WO2006115279A1

WO2006115279A1 - Composition for preservation of vascular endothelium

Info

Publication number: WO2006115279A1
Application number: PCT/JP2006/308788
Authority: WO
Inventors: Noboru Fukuda
Original assignee: Nihon University; Gentier Biosystems, Inc.
Priority date: 2005-04-21
Filing date: 2006-04-20
Publication date: 2006-11-02
Also published as: JP2008174450A

Abstract

Disclosed is a composition for the preservation of a vascular endothelium which can prevent coronary restenosis and can preserve a vascular endothelium. The composition comprises a PDGF A-chain antisense oligonucleotide or a derivative thereof as the active ingredient. The composition can inhibit the abnormal growth of vascular smooth muscle cells and preserve a vascular endothelium.

Description

Specification

Vascular endothelial preservation composition

Technical field

[0001] The present invention relates to a vascular endothelial preserving composition capable of preventing coronary restenosis and preserving endothelium in blood vessels. More specifically, the present invention relates to a vascular endothelial preservation composition comprising an antisense oligonucleotide for platelet-derived growth factor-A chain (hereinafter simply referred to as PDGF-A chain) or a derivative thereof as an active ingredient. The present invention also relates to a method for preventing or treating coronary restenosis by using this vascular endothelial preserving composition in a stent coating agent or the like.

Background art

Conventionally, as a treatment for ischemic heart diseases such as angina pectoris and myocardial infarction, a method of expanding a coronary artery with a balloon or a stent in percutaneous coronary angioplasty has been performed. Although this method is highly effective, there is a problem that coronary artery restenosis is likely to occur due to damage to the vascular endothelium caused by insertion of a balloon or stent, and abnormal proliferation of vascular smooth muscle cells at the damaged site. .

In recent years, in order to prevent coronary restenosis, drug-eluting stents have been developed in which anticoagulants, antiplatelet substances, antitumor drugs, anti-inflammatory drugs, immunosuppressive drugs, etc. are applied to the stent, and the effects of these stents have been studied. Clinical application has started (for example, see Patent Document 1).

[0003] Among them, a drug-eluting stent coated with an immunosuppressant sirolimus (trade name rapamycin) (for example, see Patent Document 2) is widely used in the United States, and has recently been approved in Japan. In 2004, clinical application was started.

Since sirolimus forcibly stops the cell cycle at the G1 phase and suppresses the proliferation of vascular smooth muscle cells, sirolimus-coated drug-eluting stent (registered trademark, CYPHER) strongly suppresses the proliferation of cells in the blood vessel, and coronary artery Effective in preventing restenosis. In large-scale clinical trials, it has been reported that the rate of coronary restenosis 6 months after stent placement has drastically decreased to less than a quarter of the restenosis rate compared to the conventional stent (for example, (See Non-Patent Documents 1 and 2).

[0004] However, since sirolimus is a drug that stops the cell cycle, only vascular smooth muscle cells It also suppresses the proliferation of vascular endothelial cells, but even though coronary restenosis can be prevented, the endothelium of the stent lumen cannot be preserved, and the metal surface of the stent is exposed. .

When using this drug-eluting stent, it is necessary to take ticlovidin hydrochloride, an antiplatelet drug, for more than 3 months after treatment, and this period is about three times that of the conventional stent.

In addition, there were approximately 300 subacute thrombosis (SAT) in the United States Food and Drug Administration (FDA) among patients who had placed sirolimus-coated drug-eluting stents by 2003, 60 of whom died As an emergency safety information, the FDA issued a recommendation to pay careful attention to SAT when using a sirolimus-coated drug-eluting stent (see Non-Patent Document 3, for example). Furthermore, ticlovidin hydrochloride has serious side effects such as severe liver damage and decreased white blood cells and platelets, and there are concerns about the risk of side effects from long-term use.

Accordingly, it is desired to provide a composition that prevents coronary restenosis, maintains the proliferation of endothelial cells in blood vessels, preserves the endothelium, and has a low risk of side effects.

Patent Document 1: Japanese Patent Publication No. 5-502179

Patent Document 2: JP-A-6-9390

Non-specific S 1: Regar E, Serruys PW, Bode C, Holubarsch C, Guermonprez JL, Wijns s W, Bartorelli A, Constantini C, Degertekin, Tanabe K, Disco C, Wuelfert E, orice MC; RAVEL Study Group. Circulation 106: 1949-1956, 2002.

