WO2022270071A1 - 肝線維化抑制のための剤及び医薬組成物 - Google Patents

肝線維化抑制のための剤及び医薬組成物 Download PDF

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WO2022270071A1
WO2022270071A1 PCT/JP2022/013322 JP2022013322W WO2022270071A1 WO 2022270071 A1 WO2022270071 A1 WO 2022270071A1 JP 2022013322 W JP2022013322 W JP 2022013322W WO 2022270071 A1 WO2022270071 A1 WO 2022270071A1
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nucleic acid
mir
hsa
precursor
seq
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PCT/JP2022/013322
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French (fr)
Japanese (ja)
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剛生 須田
直哉 坂本
直樹 川岸
隆 北潟谷
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国立大学法人北海道大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • 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
    • 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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention expresses a nucleic acid consisting of a base sequence containing a seed sequence of miR-6848, a double-stranded nucleic acid containing said nucleic acid as one strand, a precursor of said nucleic acid, and a nucleic acid encoding said nucleic acid or its precursor.
  • the present invention relates to agents and pharmaceutical compositions for suppressing hepatic fibrosis, containing at least one nucleic acid selected from the group consisting of nucleic acids that can be retained.
  • liver fibrosis is the formation of scar tissue, the excessive accumulation of connective tissue within the liver as a result of chronic hepatocyte injury, and is caused by various types of chronic liver injury, such as hepatitis virus infection and excessive alcohol consumption. It is known.
  • HSCs hepatic stellate cells
  • HSCs hepatic stellate cells
  • TGF transforming growth factor
  • extracellular matrix fibrosis-inducing factors
  • activated hepatic stellate cells are known not only to induce fibrosis, but also to participate in the regulation of inflammation and immune response, as well as in the composition of the microenvironment of liver cancer.
  • Controlling the function of activated hepatic stellate cells is considered to be an important and effective approach to suppress liver fibrosis and, in turn, hepatocarcinogenesis.
  • Agonists, CCR2/CCR5 antagonists, ASK-1 (Apoptosis signal-regulating kinase 1) inhibitors, Gallection 3 inhibitors, siRNA against HSP47, etc. are being developed).
  • canonical Wnt pathway ⁇ -catenin pathway, also called canonical Wnt pathway
  • Wnt signaling pathway that plays a role in regulating stem cell proliferation and differentiation and maintaining homeostasis
  • hepatic stellate cell activation has been shown to be epigenetically regulated by microRNAs (miRNAs), e.g., miR-29 family, miR-34 family, miR-15 family, miR-200 family, miR-199 family, miR-378 family, etc. are reported to be involved in fibrosis (eg, Non-Patent Document 2). It has been proposed that miR-29a be used as a preventive/therapeutic agent for hepatic fibrosis (Patent Document 1).
  • miRNAs microRNAs
  • the present invention provides a new means for suppressing liver fibrosis.
  • the present invention also provides new means for evaluating liver fibrosis-suppressing activity.
  • the present inventors found miRNAs that can suppress the expression of the ⁇ -SMA ( ⁇ -smooth muscle actin) gene and the activation of the canonical Wnt pathway in activated hepatic stellate cells.
  • ⁇ -SMA smooth muscle actin
  • a pharmaceutical composition for suppressing liver fibrosis comprising at least one nucleic acid selected from the group consisting of the following a) to e).
  • Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand d) a precursor of the nucleic acid of any of the above a) to c)
  • Item 2 The nucleic acid of a) above consists
  • Item 1 The pharmaceutical composition according to item 1, comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in number 1.
  • Item 3. Item 1 or 2, wherein the nucleic acid of b) consists of the base sequence shown in SEQ ID NO: 2 or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 2.
  • pharmaceutical composition. Item 4 Any of Items 1 to 3, wherein the precursor of d) consists of the base sequence shown in SEQ ID NO: 3, or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 3. or the pharmaceutical composition according to claim 1.
  • the nucleic acid of e) above can express a nucleic acid consisting of the base sequence shown in SEQ ID NO: 4 or a nucleic acid consisting of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 4.
  • Item 5 The pharmaceutical composition according to any one of Items 1 to 4, which is a nucleic acid prepared by the above method.
