JP5664992B2 - Cell specific peptides and uses thereof - Google Patents

Description

  The present invention relates to peptides and uses thereof. Specifically, the present invention relates to a peptide exhibiting specificity for a specific cell, application of the peptide to a medical device, and the like.

  The second leading cause of death among Japanese is heart disease. In the United States, heart disease has been the leading cause of death for half a century, and the number of patients suffering from ischemic heart diseases such as myocardial infarction and angina is very large worldwide. For ischemic heart disease, treatment of thrombus and stenosis is performed by placing a metal tube called a stent in a blood vessel. For normal stents, many cases of restenosis due to thrombus formation and fibrosis of surrounding tissues have been reported after placement, but a drug eluting stent (DES: Drug Eluting Stent approved by the Ministry of Health, Labor and Welfare in March 2004, for example. Non-Patent Document 1) has shown the effect of reducing the occurrence of restenosis and changed the field of heart disease treatment. However, DES has been reported to cause late stent thrombosis. The causes of this thrombosis are insufficient expansion of the stent to the inner wall of the blood vessel, insufficient proliferation of endothelial cells by cytostatic drugs, allergic reactions to polymers, etc. There is an urgent need to develop thrombotic agents.

Currently, various stents are being developed, and they are covered with an antibody (CD34) specific for the surface antigen of stents (Nobori; Terumo, Japan) and circulating endothelial progenitor cells (EPCs) using biolimus A9 as a new drug. Vascular endothelial progenitor cell capture stent (Genous ^TM Bio-engineered R stent ^TM ; Orbus Neich, Florida, USA) and PLLA (Poly-L-Lactic acid) stent (Igaki-Tamai; stock) made of biocompatible polymer Companies such as Kyoto Medical Design, Japan) are attracting attention.

  On the other hand, a technique for suppressing restenosis by specifically adsorbing and adhering endothelial progenitor cells by coating a peptide on the surface of a stent has been developed (Patent Document 1). It has also been proposed to coat the surface of the implant with a peptide (Patent Document 2).

JP 2004-344469 A JP-T-2001-526570

de la Fuente LM et al. Catheter Cardiovasc Interv. 2001 Aug; 53 (4): 480-8.

  The biggest challenge with stents is how to mimic the vascular environment. In a normal stent, the inner wall of the blood vessel of the stent insertion portion is damaged, and the portion covered with a protein called endothelial cell or extracellular matrix (ECM) is exposed. The stent itself is the source of an inflammatory response, causing cells to proliferate and cause restenosis. Drug-eluting stents that suppress cell proliferation are used as stents to improve this phenomenon, but the problem with this stent is that it suppresses even the proliferation of necessary endothelial cells, so that inactive platelets adhere. As a result, it causes a cause of stenosis called late stent thrombosis.

  A blood vessel is really well balanced, and when inserting a foreign stent into it, it does not use a simple drug, it is biocompatible and acts as a natural blood vessel. Must be applied. Inflammatory reaction does not occur and excessive cell proliferation does not occur, but control is necessary such that necessary cell proliferation occurs. In other words, the necessary cells are adhered and proliferated so that normal endothelialization occurs without causing hyperplasia of the blood vessel wall leading to restenosis or lack of endothelial cells causing thrombosis. It will be necessary. In this regard, Patent Document 1 listed above discloses a peptide effective for promoting adhesion of endothelial cells, but the peptide also exhibits adhesion to smooth muscle cells and has low cell specificity.

  The necessity of the control as described above according to the surrounding environment is not limited to stents, but is applicable to various medical devices (implantable medical devices) implanted or embedded in living bodies such as artificial small blood vessels, nerve tubes, and artificial valves. Is also true.

  Therefore, an object of the present invention is to provide a peptide excellent in cell specificity useful for the control of specific cells and its use.

  The present inventors have repeatedly studied to find a peptide exhibiting cell specificity. The first approach focused on various physical properties such as the charge, size, and hydrophobicity of the amino acids that make up the peptide. In other words, we hypothesized that there are differences in the physical properties preferred by cells, and thought that specificity might arise from the physical properties of amino acids preferred by cells, so 20 types of amino acid trimers (3-residue peptides) The experiment using was conducted. As a result, for each cell type, the tendency of amino acids preferred by the cells was clarified, and peptides showing high cell specificity were identified. In addition, when a peptide combining characteristic sequences was designed, a peptide showing high cell specificity was found.

