WO2024090800A1 - Composition pharmaceutique comprenant un inhibiteur de la lipocaline 2 pour prévenir ou traiter la fibrose hépatique - Google Patents

Composition pharmaceutique comprenant un inhibiteur de la lipocaline 2 pour prévenir ou traiter la fibrose hépatique Download PDF

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WO2024090800A1
WO2024090800A1 PCT/KR2023/014274 KR2023014274W WO2024090800A1 WO 2024090800 A1 WO2024090800 A1 WO 2024090800A1 KR 2023014274 W KR2023014274 W KR 2023014274W WO 2024090800 A1 WO2024090800 A1 WO 2024090800A1
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lipocalin
inhibitor
liver fibrosis
pharmaceutical composition
preventing
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Korean (ko)
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임승순
이은호
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계명대학교 산학협력단
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    • 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
    • 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
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis containing a lipocalin 2 inhibitor, a pharmaceutical preparation containing the composition, and a use and treatment method thereof.
  • the liver is a very important organ that is located between the digestive system and the systemic circulatory system and performs the function of defending the entire body from foreign substances that enter the digestive system. Because extracorporeal substances that enter the body pass through the liver, the liver is at greater risk of being exposed to many toxic substances in addition to nutrients than other organs, and the risk of damage is also higher than that of other organs.
  • liver tissues hepatocytes perform major liver functions such as absorption and metabolism of nutrients, storage, and plasma protein synthesis, and Kupffer cells, which are intrahepatic macrophages, synthesize connective tissue excessively when liver is damaged.
  • hepatic stellate cells lipocytes; HSC
  • endothelial cells endothelial cells
  • pit cells that cause liver fibrosis exist.
  • hepatocytes account for more than 90% of the total cells that make up liver tissue, and are parenchymal cells that perform the main functions of the liver. Deterioration of liver function during liver disease is caused by damage to these liver cells.
  • liver damage due to various causes commonly results in liver fibrosis and the mechanism can be summarized as follows.
  • Kupffer cells are activated and secrete various cytokines
  • hepatic stellate cells are activated to create connective tissue.
  • interstitial repair is triggered in which damaged liver cells are replaced by connective tissue, wounds are formed due to excessive accumulation of connective tissue in the liver tissue, and liver function deteriorates due to liver cell damage, resulting in liver fibrosis and cirrhosis.
  • HSCs hepatic stellate cells
  • Lipocalin 2 is a member of the lipocalin family that binds to and transports lipids and other hydrophobic molecules (Flower, D. R. et al. 2000, Biochim Biophys Acta 1482: 9-24).
  • LCN2 is known to be important in both apoptosis and survival of cells (Nelson, A. M. et al. 2008, J Clin Invest 118: 1468-1478), and plays a central role in inducing cell differentiation in the kidney during embryogenesis. It is known that it protects the kidney from ischemic damage (Yang, J. et al. 2002, Mol Cell 10: 1045-1056) and protects the kidney from ischemic damage (Mishra, J.
  • LCN2 facilitates mucosal regeneration by promoting cell migration in various types of gastrointestinal tract damage (Playford, R. J. et al. 2006, Gastroenterology 131: 809-817).
  • LCN2 is a liver fibrosis-inducing substance, and research on methods to prevent and treat liver fibrosis by inhibiting it has been reported to date. does not exist.
  • One aspect is to provide a pharmaceutical composition for preventing or treating liver fibrosis, which includes a lipocalin 2 inhibitor as an active ingredient.
  • Another aspect is to provide a pharmaceutical preparation for preventing or treating liver fibrosis comprising the composition.
  • Another aspect is to provide a health functional food composition for preventing or improving liver fibrosis, including a lipocalin 2 inhibitor.
  • Another aspect is to provide a pharmaceutical composition containing a lipocalin 2 inhibitor as an active ingredient for use in preventing or treating liver fibrosis.
