Systemic Administration of Peptides for the Treatment of Spinal Cord Injury and / or for Remyelination - Patent application

Abstract

The present invention relates to the treatment of nervous system injuries other than those of the brain, such as spinal cord injury and / or optic nerve injury, with SCO-spondin-derived peptides administered to patients via a systemic route. The peptides have the amino acid sequence X1-WS-A1-WS-A2-CS-A3-A4-CG-X2, where A1, A2, A3, and A4 consist of an amino acid sequence of 1 to 5 amino acids, and X1 and X2 consist of an amino acid sequence of 1 to 6 amino acids, or X1 and X2 are absent, and the N-terminal amino acid can be acetylated and the C-terminal amino acid can be amidated, or the N-terminal amino acid can be acetylated and the C-terminal amino acid can be amidated. The use of such peptides for remyelination is also described.

Description

[Technical field]

[0001] The present invention relates to the systemic administration of SCO-spondin derived peptides for the treatment of injuries of the nervous system other than the brain, such as spinal cord injury and / or optic nerve injury.The present invention also relates to the systemic administration of SCO-spondin derived peptides for remyelination in myelopathy, including spinal cord injury and other types of myelopathy related to the spinal cord or central nervous system. [Background technology]

[0002] Spinal cord injury (SCI) results in a high degree of morbidity with incomplete / complete sensorimotor paralysis. The primary injury leads to necrosis and hemorrhage, followed by secondary damage including gliosis.

[0003] Traumatic optic nerve injury is the leading cause of irreversible blindness worldwide, causing progressive visual impairment. Currently, there are insufficient pharmacological or surgical interventions to halt or reverse the progression of vision loss. Axonal regeneration is critical for functional recovery of vision after optic nerve injury. Optic nerve injury can be caused by primary and secondary mechanisms. Primary injury is due to permanent axonal damage upon impact due to mechanical shearing, crushing, and ischemic necrosis of the nerve axons. Secondary mechanisms are due to apoptosis, edema, and cell death, which encompass various mechanisms that lead to further axonal injury after the initial impact (Schmidek and Sweet, Operative Neurosurgical Techniques (Sixth Edition), 2012, pp. 2329-2338). The nervous system is divided into two parts: the peripheral nervous system and the central nervous system. While injured peripheral nerves can regenerate after injury, various inhibitory factors impede regeneration of the optic nerve and spinal cord after injury. Therefore, there is a significant unmet medical need for the treatment of spinal cord and / or optic nerve injury.

[0004] The spinal cord and optic nerves are surrounded by cerebrospinal fluid (CSF) and are separated from the blood by the blood-brain barrier (BBB), the blood-CSF barrier, and especially the blood-optic nerve barrier (hereinafter referred to as the "blood barrier").

[0005] Peptides are increasingly becoming the therapeutic modality of choice for therapeutic development in many disease conditions due to their safety, target specificity and efficacy.One of the main limitations of therapeutic peptides is their short half-life, which results in low bioavailability.In the case of therapeutic peptide development for CNS pathologies, another important problem is the low permeability across biological barriers, such as the blood-brain barrier (BBB), blood-cerebrospinal fluid barrier, and especially the blood-optic nerve barrier.

[0006] SCO-spondin-derived peptides have been described for their neuroregenerative properties, especially their ability to improve cell survival and neurite outgrowth in vitro. NX210 SCO-spondin-derived peptide has been shown to improve axonal regrowth in a rat model of SCI when administered directly to the lesion site in a collagen tube (Sakka et al., 2014, Plos One 9(3):e93179). The same authors have also shown that NX210 significantly improves functional recovery of SCI in a rat contusion model when administered intralesionally. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO-99 / 03890 [Patent Document 2] U.S. Patent No. 6,995,140 [Patent Document 3] WO2018146283 [Patent Document 4] WO 2017 / 051135 [Non-patent literature]

[0008] [Non-Patent Document 1] Schmidek and Sweet, Operative Neurosurgical Techniques (Sixth Edition), 2012, pp. 2329-2338. [Non-Patent Document 2] Sakka et al., 2014, Plos One 9(3):e93179 [Non-Patent Document 3] Liang Li et al., Frontiers in Cellular Neuroscience, April 2020, vol 14, article 109 [Non-Patent Document 4] Biophysical Journal Volume 95 November 2008 pages 4879~4889 [Non-Patent Document 5] Geoffroy CG et al. J Neurosci. 2015 Apr 22; 35(16):6413-28 [Non-Patent Document 6] Basso DM. et al., J Neurotrauma. 2006 May;23(5):635-59 [Non-Patent Document 7] Callizot N, Combes M, Steinschneider R, Poindron P (2013) Operational dissection of β-amyloid cytopathic effects on cultured neurons. J Neurosci Res 91:706–716 [Non-Patent Document 8] Leonetti et al., Molecular Neurodegeneration, 2017 Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have made an unexpected discovery that SCO-spondin derived peptides can be administered via systemic route and still remain effective for treating nervous system injury other than brain, such as spinal cord injury and optic nerve injury.This means that it has been demonstrated that SCO-spondin derived peptides can remain in blood circulation for a sufficient time, can cross the blood barrier at a sufficient level to reduce the effects of injury, particularly primary and / or secondary injury of spinal cord and / or optic nerve, and can assist or improve recovery after such injury, particularly spinal cord and / or optic nerve injury. [Means for solving the problem]

[0010] Systemic administration is unexpectedly effective for the peptides of the invention in this particular situation. This has a significant advantage over administration directly at the site of the lesion or directly into the CSF (intrathecal or intraspinal injection), since systemic administration is safer and more convenient for the patient being treated. An important or advantageous aspect of this mode of administration is that it allows repeated administration over time by medical personnel for a given patient. Another important or advantageous consequence of this method of administration is that it allows treatment very early, especially at the time of the critical care process, especially before the formation of glial scars, and also facilitates patient acceptance of treatment. The peptides can be easily administered by injection or infusion, including via perfusion (the perfusion method may or may not be dedicated to this peptide, and it is convenient to use the perfusion used for administration of other products). Early treatment is believed to reduce or prevent toxicity resulting from early cell death and secondary damage. The inventors have also found that bioavailability of these peptides at the lesion level after systemic administration does not require administration of excessively large amounts of the peptides, making systemic administration of peptides very convenient and promising for patients after such injuries.

[0011] In a mouse model of focal lesion in the corpus callosum, systemic administration of the peptide of the present invention showed beneficial effects on remyelination, as shown by myelin-associated protein levels, recruitment of Olig2-positive progenitor cells (required for the synthesis of myelin) and generation of Olig2-positive cells. Systemic administration of the SCO-spondin-derived peptide of the present invention can therefore be used for remyelination in myelopathy, including spinal cord injury and other types of myelopathy related to the spinal cord or CNS.

[0012] One object of the present invention is therefore a pharmaceutical composition comprising one or more peptides derived from the thrombospondin repeats (TR or TSR) of SCO-spondin or one or more peptides derived from the TR of SCO-spondin for use in the treatment of injuries of the nervous system other than the brain, such as spinal cord injuries and / or optic nerve injuries, wherein the peptides are administered to the patient via a systemic route.

[0013] Another object of the present invention is one or more peptides derived from TR of SCO-spondin or a pharmaceutical composition comprising one or more peptides derived from TR of SCO-spondin for use in delivery of an effective or sufficient amount of one or more peptides derived from TR of SCO-spondin to damaged nervous system other than brain, such as spinal cord and / or optic nerve, in a patient in need thereof, comprising administering said peptides to the patient via systemic route. This delivery is believed to allow the peptides to reach cerebrospinal fluid. This delivery may be further characterized by allowing the delivered peptides to treat spinal cord injury and / or optic nerve injury in a patient in need thereof.

[0014] Another object of the present invention is the use of one or more peptides derived from the TR of SCO-spondin for the preparation of a pharmaceutical composition for systemic administration to a patient for the treatment of damage to the nervous system other than the brain, such as spinal cord damage and / or optic nerve damage.

[0015] Another object of the present invention is a method for the treatment of damage to the nervous system other than the brain, such as spinal cord damage and / or optic nerve damage, in a patient in need thereof, comprising administering to said patient via a systemic route an effective or sufficient amount of one or more peptides derived from the TR of SCO-spondin, or of a pharmaceutical composition comprising a peptide.

[0016] Another object of the present invention is a method for the delivery of an effective or sufficient amount of one or more peptides derived from the TR of SCO-spondin to the damaged nervous system other than the brain, such as the spinal cord and / or the optic nerve, in particular in the cerebrospinal fluid, in a patient in need thereof, comprising administering said peptides to said patient via a systemic route.This delivery may be further characterized in that the delivered peptides allow to treat spinal cord injury and / or optic nerve injury in a patient in need thereof.

