Abstract:
The present invention provides a prophylactic, ameliorative or therapeutic medicament for vascular restenosis, ischemic disease, allergic disease, inflammatory disease, autoimmune disease, or cancer metastasis, invasion (cancer metastasis/invasion) or cachexia based on the inhibitory action on a plural of transcriptional regulatory factor. A chimera (double) decoy of the present invention has plural transcriptional regulatory factor binding sequences in a single molecule thereof. Thus, it is able to inhibit the activity of plural transcriptional regulatory factors with a single molecule. For example, stenosis of an anastomosed site of an artificial blood vessel is caused by thickening of the vascular intima, and this is mainly caused by activation of cell proliferation by an inflammatory reaction occurring at the anastomosed site. Thus, thickening of vessel walls can be inhibited by simultaneously inhibiting two transcriptional regulatory factors involved in inflammation and cell proliferation by using the chimera decoy of the present invention.

Description:
TECHNICAL FIELD  
       [0001]     The present invention relates to a decoy comprising an oligonucleotide having a plural sequence bound by transcriptional regulatory factors, and a usage method thereof. More particularly, the present invention relates to a chimera (double) decoy oligonucleotide and a usage method thereof.  
       BACKGROUND ART  
       [0002]     Artificial blood vessels are frequently used in surgical vascular reconstruction for treatment of diseases such as arteriosclerosis obliterans, aortic aneurysm and aortic dissection. In addition, balloon catheters and stents are frequently used to dilate occluded blood vessels. However, vessels frequently become reoccluded following surgery using these devices, thereby resulting in the problem of having to repeat the same procedure.  
         [0003]     For example, although surgical vascular reconstruction is used to avoid amputation of a lower limb for patients with arteriosclerosis obliterans, bypass surgery using an artificial blood vessel is the first choice of treatment for lesions above the knee. However, occlusion of the artificial vessel may occur later than one month after surgery. This occlusion is mainly caused by thickening of the vascular intima at the anastomosed site. Inhibiting this thickening would make it possible to conserve grafts for a long period of time as well as to use artificial blood vessels for vascular reconstruction at locations below the knee.  
         [0004]     The major cause of thickening of vascular intima at anastomosed sites of artificial blood vessels is the activation of cell proliferation by an inflammatory reaction occurring at the anastornosed site. Thus, inhibition of this cell proliferation is thought to be able to inhibit thickening.  
         [0005]     Gene therapy techniques have been developed in recent years for the purpose of inhibiting inflammatory reactions.  
         [0006]     Research has been conducted which is expected to demonstrate anti-inflanmatory effects by inhibiting the function of NF-κB, a transcriptional regulatory factor involved in inflammatory reactions, with a decoy nucleic acid.  
         [0007]     International Patent Publication No. (WO-A1) 96/035430 discloses that an NF-κB decoy nucleic acid can be a therapeutic agent for inflammatory diseases.  
         [0008]     In addition, gene therapy techniques for controlling cell proliferation have also been developed. For example, the proliferation of vascular media can be inhibited by inhibiting the function of E2F, a transcriptional regulatory factor that plays an important role in cell proliferation, with a decoy nucleic acid.  
         [0009]     JP-B 3392143 discloses an anticancer agent with the use of an E2F decoy nucleic acid.  
         [0010]     WO 95/11687 discloses that cell proliferation can be inhibited by using an E2F decoy nucleic acid.  
         [0011]     Previously disclosed examples of oligonucleotides having two different functions within a single molecule are indicated below.  
         [0012]     JP-A(W) 2002-515514 discloses a method for using an ICAM-1 antisense as an external skin medication, and although it is stated that it may have a DNA: RNA or RNA: DNA chimeric structure, there are no specific examples of chimera decoys indicated, including cited references. In addition, this publication discloses an invention relating to transdermal delivery of an antisense strand.  
         [0013]     US-A2003-176376discloses that a hybrid molecule of a bcl-2 antisense and CRE decoy has the potential for treating diseases caused by abnormal cell growth such as cancer.  
         [0014]     JP-A 2002-193813 indicates that a chimera decoy of NF-κB and Ets inhibits increase of aorta surface area in an aortic aneurysm model.  
       DISCLOSURE OF THE INVENTION  
       [0015]     It is necessary to simultaneously inhibit inflammation and cell proliferation in order to more effectively prevent vascular restenosis. Thus, there is a need to develop a medicament inhibiting both of them in the proper balance, while also having superior pharmacological effects and safety.  
