FR2944020A1 - New fusion peptide having neuroactive peptide having peptide containing attraction unit/unit allowing neurite growth, and peptide targeting glial scar and vector peptide, useful to treat e.g. neurodegenerative diseases - Google Patents

Abstract

The subject of the invention is fusion peptides useful for non-invasive treatments for trauma or diseases affecting the central nervous system, such as brain or medullary lesions, and more particularly for fusion peptides promoting axonal regeneration in the body. central nervous system. The said fusion peptides can themselves pass through the haematoencephalic and cerébrospinal barriers by virtue of a vector peptide, selectively bind to the lesion sites by means of a peptide targeting the glial scar, and provide in situ signals that promote guiding and / or the growth of neurons. The invention also provides conjugates comprising peptidomimetics of NGF (Nerve Growth Factor) and PDGF (Platelet-Derived Growth Factor) factors. The invention finally relates to the preparation of said fusion peptides and associated compositions.

Description

The present invention relates to fusion peptides for improving axonal regeneration in the central nervous system and their use for treating trauma or diseases affecting the system. central nervous system.

In adults, lesions of the central nervous system cause generally irreversible functional defects. Damage to the brain or spinal cord following a stroke, trauma or any other cause can lead to lasting defects in cognitive, sensory and motor functions, or even in the maintenance of vital functions. The injured nerve cells are not replaced, and the remaining nerve cells are usually unable to establish new connections. This lack of regeneration in the central nervous system is attributed to a number of factors: the presence of axonal growth-inhibiting molecules, the absence of appropriate substrate molecules for axonal growth, and the absence of trophic factors required for growth. activation of cellular processes required for axonal survival and regrowth. In contrast, in the peripheral nervous system, injured nerve fibers are able to regenerate over long distances, while recovering their function. In recent years, neurons of the central nervous system have been shown to be able to regenerate provided that the required molecular signals are present in the extracellular medium.

Recently developed strategies for treating central nervous system lesions or certain diseases of the central nervous system are aimed essentially at blocking the inhibition of axonal growth, said inhibition being induced by the interaction of inhibitory proteins with receptors present on axons. . Inhibition surveys are obtained using specific enzymes or neutralizing antibodies.

The present invention discloses an alternative means for treating lesions or diseases of the central nervous system, said means for presenting to the nerve cells of the central nervous system various signals required for axonal regeneration.

Any lesion of the central nervous system results in the activation of neuroinflammatory processes leading to the appearance of a glial scar at the site of injury. The glial scar is a barrier that inhibits the restoration of neuronal connections within the central nervous system. In the adult, the absence of axonal regeneration in the central nervous system is essentially attributed to the chondroitin sulfate proteoglycans of glial scars. Their increased expression makes the glial scar non-permissive. Some of these proteoglycans are present at significant levels in glial scars and persist for long periods. This is particularly the case of proteoglycans with chondroitin sulfate NG2, neurocane, brevicane, versicane and phosphacane. These chondroitin sulfate proteoglycans associated with glial scars hinder axonal regeneration processes in the brain and spinal cord, inhibit neurite outgrowth, and block both growth cone progression and axon regrowth. However, some injured axons succeed in bypassing these inhibitions and progressing to regions rich in proteoglycans. Inhibition due to chondroitin sulfate proteoglycans is not absolute. Various factors make it possible to compensate for this inhibition. In particular, certain substrate molecules may promote axonal regeneration at lesion sites despite the presence of chondroitin sulfate proteoglycans. A proteoglycan, the proteoglycan NG21HM, is particularly expressed in glial scars. The membrane proteoglycan NG2 / HM, so called by reference to the rat proteoglycan NG2, and its homologs, the proteoglycan NG2 of the mouse (also called AN2) and the proteoglycan HM of the human (also called MCSP), is mainly expressed during development by glial, epidermal, muscular and cartilaginous progenitor cells. The expression of the proteoglycan NG2 / HM is then stopped during the differentiation of these cells. In adults, the proteoglycan NG2 / HM is highly expressed in glial scars, and in some tumors such as chondrosarcomas, glioblastomas, astrocytomas, melanomas, and some leukemias. Proteoglycan chondroitin sulfate NG2 / HM is strongly expressed after spinal cord injury, whatever the type of lesion considered (compression, contusion, or transection), and continues for a long time at significant levels. Many NG2-positive reactive cells are initially observed at lesion sites. NG2 / HM proteoglycan deposits are then formed after secretion or cleavage by extracellular proteases. Certain domains of the NG2 / HM proteoglycan inhibit the growth of neurites and cause the resorption of growth cones. Chondroitin sulfate glycosaminoglycans, in particular, inhibit axonal regeneration in vivo. Treatments to degrade or prevent the formation of chondroitin sulfate (chondroitinase ABC) glycosaminoglycan chains significantly improve axonal regeneration after injury. Similar results are obtained by neutralizing the NG2 / HM proteoglycan inhibition sites with antibodies. The present invention describes an alternative treatment, said treatment for using neutralizing peptides as therapeutic agents in the treatment of central nervous system lesions or diseases.

Neuronal regeneration is dependent on the targeted growth of axons and dendrites, and notably involves neurite migration and guidance processes. The object of the present invention is therefore to provide fusion peptides that promote guiding and / or growth of neurites in glial scars, and thereby axonal regeneration in the central nervous system.

The fusion peptides of the present invention have the particular advantage of (i) easily passing the haematoencephalic and cerebrospinal barriers, (ii) direct targeting of the glial scar formed at the lesion site, and (iii) improving in situ the guiding and / or axonal growth.

The subject of the present invention is therefore a fusion peptide comprising: (a) a neuroactive peptide comprising: a peptide comprising a pattern of neurite attraction or a pattern for the growth of neurites, and - a peptide for targeting the glial scar, (b) a translocation vector peptide across the haematoencephalic and cerebrospinal barriers.