[0017] In one aspect, this systemic administration of the peptides of the invention has a beneficial effect on myelination or remyelination, as may be assessed, for example, using measurements of myelin-associated proteins and / or measurements of recruitment of Olig2-positive progenitor cells and / or measurements of generation of Olig2-positive cells.

[0018] Another object of the present invention is a pharmaceutical composition comprising one or more peptides derived from the thrombospondin repeats (TR or TSR) of SCO-spondin or one or more peptides derived from the TR of SCO-spondin for use in remyelination in myelopathy, including spinal cord injury and other types of myelopathy related to the spinal cord or CNS, wherein the peptides are administered to the patient via a systemic route.

[0019] Another object of the present invention is a method of treatment or remyelination in a patient in need thereof, comprising administering to said patient via a systemic route an effective or sufficient amount of one or more peptides derived from the TR of SCO-spondin or of a pharmaceutical composition comprising the peptide. This method of treatment of myelopathy includes remyelination. Myelopathy includes spinal cord injury and other types of myelopathy related to the spinal cord or CNS.

[0020] "SCO-spondin" is a glycoprotein specific to the central nervous system and is present in all vertebrates, from prochordals to humans. It is known as a molecule of the extracellular matrix secreted by the subcommissural organ, a specialized organ located in the roof of the third ventricle. It is a large molecule. It consists of more than 4,500 amino acids and has a multimodular organization with various conserved protein patterns, including in particular 26 TR or TSR patterns. Certain peptides derived from SCO-spondin, starting from the TSR pattern, are known to have biological activity in nerves or nerve cells (described in particular in WO-99 / 03890).

[0021] "TSR patterns or TR patterns" are protein domains of approximately 55-60 residues based on the alignment of the conserved amino acids cysteine, tryptophan and arginine. These patterns were first isolated in TSP-1 (thrombospondin 1), a molecule involved in coagulation. They were subsequently described in numerous other molecules, such as SCO-spondin. In fact, this thrombospondin type 1 unit (TSR) is composed of about 55-60 amino acids (AA) in all proteins studied so far and previously mentioned, some of which are highly conserved, such as cysteine ​​(C), tryptophan (W), serine (S), glycine (G), arginine (R) and proline (P).

[0022] "Systemic administration" refers to any mode of administration in which a substantial portion or sufficient amount of the administered peptide or peptide compound reaches the blood circulation after such administration. Intrathecal administration is excluded, as is any systemic administration method that does not target the peptide to the blood circulation. A systemic administration route of the present invention may qualify as a "systemic administration route that targets the blood". Furthermore, in the context of a peptide or peptide compound according to the present invention, once in the blood circulation, the peptide or peptide compound can cross a blood barrier, such as the BBB, the blood-spinal cord barrier, and / or the blood-optic nerve barrier.

[0023] The administration or use of a "peptide" or "peptides" or "peptide(s)" is a generic expression, and the present invention encompasses the administration or use of one individual peptide, or a plurality of individual peptides, i.e., at least two peptides according to the present disclosure. Thus, in the present disclosure, the singular or plural is not limiting, unless otherwise indicated, and may each time encompass one individual peptide, or at least two peptides. The same is true for the equivalent expression "peptide compound", which can be used interchangeably with "peptide".

[0024] "Spinal cord injury" means any injury caused, inter alia, by severance or compression of the spinal cord. Spinal cord severance can be caused by trauma or by surgery. Spinal cord compression can be caused by trauma or secondarily by the growth of surrounding tissues, as in the case of spinal cord tumors or spinal cord metastases. Spinal cord compression can also occur as a result of diseases that affect the spinal cord environment, such as cervical arthrosic myelopathy or Schneider's syndrome. Spinal cord compression can occur in the lumbar, thoracic and / or cervical regions, and the consequences of the injury to the patient may vary depending on the location.

[0025] The "optic nerve" is a specialized sensory nerve that transmits information from the visual world to the brain. The optic nerve originates embryologically from an outgrowth of the forebrain. It is therefore part of the central nervous system (CNS) and is composed of CNS fiber bundles. An article by Liang Li et al. (Frontiers in Cellular Neuroscience, April 2020, vol 14, article 109) describes how the mouse optic nerve crush (ONC) model has been widely used to study optic neuropathy and axonal injury and repair in the central nervous system (CNS). ONC provides a CNS neurodegeneration model that can be used to study degenerative mechanisms and to evaluate neuroprotective and regenerative therapies. Conversely, results from SCI or CNS injury models may be meaningful for optic nerve injury.

[0026] "Optic nerve injury" means a condition that occurs as a result of trauma or surgery or compression of the optic nerve and can lead to reduced or lost vision.

[0027] The "injury" may be a mild injury, a moderate injury, or a severe injury involving, inter alia, partial or complete severance of the spinal cord or optic nerve.

[0028] Myelopathies involving the spinal cord or CNS include, among others, spinal cord injury, optic nerve injury, traumatic brain injury, multiple sclerosis, post-vaccination myelopathy, infectious myelopathy, and viral myelopathy.

[0029] "Treating" or "treating" injury to the nervous system other than the brain, e.g. spinal cord injury and / or optic nerve injury, means delivering an amount of a peptide compound according to the invention to obtain a favorable effect on the primary injury and / or secondary injury in terms of suppressing one or several deleterious effects of the injury, in particular spinal cord and / or optic nerve injury, e.g. suppressing or reducing primary neuronal cell death and / or axonal degeneration, and / or reducing or reducing toxicity resulting from primary cell death, and / or reducing or reducing secondary consequences of the injury, and / or obtaining a benefit in terms of regeneration of neurons and / or axons, and / or obtaining a benefit in terms of functional recovery by the patient.

[0030] Description of peptides or peptide compounds derived from the TR of SCO-spondin useful in carrying out the present invention (for different purposes of the invention, e.g. peptides for use, methods of use, methods of treatment, use of peptides for the manufacture of medicines, etc.)

[0031] In particular, the present invention relates to a compound having the sequence X1-WS-A1-WS-A2-CS-A3-A4-CG-X2 (SEQ ID NO: 1) (In the formula, A1, A2, A3 and A4 each consist of an amino acid sequence consisting of 1 to 5 amino acids, The two cysteines may or may not form a disulfide bridge, X1 and X2 each consist of an amino acid sequence consisting of 1 to 6 amino acids, or X1 and X2 are absent; The N-terminal amino acid can be acetylated (e.g., has HCCOHN-), the C-terminal amino acid can be amidated (e.g., has -CONH), or the N-terminal amino acid can be acetylated and the C-terminal amino acid can be amidated. The peptide is used.

[0032] In one embodiment, in the formula of SEQ ID NO: 1, X1 or X2, or both X1 and X2 are absent. In one embodiment, when X1 and / or X2 are absent, the N-terminus W is acetylated and / or the C-terminus G is amidated. Preferably, both X1 and X2 are absent, the N-terminus W is acetylated, and the C-terminus G is amidated.

[0033] In particular, the present invention provides array WS-A1-WS-A2-CS-A3-A4-CG (SEQ ID NO: 2) (In the formula, A1, A2, A3 and A4 each consist of an amino acid sequence consisting of 1 to 5 amino acids, The two cysteines may or may not form a disulfide bridge. The peptide is used.

[0034] In one embodiment of the formulas of SEQ ID NO:1 and 2, the peptide is a linear peptide or the cysteines found in the peptide formulas of SEQ ID NO:1 and 2 do not form disulfide bridges (reduced form).

[0035] In another embodiment, the two cysteines found in the peptide formulas of SEQ ID NOs: 1 and 2 form a disulfide bridge (oxidized form).

[0036] Preferably, in the formulae of SEQ ID NO: 1 and 2, A1, A2, A3 and / or preferably A4 consist of preferably one or two amino acids, more preferably one amino acid.

[0037] Preferably, A1 is selected from G, V, S, P and A, more preferably from G and S.

[0038] Preferably, A2 is selected from G, V, S, P and A, more preferably from G and S.

[0039] Preferably, A3 is selected from R, A and V, more preferably from R, V.

[0040] Preferably, A4 is selected from S, T, P and A, more preferably from S and T.

[0041] Preferably, A1 and A2 are independently selected from G and S.

[0042] Preferably, A3-A4 is selected from RS or VS or VT or RT.

[0043] When X1 is an amino acid sequence of 1 to 6 amino acids, the amino acids may be any amino acid, preferably selected from V, L, A, P, and any combination thereof.

[0044] When X2 is an amino acid sequence of 1 to 6 amino acids, the amino acids may be any amino acid, preferably selected from L, G, I, F, and any combination thereof.

[0045] In one embodiment, the peptide of SEQ ID NO: 1 or 2 is such that A1 and A2 are independently selected from G and S, and A3-A4 are selected from RS or VS or VT or RT. In a particular manner, the peptide is further acetylated and / or amidated. In one embodiment, the peptide is a linear peptide or the cysteines do not form disulfide bridges. In another embodiment, the peptide has two cysteines that form disulfide bridges (C-terminal cyclization). In another embodiment, the peptides used in the present invention or administered to patients via the systemic route include both oxidized and linear peptide forms.

