JP2003159087A - Tissue fibrosing inhibitory oligonucleotide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tissue fibrosing inhibitor capable of directly controlling tissue fibrosing expression, and to provide a pharmaceutical composition for preventing or treating diseases due to tissue fibrosing by using the inhibitor. <P>SOLUTION: This inhibitor comprises an anti-sense oligonucleotide(AS-ODN) of stress protein(heat-shock protein)HSP gene, enabling tissue fibrosing expression to be inhibited directly, safely and effectively. The AS-ODN is therefore usable as the inhibitor or the pharmaceutical composition for preventing or treating diseases due to tissue fibrosing. These diseases include pulmonary fibrosis, paraquat poisoning, myocardial fibrosis after myocardial infarction, hepatic fibrosis (cirrhosis) and such diseases that a fibrosing such as vascular endothelial hyperplasia after coronary arterioplasty is associated with the morbidity development. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a tissue fibrosis expression inhibitor, and more specifically, a tissue fibrosis expression inhibitor using an antisense oligonucleotide of stress protein (heat shock protein) HSP47 gene, and the tissue fiber. The present invention relates to a pharmaceutical composition using a chemical expression inhibitor.

[0002]

2. Description of the Related Art When an individual, tissue, or cell of an organism is exposed to high temperatures, oxygen, transition metals, etc., a protein is induced as a response reaction of the cell. This is called a stress protein (heat shock protein: HSP). The stress protein shows that HSP70
There are high molecular mass and low molecular mass, and they are widely preserved and exist from bacteria to eukaryotic cells. Stress proteins control protein folding (formation of higher-order structures) and dissociation associations (called chaperone function), and play important roles in membrane permeation, localization, and degradation. HSP47 is a stress protein present in the endoplasmic reticulum and is considered to function as a molecular chaperone specific to collagen. It is known that the expression of HSP47 and collagen has a very good correlation at various cultured cell and tissue levels, as well as in the developmental process of individuals (Cell Engineering, Vol. 16, No. 9, 1282-1287,
1997).

On the other hand, for example, a disorder in which fibers are deposited in the lung collagen tissue and fibrosis of the lung occurs is known. At this time, it has been reported that HSP47 (heat shock protein) is required as a specific molecular chaperone for collagen (Eur. J. Biochem. 206, 323-329, 1992,
Cell Engineering, Vol.16, 1282-1287, 1997). Gas exchange and injury to compliance in late-stage acute lung injury (ARDS) are due to excessive fibrosis. Currently, long-term administration of steroids is performed as a treatment method before fibrosis. , Which improves lung injury and mortality (JAMA 280, 159-165, 1998). However, regarding the use of steroids, problems such as many side effects due to steroids have been pointed out. It has been pointed out that steroids cause secondary hyperproliferation of fibroblasts by suppressing the regeneration of injured alveolar epithelial cells, and rather promote lung fibrosis (Am. R.
ev. Respir. Dis. 130, 256-261, 1984).

[0004] For a long time, attempts have been made to administer antisense oligonucleotides (AS-ODN) in the body for treatment (Megumi Higaki: Modern Science Special Issue 23 "Gene Diagnosis and Gene Therapy" p251, 1994 /). 9, Maekawadaira: blood vessels
Tumorization, 40, 516-523, 2000). However, no practical examples have been seen yet. Recently, AS-ODN for pulmonary fibrosis
An attempt to use is also proposed. However, these are based on the indirect cause of pulmonary fibrosis, and the AS-ODN sequences used are also different. For example, Japanese Examined Patent Publication No. 2-152797 discloses antisense oligonucleotide regulation of the expression of the raf gene, which is one of the examples related to the abnormal growth of the raf gene and cells. However, it is not directly related to collagen that causes lung fibers.

Further, Japanese Patent Application Laid-Open No. 10-59850 discloses a platelet-derived growth factor expression inhibitor, which directly claims the inhibition and treatment of pulmonary fibrosis of interstitial pneumonia. However, it is an approach from a completely different direction of suppressing the expression of platelet-derived growth factor.
Furthermore, Japanese Patent Publication No. 8-503366 discloses an antisense oligonucleotide which inhibits the mutation of collagen and the expression of wild-type gene. This antisense oligonucleotide suppresses the expression of mutant collagen. It is a nucleotide and does not directly control the cause of lung fibrosis.

On the other hand, it has been proposed to control the expression of the stress protein HSP47 involved in the lesion accompanied by fibrosis by genetic manipulation. That is, JP-A-11
No. 243968 discloses stress protein HSP47.
280b upstream from the transcription start point in the genomic DNA of Escherichia coli
Disclosed is a method of controlling the transcriptional activity of the stress protein HSP47 gene by deleting the transcriptional activation region of the stress protein HSP47 gene consisting of a region containing p and about 100 bp in the first intron. However, this method involves genetic manipulation, and has many difficult problems when it is actually used for treating a disease associated with fibrosis. Therefore, at present, development of a method that can be safely and effectively carried out for prevention and treatment of diseases caused by tissue fibrosis is desired.