By fusion peptide is meant a chimeric or hybrid peptide combining at least two peptides. The chimeric peptides may be produced recombinantly, i.e., by fusing at least a portion of a coding sequence of a gene with at least a portion of a coding sequence of another gene. The fusion gene can then be used to transform a host organism, which then expresses the fusion peptide. Chimeric peptides can also be synthesized chemically.

More particularly, the subject of the invention is a fusion peptide comprising: (a) a neuroactive peptide comprising: - a peptide comprising a neurite attraction motif or a motif allowing the growth of neurites, and - a targeting peptide of the neurite, glial scar, (b) a vector peptide translocating across the haematoencephalic and cerebrospinal barriers, wherein the peptide comprising a neurite attraction pattern is derived from the fnC region of tenascin-C, the peptide comprising a pattern allowing neurite outgrowth is derived from the fnD region of tenascin-C, the glial scar targeting peptide specifically binds to the NG2 / HM proteoglycan, and the translocation vector peptide across the hematoencephalic and cerebrospinal barriers is a linear peptide peptide derivative of the family Protégrines or family Tachyplésines.

In a first embodiment of the present invention, the peptide comprising a motif for the growth of neurites is a peptide derived from the fnD region of tenascin-C (FIG. 1). Some extracellular matrix glycoproteins, such as tenascin-C, provide growth signals to neurites. The fnD domain of tenascin-C, in particular, facilitates neuronal extension in the presence of chondroitin sulfate proteoglycans [Meiners et al., 2001. J. Neurosci. 21 (18), 7215-7225]. A peptide derived from the fnD domain of tenascin-C, peptide ADEGVFDNFVLKIRDTKKQ (SEQ ID NO: 1), facilitates attachment of neurons, as well as formation and growth of neurites, via different sequences. The VFDNFVLK motif (SEQ ID NO: 2) is itself a signal of growth and not of guiding, facilitating the process of neuronal extension in the absence or in the presence of chondroitin sulfate proteoglycans, via different interactions with the integrin a7. (3l [Mercado et al., 2004. J Neurosci, 24 (1), 238-247] Amino acids FD and FV, in particular, are required for receptor binding and activation The same FD active site / FV is found in different species (Figure 2) .VFDNFVLK peptide (SEQ ID NO: 2), in soluble or substrate-bound form, promotes the growth of neurites and compensates for inhibitions related to chondroitin sulfate proteoglycans, Even when the peptide is added after the neurons have encountered the proteoglycans, the VFDNFVLK peptide also reduces neuronal degeneration.The VFDNFVLK peptide by itself is necessary and sufficient for the growth of the neurites. VFDNFVLK is necessary, but not sufficient for neuronal adhesion, and requires the adjacent amino acids IRDTKKQ (SEQ ID NO: 3) for adhesion activity. The N-terminal ADEG motif, in turn, optimizes the presentation of the FD / FV active site and contributes to increasing the neuronal response. The amino acids VFDNFVLK form a growth motif, while the amino acids IRDTKKQ form an adhesion pattern. The ability of the peptide ADEGVFDNFVLKIRDTKKQ (SEQ ID NO: 1) to promote neuronal attachment, as well as the formation and growth of neurites, is a most valuable property for central nervous system repair.

The use of modified biomimetic matrices incorporating this peptide is conceivable. Unfortunately, the neurosurgical implantation of such matrices presents a major invasive character. The present invention provides a non-invasive alternative method.

Combined with substances that promote neurite attraction, peptides derived from the fnD domain of tenascin-C are very useful for promoting neuronal regeneration in the central nervous system. However, their effectiveness as therapeutic agents is dependent on the expression of integrin a7. (31) Therefore, any strategy aimed at inducing optimal regrowth of neurites with these peptides must include an approach to increase expression. In the central nervous system, integrins of the f3 1 family participate in many cellular functions, including axon guidance, neurite growth, adhesion, and migration. Integrin 07 (31 in particular, is required for the growth of neurites in various populations of motor and sensory neurons.

Integrin x7 (31) provides molecular continuity between ligands (laminins, tenascin) and the cytoskeleton.The a7 subunit is responsible for ligand binding specificity and signaling, while the subunit (31 participates in the transport of α7f31 heterodimers to extracellular matrix binding sites The a7 subunit associates exclusively with the subunit (31.

A member of the neurotrophin family, NGF (Nerve Growth Factor), stimulates the coupling of integrins (31 to various neurite-mediated growth mechanisms), a member of the Platelet-Derived Growth Factor (PDGF) family. PDGF-BB makes it possible to specifically increase the expression of the? 7 integrin subunit (de novo synthesis) in different cell types, and thereby the adhesion of the? 7 integrin (31 to certain specific substrates of the extracellular matrix, such as the pattern of growth of the fnD domain neurites of tenascin-C In the peripheral nervous system, the a7 subunit is strongly expressed in motor and sensory neurons after injury. Unit A is required for regeneration of the peripheral nervous system In the central nervous system, the a7 subunit is not expressed after injury in adults. for the regeneration of the central nervous system. However, the regeneration capabilities of adult neurons can be restored in vivo by increasing expression levels of the integrin a7 subunit. The expression of the integrin a7 subunit in adult neurons no longer expressing the? 7 subunit, but only the non-limiting subunit, in fact, makes it possible to restore their motility. Such plasmids or modified viruses may be used, however, such gene transfers are hardly conceivable in vivo.The present invention presents a more suitable alternative method involving the specific activation of receptors using peptides or In this sense, the present invention particularly relates to fusion peptides incorporating peptidomimetics of NGF and PDGF-BB favoring axonal regenerations induced by the α7 integrin (31, said peptidomimetics activating signaling mechanisms usually involving these factors. .

