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WO2025225410A1 - Agent prophylactique ou thérapeutique contre la fibrose hépatique, la cirrhose et le cancer du foie, et biomarqueur de pression intrasinusoïde - Google Patents

Agent prophylactique ou thérapeutique contre la fibrose hépatique, la cirrhose et le cancer du foie, et biomarqueur de pression intrasinusoïde

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Publication number
WO2025225410A1
WO2025225410A1 PCT/JP2025/014379 JP2025014379W WO2025225410A1 WO 2025225410 A1 WO2025225410 A1 WO 2025225410A1 JP 2025014379 W JP2025014379 W JP 2025014379W WO 2025225410 A1 WO2025225410 A1 WO 2025225410A1
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WO
WIPO (PCT)
Prior art keywords
ctgf
integrin
liver
group
inhibitor
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Pending
Application number
PCT/JP2025/014379
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English (en)
Japanese (ja)
Inventor
隼人 疋田
聖也 加藤
徹郎 竹原
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Osaka NUC
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Osaka University NUC
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Filing date
Publication date
Application filed by Osaka University NUC filed Critical Osaka University NUC
Publication of WO2025225410A1 publication Critical patent/WO2025225410A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • 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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • 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
    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • 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

Definitions

  • the present invention relates to a preventive or therapeutic agent for liver fibrosis, liver cirrhosis, and liver cancer, an intrasinusoidal pressure biomarker, etc.
  • Non-Patent Document 1 There have been reported cases in which liver pathology progresses even when liver cell death is inhibited.
  • the objective of the present invention is to provide a technology for preventing or treating the progression of liver pathologies that lead to liver cancer by targeting a phenomenon different from hepatocyte death.
  • the inventors have conducted extensive research, focusing on intrasinusoidal pressure and finding that increased intrasinusoidal pressure promotes liver fibrosis and liver carcinogenesis. Based on this finding, the inventors have conducted further research and found that the above-mentioned problems can be solved by providing an agent for preventing or treating at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer in subjects suspected of having increased intrasinusoidal pressure, which agent contains at least one agent selected from the group consisting of a CTGF inhibitor and an integrin ⁇ V inhibitor. Furthermore, the inventors have identified 10 genes as intrasinusoidal pressure biomarkers. The present invention encompasses the following aspects.
  • Item 1 A preventive or therapeutic agent for at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer in a subject suspected of having increased intrasinusoidal pressure, comprising at least one agent selected from the group consisting of a CTGF inhibitor and an integrin ⁇ V inhibitor.
  • Section 1A A method for preventing or treating at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer, comprising administering at least one agent selected from the group consisting of a CTGF inhibitor and an integrin ⁇ V inhibitor to a subject suspected of having increased intrasinusoidal pressure.
  • Section 1B At least one agent selected from the group consisting of a CTGF inhibitor and an integrin ⁇ V inhibitor for use in the prevention or treatment of at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer in a subject suspected of having increased intrasinusoidal pressure.
  • Section 1C Use of at least one agent selected from the group consisting of CTGF inhibitors and integrin ⁇ V inhibitors for the manufacture of an agent for the prevention or treatment of at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer in a subject suspected of having increased intrasinusoidal pressure.
  • at least one agent selected from the group consisting of CTGF inhibitors and integrin ⁇ V inhibitors for the manufacture of an agent for the prevention or treatment of at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer in a subject suspected of having increased intrasinusoidal pressure.
  • Section 1D Use of at least one agent selected from the group consisting of CTGF inhibitors and integrin ⁇ V inhibitors as a preventive or therapeutic agent for at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer in a subject suspected of having increased intrasinusoidal pressure.
  • the CTGF inhibitor is at least one selected from the group consisting of a CTGF function inhibitor and a CTGF expression inhibitor, and/or the integrin ⁇ V inhibitor is at least one selected from the group consisting of an integrin ⁇ V function inhibitor and an integrin ⁇ V expression inhibitor.
  • the CTGF inhibitor is at least one selected from the group consisting of a low molecular weight compound, a polynucleotide targeting CTGF, an expression cassette for the polynucleotide, a peptide, a protein, and an antibody
  • the integrin ⁇ V inhibitor is at least one selected from the group consisting of a low molecular weight compound, a polynucleotide targeting integrin ⁇ V, an expression cassette for the polynucleotide, a peptide, a protein, and an antibody.
  • Item 4 The preventive or therapeutic agent according to any one of Items 1 to 3, wherein the CTGF inhibitor is an inhibitor of CTGF in hepatic sinusoidal endothelial cells, and/or the integrin ⁇ V inhibitor is an inhibitor of integrin ⁇ V in hepatic sinusoidal endothelial cells.
  • Item 5 A method for treating liver cancer, comprising the CTGF inhibitor and the disease being liver cancer, or comprising the integrin ⁇ V inhibitor. Item 5. The preventive or therapeutic agent according to any one of Items 1 to 4.
  • Item 6 The preventive or therapeutic agent according to any one of Items 1 to 5, wherein the subject has at least one condition selected from the group consisting of congestive liver damage, liver cirrhosis, Budd-Chiari syndrome, portal vein thrombosis, sinusoidal obstruction syndrome, and post-Fontan syndrome.
  • Section 7 A method for examining at least one selected from the group consisting of intrasinusoidal pressure, liver fibrosis, cirrhosis, and liver cancer, comprising the step of detecting the protein and/or mRNA of at least one gene selected from the group consisting of CTGF, ESM1, ANGPT2, PLAU, EDN1, PDGFB, ADM, CXCL9, FBLN2, and INHBB in a biological sample collected from a subject.
  • Item 9. The step (2) Item 9.
  • Item 10 The preventive or therapeutic agent according to any one of Items 1 to 6, wherein the subject is a subject who has been determined by the method described in Item 9 to have increased intrasinusoidal pressure and/or to be suffering from at least one condition selected from the group consisting of liver fibrosis, liver cirrhosis, and liver cancer.
  • At least one diagnostic agent selected from the group consisting of intrasinusoidal pressure, liver fibrosis, cirrhosis, and liver cancer, comprising a binding molecule for the protein and/or mRNA of at least one gene selected from the group consisting of CTGF, ESM1, ANGPT2, PLAU, EDN1, PDGFB, ADM, CXCL9, FBLN2, and INHBB.
  • Section 11A Use of a binding molecule for the protein and/or mRNA of at least one gene selected from the group consisting of CTGF, ESM1, ANGPT2, PLAU, EDN1, PDGFB, ADM, CXCL9, FBLN2, and INHBB in the manufacture of a diagnostic agent for at least one gene selected from the group consisting of intrasinusoidal pressure, liver fibrosis, cirrhosis, and liver cancer.
  • Section 11B Use of a binding molecule for the protein and/or mRNA of at least one gene selected from the group consisting of CTGF, ESM1, ANGPT2, PLAU, EDN1, PDGFB, ADM, CXCL9, FBLN2, and INHBB as a diagnostic agent for at least one gene selected from the group consisting of intrasinusoidal pressure, liver fibrosis, cirrhosis, and liver cancer.
  • Item 12 The test agent according to Item 11, which is a companion diagnostic agent for the preventive or therapeutic agent according to any one of Items 1 to 6.
  • the present invention can provide a technology for preventing or treating the progression of liver pathologies leading to liver cancer, targeting a phenomenon other than hepatocyte death, as well as a companion diagnostic technology for such a technology.
  • it can provide a preventive or therapeutic agent for at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer in a subject suspected of having increased intrasinusoidal pressure; a method for testing at least one disease selected from the group consisting of intrasinusoidal pressure, liver fibrosis, cirrhosis, and liver cancer; at least one diagnostic agent selected from the group consisting of intrasinusoidal pressure, liver fibrosis, cirrhosis, and liver cancer; a companion diagnostic for the above preventive or therapeutic agent; etc.
  • Test Examples 1-3 The experimental results of Test Examples 1-3 are summarized below.
  • the figure shows immunostaining results (upper left) indicating that CDH5, used as Cre, is expressed in LSECs in Test Example 1-4, RT-qPCR results for CTGF (upper right), and the protocol (lower).
  • 1 shows stained images of liver tissue sections in Test Examples 1-4.
  • 1 shows the results of serum ALT measurement (top) and the quantification results of stained areas of liver tissue sections (bottom) in Test Example 1-4.
  • 1 shows the results of portal vein pressure measurement (top) and RT-qPCR results of capillarization markers (bottom) in Test Example 1-4.
  • 1 shows the results of measuring the rate of liver tumor formation in Test Examples 1-4.
  • the protocols for Test Examples 1-6 are shown below.
  • FIG. 1 shows stained images of liver tissue sections in Test 1-6-2. 1 shows the quantification results of stained areas of liver tissue sections (top row), the measurement results of serum ALT levels, intrahepatic hydroxyproline levels, and portal vein pressure (middle row), and the RT-qPCR results of fibrosis markers and capillarization markers (bottom row) in Test 1-6-2. RT-qPCR results for 10 biomarkers in studies 2-4 are shown. 1 shows the results of a correlation analysis between the expression levels of 10 biomarkers and portal vein pressure in Tests 2-4.
  • 1 shows the results of serum ELISA for three biomarkers in Test Examples 2-6. 1 shows the calculation results of serum N-terminal CTGF levels in Test Example 2-7. 1 shows the results of single-cell analysis in Test Example 3-1-1. The images of HE staining and Sirius red staining of the surgically resected specimen in Test Example 3-1-2, as well as the results of Western blotting, are shown. 1 shows the results of single-cell analysis in Test Example 3-1-3. 1 shows the results of single-cell-based spatial gene expression analysis in Test Example 3-1-4. 1 shows HE staining images of surgically resected specimens, mRNA mapping images, and mRNA expression levels in Test Example 3-1-5.
  • Sirius red staining, mRNA mapping image, and mRNA expression level are shown for Test Example 3-1-6.
  • 1 shows the results of spatial gene expression analysis in Test Example 3-2-1.
  • 1 shows an mRNA mapping image and mRNA expression levels in Test Example 3-2-2.
  • 1 shows an mRNA mapping image and mRNA expression levels in Test Example 3-2-3.
  • 1 shows the results of serum ELISA for biomarkers in Test Example 4-1.
  • 1 shows the correlation between serum EDN1 and FBLN2 levels and liver stiffness in FALD cases in Test Example 4-2.
  • the present invention relates to an agent for preventing or treating at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer in a subject suspected of having increased intrasinusoidal pressure (sometimes referred to herein as the "agent of the present invention"), which comprises at least one agent selected from the group consisting of a CTGF inhibitor and an integrin ⁇ V inhibitor. This will be described below.
  • the target CTGF gene for inhibition is a gene encoding connective tissue growth factor.
  • the integrin ⁇ V gene is a gene encoding integrin ⁇ V.
  • the target CTGF (CTGF protein, CTGF mRNA) for inhibition is an expression product of the CTGF gene, and is CTGF protein or CTGF mRNA expressed by an organism or its cells (particularly, hepatic sinusoidal endothelial cells) to which the agent of the present invention is applied.
