Cyclic polypeptides, methods for obtaining same and use thereof in therapy

Abstract

The present invention relates to a polypeptide comprising the following amino acid sequence: W-S-X1-W-X2-X3-C-S-X4-C-G (SEQ ID NO: 59), wherein: X1, X2 and X3 independently of one another denote S or G, X4 denotes R-S or V-S or V-T or R-T, and the two cysteines form a disulfide bridge.

Description

[0001] The present invention relates to novel cyclic polypeptides, pharmaceutical compositions comprising thereof, and their use as medicines, particularly their use in the treatment of neurodegenerative diseases and in the regeneration of cells of the central and peripheral nervous systems, as well as methods for obtaining such cyclic polypeptides.

[0002] The polypeptide of the present invention has an amino acid sequence derived from one of the conserved common domains of SCO-spondin (referred to as platelet-reactive protein type 1 repeat or TSR).

[0003] It is well known that various synthetic peptides inferred from the structure of platelet-reactive proteins have interesting effects on a variety of cell types, especially their inhibition of tumors in mammals, their influence on thrombolysis and angiogenesis, and their potential to act as complement regulators or ensure the promotion of cell attachment [Sipes JM et al., J Cell Biol, 121, 469-77, 1993; Rusnati M et al., Pharmaceuticals 3, 1241-1278, 2010; Lopez-Dee Z et al., Mediators of Inflammation, Vol. 2011, Article ID 296069, 10 pages, 2011].

[0004] The general characteristics of SCO-spondin are described in particular in the articles by Meiniel et al. (Microsc ResTech. 2, 484-95, 2001) and Gobron et al. (Glia 32, 177-91, 2000).

[0005] The application of a polypeptide with the amino acid sequence WSGWSCSRSCG [SEQ ID NO:58] (which corresponds to one of the most representative amino acid sequences of the TSR motif of SCO-spondin) induced cell differentiation in B104 cells derived from rat neuroblastoma, thereby inducing axonal growth and cell aggregation [F. El-Bitar et al., Cell Tissue Res, Vol. 304, pp. 361-369, 2001]. This polypeptide does not contain a disulfide bridge between the two cysteine ​​residues.

[0006] The polypeptide, as well as the polypeptide of formula SEQ ID NO:70 and especially formula SEQ ID NO:57 in reduced form, is described and claimed in international patent applications WO 1999 / 03890 (which corresponds to US patent US 6,995,140) and WO2009027350, and can be prepared based on the specifications of these patents and methods known to those skilled in the art.

[0007] However, the stability of the peptide WGWSSCSRSCG [SEQ ID NO:58] decreases over time in aqueous solution. Therefore, it may be necessary to prepare the peptide on-the-spot at the time of treatment, which could complicate its administration and thus limit the possibility of administering the treatment by direct injection or, for example, by means of a pump (for stepwise drug delivery to the patient). Generally speaking, this low stability would be a significant drawback for developing therapeutic concepts based on this peptide.

[0008] Patent application WO 2008 / 090285 describes peptide analogues of the polypeptide WGWSSCSRSCG [SEQ ID NO:58]. However, this method has two drawbacks: these polypeptides contain non-natural amino acids, which may cause immunogenicity, and the production cost is significantly increased, as non-natural amino acids are much more expensive than natural amino acids, which reduces their commercial benefits.

[0009] One of the objectives of this invention is to provide peptide compounds that are stable and soluble in solutions, particularly compatible with intrathecal administration, wherein the properties of the polypeptide of formula SEQ ID NO:70, and especially the polypeptide of formula SEQ ID NO:57, particularly the polypeptide of sequence WSGWSSCSRSCG [SEQ ID NO:58] are maintained or improved.

[0010] This is because, for certain clinical indications under investigation, it is particularly important to provide a more soluble form of the polypeptide with the amino acid sequence WGWSSCSRSCG [SEQ ID NO:58] as described in International Patent Application WO 1999 / 03890.

[0011] This is because the choice of solution for administering the compound is very limited for intrathecal (IT) injection. In practice, only three solutions are used: physiological fluid, artificial cerebrospinal fluid, and 5% glucose solution. Other solutions known to those skilled in the art can be used if other routes of administration are employed, such as intraventricular, epidural, intraspinal, intraparenchymal, intravenous, intraluminal, intravitreal, transsphenoidal, or local routes. Furthermore, the pharmaceutical composition can be produced in any form, particularly liquids, especially injectable preparations in the form of solutions, suspensions, or emulsions, preparations for infusion, or solids; and contains any type of carrier, particularly gels, biopolymers, or biomaterials, or any medical device, such as an implant or implantable pump, that allows for controlled release or delivery systems.

[0012] Aqueous solutions can be prepared using a wide variety of solvents compatible with the route of application (ideally, isotonic solvents such as 0.9% sodium chloride or 5% glucose), but the solvent can also be water, saline solution, phosphate, citrate or acetate buffer, or any other solvent. Optionally, the preparation may also contain any excipients, auxiliaries or additives, such as propylene glycol, glycerol, polyethylene glycol, or any surfactant, wetting agent, thickener, chelating agent, isotonic agent, antioxidant or stabilizer, in combination or not.

[0013] Preparations in solution form can be administered by bolus injection or infusion.

[0014] In the case of the peptide with the amino acid sequence WSGWSSCSRSCG [SEQ ID NO:58], the solubility in physiological fluids is low, around 1-2 mg / mL, thus making it more difficult to obtain the desired maximum clinical dose of around 10 mg / mL. Solubility is particularly important for IT injections because, for example, there are limitations on injection volume, much greater than those for peripheral injections, and limitations on the solvents and solutes that can be used. The use of a 5% glucose solution improves the solubility of the peptide with the amino acid sequence WSGWSSCSRSCG [SEQ ID NO:58], bringing the solubility to approximately 10 mg / mL, while still being an isotonic solution and compatible with IT injections.

[0015] However, the presence of glucose in contact with the polypeptide is known to facilitate the formation of Schiff bases, which arise from the reaction of the aldehyde form of glucose with the primary amine of the polypeptide according to the following reaction sequence:

[0016]

[0017] In theory, these Schiff bases could complicate the preparation and use of pharmaceutical compositions. However, in the case of current peptides, these bases are reversible in vivo.

[0018] Therefore, it is very important to develop a more soluble form of the polypeptide with the amino acid sequence WSGWSSCSRSCG [SEQ ID NO:58] in solvents other than 5% glucose and preferably in physiological fluids or artificial cerebrospinal fluid.

[0019] Another aspect of the invention is to provide peptide compounds that are effective in treating neurodegenerative diseases or neurotrauma in which the regeneration of nerve cells is essential, and pharmaceutical compositions comprising them.

[0020] Another aspect of the present invention is to provide a method for obtaining these peptide compounds.

[0021] This invention relates to polypeptides comprising the following amino acid sequence:

[0022] WS-X1-W-X2-X3-CS-X4-CG(SEQ ID NO:59)

[0023] in:

[0024] X1, X2, and X3 independently represent S or G.

[0025] X4 represents RS, VS, VT, or RT, and

[0026] The two cysteine ​​residues form a disulfide bridge.

[0027] This invention particularly relates to polypeptides comprising the following amino acid sequence:

[0028] WS-X1-W-X2-X3-CS-X4-CG(SEQ ID NO:59)

[0029] in:

[0030] X1, X2, and X3 independently represent S or G.

[0031] X4 represents RS, VS, VT, or RT, and

[0032] The two cysteine ​​residues form a disulfide bridge.

[0033] The polypeptide does not actually exist in a reduced, dimer, or oligomeric form, nor in derivatives of the polypeptide wherein the thiohydroxy group is in the form of sulfoxide or sulfone.

[0034] In the sequence SEQ ID NO:59 above, X4 represents RS, VS, VT, or RT. In another preferred embodiment of the invention, X4 may also represent RS, VS, or VT.

[0035] Therefore, another objective of the present invention is the polypeptide described above, which comprises the following amino acid sequence:

[0036] WS-X1-W-X2-X3-CS-X4-CG(SEQ ID NO:1)

[0037] in:

[0038] X1, X2, and X3 have the meanings stated above, and

[0039] X4 represents RS, VS, or VT, and

[0040] The two cysteine ​​residues form a disulfide bridge.

