WO2022019303A1 - COMPOSITION PHARMACEUTIQUE À BASE DE PYRROLE-IMIDAZOLE POLYAMIDE, INHIBITEUR DU GÈNE TGFβ, COMPOSITION PHARMACEUTIQUE ET PROCÉDÉ DE PRODUCTION DE PYRROLE-IMIDAZOLE - Google Patents

COMPOSITION PHARMACEUTIQUE À BASE DE PYRROLE-IMIDAZOLE POLYAMIDE, INHIBITEUR DU GÈNE TGFβ, COMPOSITION PHARMACEUTIQUE ET PROCÉDÉ DE PRODUCTION DE PYRROLE-IMIDAZOLE Download PDF

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WO2022019303A1
WO2022019303A1 PCT/JP2021/027144 JP2021027144W WO2022019303A1 WO 2022019303 A1 WO2022019303 A1 WO 2022019303A1 JP 2021027144 W JP2021027144 W JP 2021027144W WO 2022019303 A1 WO2022019303 A1 WO 2022019303A1
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gene
hgf
polyamide
tgf
transcription
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昇 福田
蘭 陳
高浩 上野
雅紀 阿部
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学校法人日本大学
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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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A61K31/787—Polymers containing nitrogen containing heterocyclic rings having nitrogen as a ring hetero atom
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • 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

Definitions

  • the present invention relates to a pyrrole imidazole polyamide, a TGF- ⁇ gene expression inhibitor, a pharmaceutical composition, and a method for producing a pyrrole imidazole polyamide.
  • Pyrrole imidazole polyamide (hereinafter, also referred to as "PI polyamide”) is a minor groove binder that strongly hydrogen-bonds to the minor groove of double-stranded DNA in a base sequence-specific manner.
  • PI polyamide Pyrrole imidazole polyamide
  • DDS drug delivery systems
  • PI polyamide has an advantage over DNA recognition compounds such as ordinary nucleic acid drugs.
  • PI polyamide can be freely molecularly designed by targeting various genes. Since the transcriptional activity of the disease gene activated by the disease can be specifically suppressed, there are few side effects.
  • PI polyamide may be used as an orally administrable gene regulator. Therefore, many drug discovery studies using PI polyamide have been conducted so far.
  • TGF- ⁇ 1 is a responsible factor for fibrotic diseases such as progressive nephropathy, liver cirrhosis and pulmonary fibrosis.
  • the present inventors have developed a PI polyamide that binds to the TGF- ⁇ 1 gene promoter and suppresses the expression of the TGF- ⁇ 1 gene (Patent Document 1).
  • the conventional PI polyamide was designed to target the promoter of one specific gene. Therefore, conventional PI polyamides could only control the transcription of one gene. However, for example, there may be cases where it is desired to simultaneously control the transcription of two or more target genes.
  • an object of the present invention is to provide a novel pyrrole imidazole polyamide capable of simultaneously controlling the transcription of two or more genes, a pharmaceutical composition containing the pyrrole imidazole polyamide, and a method for producing a pyrrole imidazole polyamide.
  • pyrrole imidazole polyamide which increases the expression of HGF and suppresses the expression of TGF- ⁇ , which is useful as a therapeutic agent for TGF- ⁇ -related diseases or fibrotic diseases, and the TGF- ⁇ gene using the pyrrole imidazole polyamide. It is an object of the present invention to provide an expression inhibitor and a pharmaceutical composition for treating a TGF- ⁇ -related disease or a fibrous disease.
  • the present invention includes the following aspects.
  • the two or more kinds of genes include a first gene and a second gene, and the first gene is a gene encoding an expression regulator of the second gene.
  • the pyrrole imidazole polyamide according to [2] which binds to the promoter of the first gene and inhibits the binding of a transcriptional regulator to the promoter of the first gene.
  • the pyrrole imidazole polyamide according to [6] which binds to a region of the promoter of the HGF gene containing at least a part of the base sequence set forth in SEQ ID NO: 1.
  • R 1 and R 2 each independently represent a monovalent organic group. R 1 and R 2 may be interconnected to form a divalent organic group.
  • the pyrrole imidazole polyamide according to [10] which is represented by the following formula Hu-HGF-3, Hu-HGF-5 or Hu-HGF-7.
  • a TGF- ⁇ gene expression inhibitor comprising the pyrrole imidazole polyamide according to any one of [4] to [11].
  • a pharmaceutical composition comprising the pyrrole imidazole polyamide according to any one of [1] to [11].
  • a pharmaceutical composition for treating a TGF- ⁇ -related disease which comprises the pyrrole imidazole polyamide according to any one of [4] to [11].
  • a pharmaceutical composition for treating a fibrotic disease which comprises the pyrrole imidazole polyamide according to any one of [4] to [11].
  • a novel pyrrole imidazole polyamide capable of simultaneously controlling the transcription of two or more kinds of genes, a pharmaceutical composition containing the pyrrole imidazole polyamide, and a method for producing a pyrrole imidazole polyamide are provided.
  • pyrrole imidazole polyamide which increases the expression of HGF useful as a therapeutic agent for TGF- ⁇ -related diseases or fibrotic diseases and suppresses the expression of TGF- ⁇ , and TGF- ⁇ gene expression using the pyrrole imidazole polyamide.
  • Inhibitors and pharmaceutical compositions for treating TGF- ⁇ -related or fibrous disorders are provided.
  • HGF- ⁇ It is a figure explaining the relationship between HGF and TGF- ⁇ .
  • the base sequence of the human HGF promoter is shown.
  • the underlined part indicates the COUP-TF1 binding site (-108 to -96).
  • the design of two kinds of PI polyamides (Hu-HGF-1, Hu-HGF-2) targeting the vicinity of the COUP-TF1 binding site is shown.
  • the underlined part indicates the COUP-TF1 binding site.
  • Py N-methylpyrrole unit
  • Im N-methylimidazole unit
  • ⁇ ⁇ -alanine unit
  • Ac acetyl group
  • Dp dimethylaminopropyl group.
  • the design of four PI polyamides (Hu-HGF-3, Hu-HGF-4, Hu-HGF-5, Hu-HGF-6) targeting the vicinity of the COUP-TF1 binding site is shown.
  • the underlined part indicates the COUP-TF1 binding site.
  • the design of two kinds of PI polyamides (Hu-HGF-7, Hu-HGF-8) targeting around the COUP-TF1 binding site is shown.
  • the underlined part indicates the COUP-TF1 binding site.
  • the structure of Hu-HGF-3 is shown.
  • the structure of Hu-HGF-5 is shown.
  • the structure of Hu-HGF-7 is shown.
  • the base sequence of the human TGF- ⁇ 1 promoter is shown. "+1" indicates the transcription start point.
