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 application in the medical field, and is particularly intended to improve 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 injury at the implant site and is initiated by contact of non-biocompatible materials 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 activation of platelets to contain the injury and infiltration of leukocytes to remove debris and prepare the site for tissue regeneration.

[0006] However, medical equipment is not cleared.

[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 vessel wall (hemorrhage).

[0009] One such condition is caused by the presence of heart valves or vascular devices, which are viewed as 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). In 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 render 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) show that immobilization of the CD31 mimetic peptide P8RI(kwpalfvr) reduces blood component reactivity, increases in vivo endothelial cell adhesion, and enhances intravascular device integration in vivo.

[0015] Diaz-Rodriguez et al (EHJ, 2021) revealed that a soluble peptide called P8RI acts like a CD31 agonist. Therefore, the effect of CD31 mimetic metal stent coating on endothelial cell and blood component adhesion, as well as on endothelial strut coverage and foreign body neointimal growth in vivo were studied.

[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] Engagement of CD31 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, with regard to interacting domains 1 and 2 (termed “IgL1” and “IgL2”, respectively), they adopt 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 contained hydrophobic and hydrophilic interactions.

[0020] The two IgL1-2 fragments of the trans-homophilic CD31 molecule packed against each other 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 first 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 first 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 homophilicity of CD31 domains 1 and 2 promoted molecular clustering through strong cis homophilicity and lateral displacement of transmembrane and juxtamembrane extracellular sequences.

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

[0024] Phosphorylation of CD31 depended on the ability of the molecule to remain clustered in the vicinity of 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 disaggregation of CD31.

[0027] The amino acids contained in the P8RI sequence are released from the cis-cognate intermembrane portion of CD31, with which P8RI clusters and maintains 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 that have properties that mimic the trans-allogeneic (domains 1 and 2) CD31-CD31 cell-cell interaction.

[0029] According to a first object, peptides are disclosed that have the property of mimicking the trans-homophilic CD31-CD31 domains 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 peptides 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 the 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" is intended to mean mimicking natural effects.

[0044] In the present invention, the biomimetic peptides are endowed with 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 covered 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 a cell, 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 sequence of an orthologous mammalian CD31 molecule can be obtained from the NCBI ortholog database available at https: / / www.ncbi.nlm.nih.gov / gene / 5175 / ortholog / ?scope=40674. The corresponding region is determined by a skilled person based on an alignment of the CD31 orthologous amino acid sequence 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 sequences, 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 may 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 the mammalian ortholog 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 106-124aa 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) may 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 the mammalian ortholog 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 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.

[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 the mammalian ortholog 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 Group I covalently linked thereto.

[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 sequence IC: [Table 6]

[0081] For the purposes of the present invention, biomimetic peptides also include cyclic peptides. In particular, biomimetic peptides containing cysteine ​​or homocysteine ​​can be made into a cyclic form 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 of interest may be attached to the peptide termini or to a single amino acid residue.

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

[0084] For the purposes of the present invention, biomimetic peptides also include heteropeptides that contain 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 contain peptide sequences linked 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 peptide of the invention is based on the sequence [Table 9]

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

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

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

[0093] According to preferred aspects, the biomimetic peptides of the invention may include the following modifications: cysteine ​​residues may be replaced by the corresponding homocysteine ​​residues, L-amino acids may be replaced by the corresponding D-amino acids. According to preferred embodiments of the invention, the biomimetic peptides of the invention may include a spacer and / or linker and / or tail selected from the following: [Table 13] and combinations thereof, such as: [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] JPEG2024542779000017.jpg138162

[0097] For the purposes of the present invention, the biomimetic peptides of the present invention include peptides having at least 95% identity with any of the above disclosed structures, preferably at least 97%, more preferably at least 99% identity. The percent identity referred to in the present disclosure is determined after optimal global alignment of the sequences being compared, which may therefore include one or more insertions, deletions, truncations and / or substitutions. The alignment is global, meaning that the entire sequences being compared are included over their entire length. The alignment is "optimal", meaning that it is performed so that the number of insertions, deletions, truncations and / or substitutions is as small as possible. The optimal global alignment may be performed using any method of sequence analysis familiar to those skilled in the art, and the percent identity may be calculated. In addition to manual comparison, it is also possible to determine the global alignment using the algorithm of Needleman and Wunsch (1970). For nucleotide sequences, the sequence comparison may be performed using any software familiar to those skilled in the art, such as the Needle software. The parameters used are in particular: "Gap open" 10.0, "Gap extend" 0.5, and EDNAFULL matrix (NCBIEMBOSS 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] More sophisticated medical applications may involve the implantation of devices coated with the 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 peptides is disclosed.

[0106] According to an embodiment of the invention, the coating is a single layer coating, and according to another embodiment of the 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 parts 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 diarylcyclooctyne moieties (DBCO, also known as ADIBO for azadibenzocyclooctyne or DIBAC for dibenzoazacyclooctyne) with azide-labeled reaction partners, 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 a very narrow and specific reactivity towards 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 for grafting a suitable linker, and The third step is to coat the peptide with the invention.