  • Item 6. The pharmaceutical composition according to any one of Items 1 to 5, wherein the nucleic acids a) and b) are mature microRNAs, and the precursors of d) are primary microRNAs or precursor microRNAs.
  • Item 7. The pharmaceutical composition of any one of Items 1 to 6, for use in a subject undergoing or having received treatment with a therapeutic agent for hepatitis C virus.
  • Item 9 The pharmaceutical composition of any one of Items 1-8 for the treatment of hepatitis C, wherein said treatment involves the use of a hepatitis C virus therapeutic agent.
  • Item 10 A pharmaceutical for treating hepatitis C, comprising at least one nucleic acid selected from the group consisting of the following a) to e) in combination with a therapeutic drug for hepatitis C virus.
  • a nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand d) a precursor of the nucleic acid of any of the above a) to c) e) At least one kind of nucleic acid selected from the group consisting of a) to e) below in Item 11, a nucleic acid that retains an expressible nucleic acid that encodes any of the above a) to d) or a nucleic acid that encodes a precursor thereof
  • a kit for the treatment of hepatitis C comprising: , a hepatitis C virus
  • nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand d) a precursor of the nucleic acid of any of the above a) to c) e) Nucleic acid Item 12 hsa-miR-6848-5p, hsa-miR-6848-3p, or precursors thereof, carrying a nucleic acid encoding any one of the above a) to d) or a nucleic acid encoding the precursor thereof in an expressible manner
  • the method according to Item 12, comprising the step of determining that the test substance that induces expression or increases the expression level of 6848-3p or their precursors has hepatic fibrosis-suppressing activity.
  • Item 14 Incubating a test substance with a cell containing a nucleic acid having a marker gene under the control of a promoter region that controls the expression of the hsa-mir-6848 gene; Measuring the intensity of a signal derived from the marker gene in the cell; comparing the intensity of the marker gene-derived signal obtained in the presence of the test substance with the intensity of the marker gene-derived signal in the absence of the test substance; 13.
  • the method of clause 12, comprising determining that.
  • This disclosure also provides: Item 1A A liver fibrosis inhibitor containing at least one nucleic acid selected from the group consisting of a) to e) below.
  • Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand d) a precursor of the nucleic acid of any of the above a) to c)
  • Item 1A comprising a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in No. 1.
  • Item 3A Item 1A or 2A, wherein the nucleic acid of b) consists of the base sequence shown in SEQ ID NO: 2, or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 2.
  • agent. Item 4A Any of Items 1A to 3A, wherein the precursor of d) consists of the base sequence shown in SEQ ID NO: 3, or consists of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 3. or the agent according to item 1.
  • the nucleic acid of e) is capable of expressing a nucleic acid consisting of the base sequence shown in SEQ ID NO: 4, or a nucleic acid consisting of a base sequence having at least 90% identity with the base sequence shown in SEQ ID NO: 4.
  • the agent according to any one of Items 1A to 4A which is a nucleic acid obtained by Item 6A
  • Item 7A For a subject with liver fibrosis, or a subject with a disease or condition that can cause liver fibrosis, containing at least one nucleic acid selected from the group consisting of a) to e) below Pharmaceutical composition for use.
  • a nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand d) a precursor of the nucleic acid of any of the above a) to c) e)
  • Section 8A hepatitis C virus therapeutic drug that retains an expressible nucleic acid
  • a pharmaceutical for treating hepatitis C comprising at least one nucleic acid selected from the group consisting of the following a) to e) in combination with a therapeutic drug for hepatitis C virus.
  • Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand d) a precursor of the nucleic acid of any of the above a) to c)
  • nucleic acid having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases b) Nucleic acid having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand d) a precursor of the nucleic acid of any of the above a) to c) e) A nucleic acid that retains the nucleic acid encoding any of the nucleic acids a) to d) above or its precursor in an expressible manner
  • activation of hepatic stellate cells can be inhibited, thereby inhibiting liver fibrosis in a subject with liver fibrosis or with a disease or condition that can cause liver fibrosis. be able to.