  • the underlined part shows the transcription factor binding site.
  • the dotted line indicates the expected transcription factor binding site.
  • the sequence surrounded by a solid line indicates the binding site of Hu-HGF-3.
  • the sequence enclosed by the broken line indicates the Hu-HGF-7 binding site.
  • the human TGF- ⁇ 1 promoter around the Hu-HGF-3 binding site and the Hu-HGF-7 binding site is shown.
  • the sequence surrounded by a solid line indicates the binding site of Hu-HGF-3.
  • the sequence enclosed by the broken line indicates the Hu-HGF-7 binding site.
  • the broken line indicates the transcription factor binding prediction site.
  • the results of the gel shift assay for the HGF promoter of PI polyamide are shown. Hu-HGF-1 and Hu-HGF-2 were used as PI polyamides.
  • the results of the gel shift assay for the HGF promoter of PI polyamide are shown.
  • Hu-HGF-3 and Hu-HGF-4 were used as PI polyamides.
  • the results of the gel shift assay for the HGF promoter of PI polyamide are shown.
  • Hu-HGF-5 and Hu-HGF-6 were used as PI polyamides.
  • the outline of the test protocol of HGF expression level analysis and TGF- ⁇ 1 expression level analysis is shown. The operation indicated as "in the case of TGF- ⁇ 1 expression analysis" was performed only in the case of TGF- ⁇ 1 expression analysis.
  • the effect of Hu-HGF-3 on the mRNA expression level of HGF is shown.
  • HDF cells were used.
  • the effect of Hu-HGF-5 on the mRNA expression level of HGF is shown. HDF cells were used.
  • the effect of Hu-HGF-7 on the mRNA expression level of HGF is shown.
  • HDF cells were used.
  • the effect of Hu-HGF-3 on the mRNA expression level of TGF- ⁇ 1 is shown.
  • HDF cells were used.
  • the effect of Hu-HGF-5 on the mRNA expression level of TGF- ⁇ 1 is shown.
  • HDF cells were used.
  • the effect of Hu-HGF-7 on the mRNA expression level of TGF- ⁇ 1 is shown.
  • HGF cells were used.
  • the effect of Hu-HGF-5 on the mRNA expression level of TGF- ⁇ 1 is shown.
  • MC cells were used.
  • the effect of Hu-HGF-3 on the expression level of HGF protein is shown.
  • HDF cells were used.
  • the effect of Hu-HGF-5 on the expression level of HGF protein is shown.
  • HDF cells were used. The effect of Hu-HGF-7 on the expression level of HGF protein is shown. HDF cells were used. The effect of Hu-HGF-3 on the expression level of TGF- ⁇ 1 protein is shown. HDF cells were used. The effect of Hu-HGF-5 on the expression level of TGF- ⁇ 1 protein is shown. HDF cells were used. The effect of Hu-HGF-7 on the expression level of TGF- ⁇ 1 protein is shown. HDF cells were used. The effect of Hu-HGF-3 on the TGF- ⁇ 1 protein expression level in the presence of HGF siRNA is shown. HDF cells were used. # Indicates the result of the significance test for "1 DMSO" (# p ⁇ 0.05).
  • the invention provides a pyrrole imidazole polyamide that binds to the promoter regions of two or more genes and controls transcription of the two or more genes.
  • PI polyamides generally have a linker moiety (eg, a ⁇ -aminobutyric acid linker) that is entirely folded by the linker moiety to form a U-shape. In the U-shaped conformation, two chains containing Py and Im are arranged in parallel with the linker portion in between. The combination of specific pairs in this double strand binds to a specific base pair of DNA with high affinity. As a result, the PI polyamide enters the minor groove of the DNA double strand and binds to the DNA double strand in a sequence-specific manner.
  • PI polyamide In PI polyamide, Py / Im pair binds to C—G base pair, Im / Py pair binds to G—C base pair, and Py / Py pair binds to AT base pair or TA base pair. do.
  • PI polyamide may use 3-hydroxypyrrole unit (Hp) and ⁇ -alanine unit ( ⁇ ). Hp / Py pairs recognize TA base pairs, Py / Hp pairs recognize AT base pairs, ⁇ / ⁇ pairs recognize TA base pairs or AT base pairs, and ⁇ / Im pair recognizes C—G base pair, Im / ⁇ pair recognizes G—C base pair, and ⁇ / Py pair and Py / ⁇ pair recognize TA base pair or AT base pair. ..
  • Hp 3-hydroxypyrrole unit
  • ⁇ ⁇ -alanine unit
  • the number of pairs consisting of a combination of Py, Im, Hp and ⁇ constituting the PI polyamide is not particularly limited as long as it is 2 or more, but 3 to 12 is preferable, 4 to 10 is more preferable, and 4 to 10 is more preferable. Eight are more preferred.
  • the PI polyamide has 5 or more pairs, it is preferable to have 1 or more pairs containing ⁇ .
  • the methyl group bonded to nitrogen at the 1-position of Py and Im may be replaced with a hydrogen atom or an aliphatic hydrocarbon group having 2 to 10 carbon atoms.
  • the aliphatic hydrocarbon group may be linear or branched.
  • the aliphatic hydrocarbon group may be saturated or unsaturated.
  • the aliphatic hydrocarbon group is preferably an alkyl group, and examples thereof include an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group and a tert-butyl group.
  • the linker moiety of PI polyamide is a ⁇ -aminobutyric acid linker
  • it may have a substituent at the ⁇ -position or the ⁇ -position.
  • it may be an N- ⁇ -N- ⁇ -diaminobutyric acid linker in which the ⁇ -position is substituted with an amino group, or an N- ⁇ -N- ⁇ -diaminobutyric acid linker in which the ⁇ -position is substituted with an amino group. May be.
  • the amino group may be modified with a molecule such as a fluorescent group or biotin.
  • Various molecules may be introduced into the terminal portion of PI polyamide.
  • the molecule introduced into the terminal portion of PI polyamide is not particularly limited.
  • Various molecules can be introduced into the end of the PI polyamide, for example, via an amide bond.
  • Examples of such molecules include, but are not limited to, fluorescent dyes, biotins, alkylating agents and the like.
  • an acetyl group may be introduced into the N-terminal portion of PI polyamide, and a dimethylaminopropylamino group may be introduced into the C-terminal portion.
  • PI polyamide can be synthesized by a known method.
  • the PI polyamide can be produced, for example, by an automatic synthesis method by a solid phase method (solid phase Fmoc method) using Fmoc (9-fluorenylmethoxycarbonyl) (International Publication No. 03/000683).
  • solid phase Fmoc method solid phase Fmoc method
  • Fmoc (9-fluorenylmethoxycarbonyl) International Publication No. 03/000683
  • the terminal of PI polyamide can be cut out from the solid support as a carboxylic acid residue. Therefore, various functional groups can be introduced into the molecular ends to produce PI polyamide derivatives.
