WO2007034784A1 - Inhibiteur de l'expression du gène du ctgf - Google Patents

Inhibiteur de l'expression du gène du ctgf Download PDF

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WO2007034784A1
WO2007034784A1 PCT/JP2006/318512 JP2006318512W WO2007034784A1 WO 2007034784 A1 WO2007034784 A1 WO 2007034784A1 JP 2006318512 W JP2006318512 W JP 2006318512W WO 2007034784 A1 WO2007034784 A1 WO 2007034784A1
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ctgf
gene expression
pyrrole
expression inhibitor
region
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Takahiro Ueno
Noboru Fukuda
Hiroshi Sugiyama
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Nihon University
Gentier Biosystems Incorporation
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • 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
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    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
    • A61P27/00—Drugs for disorders of the senses
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • A61P9/12—Antihypertensives
    • 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

Definitions

  • the present invention relates to a connective tissue growth factor (CTGF) gene expression inhibitor and a therapeutic agent for CTGF-related diseases. More specifically, the present invention relates to a CTGF gene expression inhibitor comprising pyrrole-imidazole polyamide having a specific structure.
  • CTGF connective tissue growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • TGF- ⁇ is associated with cell proliferative diseases such as hypertensive vascular disease, neointimal formation after angioplasty, and atherosclerosis. Yes.
  • Cell growth in these pathologies is one of the forces that could be suppressed by various mechanisms of action, one of which is to suppress TGF- ⁇ expression.
  • TGF- ⁇ works to suppress the growth of most cells, but has a biphasic growth effect on mesenchymal cells such as fibroblasts and vascular smooth muscle cells (VSMCs), so it suppresses growth under normal conditions. However, when inflammation, mechanical stress, etc. are applied, it works to stimulate growth.
  • TGF- ⁇ is involved in neointimal formation after vascular injury by promoting VSMC proliferation and extracellular matrix formation.
  • TGF- ⁇ is also involved in the formation of arteriosclerotic lesions.
  • TGF- ⁇ is also thought to be involved in restenosis of the renal artery after percutaneous renal arthroplasty. Therefore, local vascular therapy aimed at regulating the effects of TGF- ⁇ is considered effective in reducing the above-mentioned vascular proliferative diseases.
  • the present inventors have developed a pyrrole-imidazole polyamide that binds to the FSE2 protein binding region of the TGF- ⁇ 1 gene and suppresses the expression of the gene, and filed a patent application in 2003 (Patent Literature). 1).
  • the present inventors are also known from subsequent studies to be associated with overexpression of the TGF- ⁇ 1 gene in various pathologies such as fibroproliferative diseases and vascular proliferative diseases.
  • Renal fibrosis refers to glomerular sclerosis and interstitial fibrosis, and is one of the pathological conditions of chronic renal failure. This fibrosis is caused by growth factors such as TGF- ⁇ 1 acting on renal fibroblasts directly or indirectly through the production of CTGF, causing extracellular matrix production and fibrosis. It is believed that. CTGF is a protein with a molecular weight of 38 kDa belonging to the CCN family. This protein is involved in fibrosis, cell proliferation, extracellular matrix metabolism, angiogenesis, arteriosclerosis, etc., and is the most important growth factor in fibrosis in the past. It is located downstream of the fiber growth cascade by TGF- ⁇ 1, which was considered (Non-patent Document 1).
  • CTGF is selectively induced by TGF- ⁇ in fibroblasts. It has been reported that the promoter region of CTGF gene has a base sequence called TGF-
  • pyrrole imidazole polyamides specifically recognize the base sequence of DNA, and have a specific gene. It has been reported that the expression of can be controlled by extracellular force.
  • a pyrrole-imidazole polyamide is a group of synthetic organic compounds, and is composed of N-methylpyrrole units (hereinafter also referred to as Py) and N-methylimidazole units (hereinafter also referred to as Im) which are aromatic rings.
  • Py and Im can take U-shaped conformation in the presence of ⁇ -aminobutyric acid by coupling and folding in succession.