  • the target integrin ⁇ V (integrin ⁇ V protein, integrin ⁇ V mRNA) for inhibition is an expression product of the integrin ⁇ V gene, and is integrin ⁇ V protein or integrin ⁇ V mRNA expressed by an organism or its cells (particularly, hepatic sinusoidal endothelial cells) to which the agent of the present invention is applied. Therefore, the target CTGF protein/mRNA and integrin ⁇ V protein/mRNA for inhibition can be changed as necessary depending on the target organism species.
  • the biological species is not particularly limited, and examples thereof include animals, such as various mammals, including humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.
  • the amino acid and nucleotide sequences of CTGF protein/mRNA and integrin ⁇ V protein/mRNA derived from various biological species are known.
  • the human CTGF gene is identified by NCBI gene ID 1490, and the amino acid sequence of the human CTGF protein is, for example, the amino acid sequence shown in SEQ ID NO: 1, and the nucleotide sequence of the human CTGF mRNA is, for example, the nucleotide sequence shown in SEQ ID NO: 2.
  • the human integrin ⁇ V gene is identified by NCBI gene ID 3685, and the amino acid sequence of the human integrin ⁇ V protein is, for example, the amino acid sequence shown in SEQ ID NO: 3, and the nucleotide sequence of the human integrin ⁇ V mRNA is, for example, the nucleotide sequence shown in SEQ ID NO: 4.
  • the amino acid and nucleotide sequences of various species can be obtained or estimated from the above information.
  • CTGF protein/mRNA and integrin ⁇ V protein/mRNA may also include the above-mentioned splicing variants.
  • the CTGF protein to be inhibited may have amino acid mutations such as substitutions, deletions, additions, and insertions, as long as it retains its original properties, i.e., the ability to proliferate and differentiate chondrocytes.
  • the CTGF mRNA to be inhibited may also have base mutations such as substitutions, deletions, additions, and insertions, as long as the protein translated from the mRNA retains its original properties, i.e., the ability to proliferate and differentiate chondrocytes.
  • the integrin ⁇ V protein to be inhibited may have amino acid mutations such as substitutions, deletions, additions, and insertions, so long as it retains its original properties, i.e., the ability to form a membrane protein (integrin) by heterodimerizing with the integrin ⁇ chain and the ability to bind to a ligand.
  • the integrin ⁇ V mRNA to be inhibited may also have base mutations such as substitutions, deletions, additions, and insertions, so long as the protein translated from the mRNA retains its original properties, i.e., the ability to form a membrane protein (integrin) by heterodimerizing with the integrin ⁇ chain and the ability to bind to a ligand.
  • Amino acid sequence mutations are preferably substitutions, more preferably conservative substitutions, from the viewpoint of being less likely to impair activity.
  • Nucleotide sequence mutations are preferably mutations that do not result in amino acid substitutions in the protein translated from the mRNA, or mutations that result in conservative amino acid substitutions.
  • CTGF protein to be inhibited is a protein that has an amino acid sequence that is 85 to 100% identical to the amino acid sequence of the wild-type CTGF protein (e.g., SEQ ID NO: 1) and that has the ability to promote chondrocyte proliferation and differentiation.
  • CTGF mRNA to be inhibited is a base sequence that has 85-100% identity to the base sequence of wild-type CTGF mRNA (e.g., SEQ ID NO: 2) and encodes a protein that has the ability to promote chondrocyte proliferation and differentiation.
  • a preferred example of the integrin ⁇ V protein to be inhibited is a protein consisting of an amino acid sequence that is 85-100% identical to the amino acid sequence of the wild-type integrin ⁇ V protein (e.g., SEQ ID NO: 3), and that has the ability to form a membrane protein (integrin) through heterodimerization with the integrin ⁇ chain and the ability to bind to a ligand.
  • SEQ ID NO: 3 wild-type integrin ⁇ V protein
  • a preferred example of the integrin ⁇ V mRNA to be inhibited is a nucleotide sequence that has 85-100% identity to the nucleotide sequence of wild-type integrin ⁇ V mRNA (e.g., SEQ ID NO: 4), and encodes a protein that has the ability to form a membrane protein (integrin) by heterodimerizing with the integrin ⁇ chain and the ability to bind to a ligand.
  • SEQ ID NO: 4 wild-type integrin ⁇ V mRNA
  • Identity is more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.
  • identity refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Thus, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences.
  • the level of identity of amino acid sequences is determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF.
  • conservative substitution refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, constitute conservative substitutions.
  • conservative substitutions include substitutions between amino acid residues having acidic side chains, such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having ⁇ -branched side chains, such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.
  • amino acid residues having acidic side chains such as aspartic acid and glutamic acid
  • amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine,
  • CTGF/integrin ⁇ V inhibitor is not particularly limited, so long as it is a component that can inhibit the function and/or expression of CTGF/integrin ⁇ V.
  • CTGF/integrin ⁇ V inhibitors preferably include low-molecular-weight compounds, polynucleotides that target CTGF/integrin ⁇ V, expression cassettes for such polynucleotides, peptides, proteins, antibodies, etc.
  • CTGF/integrin ⁇ V inhibitors can be used alone or in combination of two or more types.
  • the CTGF inhibitor is an inhibitor of CTGF in the liver (particularly hepatic sinusoidal endothelial cells) (e.g., a CTGF inhibitor targeted to the liver (particularly hepatic sinusoidal endothelial cells), a liver (particularly hepatic sinusoidal endothelial cell)-specific CTGF inhibitor), and/or the integrin ⁇ V inhibitor is an inhibitor of integrin ⁇ V in the liver (particularly hepatic sinusoidal endothelial cells) (e.g., an integrin ⁇ V inhibitor targeted to the liver (particularly hepatic sinusoidal endothelial cells), a liver (particularly hepatic sinusoidal endothelial cell)-specific integrin ⁇ V inhibitor).
  • CTGF/integrin ⁇ V function inhibitors There are no particular limitations on the CTGF/integrin ⁇ V function inhibitors, so long as they can inhibit the function of CTGF/integrin ⁇ V protein expressed in the target organism or its cells (particularly sinusoidal endothelial cells) to which the agents of the present invention are applied.
  • CTGF/integrin ⁇ V function inhibitors can be used alone or in combination of two or more.
  • CTGF function inhibitors are not particularly limited as long as they can inhibit the binding of CTGF to its receptor (integrins, including integrin ⁇ V) and its functional expression.
  • examples include substances that have binding affinity to the receptor-binding region of CTGF or other regions necessary for functional expression (e.g., regions that interact with other proteins).
  • regions include the IGFBP domain located on the N-terminus of CTGF and the CT domain located on the C-terminus.
  • the IGFBP domain is generally known to contribute to the progression of fibrosis, and the CT domain is known to interact with integrins.
  • Integrin ⁇ V function inhibitors are not particularly limited as long as they can inhibit the binding of integrins, including integrin ⁇ V, to their ligands.
  • Such function inhibitors include low molecular weight compounds (e.g., molecular weights of 1000 or less, 800 or less, 700 or less, or 600 or less; e.g., molecular weights of 100 or more, 150 or more, or 200 or more), antibodies, etc.
  • low molecular weight compounds e.g., molecular weights of 1000 or less, 800 or less, 700 or less, or 600 or less; e.g., molecular weights of 100 or more, 150 or more, or 200 or more
  • antibodies etc.
  • the binding region can be determined based on publicly known information and/or can be inferred based on publicly known information (e.g., by constructing a docking model, etc.).
  • inhibitory activity can be measured and evaluated, for example, according to the "In vitro integrin functional assays" previously reported (Nat. Med. 19(12), 1-12 (2013).).
  • CTGF/integrin ⁇ V function inhibitors are commercially available, and many have been reported in various publications.
  • Examples of integrin ⁇ V function inhibitors include CWHM12, and inhibitors currently undergoing clinical research include IDL-2965, PLN-74809, PLN-1474, JSM-6427, and AXT-107.
  • known inhibitors such as those described in known publications (Nature Reviews Drug Discovery volume 21, pages 60-78 (2022)) can be used without restriction.
  • the integrin ⁇ V function inhibitor is an RGD peptide analog.
  • Antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, and portions of the above antibodies that have antigen-binding properties, such as Fab fragments and fragments produced by an Fab expression library.
  • Antibodies of the present invention also include antibodies that have antigen-binding properties to a polypeptide consisting of at least 8 consecutive amino acids, preferably 15 amino acids, and more preferably 20 amino acids, from the amino acid sequence of CTGF/integrin ⁇ V.
  • CTGF/integrin ⁇ V function inhibitors can also be used, as long as they are molecules (e.g., peptides, proteins, artificial antibodies, aptamers, etc.) that have binding affinity (preferably specific binding affinity) to CTGF/integrin ⁇ V.
  • proteins or peptides such as antibodies as CTGF/integrin ⁇ V function inhibitors
  • their expression cassettes can also be used instead.
  • CTGF/integrin ⁇ V expression inhibitors are not particularly limited, as long as they can suppress the expression levels of CTGF/integrin ⁇ V protein and/or CTGF/integrin ⁇ V mRNA expressed by the target organism or its cells (particularly sinusoidal endothelial cells) to which the agents of the present invention are applied.
  • CTGF/integrin ⁇ V expression inhibitors can be used alone or in combination of two or more.
  • CTGF/integrin ⁇ V expression inhibitors include CTGF/integrin ⁇ V-specific small interfering RNA (siRNA), CTGF/integrin ⁇ V-specific microRNA (miRNA), CTGF/integrin ⁇ V-specific antisense nucleic acids, and expression cassettes thereof; CTGF/integrin ⁇ V-specific ribozymes; and CTGF/integrin ⁇ V gene editing agents using the CRISPR/Cas system.
  • siRNA small interfering RNA
  • miRNA microRNA
  • antisense nucleic acids and expression cassettes thereof
  • CTGF/integrin ⁇ V-specific ribozymes CTGF/integrin ⁇ V gene editing agents using the CRISPR/Cas system.
  • inhibiting expression means suppressing the expression levels of CTGF/integrin ⁇ V protein, CTGF/integrin ⁇ V mRNA, etc. to, for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1/100, 1/200, 1/300, 1/500, 1/1000, or 1/10,000 or less, and also includes reducing these expression levels to zero.
  • CTGF/integrin ⁇ V-specific siRNA, miRNA, and antisense nucleic acid are not particularly limited as long as they are double-stranded RNA molecules that specifically suppress the expression of the gene encoding CTGF/integrin ⁇ V.
  • the siRNA is preferably, for example, 18 or more bases, 19 or more bases, 20 or more bases, or 21 or more bases in length.
  • the siRNA is preferably, for example, 25 or less bases, 24 or less bases, 23 or less bases, or 22 or less bases in length. It is contemplated that the upper and lower limits of the siRNA length described herein may be combined arbitrarily.
  • the structure of the siRNA is not particularly limited.
  • the siRNA may be a small hairpin RNA (shRNA).
  • the siRNA may have additional bases at the 5' or 3' end.
  • the siRNA may have an overhang at the 3' end, such as an siRNA with dTdT (dT stands for deoxythymidine) added.
