Biomimetic coatings for intravascular stents

Abstract

Biomimetic coatings for intravascular stents The inventors have synthesized peptides that allow binding of intact CD31 molecules on all healthy endothelial cells and resting platelets and leukocytes that can come into contact with the implanted device. These cells can therefore receive the "leave-me-alone" signal conveyed by the trans-homophilic binding of CD31, essential for maintaining circulation and vascularized tissue homeostasis. The occurrence of thrombotic or life-threatening hemorrhagic or thromboembolic complications has hindered the use of intravascular devices. Devices bearing the mimetic peptides of the invention are rapidly integrated because they are recognized by platelets and leukocytes as healthy endothelium, i.e., as "self" components. Moreover, their ability to be rapidly endothelialized with a physiological endothelial cell phenotype also limits platelet and leukocyte activation at the device implantation site in the long term. Thus, the present invention relates to peptides that mimic the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction.

Description

[Technical Field]

[0001] The present invention has applications in the medical field, and is particularly aimed at improving the integration and performance of implantable medical devices. [Background technology]

[0002] Strategies are needed to improve the biocompatibility of medical devices, in order to prevent biological tissue reactions to the implantation of medical devices that may pose unintended risks to patients.

[0003] The "foreign body reaction" is caused by tissue damage at the implant site and is initiated by contact of the non-biocompatible material with blood.

[0004] Platelet adhesion and activation is followed by recruitment of inflammatory cells and an abnormal wound healing sequence characterized by chronic inflammation and the formation of granulomatous tissue.

[0005] When injury occurs, disruption of the endothelial barrier results in the activation of platelets to contain the injury and the infiltration of leukocytes to remove debris and prepare the site for tissue regeneration.

[0006] However, medical devices are not subject to clearance.

[0007] As a result, in the absence of rapid endothelialization, there is intense activation of leukocytes and platelets at the implant site.

[0008] Excessive platelet adhesion and aggregation can cause blockage of blood flow (ischemia), and proteases released by activated platelets and leukocytes can cause rupture of the blood vessel wall (hemorrhage).

[0009] One such condition is caused by the presence of heart valves or vascular devices, which are considered foreign bodies by the body.

[0010] This has hindered the use of valves and vascular devices for the prevention and treatment of serious cardiovascular conditions.

[0011] CD31 is a transmembrane glycoprotein constitutively and exclusively expressed on platelets, leukocytes, and endothelial cells (ECs). Under healthy conditions, trans-homophilic CD31-CD31 interactions allow endothelial cells, platelets, and leukocytes to recognize "self" and prevent inappropriate activation.

[0012] The favorable treatment of implant devices in contact with blood is achieved by treatments that make their surface "biocompatible," allowing the rapid formation of a functional endothelial layer and ensuring proper integration of the biomaterial.

[0013] Abundant expression of CD31 by healthy endothelium plays an important role in maintaining circulation and vascularized tissue homeostasis.

[0014] Cortese et al. (Stroke, February 2021) demonstrated that immobilization of the CD31 mimetic peptide P8RI (kwpalfvr) reduced blood component reactivity, increased endothelial cell adhesion, and enhanced the integration of intravascular devices in vivo.

[0015] Diaz-Rodriguez et al. (EHJ, 2021) demonstrated that a soluble peptide called P8RI acts as a CD31 agonist. Therefore, they investigated the effects of CD31-mimetic metal stent coatings on endothelial cell and blood component adhesion, as well as on endothelial strut coverage and neointimal growth due to foreign body reactions in vivo.

[0016] CD31 is a type I transmembrane glycoprotein composed of six extracellular Ig-like domains numbered from the membrane-distal N-terminus, a short transmembrane fragment, and a cytoplasmic tail.

[0017] CD31 engagement depended on transhomophily between domains 1 and 2 of the molecule expressed by the initiating cell, A, and the same domains expressed by the interacting cell, B.

[0018] More specifically, regarding interacting domains 1 and 2 (termed “IgL1” and “IgL2,” respectively), they adopted a classical Ig domain structure with a two-layered β-sheet, with antiparallel β-strands anchored by a pair of cysteines forming disulfide bonds.

[0019] Due to transhomophilicity between the CD31 molecules of two interacting cells, the CD31 dimer interface included hydrophobic and hydrophilic interactions.

[0020] The two IgL1-2 fragments of the transhomophilic CD31 molecule packed together in a face-to-face antiparallel pattern with one β-sheet side (IgL1 interacting with IgL2 of the opposite monomer) facing each other.

[0021] The crystal structure showed two interacting surfaces: one between IgL1 of chain A (IgL1-A, i.e., the IgL1 domain of the CD31 molecule of the initial cell A) and IgL2 of chain B (IgL2-B, i.e., the IgL2 domain of the CD31 molecule of the interacting cell B), and the other between IgL2 of chain A (IgL2-A, i.e., the IgL2 domain of the CD31 molecule of the initial cell A) and IgL1 of chain B (IgL1-B, i.e., the IgL1 domain of the CD31 molecule of the interacting cell B).

[0022] Trans-homophily of CD31 domains 1 and 2 promoted molecular clustering through strong cis-homophily and lateral displacement of transmembrane and juxtamembrane extracellular sequences.

[0023] This cis-homophily occurs at sites of cell activation and is essential to permit the regulatory function of CD31 because the protein cannot autophosphorylate.

[0024] Phosphorylation of CD31 depended on the ability of the molecule to remain clustered near activated tyrosine kinase receptors.

[0025] Upon cell activation, activation of plasma membrane proteases promoted cleavage and shedding of most of the extracellular portion of the CD31 protein.

[0026] Shedding of CD31 abolished the trans -homophilicity of the molecule because its trans -homophilic portion (contained between domains 1 and 2) was lost, resulting in disclustering of CD31.

[0027] The amino acids contained in the P8RI sequence are released from the cis-cognate membrane segment of CD31, and P8RI clusters with this sequence to maintain the regulatory signaling properties of the cleaved CD31 molecule in activated endothelial cells, platelets, and leukocytes at sites of inflammation and thrombosis. Summary of the Invention

[0028] The inventors of the present patent application have surprisingly discovered several peptides with properties that mimic trans-allogeneic (domains 1 and 2) CD31-CD31 cell-cell interactions.

[0029] According to a first object, peptides are disclosed that have properties that mimic the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction.

[0030] In a preferred aspect, these peptides can have three different general structures.

[0031] In another aspect, derivatives of these peptides are disclosed.

[0032] According to a second object, medical uses of the disclosed peptides or peptide derivatives are disclosed.

[0033] In a preferred aspect, the medical application is the prevention of complications associated with the implantation of medical devices to treat cardiac and vascular pathologies.

[0034] In another preferred aspect, the medical use is the treatment of cardiovascular pathologies.

[0035] According to a third object of the invention, a coating comprising the disclosed biomimetic peptide is disclosed.

[0036] According to a fourth object of the invention, a method for preparing a coating comprising the disclosed biomimetic peptide is disclosed.

[0037] According to a fifth object, a device is disclosed that includes a portion coated with the peptide or coating of the invention.

[0038] According to a sixth object, a method for the prevention, treatment or diagnosis of vascular pathologies is disclosed, comprising the use of the biomimetic peptides of the invention.

[0039] Such uses may include implantation of devices coated with peptides according to the invention.

[0040] According to another object, the use of the biomimetic peptides of the invention for adhering to the surface of a device is disclosed.

[0041] According to an embodiment, the use of the biomimetic peptides of the invention for adhering to vascular devices is disclosed.

[0042] According to yet another object, the use of the biomimetic peptides of the invention for promoting endothelialization of arterial blood vessels, preventing intimal proliferation, and integrating devices within target blood vessels is disclosed.

[0043] definition For purposes of the present invention, the term "biomimetic" shall mean mimicking natural effects.

[0044] In the present invention, biomimetic peptides have properties that mimic the natural effects of the endothelium, ie the layer of endothelial cells (EC) that lines the inner walls of blood vessels and especially arteries.

[0045] In particular, the natural effects of the endothelium in healthy conditions mimic those provided by trans-homophilic CD31-CD31 cell-cell interactions.

[0046] More specifically, the aforementioned mimetic activity has the effect of not activating endothelial cells (EC), platelet cells (circulating platelet cells), and leukocytes.

[0047] The aforementioned mimetic properties can also be provided to surfaces coated with the peptides of the invention.

[0048] For the purposes of this patent application, the peptides of the invention mimic the trans-homophilic CD31-CD31 domains 1 and 2 cell-cell interaction.

[0049] "Homophilic" refers to an interaction between identical molecules, and for purposes of the present invention refers to the interaction of two CD31 molecules expressed by cells, each of which interacts with one cell.

[0050] More specifically, within each CD31 molecule, interactions occur through extracellular domain 1 and domain 2.

[0051] According to a first aspect, the biomimetic peptides of the invention are designated Group I.

[0052] In a preferred embodiment, the Group I peptide covers the region of human CD31 IgL1 corresponding to His71-Ser87 or the corresponding region of an orthologous mammalian CD31 molecule. Alternatively, the peptide covers the region of human CD31 IgL1 corresponding to Gln70-Lys89 or the corresponding region of an orthologous mammalian CD31 molecule. The amino acid sequences of orthologous mammalian CD31 molecules can be obtained from the NCBI ortholog database available at https: / / www.ncbi.nlm.nih.gov / gene / 5175 / ortholog / ?scope=40674. The corresponding region can be determined by a skilled artisan based on an alignment of the amino acid sequence of the CD31 ortholog of interest with the human CD31 amino acid sequence.

[0053] In one embodiment, the Group I peptide contains mutated Gln70hCys and Met88hCys homocysteines that provide a disulfide bond and a cyclic structure.

[0054] Group I peptides include the following sequences: [Table 1]

[0055] For the purposes of the present invention, within said sequence, independently of each other, 1Q (i.e., the residue at position 1 designated Q in SEQ ID NO: 53) may be any one of Q, C, L, K, or R; 2H may be any one of H, I, V, Q or R; 5L may be any one of L, R, V, F or E; 9D may be any one of D, E or N; F may be any one of F, V, L or I; 14Y may be any one of Y, H, R or N; 15N may be either N or D; 16I can be any one of I, V, T, or A; 17S may be either S or T; 18S can be either S or T, and the other amino acid X can be any other amino acid.

[0056] The consensus sequence, SEQ ID NO:53, is based on a region corresponding to positions 70 to 89 of the amino acid sequence of human CD31 in a multiple sequence alignment of mammalian CD31 amino acid sequences retrieved from the NCBI ortholog database on December 1, 2022. Residues other than X correspond to conserved residues in mammalian orthologous CD31 amino acid sequences. Residue X corresponds to a highly variable position in the multiple alignment.

[0057] Group I peptides include: SP722, SP745, SP765, SP1374, and SP1375.

[0058] According to a second aspect, the biomimetic peptides of the invention are referred to as Group II.

[0059] In a preferred embodiment, the Group II peptide covers the region corresponding to Tyr107-Glu122 of human CD31 IgL1 or the corresponding region of an orthologous mammalian CD31 molecule. Alternatively, the peptide covers the region corresponding to aa 106-124 of human CD31 IgL1 or the corresponding region of an orthologous mammalian CD31 molecule.

[0060] In one embodiment, the mutated 106h Cys and 124h Cys homocysteines provide a disulfide bond and a cyclic structure.

[0061] Group II peptides include the following sequences: [Table 2]

[0062] For the purposes of the present invention, within said sequence, independently of each other: 2K (i.e., the residue at position 2 designated K in SEQ ID NO: 54) can be either K or R; 3S may be either C or S; 4T may be any one of T, R or S; 5V can be either V or A, 6I may be any one of I, K, V, L, T or S; 8N may be any one of N, S or D; 9N may be any one of N, S, K or R; 11E may be any one of E, Q, V, K or M; 12K may be either K or R; 13T may be any one of T, A or P; 14T may be either T or S; 16E may be any one of E, A, Q or D, and the other amino acid X may be any other amino acid.

[0063] The consensus sequence, SEQ ID NO:54, is based on a region corresponding to positions 107 to 122 of the amino acid sequence of human CD31 in a multiple sequence alignment of mammalian CD31 amino acid sequences retrieved from the NCBI ortholog database on December 1, 2022. Residues other than X correspond to conserved residues in mammalian orthologous CD31 amino acid sequences. Residue X corresponds to a highly variable position in the multiple alignment.

[0064] Group II peptides include: SP1072, SP1376.

[0065] According to a third aspect, the biomimetic peptides of the invention are designated Group III.

[0066] In a preferred embodiment, the Group III peptide is Pro1 of human CD31IgL2. Covers the region corresponding to 33-Lys158 or the corresponding region of an orthologous mammalian CD31 molecule.

[0067] In one embodiment, the Group III peptide contains mutated Val135h Cys and Cys152h Cys, thereby providing a disulfide bond and a cyclic structure.

[0068] Group III peptides include the following sequences: [Table 3]

[0069] For the purposes of the present invention, within said sequence, independently of each other: 3C (i.e., the residue at position 3 designated C in SEQ ID NO: 55) can be any one of C, V, M, or I; 4T may be any one of T, I, M or E; 5L may be either L or V; 6D may be either D or N; 7K may be either K or R; 8K may be any one of K, T, M, R or I; 11I can be any one of I, T, M, V or E; 12Q may be either Q or E; 14G may be either G or E; 16V may be either V or I; 18V can be either V or I, 19N may be any one of N, T, R, S, G or H; 22V may be any one of V, M or L; 23P may be any one of P, Q, K, E, L or R; 24E may be any one of E, G or N; 26K may be any one of K, Q, E, R or N, and the other amino acid X may be any other amino acid.