         [0016]     JP-A(W) 2002-515514 does not describe any specific therapeutic effects, and the potential for application to various diseases is completely unknown.  
         [0017]     US-A 2003-176376 does not mention the usefulness for diseases other than those listed above.  
         [0018]     In the case of JP-A 2002-193813, usefulness for diseases other than those listed above is unknown.  
         [0019]     An object of the present invention is to provide a medicament that has a decoy nucleic acid as an active ingredient and simultaneously inhibits inflammation and cell proliferation.  
         [0020]     The inventors of the present invention thought that, since thickening of vascular intima at anastomosed sites is caused by invasion of inflammatory cells and proliferation and migration of smooth muscle cells due to inflammation of vessel walls, the development of a medicament capable of simultaneously controlling these with a single molecule would be effective.  
         [0021]     As a result of extensive studies, the inventors of the present invention designed a molecule capable of simultaneously inhibiting, for example, inflammatory action controlled by NF-κB and cell proliferation action controlled by E2F. Namely, the inventors of the present invention found that a decoy having both the DNA binding sequences of NF-κB and E2F in a single molecule (chimera ((double) decoy) more effectively inhibits both transcriptional regulatory factors. A decoy refers to a double-strand oligonucleotide having a nucleic acid sequence bound by a transcriptional regulatory factor, and thereby acts as a decoy by competing with other nucleic acid sequences bound by said transcriptional regulatory factor. Although a decoy maybe DNA or RNA, DNA is preferable. In addition, in the present description, decoy, decoy oligonucleotide and decoy ODN are used synonymously.  
         [0022]     The gist of the present invention relates to: 
    (1) achimera (double) decoy containing a plural transcriptional regulatory factor binding sequences in a single molecule;     (2) the decoy described in (1), wherein the chimera decoy is comprised of a DNA or RNA oligonucleotide;     (3) the decoy described in (1) or (2), wherein the chimera decoy is comprised of a DNA oligonucleotide;     (4) the decoy described in (1) to (3), wherein the chimera decoy has transcriptional regulatory factor inhibitory action;     (5) the decoy described in (1) to (4), wherein at least one transcriptional regulatory factor binding sequence of the chimera decoy is a sequence that binds NF-κB, E2F, GATA-3, STAT-1, STAT-6, Ets or AP-1;     (6) the decoy described in any of (1) to (5), wherein an NF-κB binding sequence in the chimera decoy is GGGRHTYYHC (wherein, R represents A or G, Y represents C or T and H represents A, C or T);     (7) the decoy described in (6), wherein an NF-κB binding sequence in the chimera decoy is GGGATTTCCC or GGGACTTTCC;     (8) the decoy described in any of (1) to (5), wherein an E2F binding sequence in the chimera decoy is TTTSSCGS (wherein, S represents G or C);     (9) the decoy described in (8), wherein an E2F binding sequence in the chimera decoy is TTTCCCGC;     (10) the decoy described in any of (1) to (5), wherein a GATA-3 binding sequence in the chimera decoy is WGATAR (wherein, W represents A or T, and R represents A or G);     (11) the decoy described in (10), wherein a GATA-3 binding sequence in the chimera decoy is AGATAG;     (12) the decoy described in any of (1) to (5), wherein an STAT-1 binding sequence in the chimera decoy is TTCNNNGAA (wherein, N represents A, G, T or C);     (13) the decoy described in (12), wherein an STAT-1 binding sequence in the chimera decoy is TTCCGGGAA;     (14) the decoy described in any of (1) to (5), wherein an STAT-6 binding sequence in the chimera decoy is TTCNNNNGAA (wherein, N represents A, G, T or C);     (15) the decoy described in (14), wherein an STAT-6 binding sequence in the chimera decoy is TTCCCAAGAA;     (16) the decoy described in any of (1) to (5), wherein an Ets binding sequence in the chimera decoy is MGGAW (wherein, M represents A or C, and W represents A or T);     (17) the decoy described in (16), wherein an Ets binding sequence in the chimera decoy is CGGAA;     (18) the decoy described in any of (1) to (5), wherein an AP-1 binding sequence in the chimera decoy is TGASTMA (wherein, S represents G or C, and M represents A or C);     (19) the decoy described in (18), wherein an AP-1 binding sequence in the chimera decoy is TGAGTCA;     (20) the decoy described in any of (1) to (19), wherein the chimera decoy contains two transcriptional regulatory factor binding sequences in a single molecule thereof;     (21) the decoy described in any of (1) to (20), wherein the chimera decoy is comprised of an oligonucleotide indicated in the following formula:     5′-N(m)-Consensus 1-N(s)-Consensus 2-N(n)-3′ (wherein, N(m) represents