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WO2025127472A1 - Composition pour la prévention ou le traitement de l'obésité ou des maladies hépatiques, comprenant un peptide dérivé de la protéine de liaison à la thiorédoxine - Google Patents

Composition pour la prévention ou le traitement de l'obésité ou des maladies hépatiques, comprenant un peptide dérivé de la protéine de liaison à la thiorédoxine Download PDF

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Publication number
WO2025127472A1
WO2025127472A1 PCT/KR2024/018433 KR2024018433W WO2025127472A1 WO 2025127472 A1 WO2025127472 A1 WO 2025127472A1 KR 2024018433 W KR2024018433 W KR 2024018433W WO 2025127472 A1 WO2025127472 A1 WO 2025127472A1
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Prior art keywords
liver disease
peptide
amino acid
binding protein
metabolic
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PCT/KR2024/018433
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English (en)
Korean (ko)
Inventor
정해용
윤이주
최인표
권은수
김미선
박찬호
변재은
윤석란
최은지
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Priority claimed from KR1020240159078A external-priority patent/KR20250093160A/ko
Application filed by Korea Research Institute of Bioscience and Biotechnology KRIBB filed Critical Korea Research Institute of Bioscience and Biotechnology KRIBB
Publication of WO2025127472A1 publication Critical patent/WO2025127472A1/fr
Pending legal-status Critical Current
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents

Definitions

  • the present invention relates to a composition for preventing, treating or improving obesity or liver disease, comprising a peptide derived from thioredoxin binding protein as an active ingredient.
  • Obesity is the most common metabolic disease caused by decreased physical activity due to a high-calorie diet.
  • the average obesity rate in OECD countries is 58.7%, which is a very serious level.
  • the prevalence of obesity accounted for 13% of all adults in 2016, and it is expected to account for 20% of adults worldwide by 2025.
  • This increase in obese patients causes metabolic fatty liver disease including chronic inflammation and various immune dysfunctions, which can cause cardiovascular disease or diabetes, which are representative complications of obesity. In severe cases, it can lead to the occurrence of cancer and directly threaten life. Therefore, it is necessary to develop treatments for obesity and metabolic fatty liver disease, which can cause these serious diseases.
  • MAPKs mitogen-activated protein kinases
  • p38 MAPK p38
  • p38 MAPK is strongly activated by various environmental stresses including inflammatory cytokines and oxidative stress, and contributes to cellular homeostasis through regulation of cell differentiation, proliferation, survival, and apoptosis. It also plays an important role in regulating transcription factors and cytokine expression levels in immune and inflammatory responses, and it has been revealed that the p38 ⁇ form is mainly involved in this process.
  • p38 ⁇ In addition to anti-inflammatory effects, pharmacological inhibition of p38 ⁇ is known to have a positive effect on improving neurodegenerative diseases, heart failure-related diseases, obesity, and some metabolic diseases such as fatty liver. Regarding the association between obesity and p38, it was revealed that mice with p38 ⁇ knockout had increased body temperature and activated fat burning due to increased expression of Uncoupling Protein 1 (UCP1), a gene related to thermogenesis. This increase in energy expenditure effectively suppresses diet-induced obesity. In addition, p38 ⁇ is upregulated in hepatocytes of patients with metabolic fatty liver disease, and it has been suggested that this can be improved by inhibiting p38 ⁇ .
  • UCP1 Uncoupling Protein 1
  • TN13 is a peptide composed of 13 amino acids derived from a protein called thioredoxin-interacting protein (TXNIP).
  • TXNIP is known to bind to TRX and regulate its activity, thereby regulating oxidative stress and contributing to the establishment of a biological balance.
  • thioredoxin-interacting protein TXNIP
  • TXNIP is known as a tumor suppressor because it is an inhibitor of thioredoxin and blocks cell cycle progression, and it is known to be used as a treatment for diseases related to active oxygen by increasing resistance to active oxygen.
  • TXNIP thioredoxin-interacting protein
  • TXNIP is known as a tumor suppressor because it is an inhibitor of thioredoxin and blocks cell cycle progression, and it is known to be used as a treatment for diseases related to active oxygen by increasing resistance to active oxygen.
  • the present invention was completed by confirming the anti-obesity effect, the inhibition of fatty liver formation, and the therapeutic effect on liver disease of a peptide derived from thioredoxin binding protein.
  • the present invention provides a pharmaceutical composition for preventing or treating obesity or liver disease, comprising a peptide derived from a thioredoxin binding protein having an amino acid sequence of sequence number 1.