[0046] For the purposes of the present invention, the term "amino acid" refers to both natural and non-natural amino acids, and amino acid changes, including natural to non-natural changes, can be routinely made by those skilled in the art while maintaining the function or effectiveness of the original peptide. "Natural amino acids" refers to the L-form amino acids 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. "Non-natural amino acids" refers to the D-forms of the preceding amino acids as well as the homoforms of certain amino acids, e.g., arginine, lysine, phenylalanine and serine, or the nor-forms of leucine or valine. This definition also includes other amino acids, such as α-aminobutyric acid, agmatine, α-aminoisobutyric acid, sarcosine, statine, ornithine, deaminotyrosine, etc. The nomenclature used to describe peptide sequences is the International Nomenclature using a single letter code with the amino terminus shown on the left and the carboxy terminus shown on the right. The dash "-" represents the conventional peptide bond joining the amino acids of the sequence.

[0047] In one embodiment, a peptide according to the invention, for example any one of the peptides of sequences SEQ ID NOs: 1 to 63, comprises N-terminal acetylation, C-terminal amidation, or both N-terminal acetylation and C-terminal amidation.

[0048] In a different embodiment, the invention relates to the use of a polypeptide consisting essentially of or consisting of the following amino acid sequence (Table 1):

[0049] [Table 1A]

[0050] [Table 1B]

[0051] In one embodiment, the peptides of sequences SEQ ID NO: 3 to 34 disclosed in Table 1 are linear peptides or the cysteines do not form disulfide bridges (reduced peptides). In another embodiment, the peptides of sequences SEQ ID NO: 3 to 34 disclosed in the preceding table have two cysteines that are oxidized to form disulfide bridges (oxidized peptides). In another embodiment, the peptides used in the invention or administered to a patient via a systemic route comprise both oxidized and linear peptide forms of the same peptide sequence. In yet another embodiment, the peptides used in the invention or administered to a patient via a systemic route comprise a mixture of at least two of these different peptides selected from the sequences SEQ ID NO: 3 to 34, which mixture can be a mixture of at least two linear peptides, or a mixture of at least two oxidized peptides, or a mixture of at least one linear peptide and at least one oxidized peptide, for example, having the same amino acid sequence.

[0052] In a preferred embodiment, the peptide consists of the amino acid sequence WSGWSSCSRSCG (SEQ ID NO: 3). In one embodiment, the peptide is a linear peptide or the cysteines do not form disulfide bridges (reduced form). In another embodiment, the peptide has two cysteines that are oxidized to form disulfide bridges (oxidized form). In another embodiment, the peptides used in the present invention or administered to a patient via a systemic route include both oxidized and reduced forms.

[0053] In an embodiment of the peptide of SEQ ID NO:1, - X1 represents a hydrogen atom or P or AP or LAP or VLAP, and / or - X2 represents a hydrogen atom, L, LG, LGL, LGLI, or LGLIF.

[0054] In a different embodiment, the invention therefore relates to the use of a polypeptide consisting or consisting essentially of the following amino acid sequence (Table 2):

[0055] [Table 2]

[0056] In one embodiment, the peptides of sequences SEQ ID NO: 35-63 disclosed in Table 2 or sequences SEQ ID NO: 3-63 disclosed in Table 1 and Table 2 are linear peptides or the cysteines do not form disulfide bridges (reduced peptides). In another embodiment, the peptides have two cysteines that are oxidized to form disulfide bridges (oxidized peptides). In another embodiment, the peptides used in the invention or the peptides administered to a patient via the systemic route comprise both oxidized and linear peptide forms of the same peptide sequence. In yet another embodiment, the peptides used in the invention or the peptides administered to a patient via the systemic route comprise a mixture of at least two of these different peptides selected from sequences SEQ ID NO: 35-63 or 3-63, where the mixture can be a mixture of at least two linear peptides or a mixture of at least two oxidized peptides or a mixture of at least one linear peptide and at least one oxidized peptide, for example, having the same amino acid sequence.

[0057] Each of the peptides of the sequences of SEQ ID NOs: 3 to 63 may be acetylated, amidated, or both acetylated and amidated.

[0058] In the present invention, the peptide used in the present invention or administered to a patient via a systemic route is defined by its amino acid sequence. The peptide used may be one peptide disclosed herein or a mixture of at least two peptides disclosed herein. The mixture also includes a mixture of linear and oxidized peptides with the same or different amino acid sequences. While 100% pure peptides may be used according to the present invention, the peptides may have a purity of more than 80%, preferably 85%, more preferably 90%, even more preferably equal to or greater than 95, 96, 97, 98 or 99%, and are included in the present invention. Conventional purification methods, for example by chromatography, can be used to purify the desired peptide compound.

[0059] In one embodiment, the peptides used in the present invention or administered to a patient via a systemic route contain both oxidized peptide (Op) and linear peptide (Lp) forms, for example in comparable or different amounts, for example 10, 20, 25, 30, 40, 50, 60, 70, 80 or 90% Op (in numerical comparison %), the rest of the 100% being Lp. The oxidized and linear peptides to be combined may be of the same sequence or of different sequences. For example, oxidized and linear peptides of sequence SEQ ID NO: 3 are so combined. The same applies for any one of the peptides of sequences SEQ ID NO: 4-34 and 35-63.

[0060] The pharmaceutical compositions used in the present invention comprise a peptide as an active ingredient or a mixture of peptides as described above, e.g. peptides of different amino acid composition or peptides of the same amino acid composition in oxidized and linear form, and one or more pharma- ceutically acceptable vehicles, carriers or excipients.

[0061] The peptide compounds according to the invention can be used in pharmaceutical compositions or in the manufacture of medicaments, in which the active ingredient can be incorporated into compositions in various forms, namely in the form of a solution, generally an aqueous solution, or in a lyophilized form, or in the form of an emulsion, or in any other pharma- ceutical and physiologically acceptable form suitable for systemic administration routes.

[0062] According to an important feature of the invention, the peptide compound or a composition containing it is administered via a systemic route, of which particular mention may be made of the following injection or administration routes: intravenous, intraperitoneal, intranasal, subcutaneous, intramuscular, sublingual, oral, and combinations thereof.

[0063] According to the present invention, administration can be performed at various times following injury or suspected injury.

[0064] In one embodiment, administration is performed close to the occurrence of spinal cord and / or optic nerve injury, or close to an accident or surgery, including suspected spinal cord and / or optic nerve injury. The systemic route of administration actually allows for initial administration or initiation of treatment at a very early time, especially when medical assistance is present and as soon as spinal cord and / or optic nerve injury is diagnosed or suspected. Administration may begin at the scene of the accident or in an ambulance, helicopter, etc., or in an operating room, hospital, clinic, etc.

[0065] According to the invention, where the injury is a trauma caused by an internal cause, e.g., a tumor, administration can be performed as soon as the trauma is suspected, observed, or predicted to occur. In one embodiment, where surgery is performed to remove all or part of a tumor, administration can be performed before or after surgery or simultaneously with surgery, as described above.

[0066] Treatment can be administered or initiated within the first few days (e.g., the same day or within one week) or weeks (e.g., 1-8 weeks) or months (e.g., 2-6 months) after injury.

[0067] In one embodiment, treatment or initial administration occurs early, for example, 10 minutes, 30 minutes, 1, 2, 3, 4, 5 hours after the onset of injury, suspicion of injury, or prognosis has been determined.

[0068] In another embodiment, the treatment or first administration occurs 12, 24, 36, or 48 hours after the injury has occurred, the injury is suspected, or a prognosis has been determined.

[0069] In one embodiment, treatment according to the invention is administered to a patient who has been treated, is being treated, or will be treated with a medication that inhibits or reduces gliosis.

[0070] A single dose, expressed as the mass of peptide per patient body weight (kg), may be in the range of about 1 μg / kg to about 1 g / kg, particularly about 10 μg / kg to about 100 mg / kg, for example, about 50 μg / kg to about 50 mg / kg.

[0071] The dosing regimen may include a single dose or multiple doses. According to one embodiment, multiple doses may include administering one dose every treatment day, for example, administering one dose every day or every 2 or 3 days over the treatment period. In another embodiment, multiple doses may include administering at least two doses every treatment day, for example, administering two, three or more doses every day over the treatment period. In these embodiments, the treatment period may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.

[0072] In one embodiment, administration is by perfusion, which can last for a few minutes, tens of minutes, hours, and up to 24 hours per day.