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

[0113] Dopamine is known to self-polymerize into very adherent films on several types of substrates. Polydopamine (hereafter PDA) is a self-assembling polymer formed by the oxidation of dopamine. Indeed, PDA contains dopamine, indole, and pyrrole units. It is known that PDA offers several possibilities for the functionalization of substrates, especially in the immobilization of bioactive molecules, due to its diverse reactive groups. PDA coating can be performed by immersing the device or a part of it in a solution containing a salt of dopamine, in particular dopamine hydrochloride or dopamine ammonia. The solution can be an aqueous, alcoholic, or aqueous-alcoholic solution. The solvent of the solution is preferably alcohol, in particular absolute ethanol, if the device is sensitive to water (e.g. in case of corrosion). The device can be pretreated before immersion in the dopamine solution, for example 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 non-polydopamine coated areas and PDA aggregates. Rinsing steps can be performed with demineralized water or alcohol.

[0115] The second step corresponds to the immobilization of a linker on the polydopamine coating for the subsequent conjugation of the peptide of interest. Any linker can be used that allows the immobilization of the biomimetic peptide. For that 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 may be any suitable bifunctional reagent. The linker is preferably bioorthogonal.

[0117] The linker is preferably suitable for click chemistry as disclosed above. Thus, 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 towards polydopamine coating.

[0118] To enhance solubility in water and commonly used organic solvents with moderate polarity, the linker preferably includes a spacer. The spacer is preferably a polymer or oligomer. Potential polymers or oligomers that may be used 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. In particular, the PEG4 hydrophilic 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 part thereof obtained after the second step is contacted by suitable means with a biomimetic peptide containing a specific functional group. The grafting can be carried out by immersing the device or 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 may 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 fully or partially covered with the biomimetic peptides of the invention include coronary stents, flow diverting stents, aortic tubes, heart valves, balloon expandable stents, self-expanding scaffolds, polymer tubes of graft stents, flow diverting meshes, aortic tubes, heart valves, stent retrievers, transcatheter mitral valve devices, catheters, leaflets or portions 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 steels, cobalt chrome alloys, platinum chrome alloys, nickel titanium alloys (also known as Nitinol), cobalt chrome 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 a particular aspect, methods for the prevention or treatment of cardiac and vascular pathologies include the use of devices fully or partially coated with the inventive coatings, in particular balloon-mounted stents, heart valve bioprostheses, flow diverters, which devices can be used in interventional cardiovascular procedures such as coronary and peripheral arterial revascularization, interventional neurology, heart valve implantation, etc.

[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, a method for preventing a condition manifested by in-stent stenosis is 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 the adhesion of platelets to the surface of the foreign body, which triggers the recruitment and activation of leukocytes and subsequently the initiation of a series of reactions ultimately leading 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 leading to a reduction in the vascular lumen, e.g., the development of (re)stenosis (see, e.g., Forrester et al., J Am Coll Cardiol., 17, 758-769 (1991)).

[0132] In the context of cardiac and vascular pathology, it is for good reason that individuals implanted with medical devices such as stents or valvular bioprostheses are generally 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. The use of one, and more frequently two, antiplatelet therapy aims to eliminate the risk of platelet activation in contact with the stent, which will be exposed to the bloodstream until it is 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. Complicated therapy ultimately exposes patients to the risk of bleeding, and this observation prompted the evaluation of the safety of shortening the duration of antiplatelet therapy. Clinical trials with drug-eluting stents with reduced dual antiplatelet therapy (DAPT) to 30 days are currently ongoing. Once DAPT is completed, antiplatelet therapy typically consists of continuation with aspirin alone (without anti-P2Y12 therapy).

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

[0134] However, antiplatelet therapy can be associated with significant side effects and difficulties, especially in individuals with bleeding disorders (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). In particular, anti-P2Y12 drugs are very potent molecules that tend to cause bruising all over the body without any specific reason. Moreover, no dentist, gastroenterologist, or surgeon would want to touch a patient on anti-P2Y12 therapy to avoid the risk of uncontrollable bleeding during interventions. In some cases, this can be a serious problem, as it interferes with interventions for some diseases other than cardiovascular. These molecules also have notable 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 in contact with the device may be prevented or significantly reduced, such that antiplatelet treatment which normally follows after implantation of the device is not necessary or at least reduced to a lower dose or administered for a shorter period than usual.

[0136] Thus, following 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 heart 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 2-fold, at least 3-fold, at least 4-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 2-fold, at least 3-fold, at least 4-fold shorter) than is 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 treatment after implantation of a medical device (especially a stent) and not 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 items 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, the individual in which the medical device according to the invention is implanted takes a dose of anti-P2Y12 therapy that is significantly lower (e.g., at least 2-fold, at least 3-fold, at least 4-fold, etc.) than is recommended for drug-eluting stents in traditional dual antiplatelet therapy (DAPT). In particular, after implantation, the individual can take 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 for the intake of ticlopidine to 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 dosage of ticagrelor should be 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 (typically half the daily dosage taken twice daily).

[0143] It is desirable for the intake of prasugrel to 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 2-fold, at least 3-fold, at least 4-fold, 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 2-fold, at least 3-fold, at least 4-fold, 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 2-fold, at least 3-fold, at least 4-fold, etc.) than that recommended for a drug-eluting stent.

[0146] For example, an individual may take DAPT for a period of 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 altogether 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 strongly reduces the side effects (bleeding events) associated with antiplatelet therapy. However, it is especially 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 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 for 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 endothelialization of blood vessels, 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 recovery of their functional properties.