[0070] The consensus sequence, SEQ ID NO:53, is based on a region corresponding to positions 133 to 158 of the amino acid sequence of human CD31 in a multiple sequence alignment of mammalian CD31 amino acid sequences retrieved from the NCBI ortholog database on December 1, 2022. Residues other than X correspond to conserved residues in mammalian orthologous CD31 amino acid sequences. Residue X corresponds to a highly variable position in the multiple alignment.

[0071] Group III peptides include: SP1071, SP1380.

[0072] According to a fourth aspect, the biomimetic peptides of the invention are designated Group IVa.

[0073] In a preferred embodiment, the Group IVa peptide is a heterodimer and comprises: a) a Group II peptide covalently linked to a Group I peptide.

[0074] Group IVa peptides include SP1379;

[0075] According to a fifth aspect, the biomimetic peptides of the invention are designated Group IVb.

[0076] In a preferred embodiment, the Group IVb peptide is a heterodimer and comprises: a) a Group III peptide and a Group I peptide covalently linked together.

[0077] Group IVb peptides include: SP1383.

[0078] According to a first aspect, the biomimetic peptide of the invention is based on the following sequence IA: [Table 4]

[0079] According to a second aspect, the biomimetic peptide of the invention is based on the following sequence IB: [Table 5]

[0080] According to a third aspect, the biomimetic peptide of the invention is based on the following sequence IC: [Table 6]

[0081] For the purposes of the present invention, biomimetic peptides also include cyclic peptides, particularly biomimetic peptides containing cysteine ​​or homocysteine ​​that can be cyclicized via disulfide bonds.

[0082] For the purposes of the present invention, a biomimetic peptide may include a linker and / or a spacer and / or a tail, and may optionally be represented by an amino acid sequence, in particular, the sequence may be attached to the peptide terminus or to a single amino acid residue.

[0083] In a particularly preferred embodiment, the amino acid sequence of interest may be represented by the following sequence: [Table 7]

[0084] For the purposes of the present invention, biomimetic peptides also include heteropeptides comprising two of the peptide sequences described above.

[0085] In particular, heteropeptides may include:

[0086] A peptide having a sequence based on structure IA and a peptide having a sequence based on structure IB, or a peptide having a sequence based on structure IA and a peptide having a sequence based on structure IC.

[0087] Heteropeptides may comprise peptide sequences joined via acetylthioether bonds between amino acids of different sequences or between linkers and / or spacers.

[0088] In a preferred aspect, the peptides of the invention are based on the following sequences: [Table 8]

[0089] In a preferred aspect, the peptides of the invention are based on the sequence [Table 9]

[0090] In a preferred aspect, the peptides of the invention are based on the following structure: [Table 10]

[0091] In a preferred aspect, the peptides of the invention are based on the following structure: [Table 11]

[0092] In a particularly preferred aspect, the heteropeptide of the present invention is characterized in that it comprises the following peptide: [Table 12]

[0093] According to preferred aspects, the biomimetic peptides of the present invention may comprise the following modifications: cysteine ​​residues may be substituted with the corresponding homocysteine ​​residues, L-amino acids may be substituted with the corresponding D-amino acids. According to preferred embodiments of the present invention, the biomimetic peptides of the present invention may comprise a spacer and / or linker and / or tail selected from the following: [Table 13] and combinations thereof, e.g., [Table 14]

[0094] For purposes of the present invention, the above disclosed structures may include modifications at the C-terminus and / or N-terminus.

[0095] In particular, these changes may include: [Table 15]

[0096] According to preferred embodiments of the invention, the following biomimetic peptides are disclosed: [Table 16] JPEG2024542778000018.jpg138162

[0097] For purposes of the present invention, biomimetic peptides of the present invention include peptides having at least 95% identity to any of the above-disclosed structures, preferably at least 97%, and more preferably at least 99% identity. The percent identity referred to in this disclosure is determined after optimal global alignment of the compared sequences, which may therefore include one or more insertions, deletions, truncations, and / or substitutions. The alignment is global, meaning that the entire compared sequence is included over its entire length. The alignment is "optimal," meaning that it is performed to minimize the number of insertions, deletions, truncations, and / or substitutions. Optimal global alignment may be performed using any sequence analysis method familiar to those skilled in the art, and percent identity may be calculated. In addition to manual comparison, global alignment may also be determined using the Needleman and Wunsch (1970) algorithm. For nucleotide sequences, sequence comparison may be performed using any software familiar to those skilled in the art, such as Needle software. The parameters used are in particular: "Gap open" 10.0, "Gap extend" 0.5, and EDNAFULL matrix (NCBI EMBOSS version NUC4.4). For amino acid sequences, sequence comparison can be performed using any software familiar to those skilled in the art, such as Needle software. The parameters used are in particular: "Gap open" 10.0, "Gap extend" 0.5, and BLOSUM62 matrix.

[0098] According to a second object, medical uses of the disclosed peptides are disclosed.

[0099] According to a preferred aspect, the medical use is for the prevention of vascular pathologies.

[0100] A more sophisticated medical application may involve the implantation of devices coated with peptides according to the invention.

[0101] In particular, according to the current invention, these pathologies are selected from the group comprising heart valve pathologies, arteriosclerosis, thrombosis, ischemia, hemorrhage, restenosis, aneurysm.

[0102] In addition to the above, medical applications are disclosed for the prevention of pathological conditions represented by in-stent stenosis.

[0103] According to another preferred aspect, the medical use is for the treatment of vascular pathologies.

[0104] The above medical applications are disclosed not only for humans but also for animals in the veterinary field.

[0105] According to a third object of the invention, a coating comprising the disclosed biomimetic peptide is disclosed.

[0106] According to an embodiment of the present invention, the coating is a single layer coating, and according to another embodiment of the present invention, the coating is a multi-layer coating.

[0107] According to a fourth object of the invention, a method for preparing a coating comprising the disclosed biomimetic peptide is disclosed.

[0108] For the purposes of the present invention, the coating of the device or part thereof may be obtained by dip coating, by immersing the part to be coated in a coating bath.

[0109] A well-known method for functionalizing surfaces is click chemistry, as disclosed, for example, in US2018 / 0296732, WO2020 / 109836, or WO2021 / 239905.

[0110] Copper-free click chemistry is based on the reaction of a diarylcyclooctyne moiety (DBCO, also known as ADIBO for azadibenzocyclooctyne or DIBAC for dibenzoazacyclooctyne) with an azide-labeled reaction partner, known as strain-promoted alkyne-azide cycloaddition (SPAAC). This click chemistry is very fast at room temperature and does not require a Cu(I) catalyst. Diarylcyclooctynes ​​have very narrow and specific reactivity toward azides, leading to nearly quantitative yields of stable triazoles. The reaction can be carried out in aqueous buffered media.

[0111] According to a preferred embodiment, the method is a three-step dip coating method comprising: First step for polydopamine coating, A second step to graft a suitable linker, and The third step is to coat the inventive peptide.

[0112] The first step involves achieving a coating of a polydopamine layer on the surface of the device or a part thereof, in order to obtain a polydopamine-coated surface from dopamine.

[0113] Dopamine is known to self-polymerize into highly adherent films on several types of substrates. Polydopamine (PDA) is a self-assembling polymer formed by the oxidation of dopamine. Indeed, PDA contains dopamine, indole, and pyrrole units. Due to its diverse reactive groups, PDA offers several possibilities for substrate functionalization, particularly for the immobilization of bioactive molecules. PDA coating can be performed by immersing a device or part of it in a solution containing a dopamine salt, specifically dopamine hydrochloride or dopamine ammonia. The solution can be aqueous, alcoholic, or aqueous-alcoholic. If the device is sensitive to water (e.g., in the case of corrosion), the solvent for the solution is preferably alcohol, especially absolute ethanol. The device can be pretreated before immersion in the dopamine solution by etching with a strong acid, such as hydrofluoric acid.

[0114] Finally, a rinsing and / or ultrasonic cleaning step can be performed to remove any uncoated areas and PDA aggregates. Rinsing can be performed with demineralized water or alcohol.

[0115] The second step corresponds to immobilizing a linker on the polydopamine coating for subsequent conjugation of the peptide of interest. Any linker that allows the immobilization of a biomimetic peptide can be used. For this purpose, the polydopamine coating obtained in the first step is contacted by suitable means with a linker solution, forming a linker film on the polydopamine coating.

[0116] The linker is any suitable bifunctional reagent. The linker is preferably bioorthogonal.

[0117] The linker is preferably suitable for click chemistry as disclosed above. Accordingly, the linker contains a free triple bond, preferably a cyclooctyne moiety, more preferably a DBCO-like diallylcyclooctyne moiety. The triple bond can react with the azide group of the peptide. The linker is further functionalized with amine and / or thiol functional groups that are reactive toward polydopamine coating.

[0118] To enhance solubility in water and commonly used organic solvents with moderate polarity, the linker preferably includes a spacer. This spacer is preferably a polymer or oligomer. Potential polymers or oligomers include polyethylene glycol (PEG), polylactic acid, polylactic acid, sugars, lipids, polyglutamic acid (PGA), polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), and combinations thereof. PEG is particularly preferred. Specifically, the hydrophilic PEG4 spacer is known to reduce or eliminate aggregation and precipitation problems.

[0119] Examples of cyclooctyne PEGs include DBCO-PEG derivatives such as DBCO-PEG4-amine and DBCO-sulfo-PEG4-NH2.

[0120] If the linker is not fixed to the surface of the PDA coating, a washing step is preferably performed to remove it. The washing step can be performed with demineralized water and / or alcohol.

[0121] The third step involves grafting a biomimetic peptide onto the modified polydopamine-coated device using a suitable linker. For that purpose, the peptide preferably contains a functional group, such as an azide group, that can react with a functional group (e.g., a free triple bond) of the linker.

[0122] The device or a part thereof obtained after the second step is contacted by suitable means with a biomimetic peptide containing a specific functional group. Grafting can be carried out by immersing the device or a part thereof in an aqueous solution containing the biomimetic peptide. The reaction can be carried out at room temperature.

[0123] According to a fifth object of the invention, a device is disclosed that includes a portion coated with the biomimetic peptide or coating of the invention.

[0124] The coating can include the surface of the entire device or a portion of the device.

[0125] For the purposes of the present invention, devices that can be completely or partially coated with the biomimetic peptides of the invention include coronary stents, flow diverting stents, aortic tubing, heart valves, balloon expandable stents, self-expanding scaffolds, polymeric tubing of graft stents, flow diverting meshes, aortic tubing, heart valves, stent retrievers, transcatheter mitral valve devices, catheters, leaflets or parts thereof, in particular balloon expandable stents, self-expanding scaffolds, polymeric tubing of graft stents, flow diverting meshes, aortic tubing, heart valves, stent retrievers, transcatheter mitral valve devices, catheters, leaflets or parts thereof, and generally any medical device that can be in contact with blood for a limited or extended period of time.

[0126] The fully or partially coated device may be composed of any suitable material, such as, for example: · Metals or alloys, stainless steel, cobalt-chromium alloys, platinum-chromium alloys, nickel-titanium alloys (also known as Nitinol), cobalt-chromium-nickel alloys, magnesium or magnesium alloys such as JDBM, Mg-Nd-Zn-Zr alloys, Mg-Nd-Zn-Ca alloys, Mg-Zn-Y-Nd alloys, Mg-Al, Mg-AL-Zr, WE43, AZ31; · Synthetic polymer materials, PEBAX, PVP, PE, PP, Dacron®, Teflon®; Clinical grade biological tissues, e.g. pericardial sheets for heart valve bioprostheses.

[0127] According to a sixth object, a method for the prevention or treatment of cardiac and vascular pathologies using the biomimetic peptides of the invention is disclosed.

[0128] According to certain aspects, methods for the prevention or treatment of cardiac and vascular pathologies include the use of devices fully or partially coated with the inventive coatings, particularly balloon-mounted stents, heart valve bioprostheses, and flow diverters, which can be used in interventional cardiovascular procedures such as coronary and peripheral arterial revascularization, interventional neurology, and heart valve implantation.

[0129] In particular, the pathologies of interest are represented by the following: heart valve pathologies, stenotic vascular diseases (including arteriosclerosis, in particular atherosclerosis), atherothrombosis, ischemic heart disease and peripheral diseases, vascular remodeling with risk of bleeding, restenosis, aneurysms.

[0130] Further to the above, methods for preventing conditions manifested by in-stent stenosis are disclosed.

[0131] It is well known that one of the initial mechanisms causing pathological tissue remodeling associated with foreign bodies in contact with blood is platelet adhesion to the surface of the foreign body, which triggers the recruitment and activation of leukocytes, subsequently initiating a series of reactions that ultimately lead to the formation of a thrombus and / or the encapsulation of the foreign body by rapidly proliferating vascular cells in response to soluble factors released by activated platelets and leukocytes, ultimately resulting in a reduction in the vascular lumen, e.g., (re)stenosis (see, e.g., Forrester et al., J Am Coll Cardiol., 17, 758-769 (1991)).

