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WO2022260162A1 - Agent prophylactique et/ou thérapeutique pour la stéatohépatite non alcoolique - Google Patents

Agent prophylactique et/ou thérapeutique pour la stéatohépatite non alcoolique Download PDF

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Publication number
WO2022260162A1
WO2022260162A1 PCT/JP2022/023447 JP2022023447W WO2022260162A1 WO 2022260162 A1 WO2022260162 A1 WO 2022260162A1 JP 2022023447 W JP2022023447 W JP 2022023447W WO 2022260162 A1 WO2022260162 A1 WO 2022260162A1
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mir
nash
nucleic acid
therapeutic agent
fat diet
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Japanese (ja)
Inventor
亘 尾野
貴裕 堀江
共達 西野
紗和 宮川
聡 小比賀
勇矢 笠原
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Kyoto University NUC
National Institutes of Biomedical Innovation Health and Nutrition
University of Osaka NUC
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Osaka University NUC
Kyoto University NUC
National Institutes of Biomedical Innovation Health and Nutrition
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility

Definitions

  • the present invention relates to prophylactic and/or therapeutic agents for NASH and screening methods thereof.
  • the present invention relates to NASH model animals.
  • the present invention relates to NASH model rodents and methods for their production.
  • Non-alcoholic steatohepatitis is a type of hepatitis that can progress to cirrhosis and liver cancer, and the number of patients has been increasing in recent years, making countermeasures urgently needed.
  • NASH Non-alcoholic steatohepatitis
  • the following pathogenesis has been proposed. First, fat accumulates in the liver due to insulin resistance such as obesity, diabetes, and hyperlipidemia, leading to fatty liver, and then oxidative stress such as lipid peroxidation, cytokines, and iron progresses to NASH. .
  • miRNAs are involved in various pathological conditions and disease formation through post-transcriptional regulation.
  • miRNAs are endogenous small RNA molecules consisting of 21 to 25 nucleotides, and their existence is known from sponges in the course of evolution.
  • the number of miRNAs increases with the complexity of organisms, and more than 2,500 miRNAs are thought to exist on the human genome. diversified to.
  • SREBP sterol regulatory element-binding protein
  • SCAP SREBP cleavage activating protein
  • miR-33 is one of miRNAs, and phylogenetically, it exists from Drosophila (dme-miR-33) and exists in the intron of dSREBP. It is speculated that later, due to gene duplication, miR-33b and miR-33a remained in their respective introns when SREBP-1 and SREBP-2 were generated. Humans have SREBP-1 and SREBP-2 and therefore miR-33b and miR-33a, but only a portion of miR-33b in the intron of rodent SREBP-1. Since it remains, miR-33b is absent in rodents (Non-Patent Document 2).
  • the objective of the present invention is to provide useful means for research and development of NASH mechanisms and treatment methods.
  • Another object of the present invention is to provide a prophylactic and/or therapeutic agent for NASH.
  • nucleic acid having a specific base sequence reduces the expression level of miR-33a and/or miR-33b.
  • miR-33b knock-in rodents fed a high-fat diet developed NASH-like symptoms. It was also found that the nucleic acid ameliorated NASH-like symptoms in rodents.
  • the present invention has been completed based on such findings, and broadly includes the subjects shown in the following items.
  • a prophylactic and/or therapeutic agent for NASH containing a nucleic acid is DNA, RNA, or a nucleic acid analog that may have a crosslinked structure;
  • the prophylactic and/or therapeutic agent for NASH, wherein the nucleic acid comprises the base sequence shown in SEQ ID NO: 1 (aacnacaangca) or 2 (aacagcaangca), and the base may be modified.
  • the nucleic acid consists of a nucleotide sequence in which 1 to 4 bases are deleted, substituted, inserted or added in the nucleotide sequence shown in SEQ ID NO: 1 or 2, and is a nucleic acid that recognizes human miR33b. 2.
  • the prophylactic and/or therapeutic agent for NASH according to 1.
  • Item 3 The preventive and/or therapeutic agent for NASH according to Item 1 or 2, wherein the nucleic acid consists of the base sequence shown in SEQ ID NO: 3 or 4.
  • the nucleic acid consists of a nucleotide sequence in which 1 to 4 bases are deleted, substituted, inserted or added in the nucleotide sequence shown in SEQ ID NO: 3 or 4, and is a nucleic acid that recognizes human miR33b.
  • the prophylactic and/or therapeutic agent for NASH according to any one of 1 to 3.
  • Item 5 The preventive and/or therapeutic agent for NASH according to any one of Items 1 to 4, wherein the nucleic acid contains RNA and RNA having a crosslinked structure.
  • Item 6 The preventive and/or therapeutic agent for NASH according to any one of Items 1 to 5, wherein the nucleic acid is a compound represented by the chemical formula in FIG. 1 or 2.
  • Item 8 The rodent according to Item 7, wherein the high-fat diet contains 40% or more lipid on a calorie basis.
  • Item 9 The rodent according to Item 7 or 8, wherein the high-fat diet does not contain cholesterol.
  • Item 10 The rodent according to Item 7 or 8, wherein the high-fat diet contains cholesterol.
  • Item 11 The rodent according to any one of Items 7 to 10, wherein the high-fat diet contains sugars.
  • Item 12 The rodent according to any one of Items 7 to 11, wherein miR-33b is knocked into the 3'UTR, intron or 5'UTR of the Srebf-1 gene.
  • Item 13 The rodent according to any one of Items 7 to 10, which is a NASH model animal.
  • Item 14 A method for producing a NASH model rodent, comprising the step of feeding a miR-33b knock-in rodent with a high-fat diet.