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

[0158] [Figure 1] 1A and 1B show the structure of the octapeptide P8RI. [Diagram 2] Figure 2 shows the strategy used to analyze the biocompatibility of nitinol discs interacting with human endothelial cells. [Diagram 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. [Diagram 5] FIG. 5 shows quantitative analysis of F-actin and CD31 expression of HAECs growing on coated and bare control nitinol discs. [Figure 6] FIG. 6 shows the ratio of CD31 to F-actin expression. [Figure 7-17] 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 functional score results of the inventive peptides belonging to different groups. [Diagram 23]FIG. 23 shows a graph depicting the functional score results 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. [Diagram 25] FIG. 25 shows the ratio of CD31 to F-actin expression. [Figure 26] FIG. 26 shows the infrared spectrum of 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. [Diagram 30] Figure 30 shows the histopathological analysis of SEM cross sections 60 days after CFD stent implantation, using the SP1072 peptide coating.

[0159] Specific 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" stands for N,N'-diisopropylcarbodiimide. "DIPEA" stands for N,N-diisopropylethylamine. "DMF" refers to dimethylformamide. "Fmoc" stands for fluorenylmethyloxycarbonyl. "HOAt" refers to 1-hydroxy-7-azabenzotriazole; "HPLC" stands for high performance liquid chromatography. "LCMS" stands for liquid chromatography / mass spectrometry. "UPLC" stands for high performance chromatography. "RP-HPLC" stands for 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" stands for trifluoroacetic acid. "Trt" refers to trityl. EXAMPLES

[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] In addition, 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 in a microwave oven at 90 °C for 3 min using a 5-fold excess of activated amino acids over the free amino groups of the resin. Amino acids were activated with equimolar amounts of a 0.5 M solution of DIC in DMF and a 1 M solution of Oxyma in DMF.

[0169] The following conditions were used: Standard deprotection: 20% piperidine in DMF for 2x120 sec, 90°C Wash: 4xDMF Standard single coupling: 5 equiv. AA 0.4M / 5 equiv. DIC 1M / 5 Equivalent Oxygen 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 performed 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 into 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] Peptides were analyzed by analytical UPLC and confirmed by ESI+mass spectrometry. Crude peptides were purified by conventional preparative RP-HPLC purification using a Waters 2489 HPLC system (UV detection at 214 nm wavelength). The following solvents were used: acetonitrile + 0.1% TFA (mobile phase A) and water + 0.1% TFA (mobile phase B). Fractions containing the product were collected and lyophilized to obtain the purified product as the TFA salt. Unless otherwise stated, 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 structures of the peptides are 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 serine 87 in the C-terminal position, with two thiol groups involved in a disulfide bond.

[0180] After synthesis and cleavage according to the general procedure (Mix1), the mixture was analyzed by analytical UPLC, confirmed by ESI+mass spectrometry, and lyophilized to obtain the crude product (Y=64%). The crude material was dissolved in a mixture of 8 / 2 acetonitrile / water 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 lyophilized. The crude peptide was purified by reversed-phase HPLC using a preparative Reprosil Gold C4 (250x40mm, 120A, 5μm) column. The following gradient of eluent B was used: 20% B to 20% B in 5 min, to 40% B in 25 min, flow rate 60mL / min, wavelength 214 nm. SEQ ID NO:1 was isolated as a white lyophilized solid (Y=5%), TFA salt. The purified peptide was dissolved in a mixture of 8 / 2 acetonitrile / water at a concentration of 1 mg / mL. HCl 50 mM (10 eq) was added and the mixture was stirred for 1 h at room temperature. 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 with retention time 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 covering 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 substituted with serine.

[0182] After synthesis and cleavage according to the general procedure (Mix1), the mixture was analyzed by analytical UPLC, confirmed by ESI+mass spectrometry, and lyophilized to obtain the crude product (Y=66%). The crude peptide was purified by reversed-phase HPLC using a preparative Reprosil Gold C4 (250x40mm, 120A, 5μm) column. The following gradient of eluent B was used: 20% B for 5 min to 40% B for 20 min, flow rate 60mL / min, wavelength 214nm. SEQ ID NO:2 was isolated as an amorphous lyophilized solid (Y=4%), TFA salt. The peptide was dissolved in a mixture of acetonitrile / water 8 / 2 at a concentration of 1mg / mL, HCl 50mM (10eq) was added and the mixture was stirred for 1h at room temperature. 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 at 2.85 min of [M+3H]3+ retention time revealed a peptide mass of 989.4, consistent with the expected molecular weight of 2964.22.

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

[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 Ac2O (10eq) and DMF for 30 min. Cleavage was carried out according to the general procedure using Mix1, the mixture was analyzed by analytical UPLC, confirmed by ESI+mass spectrometry and lyophilized (Y=61%). The crude material was dissolved in a mixture of 8 / 2 acetonitrile / water at a concentration of 1mg / 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 lyophilized. The crude peptide was purified by reversed-phase HPLC using a preparative ReprosilGoldC4 (250x40mm, 120A, 5μm) column. The following gradient of eluent B was used: 25% B in 5 min to 25% B in 25 min, 40% B in 25 min, flow rate 60mL / 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, HCl 50 mM (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 with retention time 2.90 min revealed a peptide mass of 1171.0, which corresponds to 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 structures of the peptides are shown in FIG.

[0187] Seq. no. 32 (PepSP1375) covers the same region as Gln70-Lys89 of CD31IgL1-A and the long linker of the Ttds unit.

[0188] During peptide assembly on solid phase, 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 the 9th position. 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, 10um) column and a gradient of solvent B as follows, 15% B in 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 showed a peptide mass of 969.2, which matches 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 FIG.

