WO2023140664A1 - Composition pharmaceutique pour la prévention ou le traitement de la fibrose hépatique induite par la stéatohépatite non alcoolique, contenant, en tant que principe actif, un inhibiteur d'expression de la lipocaline 2 ou un récepteur de celle-ci - Google Patents

Composition pharmaceutique pour la prévention ou le traitement de la fibrose hépatique induite par la stéatohépatite non alcoolique, contenant, en tant que principe actif, un inhibiteur d'expression de la lipocaline 2 ou un récepteur de celle-ci Download PDF

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WO2023140664A1
WO2023140664A1 PCT/KR2023/000979 KR2023000979W WO2023140664A1 WO 2023140664 A1 WO2023140664 A1 WO 2023140664A1 KR 2023000979 W KR2023000979 W KR 2023000979W WO 2023140664 A1 WO2023140664 A1 WO 2023140664A1
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lipocalin
liver fibrosis
alcoholic steatohepatitis
mrna
protein
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Korean (ko)
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노구섭
김경은
이재웅
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경상국립대학교산학협력단
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    • 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
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    • 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
    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis induced by non-alcoholic steatohepatitis, containing lipocalin 2 or an expression inhibitor of its receptor as an active ingredient; a method of inhibiting activation of hepatic stellate cells (HSC) by treating with the lipocalin 2 receptor inhibitor; It relates to a method for diagnosing liver fibrosis induced by non-alcoholic steatohepatitis using lipocalin 2 and screening a substance for treating liver fibrosis.
  • HSC hepatic stellate cells
  • Fatty liver has been recognized as being related to alcoholic liver disease, which is mainly caused by high alcohol consumption.
  • Alcoholic fatty liver refers to a condition in which fat is accumulated in the liver in alcoholics, fat particles are observed in liver cells, and fat (particularly, neutral fat) is accumulated in liver cells to cause liver failure.
  • non-alcohol drinkers also have findings similar to those of alcoholic liver disease.
  • the state of fatty liver disease in non-alcohol drinkers is mostly referred to as non-alcoholic fatty liver disease (NAFLD).
  • Non-alcoholic fatty liver disease is determined by the findings of fatty liver in which deposition of neutral fat on liver cells is observed, but about 90% of patients have simple fatty liver whose symptoms can be improved by proper diet or exercise therapy. However, it is known that approximately 10% of cases of NAFLD are nonalcoholic steatohepatitis (NASH), which represents a progressive condition.
  • NAFLD nonalcoholic steatohepatitis
  • non-alcoholic steatohepatitis NASH
  • fatty hepatitis accompanied by inflammatory findings or fibrosis in liver tissue is generally observed due to degeneration or necrosis of hepatocytes in which fat is accumulated.
  • risk factors such as obesity, metabolic syndrome such as diabetes, hyperlipidemia, hypertension, hyperuricemia, etc.
  • progression from fatty liver to non-alcoholic steatohepatitis furthermore, more dangerous liver fibrosis, liver cirrhosis (liver cirrhosis)
  • liver cirrhosis liver cirrhosis
  • liver fibrosis refers to a disease in which scars occur in liver tissue due to excessive accumulation of connective tissue such as collagen in the tissue due to repeated damage and regeneration of liver tissue in a chronic inflammatory state.
  • connective tissue such as collagen
  • liver fibrosis unlike cirrhosis, is reversible, consists of thin fibrils, and does not have nodule formation.
  • this liver fibrosis mechanism continues repeatedly, crosslinking between connective tissues increases, thick fibrils accumulate, and the normal structure of the liver lobules is lost to form nodules. It progresses to irreversible liver cirrhosis.
  • Substances that inhibit hepatic fibrosis such as penicillamine, 16,16-dimethyl prostiglidin E2, biphenyl dimethyldicarboxylic acid, colchicine, glucocorticoid, malotilate, gamma-interferon, pentoxifylline, pyridine-2,4-dicarboxylic-diethylamide, pyridine-2,4-dicarboxylic-di(2-methoxy) Ethyl) amide and the like have been developed, but when applied clinically, the action is weak or the side effects are severe, so there is currently no treatment for liver fibrosis that is used clinically.
  • Korean Patent No. 2269748 discloses a kit for tracking and diagnosing progressive chronic hepatitis and the degree of hepatic fibrosis through measurement of asialo alpha-1 acid glycoprotein, a marker of liver cell damage, and its use.