  • the general structure and production method are known (Patent Documents 3-5).
  • Such a synthetic polyamide can bind with high affinity and specificity to a specific base pair in a minor groove of a double helix DNA. Specific recognition of base pairs depends on the one-to-one pairing of Py and Im. That is, in a U-shaped conformation in the minor groove of DNA, PyZlm pair targets C-G base pair, ImZPy targets G-C base pair, and PyZPy targets A-T base pair and TA Target both base pairs (Non-patent Document 45). Recent studies show that AT condensation can be overcome by preferential binding of HpZPy to the TZA pair as a result of replacing one pyrrole ring of the PyZPy pair with 3-hydroxypyrrole (Hp). The power of Gawa (Non-Patent Document 6).
  • initiation of transcription is considered to be an important point of gene regulation. Initiation of transcription requires several transcription factors that bind to specific recognition sequences in the gene promoter region.
  • the polyamide in the minor groove may interfere with gene regulation by blocking transcription factor binding if the transcription factor is important in gene expression. This hypothesis has been proven in in vitro and in vivo experiments.
  • the 8-membered Py-Im polyamide bound to the inside of the Jintafinger recognition site (TFIIIA binding site) inhibited 5SRNA gene transcription (Non-patent Document 7).
  • Patent Document 1 Japanese Patent Application No. 2003-312365
  • Patent Document 2 Japanese Patent Application No. 2004-238533
  • Patent Document 3 Patent No. 3045706
  • Patent Document 4 JP2001-136974
  • Patent Document 5 WO 03/000683 A1
  • Non-patent literature l Takigawa: Drug News Perspect "16: 11-21 (2003)
  • Non-Patent Document 2 Goldschmeding et ahNephrol. Dial Transplant, 15: 296 (2000)
  • Non-Patent Document 3 Trauger et ahNature., 382: 559-61 (1996)
  • Non-Patent Document 4 White et ahChem Biol, 4: 569-78 (1997)
  • Non-Patent Document 5 Dervan: Bioorg Med Chem., 9: 2215-35 (2001)
  • Non-Patent Document 6 White at ahNature., 391: 468-71 (1998)
  • Non-Patent Document 7 Genfeld et ahNature., 387: 202-5 (1997)
  • CTGF is important in fibroproliferative diseases where its expression is induced in response to stimulation by TGF- ⁇ 1 and its expression changes in response to stimulation by TGF- ⁇ 1 It is a protein that plays a role. Therefore, the suppression of CTGF expression can reduce the unexpected effect compared to suppressing the expression of TGF- ⁇ 1, which can be involved in more types of diseases, and is higher. It becomes possible to suppress fibrosis having safety and specificity.
  • no organic compound that specifically suppresses the expression of CTGF has been known so far.
  • CTGF gene expression inhibitors or therapeutic agents for TGF- ⁇ related diseases using pyro-l-imidazole polyamide that binds to the CTGF gene base sequence.
  • the present inventors have conducted extensive research on the development and pharmacological effect of pyrrole-imidazole polyamide that can specifically bind to a specific region of the CTGF gene promoter and inhibit the expression of the CTGF gene.
  • compounds that can inhibit gene expression and can serve as therapeutic agents among polyamides that target various fragments of the CTGF gene promoter, those in the 195 to 150 region of the promoter region home,
  • the ability to bind to the region of 164 to 150 or 195 to 174, more preferably the region of 160 to 155 or the region of 190 to 184 significantly reduces the activity of the CTGF gene promoter. Inhibiting and down-regulating the expression of the CTGF gene has led to the present invention.
  • the present invention is as follows.
  • a target region comprising a part or all of the base sequence 195 to 150 (SEQ ID NO: 2) shown below and the complementary strand thereto, ⁇ -Can be folded at the site of an aminobutyric acid unit to take a U-shaped conformation.
  • CTGF gene expression inhibitor comprising the above-mentioned pyrrolimidazole polyamide, each corresponding to a PyZPy pair.