[0191] SEQ ID NO: 33 (PepSP1374) is a sequence in which the Gln70-Lys89 region of CD31IgL1-A of SEQ ID NO: 32 has been mutated to homocysteine, and Gln70hCys and Met88hCys form a disulfide bond having 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 DMF containing 20% ​​(V / V) piperidine at 90 °C for 120 s. 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%). The crude peptide was analyzed by analytical UPLC and confirmed by ESI+mass spectrometry. The crude material was dissolved at a concentration of 1 mg / mL in a 9 / 1 mixture of DMSO / H2O, aqueous NH3 was added to reach pH 9, and the mixture was stirred at room temperature for 48 h. After this time, the formation of disulfide bridges was confirmed by UPLC, and the mixture was lyophilized. Purification of the crude material was performed on a WatersXBridgeC18 (250x50mm, 120A, 10um) column using the following gradient, 15% B for 5 min; 15% to 35% of 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 indicates 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 for all natural amino acids and Fmoc-NH-PEG4-COOH. After synthesis and cleavage performed according to the general procedure (Mix1), the mixture was analyzed by analytical UPLC and confirmed by ESI+ mass spectrometry; lyophilized to obtain the crude product (Y=70%). LCMS analysis calc. for C96H166N27O33; 2225.21 Da; found; 1114.0 (M+2)2+. The crude peptide was purified by reversed-phase HPLC using a preparative WatersXBridgeC18 (250x50mm, 120A, 10μm) column, using the following gradient of eluent B: 5% B to 5% B for 5 min, 5% B to 25% B for 20 min, flow rate 80mL / min. SEQ ID NO:34 was isolated as an amorphic 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 retention time 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 FIG.

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

[0197] Synthesis and cleavage were performed according to the general procedure (Mix1), and 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 / H2O, aqueous NH3 was added to reach pH 9, and the mixture was stirred at room temperature for 48 h. The formation of disulfide bridges was confirmed by UPLC, and the mixture was lyophilized. The crude peptide was purified by reversed-phase HPLC using an XBridgeC18 (250x50mm, 120A, 10um) column, using the following gradient of solvent B: 5% B to 5% B for 5 min, to 25% B for 20 min, with a flow rate of 80mL / min. Fractions containing the product 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). A [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 FIG.

[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 (Mix1), and the cleaved 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 reversed-phase HPLC using a preparative double Waters DeltaPack C18 cartridge (100x40mm, 300A, 15μm). The following gradient of eluent B was used: 5% B for 5 min, to 25% B for 20 min, flow rate 80mL / min. The fractions containing the product were collected and lyophilized to obtain the purified product as a TFA salt (Y = 35%). 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 1085.0, which matches 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 like SEQ ID NO: 35, but due to the mutation of homocysteine ​​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 the peptide assembly, double acylation reactions were carried out on the residues of Ser-Val-Pro, Ile-Val-Arg-Val, and Pro-Arg-homoCys-Thr-Leu fragments. Fmoc deprotection was carried out by double treatment of the resin with 20% (V / V) piperidine in DMF for 120 s at 90 °C up to Asp at position 6. After this residue, deprotection was carried out at room temperature to minimize aspartimide formation. The four Ttds and the terminal Lys-azido residues were introduced manually in DMF with HOAT (5 eq) and DIPC (5 eq) as coupling reagents. Triple coupling of the last Ttds residue was carried out to achieve full conversion. At the end of the assembly, the resin was treated with Ac2O (10 eq) and DMF for 30 min. Cleavage was performed using Mix1 according to the general procedure, and the mixture was analyzed by analytical UPLC, confirmed by ESI+mass spectrometry, and lyophilized to obtain the crude product (Y=73%). The crude material was dissolved at a concentration of 1 mg / mL in a 9 / 1 mixture of DMSO / H2O, aqueous NH3 was added to reach pH 9, and the mixture was stirred at room temperature for 72 h. After this time, the formation of disulfide bridges was confirmed by UPLC, and TFA was added to stop the reaction mixture, which was then lyophilized. The crude peptide was purified by RP-HPLC using a Reprosil C8 (250x40mm, 300A, 5μm) column, using the following gradient of solvent B: 20% B for 5 min; 20% B to 40% for 25 min; flow rate 60mL / min. Fractions containing the product were collected and lyophilized to obtain the purified product as a 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 the progress of the reaction was monitored by LCMS. TFA was added to the reaction mixture to reach pH 4, and the crude product was purified by RP-HPLC using a Reprosil C8 (250x40mm, 300A, 5μm) column. The following gradient was used, 25% B in 5 min; 25% to 45% B in 25 min; flow rate, 40mL / min. Fractions containing the product were collected and lyophilized to obtain the purified product as a 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 retention time 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 the progress of the reaction was monitored by LCMS. TFA was added to the reaction mixture to reach pH 4, and the crude product was purified by RP-HPLC using a Reprosil C8 (250x40 mm, 300 A, 5 μm) as the column. The following gradient of eluent B was used: 25% B for 5 min; 25% to 45% B for 25 min; flow rate, 40 mL / min. Fractions containing the product were collected and lyophilized to obtain the purified product as a TFA salt (Y=5%). The purified peptide was analyzed by LC / MS (Method B). The mass signal found under the peak at a retention time of [M+5H]5+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 intermediate of heterodimers sequence number 37 (SP1379) and sequence number 38 (SP1383) was prepared according to the general procedure of synthesis and cleavage, all Fmoc deprotection was performed at room temperature. The crude peptide was purified using a Reprosil C4 (250x40mm, 120A, 5μm) as column with the following gradient of eluent B: 20% B for 5 min; 20% B to 40% B for 25 min; flow rate, 60mL / min, wavelength 214nm. The fractions containing the product were collected and lyophilized to obtain the purified product as 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 procedure of synthesis. At the end of the assembly, the free amino group of the last residue (glycine) on the resin was treated with bromoacetic acid (5 equiv.) according to the DIC / HOAt method (4-fold excess relative to the resin load). The mixture was shaken for 1 h at room temperature 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 (Mix1) as reported in the general synthesis procedure. The crude peptide was purified by RP-HPLC using a Reprosil C4 (250x40mm, 120A, 5μm) column with the following gradient: 20% B for 5 min; 20% B to 40% B for 25 min; flow rate, 60mL / min, wavelength 214nm. The fractions containing the product 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 following the general synthetic procedure. The first amino acid loaded onto the resin was lysine protected with alloc at the side chain amino group. Dual acylation was performed for the following residues, SVP, IVRV, and PRVTL. After the PRVTL residue, all Fmoc deprotections were 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 min. The 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 palladium residues. Coupling of the deprotected amino group of Lys with bromoacetic acid (5 eq) was carried out in DMF by the DIC / HOAt method (4-fold excess relative to the resin loading). The mixture was shaken at room temperature for 1 h and the reaction was monitored by the Kaiser test. The resin was filtered, washed with DMF / DCM / DMF (6 times each) and treated with the cleavage mixture (Mix1) as reported in the general synthesis procedure. The crude peptide was purified using a Reprosil C4 (250x40mm, 120A, 5μm) column with the following gradient: 20% B in 5 min; 20% B to 40% B in 25 min; flow rate, 60mL / min, wavelength 214nm. The fractions containing the product were collected and lyophilized to obtain the purified product as a 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 are either untreated (bare metal) or coated with CD31 analog peptides of domain 1, domain 2, or domains 1 and 2. The tested peptides are grafted onto the Nitinol discs following a sequential dip-coating layering procedure using polydopamine, DBCO-PEG4-amine, and azide CD31 derivative peptides. After this step, all discs are incubated with human aortic endothelial cells for 48 h. The discs are then washed and the cells are fixed and stained with formalin solution. Staining is performed using phalloidin coupled to Alexa Fluor® 488 dye (green fluorescence for actin), DAPI is used for DNA staining, and CD31 antibody of domain 1 is conjugated to APC and used to stain ectodomain 1. Cell adhesion and actin / CD31 expression were assessed by image analysis of the surfaces after staining.