Non-Patent Document 2: Cohen DJ, Bakhai A, Shi C, Githiora L, Lavelle T, Berezin RH, Leon MB, Moses JW, Carrozza JP Jr, Zidar JP, Kuntz RE; SIRIUS Investigators. Circulati on. 110: 508-514 , 2004.

Non-Patent Document 3: FDA public health web notification: Food and Drug Administration. 2 003. Available at: 1113809079625— 0.% 20Accessed March 12, 2004.

Disclosure of the invention

Problems to be solved by the invention

The present invention prevents coronary restenosis, further maintains the proliferation of endothelial cells in blood vessels, It is an object of the present invention to provide a vascular endothelial preserving composition capable of preserving blood. Another object of the present invention is to provide a method for preventing or treating coronary restenosis by using this vascular endothelial preservation composition.

Means for solving the problem

[0006] As a result of diligent research to solve the above-mentioned problems, the present inventors have found that blood vessels by insertion of a composition strength stent or the like containing a PDGF-A chain antisense oligonucleotide or its derivative as an active ingredient. It was found that the abnormal proliferation of vascular smooth muscle cells associated with endothelial damage can be specifically suppressed and the endothelium in the blood vessel can be preserved.

Furthermore, by using this composition for preserving vascular endothelium as a stent coating agent or the like, it was found that the composition was effective for the prevention or treatment of coronary restenosis, and the present invention was completed.

That is, the present invention relates to the following (1) to (9).

(1) A composition for preserving vascular endothelium, comprising as an active ingredient an antisense oligonucleotide for platelet-derived growth factor-A chain or its derivative.

(2) The composition according to (1), further having an inhibitory effect on proliferation of vascular smooth muscle cells.

(3) The composition according to (1) or (2) above, wherein the derivative is chemically modified to a phosphorotype.

(4) A stent coating agent comprising a composition for preserving vascular endothelium, comprising an antisense oligonucleotide for platelet-derived growth factor-A chain or its derivative as an active ingredient.

(5) Antisense oligonucleotide for platelet-derived growth factor-A chain or its induction

A drug-eluting stent coated with a vascular endothelial preserving composition containing a conductor as an active ingredient or a stent coating agent containing the composition.

(6) The drug-eluting stent according to the above (5), which is coated with a composition for preserving a vascular endothelium or a stent coating agent containing the composition, and an idrog nore.

(7) A method for preventing or treating coronary restenosis, comprising preserving the endothelium in a blood vessel, comprising administering an antisense oligonucleotide for platelet-derived growth factor-A chain or a derivative thereof to an affected area.

(8) The coronary restenosis is after coronary artery restenosis as described in (7) above. Prevention or treatment method.

(9) A method for preventing or treating coronary restenosis, wherein administration is performed by a stent coated with an antisense oligonucleotide or a derivative thereof for platelet-derived growth factor A chain.

The invention's effect

[0008] The composition for preserving vascular endothelium of the present invention can specifically inhibit the proliferation of vascular smooth muscle cells, maintain the proliferation of the vascular endothelial cells, and preserve the endothelium in the blood vessel. By preserving the endothelium in the blood vessel, it is possible to prevent or treat coronary restenosis in angina pectoris, myocardial infarction, etc., and to recover the underlying disease.

Brief Description of Drawings

FIG. 1 is a diagram showing the base sequences of antisense ODN and non-sense ODN (Example 1)

FIG. 2 shows that antisense ODN has flowed out of a stent and has been distributed to endothelial cells of coronary arteries (Example 5).

FIG. 3 is a graph showing the presence or absence of a coronary artery lumen (Example 5).

FIG. 4 is a view showing the volume ratio of restenosis portion in a coronary stent (Example 5).

FIG. 5 shows the results of IVUS (Example 5).

FIG. 6 shows coronary artery pathological findings (Example 5).

FIG. 7 is a graph showing the area ratio of restenosis in a coronary stent (Example 5).

FIG. 8 shows the results of coronary artery hematoxin staining (Example 5).

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, “preservation of endothelium in blood vessels” refers to the suppression of excessive proliferation of vascular smooth muscle cells accompanying damage of vascular endothelium in blood vessels, thereby maintaining the proliferation of vascular endothelial cells, To preserve the endothelium in blood vessels.