[0007]

The object of the present invention is to suppress the expression of tissue fibrosis by directly suppressing the expression of tissue fibrosis, and more specifically, to suppress the expression of stress protein (heat shock protein) HSP47 gene. The present invention also provides a tissue fibrosis expression inhibitor that suppresses tissue fibrosis, and a pharmaceutical composition for preventing or treating a disease caused by tissue fibrosis using the tissue fibrosis expression inhibitor.

[0008]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventor has found that by using an antisense oligonucleotide (AS-ODN) of stress protein (heat shock protein) HSP47 gene, It was found that the expression of fibrosis can be directly and safely and effectively suppressed, and the present invention has been completed. That is, the present invention provides a tissue fibrosis expression inhibitor using an antisense oligonucleotide (AS-ODN) of the stress protein HSP47 gene, and prevention of a disease caused by tissue fibrosis using the tissue fibrosis expression inhibitor, It consists of a pharmaceutical composition for treatment. In the present invention, as a disease caused by tissue fibrosis, fibrosis such as pulmonary fibrosis, paraquat poisoning, myocardial fibrosis after myocardial infarction, liver fibrosis (cirrhosis), and thickening of vascular endothelium after coronary angioplasty are pathological conditions. Diseases associated with can be mentioned.

That is, the present invention relates to the stress protein HSP.
47. An antisense oligonucleotide for any region of the genomic DNA of 47 (claim 1) or an oligonucleotide consisting of 10 to 100 nucleotides of any region of the genomic DNA of the stress protein HSP47 of SEQ ID NO: 1. The antisense oligonucleotide according to claim 1 (claim 2) or the oligonucleotide is a nucleotide for an arbitrary region of the first exon of the genomic DNA of the stress protein HSP47. Antisense oligonucleotide (claim 3) and stress protein HSP47
4. The antisense oligonucleotide (claim 4) or the oligonucleotide according to claim 3, wherein the nucleotide for an arbitrary region of the first exon of the genomic DNA of is an oligonucleotide having 15 base pairs from the translation start point. Is a nucleotide of SEQ ID NO: 2 (Claim 5)
Or 10 to 10 of an arbitrary region of the genomic DNA of the stress protein HSP47 of SEQ ID NO: 3.
An antisense oligonucleotide according to claim 1 (claim 6), characterized in that it consists of 0 nucleotides;
The oligonucleotide is a nucleotide for an arbitrary region of the first exon of the genomic DNA of the stress protein HSP47 (claim 7) or the genomic DNA of the stress protein HSP47. The nucleotide for any region of the first exon is an oligonucleotide having 15 base pairs from the translation initiation point.
Antisense oligonucleotide according to claim (claim 8)
Or an antisense oligonucleotide characterized in that the oligonucleotide is the nucleotide of SEQ ID NO: 4 (claim 9), or one or one of the base sequences of the antisense oligonucleotide according to any one of claims 1 to 9. An oligonucleotide in which the above bases have been deleted, added and / or substituted, and the stress protein HSP
47. An oligonucleotide (claim 10) characterized by having a binding property to an arbitrary region of genomic DNA.

Further, the present invention comprises a tissue fibrosis expression inhibitor (claim 11), which comprises the oligonucleotide according to any one of claims 1 to 10 as an active ingredient, and claims 1 to 10. A pharmaceutical composition comprising the oligonucleotide according to any one of (1) to (4) as an active ingredient and a pharmacological effect for preventing or treating a disease associated with tissue fibrosis pathogenesis. 13. The pharmaceutical composition according to claim 12 (claim 13) or a disease associated with the formation of tissue fibrosis pathology is pulmonary fibrosis, paraquat poisoning, myocardial fibrosis after myocardial infarction, liver fibrosis or coronary angiogenesis. 14. The pharmaceutical composition according to claim 13 (claim 14) characterized in that it is subsequent vascular endothelial thickening, or pulmonary fibrosis is interstitial pneumonia, or liver fibrosis is cirrhosis. 14. The method according to claim 14. Consisting pharmaceutical composition (claim 15).

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to an antisense oligonucleotide (A that suppresses expression of a stress protein (heat shock protein) HSP47 gene involved in tissue fibrosis.
S-ODN). The sequence of the stress protein HSP47 gene is based on the NCBI gene database, accession number: NM009825 (from mouse: SEQ ID NO: 1 in Sequence Listing) and NM001235 (from human:
It can be searched by SEQ ID NO: 3) in the sequence listing. The AS-ODN of the present invention can be obtained as an AS-ODN for any region of the genomic DNA of the stress protein HSP47. AS-OD used in the present invention
The size of N is usually in the range of 10 to 60, but it is preferably 15 to 30 bases in consideration of intracellular uptake and the like.