[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 in 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 Hoechst33342 (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 at the cell surface), 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 formation of stress fibers, while intact CD31 expression was rather weak. In contrast, discs coated with a CD31 mimetic peptide showed strong and uniform CD31 expression at cell borders with almost no stress fiber signal, 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 of 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, important variations are observed regarding CD31 expression by HAECs. Four of the displayed CD31 analogue peptides (peptides SP1072-1374-1375 and 1380) show a clear positive effect on CD31 expression compared to other modified discs and to 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 on the disc surface (indicated on the Y-axis) and for bare discs. 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) is evident in HAECs grown on bare metal discs. Conversely, disc-like CD31-mimetic surfaces coated with SP1374 peptide 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 clearly showed higher CD31 / actin ratios, reflecting a more physiological endothelial cell phenotype.

[0230] Example 1 Functional scores and biomimetic performance Feature scores 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 disc side covered with endothelial cells, captured in the blue, green and red channels of an inverted fluorescence microscope. Functional scores of peptides belonging to different groups were calculated by multiplying the number of cells (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 the raw data from all experiments was: number of nuclei x integrated density of CD31 staining. The integrated density of F-actin staining scores were scaled from 0-1 for each experiment using the formula (x-min) / (max-min).

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

[0233] Biomimetic Performance The functional effect (biomimetic performance) of peptides belonging to the different groups (peptide numbers are indicated in brackets) was screened using the same protocol as described in the example in Figure 6. Different peptides from each group were tested in batches and the effect of each individual peptide was repeatedly evaluated in separate experiments. Data from all experiments were obtained by computer-assisted analysis of images of the disc side covered with endothelial cells, captured in the blue, green and red channels of an inverted fluorescence microscope. The functional score of peptides belonging to the different groups was calculated by multiplying the number of cells (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 Integrated density of F-actin staining The scores were scaled from 0-1 in each experiment using the formula (x-min) / (max-min) 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 of phalloidin, which binds to polymerized F-actin of the cytoskeleton. This score was used to identify the physiological / non-stressed state of arterial endothelial cells growing on the surface of the experimental discs placed at the bottom of the culture wells. A higher score indicates a more physiological state, whereas a lower score reflects a stressed state of endothelial cells and is associated with prothrombotic / pro-inflammatory activity (confirmed by the levels of soluble PAI-1 and IL-6 in the supernatants of individual culture wells). Thus, the higher the CD31 / actin score, the better.

[0237] The results of repeated experiments with different peptides are shown in Figure 25. The CD31 / actin scores obtained with the new peptides SP745, SP1374, SP1072, SP1070, SP1071, SP1379 and SP1383 according to the invention are significantly higher than those obtained with bare metal discs and discs coated with PDA alone. Although statistical significance cannot be achieved with a single measurement, it appears that sections coated with the new peptides SP547 and SP548 have CD31 / actin scores comparable to sections coated with other peptides according to the invention. Moreover, the average CD31 / actin score of all peptides according to the invention is higher than the average CD31 / actin score of 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 only and sections coated with the conventional peptide P8RI.