By preserving the endothelium in the blood vessel of the present invention, treatment of diseases in organs such as the heart and brain, particularly all diseases related to vascular stenosis and restenosis, mainly caused by excessive proliferation of vascular smooth muscle cells. And can be used for prevention. Examples of diseases mainly caused by excessive proliferation of vascular smooth muscle cells include treatment of ischemic heart diseases such as angina pectoris and myocardial infarction. Coronary restenosis in medical treatment.

[0011] Coronary restenosis, which is one of the targets for prevention or treatment of the present invention, refers to platelet aggregation caused by damage to the vascular endothelium, and platelet-derived growth factor (hereinafter sometimes simply referred to as PDGF). The process of releasing the growth factor, causing the medial smooth muscle cells to migrate to the intima side, and the bloodstream hemangioblasts to proliferate as vascular smooth muscle cells to the intima side as a repair mechanism, resulting in neointimal formation Then, it refers to the blockage of blood vessels due to abnormal proliferation of vascular smooth muscle cells. Therefore, in order to prevent or treat this coronary restenosis, the abnormal proliferation of vascular smooth muscle cells is specifically suppressed, and the proliferation of vascular endothelial cells is maintained to preserve the endothelium in the blood vessel. Is required.

The “inhibition of proliferation of vascular smooth muscle cells” in the present invention refers to specifically inhibiting the proliferation of vascular smooth muscle cells by interfering with the action of PDGF involved in the proliferation of vascular smooth muscle cells. As a result, the proliferation of vascular endothelial cells is maintained, and the endothelium in the blood vessel can be preserved.

The “antisense oligonucleotide against PDGF-A chain” of the present invention refers to an oligonucleotide complementary to the base sequence of PDGF-A chain for suppressing the expression of PDGF-A chain.

In order to suppress the expression of PDGF-A chain, it is preferable to include a sequence complementary to the transcription start codon of PDGF-A chain. It is preferable that the oligonucleotide contains a nucleotide sequence that is complementary to the base sequence of the PDGF-A chain. Alternatively, any fragment may be used. In particular, fragments having a length of about 15 to 17 bases are preferable because they are easy to handle. As such an antisense oligonucleotide, for example, the oligonucleotide of SEQ ID NO: 1 in the Sequence Listing can be mentioned.

As these antisense oligonucleotides, for example, those synthesized by a consignment contractor such as Qiagen or Proligo can be used. The base of the oligonucleotide may be either DNA or RNA.

[0013] The "derivative of an antisense oligonucleotide for PDGF-A chain" of the present invention is an oligonucleotide containing a base sequence complementary to the base sequence of PD GF-A chain. In order to enhance stability, it is a chemical modification of one or more base atoms contained in an oligonucleotide. For example, phosphorothioate, morpholino, and the like. Such thiogonucleotide derivatives are resistant to nucleolytic enzymes and are resistant to nucleolytic enzymes in tissues and living organisms with high stability. The dose will be lower as the patient improves.

The antisense oligonucleotide or derivative thereof for the PDGF-A chain of the present invention can be used as it is as long as it suppresses vascular smooth muscle cell proliferation and preserves endothelial cells in blood vessels. It can also be used as a composition together with pharmaceutically acceptable additives such as agents.

[0014] The present inventors have confirmed that the "antisense oligonucleotide for PDGF-A chain or a derivative thereof" of the present invention has an inhibitory action on proliferation of vascular smooth muscle cells in vitro using vascular smooth muscle cells. Confirm with this experiment. (For example, Fukuda N, Kubo A, Watanabe Y, Nakayama T, Soma M, Izumi Y, Kanmatsuse K: Antisense oligodeoxynucleotide com plementary to platelet-derived growth factor A-chain messenger RNA inhibits the ar terial proliferation in spontaneously hypertensive rats without alterating their blood pressure.J Hypertens 15: 1123-1136, 1997.)

[0015] The "vascular endothelial-preserving thread and composition" of the present invention refers to a composition comprising an antisense oligonucleotide for PDGF-A chain or a derivative thereof as an active ingredient. It means a composition to which an additive such as a stabilizer acceptable in the above is added.

As an additive, lipofectin, lipofectamine, polyethylenemine, HVJ-envelope and the like can be used, and it is particularly preferable to use polyethylenemine for stably storing an antisense oligonucleotide or a derivative thereof.