  • Item 15 The method for producing a rodent according to Item 14, wherein the high-fat diet contains 40% or more lipid on a calorie basis.
  • Item 16 The method for producing a rodent according to Item 14 or 15, wherein the high-fat diet does not contain cholesterol.
  • Item 17 The method for producing a rodent according to Item 14 or 15, wherein the high-fat diet contains cholesterol.
  • Item 18 The method for producing a rodent according to any one of Items 14 to 17, wherein the high-fat diet contains sugars.
  • Item 19 The method for producing a rodent according to any one of Items 14 to 18, wherein miR-33b is knocked into the 3'UTR, intron or 5'UTR of the Srebf-1 gene.
  • Item 20 The method for producing the rodent animal according to any one of Items 14 to 19, which is a NASH model animal.
  • Item 21 A method of screening for prophylactic and/or therapeutic agents for NASH, comprising the step of ingesting a high-fat diet and a test substance to miR-33b-knock-in rodents.
  • Item 22 The screening method for a preventive and/or therapeutic agent for NASH according to Item 21, wherein the high-fat diet contains 40% or more lipid on a calorie basis.
  • Item 23 The method of screening for a prophylactic and/or therapeutic agent for NASH according to Item 22 or 23, wherein the high-fat diet does not contain cholesterol.
  • Item 24 The screening method for a prophylactic and/or therapeutic agent for NASH according to Item 22 or 23, wherein the high-fat diet contains cholesterol.
  • Item 25 The screening method for a preventive and/or therapeutic agent for NASH according to any one of Items 22 to 24, wherein the high-fat diet contains sugars.
  • Item 26 The method of screening for a prophylactic and/or therapeutic agent for NASH according to any one of Items 22 to 25, wherein miR-33b is knocked into the 3'UTR, intron or 5'UTR of the Srebf-1 gene.
  • a prophylactic and/or therapeutic agent for NASH can be provided.
  • the present invention can provide high-fat diet-fed miR-33b knock-in rodents useful as NASH model animals exhibiting NASH-like symptoms. By using the model animal, candidate NASH preventive and/or therapeutic agents can be easily screened.
  • FIG. 1 shows the chemical formula of anti-miR-33a.
  • FIG. 2 shows the chemical formula of anti-miR-33b.
  • FIG. 3 shows the chemical formula of NEG-AmNA.
  • FIG. 4 shows miR-33b knock-in mice (miR-33b +/+ /miR-33b +/+ ) and wild-type mice (miR-33a +/+ /miR-33b ⁇ / ⁇ ) prepared in Example 1. and these mice prepared in Example 2 were fed with Research Diets, Inc. as a high-fat diet.
  • FIG. 5 shows hematoxylin-eosin (HE)-stained images (A) and Masson's trichrome-stained images (B) of the livers of high-fat diet-fed wild-type mice and HFD mice prepared in Example 2.
  • FIG. 6 shows single-strand DNA-stained images of livers of high-fat diet-fed wild-type mice and HFD mice prepared in Example 2.
  • FIG. 7 shows the results of lipids in the livers of the high-fat diet-fed wild-type mice and HFD mice prepared in Example 2, extracted using the Folch method, and measured by an enzymatic colorimetric method.
  • T-CHO indicates total cholesterol content
  • TG indicates triglyceride content
  • FC indicates free cholesterol content
  • PL indicates phospholipid content. * indicates P ⁇ 0.05.
  • FIG. 8 shows each gene (IL-6 and Tnf ⁇ as inflammatory cytokines, Col1a1 and ⁇ Sma as fibrosis markers, and CD68 and F4/80 as markers of inflammatory cell infiltration) in the HFD mouse liver prepared in Example 2. The results of measuring expression levels by qRT-PCR are shown.
  • FIG. 9 shows the results of Western blot analysis of the expression levels of genes (Col1a1 as a fibrosis marker) in the livers of high-fat diet-fed wild-type mice and HFD mice prepared in Example 2.
  • FIG. Figure 10 shows a liver excised from a 30 week HFD mouse.
  • A is a photographic image of the liver
  • B is an HE-stained image thereof.
  • FIG. 11 shows miR-33b knock-in mice shown in Example 5 loaded with D09103110 as a high-fat diet (herein, the mice may be referred to as GAN mice), body weight, blood tests, etc. Show the results.
  • NEG represents miR-33b knock-in mice not fed a high-fat diet subcutaneously injected with NEG-AmNA shown in FIG. 3
  • anti-33a is a mouse subcutaneously injected with NEG-AmNA shown in FIG. 1
  • anti-33b is a mouse subcutaneously injected with antimiR-33a shown in FIG.
  • Mice subcutaneously injected with -33b and anti-33a+b refer to mice subcutaneously injected with anti-miR-33a and anti-miR-33b above in GAN mice.
  • (D) is the amount of albumin (Alb) (g/dL)
  • (E) is the amount of urea nitrogen (BUN) (mg/dL)
  • (F) is creatinine (Cre) amount (mg / dL)
  • (I) is the amount of alkaline phosphatase (ALP) (IU/dL)
  • (J) is the amount of cholinesterase (ChE) (IU/dL)
  • (K) is total bilirubin (T- Bil) (mg/dL) and (L)
  • FIG. 12 shows expression levels of various genes in GAN mice and the like prepared in Example 5.
  • FIG. (A) is the expression level of miR-33a
  • (B) is the expression level of miR-33b
  • (C) is the relative value of the expression level of SREBF1 to 18S mRNA
  • (D) is the expression level of SREBF2. Shows the relative value of expression level to 18S mRNA.
  • NEG(NC), NEG(GAN), anti-33a, anti-33b and anti-33a+b in each graph are the same as in FIG.