[0073] The use according to the invention and the method of treatment according to the invention may be characterized in that it delivers an amount of a peptide compound according to the invention making it possible to obtain a favorable effect on the primary and / or secondary damage in terms of suppressing one or several of the deleterious effects of the damage, in particular on the spinal cord and / or the optic nerve. This favorable effect may include: - inhibiting or reducing primary neuronal cell death and / or axonal degeneration, - inhibiting or reducing primary neuronal death and / or axonal degeneration, - reducing or preventing toxicity resulting from primary cell death; - Reducing or preventing the secondary consequences of the injury; - obtaining benefits in terms of regeneration of neurons and / or axons and / or myelin sheaths, - To obtain benefits in terms of functional recovery by the patient.

[0074] In one embodiment, the use or method of treatment has the effect of inhibiting or reducing neuronal cell death and / or axonal degeneration, and / or inhibiting or reducing necrosis (primary damage), inhibiting or reducing secondary damage (in particular inhibiting or reducing secondary neuronal cell death and / or axonal degeneration).

[0075] In one embodiment, the use or method of treatment has the effect of inducing or promoting the restoration or regeneration of neural pathways.

[0076] In one embodiment, the use or method of treatment has the effect of aiding or inducing functional rehabilitation, meaning that the patient regains all or part of the function lost as a result of the injury.

[0077] In one embodiment, the use or method of treatment has the effect of halting or inhibiting the loss of function that occurs as a result of the injury.

[0078] The present invention also relates to one or more peptides as described herein, or a pharmaceutical composition as described herein, for use in remyelination in myelopathy, wherein the peptides are administered to a patient via a systemic route.

[0079] The present invention also relates to a method of treating myelopathy or a method of remyelination in a patient in need thereof, comprising administering to said patient via a systemic route an effective or sufficient amount of one or more peptides described herein or a pharmaceutical composition described herein. The method of treating myelopathy includes remyelination.

[0080] In one embodiment, the myelopathy is spinal cord injury or optic nerve injury, hi another embodiment, the myelopathy is another type of myelopathy involving the spinal cord or CNS, including traumatic brain injury, multiple sclerosis, post-vaccination myelopathy, infectious myelopathy, viral myelopathy, among others.

[0081] The dosing regimens, routes of administration, peptide selection and any useful features disclosed above are applicable to these two last objectives.

[0082] The invention also relates to the use of these peptides to induce myelination of neurons in vitro or in vivo, or methods of treatment using these peptides.

[0083] In one embodiment, this systemic administration of the peptides of the invention has beneficial effects on myelination or remyelination that can be measured using known methods, in particular using methods that allow measuring the levels of myelin binding protein (MBP) at the site of the lesion in a patient or animal model and / or measuring the levels of recruitment of Olig2-positive progenitor cells and generation of Olig2-positive cells at the site of the lesion in a patient or animal model.

[0084] In one embodiment, measuring the level of myelin binding protein (MBP) at the lesion site in a patient or animal model can be performed using MBP immunostaining with antibodies (see Example 4 for methods and antibodies). A vehicle control without the peptide according to the invention can be used or compared to a predefined value.

[0085] In another embodiment, measuring the level of recruitment of Olig2 positive progenitor cells and generation of Olig2 positive cells at the lesion site in patients or animal models can be done using, for example, measuring Olig2 cell amount or density by antibodies (see Example 4 for methods and antibodies). A vehicle control without the peptide according to the invention can be used or compared to a predefined value.

[0086] In another embodiment, both measurements are made.

[0087] The present invention also proposes to combine peptide treatment with measurements of remyelination based on such measurements.

[0088] In the following the invention will be explained in more detail by way of non-limiting examples with reference to the drawings. [Brief description of the drawings]

[0089] [Figure 1] Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was assessed using an open field locomotor test in vehicle-treated (n=8) and NX210-treated (n=7) mice, and BMS scores were obtained. *: p<0.05, **: p<0.01, ***: p<0.001. Two-way ANOVA followed by Bonferroni post-hoc test. [Diagram 2] Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was assessed using the open field locomotor test in vehicle-treated (n=8) and NX210-treated (n=7) mice, and BMS subscores were obtained. *: p<0.05, **: p<0.01. Two-way ANOVA followed by Bonferroni post-hoc test. [Diagram 3] Determination of myelin basic protein (MBP) staining intensity 1000 μm rostral and 1000 μm caudal to the injury following a T8 dorsal hemisection (spinal cord injury) in vehicle-treated (n=3) and NX210-treated (n=5) mice using immunofluorescence. [Figure 4]Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was assessed using an open field locomotor test in vehicle-treated mice (n=8) and mice treated with 4 mg / kg (n=9), 8 mg / kg (n=7) and 16 mg / kg (n=8) to obtain BMS scores. @ or * or #: p<0.05, ** or ##: p<0.01. Two-way ANOVA followed by Bonferroni post-hoc test. [Diagram 5] Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was assessed using an open field locomotor test in vehicle-treated mice (n=8) and mice treated with 4 mg / kg (n=9), 8 mg / kg (n=7) and 16 mg / kg (n=8) to obtain BMS subscores. * or #: p<0.05, **: p<0.01. Two-way ANOVA followed by Bonferroni post-hoc test. [Figure 6A] NX210 and NX218 (NX210 oxidized) peptides protected rat cortical neurons from glutamate-induced excitotoxicity in vitro. Primary cortical neurons isolated from E15 rat embryos were co-treated with glutamate (glu, 20 μM) and either vehicle, NX210 or NX218 (100, 250, 500 μg / mL) for 20 min at 13 days in vitro. Neurons were fixed 2 days later and immunostained with the neuronal marker microtubule-associated protein-2 (MAP-2). Neuronal survival was determined by the number of MAP-2 positive neurons. [Figure 6B] B. Neuritic networks were determined by cumulative neurite length of MAP-2 positive neurons. A, B. Data are presented as median and interquartile range. Kruskal-Wallis test followed by Dunn's test: ###p<0.001 control vs. glu; ***p<0.001, **p<0.01, *p<0.05 glu vs. glu+NX; $$p<0.01, $p<0.05 NX210 vs. NX218, n=5-6. [Figure 7]Follow-up of lesion volume after lysolecithin injection using magnetic resonance imaging (MRI) in vehicle-treated mice (n=7) and mice treated with either 5 mg / kg NX210 (n=8) or NX218 (n=8). Two-way ANOVA followed by Bonferroni post-hoc test. [Figure 8] Determination of lesion area after lysolecithin injection using myelin binding protein (MBP) immunostaining in vehicle-treated mice (n=3 per time point) and mice treated with either 5 mg / kg NX210 (n=3 per time point) or NX218 (n=3 per time point). Lesion area was expressed as a percentage of the ipsilateral corpus callosum (CC). *: p<0.05, t-test. [Figure 9] Determination of the density of Olig2 positive cells in the corpus callosum (CC) after lysolecithin injection using Olig2 immunostaining in vehicle-treated mice (n=3 per time point) and mice treated with either 5 mg / kg NX210 (n=3 per time point) or NX218 (n=3 per time point). *: p<0.05, t-test. [Figure 10] PK profile following NX210 IV administration in monkeys (bolus IV injection of 10 mg / kg NX 210). Mean monkey plasma concentrations (ng / mL) of NX218 (the cyclic form of NX210) are plotted on the y-axis on a base 10 logarithmic scale. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0090] Synthesis of NX peptide The process for the preparation of peptides of sequence SEQ ID NO: 1, 2 or of any of sequences 3-63, particularly those used in the examples section, e.g. NX210 (SEQ ID NO: 3), is based on solid-phase peptide synthesis, applying N-α-Fmoc (side chain) protected amino acids as building blocks in the construction of the peptide. The protocol employed consists of coupling of the C-terminal glycine to an N-α-Fmoc-protected amino acid attached to an MPPA linker on MBHA resin, followed by Fmoc coupling / deprotection. After construction of the peptide on the resin, the steps of cleavage of the peptide from the resin and deprotection of the amino acid side chains are carried out simultaneously.

[0091] The crude peptide is precipitated, filtered and dried. Prior to purification by preparative reverse phase chromatography, the peptide is dissolved in an aqueous solution containing acetonitrile. The purified peptide in solution is concentrated, followed by an ion exchange step to obtain the peptide in acetate form.

[0092] Those skilled in the art can refer to U.S. Pat. No. 6,995,140 and WO2018146283 for further synthetic details, and WO 2017 / 051135 for the oxidized forms of the peptides disclosed herein, all of which are incorporated herein by reference.

[0093] The skilled artisan also has available standard methods for producing any of the disclosed peptides of the present invention, including peptides modified or protected at the N-terminus and C-terminus. For acetylating and / or amidating peptides at the N-terminus and C-terminus, respectively, the skilled artisan can refer to standard methods, such as those described in Biophysical Journal Volume 95 November 2008, pages 4879-4889, which are also incorporated by reference. EXAMPLES

[0094] Functional recovery in mice treated with NX210 after dorsal hemisection (spinal cord injury) Materials and Methods Drug administration SCO-spondin derived peptide (NX210) was dissolved in vehicle (water for injection). Administration of NX210 peptide is performed via the intraperitoneal (ip) route, starting on DO as either: - 8 mg / kg, 10 minutes after injury and repeated twice weekly for 7 weeks. - 4, 8, or 16 mg / kg, 4 hours after injury and repeated twice weekly for 10 weeks.