The second approach is to establish a working hypothesis that a sequence that is specific or contained in abundance in a particular environment is functional, and under that working hypothesis, a cell-specific peptide We aimed to find out. Such a technique is significantly different from the conventional technique in which a functional sequence is searched by focusing on only the most functional part in a protein. We focused on collagen IV, which forms the basement membrane ECM closest to living endothelial cells, as a target to apply the above work hypothesis. That is, in collagen IV, it was predicted that a peptide with high adhesion to endothelial cells and low adhesion to smooth muscle would exist, and we decided to search for a trimeric sequence showing cell specificity from the sequence of collagen IV. . In addition, peptide sequences that are present in addition to collagen IV but have a high presence in collagen IV were screened to examine their cell specificity. As a result of the above studies, we succeeded in identifying a cell-specific peptide derived from collagen IV. Further studies were conducted, and a domain in which the identified peptides gathered together was found in the amino acid sequence of collagen IV and its cell specificity was confirmed.
The following invention is based on the above results.
[1] HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, GGG, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT , PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG , CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, AKG, DGY, TGP, VGP , SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, SYW, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP ( SEQ ID NO: 2), GPPGLSGPP (SEQ ID NO: 3), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGSPG (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (sequence) No. 9), any amino acid selected from the group consisting of GSPGLPGTP (SEQ ID NO: 10) and IGLSGEKG (SEQ ID NO: 11) Having a row, cell-specific peptide.
[2] HHH, VVV, TTT, TGA, NNN, KKK, AAA, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG , GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP , GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2), GPPGLSGPP (sequence) Number 3), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGSPG (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9), GSPGLPGTP (SEQ ID NO: The cell-specific peptide according to [1], which has any amino acid sequence selected from the group consisting of 10) and IGLSGEKG (SEQ ID NO: 11) and exhibits endothelial cell specificity.
[3] HHH, VVV, TTT, TGA, KKK, AAA, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP , GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY , VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2), GPPGLSGPP (SEQ ID NO: 3) ), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGSPG (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9), GSPGLPGTP (SEQ ID NO: 10) The cell-specific peptide according to [1], which has any amino acid sequence selected from the group consisting of IGLSGEKG (SEQ ID NO: 11) and exhibits endothelial cell specificity.
[4] RRR, YYY, TTT, GAT, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ The cell-specific peptide according to [1], which has any amino acid sequence selected from the group consisting of KGE, PFI, QGP, and SYW, and exhibits smooth muscle cell specificity.
[5] An implantable medical device comprising the peptide according to any one of [1] to [4] in a state of being exposed on the surface thereof.
[6] The implantable medical device according to [5], wherein the implantable medical device is a stent or an artificial blood vessel.
[7] A stent containing the peptide according to [3] in a state of being exposed on the surface thereof.
[8] An artificial blood vessel containing the peptide according to [3] in a state of being exposed on the surface of its lumen.
[9] The artificial blood vessel according to [8], containing the peptide according to [4] in a state of being exposed on the outer surface thereof.
[10] Use of the peptide according to any one of [1] to [4] for producing an implantable medical device.

The figure which shows the outline | summary of the peptide search method. A graph comparing the cell specificity of 20 amino acid trimers. HAEC represents aortic endothelial cells, SMC represents aortic smooth muscle cells, HUVEC represents venous endothelial cells, and NHDF represents skin fibroblasts. RGD was used as a control. RGD adheres and proliferates almost every cell, but some peptides have characteristic effects. The table | surface which shows the peptide which recognized high cell specificity. The figure which shows the outline | summary of the method of designing a new peptide combining a cell specific peptide. The graph which compared the cell specificity of the peptide designed by the method of FIG. TGA was found as an endothelial cell-specific peptide and GAT as a smooth muscle-specific peptide. HAEC represents aortic endothelial cells, and SMC represents aortic smooth muscle cells. A table summarizing the cell specificity of tripeptides derived from collagen IV. Tripeptides with higher specificity for endothelial cells than control (RGD) were listed. A table summarizing the cell specificity of tripeptides derived from collagen IV. Tripeptides that showed higher specificity for smooth muscle cells than control (RGD) were listed. Overview of techniques for searching for cell-specific peptides derived from collagen IV. A table summarizing the cell specificity of peptides found in collagen IV as a domain where endothelial cell-specific peptides gather collectively.