  • FIG. 13 shows the results of blood tests and expression levels of various genes in GAN mice and the like prepared in Example 5.
  • A is the amount (g/dL) of albumin (Alb)
  • B is the amount (IU/L) of aspartate aminotransferase (AST)
  • AST aspartate aminotransferase
  • C is the amount of alanine aminotransferase (ALT).
  • (D) is the amount of alkaline phosphatase (ALP) (IU/L)
  • (E) is the amount of cholinesterase (ChE) (IU/L)
  • (F) is , the amount of total bilirubin (T-Bil) (mg/dL)
  • (G) the amount of total bile acid (TBA) ( ⁇ mol/dL)
  • (H) the amount of iron (Fe) ( ⁇ g /dL)
  • (I) shows the relative value of the expression level of the Abca1 gene to 18S mRNA in the liver
  • (J) shows the relative value of the expression level of the Cpt1a gene to 18S mRNA in the liver.
  • NEG(NC), NEG(GAN), anti-33a, anti-33b and anti-33a+b in each graph are the same as in FIG. * in each graph indicates p ⁇ 0.05, ** indicates p ⁇ 0.005, *** indicates p ⁇ 0.0005 and *** indicates p ⁇ 0.0001.
  • FIG. 14 shows the results of blood tests in GAN mice produced in Example 5.
  • (A) is the amount of total cholesterol (T-CHO) in serum (mg/dL)
  • (B) is the amount of HDL cholesterol (HDL-C) in serum (mg/dL)
  • (C ) is the amount of LDL cholesterol (LDL-C) in the serum (mg / dL)
  • (D) is the amount of free cholesterol (F-CHO) in the serum (mg / dL)
  • (E) is , the amount of triglycerides (TG) in serum (mg/dL)
  • (F) the amount of free fatty acids (NEFA) in serum ( ⁇ Eq/L)
  • (G) the total liver
  • the amount of cholesterol (mg/g), (H) the amount of free cholesterol in the liver (mg/g), and (I) the amount of triglyceride (TG) in the liver (mg/g) indicates NEG(NC), NEG(GAN), anti-33a, anti-33b, and anti-33a+b
  • FIG. 15(A) shows an HE-stained image of the GAN mouse liver section prepared in Example 5.
  • FIG. NEG (NC) and NEG (GAN) are the same as in FIG. 11 respectively, anti-33a (GAN), anti-33b (GAN) and anti-33a+b (GAN) are anti-33a, anti-33b and and anti-33a+b.
  • FIG. 16(A) shows a Picrosirius-stained image of the GAN mouse liver section prepared in Example 5.
  • NEG (NC), NEG (GAN), anti-33a (GAN), anti-33b (GAN), and anti-33a+b (GAN) are the same as in FIG. 11, respectively.
  • (B) is a graph of fibrosis scores evaluated from the stained images shown in (A). * in the graph indicates p ⁇ 0.05.
  • the number of specimens in the graph is the number of marks described in the graph. 17 shows the expression levels of various genes in GAN mice and the like prepared in Example 5.
  • FIG. (A) shows the expression level of TNF ⁇
  • (B) shows the expression level of Col1a1
  • (C) shows the expression level of ⁇ SMA.
  • NEG(NC), NEG(GAN), anti-33a, anti-33b and anti-33a+b in each graph are the same as in FIG. * in each graph indicates p ⁇ 0.05, ** indicates p ⁇ 0.005, *** indicates p ⁇ 0.0005 and *** indicates p ⁇ 0.0001.
  • FIG. 18 shows the results of Example 6.
  • FIG. (A) shows a microscopic image.
  • (B) is a graph showing the ratio of cholesterol crystals calculated from the microscopic image of (A).
  • NNC, NEG, anti-miR-33a, anti-miR-33b, and anti-miR-33a+b are NEG (NC), NEG (GAN), anti-33a (GAN), anti-33b (GAN), respectively, in FIG. ), and anti-33a+b (GAN). ** indicates p ⁇ 0.01.
  • Each number of specimens corresponds to the number of marks described in each graph.
  • FIG. 19 shows a method for generating miR-33b knock-in mice prepared in Example 1, etc.
  • the preventive and/or therapeutic agent for NASH of the present invention contains a nucleic acid.
  • the nucleic acid referred to here is, for example, DNA, RNA, or a nucleic acid analogue, and may have a crosslinked structure.
  • the nucleic acid analogue is not particularly limited as long as it is a polymer that has a specific base sequence and exhibits the same function as DNA or RNA due to the sequence.
  • Specific nucleic acid analogs include GNA, LNA, BNA, PNA, AmNA, morpholinos, and the like.
  • AmNA is preferred.
  • crosslinked structure is not particularly limited as long as it stabilizes the chemical structure of DNA, RNA or nucleic acid analogues and is not decomposed by enzymes or the like.
  • cross-linking may be provided so as not to affect the conformation of base moieties in DNA, RNA or nucleic acid analogues.
  • cross-linking examples include a mode in which a cross-link is provided in the sugar moiety contained in the chemical structure of DNA, RNA or nucleic acid analogues.
  • a preferred embodiment of cross-linking includes, for example, an embodiment in which a cross-link is provided between the 2'-position and 4'-position of the sugar moiety. Specifically, a bridge between the 2'-position and the 4'-position of the ribose ring as shown in the following formula can be mentioned.
  • R 1 represents a base.
  • R 2 and R 3 are the same or different and represent a phosphate group optionally having one or more substituents.
  • Substituents in such phosphoric acid groups are not particularly limited as long as the effects of the present invention are exhibited, and examples thereof include sulfur groups.