  • Another aspect is to provide a method for preventing or treating liver fibrosis using a pharmaceutical composition containing a lipocalin 2 inhibitor as an active ingredient.
  • lipocalin 2 is a factor that induces liver cells into hepatic stellate cells, and by suppressing the expression of lipocalin 2, it suppresses the expression of hepatic stellate cells, showing the effect of preventing and treating liver diseases such as liver fibrosis. was confirmed.
  • the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising a lipocalin 2 inhibitor as an active ingredient.
  • the lipocalin-2 (LCN2) inhibitor according to the present invention can be usefully used to prevent and treat liver fibrosis.
  • the LCN2 inhibitor may include, without limitation, substances capable of achieving the purpose of LCN2 inhibition.
  • the LCN2 inhibitor may be an expression or activity inhibitor of LCN2 or an LCN2 receptor antagonist.
  • the lipocalin 2 inhibitor is an antisense nucleotide, aptamer, small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA) and It may be one or more selected from the group consisting of RNA interference (RNAi).
  • RNAi RNA interference
  • the lipocalin 2 inhibitor can inhibit the expression of lipocalin 2 by specifically binding to lipocalin 2 mRNA.
  • expression inhibition means inhibition of gene transcription and inhibition of translation into protein. In addition, it includes not only completely stopped gene expression, but also reduced gene expression.
  • the most common method of suppressing gene expression is to use antisense molecules.
  • the actions of antisense molecules to suppress the expression of target genes include inhibition of transcription initiation by triple strand formation, inhibition of transcription by hybrid formation at the site where a local open loop structure is created by RNA polymerase, and progression of synthesis. Inhibition of transcription by hybrid formation in the RNA being formed, inhibition of splicing by hybrid formation at the junction between introns and exons, inhibition of splicing by hybrid formation at the spliceosome formation site, and inhibition of splicing by hybrid formation with mRNA. These include inhibition of migration to the cytoplasm and inhibition of translation initiation by hybrid formation at the translation initiation factor binding site. They inhibit the expression of target genes by inhibiting transcription, splicing or translation processes.
  • the antisense molecules include triplicates, ribozymes, RNAi, or antisense nucleic acids.
  • Triplexer wraps around double-stranded DNA to form a three-stranded helix, thereby inhibiting transcription initiation (Maher et al., Antisense Res. and Dev., 1(3):227, 1991; Helene, C., Anticancer Drug Design, 6(6):569, 1991).
  • a ribozyme is an RNA enzyme that has the ability to specifically cleave single-stranded RNA. The ribozyme recognizes a specific nucleotide sequence within the target RNA molecule and cleaves it site-specifically, thereby suppressing protein expression of the target gene (Cech, J.Amer.
  • RNA interference is a method of suppressing gene expression at the transcriptional or post-transcriptional level using hairpin-shaped small molecule RNA that acts specifically on the base sequence (Mette et al., EMBO J., 19: 5194- 5201, 2000).
  • the small molecule RNA used in the RNAi method is a double-stranded RNA molecule that has homology to the target gene.
  • known chemical synthesis methods and enzymatic methods can be used. For example, chemical synthesis of RNA molecules can be done using methods disclosed in the literature (Verma and Eckstein, Annu.
  • Antisense nucleic acid refers to a DNA or RNA molecule that is at least partially complementary to a target mRNA molecule (Weintraub, Scientific American, 262:40, 1990).
  • antisense nucleic acids hybridize with the corresponding mRNA to form a double-stranded molecule, thereby inhibiting mRNA translation of the target gene and suppressing protein expression (Marcus-Sakura, Anal. Biochem., 172:289, 1988).
  • the antisense nucleic acid is preferably in the form of an oligonucleotide and can be prepared by any suitable method known in the art.
  • the antisense oligonucleotides can be synthesized by chemical synthesis, for example, by phosphoamidite chemistry by sulfurization with tetraethylthiuram disulfide in acetonitrile as described in Tetrahedron Lett., 1991, 32, 30005-30008. It can be manufactured very easily.