  • compounds having an alkylating ability for DNA such as duocarmycin, pyrolobenzodiazepine, bleomycin, enediyne compound, nitrogen mustard, and derivatives thereof can be introduced into the terminal of PI polyamide.
  • the solid-phase Fmoc method is an automatic synthesis method using a commercially available protein (peptide) synthesizer, it is also possible to synthesize a conjugate of a naturally occurring protein or an unnatural protein and a pyrrole imidazole polyamide.
  • the reaction conditions of the solid-phase Fmoc method are more relaxed than those of the t-BOC method, it is possible to introduce organic compounds other than proteins (including compounds having unstable functional groups under acidic conditions). ..
  • PI polyamide having a carboxy group at the terminal can be synthesized.
  • Specific examples thereof include PI polyamide having a ⁇ -alanine residue ( ⁇ -aminopropionic acid residue) or a ⁇ -aminobutyric acid residue at the terminal.
  • PI polyamides having ⁇ -alanine residues or ⁇ -aminobutyric acid residues at the ends include, for example, aminopyrol carboxylic acid, aminoimidazole carboxylic acid, ⁇ -alanine or ⁇ -aminobutyric acid, each of which has an amino group protected by Fmoc.
  • the supported solid phase carrier can be used for synthesis by the solid phase Fmoc method using a peptide synthesizer.
  • aminopyrrole carboxylic acid examples include 4-amino-2-pyrrole carboxylic acid, 4-amino-1-methyl-2-pyrrole carboxylic acid, 4-amino-1-ethyl-2-pyrrole carboxylic acid, and the like. Examples thereof include 4-amino-1-propyl-2-pyrrolecarboxylic acid and 4-amino-1-butyl-2-pyrrolecarboxylic acid.
  • aminoimidazole carboxylic acid include 4-amino-2-imidazole carboxylic acid, 4-amino-1-methyl-2-imidazole carboxylic acid, 4-amino-1-ethyl-2-imidazole carboxylic acid, and the like. Examples thereof include 4-amino-1-propyl-2-imidazole carboxylic acid and 4-amino-1-butyl-2-imidazole carboxylic acid.
  • a conjugate of PI polyamide and FITC fluorescein isothiocyanate
  • FITC fluorescein isothiocyanate
  • the conjugate of PI polyamide and FITC can be used to prove that the PI polyamide recognizes a particular DNA sequence.
  • the PI polyamide of this embodiment binds to the promoters of two or more kinds of genes and controls the transcription of the two or more kinds of genes.
  • PI polyamides have been designed to target the promoter of the particular gene for the purpose of controlling the expression of that particular gene.
  • the PI polyamide of this embodiment is designed based on a completely new concept of controlling the expression of two or more genes at the same time.
  • the PI polyamide of this embodiment is designed to bind to the promoters of two or more genes. By binding to the promoters of two or more genes, it is possible to control the transcription of two or more genes with one type of PI polyamide.
  • the PI polyamide of this embodiment is preferably designed to bind to a transcription promoting region or a transcription repressing region in the promoters of two types of genes.
  • promoter means a region located upstream of a gene and regulating transcription of the gene.
  • transcription promoting region means a region located upstream of a gene and in which transcription of the gene is promoted by binding to a transcription factor, a complex protein, or the like. Transcription-promoting regions can usually be present in the promoter.
  • transcription-suppressing region means a region located upstream of a gene and in which transcription of the gene is suppressed by binding to a transcription factor, a complex protein, or the like. The transcriptional repression region can usually be present in the promoter.
  • transcriptional regulatory factor means a protein (including a transcription factor, a complex protein, etc.) that binds to a transcription promoting region or a transcriptional repressing region of a specific gene and promotes or suppresses the transcription of the gene.
  • expression control factor means a protein (including a transcription factor, a complex protein, etc.) that promotes or suppresses the expression of a specific gene.
  • the expression regulator may be any protein whose expression of the specific gene is upregulated or downregulated by the presence of the protein, and includes a transcriptional regulator.
  • a transcriptional regulator When a transcriptional regulator binds to the transcriptional promoting region of the promoter, gene transcription is initiated or promoted. When a transcriptional regulator binds to the transcriptional repressive region of a promoter, gene transcription is repressed. Therefore, it is possible to inhibit the transcription of a gene by inhibiting the binding of a transcriptional regulatory factor to the transcription promoting region in the promoter. In addition, gene transcription can be promoted by inhibiting the binding of transcriptional regulators to the transcriptional repressive region of the promoter. Therefore, when PI polyamide binds to the transcription promoting region in the promoter, the binding of the transcriptional regulator to the transcription promoting region is inhibited, and the expression of the gene is inhibited. When the PI polyamide binds to the transcriptional repressor region of the promoter, the binding of the transcriptional regulator to the transcriptional repressor region is inhibited, and gene expression is initiated or promoted.
  • controlling the transcription of a gene means changing the transcriptional activity of the gene. Transcription control may be upward control or downward control.
  • Transcription control may be upward control or downward control.
  • PI polyamide binds to the transcription-promoting region of the promoter, the binding of transcriptional regulators to the transcription-promoting region is inhibited and gene expression is down-regulated.
  • the PI polyamide binds to the transcriptional repressive region in the promoter, the binding of the transcriptional regulator to the transcriptional repressor region is inhibited and the gene expression is upwardly regulated.
  • PI polyamide that binds to the promoters of two or more genes can be performed, for example, as follows.
  • Two or more kinds of genes can be arbitrarily selected according to the purpose.
  • Examples of the two or more genes include a combination of two or more genes whose expression is upregulated or downregulated in a specific disease; a combination of a gene whose expression is upregulated in a specific disease and a gene whose expression is downregulated in the specific disease, and the like. Will be.
  • oncogenes whose expression is enhanced in a cancer-specific manner
  • genes whose expression is enhanced in a cancer-specific manner include CA125, CEA, CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD70, CD8, CLL-1, c-Met, PSA, PSMA, ROR1, HER2, MAGE, p53 and the like can be mentioned.
  • PI polyamide When PI polyamide is used for cancer treatment, two or more of these oncogenes may be selected as target genes.
  • an immune checkpoint molecule gene PD1, PD-L1, CTLA-4, etc.
  • an oncogene may be selected as two or more target genes.
  • Examples of the combination of the gene whose expression is enhanced in a specific disease and the gene whose expression is decreased in the specific disease include a combination of a TGF- ⁇ gene and an HGF gene.
  • the promoter sequences of two or more target genes are analyzed to identify the transcription-promoting region and the transcription-suppressing region. For example, for a gene whose expression is suppressed, a transcription promoting region is acquired. For genes that promote expression, a transcriptional repression region is acquired.