  • Korean Patent No. 2112760 discloses a method for diagnosing liver disease using TM4SF5 protein expression change and a method for screening a therapeutic agent for liver disease, and Korean Patent No.
  • a therapeutic composition for preventing or treating liver fibrosis induced by non-alcoholic steatohepatitis containing the expression inhibitor of lipocalin 2 or its receptor of the present invention as an active ingredient, a method for inhibiting activation of hepatic cells by treating with the lipocalin 2 receptor inhibitor, a method for diagnosing liver fibrosis induced by non-alcoholic steatohepatitis using lipocalin 2, and a method for screening a substance for treating liver fibrosis have not been disclosed.
  • the present invention was derived from the above needs, and after inducing leptin-deficient non-alcoholic steatohepatitis and hepatic fibrosis by consuming a high-fat diet in ob/ob mice lacking leptin, it was confirmed that lipocalin 2 (LCN2) content in blood and LCN2, ⁇ -SMA, MMP9, and pSTAT3 content in liver tissue were increased, and recombinant LCN2 protein was injected into ob/ob mice lacking leptin.
  • LCN2 lipocalin 2
  • the contents of MMP9 and pSTAT3 were increased, and in an animal model lacking both leptin and LCN2, it was confirmed that the contents of MMP9 in blood, MMP9, and pSTAT3 in liver tissue were reduced, and hepatic fibrosis was reduced.
  • LPS was treated in mouse macrophages and cultured medium (LTM) was treated with hepatic stellate cells, the expression of hepatic fibrosis-related factors increased. By confirming that it is inhibited, the present invention was completed.
  • the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis induced by non-alcoholic steatohepatitis, containing lipocalin 2 or an expression inhibitor of its receptor as an active ingredient.
  • the present invention provides a method for inhibiting the activation of hepatic stellate cells, including the step of treating a lipocalin 2 receptor inhibitor in a non-human subject.
  • step 2) treating the activated liver stellate cells of step 1) with a candidate substance for liver fibrosis treatment;
  • step 2) measuring the expression level of lipocalin 2 protein or mRNA
  • step 3) after step 3), comparing the candidate substance of step 2) to the untreated control group, selecting a candidate substance for treating liver fibrosis that further reduces the expression of lipocalin 2 protein or mRNA;
  • the present invention provides a composition for diagnosing liver fibrosis induced by non-alcoholic steatohepatitis lacking leptin containing an antibody that specifically binds to lipocalin 2 protein as an active ingredient, and a kit for diagnosing liver fibrosis induced by non-alcoholic steatohepatitis lacking leptin containing the composition.
  • the present invention provides a kit for diagnosing hepatic fibrosis induced by non-alcoholic steatohepatitis lacking leptin, including an agent for quantitatively detecting lipocalin 2 mRNA.
  • step 2) comparing the amount of protein or mRNA measured in step 1) with a normal control group; providing information on the amount of lipocalin 2 protein or mRNA for diagnosis of hepatic fibrosis induced by non-alcoholic steatohepatitis lacking leptin.
  • the present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis induced by non-alcoholic steatohepatitis containing lipocalin 2 or its receptor expression inhibitor as an active ingredient, a method for inhibiting activation of hepatic stellate cells by treating the lipocalin 2 receptor inhibitor, and a method for diagnosing non-alcoholic steatohepatitis-induced liver fibrosis using lipocalin 2 and screening for a substance for treating liver fibrosis,
  • MMP9 and pSTAT3 contents are increased to induce hepatic fibrosis
  • DKO animal model lacking leptin and lipocalin 2 genes
  • the contents of MMP9 and pSTAT3 were reduced, and hepatic fibrosis was improved.
  • Figure 1 shows changes in liver tissue according to diet (A), staining results (B) and quantification results (C) to confirm the degree of adipogenesis and liver fibrosis in wild-type mice (WT) and ob/ob mice lacking leptin.
  • WT wild-type mice
  • HFD high fat diet mouse.
  • *** means that there is a statistically significant difference from each other, p ⁇ 0.001.
  • Figure 2 shows changes in LCN2 mRNA expression in liver tissue and LCN2 mRNA expression in liver tissue according to diet in wild-type mice (WT) and ob/ob mice lacking leptin, and changes in LCN2 and ⁇ -SMA protein expression in liver tissue (B).