In the present invention, the stress protein HSP
Oligonucleotides to any region of the first exon of the 47 genomic DNA, particularly SEQ ID NO: 2 or SEQ ID NO: 4
An oligonucleotide of 15 base pairs from the translation initiation point shown in (3) can be effectively used. The AS-ODN used in the present invention is one in which one or more bases in the base sequence of the AS-ODN is deleted or added, as long as the AS-ODN binds to the stress protein HSP47 gene and retains the activity of suppressing the transcription of the gene. And / or replaced sequences.

When the AS-ODN of the present invention is used as a medicine for preventing or treating a disease associated with tissue fibrosis pathological condition formation, it can be carried out using a means usually used in this field. For example, the AS of the present invention
-To prepare ODN, it can be synthesized by a commonly used nucleotide synthesis method such as a commercially available automatic synthesizer. Further, the purification of the oligonucleotide can also be carried out by a method usually used in this field, for example, high performance liquid chromatography, polyacrylamide gel electrophoresis, solvent extraction, salting out and the like. Furthermore, for the drug delivery system for administering the AS-ODN of the present invention as a medicine, the methods usually used in this field can be applied. For example, A of the present invention
S-ODN can be used after being appropriately modified. AS
Examples of the modification of ODN include a phosphorothioate type in which an oxygen atom of a phosphoric acid group of a phosphodiester bond is replaced with a sulfur atom, a methylphosphonate type in which an oxygen atom of the phosphoric acid group is replaced with a methyl group, and a phosphoric acid bond of α -Α-oligo type instead of binding, and modification by binding of acridine or polylysine (Modern Gakugaku Special Edition 23 “Gene Diagnosis and Gene Therapy”, P251-257, 1994).

Diseases associated with the formation of tissue fibrosis pathological conditions to which the antisense oligonucleotide of the present invention is applied as a pharmaceutical composition include pulmonary fibrosis, paraquat poisoning, myocardial fibrosis after myocardial infarction, and liver fibrosis (cirrhosis). Or the thickening of vascular endothelium after coronary artery formation. The tissue fibrosis expression inhibitor of the present invention is capable of directly suppressing the expression of tissue fibrosis, and is a pharmaceutical composition which can be used safely and effectively for the prevention and treatment of diseases caused by tissue fibrosis. It is possible to provide.

[0015]

The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples. Example 1 Experimental Method and Preparation of Lung Injury Model Rats Fibers are deposited in lung collagen tissue and pulmonary fibrosis occurs. At this time, HSP47 (heat shock protein) is known to be required as a collagen-specific molecular chaperone. (Eur. J. Biochem. 206, 323-329, 1992, Cell Engineering Vol. 16, 1281, 1997). In the present invention, it is considered that lung fibrosis can be prevented by suppressing the expression of HSP47, which is the direct cause of this lung fibrosis, and the nucleotide sequence of the gene related to the translation initiation site of HSP47 protein is AS-
As an ODN, an experiment for suppressing lung fibrosis was conducted.

First, using rat, an acute lung injury model by administration of lipopolysaccharide (LPS) and a lung fibrosis model by bleomycin were constructed (in both models, lung fibrosis is greatly involved in the pathogenesis). It has been known). It is possible to directly administer AS-ODN corresponding to 15 base pairs from the translation initiation point of the HSP47 gene into the body of a model rat in which this lung disorder has occurred to suppress the expression of the HSP47 protein and suppress acute lung fibrosis. Proven. For administration of AS-ODN, a phosphotinated derivative was used.
It is irrelevant to the suppression of P47 protein expression, and any modification method can be used as long as it is an AS-ODN introduction method into cells.

Example 2 AS-ODN for lung fibrosis
Administration test (experimental method) When bleomycin is administered to rats, fibrosis occurs in the lungs.
The effect of S-ODN was examined. Rats (6 weeks old) were divided into the following 3 groups. There were 1 group (a group administered with bleomycin only), 2 groups (a group administered with bleomycin + AS-ODN), and 3 groups (a group administered with bleomycin + sense oligonucleotide). Bleomycin 10 units / kg
Was intratracheally administered (all groups) to induce lung fibrosis experimentally. In group 2, AS-ODN was further intratracheally administered at 30 nmol / animal immediately after administration of bleomycin.
In the 3 groups, for control, ODN of random sequence was intratracheally administered at 30 nmol / animal immediately after the administration of bleomycin. Change in survival rate in each group after administration, tissue examination after 4 weeks, HSP
Changes in 47 protein expression were examined.