  • Fig. 4 is a graph showing the ⁇ SMA gene expression level in hepatic stellate cells introduced with mock miRNA (N.C.), miR-29, miR-342, miR-6848 or miR-449.
  • Fig. 4 is a graph showing the expression level of the collagen I gene in hepatic stellate cells introduced with mock miRNA (N.C.), miR-29, miR-342, miR-6848 or miR-449.
  • Fig. 10 is a graph showing the activity of TCF/LEF promoter, NFAT promoter or AP-1 promoter in hepatic stellate cells introduced with mock miRNA (N.C.), miR-29, miR-342, miR-6848 or miR-449.
  • Fig. 2 is a schematic diagram showing an outline of an administration schedule in an animal test for evaluating suppression of liver fibrosis by a lipid membrane structure encapsulating miRNA.
  • Normal mouse normal
  • carbon tetrachloride-administered mouse CCl4
  • carbon tetrachloride plus negative control mimic-encapsulated lipid membrane construct NC
  • carbon tetrachloride plus miRNA-encapsulated lipid membrane construct is a graph showing Sirius-Red stained areas of liver tissue sections taken from mice (342, 6848) administered with
  • miRNAs are non-coding RNAs with a length of about 16 to 29 bases, mostly around 22 bases, encoded on the genome of eukaryotes, and play an important role in regulating gene expression.
  • pri-miRNA primary miRNA
  • miRNA is processed by Drosha in the nucleus to form a precursor miRNA with a hairpin structure.
  • pre-miRNA pre-miRNA moves into the cytoplasm and is further processed by Dicer to become double-stranded miRNA.
  • Double-stranded miRNA is incorporated into Ago protein, and one RNA strand is complexed with Ago protein to form an RNA-protein complex called RISC (RNA-induced silencing complex).
  • This RNA strand is called mature miRNA, and has a base sequence called a seed sequence at the 2nd to 8th positions from the 5' end.
  • the seed sequence of mature miRNA exerts the function of inhibiting the translation of the target gene by binding with the complementary sequence present in the 3' untranslated region of the target gene mRNA.
  • miRNAs are defined in miRBase (http://www.miRbase.org/), which is a database that collects information such as base sequences of miRNAs. be.
  • the nucleotide sequences of each miR are as registered in miRBase, and shown in the sequence listing as necessary.
  • At least one nucleic acid selected from the group consisting of a) to e) below can be used for suppressing liver fibrosis.
  • RNA having a nucleotide sequence consisting of GGGGGCU at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases b) RNA having a nucleotide sequence consisting of UGGUCUC at the 2nd to 8th positions from the 5' end and having a sequence length of 16 to 29 bases c) a double-stranded nucleic acid containing the nucleic acid of a) or b) above as one strand d) a precursor of the nucleic acid of any of the above a) to c) e)
  • the nucleic acid a) is a nucleic acid having a base sequence consisting of GGGGGCU as a seed sequence, and is a mature miRNA or a nucleic acid having functions equivalent thereto.
  • the chain length of the nucleic acid a) may be 16 to 29 bases, preferably 18 bases or more, and preferably 25 bases or less.
  • nucleic acid a examples include RNA consisting of the nucleotide sequence shown in SEQ ID NO: 1; RNA consisting of a nucleotide sequence having identity; and having a nucleotide sequence consisting of GGGGGCU and having 1 or 2, preferably 1 base deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1 RNA consisting of a base sequence can be mentioned.
  • the RNA consisting of the base sequence shown in SEQ ID NO: 1 is hsa-miR-6848-5p registered with miRBase under accession number MIMAT0027596.
  • the RNA in b) is a nucleic acid having a nucleotide sequence consisting of UGGUCUC as a seed sequence, and is a mature miRNA or a nucleic acid having functions equivalent thereto.
  • the chain length of the nucleic acid b) may be 16 to 29 bases, preferably 18 bases or more, and preferably 25 bases or less.
  • Preferred examples of the nucleic acid b) include RNA consisting of the nucleotide sequence shown in SEQ ID NO: 2; RNA consisting of a base sequence having identity; and having a base sequence consisting of UGGUCUC, and having 1 or 2, preferably 1 base deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 RNA consisting of a base sequence can be mentioned.
