WO2007034784A1

WO2007034784A1 - Ctgf gene expression inhibitor

Info

Publication number: WO2007034784A1
Application number: PCT/JP2006/318512
Authority: WO
Inventors: Takahiro Ueno; Noboru Fukuda; Hiroshi Sugiyama
Original assignee: Nihon University; Gentier Biosystems Incorporation
Priority date: 2005-09-20
Filing date: 2006-09-19
Publication date: 2007-03-29
Also published as: JPWO2007034784A1

Abstract

Disclosed is an inhibitor of the expression of CTGF gene, which comprises a pyrrole-imidazole polyamide. The pyrrole-imidazole polyamide has an N-methylpyrrole unit (hereinafter, referred to as “Py”), an N-methylimidazole unit (hereinafter, referred to as “Im”) and a Ϝ-aminobutanoic acid unit. In the pyrrole-imidazole polyamide, the region of the Ϝ-aminobutanoic acid unit is folded to form an U-shaped conformation in a miner groove of a double-helical region having a part or the entirety of a nucleotide sequence lying between position -195 to position -150 (SEQ ID NO:2) in a connective tissue growth factor (hereinafter, referred to as “CTGF”) promoter or a sequence complementary to the nucleotide sequence (hereinafter, the double-helical region is referred to a 'target region'). In the pyrrole-imidazole polyamide, a Pm/Im pair corresponds to a C-G base pair, an Im/Py pair corresponds to a G-C base pair, and a Py/Py pair corresponds to both an A-T base pair and a T-A base pair.

Description

Specification

CTGF gene expression inhibitor

Technical field

[0001] 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.

Background art

[0002] It is known that the transforming growth factor TGF-β 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-β expression. TGF-β 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. These results suggest that TGF-β is involved in neointimal formation after vascular injury by promoting VSMC proliferation and extracellular matrix formation. TGF-β is also involved in the formation of arteriosclerotic lesions. TGF-β 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-β is considered effective in reducing the above-mentioned vascular proliferative diseases.

[0003] The method of inactivation of gene function by reverse genetics is used to pray for the function of a specific gene. On the other hand, it is used for viral infection, cancer, and abnormal expression of a gene. There is also great potential for the treatment of other diseases based. That is, it is known that inactivation of gene function can be performed at the DNA level by homologous recombination, or at the RNA level by antisense oligonucleotides, ribozymes, siRNA, and the like. However, antisense oligonucleotide and ribozyme methods However, there is a problem that the target sequence is limited, and is easily degraded by ribonuclease, which is poor in thread and texture and migration into cells.

[0004] The present inventors have developed a pyrrole-imidazole polyamide that binds to the FSE2 protein binding region of the TGF-β1 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-β1 gene in various pathologies such as fibroproliferative diseases and vascular proliferative diseases. Developed pyrrole-imidazole polyamide that suppresses the expression of TGF-β1 gene by binding to the binding region as a target and suppressing the binding of AP-1 protein to the relevant region, and applied for a patent in 2004. (Patent Document 2).

[0005] 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-β1 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-β1, which was considered (Non-patent Document 1). Increased CTGF expression has been observed in various fibroproliferative diseases, and CTGF is selectively induced by TGF-β in fibroblasts. It has been reported that the promoter region of CTGF gene has a base sequence called TGF- | 8 responsive element, and TGF-β directly induces CTGF mRNA (Non-patent Document 2). That is, TGF-β force SCTGF is induced, and it is inferred that this CTGF promotes fibrosis downstream of TGF-β1 in the mechanism of fibrosis of organs and tissues involving TGF-β1. This could be a new therapeutic target.

On the other hand, unlike nucleic acid reagents such as antisense reagents such as ribozyme, pyrrole imidazole polyamides (hereinafter also referred to as Py-Im 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. [0007] 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. Patent Document 3-5). Py and Im can take U-shaped conformation in the presence of γ-aminobutyric acid by coupling and folding in succession. In the pyrrole-imidazole polyamide according to the present invention, the Ν-methylpyrrole unit (Py), the N-methylimidazole unit (Im) and the γ-aminobutyric acid unit (also referred to as γ-linker) are mutually connected with an amide bond (C (= Ο ) The general structure and production method are known (Patent Documents 3-5).

[0008] 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).

[0009] In general, 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).