(Experimental results) 1) Change in survival rate (FIG. 1): In 1 group of bleomycin-treated model rats, 7 out of 10 rats died 6 to 9 days after administration. On the other hand, two groups of HSP47 AS-OD
In the N-administered group, all 10 animals survived. The random sequence ODN administration group of the 3 groups had the same loss ratio as that of the 1 group, and therefore the data are not shown (the same applies hereinafter). This makes H
It was confirmed that SP47 AS-ODN improves the survival rate of bleomycin pulmonary fibrosis.

2) Histological findings (Fig. 2 [Reference photograph 1]: Left column shows lungs treated with bleomycin, upper row x40, lower row x200.
×; bleomycin + AS-ODN lungs, upper row x 4 in right column
(0x, lower x200): The tissue findings 4 weeks after the administration of bleomycin were shown. The left column shows the lung tissue of group 1, and the upper row shows the magnification of 40 times, and the lower row shows the magnification of 200 times. Marked lung fibrosis is observed. On the other hand, it was confirmed that the lung tissue was maintained in the lungs of the two groups in the right column and fibrosis was prevented. In the results of macroscopic findings, the lungs treated with bleomycin showed hardening of the lungs and fibrosis, whereas those treated with bleomycin + AS-ODN showed that
It was observed that fibrosis did not occur (Fig. 3 [Reference Photo 2]: left A figure shows macroscopic findings of bleomycin-administered lungs, and right B figure shows macroscopic findings of bleomycin + AS-ODN-administered lungs).

3) Protein expression (Fig. 4: 1 is a control;
2 for bleomycin 2 weeks; 3 for bleomycin + A
S-ODN 2 weeks; 4 is bleomycin 8 weeks; 5
Bleomycin + AS-ODN 8 weeks): HSP47 protein expression in lung tissue. The first control is the expression level in normal lung without treatment. It can be seen that the expression of HSP47 is increased 2 weeks after the administration of bleomycin (group 1). In contrast, AS-ODN administration (2
It is understood that the expression of HSP47 is suppressed in the group). After 8 weeks, there is no difference between Group 1 and Group 2, and as a result, the effect of AS-ODN disappears after 8 weeks, and it remains in the lung continuously, and no side effects are expressed.

(Evaluation) From the above results, suppression of the expression of HSP47 protein was carried out by intratracheally administering HSP47 AS-ODN to a rat model of interstitial pneumonia and pulmonary fibrosis induced by bleomycin administration. The histological findings showed the disappearance of lung fibrotic lesions. This fact is considered to be that AS-ODN effectively acted to almost completely suppress pulmonary inflammation and fibrosis induced by bleomycin through suppression of HSP47 expression, and the survival rate was markedly improved. That is, the result of this experiment is AS-O of HSP47.
It strongly suggests that DN is effective as a therapeutic agent for lung fibrosis. Furthermore, considering that the mechanism of tissue fibrosis is all mediated by HSP47, when not only lung tissue but fibrosis of various tissues forms a pathological condition, HSP47 AS-ODN is a fiber of various tissues. It is strongly suggested that it is effective as a therapeutic drug for aging.

Example 3 AS-OD for acute lung injury
N administration test (experimental method) Acute respiratory distress syndrome (ARDS) in human
Is a refractory disease, and one of the causes is considered to be pulmonary fibrosis in the late period. As a model close to this ARDS, a rat intrapulmonary lipopolysaccharide (LPS) administration model was prepared. Using this experimental system, the AS-O of the present invention
The effect of DN was examined. Rats (6 weeks old)
It was divided into groups. Group 1 (group administered only LPS), group 2 (LPS + AS-ODN administration group), and group 3 (LPS +
Random sequence ODN administration group). LPS 20 mg /
Intratracheal administration of kg (all groups) lung inflammation,
Fibrosis was induced experimentally. In 2 groups, 30 nmol / animal was added intratracheally to AS-ODN immediately after LPS administration.
Random sequence ODNs were intratracheally administered at 30 nmol / animal immediately after the administration of bleomycin in the 3 groups for control. Histological examination in each group after administration, expression of collagen fibers in tissues, HS
Changes in protein expression of P47 were examined.

(Experimental results) 1) Histological findings (Fig. 5 [Reference photograph 3]: Left column shows lungs treated with LPS on the 7th day, upper row × 40 times, lower row × 200 times; central row shows LP
S-administered lung on day 28, upper row x 40 times, lower row x 200 times; right column shows LPS + AS-ODN administration lung day 28, upper row x 40.
(Fold, lower row × 200 times): The left column is 7 days after LPS administration in the first group, and the central column is 28 days after LPS administration, and significant inflammation and fibrosis of lung tissue occur. On the other hand, the right column shows two groups administered with AS-ODN, and inflammation and fibrosis are improved. Data are not shown since the three random sequence ODN administration groups were similar to the one group. (Same below)

2) Expression of collagen fibers in tissues (Fig. 6)
[Reference Photo 4]: Azan staining of tissue, left column shows lungs treated with LPS, right column shows lungs treated with LPS + AS-ODN): Expression of collagen fibers in tissues is observed by Azan staining. It can be seen that deposition of blue-colored collagen fibers is prominently occurring in the left group 1. On the other hand, it can be seen that the collagen fiber deposition is suppressed in the second group.