  • the crosslinked structure does not need to be provided in all the monomers that constitute the nucleic acid, and can be provided in some of the monomers.
  • the base sequence in the above nucleic acid consists of the base sequence of SEQ ID NO: 1 as aacnacaangca or the base sequence of SEQ ID NO: 2 as aacagcaangca in order from the 3' end.
  • the base sequence shown in SEQ ID NO: 2 is preferable in view of exhibiting the effects of the present invention.
  • a is adenine
  • c is cytosine
  • g is guanine
  • n is thymine or uracil. It is preferable that n is thymine in view of exhibiting the effect of the present invention more.
  • the base sequence of the above nucleic acid can tolerate mutation to a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 1 or 2 within the range that recognizes human miR33b, preferably 85% or more. more preferably 90% or more homology, most preferably 95% or more homology.
  • the above mutations can include deletion, substitution, insertion or addition.
  • the nucleotide sequence introduced with the above mutation is 4 nucleotides, more preferably 3 nucleotides, still more preferably 2 nucleotides, most preferably 1 It can be a nucleotide sequence of a nucleic acid that recognizes human miR33b, which consists of a nucleotide sequence in which 10 nucleotides are deleted, substituted, inserted or added.
  • recognizing human miR33b can be rephrased as binding to human miR33b, and is generally interpreted as binding of a nucleic acid consisting of the above base sequence to human miR33b. Specifically, it can be confirmed by a known method whether the two bind to each other, and the binding of the nucleic acid to human miR33b reduces the expression level of human miR33b. ) can be confirmed by the experiment shown in
  • the bases in the base sequence that constitutes the above nucleic acid can be modified. Such modifications are not particularly limited as long as the effect of the present invention is exhibited, and examples thereof include alkylation (methylation, ethylation, propylation, butylation, etc.), deamination, hydroxylation, halogenation, etc. can be mentioned. In view of exhibiting the effect of the present invention more, it is preferable that the above base is modified by methylation.
  • the type of base to be modified is not particularly limited as long as the effect of the present invention is exhibited. In view of exhibiting the effects of the present invention more effectively, it is preferable to modify cytosine, and it is more preferable to modify the 5-position of cytosine by methylation.
  • a more preferable base sequence of the above nucleic acid is the base sequence of SEQ ID NO: 3 as aactacaatgca or the base sequence of SEQ ID NO: 4 as aacagcaatgca in order from the 3' end.
  • the 5-position of cytosine in the above SEQ ID NOs: 3 and 4 is methylated.
  • the base sequence shown in SEQ ID NO: 4 is preferable in view of exhibiting the effect of the present invention.
  • the base sequence of the above nucleic acid can allow mutation to a base sequence having 80% or more homology with the base sequence shown in SEQ ID NO: 3 or 4 within the range of recognizing human miR33b.
  • the nucleotide sequence has a homology of 85% or more, more preferably a homology of 90% or more, and most preferably a homology of 95% or more.
  • the above mutations can include deletion, substitution, insertion or addition.
  • the nucleotide sequence into which the mutation is introduced is 4 bases, more preferably 3 bases, still more preferably 2 bases, most preferably 1 base in the base sequence shown in SEQ ID NO: 3 or 4. It can be a nucleotide sequence of a nucleic acid that recognizes human miR33b, consisting of a nucleotide sequence in which one base is deleted, substituted, inserted or added.
  • Preferred embodiments of the above-described nucleic acids include antisense oligonucleotides, siRNA, shRNA, and the like, more preferably antisense oligonucleotides, and still more preferably compounds represented by the chemical formulas of FIGS. can be mentioned. In view of the effect of the present invention being exhibited more effectively, the compound represented by the chemical formula in FIG. 2 is most preferable.
  • nucleic acids can be synthesized by conventional methods, and can be easily synthesized, for example, using a commercially available nucleic acid synthesizer.
  • AmNA in which nucleotides are sugar-modified which is a preferred embodiment of the above nucleic acid, can be synthesized by the method disclosed in WO11/052436.
  • siRNA and shRNA can be artificially chemically synthesized.
  • siRNA and shRNA can be used, for example, to synthesize antisense strand and sense strand RNA from template DNA in vitro using T7 RNA polymerase, T7 promoter, and the like.
  • the prophylactic and/or therapeutic agent for NASH may contain substantially only the nucleic acid, or may contain other components.
  • the above-mentioned other components are not particularly limited as long as the effects of the present invention can be exhibited.
  • examples include pharmaceutically acceptable bases, carriers, additives (eg, excipients, solvents, surfactants, preservatives, pH adjusters, thickening agents, etc.).
  • bases e.g, excipients, solvents, surfactants, preservatives, pH adjusters, thickening agents, etc.
  • additives eg, excipients, solvents, surfactants, preservatives, pH adjusters, thickening agents, etc.
  • Such base materials, carriers, additives and the like are described, for example, in Dictionary of Pharmaceutical Excipients, etc., and these can be employed as appropriate.
  • the dosage form of the prophylactic and/or therapeutic agent for NASH is not particularly limited as long as the effect of the present invention can be exhibited, and can be, for example, a solution, a suspension, or the like.
  • Such a dosage form can be appropriately prepared by mixing the nucleic acid as an active ingredient and other ingredients in a conventional manner.
  • the administration method of the preventive and/or therapeutic agent for NASH is not particularly limited as long as it is a known administration method optimized for the above various dosage forms within the range where the effects of the present invention can be exhibited.
  • oral administration intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, intradermal administration, intraperitoneal administration, intranasal administration, intrapulmonary administration, intraocular administration, intravaginal administration, intracervical administration Administration, rectal administration, subcutaneous administration, and the like can be mentioned.