  • ND is a normal diet mouse
  • HFD is a high fat diet mouse.
  • *, **, *** means that there is a statistically significant difference from each other, * means p ⁇ 0.05, ** means p ⁇ 0.01, *** p ⁇ 0.001.
  • Figure 3 shows changes in MMP9 protein expression and STAT3 phosphorylation in liver tissue according to diet in wild-type mice (WT) and ob/ob mice lacking leptin (A), results of LCN2+MMP9 Duolink analysis (B), and results of quantifying the area of LCN2+MMP9-positive cells (C).
  • WT wild-type mice
  • A ob/ob mice lacking leptin
  • B results of LCN2+MMP9 Duolink analysis
  • C results of quantifying the area of LCN2+MMP9-positive cells
  • ND is a normal diet mouse
  • HFD is a high fat diet mouse.
  • *, **, *** means that there is a statistically significant difference from each other, * means p ⁇ 0.05, ** means p ⁇ 0.01, *** p ⁇ 0.001.
  • Figure 4 shows the results of confirming the role of LCN2 in wild-type mice (WT) and leptin-deficient ob/ob mice by treating them with recombinant LCN2 (rLCN2).
  • A is the result of confirming the LCN2, MMP9 protein expression change and the degree of STAT3 phosphorylation in liver tissue by Western blot
  • B is the result of quantification thereof.
  • ND is a normal diet mouse
  • HFD is a high fat diet mouse.
  • *, **, *** means that there is a statistically significant difference from each other, * means p ⁇ 0.05, ** means p ⁇ 0.01, *** p ⁇ 0.001.
  • FIG. 5 is a result confirming the effect of LCN2 deficiency and leptin treatment in ob/ob mice.
  • A is the result of confirming the content of leptin and LCN2 in the blood
  • B is the result of confirming the expression of liver fibrosis-related factors in liver tissue by Western blot
  • C is the result of quantifying them.
  • D is a photograph confirming the result of staining the liver tissue with Nile red
  • (E) is the result of quantifying the stained area with Nile red.
  • DKO is a mouse doubly lacking leptin and LCN2. *** means that there is a statistically significant difference from each other, p ⁇ 0.001.
  • FIG. 6 is a result confirming the effect of LCN2 deficiency on liver fibrosis.
  • A is the result of confirming the protein expression of LCN2 and liver fibrosis-related factors in liver tissue
  • (B) is the result of analyzing the content of MMP9 in blood
  • (C) is the result of confirming the degree of liver fibrosis by staining with Sirius red.
  • HFD means high fat diet administration. *, *** means that there is a statistically significant difference between each other, * is p ⁇ 0.05, *** is p ⁇ 0.001.
  • Figure 7 confirms changes in the expression of liver fibrosis-related factors in human cell line LX-2 cells according to LPS-treated medium (LTM) treatment of RAW 264.7 cells treated with LPS.
  • LTM LPS-treated medium
  • A confirms changes in LCN2 and TNF ⁇ content of LTM
  • B is a schematic diagram of LTM treatment of LX-2 cells
  • C confirms changes in ⁇ -SMA expression in LTM-treated LX-2 cell lysates
  • (D) is the result of confirming the content of MMP9 secreted from LTM-treated LX-2 cells.
  • *** means that there is a statistically significant difference from each other, p ⁇ 0.001.
  • Figure 8 confirms changes in the expression of liver fibrosis-related factors according to LTM treatment in ob/ob mouse primary hepatic stellate cells (mHSC).
  • A is the result of confirming the mRNA expression of LCN2 and collagen 1 ⁇ in liver tissue
  • B is the result of confirming the change in protein expression in the lysate and culture medium of LTM-treated mHSC.
  • *, **, *** means that there is a statistically significant difference from each other, * means p ⁇ 0.05, ** means p ⁇ 0.01, *** p ⁇ 0.001.
  • FIG. 9 shows the results of confirming the effect of suppressing the expression of the LCN2 receptor 24p3R in ob/ob mouse primary hepatic stellate cells (mHSC) activated by LTM treatment.
  • A is the result of confirming the protein expression change according to LTM and si24p3R treatment in the cell lysate and culture medium
  • B is the result of confirming the degree of fat droplet reduction by staining with Nile red.