a 5′-terminal flanking sequence, N(s) represents a spacer, and N(n) represents a 3′-terminal flanking sequence; Consensus 1 and Consensus 2 represent transcriptional regulatory factor binding sequences; m, s and n respectively and independently represent 0 or an integer of 1 to 20; and N represents a nucleotide A, G, T or C);     (22) the decoy described in any of (1) to (21), wherein Consensus 1 and/or Consensus 2 are sequences selected from the group consisting of GGGRHTYYHC (wherein, R represents A or G, Y represents C or T, and H represents A, C or T), TTTSSCGS (wherein, S represents G or C), WGATAR (wherein, W represents A or T, and R represents A or G), TTCNNNGAA (wherein, N represents A, G, T or C), TTCNNNNGAA (wherein, N represents A, G, T or C), MGGAW (wherein, M represents A or C, and W represents A or T) and TGASTMA (wherein, S represents G or C, and M represents A or C);     (23) the decoy described in any of (1) to (22), wherein Consensus 1 and/or Consensus 2 are sequences selected from the group consisting of GGGATTTCCC, GGGACTTTCC, TTTCCCGC, AGATAG, TTCCGGGAA, TTCCCAAGAA, CGGAA and TGAGTCA;     (24) the decoy described in any of (21) to (23), wherein (m) is 0 or an integer of 1 to 20 in the formula of (21);     (25) the decoy described in any of (21) to (24), wherein (m) is 0 or an integer of 1 to 10 in the formula of (21);     (26) the decoy described in any of (21) to (25), wherein (m) is 0 or an integer of 1 to 5 in the formula of (21);     (27) the decoy described in any of (21) to (26), wherein (s) is 0 or an integer of 1 to 20 in the formula of (21);     (28) the decoy described in any of (21) to (27), wherein (s) is 0 or an integer of 1 to 10 in the formula of (21);     (29) the decoy described in any of (21) to (28), wherein (s) is 0 or an integer of 1 to 5 in the formula of (21);     (30) the decoy described in any of (21) to (29), wherein (n) is 0 or an integer of 1 to 20 in the formula of (21);     (31) the decoy described in any of (21) to (30), wherein (n) is 0 or an integer of 1 to 10 in the formula of (21);     (32) the decoy described in any of (21) to (31), wherein (n) is 0 or an integer of 1 to 5 in the formula of (21);     (33) the decoy described in any of (1) to (32), wherein the chimera decoy is a chimera decoy of NF-κB and another transcriptional regulatory factor;     (34) the decoy described in any of (1) to (33), wherein the chimera decoy is a chimera decoy of NF-κB and E2F;     (35) the decoy described in any of (1) to (34), wherein the chimera decoy is the chimera decoy represented by Sequence No. 1;     (36) the decoy described in any of (1) to (35), wherein the chimera decoy is composed of double-strand DNA;     (37) a pharmaceutical composition containing the decoy described in any of (1) to (36);     (38) a prophylactic, ameliorative or therapeutic medicament for an ischemic disease, allergic disease, inflammatory disease, autoimmune disease, or cancer metastasis, invasion (cancer metastasis/invasion) or cachexia, comprising the decoy described in any of (1) to (37);     (39) a prophylactic, ameliorative or therapeutic medicament for vascular restenosis, acute coronary syndrome, cerebral ischemia, myocardial infarction, reperfusion injury of ischemic diseases, atopic dermatitis, psoriasis vulgaris, contact dermatitis, kelloids, bedsores, ulcerative colitis, Crohn&#39;s disease, nephropathia, glomerular sclerosis, albuminuria, nephritis, renal insufficiency, chronic rheumatoid arthritis, osteoarthritis, asthma or chronic obstructive pulmonary disease (COPD), comprising the decoy described in any of (1) to (37);     (40) a prophylactic, ameliorative or therapeutic medicament for vascular restenosis following percutaneous transluminal coronary angioplasty (PTCA), percutaneous transluminal angioplasty (PTA), bypass surgery, organ transplant or organ surgery, comprising the decoy described in any of (1) to (37);     (41) the prophylactic, ameliorative or therapeutic medicament comprising the decoy described in any of (1) to (37), wherein the vascular restenosis is restenosis caused by the use of an artificial blood vessel, catheter or stentor by vein transplant;     (42) the prophylactic, ameliorative or therapeutic medicament comprising the decoy described in any of (1) to (37), wherein the vascular restenosis is caused by surgical treatment for arteriosclerosis obliterans, aneurysm, aortic dissection, acute coronary syndrome, cerebral ischemia, marfan syndrome or plaque rupture;     (43) use of the decoy described in any of (1) to (37) for producing a prophylactic, ameliorative or therapeutic medicament for an ischemic disease, allergic disease, inflammatory disease, autoimmune disease, or cancer metastasis, invasion (cancer metastasis/invasion) or cachexia; and     (44) a method for prophylaxis, amelioration or treatment for an ischemic disease, allergic disease, inflammatory disease, autoimmune disease, or cancer metastasis, invasion (cancer metastasis/invasion) or cachexia, which comprises using the decoy according to any of claims  1  to 37.    
 