  • dT stands for deoxythymidine
  • siRNA Target Finder provided by Ambion (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html)
  • Insert Design Tool for pSilencer registered trademark
  • Expression Vector http://www.ambion.com/jp/techlib/misc/psilencer_converter.html
  • RNAi GeneSeer provided by Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi).
  • the CTGF/integrin ⁇ V-specific miRNA can be any type as long as it inhibits the translation of the gene encoding CTGF/integrin ⁇ V.
  • the miRNA instead of cleaving the target mRNA like siRNA, the miRNA may inhibit its translation by pairing with the target's 3' untranslated region (UTR).
  • the miRNA may be a pri-miRNA (primary miRNA), a pre-miRNA (precursor miRNA), or a mature miRNA.
  • the length of the miRNA is not particularly limited; the length of a pri-miRNA is typically several hundred to several thousand bases, the length of a pre-miRNA is typically 50 to 80 bases, and the length of a mature miRNA is typically 18 to 30 bases.
  • the CTGF/integrin ⁇ V-specific miRNA is preferably a pre-miRNA or a mature miRNA, and more preferably a mature miRNA.
  • Such CTGF/integrin ⁇ V-specific miRNA may be synthesized by known techniques or purchased from a company that provides synthetic RNA.
  • CTGF/integrin ⁇ V-specific antisense nucleic acids are nucleic acids containing a base sequence complementary or substantially complementary to the base sequence of the mRNA of the gene encoding CTGF/integrin ⁇ V, or a portion thereof, and function to inhibit CTGF/integrin ⁇ V protein synthesis by binding to the mRNA and forming a specific and stable duplex.
  • Antisense nucleic acids may be DNA, RNA, or DNA/RNA chimeras.
  • antisense nucleic acids are DNA, the RNA:DNA hybrid formed by the target RNA and antisense DNA is recognized by endogenous ribonuclease H (RNase H), causing selective degradation of the target RNA.
  • RNase H endogenous ribonuclease H
  • the target sequence may be not only a sequence in the mRNA, but also a sequence in the intron region of the initial translation product of the CTGF/integrin ⁇ V gene.
  • Intron sequences can be determined by comparing the genomic sequence with the cDNA base sequence of the CTGF/integrin ⁇ V gene using homology search programs such as BLAST and FASTA.
  • the length of the target region of a CTGF/integrin ⁇ V-specific antisense nucleic acid is not limited, as long as hybridization of the antisense nucleic acid results in inhibition of translation into CTGF/integrin ⁇ V protein.
  • the CTGF/integrin ⁇ V-specific antisense nucleic acid may be the entire sequence or a partial sequence of the mRNA encoding CTGF/integrin ⁇ V.
  • oligonucleotides consisting of approximately 10 to approximately 40 bases, particularly approximately 15 to approximately 30 bases, are preferred, but are not limited to these.
  • preferred target regions of the CTGF/integrin ⁇ V gene include, but are not limited to, the 5'-end hairpin loop, 5'-end untranslated region, translation initiation codon, protein coding region, ORF translation termination codon, 3'-end untranslated region, 3'-end palindrome region, and 3'-end hairpin loop.
  • “complementary” does not only refer to binding based on a perfect complementary relationship (A and T, and G and C), but also to binding based on a complementary relationship to the extent that hybridization is possible under stringent conditions.
  • Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe binds, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA).
  • washing conditions after hybridization typically include approximately "1x SSC, 0.1% SDS, 37°C.” It is preferable that the hybridized state is maintained even when washed under such conditions.
  • examples of more stringent hybridization conditions include approximately "0.5x SSC, 0.1% SDS, 42°C,” and examples of even more stringent hybridization conditions include washing conditions of approximately "0.1x SSC, 0.1% SDS, 65°C.”
  • CTGF/integrin ⁇ V-specific siRNA, CTGF/integrin ⁇ V-specific miRNA, and CTGF/integrin ⁇ V-specific antisense nucleic acids can be prepared by determining the target sequence of the mRNA or initial transcription product based on the cDNA sequence or genomic DNA sequence of the CTGF/integrin ⁇ V gene, and then synthesizing a complementary sequence using a commercially available automated DNA/RNA synthesizer.
  • Antisense nucleic acids containing various modifications can also be chemically synthesized using known techniques.
  • the expression cassette for CTGF/integrin ⁇ V-specific siRNA, CTGF/integrin ⁇ V-specific miRNA, or CTGF/integrin ⁇ V-specific antisense nucleic acid is not particularly limited, so long as it is a polynucleotide into which CTGF/integrin ⁇ V-specific siRNA, CTGF/integrin ⁇ V-specific miRNA, or CTGF/integrin ⁇ V-specific antisense nucleic acid has been incorporated in an expressible state.
  • the expression cassette comprises a polynucleotide comprising a promoter sequence and a coding sequence for the CTGF/integrin ⁇ V-specific siRNA, CTGF/integrin ⁇ V-specific miRNA, or CTGF/integrin ⁇ V-specific antisense nucleic acid (and, if necessary, a transcription termination signal sequence), as well as other sequences as necessary.
  • nucleic acid and “polynucleotide” are not particularly limited and encompass both natural and artificial nucleic acids. Specifically, in addition to DNA, RNA, and the like, they may also be chemically modified as described below. To prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithioate phosphorodithioate
  • the hydroxyl group at the 2nd position of the sugar (ribose) of each ribonucleotide may be replaced with -OR (where R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN).
  • R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN.
  • the base moiety pyrimidine, purine
  • phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group.
  • BNA LNA
  • the conformation of the sugar moiety of the nucleotide is fixed to the N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety, can also be used.
  • CTGF/integrin ⁇ V gene editing agent is not particularly limited, as long as it is capable of suppressing expression of the CTGF/integrin ⁇ V gene using a target sequence-specific nuclease system (e.g., a CRISPR/Cas system).
  • CTGF/integrin ⁇ V gene expression can be suppressed, for example, by disrupting the CTGF/integrin ⁇ V gene or by modifying the CTGF/integrin ⁇ V gene promoter to suppress promoter activity.
  • a vector (CTGF/integrin ⁇ V gene editing vector) containing a guide RNA expression cassette targeting the CTGF/integrin ⁇ V gene or its promoter and a Cas protein expression cassette can typically be used as a CTGF/integrin ⁇ V gene editing agent, but is not limited to this.
  • a combination of a vector containing a guide RNA and/or its expression cassette targeting the CTGF/integrin ⁇ V gene or its promoter, and a vector containing a Cas protein and/or its expression cassette can also be used as a CTGF/integrin ⁇ V gene editing agent.
  • guide RNA there are no particular limitations on the guide RNA as long as it is one that can be used in the CRISPR/Cas system.
  • various types of guide RNAs can be used that can bind to a target site in genomic DNA (e.g., the CTGF/integrin ⁇ V gene, its promoter, etc.) and guide the Cas protein to the target site in genomic DNA by binding to the Cas protein.
  • a target site refers to a site on genomic DNA consisting of a DNA strand (target strand) consisting of a PAM (Proto-Spacer Adjacent Motif) sequence and a sequence adjacent to the 5' side that is approximately 17 to 30 bases long (preferably 18 to 25 bases long, more preferably 19 to 22 bases long, and particularly preferably 20 bases long) and its complementary DNA strand (non-target strand).
  • PAM Proto-Spacer Adjacent Motif
  • the guide RNA has a sequence involved in binding to the target site in genomic DNA (sometimes referred to as the crRNA (CRISPR RNA) sequence), and this crRNA sequence binds complementary (preferably complementary and specific) to a sequence excluding the PAM sequence complementary sequence of the non-target strand, allowing the guide RNA to bind to the target site in genomic DNA.
  • the guide RNA also has a sequence involved in binding to the Cas protein (sometimes referred to as the tracrRNA (trans-activating crRNA) sequence), and this tracrRNA sequence binds to the Cas protein, allowing the Cas protein to be guided to the target site in genomic DNA.
  • the tracrRNA sequence is not particularly limited.
  • a tracrRNA sequence is typically an RNA consisting of a sequence of approximately 50 to 100 bases in length that can form multiple (usually three) stem-loops, and its sequence varies depending on the type of Cas protein used.
  • Various known sequences can be used as the tracrRNA sequence, depending on the type of Cas protein used.
  • the guide RNA typically contains the crRNA sequence and tracrRNA sequence described above.
  • the guide RNA may be a single-stranded RNA (sgRNA) containing the crRNA sequence and the tracrRNA sequence, or an RNA complex formed by complementary binding of an RNA containing the crRNA sequence and an RNA containing the tracrRNA sequence.
  • sgRNA single-stranded RNA
  • Cas protein there are no particular limitations on the Cas protein as long as it is one that can be used in the CRISPR/Cas system; for example, various proteins can be used that can bind to a target site in genomic DNA in a complex with a guide RNA and cleave the target site.
  • Cas proteins derived from various organisms are known, such as the Cas9 protein, and more preferably the Cas9 protein endogenously contained in bacteria belonging to the genus Streptococcus. Information on the amino acid sequences of various Cas proteins and their coding sequences can be easily obtained from various databases such as NCBI.
  • CTGF/integrin ⁇ V gene editing agents can be easily produced using known genetic engineering techniques. For example, they can be produced using PCR, restriction enzyme digestion, DNA ligation techniques, in vitro transcription/translation techniques, recombinant protein production techniques, etc.
  • At least one selected from the group consisting of CTGF inhibitors and integrin ⁇ V inhibitors can be used as an active ingredient in an agent for preventing or treating at least one disease selected from the group consisting of liver fibrosis, liver cirrhosis, and liver cancer in subjects suspected of having increased intrasinusoidal pressure.
  • the agent of the present invention is used for administration to a subject suspected of having increased intrasinusoidal pressure (preferably a subject with increased intrasinusoidal pressure).
  • a "subject suspected of having increased intrasinusoidal pressure” is, for example, a subject with a disease/disorder/syndrome that causes or has the potential to cause increased intrasinusoidal pressure. More specifically, it can be a subject with at least one condition selected from the group consisting of congestive liver disease, liver cirrhosis, Budd-Chiari syndrome, portal vein thrombosis, sinusoidal obstruction syndrome, and post-Fontan syndrome.
  • a "subject suspected of having increased intrasinusoidal pressure” can be a subject who has been determined to have increased intrasinusoidal pressure and/or to be suffering from at least one condition selected from the group consisting of liver fibrosis, liver cirrhosis, and liver cancer by the testing method of the present invention described below.
  • the agent of the present invention can also exert a preventive or therapeutic effect against liver fibrosis, cirrhosis, and liver cancer that are caused independently of hepatocyte death (i.e., liver cell death is not the cause of the disease, and in one embodiment, does not involve hepatocyte death). Therefore, in one embodiment, the target diseases for the agent of the present invention are liver fibrosis, cirrhosis, and liver cancer that are caused independently of hepatocyte death.
  • treatment can include concepts such as curing, remission, alleviation, mitigation, and suppression of progression of symptoms.
  • prevention can include not only preventing the onset of a disease, but also delaying its onset and suppressing symptoms if it does occur.
