme inhibitor may be a siRNA composed of the nucleotide represented by SEQ ID NO: 3, and the siRNA composed of the nucleotide represented by SEQ ID NO: 3 may inhibit the expression of TRABID .
  • 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.
  • 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
  • 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.
  • LNA locked nucleic acid
  • UNA locked nucleic acid
  • morpholino peptide nucleic acid
  • PNA peptide nucleic acid
  • 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.
  • 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.
  • the deubiquitination enzyme inhibitor may promote degradation of endoplasmic reticulum stress proteins as a mechanism for preventing and treating diabetes.
  • the deubiquitination enzyme inhibitor may promote degradation of endoplasmic reticulum stress proteins as a mechanism for inhibiting fat synthesis and accumulation in the liver.
  • 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.
  • 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.
  • the endoplasmic reticulum refers to a very important organelle that folds during protein biosynthesis.
  • proteins in an unfolded state accumulate in the endoplasmic reticulum, which can cause various pathological conditions.
  • 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.
  • the pharmaceutical composition may exhibit effects of improving, preventing, and treating diabetes symptoms.
  • “liver disease” refers to a group consisting of fatty liver, liver fibrosis, hepatitis, and liver cancer. It may be one or more selected from.
  • 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.
  • NAFLD -alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • 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.
  • 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.
  • 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, ⁇ -1-Antitrypsin deficiency And it may be one selected from the group consisting of primary biliary cirrhosis, but is not limited thereto.
  • the liver fibrosis or liver cirrhosis may be caused by non-alcoholic steatohepatitis.
  • prevention refers to all activities of suppressing or delaying the onset of bone diseases by the pharmaceutical composition according to the present invention.
  • 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.
  • 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).
  • LSECs liver sinusoidal endothelial cells
  • 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.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • TG triglycerides
  • total Cholesterol, TC total Cholesterol
  • 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), PPAR ⁇ (peroxisome proliferator-activated receptors ⁇ ), or FAS (fatty acid synthase).
  • SREBP1c sterol regulatory element-binding transcription factor-1c
  • ACC acetyl-CoA carboxylase
  • PPAR ⁇ peroxisome proliferator-activated receptors ⁇
  • 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-1 ⁇ (interleukin 1 ⁇ ), IL-1 ⁇ (interleukin 1 ⁇ ), TNF- ⁇ (tumor necrosis factor- ⁇ ), IL-6 or IL-8.
  • pro-inflammatory factors which refer to those conventionally known as inflammatory factors, for example, F4/80, MCP-1 (monocyte chemoattractant protein-1) , IL-1 ⁇ (interleukin 1 ⁇ ), IL-1 ⁇ (interleukin 1 ⁇ ), TNF- ⁇ (tumor necrosis factor- ⁇ ), 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.
  • a pro-fibrotic factor refers to a factor conventionally known as a factor involved in fibrosis.
  • it may be ⁇ -smooth muscle actin ( ⁇ -SMA), collagen 1 ⁇ (Col1 ⁇ ), collagen 4 ⁇ (Col4 ⁇ ), or transforming growth factor- ⁇ (TGF- ⁇ ).
  • 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).
  • endothelial adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1)
  • IAM-1 intercellular adhesion molecule-1
  • VCAM-1 vascular cell adhesion molecule-1
  • CD31 cluster of differentiation 31
  • 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.
  • 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.
  • 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.
  • it can be administered daily or every other day, or divided into 1 to 3 times a day.
  • the dosage is not limited to the scope of the present invention in any way.
  • the deubiquitination enzyme is 0.1 ⁇ g to 3.0 ⁇ g, 0.1 ⁇ g to 2.5 ⁇ g, 0.1 ⁇ g to 2.0 ⁇ g, 0.15 ⁇ g to 2.0 ⁇ g, 0.2 ⁇ g to 2.0 ⁇ g, 0.25 ⁇ g to 0.25 ⁇ g to the pharmaceutical composition. ⁇ g to 2.0 ⁇ g, 0.25 ⁇ g to 1.5 ⁇ g or 0.25 ⁇ g to 1.0 ⁇ g.
  • the compounds 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.
  • 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.
  • excipients such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.
  • 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.
  • 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.
  • 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.
  • 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.
  • injectable ester such as ethyl oleate
  • witepsol macrogol, tween 61, cacao butter, laurin fat, and glycerogeratin may be used.
  • the present invention provides a health functional food composition for preventing or improving endoplasmic reticulum stress-related diseases comprising a deubiquitination enzyme inhibitor.
  • 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.
  • 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.
  • 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).
  • 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.
  • the amount may be less than the above range.
  • the health functional food composition of the present invention may contain other ingredients as essential ingredients without particular limitation.