[0132] In the context of cardiac and vascular pathologies, individuals implanted with medical devices such as stents or valvular bioprostheses are typically administered antiplatelet therapy (aspirin and / or anti-P2Y12 therapy, e.g., clopidogrel, ticlopidine, ticagrelor, or prasugrel) for at least one to several months after stent implantation for a number of reasons. The use of single, and more frequently dual, antiplatelet therapy aims to eliminate the risk of platelet activation in contact with the stent, which is exposed to blood flow until fully reendothelialized, thus eliminating the risk of stent thrombosis and limiting the rate of restenosis. In most cases, dual antiplatelet therapy (DAPT), including aspirin and anti-P2Y12 therapy, is administered for 6–12 months. Prolonged therapy ultimately exposes patients to the risk of bleeding, and this observation has prompted the evaluation of the safety of shortening the duration of antiplatelet therapy. Clinical trials using drug-eluting stents with DAPT shortened to 30 days are currently underway. Once the DAPT period is over, antiplatelet therapy typically consists of continuation with aspirin alone (without anti-P2Y12 therapy).

[0133] When using a drug-eluting stent, the recommended human dose of aspirin (acetylsalicylic acid or its salt) is 50-100 mg / day, e.g., 75 mg / day. Regarding anti-P2Y12 drugs, the recommended human dose varies depending on the specific anti-P2Y12 drug used. The recommended dose of clopidogrel is 50-100 mg / day, particularly 75 mg / day (generally 75 mg once daily). The recommended dose of ticlopidine is 300-300 mg / day, particularly 250 mg / day (generally 250 mg once daily). The recommended dose of ticagrelor is 160-300 mg / day, particularly 180 mg / day (generally 90 mg twice daily). The recommended dose of prasugrel is 5-20 mg / day, particularly 10 mg / day (generally 10 mg once daily).

[0134] However, antiplatelet therapy can be associated with significant side effects and complications, especially in individuals with bleeding disorders, such as hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency), von Willebrand disease, and rare factor deficiencies, including factor I, II, V, VII, X, XI, XII, and XIII. Anti-P2Y12 drugs, in particular, are highly potent and prone to bruising throughout the body for no apparent reason. Furthermore, no dentist, gastroenterologist, or surgeon would want to touch a patient undergoing anti-P2Y12 therapy to avoid the risk of uncontrollable bleeding during intervention. In some cases, this can be a serious problem, as it can interfere with interventions for some non-cardiovascular diseases. These drugs also have significant side effects, such as diarrhea, itching, nausea, skin rash, and stomach pain.

[0135] Based on the physiological, non-activating, endothelial tissue mimetic properties of the coating made of CD31-derived peptides on the medical device according to the invention, the initial adhesion / activation of platelets coming into contact with the device may be prevented or significantly reduced, making the antiplatelet treatment that usually follows after implantation of the device unnecessary or at least reduced to a lower dose or administered for a shorter period than usual.

[0136] Thus, after implantation of a medical device according to the invention, in the context of either a method for the prevention or treatment of cardiac and vascular pathologies, including cardiac valve pathologies, arteriosclerosis, atherothrombosis, ischemia, hemorrhage, restenosis, aneurysms, or a method for the prevention of conditions represented by in-stent stenosis, the recipient individual preferably:

[0137] a) not receiving anti-P2Y12 therapy (individuals may not receive antiplatelet therapy at all); b) taking a dose of anti-P2Y12 therapy (and optionally also a dose of aspirin that is significantly lower, e.g., at least two-fold, at least three-fold, at least four-fold lower) than that recommended for drug-eluting stents within traditional dual antiplatelet therapy (DAPT); c) taking anti-P2Y12 therapy (and optionally aspirin) for a period significantly shorter (e.g., at least two-fold, at least three-fold, at least four-fold shorter) than recommended for drug-eluting stents within traditional dual antiplatelet therapy (DAPT); or d) Any combination of b) and c).

[0138] All items a) to d) above relate only to treatments after implantation of a medical device (especially a stent) and do not relate to medications that may be administered during implantation of a medical device. These medications are chosen by the physician according to clinical recommendations.

[0139] In the above sections b), c), and d), "drug-eluting stent" or "DES" refers to a stent that slowly releases drugs, such as sirolimus, that inhibit smooth muscle cell (SMC) proliferation. Examples of commercially available drug-eluting stents include HT Supreme®, XienceV®, Promus®, Cypher®, Taxus®, and Endeavor®.

[0140] In item b) above, after implantation, individuals implanted with a medical device according to the invention receive a significantly lower (e.g., at least two-fold, at least three-fold, or at least four-fold) dose of anti-P2Y12 therapy than that recommended for drug-eluting stents in traditional dual antiplatelet therapy (DAPT). In particular, after implantation, individuals may receive less than 50 mg per day of clopidogrel, preferably less than 40 mg per day, less than 35 mg per day, less than 30 mg per day, less than 25 mg per day, or less than 20 mg per day.

[0141] It is desirable that the intake of ticlopidine be less than 300 mg per day, preferably less than 175 mg per day, less than 150 mg per day, less than 125 mg per day, or less than 100 mg per day.

[0142] The recommended dose of ticagrelor is less than 160 mg per day, preferably less than 150 mg per day, less than 140 mg per day, less than 130 mg per day, less than 120 mg per day, less than 110 mg per day, less than 100 mg per day, less than 90 mg per day, or less than 80 mg per day (usually half the daily dose taken twice daily).

[0143] It is desirable that the intake of prasugrel be less than 5 mg per day, preferably less than 4 mg per day, less than 3 mg per day, less than 2.5 mg per day, or less than 2 mg per day.

[0144] The individual can further receive a significantly lower (e.g., at least twofold, at least threefold, at least fourfold, etc.) dose of aspirin after implantation. For example, the individual can receive less than 50 mg per day, preferably less than 40 mg per day, less than 35 mg per day, less than 30 mg per day, less than 25 mg per day, or less than 20 mg per day of aspirin.

[0145] In item c) above, an individual implanted with a medical device according to the invention receives anti-P2Y12 therapy for a period significantly shorter (e.g., at least two times, at least three times, at least four times, etc.) than that recommended for a drug-eluting stent. The individual may further take aspirin after implantation for a period significantly shorter (e.g., at least two times, at least three times, at least four times, etc.) than that recommended for a drug-eluting stent.

[0146] For example, an individual may take DAPT for less than 3 months, preferably less than 2 months, less than 1 month, or less than 4 weeks, less than 3 weeks, less than 2 weeks, or less than 1 week. A shorter antiplatelet treatment may also mean that an individual stops anti-P2Y12 therapy entirely after a period of treatment.

[0147] Item d) above is any combination of items b) and c) above.

[0148] This revised protocol with no or less antiplatelet therapy is useful for any individual because it prevents or significantly reduces the side effects (bleeding events) associated with antiplatelet therapy, but it is particularly useful for individuals with bleeding disorders such as hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency), von Willebrand disease, and rare factor deficiencies, including factor I, II, V, VII, X, XI, XII, and XIII.

[0149] The methods of prevention or treatment disclosed above are disclosed for humans as well as animals in the veterinary field.

[0150] According to another object, the use of the biomimetic peptides of the invention for adhering to the surface of a device is disclosed.

[0151] According to one embodiment, the use of the biomimetic peptides of the invention for adhesion to vascular devices is disclosed.

[0152] According to yet another object, the use of the biomimetic peptides of the invention is disclosed to promote vascular endothelialization, prevent neointimal growth, and integrate devices within target vessels.

[0153] In particular, the target vessel is represented by a coronary vessel, a peripheral vessel, or a cerebral vessel.

[0154] According to yet another object, the use of the biomimetic peptides of the present invention to improve the biocompatibility of devices is disclosed.

[0155] According to yet another object, the use of the biomimetic peptides of the present invention to impart the anti-inflammatory and anti-thrombotic properties of restored endothelium to stent segments is disclosed.

[0156] According to yet another object, it is disclosed that the biomimetic peptides of the present invention can be used to improve adaptive remodeling of stent segments, allowing the restoration of their functional properties.

[0157] The invention is further disclosed in the following non-limiting examples. [Brief explanation of the drawings]

[0158] [Figure 1A-1B] 1A and 1B show the structure of the octapeptide P8RI. [Figure 2] Figure 2 shows the strategy used to analyze the biocompatibility of nitinol discs interacting with human endothelial cells. [Figure 3] Figure 3 shows representative images and quantification of F-actin staining of HAECs growing on bare and coated discs. [Figure 4] Figure 4 shows representative images and quantification of CD31 staining of HAECs growing on bare and coated discs. [Figure 5]Figure 5 shows quantitative analysis of F-actin and CD31 expression of HAECs growing on coated discs and bare control nitinol discs. [Figure 6] FIG. 6 shows the ratio of CD31 to F-actin expression. [Figure 7-17] Figures 7 to 17 show the structures of preferred peptides of the invention. [Figure 18] FIG. 18 shows the structure of intermediate A. [Figure 19] FIG. 19 shows the structure of intermediate B. [Figure 20] FIG. 20 shows the structure of intermediate C. [Figure 21] FIG. 21 shows the structure of a linker according to the present invention. [Figure 22] FIG. 22 shows the results of the functional scores of the inventive peptides belonging to different groups. [Figure 23] FIG. 23 shows a graph depicting the results of the functional scores of the peptides of the invention belonging to different groups. [Figure 24] FIG. 24 shows a graph illustrating the biomimetic performance of the inventive peptides belonging to different groups. [Figure 25] FIG. 25 shows the ratio of CD31 to F-actin expression. [Figure 26] FIG. 26 shows the infrared spectrum of the eG™ NTMA film. [Figure 27] FIG. 27 shows the shape of a water droplet on a bare surface and on an eG™ NTMA surface. [Figure 28] FIG. 28 shows SEM cross sections 7 days after CFD stent implantation comparing eGNTMA and SP1072 peptide coatings. [Figure 29] FIG. 29 shows histopathological analysis of SEM cross sections 7 days after CFD stent implantation, comparing eGNTMA and SP1072 peptide coatings. [Figure 30]Figure 30 shows the histopathological analysis of SEM cross sections 60 days after CFD stent implantation, using the SP1072 peptide coating.

[0159] Certain abbreviations used in the examples and text are as follows: "AA" stands for amino acid, "Alloc" refers to allyloxycarbonyl, "Boc" stands for tert-butyloxycarbonyl, "tBu" stands for tertiary butyl, "DCM" refers to dichloromethane "DIC" refers to N,N'-diisopropylcarbodiimide, "DIPEA" refers to N,N-diisopropylethylamine, "DMF" refers to dimethylformamide, "Fmoc" refers to fluorenylmethyloxycarbonyl, "HOAt" refers to 1-hydroxy-7-azabenzotriazole; "HPLC" stands for high performance liquid chromatography, "LCMS" refers to liquid chromatography / mass spectrometry; "UPLC" stands for high performance chromatography. "RP-HPLC" refers to reversed-phase high-performance liquid chromatography, "MS" refers to mass spectrometry; "OtBu" stands for O-tertiary butyl, "Oxyma" refers to ethyl cyanohydroxyiminoacetate. "Pbf" refers to 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl "TIPS" stands for triisopropylsilane. "TFA" refers to trifluoroacetic acid, "Trt" refers to trityl. [Example]

[0160] Materials and Methods Peptides were synthesized by standard solid-phase peptide synthesis (SPPS) using Fmoc / t-Bu chemistry. DMF was used as the solvent. The following starting materials and methods were used in the synthetic procedures described in the examples.

[0161] Fmoc-protected natural amino acids were purchased from Novabiochem, Iris Biotech, Bachem, or Chem-Impex International. The following standard amino acids were used in the synthesis: Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp(OMpe)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Tyr(tBu)-OH, and Fmoc-L-Val-OH.

[0162] Additionally, the following amino acids were purchased from the same supplier: Fmoc-K-(N3)OH; Fmoc-K(alloc)OH; Fmoc-c-OH, Fmoc-hC-OH, Fmoc-Ttds-COOH, Fmoc-NH-PEG4-COOH.

[0163] Analytical characterization Crude and purified peptides were analyzed by ultra-performance liquid chromatography with UV and mass spectrometry detection (UPLC-UV-MS). Analytical UPLC was performed according to one of the following methods: Method A: Detection at 214 nm Column: Acquity Waters BEH130 C4, 1.7 μm (2 .1x100mm) 45℃ Solvent: HO + 0.1% TFA, ACN + 0.1% TFA (flow rate 0.4 ml / min) Gradient: 85, 15 (0 min) to 85, 15 (1 min) to 65, 35 (5 min) to 10, 90 (5.2 min) to 10, 90 (5.5 min) to 85, 15 (5.7 min) to 85, 15 (6 min) Mass spectrometer: Waters SQ detector using electrospray ionization in positive ion detection mode

[0164] Method B: Detection at 214 nm Column: Acquity Waters BEH130 C4, 1.7 μm (2.1 x 100 mm) 45°C Solvent: HO + 0.1% TFA, ACN + 0.1% TFA (flow rate 0.4 ml / min) Gradient: 80, 20 (0 min) to 80, 20 (1 min) to 60, 40 (5 min) to 10, 90 (5.2 min) to 10, 90 (5.5 min) to 80, 20 (5.7 min) to 80, 20 (6 min)

[0165] Method C: Detection at 214 nm Column: Acquity Waters BEH130 C4, 1.7 μm (2.1 x 100 mm) 45°C Solvent: HO + 0.1% TFA, ACN + 0.1% TFA (flow rate 0.4 ml / min) Gradient: 75, 25 (0 min) to 75, 25 (1 min) to 55, 45 (4 min) to 10, 90 (4.2 min) to 10, 90 (4.5 min) to 80, 20 (4.7 min) to 75, 25 (5 min) Mass spectrometer: Waters SQ detector using electrospray ionization in positive ion detection mode

[0166] Mass spectrometry was performed using a Waters SQ detector with electrospray ionization in positive ion detection mode, with a mass-to-charge ratio scan range of 400-1800. [Table 17]

[0167] General procedure for peptide synthesis on solid support All peptides were synthesized by standard Fmoc sequential solid-phase synthesis (SPPS) using a Liberty Blue microwave synthesizer (CEM). Assembly was performed using ProtideRink-amide (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin, CEM, 300 μmol, 100-300 mesh; loading 0.2 mmol / g) on ​​a 0.2 mmol scale with DIC / Oxyma activation. DMF was used as the solvent.