[0041] Within the scope of this invention, "amino acid" refers to both natural and non-natural amino acids.

[0042] "Natural amino acids" refer to amino acids in the L form that can be found in natural proteins, namely alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0043] "Non-natural amino acids" refer to amino acids preceding the D form, as well as high amino acid forms of certain amino acids (such as arginine, lysine, phenylalanine, and serine) or positive amino acid forms of leucine or valine.

[0044] The definition also includes other amino acids, such as α-aminobutyric acid, spermine, α-aminoisobutyric acid, sarcosine, statin, ornithine, and deaminated tyrosine.

[0045] The nomenclature used to describe peptide sequences is the International Nomenclature, which uses single-letter codes in which the amino terminus is presented on the left and the carboxyl terminus on the right.

[0046] The hyphen “-” indicates a normal peptide bond that connects the amino acids in the sequence.

[0047] Within the scope of this invention, a polypeptide refers to any polymer of amino acids linked together by peptide bonds, regardless of its length. Therefore, within the scope of this invention, the term "polypeptide" also includes peptides and proteins.

[0048] This invention relates to polypeptides comprising the following amino acid sequence:

[0049] WS-X1-W-X2-X3-CS-X4-CG(SEQ ID NO:59)

[0050] in:

[0051] X1, X2, and X3 independently represent S or G.

[0052] X4 represents RS, VS, VT, or RT, and

[0053] The two cysteine ​​residues form a disulfide bridge.

[0054] Therefore, product SEQ ID NO:59 has the following structure:

[0055]

[0056] It can also be represented using the international nomenclature system with three-letter codes, as follows:

[0057]

[0058] Or it can be expressed as follows:

[0059]

[0060] In this embodiment, the present invention relates to polypeptides comprising the following amino acid sequence:

[0061]

[0062]

[0063] In these sequences, two cysteine ​​residues form a disulfide bridge with each other.

[0064] According to an advantageous embodiment, the present invention relates to polypeptides defined above comprising the following amino acid sequence:

[0065] WSGWSSCSRSCG(SEQ ID NO:2)

[0066] The two cysteine ​​residues form a disulfide bridge.

[0067] Therefore, product SEQ ID NO:2 has the following structure:

[0068]

[0069] It can also be represented using the international nomenclature system with three-letter codes, as follows:

[0070]

[0071] Or it can be expressed as follows:

[0072]

[0073] Surprisingly, the inventors discovered that the formation of a disulfide bridge between two cysteine ​​residues in a polypeptide with the amino acid sequence WSGWSSCSRSCG [SEQ ID NO:2] not only maintains the polypeptide's efficacy in inducing increased axonal growth and synaptic connections, but also yields a polypeptide with better stability and solubility in solutions compatible with application (especially intrathecal application).

[0074] What is even more surprising about this invention is that, in the protein SCO-spondin from which this peptide sequence is derived, the two cysteine ​​residues present in the sequence WGWSSCSRSCG [SEQ ID NO:2] above do not form disulfide bridges with each other, but instead participate in disulfide bridges with other cysteine ​​residues present in the protein.

[0075] According to a particular embodiment, the present invention relates to a polypeptide as defined above, which consists of the following amino acid sequence:

[0076] X7-WS-X1-W-X2-X3-CS-X4-CG-X8(SEQ ID NO:68)

[0077] in:

[0078] X1, X2, and X3 independently represent S or G.

[0079] X4 represents RS, VS, VT, or RT.

[0080] The two cysteine ​​residues form a disulfide bridge.

[0081] X7 represents a hydrogen atom or an amino acid chain with 0 to 4 amino acids, and

[0082] X8 represents a hydrogen atom or an amino acid chain with 0 to 5 amino acids.

[0083] According to another particular embodiment, the present invention relates to a polypeptide as defined above, which consists of the following amino acid sequence:

[0084] X5-WS-X1-W-X2-X3-CS-X4-CG-X6(SEQ ID NO:69)

[0085] in:

[0086] X1, X2, and X3 independently represent S or G.

[0087] X4 represents RS, VS, VT, or RT.

[0088] The two cysteine ​​residues form a disulfide bridge.

[0089] X5 represents a hydrogen atom or P or AP or LAP or VLAP, and

[0090] X6 represents a hydrogen atom or L or LG or LGL or LGLI or LGLIF.

[0091] In the sequences SEQ ID NO:68 and SEQ ID NO:69 above, X4 represents RS, VS, VT, or RT.

[0092] In another preferred embodiment of the invention, X4 may also represent RS, VS, or VT. The corresponding sequences are SEQ ID NO:8 and SEQ ID NO:3.

[0093] In this embodiment, the present invention particularly relates to polypeptides with the following sequence:

[0094] WSGWSSCSRSCG(SEQ ID NO:2)

[0095] And a polypeptide with the following amino acid sequence:

[0096] P-W-S-G-W-S-S-C-S-R-S-C-G SEQ ID NO:28 A-P-W-S-G-W-S-S-C-S-R-S-C-G SEQ ID NO:29 L-A-P-W-S-G-W-S-S-C-S-R-S-C-G SEQ ID NO:30 V-L-A-P-W-S-G-W-S-S-C-S-R-S-C-G SEQ ID NO:31 W-S-G-W-S-S-C-S-R-S-C-G-L SEQ ID NO:32 W-S-G-W-S-S-C-S-R-S-C-G-L-G SEQ ID NO:33 W-S-G-W-S-S-C-S-R-S-C-G-L-G-L SEQ ID NO:34 W-S-G-W-S-S-C-S-R-S-C-G-L-G-L-I SEQ ID NO:35 W-S-G-W-S-S-C-S-R-S-C-G-L-G-L-I-F SEQ ID NO:36 P-W-S-G-W-S-S-C-S-R-S-C-G-L SEQ ID NO:37 P-W-S-G-W-S-S-C-S-R-S-C-G-L-G SEQ ID NO:38 P-W-S-G-W-S-S-C-S-R-S-C-G-L-G-L SEQ ID NO:39 P-W-S-G-W-S-S-C-S-R-S-C-G-L-G-L-I SEQ ID NO:40 P-W-S-G-W-S-S-C-S-R-S-C-G-L-G-L-I-F SEQ ID NO:41 A-P-W-S-G-W-S-S-C-S-R-S-C-G-L SEQ ID NO:42 A-P-W-S-G-W-S-S-C-S-R-S-C-G-L-G SEQ ID NO:43 A-P-W-S-G-W-S-S-C-S-R-S-C-G-L-G-L SEQ ID NO:44 A-P-W-S-G-W-S-S-C-S-R-S-C-G-L-G-L-I SEQ ID NO:45 A-P-W-S-G-W-S-S-C-S-R-S-C-G-L-G-L-I-F SEQ ID NO:46 L-A-P-W-S-G-W-S-S-C-S-R-S-C-G-L SEQ ID NO:47 L-A-P-W-S-G-W-S-S-C-S-R-S-C-G-L-G SEQ ID NO:48 L-A-P-W-S-G-W-S-S-C-S-R-S-C-G-L-G-L SEQ ID NO:49 L-A-P-W-S-G-W-S-S-C-S-R-S-C-G-L-G-L-I SEQ ID NO:50 LAPWSGWSSCSRSCGLGLIF SEQ ID NO:51 VLAPWSGWSSCSRSCGL SEQ ID NO:52 VLAPWSGWSSCSRSCGLG SEQ ID NO:53 VLAPWSGWSSCSRSCGLGL SEQ ID NO:54 VLAPWSGWSSCSRSCGLGLI SEQ ID NO:55 VLAPWSGWSSCSRSCGLGLIF SEQ ID NO:56

[0097] The sequence in which the two cysteine ​​residues form a disulfide bridge.

[0098] According to a more advantageous embodiment, the present invention relates to a polypeptide as defined above, which consists of the following amino acid sequence:

[0099] WSGWSSCSRSCG(SEQ ID NO:2)

[0100] The two cysteine ​​residues form a disulfide bridge.