  The first aspect of the present invention relates to a peptide exhibiting cell specificity. “Cell specificity” refers to specific adhesion (high or low adhesion) to specific cells. It is preferable to determine “cell specificity” in consideration of specific proliferation ability (high or low proliferation ability) for a specific cell. Accordingly, among the peptides exhibiting “cell specificity”, those exhibiting high adhesion and proliferation to specific cells or low adhesion and proliferation to specific cells are preferred. Examples of “specific cells” here are endothelial cells, smooth muscle cells, and fibroblasts. A peptide exhibiting cell specificity is referred to herein as “cell-specific peptide”.

  The cell-specific peptides of the present invention are HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, GGG, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS , CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR , PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, AKG , DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, SYW, LPGFPGLK (array Number 1), LPGFPGTP (SEQ ID NO: 2), GPPGLSGPP (SEQ ID NO: 3), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGSPG (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8) Selected from the group consisting of FPGSPGFPG (SEQ ID NO: 9), GSPGLPGTP (SEQ ID NO: 10) and IGLSGEKG (SEQ ID NO: 11) Having any one of amino acid sequences. Among the above peptides, HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, and GGG have been found by an approach that focuses on the physical properties of amino acids. As shown in Examples below, HHH, VVV, TTT, TGA, KKK and AAA (hereinafter collectively referred to as “group 1 peptides”) and NNN are endothelial cell-specific peptides, RRR, YYY, TTT and GAT (hereinafter collectively referred to as “group 2 peptides”) are smooth muscle cell-specific peptides, and GGG is a fibroblast-specific peptide. On the other hand, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, SYW, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2), GPPGLSGPP (SEQ ID NO: 3), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5) , FPGLPGPP (SEQ ID NO: 6), GPPGPPGSPG (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9), GSPGLPGTP (SEQ ID NO: 10) and IGLSGEKG (SEQ ID NO: 11) are peptide sequences derived from collagen IV. is there. Of these, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS , GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP , NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2), GPPGLSGPP (SEQ ID NO: 3), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (sequence) No. 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGSPG (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9), GSPGLPGTP (SEQ ID NO: 10) and IGLSGEKG (SEQ ID NO: 11) (hereinafter referred to as these (Also collectively referred to as “Group 3 peptides”) are endothelial cell specific peptides, including AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP and S YW (hereinafter collectively referred to as “group 4 peptides”) is a smooth muscle-specific peptide.

  Group 1 and 3 peptides are effective for endothelial cell adhesion and proliferation, and are useful for applications where it is desired to capture and proliferate endothelial cells. On the other hand, NNN is effective for non-adhesion (non-adhesion) and non-proliferation (non-growth) of endothelial cells, and is useful for applications where it is desired to eliminate endothelial cells. Moreover, the peptides of groups 2 and 4 are effective for adhesion and proliferation of smooth muscle cells, and are useful for applications where it is desired to capture and proliferate smooth muscle cells. GGG is effective for non-adhering (not allowing adhesion) and non-proliferation (not allowing proliferation) of fibroblasts, and is useful for applications where it is desired to eliminate fibroblasts.

  Among the peptides of group 3, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, and LGL have particularly high specificity for endothelial cells. Therefore, in a preferred embodiment, any amino acid sequence selected from the group consisting of PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG and LGL An endothelial cell specific peptide is provided. Similarly, among group 4 peptides, AKG, DGY, and TGP have particularly high specificity for smooth muscle cells. Therefore, in a preferred embodiment, a smooth muscle cell-specific peptide having any amino acid sequence selected from the group consisting of AKG, DGY and TGP is provided.

  The peptide of the present invention can be prepared by a known peptide synthesis method (for example, solid phase synthesis method, liquid phase synthesis method). In addition, if an automatic peptide synthesizer is used, the target peptide can be synthesized easily and quickly.

  The target peptide may be prepared using genetic engineering techniques. That is, the target peptide may be obtained by introducing a nucleic acid encoding the peptide of the present invention into a suitable host cell and recovering the peptide expressed in the transformant. The recovered peptide is purified as necessary. The recovered peptide can be subjected to an appropriate substitution reaction and converted into a desired modified peptide.