[0168] All amino acids were dissolved in DMF at a concentration of 0.4 M. Acylation reactions were carried out using a 5-fold excess of activated amino acid relative to the free amino groups on the resin at 90 °C for 3 min under microwave irradiation. Amino acids were activated with equimolar amounts of 0.5 M DIC solution in DMF and 1 M Oxyma solution in DMF.

[0169] The following conditions were used: Standard deprotection: 20% piperidine in DMF 2x120 sec, 90°C Wash: 4xDMF Standard single coupling: 5 equiv. AA 0.4M / 5 equiv. DIC 1M / 5 Equivalent of Oxyma 1M, 120 seconds, 90℃ Standard double coupling: Standard single coupling repeated twice Wash: 4xDMF

[0170] At the end of the assembly, the resin was washed with DMF, MeOH, DCM, and Et2O.

[0171] Cleavage of the peptide from the resin was carried out using the following cleavage cocktail:

[0172] Mix 1) 87.5% TFA, 5% phenol, 5% water, 2.5% TIPS at room temperature for 1.5 to 2.5 h (30 ml).

[0173] Mix 2) 87.5% TFA, 5% phenol, 5% water, 2.5% thioanisole at room temperature for 1.5 to 2.5 h (20 ml).

[0174] The resin used in the synthesis was such that the C-terminus was cleaved from the resin as a primary amide.

[0175] The cleavage mixture was collected by filtration, and the crude peptide was precipitated in methyl tert-butyl ether, centrifuged, the supernatant removed, and fresh diethyl ether was added to the peptide and recentrifuged twice, after which the crude peptide was lyophilized.

[0176] The peptides were analyzed by analytical UPLC and confirmed by ESI+mass spectrometry. The crude peptides were purified by conventional RP-HPLC purification using a Waters 2489 HPLC system (UV detection at 214 nm). The following solvents were used: acetonitrile + 0.1% TFA (mobile phase A) and water + 0.1% TFA (mobile phase B). The product-containing fractions were collected and lyophilized to obtain the purified product as the TFA salt. Unless otherwise noted, the compounds were tested as the TFA salt.

[0177] Synthesis procedure for SEQ ID NO: 29: Ac-c*HQMLFYKDDVLFYNISSC*-GGSGGSGG-K(N 3 )-CONH 2

[0178] The structure of the peptide is shown in FIG.

[0179] SEQ ID NO: 29 (PepSP722) covers the His71-Ser87 region of CD31IgL1-A with an acetylated N-terminal D-cysteine ​​and a cysteine ​​at C-terminal position serine 87, with two thiol groups involved in a disulfide bond.

[0180] After synthesis and cleavage according to the general procedure (Mix 1), the mixture was analyzed by analytical UPLC, confirmed by ESI+MS, and lyophilized to obtain the crude product (Y = 64%). The crude material was dissolved in an 8 / 2 acetonitrile / water mixture at a concentration of 1 mg / mL. Aqueous NH3 was added to reach pH 9, and the mixture was stirred at room temperature for 72 hours. TFA was added to the mixture, and the mixture was lyophilized. The crude peptide was purified by reverse-phase HPLC using a preparative Reprosil Gold C4 (250 x 40 mm, 120 A, 5 μm) column. The following gradient of eluent B was used: 20% B to 20% B in 5 min, then to 40% B in 25 min, with a flow rate of 60 mL / min and a wavelength of 214 nm. SEQ ID NO: 1 was isolated as a white lyophilized solid (Y = 5%), the TFA salt. The purified peptide was dissolved in an 8 / 2 acetonitrile / water mixture at a concentration of 1 mg / mL. 50 mM HCl (10 eq) was added, and the mixture was stirred at room temperature for 1 h. After lyophilization, the peptide was dissolved in acetonitrile / water (8 / 2) and lyophilized again. The peptide was analyzed by LC / MS (Method C). A [M+3H]3+ mass signal was found under the peak at a retention time of 3.27 min, revealing a peptide mass of 1013.5, which is consistent with the expected molecular weight of 3036.37.

[0181] Procedure for synthesis of SEQ ID NO: 30: The structure of the SHQMLFYKDDVLFYNISSS-GGSGGSGG-K(N3)-CONH2 peptide is shown in Figure 8. SEQ ID NO: 30 (PepSP745) is a linear sequence that covers the His71-Ser87 region of CD31IgL1-A in common with SEQ ID NO: 29, but with the two cysteine ​​residues of SEQ ID NO: 1 replaced by serine.

[0182] After synthesis and cleavage according to the general procedure (Mix 1), the mixture was analyzed by analytical UPLC, confirmed by ESI+MS, and lyophilized to obtain the crude product (Y = 66%). The crude peptide was purified by reverse-phase HPLC using a preparative Reprosil Gold C4 (250 x 40 mm, 120 A, 5 μm) column. The following gradient of eluent B was used: 20% B for 5 min, then 40% B in 20 min, at a flow rate of 60 mL / min and a wavelength of 214 nm. SEQ ID NO: 2 was isolated as an amorphous lyophilized solid (Y = 4%), the TFA salt. The peptide was dissolved at a concentration of 1 mg / mL in a mixture of acetonitrile / water (8:2). 10 eq of 50 mM HCl was added, and the mixture was stirred at room temperature for 1 h. After lyophilization, the peptide was dissolved in acetonitrile / water (8:2) and lyophilized again. The peptide was analyzed by LC / MS (Method C). The mass signal found under the peak with a retention time of 2.85 min [M+3H]3+ revealed a peptide mass of 989.4, consistent with the expected molecular weight of 2964.22.

[0183] Procedure for synthesis of SEQ ID NO: 31: The structure of the Ac-KKKc*HQMLFYKDDVLFYNISSC*-Ttds-Ttds-K(N3)-CONH2 peptide is shown in Figure 9.

[0184] SEQ ID NO: 31 (PepSP765) covers the same region as His71-Ser87 of CD31IgL1-A and has a disulfide bond similar to SEQ ID NO: 29, but has a solubilizing tail of three lysine residues at the N-terminus.

[0185] The synthesis was carried out according to the general procedure. At the end of the assembly, the resin was treated with AcO (10 eq) and DMF for 30 min. Cleavage was carried out using Mix 1 according to the general procedure, and the mixture was analyzed by analytical UPLC, confirmed by ESI+mass spectrometry, and lyophilized (Y = 61%). The crude material was dissolved in an 8 / 2 acetonitrile / water mixture at a concentration of 1 mg / mL. Aqueous NH3 was added to reach pH 9, and the mixture was stirred at room temperature for 72 h. TFA was added to the mixture, and it was lyophilized. The crude peptide was purified by reverse-phase HPLC using a preparative Reprosil Gold C4 (250 x 40 mm, 120 A, 5 μm) column. The following gradient of eluent B was used: 25% B in 5 min, 25% B in 25 min, 40% B in 25 min, with a flow rate of 60 mL / min. SEQ ID NO:3 was isolated as an amorphous lyophilized solid (Y = 2%), TFA salt. The peptide was dissolved at a concentration of 1 mg / mL in a mixture of acetonitrile / water (8:2). 50 mM HCl (10 eq) was added, and the mixture was stirred at room temperature for 1 h. After lyophilization, the peptide was dissolved in acetonitrile / water (8:2) and lyophilized again. The peptide was analyzed by LC / MS (Method C). The [M+3H]3+ mass signal found under the peak at retention time 2.90 min revealed a peptide mass of 1171.0, consistent with the expected molecular weight of 3509.17.

[0186] Procedure for synthesis of SEQ ID NO: 32: QHQMLFYKDDVLFYNISSMK-Ttds-Ttds-Ttds-Ttds-K(N3)-CONH2 The structure of the peptide is shown in FIG.

[0187] Seq number 32 (PepSP1375) covers the same region as Gln70-Lys89 of CD31IgL1-A and the long linker of the Ttds unit.

[0188] During solid-phase peptide assembly, a double acylation reaction of Val and Leu was performed. Fmoc deprotection was performed by double treatment of the resin with 20% (V / V) piperidine in DMF for 120 min at 90 °C until an Asp residue was reached at position 9. After this residue, deprotection was performed at room temperature to minimize aspartimide formation. Cleavage was performed according to the general procedure using Mix2. The mixture was analyzed by analytical UPLC, confirmed by ESI+mass spectrometry, and lyophilized to obtain the crude product (Y = 70%).

[0189] Purification of the crude material was performed using an XBridge C18 (250x50mm, 120A, 10µm) column and a gradient of solvent B as follows: 15% B for 5 min; 15% B to 35% B in 20 min; flow rate: 80mL / min. Fractions containing the product were collected and lyophilized to obtain the purified product as a TFA salt (Y = 10%). The purified peptide was analyzed by LC / MS (Method B). The mass signal found under the [M+4H]4+ peak at retention time 4.20 min indicated a peptide mass of 969.2, consistent with the expected molecular weight of 3870.58.

[0190] Procedure for synthesis of SEQ ID NO: 33: The structure of the hCHQMLFYKDDVLFYNISShCK-Ttds-Ttds-Ttds-Ttds-K(N3)-CONH2 peptide is shown in Figure 11.

[0191] SEQ ID NO: 33 (PepSP1374) is obtained by mutating the Gln70-Lys89 region of CD31IgL1-A of SEQ ID NO: 32 to homocysteine, and Gln70hCys and Met88hCys form a disulfide bond with a cyclic structure.

[0192] During peptide assembly, a double acylation reaction of Val and Leu was performed. Fmoc deprotection was performed up to the Asp residue at position 9 by double treatment of the resin with 20% (V / V) piperidine in DMF for 120 seconds at 90 °C. After this residue, deprotection was performed at room temperature to minimize aspartimide formation. Cleavage was performed according to the general procedure using Mix 2. The mixture was analyzed by analytical UPLC, confirmed by ESI+MS, and lyophilized to obtain the crude product (Y = 70%). The crude peptide was analyzed by analytical UPLC and confirmed by ESI+MS. The crude material was dissolved at a concentration of 1 mg / mL in a 9 / 1 mixture of DMSO / HO. Aqueous NH3 was added to reach pH 9, and the mixture was stirred at room temperature for 48 hours. After this time, disulfide bridge formation was confirmed by UPLC, and the mixture was lyophilized. Purification of the crude material was performed on a Waters XBridge C18 (250x50mm, 120A, 10µm) column using the following gradient: 15% B in 5 min; 15% to 35% B in 20 min; flow rate 80mL / min. Fractions containing the product were collected and lyophilized to obtain the purified product as a TFA salt (Y = 10%). The purified peptide was analyzed by LC / MS (Method B). The [M+4H]4+ mass signal found under the peak at retention time 4.05 min indicated a peptide mass of 962.4, consistent with the expected molecular weight of 3843.58.

[0193] Procedure for synthesis of SEQ ID NO: 34: The structure of the YKSTVIVNNKEKTTAE-PEG4-K(N3)-CONH2 peptide is shown in Figure 12. SEQ ID NO: 34 (PepSP1072) covers the Tyr107-Glu122 region of CD31IgL1-A.

[0194] During assembly, double acylation reactions were performed on all natural amino acids and Fmoc-NH-PEG-COOH. After synthesis and cleavage, which were performed according to the general procedure (Mix 1), the mixture was analyzed by analytical UPLC and confirmed by ESI+ mass spectrometry; it was lyophilized to obtain the crude product (Y = 70%). LCMS analysis calculated: for C96H166N27O33, 2225.21 Da; found; 1114.0 (M+2)2+. The crude peptide was purified by reverse-phase HPLC using a preparative Waters XBridge C18 (250 x 50 mm, 120 A, 10 µm) column using the following gradient of eluent B: 5% B to 5% B for 5 min, then 5% B to 25% B for 20 min, at a flow rate of 80 mL / min. SEQ ID NO: 34 was isolated as an amorphous lyophilized solid (Y = 25%) as the TFA salt. The purified peptide was analyzed by LC / MS (Method A). A [M+3H]3+ mass signal was found under the peak at a retention time of 2.97 min, revealing a peptide mass of 743.0, which is consistent with the expected molecular weight of 2226.54.

[0195] Procedure for synthesis of SEQ ID NO: 23: The structure of the hCYKSTVIVNNKEKTTAEYhC-(Ttds)4-K(N3)-NH2 peptide is shown in Figure 13.

[0196] SEQ ID NO: 23 (PepSP1376) covers the region 106-124, in which positions 106 and 124 are mutated to homocysteine, which forms a disulfide bond with a cyclic structure.

[0197] Synthesis and cleavage were performed according to the general procedure (Mix 1). The cleaved mixture was analyzed by analytical UPLC, confirmed by ESI+mass spectrometry, and lyophilized to obtain the crude product (Y = 65%). The crude material was dissolved at a concentration of 1 mg / mL in a 9 / 1 mixture of DMSO / HO. Aqueous NH3 was added to reach pH 9, and the mixture was stirred at room temperature for 48 h. Disulfide bridge formation was confirmed by UPLC, and the mixture was lyophilized. The crude peptide was purified by reverse-phase HPLC using an XBridge C18 (250 x 50 mm, 120 A, 10 μm) column with the following solvent B gradient: 5% B for 5 min, then 25% B for 20 min, at a flow rate of 80 mL / min. The product-containing fractions were collected and lyophilized to obtain the purified product as a TFA salt (Y = 28%). The purified peptide was analyzed by LC / MS (Method A). The [M+3H]3+ mass signal was found under the peak at retention time 3.57 min, revealing a peptide mass of 1195.4 consistent with the expected molecular weight of 3583.22.