  • *, **, *** means that there is a statistically significant difference from each other, * means p ⁇ 0.05, ** means p ⁇ 0.01, *** p ⁇ 0.001.
  • the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis induced by non-alcoholic steatohepatitis, containing lipocalin 2 or an expression inhibitor of its receptor as an active ingredient.
  • the expression inhibitor of lipocalin 2 or its receptor may be an antibody or aptamer specific to lipocalin 2 or its receptor; And siRNA, shRNA, or miRNA specific for lipocalin 2 or its receptor gene; It may be one or more selected from the group consisting of, preferably, siRNA specific for the lipocalin 2 receptor gene, more preferably one or more si24p3R selected from the group consisting of SEQ ID NOs: 1 to 4, but is not limited thereto.
  • the liver fibrosis induced by nonalcoholic steatohepatitis is preferably induced by nonalcoholic steatohepatitis lacking leptin, but is not limited thereto.
  • the pharmaceutical composition of the present invention may include pharmaceutically acceptable carriers, excipients, or diluents in addition to active ingredients, and these carriers are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, and methylhydroxybenzoate. ethyl alcohol, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto.
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
  • a suitable dosage of the pharmaceutical composition according to the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and response sensitivity.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. may be administered.
  • composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the inhibition and treatment of hepatic fibrosis.
  • the concentration of the active ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the required period, etc., and is not limited to a specific range of concentration.
  • the pharmaceutical composition of the present invention can be prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier or excipient according to a method that can be easily performed by those skilled in the art, or by placing it in a multi-dose container.
  • the formulation may be prepared as any one formulation selected from injections, creams, sprays, ointments, warnings, lotions, liniments, pastas, and cataplasmas, and may additionally include a dispersing agent or a stabilizer.
  • the present invention provides a method for inhibiting the activation of hepatic stellate cells, including the step of treating a lipocalin 2 receptor inhibitor in a non-human subject.
  • the lipocalin 2 receptor inhibitor may be an antibody or aptamer specific for the lipocalin 2 receptor; siRNA, shRNA or miRNA specific for the lipocalin 2 receptor gene; And it may be at least one selected from the group consisting of a receptor antagonist, preferably a siRNA specific to the lipocalin 2 receptor gene, but is not limited thereto.
  • Inhibiting the activation of hepatic stellate cells has the effect of suppressing the progress of hepatic fibrosis induced by the activation of hepatic stellate cells.
  • step 2) treating the activated liver stellate cells of step 1) with a candidate substance for liver fibrosis treatment;
  • step 2) measuring the expression level of lipocalin 2 protein or mRNA
  • step 3) after step 3), comparing the candidate substance of step 2) to the untreated control group, selecting a candidate substance for treating liver fibrosis that further reduces the expression of lipocalin 2 protein or mRNA;
  • the culture medium after treating the mouse macrophages in step 1) with LPS is preferably obtained by treating RAW 264.7 cells, which are mouse macrophages, with LPS for 20 to 28 hours, and more preferably RAW 264.7 cells. It is obtained by treating LPS for 24 hours, but is not limited thereto.
  • step 3 in addition to lipocalin 2 protein or mRNA expression, MMP9 and ⁇ -SMA protein and mRNA expression; And the level of one or more selected from the degree of STAT3 phosphorylation can be measured and compared with the control group.
  • the substance for treating liver fibrosis is a substance for treating liver fibrosis induced by non-alcoholic steatohepatitis lacking leptin, but is not limited thereto.
  • the present invention provides a composition for diagnosing hepatic fibrosis induced by non-alcoholic steatohepatitis lacking leptin, comprising an antibody that specifically binds to lipocalin 2 protein as an active ingredient.
  • the antibody may include both monoclonal antibodies and polyclonal antibodies.
  • the diagnostic composition of the present invention may be provided in an immobilized state on a suitable carrier or support using various methods known in the art.
  • suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposomes, carboxymethyl cellulose, polyacrylamide, polysterine, gabbro, filter paper, ion exchange resins, plastic films, plastic tubes, glass, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylon, cups, flat packs.
  • Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes.
  • the support may have any conceivable shape, for example spherical (bead), cylindrical (inside a test tube or well), planar (sheet, test strip).
  • the present invention provides a kit for diagnosing liver fibrosis induced by non-alcoholic steatohepatitis lacking leptin comprising the composition.