The nucleic and peptide sequences of human tenascin-C can be found in Genbank with the identification number NM-002160. According to a first embodiment, the peptide comprising a unit for the growth of neurites is a peptide of at most 30 amino acids comprising one of the following units: VFDNFVLK (SEQ ID NO: 2), VFDSFVLK (SEQ ID NO: 4) or IFDSFVIR (SEQ ID NO: 5). Said peptide comprising a unit for the growth of neurites may also be a peptide of at most 30 amino acids comprising one of the following sequences: VFDNFVLKIRDTKKQ (SEQ ID NO: 6), VFDSFVLKIRDTKKQ (SEQ ID NO: 7), IFDSFVIRIRDTKKQ ( SEQ ID NO: 8), VFDSFVLKIRDTKRK (SEQ ID NO: 9), VFDNFVLKIRDTKRK (SEQ ID NO: 10), or IFDSFVIRIRDTKRK (SEQ ID NO: II). Said peptide comprising a unit for the growth of neurites may also be a peptide of at most 30 amino acids comprising one of the following sequences: ADEGVFDNFVLKIRDTKKQ (SEQ ID NO: 1), ADEGVFDSFVLKIRDTKKQ (SEQ ID NO: 12), ADEGIFDSFVIRIRDTKKQ ( SEQ ID NO: 13), ADDGVFDSFVLKIRDTKRK (SEQ ID NO: 14), ADEGVFDNFVLKIRDTKRK (SEQ ID NO: 15), ADDGVFDSFVLKIRDTKRK (SEQ ID NO: 16), ADEGIFDSFVIRIRDTKRK (SEQ ID NO: 17), ADDGVFDNFVLKIRDTKKQ (SEQ ID NO: 18). ), ADDGVFDSFVLKIRDTKKQ (SEQ ID NO: 19), ADDGIFDSFVIRIRDTKKQ (SEQ ID NO: 20), ADDGVFDNFVLKIRDTKRK (SEQ ID NO: 21), ADDGIFDSFVIRIRDTKRK (SEQ ID NO: 22). According to another embodiment of the invention, the peptide comprising a unit for the growth of neurites is a peptide of at most 20 amino acids comprising one of the units SEQ ID NO: 1 to 22.

According to another embodiment of the invention, the peptide comprising a unit for the growth of neurites is a peptide of at most 16 amino acids comprising one of the units SEQ ID NO: 2, 4, 5 and 6 to 11 .

In a second embodiment of the invention, the peptide comprising a pattern of neurite attraction is a peptide derived from the fnC region of tenascin-C (Fig. 1). Some extracellular matrix glycoproteins, such as tenascin-C, provide guidance signals to neurites. The fnC domain of tenascin-C, in particular, attracts and retains growing neurites. In vitro, the neurites preferentially travel to the fnC (neurite attraction) domain and remain on fnC (neurite retention) in the absence or in the presence of chondroitin sulfate proteoglycans [Liu et al., 2005. Eur. J. Neurosci. 22, 1863-1872]. A peptide derived from the fnC domain of tenascin-C, of sequence DINPYGFTVSWMASE (SEQ ID NO: 23), alone attracts and retains the neurites via two different sequences. The NPYG and ASE motifs contribute to the attraction and retention of neurites, respectively. The ASE peptide, by itself, is necessary and sufficient for the retention of neurites. The NPYG peptide is necessary but not sufficient for neurite attraction and requires the adjacent amino acids FTVSWM for attraction activity. The amino acids NPYGFTVSWM form a pattern of attraction, while the amino acids ASE form a retention pattern. The same patterns are found in different species (Figure 3).

The ability of peptide DINPYGFTVSWMASE (SEQ ID NO: 23) to attract neurites is a property of the most interest for the repair of the central nervous system. Peptide NPYGFTVSWM (SEQ ID: 26) lacking ASE retention pattern attracts neurites, but does not retain them. Combined with substances promoting the neuronal extension process, peptides derived from the fnC domain are very useful for promoting guided neuronal regeneration in the central nervous system. According to the second embodiment, the peptide comprising a neurite attraction pattern is a peptide of at most 30 amino acids comprising one of the following units: DINPYGFTVSWM (SEQ ID NO: 24), DTNPYGFTVSWT (SEQ ID NO: 25), NPYGFTVSWM (SEQ ID NO: 26), NPYGFTVSWT (SEQ ID NO: 27). Said peptide comprising a neurite attraction motif may also be a peptide of at most 30 amino acids comprising one of the following sequences: DINPYGFTVSWMASE (SEQ ID NO: 23), DTNPYGFTVSWTASE (SEQ ID NO: 28), NPYGFTVSWMASE ( SEQ ID NO: 29), NPYGFTVSWTASE (SEQ ID NO: 30).

According to another embodiment of the invention, the peptide comprising a neurite attraction motif is a peptide of at most 20 amino acids comprising one of the units SEQ ID NO: 23 to 30. According to another embodiment of Embodiment of the invention, the peptide comprising a neurite attraction pattern is a peptide of at most 15 amino acids comprising one of SEQ ID NO: 23-30.

Variants of the amino acid sequences SEQ ID NOs 1 to 30 of the present invention may be prepared. Such variants include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence. The present invention relates to any variant obtained from the sequences presented in this text since the expression of the variant has the property of attraction of neurites or allows the growth of neurites.

To analyze the consequences of a mutation, the variants must be tested for the desired activity. In general, the sequences of the variants of the present invention have at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90% or even 95% sequence identity, with the original sequences peptides derived from the fnC or fnD regions of tenascin C. The sequence identity is defined here as the percentage of amino acids in the sequence of the variant identical to those of the original sequence, after alignment of the sequences and introduction of spaces , if necessary, to obtain a maximum of sequence identity, and without considering the conservative substitutions (Table 1) as part of the sequence identity. Sequence identity can be determined by standard methods using programs such as BLAST or FASTA.