[0198] Procedure for synthesis of SEQ ID NO: 35: The structure of the K(N3)-PEG4-PRVTLDKKEAIQGGIVRVNSSVPEEK-CONH2 peptide is shown in Figure 14.

[0199] SEQ ID NO: 35 (PepSP1071) covers the Pro133-Lys158 region of CD31IgL2-A.

[0200] The synthesis and cleavage were performed according to the general procedure (Mix 1). The cleavage mixture was analyzed by analytical UPLC, confirmed by ESI+ mass spectrometry, and lyophilized to obtain the crude product (yield = 54%). The crude peptide was purified by reverse-phase HPLC using a preparative double Waters DeltaPack C18 cartridge (100 x 40 mm, 300 A, 15 µm). The following gradient of eluent B was used: 5% B over 5 min, then 25% B over 20 min, at a flow rate of 80 mL / min. The product-containing fractions were collected and lyophilized to obtain the purified product as a TFA salt (yield = 35%). The purified peptide was analyzed by LC / MS (Method A). A [M+3H]3+ mass signal was found under the peak at a retention time of 3.57 min, revealing a peptide mass of 1085.0, consistent with the expected molecular weight of 3251.76.

[0201] Procedure for synthesis of SEQ ID NO: 36: The structure of the Ac-K(N3)-(Ttds)4-PRhCTLDKKEAIQGGIVRVNhCSVPEEK-CONH2 peptide is shown in Figure 15.

[0202] SEQ ID NO: 36 (PepSP1380) covers the Pro133-Lys158 region of CD31IgL2-A, similar to SEQ ID NO: 35, but due to the mutation of homocysteines at positions 135 and 152, disulfide bonds with a cyclic structure are formed.

[0203] The synthesis was carried out according to the general procedure. During peptide assembly, double acylation reactions were carried out on the residues of the Ser-Val-Pro, Ile-Val-Arg-Val, and Pro-Arg-homoCys-Thr-Leu fragments. Fmoc deprotection was performed by double treatment of the resin with 20% (V / V) piperidine in DMF at 90 °C for 120 s up to Asp at position 6. After this residue, deprotection was carried out at room temperature to minimize aspartimide formation. The four Ttds and terminal Lys-azide residues were introduced manually in DMF using HOAT (5 eq) and DIPC (5 eq) as coupling reagents. Triple coupling of the final Ttds residue was performed to achieve complete conversion. At the end of assembly, the resin was treated with AcO (10 eq) and DMF for 30 min. Cleavage was performed using Mix 1 according to the general procedure. The mixture was analyzed by analytical UPLC, confirmed by ESI+MS, and lyophilized to obtain the crude product (Y = 73%). The crude material was dissolved at 1 mg / mL in a 9 / 1 mixture of DMSO / HO. Aqueous NH3 was added to reach pH 9, and the mixture was stirred at room temperature for 72 h. After this time, disulfide bridge formation was confirmed by UPLC, and TFA was added to quench the reaction mixture, which was then lyophilized. The crude peptide was purified by RP-HPLC using a Reprosil C8 (250 x 40 mm, 300 A, 5 µm) column with a gradient of solvent B: 20% B over 5 min; 20% B to 40% over 25 min; flow rate 60 mL / min. Fractions containing the product were collected and lyophilized to obtain the purified product as the TFA salt (Y = 5%). The purified peptide was analyzed by LC / MS (Method B). The mass signal found at a retention time of 3.23 min for the [M+4H]4+ peak indicated a peptide mass of 1076.8, consistent with the expected molecular weight of 4301.09.

[0204] Synthesis of SEQ ID NO: 37: The structure of the [#CH2CONH2GTYKSTVIVNNKEKTTAEYQ-(Ttds)4-K(N3)-NH2]-[QHQMLFYhC#DDVLFYNISSMK-CONH2] peptide is shown in Figure 16.

[0205] SEQ ID NO: 37-38 (SP1379) is a heterodimer obtained via a thioether bond between hCys8 of intermediate A (INTA) (QHQMLFYhC#DDVLFYNISSMK-CONH2) and the bromoacetyl group of intermediate B (INTB) [BrCH2CO-G#TYKSTVIVNNKEKTTAEYQ-(Ttds)4-K(N3)-NH2].

[0206] A 5 mM solution of INTB (1 eq) in DMSO was added dropwise to a 2 mM solution of INTA (1 eq) in DMSO in the presence of DIPEA (5 eq). The reaction was stirred at room temperature for 1 h, during which time the reaction progress was monitored by LC / MS. TFA was added to the reaction mixture to reach pH 4, and the crude product was purified by RP-HPLC using a Reprosil C8 (250 x 40 mm, 300 A, 5 μm) column. The following gradient was used: 25% B in 5 min; 25% to 45% B in 25 min; flow rate: 40 mL / min. Fractions containing the product were collected and lyophilized to obtain the purified product as the TFA salt (Y = 21%). The purified peptide was analyzed by LC / MS (Method B). A [M+4H]4+ mass signal was found under the peak at a retention time of 4.35 min, revealing a peptide mass of 1545.5, consistent with the expected molecular weight of 6173.13.

[0207] Synthesis of SEQ ID NO: 38: The structure of the [Ac-K(N3)-(Ttds)4-PRVTLDKKEAIQGGIVRVNSSVPEEK-Ttds-K#(CH2CO)-CONH2]-[QHQMLFYhC#DDVLFYNISSMK-CONH2] peptide is shown in Figure 17.

[0208] SEQ ID NO: 38 is a heterodimer obtained through a thioether bond between hCys8 of intermediate A (INTA) (QHQMLFYhCDDVLFYNISSMK-CONH2) and the bromoacetyl group of intermediate C (INTC) [Ac-K(N3)-(Ttds)4-PRVTLDKKEAIQGGIVRVNSSVPEEK-Ttds-K(BrCH2CO)-CONH2].

[0209] A 5 mM solution of INTC (1 eq) was added dropwise to a 2 mM solution of INTA (1 eq) in DMSO in the presence of DIPEA (5 eq). The reaction was stirred at room temperature for 1 h, during which time the reaction progress was monitored by LC / MS. TFA was added to the reaction mixture to reach pH 4, and the crude product was purified by RP-HPLC using a Reprosil C8 (250 x 40 mm, 300 A, 5 µm) column. The following gradient of eluent B was used: 25% B in 5 min; 25% to 45% B in 25 min; flow rate: 40 mL / min. The product-containing fractions were collected and lyophilized to obtain the purified product as the TFA salt (Y = 5%). The purified peptide was analyzed by LC / MS (Method B). The mass signal found beneath the [M+5H] peak at a retention time of 4.59 min indicated a peptide mass of 1445.9, consistent with the expected molecular weight of 7221.47.

[0210] Synthesis of SEQ ID NO: 1: Intermediate A:QHQMLFYhCDDVLFYNISSMK-CONH2

[0211] The structure of intermediate A is shown in FIG.

[0212] The common linear precursor intermediates of the heterodimers SEQ ID NO:37 (SP1379) and SEQ ID NO:38 (SP1383) were prepared according to the general synthesis and cleavage procedures. All Fmoc deprotection was performed at room temperature. The crude peptide was purified using a Reprosil C4 column (250 x 40 mm, 120 A, 5 µm) with a gradient of 20% B over 5 min, then 20% B to 40% B over 25 min; flow rate: 60 mL / min, wavelength: 214 nm. The product-containing fractions were collected and lyophilized to obtain the purified product as a TFA salt (Y = 2%).

[0213] Synthesis of SEQ ID NO:2: Intermediate B: (BrCH2CO-GTYKSTVIVNNKEKTTAEYQ-(Ttds)4-K(N3)-NH2)

[0214] The structure of intermediate B is shown in FIG.

[0215] The linear precursor of the heterodimer, SEQ ID NO: 37-38 (SP1379), was prepared according to the general synthetic procedure. At the end of the assembly, the free amino group of the last residue (glycine) on the resin was treated with bromoacetic acid (5 equivalents) according to the DIC / HOAt method (4-fold excess relative to the resin load). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by Kaiser test. The resin was filtered, washed with DMF / DCM / DMF (6 times each), and treated with the cleavage mixture (Mix 1) as reported in the general synthetic procedure. The crude peptide was purified by RP-HPLC using a Reprosil C4 (250 x 40 mm, 120 A, 5 µm) column with the following gradient: 20% B for 5 min; 20% B to 40% B for 25 min; flow rate: 60 mL / min; wavelength: 214 nm. The product-containing fractions were collected and lyophilized to obtain the purified product as a TFA salt (Y = 30%).

[0216] Synthesis of SEQ ID NO:3:

[0217] INTC, Ac-K(N3)-(Ttds)4-PRVTLDKKEAIQGGIVRVNSSVPEEK-Ttds-K(BrCH2CO)-CONH2

[0218] The structure of intermediate C is shown in FIG.

[0219] The linear precursor C of the heterodimer SEQ ID NO: 39-40 (SP1383) was prepared according to the general synthetic procedure. The first amino acid loaded onto the resin was lysine, with the side chain amino group protected with alloc. Double acylation was performed on the following residues: SVP, IVRV, and PRVTL. After the PRVTL residue, all Fmoc deprotection was performed at room temperature.

[0220] At the end of the assembly, the resin was suspended in DCM, phenylsilane (24 eq) and Pd(PPh3)4 (0.25 eq) were added, and the resin was shaken for 30 minutes. This cycle was repeated twice. At the end of the second cycle, a solution of 0.5% DIPEA and 0.5% sodium dithiocarbamate in DMF was passed through the resin to remove any palladium residues. Coupling of the deprotected amino group of Lys with bromoacetic acid (5 eq) was carried out in DMF using the DIC / HOAt method (a 4-fold excess relative to the resin loading). The mixture was shaken at room temperature for 1 hour, and the reaction was monitored by Kaiser test. The resin was filtered, washed with DMF / DCM / DMF (6 times each), and treated with the cleavage mixture (Mix 1) as reported in the general synthetic procedure. The crude peptide was purified using a Reprosil C4 column (250 x 40 mm, 120 A, 5 µm) with the following gradient: 20% B in 5 min; 20% B to 40% B in 25 min; flow rate: 60 mL / min, wavelength: 214 nm. The product-containing fractions were collected and lyophilized to obtain the purified product as the TFA salt (Y = 30%).

[0221] Synthesis of SEQ ID NO: 41: Ac-K(N3)-GGSGGSGG-YKDDVLFYNISSMKST-NH2 (SP547) sequence number 41 (PepSP547) is a linear peptide covering the Tyr76-Thr91 region of CD31IgL1-A, with the azide linker positioned at the N-terminus.

[0222] Synthesis of SEQ ID NO: 42: Ac-YKDDVLFYNISSMKST-GGSGGSGG-K(N3)-NH2 (SP548) sequence number 42 (PepSP548) is also a linear peptide covering the Tyr76-Th91 region of CD31IgL1-A, but the azide linker is located at the C-terminus.

[0223] protocol Nitinol discs were either untreated (bare metal) or coated with CD31 analog peptides for domain 1, domain 2, or domains 1 and 2. The tested peptides were grafted onto the nitinol discs using a sequential dip-coating procedure using polydopamine, DBCO-PEG4-amine, and azide CD31 derivative peptides. After this step, all discs were incubated with human aortic endothelial cells for 48 hours. The discs were then washed, and the cells were fixed and stained with formalin solution. Staining was performed using phalloidin conjugated to Alexa Fluor® 488 dye (green fluorescence of actin), DAPI was used for DNA staining, and a CD31 antibody for domain 1 conjugated to APC was used to stain ectodomain 1. Cell adhesion and actin / CD31 expression were assessed by image analysis of the stained surfaces.

[0224] The results are shown in Figures 2 to 6.

[0225] Specifically, as shown in Figure 2, bare or coated nitinol discs were placed on the bottom of culture wells. Primary human aortic endothelial cells (HAECs) were allowed to grow on the discs for a period of 48 h. The discs were then washed with phosphate-buffered saline, fixed in formalin, and processed for fluorescence microscopy. Cells attached to the discs were immersed for 1 h at 37 °C in a solution containing Hoechst 33342 (1 μg / ml, to stain nuclei), phalloidin conjugated to AlexaFluor® 488 dye (0.5 μM, to stain F-actin fibers), and a monoclonal antibody against CD31 domain 1, conjugated to allophycocyanin (to detect the presence and localization of intact CD31 on the cell surface). Then, they were washed, mounted upside down in a glass-bottom imaging chamber, and imaged with an inverted fluorescence microscope. Representative images show that HAECs growing on bare material exhibited strong F-actin staining, reflecting enhanced stress fiber formation, while intact CD31 expression was rather weak. In contrast, discs coated with the CD31 mimetic peptide showed little stress fiber signal and strong, uniform CD31 expression at cell boundaries, reflecting a more physiological endothelial phenotype.

[0226] Figure 3 shows the quantitative analysis of the signal in individual disc images, expressed as "integrated density" (reported by ImageJ analysis open software as signal density multiplied by the area of ​​positive staining). Surface types are indicated on the Y-axis, and data are expressed as mean ± SD (N = 6 / surface type). Each dot corresponds to a specific surface modification. F-actin expression is related to cellular stress in cells in the presence of foreign materials. All surface modifications result in a decrease in cellular stress compared to bare metal, except for the SP1070 peptide, which shows no effect on cellular stress compared to bare metal.