  • the RNA consisting of the base sequence shown in SEQ ID NO: 2 is hsa-miR-6848-3p registered in miRBase under accession number MIMAT0027597.
  • the double-stranded nucleic acid of c) has the nucleic acid of a) or b) on one strand.
  • the precursor of d) is a nucleic acid capable of producing any of the nucleic acids of a) to c) in cells, for example, pri-miRNA or pre-miRNA.
  • Preferred examples of the precursor of d) include RNA consisting of the nucleotide sequence shown in SEQ ID NO: 3; RNA consisting of a nucleotide sequence having 95% identity; can include RNA consisting of a nucleotide sequence in which 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2 nucleotides have been deleted, substituted or added.
  • the RNA consisting of the base sequence shown in SEQ ID NO: 3 is hsa-miR-6848 registered with miRBase under accession number MI0022694.
  • the nucleic acid of e) holds any nucleic acid of a) to c) or a nucleic acid encoding the precursor of d) in an expressible manner, and as a result of transcription and processing, any of a) to c) or a nucleic acid capable of expressing the precursor of d).
  • Preferred examples of the nucleic acid of e) include DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4; and a DNA consisting of a base sequence with 100% identity; A nucleic acid that retains a DNA consisting of a base sequence in which 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2 bases have been deleted, substituted, or added so as to be expressible can be mentioned.
  • DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 is DNA encoding hsa-miR-6848.
  • the nucleic acid of e) is preferably in the form of an expression vector into which the nucleic acid of any one of a) to c) or the nucleic acid encoding the precursor of d) is integrated under the control of an appropriate promoter sequence.
  • the expression vector may further contain any functional base sequence that regulates transcriptional expression, such as operator sequences, enhancers, and the like. Any promoter sequence may be used as long as it can function in mammalian cells, and a promoter sequence having a high ability to induce expression in hepatic stellate cells is particularly preferred.
  • Nucleotide sequence identity refers to an optimal alignment calculated using an algorithm known in the art (preferably, the algorithm takes into account the introduction of gaps in one or both of the sequences for optimal alignment. It means the ratio (%) of identical bases to all overlapping bases in the total number of overlapping bases.
  • Identity can be calculated, for example, by aligning two base sequences using NCBI BLAST-2 (National Center for Biotechnology Information Basic Local Alignment Search Tool) with default conditions.
  • the nucleic acids a) to e) may be chimeric nucleic acids containing deoxyribonucleotides and ribonucleotides in their base sequences, or hybrid nucleic acids consisting of a DNA strand and an RNA strand.
  • nucleic acids a) to e) may be chemically modified nucleic acids.
  • Chemical modifications of nucleic acids may be performed on any of the base, phosphate, and sugar moieties, and examples of base moieties include 5-methylcytosine, 5-propynylcytosine, phenoxazine, 9-( aminoethoxy)phenoxazine and the like, and examples of modification of the phosphate moiety include phosphodiester, phosphorothioate, boranophosphate, phosphoramidate, methylphosphonate, phosphotriester, and the like. Modification examples include 2'-OMe conversion, 2'-F conversion, 2'-MOE conversion, and cross-linking such as LNA or BNA.
  • the type and number of modified bases in the modified nucleic acid are not particularly limited as long as the effect of suppressing hepatic fibrosis is not lost.
  • the nucleic acids a) to e) can be prepared using genetic recombination technology or chemical synthesis technology. Various techniques such as gene recombination, chemical synthesis of nucleic acids, and synthesis of modified nucleic acids are well known to those skilled in the art.
  • Nucleic acids a) to e) can be introduced by various known methods capable of introducing nucleic acids into cells, for example, nucleic acid introduction methods using viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, etc., jetPEI (PolyPlus-transfection), a nucleic acid transfection method using a non-viral carrier such as lipofectamine, a hydrodynamics method, or the like, can be used for introduction into the cells of the target animal.
  • the invention also provides, in one aspect, the nucleic acids of a)-e) in combination with a viral vector or non-viral carrier.
  • the nucleic acids a) to e) can suppress the expression of the ⁇ SMA gene in activated hepatic stellate cells and can also suppress the activation of the canonical Wnt pathway.