  • the present invention provides a food composition for preventing or improving obesity or liver disease, comprising a peptide derived from a thioredoxin binding protein having an amino acid sequence of sequence number 1.
  • the present invention provides a method for preventing or treating obesity or liver disease, comprising a step of administering to a subject in need thereof a composition comprising a peptide derived from a thioredoxin binding protein comprising an amino acid sequence of SEQ ID NO: 1.
  • the present invention seeks to provide a use of a peptide derived from a thioredoxin binding protein comprising an amino acid sequence of sequence number 1 for the manufacture of a medicament for the prevention or treatment of obesity or liver disease.
  • the present invention provides a pharmaceutical composition comprising a peptide derived from a thioredoxin binding protein comprising an amino acid sequence of SEQ ID NO: 1 for use in the prevention or treatment of obesity or liver disease.
  • the inventor of the present invention discovered that the TN13 peptide derived from thioredoxin binding protein has a therapeutic effect on obesity and metabolic liver disease, thereby completing the present invention.
  • a pharmaceutical composition and a food composition for preventing or treating obesity or liver disease each comprising a peptide derived from a thioredoxin binding protein of the present invention, and a method for preventing or treating obesity or liver disease using the same are specifically described.
  • composition and treatment method for preventing or treating obesity or liver disease comprising peptide derived from thioredoxin binding protein according to the present invention
  • a pharmaceutical composition for preventing or treating obesity or liver disease comprising a peptide derived from a thioredoxin binding protein having an amino acid sequence of sequence number 1.
  • a thioredoxin-binding protein-derived peptide comprising an amino acid sequence of sequence number 1 may be, specifically, a thioredoxin-binding protein-derived peptide consisting essentially of the amino acid sequence of sequence number 1. Specifically, it may be a thioredoxin-binding protein-derived peptide consisting of the amino acid sequence of sequence number 1.
  • TXNIP thioredoxin binding protein
  • TN13 To enable TN13 to efficiently penetrate cells, a cell-penetrating peptide (CPP) derived from the human immunodeficiency virus (HIV) trans-activator protein (TAT) sequence was conjugated to TN13 to produce TAT-TN13.
  • CPP cell-penetrating peptide
  • HAV human immunodeficiency virus
  • TAT trans-activator protein
  • TN13 has high safety compared to existing known chemical p38 inhibitors, and while existing inhibitors must be dissolved in organic solvents that are toxic to cells, TN13 is well soluble in water, so there is no solvent-induced toxicity during processing, and it shows high absorption.
  • TN13 unlike existing inhibitors that simultaneously inhibit p38 ⁇ and p38 ⁇ isomers, TN13 selectively inhibits only p38 ⁇ with a short amino acid sequence, so it is expected to have fewer side effects.
  • Chemical drugs are likely to cause side effects because their residual period in the body is uncertain, but peptide drugs are relatively less likely to cause side effects due to their short residual period in the body.
  • the peptide derived from thioredoxin binding protein according to the present invention has anti-inflammation, an effect of inhibiting lipogenesis in liver cancer cell lines, an anti-obesity effect, an effect of inhibiting metabolic fatty liver formation, and a minor action effect. Through these action effects, it is possible to prevent and/or treat obesity or fatty liver disease, especially liver diseases such as metabolic fatty liver disease.
  • a peptide derived from a thioredoxin binding protein comprising the amino acid sequence of sequence number 1 of the present invention may be composed of a sequence in which one or several amino acids of the protein are added, deleted, or substituted, as long as it has the same activity as the protein or has the same gene location encoding the thioredoxin binding protein on the chromosome.
  • the above thioredoxin binding protein-derived peptide is comprised of a sequence having at least 80% homology to the amino acid sequence of SEQ ID NO: 1, more specifically at least 90% homology, and most specifically at least 95%, 96%, 97%, 98%, 99% or 99.5% homology, but is not limited thereto.
  • a peptide derived from a thioredoxin binding protein comprising an amino acid sequence of SEQ ID NO: 1 according to the present invention may have an amino acid sequence of 30 or less and may include an amino acid sequence of SEQ ID NO: 1.
  • amino acid sequence of sequence number 1 may further include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 amino acids at the N-terminus and/or C-terminus as the Core sequence.
  • the peptide derived from the thioredoxin binding protein can be fused with a cell-penetrating peptide as needed.