  • the target region is a double helix region comprising a part or all of the base sequence shown below of the CTGF promoter—164 to 150 (SEQ ID NO: 3) and a complementary strand thereto. 2)
  • CTGF gene expression inhibitor which is an amide with the above amidecylaminopropylamine or N, N-dimethylaminopropylamine.
  • a CTGF gene expression inhibitor comprising a pyrrole-imidazole polyamide represented by the following formula:
  • a CTGF gene expression inhibitor comprising a pyrrole-imidazole polyamide represented by the following formula:
  • CTGF gene expression inhibitor that does not have the disadvantage of being decomposed by ribonuclease because it is an organic compound and has no side effects such as legal agents.
  • the CTG F gene expression inhibitor of the present invention is effective as a therapeutic agent for the above-mentioned various fibroproliferative diseases, vascular proliferative diseases, and arteriosclerotic diseases. Since CTGF acts on fibrosis in the downstream region rather than involving TGF- ⁇ 1, the gene expression inhibitor of CTGF can lead to more safe and specific inhibition of high fibrosis.
  • N-methylpyrrole unit hereinafter also referred to as Py
  • N-methylimidazole unit hereinafter also referred to as Im
  • ⁇ -aminobutyric acid unit ⁇ linker
  • pyrrole-imidazole polyamide can be easily produced by an automatic synthesis method using a solid phase method (solid phase Fmoc method) using Fmoc (9-fluorenylmethoxycarbon) (Patent Document 5).
  • solid phase Fmoc method solid phase Fmoc method
  • Fmoc (9-fluorenylmethoxycarbon) Patent Document 5
  • the end of pyrrole-imidazole polyamide can be cut out as a solid carrier by using a carboxylic acid residue, so that various functional groups can be introduced into the end of the molecule to create derivatives of pyrrole-imidazole polyamide. it can .
  • a compound having an alkylotrophic ability with respect to DNA such as duo force noremycin, pyro-benzodiazepine, bleomycin, enediyne compound, nitrogen mustard, and derivatives thereof, can be introduced as necessary.
  • the solid-phase Fmoc method is an automated synthesis method using a commercially available protein (peptide) synthesizer, it is also possible to synthesize conjugates (conjugates) of naturally occurring proteins and non-natural proteins and pyrrole-imidazole polyamides. .
  • the Fmoc method has milder reaction conditions than the t-BOC method, it is also possible to introduce organic compounds other than proteins (including compounds having functional groups that are unstable under acidic conditions). For example, it is possible to automatically synthesize conjugates of pyrrole-imidazole polyamide and DNA or RNA (or their derivatives).
  • a pyrrole imidazole polyamide having a carboxyl group at the terminal can be synthesized.
  • Specific examples thereof include, for example, a pyrrolimine having a j8-alanine residue (j8-aminopropionic acid residue) or a ⁇ -aminobutyric acid residue at the terminal.
  • a dazole polyamide etc. are mentioned.
  • the pyrrole imidazole polyamide having a terminal j8-alanine residue or ⁇ -aminobutyric acid residue is, for example, an aminopyrrole carboxylic acid, an amino imidazole carboxylic acid, j8-alanine or y-amino having an amino group protected by Fmoc, respectively. It can be synthesized by solid phase Fmoc method using a peptide synthesizer using a solid phase carrier carrying butyric acid.
  • aminopyrrole carboxylic acid examples include, for example, 4 amino-2-pyrrole carboxylic acid, 4 amino-1-methyl-2-pyrrole carboxylic acid, 4 amino-1-ethyl-2-pyrrole carboxylic acid, 4 amino-1-propyl.
  • aminoimidazole carboxylic acid examples include, for example, 4 amino-2 imidazole carboxylic acid, 4-amino 1-methyl-2 imidazole carboxylic acid, 4 amino 1 ethyl 2 imidazole carboxylic acid, 4 amino-1 propyl 2 imidazole carboxylic acid, 4 amino 1 Butyl-2-imidazolecarboxylic acid and the like.
  • a conjugate of pyrrole imidazole polyamide and FITC fluorescein isothiocyanate
  • FITC fluorescein isothiocyanate
  • the resulting conjugate can be used to prove that the pyrrole imidazole polyamide recognizes a specific DNA sequence.