  • a PI polyamide that binds to a specific sequence in the acquired transcriptional control region is designed.
  • the designed PI polyamide can bind to the transcriptional control region of the remaining target genes. If the PI polyamide is capable of binding to the transcriptional control regions of all target genes, then that PI polyamide is selected. If there is a target gene that is not bindable to the transcriptional control region, select other sequences in the transcriptional control region and redesign the PI polyamide. It is confirmed whether the redesigned PI polyamide can bind to the transcriptional control region of the remaining target genes. Hereinafter, the above steps are repeated until a PI polyamide capable of binding to the transcriptional control region of all target genes can be obtained.
  • PI polyamide that binds to the transcriptional control regions of two or more genes can be obtained.
  • the number of target genes of PI polyamide is not particularly limited, but is, for example, 2 to 5, 2 to 4, 2 to 3, or 2.
  • PI polyamide can control the transcription of both the first gene and the second gene.
  • the first gene is the gene encoding the transcriptional regulator of the second gene.
  • the second gene is promoted by PI polyamide by promoting the transcription of the first gene and inhibiting the transcription of the second gene. Transcription of the gene can be suppressed more strongly.
  • the PI polyamide is preferably bound to the transcriptional repression region in the promoter of the first gene.
  • PI polyamide preferably binds to the transcription promoting region in the promoter of the second gene.
  • the PI polyamide suppresses the transcription of the first gene and promotes the transcription of the second gene. It can more strongly promote the transcription of the second gene.
  • the PI polyamide preferably binds to the transactivation region in the promoter of the first gene.
  • PI polyamide preferably binds to the transcriptional repression region in the promoter of the second gene.
  • the second gene is promoted by promoting the transcription of the first gene by PI polyamide and promoting the transcription of the second gene. Transcription of the gene can be promoted more strongly.
  • the PI polyamide is preferably bound to the transcriptional repression region in the promoter of the first gene.
  • PI polyamide preferably binds to the transcriptional repression region in the promoter of the second gene.
  • the PI polyamide suppresses the transcription of the first gene and suppresses the transcription of the second gene. Transcription of the second gene can be suppressed more strongly.
  • the PI polyamide preferably binds to the transactivation region in the promoter of the first gene.
  • PI polyamide preferably binds to the transcription promoting region in the promoter of the second gene.
  • the first gene and the second gene are not particularly limited, and any gene can be used.
  • any combination may be selected depending on the purpose from those listed as the combinations of the above two or more target genes.
  • Examples of the first gene include the HGF gene.
  • Examples of the second gene include the TGF- ⁇ gene.
  • PI polyamide may have a function of promoting the transcription of the HGF gene and inhibiting the transcription of the TGF- ⁇ gene. preferable.
  • PI polyamide (P) a PI polyamide in which the first gene is an HGF gene and the second gene is TGF- ⁇ may be referred to as “PI polyamide (P)”.
  • FIG. 1 is a diagram illustrating the relationship between HGF and TGF- ⁇ .
  • TGF- ⁇ has actions such as fibroblast activation, mesangial cell activation, and epithelial-mesenchymal transition, and promotes tissue fibrosis.
  • HGF exhibits angiogenic action, anti-apoptotic action, anti-fibrotic action, and has an action of suppressing the expression of TGF- ⁇ . Therefore, by increasing the expression level of HGF, the expression level of TGF- ⁇ is suppressed, and the progress of fibrosis is suppressed.
  • PI polyamide (P) promotes the transcription of the HGF gene and inhibits the transcription of the TGF- ⁇ gene.
  • the inhibitory effect of HGF on the expression of TGF- ⁇ becomes stronger.
  • PI polyamide (P) directly inhibits the transcription of the TGF- ⁇ gene, it strongly suppresses the expression of the TGF- ⁇ protein by the additive effect with the TGF- ⁇ expression inhibitory action of HGF. Therefore, according to PI polyamide (P), the progress of fibrosis due to TGF- ⁇ can be efficiently suppressed.
  • PI polyamide (P) is an HGF-mediated expression suppression mechanism of TGF- ⁇ (hereinafter, also referred to as “expression suppression mechanism 1”) by promoting transcription of the HGF gene, and TGF- ⁇ by inhibiting the transcription of the TGF- ⁇ gene.
  • expression suppression mechanism 1 an HGF-mediated expression suppression mechanism of TGF- ⁇
  • TGF- ⁇ by promoting transcription of the HGF gene
  • TGF- ⁇ by inhibiting the transcription of the TGF- ⁇ gene.
  • the expression of TGF- ⁇ can be suppressed by two expression suppression mechanisms of the expression suppression mechanism (hereinafter, also referred to as “expression suppression mechanism 2”). Therefore, the PI polyamide (P) can suppress the expression of TGF- ⁇ at a lower concentration as compared with the conventional PI polyamide that suppresses the expression of TGF- ⁇ only by the expression suppression mechanism 2.
  • PI polyamide (P) inhibits the binding of the transcriptional regulatory factor to the transcriptional repressive region in the HGF promoter by binding to the transcriptional repressive region in the promoter of the HGF gene (hereinafter, also referred to as “HGF promoter”). ..
  • HGF promoter the transcriptional repressive region in the promoter of the HGF gene
  • the PI polyamide (P) is preferably bound to a region of the HGF promoter containing at least a part of the base sequence (AGGTGACCTTTTC) shown in SEQ ID NO: 1.
  • the nucleotide sequence shown in SEQ ID NO: 1 is a sequence predicted as a binding site of COUP-TF1 by analysis of the human HGF promoter. It has been reported that the binding site of COUP-TF1 is often a transcriptional repression region. It has been reported that COUP-TF1 often acts as a transcriptional repressor by binding to the binding site.
  • sequence to which PI polyamide (P) binds in the HGF promoter include a region containing any of the following sequences 1 to 3. As shown in Examples below, PI polyamides (Hu-HGF-3, Hu-HGF-5, Hu-HGF-7) designed to bind to the following sequences 1-3 are the expression of TGF- ⁇ 1. Can be effectively suppressed. Sequence 1: AGGTGAC Sequence 2: ACCTTTT Sequence 3: CTTTTCT
  • PI polyamide (P) inhibits the transcription of the TGF- ⁇ gene by binding to the transcriptional repression region in the promoter of the TGF- ⁇ gene (hereinafter referred to as "TGF- ⁇ promoter").
  • TGF- ⁇ promoter the transcriptional repression region in the promoter of the TGF- ⁇ gene
  • PI polyamide (P) inhibits transcription of the TGF- ⁇ gene by inhibiting the binding of transcriptional regulators to the transcriptional repressor region of the TGF- ⁇ promoter.