  • the ⁇ SMA gene is a fibroblast marker gene, and activation of the canonical Wnt pathway is involved in tissue fibrosis. Therefore, the nucleic acids a) to e) can be used as active ingredients of agents and pharmaceutical compositions for suppressing activation of hepatic stellate cells and suppressing hepatic fibrosis.
  • the pharmaceutical composition should be used as a medicament for suppressing liver fibrosis in subjects with or at risk of liver fibrosis, particularly subjects with diseases or conditions that can cause liver fibrosis. can be done.
  • subject includes humans and non-human animals.
  • non-human animals include rodents including mice, rats, hamsters and guinea pigs, primates including chimpanzees and rhesus monkeys, domestic animals including pigs, cattle, goats, horses and sheep, and pet animals including dogs and cats. can be mentioned.
  • a preferred subject is a human.
  • Hepatic fibrosis and its suppression are detected by immunohistological staining of liver tissue; measurement of liver fibrosis markers such as blood hyaluronic acid or type IV collagen; ultrasonography, CT, MRI, ultrasonic elastography, magnetic resonance It can be assessed using techniques such as elastography or imaging studies such as acoustoradiography.
  • liver fibrosis diseases or conditions that can cause liver fibrosis include conditions that directly affect the liver, such as bacterial infections (such as brucellosis), parasitic infections (such as echinococcosis), viral infections (such as hepatitis B, hepatitis C, etc.), nonalcoholic steatohepatitis (NASH), primary biliary cirrhosis, primary sclerosing cholangitis, congenital liver fibrosis, autoimmune hepatitis, ⁇ 1-antitrypsin deficiency, copper Storage disease, fructoseemia, galactosemia, glycogen storage disease, iron overload syndrome, dyslipidemia, peroxisome disease, tyrosinemia, etc.; conditions affecting hepatic blood flow, such as Budd-Chiari syndrome, heart failure, central hepatic vein Obstruction, portal vein thrombosis, etc.; intake of drugs or chemicals that can cause fibrosis, such as alcohol, amiodarone
  • suppression of liver fibrosis refers to improvement of the fibrosis state of the liver. including regression or disappearance of “Inhibition of liver fibrosis” is used interchangeably with prevention and/or treatment of liver fibrosis.
  • the pharmaceutical composition is preferably used in a subject suffering from hepatitis C, a subject suffering from hepatitis C and being treated with an HCV therapeutic agent, or a subject suffering from hepatitis C and treated with an HCV therapeutic agent. It can be used to suppress liver fibrosis in subjects who have achieved SVR with treatment.
  • SVR is also called sustained virologic negative change, and means that blood HCV RNA becomes negative both in the test at the end of anti-HCV treatment and at 12 or 24 weeks after the end of treatment. .
  • the pharmaceutical composition contains an effective amount of at least one nucleic acid selected from the group consisting of a) to e), and pharmaceutically acceptable substances such as buffers, antioxidants, preservatives, excipients, carriers, and the like. and other pharmaceutically active ingredients.
  • pharmaceutically acceptable ingredients are well known to those skilled in the art, and can be appropriately selected and used within the scope of ordinary ability of those skilled in the art according to the properties of the active ingredient and the form of the formulation.
  • the pharmaceutical composition is preferably administered using a drug delivery system (DDS) that can selectively introduce nucleic acids into hepatic stellate cells.
  • DDS drug delivery system
  • examples of such DDS include DDS having tropism to the liver, especially hepatic stellate cells, lipid membrane structures such as viral vectors, liposomes or micelles, particularly cationic lipid membrane structures suitable for intracellular delivery of nucleic acids. , which utilize DDS materials such as magnetic microparticles.
  • the present invention provides a pharmaceutical composition in which at least one nucleic acid selected from the group consisting of a) to e) is carried or encapsulated in such a DDS material, and a DDS material carries or encapsulates the nucleic acid. Also provided is said nucleic acid.
  • the pharmaceutical composition is preferably used in the form of parenteral preparations such as injections and infusions.
  • the administration method of the pharmaceutical composition is not particularly limited. topical administration to .