  • a cell-penetrating peptide is a type of signal peptide, which is a peptide sequence that aims to transport substances into cells. It is mainly composed of a peptide sequence of about 7 to 30 amino acids, and any sequence that has a signal peptide that can be transported into cells may be included in the present invention.
  • cell-penetrating peptides mainly contain basic amino acid residues such as lysine/arginine, and thus play a role in allowing proteins fused with them to penetrate the cell membrane and enter the cell.
  • the cell-penetrating peptides include, but are not limited to, HIV-1 Tat protein, the homeodomain of Drosophila antennapedia (penetratin), HSV VP22 transcription regulatory protein, MTS peptide derived from vFGF, PTD-5, sequences derived from transporters or Pep-1 peptides, etc.
  • CPPs e.g., TAT48-60, penetratin (pAntp)43-58, polyarginine, Pep-1, transporters, etc.
  • CPPs e.g., TAT48-60, penetratin (pAntp)43-58, polyarginine, Pep-1, transporters, etc.
  • HIV-1 Tat (SEQ ID NO: 2) YGRKKRRQRRR Penetratin (sequence number 3) RQIKIWFQNRRMKWKK HSV VP22 (SEQ ID NO: 4) DAATATRGRSAASRPTERPRAPARSASRPRRPVE MTS (SEQ ID NO: 5) AAVALLPAVLLAAP Transportan (sequence number 6) GWTLNSAGYLLGKINLKALAALAKKIL PEP-1 (SEQ ID NO: 7) KETWWETWWTEWSQPKKKRKV Poly arginine (SEQ ID NO: 8) RRRRRRR PTD-5 (SEQ ID NO: 9) RRQRRTSKLMKR
  • the peptide derived from the thioredoxin binding protein may be linked to any one cell-penetrating peptide selected from the group consisting of SEQ ID NOs: 2 to 9. More specifically, the cell-penetrating peptide may be an amino acid sequence of SEQ ID NO: 2, and the TN13 peptide according to the present invention may be fused with the cell-penetrating peptide to form a fusion peptide.
  • the cell penetrating peptide may be a TAT peptide, and is preferably bound to a terminus of a peptide comprising an amino acid sequence of SEQ ID NO: 1, wherein the terminus may be either the amino terminus (5' terminus, N-terminus) or the carboxy terminus (3' terminus, C-terminus), but is most preferably the amino terminus (5' terminus, N-terminus).
  • the above peptide may additionally undergo modifications such as phosphorylation, acetylation, methylation, and glycosylation, and may bind to other proteins; however, as long as this does not change the protein to the extent that its function is lost, it can be considered to be the same as the protein before modification.
  • the peptide derived from the thioredoxin binding protein of the present invention may have a cell-penetrating peptide linked to the N-terminus. More specifically, it is preferred that a TAT peptide described in SEQ ID NO: 2 is linked thereto, but is not limited thereto.
  • connection between the above cell-penetrating peptide and the TN13 peptide may be direct or may be connected via a linker or spacer peptide.
  • linker refers to a short amino acid sequence used to separate two peptides with different functions in the construction of a fusion protein.
  • the absence of a linker between two or more individual domains in a protein can result in reduced or inappropriate function of the protein domains due to steric hindrance, such as reduced catalytic activity or binding affinity for a receptor/ligand.
  • the use of an artificial linker to connect protein domains in a chimeric protein can increase the spacing between the domains.
  • the linker or spacer is not particularly limited to the effect of enhancing the activity of the conjugate of a cell-penetrating peptide and a vesicle-transporting peptide. While it will not have any specific biological activity other than to join the domains or to preserve some minimal distance or other spatial relationship between them, the constituent amino acids can be selected to affect some property of the molecule, such as folding, net charge, or hydrophobicity.
  • the linker comprises a sequence recognized by a signal peptidase enzyme and thereby be cleaved. That is, a sequence recognizable by a signal peptidase enzyme can be added between the cell-penetrating peptide and the TN13 peptide sequence.
  • the term “signal peptidase cleavage peptide” refers to a linker sequence that is recognized by a signal peptidase and can provide cleavage between a TN13 peptide and a cell penetrating peptide sequence.
  • the “signal peptidase cleavage peptide” can recognize the C-terminus of the cell penetrating peptide sequence and the above cleavage peptide sequence and provide peptide cleavage between them. Since the cell penetrating peptide sequence is derived from the signal sequence, it can have “AXA” or “VXA” (wherein X is any amino acid sequence) at the C-terminus.