Examples of variants are: insert-derived variants: the amino acid insertion comprises N- or C-terminal fusions as well as insertions within the sequence of one or more amino acids. variants obtained by substitutions: these variants are for example obtained by substitutional mutagenesis. Conservative substitutions are shown in the following table. Original Amino Acid Substitutions Preferred Substitutions Ala (A) val; leu; val val (R) lys; gln; asn lys Asn (N) gln; his; asp; lily; arg arg Asp (D) glu; asn glue Cys (C) ser; ala ser Gin (Q) asn; to the ; ser ser Glu (E) asp; gln asp Gly (G) ala His (H) asn; gln; lily; arg Ile (I) leu; val; puts; to the ; leu phe; norleucine norleucine Leu (L) norleucine; Isle ; val ile met; to the ; phe Lys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; Isle ; to the ; tyr Pro (P) ala Ser (S) thr Thr (T) ser ser Trp (W) tyr; Tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; puts; phe; leu ala; norleucine The natural residues can be divided into different groups according to their properties: (1) hydrophobic residues: norleucine, met, ala, val, leu, ile; (2) polar neutral residues: cys, ser, thr; (3) acid residues: asp, glu; (4) basic residues: asn, gin, his, lys, arg; (5) residues influencing the orientation of the chain: gly, pro; and (6) aromatic residues: trp, tyr, phe.

Conservative substitutions involve the replacement of an amino acid with another member of the same group, while non-conservative substitutions involve the replacement of one amino acid by a member of another group.

In one embodiment of the invention, said glial scar targeting peptide is a peptide that specifically binds to NG2 / HM proteoglycan. Two peptides, the decapeptides TAASGVRSMH (SEQ ID NO: 31) and LTLRWVGLMS (SEQ ID NO: 32) bind to the extracellular portion of the proteoglycan NG2 / HM. Injected intravenously, the two peptide sequences TAASGVRSMH (SEQ ID NO: 31) and LTLRWVGLMS (SEQ ID NO: 32) make it possible to target in vivo therapeutic agents on the tumor vascular walls expressing the NG2 / HM marker (tumor neoangiogenesis) [ Burg et al., 1999. Cancer Res. 59, 2869-2874]. The present invention describes a particular use of these two peptides as targeting peptides for glial scars bordering lesions sites of the central nervous system. The peptide KKEKDIMKKTI (SEQ ID NO: 33) derived from the SGI motif of the integrin α4 subunit binds specifically to the MCSP proteoglycan and inhibits the adhesion of the α4P1 integrin to human melanoma cells expressing this proteoglycan [Iida et al. ., 1998. J. Biol. Chem. 273 (10), 5955-5962]. The cell surface proteoglycan MCSP interacts directly with the α4 [31 integrin via the SG1 motif and activates various adhesion and signaling processes associated with this integrin. Treatments to degrade chondroitin sulfate (chondroitinase ABC) glycosaminoglycan chains inhibit α4f31 integrin adhesion. The α1 integrin SG1 motif (31) is a specific site for binding to chondroitin sulfate glycosaminoglycan chains.The same motif is found in different species (Figure 4) .The present invention describes a particular use of these peptides as The present invention furthermore discloses a particular use of these peptides as neutralizing peptides, said neutralizing peptides blocking certain NG2 / HM proteoglycan inhibition sites Fourteen other peptides, the peptides GGGTRAGMKY (SEQ ID NO: 34), WGKIEDPLRA (SEQ ID NO: 35), AGQTLTASGD (SEQ ID NO: 36), DLLAVSWLRA (SEQ ID NO: 37), SAERGVVAMS (SEQ ID NO: 38), AIHSELMWVS (SEQ ID NO: 39), FWTERAGWAY (SEQ ID NO: 40), MVWSKGPLFL (SEQ ID NO: 41), AGTRMSWEVL (SEQ ID NO: 42), VSRSSRWGSI (SEQ ID NO: 43), DAHVLVPRTP (SEQ ID NO: 44), AQGIVLQLAL (SEQ ID NO : 45), LSPLLSPATA (SEQ ID NO: 46), CLSGSLSC (SEQ ID NO: 47), can bind to proteoglycol NG2 / HM gene [Burg et al., 1999. Cancer Res. 59, 2869-2874].

The present invention describes a particular use of these fourteen peptides as glial scarring targeting peptides.

In one embodiment of the invention, said NG2 / HM proteoglycan binding peptide is a peptide of at most 30 amino acids comprising one of the following: TAASGVRSMH (SEQ ID NO: 31), LTLRWVGLMS (SEQ ID NO: 31), ID NO: 32), KKEKDIMKKTI (SEQ ID NO: 33), KKEKDIIKKTI (SEQ ID NO: 48), KKEKDIIEKTI (SEQ ID NO: 49), KKERDIIEKTI (SEQ ID NO: 50), KKEKDIIRKTI (SEQ ID NO: 51) , KKEKDVIRKMI (SEQ ID NO: 52), KKEKDIIREKI (SEQ ID NO: 53), GGGTRAGMKY (SEQ ID NO: 34), WGKIEDPLRA (SEQ ID NO: 35), AGQTLTASGD (SEQ ID NO: 36), DLLAVSWLRA (SEQ ID NO: 37), SAERGVVAMS (SEQ ID NO: 38), AIHSELMWVS (SEQ ID NO: 39), FWTERAGWAY (SEQ ID NO: 40), MVWSKGPLFL (SEQ ID NO: 41), AGTRMSWEVL (SEQ ID NO: 42), VSRSSRWGSI (SEQ ID NO: 43), DAHVLVPRTP (SEQ ID NO: 44), AQGIVLQLAL (SEQ ID NO: 45), LSPLLSPATA (SEQ ID NO: 46), CLSGSLSC (SEQ ID NO: 47).

In another embodiment of the invention, the glial scar targeting peptide is a peptide of at most 30 amino acids comprising one of the following motifs: KKEKDIMKKTI (SEQ ID NO: 33), KKEKDIIKKTI (SEQ ID NO: 48), KKEKDIIEKTI (SEQ ID NO: 49), KKERDIIEKTI (SEQ ID NO: 50), KKEKDIIRKTI (SEQ ID NO: 51), KKEKDVIRKMI (SEQ ID NO: 52), KKEKDIIREKI (SEQ ID NO: 53), said peptide further blocking certain NG2 / HM proteoglycan inhibition sites. In another embodiment of the invention, the glial scar targeting peptide is a peptide of at most 20 amino acids comprising one of SEQ ID NO: 31 to 53. In another embodiment of the invention, In the invention, the glial scar targeting peptide is a peptide of at most 15 amino acids comprising one of SEQ ID NOs: 31 to 53.