  • intravenous administration is preferred.
  • the dose of the preventive and/or therapeutic agent for NASH described above is not particularly limited as long as the effects of the present invention can be exhibited.
  • administering 0.001 to 1000 mg/m 2 of a NASH prophylactic and/or therapeutic agent to an adult with a body weight of 60 kg can be mentioned.
  • the dose is 0.01 to 500 mg/m 2 , more preferably 0.1 to 100 mg/m 2 , and still more preferably 1 to 50 mg/m 2 .
  • a dose of ⁇ 40 mg/m 2 is most preferred.
  • Such doses can be administered once a day, or can be divided into several administrations per day.
  • High-fat diet-fed miR-33b knock-in rodents miR-33b ⁇ / ⁇ indicate wild-type mice and miR-33b +/+ miR-33b knock-in mice.
  • the knock-in rodent of the present invention is a rodent in which miR-33b is knocked in and fed with a high-fat diet (high-fat diet-loaded miR-33b knock-in rodent).
  • mice are not particularly limited, and examples include mice, rats, rats such as Chinese hamsters (), rabbits, and squirrels. Among them, mice, rats and the like are preferable.
  • the miR-33b knocked in in the above knock-in mouse is an RNA consisting of the base sequence of gugcauugcuguugcauugc (SEQ ID NO: 5). Knock-in of miR-33b is, for example, integration of DNA expressing the RNA into the genome.
  • Knock-in of miR-33b into rodents can be performed by known methods. For example, there is a method in which a gene modification (knock-in) vector is prepared, introduced into pluripotent stem cells (eg, ES cells, iPS cells, etc.), and miR-33b is knocked in by homologous recombination.
  • miR-33b can also be knocked in using genome editing technology (eg, CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) technology).
  • the miR-33b knock-in rodent of the present invention may have miR-33b heterozygous or homozygous, but is preferably homozygous.
  • the location on the genome to be knocked-in is preferably a location where knock-in miR-33b is expected to be expressed in rodents at the same time as miR-33b is expressed in humans. Since the location of miR-33b in humans is the intron (intron 16) of the sterol regulatory element-binding transcription factor 1 (srebf-1) gene that encodes SREBP-1, the expression of miR-33b in humans is It is thought to synchronize with the expression of the srebf-1 gene. Therefore, sites other than exons of the srebf-1 gene are preferred sites for miR-33b knock-in in rodents.
  • srebf-1 sterol regulatory element-binding transcription factor 1
  • Examples of such sites include 3'UTR (untranslated region), intron 5'UTR (untranslated region), and the like. Among them, intron is preferable.
  • the Srebf-1 gene has 19 exons. These exons (exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19), miR-33b may be knocked in in the intron between exons 16 and 17 of srebf-1. Since it is between exons 16 and 17, it is more preferable to knock in intron 16.
  • Gene ID 20787 for mouse srebf-1 gene, Gene ID 78968 for rat srebf-1 gene, and Gene ID 100689018 for Chinese hamster can be mentioned. is. Such genetic information can be retrieved, for example, from the NCBI web page.
  • the high-fat diet in the high-fat diet-loaded miR-33b knock-in rodent is not particularly limited. can.
  • the calorie base is preferably 45% or more, more preferably 50% or more, even more preferably 55% or more, and particularly preferably 60% or more.
  • a high-fat diet may or may not contain cholesterol.
  • Cholesterol-containing high-fat diets tend to result in rodents characterized by high accumulation of free cholesterol and triglycerides in the liver, whereas cholesterol-free high-fat diets result in accumulation of triglycerides in the liver.
  • Rodents tend to be obtained that are characterized by an abundance of
  • the content of cholesterol in the high-fat diet is not particularly limited as long as the effects of the present invention can be exhibited. preferably 0.01 to 5 parts by weight, more preferably 1 to 4 parts by weight, most preferably 1 to 3 parts by weight.
  • the high-fat diet may contain sugar.
  • Specific sugars are not particularly limited as long as the effects of the present invention can be exhibited. , fucose, fucrose, rhamnose, sucrose, lactose, maltose, trehalose, turanose, cellobiose and the like. It is preferable to contain fructose in view of exhibiting the effect of the present invention more.
  • it can be a high-fat diet containing palm oil.
  • Such a high-fat diet can be used by purchasing a commercially available product, for example Research Diets, Inc. , Japan SLC Co., Ltd., etc. Among them, product number: D12451 (45 kcal%) or product number D09103110 (40 kcal%) is preferable.
  • High-fat diet loading can be performed by having miR-33b knock-in rodents ingest the above-mentioned high-fat diet.
  • the period of ingestion is not particularly limited, and for example, it may be ingested until NASH-like symptoms are exhibited.
  • miR-33b knock-in rodents can be ingested for 8 weeks or longer, preferably 10 weeks or longer, and more preferably 12 weeks or longer.
  • the method of ingestion is not particularly limited, and for example, free ingestion can be mentioned.
  • High-fat diet-fed miR-33b knock-in rodents thus obtained exhibit NASH-like symptoms.
  • NASH-like symptoms for example, transaminase (AST, ALT) levels, alkaline phosphatase (ALP) levels, bilirubin levels, etc. in the liver become higher (preferably significantly higher) than wild-type.
  • findings such as an increase in lipid droplets in the liver and fibrosis of the liver tissue can be confirmed. Therefore, high-fat diet-fed miR-33b knock-in rodents are useful as NASH model animals.
  • miR-33b knock-in rodents can develop liver cancer by continuing to be fed a high-fat diet even after exhibiting NASH-like symptoms.