  • the pharmaceutical composition is applied to the subject by intravenous administration or by topical administration at or near the fibrotic site of the liver.
  • the pharmaceutical composition can be used as a combination drug in combination with other drugs for diseases or conditions that can cause liver fibrosis.
  • a combination medicament means a combination of medicaments intended to be administered together or separately, simultaneously or sequentially to a subject in need thereof, i.e. a subject with or at risk of liver fibrosis .
  • Intended modes of administration include administration of a formulation containing at least one nucleic acid selected from the group consisting of a) to e) above and other pharmaceutically active ingredients in one formulation, or administration of separate formulations. It can include administration of at least one nucleic acid selected from the group consisting of a) to e) formulated above and other pharmaceutical agents.
  • RNA polymerase inhibitors such as ribavirin, protease inhibitors such as asunaprevir, nonstructural protein 5A (NS5A) inhibitors such as daclatasvir, and nonstructural protein 5A (NS5A) inhibitors such as sofosbuvir.
  • RNA polymerase inhibitors of protein 5B (NS5B), interferons, and pharmaceutical combinations thereof can be mentioned.
  • the combination pharmaceutical can be a kit for treating hepatitis C, containing at least one nucleic acid selected from the group consisting of a) to e) above and a therapeutic agent for HCV.
  • the at least one nucleic acid and HCV therapeutic agent contained in the kit are administered together or separately, simultaneously or sequentially to a subject in need of treatment for hepatitis C, and administration of each formulation There are no particular restrictions on the order and timing of administration.
  • the present invention also provides, in one aspect, a pharmaceutical composition for the treatment of hepatitis C, comprising at least one nucleic acid selected from the group consisting of a) to e) above, wherein the treatment comprises C
  • a pharmaceutical composition for the treatment of hepatitis C comprising at least one nucleic acid selected from the group consisting of a) to e) above, wherein the treatment comprises C
  • the pharmaceutical composition is provided, comprising the use of a therapeutic agent for hepatitis virus.
  • the present invention provides a method of suppressing liver fibrosis in a subject by administering an effective amount of a pharmaceutical composition for suppressing liver fibrosis.
  • the present invention provides at least one nucleic acid selected from the group consisting of a) to e) for use in suppressing liver fibrosis in a subject, and in one aspect, liver fibrosis Provided is the use of at least one nucleic acid selected from the group consisting of a) to e) above in the manufacture of a pharmaceutical composition for inhibiting catalysis. Definitions and other explanations for each term are as described above.
  • an effective amount used herein with respect to agents and pharmaceutical compositions for suppressing liver fibrosis means an amount sufficient to suppress liver fibrosis in a subject, for example, suppressing progression of liver fibrosis It means an amount sufficient to reduce the size of the fibrotic site.
  • the term “effective amount” used herein with respect to agents and pharmaceutical compositions for suppressing activation of hepatic stellate cells means suppressing activation of hepatic stellate cells or inactivating activated hepatic stellate cells. means an amount sufficient to Such an effective amount is appropriately adjusted depending on the subject to be administered, degree of fibrosis and other medical factors.
  • a A method for evaluating the hepatic fibrosis-suppressing activity of a test substance as an index is provided.
  • the precursor is typically the precursor microRNA (hsa-mir-6848, SEQ ID NO: 3) or primary microRNA of hsa-miR-6848-5p and hsa-miR-6848-3p.
  • hsa-miR-6848-5p, hsa-miR-6848-3p, or substances capable of inducing or enhancing the expression of these precursors can induce or enhance the expression of these nucleic acids in hepatic stellate cells. It is expected that the activation of hepatic stellate cells can be suppressed and an inhibitory effect on hepatic fibrosis can be exhibited.
  • a method for evaluating the hepatic fibrosis-suppressing activity of a test substance comprises the step of incubating the test substance with hepatic stellate cells; hsa-miR-6848-5p, hsa- measuring the expression level of miR-6848-3p or their precursors; and a test substance that induced the expression of hsa-miR-6848-5p, hsa-miR-6848-3p or their precursors or increased the expression level of liver fibrosis suppression It includes the step of determining that it has activity.