  • the content of the active ingredient in the agent of the present invention can be appropriately determined taking into consideration the type of disease being treated, the desired therapeutic effect, the method of administration, the treatment period, the patient's age, and the patient's weight.
  • the content of the active ingredient in the agent of the present invention can be approximately 0.0001 to 100 parts by weight, with the total amount of the agent of the present invention being 100 parts by weight.
  • the administration form of the agent of the present invention is not particularly limited as long as the desired effect is achieved, and it can be administered to mammals, including humans, via either oral or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, or topical administration).
  • the preferred administration form is parenteral administration.
  • Dosage forms for oral and parenteral administration and methods for their preparation are well known to those skilled in the art, and can be prepared in accordance with standard methods by mixing the active ingredient with a pharmaceutically acceptable carrier, etc.
  • Dosage forms for parenteral administration include injectable preparations (e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), topical preparations (e.g., ointments, poultices, lotions, creams, gels), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, liposomes, and LNP (Lipid Nano Particle) preparations.
  • injectable preparations are prepared by dissolving the active ingredient in distilled water for injection, and solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, etc. can be added as needed.
  • the agent of the present invention can also be made into a freeze-dried preparation for preparation immediately before use.
  • the agent of the present invention may further contain other drugs that are effective in treating or preventing diseases.
  • the agent of the present invention may contain any carrier or additive, such as a pharmaceutically acceptable carrier or additive.
  • Pharmaceutically acceptable carriers and additives include, but are not limited to, excipients such as sucrose and starch; binders such as cellulose and methylcellulose; disintegrants such as starch and carboxymethylcellulose; lubricants such as magnesium stearate and aerosil; flavorings such as citric acid and menthol; preservatives such as sodium benzoate and sodium bisulfite; stabilizers such as citric acid and sodium citrate; suspending agents such as methylcellulose and polyvinylpyrrolide; dispersing agents such as surfactants; diluents such as water and saline; base waxes, etc.
  • excipients such as sucrose and starch
  • binders such as cellulose and methylcellulose
  • disintegrants such as starch and carboxymethylcellulose
  • lubricants such as magnesium stearate and aerosil
  • flavorings such as citric acid and menthol
  • preservatives such as sodium benzoate and sodium bisulfite
  • stabilizers
  • the dosage of the agent of the present invention can be determined based on various factors, such as the route of administration, type of disease, severity of symptoms, the patient's age, sex, and body weight, severity of the disease, pharmacological knowledge such as pharmacokinetic and toxicological characteristics, whether a drug delivery system is used, and whether the agent is administered as part of a combination of other drugs.
  • the dosage of the agent of the present invention can be, for example, approximately 1 ⁇ g/kg (body weight) to 10 g/kg (body weight) per day.
  • the administration schedule of the agent of the present invention can also be determined taking into account the same factors as the dosage. For example, the above daily dosage can be administered 1 to 5 times per day to 1 month.
  • the present invention relates to (1) a method for testing at least one kind selected from the group consisting of intrasinusoidal pressure, liver fibrosis, liver cirrhosis, and liver cancer (also referred to herein as the "testing method of the present invention"), which comprises the step of detecting the protein and/or mRNA of at least one gene selected from the group consisting of CTGF, ESM1, ANGPT2, PLAU, EDN1, PDGFB, ADM, CXCL9, FBLN2, and INHBB in a biological sample collected from a subject (also referred to herein as the "target molecule of the present invention”). This method is described below.
  • the subject may be any mammal, but in one embodiment, the subject is a mammal suspected of having increased intrasinusoidal pressure (see 1-2 above).
  • mammals include rodents such as mice, rats, hamsters, and guinea pigs, laboratory animals such as rabbits, pets such as dogs and cats, livestock such as cows, pigs, goats, horses, and sheep, primates such as monkeys, orangutans, and chimpanzees, and humans, with humans being particularly preferred.
  • the biological sample is not particularly limited as long as it can contain the target molecule of the present invention.
  • biological samples include body fluids such as whole blood, serum, plasma, follicular fluid, menstrual blood, saliva, cerebrospinal fluid, synovial fluid, urine, interstitial fluid, sweat, tears, and saliva, as well as samples derived from these body fluids.
  • Biological samples can also be body tissues, preferably liver tissues (particularly preferably sinusoidal endothelial cells), or samples derived from these tissues.
  • Samples derived from body fluids/tissues are not particularly limited as long as they are samples prepared from body fluids/tissues, and examples include samples obtained by concentrating and purifying proteins or nucleic acids contained in the body fluids/tissues.
  • Preferred examples of body fluids include whole blood, serum, and plasma.
  • Biological samples may be used alone or in combination of two or more types.
  • Biological samples can be collected from subjects using methods known to those skilled in the art. For example, whole blood can be collected using a syringe or similar device. It is desirable that blood collection be performed by a medical professional such as a doctor or nurse. Serum is the portion of blood from which blood cells and specific blood clotting factors have been removed, and can be obtained, for example, as the supernatant after blood has been allowed to clot. Plasma is the portion of blood from which blood cells have been removed, and can be obtained, for example, as the supernatant when blood is centrifuged under conditions that do not cause blood to clot.
  • CTGF gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 1490.
  • CTGF genes of other species can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/nucleotide sequence of a known CTGF gene.
  • the amino acid sequence/nucleotide sequence of CTGF can be easily identified in accordance with publicly known information and/or based on identity analysis with the known amino acid sequence/nucleotide sequence of CTGF.
  • An example of the amino acid sequence of human CTGF is the amino acid sequence shown in SEQ ID NO: 1
  • an example of the nucleotide sequence of human CTGF mRNA is the nucleotide sequence shown in SEQ ID NO: 2.
  • the ESM1 gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 71690. ESM1 genes of other species can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/nucleotide sequence of a known ESM1 gene. The amino acid sequence/nucleotide sequence of ESM1 can be easily identified in accordance with publicly known information and/or based on identity analysis with the known ESM1 amino acid sequence/nucleotide sequence.
  • An example of the amino acid sequence of human ESM1 is the amino acid sequence shown in SEQ ID NO: 5, and an example of the nucleotide sequence of human ESM1 mRNA is the nucleotide sequence shown in SEQ ID NO: 6.
  • the ANGPT2 gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 285.
  • ANGPT2 genes of other species can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/nucleotide sequence of the publicly known ANGPT2 gene.
  • the amino acid sequence/nucleotide sequence of ANGPT2 can be easily identified in accordance with publicly known information and/or based on identity analysis with the publicly known ANGPT2 amino acid sequence/nucleotide sequence.
  • An example of the amino acid sequence of human ANGPT2 is the amino acid sequence shown in SEQ ID NO: 7, and an example of the nucleotide sequence of human ANGPT2 mRNA is the nucleotide sequence shown in SEQ ID NO: 8.
  • the PLAU gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 5328.
  • PLAU genes of other species can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/nucleotide sequence of a known PLAU gene.
  • the amino acid sequence/nucleotide sequence of PLAU can be easily identified in accordance with publicly known information and/or based on identity analysis with the known PLAU amino acid sequence/nucleotide sequence.
  • An example of the amino acid sequence of human PLAU is the amino acid sequence shown in SEQ ID NO: 9
  • an example of the nucleotide sequence of human PLAU mRNA is the nucleotide sequence shown in SEQ ID NO: 10.
  • the EDN1 gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 1906.
  • EDN1 genes of other species can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/nucleotide sequence of known EDN1 genes.
  • the amino acid sequence/nucleotide sequence of EDN1 can be easily identified in accordance with publicly known information and/or based on identity analysis with the known EDN1 amino acid sequence/nucleotide sequence.
  • An example of the amino acid sequence of human EDN1 is the amino acid sequence shown in SEQ ID NO: 11, and an example of the nucleotide sequence of human EDN1 mRNA is the nucleotide sequence shown in SEQ ID NO: 12.
  • the PDGFB gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 5155.
  • PDGFB genes of other organisms can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/nucleotide sequence of a known PDGFB gene.
  • the amino acid sequence/nucleotide sequence of PDGFB can be easily identified in accordance with publicly known information and/or based on identity analysis with the known PDGFB amino acid sequence/nucleotide sequence.
  • An example of the amino acid sequence of human PDGFB is the amino acid sequence shown in SEQ ID NO: 13
  • an example of the nucleotide sequence of human PDGFB mRNA is the nucleotide sequence shown in SEQ ID NO: 14.
  • the ADM gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 133.
  • ADM genes of other species can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/base sequence of known ADM genes.
  • the amino acid sequence/base sequence of ADM can be easily identified in accordance with publicly known information and/or based on identity analysis with the known ADM amino acid sequence/base sequence.
  • An example of the amino acid sequence of human ADM is the amino acid sequence shown in SEQ ID NO: 15, and an example of the base sequence of human ADM mRNA is the base sequence shown in SEQ ID NO: 16.
  • the CXCL9 gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 4283.
  • CXCL9 genes of other organisms can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/nucleotide sequence of a known CXCL9 gene.
  • the amino acid sequence/nucleotide sequence of CXCL9 can be easily identified in accordance with publicly known information and/or based on identity analysis with the known CXCL9 amino acid sequence/nucleotide sequence.
  • An example of the amino acid sequence of human CXCL9 is the amino acid sequence shown in SEQ ID NO: 17, and an example of the nucleotide sequence of human CXCL9 mRNA is the nucleotide sequence shown in SEQ ID NO: 18.
  • the FBLN2 gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 2199.
  • FBLN2 genes of other species can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/nucleotide sequence of a publicly known FBLN2 gene.
  • the amino acid sequence/nucleotide sequence of FBLN2 can be easily identified in accordance with publicly known information and/or based on identity analysis with the publicly known FBLN2 amino acid sequence/nucleotide sequence.
  • An example of the amino acid sequence of human FBLN2 is the amino acid sequence shown in SEQ ID NO: 19, and an example of the nucleotide sequence of human FBLN2 mRNA is the nucleotide sequence shown in SEQ ID NO: 20.
  • the INHBB gene is publicly known, and in the case of humans, it is, for example, the gene with NCBI Gene ID 3625. INHBB genes of other species can also be easily identified in accordance with publicly known information and/or based on identity analysis with the amino acid sequence/nucleotide sequence of known INHBB genes. The amino acid sequence/nucleotide sequence of INHBB can be easily identified in accordance with publicly known information and/or based on identity analysis with the known INHBB amino acid sequence/nucleotide sequence.
  • An example of the amino acid sequence of human INHBB is the amino acid sequence shown in SEQ ID NO: 21, and an example of the nucleotide sequence of human INHBB mRNA is the nucleotide sequence shown in SEQ ID NO: 22.
  • CTGF in one embodiment of the present invention, from the viewpoint of test accuracy, CTGF, ESM1, ANGPT2, PLAU, EDN1, PDGFB, and ADM are preferred. Furthermore, in another embodiment of the present invention, from the same viewpoint, CTGF, ESM1, ANGPT2, PLAU, EDN1, PDGFB, ADM, and CXCL9 are preferred.
  • CTGF, ANGPT2, EDN1, and PDGFB are preferred, as they are particularly suitable for analysis using blood samples (whole blood, serum, plasma).