[0101] According to an advantageous embodiment, the present invention relates to a polypeptide as defined above, which consists of the following amino acid sequence:

[0102] WS-X1-W-X2-X3-CS-X4-CG(SEQ ID NO:59)

[0103] in:

[0104] X1, X2, and X3 independently represent S or G.

[0105] X4 represents RS, VS, VT, or RT, and

[0106] The two cysteine ​​residues form a disulfide bridge.

[0107] The polypeptide does not actually exist in a reduced form, a dimer form, or an oligomer form, nor in derivatives of the polypeptide wherein the thiohydroxy group is in the form of a sulfoxide or a sulfone.

[0108] The statement "actually none" means that the polypeptide according to the preferred embodiment shown above contains very small amounts of impurities identified by the inventors of this application. These impurities include the reduced structure in which the two thiohydroxy groups of the two cysteines are in free form, a dimer (or polymer), and an oxidized form in which the thiohydroxy groups are in sulfoxide or sulfone form. This actually pure form is preferably obtained by performing the methods described below. Under these conditions, the purity of the obtained product is at least about 80%. This purity can reach 85%, 90%, or even 95%.

[0109] Therefore, according to another advantageous embodiment, the present invention relates to polypeptides consisting of the following amino acid sequence:

[0110] WS-X1-W-X2-X3-CS-X4-CG(SEQ ID NO:59)

[0111] in:

[0112] X1, X2, and X3 independently represent S or G.

[0113] X4 represents RS, VS, VT, or RT, and

[0114] The two cysteine ​​residues form a disulfide bridge.

[0115] The polypeptide has a purity of more than 80%, preferably 85%, more preferably 90%, and even more preferably equal to or greater than 95%.

[0116] According to a more particular embodiment, the present invention relates to polypeptides consisting of the following amino acid sequence:

[0117] WSGWSSCSRSCG(SEQ ID NO:2)

[0118] The two cysteine ​​residues form a disulfide bridge.

[0119] The polypeptide does not actually exist in a reduced, dimer, or oligomeric form, nor in derivatives of the polypeptide wherein the thiohydroxy group is in the form of sulfoxide or sulfone.

[0120] Conventional purification methods (such as chromatography) can be used to purify products containing the desired disulfide bridge.

[0121] For example, the synthesis of the compound corresponding to sequence SEQ ID NO:2 in the absence of albumin, using methods known to those skilled in the art, yielded a mixture of approximately 60 products, in which the desired product was present, having a purity of 44.51% (as determined by HPLC). Several successive HPLC runs using a gradient of 0.1% TFA / water / acetonitrile followed by equal parts of the mixture (pool) enabled the separation of the compound corresponding to sequence SEQ ID NO:2 with a final purity of 99%.

[0122] Therefore, according to a more particular embodiment, the present invention relates to the polypeptide defined above, which consists of the following amino acid sequence:

[0123] WSGWSSCSRSCG(SEQ ID NO:2)

[0124] The two cysteine ​​residues form a disulfide bridge.

[0125] The polypeptide has a purity of more than 80%, preferably 85%, more preferably 90%, and even more preferably equal to or greater than 95%.

[0126] According to another aspect, the present invention relates to a method for obtaining a polypeptide with the amino acid sequence SEQ ID NO:59:

[0127] WS-X1-W-X2-X3-CS-X4-CG(SEQ ID NO:59)

[0128] in:

[0129] X1, X2, and X3 independently represent S or G.

[0130] X4 represents RS, VS, VT, or RT, and

[0131] The two cysteine ​​residues form a disulfide bridge.

[0132] The method includes the step of forming a disulfide bridge in the polypeptide with the sequence SEQ ID NO:70 in the presence of albumin:

[0133] WS-X1-W-X2-X3-CS-X4-CG(SEQ ID NO:70)

[0134] in:

[0135] X1, X2, and X3 independently represent S or G.

[0136] X4 represents RS, VS, VT, or RT.

[0137] The polypeptide with the sequence SEQ ID NO:70 is in solution, preferably in aqueous solution.

[0138] The polypeptide with the sequence SEQ ID NO:70 does not have a disulfide bridge.

[0139] In this embodiment of the invention, the formation of a disulfide bridge between two cysteine ​​residues in the amino acid sequence is achieved by oxidizing cysteine.

[0140] In the sequence SEQ ID NO:70 above, X4 represents RS, VS, VT, or RT.

[0141] In another preferred embodiment of the invention, the above method may also be implemented starting with a product having the sequence SEQ ID NO:57 (where X4 represents RS, VS, or VT).

[0142] According to another aspect, the present invention relates to a method for obtaining a polypeptide with the amino acid sequence SEQ ID NO:2:

[0143] WSGWSSCSRSCG(SEQ ID NO:2)

[0144] in:

[0145] The two cysteine ​​residues form a disulfide bridge.

[0146] The method includes the step of forming a disulfide bridge in the polypeptide with the sequence SEQ ID NO:58 in the presence of albumin:

[0147] WSGWSSCSRSCG(SEQ ID NO:58).

[0148] The inventors of this application have discovered an inventive and unexpected method for forming disulfide bridges. This method involves using albumin as an oxidation catalyst to reduce or avoid the formation of impurities and also accelerates the reaction kinetics, a result that is always beneficial for the industrial production of peptides.

[0149] The presence of albumin in this reaction enables, in particular, the accelerated oxidation of the starting product. In oxidation reactions, it is often desirable to gently accelerate the reaction to avoid the formation of undesirable products from uncontrolled oxidation. The results presented in the experimental section show that the reaction in the presence of albumin allows for better control and therefore higher purity of the product formed (more specifically, the polypeptide with the sequence SEQ ID NO:2).

[0150] More specifically, the present invention relates to a method for obtaining a polypeptide with the sequence SEQ ID NO:1 or a polypeptide with the sequence SEQ ID NO:2 as defined above, characterized in that albumin and a polypeptide with the sequence SEQ ID NO:70 or a polypeptide with the sequence SEQ ID NO:58 are present in an albumin:polypeptide ratio of 1:1 to 1:100, preferably 1:1 to 1:10, and more preferably 1:1.

[0151] According to another particular embodiment, the present invention relates to a method for obtaining a polypeptide with the sequence SEQ ID NO:59 or a polypeptide with the sequence SEQ ID NO:2 as defined above, characterized in that the step of forming a disulfide bridge in the polypeptide with the sequence SEQ ID NO:70 or a polypeptide with the sequence SEQ ID NO:58 in the presence of albumin is carried out in ambient air.

[0152] According to another particular embodiment, the present invention also relates to the method for obtaining the polypeptide with the sequence SEQ ID NO:1 as defined above, characterized in that the step of forming a disulfide bridge in the polypeptide with the sequence SEQ ID NO:57 in the presence of albumin is carried out in ambient air.

[0153] According to an advantageous embodiment, the present invention relates to a method for obtaining a polypeptide with the sequence SEQ ID NO:59 or a polypeptide with the sequence SEQ ID NO:2 as defined above, characterized in that the step of forming a disulfide bridge in the polypeptide with the sequence SEQ ID NO:70 or the polypeptide with the sequence SEQ ID NO:58 in the presence of albumin is performed without detaching the polypeptide with the sequence SEQ ID NO:70 or the polypeptide with the sequence SEQ ID NO:58 from the resin used for peptide synthesis of these polypeptides, and then the polypeptide with the sequence SEQ ID NO:59 or the polypeptide with the sequence SEQ ID NO:2 is obtained by separating the polypeptide from the resin after the step of forming the disulfide bridge.

[0154] The polypeptide with sequence SEQ ID NO:1 can also be obtained by starting with the polypeptide with sequence SEQ ID NO:57 under the same conditions.

[0155] The method for preparing the polypeptide of sequence SEQ ID NO:58 is based on solid-phase peptide synthesis, wherein N-α-Fmoc-protected amino acids are used as synthons for polypeptide construction.