  • Incubation of the hepatic stellate cells with the test substance is carried out in an appropriate medium in which the hepatic stellate cells can survive, typically in a physiological buffer such as PBS or a cell culture medium such as DMEM.
  • the test can be performed by coexisting the hepatic stellate cells and the test substance under temperature and oxygen conditions, for example, a temperature of 37° C. and 5% CO 2 .
  • hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors in hepatic stellate cells can be determined by hybridization, quantitative It can be carried out by PCR, RNA-Seq and other common methods capable of detecting and quantifying specific gene expression.
  • hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors is not detected in hepatic stellate cells not incubated with test substance but is detected in hepatic stellate cells incubated with test substance or expression levels of hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors in hepatic stellate cells incubated with the test article compared to hepatic stellate cells not incubated with the test article When it is high, the test substance can be determined to have hepatic fibrosis inhibitory activity.
  • the method for evaluating the hepatic fibrosis-suppressing activity of a test substance includes the method of evaluating the hepatic fibrosis-suppressing activity of a test substance, comprising: measuring the intensity of the marker gene-derived signal in the cells; comparing the measured intensity of the marker gene-derived signal with the intensity of the marker gene-derived signal in the absence of the test substance; and a marker A step of determining that the test substance that increased the intensity of the gene-derived signal has hepatic fibrosis-suppressing activity.
  • This method evaluates the effect of inducing or enhancing the expression of hsa-miR-6848-5p, hsa-miR-6848-3p, or their precursors using a reporter assay.
  • the marker gene is not particularly limited as long as the expression induction of the gene can be detected, for example, a gene encoding a fluorescent protein such as GFP or a chemiluminescent protein such as luciferase, or a drug resistance gene or auxotrophy of cells Genes that confer a phenotypic change on cells, such as genes that can complement , can be used.
  • Cells containing a nucleic acid having a marker gene under the control of the hsa-mir-6848 promoter region can be obtained, for example, by combining the nucleotide sequence of the genomic region encoding hsa-mir-6848 in hepatic stellate cells with the nucleotide sequence encoding the marker gene. It can be produced by recombination with Also, based on the human genome nucleotide sequence information, construct an expression vector containing a human hsa-mir-6848 promoter region and a marker gene recombined under its control, and introduce the expression vector into cells. can also
  • Incubation can be performed by coexisting the cells and the test substance in a medium or buffer solution in an appropriate container. Conditions such as incubation temperature and time may be conditions that allow the cell to express the marker gene. In addition, marker gene-derived signals can be measured by a method suitable for the marker gene used.
  • Example 1 Suppression of ⁇ SMA Expression by Introduction of miRNA 1 miRNA
  • the following miRNA manufactured by Bioneer (BIN) was purchased from Cosmo Bio and used. These miRNAs are double-stranded RNAs consisting of the base sequences shown below and their complementary base sequences.
  • Real-time PCR was performed using Platinum SYBER Green qPCR SuperMix-UDG with ROX (Thermo) and the following primers.
  • ⁇ -SMA When the expression level in mock miRNA-introduced hepatic stellate cells without TGF- ⁇ 1 was set to 1, the relative expression level of ⁇ -SMA in each cell is shown in Fig. 1, and the relative expression level of collagen I is shown in Fig. 2. .
  • miR-6848 was also confirmed to suppress the expression of collagen I that was particularly enhanced by TGF- ⁇ 1, demonstrating its ability to suppress fibrosis at the molecular level.
  • Example 2 Suppression of Activation of Wnt Signaling Pathway by Introduction of miRNA DMEM containing 2% FBS, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin was dispensed into a 6-well plate to give 1 ⁇ 10 5 cells/well.
  • Human hepatic stellate cells (LX-2 hepatic stellate cells, purchased from Millipore) were seeded and cultured.
  • the NFAT reporter vector and the AP-1 reporter vector activate the non-canonical Wnt pathway and NFAT/AP-1 It is an expression vector into which a firefly luciferase gene has been introduced under the TCF/LEF, NFAT or AP-1 promoter so that luciferase expression is induced when the expression of is increased.