  As long as the desired cell specificity is maintained, the peptide may be modified in some way. That is, one embodiment of the present invention provides a modified form of the above peptide (hereinafter referred to as “modified peptide”). “Modified peptide” in the present invention means that a part of a specific peptide (may be a plurality of positions) as a basic structure is replaced with another atomic group, or other molecules are added. A compound having a structure different from that of the peptide at least partially by modification. A person skilled in the art can design modifications such as substitutions based on the above peptides using well-known or conventional means. In addition, based on such a design, a desired modified product can be prepared using known or conventional means.

  As a typical example of the modified peptide, a peptide derivative in which a part of the side chain (atom or atomic group) is substituted with another atom or atomic group in the amino acid residue constituting the peptide can be exemplified. Such peptide derivatives can be prepared by any manufacturing process designed to obtain the peptide derivative as a final product. Therefore, when a target peptide derivative is a peptide in which a part (for example, an atomic group that is a part of a side chain) is apparently substituted by a specific atomic group, the target peptide derivative is Even if it is produced by a substitution reaction using the specific peptide as a starting material, which is an apparently basic peptide, or an appropriate substitution reaction using a peptide having another structure as a starting material, It may be manufactured by a process). Other atoms or atomic groups herein include hydroxyl groups, halogens (fluorine, chlorine, bromine, iodine, etc.), alkyl groups (methyl groups, ethyl groups, n-propyl groups, isopropyl groups, etc.), hydroxyalkyl groups ( Examples thereof include hydroxymethyl group, hydroxyethyl group, etc., alkoxy groups (methoxy group, ethoxy group, etc.), acyl groups (formyl group, acetyl group, malonyl group, benzoyl group, etc.) and the like.

  The modified peptides include those in which the functional group in the constituent amino acid residue is protected by an appropriate protecting group. As the protecting group used for such a purpose, an acyl group, an alkyl group, a monosaccharide, an oligosaccharide, a polysaccharide and the like can be used. Such a protecting group is linked by an amide bond, an ester bond, a urethane bond, a urea bond, or the like according to the peptide site to which the protecting group is bound, the kind of the protecting group to be used, or the like.

  Other examples of modified peptides include those that have been modified by the addition of sugar chains. Various peptide derivatives classified as alkylamines, alkylamides, sulfinyls, sulfonylamides, halides, amides, aminoalcohols, esters, aminoaldehydes, etc. are also modified by replacing the N-terminal or C-terminal with other atoms. One of the peptides.

  A further example of a modified peptide is a labeled peptide. For example, a peptide labeled with biotin or FITC at the N-terminus, a peptide labeled with a fluorescent dye, or the like corresponds to the labeled peptide.

  In addition, the peptide derivative comprised by combining the various modification methods demonstrated above is good also as the modified peptide of this invention.

  The second aspect of the present invention relates to the use of the peptide. The peptide of the present invention is used for applications in which it is desired to induce, capture, proliferate or eliminate specific cells using its cell specificity. A specific example of the application is an implantable medical device. "In-vivo implantable medical device" refers to a stent, catheter, balloon, artificial blood vessel, nerve tube, artificial valve, artificial heart-lung machine, implant (such as artificial bone) that is used implanted in the body. It refers to medical devices such as In the implantable medical device according to the present invention, the peptide of the present invention is contained in a state of being exposed on the surface thereof. Due to this feature, cell specificity is imparted to the contact surface with the living body, and the specificity is exhibited with respect to specific cells. For example, when the peptide of group 1 or 3 (endothelial cell-specific peptide) is applied, a surface having affinity for endothelial cells is formed, and adhesion and proliferation of the endothelial cells are promoted there (if the view is changed). It can be said that it can suppress the capture and proliferation of cells other than endothelial cells).

  As a preferred embodiment, a stent to which the peptide of the present invention is applied is provided. A stent is a medical device used for the purpose of treatment / improvement of obstruction or stenosis of blood vessels, airways, esophagus, etc., and is usually cylindrical. Various types of stents have been developed and proposed (see the column of background art, JP-T-8-502428, JP-A-8-507243, JP-T-10-503676, etc.).