[0010] 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: Gottesfeld et ahNature., 387: 202-5 (1997)

Disclosure of the invention

Problems to be solved by the invention

[0011] CTGF is important in fibroproliferative diseases where its expression is induced in response to stimulation by TGF-β1 and its expression changes in response to stimulation by TGF-β1 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-β1, which can be involved in more types of diseases, and is higher. It becomes possible to suppress fibrosis having safety and specificity. However, no organic compound that specifically suppresses the expression of CTGF has been known so far. In particular, there were no reports of CTGF gene expression inhibitors or therapeutic agents for TGF-β related diseases using pyro-l-imidazole polyamide that binds to the CTGF gene base sequence.

In addition, conventional methods for suppressing the expression of TGF-β1 gene and CTGF gene using antisense oligonucleotides and ribozymes have limited target sequences, and transfer to drug tissues and cells. There is a problem that it is bad and easily broken down by ribonuclease.

Means for solving the problem

[0012] 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. To obtain 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, Preferably, 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.

That is, the present invention is as follows.

(1) A pyrrole-imidazole polyamide containing N-methylpyrrole unit (hereinafter also referred to as Py), N-methylimidazole unit (hereinafter also referred to as Im) and γ-aminobutyric acid unit, and connective tissue growth factor (hereinafter also referred to as CTGF). ) In the minor groove of a double helix region (hereinafter also referred to as 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, Ύ -Can be folded at the site of an aminobutyric acid unit to take a U-shaped conformation. Py Zlm pair for CG base pair, ImZPy counter force A for G-C base pair — For T base pairs and TA base pairs! CTGF gene expression inhibitor comprising the above-mentioned pyrrolimidazole polyamide, each corresponding to a PyZPy pair.

(2) The CTGF gene expression inhibitor according to (1), further comprising a β-alanine unit.

(3) The above (1) or (3) wherein 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) The CTGF gene expression inhibitor described in 2).

(4) The above target region (1) or (2) wherein the target region is a double helix region comprising part or all of the base sequence 160 to 155 (SEQ ID NO: 4) shown below of the CTGF promoter and a complementary strand thereto: ) CTGF gene expression inhibitor as described in the above.

(5) The above target region (1) or (1), wherein the target region is a double helix region comprising part or all of the base sequence shown below of the CTGF promoter—195 to 174 (SEQ ID NO: 5) and a complementary strand thereto: 2) The CTGF gene expression inhibitor described in 2).

(6) The above target region (1) or (1), wherein the target region is a double helix region comprising part or all of the base sequence shown below of the CTGF promoter: 190 to 184 (SEQ ID NO: 6) and a complementary strand thereto. 2) The CTGF gene expression inhibitor described in 2).

(7) The carboxyl group at the end of the pyrrole-imidazole polyamide forms an amide. V, The CTGF gene expression inhibitor according to any one of (1) to (6) above.

(8) The CTGF gene expression inhibitor according to the above (7), which is an amide with the above amidecylaminopropylamine or N, N-dimethylaminopropylamine.

Expression inhibitor.

(9) A CTGF gene expression inhibitor comprising a pyrrole-imidazole polyamide represented by the following formula:

[Chemical 1]

(10) A CTGF gene expression inhibitor comprising a pyrrole-imidazole polyamide represented by the following formula:

[Chemical 2]

ol. Wt .: 1912.00 Effects of the Invention

According to the present invention, since specific gene expression can be specifically suppressed, It is possible to obtain a 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-β1, the gene expression inhibitor of CTGF can lead to more safe and specific inhibition of high fibrosis.

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] In the pyrrole-imidazole polyamide according to the present invention! /, N-methylpyrrole unit (hereinafter also referred to as Py), N-methylimidazole unit (hereinafter also referred to as Im) and γ-aminobutyric acid unit (γ linker). Are also linked to each other by an amide bond (—C (= 0) —ΝΗ—), and their general structure and production method are known (see, for example, Patent Documents 3 to 5).

[0016] For example, 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). ). According to the solid-phase Fmoc method, 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 . For example, 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. Since 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. . In addition, since 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).

[0017] According to the known Fmoc method and the like, 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 γ-aminobutyric acid residue at the terminal. A dazole polyamide etc. are mentioned. The pyrrole imidazole polyamide having a terminal j8-alanine residue or γ-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.