[0227] In Figure 4, the surface type is indicated on the Y-axis. Each dot corresponds to a specific surface modification. Data are expressed as "integrated density" mean ± SD (N = 6 / surface type). CD31 expression is related to the ability of endothelial cells to adopt a physiological phenotype when growing on a specific substrate. Depending on the surface modification of the nitinol discs, significant variations are observed in CD31 expression by HAECs. Four of the displayed CD31 analog peptides (peptides SP1072-1374-1375 and 1380) show a clear positive effect on CD31 expression compared to other modified discs and the bare control.

[0228] The "mirror" bars in Figure 5 report the average integrated density of F-actin (left) and CD31 (right) staining for each peptide type coated onto the disc surface (indicated on the Y-axis) and for bare discs. The data are sorted (top to bottom) according to increasing cellular stress (actin expression) for each coating. As shown in the figure, a degree of cellular stress (detected by high actin expression) and impaired endothelial phenotype (documented by low CD31 expression) are evident in HAECs grown on bare metal discs. Conversely, discs coated with the SP1374 peptide, like the CD31-mimetic surface, result in low cellular stress (low actin expression) and a consistent functional endothelial phenotype (high CD31 expression).

[0229] The data shown in Figure 6 are expressed as the ratio of CD31 to F-actin expression. Compared to bare metal and PDA-only coated discs, cells growing on surfaces coated with peptides SP1072, 1074, 1075, and 1080 displayed a significantly higher CD31 / actin ratio, reflecting a more physiological endothelial cell phenotype.

[0230] Example 1 Functional scores and biomimetic performance Functional Score Following the same protocol as in Figure 6, the effects of different peptides belonging to different groups were tested in batches. Different peptides from each group were tested in batches, and the effects of individual peptides were repeatedly evaluated in separate experiments. Data from all experiments were obtained by computer-assisted analysis of images of the endothelial cell-covered disc side, captured in the blue, green, and red channels of an inverted fluorescence microscope. The functional scores of peptides belonging to different groups were calculated by multiplying the cell count (blue, detected by the number of Dapi+ nuclei) by the integrated density of CD31 expression (red signal due to mouse anti-human CD31 monoclonal antibody, clone 9G11, allophycocyanin-conjugated) and dividing the product by the integrated density of F-actin polymerization (phalloidin-conjugated to a green fluorescent dye).

[0231] The formula used to calculate the score using raw data from all experiments was: number of nuclei x integrated density of CD31 staining. The integrated density of F-actin staining score was scaled from 0-1 using the formula (x-min) / (max-min) for each experiment.

[0232] The data are reported in the table shown in FIG. 22 and the graph in FIG.

[0233] Biomimetic Performance The functional effects (biomimetic performance) of peptides belonging to different groups (peptide numbers are shown in parentheses) were screened using the same protocol as described in the example in Figure 6. Different peptides from each group were tested in batches, and the effects of individual peptides were repeatedly evaluated in separate experiments. Data from all experiments were obtained by computer-assisted analysis of images of discs covered with endothelial cells, captured in the blue, green, and red channels of an inverted fluorescence microscope. The functional scores of peptides belonging to different groups were calculated by multiplying the cell count (blue, detected by the number of Dapi+ nuclei) by the integrated density of CD31 expression (red signal due to mouse anti-human CD31 monoclonal antibody, clone 9G11, allophycocyanin-conjugated) and dividing the product by the integrated density of F-actin polymerization (phalloidin-conjugated to a green fluorescent dye).

[0234] The formula used to calculate the score using the raw data from all experiments was: Number of nuclei x Integrated density of CD31 staining x Integrated density of F-actin staining. The scores were scaled from 0-1 using the formula (x-min) / (max-min) for each experiment, and the scaled scores from all experiments are shown in Figure 24.

[0235] Example 2 Further CD31 / actin scores of the peptides according to the invention compared with the prior art peptide P8RI Materials and Methods Following the same protocol as in Example 1, the peptides according to the invention defined above (SP547, SP548, SP745, SP1374, SP1072, SP1070, SP1071, SP1379 and SP1383) and the prior art peptide P8RI of sequence KWPALFVR (SEQ ID NO: 52) were used.

[0236] result The "CD31 / actin" score is the integrated density of the CD31 immunostaining signal divided by the signal from phalloidin, which binds to polymerized F-actin in the cytoskeleton. This score was used to identify the physiological / non-stressed state of arterial endothelial cells growing on the surface of experimental discs placed at the bottom of culture wells. A higher score indicates a more physiological state, while a lower score reflects a stressed state of endothelial cells, which is associated with prothrombotic / pro-inflammatory activity (as confirmed by the levels of soluble PAI-1 and IL-6 in the supernatants of individual culture wells). Therefore, the higher the CD31 / actin score, the better.

[0237] The results of repeated experiments using different peptides are shown in Figure 25. The CD31 / actin scores obtained with the novel peptides SP745, SP1374, SP1072, SP1070, SP1071, SP1379, and SP1383 are significantly higher than those obtained with bare metal discs or discs coated with PDA alone. Although statistical significance cannot be achieved with a single measurement, sections coated with the novel peptides SP547 and SP548 appear to have CD31 / actin scores comparable to those coated with the other peptides of the invention. Furthermore, the average CD31 / actin scores for all of the peptides of the invention are higher than the average CD31 / actin score for the prior art peptide P8RI.

[0238] conclusion The above results confirm that discs coated with the new peptide according to the invention show higher and therefore better CD31 / actin scores than sections coated with PDA alone and sections coated with the conventional peptide P8RI.

[0239] Example 3 In vivo efficacy of flow diverter stents (CFDs) coated with the peptide SP1072 according to the present invention compared with stents coated only with the hydrophilic polymer NTMA

[0240] Materials and Methods stents Flow diverter stents (CFD) are woven nitinol mesh stents manufactured by Sinomed. They are coated with one of the following coatings: 1. The SP1072 peptide according to the invention, or 2. Hydrophilic polymer eGNTMA (obtained from electrografting of N-[tris(hydroxymethyl)methyl]acrylamide). [ka]

[0241] For the SP1072-coated flow diverter stents, the coating was performed using the following materials and protocol:

[0242] material: Desalinated water Tris 10 mM (molecular weight 121.135 or 1.21 mg / mL) pH 8.5 buffer 0.6g Trizma Base (CAS77-86-1) 500ml demineralized water Adjust pH to 8.5 with HCl Filter through a 0.45 μm filter and store at room temperature. Polydopamine (PDA) 2 mg / mL Dopamine hydrochloride 99% (CAS 62-31-7, molecular weight 189.64, Store at 4°C. Dissolve an appropriate amount of dopamine hydrochloride in 10 mM Tris buffer pH 8.5 to obtain a solution with a final concentration of 2 mg / mL (store in the dark). DBCO (molecular weight, 678.79, DBCO-sulfo-PEG(4)-NH2 (reference, RL-2421, IRISbiotech) Upon receiving the compound, prepare a stock weight of 15-20 mg. Dissolve the powder and Create a 20 mg / mL stock solution in 10 mM Tris buffer pH 8.5. Prepare the required volume for the coating process at a final concentration of 300 µM (203 µg / mL).

[0243] SP1072 peptide is used at 50 μg / mL.

[0244] protocol, 1) Transfer each stent to a 5 mL polypropylene round-bottom tube (BdFalcon Ref 352063). Ensure that the stent falls to the bottom of the tube. 2) Add 4.5 mL of 2 mg / mL PDA solution to each tube. Homogenize each tube overnight (18 + / - 2 hours) at room temperature on a rotating wheel (approximately 20 rpm) and protect from light. Ensure the stent moves within the tube and does not get caught on the cap. The stent color should change from silver to brown / black. After the incubation period, the solution will turn black with small PDA aggregates. 3) Transfer each stent to a new tube and rinse three times with demineralized water. Homogenize on a rotating wheel for 5 minutes after each rinse. 4) Leave the final rinse and sonicate the tube for at least 30 seconds to remove any remaining clumps of PDA. 5) Transfer each stent into a 2 mL Eppendorf tube and include 1.8 mL of DBCO (300 µM) per tube. Homogenize each tube on a rotating wheel (approximately 20 rpm) overnight at room temperature, protected from light. 6) Transfer each stent to a new tube and rinse three times with demineralized water. Homogenize on a rotating wheel for 5 minutes after each rinse. 7) Transfer each stent into a 2 mL Eppendorf tube and include 1.8 mL of CD31 peptide (50 µg / mL) per tube. Homogenize each tube on a rotating wheel (approximately 20 rpm) for 2 h at room temperature, protected from light. 8) Transfer each stent to a new tube and rinse three times with demineralized water. Homogenize on a rotating wheel for 5 minutes after each rinse. 9) Immerse each stent in absolute ethanol for 5 seconds and then allow to dry.

[0245] The comparative hydrophilic polymer eGNTMA-coated flow diverter stents were obtained according to the following protocol: (1) Pre-cleaning treatment: the stent frame was sonicated in acetone, ethanol, and water for injection for 10 min each.

[0246] (2) Preparation of electrografting solution: N-[tris(hydroxymethyl)methyl]acrylamide, concentration 0.30 M; NaNO3, concentration 0.05 M; 4-nitrophenyltetrafluoroborate diazonium salt, 0.005 M; and the remaining amount is DMSO solvent.

[0247] (3) Electrografting process, the pretreated stent was used as the working electrode and platinum foil was used as the counter electrode, which was immersed in the electrografting solution, and a linear sweep voltage from −0.1 V to −3.0 V was applied between the two electrodes at a voltage scan rate of 0.05 V / s for 10 cycles.

[0248] (4) Smoothing and drying of the coating, the electrografted scaffolds were smoothed with acetone (2 L) with nitrogen bubbling (rate 0.5–10 L / min) for 10 min and then placed in a vacuum drying oven at 40 °C for 2 h.

[0249] The same protocol was applied to a knitted mesh of the same composition and to a coupon of the same composition. A 125 nm film was obtained, as measured by profilometry (KLA-Tencor), at the step of scratching the electrografted coupon with a wooden stick.

[0250] Animal procedures A total of eight healthy New Zealand White rabbits (including two reserve animals), male and female, weighing approximately 3 kg, were included in this study. Three observation time points were set: 7, 30, and 60 days after surgery. The experiment was divided into two steps: 1) Creation of an aneurysm animal model; 2) Implant a dense mesh stent into the aneurysm-bearing artery and abdominal aorta of each animal. Stent implantation into the aneurysm-bearing artery was used to examine the safety and efficacy of the stent in treating aneurysms, while stent implantation into the abdominal aorta was used to examine the safety of the tested stent by covering a pair of lumbar arteries. See Table 1 below for details. [Table 18]

[0251] For steps 1) and 2), anesthesia was performed as follows: all surgical procedures were performed using aseptic technique, and experimental animals were under general anesthesia during the implantation surgery.

[0252] On the day of surgery, the animals are sedated and anesthesia is induced with an intramuscular injection of 6 mg / kg of Sutex® 50. If necessary, animals can be anesthetized with isoflurane via inhalation using a breathing mask.

[0253] After successful induction of anesthesia, the experimental animal is intubated through an intraoral thoracic tube to establish respiratory access and connected to a ventilator device to maintain anesthesia through continuous inhalation of an anesthesia-oxygen mixture. It may be necessary to administer atropine to the experimental animal before surgery to prevent vomiting and prevent choking on vomit.

[0254] The anesthetized and intubated experimental animal was placed on the operating table in a lateral or supine position and immobilized using restraint bands. Its position was photographed and recorded (to facilitate the same position and angle for later observation). The surgical site on the right hind leg was prepared, disinfected, and covered with a sheet. If the animal's position was changed, a new sheet and surgical site preparation were required.

[0255] An intravenous needle is inserted into one of the peripheral veins, and medications and fluids are administered as needed through the catheter.

[0256] Step 1) (creation of an aneurysm animal model) was carried out as follows: (a) The rabbit is placed supine on the operating table, and the neck is shaved. The area is usually disinfected with iodoform and alcohol and covered with a towel. (b) Locate the right common carotid artery. Make a midline neck incision (1.5 cm above and below the superior sternal fossa), incise the skin, and separate the right sternocleidomastoid muscle along the lateral side. Locate and release the right common carotid artery, taking care to protect the vagus nerve so that the rabbit's heart rate and breathing do not slow or stop. Every 20 minutes, inject saline into the rabbit's cervical vessels and vagus nerve to keep them moist. Two No. 1 silk wires were wrapped around the right common carotid artery. (c) To completely expose the beginning of the right common carotid artery, open a portion of the right pectoral muscle with tissue scissors and separate it proximally along the right common carotid artery, carefully separating the right common carotid artery and a portion of the right subclavian artery. (d) To create an occluded lumen at the beginning of the right common carotid artery, tie off a No. 1 wire approximately 2.5 cm from the beginning of the right common carotid artery, wrap another No. 1 wire around the knot but do not tie it, and clamp the aneurysm clip at the beginning of the right common carotid artery near the right subclavian artery, ensuring that the inside of the aneurysm clip is below the junction of the right common carotid artery and the right subclavian artery. (e) Injection of elastase: Cut the side wall of the right common carotid artery 1.5 cm from its beginning with ophthalmic scissors, insert a cannula needle (a 22G cannula needle with a syringe attached to the end) containing elastase, and position the tip of the needle as close as possible to the aneurysm clip. Ligate the insertion point of the cannula needle with silk thread to prevent fluid leakage, and inject approximately 75 U of porcine pancreatic elastase into the lumen. (f) Vascular ligation, 20 minutes after elastase ablation, remove the trocar needle, ligate the puncture site, carefully loosen the aneurysm clip, and moisten it with saline drip if necessary. (g) Suture the wound, suturing the muscle and skin layers layer by layer with gauze soaked in dried accumulated blood, and disinfect the incision with iodophor. (h) Immediately after surgery, administer 300 U / kg of heparin sodium and 0.3 g / kg of ceftriaxone sodium solution intravenously. Carefully monitor the animals' vital signs until the procedure is complete. Keep the animals in separate cages. Continue antibiotic use for 3-5 days after surgery.