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

[0240] Materials and Methods Stents Flow Diverter Stents (CFD) are a woven mesh 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 eGNTMA (derived from electrografting of N-[tris(hydroxymethyl)methyl]acrylamide). [ka]

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

[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) 2mg / mL Dopamine hydrochloride 99% (CAS62-31-7, molecular weight, 189.64, Store at 4℃) 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 (keep 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 Make a stock solution of 20 mg / mL 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] The SP1072 peptide is used at 50 μg / mL.

[0244] protocol, 1) Transfer each stent into a 5 mL polypropylene round-bottom tube (BdFalcon Ref352063). 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 on a rotating wheel (approximately 20 rpm) overnight (18 + / - 2 hours) at room temperature and protect from light. Do not allow the stent to move within the tube and get caught on the cap. The stent should change color 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 min after each rinse. 4) Leave out 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 contain 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 3 times with demineralized water. Homogenize on a rotating wheel for 5 min 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 min after each rinse. 9) Immerse each stent in absolute ethanol for 5 seconds and then dry.

[0245] Comparative hydrophilic polymer eGNTMA-coated flow diverter stents were obtained according to the following protocol: (1) cleaning pretreatment, the stent frame was ultrasonicated 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; 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 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 step of scratching the electrografted coupon with a wooden stick resulted in a film of 125 nm, as measured by profilometry (KLA-Tencor).

[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 days, 30 days, 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 aneurysmal artery and abdominal aorta of each animal. Stent implantation into the aneurysmal 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 the 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 Sutex® 50. If necessary, animals can be anesthetized with isoflurane by inhalation using a breathing mask.

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

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

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

[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, usually disinfected with iodoform and alcohol, and covered with a towel. (b) Locate the right common carotid artery, make a midline cervical incision (1.5 cm above and below the superior sternal fossa), incise the skin and separate it along the lateral side of the right sternocleidomastoid muscle, locate and release the right common carotid artery, paying attention to protecting 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 moisten them. Two No. 1 silk wires were wrapped around the right common carotid artery. (c) To completely expose the starting part of the right common carotid artery, open a part of the right pectoral muscle with tissue scissors and separate it proximally along the right common carotid artery, and carefully separate the right common carotid artery and a part of the right subclavian artery. (d) Create an occluded lumen at the beginning of the right common carotid artery by ligating a No. 1 wire approximately 2.5 cm from the beginning of the right common carotid artery and wrapping another No. 1 wire around the knot but not ligating it, and clamping an aneurysm clip to the beginning of the right common carotid artery near the right subclavian artery, making sure that the medial side 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 containing elastase (a 22 G cannula needle with a syringe attached to the end) so that the tip of the needle is as close as possible to the aneurysm clip, tie off the insertion part of the cannula needle with silk thread to prevent any liquid from leaking, and inject approximately 75 U of porcine pancreatic elastase into the lumen. (f) Vascular ligation. Twenty 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 in layers with gauze soaked in dried accumulated blood, and disinfecting the incision with iodophor. (h) Immediately after surgery, heparin sodium 300 U / kg and ceftriaxone sodium solution 0.3 g / kg were injected intravenously, and the animals' vital signs were closely observed until cleaning, and they were kept in separate cages. Continue the use of antibiotics 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 at the carrier artery position, 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, the push rod is further pushed to push the stent into the carrier artery, and the stent position is adjusted so that the tumor neck is near the center of the stent. The push rod is fixed, and the microcatheter is pulled back to partially release the stent and bring it into the position of the retrieval point. If the stent position is satisfactory, the microcatheter is continued to be pulled back 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 position is adjusted and released again to completely cover the aneurysm neck. After the stent placement is completed, the delivery system and the microcatheter are withdrawn. (f) Stent implantation in abdominal aorta, test article at carrier artery position After the release of the stent is completed, pull back the guide catheter and place it in the abdominal aorta position, and place the second stent in the same way. The stent should be placed in the abdominal aorta through the beginning of at least a pair of lumbar arteries. After the release is completed, pull back the delivery system, microcatheter, and guide catheter, and suture the femoral artery incision. (g) Wait for the animal to wake up and feed as usual.

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

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

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

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

[0262] Scanning electron microscope (SEM) analysis One animal each was randomly selected at 7D (7 days), 30D (30 days), and 60D (60 days), and the aorta with the implanted aneurysm and abdominal aorta 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 Confirmation 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 on 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 in the vessel, with good stent-vessel coaptation and patency of the vessel lumen. Small amounts of thrombus were scattered in partial areas. In the small stent, there was an endothelial coating on the stent surface, and in the areas without endothelial coating, there were different degrees of red blood cells, inflammatory cells, and platelet aggregates attached to the stent surface. No obstruction was observed in the caliber of the collateral vessel, indicating patency of blood flow in the collateral vessel.

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

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

[0271] In the eGTMNTMA-coated stent (see Fig. 29A), the stent in front of the neck of the aneurysm (see right side) is covered with new wall. However, the latter appears to be infiltrated with leukocytes (black nuclei in the muscle part and near the stent struts, see bottom right picture), layered with fresh fibrin layer on the inside and packed with erythrocytes and platelets on the outside (dark grey areas of fibrin layer, see black circle on bottom left picture). Moreover, the thrombus in the aneurysm sac appears unorganized (no evidence of the presence of polymerized extracellular matrix sheets) and fresh blood (erythrocytes, platelets, leukocytes) is still inside the thrombus. This is important because the fact that fresh blood still enters and is trapped in the sac may increase the risk of delayed aneurysm rupture. The continuous arrival of fresh blood will generate new waves of thrombus formation / fibrinolysis, which will eventually allow enzymes of the two systems (thrombin, plasmin, etc.) to promote the degradation of the aneurysm wall. Collectively, these data indicate that the neotissue covering the neck of the aneurysm is hyperreactive (thrombotic and inflamed), possibly to metal (foreign body), and prone to leaking.