3) Protein expression of HSP47 (Fig. 7: 1 shows A
RDS 2 weeks; 2 ARDS + AS-ODN 2 weeks; 3 ARDS 8 weeks; 4 ARDS + AS-OD
N 8 weeks; 5 is control;): HPS47 protein expression is clearly observed in the ARDS model 2 weeks after LPS administration, but it is found that this expression is suppressed in the 2 groups administered AS-ODN. . In addition, it was found that the expression levels were similar in both groups after 8 weeks from the administration, and the effect of AS-ODN disappeared after 8 weeks, remained in the lung continuously, and did not cause side effects.

(Evaluation) From the above results, it is possible to suppress the expression of HPS47 protein by administrating HPS47 AS-ODN to the respiratory tract against interstitial pneumonia and pulmonary fibrosis which were also induced in the ARDS model by LPS administration. Histopathological findings showed disappearance of lung fibrosis lesions and suppression of collagen fiber deposition. This fact suggests that AS-ODN effectively acts to suppress the expression of HSP47, and therefore collagen fibers cannot be deposited in the tissue, and as a result, LPS-induced lung inflammation and fibrosis are almost completely suppressed. To be
That is, the results of this experiment show that AS-ODN of HSP47 is effective as a therapeutic agent for lung fibrosis. Moreover, the mechanism of tissue fibrosis is HP
Considering that it is caused by collagen deposition via S47, it is shown that HSP47 AS-ODN can be used as a therapeutic agent for fibrosis of various tissues.

Example 4 Experimental Method and Preparation of Cirrhosis Model Rat (Experimental Method) In liver cirrhosis, collagen fibers are deposited in the liver tissue to cause liver cirrhosis. In this case as well, HSP47 is used.
Is required as a specific molecular chaperone for collagen (J. Clin. Invest. 94, 2481-248).
8, 1994). In the present invention, it is considered that liver fibrosis and cirrhosis can be prevented by suppressing the expression of HSP47, which is the direct cause of this fibrosis, and the nucleotide sequence of the gene related to the translation initiation site of HSP47 protein is AS- As an ODN, experiments were conducted to prevent liver fibrosis and liver cirrhosis.

Since administration of carbon tetrachloride to rats causes liver fibrosis and liver cirrhosis, the effect of AS-ODN of the present invention was examined by this experimental system. 1 group (carbon tetrachloride +
The group was a saline-administered group), a second group (carbon tetrachloride + AS-ODN administration group), and a third group (carbon tetrachloride + sense oligonucleotide administration group). Carbon tetrachloride was mixed with olive oil in an equal amount (50% carbon tetrachloride olive oil solution), and converted into a carbon tetrachloride stock solution by intraperitoneally administering 1 mL / kg three times a week (total). Group) Liver fibrosis and liver cirrhosis were experimentally induced. The day after the first intraperitoneal administration of carbon tetrachloride in all groups, the abdomen was opened under general anesthesia, and saline was punctured from the portal vein in one group with AS-ODN and an equivalent volume 27 G needle. In group 2, AS-ODN was injected by puncture through the portal vein at 100 nmol / kg. For 3 groups, a random sequence ODN was injected by puncture from 100 nmol / kg portal vein for control.
The injection from the portal vein was once in all groups, and thereafter, the intraperitoneal administration of 1 mL / kg of carbon tetrachloride once a day was repeated three times a week.
It continued for a week. After the administration, changes in survival rate in each group, tissue examination after 4 weeks, and protein expression of HSP47 were examined.

(Experimental Results) 1) Change in survival rate (FIG. 8): In 12 animals in the carbon tetrachloride + saline group, the survival rate after 4 weeks decreased to 33%, 8
After a week, the survival rate was 0%. On the other hand, in the carbon tetrachloride + AS-ODN administration group of 2 groups, the survival rate after 4 weeks was 75% of 16 animals, and the survival rate after 8 weeks was 62.5.
%Met. The survival rate of the carbon tetrachloride + sense oligonucleotide administration group of the 3 groups was almost the same as that of the 1 group. From the above, the effect of improving the survival rate of AS-ODN administration was confirmed.