         [0068]     A chimera decoy of the present invention is a nucleic acid-based medicament that simultaneously inhibits a plural transcriptional regulatory factors as a single molecule. This chimera decoy can be used for, for example, an ischemic disease, allergic disease, inflammatory disease, autoimmune disease, or cancer metastasis, invasion (cancer metastasis/invasion) or cachexia. More preferably, this chimera decoy can be used for restenosis following percutaneous transluminal coronary angioplasty (PTCA), percutaneous transluminal angioplasty (PTA) or bypass surgery used for the surgical treatment of arteriosclerosis obliterans, aneurysm, aortic dissection, marfan syndrome or plaque rupture.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0069]     The present invention provides a decoy nucleic acid (chimera (double) decoy) having plural transcriptional regulatory factor binding sequences as a single molecule thereof, and a pharmaceutical composition having this decoy nucleic acid as an active ingredient thereof.  
         [0070]     In the present description, a chimera decoy refers to a decoy having two or more transcriptional regulatory factor binding sequences, while a double decoy refers to a decoy having two transcriptional regulatory factor binding sequences. Thus, a double decoy is included in the concept of a chimera decoy.  
         [0071]     Although examples of decoys of the present invention include decoys of NF-κB, STAT-1, GATA-3, STAT-6, AP-1, Ets and E2F, a chimera decoy combining, for example, NF-κB and E2F is preferable. A combined chimera decoy can be produced by selecting the NF-κB binding sequence from the consensus sequence GGGRHTYYHC (R (A, G); Y(C,T);H(A C,T)) (Sequence No. 2), and selecting the E2F binding sequence from the consensus sequence TTTSSCGS (S (G,C)) (Sequence No. 3). For example, preferable examples of a chimera decoy include, but are not limited to, GAAGGGATTTCCCTCCATTTCCCGCGGA (Sequence No. 1) (chimera decoy of NF-κB and E2F) having for core sequences thereof GGGATTTCCC (Sequence No. 9) of NF-κB and TTTCCCGC (Sequence No. 11) of E2F.  
         [0072]     In addition, sequences selected from the GATA-3 consensus sequence WGATAR (W(A,T); R(A,G)) (Sequence No. 4), the STAT-1 consensus sequence TTCNNNGAA (N(A,G,T,C)) (Sequence No.5), the STAT-6 consensus sequence TTCNNNNGAA (N (A, G, T, C)) (Sequence No. 6), the Ets consensus sequence MGGAW (M (A, C); W (A, T) ) (Sequence No.7), and the AP-1 consensus sequence TGASTMA (S(G,C); M(A,C)) (Sequence No. 8) can also be used in a suitable combination thereof. Examples of such combinations include, but are not limited to, suitable combinations of NF-κB binding sequence GGGACTTTCC (Sequence No. 10), GATA-3 binding sequence AGATAG (Sequence No. 12), STAT-1 binding sequence TTCCGGGAA (Sequence No.13), STAT-6 binding sequence TTCCCAAGAA (Sequence No. 14), Ets binding sequence CGGAA (Sequence No. 15) and AP-1 binding sequence TGAGTCA (Sequence No. 16).  
         [0073]     A chimera decoy as claimed in the present invention can be defined with the following formula: 
 