  • the diagnostic kit may be provided in the form of, for example, a lateral flow assay kit based on immunochromatography to detect the LCN2 protein in serum samples.
  • the lateral flow assay kit may be provided with a sample pad to which a serum sample is applied, a release pad coated with an antibody for detection, a membrane (e.g., nitrocellulose) or strip for development in which the sample is moved and separated and an antigen-antibody reaction occurs, and an absorption pad.
  • the present invention provides a kit for diagnosing hepatic fibrosis induced by non-alcoholic steatohepatitis lacking leptin, including an agent for quantitatively detecting lipocalin 2 mRNA.
  • the mRNA quantitative detection is preferably performed through a method selected from among RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting, and DNA chip, but is not limited thereto.
  • the preparation may include, but is not limited to, primers for preparing cDNA corresponding to lipocalin 2 mRNA, reverse transcriptase, Taq polymerase, PCR primers, and dNTPs.
  • step 2) comparing the amount of protein or mRNA measured in step 1) with a normal control group; providing information on the amount of lipocalin 2 protein or mRNA for diagnosis of hepatic fibrosis induced by non-alcoholic steatohepatitis lacking leptin.
  • the sample is preferably any one selected from the group consisting of liver tissue, serum, plasma, blood and urine, but is not limited thereto.
  • step 1) the amount of MMP9 protein or mRNA may be additionally measured in addition to lipocalin 2, but is not limited thereto.
  • mice Male wild-type (WT) C57BL/6J and leptin-deficient ob/ob mice were purchased from Central Laboratory Animal, Inc. (Seoul, Korea).
  • heterozygous ob/ob mice were mated with LCN2 knockout mice (Jackson Laboratory, Maryland, USA) to obtain heterozygous LCN2+/-/ob+/- mice, which were further mated to construct DKO mice, and gDNA (genomic DNA) isolated from the tail end of 4-week-old mice was used to confirm genotype through polymerase chain reaction (PCR). .
  • LCN2 knockout mice Jackson Laboratory, Maryland, USA
  • mice All mice were housed in a virus-free facility with a 12-hour light/dark cycle, and animal experiments were performed according to the guidelines for the use of laboratory animals at the National Institute of Health.
  • HFD high-fat diet
  • mice at least 5 weeks of age were fed a high-fat diet for 20 weeks, and were fed a normal diet (ND) as a control group.
  • ND normal diet
  • rLCN2 lipocalin 2
  • 22-week-old male WT and ob/ob mice were intraperitoneally injected with rLCN2 (2.2 mg/mL) for 3 days prior to sacrifice.
  • Restriction enzymes (Nde1, EcoR1 and Nhe1) were purchased from New England BioLabs (Ipswich, MA, USA). Agarose, kanamycin, isopropyl- ⁇ -D-thiogalactoside, leupeptin and trypsin inhibitors were purchased from Sigma-Aldrich.
  • BL21(DE3) E. coli competent cells and BCA protein assay kits were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). LB broth was purchased from Difco (Sparks, MD, USA).
  • the pET28a-mCherry-LCN2 vector was constructed by digesting the 'mCherry and LCN2'-encoding genes (1314 bp; inserted into the pUC57-mini vector obtained from GenScript, Piscataway, NJ, USA) with Nde1 and EcoR1 and ligation into the linearized pET28a vector (Merck Millipore, Billerica, MA, USA).
  • pET28a-SUMO-leptin-ABD a SUMO-tagged leptin-ABD expression vector named pET28a-SUMO-leptin-ABD was prepared by digesting the 'human leptin' gene (510 bp) from pUC-57 mini-leptin (GenScript) with Nde1 and Nhe1 and ligation to the linearized pET28a-SUMO-ABD vector.
  • starter cultures were prepared by inoculating colonies of pET28a-mCherry-LCN2-transformed BL21(DE3) E. coli cells into 50 mL LB medium (containing 80 ⁇ g/mL kanamycin), and incubated at 37° C. for at least 16 hours with shaking at 250 rpm. After cultivation, the starter culture was added to 1 L of fresh LB medium (containing 80 ⁇ g/mL kanamycin) and cultured under the same culture conditions. UV absorbance was measured at 600 nm (OD600), and when the absorbance value reached 1, isopropyl- ⁇ -D-thiogalactoside was added (final concentration: 0.5 mM) to induce protein expression.