[0095] Surgical procedure animal Female C57Bl / 6 mice, 6-8 weeks old and weighing approximately 18-20 g, were group-housed with free access to food and water. Mice were housed in a temperature- and humidity-controlled animal facility (temperature 22°C, relative humidity 52%) under a 12-h light / 12-h dark cycle. Mice were numbered by ear tag.

[0096] T8 dorsal hemisection of the spinal cord: The dura was punctured bilaterally with a 30G needle at the appropriate location (Geoffroy CG et al., J Neurosci. 2015 Apr 22; 35(16):6413-28). The spinal cord was then cut using a pair of ultra-precise iridectomy scissors: the dorsal hemisection of the spinal cord, 0.8 mm deep for the dorsal hemisection. Finally, a MICRO FEATHER scalpel was used to retrace the lesion to ensure its completeness. The muscles were sutured with 5.0 sutures, the skin was secured with 5.0 sutures, and the skin was attached using Dermabond.

[0097] Randomization of animals Mice were randomly distributed into cages (maximum n=5 per cage) by a blinded observer. Surgeons were provided with blinded, labeled syringes to prevent blinding to the contents. Mice were tested in a randomized and double-blind fashion: all behavioral tests were performed by an observer blinded to drug treatment and quantified by a different observer who was also blinded to drug treatment groups.

[0098] Animal mortality / observations Mice were examined daily for health status. Mice were monitored daily for general appearance and activity, and body weights were monitored weekly. Acute and delayed mortality were assessed.

[0099] Behavioral testing Open Field-BMS For the BMS score (Basso Mouse Scale, a modified version of the widely used BBB scoring system for rats, Basso DM. et al., J Neurotrauma. 2006 May;23(5):635-59), mice were placed in an open field and observed for 5 min by two observers blinded to treatment (Geoffroy et al., 2015). A number of features were recorded, including ankle movement, gait pattern, frequency, coordination, limb placement, trunk instability and tail position. A BMS score was calculated ranging from 0 (no movement) to 9 (normal gait movement). Mice were tested weekly for the open field-BMS.

[0100] Rotarod For the rotarod test, mice were placed on a rod (Ugo Basile) which rotated at a constant acceleration from 5 rpm to 50 rpm for 3 min. The latency to fall (in seconds) was averaged over two trials per session. Mice were first acclimated to the test for 5 days (2 sessions each) 1 week prior to injury, with an additional session performed 1 day prior to injury (baseline). Mice were tested weekly for the rotarod assay (Geoffroy et al., 2015).

[0101] Activity Chamber Locomotor activity was recorded by placing mice in an open field arena equipped with light beam arrays on the horizontal X and Y axes. Hardware detects beam paths interrupted by the mouse and determines the mouse's location within the cage. The chamber provides information about the general activity (e.g., total number of movements) of the mouse within the chamber. Mice were trained twice prior to testing, then tested on day -1 and weekly thereafter. During each session, mouse activity was recorded for 10 min.

[0102] Euthanasia and tissue sample collection Mice were euthanized on day 56 (experiment 1) or day 73 (experiment 2). Mice were administered a lethal dose of Fatal plus (pentobarbital) and perfused transcardially with PBS-heparin (10,000 units / L, 20 ml, 5 ml / min) followed by 4% paraformaldehyde (30-40 ml / animal, 5 ml / min). After removal of the spinal cord, the tissue was post-fixed overnight at 4°C in the same fixative. The tissue was incubated in 30% sucrose for 3 days for cryoprotection. Different segments of the spinal cord (4 mm rostral and 4 mm caudal to the injury in the dorsal hemisection group, plus a segment 4 mm above the injury) were embedded in OCT compound and snap frozen on dry ice. For further processing, spinal cords were sectioned at 25 μm thickness on a cryostat (Thermofisher Microm HM550) and stored in cryoprotectant solution (30% sucrose, ethylene glycol, PBS) at −20° C. All tissue processing, staining and quantification were performed by an observer blinded to the group treatments.

[0103] analysis immunohistochemistry Free-floating serial sagittal sections centered on the injury site were blocked in PBS-Tx-0.4% plus 5% horse serum for 2 h at room temperature and then stained for myelin basic protein (MBP) using monoclonal rat anti-MBP (overnight incubation, MAB386 Millipore). The next day, sections were washed and anti-rat secondary antibody Alexa fluor 488 (1:500, A-11006, Thermofisher) was added for 1 h at room temperature. After several washes with PBS, all sections were stained with DAPI and then coverslipped with Fluoromount-G (Southern Biotech).

[0104] Immunoreactivity measurements Lesion severity was determined by measuring the MBP-negative area and the injury size, as determined by the maximum depth of the injury. The effect on myelin was assessed by measuring the MBP staining intensity at various distances rostral and caudal to the injury. After MBP immunostaining, a series of 100 μm wide rectangles covering the entire dorsoventral axis of the spinal cord, starting at the injury site and extending all the way to 1.0 mm rostral to the injury, were superimposed on the sagittal sections. After background subtraction, the MBP intensity was measured for all rectangles using ImageJ and normalized to the intensity at 1.0 mm away from the injury. This ratio was the staining intensity ratio and was plotted as a function of distance to the injury. For each mouse, an average of three spinal cord sections was taken.

[0105] statistical analysis All values ​​were expressed as mean ± SEM. Statistical analysis was performed for different conditions using two-way ANOVA followed by Bonferroni post-hoc test for behavior. A value of p<0.05 was considered statistically significant.

[0106] result Model of spinal cord injury - T8 dorsal hemisection - Experiment 1 After T8 dorsal hemisection in mice, NX210 peptide (8 mg / kg) was administered via intraperitoneal (ip) route twice weekly, with the first injection performed 10 min after injury. Locomotor function in NX210- or vehicle-treated mice was assessed weekly using the Basso Mouse Scale (BMS) open field test (Table 1, Table 2 and Table 3 - Figures 1 and 2) and the rotarod test to analyze performance under forced exercise (Table 4). Postmortem analysis was also performed using immunostaining (especially MBP labeling, Table 5 and Figure 3).

[0107] Analysis of locomotor activity using the BMS open field test revealed a significant increase in recovery in NX210-treated mice when compared to vehicle-injected mice (Table 1 and Figure 1). BMS scores were significantly higher in NX210-treated mice from day 7 to the end of the study (day 42 post-injury).

[0108] Similar results were obtained for BMS subscores (Table 2 and Figure 2), which may detect more subtle differences in gait that may not be evident in the overall BMS score.

[0109] The percentage of mice that achieved a BMS score of 5 or greater corresponds to plantar walking and some degree of coordination. Interestingly, all NX210-treated mice (100%) had a BMS score of 5 or greater at the end of the study (day 42 post-injury), demonstrating functional recovery, compared to only 37.5% of vehicle-treated mice (Table 3).

[0110] Analysis of locomotor activity using the rotarod test demonstrated that NX210 treatment increased duration on the rod and latency to fall (Table 3).

[0111] Spinal cord injury, especially at the secondary injury stage, induces strong demyelination throughout the entire rostral-caudal axis of the lesion. Eight weeks after injury, spinal cord sections (1000 μm rostral and 1000 μm caudal to the injury) were stained for myelin basic protein (MBP), which detects myelin proteins and myelin sheaths. After quantification, MBP immunostaining revealed a significant increase in MBP intensity levels both at the lesion site and throughout 1000 μm rostral and caudal distances in NX210-treated mice compared to vehicle treatment (Table 5 and Figure 3).

[0112] Taken together, these locomotor and histological data strongly suggest improved functional and biological recovery and demonstrate the benefit of administration of NX210, a SCO-spondin-derived peptide, following SCI.

[0113] [Table 3]

[0114] Table 1: Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was assessed using the open field locomotor test and BMS scores were obtained in vehicle-treated (n=8) and NX210-treated (n=7) mice. *: p<0.05, **: p<0.01, ***: p<0.001. Two-way ANOVA followed by Bonferroni post-hoc test.

[0115] [Table 4]

[0116] Table 2: Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was assessed using the open field locomotor test to obtain BMS subscores in vehicle-treated (n=8) and NX210-treated (n=7) mice. *: p<0.05, **: p<0.01. Two-way ANOVA followed by Bonferroni post-hoc test.

[0117] [Table 5]

[0118] Table 3: Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was determined using the open field locomotor test in vehicle-treated (n=8) and NX210-treated (n=7) mice. Percentage of mice with a BMS score ≧5.

[0119] [Table 6]

[0120] Table 4: Motor performance after T8 dorsal hemisection (spinal cord injury) using the rotarod test (residence time on the rotarod) in vehicle-treated (n=8) and NX210-treated (n=7) mice. Two-way ANOVA followed by Bonferroni post-hoc test.