  • CTGF, ESM1, ANGPT2, PLAU, CXCL9, EDN1, and FBLN2 are preferred, and CTGF, ESM1, ANGPT2, PLAU, and CXCL9 are more preferred.
  • CTGF is composed of four domains, Module 1-Module 4 from the N-terminus.
  • Module 1-Module 4 from the N-terminus.
  • N-terminal region from the full-length CTGF
  • C-terminal region from the C-terminal region
  • the C-terminal region is susceptible to degradation, and it is thought that the full-length and N-terminal regions are mainly present in the blood. For this reason, when detecting CTGF protein in blood samples, it is preferable to detect a fragment of the N-terminal region of CTGF.
  • the target molecules of the present invention that are the subject of detection in step (1) also include isoforms and those that contain mutations that occur between individuals.
  • the method for detecting the target molecule of the present invention is not particularly limited, as long as it is a method that can detect the target molecule of the present invention.
  • examples of such methods include immunoassays. Immunoassays can be widely used, regardless of whether they are direct, indirect, homogeneous, heterogeneous, competitive, or non-competitive.
  • immunoassays include ELISA (e.g., direct, indirect, sandwich, competitive, etc.), radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme immunoassay (EIA), sandwich EIA, immunochromatography, Western blot, immunoprecipitation, slot or dot blot assay, immunohistochemical staining, fluorescent immunoassay, immunoassays using avidin-biotin or streptavidin-biotin systems, and immunoassays using surface plasmon resonance (SPR).
  • ELISA e.g., direct, indirect, sandwich, competitive, etc.
  • RIA radioimmunoassay
  • IRMA immunoradiometric assay
  • EIA enzyme immunoassay
  • sandwich EIA sandwich EIA
  • immunochromatography Western blot
  • Western blot Western blot
  • immunoprecipitation slot or dot blot assay
  • SPR surface plasmon resonance
  • the target molecule of the present invention can be detected by immunoassay, for example, by directly or indirectly contacting a labeled antibody with a molecule binding to the target molecule of the present invention that has bound to the target molecule of the present invention, and quantifying the signal derived from the label of the bound labeled antibody.
  • the labeled antibody used in this case and the antibody that mediates between the labeled antibody and the molecule binding to the target molecule of the present invention or the target molecule of the present invention are not particularly limited, and examples that can be used include antibodies against antibody constant regions and anti-idiotype antibodies.
  • binding molecules to the target molecule of the present invention include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, or molecules containing portions of the above antibodies that have antigen-binding ability, such as Fab fragments and fragments produced by an Fab expression library.
  • Binding molecules to the target molecule of the present invention that have antigen-binding ability to a polypeptide consisting of at least 8 consecutive amino acids, preferably 15 amino acids, and more preferably 20 amino acids, from the amino acid sequence of the target molecule of the present invention are also included in the binding molecules to the target molecule of the present invention.
  • labeled antibody used to detect the target molecule of the present invention
  • labeled antibody e.g., labeled antibody
  • examples of labels include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, and radioisotopes.
  • enzyme labels such as peroxidase and alkaline phosphatase are preferred from the standpoints of safety, economy, detection sensitivity, etc.
  • the method for detecting the target molecule of the present invention (preferably a method for measuring the amount or concentration of the target molecule of the present invention) is not particularly limited, as long as it is a method that can detect the target molecule of the present invention.
  • examples of such methods that can be used include RNA-Seq, Northern blotting, RNase protection assay, reverse transcription polymerase chain reaction (RT-PCR) (Weis JH et al., Trends in Genetics 1992;8:263-264), and quantitative real-time RT-PCR (Held CA et al., Genome Research 1996;6:986-994).
  • the above methods can use molecules (e.g., primers, probes, etc.) that bind to the target molecule of the present invention.
  • Primer pairs and probes contain sequences that can complementarily bind to the nucleotide sequence of the target molecule mRNA of the present invention or its complementary sequence, and can be synthesized based on known base sequences.
  • the base lengths of the primers and probes are not particularly limited.
  • the primers can each be 10 to 50 nucleotides in length, preferably 15 to 30 nucleotides in length.
  • the probes can each be 10 nucleotides in length to the full length of the nucleotide sequence complementary to the nucleotide sequence of the mRNA of the molecule of interest of the present invention, preferably 20 to 150 nucleotides in length.
  • Primer pairs and probes may be made of natural nucleic acids such as RNA and DNA, or, if necessary, natural nucleic acids may be combined with chemically modified nucleic acids or pseudonucleic acids.
  • chemically modified nucleic acids and pseudonucleic acids include PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid; registered trademark), methylphosphonate DNA, phosphorothioate DNA, and 2'-O-methyl RNA.
  • primers and probes may be labeled or modified with fluorescent substances and/or quenchers, radioisotopes (e.g., 32P , 33P , 35S ), or modifying substances such as biotin, (strept)avidin, or magnetic beads.
  • Labeling substances are not limited, and commercially available ones can be used.
  • fluorescent substances such as FITC, Texas, Cy3, Cy5, Cy7, Cyanine3, Cyanine5, Cyanine7, FAM, HEX, VIC, fluorescamine and its derivatives, and rhodamine and its derivatives can be used.
  • Quenchers that can be used include AMRA, DABCYL, BHQ-1, BHQ-2, and BHQ-3.
  • the labeling position of the labeling substance in the primer and probe can be determined appropriately depending on the properties of the modifying substance and the intended use. Generally, the 5' or 3' end is often modified.
  • a single primer and probe molecule may be labeled with one or more types of labeling substances.
  • a single detection method may be used, or two or more may be used in combination.
  • the testing method of the present invention which includes step (1), can provide the amount and/or concentration of the target molecule of the present invention, which is an indicator of at least one test selected from the group consisting of intrasinusoidal pressure, liver fibrosis, cirrhosis, and liver cancer, thereby assisting in testing intrasinusoidal pressure, etc.
  • the testing method of the present invention preferably further comprises the step of (2) determining whether or not the subject has increased intrasinusoidal pressure and/or whether or not the subject has at least one disease selected from the group consisting of hepatic fibrosis, cirrhosis, and liver cancer, based on the amount or concentration of the protein and/or the mRNA detected in step (1).
  • step (2) comprises: (2A) determining that the subject has increased intrasinusoidal pressure and/or is suffering from at least one disease selected from the group consisting of hepatic fibrosis, cirrhosis, and liver cancer when the amount or concentration of the protein and/or mRNA detected in step (1) is equal to or greater than a cutoff value; and/or (2B) determining that the subject does not have increased intrasinusoidal pressure and/or is not suffering from at least one disease selected from the group consisting of hepatic fibrosis, cirrhosis, and liver cancer when the amount or concentration of the protein and/or mRNA detected in step (1) is equal to or less than a cutoff value.
  • the cutoff value is set in advance, for example, based on a statistical analysis or ROC analysis of the data on the amount or concentration of each target molecule of the present invention in subjects with elevated intrasinusoidal pressure and subjects without elevated intrasinusoidal pressure, by preparing a database that tracks the amount or concentration of each target molecule of the present invention and the presence or absence of at least one disease selected from the group consisting of liver fibrosis, cirrhosis, and liver cancer for the evaluation population.
  • the cutoff value can be set each time.
  • the cutoff value is determined by statistical analysis, for example, the median, arithmetic mean, or other average value of the data on the amount or concentration of the target molecule of the present invention in the evaluation population can be used.
  • the cutoff value based on ROC analysis can be the amount or concentration of the target molecule of the present invention at the point on the ROC curve where the distance between the point on the vertical axis (sensitivity or true positivity) of the ROC curve graph and the point on the horizontal axis (1 - specificity) is 1.0 is the smallest, or the cutoff value can be derived from the Youden index of the ROC curve (Cancer 1950;3:32-35.).
  • the database of the evaluation population may be used to set the cutoff value in the method of evaluating the presence or absence of increased intrasinusoidal pressure in a subject of the present invention without any changes.
  • new subjects including the subject of the present invention may be incorporated into the evaluation population, and the database of the evaluation population for intrasinusoidal pressure may be updated as appropriate, and used to set the cutoff value in the method of evaluating the presence or absence of increased intrasinusoidal pressure in a subject of the present invention.
  • the cutoff value can be, for example, a percentile value of the amount or concentration value of the target molecule of the present invention in a biological sample from a reference subject group, such as any of the 10th to 90th percentile values, any of the 30th to 70th percentile values, or any of the 40th to 60th percentile values.
  • a subject determined to have increased intrasinusoidal pressure in step (2) can be determined to be a candidate for administration of the agent of the present invention.
  • the testing method of the present invention can be used as a companion diagnostic for the agent of the present invention.
  • At least one diagnostic agent selected from the group consisting of intrasinusoidal pressure, liver fibrosis, liver cirrhosis, and liver cancer relates to at least one diagnostic agent selected from the group consisting of intrasinusoidal pressure, liver fibrosis, liver cirrhosis, and liver cancer (the diagnostic agent of the present invention), which comprises a molecule capable of binding to a target molecule of the present invention. This will be described below.
  • the diagnostic agent of the present invention is a drug that tests for at least one condition selected from the group consisting of intrasinusoidal pressure, liver fibrosis, liver cirrhosis, and liver cancer (specifically, tests for the presence or absence of increased intrasinusoidal pressure and/or the presence or absence of at least one condition selected from the group consisting of liver fibrosis, liver cirrhosis, and liver cancer).
  • the diagnostic agent of the present invention can be used in the testing method of the present invention.
  • the diagnostic agent of the present invention can be used as a companion diagnostic agent for the agent of the present invention.
  • the test agent of the present invention may be in the form of a composition containing a molecule capable of binding to the target molecule of the present invention.
  • the composition may contain other components as necessary. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.
  • the test agent of the present invention may be in the form of a kit containing a molecule that binds to the target molecule of the present invention.
  • the kit may also include instruments, reagents, etc. that can be used to carry out the test method of the present invention.
  • the binding molecules of the present invention to the target molecules can also be immobilized on any solid phase. Therefore, the test agent of the present invention can be provided in the form of a substrate on which the binding molecules of the present invention to the target molecules are immobilized (for example, a microarray chip on which probes are immobilized, or another example, an ELISA plate on which antibodies are immobilized).
  • a substrate on which the binding molecules of the present invention to the target molecules are immobilized for example, a microarray chip on which probes are immobilized, or another example, an ELISA plate on which antibodies are immobilized.
  • the solid phase used for immobilization is not particularly limited as long as it is capable of immobilizing antibodies, etc., and examples include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. There are no particular limitations on the immobilization of the detection agent to the solid phase.
  • test tubes examples include test tubes, microtiter plates, agarose particles, latex particles, purification columns, epoxy-coated glass slides, and gold colloid-coated glass slides.
  • Reagents include, for example, labeled antibodies and standard samples (positive and negative controls).
  • a variety of commercially available labeled antibodies can be used depending on the type of molecule (e.g., isotype) that binds to the target molecule of the present invention.
  • the target molecule of the present invention is used as the standard sample.
  • the target molecule of the present invention can be obtained, for example, by culturing cells into which an expression vector for the target molecule of the present invention has been introduced, and purifying the cells or the culture supernatant.