  • the siRNA against lipocalin 2 of the present invention consists of a sense sequence of 15 to 30 mers selected from the base sequence of the mRNA of the gene encoding the LCN2 protein and an antisense sequence that binds complementary to the sense sequence.
  • the sense sequence is not particularly limited thereto, but is preferably composed of 25 bases.
  • the lipocalin 2 inhibitor may be one or more selected from the group consisting of compounds that specifically bind to the lipocalin 2 protein, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies.
  • the lipocalin 2 inhibitor can inhibit the activity of LCN2 by specifically binding to the LCN2 protein.
  • the lipocalin 2 inhibitor may be one or more selected from the group consisting of compounds, peptides, peptide mimetics, and substrate analogs that specifically bind to the lipocalin 2 receptor (24p3R) protein.
  • the lipocalin 2 inhibitor can act antagonistically with lipocalin 2 by specifically binding to the lipocalin 2 receptor protein, thereby inhibiting the activity of lipocalin 2.
  • the peptide mimetics may inhibit the activity of LCN2 protein by acting antagonistically on lipocalin 2 protein expression or the binding domain of lipocalin 2.
  • Peptidomimetics may be peptides or non-peptides and may be composed of amino acids linked by non-peptide bonds, such as psi bonds.
  • the peptide mimetic is structured similarly to the secondary structure characteristics of the LCN2 protein, can mimic the inhibitory properties of large molecules such as antibodies or soluble receptors, and is a novel antibody that can act with an effect equivalent to that of a natural antagonist. It may be a small molecule.
  • the aptamer is a single-stranded DNA or RNA molecule that can be converted to a specific chemical or biological molecule by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). It can be obtained by separating oligomers that bind with high affinity and selectivity. Aptamers can specifically bind to a target and modulate the target's activity, for example, they can block the target's function through binding.
  • SELEX systematic evolution of ligands by exponential enrichment
  • the anti-lipocalin 2 antibody may be a polyclonal antibody or a monoclonal antibody.
  • the antibody of the present invention can be produced by a conventional method widely known in the field of immunology using LCN2 protein as an antigen.
  • the lipocalin 2 antibody may be an anti-LCN2 antibody capable of neutralizing the activity of LCN2.
  • Polyclonal antibodies can be prepared from various warm-blooded animals such as horses, cattle, goats, sheep, dogs, chickens, turkeys, rabbits, mice, or rats using any of the techniques conventional in the art. That is, the animal is immunized through intraperitoneal, intramuscular, intraocular, or subcutaneous injection of the antigen. Immunity to the antigen can be increased using adjuvants, such as Freund's complete adjuvant or incomplete adjuvant. Following booster immunization, a small sample of serum is collected and tested for reactivity to the antigen of interest.
  • adjuvants such as Freund's complete adjuvant or incomplete adjuvant.
  • Monoclonal antibodies can also be produced using known techniques (Kennettm McKearn and Bechtol (eds.), Monoclonal Antibodies, Hybridomas; A New Dimension in Biological Analyses, Plenum Press, 1980).
  • the monoclonal antibody immunizes animals using the LCN2 protein as an immunogen, creates hybridomas by fusing the spleen cells of the immunized animal with myeloma cells, selects hybridomas that selectively recognize the LCN2 protein, and selects the selected high It can be produced by culturing hybridomas and isolating antibodies from the hybridoma culture medium.
  • the monoclonal antibody of the present invention can be prepared by injecting the above hybridoma that produces an anti-LCN2 antibody that selectively recognizes the LCN2 protein into an animal and isolating it from the ascites of the animal recovered after a certain period of time after the injection. You can.
  • the lipocalin 2 inhibitor may inhibit the induction of lipocalin 2 from liver cells to hepatic stellate cells.
  • liver fibrosis refers to liver cirrhosis, hepatorenal syndrome, peliosis hepatis, metabolic liver disease, chronic liver disease, hepatitis B virus infection, and hepatitis C virus infection.