The “vascular endothelial preserving composition” of the present invention can be used for any pharmaceutical composition, pharmaceutical, and medical device as long as it suppresses the proliferation of vascular smooth muscle cells and preserves endothelial cells in the blood vessels. .

For example, when preventing or treating vascular restenosis, depending on the target site, the mode of drug delivery, etc., in addition to using it as an active ingredient of a stent coating agent, an effective pharmaceutical composition such as a catheter or injection It can also be used as a component. [0016] The "stent coating agent" of the present invention is a combination of an endothelium preserving composition containing an antisense oligonucleotide for PDGF-A chain or a derivative thereof as an active ingredient, and a pharmaceutically acceptable carrier. It refers to a drug prepared so that it can be applied to a stent.

Pharmaceutically acceptable carriers can be used in solid or liquid form. For example, as a solid carrier, additives such as calcium phosphate, magnesium stearate, talc, glucose, sucrose, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, carboxymethyl cellulose-sodium and polyvinylpyrrolidone are used. Can do. As the liquid carrier, water, a solution partially containing an additive, alcohols and derivatives thereof, fats and oils, organic solvents, and the like can be used.

[0017] These carriers and the vascular endothelial preserving composition are combined, and further mixed with excipients such as cell mouth, glycerin fatty acid ester, calcium phosphate, etc. as necessary, and compressed into tablets, powders, granules, capsules It may be in the form of chewable, cream, ointment, paste, gel, viscous liquid. The stent coating agent formed in a solid state can be prepared in a liquid form or a cream form for easy application when applied to the stent.

[0018] The "drug-eluting stent" of the present invention means that the drug applied to the stent dissolves into the inner wall of the blood vessel and suppresses abnormal growth of vascular smooth muscle cells, whereby the proliferation of vascular endothelial cells is maintained. A stent that has the effect of preventing coronary restenosis while preserving the inner endothelium.

For the drug-eluting stent of the present invention, it is preferable to use a stent coating agent containing an antisense oligonucleotide for PDGF-A chain or a derivative thereof as an active ingredient, for example, a 3.5 mm × 20 mm stent. In this case, it is preferable to use 10 μg to 100 / ig, preferably 100 μg, of a stent coating agent per stent.

[0019] The stent to which the stent coating agent of the present invention is applied is a stent that is inserted into a blood vessel and held in a place to be treated or prevented, and is capable of eluting the drug with blood vessel expansion. You can also use a good stent. Furthermore, in the case of a metal stent, it is preferable to use a stent capable of carrying a drug-supporting coating covering at least the metal portion.

[0020] As a method of applying the stent coating agent of the present invention to a stent, a Hyde mouth gel coating is used. For example, it can be applied by hide-mouth gel coating.

[0021] "An anti-sense oligonucleotide for PDGF-A chain or a derivative thereof is administered to the affected area" means, for example, that the drug-eluting stent of the present invention is inserted into a site for prevention or treatment of coronary artery restenosis. The stent is placed so that the drug applied to the stent gradually dissolves into the inner wall of the blood vessel and acts locally. In addition to stents, administration to the affected area can be performed by injection, catheter insertion, and injection.

In the administration route of the drug-eluting stent of the present invention, the insertion site can be placed at a site where coronary restenosis can be prevented or treated by force that varies depending on the degree of symptoms of the patient, for example, intravenous administration.

In addition, this drug-eluting stent has an inhibitory effect on abnormal proliferation of vascular smooth muscle cells for 1 to 60 days after insertion, depending on the amount of drug applied.

[0022] Thus, coronary arteries in the treatment of ischemic heart diseases such as angina pectoris and myocardial infarction by using the vascular endothelial preserving composition of the present invention and suppressing the excessive proliferation of vascular smooth muscle cells. With restenosis, the proliferation of vascular endothelial cells is maintained and the endothelium within the blood vessels can be preserved. Furthermore, by using a stent coating agent containing the vascular endothelial preserving composition of the present invention as an active ingredient and using a drug-eluting stent to which this is applied, coronary artery restenosis can be prevented or treated.