  • the signal peptidase enzyme can recognize the cleavage site between the above “AXA” or “VXA” and the “EA” sequence and cleave it. Through this, the target TN13 can be successfully delivered into cells, thereby enhancing drug action.
  • liver disease may be any one selected from the group consisting of hepatic fibrosis, cholesterolosis, cirrhosis, viral and alcoholic hepatitis, Wilson's disease, hemochromatosis, steatosis, metabolic fatty liver disease (MASLD) and metabolic associated steatohepatitis (MASH).
  • metabolic dysfunction-associated steatotic liver disease is a disease in which fat accumulates in liver tissue regardless of significant alcohol intake. Metabolic dysfunction-associated steatotic liver disease is divided into primary and secondary depending on the cause. Primary MASLD is caused by hyperlipidemia, diabetes, or obesity, which are characteristics of metabolic syndrome, and secondary MASLD is caused by nutritional causes (rapid weight loss, starvation, intestinal bypass, etc.), various drugs, toxic substances (poisonous mushrooms, bacterial toxins, etc.), metabolic causes, and other factors. Metabolic dysfunction-associated steatotic liver disease in the present invention includes both primary and secondary metabolic dysfunction fatty liver diseases.
  • “simple fatty liver” is also called simple hepatic steatosis, and means a state of steatosis in which fat exceeds 5%, which is the proportion of fat in a normal liver.
  • Simple fatty liver is not accompanied by inflammation and thus does not cause liver cell damage, and is therefore distinguished from steatohepatitis accompanied by inflammation.
  • metabolic dysfunction-associated steatohepatitis is an advanced type of metabolic dysfunction fatty liver disease, which means a state in which inflammation of hepatocytes is induced by fatty liver, and exhibits tissue changes such as inflammation and necrosis, which are histologically similar to alcoholic liver disease in people who do not consume significant alcohol.
  • Metabolic dysfunction-associated steatohepatitis is accompanied by abnormal fat accumulation or deposition (steatosis) in the liver, hepatocyte inflammation and necrosis, and fibrosis due to tissue damage, and is a progressive disease, so it can develop from fatty liver to steatohepatitis, liver fibrosis, cirrhosis, and even liver cancer.
  • liver fibrosis is also called hepatic fibrosis, and refers to a state in which extracellular matrix proteins including collagen are excessively accumulated in liver tissue.
  • Liver fibrosis is a fibrosis phenomenon that occurs due to continuous destruction of liver cells and damage to liver tissue caused by various causes, and is caused by an imbalance in which the production of extracellular matrix increases but its decomposition relatively decreases. If liver fibrosis persists, it can develop into cirrhosis.
  • liver cirrhosis is also called liver cirrhosis, and means that normal liver tissue changes into fibrous tissue such as regenerative nodules due to chronic inflammation, and the liver function deteriorates as the original function cannot be properly performed.
  • symptoms are not clear, but if liver damage progresses significantly, complications such as jaundice, ascites, hepatic encephalopathy, and variceal bleeding may occur.
  • the liver disease may be any one selected from the group consisting of, for example, liver fibrosis, cirrhosis, metabolic fatty liver disease (MASLD), and metabolic associated steatohepatitis (MASH).
  • MASLD metabolic fatty liver disease
  • MASH metabolic associated steatohepatitis
  • prevention means any act of suppressing or delaying the onset of obesity or liver disease by administering a composition.
  • treatment means any act by which the symptoms of obesity or liver disease are improved or beneficially changed by administration of the composition.
  • a diluent, a dispersant, a surfactant, a binder, and a lubricant may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule, or a tablet.
  • an injectable formulation such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule, or a tablet.
  • the pharmaceutical composition of the present invention may be a patch, a solution, a pill, a capsule, a granule, a tablet, a suppository, or the like.
  • These preparations can be prepared by conventional methods used in formulation in the art or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, and can be formulated into various preparations depending on each disease or ingredient.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be determined according to factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the excretion rate, the treatment period, the drugs used in combination or simultaneously, and other factors well known in the medical field.
  • the composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses.
  • the daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to administer it once or several times a day.
  • administration means introducing a predetermined substance into a patient by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue.
  • the pharmaceutical composition of the present invention may be administered by any device through which the active substance can move to the target tissue.
  • it may be administered by transdermal administration, oral administration, intrathecal administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, inner ear administration, intrauterine epidural administration, sublingual administration, and intracerebrovascular injection, but is not limited thereto.