5′-N(m)-Consensus 1-N(s)-Consensus 2-N(n)-3′
 
 (wherein, N(m) represents a 5′-terminal flanking sequence, N(s) represents a spacer, and N(n) represents a 3′-terminal flanking sequence; Consensus 1 and Consensus 2 represent transcriptional regulatory factor binding sequences; m, s and n respectively and independently represent 0 or an integer of 1 to 20; and N represents a nucleotide A, G, T or C). 
 
         [0074]     With respect to the 5′-terminal flanking sequence of the formula, although (m) is normally 0 or an integer of 1 to 20, (m) is preferably 0 or an integer of 1 to 10, and more preferably 0 or an integer of 1 to 5. In addition, although (s) of the spacer in the formula is normally 0 or an integer of 1 to 20, (s) is preferably 0 or an integer of 1 to 10, and more preferably 0 or an integer of 1 to 5. With respect to the 3′-terminal flanking sequence in the formula, although (n) is normally 0 or an integer of 1 to 20, (n) is preferably 0 or an integer of 1 to 10, and more preferably 0 or an integer of 1 to 5.  
         [0075]     Although a DNA or RNA oligonucleotide is normally used for a chimera decoy of the present invention, double-strand DNA is preferable. In addition, oligonucleotides containing a complement thereto, mutants thereof, or compounds containing these in a molecule thereof can also be used. In addition, modified nucleic acids and/or pseudo nucleic acids may also be contained in these oligonucleotides. Examples of these nucleic acid-based medicaments include double-strand oligonucleotides or mutants thereof containing two or more of the above-mentioned nucleic acid sequences.  
         [0076]     Furthermore, a chimera decoy as claimed in the present invention can be produced according to ordinary methods such as the use of a DNA synthesizer.  
         [0077]     In addition, although varying according to age, body weight, symptoms, therapeutic effects, administration method and soon, the dose of a chimera decoy as claimed in the present invention is administered by introducing a decoy solution having a concentration of normally 0.1 to 1000 μmol/L, preferably 1 to 100 μmol/L and more preferably 10 to 80 μmol/L to a treatment site by, for example, cannulation. A chimera decoy as claimed in the present invention can be administered normally at 0.1 to 10,000 nmol, preferably 1 to 1,000 nmol and more preferably 10 to 100 nmol, as the daily adult dose using these solutions. A decoy nucleic acid solution is normally introduced at a pressure of 25 to 250 mmHg, preferably 50 to 200 mmHg, and more preferably 100 to 175 mmHg.  
       INDUSTRIAL APPLICABILITY  
       [0078]     The present invention provides a prophylactic, ameliorative or therapeutic medicament for vascular restenosis, ischemic disease, allergic disease, inflammatory disease, cancer metastasis, invasion (cancer metastasis/invasion) or cachexia based on action that inhibits plural transcriptional regulatory factors. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0079]      FIG. 1  is a graph showing the results of a vascular smooth muscle cell proliferation assay (wherein, “-PDGF” indicates normal (not stimulated with PDGF), hereinafter the same).  
         [0080]      FIG. 2  is a graph showing the results of a vascular epithelial cell proliferation assay.  
         [0081]      FIG. 3  shows micrographs of cells following a vascular smooth muscle cell migration assay (magnification: ×400).  
         [0082]      FIG. 4  is a graph showing the results of a vascular smooth muscle cell migration assay.  
         [0083]      FIG. 5  is a graph comparing introduction rates of a decoy ODN to rabbit vascular wall.  