The variants of the sequences SEQ ID Nos. 31 to 53 follow the same definition described above and are also part of the present invention. In one embodiment of the invention, said translocating vector peptide across the blood brain barrier is a linear peptide derived from peptides of the family Protégrines or family Tachyplésines. according to one of the following formulas or to a fragment of one of the following formulas: RX1X1RX1X2X1X2RRRX1X2XIX2X1X1R (SEQ ID NO: 54) RX1X2X1RX1X2X1RX1X1X2XIRRX2R (SEQ ID NO: 55) in which R represents arginine, X groups 1 identical or different represent a natural amino acid or not (including amino acids in configuration D) aliphatic or aromatic, such as glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteineA® m, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, amino-butyric acid, acid amino-1-cyclohexane carboxylic acid, aminoisobutyric acid, 2-aminotetralin carboxylic acid, 4-bromophenylalanine, tert-leucine, 4-chlorophenylalanine, beta-cyclohexylalanine, 3,4-dichlorophenylalanine, 4-fluorophenylalanine, homoleucine, beta-homoleucine homophenylalanine, 4-methylphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2-thienyl] alanine. and X2 represents serine or threonine.

In vertebrates, the central nervous system is protected by the haematoencephalic and cerebrospinal barriers, two membranes separating the blood circulation and the cerebrospinal fluid, the fluid in which the brain and the spinal cord bathe. These two barriers constitute an obstacle to the transport of many therapeutic molecules. Strategies for using vector peptides to deliver therapeutic molecules to the central nervous system beyond the blood brain and cerebrospinal barriers have been developed. The vectors of the SynB type derived from the antimicrobial peptide protectrin 1 (PG-1) or the antimicrobial peptide tachyplesin 1 (T-1), in particular, are of major interest (FIG. 5). In its native form, the PG-1 peptide has a structure constrained by two disulfide bridges. This peptide destabilizes the bacterial membranes by forming pores. Many linear analogs of PG-1 peptide lacking cysteine residues have been described. These linear peptides cross membranes via an energy-dependent endocytosis mechanism without any cytolytic effect [Drin et al., 2002. AAPS Pharm. Sci. 4 (4), art. 26]. Injected intravenously, the translocation vectors SynB 1 and SynB3 allow the transport of therapeutic molecules beyond the blood-brain barrier. The conjugation of therapeutic molecules to SynB vectors leads to a considerable accumulation of these molecules in the central nervous system after intravenous injection. The SynB1 and SynB3 peptides do not exhibit significant lytic activity even at high concentrations. However, high concentrations of SynB5 can disrupt membrane integrity. The enantiomeric forms D in which all the amino acids are in configuration D and the retro-inverted sequences have identical penetrations. The enantiomeric forms D confer resistance to the activity of proteases. Linear analogues obtained by substitution and / or permutation of amino acids have been described. In its native form, the T-1 peptide has a structure constrained by two disulfide bridges similar to that of the protegrins. This peptide destabilizes the membranes by forming pores. Linear analogs of peptide T-1 lacking cysteine residues have been described. The SynB4 peptide has properties similar to those of the other previously described SynB vectors.

In a preferred embodiment of the invention, said translocation vector peptide across the haematoencephalic and cerebrospinal barriers is a peptide having one of the following sequences: RGGRLSYSRRRFSTSTGR (SEQ ID NO: 56), RRLSYSRRRF (SEQ ID NO: 57 ), RGGRLAYLRRRWAVLGR (SEQ ID NO: 58), AWSFRVSYRGISYRRSR (SEQ ID NO: 59) or their retroinversed sequences: RGTSTSFRRRSYSLRGGR (SEQ ID NO: 60), FRRRSYSLRR (SEQ ID NO: 61), RGLVAWRRRLYALRGGR (SEQ ID NO: 62) , RSRRYSIGRYSVRFSWA (SEQ ID NO: 63).

In particular, the sequences RGGRLSYSRRRFSTSTGR (SEQ ID NO: 56), RRLSYSRRRF (SEQ ID NO: 57) and RGGRLAYLRRRWAVLGR (SEQ ID NO: 58) are in an enantiomeric form D.

According to one embodiment of the invention, the peptide comprising a neurite attraction motif or a pattern for the growth of neurites and the glial scar targeting peptide are linked by a flexible peptide linker. Preferably, said linker is stable in plasma and cerebrospinal fluid, the liquid in which the brain and spinal cord bathe.

A linker may for example be a peptide sequence comprising glycine and serine residues. In a preferred embodiment of the invention, said linker is the peptide (Gly4Ser) 3 (SEQ ID NO: 64). In another preferred embodiment of the invention, said linker comprises 3, 4, 5 or 6 repeats of the GGGGS motif. Preferably, said linker is selected from the peptide sequences SEQ ID NO: 64 to 67. According to one embodiment of the invention, the vector peptide is linked directly or indirectly to the neuroactive peptide via a linker specifically cleaved when the fusion peptide of the invention reaches the central nervous system, the linker binding the peptide vector either to the peptide comprising a pattern of neurite attraction or a pattern for the growth of neurites or the targeting peptide of the glial scar. The neuroactive peptide may be indirectly linked to the vector peptide through a functional group that is naturally present or inserted into either the vector or the neuroactive peptide, or both. Functional groups such as OH, SH, COOH, C (O) H, C (O), NH 2 may be present naturally or may be inserted into the vector peptide or into the neuroactive peptide or both. The coupling between the peptide vector and the neuroactive peptide may be carried out using any acceptable coupling method taking into account the chemical nature of both the vector and the neuroactive peptide.

Thus, the linkage between the neuroactive peptide and the vector peptide may be chosen from groups comprising a covalent bond, a hydrophobic bond, an ionic bond, a linker which is cleavable or non-cleavable in a physiological medium or inside the cells.