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

[0273] Black circle in the upper right image, new arterial wall, extracellular matrix abundant and compact (dark grey), without inflammation (no black nuclei) or thrombosis 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 large aneurysm sac, it cannot enter the thrombus or clot on the new wall, even through certain parts of the stent, as shown in the examples, allowing for a complete healing of the thrombus and occlusion in a short period of time, compared to the situation where blood can contact a fresh thrombus and a bare stent. In fact, once the cavity is virtual, the CD31-mimetic coating on the bare stent (parts without new wall) promotes the migration and growth of neighboring endothelial cells on and between the device, completely covering the organized thrombus and thereby occluding the neck of the aneurysm.

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

[0276] conclusion The above results show that the SP1072 coating exhibits: - 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 the formation of an organized, noninflammatory thrombus within the sac; and - At sites other than the aneurysm entrance, endothelialization of the device was rapid and complete, 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 be expected by the skilled artisan.

[0278] Example 4 To compare the biological effectiveness of a flow diverter stent (CFD) coated with the peptide SP1072 according to the invention with a stent coated only with the hydrophilic polymer NTMA in the absence of administration of an antiplatelet compound.

[0279] The positive results obtained in Example 3 suggest that a stent 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 flow-diverting stents coated with SP1072 were alive and thrombus-free after 7 days (n=3). In contrast, all animals implanted with eGTMNTMA-coated stents died of thrombosis by day 7 (n=3).

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

[0284] This result is very significant as antiplatelet compounds, especially anti-P2Y12 therapy, cannot be safely used in individuals at risk for bleeding. Moreover, even in individuals without bleeding risk, antiplatelet compounds can cause significant side effects (e.g., peptic ulcers with aspirin, neutropenia with anti-P2Y12), and the use of medical devices coated with the peptides according to the invention may prevent these side effects when antiplatelet compounds are not needed or can be used in 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 and a stent coated only with the hydrophilic polymer NTMA.

[0286] Materials and Methods Stents Self-expanding stents (ISS) are woven mesh 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 by 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 are selected as experimental animals, and two reserve animals are added. There are three observation time points: 7 days, 14 days, and 28 days after surgery, with two animals at the 7th day time point, one at the 14th day time point, and two at the 28th day time point. A total of four stents are implanted in each experimental animal (two in test article A and two in test article B), and the abdominal aorta and bilateral iliac arteries are selected as the stent implantation sites for each animal, with two stents implanted in the abdominal aorta and one in the bilateral iliac arteries. The postoperative observation period is followed up with imaging diagnosis, and one randomly selected sample from each animal in groups A and B is used for SEM, and the remaining samples are analyzed histopathologically. The general design of the experiment is as follows: [Table 19] NOTE: Test articles Group A and Group B are two different coatings of self-expanding stents.

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

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

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

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

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

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

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

[0296] (5) Implantation of the iliac artery stent: the system enters the microcatheter through the introducer sheath, advances the push rod to the appropriate position, withdraws the introducer sheath, pushes the push rod further to push the stent into the iliac artery, and adjusts the position of the stent to ensure that the stent is in the desired position in the blood vessel. Fix the push rod and pull back the microcatheter to start the partial release of the stent and place it at the retrieval point position. If the position of the stent is satisfactory, continue to pull back the microcatheter to fully release the stent. If the position is not satisfactory, push the microcatheter backward to retrieve the stent into the microcatheter, adjust the position and release again. After the stent placement is completed, pull back the delivery system, push the microguidewire into the microcatheter again, and push the microguidewire and microcatheter together into the opposite iliac artery to complete the implantation of the stent on the opposite side in the same way.

[0297] (6) Implantation of the stent in the abdominal aorta, positioning the test pin in the iliac artery. After the release of the stent is completed, pull back the microcatheter and place it in the abdominal aorta position, and 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 animals.

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

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

[0300] Histopathological analysis Tissues collected from stented segments of carrier arteries were immersion fixed in 10% neutral formalin for at least 48 h, dehydrated in alcohol gradients, processed for xylene clearing, and stored for histopathological analysis.

[0301] Resin-embedded stented segments of the iliac arteries were transversely sectioned and stained with HE, whereas non-stented segments were embedded in paraffin and sectioned once at the proximal end and once at the distal end, and stained with HE 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 aorta at the above time points to observe by SEM to confirm endothelialization of the ISS stent.

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

[0304] Example 6 The biological effectiveness of a balloon-expandable CoCr stent coated with the peptide SP1072 according to the present invention is compared with a bare metal stent and a 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-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 were selected. The animals were divided into three observation time points, 7 days, 14 days, and 28 days after surgery, with three animals at each time point. Four stents were implanted in each animal, and the abdominal aorta and bilateral iliac arteries of each animal were selected as the stent implantation sites. Two stents were implanted in the abdominal aorta, and one stent was implanted in each bilateral iliac artery. At the end of the postoperative observation period, two samples of each animal with different coatings were randomly selected to be subjected to SEM, and the remaining samples were analyzed histopathologically. The general design of the experiment 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 the implantation procedure. On the day of surgery, the animals were sedated and anesthesia was induced with an intramuscular injection of 6 mg / kg Sutex® 50. If necessary, the animals could be anesthetized with isoflurane using an inhalation mask.