[0257] Step 2) (implantation of high-density mesh stents into the aneurysmal artery and abdominal aorta of each animal) was performed as follows: (a) Anesthesia, as above. (b) The right femoral artery was dissected and a 5F protective vascular puncture sheath was inserted. (c) A 5F introducer catheter was placed in the aortic arch, and DSA angiography was performed. (d) Microcatheter placement. Intravenous heparin was treated with systemic heparinization, and the microcatheter was pushed through the microguidewire from the subclavian artery to the distal end of the vessel and withdrawn. (e) Stent implantation in the carrier artery. The high-density mesh stent system is inserted into the microcatheter through the introducer sheath, the push rod is advanced to the appropriate position, the introducer sheath is withdrawn, and the push rod is further pushed to push the stent into the carrier artery, adjusting the stent position so that the tumor neck is near the center of the stent. The push rod is fixed and the microcatheter is withdrawn to partially release the stent and position it at the retrieval point. If the stent position is satisfactory, the microcatheter is continued to be withdrawn to fully release the stent. If the position is not satisfactory, the microcatheter is pushed backward to retrieve the stent into the microcatheter, and the stent is then adjusted and released again to completely cover the aneurysm neck. After stent placement is complete, the delivery system and microcatheter are withdrawn. (f) Stent implantation in the abdominal aorta. After the release of the test article stent into the carrier artery, the guide catheter is retracted and positioned in the abdominal aorta. A second stent is placed in the same manner. The stent must be positioned in the abdominal aorta through the beginning of at least one lumbar artery. After release is complete, the delivery system, microcatheter, and guide catheter are retracted, and the femoral artery incision is sutured. (g) Wait for the animal to wake up and feed as usual.

[0258] The test group received a dense mesh stent system implanted in the aneurysm location (common carotid artery) and abdominal aorta of the animal model, while the control group received a dense mesh stent system implanted in the aneurysm-bearing artery. The device was implanted in the abdominal aorta to evaluate the effect of the stent on penetrating and branching vessels.

[0259] In addition to 150 U / kg of IV heparin sodium injection during implantation, animals received antiplatelet therapy (aspirin and clopidogrel) orally at 5 mg / kg once daily starting 3 days before surgery and continued orally at 5 mg / kg postoperatively until the endpoint.

[0260] Histopathological analysis Tissues collected from the stented segments of the carrier arteries were immersion fixed in 10% neutral formalin for at least 48 hours, dehydrated in alcohol gradients, and subjected to xylene clearing for histopathological analysis.

[0261] Resin-stented segments of aneurysmal arteries were transversely sectioned through the aneurysmal neck, with one aneurysmal and two non-aneurysmal sections cut and stained with HE. Non-stented segments were paraffin-embedded, with one proximal and one distal section cut and stained with HE for histopathological evaluation.

[0262] Scanning electron microscope (SEM) analysis One animal was randomly selected at 7 days, 30 days, and 60 days, and the aorta and abdominal aorta with the implanted aneurysm were collected at the above time points and subjected to SEM to observe the endothelialization of the flow-directed stent at the neck of the aneurysm and the opening of the branch vessels of the abdominal aorta.

[0263] NOTE: At the 7 D time point, only the abdominal aorta was examined by SEM, at the 30 D time point, the SEM of the carrier arteries was determined, and at the 60 D time point, the carrier arteries and abdominal aorta were examined by SEM.

[0264] result Verification of eG™ NTMA Film Graft on Electrografted Coupons The infrared spectrum of the eG™ NTMA film is shown in FIG.

[0265] Furthermore, the comparison of the hydrophilicity of the samples can be visualized by the shape of the water droplet (Figure 27). On the bare surface, the water droplet stands on the surface, whereas on the eG™ NTMA surface, the water droplet is completely flat and the water contact angle is barely measurable, indicating that the surface is superhydrophilic.

[0266] These results confirm the efficient electrografting of eG™ NTMA films onto comparative CFD stents.

[0267] SEM results Representative SEM cross sections of stents coated with eGTMNTMA or SP1072 peptides according to the invention at 7 days are shown in FIG.

[0268] In the case of the eGTMNTMA-coated stent (see left), the stent was fully expanded within the vessel, demonstrating good stent-vessel coaptation and vascular lumen patency. Small amounts of thrombus were scattered in localized areas. In the small stent, the stent surface had endothelial coverage, while in the non-endothelialized areas, red blood cells, inflammatory cells, and platelet aggregates adhered to the stent surface to varying degrees. No obstruction was observed in the caliber of the collateral vessel, indicating patency of blood flow within the collateral vessel.

[0269] In the SP1072-coated stent (see right), the stent was fully expanded within the vessel, demonstrating good stent-vessel conformance and vascular lumen patency. No obvious thrombus was observed, in contrast to the eGTMNTMA-coated stent. Partial endothelial coverage was observed on the stent surface (see particularly the good coverage in areas B and D in Figure 26B), and in areas without endothelial coverage, red blood cells, inflammatory cells, and platelets adhered to the stent surface. No obstruction was observed in the caliber of the collateral vessels, indicating patency of blood flow within the collateral vessels.

[0270] Histopathological analysis Representative pathology results on day 7 are shown in FIG.

[0271] In the eGTMNTMA-coated stent (see Figure 29A), the stent in front of the aneurysm neck (see right) is covered with a new wall. However, the latter appears to be infiltrated with leukocytes (black nuclei within the muscle area and near the stent struts, see the bottom right image), with a fresh fibrin layer on the inside and packed with erythrocytes and platelets on the outside (dark gray areas of the fibrin layer, see the black circles in the bottom left image). Furthermore, the thrombus within the aneurysm sac appears disorganized (no evidence of a polymerized extracellular matrix sheet), and fresh blood (erythrocytes, platelets, and leukocytes) is still present within the thrombus. This is important because the fact that fresh blood is still entering and trapped within the sac may increase the risk of delayed aneurysm rupture. The continued arrival of fresh blood generates new waves of thrombus formation / fibrinolysis, ultimately leading to the enzymes of the two systems (thrombin, plasmin, etc.) promoting the degradation of the aneurysm wall. Collectively, these data indicate that the neotissue covering the neck of the aneurysm is hyper-reactive (thrombus-forming and inflamed), possibly to metal (foreign body), and leaky.

[0272] In contrast, in the case of the SP1072-coated stent (see Figure 29A), the thrombus within the aneurysm sac was "organized" and detected as a long, wavy, dark gray line due to the visible, well-defined, aligned extracellular matrix sheets. It is noteworthy that the space between the new wall and the thrombus within the aneurysm sac does not contain amorphous gray material (fresh fibrin or red blood cells), indicating that fresh blood no longer enters and becomes trapped there, thus reflecting a low risk of aneurysm wall degradation by blood enzymes. The thick, regular shape of the arterial wall around the sac supports this assumption, as does the lack of amorphous material / dark core within the thrombus, which supports the assumption that it is impermeable to fresh platelets and coagulation factors.

[0273] The black circle in the upper right image, new arterial wall, is dense and compact (dark gray) with abundant extracellular matrix, and no inflammation (no black nuclei) or thrombosis has formed beneath the stent.

[0274] The presence of an organized thrombus on the outside and an organized new wall on the inside supports the fact that, even though blood may still enter the sac of a large aneurysm, it cannot enter the thrombus or clot on the new wall, even through certain parts of the stent, as shown in the examples. This allows for complete healing and occlusion of the thrombus in a short period of time, compared to situations where blood can contact fresh thrombus or a bare stent. In fact, once the cavity is virtually formed, the application of a CD31-mimetic coating to the bare stent (the part without the new wall) promotes the migration and growth of neighboring endothelial cells onto and between the device, completely covering the organized thrombus and thereby occluding the neck of the aneurysm.

[0275] Representative pathology results for an SP1072-coated stent aneurysm at 60 days are shown in Figure 30. Despite the very large aneurysmal sac, the thrombus is very well organized (detectable by a long, orderly sheet of extracellular matrix spanning the entire thrombus, impermeable to fresh blood, and the absence of leukocyte / platelet / erythrocyte infiltration into the thrombus). As soon as the front of the organized thrombus reaches the stent, the neck is completely covered by a new arterial wall. The latter is likely well organized, as suggested by the lack of inflammation / thrombosis (lack of reaction to a foreign body) of the arterial wall already in contact with the stent struts.

[0276] conclusion The above results show that the SP1072 coating exhibits the following: - Promotes early endothelialization on the inner surface of flow diverter stents, promotes the growth of an organized, blood-impermeable new arterial wall at the entrance to the aneurysmal sac, allowing for the formation of an organized, non-inflammatory thrombus within the sac; and - Endothelialization of the device was rapid and complete at sites other than the aneurysm entrance, with no obvious inflammation or neointima formation.

[0277] Considering the comparable in vitro results obtained with other peptides according to the invention, similar in vivo results would also be expected by the skilled artisan.

[0278] Example 4 The biological effects of flow diverter stents (CFDs) coated with the peptide SP1072 according to the present invention are compared with those coated with the hydrophilic polymer NTMA alone in the absence of administration of antiplatelet compounds.

[0279] The positive results obtained in Example 3 suggest that stents coated with SP1072 (or another peptide according to the invention) may allow a sufficiently rapid re-endothelialization and a sufficient lack of inflammation to prevent the administration of antiplatelet compounds, especially anti-P2Y12 agents, after implantation.

[0280] A pilot experiment was conducted to test this hypothesis.

[0281] Materials and Methods The same materials and protocols as in Example 3 were used, with the following exceptions: Only three animals were used for each condition. · Animals were not given aspirin or clopidogrel after transplantation.

[0282] result Preliminary results indicate that: Animals implanted with SP1072-coated flow-diverting stents remained thrombus-free after 7 days (n=3). In contrast, all animals implanted with eGTMNTMA-coated stents died of thrombosis on day 7 (n=3).

[0283] conclusion Although preliminary, this experiment suggests that patients may not need the use of anti-P2Y12, and more generally antiplatelet therapy (including aspirin), during post-implantation treatment with a medical device coated with the peptide according to the invention.

[0284] "This finding is extremely significant, as it indicates that antiplatelet compounds, particularly anti-P2Y12 therapy, cannot be safely used in individuals at risk for bleeding. Furthermore, even in individuals without a bleeding risk, antiplatelet compounds can cause serious side effects (e.g., peptic ulcers with aspirin and neutropenia with anti-P2Y12), and the use of medical devices coated with the peptides of the invention may prevent these side effects when antiplatelet compounds are not needed or can be used at much lower concentrations."

[0285] Example 5 Comparison of the biological effects of a self-expanding stent coated with the peptide SP1072 according to the present invention with a stent coated only with the hydrophilic polymer NTMA

[0286] Materials and Methods stents Self-expanding stents (ISS) are woven mesh stents made of Nitinol manufactured by Sinomed. They are coated with one of the following coatings: 1. The SP1072 peptide according to the invention, or 2. Hydrophilic polymer eGTMNTMA (obtained from electrodeposition of N-[tris(hydroxymethyl)methyl]acrylamide). SP1072 peptide coating is performed as described in Example 3 above.

[0287] Hydrophilic polymer eGTMNTMA coated stents are obtained as described in Example 3 above.

[0288] Animal procedures In this study, five healthy New Zealand White rabbits were selected as experimental animals, with two additional reserve animals. Three observation time points were performed: 7, 14, and 28 days after surgery. Two animals were observed at the 7-day time point, one at the 14-day time point, and two at the 28-day time point. A total of four stents were implanted in each experimental animal (two in Test Article A and two in Test Article B). The abdominal aorta and both iliac arteries were selected as the stent implantation sites for each animal, with two stents implanted in the abdominal aorta and one in each of the iliac arteries. The postoperative observation period was followed up with imaging diagnostics. One randomly selected sample from each animal in Groups A and B was used for SEM, and the remaining samples were analyzed histopathologically. The general experimental design was as follows: [Table 19]

[0289] surgical procedures, (1) Anesthesia was administered as follows: All surgical procedures are performed using aseptic technique, and the animals are under general anesthesia during the implantation procedure. On the day of surgery, the animals are sedated and anesthesia is induced with an intramuscular injection of 6 mg / kg of Sutex® 50. If necessary, the animals can be anesthetized with isoflurane via inhalation using a breathing mask.

[0290] After successful induction of anesthesia, the experimental animal is intubated through the oro-tracheal plane to establish respiratory access and connected to a ventilator to maintain anesthesia through continuous inhalation of an anesthesia-oxygen mixture. It may be necessary to administer atropine to the experimental animal before surgery to prevent vomiting and prevent choking on vomit.

[0291] The anesthetized, intubated experimental animal is placed on the operating table in a lateral or supine position, and the animal is immobilized using restraining bands. Its position is photographed and recorded (to ensure the same position and angle are used for subsequent observations). The surgical site on the right hind leg is prepared, disinfected, and sheeted. If the animal's position is changed, the surgical site must be sheeted and prepared again.