If the coupling is conducted indirectly, a linker is advantageously used. Bi- or multifunctional linkers containing alkyl, aryl, alkylaryl or peptide groups, esters, aldehydes or alkyl, aryl or alkylaryl acids, anhydride, sulfhydryl, carboxyl, maleimidyl or succinimidyl groups, groups derived from bromide or cyanogen chloride, carbonyldiimidazole, succinimide esters or sulfonic halides can be used. In a preferred embodiment of the invention, said linker is a linker capable of forming a disulfide bridge. In fact, this type of binding is characterized by its stability in the plasma after injection of the fusion peptide, and then once the fusion peptide has crossed the blood-brain barrier, said disulfide bridge is reduced and releases the neuroactive peptide / peptide-targeting peptide complex. the glial scar. Such linkers are well known to those skilled in the art. For example, a linker may be: N-hydroxysuccinimidyl 3- (2-pyridyldithio) -propionate (SPDP) ON * S'S \ / ON ~ o ~ O or succinimidyl 6- [3- (2-pyridyldithio) -propionamido] - hexanoate. In a preferred embodiment of the invention, the neuroactive peptide can be linked to a SynB-type vector peptide by a specifically cleaved disulfide bond when the fusion peptide reaches the central nervous system (FIGS. 6 and 7). . An example of a disulfide bond is shown below: ## STR1 ## This type of bond can be obtained by treating both peptides in such a way that a N-succinimidyl-3 linker is obtained. (2-Pyridyl-dithio) -propionate (SPDP) can be coupled at their N-terminus to form a 2-pyridyl disulfide structure. The neuroactive peptide thus modified is then converted into an aliphatic thiol by reaction with dithiothreitol (DIT). The thiol peptide thus formed is finally coupled to the SynB-2-pyridyl disulfide derivative by thiol disulfide exchange (FIG. 8).

An object of the invention is a fusion peptide comprising (a) a neuroactive peptide comprising: a NGF peptidomimetic (Nerve Growth Factor), and optionally a glial scar targeting peptide, (b) a translocation vector peptide through the hematoencephalic and cerebrospinal barriers, wherein the glial scar targeting peptide specifically binds to the NG2 / HM proteoglycan as described above, and the translocation vector peptide across the hematoencephalic and cerebrospinal barriers is a linear peptide derived from peptides of the family Protégrines or family Tachyplesins as described above.

Axonal growth implements highly coordinated cellular mechanisms that require different extracellular signals. Soluble or substrate-bound molecular signals regulate the rate and direction of growth. A specialized structure, the growth cone, and its mobile extensions, the filopodia, are key elements in the detection and transduction of these signals. The simple contact of a filopod with a specific substrate can reorient a growth cone and its ramifications in a different direction. A member of the neurotrophin family, the Nerve Growth Factor (NGF) plays a major role in guiding growth cones. NGF induces the formation of filopodia and stimulates the transport of non-substrate-bound X31 integrins at the end of these filopodia, thus modulating their presentation to the environment. In addition, NGF activates the retrograde transport of integrins 1 bound to a substrate. Coupling integrins (31 bound to the extracellular matrix to the retrograde transport mechanism creates traction promoting growth cone progression and neurite growth (Figure 9).

These cellular mechanisms are activated by the binding of NGF to TrkA receptors (Tropomyosin receptor kinase A). NGF binding induces TrkA receptor dimerization, transient activation of their intrinsic kinase activity, and internalization of NGF-TrkA complexes required for NGF signal transfer by signaling endosomes to the neuronal cell body. This process results in the activation of specific signaling pathways. The integrin a7 (31 is a major player in NGF-mediated TrkA-dependent axonal regeneration processes.) The absence of a7 signaling limits the growth of NGF-induced neurites The NGF / TrkA system is mainly involved in the growth neurons and, potentially, in various pathologies of the central nervous system, however, problems of stability, penetration of blood and brain barriers, and various adverse effects limit the use of NGF as a therapeutic agent.The present invention describes an alternative treatment, said treatment aimed at using peptidomimetic compounds having more favorable stability, tissue penetration and biological action profiles, three P-turn structures of NGF (loops 1, 2 and 4) are involved in the TrkA binding. The DEKQ motif of NGF loop 4 (mouse), in particular, plays a predominant role in the linking and activation processes. The R-turn structure formed by the D-Q residues and the orientation of the side chains determine the binding specificity of NGF to the TrkA receptor [Beglova et al., 1998. J. Biol. Chem. 273 (37), 23652-23658]. The same structure is found in different species (Figure 10). Various peptides incorporating the DEKQ motif have been tested. Only certain cyclic peptides have been shown to be active ligands of the TrkA receptor. A disulfide-bonded N-acetylated (N-Ac) cyclic peptide, N-Ac-YCTDEKQCY peptide, has been particularly studied. The peptide oscillates in solution between two structures (3-tum (type I / type yL-aR) and adapts to the TrkA receptor by a specific adjustment mechanism.By stabilizing TrkA in a conformation promoting TrkATrkA interactions, this peptide facilitates the activation of the receptor and thus the induction of trophic signals and differentiation A second cyclic peptide integrating the sequence TDEKQ, a dimer called P92, has similar properties, however, the presence of disulfide bridges limits the use of these peptides as therapeutic agents To overcome this drawback, cyclic peptidomimetics devoid of disulfide bridges have been developed, various 13-turn cyclic structures incorporating di- or tripeptide sequences have been synthesized, and most of these peptidomimetics have been synthesized. proved to be non-selective TrkA receptor antagonists or agonists, however, a partial agonist of the receiver TrkA called D3 or (EKHse) G-OH has been the subject of particular attention [Maliartchouk et al., 2000. Mol. Pharmacol. 57 (2), 385-391]. Although having low intrinsic agonist activity, peptidomimetic D3 promotes selective activation of TrkA receptors. By positioning TrkA receptors in a conformation facilitating the binding of NGF, peptidomimetic D3 enhances the action of low concentrations of NGF (synergistic action). The addition of D3 at suboptimal levels of NGF does not allow maximum survival, enhances the effects of NGF and greatly enhances neuronal survival. The peptidomimetic D3 not only protects neurons expressing TrkA (selective neuroprotection), but also promotes their extension (TrkA-dependent growth). The peptidomimetic D3 can remain associated with TrkA receptors for long periods. The present invention describes two other cyclic peptidomimetics, designated (DKHse) G-OH and (GKHse) G-OH, incorporating DK and GK dipeptide sequences respectively. The peptidomimetics (EKHse) G-OH (D3), (DKHse) G-OH and (GKHse) G-OH are very useful for promoting neuronal regeneration in the central nervous system. The cyclic peptidomimetics (EKHse) G-OH (D3), (DKHse) G-OH and (GKHse) G-OH can be prepared by chemical synthesis using previously described methods for such compounds. The synthesis method generally used consists of macrocycling linear precursors via a SNAr reaction on resin. In one embodiment of the invention, said NGF peptidomimetic is a TrkA receptor agonist named D3 or (EKHse) G-OH having the formula below: Glutamic acld (GIu, E) Lysine OH (Lys, K ## STR2 ## or one of the following cyclic compounds: (DKI-Ise) G-OH having the formula below: Aspartic acid Lysine (Asp, D) (Lys, K) O OHO - NH2 L-Homoserine (Hse)