Examples of the present invention are shown below, but the present invention is not limited by these. Example 1

[0023] Preparation of Gusal Endothelial Conserving Composition>

1. Preparation of antisense oligonucleotide

Antisense oligodeoxynucleotides (hereinafter referred to as "antisense oligodeoxynucleotides"), which include the complementary base sequence of the start codon from the human and rat PDGF—A chain sequences, and the total length of the front and rear IJs is 15 bases. h Simply ODN) ¾ sS: f "7 ODN was synthesized using Applied Biosy stems DNA synthesizer 394 (Foster) and purified by OPC column (Applied Biosystems). The base sequence of the purified antisense oligonucleotide is shown in SEQ ID NO: 1 in the Sequence Listing. In addition, nonsense oligonucleotides (nons ense oligodeoxynucleotides) were designed and synthesized and purified in the same manner as antisense oligonucleotides. The base sequence of the purified non-sense oligonucleotide is shown in SEQ ID NO: 2 in the Sequence Listing. These base sequences are shown in FIG.

The resulting ODN was modified as a phosphorothioate by oxidation of the phosphate bond of 3H-1,2-benzodithiol-3-one 1,1-dioxide.

2. Creation of vascular endothelial preservation composition

Vascular endothelium-preserving, anomaly was prepared by the following method.

1) An antisense oligonucleotide solution (1 ug / β β) prepared by dissolving the antisense oligonucleotide prepared above in physiological saline is used as a stock solution and diluted with physiological saline to obtain an antisense oligonucleotide solution ( 50 g / 1.25 inl) was prepared and used as solution A.

2) Polyethylenemine solution (Ex Gen500) dissolved in physiological saline is used as a stock solution and diluted with physiological saline to prepare a polyethylenelenine solution (250 1 / 1.25 ml). It was.

3) Solution B was added to solution A, vortexed for 10 seconds, and incubated at room temperature for 10 minutes.

4) The obtained solution was mixed with the matrix solution to obtain a vascular endothelial preservation composition.

Example 2

[0024] <Creation of stent coating agent>

100 _μg of hydrogenore (manufactured by Nichiban) was mixed with 100 _g of the vascular endothelial preservation composition prepared above to prepare a stent coating agent.

Example 3

[0025] <Production of drug eluting stent>

A stent coating agent prepared by the same method as in Examples 1 and 2 was applied to a stent (diameter 3.5 mm, length 20 mm, manufactured by Interventional Radiology) to prepare a drug eluting stent. A stent coating agent prepared using an antisense oligonucleotide solution (20 g / ml) as solution A and a polyethylenelenine solution (100 β 1 / ml) as a liquid solution was used. The stent coating agent was applied to the stent in the same manner as in Example 3. In addition to the stent of the present invention, a stent containing non-sense ODN was prepared as a control, and a physiological stent was applied as a control to a conventional stent. A drug eluting stent was created. The stent coating agent was applied to the stent by the following method. All operations were performed under aseptic conditions.

1) Place the stent in a tube (containing 2.5 ml of antisense + polyethylenemine solution).

2) After completely immersing the stent, the stent was removed from the tube and allowed to air dry.

3) After completely drying the stent, put it in the tube once more.

4) Repeat the above process 6 times.

5) After completely immersing the stent, remove the stent from the tube and air dry.

6) After completely drying the stent, place it in the tube once more.

The above process was repeated 6 times to produce a drug eluting stent.

Example 4

[0026] <Examination of preservation of endothelium in blood vessels using drug-eluting stent>

Example 5

[0027] Study on prevention of coronary restenosis using drug eluting stent>

Nineteen boars (BW: 22-28kg: procedure success = 100%) were divided into three groups: control (n = 6), control (n = 4), and present invention (n == 9). Three types of stents prepared by the described method were transplanted to each group. One group was used as a control group, two groups as control groups, and three groups as the groups of the present invention. In addition, as a comparison with the present invention, two boars were introduced and observed in the same manner as in the sirolimus-coated drug-eluting stent (Cypher ™, Corids, Johnson & Johnson company).

In the stent implantation, each pig was given aspirin (325 mg) one day before implantation and fasted for 1 kg, followed by intramuscular injection of 25 mg / kg pentovanolebital sodium as a premedication, followed by lm _g Anesthesia was performed by intravenous injection of / kg / h ketamine chloride. Henoline was injected intravenously as a 5000 IU bolus.

After endotracheal intubation, controlled mechanical aeration was performed by cyclic aeration of 10-15 ml / kg in a volumetric cycle ventilator (Servo 900-E, Siemems—Elema Inc.). The stent was inserted into the right carotid artery with a 6F sheath.