[0018] Specific examples of the aminopyrrole carboxylic acid 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. — 2-pyrrole carboxylic acid, 4-amino 1 butyl 2-pyrrole carboxylic acid and the like. Specific examples of the aminoimidazole carboxylic acid 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.

[0019] According to the solid phase Fmoc method, for example, a conjugate of pyrrole imidazole polyamide and FITC (fluorescein isothiocyanate) can also be synthesized. Since FITC is conventionally known as a fluorescent labeling reagent for antibodies, the resulting conjugate can be used to prove that the pyrrole imidazole polyamide recognizes a specific DNA sequence.

[0020] 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 γ-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 γ-aminobutyric acid unit and is U-shaped. Type conformation, PyZlm counterforce for CG base pairs, ImZPy counterforce for G—C base pairs, A—T base pairs and TA base pairs, the difference is PyZPy pair Each contains the corresponding pyrrole-imidazole polyamide.

[0021] Usually, 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). Where 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. In this case, 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, and the AT base pair and TA base pair are not. PyZPy pairs correspond to each other. Then, the molecule is folded at the site of the γ-aminobutyric acid unit in the pyrrole-imidazole polyamide molecule to take a U-shaped conformation.

[0022] If 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. Thus, 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.

[0023] 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.

[Chemical 3]

G B P 2 1 0 or

[Chemical 4]

ol. Wi: 1912.00

G B P 2 1 0 2

[0024] GBP2101 has the molecular formula C H N O, molecular weight 1667.75, and its target sequence is

76 94 30 15

Of the 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 (— 160--155) Suppresses the expression of CTGF gene by binding to 6 bases of (SEQ ID NO: 4).

[0025] GBP2102 has the molecular formula C H N O, molecular weight 1912.00, and its target sequence is

88 106 34 17

Among the regions of the CTGF gene regulatory region — 195 to 150 (SEQ ID NO: 2), 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).

[0026] 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. (Janssen et ahMol Cell. 2000; 6: 1013-24, Janssen at al: Mol Cell. 2000; 6: 999-1011.). We have synthesized Py-Im polyamides that target specific regions of the CT GF promoter. These polyamides stayed stably in the core for 48 hours without disappearing. 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.

[0027] Until recently, the development of Py-Im polyamides was based on the structural properties of transcription factor DNA complexes in the promoter sequence. An efficient way to target sequences in a TATA box-containing promoter would be to design a base pair adjacent to the TATA box. The TATA box is located 25 to 35 base pairs upstream of the transcription start site of most protein-coding genes. Transcription mediator D (TAF D) is

II

It contains a TATA box binding protein (TBP) that binds specifically to tuss, and employs other transcription factors in the core promoter to form a pre-initiator complex (PIC). PIC initiates gene transcription and interacts with activators or sublessors to regulate gene expression. 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. Of the successful examples of polyamides designed with various promoters, those targeting the TATA box are known to always work.

[0028] There are various types of promoters, including TATA boxes and initiator regions (Inr)!

(Javahery et al: Mol Cell Biol.l994; 14: 116-27; Lo et al: Gene.l996 Dec 5; 182 (1-2): 1 3-22; Romeo et al: Gene.l997; 189: 289 -95.). Promoters of highly expressed and specialized genes tend to have a TATA box! /, But promoters of housekeeping genes tend to lack it. Promoters without TATA may not be necessary for genes that are expressed at low levels or require strict down-regulation during growth, but the mechanism still requires further study. The hTGF-β 1 promoter belongs to this class. It contains a number of positive and negative sequences in various regions upstream of the transcription start site (Kim et al: J Biol Chem. 1989; 264: 402-8.) 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! Several substances such as TPA, V-src gene product and hTGF-β1 itself stimulate the expression of hTGF-β1 gene via the AP-1 sequence (Kim et al: J Biol Chem.l989). ; 264: 7041-5, Kim et al: J Biol Chem.l989; 264: 19 373-8, Kim et al: Mol Cell Biol.l990; 10: 1492- 7, BirchenaU- Roberts et al: Mol Cell B iol l990; 10: 4978-83).