  • Test Example 1 Development of preventive/therapeutic drugs 1 Test Example 1-1. Creation of a mouse model of increased intrasinusoidal pressure ⁇ Test 1-1-1> Partial IVC ligation (pIVCL) was performed on wild-type male C57BL/6J mice (CLEA Japan) aged 8-10 weeks as previously described (Simonetto DA, et al. Hepatology 2015). Under triple anesthesia, the IVC between the diaphragm and liver was partially ligated using a 0.6 mm spacer to induce hepatic congestion. All procedures except for the partial IVC ligation were performed in sham surgery.
  • pIVCL Partial IVC ligation
  • HE staining images and serum ALT levels were obtained 6 weeks after pIVCL and sham surgery.
  • HE staining revealed hemorrhagic necrosis around the central vein and dilated sinusoids in the pIVCL group, but no elevation of serum ALT or signs of liver inflammation were observed.
  • Portal vein pressure was measured directly using a 1.2 Fr microcatheter (Transonic, FTH-1211B-0018) inserted into the superior mesenteric vein and analyzed using the dedicated software LabScribe4 software chart 5.5.6 (Transonic, ADInstruments). Portal vein pressure was measured 2 and 6 weeks after surgery, and it was found to be elevated from the early postoperative period. The resulting model mice allow for analysis of the effects of elevated intrasinusoidal pressure without being affected by hepatocyte death.
  • PSI Picrosirius Red Stain Kit
  • Test Example 1-2 Analysis of intrasinusoidal hypertension model mice (pIVCL) ⁇ Test 1-2-1> Single-cell analysis was performed on hepatic nonparenchymal cells 2 weeks after pIVCL and sham surgery.
  • Cell suspensions were prepared by perfusion of pronase E (Sigma-Aldrich, 107433) and collagenase (Sigma-Aldrich, C5138) through the portal vein.
  • Hepatocytes were removed by low-speed centrifugation (50 g). Dead cells and red blood cells were removed by density gradient centrifugation using Percoll PLUS (Cytiva, GE17-5445-02).
  • Whole-transcriptome analysis (BD Rhapsody) was performed.
  • CTGF connective tissue growth factor
  • RT-qPCR revealed elevated CTGF expression in LSECs isolated 2 days after pIVCL and sham surgery.
  • Cells were isolated by perfusion with pronase (Sigma-Aldrich, 107433) and collagenase (Sigma-Aldrich, C5138) via the portal vein. Hepatocytes were removed by low-speed centrifugation (50 g).
  • Density gradient centrifugation using Percoll PLUS removed dead cells and red blood cells to prepare a cell suspension of nonparenchymal hepatocytes.
  • LSECs were then positively selected by MACS using CD146 microbeads (Miltenyi, 132-092-007).
  • the primers used were Actb (Mm00607939_s1) and Ctgf (Mm01192933_g1).
  • Protein was extracted from liver tissue 2 days, 2 weeks, 6 weeks, and 12 weeks after pIVCL and sham surgery, and Western blotting was performed, showing increased expression of ⁇ -SMA and CTGF in the pIVCL group. Immunostaining for ⁇ -SMA and CTGF revealed positive cells primarily in Zone 3. The primary antibodies used were ⁇ -SMA (abcam, ab5694), CTGF (Santa Cruz, sc-14939), and b-Actin (CST, #4967).
  • ⁇ Test 1-2-4> We isolated mRNA from LSECs isolated from pIVCL and 2 weeks after sham surgery, and performed RT-qPCR. The results showed that YAP/TAZ target gene expression was elevated in the pIVCL group.
  • Cells were isolated by perfusion of pronase (Sigma-Aldrich, 107433) and collagenase (Sigma-Aldrich, C5138) via the portal vein. Hepatocytes were removed by low-speed centrifugation (50 g). Density gradient centrifugation using Percoll PLUS (Cytiva, GE17-5445-02) removed dead cells and red blood cells to prepare a non-parenchymal cell suspension.
  • LSECs were then positively selected by MACS using CD146 microbeads (Miltenyi, 132-092-007).
  • the primers used were Actb (Mm00607939_s1), Tgfb1 (Mm01178820_m1), Ctgf (Mm01192933_g1), Cyr61 (Mm00487498_m1), Edn1 (Mm00438656_m1), and Myc (Mm00487804_m1).
  • Protein was extracted from pIVCL and LSECs isolated two weeks after sham surgery, and Western blotting demonstrated activation of YAP/TAZ in the pIVCL group.
  • the primary antibodies used were phospho-YAP (abcam, ab76252), YAP (abcam, ab205270), TAZ (CST, #72804), CTGF (Santa Cruz, sc-14939), and b-Actin (CST, #4967).
  • YAP and phospho-YAP were quantified using the image analysis software Fusion, and YAP/phospho-YAP was calculated.
  • YAP/TAZ staining was primarily observed in the nuclei of LSECs in the pIVCL group.
  • the primary antibodies used were YAP (Abcam, ab205270) and TAZ (CST, #72804).
  • liver tissue was cut into 1mm3 sections, fixed in 2.5% glutaraldehyde, and prepared for electron microscopy. Transmission electron microscopy revealed the appearance of a basement membrane along the sinusoidal wall 6 weeks after pIVCL.
  • MRNA was extracted from liver tissue samples 6 weeks after pIVCL and sham surgery, and RT-qPCR showed an increase in capillarization markers. Furthermore, mRNA was extracted from LSECs isolated 2 weeks after pIVCL and sham surgery, and RT-qPCR showed an increase in basement membrane-related markers.
  • the primers used were Actb (Mm00607939_s1), Pecam1 (Mm01242584_m1), Cd34 (Mm00519283_m1), Vwf (Mm00550376_m1), Plvap (Mm00453379_m1), Col4a1 (Mm01210125_m1), Lamc1 (Mm00711820_m1), Fn1 (Mm01256744_m1), Nid1 (Mm00477827_m1), and Hspg2 (Mm01181173_g1).
  • Test Example 1-3 Analysis of in vitro/ex vivo models ⁇ Test 1-3-1>
  • To isolate LSECs we perfused pronase (Sigma-Aldrich, 107433) and collagenase (Sigma-Aldrich, C5138) into the portal vein to prepare a cell suspension. After removing hepatocytes by low-speed centrifugation (50 g), we performed positive selection of LSECs by MACS using CD146 microbeads (Miltenyi, 132-092-007). Mouse LSECs used in subsequent in vitro experiments were isolated in a similar manner.
  • LSECs isolated from wild-type mice were plated overnight on collagen-coated 4 cm2 silicon chambers (STREX, STB-CH-04) and allowed to stand for 6 hours without serum. After 6 hours of serum-free incubation, LSECs were stimulated for 4 hours with a growth stimulator (STREX, STB-150) at 120% saturation and 0.5 Hz. mRNA was extracted and analyzed by RT-qPCR. Upregulation of CTGF was confirmed using primers (Applied Bio Systems) for Actb (Mm00607939_s1) and Ctgf (Mm01192933_g1).
  • the human immortalized hepatic endothelial cell line TMNK-1 was also cultured under stretch stimulation in the same manner as above, and mRNA and protein extraction was performed. It was shown that stretch stimulation of TMNK-1 increases CTGF expression, accompanied by YAP activation.
  • the primers used were Actb (Hs01060665_g1) and Ctgf (Hs00170014_m1).
  • the primary antibodies used were phospho-YAP (abcam, ab76252), YAP (abcam, ab205270), CTGF (Santa Cruz, sc-14939), and b-Actin (CST, #4967).
  • TMNK-1 The human immortalized hepatic endothelial cell line TMNK-1 was treated with siRNA to suppress YAP (Ambion, Silencer TM Select, s20367), TAZ (Ambion, Silencer TM Select, s24787), or both YAP and TAZ.
  • siRNA was extracted and RT-qPCR demonstrated that siRNA suppressed YAP and TAZ expression and reduced CTGF expression.
  • the YAP inhibitor Verteporfin RV&D SYSTEMS, #5305
  • TMNK-1 cells were plated overnight onto collagen-coated 4cm2 silicon chambers (STREX, STB-CH-04) after YAP/TAZ suppression and 48 hours after siRNA addition. After 6 hours of serum-free incubation, cells were stretched for 4 hours at 120% saturation and 0.5 Hz. mRNA was extracted and CTGF expression was analyzed by RT-qPCR. TMNK-1 cells were plated overnight onto collagen-coated 4cm2 silicon chambers. After 4 hours of serum-free incubation, cells were stretched for 4 hours at 120% saturation and 0.5 Hz. mRNA was extracted and CTGF expression was analyzed by RT-qPCR. These results demonstrate that stretch-induced CTGF expression in TMNK-1 cells is YAP/TAZ-dependent.
  • the primers used were Actb (Hs01060665_g1), Yap (Hs00902712_g1), Taz (Wwtr1; Hs00210007_m1), and Ctgf (Hs00170014_m1).
  • LSECs isolated from wild-type mice were plated onto collagen-coated wells overnight, left serum-free for 6 hours, and then subjected to hydrostatic pressure stimulation (101/135 kPa, 0.01 Hz) for 8 hours using a hydrostatic pressure device.
  • mRNA was extracted and RT-qPCR was performed, showing an increase in YAP/TAZ target genes and basement membrane-related markers.
  • TMNK-1 The immortalized human hepatic endothelial cell line TMNK-1 was plated on collagen-coated wells overnight, serum-free for 6 hours, and then subjected to hydrostatic pressure (101/135 kPa, 0.01 Hz) for 8 hours. mRNA was extracted and RT-qPCR demonstrated upregulation of YAP/TAZ target genes. Primers (Applied Bio Systems) were used: Actb (Hs01060665_g1), Tgfb1 (Hs00998133_m1), Ctgf (Hs00170014_m1), Cyr61 (Hs00155479_m1), Edn1 (Hs00174961_m1), and Myc (Hs00153408_m1).
  • CTGF levels in the supernatant after the above culture were measured using ELISA (FUJIFILM, 290-84701), and the results showed that hydrostatic pressure stimulation also increased CTGF secretion by TMNK-1.
  • TMNK-1 The human immortalized hepatic endothelial cell line TMNK-1 was subjected to hydrostatic pressure (HPS) stimulation with the pan-integrin ⁇ V inhibitor CWHM-12 (MCE, HY-18644). TMNK-1 cells were plated onto collagen-coated wells overnight and incubated serum-free for 4 hours. CWHM-12 was then added at 1 ⁇ M. After 2 hours, HPS (101/135 kPa, 0.01 Hz) was added for 4 hours, and protein extraction was performed. Western blotting demonstrated that CWHM-12 inhibited YAP/TAZ activity.
  • HPS pan-integrin ⁇ V inhibitor
  • TMNK-1 cells were treated with 1 ⁇ M CWHM-12 or 2 ⁇ g/mL of the YAP inhibitor Verteporfin (R&D SYSTEMS, #5305) and subjected to HPS (101/135 kPa, 0.01 Hz) for 8 hours, followed by mRNA extraction.