  • the CTGF gene expression inhibitor of the present invention is a pyrrole-imidazole polyamide containing an N-methylpyrrole unit (Py), an N-methylimidazole unit (Im), and a ⁇ -aminobutyric acid unit, wherein the base sequence of the CTGF promoter is from 195 to In a minor groove of a double helix region (hereinafter also referred to as a target region) containing part or all of 150 (SEQ ID NO: 2) and a complementary strand thereto, it is folded at the site of the ⁇ -aminobutyric acid unit and is U-shaped.
  • a target region containing part or all of 150 (SEQ ID NO: 2) and a complementary strand thereto
  • the DNA double helix skeleton forms two rows of grooves.
  • the wide and deep groove is called the main groove (major group), and the narrow and shallow groove is called the minor groove (minor group).
  • the above pillow Ruimidazole polyamides can bind in a non-conjugated manner with high affinity and specificity to minor grooves created by specific base pairs.
  • the PyZlm pair of pyrrole imidazole polyamide is against the CG base pair in the minor groove
  • the ImZPy pair is against the G-C base pair
  • the AT base pair and TA base pair are not. PyZPy pairs correspond to each other.
  • the molecule is folded at the site of the ⁇ -aminobutyric acid unit in the pyrrole-imidazole polyamide molecule to take a U-shaped conformation.
  • the base pair of the minor groove and the Py and Im pair of pyrrole imidazole polyamide do not correspond as described above, the bond between the minor groove and the pyrrole imidazole polyamide becomes insufficient.
  • the pyrrole imidazole polyamide is called mismatch or mismatch polyamide, where the minor groove base pair and Py-Im pair correspond to each other as described above.
  • the nucleotide sequence of the CTGF gene regulatory region is as shown in Fig. 1 and SEQ ID NO: 1 (Xine tal, J. Clin, Pathol. 49, 91-97 (1996)).
  • the pyrrole-imidazole polyamides GBP2101 and GBP2102 of the present invention are as shown below.
  • GBP2101 has the molecular formula C H N O, molecular weight 1667.75, and its target sequence is
  • CTGF gene regulatory region — 195 to 150 SEQ ID NO: 2
  • the region downstream from the Smad binding region is 164 to 150 (SEQ ID NO: 3)
  • directly gctga g Suppresses the expression of CTGF gene by binding to 6 bases of (SEQ ID NO: 4).
  • GBP2102 has the molecular formula C H N O, molecular weight 1912.00, and its target sequence is
  • the region upstream of the Smad binding region — 195 to 174 (SEQ ID NO: 5), which is directly gagtgt g ( — 190 to 184) Suppresses the expression of the CTGF gene by binding to 7 bases of (SEQ ID NO: 6).
  • Py-Im polyamides are effective inhibitors or activators of transcription factors that are common or tissue-specific in in vitro studies.
  • the growth of Dro sophila under specific polyamide administration is the ability to gain or lose a functional phenotype with no particular toxicity. This is the result of polyamides specifically controlling gene expression.
  • polyamides Compared to antisense oligonucleotides and ribozymes, polyamides have better permeability (low concentration, no transfection medium required) and higher stability in cultured human mesangial cells. It showed. The high permeability and stability of polyamides provides an ideal drug approach to the nucleus of eukaryotic cells for gene therapy.
  • TATA box D Transcription mediator D
  • TBP TATA box binding protein
  • PIC pre-initiator complex
  • TBP also binds to the minor groove of the double helix DNA (Lee et al: Cell.l991 Dec 20; 67 (6): 1241-50, Starr et al: Cell.l991; 67: 1231- 40, Courey et al: Cell.l988; 55: 887-98), synthetic polyamides competitively occupy the binding site of the TATA binding protein and interfere with gene transcription.
  • polyamides designed with various promoters those targeting the TATA box are known to always work.