[0028] [Table 1] Group Number Sample used (drug-eluting stent)

1 6 Conventional saline applied to a stent

2 4 100 g of non-sense stent coating applied to a conventional stent

3 9 \ 0 μg of PDGF-A chain antisense ODN * ² ) (phosphorothioate) containing a stent coating agent applied to a conventional stent

Nonsense OD is an ODN with the same number of bases in antisense ODN and ACGT, but in a completely different order. As a control, the base sequence described in SEQ ID NO: 2 was used.

* Using the nucleotide sequence of ^¾ SEQ ID NO: 1.

[0029] <Confirmation of anti-sense ODN distribution>

Three groups of stents coated with 100 μg of antisense ODN labeled with FITC (fulorescein-isothiocyanate) implanted in the porcine coronary artery showed the distribution of antisense ODN applied to the stent in the coronary artery. Examined. At 24 hours after transplantation, the heart is removed from the pig, and the coronary artery including the stent is removed. After fixation with 4% paraformaldehyde, the fluorescence emission microscope confirms the emission of FITC, and antisense ODN is distributed. The site was examined. FIG. 2 is a view showing a coronary artery including a stent before and after confirming luminescence. When the coronary artery shown in FIG. 2A was confirmed to emit light with a fluorescence microscope, it was confirmed that the antisense ODN labeled with FITC flowed out of the stent and distributed to the endothelial cells of the coronary artery, as shown in FIG. 2B.

<0030> <Detection of in-stent restenosis by angiography and IVUS>

During the above process, blood flow and body surface electrocardiogram (ECG) were monitored. After standard coronary angiography, mechanical intravascular ultrasound aVUS: registered trademark Atlantis 40 MHz, Boston Scientific Corporation) was performed in the anterior descending (LAD) artery. The IVUS catheter was automatically retracted 0.5 mm every 2 seconds and IVUS images were continuously recorded on SV HS video for analysis. In order to demonstrate the implantation of the stent, a fragment with an IVUS image power diameter of approximately 3.0 mm was selected. A 3.5 mm x 20 mm coated stent was used with a stent-artery ratio of 1.3: 1 with an inflation pressure of 10-12 atm. After stent implantation, IVUS was performed immediately to confirm the stent artery ratio. If the ratio was not reached, the stent balloon was inflated again with high pressure at the site where the stent was inserted.

In addition, coronary angiography and IVUS were performed 28 days later, and the coronary artery where the stent was placed was removed for pathological analysis.

[0031] Results The stent / artery ratio in the initial procedure was 1.3 ± 0.1, 1.3 ± 0.1, and 1.4 ± 0.1 in groups 1, 2, and 3, respectively. These were powerful with no difference between the three groups. FIG. 3 shows the presence or absence of lumens in groups 1 and 3. The arrows in Fig. 3 indicate the stent, and * indicates the neointima. As shown in FIGS. 3C and D, in group 1, the neointima due to occlusion crammed the lumen and surrounded the ultrasound catheter. On the other hand, as shown in FIGS. 3A and 3B, the three groups had very clear lumens.

The volume ratios of restenosis in coronary stents were 63.7 ± 11.9, 4 4.4 ± 3.4, and 25.5 ± 3 _: 8% in groups 1, 2, and 3, respectively. As shown in Fig. 4, the volume ratio of restenosis in the stent was significantly lower in group 3 compared to groups 1 and 2.

[0032] IVUS Measurement and Pathological Analysis>

The IVUS image recorded on the SVHS tape was analyzed by a computer-based contour detection program (registered trademark, NetralVUS software package for Windows NT, Sclmage Corporation. USA) for three-dimensional reconstruction and volume measurement. The cross grid recorded in the IVUS image was used as a scale. The interface between the neointima and lumen was manually tracked, and outside the outer elastic membrane boundary. As a standard for manual tracking, the measurement parameters were determined by computer software as follows.

LV: Lumen volume, W: Vessel volume.

The volume ratio of the restenosis portion in the stent was determined by (W—LV) / W.

[0033] For pathological analysis, one fragment was cut from each of the central, distal, and proximal portions along each stent. Each fragment was about 50 m thin and stained with hematoxin monoeosin. A computer-based image system (ImageJ 1.30) was used for the pathological analysis of the following parameters.

IEL: internal elastic lamina

The lumen area was defined as the area limited by the neointima-luminal border, and the neointima area was defined as the area between the lumen door IELs.

Restenosis in the stent was evaluated as neointima region / IEL region.