  • TGF- ⁇ may be any of TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3, but TGF- ⁇ 1 is preferable.
  • PI polyamide (P) changes the three-dimensional structure of the TGF- ⁇ promoter by binding to the peripheral region of the TGF- ⁇ promoter, and inhibits the binding of transcriptional regulators to the transcriptional repressor region of the TGF- ⁇ promoter. May be.
  • PI polyamide (P) A suitable example of PI polyamide (P) is given below.
  • R 1 and R 2 each independently represent a monovalent organic group.
  • R 1 and R 2 may be interconnected to form a divalent organic group.
  • R 1 and R 2 each independently represent a monovalent organic group.
  • R 1 and R 2 are not particularly limited and may be any organic group.
  • Any molecule may be introduced into R 1 and R 2 , for example, via an amide bond. Examples of the introduced molecule include those listed as the introduced molecule to the terminal of the PI polyamide.
  • R 1 may be, for example, a group represented by -NHCO-R 11 (R 11 is a monovalent organic group).
  • R 11 is not particularly limited, and examples thereof include an aliphatic hydrocarbon group.
  • the aliphatic hydrocarbon group may be linear, may be branched, or may be cyclic.
  • the aliphatic hydrocarbon group may be saturated or unsaturated. Examples of the number of carbon atoms of the aliphatic hydrocarbon group include 1 to 10 carbon atoms, 1 to 5 carbon atoms, 1 to 3 carbon atoms, 1 or 2 carbon atoms, and the like.
  • the R 11 is preferably an alkyl group, and examples thereof include a methyl group and an ethyl group.
  • R 2 may be, for example, a group represented by —CONH-R 21 (R 21 is a monovalent organic group).
  • the R 21 is not particularly limited, and examples thereof include an aliphatic hydrocarbon group which may have a substituent.
  • the aliphatic hydrocarbon group may be linear, may be branched, or may be cyclic.
  • the aliphatic hydrocarbon group may be saturated or unsaturated. Examples of the number of carbon atoms of the aliphatic hydrocarbon group include 1 to 15 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, and the like.
  • Examples of the substituent that the aliphatic hydrocarbon group may have include, but are not limited to, an amino group, a hydroxy group, a carboxy group and the like.
  • the aliphatic hydrocarbon group may have a part of carbon atoms constituting the carbon chain substituted with —O—, —CO—, —COO—, —CONH—, or the like.
  • R 21 may be a group represented by ⁇ -R 22 ( ⁇ is a ⁇ -alanine unit; R 22 is a monovalent organic group).
  • the R 22 is not particularly limited, and examples thereof include an aliphatic hydrocarbon group which may have a substituent.
  • the aliphatic hydrocarbon group may have the substituent include the same ones as exemplified in R 21.
  • Examples of R 22 include a dimethylaminoalkyl group and an aminoalkyl group. Specific examples of R 22 include, for example, a dimethylaminopropyl group and an aminopropyl group.
  • R 1 and R 2 may be interconnected to form a divalent organic group.
  • the PI polyamide represented by the formula (P-3), (P-5) or (P-7) forms a cyclic structure.
  • Cyclic peptides are known to be suitable for oral administration (Keiichi Masuya, Journal of Pharmacology, 148.322-328 (2016)). It has also been reported that the binding properties of PI polyamide do not change significantly even when PI polyamide is used as a cyclic structure (David M. Chenoweth, J Am Chem Soc. 2009 May 27; 131 (20): 7182-7188.). .. Therefore, the cyclic structure PI polyamide is promising as an orally administered drug.
  • the invention provides a TGF- ⁇ gene expression inhibitor comprising PI polyamide (P).
  • PI polyamide (P) can effectively suppress TGF- ⁇ gene expression as described above. Therefore, PI polyamide (P) can be used as a TGF- ⁇ gene expression inhibitor.
  • gene expression means that the protein encoded by the gene is produced by transcription and translation of the gene.
  • gene expression inhibitor means an agent having an action of suppressing the production of a protein encoded by the gene.
  • the TGF- ⁇ gene expression inhibitor of this embodiment may be used in vitro or in vivo. When used in vitro, it is preferably used for human cells or common marmoset cells. When used in vivo, it is preferably administered to humans or common marmosets. When used in vivo, it may be formulated as a pharmaceutical composition described later.
  • the invention provides a pharmaceutical composition comprising said PI polyamide.
  • the PI polyamide of the above embodiment can control the transcription of two or more target genes, it can be used as a pharmaceutical composition for treating a disease associated with abnormal gene expression. Two or more target genes can be appropriately selected depending on the disease to be treated.
  • the invention provides a pharmaceutical composition for treating a TGF- ⁇ related disease, comprising PI polyamide (P). In one aspect, the invention provides a pharmaceutical composition for treating a fibrotic disease, comprising PI polyamide (P).
  • TGF- ⁇ -related disease means a disease caused by an increase in TGF- ⁇ .
  • TGF- ⁇ -related diseases include, but are not limited to, fibrotic diseases, various renal diseases, and male-type frontal hair loss.
  • Fibrotic disease means a disease that occurs with fibrosis of a tissue or organ. The fibrotic disease is not particularly limited, and is, for example, liver cirrhosis, pulmonary fibrosis, renal fibrosis, pancreatic fibrosis, myocardial fibrosis, myeloid fibrosis, retroperitoneal fibrosis, mesenteric fibrosis, mammary fibrosis, cyst.
  • Examples include sexual fibrosis, gastrointestinal fibrosis, adipose tissue fibrosis, systemic fibrosis, localized fibrosis, keroids, hypertrophic scars, post-skin wound or post-skin ulcer scars, skin fibrosis, etc. , Not limited to these.
  • Preferred examples of the fibrotic disease include various liver diseases, various renal diseases, pancreatic fibrosis, myocardial fibrosis, and various skin fibrosis diseases described later.
  • hepatic stellate cells play an important role in extracellular matrix production (Bataller R et al, Gastroenterology 118,1149,2000). Activation of stellate cells is carried out by TGF- ⁇ 1, and further activated stellate cells cause the secretion of TGF- ⁇ 1 from inflammatory cells in the injured liver. At the same time, TGF- ⁇ 1 receptor expression in activated stellate cells is enhanced, and extracellular matrix protein is increased by the autocrine mechanism by TGF- ⁇ 1 (Hisatake Watanabe et al., Hyundai Medical, Vol. 35 (No. 2), 2003). From these facts, the above-mentioned various liver diseases can be exemplified as TGF- ⁇ -related diseases.
  • model animals for renal diseases such as IgA nephropathy, focal glomerulonephritis, crescentic nephritis, focal sclerosing lupus nephritis, diffuse proliferative lupus nephritis, diabetic nephropathy, and hypertensive nephropathy.