  • prevention used in the present invention refers to all actions that suppress or delay the onset of bone disease by the pharmaceutical composition according to the present invention.
  • treatment refers to any action in which the symptoms of a bone disease are improved or beneficially changed by the pharmaceutical composition according to the present invention.
  • the pharmaceutical composition can reduce the expression of pro-fibrotic (fibrosis-related) factors, which refer to factors conventionally known to be involved in fibrosis.
  • pro-fibrotic (fibrosis-related) factors which refer to factors conventionally known to be involved in fibrosis.
  • transforming growth factor- ⁇ TGF- ⁇
  • alpha smooth muscle actin ⁇ -SMA
  • tissue inhibitor matrix metalloproteinase-1 tissue inhibitor matrix metalloproteinase-1
  • TIMP-1 tissue inhibitor matrix metalloproteinase-1
  • collagen type I alpha 1 chain, COL1A1, collagen type III alpha 1 chain, COL3A1, collagen type V alpha 2 chain It may be COL5A2), collagen type VI alpha 1 chain (COL6A1), matrix metallopeptidase 2 (MMP2), or matrix metallopeptidase 9 (MMP9).
  • the pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, intranasally, inhaled, or applied topically) according to the desired method, and the dosage depends on the patient's condition. It varies depending on body weight, disease severity, drug type, administration route and time, but may be appropriately selected by a person 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 with a reasonable benefit/risk ratio applicable to medical treatment or diagnosis, and the effective dose level is determined by the type of disease, severity, and drug of the patient. It can be determined based on factors including activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, 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 singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, condition, body weight, absorption, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and drug used in combination.
  • it can be administered every day or every other day, or divided into 1 to 3 times a day.
  • the above dosage does not limit the scope of the present invention in any way.
  • the lipocalin 2 inhibitor is added to the pharmaceutical composition in an amount of 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 It may be included in ⁇ g to 2.0 ⁇ g, 0.25 ⁇ g to 1.5 ⁇ g or 0.25 ⁇ g to 1.0 ⁇ g.
  • the lipocalin 2 inhibitor When administered as a composition, it may be formulated with an appropriate amount of a pharmaceutically acceptable vehicle or carrier to provide an appropriate dosage form.
  • composition may further include a carrier, excipient, and diluent used in the preparation of the pharmaceutical composition.
  • the carrier is commonly used and includes, but is not limited to, saline solution, sterilized water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc., and if necessary, antioxidant. , and other common additives such as buffer solutions 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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Examples include, but are not limited to, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.
  • the formulations can be preferably formulated according to each ingredient using the method disclosed in Remington's literature.
  • the pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection formulation, an injection formulation, a spray formulation, an inhalation formulation, or an external application for the skin.
  • composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions.
  • Solid preparations for oral administration can be tablets, pills, powders, granules, capsules, etc., and the solid preparations include the tectorigenin and its fractions with at least one excipient, such as starch, calcium carbonate, and sucrose. It can be prepared by mixing , lactose, or gelatin. Additionally, in addition to the above excipients, lubricants such as magnesium styrate and talc may be used.
  • Liquid preparations for oral administration may include suspensions, oral solutions, emulsions, and syrups.
  • simple diluents such as water and liquid paraffin
  • excipients such as wetting agents, sweeteners, fragrances, and preservatives may be used.
  • Preparations for parenteral administration can be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • the non-aqueous solvent or suspension may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate.
  • injectable ester such as ethyl oleate.
  • witepsol, macrogol, tween 61, cacao, laurel, and glycerogenatin can be used as a base for the suppository.
  • the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising a polynucleotide encoding a lipocalin 2 inhibitor as an active ingredient.
  • the polynucleotide encoding the lipocalin 2 inhibitor may include DNA or RNA.
  • the polynucleotide is introduced into an expression vector such as a plasmid or a viral vector by a known method, and then the expression vector is targeted by infection or transduction by various methods known in the art. It can be introduced into cells.