         [0084]      FIG. 6  shows micrographs of distal anastomosed sites of rabbit blood vessels having undergone bypass surgery (magnification: ×100). The upward direction in the micrographs indicates the vascular lumen side, the anastomosis sites of artificial blood vessel are indicated by the arrows, and it is observed that chimera decoy inhibited thickening of the anastomosed site more than the control and the scrambled decoy.  
         [0085]      FIG. 7  are graphs showing the thicknesses of the intima and media of distal anastomosed sites of rabbit blood vessels having undergone bypass surgery.  
         [0086]      FIG. 8  is a graph showing a ratio of the thicknesses of the intima and media of anastomosed sites of rabbit blood vessels having undergone bypass surgery.  
         [0087]      FIG. 9  shows micrographs of proximal anastomosed sites of rabbit blood vessels having undergone bypass surgery (magnification: ×100) The upward direction in the micrographs indicates the vascular lumen side, the anastomosis sites of artificial blood vessel are indicated by the arrows, and it is observed that chimera decoy inhibited thickening of the anastomosed site more than the control and the scrambled decoy.  
         [0088]      FIG. 10  are graphs showing the thicknesses of intima and media of proximal anastomosed sites of rabbit blood vessels having undergone bypass surgery.  
         [0089]      FIG. 11  is a graph showing the ratio of intima to media of proximal anastomosed sites of rabbit blood vessels having undergone bypass surgery.  
         [0090]      FIG. 12  shows micrographs of immunohistochemical staining of macrophages of graft anastomosed sites excised from a rabbit bypass model (magnification: ×400) and a graph showing the proportions of macrophages among all cells.  
         [0091]      FIG. 13  shows micrographs of immunohistochemical staining of proliferating cell nuclear antigen (PCNA) -positive cells of graft anastomosed sites excised from a rabbit bypass model (magnification: ×400) and a graph showing the proportions of PCNA-positive cells among all cells. 
     
    
     EXAMPLES  
       [0092]     Although the following indicates examples of the present invention to provide a detailed explanation thereof, the present invention is not limited to these examples.  
         [0000]     1. Vascular Smooth Muscle Cell Proliferation Assay  
         [0093]     Vascular smooth muscle cells having undergone 5 to 6 rounds of subculturing (VSMC, Sanko Junyaku, cryo AOSMC, Cat. No. CC-2571) were seeded onto a  96 -well plate at 5×103 cells/well followed by culturing for 48 hours in serum-free medium. After introducing decoy ODN (20 nM or 600 nM) into the cells using Oligofectamine (Oligofectamine Reagent, Invitrogen, Cat. No. 12252-011), the cells were additionally cultured for 24 hours in serum-free medium. The sequence of the decoy ODN are shown below.  
                                   Chimera decoy   5′-GAAGGGATTTCC   (Sequence No. 1)               CTCCATTTCCCGCGG           A-3′           3′-CTTCCCTAAAGG           GAGGTAAAGGGCGCC           T-5′               Scrambled decoy   5′-CGTACCTGACTT   (Sequence No. 17)           AGCCATTTCGAGCGG           A-3′           3′-GCATGGACTGAA           TCGGTAAAGCTCGCC           T-5′               NF-κB decoy   5′-CCTTGAAGGGAT   (Sequence No. 18)           TTCCCTCC-3′           3′-GGAACTTCCCTA           AAGGGAGG-5′               E2F decoy   5′-CTAGATTTCCCG   (Sequence No. 19)           CG-3′           3′-TAAAGGGCGCCT   (Sequence No. 20)           AG-5′          
 