[0156] Glycyl residues at the C-terminus are coupled to the resin MBHA as a component of the linker Fmoc-Gly-MPPA-OH. Other amino acid residues are incorporated through a series of cycles of Fmoc group deprotection and amino acid coupling, resulting in a protected polypeptide linked to the resin. After solid-phase assembly of the polypeptide, the cleavage of the polypeptide from the resin and the deprotection of the polypeptide are carried out simultaneously in one step using a trifluoroacetic acid (TFA) / water mixture, yielding a crude polypeptide, which is precipitated using an MTBE / hexane mixture, followed by filtration and drying.

[0157] Prior to purification, the crude polypeptide with sequence SEQ ID NO:58 was dissolved in an acetonitrile / water / acetic acid (AcOH) mixture. Purification was performed by preparative reversed-phase chromatography, using trifluoroacetic acid (TFA) as the eluent followed by acetate. The purified polypeptide obtained (in acetate form) in solution was diluted with water and concentrated. After adding 5% acetonitrile, the resulting solution was filtered and lyophilized to yield the polypeptide with sequence SEQ ID NO:58 in its pharmaceutical form.

[0158] This method has several advantages. Direct work on the resin is more controlled and therefore cleaner. It produces fewer impurities and avoids several intermediate steps (including complex purification of peptides with sequences of SEQ ID NO:70 or SEQ ID NO:58, or also SEQ ID NO:57). Optimized experimental conditions regarding concentration, solvent, and incubation time can be determined by those skilled in the art.

[0159] The present invention also relates to a polypeptide with the sequence SEQ ID NO:59 obtained by the methods defined above.

[0160] The present invention also relates to a polypeptide with the sequence SEQ ID NO:1 obtained by the methods defined above.

[0161] The present invention also relates to a polypeptide with the sequence SEQ ID NO:2 obtained by the methods defined above.

[0162] According to another aspect, the present invention relates to pharmaceutical compositions comprising the aforementioned polypeptide as an active ingredient, and optionally, one or more pharmaceutically acceptable excipients.

[0163] The peptide compounds according to the invention can be used in pharmaceutical compositions or for the preparation of pharmaceuticals. In these compositions or pharmaceuticals, the active ingredient can be incorporated into the composition in various forms (i.e., in solution (usually aqueous solution), or in lyophilized form, or in gel or hydrogel form, or in emulsion form, or in any other pharmaceutically and physiologically acceptable form).

[0164] The dosage to be used can vary from 1 μg / kg to 1000 μg / kg, and the routes of administration can be selected from intraspinal, intrathecal, intraventricular, epidural, intraparenchymal, intravitreal, transsphenoidal, or local routes.

[0165] Other routes of administration known to those skilled in the art may also be used, such as subcutaneous, intravenous, intraluminal, or intranasal routes. When using one or more of these routes of administration, the dose to be used can vary from 1 μg / kg to 50 mg / kg.

[0166] These drugs or compositions are specifically intended for the treatment of neurodegenerative diseases and / or the regeneration of cells in the central nervous system (brain, spinal cord) or peripheral nerves following trauma. Their use in the regeneration of nervous system cells can be performed either by direct administration to the patient or in a non-invasive manner. These drugs or compositions are particularly suitable for the treatment of Alzheimer's disease, multiple sclerosis, Parkinson's disease, or any other neurodegenerative pathological condition, or accident- or traumatic pathological conditions such as spinal cord injury, head trauma, or stroke. These drugs or compositions can also be used to treat injuries to the auditory nerve, optic nerve, olfactory nerve, or any cranial or peripheral nerve, whether of traumatic, accidental, or degenerative origin.

[0167] According to another aspect, the present invention relates to the polypeptides described above for use in treating neurodegenerative pathological conditions or trauma in which central nervous system regeneration is sought. The polypeptides according to the invention can be used to treat traumatic or non-traumatic injuries of the peripheral nervous system.

[0168] According to another advantageous embodiment, the present invention relates to the polypeptides described above for use in treating accidental, traumatic, or degenerative pathological conditions, particularly Alzheimer's disease, multiple sclerosis, Parkinson's disease, stroke, spinal cord injury, cranial trauma, or injury to the optic nerve, olfactory nerve, auditory nerve, or any cranial or peripheral nerve.

[0169] Attached Figure Description

[0170] Figure 1 : Figure 1Optical microscopic observations of droplets of solutions of peptide with sequence SEQ ID NO:58 (A, magnification x 25; B, magnification x 100) and peptide with sequence SEQ ID NO:2 (C, magnification x 25) in 0.9% NaCl at a concentration of 29.4 mg / ml are shown.

[0171] Figure 2 : Figure 2 The superimposed chromatograms show the reaction of cyclization of the polypeptide with sequence SEQ ID NO:58 to the polypeptide with sequence SEQ ID NO:2 in the presence of human albumin, prepared at the start of the reaction (T0 min) and after 60 min of cyclization reaction (T60 min).

[0172] Figure 3 : Figure 3 Phase-contrast optical microscopy (magnification x 400) shows the effect of the cyclic peptide with sequence SEQ ID NO:2 on B104 cells after 48 hours. Cells were cultured for 2 days in serum-free medium and in the absence (A) or presence (B) of 1 mg / mL of the cyclic peptide with sequence SEQ ID NO:2.

[0173] Figure 4 : Figure 4 Phase-contrast optical microscopy (magnification x 400) shows the effect of the cyclic peptide with sequence SEQ ID NO:2 on B104 cells after 72 hours. Cells were cultured for 3 days in serum-free medium and in the absence (A) or presence (B) of 500 μg / mL of the cyclic peptide with sequence SEQ ID NO:2.

[0174] Figure 5 : Figure 5 The effect of the cyclic polypeptide with sequence SEQ ID NO:2 on cell number was quantified. The mean number of B104 cells was analyzed after 1, 2, or 3 days of culture. Values ​​are expressed as mean ± SEM (n = 3). Cells were cultured in serum-free medium alone (control) or in serum-free medium containing 1 mg / mL or 500 μg / mL of the cyclic polypeptide with sequence SEQ ID NO:2.

[0175] The term "SEM" refers to the standard error of the mean.

[0176] Figure 6 : Figure 6The effect of the cyclic peptide with sequence SEQ ID NO:2 on axon number was quantified. The mean budding number / B104 cells was analyzed after 1, 2, or 3 days of culture. Values ​​are mean ± SEM (n = 3). Cells were cultured in serum-free medium alone (control) or in serum-free medium containing 1 mg / mL or 500 μg / mL of the cyclic peptide with sequence SEQ ID NO:2.

[0177] Figure 7 : Figure 7 The effect of the cyclic peptide with sequence SEQ ID NO:2 on axon length is shown. Mean axon length / B104 cells was analyzed after 1, 2, or 3 days of culture. Values ​​are mean ± SEM (n=3). Cells were cultured in serum-free medium alone (control) or in serum-free medium containing 1 mg / mL or 500 μg / mL of the cyclic peptide with sequence SEQ ID NO:2.

[0178] Figure 8 : Figure 8 The results of the study of motor function are shown using the Basso-Beattie-Bresnahan (BBB) ​​scale (Basso DM et al., J Neurotrauma, 12, 1-21, 1995; Basso DM et al., Exp Neurol, 139, 244-256, 1996). Values ​​are expressed as mean ± SEM. BBB scores are graded from 0 (no hind paw movement) to 21 (normal walking with coordinated paws and parallel paw placement).

[0179] Figure 9 : Figure 9 The percentage of rats with a BBB score of 14 or higher is shown.

[0180] Figure 10 : Figure 10 The percentage of rats with BBB scores between 8 and 14 is shown.

[0181] Figure 11 : Figure 11 The results of the evaluation of the toe-separation reflex activity are shown. The scores considered are: 0 = no reflex activity, 1 = significantly below normal, 2 = slightly below normal, 3 = normal.

[0182] Figure 12 : Figure 12 The results of the evaluation of hind paw placement are shown. The scores considered are: 0 = no reflex activity, 1 = significantly below normal, 2 = slightly below normal, 3 = normal.

[0183] Figure 13 : Figure 13 The percentage of rats with a hind paw placement score of 3 (normal) is shown.

[0184] Figure 14 : Figure 14 The average number of days it took for rats to regain bladder control is shown. Values ​​are average ± SEM. Example

[0185] Example 1: Method for synthesizing a polypeptide whose sequence is WGWSSCSRSCG (SEQ ID NO:2), in which two cysteine ​​residues are linked by a disulfide bridge.