  Two or more kinds of peptides may be used in combination. When used in combination, the same type of peptides may be combined in terms of cell specificity, or different types of peptides may be combined. An example of the former is a case where two or more peptides selected from the group 1 or 3 are selected and used together. An example of the latter is a peptide (1 or more) included in the group 1 and a peptide included in the group 2 (1 or 2 or more) is used together. When combining different types of peptides, in principle, a peptide-containing region is set for each cell specificity. That is, a peptide having different cell specificities is not mixed. In this way, for example, two or more types of cells can be controlled, such as capturing and growing endothelial cells in one region and simultaneously capturing and growing smooth muscle cells in another region. If the specific example of the said control is given, while containing the peptide (1 or 2 or more) contained in the group 1 or 3 in the state exposed to the lumen | bore surface, the peptide (1 or 2 or more) contained in the group 2 or 4 ) In an exposed state on the outer surface. In such an artificial blood vessel, endothelial cells can be induced / captured on the inside to promote proliferation therein, and smooth muscle cells can be induced / captured on the outside to promote proliferation there. This can be expected to improve the rate of engraftment on the living body and to suppress side effects such as stenosis.

The peptide inclusion mode is not particularly limited. For example, a desired peptide is attached or fixed to the surface of a medical device directly or via a linker (spacer). Examples of linkers include PEG (polyethylene glycol), PEG derivatives (diethylene glycol, tetraethylene glycol, etc.), compounds having a C _{10 to} C ₁₈ carbon chain, and biological constituent lipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Can do. On the other hand, the surface of the medical device may be coated with a material containing a peptide. Or you may decide to form the surface of a medical device with the material containing a peptide (a peptide is mixed in a forming material).

The amount of peptide used is not particularly limited, and for example, the peptide can be contained at a density of 100 pmol / cm ^{2 to} 100 mmol / cm ² . It should be noted that the density of the peptide may not be uniform in the region containing the peptide. For example, providing a peptide-containing region so that a density gradient is formed can also be expected to induce cells to a specific site.

  The material of the implantable medical device (particularly, the material containing the peptide of the present invention) is not particularly limited. Examples of materials include metal materials such as stainless steel (for example, SUS316L), tantalum, cobalt chromium alloy nickel / titanium alloy, magnesium alloy, polymer materials such as polyethylene, polypropylene, polyethylene terephthalate, and cellulose acetate, and ceramics. Biodegradable materials such as polylactic acid, polyglycolic acid, and polycaprolactone can also be used.

  Note that matters not specifically mentioned in the present specification (conditions, operation methods, etc.) may follow conventional methods, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987), Current protocols in Immunology, John Wiley & Sons Inc, and the like.

<Search for cell-specific peptides focusing on the physical properties of amino acids>
Cell-specific peptides were searched by paying attention to various physical properties such as charge, size, and hydrophobicity of amino acids constituting the peptides. The physical properties of amino acids are quantified and reported as AA index (Amino Acid Index) (Nucleic Acids Res. 2008 Jan; 36 (Database issue): D202-5. Epub 2007 Nov 12). High adhesion peptide sequences have been screened by studies using such physical properties and information processing analysis (Biotechniques. 2008 Mar; 44 (3): 393-402.). Based on the hypothesis that there are differences in the physical properties preferred by the cells, we thought that specificity might arise from the physical properties of the amino acids preferred by the cells, and conducted experiments using 20 amino acid trimers.

1. Method (Figure 1)
Trimers (tripeptides) were synthesized for 20 types of amino acids. Using the Fmoc solid phase synthesis method, each trimer was synthesized and extended on the surface of the array one residue at a time to obtain a peptide array. Each spot of the peptide array was punched out and attached to each well of the culture plate. Each well was seeded with cells (aortic endothelial cells (HAEC), venous endothelial cells (HUVEC), aortic smooth muscle cells (SMC), skin fibroblasts (NHDF)). Non-specifically adsorbed cells on the surface were washed away by a washing step, followed by calcein staining (30 minutes). After confirming that living cells were stained with calcein, the cells were washed three times with a phosphate buffer, and fluorescence in each well was detected. Estimated number of cells attached based on fluorescence value

  On the other hand, after conducting the same cell seeding experiment, cell culture was performed in an incubator with the cells adhering to the array for 1 day or 3 days to promote cell proliferation. One day or three days later, nonspecifically adsorbed cells on the surface were washed away by a washing step, followed by calcein staining (30 minutes). After confirming that living cells were stained with calcein, the cells were washed three times with a phosphate buffer, and fluorescence in each well was detected. Based on the fluorescence value, the number of adhered cells was estimated.