[0029] It has been postulated that SP-1 and Z or AP-1 sequences mediate activation of hTGF-β1 gene expression. Important polyamides are designed to target base pairs adjacent to different AP-1 and SP-1 sequences to block the binding of responsive transcription factors. However, there are insufficient data to show whether these polyamides can inhibit or activate the hTGF-β1 promoter activity. These results cannot be applied because the position of the designed polyamide in the minor groove such as DNA is inappropriate. We extended the target sequence upstream of the hTGF-β1 promoter. One polyamide targets the hTGF-β1 promoter, which has been shown to inhibit promoter activity in vitro and hTGF-β1 mRNA and protein in cultured hVSMC. 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.

[0030] In addition to the regulation of transcription factors in the promoter region, other factors may affect gene expression. These factors include chromatin packing, polyaddition, splicing, mRNA stability, translation initiation, etc. (Berger et al: Mol Cell. 2001; 5: 263-8, McKeown Annu Rev Cell Biol. L992 ; 8: 133- 55, Decker et al: Tr ends Biochem Sci.l994; 19: 336-40, Kozak Annu Rev Cell Biol.l992; 8: 197-225). 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.

[0031] CTGF is a renal disease (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 J Pathol 165 (6) 2033-43, 2004, Crean JK et al: 18 (13) 1541-3, 2004) _{0 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 the above-mentioned various renal diseases.

[0032] 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.

[0033] 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.

[0034] During liver fibrosis, hepatic stellate cells play an important role in extracellular matrix production (Bataller R et al: Gastroenterology 118: 1149, 2000). 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.

[0035] Currently, treatments for chronic renal failure are roughly divided into hemodialysis and CAPD. 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.

[0036] 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-β (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.

[0037] The force proposed by TGF-β 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.

[0038] 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.

Example

[0039] I. Materials and Methods

(1) Py—Im polyamide design for CTGF promoter

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.

[0040] (2) Machine-assisted (machine-assisted) automatic synthesis of Py—Im polyamide using Fmoc method

Machine-assisted automated synthesis of pyrrole-imidazole polyamides using a continuous flow peptide synthesizer Pioneer ™ (Applied Biosystems) on a 0.1 mmol scale (20 Omg of Fmoc-β-alanine mono-CLEAR acid resin, 0.50 meqZg, Peptide Institute, Inc.). Automated solid phase synthesis requires DMF wash, removal of Fmoc group with 20% piperidine Z DMF, methanol wash, 60 min coupling with monomer in the presence of HATU and DIEA (each 4 equivalents), methanol wash required Depending on the condition, it is protected by acetic anhydride Z-pyridine and the final DMF detergency. Py—Im polyamides were generally obtained in moderate yields (10-30%).

[0041] 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.

General procedure: After removing the Fmoc group of Fmoc-β-alanine-Wang resin, the resin was washed successively with methanol. The coupling step was performed with Fmoc amino acid, followed by washing with methanol. These steps were repeated many times until the entire sequence was introduced. After completing the coupling step, the N-terminal amino group was protected as necessary or coupled with FITC, washed with DMF, and the reaction vessel was removed.

[0042] Decomposition as carboxylic acid: Decomposition process of synthetic polyamide by cold ether precipitation (91% TFA—3% ZTIS—3% DMS—3% water mixture 5 ml Z resin 0.1 mmol).

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, 晚).

Purification: Final purification was performed on an analytical RP-HPLC with a flow rate of lOmlZmin using a linear gradient of B (acetonitrile) in buffer A (0.1% TFAZ water or 0.1% ACOHZ water) at 35 Onm. This was done by UV detection.

[0043] (3) Human mesangial cell culture

Human mesangial cells (Clonetics) were cultured in Dulbecco's modified Eagle medium (DM EM) containing 10% offspring serum (Invitrogen), lOOUZml persilin, and lOOmgZml streptomycin. Cells were passaged by trypsinization with 0.05% trypsin (Invitrogen) in Ca ²⁺ , Mg ²⁺ free phosphate buffered saline (PBS) and cultured in 75-cm ² tissue culture flasks. The medium was changed every 4-5 days and experiments were performed on cells between passages 5-10.

(4) Incubation of FITC-labeled polyamides in cultured human mesangial cells Passage human mesangial cells were cultured in 24-well flasks at a density of 10 ⁵ Zcm ² for 24 hours. FITC-labeled polyamide was added directly to the medium at a concentration of ^10_9 M, washed 2 hours later, and thereafter observed with a fluorescence microscope every hour.

[0044] (5) Genore Shift Atssey

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 [γ- 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

Incubated with polyamide or mismatched polyamide in 1). The resulting complex was electrophoresed on a 20% polyacrylamide gel and visualized by autoradiography.