OH (Gy, G) DKHseG-OH and (GKHse) G-OH having the following formula: GKHse (3-OH) In one embodiment of the invention, the peptidomimetic of NGF can be directly or indirectly to the vector peptide via a linker specifically cleaved when the fusion peptide of the invention reaches the central nervous system, said linker binding the peptide vector either to the peptidomimetic of NGF or to the targeting peptide of the glial scar when Preferably, said linker is a linker capable of forming a disulfide bridge, for example a linker may be N-hydroxysuccinimidyl 3- (2-pyridyldithio) propionate (SPDP) or succinimidyl 6- [3- ( 2-pyridyldithio) -propionamido] -hexanoate.

In a preferred embodiment, the NGF peptidomimetic can be directly or indirectly linked to a SynB-type peptide by a disulfide bridge, said disulfide bridge linking the SynB peptide to either the NGF peptidomimetic or the glial scar targeting peptide. when present (Figure II). In one embodiment of the invention, the cyclic peptidomimetic compounds (EKHse) G-OH (D3), (DKHse) G-OH and (GKHse) G-OH are linked to a SynB-type vector peptide by a disulfide bridge. specifically cleaved when the fusion peptide of the invention reaches the central nervous system. According to this embodiment, the peptidomimetics are chemically modified so that the nitro group of the compounds (EKHse) G-OH (D3), (DKHse) G-OH and (GKHse) G-OH is replaced by a structure 2 pyridyl-disulfide. The peptidomimetics thus modified have the same activity as their unmodified counterparts. The modified peptidomimetics correspond to the following cyclic compounds: [(EKHse) G-OH] -SS-2-pyridyl or D3-SS-2-pyridyl having the formula below: HHNN O "S [(EKHse) G • OH] • SS-2-pyridyl [(DKHse) G-OH] -SS-2-pyridyl having the formula below: [(DKHse) G OH] -SS-2-pyrldyl or [(GKHse) G-OH ] -SS-2-pyridyl having the formula below: [(G KHse) G-OHJ • SS-2-pyridyl Cyclic peptidomimetics incorporating a 2-pyridyl disulfide structure are prepared according to the same method as that used for peptidomimetics unmodified cyclic (EKHse) G-OH (D3), (DKHse) G-OH and (GKHse) G-OH, except that after the cyclization step the nitro group of cyclic peptidomimetics (EKHse) G-OH (D3 ), (DKHse) G-OH and (GKHse) G-OH is reduced by treatment with SnC12 so that an N-succinimidyl-3- (2-pyridyl-dithio) -propionate linker (SPDP) can be coupled to the new arylamine formed to form a 2-pyridyl disulfide structure. It is cleaved from the resin and purified. The peptidomimetics thus modified (D3-S-S-2-pyridyl or analogous compound) are then converted to aliphatic thiols by reaction with dithiothreitol (DTT). The thiol peptide (D3-SH or similar compound) thus formed is coupled to the SynB-2-pyridyl disulfide derivative (SynB-S-S-2-pyridyl) by a thiol disulfide exchange.

In one embodiment of the invention, the NGF peptidomimetic can be ligated to the glial scar targeting peptide by a flexible peptide linker as previously described. In a preferred embodiment, the C-terminal glycine residue of the previously modified peptidomimetics is bound to the N-terminal residue of a flexible linker (Gly4Ser) n such that the C-terminal residue of the flexible peptide linker can be bound to the N-terminal residue of the glial scar binding peptide. These modified peptidomimetics correspond to the following cyclic compounds: [(EKHse) G- (GGGGS) n] -SS-2-pyridyl or [D3- (GGGGS) n] -SS-2-pyridyl having the formula below: OH Oj ## STR2 ## wherein R 1 is a mixture of IInker (GGGGS) n 1 (E KHse) G- (GGGGS) n) -SS-2-pyridyl [(DKHse) G- (GGGGS) n] -SS-2-pyridyl having the formula below: NHz HN,, 0 HN. ## STR2 ## ## STR2 ## ## STR2 ## where ## STR2 ## [0001] An object of the invention is a fusion peptide comprising (a) a neuroactive peptide comprising: a peptidomimetic of PDGF (Platelet-Derived Growth Factor) and, optionally, a glial scar targeting peptide, (b) a translocation vector peptide across the haematoencephalic and cerebrospinal barriers, wherein the glial scar targeting peptide specifically binds to the NG2 / HM proteoglycan As described above, the translocation vector peptide across the blood brain and cerebrospinal barriers is a linear peptide derived from peptides of the family Protégrines or family Tachyplésines as described above. A member of the PDGF (Platelet-Derived Growth Factor) family, the PDGF-BB factor, makes it possible specifically to increase the expression of the flexible Ilnker subunit (GGGGS) n HNN Q 'v S'sN integrin a7 (de novo synthesis) in different cell types [Chao et al., 2006. Am. J. Physiol. Cell Physiol. 290, 972-980], thus promoting the adhesion of the integrin a7P 1 to certain specific substrates of the extracellular matrix, such as the pattern of growth of neurites of the fnD region of tenascin-C or certain laminin motifs. The PDGF-BB factor further promotes neurite growth and neuron survival. This neuroprotective effect of PDGF-BB depends directly on the PDGFR-f3 receptors expressed by the neurons. The PDGF-B / PDGFR-P system plays a crucial neuroprotective role in CNS lesions. Peptidomimetics of PDGF-BB or specific agonists can also activate the PDGFR-3 receptors present, and thereby increase the expression of the u7 integrin subunit Two N-acetylated peptides, the N-Ac peptides -ISRRLIDRTNANFLC (N-Ac-SEQ ID NO: 68) and N-Ac-RKIEIVRKKC (N-Ac-SEQ ID NO: 69) exhibit agonist activity, essentially when presented as homodimers [Jehanli et al., 1999. US6001802] These two peptides each have a C-terminal cysteine residue for the formation of disulfide bridges, and other more suitable binding methods may be used. Embodiment of the invention, said PDGF peptidomimetic is an N-acetylated N-Ac-ISRRLIDRTNANFLC (N-Ac-SEQ ID NO: 68) or an N-acetylated N-Ac-RKIEIVRKKC ( N-Ac-SEQ ID NO: 69), preferably presented in In one embodiment of the invention, the peptidomimetic of PDGF can be linked to the glial scar targeting peptide by a flexible peptide linker as described above.