  • it may contain various flavoring agents or natural carbohydrates as additional ingredients like a normal beverage.
  • 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.
  • natural flavoring agents thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the ratio of the natural carbohydrates can be appropriately determined by a person skilled in the art.
  • 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.
  • the present invention provides a pharmaceutical formulation for preventing or treating endoplasmic reticulum stress-related diseases, including the pharmaceutical composition.
  • the present invention provides a method for preventing or treating endoplasmic reticulum stress-related diseases comprising administering the pharmaceutical composition to a subject.
  • subject 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.
  • the present invention provides a use of the pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases.
  • the present invention provides a use of a deubiquitination enzyme inhibitor for preparing a drug for treating endoplasmic reticulum stress-related diseases.
  • 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.
  • Nanoparticles were attached to the deubiquitination enzymes USP42 siRNA (50 ⁇ g/1 mice) and TRABID siRNA (50 ⁇ g/1 mice) and intravenously injected 3 times through the tail of mice (C57BL/6, black mice) to obtain deubiquitin An animal model deficient in the enzyme gene was constructed (see Table 1).
  • siRNAs UUCUCCGAACGUGUCACGU SEQ ID NO: 1
  • USP42 siRNA AUACAGUCUACCUCGAACG SEQ ID NO: 2
  • TRABID siRNAs GAGGAAGAAUCUCCCAUUA SEQ ID NO: 3
  • DNA (30 ⁇ g/1 mouse) of deubiquitination enzymes (USP42 and TRABID) was reacted with in vivo-jetPEI and then intravenously injected three times through the mouse tail to construct an animal model overexpressing the deubiquitination enzyme gene.
  • tunicamycin (1 mg/kg) was intraperitoneally injected twice to induce fatty liver formation in mice (see FIG. 3A ).
  • 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 suppressed in the USP42 deficient mouse model (see Fig. 3D).
  • H&E staining hematoxylin and eosin 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).
  • 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).
  • 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).
  • 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.
  • 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).
  • HFHS High Fat High Sucrose
  • liver tissue was confirmed in the steatohepatitis mouse model by hematoxylin and eosin staining (H&E staining) and Oil red O staining.
  • H&E staining hematoxylin and eosin staining
  • Oil red O staining oil red O staining
  • BMI1 protein expression was analyzed in the liver tissue of the BMI1 deficiency model (see FIG. 11E).
  • mouse adipocytes, 3T3-L1 were treated with siRNA. It was confirmed that the induction of endoplasmic reticulum stress by tunicamycin was inhibited by TRABID siRNA (see FIG. 12).
  • tunicamycin (1 mg/kg) was intraperitoneally injected twice to induce fatty liver formation in mice (see FIG. 13A).
  • fatty liver induced by tunicamycin was suppressed in the TRABID-deficient animal model, and the weight of the reduced liver also increased again (see FIGS. 13B and 13C), and the increased triglyceride in the fatty liver model induced by tunicamyin was It was confirmed that it was suppressed in the TRABID deficient mouse model (see FIG. 13D).
  • H&E staining hematoxylin and eosin 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 TRABID deficient mouse model (see FIG. 13E).
  • cancer cells were transfected with TRABID DNA and changes in tunicamycin-induced endoplasmic reticulum stress were examined.
  • tunicamycin was injected intraperitoneally twice to induce fatty liver formation in mice. As a result, it was confirmed that fatty liver was induced only by TRABID gene overexpression. On the other hand, in an animal model in which the cysteine 443 mutation type of TRABID was overexpressed, fatty liver was not induced even when tunicamycin was administered (see FIG. 16A). In addition, it was confirmed that triglycerides were increased in the TRABID overexpression animal model and suppressed in the TRABID mutant type overexpression animal model (see FIG. 16B).
  • liver tissue Thereafter, morphological changes in liver tissue were confirmed by hematoxylin and eosin staining (H&E staining). As a result, fat (vacuole) produced by tunicamycin was suppressed in the TRABID mutant type overexpressing animal model (see FIG. 16C ).
  • Thapsigargin is an intracellular calcium pump inhibitor known to promote insulin secretion from pancreatic beta cells. Accordingly, the association of TRABID with insulin resistance in pancreatic beta cells as well as hepatocytes was confirmed.
  • TRABID is involved not only in inducing fatty liver but also in forming hepatocarcinoma.
  • tunicamycin was administered to create an acute fatty liver induction model. It was confirmed that the TRABID inhibitor inhibited the induction of fatty liver by tunicamycin (see FIG. 19A). In addition, it was confirmed that triglyceride increased by tunicamycin was suppressed when the TRABID inhibitor was administered (see FIG. 19B).
  • liver tissue Thereafter, morphological changes in liver tissue were confirmed by hematoxylin and eosin staining (H&E staining). As a result, it was confirmed that the fat (vacuole) produced by tunicamycin was suppressed in the animal model to which the TRABID inhibitor was administered (see FIG. 19C).
  • composition comprising Inhibitor of deubiquitinating enzyme for Preventing or Treating Endoplasmic Reticulum Stress-related Diseases