2) Histological findings: HE magnification 40 times (FIG. 9) shows the tissue findings at 40 magnification by HE staining. The left end (A) showed normal liver tissue. Center (B)
Is a histological finding at 4 weeks in the group 1 carbon tetrachloride + saline group. The hepatic lobule structure is destroyed, and hepatocyte necrosis, inflammatory cell infiltration, hepatocyte steatosis, and liver fibrosis are observed.
The right end (C) is a tissue finding at 4 weeks of the group 2 carbon tetrachloride + AS-ODN administration group. Hepatocyte steatosis is observed, but hepatic lobule structure is relatively well maintained, fibrosis,
Infiltration of inflammatory cells is also mild. HE magnification 100 times (Fig. 1
0) is the magnification increased to 100 times. The left end (A) is normal liver, and the center (B) is group 1 carbon tetrachloride +
The raw diet group, the right end (C) is the group 2 carbon tetrachloride + AS-ODN administration group.

(Evaluation) From the above results, the progression to liver fibrosis and liver cirrhosis caused by the administration of carbon tetrachloride was found to be HSP.
By administrating 47 AS-ODN periportally,
Inhibition of HSP47 protein expression, liver fibrosis in histological findings,
Suppression of progression to cirrhosis and improvement of mortality were observed. This fact shows that AS-ODN effectively acts to suppress the expression of HSP47, and therefore collagen fibers cannot be deposited in the liver tissue, resulting in suppression of carbon tetrachloride-induced liver fibrosis. It is thought that a significant improvement in survival rate was obtained. That is, the result of this experiment is AS- of HSP47.
It strongly suggests that ODN is effective as a therapeutic agent for not only the above-mentioned lung fibrosis but also liver fibrosis and cirrhosis. Furthermore, considering that the mechanism of tissue fibrosis is all caused by HSP47-mediated collagen deposition, it is suggested that HSP47 AS-ODN can be used as a therapeutic agent for fibrosis of various tissues. .

[0032]

EFFECTS OF THE INVENTION For tissue fibrosis for which there is currently no effective preventive / therapeutic method, the tissue fibrosis expression inhibitor of the present invention and its pharmaceutical composition can provide an effective preventive / therapeutic method. In particular, since the pharmaceutical composition of the present invention is administered as a pharmaceutical composition containing an antisense oligopeptide as an active ingredient, its pharmaceutical effect can be expected safely and effectively. Furthermore, since the mechanism of tissue fibrosis is caused by the deposition of collagen via HPS47, which is the target of direct expression suppression control in the present invention, the HSP47 AS-ODN of the present invention treats fibrosis of various tissues. It can be used as a drug and is known as a disease associated with tissue fibrosis pathogenesis,
Pulmonary fibrosis, paraquat poisoning, myocardial fibrosis after myocardial infarction,
It can be universally applied as a method for preventing or treating fibrotic diseases in a wide range of tissues such as liver cirrhosis or thickening of vascular endothelium after coronary artery formation.

[0033]