  • SP-1 sequences O In the vicinity of the transcription start site There are several SP-1 sequences and two AP-1 sequences. Since various viral and cellular promoters are activated by SP-1 protein, a single SP-1 sequence is sufficient for the promoter to be stimulated by SP-1. (Kadonaga et al: Cell.l987; 51: 1079-90, Courey et al: Cell.l988; 55: 887-98). AP-1 sequence responds to AP-1 transcription factor, which also acts as an unhomodimer or FosZjun heterodimer complex!
  • Transcriptional repressors that suppress transcription from core promoters containing a TATA box have been found. Forces have not been found from promoters without TATA (Aso et ahEMBO J.1994; 13: 435-45, Mack et al: Nature.l993; 3 63: 281-3, Merino et al: Nature.l993; 365: 227-32). Since the expression of most mammalian genes tends to depend on a combination of the actions of a large number of proteins bound to a promoter and an enzyme sequence, the simplest model to explain this result is the present model. The bright pyrrole-imidazole polyamide blocks transcription factor-DNA interactions and has an inhibitory effect on CTGF promoter activity.
  • Synthetic polyamides can approach the target site due to the position of the nucleonome, and may influence the condensation / decondensation structure of chromatin by targeting specific sequences (Gottesfeld et al: J Mol Biol. 2002; 321: 249-63; Gottesfeld et al: J Mol Biol.20 01; 309: 615-29.). It has been demonstrated that pyrrole-imidazole polyamides open heterochromatin brown satellites and allow GAF binding, resulting in phenotypic changes in drosophila melanogaster. Because pyrrole-imidazole polyamides can be designed to target sequences of interest, functional genomic studies are ultimately useful for gene therapy such as CTGF gene inhibition and activity.
  • the Py-Im polyamide according to the present invention can be designed upstream from the transcription initiation region, which shows an inhibitory effect on the expression of the CTGF gene.
  • CTGF is a renal disease (IgA nephropathy, focal glomerulosclerosis, crescent nephritis, focal sclerosis lupus nephritis, diffuse proliferative lupus nephritis, diabetic nephropathy, hypertensive nephropathy)
  • IgA nephropathy, focal glomerulosclerosis, crescent nephritis, focal sclerosis lupus nephritis, diffuse proliferative lupus nephritis, diabetic nephropathy, hypertensive nephropathy are reported to be deeply involved in fibrosis (Qi W et al: Am J Physio 1 Renal Physiol Nov 9, 2004, Okada H et al: J Am Soc Nephrol 16 (1) 133-43, 2004, Kanemoto K et al: Lab Invest 83 (11) 1615-25, 2003, Zhou G et al: Am
  • CTGF is also expressed in vascular endothelial cells and vascular smooth muscle cells, and has been shown to play an important role in angiotensin II vascular cell proliferation and sclerosis in vascular proliferative diseases ( Ruperez M et al: Circulation 108 (12) 1499-505, 2003, Kaji T et al: Biochem Biophys Res Commun 322 (1) 22-8, 2004, Chowdhury I et al: Eur J Bio chem 271 (22) 4436- 50, 2004). From these facts, it can be reasonably considered that the CTGF gene expression inhibitor of the present invention is effective as a therapeutic agent for vascular proliferative diseases.
  • CTGF has also been shown to play an important role in myocardial hypertrophy and fibrosis in heart disease (Ahmed MS et al: J Mol Cell Cardiol 36 (3) 393-404, 2004, Matsui Y et al: JM ol Cell Cardiol 37 (2) 477-81, 2004). From these facts, it can be reasonably considered that the CTGF gene expression inhibitor of the present invention is effective as a therapeutic agent for cardiomyopathy.
  • CTGF plays an important role in stellate cell activation and adhesion (Gao R et al: J Biol Chem 279 (10) 8848-5 5), and CTGF suppression is known to suppress liver fibrosis. (Uchio K et al: Wo und Repair Regen 12 (1) 60-6). From these facts, it can be reasonably considered that the CTGF gene expression inhibitor of the present invention is effective as a therapeutic agent for liver fibroproliferative diseases.