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Abstract

Un inhibiteur de la désubiquitinase de la présente invention peut inhiber l'expression des enzymes de désubiquitination USP42 et TRABID pour augmenter la dégradation des protéines de stress du réticulum endoplasmique, présentant ainsi des effets de prévention et de traitement du diabète et de maladies hépatiques telles que la stéatose hépatique et, par conséquent, on s'attend à ce qu'une composition pharmaceutique contenant l'inhibiteur de la désubiquitinase puisse être utilisé dans un agent thérapeutique ou similaire présentant un excellent effet de traitement sur des maladies associées au stress du réticulum endoplasmique.
PCT/KR2023/001666 2022-02-07 2023-02-06 Composition pharmaceutique comprenant un inhibiteur de la désubiquitinase pour la prévention ou le traitement de maladies associées au stress du réticulum endoplasmique WO2023149768A1 (fr)

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KR10-2022-0015578 2022-02-07
KR20220015578 2022-02-07
KR10-2023-0014779 2023-02-03
KR10-2023-0014865 2023-02-03
KR1020230014865A KR20230120583A (ko) 2022-02-07 2023-02-03 탈유비퀴틴화효소 억제제를 포함하는 소포체 스트레스 관련 질환 예방 또는 치료용 약학적 조성물
KR1020230014779A KR20230120580A (ko) 2022-02-07 2023-02-03 탈유비퀴틴화효소 억제제를 포함하는 소포체 스트레스 관련 질환 예방 또는 치료용 약학적 조성물

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Citations (4)

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Publication number Priority date Publication date Assignee Title
KR100794706B1 (ko) * 2006-07-14 2008-01-14 (주) 차바이오텍 탈유비퀴틴화 효소를 코딩하는 유전자로부터 유래되는 비만또는 당뇨병 진단용 프로브, 프라이머, 또는 이를 이용한비만 또는 당뇨병의 진단 방법
US20170088520A1 (en) * 2014-06-02 2017-03-30 Pharmakea, Inc. Deubiquitinase inhibitors
WO2018234775A1 (fr) * 2017-06-20 2018-12-27 Mission Therapeutics Limited Cyanopyrrolidines substituées présentant une activité en tant qu'inhibiteurs de dub
US20200263159A1 (en) * 2017-11-06 2020-08-20 The Trustees Of Columbia University In The City Of New York Compositions and methods for using engineered deubiquitinases for probing ubiquitin-dependent cellular processes

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100794706B1 (ko) * 2006-07-14 2008-01-14 (주) 차바이오텍 탈유비퀴틴화 효소를 코딩하는 유전자로부터 유래되는 비만또는 당뇨병 진단용 프로브, 프라이머, 또는 이를 이용한비만 또는 당뇨병의 진단 방법
US20170088520A1 (en) * 2014-06-02 2017-03-30 Pharmakea, Inc. Deubiquitinase inhibitors
WO2018234775A1 (fr) * 2017-06-20 2018-12-27 Mission Therapeutics Limited Cyanopyrrolidines substituées présentant une activité en tant qu'inhibiteurs de dub
US20200263159A1 (en) * 2017-11-06 2020-08-20 The Trustees Of Columbia University In The City Of New York Compositions and methods for using engineered deubiquitinases for probing ubiquitin-dependent cellular processes

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Title
DATABASE NUCLEOTIDE ANONYMOUS : "PREDICTED: Arvicanthis niloticus ubiquitin specific peptidase 42 (Usp42), transcript variant X3, mRNA", XP093082317, retrieved from NCBI *
DATABASE NUCLEOTIDE ANONYMOUS : "PREDICTED: Carcharodon carcharias ubiquitin thioesterase ZRANB1 (LOC121289719), transcript variant X7, mRNA", XP093082319, retrieved from NCBI *

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