  • an expression vector such as a plasmid or a viral vector
  • the plasmid expression vector is an FDA-approved gene delivery method for use in humans and is a method of delivering plasmid DNA directly to human cells (Nabel, E. G., et al., Science, 249:1285-1288, 1990). Unlike viral vectors, plasmid DNA has the advantage of being able to be purified homogeneously.
  • a mammalian expression plasmid known in the art can be used as a mammalian expression plasmid known in the art.
  • pRK5 European Patent No. 307,247)
  • pSV16B International Patent Publication No. 91/08291
  • pVL1392 are representative examples.
  • the plasmid expression vector containing the polynucleotide according to the present invention can be used by methods known in the art, such as, but not limited to, transient transfection, microinjection, and transduction. , cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection ), electroporation, gene gun, and other known methods for introducing DNA into cells.
  • An expression vector capable of expressing the lipocalin 2 inhibitor can be administered by a known method.
  • it can be administered topically parenterally, orally, intranasally, intravenously, intramuscularly, subcutaneously, or by other suitable means.
  • the present invention can provide a pharmaceutical composition for preventing or treating liver fibrosis, which contains an expression vector containing a polynucleotide encoding a lipocalin 2 inhibitor as an active ingredient.
  • the present invention provides a pharmaceutical formulation for preventing or treating liver fibrosis, comprising the composition.
  • the present invention provides a health functional food composition for preventing or improving liver fibrosis containing a lipocalin 2 inhibitor.
  • the term “improvement” means any action that reduces at least the severity of a parameter related to the condition being treated, such as a symptom.
  • the health functional food composition can be used simultaneously or separately with a drug for treatment before or after the onset of the disease in order to prevent or improve the disease.
  • the active ingredient can be added as is to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.
  • the mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement).
  • the composition of the present invention may be added in an amount of preferably 15% by weight or less, preferably 10% by weight or less, based on the raw materials.
  • the amount may be below the above range.
  • the health functional food composition of the present invention may contain other ingredients as essential ingredients without any particular restrictions.
  • it may contain various flavoring agents or natural carbohydrates as additional ingredients.
  • natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.
  • the health functional food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, It may contain alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used independently or in combination, and the proportions of these additives can also be appropriately selected by those skilled in the art.
  • Lipocalin 2 inhibitor “liver fibrosis,” “prevention,” etc. may be within the aforementioned scope.
  • the present invention provides a method for preventing or treating liver fibrosis, comprising administering the pharmaceutical composition to a subject.
  • “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, and cows. It means mammal.
  • Lipocalin 2 inhibitor “Lipocalin 2 inhibitor,” “liver fibrosis,” “administration,” “prevention,” “treatment,” etc. may be within the aforementioned scope.
  • the present invention provides a use of the pharmaceutical composition for preventing or treating liver fibrosis.
  • the present invention provides the use of a lipocalin 2 inhibitor for manufacturing a drug for treating liver fibrosis.
  • the present invention also provides the use of a composition comprising a lipocalin 2 inhibitor for preparing a medicament for treating liver disease.
  • the pharmaceutical composition for preventing or treating liver fibrosis containing the lipocalin 2 inhibitor of the present invention as an active ingredient prevents and treats liver fibrosis by suppressing the induction of liver cells into hepatic stellate cells by suppressing the expression of lipocalin 2. It can be expressed.
  • Figure 1 is a diagram confirming liver fibrosis due to the expression of lipocalin 2 using mice in which liver fibrosis was induced.
  • Figures 1A to 1C show results confirming that liver fibrosis progresses less in SREBP-1c knockout mice compared to the normal mouse control group.
  • Figure 1D shows the results confirming that when lipocalin 2 expression is decreased in a liver fibrosis mouse model, the expression of liver fibrosis-inducing factors is also decreased.
  • Figure 2 is a diagram confirming that liver fibrosis increases when lipocalin 2 is overexpressed in SREBP-1c knockout mice.