[Sequence list]                                SEQUENCE LISTING <110> JAPAN SCIENCE AND TECHNOLOGY CORPORATION <120> Fibrosis-suppressing oligonucleotide <130> Y2002-P061 <140> <141> <150> JP P2001-277929 <151> 2001-09-13 <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 1254 <212> DNA <213> Homo sapiens <400> 1 atgcgctctc tccttctggg caccttatgc ctcctggctg tggccctggc agccgaggtg 60 aagaaacctg tagaggccgc agcccctggt actgcggaga agctgagttc caaggcgacc 120 acactggcag agcccagcac aggcctggcc ttcagcctgt atcaggcaat ggccaaggac 180 caggcagtgg agaacatcct ggtgtcaccc gtggtggtgg cctcgtcgct gggtctcgtg 240 tcgctgggcg gcaaggcgac cacggcgtcg caggccaagg cagtgctgag cgccgagcag 300 ctgcgcgacg aggaggtgca cgccggcctg ggtgagctgc tgcgctcact cagcaactcg 360 acggcgcgca acgtgacctg gaagctgggc agccgactgt acggacccag ctcagtgagc 420 ttcgctgatg acttcgtgcg cagcagcaag cagcactaca actgcgagca ctccaagatc 480 aacttcccgg acaagcgcag cgcgctgcag tccatcaacg agtgggccgc gcagaccacc 540 gacggcaagc tgcccgaggt caccaaggac gtggagcgca cggacggcgc cctgctagtc 600 aacgccatgt tcttcaagcc acactgggat gagaaattcc accacaagat ggtggacaac 660 cgtggcttca tggtgactcg gtcctatact gtgggtgtta cgatgatgca ccggacaggc 720 ctctacaact actacgacga cgagaaggag aagctgcagc tggtggagat gcccctggct 780 cacaagctct ccagcctcat catcctcatg ccccatcacg tggagcctct cgagcgcctt 840 gaaaagctgc taaccaaaga gcagctgaag atctggatgg ggaagatgca gaagaaggct 900 gttgccatct ccttgcccaa gggtgtggtg gaggtgaccc atgacctgca gaaacacctg 960 gctgggctgg gcctgactga ggccattgac aagaacaagg ccgacttatc acgcatgtct 1020 ggcaagaagg atctgtacct ggccagtgtg ttccacgcca ccgcctttga gttggacaca 1080 gatggcaacc cctttgacca ggacatctac gggcgcgagg agctgcgcag ccccaagctg 1140 ttctacgccg accacccctt catcttcctg gtgcgggaca cccaaagcgg ctccctgcta 1200 ttcattgggc gcctggtccg gctcaagggt gacaagatgc gagacgagtt atag 1254 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Antisense       oligonucleotide against collagen-binding stress       protein HSP47 <400> 2 tacgcgagag aggaa 15 <210> 3 <211> 2047 <212> DNA <213> Homo sapiens <400> 3 gtcgcggctc gagagcgaga gtcacgtccc ggcgctagcc cagcccgacc caggcccacc 60 gtggtgcacg caaaccactt cctggccatg cgctccctcc tgcttctcag cgccttctgc 120 ctcctggagg cggccctggc cgccgaggtg aagaaacctg cagccgcagc agctcctggc 180 actgcggaga agttgagccc caaggcggcc acgcttgccg agcgcagcgc cggcctggcc 240 ttcagcttgt accaggccat ggccaaggac caggcagtgg agaacatcct ggtgtcaccc 300 gtggtggtgg cctcgtcgct ggggctcgtg tcgctgggcg gcaaggcgac cacggcgtcg 360 caggccaagg cagtgctgag cgccgagcag ctgcgcgacg aggaggtgca cgccggcctg 420 ggcgagctgc tgcgctcact cagcaactcc acggcgcgca acgtgacctg gaagctgggc 480 agccgactgt acggacccag ctcagtgagc ttcgctgatg acttcgtgcg cagcagcaag 540 cagcactaca actgcgagca ctccaagatc aacttccgcg acaagcgcag gccgctgcag 600 tccatcaacg agtgggccgc gcagaccacc gacggcaagc tgcccgaggt caccaaggac 660 gtggagcgca cggacggcgc cctgttagtc aacgccatgt tcttcaagcc acactgggat 720 gagaaattcc accacaagat ggtggacaac cgtggcttca tggtgactcg gtcctatacc 780 gtgggtgtca tgatgatgca ccggacaggc ctctacaact actacgacga cgagaaggaa 840 aagctgcaaa tcgtggagat gcccctggcc cacaagctct ccagcctcat catcctcatg 900 ccccatcacg tggagcctct cgagcgcctt gaaaagctgc taaccaaaga gcagctgaag 960 atctggatgg ggaagatgca gaagaaggct gttgccatct ccttgcccaa gggtgtggtg 1020 gaggtgaccc atgacctgca gaaacacctg gctgggctgg gcctgactga ggccattgac 1080 aagaacaagg ccgacttgtc acgcatgtca ggcaagaagg acctgtacct ggccagcgtg 1140 ttccacgcca ccgcctttga gttggacaca gatggcaacc cctttgacca ggacatctac 1200 gggcgcgagg agctgcgcag ccccaagctg ttctacgccg accacccctt catcttccta 1260 gtgcgggaca cccaaagcgg ctccctgcta ttcattgggc gcctggtccg gcctaagggt 1320 gacaagatgc gagacgagtt atagggcctc agggtgcaca caggatggca ggaggcatcc 1380 aaaggctcct gagacacatg ggtgctattg gggttggggg ggaggtgagg taccagcctt 1440 ggatactcca tggggtgggg gtggaaaagc agaccggggt tcccgtgtgc ctgagcggac 1500 cttcccagct agaattcact ccacttggac atgggcccca gataccatga tgctgagccc 1560 ggaaactcca catcctgtgg gacctgggcc atagtcattc tgcctgccct gaaagtccca 1620 gatcaagcct gcctcaatca gtattcatat ttatagccag gtaccttctc acctgtgaga 1680 ccaaattgag ctaggggggt cagccagccc tcttctgaca ctaaaacacc tcagctgcct 1740 ccccagctct atcccaacct ctcccaacta taaaactagg tgctgcagcc cctgggacca 1800 ggcaccccca gaatgacctg gccgcagtga ggcggattga gaaggagctc ccaggagggg 1860 cttctgggca gactctggtc aagaagcatc gtgtctggcg ttgtggggat gaactttttg 1920 ttttgtttct tcctttttta gttcttcaaa gatagggagg gaagggggaa catgagcctt 1980 tgttgctatc aatccaagaa cttatttgta catttttttt ttcaataaaa cttttccaat 2040 gacattt 2047 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 4 tacgcgaggg aggac 15

[Brief description of drawings]

FIG. 1 is a graph showing changes in the survival rate when the AS-ODN of the present invention was administered to bleomycin-treated model rats in the examples of the present invention.