Immunohistochemical labeling of vascular endothelial cells was pre-treated with 0.25% pepsin—HC1 solution and 1: 250 Usagi anti-vWf ^ rectal antibody (DAKO Diagnostika, Hamburg, Germany) It went by. The value obtained by the analysis was expressed as mean soil SE, and the mean value of the variables was compared by a twoside d unpaired t test. A value of 0.05 was considered significant.

[0034] Results The results of IVUS are shown in FIG. As shown in Figs. 5A to 5C, in each of the 1 to 3 groups, there was no stenosis in the coronary artery where the stent was placed, and the lumen surface was slippery. Met.

In addition, as shown in Fig. 5C, it was observed that the metal surface of the stent coated with 3 groups of antisense ODN was embedded in the neointima. It was not observed in the other stents, and it was confirmed that neointima formation was reduced by antisense ODN. On the other hand, as shown in Fig. 5D, when using a sirolimus-coated drug-eluting stent, exposure of the metal surface of the stent was observed in the lumen, and several red thrombi were observed on the surface of the lumen. It was shown to be attached.

Fig. 6 shows the pathological findings of the coronary artery in which groups 2 or 3 were placed. Compared to using two groups of non-sense ODN-coated stents as shown in Fig. 6A, the three-group anti-sense ODN-coated stents were used in the middle, as shown in Fig. 6B. The formation of restenosis was suppressed even if the tip or the proximal part was shifted or distorted.

FIG. 7 shows the area ratio of the restenosis portion in the stent in the central and distal portions and the proximal portion of the stent in the groups 1-3. It was confirmed that group 3 reduced restenosis in the stent at all of proximal, central and distal compared to group 1. Therefore, it was shown that restenosis is prevented by using a stent coated with phosphorotype anti-sense ODN.

In addition, Fig. 8 shows the results of hematoxin staining of the site where the stents coated with antisense QDN of 3 groups or sirolimus-coated drug eluting stents were placed. As shown in FIG. 8A, it was observed that the endothelial cells were completely and continuously aligned when using three groups of stents. On the other hand, as shown in FIG. 8B, when the sirolimus-coated drug-eluting stent was used, suspended endothelial cells were observed along the surface of the coronary artery. These results suggested that the antisense ODN of the present invention does not interfere with the endothelialization process.

Industrial applicability

[0035] The vascular endothelial preserving composition of the present invention specifically suppresses the proliferation of vascular smooth muscle cells, The proliferation of vascular endothelial cells is maintained and the endothelium within the blood vessels can be preserved. Furthermore, by using a stent coating agent containing the vascular endothelial preserving composition of the present invention as an active ingredient and a drug-eluting stent, coronary arteries in angina or myocardial infarction due to preservation of the endothelium in the blood vessel is used. It is possible to prevent or treat restenosis and to restore the underlying disease.

The drug-eluting stent containing this vascular endothelial preserving composition does not require the use of ticlovidin hydrochloride or the like, and therefore eliminates the risk of side effects caused by the combined drug.

Claims

The scope of the claims

[1] A composition for preserving vascular endothelial, comprising an antisense oligonucleotide for platelet-derived growth factor-A chain or its derivative as an active ingredient.

[2] The composition according to claim 1, which further has an inhibitory effect on proliferation of vascular smooth muscle cells.

[3] The composition according to claim 1 or 2, wherein the derivative is chemically modified to a phosphorotype.

[4] A stent coating agent comprising a composition for preserving vascular endothelial, comprising an antisense oligonucleotide for platelet-derived growth factor-A chain or a derivative thereof as an active ingredient.

[5] A drug-eluting stent coated with a vascular endothelial preserving composition containing an antisense oligonucleotide for platelet-derived growth factor-A chain or its derivative as an active ingredient, or a stent coating agent containing the same.

6. The drug-eluting stent according to claim 5, wherein the vascular endothelial preserving composition or the stent coating agent containing the composition is coated with a hyde mouth gel.

[7] A method for preventing or treating coronary restenosis, comprising preserving the endothelium in a blood vessel, comprising administering an antisense oligonucleotide for platelet-derived growth factor-A chain or a derivative thereof to an affected area.

8. The method for preventing or treating coronary restenosis according to claim 7, wherein the coronary restenosis is after stent placement.

[9] A method for preventing or treating coronary restenosis, wherein administration is performed by a stent coated with an antisense oligonucleotide or derivative thereof for platelet-derived growth factor-A chain.