[0045] (6) RNA extraction and reverse transcription reaction for growth factor mRNA, polymerase chain reaction (R T-PCR)

The cultured cells are washed with PBS, lysed in 1000 / z L Trizol (Invitrogen), mixed with 100 / z L Kroform form, centrifuged, and the upper aqueous phase is mixed with an equal volume of isopropanol to obtain RNA. Precipitated. The RNA pellet was washed twice with 500 μL of 75% ethanol, dried, and then dissolved in 10 1 buffer solution. After denaturation at 65 ° C for 15 min, the 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

2

Equal volume of 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

2

5U / 20 μL avian myeloblastoma virus reverse transcriptase (Takara Biochemicals, Osaka, Japan) was used for reverse transcription into single-stranded cDNA. Add 2 μL of diluted cDNA product to 10 mM Tris—HCl (pH 8.3), 50 mM KC1, 4 mM MgCl 2, 0.025 U / μL Ta

2

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 μL. 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. For human 18S ribosomal RNA Sense primer (5, -TCAAGAACGAAAGTCGGACG-3 ') (SEQ ID NO: 9) and antisense primer (5,-GGACATCTAAGGGCATCACA-3,) (SEQ ID NO: 10) were used as internal controls. 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. To confirm that genomic DNA was not amplified by PCR, a controlled RT-PCR experiment with a primer set without reverse transcriptase was performed. No product was amplified in any of the reactions. PCR for semi-quantitative analysis of mRNA The number of cycles at which the product was detectable on the gel was compared between the various samples. Consecutive

Ten-fold diluted cDNA (100, 10 and lng) was amplified. PCR products became detectable at earlier cycles as the amount of cDNA was increased. The amount of PCR product corresponding to each target mRNA increased linearly over 20-35 cycles. PCR product quantification

This was performed using Analyzer (Agilent).

[0047] (7) Statistical analysis

The results were expressed in mean valuer SEM. The significance of the difference between the mean values was evaluated by Student's t test. A p value less than 0.05 was considered significant.

[0048] II. Results

(1) Binding of synthetic polyamide to double-stranded oligonucleotide

The binding of GBP2101 and GBP2102 to the target sequence was examined by gel shift assay. 50 base sense and antisense oligonucleotides containing the target sequence of each pyrrole-imidazole polyamide

Sense ctgtgagctggagtgtgccagctttttcagacggaggaatgctgagtgtc (歹 U¾ ^ l 1) GCTCACAG (SEQ ID NO: 12)

And was annealed to create double-stranded DNA at the target site and incubated with each pyrrole-imidazole polyamide. These were electrophoresed on a polyacrylamide gel to examine the binding between pyrroleimidazole and the target sequence. When pyrrole-imidazole polyamide (Py Im) was added to double-stranded DNA (DS) for both GBP2 101 and GBP2102, it was suggested that the mobility decreased compared to the lane containing only DS, and that the polymer was polymerized. The bond with was proved. The result is shown in figure 2.

[0049] (2) Inhibition of CTGF messenger RNA expression by GBP2101

Culturing the cultured Hitomesangiumu cells derived from kidney, 1χ10 ^_6 Μ of PMA to (12- 0- tetradec anoylpholbol- 13 -acetate) and stimulated添Ka卩, the CT GF messenger RNA by the addition of Py-Im (GBP2101) The expression suppression effect was examined.

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. Messenger RNA was isolated by the anidium thiocyanate method, reverse-transcribed by avian myeloblastoma virus reverse transcriptase, and amplified by PCR. PCR products were quantified by Agilent Bioanalyzer. A specific primer for the CTGF gene and 18S rRNA 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 1χ10 ^_6 Μ, was inhibited up to 50% or less 1χ10 ^_6 Μ by GBP2101 added pressure (Fig. 3).

[0050] (3) Inhibition of CTGF protein expression by GBP2101

Culturing the cultured Hitomesangiumu cells derived from kidney, 1χ10 ^_6 Μ of PMA (12- 0- tetrade canoylpholbol - 13 - acetate) stimulated by添Ka卩a, Py- Im the addition of the C TGF proteins (GBP2101) The expression suppression effect was examined.

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 1χ10 ^_6 Μ, was inhibited up to 50% or less 1χ10 ^_6 Μ by GBP2101 added Caro (Figure 4).