  RGD was used as a reference peptide (control), and the cell adhesion / proliferation of each peptide (verification peptide) was evaluated. For example, when the fluorescence measurement value of the live cell on the peptide array on which the RGD peptide as a control is placed is 100, if the fluorescence measurement value of the live cell on the peptide array on which the verification peptide is placed is 200, 200/100 was calculated, and the adhesive / proliferative ratio was set to 2.0.

2. Results and Discussion FIG. 2 shows the cell specificity of each peptide. The control RGD generally showed strong adhesion and proliferation to any cell, whereas some peptides exhibited strong adhesion and proliferation to specific cells. The peptides with high cell specificity are summarized in the table of FIG. As shown in the table, peptides preferred by aortic endothelial cells were HHH, VVV and TTT. On the other hand, the peptide that aortic endothelial cells repel was NNN. Peptides preferred by venous endothelial cells were KKK, AAA and VVV. Similarly, peptides preferred by aortic smooth muscle cells were RRR, YYY and TTT. The peptide that skin fibroblasts repel was GGG.

  As described above, peptides specific for endothelial cells (HHH, VVV, TTT, KKK, AAA, NNN), peptides specific for smooth muscle cells (RRR, YYY and TTT), and fibroblast specific A peptide (GGG) was found. HHH, VVV, TTT, KKK, AAA for specific capture and proliferation of endothelial cells (specific culture), NNN for specific removal of endothelial cells, RRR, YYY and TTT for specific capture and proliferation of smooth muscle cells It can be said that GGG is useful for specific removal of fibroblasts (specific culture).

  After receiving the above results, a peptide was designed by combining peptide sequences that showed specificity for specific cells, and the cell specificity was examined (FIG. 4). As a result, TGA was found as an endothelial cell-specific peptide and GAT as a smooth muscle-specific peptide (FIG. 5). The former is useful for specific capture and proliferation of endothelial cells (specific culture), and the latter is useful for specific capture and proliferation of smooth muscle cells (specific culture).

<Search for cell-specific peptides derived from collagen IV>
Under the working hypothesis that “specific or abundant sequences contained in abundant proteins in a specific environment are functional,” it was decided to search for cell-specific peptides. This working hypothesis is that (1) in general, there are many sequences in the protein that have an adhesion ability above a certain level, and (2) the protein itself has cell specificity, but it has been reported in the past. Since peptides do not have cell specificity (there is cell specificity as a whole, but only one specific domain has not been found), “There are multiple adhesion peptides among proteins showing adhesion, It is based on the idea that it is involved in multiple ways to form the characteristics and atmosphere of the protein.

  Focusing on collagen IV, which constitutes the basement membrane ECM closest to living endothelial cells, as an object to apply the above work hypothesis, “In collagen IV, it adheres to endothelial cells and has low adhesion to smooth muscle cells. Under the expectation that a peptide is present, it was decided to search for a trimeric sequence exhibiting cell specificity from the sequence of collagen IV. In addition, peptide sequences that are present in addition to collagen IV but have a high presence in collagen IV were screened to examine their cell specificity.

1. Method Compare the amino acid sequence of collagen IV with the amino acid sequences of four types of collagens I, II, III, and V, and among the 3319 tripeptides in five types of collagen, characteristic tripeptides inherent in collagen IV Sequences (907 types) were extracted, and 115 sequences were analyzed from the number of thoughts present. On the other hand, among the tripeptide sequences (1071 types) having a higher abundance ratio in collagen IV compared to the four types of collagens I, II, III, and V, 139 sequences having a higher abundance ratio were analyzed. As a result, a total of 254 sequences were assayed and the cell specificity of the tripeptide was examined by the following method. First, each synthesized peptide was stretch-synthesized on a cellulose membrane using the Fmoc solid phase synthesis method to obtain a peptide array (see FIG. 1). Each spot of the peptide array was punched out and attached to each well of the culture plate. Cells (aortic endothelial cells (HAEC), aortic smooth muscle cells (SMC)) were added to each well. After calcein staining (30 minutes), it was allowed to stand for 1 hour. Thereafter, the wells were washed three times with a phosphate buffer, and fluorescence in each well was detected. The number of adhered cells was estimated from the fluorescence value. The value obtained by the following calculation formula was used as an index of cell specificity. RGD was used as a reference peptide (control), and the cell adhesion / proliferation of each peptide (verification peptide) was evaluated. For example, when the fluorescence measurement value of the live cell on the peptide array on which the RGD peptide as a control is placed is 100, if the fluorescence measurement value of the live cell on the peptide array on which the verification peptide is placed is 200, 200/100 was calculated, and the adhesive / proliferative ratio was set to 2.0.