  • the ability to develop liver cancer is also a feature similar to NASH, so it can be understood that the high-fat diet-fed miR-33b knock-in rodent of the present invention is an excellent model animal for NASH.
  • a screening method for a prophylactic and/or therapeutic agent for NASH comprises: (1) a screening method for a NASH preventive or therapeutic agent, which comprises the step of ingesting a test substance to miR-33b knock-in rodents loaded with a high-fat diet; And a screening method for a NASH preventive or therapeutic agent, comprising the step of ingesting a test substance, encompasses
  • the test substance is not particularly limited, and examples include compounds and compositions.
  • examples of the above compounds include low-molecular-weight compounds, nucleic acids (eg, DNA, RNA, etc.), proteins (eg, antibodies or portions thereof, etc.), and macromolecular compounds such as polymers.
  • the composition can include extracts obtained from organisms (eg, animals, plants, microorganisms, etc.), and can also be a combination of two or more of the above compounds.
  • the method (route) of ingesting the test substance is not particularly limited, and can be selected as appropriate in consideration of the properties of the test substance to be studied.
  • the test substance can be administered to miR-33b knock-in rodents by oral administration, transvascular administration (intravenous administration, arterial administration), transdermal administration, and the like.
  • a device for administration such as a sonde or the like for oral administration and a syringe or the like for transvascular administration.
  • the test substance can be mixed with a pharmaceutically or food hygienically acceptable carrier, food, or the like, and administered as a composition suitable for each administration route.
  • the administered test substance can be selected as a NASH prophylactic or therapeutic agent candidate.
  • the administered test substance can be selected as a NASH prophylactic or therapeutic agent candidate.
  • the timing of ingestion of the high-fat meal and the test substance is not particularly limited. For example, both may be ingested at the same time, or the test substance may be ingested between meals.
  • the steps in the above screening method can be performed with one test animal and one control animal, it is preferable to examine groups of two or more animals (for example, 5 to 10 animals).
  • the expression “comprising” or “contains” a certain component means that the component is included and may further contain other components, and that only the component is included Also included is the concept of "consisting only of” in the sense of and “consisting essentially of” in the sense of comprising essentially the component.
  • Example 1 Preparation of miR-33b knock-in mice According to the method described in Non-Patent Document 1, human miR-33b (nucleotide sequence: GUGCAUUGCUGUUGCAUUGC; SEQ ID NO: 5) expressing human miR-33b (nucleotide sequence: GUGCAUUGCUGUUGCAUUGC; SEQ. was used to knock-in and generate male miR-33b knock-in mice (homologous: miR-33b +/+ ). As shown in FIG. 19, it was confirmed that the human miR-33b gene was integrated at the site of interest in the knock-in mouse (KI++), and that the human miR-33b gene was expressed.
  • the knock-in mouse is a C57BL/6J background that expresses miR-33a, and is a mouse that expresses miR-33b at the same time as humans express SREBF1, so miR-33a +/+ /miR-33b +/+ .
  • miR-33b knock-in mice miR-33a +/+ /miR-33b +/+
  • wild-type mice are referred to as miR-33b ⁇ / ⁇ .
  • Example 2 Preparation of HFD mice
  • the 8-week-old knock-in mice prepared in Example 1 were given 45% (45 kcal%) high-fat diet (D12451: Research Diets, Inc.) on a calorie basis ad lib (ad libitum). , food-challenged until 20 weeks of age.
  • FIG. 4 shows the body weight and high-fat diet intake of the mice at this time.
  • food-loaded knock-in mice (miR-33b +/+ ) were used as HDF mice
  • food-loaded wild-type mice (miR-33b ⁇ / ⁇ ) were used as comparative examples for various tests.
  • Example 3 Examination of HFD Mice (1) Serum Findings 20-week-old knock-in mice and wild-type mice loaded with food under the above conditions were fasted for 4 to 6 hours, blood was collected from the inferior vena cava under anesthesia, and serum fractions were collected. Serum biochemical findings were determined by standard methods on a Hitachi 7180 automated analyzer (Nagahama Life Science Laboratory, Nagahama, Japan). The results are shown in Table 1 below. In the table, AST, ALT, ALP and T-BIL in particular are known as hepatic dysfunction markers. *, **, and *** indicate P ⁇ 0.05, P ⁇ 0.01, and P ⁇ 0.001, respectively. These results suggested that HDF mice may have liver dysfunction.
  • FIG. 5 shows the results of hematoxylin and eosin staining.
  • FIG. 5(A) fatty deposits, inflammatory cell infiltration, hepatocyte balloon-like degeneration, etc. were observed in the liver of knock-in mice.
  • FIG. 5(B) many lipid droplets were observed in the liver of the knock-in mouse, and liver fibrosis was also observed.
  • Figure 6 shows the results of evaluating the degree of hepatocyte apoptosis using single-strand DNA staining. These results confirmed that HFD mice exhibited NASH-like symptoms.
  • FIG. 7 shows the results of measurement of these lipids (T-CHO, TG, FC and PL) by an enzymatic colorimetric method. Cholesterol and triglycerides were accumulated in the liver of HFD mice, demonstrating NASH-like symptoms.
  • qRT-PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo). The results are shown in FIG. 8 as a graph. Expression was normalized using the housekeeping gene ⁇ -actin. Table 2 shows the base sequences of the primers used.
  • the graph shown in FIG. 8 shows relative values of each gene expression level in the HFD mouse liver when each gene expression level by qRT-PCR in wild-type mouse (miR33b ⁇ / ⁇ ) is set to 1.
  • FIG. 9 shows the results of analyzing the expression of the fibrosis marker protein Col1a1 in the liver of HFD mice by Western blotting.