  • the pharmaceutical composition may appropriately include a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc., depending on the administration method or formulation.
  • a suspending agent e.g., a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc.
  • Oral liquid preparations include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin, glycerogelatin, and the like.
  • injections may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.
  • the route of administration of the pharmaceutical composition of the present invention may be through any general route as long as it can reach the target tissue, but may be through subcutaneous injection using an osmotic pump, intradermal injection, intravein injection, intraperitoneal injection, or intravitreal injection.
  • composition of the present invention may additionally contain one or more active ingredients exhibiting the same or similar function.
  • the composition of the present invention contains 0.0001 to 10 wt% of the protein, preferably 0.001 to 1 wt%, based on the total weight of the composition.
  • the present invention also provides a method for preventing or treating obesity or liver disease, comprising administering to a subject in need thereof a composition comprising a peptide derived from a thioredoxin binding protein comprising an amino acid sequence of SEQ ID NO: 1.
  • subject of the present invention means any animal that has developed or may develop obesity or liver disease, and typically may be an animal that can exhibit a beneficial effect by treatment with a peptide derived from a thioredoxin binding protein comprising the amino acid sequence of SEQ ID NO: 1 of the present invention, but includes without limitation any subject that has symptoms of obesity or liver disease or may have such symptoms.
  • the pharmaceutical composition of the present invention can be administered as an individual therapeutic agent, or can be administered in combination with existing obesity or liver disease therapeutic agents, and can be administered sequentially or simultaneously with the existing therapeutic agents.
  • administration means introducing a predetermined substance into a patient by an appropriate method, and the route of administration of the composition may be administered through any general route as long as it can reach the target tissue.
  • the pharmaceutical composition of the present invention may be administered by any device through which the active substance can move to the target tissue.
  • it may be administered by oral administration, intrathecal administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, inner ear administration, intrauterine epidural administration, sublingual administration, and intracerebrovascular injection, but is not limited thereto.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking all of the above factors into consideration, it may be administered in an amount that can obtain the maximum effect with the minimum amount without causing side effects, which can be easily determined by those skilled in the art.
  • the present invention also provides a use of a peptide derived from a thioredoxin binding protein comprising an amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament for the prevention or treatment of obesity or liver disease.
  • the peptide derived from thioredoxin binding protein comprising the amino acid sequence of the above-mentioned sequence number 1 for the manufacture of a pharmaceutical agent can be mixed with acceptable excipients, diluents, carriers, etc., and can be manufactured into a complex preparation together with other active agents to have a synergistic effect of the active ingredients.
  • Food composition for preventing or improving obesity or liver disease comprising a peptide derived from a thioredoxin binding protein according to the present invention
  • a food composition for preventing or improving obesity or liver disease comprising a peptide derived from a thioredoxin binding protein having an amino acid sequence of sequence number 1.
  • thioredoxin-binding protein-derived peptide In the above food composition, “thioredoxin-binding protein-derived peptide”, “obesity”, “fatty liver disease”, “prevention”, “treatment” and “composition” are as described above.
  • composition may contain, in addition to the active ingredient, a food additive acceptable from a food science perspective.
  • food supplement additive used in the present invention means a component that can be added to food as an auxiliary, and can be appropriately selected and used by those skilled in the art as added in the manufacture of health functional foods of each formulation.
  • food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like, but the types of food supplement additives of the present invention are not limited by the above examples.
  • the food composition of the present invention may include a health functional food composition.
  • the term "health functional food” used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients having useful functionality for the human body.
  • “functionality” means obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.
  • the health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process.
  • the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food.
  • the food composition of the present invention can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long period of time using food as a raw material, and is excellent in portability, so the health functional food of the present invention can be taken as a supplement to enhance the anti-obesity effect or the effect of preventing or improving fatty liver disease.
  • the health functional food composition is preferably manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.
  • the health functional food composition of the present invention can be manufactured by adding the effective ingredient as it is or mixing it with other foods or food ingredients, and can be manufactured appropriately according to a conventional method.
  • foods to which the mixed extract of elderberry and tangerine peel can be added include dairy products including caramel, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense. That is, there is no particular limitation on the type of the food.
  • the above health functional food composition may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.
  • it may contain fruit pulp for the production of natural fruit juice and vegetable beverages.
  • the above components may be used independently or in combination.
  • the health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional components, and the natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • Sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame.