  • Figure 3 shows the results confirming that there is a correlation between the expression level of lipocalin 2 and the development stage of liver fibrosis.
  • liver fibrosis induction by CCl 4 (Carbon tetrachloride) administration
  • 12 8-week-old C57BL/6J male mice and 12 SREBP-1c gene knockout transgenic mice were prepared and then divided into 4 groups of 6 mice each, into a CCl 4 administration group and a non-administration group.
  • the breeding room environment is at 23°C and 60 to 70% humidity with a 12:12 hour light/dark cycle (light from 6 am to 6 pm, dark from 6 pm to 6 pm) with free access to water, and mouse facilities free of specific pathogens. Accepted.
  • Liver fibrosis was induced for 5 weeks by administering 2 ml/kg CCl 4 twice a week by intramuscular injection into the legs of normal mice and SREBP-1c gene knockout transgenic mice, and then the degree of liver fibrosis was verified. Mice were anesthetized with isoflurane (Gyeonggi-do Hana Pharmaceutical) and sacrificed at 9 AM during the photoperiod. The present inventors performed all animal experiments at Keimyung University College of Medicine, Daegu, South Korea, according to protocols approved by the Keimyung University Animal Experiment and Use Committee (KM-2022-15R1).
  • mice A total of four groups of mice, each transgenic and normal, administered and not administered CCl 4 for 5 weeks, were sacrificed by anesthetizing with isoflurane, blood was collected, the liver was incised, and the separated liver tissue was placed in formalin. It was fixed and cut to a thickness of 10 ⁇ m using a microtome to create tissue slides.
  • liver fibrosis The morphology of liver tissue and liver fibrosis were confirmed through H&E (hematoxylin & eosin), Masson's trichrome, and Sirius red staining, and tissue staining was performed through an experimental procedure widely known in the art. As a result, it was confirmed that liver fibrosis progressed less in SREBP-1c knockout mice compared to the normal mouse control group (see Figures 1A to 1C).
  • fibrosis-related genes were analyzed to investigate the degree of liver fibrosis induction by CCl 4 administration.
  • qPCR was performed with a CFX96TM real-time PCR system (Bio-Rad Laboratories). mRNA levels were normalized to the expression of ribosomal protein L32 by calculating the delta-delta threshold cycle method. Primer sequences of genes used in qPCR are shown in [Table 1].
  • lipocalin 2 is an important factor in causing liver fibrosis.
  • lipocalin 2 was overexpressed in SREBP-1c knockout mice again and the degree of liver fibrosis was confirmed.
  • SREBP-1c knockout mice were injected with LCN2 adenovirus through the tail vein and then treated with CCl 4 for 5 weeks to induce liver fibrosis.
  • the degree of liver fibrosis was measured using the Sirius Red staining method.
  • total RNA was prepared using liver tissue, and cDNA was synthesized using the iScriptTM cDNA synthesis kit. Primer sequences of genes used in qPCR are shown in [Table 2].
  • mice overexpressing lipocalin 2 had an increased degree of liver fibrosis compared to SREBP-1c knockout mice (see Figure 2A).
  • liver fibrosis-related genes The expression levels of ‘Tgfb1, Col1a1, Col3a1, Col5a2, Col6a1, Mmp2, Mmp9, and Timp1’, mRNAs of liver fibrosis-related genes, were measured (see Figures 2B to 2I). As a result, it was confirmed that the expression level of liver fibrosis-related genes was increased in mice overexpressing lipocalin 2 compared to SREBP-1c knockout mice. This indicates that lipocalin 2 is an important factor in the progression of liver fibrosis.
  • hepatocytes were isolated from normal mice and SREBP-1c gene knockout transgenic mice and incubated with 10% FBS and 1% (v/v) penicillin/streptomycin (Gibco). The cells were cultured in high-glucose DMEM (HyClone) under 5% CO 2 humidified culture conditions and at a temperature of 37°C. Afterwards, the cells were dispensed into a 6 well (2 To measure the expression level of genes, total RNA was prepared by the TriZol procedure (Invitrogen), and cDNA was synthesized using the iScriptTM cDNA synthesis kit. Primer sequences of genes used in qPCR are shown in [Table 3].