  • human TGF- ⁇ 1 (PeproTech) was added to 2 ng/mL in some wells, and human Wnt3A (biotechne) was added to 100 ng/mL in some wells. It was added to the medium and cultured for an additional 24 hours. After culturing, the cells were harvested and whole cell lysates were prepared, and changes in each promoter activity were evaluated by dual luciferase assay. Luciferase activity was measured using a Dual Luciferase Reporter Assay System (Promega) with a luminometer (GloMax®-Multi Detection System, Promega). Corrected and taken as promoter activity.
  • Figure 3 shows the relative promoter activity values in each cell when the promoter activity in mock miRNA-introduced hepatic stellate cells to which TGF- ⁇ 1 and Wnt3A are not added is set to 1.
  • TGF- ⁇ 1 and Wnt3A are not added.
  • Wnt3A-induced suppression of TCF/LEF promoter activity was observed, but no such tendency was observed for NFAT and AP-1 promoter activities. This suggests that miR-6848 suppresses canonical Wnt pathway activation, but not non-canonical Wnt pathway activation.
  • Example 3 Inhibition of liver fibrosis by introduction of miRNA into liver fibrosis model mice 1) Preparation of miR-6848-encapsulated MEND It was manufactured according to the method described in JP 2016-141664. Specifically, to a tert-butanol solution containing a cationic lipid, cholesterol, and methoxyethylene glycol 2000 dimethylglycerol, an aqueous solution containing miR-6848 was added under stirring to obtain a lipid-miR-6848 mixed solution, and then quenched.
  • MENDs encapsulating miR-6848 were obtained by injecting the lipid-miR-6848 mixed solution into an acid buffer (pH 4.0) under agitation, followed by addition of PBS followed by ultrafiltration purification. 6848 encapsulated MEND) were manufactured. Similarly, miR-342-encapsulated MENDs (miR-342-encapsulated MENDs) and negative control mimics (AccuTarget TM miRNA mimic Negative control #1, Purification: BioRP, Bionner) were also encapsulated in MENDs (negative control mimic Encapsulated MEND) was manufactured. A 20% PBS solution of each manufactured MEND was used for administration to mice.
  • a tissue section was created from the collected liver and stained with Sirius Red. For sections after staining, the area of the region stained red with Sirius Red was measured using image analysis software WinROOF2018 Ver 4.15.0 (MITANI) and divided by the area of the entire observation field.

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JP2012532613A (ja) * 2009-07-09 2012-12-20 アブラクシス バイオサイエンス リミテッド ライアビリティー カンパニー Ｓｐａｒｃアンチセンス組成物及びその使用
JP2013531487A (ja) * 2010-06-02 2013-08-08 アルナイラム ファーマシューティカルズ， インコーポレイテッド 肝線維症治療用組成物および肝線維症の治療法
JP2013532952A (ja) * 2010-03-24 2013-08-22 アールエックスアイ ファーマシューティカルズ コーポレーション 皮膚および線維症適用におけるｒｎａ干渉
JP2017145222A (ja) * 2016-02-18 2017-08-24 公立大学法人大阪市立大学 肝線維化予防・治療剤
WO2019222693A1 (en) * 2018-05-17 2019-11-21 Lifesplice Pharma Llc Splice modulating oligonucleotides targeting receptor for advanced glycation end products and methods of use

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012532613A (ja) * 2009-07-09 2012-12-20 アブラクシス バイオサイエンス リミテッド ライアビリティー カンパニー Ｓｐａｒｃアンチセンス組成物及びその使用
JP2013532952A (ja) * 2010-03-24 2013-08-22 アールエックスアイ ファーマシューティカルズ コーポレーション 皮膚および線維症適用におけるｒｎａ干渉
JP2013531487A (ja) * 2010-06-02 2013-08-08 アルナイラム ファーマシューティカルズ， インコーポレイテッド 肝線維症治療用組成物および肝線維症の治療法
JP2017145222A (ja) * 2016-02-18 2017-08-24 公立大学法人大阪市立大学 肝線維化予防・治療剤
WO2019222693A1 (en) * 2018-05-17 2019-11-21 Lifesplice Pharma Llc Splice modulating oligonucleotides targeting receptor for advanced glycation end products and methods of use

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