[0121] [Table 7]

[0122] Table 5: Determination of myelin basic protein (MBP) staining intensity following T8 dorsal hemisection (spinal cord injury), 1000 μm rostral and 1000 μm caudal to the injury, in vehicle-treated (n=3) and NX210-treated (n=5) mice using immunofluorescence.

[0123] Model of spinal cord injury - T8 dorsal hemisection - experiment 2 After T8 dorsal hemisection in mice, NX210 peptide was administered twice weekly at different doses (4, 8 and 16 mg / kg) via intraperitoneal (ip) route, with the first injection performed 4 hours after hemisection (4 hours after injury in mice may correspond to a therapeutic window of 1-day to several days in humans). Locomotor function in NX210- or vehicle-treated mice was assessed weekly using the Basso Mouse Scale (BMS) open field test (Tables 6, 7 and 8 - Figures 4 and 5), the rotarod test to analyze performance under forced exercise (Table 8) and the activity chamber test to investigate spontaneous locomotor activity (Tables 10 and 11). Postmortem analysis was also performed using immunostaining.

[0124] Analysis of locomotor activity using the BMS open field test revealed a significant increase in recovery in mice treated with NX210 when compared to vehicle-injected mice (Table 6 and FIG. 6), with BMS scores being significantly higher in mice treated with 8 mg / kg or 16 mg / kg NX210 from days 7 or 21, respectively, until the end of the study (day 56 post-injury). Mice treated with 4 mg / kg also exhibited higher BMS scores than vehicle-injected mice.

[0125] Similarly, BMS subscores were significantly higher in NX210 treated groups at 8 mg / kg or 16 mg / kg from day 21 to the end of the study (day 56 post-injury) (Table 7 and FIG. 7). Mice treated with 4 mg / kg also exhibited higher BMS subscores than vehicle-injected mice.

[0126] Twenty-five percent (25%) of vehicle-treated mice exhibited a BMS score of 5 or greater at the end of the study (day 56 post-injury), whereas 86% of NX210-treated mice (8 mg / kg) achieved a BMS score of 5 or greater as early as day 21 post-injury and maintained that score until the end of the study, demonstrating robust functional recovery (Table 7). Forty-four and 75% of mice treated with 4 mg / kg or 16 mg / kg NX210, respectively, achieved a BMS score of 5 or greater.

[0127] Analysis of locomotor activity using the rotarod test showed that administration of 8 mg / kg NX210 significantly increased the duration on the rod, with significant improvements as early as day 8 post-injury and maintained until the end of the study (day 57 post-injury) (Table 9). Mice treated with 16 mg / kg NX210 also exhibited increased duration on the rotarod in contrast to vehicle-treated mice.

[0128] Locomotor activity was assessed weekly using an activity chamber test. NX210-treated mice (especially the 8 mg and 16 mg / kg groups) showed a significant increase in total distance traveled and average speed compared to vehicle-treated mice (Tables 10 and 11), further demonstrating the functional recovery of NX210-treated mice.

[0129] The body weight of NX210-treated mice increased from day 2 after injury, reached pre-injury values ​​between days 20 and 27, and continued to increase until the end of the study, whereas the body weight of vehicle-treated mice only increased (slowly) from day 27 onwards, but had not yet reached pre-injury values ​​at the end of the study (day 58 after injury), and the mice regained weight more quickly and appeared generally healthier (data not shown).

[0130] Taken together, these data confirm the potent improvement of functional recovery following administration of NX210, a SCO-spondin-derived peptide, after spinal cord injury.

[0131] [Table 8]

[0132] Table 6: Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was assessed using the open field locomotor test and BMS scores were obtained in vehicle-treated mice (n=8) and mice treated with 4 mg / kg (n=9), 8 mg / kg (n=7) and 16 mg / kg (n=8). *: p<0.05, **: p<0.01. Two-way ANOVA followed by Bonferroni post-hoc test.

[0133] [Table 9]

[0134] Table 7: Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was assessed using the open field locomotor test and BMS subscores were obtained in vehicle-treated mice (n=8) and mice treated with 4 mg / kg (n=9), 8 mg / kg (n=7) and 16 mg / kg (n=8). *: p<0.05, **: p<0.01. Two-way ANOVA followed by Bonferroni post-hoc test.

[0135] [Table 10]

[0136] Table 8: Recovery of hindlimb function after T8 dorsal hemisection (spinal cord injury) was determined using the open field locomotor test in vehicle-treated mice (n=8) and mice treated with 4 mg / kg (n=9), 8 mg / kg (n=7) and 16 mg / kg (n=8). Percentage of mice with a BMS score ≧5.

[0137] [Table 11]

[0138] Table 9: Motor performance after T8 dorsal hemisection (spinal cord injury) using the rotarod test (dwell time on the rod) in vehicle-treated mice (n=8) and mice treated with 4 mg / kg (n=9), 8 mg / kg (n=7) and 16 mg / kg (n=8). *: p<0.05, **: p<0.01, ***: p<0.001. Two-way ANOVA followed by Bonferroni post-hoc test.

[0139] [Table 12]

[0140] Table 10: Total distance traveled (m) after T8 dorsal hemisection using the open field test (chamber activity) in vehicle-treated mice (n=8) and mice treated with 4 mg / kg (n=9), 8 mg / kg (n=7) and 16 mg / kg (n=8). *p<0.05. Two-way ANOVA followed by Bonferroni post-hoc test.

[0141] [Table 13]

[0142] Table 11: Mean speed (m / min) after T8 dorsal hemisection using the open field test (chamber activity) in vehicle treated mice (n=8) and mice treated with 4 mg / kg (n=9), 8 mg / kg (n=7) and 16 mg / kg (n=8). *p<0.05, **p<0.01. Two-way ANOVA followed by Bonferroni post-hoc test. EXAMPLES

[0143] Protection of rat primary cortical neurons against glutamate-induced excitotoxicity by NX210 and NX218 Neuronal death due to glutamate excitotoxicity is a common pathological feature in SCI. To investigate the neuroprotective potential of NX210 and NX218 (the oxidized or cyclic forms of the NX210 peptide), we performed co-treatment experiments with glutamate on in vitro cultured rat cortical neurons, and neuronal survival and neurite networks were assessed by immunohistochemistry.

[0144] Primary culture of rat neurons: Cortical neurons were cultured as previously described (Callizot N, Combes M, Steinschneider R, Poindron P (2013) Operational dissection of β-amyloid cytopathic effects on cultured neurons. J Neurosci Res 91:706-716). Briefly, fetuses were isolated from Wistar rats on day 15 of gestation and immediately placed in ice-cold L15 Leibovitz medium containing 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) (PS) solution and 1% bovine serum albumin (Dutscher). Tissues were enzymatically dissociated using 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (Dutscher) for 20 min at 37°C. The action of trypsin was neutralized by the addition of fresh medium containing Dulbecco's modified Eagle's medium, 4.5 g / liter glucose, 0.5 mg / ml DNase I grade II, and 10% fetal calf serum (FCS; Dutscher). Cells were then mechanically dissociated by forced passage three times through a 10 mL tip, followed by centrifugation at 515 g for 10 min at 4° C. The pellet was resuspended in Neurobasal™ medium containing 2% B27 supplement (Fisher Scientific), 2 mM L-glutamine (Dutscher), 2% PS solution, and 10 ng / mL brain-derived neurotrophic factor (Dutscher). Finally, neurons were seeded at a density of 25,000 cells per well on 96-well plates previously coated with poly-L-lysine and cultured at 37° C. in a 5% CO2 incubator. Medium was changed every other day. On day 13 of culture, neurons were simultaneously exposed to 20 μM glutamate (Sigma-Aldrich) and either 100, 250 or 500 μg / mL vehicle (sterile water for cell culture; Dutscher), NX210 or NX218 for 20 min.

[0145] Immunofluorescence of rat cortical neurons: Forty-eight hours after glutamate exposure, neurons were fixed with a cold solution of ethanol (95%) and acetic acid (5%) for 5 min at -20°C and permeabilized with a solution of 0.1% saponin (VWR) in phosphate-buffered saline (PBS; Dutscher), then incubated for 2 h at room temperature with mouse monoclonal anti-microtubule associated protein-2 (MAP-2, 1 / 400; Sigma-Aldrich) primary antibody diluted in PBS containing 1% FCS and 0.1% saponin.

[0146] The number of MAP-2 positive neurons was measured to assess neuronal survival, while the cumulative length of MAP-2 positive neurites was measured to assess neurite networks. The results are presented in Table 12 below and in FIG. 6.