  • the peptide derived from thioredoxin-interacting protein (TXNIP) inhibits fat production, exhibits an anti-obesity effect, and has the effect of inhibiting fatty liver production and liver fibrosis related to metabolic disorders, so that it can be used for the prevention or treatment of obesity or liver disease.
  • Figure 2 shows the results of Western blotting analysis on the p38-NF ⁇ B signal pathway inhibitory effect of TN13 of the present invention upon palmitic acid treatment in RAW264.7 cells (statistical analysis was performed using unpaired two-tailed t-test. *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001).
  • Figure 3 shows the results of a CCK-8 assay for the viability of HepG2 cells treated with TN13 according to the present invention (A), a flow cytometry analysis result (B), and an analysis result of cell permeability of FITC-TAT-TN13 using an optical microscope (C).
  • Figure 4 shows the results of analyzing the time-dependent activation of p38 induced by palmitic acid (PA) by western blotting (A), the results of analyzing the inhibitory effect of TN13 of the present invention on p38 MAPK by western blotting (B), and the results of evaluating adipogenesis by palmitic acid in HepG2 cells using Oil Red O staining (C).
  • PA palmitic acid
  • FIG. 5 is a schematic diagram showing the timeline of high-fat diet (HFD) and TN13 treatment in a mouse model experiment.
  • HFD high-fat diet
  • Figure 6 shows the results of body weight measurements (A, B) and photographs of the mice (C ) for each of the normal diet group (ND), high-fat diet group (HFD), and TN13-administered high-fat diet group (HFD+TN13) over 15 weeks in an in vivo experiment using a mouse model.
  • Figure 7 shows the results of blood glucose level analysis of the normal diet group (ND), high-fat diet group (HFD), and TN13-administered high-fat diet group (HFD+TN13) in an in vivo experiment using a mouse model.
  • Figure 8 shows the results of measuring the mass of eWAT (epididymal white adipose tissue) in the normal diet group (ND), high-fat diet group (HFD), and TN13-administered high-fat diet group (HFD+TN13) in an in vivo experiment using a mouse model (A), the results of photographing the appearance of eWAT (B), and the results of analyzing the size of adipocytes in eWAT using H&E staining (C).
  • eWAT epididymal white adipose tissue
  • Figure 9 shows the results of the analysis of the inflammatory response in the body due to a high -fat diet in the normal diet group (ND), high-fat diet group (HFD), and high-fat diet group administered TN13 (HFD+TN13) in an in vivo experiment using a mouse model, including the results of CBC analysis for inflammatory immune cells (A, B, C) and the results of measuring the level of pro-inflammatory cytokines in serum by ELISA analysis (D, E, F).
  • Figure 10 shows the results of imaging the liver of each group of the normal diet group (ND), high-fat diet group (HFD), and TN13-administered high-fat diet group (HFD+TN13) in an in vivo experiment using a mouse model (A), the result of measuring liver mass (B), the result of measuring TG levels in the liver to analyze fatty liver (C), the result of examining the liver tissue through H&E staining and Oil Red O staining (D), and the result of evaluating the protein levels related to lipogenesis and p38 by Western blotting (E) (Statistical analysis was performed using an unpaired two-tailed t-test. *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001).
  • Figure 11 shows the results of measuring the levels of proinflammatory cytokines in the livers of each group of the normal diet group (ND), high -fat diet group (HFD), and TN13-administered high-fat diet group (HFD+TN13) by ELISA analysis in an in vivo experiment using a mouse model (statistical analysis was performed using an unpaired two-tailed t-test. *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001).
  • Figure 12 shows the results of IHC staining analysis ( D) and cell counting (E, F) of macrophages and neutrophils in the liver in each group of the normal diet group (ND), high-fat diet group (HFD), and high-fat diet group administered TN13 (HFD+TN13) in an in vivo experiment using a mouse model (statistical analysis was performed using an unpaired two-tailed t-test. *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001).
  • Figure 13 is an image confirming liver fibrosis using Sirius Red staining (A), and the results of measuring the levels of fibrosis-activating cytokines in each group of the normal diet group (ND), high-fat diet group (HFD), and TN13-administered high-fat diet group (HFD+TN13) in an in vivo experiment using a mouse model by ELISA analysis (B), and the results of evaluating the levels of proteins related to fibrosis by Western blotting (C) are shown (statistical analysis was performed using an unpaired two-tailed t-test. *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001).
  • Figure 14 shows the results of confirming liver damage by measuring ALT and AST levels in each group of the normal diet group (ND), high -fat diet group (HFD), and TN13-administered high-fat diet group (HFD+TN13) in an in vivo experiment using a mouse model (statistical analysis was performed using an unpaired two-tailed t-test. *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001).