[0186] 1) Oxidation of the polypeptide with the sequence WGWSSCSRSCG (SEQ ID NO:58) in the presence of human albumin

[0187] To perform this synthesis, we combined the polypeptide with the sequence WGWSSCSRSCG (SEQ ID NO:58) with different amounts of albumin so that the ratio of "polypeptide with sequence SEQ ID NO:58: human albumin (HSA)" was approximately 1:100, 1:10, and 1:1.

[0188] Starting with a polypeptide of sequence WSGWSSCSRSCG (SEQ ID NO: 58) that was incubated with HSA at a 1:1 ratio in air with stirring at ambient temperature for 1 to 3 hours, we observed by HPLC the corresponding sequence WSGWSSCSRSCG (SEQ ID NO: 58) where two cysteine ​​residues are linked by disulfide bridges.

[0189] 2) Formation of the peptide peak. After removing albumin by precipitation, the product was purified and analyzed by HPLC. The use of different ratios of albumin to the peptide corresponding to sequence SEQ ID NO:58 can affect the cyclization rate and the final cyclization yield, with lower amounts of albumin being easier to remove.

[0190] 2) Another method for preparing the polypeptide corresponding to the sequence SEQ ID NO:2, which avoids the prior separation and purification of the linear polypeptide (the polypeptide with the sequence SEQ ID NO:58).

[0191] Similar to the conventional method described in International Patent Application WO 1999 / 03890 (but without cleaving or removing it from the resin), a polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:58) is synthesized on the resin. This allows for the avoidance of two time- and financially costly steps involving the separation and purification of the polypeptide before it is resuspended in solution to oxidize it to the desired product corresponding to sequence SEQ ID NO:2. Oxidation in the presence of albumin is carried out directly with the polypeptide having the sequence WSGWSSCSRSCG (SEQ ID NO:58) attached to the resin. Purification is similar to that described in the examples above, but the albumin separation step is easier as it is performed by simple washing while the polypeptide remains attached to the resin. Once the albumin is removed, the polypeptide can then be removed from the resin using conventional methods.

[0192] Example 2: The effect of albumin on the cyclization of a polypeptide with the sequence WGWSSCSRSCG (SEQ ID NO:58) (i.e., its conversion to a polypeptide with the sequence WGWSSCSRSCG (SEQ ID NO:2) in which two cysteine ​​residues are linked by disulfide bridges).

[0193] We investigated the behavior of a polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:58) in the absence and presence of human albumin (HSA), and the resulting formation of a polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:2) in which two cysteine ​​residues are linked by disulfide bridges.

[0194] The method is as follows:

[0195] A solution containing 50 μg / mL of the peptide with the sequence WSGWSSCSRSCG (SEQ ID NO:58) was prepared in PBS and 5% glucose in the presence or absence of 2.5 mg / mL HSA.

[0196] The ratio of "peptide with sequence WGWSSCSRSCG (SEQ ID NO:58):HSA" is approximately 1:1, and therefore they are equimolar.

[0197] Mix the solutions and follow the instructions for use. The method involves using a Phenomenex Jupiter column and an elution solvent consisting of HPLC water containing 0.1% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid, and directly injecting the mixture by reversed-phase HPLC, thereby enabling the separation of peaks of HSA, a polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:58), and a polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:2) in which two cysteine ​​residues form a disulfide bridge.

[0198] Repeat this operation at T=0 minutes and at T=60 minutes.

[0199] The chromatographic characteristic curves enable the establishment of the following observations:

[0200] - Within 60 minutes, in the absence of albumin (HSA) and thus through oxidation in air, the peak of the polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:2), in which two cysteine ​​residues form a disulfide bridge, increased by 9.1% relative to the initial peak of the polypeptide with the sequence SEQ ID NO:58.

[0201] - Under the same conditions, but in the presence of albumin, at T=60 minutes, the peak of the polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:2), in which two cysteine ​​residues form a disulfide bridge, increased by 38% relative to the initial peak of the polypeptide with the sequence SEQ ID NO:58.

[0202] The results demonstrate the accelerating effect of albumin on the cyclization of a polypeptide with the sequence SEQ ID NO:58, which leads to the formation of a polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:2), in which two cysteine ​​residues form a disulfide bridge.

[0203] - In the absence of albumin, within 60 minutes, 25% of the peak of the peptide with sequence SEQ ID NO:58 was converted into the peptide with sequence WSGWSSCSRSCG (SEQ ID NO:2) in which two cysteine ​​residues form a disulfide bridge.

[0204] - In the presence of albumin and under the same conditions, within 60 minutes, 54% of the polypeptide with sequence SEQ ID NO:58 was converted into the polypeptide with sequence WSGWSSCSRSCG (SEQ ID NO:2) in which two cysteine ​​residues form a disulfide bridge.

[0205] The results show that, in the presence of albumin, a greater amount of the product corresponding to SEQ ID NO:2 is formed compared to oxidation by air, which is in line with the higher specificity and purity of albumin.

[0206] discuss:

[0207] The two experiments described in this embodiment demonstrate the important role of albumin in generating a polypeptide from the sequence WGWSSCSRSCG (SEQ ID NO: 2) with two cysteine ​​residues forming a disulfide bridge, starting from the polypeptide with the sequence WGWSSCSRSCG (SEQ ID NO: 58), compared to oxidation in air. This is because, in air, the reaction is at least 2 times faster, preferably 5 times faster, and more preferably 10 times faster, depending on the "peptide / albumin" ratio. In the embodiment shown above, a formation of 9.1% was observed without albumin, compared to 38% with albumin, thus the reaction is approximately 4 times faster here, and the reaction also proceeds with better specificity (25% without albumin, compared to 54% with albumin), which corresponds to higher purity. This is because, within 60 minutes, in the absence of albumin, a 25% conversion of the polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:58) corresponds to the possible formation of 75% impurities, while in the presence of albumin, a 54% conversion of the polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:58) corresponds to the formation of only 46% impurities.

[0208] Example 3: Effect of the ratio of the polypeptide with the sequence WGWSSCSRSCG (SEQ ID NO:58) to albumin on the efficiency of cyclizing the polypeptide with the sequence WGWSSCSRSCG (SEQ ID NO:58) into a polypeptide with the sequence WGWSSCSRSCG (SEQ ID NO:2) in which two cysteine ​​residues are linked by disulfide bridges.

[0209] Example 2 has shown how equimolar ratios (the same molar ratio) of albumin and peptide enable the production of larger quantities of the peptide corresponding to sequence SEQ ID NO:2, starting with the peptide of sequence SEQ ID NO:58, with higher purity. The examples described below illustrate how different ratios of albumin and peptide affect the disulfide bridge formation rate and the purity of the peptide of sequence SEQ ID NO:2.

[0210] Three amounts of the peptide with the sequence WGWSSCSRSCG (SEQ ID NO:58) were incubated at 37°C in the same volume of rat cerebrospinal fluid (containing a constant physiological amount of albumin) for approximately 1 hour. The formation rate of the cyclic peptide WGWSSCSRSCG (SEQ ID NO:2), in which two cysteine ​​residues form a disulfide bridge, was observed by HPLC. This study sought to demonstrate the effect of the peptide:albumin ratio on the cyclization rate (measured by Tmax) of the linear peptide with the sequence SEQ ID NO:58.

[0211] Table 1 reports Tmax, which corresponds to the time when the cyclic polypeptide WGWSSCSRSCG (SEQ ID NO:2) with the maximum amount of sequence formed is formed when two cysteine ​​residues form a disulfide bridge.

[0212] Table 1 shows the proportions of the polypeptide with sequence SEQ ID NO:58 corresponding to different dosages added.

[0213] Table 1. Effect of the ratio of "peptide with sequence SEQ ID NO:58:albumin" on the Tmax of the reaction of cyclization of peptide with sequence SEQ ID NO:58 to peptide with sequence SEQ ID NO:2.