  • the supernatant containing the solubility mCherry-lcn2 loads the talon ® metal affinity resin (Clontech, Mountain View, CA, USA) and washing with a washing charging buffer (20mm PBS and 300mm nacl; pH 7), and then the combined mcherry-lcn2 is a dissolution buffer (20mm) PBS, 300mm NACL, and 300mm imidazole; pH 7) were fulfilled.
  • mCherry-LCN2 and leptin-ABD were expressed as soluble proteins from E. coli , and the average production yields of the final products were 25 and 5.7 mg/L, respectively.
  • Serum LCN2, MMP9, and leptin were measured using mouse LCN2, MMP9, and leptin (R&D Systems, Minneapolis, MN, USA) ELISA kits according to the manufacturer's protocol.
  • mice were anesthetized with zoletil and perfused with 4% paraformaldehyde diluted in 0.1 M PBS. Tissues including liver and pancreas were excised, fixed in 4% paraformaldehyde at 4° C. for 12 hours, embedded in paraffin, and cut into 5 ⁇ m sections.
  • Nile red (Sigma-Aldrich) staining to check liver triglyceride (TG), Picro-Sirius red (SigmaAldrich) staining and hematoxylin and eosin (H&E) staining to check histological fibrosis were performed on frozen and paraffin-embedded liver sections, respectively.
  • Nile red positive area 250 ⁇ 250 ⁇ m 2
  • IMT i-Solution, Inc. Vancouver, BC, Canada
  • rehydrated sections were stained with hematoxylin for 8 minutes and then with Sirius red solution for 1 hour. After staining, the sections were directly dehydrated in 100% alcohol, removed with xylene, and mounted in a resin medium. Sections were visualized with a BX53 light microscope (Olympus, Tokyo, Japan).
  • Sirius red-positive areas in 5-14 fields were quantified with ImageJ software (version 1.52a, NIH, Bethesda, MD, USA). Data are expressed as percentage area stained positive for Sirius red.
  • NAFLD Nonalcoholic Fatty Liver Disease
  • the NAFLD activity score was defined as the unweighted sum of the scores for hepatic steatosis (0-3), lobular inflammation (0-3) and hepatocyte expansion (0-2).
  • non-alcoholic fatty liver disease score criteria score steatosis (steatosis) ⁇ 5% hepatocyte involved 0 5-33% hepatocyte involved +1 34-66% hepatocyte involved +2 >66% hepatocyte involved +3 lobular inflammation (lobular inflammation) No foci 0 1 foci per X 200 fields +1 2 to 4 foci per X 200 field +2 >4 foci per X 200 fields +3 Hepatocellular balloon degeneration (hepatocellular ballooning) None 0 few ballooned cells +1 many cells/prominent ballooning +2
  • Duolink proximity ligation assay (PLA; Sigma-Aldrich) was used to analyze the interaction between LCN2 and MMP9 in paraffin-embedded liver sections of ob/ob mice.
  • the sections were reacted with goat anti-LCN2 (ab31289) and rabbit anti-MMP9 (ab38898) or anti-LCN2 (R&D 1857) and anti-24p3R (PA5-20543). Then, the sections were reacted with PLA probe anti-rabbit MINUS (DUO92005) and PLA probe anti-goat Plus (DUO92003) at 37° C. for 1 hour. After performing Duolink analysis, sections were stained with DAPI (Invitrogen), washed, and mounted on cover slips using mounting solution. Slice images were confirmed with a BX51-DSU microscope (Olympus).
  • RAW 264.7 a macrophage cell line, and LX-2 (SigmaAldrich), a human hepatic stellate cell (HSC), were cultured in a DMEM medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C, 5% CO 2 in a humidified incubator.
  • DMEM medium Gibco, Grand Island, NY, USA
  • fetal bovine serum Gibco
  • penicillin/streptomycin Gabco
  • LPS lipopolysaccharide
  • mHSC primary mouse HSC
  • Hepatocytes and mHSCs were isolated from ob/ob mice by enzymatic digestion and density gradient centrifugation. Briefly, cells were isolated from liver tissue by density gradient centrifugation in a solution containing DNase I (Thermo Fisher Scientific) after in situ liver perfusion with EGTA, pronase (Roche Diagnostics GmbH, Mannheim, Germany) and collagenase NB4G (Nordmark Pharma GmbH, Uetersen, Germany).