         [0094]     After stimulating for 24 hours with platelet-derived growth factor (PGDF)-BB (10 ng/ml) (PeproTechECLtd., Cat. No. 100-14B), the medium was replaced with serum-free medium and 24 hours later, the number of cells were counted using a WST-1 cell counting kit (Cell Counting Kit, Dojindo Laboratories).  
         [0095]     Results: The chimera decoy significantly inhibited vascular smooth muscle cell proliferation in comparison with the scrambled decoy (see Table 1,  FIG. 1 ).  
                                           TABLE 1                           (Abs450)                20 nM ODN   600 nM ODN                        Normal (not stimulated with PDGF)   0.21 ± 0.008           Control (stimulated with PDGF)   0.38 ± 0.018       Scrambled decoy   0.35 ± 0.007       Chimera decoy   0.31 ± 0.015   0.208 ± 0.013       NF-kB decoy   0.35 ± 0.011   0.253 ± 0.009       E2F decoy   0.33 ± 0.011   0.233 ± 0.009                  
 
 2. Vascular Endothelial Cell Proliferation Assay 
 
         [0096]     Vascular endothelial cells having undergone 5 to 6 rounds of subculturing (EC, Sanko Junyaku, cryo HAEC, Cat. No. CC-2535) were seeded onto a 96-well plate at 1×10 4  cells/well followed by culturing for 48 hours in 0.5% serum medium. After introducing decoy ODN (600 nM) into the cells using Oligofectamine, the cells were additionally cultured for 24 hours in 0.5% serum medium. After stimulating for 24 hours with 5% serum medium, the medium was replaced with0.5% serum medium and 24 hours later, the number of cells were counted using a WST-1 cell counting kit.  
         [0097]     Results: None of the ODN, including the chimera decoy, inhibited proliferation of vascular end othelial cells (see Table 2,  FIG. 2 ).  
                                       TABLE 2                           (Abs450)                600 nM ODN                            Normal (not stimulated with PDGF)   0.28 ± 0.006           Control (stimulated with PDGF)   0.51 ± 0.016           Scrambled decoy   0.55 ± 0.022           Chimera decoy   0.54 ± 0.008           NF-kB decoy   0.54 ± 0.018           E2F decoy   0.51 ± 0.017                      
 
 3. Vascular Smooth Muscle Cell Migration Assay 
 
         [0098]     VSMC having undergone 5 to 6 rounds of subculturing were seeded onto a 6-well plate to 50% confluence followed by culturing for 48 hours in serum-free medium. After introducing decoy ODN (600 nM) into the cells using Oligofectamine, the cells were additionally cultured for 24 hours in serum-free medium. 2.5×10 4  of the VSMC were then seeded on the upper chamber of a 24-well Matrigel Invasion Chamber, and PDGF-BB (50 ng/ml) were added to the lower chamber to stimulate the cells for 48 hours. Subsequently, the number of migrating cells were then stained with Diff Quik stain and counted.  
         [0099]     Results: The chimera decoy, the NF-κB decoy and the E2F decoy significantly inhibited cell migration as compared with the scrambled decoy, and the chimera decoy demonstrated the most potent activity (see Table  3 ,  FIGS. 3 and 4 ).  
                                       TABLE 3                           (No. of cells/Field)                600 nM ODN                            Normal (not stimulated with PDGF)     8 ± 1.1           Scrambled decoy   38.4 ± 6.9           Chimera decoy   12.8 ± 5.0           NF-κB decoy   21.8 ± 5.5           E2F decoy   15.8 ± 2.7                      
 
 4. Rabbit Bypass Model Experiment 
 
         [0100]     The most suitable pressure for introducing decoy ODN into the artery wall was determined. FITC-ODN (40 μmol/L) was introduced at an arbitrary pressure into an artery anastomosed site. Measurement of the introduction efficiency clearly demostrated the most suitable pressure to be 150 mmHg (see Table 4,  FIG. 5 ).  
                                         TABLE 4                                   Pressure (mmHg)   Introduction efficiency (%)                                        50   39.6 ± 3.8           100   34.1 ± 2.8           150   60.0 ± 4.7           200   51.8 ± 3.9                      
 