[0214]

[0215] Conclusion: A catalytic effect was observed in albumin, as illustrated by the proportions present in Table 1, demonstrating that albumin is involved in accelerating cyclization. This is because very slight differences in the amount of albumin present with the peptide (as represented by the proportions of 20:1 and 200:1) enabled accelerated cyclization, thus reducing its Tmax from 120 minutes to 30 minutes.

[0216] In summary, the ratio of the polypeptide with the sequence WSGWSSCSRSCG (SEQ ID NO:58) to albumin is correlated with cyclization kinetics.

[0217] This is because the smaller the ratio of peptide to albumin, the faster the reaction. In other words, the closer the ratio of peptide to albumin is to 1:1, the faster the reaction. Finally, a small amount of albumin is sufficient to accelerate the cyclization reaction and the formation of disulfides (catalytic effect).

[0218] Alternatively, cyclization and disulfide formation in the presence of albumin can be carried out in the complete absence of air or in the presence of very small amounts of air and therefore oxygen, which can prevent the formation of polluting products from uncontrolled oxidation reactions.

[0219] Example 4: Study on the solubility of the cyclic polypeptide WSGWSSCSRSCG (SEQ ID NO:2) with two cysteine ​​residues forming a disulfide bridge in NaCl aqueous solution.

[0220] For peptides

[0221] WSGWSSCSRSCG(SEQ ID NO:58)

[0222] and peptides

[0223] (SEQ ID NO:2),

[0224] Weigh 0.5 mg twice in a 1.5 mL microtube.

[0225] 17 μL of a pre-prepared 0.9% NaCl solution was added to each tube to produce a peptide solution of 29.4 mg / mL. The tubes were then vortexed for a few seconds, and a drop of each obtained peptide solution was placed on a glass slide for observation under an optical microscope. Under these conditions, only the peptide with sequence SEQ ID NO:2 appeared to be soluble, without any visible particles or aggregates. Figure 1 The polypeptide with sequence SEQ ID NO:58 has a large number of particles.

[0226] Under a microscope and by visual inspection, the solubility limit of the polypeptide with sequence SEQ ID NO:58 in 0.9% NaCl aqueous solution was determined to be approximately 2 mg / mL.

[0227] Visual inspection revealed that the solubility of the polypeptide with sequence SEQ ID NO:2 in 0.9% NaCl is estimated to be greater than 29 mg / mL.

[0228] Therefore, the solubility limit of the polypeptide with sequence SEQ ID NO:2 in 0.9% NaCl solution is at least 15 times that of the polypeptide with sequence SEQ ID NO:58 in the same solution.

[0229] Example 5: The peptide WGWSSCSRSCG (SEQ ID NO:2) with two cysteine ​​residues linked by disulfide bridges exhibits better stability than WGWSSCSRSCG (SEQ ID NO:58) in the presence of an enzyme.

[0230] Enzymatic digestion was performed using a neutral lysozyme incubated with either the purified form of the target polypeptide (SEQ ID NO:58) or its cyclic form (SEQ ID NO:2). Time parameters (t=0 and t=30 min) were probed using the enzyme diluted to one-tenth in water. So-called "control" samples, prepared in parallel without the enzyme, were used to establish analytical references when probing each parameter.

[0231] The peptides were diluted in deoxygenated water to maintain the stability of both forms over time. Digestion was analyzed using LC / MS / MS mass spectrometry.

[0232] The results (area under the curve and percentage of digestion) are presented in Table 2. For a given peptide, the percentage of digestion corresponds to the percentage of the area under the curve in the presence of the enzyme relative to the area under the curve of the control for the same peptide.

[0233] Table 2: Area under the curve (AUC) measured by LC / MS / MS after analysis of peptide digestion with neutral lysozyme, and percentage of digestion for peptides with sequence SEQ ID NO:58 and sequence SEQ ID NO:2.

[0234]

[0235] Conclusion: At t=0, both peptides appeared to be stable in the presence of neutral lysozyme. At t=30 min, the cyclic peptide (SEQ ID NO:2) was more stable than the linear peptide (SEQ ID NO:58) (28% digestion vs. 8.5%).

[0236] Example 6: The separation of the two peaks corresponding to the sequences SEQ ID NO:2 and SEQ ID NO:58 indicates a conformational difference.

[0237] A polypeptide with the sequence SEQ ID NO:2, starting from the polypeptide of SEQ ID NO:58, was synthesized in the presence of human albumin. Two HPLC analyses of the present compounds were performed during the synthesis reaction: the first at T=0 min and the second at T=60 min. The obtained chromatograms were superimposed and presented on [the image / platform]. Figure 2 middle.

[0238] The analytical conditions applied to separate the compounds are as follows:

[0239] ·Pillar: C18, 2.1 x 100 mm, 3.5 μm

[0240] • Mobile phase: A: Acetonitrile containing 0.1% TFA (trifluoroacetic acid)

[0241] B: Distilled water containing 0.1% TFA

[0242] • Washing rate: 0.500 mL / min

[0243] ·gradient:

[0244] Time (minutes) A％ B％ 0 10 90 11 25 75 21 10 90

[0245] Figure 2 The results show that the peaks of the peptides corresponding to the sequences SEQ ID NO 2 (cyclic) and SEQ ID NO:58 (linear, acyclic) can be easily separated.

[0246] Example 7: In vitro pharmacology

[0247] At 75cm 2 Neuroblastoma B104 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 2 mM glutamine, 50 U / mL penicillin G, 50 μg / mL streptomycin sulfate, and 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells were seeded into 48-well plates coated with 10 μg / mL poly-D-lysine to achieve a final cell density of 5000 cells / well. Four hours after seeding, the medium was replaced with FBS-free medium at 200 μL / well (with or without the cyclic polypeptide of SEQ ID NO:2 at concentrations of 1000, 500, 375, or 150 μg / mL). The medium was not changed throughout the duration of the experiment. In this case, the absence of serum induced the initiation of differentiation in B104 cells, with the appearance of more or less significant axonal elongation.

[0248] To quantify the effect of the cyclic polypeptide with sequence SEQ ID NO:2 on the culture, cells were observed under a phase-contrast microscope in randomly selected fields of view after 1, 2, and 3 days of culture, using a gridded objective (1 × 1 cm, divided into 10 × 10 squares) at a magnification of 400. For each experiment, each well was counted in triplicate, and 3 fields / well were examined. The parameters analyzed were cell number, number of axons (cell extensions at least as large as the diameter of the cell body) per cell, and the average length of the 10 longest axons.

[0249] Under these conditions, a cyclic peptide with the sequence SEQ ID NO:2 was observed. The number of B104 cells has effect :

[0250] Under standard conditions, i.e., in the absence of the polypeptide with sequence SEQ ID NO:2, the growth of B104 cells increased for 48 hours, before the cell number began to decline due to the absence of serum and depletion of culture medium. However, in the presence of the cyclic polypeptide with sequence SEQ ID NO:2, the cell number increased from 24 hours onwards, with 5-fold more cells compared to the control conditions.

[0251] Examples of B104 cell growth are presented. Figure 2 , 3 , 4 and 5.

[0252] • A cyclic polypeptide with the sequence SEQ ID NO:2 It has an effect on the morphology of B104 cells. :

[0253] Although B104 cells exhibit fibroblast-type morphology when cultured in the presence of serum, they participate in neuronal-type differentiation when cultured in the absence of serum. At 24 hours, in serum-free cultures and with or without differentiation factors, B104 cells showed unipolar or bipolar extensions as well as budding (small extensions smaller than the diameter of the cell body) and axons (cell extensions at least as large as the diameter of the cell body), which are typical morphologies under these conditions (Schubert D et al., Nature 249(454), 224-2271974).

[0254] Regardless of concentration, the cyclic polypeptide with sequence SEQ ID NO:2 had no effect on budding number. However, the presence of the cyclic polypeptide with sequence SEQ ID NO:2 increased the axon number / cell ratio. Figure 6 ) and its length ( Figure 7 ).

[0255] After 3 days of culture, the treated B104 cells developed significant axonal elongation that increased with culture time. This effect on axonal length appeared to be dose-dependent.