  • the expression of TGF- ⁇ is increased in parallel with the extracellular substrate in the renal biopsy tissue of patients with glomerulonephritis and diabetic nephropathy (Yamamoto T et al: Kidney Int 49: 461, 1996, Border WA). et al: Kidney Int 51: 1388, 1997).
  • TGF- ⁇ is continuously enhanced in the infarct lesion during the scar formation stage and is involved in the promotion of myocardial fibrosis (Ono et al: Circulation 98: 149, 1998). From these facts, myocardial fibrosis after myocardial infarction can be exemplified as a TGF- ⁇ -related disease.
  • pulmonary fibrosis is improved by administering an anti-TGF- ⁇ antibody or TGF- ⁇ soluble receptor to a pulmonary fibrosis model animal (Giri SN al: Thorax 1993). From these facts, pulmonary fibrosis can be exemplified as a TGF- ⁇ related disease.
  • TGF- ⁇ was proposed as a cause of scleroderma, and Mori et al. Reported that TGF- ⁇ induces skin fibrosis in skin fibrosis model mice (Mori et al: J Cell Physiol 181: 153). , 1999). From these facts, various skin fibrosis diseases can be exemplified as TGF- ⁇ related diseases.
  • TGF- ⁇ mRNA is enhanced in megakaryocytes of patients with myeloid fibrosis (Reilly Jet al: Clin Haematol: 11751-767, 1998), and the TGF- ⁇ concentration in platelets is high (Martyre MC). It has been reported that the plasma TGF- ⁇ concentration in patients is significantly high (Rameshwar Pet al: Am J Haematol 59: 133-142, 1998) in et al: Br J Haematol 77: 80-86, 991).
  • TGF- ⁇ 1 TGF- ⁇ 1
  • FASEB J 16 1967-1969, 2002
  • male-type frontal hair loss can be exemplified as a TGF- ⁇ -related disease.
  • the administration target of the pharmaceutical composition of this embodiment is preferably human.
  • the pharmaceutical composition of this embodiment is preferably administered to a human or common marmoset.
  • the effect of the pharmaceutical composition of this embodiment can be confirmed by animal experiments using primates.
  • Common marmosets can be preferably used as primates.
  • the pharmaceutical composition of this embodiment may contain an arbitrary component in addition to PI polyamide.
  • the optional component include a pharmaceutically acceptable carrier.
  • the "pharmaceutically acceptable carrier” means a carrier that does not inhibit the physiological activity of the active ingredient and does not show substantial toxicity to the administration subject thereof.
  • “Not substantially toxic” means that the ingredient is not toxic to the subject at the dose normally used.
  • the pharmaceutically acceptable carrier is a carrier that does not inhibit the binding of the PI polyamide to the promoter and does not show substantial toxicity to the administration subject thereof. ..
  • the pharmaceutically acceptable carrier does not inhibit the HGF gene transcription promoting ability and the TGF- ⁇ gene transcription inhibitory ability of PI polyamide (P), and the carrier thereof is to be administered.
  • the pharmaceutically acceptable carrier includes any known pharmaceutically acceptable ingredient, which is typically considered an inactive ingredient.
  • the pharmaceutically acceptable carrier is not particularly limited, and is, for example, a solvent, a diluent, a vehicle, an excipient, a flow accelerator, a binder, a granulator, a dispersant, a suspending agent, a wetting agent, and the like.
  • Lubricants disintegrants, solubilizers, stabilizers, emulsifiers, fillers, preservatives (eg antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc.
  • preservatives eg antioxidants
  • chelating agents eg antioxidants
  • flavoring agents sweeteners, thickeners, buffers, colorants, etc.
  • the pharmaceutical composition of this embodiment may contain an arbitrary component other than a pharmaceutically acceptable carrier.
  • the arbitrary ingredient is not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation.
  • the pharmaceutical composition of this embodiment may contain an active ingredient other than PI polyamide. Examples of the active ingredient include vitamins and their derivatives, anti-inflammatory agents, anti-inflammatory agents, blood circulation promoters, stimulants, hormones, stimulants, analgesics, cell activators, plant / animal / microbial extracts, and antipruritics.
  • Agents anti-inflammatory analgesics, antifungal agents, antihistamines, hypnotic sedatives, tranquilizers, antihypertensive agents, antihypertensive diuretics, antibiotics, anesthetics, antibacterial agents, antiepileptic agents, coronary vasodilators, crude drugs, cessation Examples include, but are not limited to, antipruritics, keratin softening and stripping agents. As for the other components, one kind may be used alone, or two or more kinds may be used in combination.
  • the dosage form of the pharmaceutical composition of this embodiment is not particularly limited, and can be a dosage form generally used as a pharmaceutical preparation.
  • the pharmaceutical composition of this embodiment may be an oral preparation or a parenteral preparation.
  • the oral preparation include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drop loves, emulsions and the like.
  • the parenteral preparation include injections, suppositories, ointments, sprays, external solutions, ear drops, eye drops, nasal drops, inhalants and the like.
  • Pharmaceutical compositions of these dosage forms can be formulated according to a conventional method (for example, the method described in the Japanese Pharmacopoeia).
  • the administration route of the pharmaceutical composition of this embodiment is not particularly limited, and can be administered by an oral or parenteral route.
  • Parenteral routes include all non-oral routes of administration, such as intravenous, intramuscular, subcutaneous, intranasal, intradermal, eye drops, intracerebral, rectal, intravaginal and intraperitoneal administration.
  • the administration may be local administration or systemic administration.
  • Preferred routes of administration of the pharmaceutical composition of this embodiment include, for example, intravenous injection or intramuscular injection.
  • the PI polyamide (P) contained in the pharmaceutical composition of this embodiment preferably has a cyclic structure.
  • the pharmaceutical composition of this embodiment can be administered with a therapeutically effective amount of PI polyamide.
  • “Therapeutically effective amount” means the amount of a drug effective for the treatment or prevention of a target disease.
  • a therapeutically effective amount of PI polyamide (P) can be an amount that can delay the onset and / or progression of TGF- ⁇ -related or fibrotic disease.
  • the therapeutically effective amount may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc., the dosage form of the pharmaceutical composition, the administration method, and the like.
  • the pharmaceutical composition of this embodiment can have a single dose of PI polyamide of 0.001 to 1000 mg per 1 kg of body weight of the subject to be administered.
  • the dose may be 0.01 to 800 mg / kg, 0.1 to 500 mg / kg, 1 to 100 mg / kg, or 1 to 50 mg / kg. May be good.
  • the pharmaceutical composition of this embodiment may contain a therapeutically effective amount of PI polyamide per unit dosage form.