  • TMNK-1 cells were also treated with siRNA to inhibit YAP (Ambion, Silencer TM Select, s20367) and TAZ (Ambion, Silencer TM Select, s24787). After 48 hours of siRNA addition, cells were plated onto collagen-coated wells overnight, incubated serum-free for 6 hours, and then subjected to HPS (101/135 kPa, 0.01 Hz) for 8 hours.
  • RT-qPCR demonstrated that HPS-induced upregulation of CTGF expression in TMNK-1 cells was significantly suppressed by CWHM-12, Verteporfin, and siYAP/TAZ.
  • the primers used were Actb (Hs01060665_g1) and Ctgf (Hs00170014_m1).
  • TMNK-1 CTGF KO TMNK-1 CTGF knockout cells
  • CTGF knockout was confirmed by Western blotting (Santa Cruz, sc-14939) and ELISA (FUJIFILM, 290-84701).
  • the effects of human recombinant (rh)CTGF (FUJIFILM, 036-19471) on these CTGF knockout cells were examined with or without the addition of the pan-Integrin ⁇ V inhibitor CWHM-12 (MCE, HY-18644).
  • TMNK-1 CTGF KO cells were plated overnight on collagen-coated wells, and after 4 hours in a serum-free environment, CWHM-12 was added at 1 ⁇ M. After 2 hours, 1 ⁇ g/mL rhCTGF was added, and protein was extracted 6 hours later.
  • human hepatic stellate cells SCR, #5300
  • SCR hepatic stellate cells
  • rhCTGF was added at 1 ⁇ g/mL.
  • mRNA was extracted and RT-qPCR was performed, demonstrating that the addition of rhCTGF increased Col1a1 expression.
  • the primers used were Actb (Hs01060665_g1) and Ctgf (Hs00170014_m1).
  • a WST assay performed 48 hours after the addition of rhCTGF demonstrated increased cell viability.
  • ⁇ Test 1-3-7> We generated tamoxifen-inducible endothelial cell-specific YAP/TAZ-deficient mice (iCdh5-Cre; YAP/TAZ fl/fl ). Six-week-old iCdh5-Cre; YAP/TAZ fl/fl and YAP/TAZ fl/fl mice were intraperitoneally administered tamoxifen (Sigma, T5648-5G) at 0.1 mg/g/day for 5 days. At 9 weeks of age, LSECs were isolated and subjected to hydrostatic pressure stimulation (HPS).
  • HPS hydrostatic pressure stimulation
  • Isolated LSECs were plated overnight, serum-free for 6 hours, and then subjected to HPS (101/135 kPa, 0.01 Hz) for 8 hours.
  • mRNA was extracted and analyzed for CTGF and COL4A1 expression by RT-qPCR.
  • HPS-induced upregulation of Ctgf and Col4a1 in LSECs was shown to be YAP/TAZ-dependent.
  • the primers used were Actb (Mm00607939_s1), Ctgf (Mm01192933_g1), and Col4a1 (Mm01210125_m1).
  • Test Example 1-4 Analysis of CTGF Knockout Mice .
  • Tamoxifen-inducible endothelial cell-specific CTGF-deficient mice iCdh5-Cre; CTGF fl/fl
  • CTGF i ⁇ EC CTGF i ⁇ EC
  • CTGF fl /fl mice aged 6-7 weeks were intraperitoneally administered tamoxifen (Sigma, T5648-5G) at 0.1 mg/g/day for 5 days. At 9-10 weeks of age, they underwent pIVCL/sham angioplasty and were analyzed 6 weeks later.
  • mRNA was extracted from LSECs isolated from CTGF i ⁇ EC and CTGF fl/fl mice at 9-10 weeks of age, and RT-qPCR confirmed that CTGF expression in LSECs was knocked out (Figure 2).
  • Primers (Applied Bio Systems) for Actb (Mm00607939_s1) and Ctgf (Mm01192933_g1) were used.
  • Hepatic tissue sections from the CTGF- i ⁇ EC and CTGF -fl/fl pIVCL groups and the sham group were stained with HE, Sirius red, ⁇ -SMA, and type IV collagen (Figure 3).
  • the image analysis software HALO was used to quantify Sirius red (PSI, Picrosirius Red Stain Kit), ⁇ -SMA (Abcam, ab5694), and type IV collagen (Abcam, ab6586) staining.
  • Knockout of CTGF in endothelial cells improved pIVCL-induced liver fibrosis ( Figure 4).
  • liver tumor formation in CTGF i ⁇ EC and CTGF fl/fl mice was evaluated 48 weeks after pIVCL. Liver tumor formation was 6/15 (40%) in wild-type (CTGF fl/fl ) mice and 0/16 (0%) in knockout (CTGF i ⁇ EC ) mice, demonstrating a significant reduction in liver tumor formation in the knockout mice (Fig. 6).
  • Test Example 1-5 Analysis of clinical samples ⁇ Test 1-5-1> HE-stained and Sirius red-stained images were obtained from surgically resected specimens of a normal liver (donor of a living donor liver transplant), a patient with chronic hepatitis (chronic hepatitis C, after viral eradication), and two patients with Fontan-associated liver disease (FALD).In FALD, perisinusoidal fibrosis (fibrosis of the sinusoidal wall) was prominent in areas of congestion and sinusoidal dilation.
  • RNA profiling (10x Genomic) was performed on surgically frozen specimens from two normal livers and three FALD patients. Primary quality control was performed using Cell Ranger software using the FASTQ files and human genome annotation file (GRCh38) generated by next-generation sequencing. Secondary quality control was performed using the single-cell analysis software SeqGeq (BD Rhapsody) by excluding cells with counts ⁇ 2000, gene counts ⁇ 500, and mitochondrial gene ratios >20%. After normalization and batch effect correction, the resulting cells were integrated and clustered using Seurat. The results were displayed as a UMAP. Cell populations within each cluster were identified by extracting marker genes. Gene expression analysis of endothelial cell clusters, including LSECs, revealed significantly elevated CTGF expression in the FALD group (fold change: 3.34, q-value ⁇ 0.0001).
  • Test Example 1-6 Analysis of the effect of an integrin ⁇ V inhibitor The protocol for Test Example 1-6 is shown in FIG.
  • ⁇ Test 1-6-2> The images of liver tissue sections from the control (DMSO) and treatment (CWHM-12) groups were stained with HE, Sirius red (PSI, Picrosirius Red Stain Kit), ⁇ -SMA immunostaining (Abcam, ab5694), and type IV collagen staining (Abcam, ab6586) (Figure 9).
  • the image analysis software HALO was used to quantify the areas positive for Sirius red staining, ⁇ -SMA staining, and type IV collagen staining, demonstrating that liver fibrosis was improved in the treatment group (Figure 10, top).
  • Serum ALT levels (FUJIFILM, DRI-CHEM NX700) and intrahepatic hydroxyproline levels (CBO, Hydroxyproline Assay Kit) were measured (Figure 10, middle).
  • Portal vein pressure was measured directly using a 1.2 Fr microcatheter (Transonic, FTH-1211B-0018) inserted into the superior mesenteric vein and analyzed using the dedicated software LabScribe4 software chart 5.5.6 (ADInstruments), showing improvement in portal hypertension in the treatment group ( Figure 10, middle).
  • Test Example 2 Biomarker Development 1 ⁇ Test 2-1> The results of Test 1-2-1 are shown below.
  • the top 10 secreted proteins that were significantly elevated in the pIVCL group in the Zone 3 LSEC cluster were Ctgf, Esm1, Angpt2, Plau, Edn1, Pdgfb, Cxcl9, Fbln2, Inhbb, and Adm.
  • ⁇ Test 2-2> We examined the expression distribution of these genes (Ctgf, Esm1, Angpt2, Plau, Edn1, Pdgfb, Cxcl9, Fbln2, Inhbb, and Adm) on the UMAP to clarify which clusters they are expressed in. Ctgf and Adm were elevated in both LSECs and hepatic stellate cells, whereas the other genes were elevated specifically in LSECs.
  • ⁇ Test 2-4> Portal vein pressure was measured at 2 and 6 weeks after pIVCL/sham surgery, and mRNA was extracted from liver tissue and analyzed by RT-qPCR. Portal vein pressure was measured directly using a 1.2 Fr microcatheter (Transonic, FTH-1211B-0018) inserted into the superior mesenteric vein. Analysis was performed using the dedicated software LabScribe4 software chart 5.5.6 (ADInstruments). Expression of both genes was significantly elevated in the pIVCL group from 2 weeks after surgery ( Figure 11).
  • the primers used were Actb (Mm00607939_s1), Ctgf (Mm01192933_g1), Esm1 (Mm00469953_m1), Angpt2 (Mm00545822_m1), Plau (Mm00447054_m1), Edn1 (Mm00438656_m1), Pdgfb (Mm00440677_m1), Cxcl9 (Mm00434946_m1), Fbln2 (Mm00484266_m1), Inhbb (Mm03023992_m1), and Adm (Mm00437438_g1).
  • LSECs isolated from wild-type mice were plated overnight, serum-free for 6 hours, and then subjected to hydrostatic pressure (101/135 kPa, 0.01 Hz) for 8 hours. RNA was extracted and RNA-seq was performed. Expression of the 10 genes listed above (Ctgf, Esm1, Angpt2, Plau, Edn1, Pdgfb, Cxcl9, Fbln2, Inhbb, and Adm) was significantly increased by hydrostatic pressure.
  • ELISA was performed on serum ANGPT2 (R&D SYSTEMS, MANG20), EDN1 (R&D SYSTEMS, DET100), and PDGFB (R&D SYSTEMS, MBB00) at 2 and 6 weeks after pIVCL/sham transplantation. Serum ANGPT2 and PDGFB levels were significantly elevated in the pIVCL group ( Figure 13).
  • Test Example 3-1 Elucidation of the mechanism of progression of liver pathology due to increased intrasinusoidal pressure and its effects ⁇ Test 3-1-1: Figure 15> Single-cell analysis was performed on hepatocytes from the control (DMSO) and treatment (CWHM-12) groups 6 weeks after pIVCL. Pronase (Sigma-Aldrich, 107433) and collagenase (Sigma-Aldrich, C5138) were perfused via the portal vein to prepare a cell suspension. Hepatocytes were isolated by low-speed centrifugation (50 g).
  • Nonparenchymal hepatocytes were then isolated by density gradient centrifugation using Percoll PLUS (Cytiva, GE17-5445-02) to remove dead cells and red blood cells.
  • Percoll PLUS Cytiva, GE17-5445-02
  • a cell suspension with a hepatocyte:nonparenchymal hepatocyte ratio of 1:9 was prepared, and whole transcriptome analysis (BD Rhapsody) was performed.
  • Primary quality control was performed using the Seven Bridge pipeline using FASTQ files obtained by next-generation sequencing and the mouse genome annotation file (GRCm39). Secondary quality control was then performed using R to extract cells with a count of 500 ⁇ 30,000, a gene count of 250 ⁇ 4,000, and a mitochondrial gene ratio of ⁇ 25%.
  • the resulting cells were normalized and corrected for batch effects before being integrated for dimensionality reduction and clustering.
  • the results were displayed as a UMAP.