  • DNase I Thermo Fisher Scientific
  • the dispersed cell suspension was filtered through a 70 ⁇ m cell strainer, centrifuged at 100 ⁇ g for 3 minutes, and the cell pellet and supernatant were fractionated into parenchymal (PC) and non-parenchymal (NPC), respectively.
  • PC parenchymal
  • NPC non-parenchymal
  • the PC fraction was washed 4 times in HBSS (Hank's Balanced Salt Solution; Gibco) and lysed in RIPA lysis buffer (50 mM Tris-HCl; pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) containing protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific) for hepatocyte purification.
  • HBSS Hank's Balanced Salt Solution
  • RIPA lysis buffer 50 mM Tris-HCl; pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA
  • protease and phosphatase inhibitor cocktails Thermo Fisher Scientific
  • the NPC fraction was centrifuged at 580 ⁇ g for 10 minutes, the pellet was washed, resuspended in 15% OptiPrep (Sigma-Aldrich), thoroughly mixed, and transferred to a 15mL centrifugal tube. Then, 5 mL of 11.5% OptiPrep and 2 mL HBSS were carefully layered individually on the cell suspension. The cell fraction was then centrifuged at 1400 x g for 20 minutes and the tube was divided into two layers (mHSC fraction in the first layer and Kupffer cell fraction in the second layer). The Kupffer cell fraction was purified by washing with HBSS and lysing in RIPA lysis buffer.
  • the mHSC fraction was divided in half, half was lysed in RIPA lysis buffer, and the other half was suspended and maintained in DMEM supplemented with 10% fetal bovine serum (Gibco) and antibiotics (Gibco).
  • the purity of mHSC was evaluated and estimated by RT-PCR one day after isolation, and the purity was maintained at 98% or higher.
  • mHSCs were cultured for 24 hours after treatment with 1, 3 or 5 ⁇ g/mL rLCN2.
  • siRNA targets of human or mouse 24p3R were purchased from Bioneer Corp. (Daejeon, Korea). siRNA and scrambled RNA sequences are as set forth in Table 2 below. Cells were transfected with 24p3R siRNA or control scrambled siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.
  • LX-2 cell line is a human-derived cell line
  • human si243R sequence was used, and mHSC cells were ob/ob mouse-derived cells, so mouse si243R sequence was used.
  • mHSC transfected with 24p3R siRNA were seeded into culture inserts (ibidi culture insert 2 well, ibidi GmbH, Martinsried, Germany) at a density of 1 ⁇ 10 5 cells per well. Thereafter, the cells were allowed to attach for 16 hours, the culture insert was removed, and the cells were cultured for 24 hours with serum-starved medium or LTM.
  • LX-2 cells were cultured in 6-well plates at 37° C. in a 5% CO 2 humidified incubator, and upon reaching confluency, cells were transfected with 20 nM 24p3R siRNA. After 24 hours, cells were starved for 16 hours before monolayer scraping. Wounded monolayers were then cultured in serum-starved medium or treated with LTM for 24 hours. Wounds or scrapes were monitored with an Olympus IX71 phase-contrast inverted microscope and areas were calculated using ImageJ software (version 1.52a).
  • RNA isolation and real-time polymerase chain reaction RT-PCR
  • livers were homogenized in T-PER lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for protein extraction.
  • livers were lysed in ice-cold lysis buffer (10 mM HEPES-KOH; pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, protease inhibitors) and high salt extraction buffer (20 mM HEPES-KOH; pH 7.9, 1.5 mM MgCl 2 , 420 mM NaCl, 0.2 mM EDTA, 25% glycerol , protease inhibitor, 0.5 mM DTT), respectively. Protein concentration was determined by BCA assay (Thermo Fisher Scientific).
  • WB western blot
  • IF immunofluorescence
  • IHC immunohistochemistry
  • LCN2 expression of LCN2 was confirmed in non-alcoholic steatohepatitis and liver fibrosis caused by it.
  • Figure 1 when the degree of fat formation was confirmed by staining with Nile red, fatty liver was induced by a high fat diet (HFD), and ob / ob mice, which are leptin-deficient mice, had a normal diet (ND) and high fat diet. Fatty liver was induced in both (HFD).
  • liver fibrosis was significantly progressed in ob/ob mice fed a high-fat diet compared to wild-type mice (WT) and ob/ob mice fed a normal diet.