  • CAPD unlike hemodialysis, is established as a very effective treatment for rehabilitation of patients without requiring frequent visits because patients are performed at home or work. . If the patient undergoes CAPD, the fibrosis of the peritoneum progresses, and the peritoneal sclerosis develops. If the efficiency of CAPD decreases and it becomes difficult to continue CAPD, The development of encapsulated peritoneal sclerosis, which can lead to bad habits, is a problem. In this encapsulated peritoneal sclerosis, the expression of CTGF was excessive, and it was reported that it was deeply involved in its onset and progression. Based on these facts, it can be reasonably considered that the CTGF gene expression inhibitor of the present invention is effective as a therapeutic agent for encapsulating peritoneal sclerosis.
  • CTGF has been reported to play an important role in the development and progression of pulmonary fibrosis model animals (Xie S et al: Am J Physiol Lung Cell Mol Physiol 288). (1) L68-76, 2004) o It has also been reported that CTGF expression in airway cells is related to TGF- ⁇ (Bonniaud P et al: Am J Respir Cell Mol Biol 31 (5) 510 -6, 2004). From these facts, it can be reasonably considered that the present invention, which is a CTGF gene expression inhibitor of the present invention, is effective as a therapeutic agent for pulmonary fibrosis.
  • TGF- ⁇ As the etiology of scleroderma CTGF has also been reported to play an important role in the fibroblasts of this disease (Leask A: Keio J Med 53 (2) 74-7, Chujo S et al: J Cell Phisiol Dec 16 2004) 0
  • the present invention which is a gene expression inhibitor, can be reasonably considered to be effective as a therapeutic agent for skin fibrosis.
  • CTGF has been reported to play an important role in the development and progression of diabetic retinopathy (Kuiper EJ et al: Br J Ophthalmol 88 (8 1082-7, 2004) o From these facts, it can be reasonably considered that the CTGF gene expression inhibitor of the present invention is effective as a therapeutic agent for diabetic retinopathy.
  • GBP2101 or GBP2102 as described above was designed to bind to 160-1155 or 190-1184 base pairs of the CTGF promoter as Py-Im polyamide.
  • FITC coupling A 4-fold excess of fluorescein (0.40 mmol) and DIEA (without HATU) dissolved in DMF was flushed through the column for 60 minutes.
  • Decomposition as amine The synthetic polyamide was isolated by cold ethyl ether precipitation after the decomposition step (N, N-dimethylaminopropylamine 5 ml Z resin 0.1 mmol, 50 ° C, ⁇ ).
  • DM EM Dulbecco's modified Eagle medium
  • Ivitrogen 10% offspring serum
  • PBS free phosphate buffered saline
  • Oligonucleotides were synthesized and annealed into 12 double-stranded oligonucleotides corresponding to the CTGF promoter base pairs. Double-stranded DNA was labeled with T4 polynucleotide kinase using [ ⁇ - 32 P] -ATP, and binding buffer (40 mM Tris, pH 7.9, 250 mM NaCl, 25 mM EDTA, 25 mM DTT for 15 minutes at 37 ° C) , lOOmM KC
  • RNA extraction and reverse transcription reaction for growth factor mRNA RNA extraction and reverse transcription reaction for growth factor mRNA, polymerase chain reaction (R T-PCR)
  • RNA sample is 0.5 U in 0.5 ml DNase buffer (20 mM Tris—HC1 pH 8.3, 50 mM KC1, 2.5 mM MgCl 2) for 45 min at room temperature. Treated with DNase (Gibco). DNase
  • RNA (l i u gZ20 i u L) inactivated by adding 0.5 mL 0.5 M EDTA and heating at 98 ° C for 10 min was added to 10 mM Tris—HCl (pH 8.3 ), 5 mM MgCl 2, 50 mM KC1, ImM Deoxy NTPs, and 2.5 M in random hexamers, 2
  • avian myeloblastoma virus reverse transcriptase (Takara Biochemicals, Osaka, Japan) was used for reverse transcription into single-stranded cDNA.
  • q DNA polymerase (Takara Biochemicals, Osaka, Japan) and upstream sense primer and downstream antisense primer were mixed with 0.2 M each to make a total of 25 ⁇ L.