[0256] Example 8: In vivo pharmacology – evaluation in a spinal cord injury model

[0257] The contusion model is well known and used to simulate spinal cord injury observed in humans (Young W., ProgBrain Res., 137, 231-55, 2002).

[0258] This model was used to evaluate the efficacy of the active ingredient in restoring function. Experiments were conducted on approximately 250g female adult Sprague-Dawley rats. Surgical procedures were performed under general anesthesia with 5% isoflurane (in 70% N₂O and 30% O₂; flow rate: 300 ml / min).

[0259] Following laminectomy at the T9 and T10 thoracic vertebrae, spinal cord injury was obtained by using an electromagnetically controlled device (PinPoint system, Hatteras Inc., USA) that enables the acquisition of calibrated contusions (2 mm depth, 2 mm impactor diameter, and 85 ms advance at 1.5 m / s) (according to Bilgen M. Neurorehab and Neuralre. 19(3), 2005).

[0260] Within fifteen minutes of trauma, 3 μl of a cyclic polypeptide or carrier with the sequence SEQ ID NO:2 at a concentration of 7 μg / mL was injected using a Hamilton syringe (10pi, model 1701) and a microinfusion system (Harvard Apparatus) that allows injection into the spinal space. After injection, the needle was left in place for 5 minutes. Once the needle was withdrawn, a cell adhesive was applied to the injection site to seal the dura mater and prevent any leakage of biological fluid. After skin suturing, rats were housed individually in standard cages at 22°C ± 1°C under controlled lighting (12 hours / day) with free access to water and food. A second injection was given after 2 days. Behavioral tests were performed on each animal individually in a blinded manner.

[0261] Motor function was studied using the Basso-Beattie-Bresnahan (BBB) ​​scale (Basso DM et al., J Neurotrauma, 12, 1-21, 1995; Basso DM et al., Exp Neurol, 139, 244-256, 1996).

[0262] Each animal was evaluated before trauma and then weekly. In parallel, each animal was weighed before trauma and then weekly.

[0263] In the BBB score, statistical data are expressed as mean ± SEM.

[0264] BBB scores are graded from 0 (no movement of the hind paws) to 21 (normal walking with coordinated paws and parallel placement). Scores of 0 to 7 indicate the recovery of isolated movement of the three joints of the hind paws (ankle, knee, hip), scores of 8 to 13 indicate the recovery of hind paw placement and coordination with the forepaws, and scores of 14 to 21 indicate the recovery of toe separation, paw and tail position, and trunk stability.

[0265] BBB scores were assessed by two independent observers before injury, and then on days 1, 7, 14, 21, and 28 after treatment. Figure 8 , 9 10).

[0266] Analysis of toe separation was performed on days 1, 7, 14, 21, and 28 post-treatment. Figure 11 ) and hind paw placement ( Figure 12 and 13 The reflex activity is considered. The scores are: 0 = no reflex activity, 1 = significantly below normal, 2 = slightly below normal, 3 = normal.