  • the content of PI polyamide in the pharmaceutical composition of this embodiment may be 0.01 to 90% by mass, 0.1 to 80% by mass, or 1 to 50% by mass. May be good.
  • the administration interval of the pharmaceutical composition of this embodiment may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc., the dosage form of the pharmaceutical composition, the administration method, and the like.
  • the administration interval may be, for example, every few hours, once a day, once every two to three days, once a week, or the like.
  • the pharmaceutical composition of this embodiment may be used in combination with other pharmaceuticals.
  • it can be used in combination with other therapeutic agents for fibrotic diseases.
  • the present invention provides a method for producing a pyrrole imidazole polyamide.
  • the methods of this embodiment include (a) a step of designing a pyrrole imidazole polyamide that binds to promoters of two or more genes, (b) a step of synthesizing the pyrrole imidazole polyamide, and (c) the step of synthesizing the synthesized pyrrole imidazole imidazole.
  • a step of selecting a pyrrole imidazole polyamide that controls the transcription of the two or more kinds of genes is included.
  • Step (a)> a PI polyamide that binds to the promoters of two or more genes is designed.
  • the step (a) may include, for example, the following (i) to (iv).
  • (I) Select two or more genes targeted by PI polyamide.
  • a transcription promoting region or a transcription repressing region is specified for the two or more kinds of genes.
  • a PI polyamide that binds to the transcription promoting region or the transcription suppression region is designed.
  • (Iv) From the designed PI polyamide, a PI polyamide that binds to a transcription promoting region or a transcription repressing region of a gene other than the first gene among the two or more kinds of genes is selected.
  • the promoter sequence is analyzed to identify the transcription-promoting region or transcription-suppressing region. For example, for a gene whose expression is suppressed, a transcription promoting region is acquired. For genes that promote expression, a transcriptional repression region is acquired.
  • the promoter sequences of the two or more target genes are analyzed to identify the transcription-promoting region and the transcription-suppressing region. For example, for a gene whose expression is suppressed, a transcription promoting region is acquired. For genes that promote expression, a transcriptional repression region is acquired. For example, the promoter sequences of the first gene and the second gene are analyzed to identify the transcription-promoting or transcription-repressing region of the first gene and the transcription-promoting or transrepression region of the second gene.
  • a PI polyamide that binds to a transcription promoting region or a transcription repressing region is designed.
  • a PI polyamide that binds to the transcription promoting region is designed.
  • a PI polyamide that binds to the transcriptional repression region is designed. At this time, it is preferable to design a plurality of PI polyamides.
  • a method of designing a plurality of PI polyamides a method of designing a PI polyamide for each of a plurality of transcription-promoting regions or transcription-suppressing regions; the target sequence is shifted with respect to one transcription-promoting region or transcription-suppressing region.
  • Methods for designing multiple PI polyamides and combinations thereof.
  • a PI polyamide that binds to a transcription promoting region or a transcription repressing region of a target gene other than the first gene among the two or more target genes is selected.
  • a sequence to which the PI polyamide designed in (iii) can be bound (hereinafter, also referred to as “candidate PI polyamide binding sequence”) is acquired.
  • candidate PI polyamide binding sequence is acquired.
  • a sequence of a transcription promoting region or a transcription repressing region of a target gene other than the first gene is searched.
  • the PI polyamide targeting the candidate PI polyamide-binding sequence is selected. ..
  • a PI polyamide capable of binding to the transcription promoting region or the transcription suppression region of the second gene is selected.
  • step (a) may include, for example, the following (iii') and (iv') in place of the above (iii) and (iv).
  • (Iii') Search for a target sequence to which the same PI polyamide can bind in the transcription-promoting region or transcription-suppressing region of the two or more genes.
  • (Iv') Design a PI polyamide capable of binding to the target sequence detected by (iii').
  • a target sequence to which the same PI polyamide can bind is searched for in the transcription promoting region or the transcription suppression region of all target genes. For example, a target sequence to which the same PI polyamide can bind is searched for in the transcription-promoting region or the transcription-suppressing region of the first gene and the transcription-promoting region or the transcription-suppressing region of the second gene.
  • Step (b)> the PI polyamide designed in the step (a) is synthesized.
  • the PI polyamide can be synthesized by a known method such as the solid phase Fmoc method.
  • Step (c)> among the PI polyamides synthesized in the step (b), the PI polyamide that controls the transcription of the two or more kinds of genes is selected.
  • PI polyamide synthesized in step (b) cells expressing all target genes are cultured.
  • the expression of the target gene is promoted or suppressed as compared with the cells cultured in the absence of PI polyamide, it can be determined that PI polyamide regulates the transcription of the target gene.
  • the PI polyamide is designed to bind to the transcription-promoting region of the first gene and to the transcription-repressing region of the second gene.
  • transcription of the first gene is suppressed and transcription of the second gene is promoted.
  • Gene expression may be confirmed by mRNA or protein.
  • the transcription amount of mRNA can be measured by, for example, Northern blotting, RT-qPCR, or the like.
  • the expression level of the protein can be measured by, for example, Western blotting, ELISA or the like.
  • the method according to this embodiment it is possible to produce a PI polyamide that binds to the promoters of two or more kinds of genes and controls the transcription of the two or more genes.
  • the PI polyamide exemplified above can be produced.
  • the invention provides the use of PI polyamide (P) in the manufacture of pharmaceutical compositions for treating or preventing TGF- ⁇ related or fibrotic diseases.
  • the invention provides PI polyamide (P) for use in the treatment or prevention of TGF- ⁇ related or fibrotic diseases.
  • the invention comprises administering PI polyamide (P) to a subject (eg, a patient suffering from a TGF- ⁇ related disease or fibrous disease, etc.), comprising administering the PI polyamide (P) to a TGF- ⁇ related disease or fibrous.
  • a subject eg, a patient suffering from a TGF- ⁇ related disease or fibrous disease, etc.
  • the invention provides PI polyamide (P) for treating or preventing TGF- ⁇ related or fibrotic diseases.
  • PI polyamide (Analysis of human HGF gene promoter) The promoter of the human HGF gene was analyzed using the gene analysis software PROMO to predict the binding site of the transcriptional regulator. As a result, a binding site (-108 to -96; AGGTGACCTTTTC: SEQ ID NO: 1) of COUP-TF1 functioning as a transcriptional regulator was detected (see FIG. 2). Eight types of PI polyamides were designed targeting the peripheral region of this COUP-FT1 binding site (Hu-HGF-1, Hu-HGF-2: FIG. 3; Hu-HGF-3, Hu-HGF-4, Hu). -HGF-5, Hu-HGF-6: FIG. 4; Hu-HGF-7, Hu-HGF-8: FIG. 5). Assuming the use of common marmosets in in vivo tests, regions homologous to humans and common marmosets were selected as target regions for PI polyamides.