[0308] After successful induction of anesthesia, the experimental animal is intubated through an intraoral thoracotracheal in order to establish respiratory access and is connected to a ventilator device to maintain anesthesia through continuous inhalation of anesthesia-oxygen mixture. It may be necessary to administer atropine to the experimental animal before surgery to stop vomiting. This is to 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 restraining 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 through the catheter as needed.

[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 descending aorta of the heart 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, pull out the contrast catheter, prepare the stent delivery system, evacuate the air in the balloon of the delivery system until a negative pressure is reached, and then let the balloon aspirate a mixture of contrast and heparinized saline. The stent delivery system is guided by a 0.014 guidewire to deliver the stent to the selected stent implantation site. After the stent is delivered to the iliac artery vascular implantation site, the balloon expansion pressure pump pressurizes and opens the stent, and saves the fluoroscopic image to record the stent implantation information. The balloon is expanded with an appropriate pressure of 1.10-1.20, the ratio of the stent diameter to the target vessel diameter at the implantation site is 1, 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 from the body and another angiogram was performed to evaluate the implanted stent. Stenting of the other iliac artery was completed in the same manner.

[0315] (6) After completing the implantation of the stent in the abdominal aorta and the stent release at the iliac artery site, 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 sodium heparin injections IV during implantation, animals received antiplatelet therapy (aspirin and clopidogrel) orally at 5 mg / kg once daily starting 3 days before surgery and then orally at 5 mg / kg postoperatively until the endpoint.

[0318] Histopathological analysis Tissues collected from stented segments of carrier arteries were immersion fixed in 10% neutral formalin for at least 48 h, dehydrated in alcohol gradients, processed for xylene clearing, and stored for histopathological analysis.

[0319] Resin-embedded stented segments of the iliac arteries were transversely sectioned and stained with HE, whereas non-stented segments were embedded in paraffin and sectioned once at the proximal end and once at the distal end, and stained with HE 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 aorta at the above time points to observe by SEM to confirm endothelialization of the ISS stent.

[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 show 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 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 that 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 invention are rapidly integrated because they are recognized by platelets and leukocytes as healthy endothelium, i.e., as "self" components.

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

Claims

1. Trans-homophilic CD31 - a peptide that mimics the cell-cell interaction of CD31 domains 1 and 2.

2. 2. A peptide mimicking the trans-homophilic CD31-CD31 domain 1 cell-cell interaction, comprising 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; or b) Table 2 where, independently of each other, 2K may be either K or R; 3S may be either S or C; 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; or 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. A peptide according to claim 1 or 2, which mimics the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction, and which comprises one of the following sequences: Table 4

4. A peptide according to claim 3 that mimics the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction, comprising two of said sequences.

5. 2. The peptide of claim 1, which mimics the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction, 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 A peptide comprising:

6. 2. The peptide of claim 1, which mimics the intercellular interaction of transhomophilic CD31-CD31 domains 1 and 2, characterized in that it comprises the following sequence: Table 9

7. 3. The peptide according to claim 1 or 2, which mimics the cell-cell interaction of trans-homophilic CD31-CD31 domains 1 and 2, and which is a cyclic peptide.

8. A peptide according to claim 1 or 2, which mimics the cell-cell interaction of trans-homophilic CD31-CD31 domains 1 and 2, and which comprises a linker and / or spacer and / or tail at any terminus or is attached to an amino acid residue.

9. A peptide mimicking the cell-cell interaction of trans-homophilic CD31-CD31 domains 1 and 2 according to claim 1 or 2, wherein the linker and / or spacer and / or tail are represented by the following or a combination thereof: Table 10

10. 3. A peptide according to claim 1 or 2, which mimics the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction, and which 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. A peptide according to claim 1 or 2, which mimics the trans-homophilic CD31-CD31 domain 1 and 2 cell-cell interaction, comprising 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. 2. The peptide of claim 1, which mimics the cell-cell interaction of trans-homophilic CD31-CD31 domains 1 and 2, characterized in that it has one of the following structures: Table 12

14. A pharmaceutical composition comprising a peptide mimicking the cell-cell interaction of trans-homophilic CD31-CD31 domains 1 and 2 according to any one of claims 1, 2 and 13, for use in the prevention or treatment of vascular lesions.

15. The pharmaceutical composition described in claim 14, wherein the vascular lesion is selected from the group consisting of cardiac valve lesion, atherosclerosis, thrombosis, ischemia, hemorrhage, restenosis, and aneurysm.

16. The pharmaceutical composition according to claim 14, for preventing a pathological condition represented by in-stent stenosis.

17. 15. The pharmaceutical composition of claim 14 for use in human or veterinary medical treatment.

18. A coating comprising one or more peptides according to any one of claims 1, 2 and 13.

19. 20. The coating of claim 18, which is a single layer or multi-layer coating.

20. 19. Use of a biomimetic peptide according to any one of claims 1, 2 and 13 for adhesion to a vascular device.

21. 19. Use of a biomimetic peptide according to any one of claims 1, 2 and 13 for promoting endothelialization of blood vessels, preventing neointimal growth or integrating a vascular device in a target vessel.

22. 19. Use of a biomimetic peptide according to any one of claims 1, 2 and 13 for improving adhesion of vascular devices.

23. 19. Use of a biomimetic peptide according to any one of claims 1, 2 and 13 to confer the anti-inflammatory and anti-thrombotic properties of restored endothelium to a stent segment.

24. Use of a biomimetic peptide according to any one of claims 1, 2 and 13 is disclosed to improve adaptive remodelling in a stent segment and allow the recovery of its functional properties.

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