  • lipocalin 2 and inflammation-related gene mRNAs ‘F4/80, Tnf- ⁇ , Il-6, and Mcp1’ were measured (see Figures 3A to 3E).
  • the expression levels of lipocalin 2 and inflammation-related genes in primary hepatocytes were decreased in SREBP-1c gene knockout transgenic mice treated with fatty acid compared to normal mice treated with fatty acid (see Figures 3A to 3E).

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Abstract

Un inhibiteur de lipocaline 2 selon la présente invention peut présenter les effets de prévention et de traitement de la fibrose hépatique par inhibition de l'induction de cellules hépatiques dans des cellules stellaires hépatiques par l'inhibition de l'expression de la lipocaline 2, et ainsi une composition pharmaceutique le comprenant est censée être utilisée dans des agents thérapeutiques et analogues pour présenter d'excellents effets de traitement sur la fibrose hépatique.
PCT/KR2023/014274 2022-10-27 2023-09-20 Composition pharmaceutique comprenant un inhibiteur de la lipocaline 2 pour prévenir ou traiter la fibrose hépatique WO2024090800A1 (fr)

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KR10-2022-0140726 2022-10-27
KR1020220140726A KR20240083161A (ko) 2022-10-27 2022-10-27 리포칼린 2 억제제를 포함하는 간섬유화 예방 또는 치료용 약학적 조성물

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WO2024090800A1 true WO2024090800A1 (fr) 2024-05-02

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Citations (2)

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KR20150040185A (ko) * 2013-10-04 2015-04-14 경북대학교 산학협력단 리포칼린 2 억제제를 유효성분으로 함유하는 염증성 통증의 예방 또는 치료용 조성물
KR20230112257A (ko) * 2022-01-20 2023-07-27 경상국립대학교산학협력단 리포칼린 2 또는 이의 수용체의 발현 억제제를 유효성분으로 함유하는 비알코올성지방간염에 의해 유도된 간섬유화 예방 또는 치료용 약학 조성물

Patent Citations (2)

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KR20150040185A (ko) * 2013-10-04 2015-04-14 경북대학교 산학협력단 리포칼린 2 억제제를 유효성분으로 함유하는 염증성 통증의 예방 또는 치료용 조성물
KR20230112257A (ko) * 2022-01-20 2023-07-27 경상국립대학교산학협력단 리포칼린 2 또는 이의 수용체의 발현 억제제를 유효성분으로 함유하는 비알코올성지방간염에 의해 유도된 간섬유화 예방 또는 치료용 약학 조성물

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ANONYMOUS: "Identification of the mechanism controlling ‘fibrosis of non-alcoholic steatohepatitis’ caused by obesity and diabetes", GNU TODAY PRESS RELEASE, 17 May 2022 (2022-05-17), XP093163272, Retrieved from the Internet <URL:https://www.gnu.ac.kr/main/na/ntt/selectNttInfo.do?mi=1070&bbsId=1021&nttSn=2134953> *
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KIM KYUNG EUN, LEE JAEWOONG, SHIN HYUN JOO, JEONG EUN AE, JANG HYE MIN, AHN YU JEONG, AN HYEONG SEOK, LEE JONG YOUL, SHIN MEONG CH: "Lipocalin‐2 activates hepatic stellate cells and promotes nonalcoholic steatohepatitis in high‐fat diet–fed Ob/Ob mice", HEPATOLOGY, JOHN WILEY & SONS, INC., US, vol. 77, no. 3, 1 March 2023 (2023-03-01), US , pages 888 - 901, XP093079160, ISSN: 0270-9139, DOI: 10.1002/hep.32569 *
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