[0267] The number of days it takes for bladder control to recover is also one of the parameters being measured. Figure 14 ). sequence list <110> Neuronax <120> Cyclic peptides, their acquisition methods, and their therapeutic applications <130> IFB 15 BR NEU N218 <141> 2016-08-01 <150> FR15 / 59084 <151> 2015-09-25 <160> 70 <170> SIPOSequenceListing 1.0 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> misc_feature <222> (3)..(3) <223> Xaa = Ser or Gly <220> <221> misc_feature <222> (5)..(5) <223> Xaa = Ser or Gly <220> <221> misc_feature <222> (6)..(6) <223> Xaa = Ser or Gly <220> <221> DISULFID <222> (7)..(10) <220> <221> misc_feature <222> (9)..(9) <223> Xaa = Arg-Ser or Val-Ser or Val-Thr <400> 1 Trp Ser Xaa Trp Xaa Xaa Cys Ser Xaa Cys Gly 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 2 Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> misc_feature <222> (1)..(1) <223> Xaa = hydrogen atom or Pro or Ala-Pro or Leu-Ala-Pro or Val-Leu-Ala-Pro <220> <221> misc_feature <222> (4)..(4) <223> Xaa = Ser or Gly <220> <221> misc_feature <222> (6)..(6) <223> Xaa = Ser or Gly <220> <221> misc_feature <222> (7)..(7) <223> Xaa = Ser or Gly <220> <221> DISULFID <222> (8)..(11) <220> <221> misc_feature <222> (10)..(10) <223> Xaa = Arg-Ser or Val-Ser or Val-Thr <220> <221> misc_feature <222> (13)..(13) <223> Xaa = hydrogen atom or Leu or Leu-Gly or Leu-Gly-Leu or Leu-Gly-Leu-Ile or Leu-Gly-Leu-Ile-Phe <400> 3 Xaa Trp Ser Xaa Trp Xaa Xaa Cys Ser Xaa Cys Gly Xaa 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 4 Trp Ser Ser Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 5 Trp Ser Ser Trp Ser Gly Cys Ser Arg Ser Cys Gly 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 6 Trp Ser Ser Trp Gly Ser Cys Ser Arg Ser Cys Gly 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 7 Trp Ser Ser Trp Gly Gly Cys Ser Arg Ser Cys Gly 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa = hydrogen atom or an amino acid chain with 0 to 4 amino acids. <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa = Ser or Gly <220> <221> DISULFID <222> (8)..(11) <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Arg-Ser or Val-Ser or Val-Thr <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa = hydrogen atom or an amino acid chain with 0 to 5 amino acids. <400> 8 Xaa Trp Ser Xaa Trp Xaa Xaa Cys Ser Xaa Cys Gly Xaa 1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 9 Trp Ser Gly Trp Ser Gly Cys Ser Arg Ser Cys Gly 1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 10 Trp Ser Gly Trp Gly Ser Cys Ser Arg Ser Cys Gly 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 11 Trp Ser Gly Trp Gly Gly Cys Ser Arg Ser Cys Gly 1 5 10 <210> 12 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 12 Trp Ser Ser Trp Ser Ser Cys Ser Val Ser Cys Gly 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 13 Trp Ser Ser Trp Ser Gly Cys Ser Val Ser Cys Gly 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 14 Trp Ser Ser Trp Gly Ser Cys Ser Val Ser Cys Gly 1 5 10 <210> 15 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 15 Trp Ser Ser Trp Gly Gly Cys Ser Val Ser Cys Gly 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 16 Trp Ser Gly Trp Ser Ser Cys Ser Val Ser Cys Gly 1 5 10 <210> 17 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 17 Trp Ser Gly Trp Ser Gly Cys Ser Val Ser Cys Gly 1 5 10 <210> 18 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 18 Trp Ser Gly Trp Gly Ser Cys Ser Val Ser Cys Gly 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 19 Trp Ser Gly Trp Gly Gly Cys Ser Val Ser Cys Gly 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 20 Trp Ser Ser Trp Ser Ser Cys Ser Val Thr Cys Gly 1 5 10 <210> twenty one <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> twenty one Trp Ser Ser Trp Ser Gly Cys Ser Val Thr Cys Gly 1 5 10 <210> twenty two <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> twenty two Trp Ser Ser Trp Gly Ser Cys Ser Val Thr Cys Gly 1 5 10 <210> twenty three <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> twenty three Trp Ser Ser Trp Gly Gly Cys Ser Val Thr Cys Gly 1 5 10 <210> twenty four <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> twenty four Trp Ser Gly Trp Ser Ser Cys Ser Val Thr Cys Gly 1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 25 Trp Ser Gly Trp Ser Gly Cys Ser Val Thr Cys Gly 1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 26 Trp Ser Gly Trp Gly Ser Cys Ser Val Thr Cys Gly 1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 27 Trp Ser Gly Trp Gly Gly Cys Ser Val Thr Cys Gly 1 5 10 <210> 28 <211> 13 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (8)..(12) <400> 28 Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 <210> 29 <211> 14 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (9)..(13) <400> 29 Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 <210> 30 <211> 15 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (10) (14) <400> 30 Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 15 <210> 31 <211> 16 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (11) (15) <400> 31 Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 15 <210> 32 <211> 13 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 32 Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu 1 5 10 <210> 33 <211> 14 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 33 Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly 1 5 10 <210> 34 <211> 15 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 34 Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu 1 5 10 15 <210> 35 <211> 16 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 35 Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu Ile 1 5 10 15 <210> 36 <211> 17 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 36 Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu Ile 1 5 10 15 Phe <210> 37 <211> 14 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (8)..(12) <400> 37 Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu 1 5 10 <210> 38 <211> 15 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (8)..(12) <400> 38 Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly 1 5 10 15 <210> 39 <211> 16 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (8)..(12) <400> 39 Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu 1 5 10 15 <210> 40 <211> 17 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (8)..(12) <400> 40 Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu 1 5 10 15 Ile <210> 41 <211> 18 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (8)..(12) <400> 41 Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu 1 5 10 15 Ile Phe <210> 42 <211> 15 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (9)..(13) <400> 42 Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu 1 5 10 15 <210> 43 <211> 16 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (9)..(13) <400> 43 Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly 1 5 10 15 <210> 44 <211> 17 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (9)..(13) <400> 44 Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly 1 5 10 15 Leu <210> 45 <211> 18 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (9)..(13) <400> 45 Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly 1 5 10 15 Leu Ile <210> 46 <211> 19 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (9)..(13) <400> 46 Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly 1 5 10 15 Leu Ile Phe <210> 47 <211> 16 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (10) (14) <400> 47 Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu 1 5 10 15 <210> 48 <211> 17 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (10) (14) <400> 48 Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu 1 5 10 15 Gly <210> 49 <211> 18 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (10) (14) <400> 49 Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu 1 5 10 15 Gly Leu <210> 50 <211> 19 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (10) (14) <400> 50 Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu 1 5 10 15 Gly Leu Ile <210> 51 <211> 20 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (10) (14) <400> 51 Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu 1 5 10 15 Gly Leu Ile Phe 20 <210> 52 <211> 17 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (11) (15) <400> 52 Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 15 Leu <210> 53 <211> 18 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (11) (15) <400> 53 Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 15 Leu Gly <210> 54 <211> 19 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (11) (15) <400> 54 Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 15 Leu Gly Leu <210> 55 <211> 20 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (11) (15) <400> 55 Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 15 Leu Gly Leu Ile 20 <210> 56 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (11) (15) <400> 56 Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 15 Leu Gly Leu Ile Phe 20 <210> 57 <211> 11 <212> PRT <213> Homo sapiens <220> <221> misc_feature <222> (3)..(3) <223> Xaa = Ser or Gly <220> <221> misc_feature <222> (5)..(5) <223> Xaa = Ser or Gly <220> <221> misc_feature <222> (6)..(6) <223> Xaa = Ser or Gly <220> <221> misc_feature <222> (9)..(9) <223> Xaa = Arg-Ser or Val-Ser or Val-Thr <400> 57 Trp Ser Xaa Trp Xaa Xaa Cys Ser Xaa Cys Gly 1 5 10 <210> 58 <211> 12 <212> PRT <213> Homo sapiens <400> 58 Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly 1 5 10 <210> 59 <211> 11 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa = Ser or Gly <220> <221> DISULFID <222> (7)..(10) <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa = Arg-Ser or Val-Ser or Val-Thr or Arg-Thr <400> 59 Trp Ser Xaa Trp Xaa Xaa Cys Ser Xaa Cys Gly 1 5 10 <210> 60 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 60 Trp Ser Ser Trp Ser Ser Cys Ser Arg Thr Cys Gly 1 5 10 <210> 61 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 61 Trp Ser Ser Trp Ser Gly Cys Ser Arg Thr Cys Gly 1 5 10 <210> 62 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 62 Trp Ser Ser Trp Gly Ser Cys Ser Arg Thr Cys Gly 1 5 10 <210> 63 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 63 Trp Ser Ser Trp Gly Gly Cys Ser Arg Thr Cys Gly 1 5 10 <210> 64 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 64 Trp Ser Gly Trp Ser Ser Cys Ser Arg Thr Cys Gly 1 5 10 <210> 65 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 65 Trp Ser Gly Trp Ser Gly Cys Ser Arg Thr Cys Gly 1 5 10 <210> 66 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 66 Trp Ser Gly Trp Gly Ser Cys Ser Arg Thr Cys Gly 1 5 10 <210> 67 <211> 12 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> DISULFID <222> (7)..(11) <400> 67 Trp Ser Gly Trp Gly Gly Cys Ser Arg Thr Cys Gly 1 5 10 <210> 68 <211> 13 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa = hydrogen atom or an amino acid chain with 0 to 4 amino acids. <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa = Ser or Gly <220> <221> DISULFID <222> (8)..(11) <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Arg-Ser or Val-Ser or Val-Thr or Arg-Thr <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa = hydrogen atom or an amino acid chain with 0 to 5 amino acids. <400> 68 Xaa Trp Ser Xaa Trp Xaa Xaa Cys Ser Xaa Cys Gly Xaa 1 5 10 <210> 69 <211> 13 <212> PRT <213> Artificial sequence <220> <223> A clip from SCO-Spondin <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa = hydrogen atom or Pro or Ala-Pro or Leu-Ala-Pro or Val-Leu-Ala-Pro <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa = Ser or Gly <220> <221> DISULFID <222> (8)..(11) <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Arg-Ser or Val-Ser or Val-Thr or Arg-Thr <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa = hydrogen atom or Leu or Leu-Gly or Leu-Gly-Leu or Leu-Gly-Leu-Ile or Leu-Gly-Leu-Ile-Phe <400> 69 Xaa Trp Ser Xaa Trp Xaa Xaa Cys Ser Xaa Cys Gly Xaa 1 5 10 <210> 70 <211> 11 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa = Ser or Gly <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa = Arg-Ser or Val-Ser or Val-Thr or Arg-Thr <400> 70 Trp Ser Xaa Trp Xaa Xaa Cys Ser Xaa Cys Gly 1 5 10

Claims

1. A polypeptide, which consists of the following amino acid sequence: WSGWSSCSRSCG (SEQ ID NO: 2) The two cysteine ​​residues form a disulfide bridge.

2. A method for obtaining a polypeptide with the amino acid sequence SEQ ID NO: 2: WSGWSSCSRSCG (SEQ ID NO: 2) in: The two cysteine ​​residues form a disulfide bridge. The method includes the step of forming a disulfide bridge in the polypeptide with the sequence SEQ ID NO: 58 in the presence of albumin: WSGWSSCSRSCG (SEQ ID NO: 58).

3. The method according to claim 2, characterized in that, Albumin and the polypeptide with sequence SEQ ID NO: 58 are present in a ratio of 1:1 to 1:

10.

4. The method of claim 3, wherein the ratio is 1:

1.

5. The method according to any one of claims 2-4, characterized in that, The step of forming disulfide bridges in the presence of albumin is carried out in ambient air.

6. The method according to any one of claims 2-4, characterized in that, The step of forming a disulfide bridge in the polypeptide with sequence SEQ ID NO: 58 in the presence of albumin is performed without detaching the polypeptide from the resin used for peptide synthesis of the polypeptide, and then the polypeptide with sequence SEQ ID NO: 2 is obtained by separating the polypeptide from the resin after the step of forming the disulfide bridge.

7. A pharmaceutical composition comprising the polypeptide described in claim 1 as an active ingredient and one or more pharmaceutically acceptable excipients.

8. Use of the polypeptide of claim 1 in the manufacture of a medicament for use in the treatment of neurodegenerative pathological conditions, wherein the treatment seeks regeneration of the central nervous system.

9. The neurodegenerative pathological condition according to claim 8 is Alzheimer's disease, multiple sclerosis, or Parkinson's disease.

10. Use of the polypeptide of claim 1 in the manufacture of a medicament for use in the treatment of trauma, wherein the treatment seeks regeneration of the central nervous system.

11. The use according to claim 10, wherein the trauma is spinal cord injury, cranial trauma, or stroke.

12. Use of the polypeptide of claim 1 in the manufacture of a medicament for use in the treatment of any injury to a cranial nerve or peripheral nerve.

13. The use according to claim 12, wherein the injury includes damage to the optic nerve, olfactory nerve, or auditory nerve.

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