  • the primary antibodies used for Western blotting are anti-Col1a1 1/5000 (ab34710, Abcam, Cambridge, UK) and anti-Gapdh 1/3000 (14C10; no.2118S, Cell Signaling Technology, Beverly, MA, USA). .
  • liver cancer due to continued high-fat diet loading Livers were excised from 38-week-old HFD mice fed with the diet under the conditions described in Example 2 (ie, 30 weeks after the start of high-fat diet loading). A photograph of the liver (for 2 animals) is shown in FIG. 8(A). Along with the characteristics of fatty liver, liver cancer was observed at several sites (indicated by triangles in FIG. 10(A)). Further, the excised liver was subjected to HE staining in the same manner as described in "(2) Liver tissue findings" above, and the results are shown in FIG. 10(B). Since many lipid droplets were observed on the left side of (B), it was confirmed that the excised liver was fatty liver. In addition, on the right side of (B), it was also confirmed that the central reddish round area was liver cancer tissue, and that the excised liver had liver cancer.
  • NASH model mice by feeding them with a high-fat diet containing cholesterol or a methionine/choline-deficient (MCD) diet (References 3-6 below).
  • MCD methionine/choline-deficient
  • the diet fed to miR-33b knock-in mice is not MCD diet and cholesterol-free diet.
  • Such miR-33b knock-in mice are thought to be different from the NASH model mice described in references 3-6.
  • model animals produced on a methionine/choline deficient (MCD) diet may differ from model animals produced on a cholesterol-free, high-fat diet. Conceivable.
  • miR-33b knock-in mice of the present invention exhibiting phenotypes similar to clinical NAFLD pathology are very useful.
  • Example 4 Synthesis and Purification of Antisense Oligonucleotides AmNA amidites were obtained from Osaka Synthetic Organic Chemistry Laboratory Co., Ltd. Antisense oligonucleotides containing AmNA were synthesized and purified at Ajinomoto Bio-Pharma Services/Gene Design Co., Ltd.
  • the synthesized antisense oligonucleotides are shown in Figures 1 to 3.
  • 33a-2-AmNA (12) shown in FIG. 1 indicates an oligonucleotide against miR-33a
  • 33b-2-AmNA (12) shown in FIG. 2 shows an oligonucleotide against miR-33b
  • NEG-AmNA (12) represents a control antisense oligonucleotide.
  • the nucleosides having a bridge structure (notation of Y) are described as A(Y) for adenine, T(Y) for thymine, G(Y) for guanine, and 5 (Y) represents 5-methylcytosine.
  • a adenine
  • t thymine
  • g guanine
  • c cytosine, which are described as nucleosides having no cross-linking structure.
  • Phosphorothioate which indicates linkage between nucleosides, is represented by ⁇ .
  • Example 5 Preparation of GAN mice The 8-week-old knock-in mice prepared in Example 1 were fed a high-fat diet containing 40% (40kcal%) fructose, palm oil and 2% cholesterol on a calorie basis (D09103110: Research Diets, Inc.) was fed ad lib (ad libitum) until 20 weeks of age.
  • NEG-AmNA 10 mg/kg antimiR-33a, 10 mg/kg antimiR-33b, 10 mg/kg antimiR-33a+b (5 mg/kg antimiR-33a and 5 mg/kg of anti-miR-33b) and 10 mg/kg of NEG-AmNA were administered by subcutaneous injection every two weeks.
  • a miR-33b knock-in mouse not fed a high-fat diet was administered with 10 mg/kg NEG-AmNA (hereinafter referred to as "NEG (NC)").
  • NEG 10 mg/kg NEG-AmNA
  • anti-miR-33a may be referred to as anti-33a
  • anti-miR-33b may be referred to as anti-33b
  • anti-miR-33a+b may be referred to as anti-33a+b.
  • miR-33a tended to increase about 7.0-fold in the GAN feed-administered group compared to the normal feed group, and miR-33b tended to increase about 3.0-fold. A significant 9-fold increase was found.
  • miR-33a showed a significant decrease of about 0.19 times in the anti-33a administration group and about 0.14 times in the anti-33a+b administration group compared to the NEG-AmNA administration group.
  • the expression of miR-33b was significantly reduced by about 0.010 times in the anti-33b administration group and by about 0.12 times in the anti-33a+b administration group compared to the NEG-AmNA administration group.
  • the expression of miR-33b host gene Srebf1 was significantly increased by about 6.6 times in the GAN feed administration group compared to the normal feed administration group. It became clear that in the GAN feed administration group, the expression of Srebf1 showed a significant decrease of approximately 0.50 times in the anti-33b administration group. The expression of Srebf2, the host gene of miR-33a, did not change significantly between groups.
  • miR-33b is known to be expressed at a level 5 to 7 times higher than that of miR-33a. From the above results, miR-33a, like miR-33b, is thought to affect the expression of ABCA1 to some extent. , ALT and TBA tend to decrease.
  • GAN diet increases Srebf1 and miR-33b compared to normal diet due to the effects of insulin and hyperglycemia. From the above results, administration of anti-miR-33b is considered to significantly increase Abca1 and Cpt1a, which are target genes of miR-33b.
  • the anti-33b administration group Under the GAN diet, compared with the NEG administration group, the anti-33b administration group exhibited a significant increase of approximately 1.9 times, and the anti-33a+b administration group exhibited a significant increase of approximately 1.5 times.
  • the anti-33b administration group compared with the NEG administration group under the GAN diet, the anti-33b administration group showed a significant increase of about 1.6 times and the anti-33a+b administration group about 1.5 times.
  • the anti-33b administration group exhibited a significant increase of about 3 times compared to the NEG administration group under the GAN diet.