[0147] [Table 14]

[0148] Table 12: NX210 and NX218 peptides protect rat cortical neurons against glutamate-induced excitotoxicity in vitro

[0149] NX218 at 250 and 500 μg / mL protected rat cortical neurons from glutamate-induced neuronal death (29.40% cell death in glu-treated neurons vs. 14.72% in NX218 / glu-treated neurons at 250 μg / mL, p=0.0101) and completely restored the neurite network at all doses used (-36.93% total neurite network length in glu-treated neurons vs. -5.12% in NX218 / glu-treated neurons at 250 μg / mL, p=0.0002). NX210 did not exert any protective effect on the neurite network (p=0.6602, 0.0617, and 0.1487 between glu-treated neurons and 100, 250, and 500 μg / mL NX210 / glu-treated neurons), but the highest dose increased neuronal survival (29.40% cell death in glu-treated neurons vs. 19.00% in NX210 / glu-treated neurons, p=0.0498). Thus, the neurite network was significantly more preserved with NX218 at 100 and 250 μg / mL than with NX210 (p=0.0071, 0.0467, and 0.1419 between glu-treated neurons exposed to 100, 250, and 500 μg / mL NX210 or NX218, respectively). EXAMPLES

[0150] Effects of NX210 and NX218 on white matter remyelination following focal lesions within the corpus callosum in mice The therapeutic effects of treatment with two subcommissural organ (SCO)-spondin-derived peptides, NX210 and its cyclic form NX218, on white matter lesion progression and remyelination following focal lesions of the corpus callosum (CC) in male adult C57BL / 6J mice were assessed using magnetic resonance imaging (MRI) and immunohistochemical analysis.

[0151] A focal unilateral lesion of the right CC was induced by stereotactic injection of lysolecithin (LPC) in adult male C57BL / 6J mice. In this model, LPC injection induces axonal demyelination, resulting in reproducible lesions that persist for more than 21 days in mice (Leonetti et al., Molecular Neurodegeneration, 2017, incorporated herein by reference). The demyelinating volume progressively decreases over time as new oligodendrocyte progenitor cells (OPCs) proliferate, migrate, and differentiate into mature oligodendrocytes to remyelinate the lesion.

[0152] NX210 and NX218 were administered via intraperitoneal route at a dose of 5 mg / kg every other day from day 2 (D2) up to D21. Lesion volumes were measured via longitudinal magnetic resonance imaging (MRI) studies obtained on D1, D3, D7, D14, and D21 in 7-8 mice per group. Before the start of treatment on D1, the mean lesion volumes of all groups and subgroups were similar, ranging from 0.8 mm 3 During the first week of LPC injection, the mean lesion volume increased in the vehicle-treated group until at least D7 and in the NX210-treated group only at D3, whereas in the NX218-treated group it remained stable until D3 and then decreased from D3 to D7: at day 7, the mean lesion volume was 0.73 mm in mice treated with NX218 or NX210, respectively. 3 and 0.75 mm 3 in vehicle-treated mice, compared with 0.84 mm 3 After the first week, the mean lesion volume continued to decrease in all groups until D21 (Table 13 and Figure 7).

[0153] [Table 15]

[0154] Table 13: Follow-up of lesion volumes using magnetic resonance imaging (MRI) after lysolecithin injection in vehicle-treated mice (n=7) and mice treated with either 5 mg / kg NX210 (n=8) or NX218 (n=8). Two-way ANOVA followed by Bonferroni post-hoc test.

[0155] Myelin binding protein (MBP) immunohistochemistry (using the method and antibodies described by Leonetti above) was performed on three vehicle mice at D1 and on three mice per group at D3, D7, D14 and D21 to visualize myelin and measure lesion volume. Oligodendrocyte transcription factor 2 (Olig2) labeling and positive cell counting in the lesions was also performed on D7 on three mice per group.

[0156] For lesion area (percentage of ipsilateral CC) using MBP immunostaining, the temporal evolution was similar to that observed for MRI lesion evolution, with lesion area increasing until D7 in vehicle-treated groups, whereas it decreased already after D3 in NX218-treated groups. There was a trend towards smaller lesion area in NX218-treated mice. When analyzed over time, lesion area was significantly smaller in NX218-treated groups compared to vehicle-treated groups at D7 (t-test; p=0.0104), confirming the trend observed with MRI (Table 14 and Figure 8).

[0157] [Table 16]

[0158] Table 14: Determination of lesion area after lysolecithin injection using myelin binding protein (MBP) immunostaining in vehicle-treated mice (n=3 per time point) and mice treated with either 5 mg / kg NX210 (n=3 per time point) or NX218 (n=3 per time point). Lesion area is expressed as a percentage of the ipsilateral corpus callosum (CC). *: p<0.05, t-test.

[0159] Analysis of Olig2 positive cells was also performed using the methods and reagents described by Leonetti, supra.

[0160] At D7, the density of Olig2-positive cells was significantly higher in the lesions of NX218 mice compared to vehicle mice (t test, p = 0.028), with each 1 mm 2 The mean density per 1 mm lesion was 8085.9±933.9 and 5007.2±618.4 (mean±SEM), indicating greater recruitment of Olig2 progenitor cells to the lesion in NX218-treated mice compared to vehicle-treated mice. These data indicate that NX218 induces recruitment or proliferation of OPCs or facilitates their migration to the lesion following demyelination of the CC. NX210 tended to increase this parameter compared to vehicle (1 mm 2 In addition, there were no significant differences in Olig2 cell density between vehicle, NX210, and NX218 in the entire ipsilateral or contralateral CC (Table 15 and FIG. 9).

[0161] [Table 17]

[0162] Table 15: Determination of Olig2 positive cell density in the corpus callosum (CC) after lysolecithin injection using Olig2 immunostaining in vehicle treated mice (n=3 per time point) and mice treated with either 5 mg / kg NX210 (n=3 per time point) or NX218 (n=3 per time point). *: p<0.05, t-test. EXAMPLES

[0163] Pharmacokinetics in animals Preliminary in vitro experiments demonstrated that NX210 is rapidly converted to NX218 by oxidation in rat plasma. Therefore, NX210 PK in animals is tracked by measuring its cyclic form, NX218. First, preliminary PK studies in rats were performed to validate the method for detecting NX218 in plasma, followed by moving to monkeys by performing more robust PK studies with replicate experiments. All PK studies were performed using NaCl 0.9% as vehicle.

[0164] Preliminary PK study in rats: In the four rats tested, after slow bolus IV injection of 49 mg / kg NX210, NX218 concentrations declined rapidly and were unquantifiable after 3 hours (data not shown). This study demonstrated that identification of NX218 in animal plasma is feasible.

[0165] PK studies in monkeys: Data were reproducible after repeated testing on different days (D22, D37 and D51) following a bolus IV injection of 10 mg / kg NX210 in the three monkeys tested. NX218 concentrations declined rapidly by 30 minutes after injection (Figure 10). The half-life was determined to be approximately 12 minutes (Table 16), in the same range as observed for rats and dogs (data not shown), with high consistency with repeated dosing.

[0166] [Table 18] EXAMPLES

[0167] NX218 protection of human primary cortical neurons against glutamate-induced excitotoxicity To confirm the neuroprotective ability of NX218 (the oxidized or cyclic form of the NX210 peptide) on human cortical neurons, co-treatment experiments with glutamate were performed on human neuronal cell cultures, and neuronal survival and neurite networks were determined using several assays. The results are presented in Table 17 to Table 19 below.

[0168] Materials and Methods: Primary culture of human cortical neurons: Human fetal cortical neurons (Sciencell Research Laboratories) were seeded at a density of 30,000 cells per well on 96-well plates pre-coated with 1 mg / mL poly-L-lysine (Sigma-Aldrich) and cultured in Neurobasal™ medium containing 2% B27 supplement (ThermoFisher) at 37°C in a 5% CO2 incubator. The next day, the medium was changed to remove residual dimethylsulfoxide (Sigma-Aldrich) and unattached cells. Thereafter, the medium was changed every other day and left in vitro for up to 7 days (div). At 8 div, neurons were exposed simultaneously to 100 μM glutamate (Sigma-Aldrich) and either vehicle or NX218 at 100, 250 or 500 μg / mL in medium minus B27 supplement for 15 min. Different culture plates were used to perform lactate dehydrogenase (LDH) and neuron-specific class III β-tubulin (Tuj1) immunostaining on the one hand, and WST-8 assay and caspase 3 / 7 staining on the other hand, as described below.

[0169] WST-8 assay: 24 hours after glutamate exposure, human neuronal viability was determined by measuring the reduction of WST-8 relative to formazan (Sigma-Aldrich). For this purpose, neurons were incubated with 10 μL of CCK-8 reagent (WST-8) for 1 hour at 37° C., after which the absorbance at 450 nm was quantified using a Synergy II microplate reader. Data are expressed as a percentage of the absorbance of the cell layer of the vehicle control.