  • Hu-HGF-3, Hu-HGF-5, and Hu-HGF-7 are shown in FIGS. 6 to 8, respectively.
  • the sequences to which Hu-HGF-3, Hu-HGF-5, and Hu-HGF-7 can bind are shown in Table 1.
  • Table 1 the underlined sequences are the sequences present in the TGF- ⁇ 1 promoter.
  • FIG. 9 (Analysis of human TGF- ⁇ 1 promoter) The sequence of the human TGF- ⁇ 1 promoter is shown in FIG. In FIG. 9, the sequence surrounded by a solid line shows the binding sequence of Hu-HGF-3, and the sequence surrounded by a broken line shows the binding sequence of Hu-HGF-7.
  • FIG. 10 shows a transcriptional regulatory factor binding prediction site around the binding region of PI polyamide. There are multiple transcriptional regulator binding prediction sites around the binding regions of Hu-HGF-3 and Hu-HGF-5.
  • FIG. 14 shows an outline of the test protocol.
  • Human skin-derived fibroblasts (HDF cells) were maintained in 10% FBS-DMEM medium. The medium was replaced with 0.5% FBS-DMEM medium and cultured for 24 hours. 10-11 to 10-7 M PI polyamide was added. In the negative control, DMSO was added instead of PI polyamide. After the addition of PI polyamide or DMSO, the cells were cultured for 15 hours and the cells were collected.
  • FIG. 14 shows an outline of the test protocol.
  • the cells were cultured and recovered in the same manner as above, except that 0.1 ⁇ M PMA (phorbol 12-myristate 13-acetylate) was added 3 hours after the addition of PI polyamide or DMSO.
  • PMA phorbol 12-myristate 13-acetylate
  • FIG. 21 shows the results when human renal mesandium (MC) cells were used instead of HDF cells.
  • Hu-HGF-5 was used as the PI polyamide. It was also confirmed that the expression of TGF- ⁇ 1 mRNA stimulated by PMA was suppressed by the addition of PI polyamide in MC cells.
  • HGF protein expression level The cells were cultured with the addition of PI polyamide, and the effect of PI polyamide on the expression level of HGF protein was evaluated. The cells were cultured and recovered in the same manner as in [Evaluation of HGF mRNA expression level]. The cells were disrupted and Western blotting was performed using an anti-HGF antibody (Catalog (MAB294), Monoclonal Mouse, R & D System). The expression level of HGF protein was leveled by the expression level of ⁇ -actin.
  • TGF- ⁇ 1 protein expression level The cells were cultured with the addition of PI polyamide, and the effect of PI polyamide on the expression level of TGF- ⁇ 1 protein was evaluated. The cells were cultured and recovered in the same manner as in [Evaluation of TGF- ⁇ 1 mRNA expression level]. The cells were disrupted and subjected to Western blotting using an anti-TGF- ⁇ antibody (Catalog (Y241), Polyclonal, Rabbit, Peptide Research Institute, Inc.). The expression level of TGF- ⁇ protein was leveled by the expression level of ⁇ -actin.
  • HGF siRNA As HGF siRNA, HSS179212 (Thermo Fisher SCIENTIFIC) was used. It was confirmed that HGF siRNA specifically suppresses the expression of HGF mRNA (data not shown).
  • the cells were cultured and recovered in the same manner as in [Evaluation of HGF mRNA expression level] except that 10 nM HGF siRNA was added.
  • the cells were disrupted and Western blotting was performed using an anti-HGF antibody (Catalog (MAB294), Monoclonal Mouse, R & D System).
  • the expression level of HGF protein was leveled by the expression level of ⁇ -actin.
  • FIGS. 28 to 29 The results of using Hu-HGF-3 and Hu-HGF-5 as PI polyamides are shown in FIGS. 28 to 29, respectively.
  • the numbers on the horizontal axis indicate the following.
  • the concentration of PMA was 0.1 ⁇ M, and the concentration of HGF siRNA was 10 nM.
  • DMSO 2 DMSO + PMA + HGF siRNA 3: DMSO + PMA + HGF siNRA + 10 -11 M PI polyamide 4: DMSO + PMA + HGF siNRA + 10 -10 M PI polyamides
  • [Evaluation of TGF- ⁇ 1 mRNA expression level using mismatched PI polyamide] A mismatched PI polyamide (HGF mismatch) that was not set to bind to the transcriptional repression region of the HGF gene and the transcription promotion region of the TGF- ⁇ 1 gene was synthesized. The cells were cultured in the same manner as in [Evaluation of TGF- ⁇ 1 mRNA expression level] except that the mismatched PI polyamide was used, and the TGF- ⁇ 1 mRNA expression level was evaluated. The structure of HGF mismatch is shown below.
  • a formula pyrrole imidazole polyamide capable of simultaneously controlling transcription of two or more kinds of genes, and a pharmaceutical composition containing the pyrrole imidazole polyamide.
  • pyrrol imidazole polyamide useful as a therapeutic agent for TGF- ⁇ -related diseases or fibrous diseases, TGF- ⁇ gene expression inhibitor using the pyrrol imidazole polyamide, and TGF- ⁇ -related diseases or fibrous diseases are treated.
  • a pharmaceutical composition for this is provided.

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Abstract

L'invention concerne un polyamide pyrrole-imidazole qui se lie aux promoteurs d'au moins deux gènes et régule simultanément la transcription des au moins deux gènes. L'invention concerne également une composition pharmaceutique comprenant le polyamide pyrrole-imidazole. L'invention concerne en outre : un polyamide pyrrole-imidazole qui régule simultanément la transcription du gène HGF et du gène TGF-β ; un inhibiteur d'expression du gène TGF-β comprenant le polyamide pyrrole-imidazole ; et une composition pharmaceutique pour le traitement d'une maladie associée au TGF-β ou d'une maladie fibreuse.
PCT/JP2021/027144 2020-07-20 2021-07-20 COMPOSITION PHARMACEUTIQUE À BASE DE PYRROLE-IMIDAZOLE POLYAMIDE, INHIBITEUR DU GÈNE TGFβ, COMPOSITION PHARMACEUTIQUE ET PROCÉDÉ DE PRODUCTION DE PYRROLE-IMIDAZOLE WO2022019303A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023200009A1 (fr) * 2022-04-15 2023-10-19 国立大学法人 東京大学 Inhibiteur du facteur de transcription protozoaire
WO2023204300A1 (fr) * 2022-04-22 2023-10-26 国立大学法人 東京大学 Inhibiteur transcriptionnel pour un gène lié au système immunitaire

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WO2023204300A1 (fr) * 2022-04-22 2023-10-26 国立大学法人 東京大学 Inhibiteur transcriptionnel pour un gène lié au système immunitaire

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