FIG. 2 is a set of photographs showing the results of histological findings when the AS-ODN of the present invention was administered to bleomycin-treated model rats in the examples of the present invention.

FIG. 3 is a set of photographs showing the results of macroscopic findings in the lungs when the AS-ODN of the present invention was administered to bleomycin-treated model rats in the examples of the present invention.

FIG. 4 is a graph showing the results of protein expression when the AS-ODN of the present invention was administered to bleomycin-treated model rats in the examples of the present invention.

FIG. 5 is a diagram showing photographs of histological findings when an AS-ODN of the present invention was administered to a rat intrapulmonary lipopolysaccharide (LPS) administration model in an example of the present invention.

FIG. 6 is a diagram showing photographs of collagen fiber expression in tissues when the AS-ODN of the present invention was administered to a rat intrapulmonary lipopolysaccharide (LPS) administration model in an example of the present invention. Is.

FIG. 7 is a view showing a photograph of protein expression of HSP47 when the AS-ODN of the present invention was administered to a rat intrapulmonary lipopolysaccharide (LPS) administration model in an example of the present invention.

FIG. 8 is a graph showing changes in the survival rate when the AS-ODN of the present invention was administered to a model rat in which liver fibrosis and liver cirrhosis were caused by carbon tetrachloride treatment in the example of the present invention.

FIG. 9 shows the results of histological findings when the AS-ODN of the present invention was administered to a model rat in which liver fibrosis and liver cirrhosis were caused by carbon tetrachloride treatment in the example of the present invention.
It is a figure which shows the photograph of 40 times by E staining.

FIG. 10 shows the results of histological findings when the AS-ODN of the present invention was administered to a model rat in which liver fibrosis and liver cirrhosis were caused by carbon tetrachloride treatment in Examples of the present invention.
It is a figure which shows the photograph of 100 times by HE staining.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 11/00 A61P 39/02 39/02 43/00 43/00 111 111 C12N 15/00 ZNAA

Claims (15)

[Claims] 1. Genomic DN of stress protein HSP47
Antisense oligonucleotide to any region of A. 2. The antisense oligonucleotide according to claim 1, wherein the oligonucleotide consists of 10 to 100 nucleotides in an arbitrary region of the genomic DNA of the stress protein HSP47 of SEQ ID NO: 1. 3. The oligonucleotide is a nucleotide for an arbitrary region of the first exon of the genomic DNA of the stress protein HSP47.
Or the antisense oligonucleotide according to 2. 4. Genomic DN of stress protein HSP47
The antisense oligonucleotide according to claim 3, wherein the nucleotide for an arbitrary region of the first exon of A is an oligonucleotide having 15 base pairs from the translation start point. 5. An antisense oligonucleotide, wherein the oligonucleotide is the nucleotide of SEQ ID NO: 2. 6. The antisense oligonucleotide according to claim 1, wherein the oligonucleotide comprises 10 to 100 nucleotides in an arbitrary region of the genomic DNA of the stress protein HSP47 of SEQ ID NO: 3. 7. The oligonucleotide is a nucleotide for an arbitrary region of the first exon of the genomic DNA of the stress protein HSP47.
The described antisense oligonucleotide. 8. Genomic DN of stress protein HSP47
The antisense oligonucleotide according to claim 7, wherein the nucleotide for any region of the first exon of A is an oligonucleotide having 15 base pairs from the translation start point. 9. An antisense oligonucleotide, characterized in that the oligonucleotide is the nucleotide of SEQ ID NO: 4. 10. An oligonucleotide in which one or more bases in the base sequence of the antisense oligonucleotide according to any one of claims 1 to 9 is deleted, added and / or substituted, and the stress protein HSP4.
7. An oligonucleotide having a binding property to any region of the genomic DNA of 7. 11. An agent for suppressing the expression of tissue fibrosis, which comprises the oligonucleotide according to any one of claims 1 to 10 as an active ingredient. 12. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1 to 10 as an active ingredient. 13. The pharmaceutical composition according to claim 12, wherein the pharmacological effect is to prevent or treat a disease associated with tissue fibrosis pathogenesis. 14. The disease associated with tissue fibrosis pathogenesis is pulmonary fibrosis, paraquat poisoning, myocardial fibrosis after myocardial infarction, liver fibrosis, or vascular endothelial thickening after coronary angioplasty. 13. The pharmaceutical composition according to 13. 15. The pharmaceutical composition according to claim 14, wherein the lung fibrosis is interstitial pneumonia or the liver fibrosis is cirrhosis.