2. Results / Discussion Peptides showing higher cell specificity than the control (RGD) are shown in FIGS. The peptide shown in FIG. 6 is specific to endothelial cells (having high adhesion to endothelial cells) and can be said to be useful for specific capture and proliferation (specific culture) of endothelial cells. On the other hand, the peptide shown in FIG. 7 is specific to smooth muscle cells (having high adhesion to smooth muscle cells) and can be said to be useful for specific capture and proliferation (specific culture) of smooth muscle cells. Here, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, and LGL have a specificity of 1.5 or more (FIG. 6) and are particularly useful. High nature. Similarly, AKG, DGY, and TGP have a specificity of −1.5 or less (FIG. 7), and are particularly useful.

It was examined whether the domain in which the identified endothelial cell-specific peptides gathered together was present in the amino acid sequence of collagen IV (FIG. 8). As a result, since a total of 11 types of sequences listed below were found, their cell specificity was determined according to the above-described method.
LPGFPGLK (SEQ ID NO: 1)
LPGFPGTP (SEQ ID NO: 2)
GPPGLSGPP (SEQ ID NO: 3)
FPGPPGPP (SEQ ID NO: 4)
LPGLPGPP (SEQ ID NO: 5)
FPGLPGPP (SEQ ID NO: 6)
GPPGPPGSPG (SEQ ID NO: 7)
LPGPPGPP (SEQ ID NO: 8)
FPGSPGFPG (SEQ ID NO: 9)
GSPGLPGTP (SEQ ID NO: 10)
IGLSGEKG (SEQ ID NO: 11)

  The examination result of cell specificity is shown in FIG. Both peptides showed high specificity for endothelial cells. Therefore, it can be said that these peptides are useful for specific capture and proliferation (specific culture) of endothelial cells.

  The peptide of the present invention is excellent in cell specificity. Adhesion and proliferation of specific cells can be controlled by using the peptide of the present invention. Therefore, the peptide of the present invention can be used as a material for treating the surface of an implantable medical device such as a stent. It is also assumed that a plurality of types of cells may be used as a material for constructing an organization in which two-dimensional or three-dimensional aggregates are in a specific localized state. Thus, it can be expected to be used not only in the field of medical equipment but also in the field of tissue engineering.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

  SEQ ID NOs: 1-11: Description of artificial sequence: Peptide derived from collagen IV

Claims (6)

HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, GGG, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, AKG, DGY, TGP, VGP, SLW, An amino acid sequence selected from the group consisting of AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP and SYW An implantable medical device comprising a cell-specific peptide that is exposed on its surface. The implantable medical device according to claim 1 , wherein the implantable medical device is a stent or an artificial blood vessel. HHH, VVV, TTT, TGA, KKK, AAA, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, A peptide having an amino acid sequence selected from the group consisting of DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP and GNS, and showing endothelial cell specificity A stent containing the surface exposed on the surface. HHH, VVV, TTT, TGA, KKK, AAA, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, A peptide having an amino acid sequence selected from the group consisting of DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP and GNS, and showing endothelial cell specificity An artificial blood vessel that contains the exposed surface of the lumen. RRR, YYY, TTT, GAT, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, The artificial blood vessel according to claim 4 , comprising a peptide having any amino acid sequence selected from the group consisting of PFI, QGP, and SYW and having a smooth muscle cell specificity exposed on its outer surface. For manufacturing implantable medical devices, HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, GGG, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, From GNS, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP and SYW Use of a cell-specific peptide having any amino acid sequence selected from the group consisting of:

Images