  • TN13 a domain fragment of TXNIP containing the amino acid sequence of sequence number 1, was prepared.
  • sequence number 1 is as disclosed in Table 1 below.
  • TN13 a domain fragment of TXNIP including the amino acid sequence of sequence number 1 according to Example 1-1, was produced, and the HIV TAT transduction domain sequence (sequence number 2) was linked to the N-terminus of TN13 to produce a TAT-TN13 peptide.
  • sequence numbers 1 and 2 are as disclosed in Table 2 below.
  • the mouse macrophage cell line RAW264.7 was cultured in RPMI (Welgene Inc., Gyeongsan, Korea) containing 10% FBS (R&D Systems, Minneapolis, MN, USA).
  • the human hepatoma cell line HepG2 was cultured in DMEM (Welgene Inc., Gyeongsan, Korea) containing 10% FBS, and all cells were cultured at 37°C in an atmosphere of 5% CO2 .
  • CCK-8 Cell viability was measured using the Cell Counting kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan).
  • CCK-8 Cell Counting kit
  • RAW264.7 cells or HepG2 cells were seeded at 2 ⁇ 10 4 cells/well in a 96-well plate and stabilized in a 5% CO 2 incubator at 37°C, and then treated with TN13 at various concentrations.
  • CCK-8 solution (10 ⁇ l per well) was treated and additionally incubated for 30 min to 1 h at 37°C and 5% CO 2 , and then the absorbance at 450 nm was measured using a microplate reader.
  • liver tissues isolated from mice were lysed using radioimmunoprecipitation assay (RIPA) cell lysis buffer containing protease and phosphatase inhibitors and 1 mM PMSF.
  • ELISA analysis was performed using liver tissue samples obtained through this method.
  • mice Five-week-old male C57BL/6 mice were purchased from Dooyeolbiotech (Seoul, Korea). All mice were housed in a Specific Pathogen Free Animals (SPF) environment with a 12-h light/dark cycle and constant temperature and humidity. After a 1-week adaptation period, the mice were randomly divided into negative control, positive control, and experimental groups for the experiment. The negative control group was fed a normal diet (Inotiv, #2018s), and the positive control and experimental groups were fed an adjusted calorie 60% HFD (Inotiv, #TD.06414). The experimental group was administered 25 mg/kg of TN13 by intraperitoneal injection twice a week along with the high-fat diet. At the 15th week, the mice were sacrificed, and the blood, epididymal fat (eWAT), and liver were separated for analysis.
  • SPF Specific Pathogen Free Animals
  • CBC Blood glucose and Complete Blood Count
  • Adipose tissue and liver tissue were prepared as paraffin blocks, respectively, and then cut to prepare paraffin sections.
  • Adipose tissue sections were stained with hematoxylin and eosin (H&E).
  • Liver tissue sections were stained with H&E, Sirius Red, and Immunohistochemistry (IHC) staining using F4/80 (CST, #70076) and Neutrophil (Abcam, #ab2557) antibodies. Additionally, liver tissue was prepared as frozen sections and Oil Red O staining was performed.
  • Liver tissue was pulverized with 5% TritonX-100 solution, and triglyceride (TG) was dissolved in a constant temperature water bath at 80-100°C for 3-5 minutes. The supernatant was centrifuged at 13,000 rpm for 5 minutes, and the TG value was measured.
  • TG assay kit Bioassay system, #ETGA-200 was used, and the measurement method was performed according to the instructions provided by the manufacturer.
  • TAT-TN13 (TN13) manufactured according to Example 1-1 was treated to cells, and cell viability was confirmed 24 hours later through a CCK8 assay.
  • TN13 on p38 phosphorylation (phospho p38, p-p38) in RAW246.7 cells
  • cells were pretreated with various concentrations of TN13 for 1 hour, and then treated with 100 ng/ml LPS for 30 minutes, and protein expression was confirmed through Western blotting.
  • TN13 according to the present invention has an anti-inflammatory effect.
  • Palmitic Acid a saturated fatty acid
  • Palmitic Acid is a common saturated fatty acid found in fats and waxes, including olive oil, palm oil, and body fat, and is known to induce liver damage, fatty liver disease, insulin resistance, and metabolic abnormalities, depending on the dose.