[0170] LDH assay: 24 hours after glutamate exposure, the plasma membrane integrity of human neurons was determined by measuring LDH release into the culture supernatant using the "Cytotoxicity Detection Kit (LDH)" (Roche). For this purpose, neurons were incubated with sodium pyruvate in the presence of nicotinamide adenine dinucleotide hydrogen (NADH). Pyruvate is metabolized to lactate by free LDH with the concomitant oxidation of NADH to NAD+. The rate of oxidation of NADH to NAD+ was measured using a Synergy II microplate reader at 490 nm. Data are expressed as a percentage of the LDH content in the medium of the vehicle control.

[0171] Immunofluorescence of human neurons: 24 hours after glutamate exposure, neurons were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS. Non-specific sites were subsequently blocked with 3% BSA in PBS (Santa Cruz). Cells were incubated with mouse anti-Tuj1 antibody (1 / 1000; Abcam) diluted in blocking buffer for 1 hour at room temperature. After several washes, cells were incubated with anti-mouse Alexa fluor-488-conjugated secondary antibody (1 / 100; Abcam) diluted in 0.5% BSA in PBS for 1 hour at room temperature. Four pictures per well for each condition were acquired at 10x magnification using a CellInsight CX7 fluorescence microscope (ThermoFisher). Image analysis was performed using a Cellomics analyzer system (ThermoFisher) to measure several neurite outgrowth parameters, including the average neurite length and the number of roots and ends. Data are expressed as a percentage of vehicle control.

[0172] Caspase 3 / 7 assay: 24 h after glutamate exposure, activation of caspases 3 and 7 was determined by addition of a fluorogenic substrate for caspase 3 / 7 to the culture medium (Cell event Caspase 3 / 7 green detection kit; ThermoFisher). After several washes, four images per well were acquired at 10x magnification using a CellInsight CX7 fluorescence microscope and analyzed using a Cellomics analyzer system. Data are expressed as the percentage of caspase 3 / 7 positive neurons relative to the total number of nuclei.

[0173] [Table 19]

[0174] [Table 20]

[0175] [Table 21]

[0176] Tables 17-19: Assessment of neuronal survival, death and apoptosis and neurite network in human primary cortical neuron cultures 2 days after 8 days of in vitro co-exposure with glutamate (glu, 100 μM) and either vehicle or NX218 (100, 250, 500 μg / mL) for 15 min each. Primary cortical neurons isolated from human fetuses were co-treated (div) with glutamate (glu, 100 μM) and either vehicle (control) or NX218 (100, 250, 500 μg / mL) for 15 min at 8 days in vitro. After 1 day, cultures were subjected to either biochemical assays (WST-8 for cell layers and lactate dehydrogenase (LDH) for culture medium) or staining for neuronal marker neuron-specific class III beta-tubulin (Tuj1) and staining for caspases 3 and 7 (apoptotic markers). A. In the cell layer, neuronal viability was determined by WST-8 biochemical assay. B. Neuronal cell death was determined by measuring LDH content in the culture medium. C. The number of apoptotic cells was determined by measuring the activity of caspases 3 and 7. Results are expressed as the percentage of apoptotic neurons relative to the total number of nuclei. D-F. Neurite outgrowth was determined by measuring the average length of neurites (D) and the number of roots (E) and ends (F) of Tuj positive neurons. A-F. Data are presented as median and interquartile range. One-way ANOVA followed by Tukey's multiple comparison test: ###p<0.001 control vs. glu; ***p<0.001, **p<0.01, *p<0.05 glu vs. NX218 / glu, n=6 (A-E) and n=5-6 (F).

[0177] The neuroprotective effect of NX218 was confirmed in human cortical neurons exposed to glutamate. Indeed, it was shown that NX218 fully preserved neuronal viability at 500 μg / mL as assessed by WST-8 colorimetric assay (neuronal viability -22.59% in glu-treated neurons vs. -3.06% in NX218 / glu-treated neurons, p<0.0001; p=0.9009 between control and NX218 / glu-treated neurons). Furthermore, at all doses used, neurons co-treated with glutamate and NX218 released significantly less LDH than glutamate alone (LDH in NX218 / glu-treated neurons at 250 μg / mL compared to glu-treated neurons -52.70%, p<0.0001). The beneficial effect of NX218 against necrosis was accompanied by normal basal levels of apoptotic cells (14.27% apoptotic cells in control neurons vs. 27.83% in glu-treated neurons and 12.84% in 250 μg / mL NX218 / glu-treated neurons; p<0.0001 between control and glu-treated neurons, p<0.0001 between glu-treated and NX218 / glu-treated neurons, and p=0.9170 between control and NX218 / glu-treated neurons), revealing a strong neuroprotective effect of NX218 against both necrosis and apoptosis in human cortical neurons.

Claims

1. 1. A composition for use in treating injury to the nervous system other than the brain, comprising: Amino acid sequence X1-WS-A1-WS-A2-CS-A3-A4-CG-X2 (SEQ ID NO: 1) (In the formula, - A1 and A2 are independently selected from G and S, and / or A3-A4 are selected from RS or VS or VT or RT; - X1 and X2 consist of an amino acid sequence consisting of 1 to 6 amino acids, or X1 and X2 are absent; - the N-terminal amino acid can be acetylated, the C-terminal amino acid can be amidated, or the N-terminal amino acid can be acetylated and the C-terminal amino acid can be amidated. and a composition comprising the peptide of formula (I) and administered to a patient via a systemic route.

2. The composition according to claim 1, wherein the damage to the nervous system other than the brain is spinal cord damage and / or optic nerve damage.

3. - X1 represents a hydrogen atom or P or AP or LAP or VLAP, and / or 3. The composition according to claim 1, wherein X2 represents a hydrogen atom or L or LG or LGL or LGLI or LGLIF.

4. The peptide has an amino acid sequence WS-A1-WS-A2-CS-A3-A4-CG (SEQ ID NO: 2) The composition according to any one of claims 1 to 3, which is a peptide of the formula:

5. 5. The composition of any one of claims 1 to 4, wherein the peptide is a linear peptide or an oxidized peptide, or a mixture of both linear and oxidized peptides, with the cysteines found in the peptide formulas of SEQ ID NOs: 1 and 2 forming disulfide bridges.

6. The composition according to any one of claims 1 to 5, wherein the peptide is a peptide of a sequence selected from the group consisting of the sequences SEQ ID NO: 3 to 63.

7. 7. The composition of any one of claims 1 to 6, administered to a patient via subcutaneous, intravenous, intraperitoneal, intranasal, intramuscular, sublingual, or oral routes.

8. The composition of any one of claims 1 to 7, wherein the injury is a traumatic injury or an injury resulting from the growth of surrounding cells.

9. 9. A composition according to any one of claims 1 to 8, which induces inhibition or reduction of neuronal cell death and / or axonal degeneration, and / or inhibition or reduction of necrosis (primary damage), inhibition or reduction of secondary damage.

10. 10. A composition according to any one of claims 1 to 9, which induces myelination.

11. 11. A composition according to any one of claims 1 to 10, which induces functional recovery.

12. 12. A composition according to any one of claims 1 to 11, which induces an increase in the level of myelin binding protein (MBP) at the site of the lesion in a patient or animal model.

13. A composition according to any one of claims 1 to 12, which induces mobilisation of Olig2-positive progenitor cells and / or increased production of Olig2-positive cells.

14. 7. A composition according to any one of claims 1 to 6 for treating damage to the nervous system other than the brain in a subject, the composition further comprising a pharma- ceutically acceptable vehicle or excipient, the composition being administered to the subject via a systemic route.

15. The composition according to claim 14, wherein the damage to the nervous system other than the brain is spinal cord damage and / or optic nerve damage.

16. 16. The composition according to claim 14 or 15, which induces inhibition or reduction of neuronal cell death and / or axonal degeneration, and / or inhibition or reduction of necrosis (primary damage), inhibition or reduction of secondary damage.

17. 17. The composition of any one of claims 14 to 16, which induces functional recovery.

18. 18. The composition of any one of claims 14 to 17, which induces an increase in the level of myelin binding protein (MBP) at the site of the lesion in a patient or animal model.

19. A composition described in any one of claims 14 to 18, which induces mobilization of Olig2-positive progenitor cells and / or increased production of Olig2-positive cells.

20. 20. A composition according to any one of claims 14 to 19, which induces myelination or remyelination.

21. 7. A composition according to any one of claims 1 to 6 for use in remyelination in myelopathy, administered to a patient via a systemic route.

22. 7. The composition of any one of claims 1 to 6 for the treatment of myelopathy in a patient in need thereof, wherein the composition is administered to the patient via a systemic route.

23. 13. Use of a composition according to any one of claims 1 to 6 for the manufacture of a medicament for treating a nervous system injury other than the brain or for treating a myelopathy in a subject, wherein the composition is administered to the subject via a systemic route.

24. 24. The use according to claim 23, wherein the damage to the nervous system other than the brain is spinal cord damage and / or optic nerve damage.

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