  • the sense primer (5, -CCTGGTCCAGACCACAGAGT-3) (SEQ ID NO: 7) and the antisense primer (5, -TGGAGATTTTGGGAGTACGG-3,) (SEQ ID NO: 8) were used for PCR amplification of CTGF mRNA.
  • RNA Sense primer 5, -TCAAGAACGAAAGTCGGACG-3 '
  • antisense primer 5,-GGACATCTAAGGGCATCACA-3,
  • PCR was performed on a thermal cycler (Perkin Elmer, Foster, CA). PCR conditions were 94 ° C for 2 min, followed by 30 cycles of denaturation at 94 ° C for 1 min, annealing at 58 ° C for 1 min, extension at 72 ° C for 1 min, and finally an extension reaction at 72 ° C for 10 min. It was. PCR with primers for 18S rRNA was included in each reaction as an internal control.
  • CTGF gene expression level was increased 20% by PMA stimulation 1 ⁇ 10 _6 ⁇ , was inhibited up to 50% or less 1 ⁇ 10 _6 ⁇ by GBP2101 added pressure (Fig. 3).
  • the cells were subconfluent and replaced with serum-free medium and cultured for 24 hours, and 1x10 "6M PMA and various concentrations of Py-Im were added, and the cells were further cultured for 12 hours without serum. It was dissolved and electrophoresed on a polyacrylamide gel and transferred to a trocellulose membrane.
  • the protein transferred to the nitrocellulose membrane consists of an anti-human CTGF antibody as the primary antibody and an anti-goat IgG antibody as the secondary antibody.
  • Western blot was performed using ECL (Amersham-Pharmacia), and the color was quantified using NIH Image.
  • CTGF protein expression level was increased 20% by PMA stimulation 1 ⁇ 10 _6 ⁇ , was inhibited up to 50% or less 1 ⁇ 10 _6 ⁇ by GBP2101 added Caro ( Figure 4).
  • the cells were subconfluent and replaced with serum-free medium for 24 hours, and 1x10 "6M TGF- ⁇ 1 and each concentration of Py-Im were added and cultured for 12 hours without serum.
  • messenger RNA was isolated by guanidium thiocyanate method, reverse transcribed by avian myeloblastoma virus reverse transcriptase, and amplified by PCR method. The PCR product was quantified by A gilent Bioanalyzer. An image was prepared and PCR was performed, and the CTGF gene expression level was corrected at 18 s. CTGF gene expression level was increased 20% by PMA stimulation 1 ⁇ 10 _6 ⁇ , was inhibited up to 50% or less 1 ⁇ 10 _6 ⁇ by GBP2102 added pressure (Figure 5).
  • the cells were subconfluent and replaced with serum-free medium and cultured for 24 hours, and 1x10 "6M PMA and various concentrations of Py-Im were added, and the cells were further cultured for 12 hours without serum. It was dissolved and electrophoresed on a polyacrylamide gel and transferred to a trocellulose membrane.
  • the protein transferred to the nitrocellulose membrane consists of an anti-human CTGF antibody as the primary antibody and an anti-goat IgG antibody as the secondary antibody.
  • Western blot was performed using ECL (Amersham-Pharmacia), and the color was quantified using NIH Image.
  • CTGF protein expression level was increased 20% by PMA stimulation 1 ⁇ 10 _6 ⁇ , was inhibited up to 50% or less 1 ⁇ 10 _6 ⁇ by GBP2102 added Caro ( Figure 6).
  • CTGF gene expression inhibitor of the present invention can be used as a therapeutic agent for diseases associated with the production of TGF- ⁇ and thus CTGF.
  • Fig. 1 shows the base sequence of the regulatory region of the human CTGF gene.
  • FIG. 2 shows gel shift assembly of Py-Im polyamide oligonucleotide complex.
  • FIG. 3 is a graph showing the inhibitory effect of GBP2101 on the expression of CTGF messenger RNA.
  • FIG. 4 is a graph showing the effect of suppressing the expression of CTGF protein by GBP2101.