  • LCN2 expression was increased in ob/ob mice lacking leptin compared to wild-type mice, and the expression of LCN2 was markedly increased in ob/ob mice fed a high-fat diet with advanced hepatic fibrosis.
  • LCN2 expression was significantly increased in liver tissue of ob/ob mice fed a high-fat diet, similar to the change in LCN2 expression in blood.
  • LCN2 plays an important role in ⁇ -SMA-related liver fibrosis signaling in non-alcoholic steatohepatitis lacking leptin.
  • LCN2- and MMP9-positive cells were significantly increased in ob/ob mice fed a high-fat diet.
  • LCN2-related MMP9/STAT3 signaling plays an important role in the progression of liver fibrosis in non-alcoholic fatty liver.
  • FIG. 6A it was confirmed that the phosphorylation of MMP9 and STAT3 was significantly reduced by the lack of LCN2 in liver tissue, and as shown in FIG. 6B, it was confirmed that the content of MMP9 in the blood was also reduced.
  • LPS-treated mouse macrophage RAW 264.7 culture medium LPS-treated medium, LTM
  • FIG. 7A when RAW 264.7 cells were treated with LPS and cultured, it was confirmed that the contents of LCN2, TNF ⁇ , and IL-6 in the culture medium increased.
  • LX-2 cells As a result of treating the culture medium with LX-2 cells, a hepatic cell line, as shown in FIG. Secretion increased.
  • hepatic stellate cells by LTM treatment also occurs in primary mouse hepatic stellate cells (mHSCs) derived from ob/ob mice.
  • mHSCs primary mouse hepatic stellate cells
  • FIG. 8A it was confirmed that the expression of LCN2 in liver tissue and the mRNA expression of collagen 1 ⁇ were increased, and as shown in FIG. 8B, LTM treatment increased the expression of LCN2, its receptor 247p3R, ⁇ -SMA and STAT3 protein and the degree of phosphorylation of STAT3 in the cell lysate, and it was confirmed that MMP9 and LCN2 protein expression increased in the cell culture medium.
  • LCN2 directly regulates the activation of hepatic cells through STAT3-related signaling and secretion of MMP9.
  • Example 7 In hepatic stellate cells activated by LTM treatment, cell migration promotion effect of hepatic stellate cells through 24p3R of LCN2
  • hepatic stellate cells migrate to damaged liver tissue and secrete MMP9 to promote hepatic fibrosis. As shown in FIG. 10 , it was confirmed that the increased cell migration by LTM treatment was reduced by si24p3R treatment.
  • LCN2 activates hepatic cells through LCN2/24p3R in the absence of leptin, promotes cell migration, and suppresses 24p3R expression to inhibit liver stellate cell activation.

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Abstract

La présente invention concerne : une composition pharmaceutique pour la prévention ou le traitement de la fibrose hépatique induite par la stéatohépatite non alcoolique, contenant, en tant que principe actif, un inhibiteur d'expression de la lipocaline 2 ou un récepteur de celle-ci ; un procédé d'inhibition de l'activation de cellules stellaires hépatiques par traitement avec l'inhibiteur du récepteur de la lipocaline 2 ; et un procédé de criblage d'agents pour le diagnostic de la fibrose hépatique induite par la stéatohépatite non alcoolique et le traitement de la fibrose hépatique à l'aide de la lipocaline 2. La composition contenant, en tant que principe actif, un inhibiteur du récepteur de la lipocaline 2 ou un inhibiteur de l'expression de la lipocaline 2, de la présente invention, peut être utilisée en tant qu'agent thérapeutique pour la fibrose hépatique induite par la stéatohépatite non alcoolique. La présente invention peut fournir un procédé de criblage d'agents pour le diagnostic de la fibrose hépatique induite par la stéatohépatite non alcoolique et le traitement de la fibrose hépatique à l'aide de la lipocaline 2, et peut fournir un procédé d'inhibition de l'activation de cellules stellaires hépatiques par traitement avec un inhibiteur du récepteur de la lipocaline 2.
PCT/KR2023/000979 2022-01-20 2023-01-19 Composition pharmaceutique pour la prévention ou le traitement de la fibrose hépatique induite par la stéatohépatite non alcoolique, contenant, en tant que principe actif, un inhibiteur d'expression de la lipocaline 2 ou un récepteur de celle-ci WO2023140664A1 (fr)

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