  • the neutral fat shown in FIG. 14(E) shows a significant decrease of about 0.5 times in the anti-33a+b administration group compared to the NEG administration group under the GAN diet. No significant difference in NEFA shown in FIG. 14(F) was observed between the groups.
  • Hepatic lipids were quantified using the Folch method described above. It was revealed that the total cholesterol shown in FIG. 14(G) was significantly increased by about 9.5 times in the GAN diet compared to the normal diet. Under the GAN diet, the anti-33b-administered group tended to decrease about 0.78-fold compared to the NEG-AmNA-administered group. It was revealed that the free cholesterol shown in FIG. 14(H) significantly increased by about 2.4 times in the GAN feed administration group compared to the normal feed administration group. Under the GAN diet, a significant decrease of approximately 0.84 times was observed in the anti-33b administration group compared to the NEG-AmNA administration group. It was revealed that neutral fat shown in FIG.
  • administration of anti-33b increases Abca1, a cholesterol transporter, and as a result, free cholesterol in the liver is excreted, resulting in a decrease in the liver and an increase in the blood. Conceivable. It is considered that the administration of anti-miR-33b improves liver function by improving inflammation and reducing free cholesterol, thereby promoting lipid decomposition and reducing accumulation of triglycerides in the liver.
  • liver evaluation of the livers of the GAN mice and the like was performed by hematoxylin-eosin (HE) staining. The results are shown in FIG. 15(A). Moreover, based on the stained image of FIG. 15(A), evaluation of severity was scored using NAFLD activity score (NAS). NAS scores the degree of steatosis (0 to 3 points), parenchymal inflammatory stage (0 to 3 points), and balloon-like hepatocyte enlargement (0 to 2 points). The results are shown in the graph of FIG. 15(B).
  • NAS NAFLD activity score
  • NAS was found to show a significant increase in the GAN feed administration group compared to the normal feed (normal feed: average 0.83 points, GAN feed: average 6.89 points).
  • the anti-33b-administered group under the GAN diet averaged 2.67 points, and the anti-33a+b-administered group averaged 4.29 points, showing a significant improvement in NAS compared to the NEG-AmNA-administered group.
  • Fibrosis of the liver of the above GAN mice, etc. was evaluated by Picrosirius staining. The results are shown in FIG. 16(A). Based on the stained image of FIG. 16(A), the fibrosis area of the liver was quantitatively evaluated using Image J (NIH). The results are shown in the graph of FIG. 16(B).
  • the fibrosis area was significantly increased by about 9.1 times in the GAN feed administration group compared to the normal feed administration group.
  • the anti-33b administration group significantly decreased by about 0.33 times compared to the NEG-AmNA administration group under the GAN diet.
  • the anti-33a+b administration group showed a decreasing tendency of about 0.46 times.
  • the NEG-AmNA administration group averaged 2.1 points
  • the anti-33b administration group had a mean of 0.25 points
  • the anti-33a+b administration group had an average of 0.75 points. admitted.
  • cDNA was prepared from the obtained total RNA using Verso cDNA synthesis kit (Thermo Fisher).
  • primers specific for TNF ⁇ , Col1a1, and ⁇ SMA in the liver shown in Table 2 above and TUNDERBIRD Syber q-PCR mix (Toyobo) were used in a StepOnePlus real-time PCR system (Thermo Fisher). detected.
  • Gene expression was corrected for expression of 18S ribosomal RNA (specific primer sequences are shown in Table 7). The results are shown in FIGS. 17(A) to 17(C).
  • FIG. 17(A) it was revealed that the expression level of TNF ⁇ (approximately 7.9 times) was significantly increased in the GAN feed administration group compared to the normal feed administration group.
  • FIG. 17(B) it was revealed that the expression level of Col1a1 was also significantly increased by about 28.3 times in the GAN feed administration group compared to the normal feed administration group.
  • FIG. 17(C) it was revealed that the expression level of ⁇ SMA also significantly increased to about 3.9 in the GAN feed administration group compared to the normal feed administration group.
  • TNF ⁇ (about 0.22 times), Col1a1 (about 0.074 times), and ⁇ SMA (about 0.087 times) were significant in the anti-33b administration group compared to the NEG-AmNA administration group under the GAN diet. showed a significant decline. Under the GAN diet, there was a significant reduction in TNF ⁇ (approximately 0.37-fold) and Col1a1 by approximately 0.16-fold and ⁇ SMA by approximately 0.29-fold in the anti-33a+b-treated group compared to the NEG-AmNA-treated group. showed a downward trend.
  • NLRs nucleotide-binding domains and leucine-rich repeats
  • NLRP3 NLR family pyrin domain-containing protein 3
  • NLRP3 in intrahepatic macrophages has been associated with many, including NASH. known to play a particularly important role in the development of chronic inflammatory diseases in humans. Therefore, cholesterol crystals were quantified in the above samples.

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Abstract

La présente invention aborde le problème de la fourniture d'un moyen qui est utile pour la recherche et le développement du mécanisme de la stéatohépatite non alcoolique (SHNA) et d'un procédé thérapeutique. En outre, la présente invention aborde le problème de la fourniture d'un agent prophylactique et/ou thérapeutique pour la SHNA. Pour résoudre ces problèmes, l'invention concerne un agent prophylactique et/ou thérapeutique pour la SHNA qui contient un acide nucléique ayant une séquence de base spécifique. L'invention concerne également un rongeur knock-in miR-33b nourri avec un régime riche en graisses.
PCT/JP2022/023447 2021-06-11 2022-06-10 Agent prophylactique et/ou thérapeutique pour la stéatohépatite non alcoolique Ceased WO2022260162A1 (fr)

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