  • FIG. 5 is a graph showing the effect of suppressing the expression of CTGF messenger RNA by GBP2102.
  • FIG. 6 is a graph showing the effect of suppressing the expression of CTGF protein by GBP2102.

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Abstract

L'invention concerne un inhibiteur de l'expression du gène du CTGF, lequel comprend un polyamide de pyrrole-imidazole. Le polyamide de pyrrole-imidazole a une unité N-méthylpyrrole (ci-après appelée 'Py'), une unité N-méthylimidazole (ci-après appelée 'Im') et une unité acide Ϝ-aminobutanoïque. Dans le polyamide de pyrrole-imidazole, la région de l'unité acide Ϝ-aminobutanoïque est pliée pour former une conformation en U dans un petit sillon d'une région en double hélice ayant une partie ou la totalité d'une séquence de nucléotides placée entre la position 195 et la position 150 (SEQ ID n° : 2) dans un promoteur de facteur de croissance du tissu conjonctif (ci-après appelé 'CTGF') ou une séquence complémentaire à la séquence de nucléotides (ci-après, la région en double hélice est appelée 'région cible'). Dans le polyamide de pyrrole-imidazole, une paire Pm/Im correspond à une paire de bases C-G, une paire Im/Py correspond à une paire de bases G-C et une paire Py/Py correspond à la fois à une paire de bases A-T et à une paire de bases T-A.
PCT/JP2006/318512 2005-09-20 2006-09-19 Inhibiteur de l'expression du gène du ctgf WO2007034784A1 (fr)

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WO2009066774A1 (fr) * 2007-11-22 2009-05-28 Nihon University Inhibiteur d'expression sélectif pour les gènes de la kinase aurora a et de la kinase aurora b
JP2014505704A (ja) * 2011-02-02 2014-03-06 エクスカリアード・ファーマシューティカルズ，インク 結合組織増殖因子（ｃｔｇｆ）をターゲティングするアンチセンス化合物を用いた、ケロイドまたは肥厚性瘢痕の治療法
JP5685081B2 (ja) * 2008-04-17 2015-03-18 浩喜 永瀬 マトリックスメタロプロテネース９遺伝子選択的発現抑制剤
JP5865347B2 (ja) * 2011-03-31 2016-02-17 隆光 矢野 ポリアミド化合物及びミトコンドリア遺伝子疾患治療用医薬組成物
WO2022019303A1 (fr) * 2020-07-20 2022-01-27 学校法人日本大学 COMPOSITION PHARMACEUTIQUE À BASE DE PYRROLE-IMIDAZOLE POLYAMIDE, INHIBITEUR DU GÈNE TGFβ, COMPOSITION PHARMACEUTIQUE ET PROCÉDÉ DE PRODUCTION DE PYRROLE-IMIDAZOLE

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009066774A1 (fr) * 2007-11-22 2009-05-28 Nihon University Inhibiteur d'expression sélectif pour les gènes de la kinase aurora a et de la kinase aurora b
JP5685081B2 (ja) * 2008-04-17 2015-03-18 浩喜 永瀬 マトリックスメタロプロテネース９遺伝子選択的発現抑制剤
JP2014505704A (ja) * 2011-02-02 2014-03-06 エクスカリアード・ファーマシューティカルズ，インク 結合組織増殖因子（ｃｔｇｆ）をターゲティングするアンチセンス化合物を用いた、ケロイドまたは肥厚性瘢痕の治療法
JP5865347B2 (ja) * 2011-03-31 2016-02-17 隆光 矢野 ポリアミド化合物及びミトコンドリア遺伝子疾患治療用医薬組成物
WO2022019303A1 (fr) * 2020-07-20 2022-01-27 学校法人日本大学 COMPOSITION PHARMACEUTIQUE À BASE DE PYRROLE-IMIDAZOLE POLYAMIDE, INHIBITEUR DU GÈNE TGFβ, COMPOSITION PHARMACEUTIQUE ET PROCÉDÉ DE PRODUCTION DE PYRROLE-IMIDAZOLE

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