  • TN13 effectively acts in the inflammatory response induced by saturated fatty acids by inhibiting the phosphorylation of p38, which is activated by the MAPK pathway that activates the inflammatory response, and p65, which is activated by the NF- ⁇ B pathway.
  • TNXIP-derived peptide TN13
  • FITC-TAT-TN13 of Example 1-2 was treated to cells for 2 hours, and the results were confirmed using a flow cytometer and a fluorescence microscope.
  • Example 5 Analysis of the lipogenesis inhibitory effect of TNXIP-derived peptides in liver cancer cell lines
  • TNXIP-derived peptide (TN13) affects the reduction of adipogenesis through p-p38 inhibition in HepG2 induced fat.
  • cells were treated with 100 ⁇ M PA, and cells were harvested at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and western blotting was performed.
  • HepG2 cells were pretreated with TN13 for 1 hour and then treated with PA for 15 minutes to confirm changes in p-p38 levels.
  • mice were divided into three experimental groups and an experiment was conducted.
  • Six-week-old C57BL/6J mice were randomly divided into a normal diet group (negative control group), a high-fat diet group (positive control group), and a TN13-administered high-fat diet group (experimental group), and were supplied with regular feed or a high-fat diet (60% fat) for 15 weeks, and their body weights were measured every week.
  • the epididymal fat of the experimental group was found to be reduced in weight compared to the high-fat diet group (Fig. 8A, 8B).
  • epididymal white adipose tissue eWAT
  • H&E staining was performed, and the results showed that the size of fat cells in the experimental group was significantly reduced (Fig. 8C).
  • Example 6 Using the animal model of Example 6, the inhibitory effect on metabolic abnormality steatohepatitis and liver fibrosis was confirmed. The worsening of fatty liver can progress to steatohepatitis or liver fibrosis. An experiment was conducted to confirm the inhibitory effect of TN13 on this.
  • TN13 the number of macrophages and neutrophils actually distributed in the liver tissue was quantified through IHC staining, which can determine the presence or absence of specific proteins in tissue sections, and it was confirmed that the number of these immune cells was significantly reduced in the experimental group administered TN13 (HFD+TN13) (Fig. 12A, B, C). From these results, it can be expected that TN13 will be effective not only in simple hepatic steatosis but also in steatohepatitis.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase

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Abstract

La présente invention concerne une composition pour prévenir, traiter ou soulager l'obésité ou les maladies hépatiques, comprenant un peptide dérivé de la protéine interagissant avec la thiorédoxine (TXNIP) en tant que principe actif. Le peptide dérivé de TXNIP selon la présente invention inhibe l'adipogenèse, présente un effet anti-obésité, et présente les effets d'inhibition du développement de la stéatose hépatique associée à un dysfonctionnement métabolique et de la fibrose hépatique, et peut ainsi être utilisé pour la prévention ou le traitement de l'obésité ou des maladies hépatiques.
PCT/KR2024/018433 2023-12-14 2024-11-20 Composition pour la prévention ou le traitement de l'obésité ou des maladies hépatiques, comprenant un peptide dérivé de la protéine de liaison à la thiorédoxine Pending WO2025127472A1 (fr)

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KR10-2023-0181440 2023-12-14
KR20230181440 2023-12-14
KR1020240159078A KR20250093160A (ko) 2023-12-14 2024-11-11 티오레독신 결합단백질 유래 펩타이드를 포함하는 비만 또는 간질환 예방 또는 치료용 조성물
KR10-2024-0159078 2024-11-11

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Citations (3)

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Publication number Priority date Publication date Assignee Title
KR20140009079A (ko) * 2012-07-12 2014-01-22 한국생명공학연구원 Ｔｒｘ-ｔｘｎｉｐ 복합체 변형 단백질의 결정화 방법 및 그의 입체구조
KR20200067745A (ko) * 2018-12-04 2020-06-12 주식회사 시그넷바이오텍 Fas 신호전달 억제용 펩티드를 포함하는 비만, 지방간 또는 지방간염의 예방 또는 치료용 약학적 조성물
KR20210063198A (ko) * 2019-11-22 2021-06-01 중앙대학교 산학협력단 Lgi3 유래 펩타이드를 유효성분으로 포함하는 고지혈증 및 지방간의 예방, 치료, 또는 개선용 조성물

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KR20200067745A (ko) * 2018-12-04 2020-06-12 주식회사 시그넷바이오텍 Fas 신호전달 억제용 펩티드를 포함하는 비만, 지방간 또는 지방간염의 예방 또는 치료용 약학적 조성물
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