  • Cell populations within each cluster were identified by extracting marker genes, and clusters of LSECs around the central vein (pericentral LSECs) and hepatic stellate cells (HSCs) were identified.
  • the pericentral LSEC cluster showed decreased expression of YAP/TAZ target genes, Ctgf, and type 4 collagen (Col4a1, Col4a2), while the HSC cluster showed decreased expression of Col1a1 and Col4a1.
  • ⁇ Test 3-1-2 Figure 16> HE-stained and Sirius red-stained images of surgically resected specimens from a normal liver (donor of a living donor liver transplant) and two cases of Fontan-associated liver disease (FALD) were shown. Perisinusoidal fibrosis (fibrosis of the sinusoidal wall) was prominent in areas of congestion and sinusoidal dilation in FALD. Immunostaining for CTGF (Santa Cruz, sc-14939), integrin ⁇ V (Abcam, ab179475), and YAP (Abcam, ab205270) was also observed. In FALD, CTGF and integrin ⁇ V stained primarily along the sinusoidal wall in areas of congestion and sinusoidal dilation, while YAP stained primarily in the nuclei of LSECs.
  • Protein was extracted from frozen samples, and Western blotting demonstrated activation of YAP and increased expression of CTGF and type IV collagen in FALD.
  • the primary antibodies used were phospho-YAP (abcam, ab76252), YAP (abcam, ab205270), TAZ (CST, #72804), CTGF (Santa Cruz, sc-14939), Collagen IV (abcam, ab6586), and b-Actin (CST, #4967).
  • mRNA was extracted from frozen samples, and increased expression of CTGF in FALD was demonstrated.
  • Primers used were Actb (Hs01060665_g1) and Ctgf (Hs00170014_m1).
  • Total serum CTGF full-length + N-terminal (FUJIFILM, 292-84901) and full-length CTGF (FUJIFILM, 290-84701) were measured by ELISA, and the serum N-terminal CTGF level was calculated by calculating the difference. Increased serum N-terminal CTGF levels were observed in FALD.
  • RNA profiling (10x Genomic) was performed on surgically frozen specimens from three normal livers (two donors from living donor liver transplants and one liver with underlying hemangioma) and four FALD cases.
  • Primary quality control was performed using Cell Ranger using FASTQ files and the human genome annotation file (GRCh38) obtained by next-generation sequencing.
  • Secondary quality control was performed using R to extract cells with a count value of 500 ⁇ 30,000, a gene count value of 250 ⁇ 4,000, and a mitochondrial gene ratio of ⁇ 20%. After normalization and batch effect correction, the resulting cells were integrated for dimensionality reduction and clustering. The results were displayed as a UMAP. Marker gene extraction identified cell populations within each cluster.
  • Endothelial cell clusters including LSECs
  • LSECs Gene expression analysis of endothelial cell clusters, including LSECs, revealed significantly elevated CTGF expression in the FALD group.
  • elevated YAP/TAZ target genes and basement membrane-related genes were observed in endothelial cells from the FALD group.
  • Analysis of intercellular communication using CellChat revealed an increase in the number of signals from endothelial cells to other cells in the FALD group, particularly signals from basement membrane components of endothelial cells, suggesting that basement membrane formation by LSECs may contribute to the progression of FALD pathology.
  • ⁇ Test 3-1-4 Thin sections were prepared from FFPE blocks of normal liver (living donor liver transplants) and surgically resected specimens of patients with Fontan-associated liver disease (FALD). Single-cell spatial gene expression analysis was performed using a CosMx Spatial Molecular Imager (Bruker). The analysis area (FOV: 0.5 mm x 0.5 mm) was selected based on HE staining of serial sections. Cell segmentation was performed using anti-B2M/CD298, anti-PanCK, anti-CD45, anti-CK8/18 antibodies, and DAPI staining.
  • mRNA was detected in the tissue using nine custom gene probes, including CCN2 (CTGF), in the CosMx Human Universal Cell Characterization RNA Panel (1000-plex). Analyzed data were subjected to normalization, batch effect correction, dimensionality reduction, and clustering on the dedicated analysis platform, AtoMx. Clustering was performed using the liver reference file used for single-cell analysis. The data was converted into Seurat objects and clustered using UMAP in R to show the distribution of CTGF expression.
  • CCN2 CCN2
  • AtoMx dedicated analysis platform
  • ⁇ Test 3-1-5 Figure 19> (Top) HE-stained images and corresponding mapping images (created with AtoMx) of periportal LSECs and pericentral LSECs in normal and FALD livers, as well as merged images of CTGF mRNA (created with AtoMx) are shown. CTGF expression was compared between periportal and pericentral LSECs in normal and FALD livers. While no difference in expression was observed between the two in normal livers, CTGF expression was significantly elevated in pericentral LSECs in FALD livers.
  • ⁇ Test 3-1-6 Figure 20> In FALD livers, the areas around the central vein were classified into mild fibrosis (perisinusoidal fibrosis) and severe fibrosis (bridging fibrosis) by Sirius red staining in each region analyzed with CosMx (Field of View: 0.5 mm x 0.5 mm). Mapping images (generated with AtoMx) of pericentral LSECs, hepatic stellate cells (HSCs), and CTGF, COL1A1, COL1A2, COL4A1, and COL4A2 mRNA in normal livers, FALD liver perisinusoidal fibrosis regions, and FALD liver bridging fibrosis regions are shown.
  • HSCs hepatic stellate cells
  • CTGF CTGF mRNA
  • HSCs pericentral LSECs
  • type IV collagen COL4A1 and COL4A2
  • HSCs there was no difference in the expression of type IV collagen (COL4A1, COL4A2)
  • type I collagen COL1A1, COL1A2
  • the proportion of hepatic stellate cells was increased in the bridging fibrosis region.
  • CTGF levels increase in LSECs from the early stages of congestive liver injury, and that this, along with increased expression of type IV collagen in LSECs and increased expression of type I collagen in HSCs, may contribute to the progression from perisinusoidal fibrosis to bridging fibrosis.
  • Test Example 3-2 Application to diseases with increased intrasinusoidal pressure and development of new treatments ⁇ Test 3-2-1: Figure 21> Thin sections were prepared from FFPE blocks of surgically resected normal livers (living donors), Fontan-associated liver disease (FALD), and cirrhotic livers (non-HBV/HCV), and single-cell spatial gene expression analysis was performed using a CosMx Spatial Molecular Imager (Bruker). The analysis area (FOV: 0.5 mm x 0.5 mm) was selected based on HE staining of serial sections. Cell segmentation was performed using anti-B2M/CD298, anti-PanCK, anti-CD45, anti-CK8/18 antibodies, and DAPI staining.
  • mRNAs were detected in the tissue using nine custom gene probes, including CCN2 (CTGF), in the CosMx Human Universal Cell Characterization RNA Panel (1000-plex). Analysis data were subjected to normalization, batch effect correction, dimensionality reduction, and clustering on the dedicated analysis platform, AtoMx. Clustering was performed using the liver reference file used for single-cell analysis. The data was converted into Seurat objects and clustered using UMAP in R.
  • CCN2 CCN2
  • AtoMx dedicated analysis platform
  • ⁇ Test 3-2-2 Figure 22> Mapping images of COL1A1, COL1A2, COL4A1, and COL4A2 mRNA in pericentral LSECs (CV.LSECs) and hepatic stellate cells (HSCs) from FALD livers (generated by AtoMx) are shown, along with a merged image of CTGF mRNA (generated by AtoMx). Violin plots are shown for CTGF, COL1A1, COL1A2, COL4A1, and COL4A2 expression in periportal LSECs (PV.LSECs), pericentral LSECs (CV.LSECs), and hepatic stellate cells (HSCs). Type I collagen (COL1A1 and COL1A2) was highly expressed in HSCs, while CTGF and type IV collagen (COL4A1 and COL4A2) were highly expressed not only in HSCs but also in CV.LSECs.
  • PV.LSECs periportal LSEC
  • ⁇ Test 3-2-3 Figure 23> Mapping images of COL1A1, COL1A2, COL4A1, and COL4A2 mRNAs (generated by AtoMx) in cirrhotic liver periportal LSECs (PV.LSECs), pericentral LSECs (CV.LSECs), and hepatic stellate cells (HSCs) are shown, along with a merged image of CTGF mRNA (generated by AtoMx).
  • ITGAV integrated ⁇ V
  • Type I collagen (COL1A1 and COL1A2) was highly expressed in HSCs
  • type IV collagen (COL4A1 and COL4A2) was highly expressed in both PV.LSECs and CV.LSECs, as well as HSCs.
  • Test Example 4 Biomarker Development 2 ⁇ Test 4-1: Figure 24> The top 10 secreted proteins elevated in LSECs by single-cell analysis of the pIVCL model: Ctgf, Esm1, Angpt2, Plau, Edn1, Pdgfb, Cxcl9, Fbln2, Inhbb, and Adm. Serum concentrations of these proteins were measured in normal livers, FALD, and patients with hepatitis C virus cirrhosis.
  • ESM1 serum ESM1
  • ANGPT2 R&D SYSTEMS, DANG20
  • PLAU R&D SYSTEMS, DUPA00
  • EDN1 R&D SYSTEMS, DET100
  • PDGFB R&D SYSTEMS, DBB00
  • CXCL9 R&D SYSTEMS, DCX900
  • FBLN2 Abbexa, abx350725)
  • INHBB INHBB
  • AssayGenie, HUFl02589) ADM (AssayGenie, HUFl00565) was performed in 7 normal livers (healthy individuals hospitalized for colon polypectomy), 58 FALD cases (provided by the University of Tokyo), and 15 type C liver cirrhosis cases.
  • Serum N-terminal CTGF was calculated by measuring serum total CTGF (full-length + N-terminal (FUJIFILM, 292-84901) and full-length CTGF (FUJIFILM, 290-84701) by ELISA and calculating the difference. Serum N-terminal CTGF, ESM1, ANGPT2, PLAU, and CXCL9 levels were significantly elevated in patients with FALD and cirrhosis compared with controls (normal liver).
  • liver stiffness is a non-invasive test that reflects hepatic congestion (intrasinusoidal hypertension) and liver fibrosis.
  • serum EDN1 and FBLN2 levels showed a significant positive correlation with liver stiffness.

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Abstract

La présente invention concerne une technologie qui est destinée à prévenir des affections hépatiques conduisant au cancer du foie, ou à en traiter la progression, et cible un phénomène autre que la mort des hépatocytes. La solution selon l'invention porte sur un agent prophylactique ou thérapeutique qui agit contre au moins une maladie choisie dans le groupe constitué par la fibrose hépatique, la cirrhose et le cancer du foie, contient au moins une substance choisie dans le groupe constitué par les inhibiteurs de CTGF et les inhibiteurs de l'intégrine αV, et est destiné à des patients suspectés d'avoir une pression intrasinusoïde accrue.
PCT/JP2025/014379 2024-04-22 2025-04-10 Agent prophylactique ou thérapeutique contre la fibrose hépatique, la cirrhose et le cancer du foie, et biomarqueur de pression intrasinusoïde Pending WO2025225410A1 (fr)

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