         [0101]     After ablating the common carotid artery of hyperlipemic domestic rabbits and cannulating the artery, decoy ODN (40 μmol/L) introduced into the artery wall at a pressure of 150 mmHg. A 2 mm expanded polytetrafluoroethylene (ePTFE) graft (2 cm) was replaced at the introduction site to create a bypass model. Four weeks later, thickening of the intima at the anastomosed site was measured and evaluated by Elastica Van Gieson (EVG) staining.  
         [0102]     Results: The chimera decoy significantly inhibited thickening of the intima and media at the anastomosed site as compared with the scrambled decoy. The chimera decoy demonstrated similar inhibitory activity regardless of the whether the anastomosed site was distal (location far from the body center) or proximal (location close to the body center) (see Tables 5 and 6, FIGS.  6  to  11 ).  
                                                   TABLE 5                           Distal Anastomosed Site (μm)                Intima   Media   Intima/Media ratio                        Control   109.03 ± 11.87   130.38 ± 10.09   0.929 ± 0.125       Scrambled decoy   129.49 ± 15.51   161.79 ± 12.69   0.822 ± 0.081       Chimera decoy   52.63 ± 8.57   112.58 ± 5.10    0.447 ± 0.069                  
 
         [0103]                                                        TABLE 6                           Proximal Anastomosed Site (μm)                Intima   Media   Intima/Media ratio                        Control   152.41 ± 10.06   136.66 ± 7.42   1.17 ± 0.17       Scrambled   170.92 ± 17.64    146.26 ± 10.53   1.21 ± 0.12       decoy       Chimera decoy    89.50 ± 10.06   128.26 ± 7.49   0.73 ± 0.09                    
 5. Macrophage and Proliferating Cell Nuclear Antigen (PCNA) Staining 
 
         [0104]     The proportions of macrophages and PCNA among cells at the graft anastomosed site excised from the rabbit bypass model used in 4 above were investigated. Immunohistochemical staining was carried out using peroxidase and an avidin-biotin complex system.  
         [0105]     The paraffin from paraffin sections having a thickness of 5 μm was removed followed by rehydration. Endogenous peroxidase was blocked by using 3% hydrogen peroxide. Blocking was carried out for 30 minutes using phosphate-buffered saline (PBS) containing 5% horse serum. Primary antibody diluted by 1:50 was added to the sections followed by incubating overnight at 4° C. RAM 11  antibody (Dako, USA) was used to detect macrophages, while PCNA antibody (Clone: PC10) (Dako, USA) was used to detect PCNA. PBS containing biotinated anti-mouse IgG (Vector Laboratories, Burlingame, Calif., USA) was added to the sections followed by incubation for 30 minutes while being suitably washed with PBS. Then, PBS containing avidin-biotinated horseradish peroxidase was added followed by further incubation for 30 minutes. This was carried out using the Vectastain Elite ABC Kit (Vector Laboratories) according to the instructions provided with the kit. Immune complex was detected using 0.05% 3,3′-diaminobenzidine (DAB, Vector Laboratories), after which the sections were counter-stained with hematoxylin.  
         [0106]     Results: The chimera decoy significantly inhibited invasion of macrophages as compared with the scrambled decoy (see Table 7,  FIG. 12 ). In addition, since the chimera decoy decreased the number of PCNA-positive cells as compared with the scrambled decoy, the chimera decoy was indicated to inhibit cell proliferation (see Table 8,  FIG. 13 ).  
                           TABLE 7                                   Macrophage   (% among all cells)                           Control   25.88 ± 4.71           Scrambled decoy   22.95 ± 3.51           Chimera decoy   10.69 ± 1.88                      
 
         [0107]                                TABLE 8                                   PCNA-positive cells   (% among all cells)                           Control   36.09 ± 6.56           Scrambled decoy   35.55 ± 6.16           Chimera decoy   12.09 ± 2.08                        
 6. All Statistical Analysis Data are Shown as the Mean±Standard Error (SEM).