[0292] An intravenous needle is inserted into one of the peripheral veins, and medications and fluids are administered as needed through the catheter.

[0293] (2) Establish vascular access by inserting a 5F vascular sheath into the left or right common carotid artery.

[0294] (3) Under the guidance of a guidewire, a 5F catheter is introduced into the descending aorta through vascular access and angiography of the iliac arteries is performed. After angiography, quantitative arterial vascular measurements are performed to guide the selection of stent implantation sites.

[0295] (4) Microcatheter placement: After intravenous heparinization with systemic heparinization, the microcatheter is placed. The microcatheter is advanced through the microguidewire to the site where the stent will be implanted in the iliac artery of the experimental animal, and the microguidewire is then withdrawn.

[0296] (5) Implantation of the iliac artery stent: The system passes through the introducer sheath into the microcatheter, advances the pushrod to the appropriate position, withdraws the introducer sheath, and further advances the pushrod to push the stent into the iliac artery. Adjust the stent position and confirm that the stent is in the desired position within the vessel. Fix the pushrod and retract the microcatheter to begin partial release of the stent and place it at the retrieval point. If the stent position is satisfactory, continue to retract the microcatheter to fully release the stent. If the position is not satisfactory, push the microcatheter back to retrieve the stent into the microcatheter, adjust the position, and release it again. After stent placement is complete, retract the delivery system, push the microguidewire back into the microcatheter, and push the microguidewire and microcatheter together into the opposite iliac artery. Stent implantation on the other side is completed in the same manner.

[0297] (6) Implantation of the stent in the abdominal aorta and the iliac artery. After the release of the test pin stent is complete, pull back the microcatheter and place it in the abdominal aorta. Then, place two sets of stents in the abdominal aorta in the same way. At the end of the procedure, all instruments and equipment are removed from the experimental animal.

[0298] (7) Wait for the animal to wake up and keep it as usual until the end of the experiment.

[0299] In addition to IV heparin sodium injection at 150 U / kg during implantation, oral aspirin at 5 mg / kg once daily starting 3 days before surgery and 5 mg / kg orally after surgery until endpoint was administered, and oral clopidogrel at 18.75 mg once daily starting 3 days before surgery and 18.75 mg orally after surgery until endpoint was administered.

[0300] Histopathological analysis Tissue collected from the stented segment of the carrier artery is immersion fixed in 10% neutral formalin for at least 48 hours, dehydrated in alcohol gradients, processed for xylene clearing, and preserved for histopathological analysis.

[0301] Resin-embedded stented segments of the iliac arteries were transversely sectioned and stained with HE. Non-stented segments were embedded in paraffin and sectioned, one at the proximal end and one at the distal end. HE staining was performed for histopathological evaluation.

[0302] Scanning electron microscope (SEM) analysis Randomly select one animal each at 7D (7 days), 14D (14 days), and 28D (28 days), and collect the implanted stented iliac arteries and abdominal aortas at the above time points and observe them by SEM to confirm endothelialization of the ISS stents.

[0303] result Based on the results already obtained with the CFD stents in Example 3, the SP1072 peptide-coated ISS stents are expected to have better endothelialization and limited neointima formation than the eGTMNTMA electrografted ISS stents.

[0304] Example 6 The biological effects of a balloon-expandable CoCr stent coated with the peptide SP1072 according to the present invention are compared with those of a bare metal stent and an HT Supreme stent.

[0305] Materials and Methods stents Three types of balloon-expandable stents are used: CoCr stents coated with SP1072 peptide, Bare metal stents, and The FTSupreme stent, commercialized by Sinomed, contains an eGBuMA primer layer and a 10 μm thick PLGA (poly(lactic acid-succinic acid)) layer, with a sirolimus concentration of 1.2 μg / mm².

[0306] Animal procedures In this study, nine healthy New Zealand White rabbits were selected as experimental animals and two reserve animals. The animals were divided into three observation time points: 7, 14, and 28 days after surgery, with three animals at each time point. Four stents were implanted into each animal, with the abdominal aorta and both iliac arteries selected as stent implantation sites. Two stents were implanted in the abdominal aorta and one stent in each iliac artery. At the end of the postoperative observation period, two samples from each animal with different coatings were randomly selected and subjected to SEM, while the remaining samples were analyzed histopathologically. The general experimental design is shown in Table 4 below. [Table 20]

[0307] Surgical procedure (1) Anesthesia, All surgical procedures were performed using aseptic technique, and the animals were under general anesthesia during implantation. On the day of surgery, the animals were sedated and anesthesia was induced with an intramuscular injection of 6 mg / kg of Sutex® 50. If necessary, animals could be anesthetized with isoflurane using an inhalation mask.

[0308] After successful induction of anesthesia, the experimental animal is intubated through an intraoral thoracic tube to establish respiratory access and connected to a ventilator device to maintain anesthesia through continuous inhalation of an anesthesia-oxygen mixture. It may be necessary to administer atropine to the experimental animal before surgery to prevent vomiting and prevent choking on vomit.

[0309] The anesthetized and intubated animal is placed on the operating table in a lateral or supine position and immobilized using restraint bands. The surgical site is prepped, disinfected, and sheeted. If the animal's position is changed, the surgical site must be re-sheeted and prepped.

[0310] An intravenous needle is inserted into one of the peripheral veins, and medications and fluids are administered as needed through the catheter.

[0311] (2) Establish vascular access by inserting a 6F vascular sheath into the left or right common carotid artery.

[0312] (3) Under the guidance of a guidewire, a 5F catheter was introduced into the cardiac descending aorta via vascular access, and angiography of the abdominal aorta and iliac arteries was performed. After angiography, quantitative arterial vascular measurements were performed to guide the selection of the stent implantation site.

[0313] (4) Implantation of the iliac artery stent. The contrast catheter is withdrawn, the stent delivery system is prepared, and the air in the delivery system's balloon is evacuated until negative pressure is reached. The balloon is then filled with a mixture of contrast agent and heparinized saline. The stent delivery system is guided by a 0.014 guidewire and the stent is delivered to the selected stent implantation site. After the stent is delivered to the iliac artery vascular implantation site, the balloon inflation pressure pump pressurizes and opens the stent, and fluoroscopic images are saved to record the stent implantation information. The balloon is inflated to an appropriate pressure, with a stent diameter to target vessel diameter ratio of 1.10-1.20, and the ballast stent is released and held for 30 seconds to ensure good contact of the implanted stent to the wall.

[0314] (5) After implantation of the stent on one side, the delivery balloon was withdrawn and another angiogram was performed to evaluate the implanted stent. Stenting of the other iliac artery was similarly completed.

[0315] (6) After completing the stent implantation in the abdominal aorta and stent release in the iliac artery, two sets of stents were placed in the abdominal aorta in the same manner. After the operation, all instruments and devices were removed from the experimental animals.

[0316] (7) Wait for the animal to wake up and keep it as usual until the end of the experiment.

[0317] In addition to 150 U / kg of IV heparin sodium injection during implantation, animals received antiplatelet therapy (aspirin and clopidogrel) orally at 5 mg / kg once daily starting 3 days before surgery and continued orally at 5 mg / kg postoperatively until the endpoint.

[0318] Histopathological analysis Tissue collected from the stented segment of the carrier artery is immersion fixed in 10% neutral formalin for at least 48 hours, dehydrated in alcohol gradients, processed for xylene clearing, and preserved for histopathological analysis.

[0319] Resin-embedded stented segments of the iliac arteries were transversely sectioned and stained with HE. Non-stented segments were embedded in paraffin and sectioned, one at the proximal end and one at the distal end. HE staining was performed for histopathological evaluation.

[0320] Scanning electron microscope (SEM) analysis Randomly select one animal each at 7D (7 days), 14D (14 days), and 28D (28 days), and collect the implanted stented iliac arteries and abdominal aortas at the above time points and observe them by SEM to confirm endothelialization of the ISS stents.

[0321] result Based on the results already obtained with the CFD stents in Example 3, the SP1072 peptide-coated balloon-expandable stents are expected to have better endothelialization and limited neointima formation than the balloon-expandable bare metal stents and the HT Supreme stents.

[0322] From the above description, the advantages of the present invention will be apparent to those skilled in the art.

[0323] While the P8RI sequence can maintain the clustering of cleaved CD31 molecules expressed by activated cells, the presence of the peptide of the present invention allows engagement of intact CD31 molecules on all healthy endothelial cells and resting platelets and leukocytes that can come into contact with the implanted device.

[0324] Thus, these cells can receive a "leave-me-alone" signal delivered by trans-homophilic binding of CD31, which is essential for maintaining circulatory and vascularized tissue homeostasis.

[0325] The occurrence of thrombotic or life-threatening hemorrhagic or thromboembolic complications has hindered the use of intravascular devices.

[0326] Devices bearing the mimetic peptides of the present invention are rapidly integrated because they are recognized by platelets and leukocytes as healthy endothelium, i.e., as "self" components.

[0327] Furthermore, their ability to rapidly endothelialize with a physiological endothelial cell phenotype also limits platelet and leukocyte activation at the device implantation site in the long term.

Claims

1. A medical device comprising a coating comprising a peptide that mimics the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction.

2. 2. The medical device of claim 1, wherein the peptide mimicking the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction comprises one of the following sequences: a) Table 1 where, independently of each other, 1Q may be any one of Q, C, L, K, or R; 2H may be any one of H, I, V, Q or R; 5L may be any one of L, R, V, F or E; 9D may be any one of D, E or N; F may be any one of F, V, L or I; 14Y may be any one of Y, H, R or N; 15N may be either N or D; 16I may be any one of I, V, T, or A; 17S may be either S or T; 18S may be either S or T, and the other amino acid X may be any other amino acid; b) Table 2 where, independently of each other, 2K may be either K or R; 3S may be either C or S; 4T may be any one of T, R or S; 5V may be either V or A; 6I may be any one of I, K, V, L, T or S; 8N may be any one of N, S or D; 9N may be any one of N, S, K or R; 11E may be any one of E, Q, V, K or M; 12K may be either K or R; 13T may be any one of T, A or P; 14T may be either T or S; 16E may be any one of E, A, Q or D, and the other amino acid X may be any other amino acid; c) Table 3 where, independently of each other, 3C may be any one of C, V, M or I; 4T may be any one of T, I, M or E; 5L may be either L or V; 6D may be either D or N; 7K may be either K or R; 8K may be any one of K, T, M, R or I; 11I may be any one of I, T, M, V, or E; 12Q may be either Q or E; 14G may be either G or E; 16V may be either V or I; 18V may be either V or I; 19N may be any one of N, T, R, S, G or H; 22V may be any one of V, M or L; 23P may be any one of P, Q, K, E, L or R; 24E may be any one of E, G or N; 26K may be any one of K, Q, E, R or N, and the other amino acid X may be any other amino acid; or d) Any combination of a) to c).

3. The medical device of claim 1, wherein the peptide that mimics the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction comprises one of the following sequences: Table 4

4. The medical device of claim 3 , wherein the peptide mimicking the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction comprises two of the sequences.

5. The peptide mimicking the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction is A peptide based on structure (IA): Table 5 and a peptide based on structure (IB): Table 6 or A peptide based on structure (IA): Table 7 and peptides based on structure (IC) Table 8 The medical device of claim 1 , comprising:

6. The medical device of claim 1, wherein the peptide mimicking the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction comprises the following sequence: Table 9

7. The medical device of claim 1 or 2, wherein the peptide that mimics the cell-cell interaction of trans-homophilic CD31-CD31 domains 1 and 2 is a cyclic peptide.

8. The medical device of claim 1 or 2, wherein the peptide mimicking the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction comprises a linker and / or spacer and / or tail at any terminus or is attached to an amino acid residue.

9. 9. The medical device of claim 8, wherein the linker and / or spacer and / or tail is represented by the following or a combination thereof: Table 10

10. The medical device of claim 1 or 2, wherein the peptide that mimics the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction comprises modifications at the C-terminus and / or N-terminus.

11. The medical device of claim 10 , wherein the modification is selected from the following: Table 11

12. 3. The medical device of claim 1 or 2, wherein the peptide mimicking the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction comprises any one of the following modifications: - Cysteine ​​residues may be replaced by the corresponding homocysteine; L-amino acid residues may be replaced with the corresponding D-amino acid residues.

13. The medical device of claim 1, wherein the peptide mimicking the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction has one of the following structures: Table 12

14. A medical device described in any one of claims 1, 2 and 13 for the prevention or treatment of vascular lesions.

15. A medical device as described in claim 14 for the prevention or treatment of vascular lesions selected from the group consisting of cardiac valve lesions, atherosclerosis, thrombosis, ischemia, hemorrhage, restenosis and aneurysms.

16. A medical device as described in claim 14 for medical use to prevent pathological conditions represented by in-stent stenosis.

17. A medical device as described in claim 14 for the prevention or treatment of a disease in humans or animals.

18. The medical device of any one of claims 1, 2 and 13, which is partially or completely coated with a coating comprising a peptide that mimics the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction.

19. 20. The medical device of claim 18, selected from the group comprising a balloon expandable stent, a self-expanding scaffold, a polymeric tube of a graft stent, a flow shunt mesh, an aortic tube, a heart valve, a stent retriever, a transcatheter mitral valve device, a catheter valve leaflet, or any portion thereof.

20. A medical device as described in any one of claims 1, 2 and 13, wherein the subject to be treated is suffering from a bleeding disorder selected from the group consisting of hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency), von Willebrand disease, and rare factor deficiencies including I, II, V, VII, X, XI, XII and XIII.

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