[0051] (4) Inhibition of CTGF messenger RNA expression by GBP2102

Culturing the cultured Hitomesangiumu cells derived from kidney, were stimulated by added Caro the TGF-beta 1 in 1χ10 ^_6 Μ, was to examine the inhibition effects on expression of Py-Im (GBP2102) mosquitoes卩Ete CTGF messenger RNA.

The cells were subconfluent and replaced with serum-free medium for 24 hours, and 1x10 "6M TGF-β1 and each concentration of Py-Im were added and cultured for 12 hours without serum. In addition, 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 1χ10 ^_6 Μ, was inhibited up to 50% or less 1χ10 ^_6 Μ by GBP2102 added pressure (Figure 5).

[0052] (5) Inhibition of CTGF protein expression by GBP2102

Culturing the cultured Hitomesangiumu cells derived from kidney, 1χ10 ^_6 Μ of PMA to (12- 0- tetradec anoylpholbol- 13 -acetate) and stimulated添Ka卩, Py- Im the addition of CT GF protein (GBP2102) The expression suppression effect was examined.

CTGF protein expression level was increased 20% by PMA stimulation 1χ10 ^_6 Μ, was inhibited up to 50% or less 1χ10 ^_6 Μ by GBP2102 added Caro (Figure 6).

Industrial applicability

[0053] The CTGF gene expression inhibitor of the present invention can be used as a therapeutic agent for diseases associated with the production of TGF-β and thus CTGF.

Brief Description of Drawings

[0054] 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.

Sequence listing free text

[0055] SEQ ID NO: 7 sense primer SEQ ID NO: 8 antisense primer

SEQ ID NO: 9 sense primer

SEQ ID NO: 10 antisense primer

SEQ ID NO: 11 Gel shift assembly antisense oligonucleotide SEQ ID NO: 12 Gel shift assembly antisense oligonucleotide

Claims

The scope of the claims

[1] A pyrrole-imidazole polyamide containing an N-methylpyrrole unit (hereinafter also referred to as Py), an N-methylimidazole unit (hereinafter also referred to as Im), and a γ-aminobutyric acid unit, which is also referred to as connective tissue growth factor (hereinafter also referred to as CTGF). ) In the minor groove of a double helix region (hereinafter referred to as a target region) containing part or all of the base sequence 195 to 150 (SEQ ID NO: 2) shown below and the complementary strand thereto, the above 0-aminobutyric acid It can be folded at the unit site to form a U-shaped conformation, PyZlm counterforce for C—G base pairs, ImZPy counterforce A—T base for C—G base pairs A CTGF gene expression inhibitor comprising the pyrrole-imidazole polyamide, wherein the pair and the TA base pair correspond to the PyZPy pair, respectively.

[2] The CTGF gene expression inhibitor according to claim 1, further comprising a j8-alanine unit.

[3] The target region is a double helix region comprising a part or all of the following base sequence of the CTGF promoter: 164 to 150 (SEQ ID NO: 3) and a complementary strand thereto. CTGF gene expression inhibitor.

[4] The target region is a double helix region comprising part or all of the base sequence shown below of the CTGF promoter: 160 to 155 (SEQ ID NO: 4) and a complementary strand thereto. CTGF gene expression inhibitor.

[5] The target region according to claim 1 or 2, wherein the target region is a double helix region comprising part or all of the base sequences 195 to 174 (SEQ ID NO: 5) shown below of the CTGF promoter and a complementary strand thereto: CTGF gene expression inhibitor.

[6] The target region is a double helix region comprising a part or all of the following nucleotide sequence of the CTGF promoter: 190 to 184 (SEQ ID NO: 6) and a complementary strand thereto. CTGF gene expression inhibitor.

7. The CTGF gene expression inhibitor according to any one of claims 1 to 6, wherein a carboxyl group at a terminal of the pyrrole-imidazole polyamide forms an amide.

[8] The CTGF gene expression inhibitor according to [7], which is an amide with said amidecylaminopropylamine or N, N dimethylaminopropylamine.

[9] CTGF gene expression suppression comprising pyrrole-imidazole polyamide represented by the following formula: Formulation.

[Chemical 1]

A CTGF gene expression inhibitor comprising pyrrole-imidazole polyamide represented by the following formula.

[Chemical 2]

Mol. Wt: 1912.00