In one embodiment of the invention, the PDGF peptidomimetic may be directly or indirectly linked to the vector peptide via a specifically cleaved linker when the fusion peptide of the invention reaches the central nervous system, said linker binding the peptide vector either to the peptidomimetic of PDGF or to the glial scar targeting peptide when present. Preferably, said linker is a linker capable of forming a disulfide bridge as described above. In a preferred embodiment, the PDGF peptidomimetic can be directly or indirectly linked to a SynB-type peptide by a disulfide bridge, said disulfide bridge linking the SynB peptide to either the PDGF peptidomimetic or the glial scar targeting peptide. when present (Figure 12).

An object of the invention is a fusion peptide useful as an agent for imaging glial scars comprising: (a) a labeled neuroactive peptide comprising: a glial scar targeting peptide, a molecule detectable by an imaging device, in optionally, a peptide comprising a neurite attraction pattern or a pattern for neurite outgrowth, a NGF peptidomimetic or a PDGF peptidomimetic, (b) a translocation vector peptide across the blood brain and cerebrospinal barriers. wherein, the glial scar targeting peptide specifically binds to NG2 / HM proteoglycan, said detectable molecule by an imaging device is a radionuclide, a contrast agent or a light emitting molecule, the peptide vector of Translocation across the blood brain and cerebrospinal barriers is a linear peptide derived from peptides of the family of protegrins or the family of tachyplines.

In one embodiment of the invention, the labeled neuroactive peptide may be linked to the vector peptide via a specifically cleaved linker when the fusion peptide of the invention reaches the central nervous system as previously described. In one embodiment of the invention, the labeled neuroactive peptide is followed by an imaging device.

According to one embodiment of the invention, the fusion peptide of the invention can be provided in the form of inorganic ion salts (chloride, bromide, iodide, fluoride) or organic ion salts (acetate, formate, benzoate).

The present invention also relates to the nucleic sequences encoding the fusion peptides of the invention.

The subject of the present invention is also the vectors comprising the nucleic sequences coding for the fusion peptides of the invention, as well as the host cells, prokaryotic or eukaryotic, transformed with said nucleic sequences or said vectors. The present invention includes all recombinant vectors containing coding sequences for transformation, transfection or eukaryotic or prokaryotic gene therapy. Such vectors may be prepared according to conventional molecular biology techniques and will further include a suitable promoter, optionally a signal sequence for export or secretion, and regulatory sequences necessary for transcription of the nucleotide sequence.

The subject of the present invention is a composition comprising at least one fusion peptide according to the invention.

The present invention also relates to a pharmaceutical composition comprising at least one fusion peptide according to the invention and at least one pharmaceutically acceptable carrier.

The term "vehicle" means a non-toxic material that does not affect the effectiveness of the active ingredients of the composition. Pharmaceutically acceptable expression refers to non-toxic material compatible with a biological system such as a cell, cell culture, tissue or organism. The characteristics of the vehicle will depend on the mode of administration.

Examples of pharmaceutically acceptable carriers are aqueous solutions such as water or saline solutions. Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, or injectable organic esters such as ethyl oleate and, as examples of aqueous vehicles, water, alcohol / water solutions, emulsions or suspensions.

Examples of pharmaceutically acceptable carriers include negatively charged molecules such as heparin or heparin-like injectable substances. Negatively charged molecules such as heparin make it possible to increase the protection and diffusion of the fusion peptides of the invention.

In one embodiment of the invention, the composition or pharmaceutical composition as described above also comprises a fusion peptide comprising a NGF peptidomimetic as described above and / or a peptide fusion comprising a PDGF peptidomimetic.

The action of the fusion peptide of the invention may be potentiated by the presence of one or more peptide (s) fusion comprising a peptidomimetic of NGF and / or a peptidomimetic of PDGF. In fact, in an elderly person, the number of integrins decreases. An NGF peptidomimetic can induce the formation of filopodia at the growth cones and regulate the accumulation of non-substrate-bound integrins at the end of the filopodia, i.e. at the level of the neurite growth zone; while a peptidomimetic of PDGF can activate the PDGFR-13 receptors present, and thereby induce the expression of the integrin a7 subunit (de novo synthesis), thus promoting the adhesion of the integrin x7 (31 to specific substrates of the extracellular matrix, such as the aforementioned fnD neurite growth pattern of tenascin-C or certain laminin motifs.The conjugated action of NGF peptidomimetic and PDGF peptidomimetic can promote growth and neurite survival.

The present invention also relates to the use of at least one fusion peptide according to the invention for the manufacture of a pharmaceutical composition as described above for the treatment of trauma or diseases affecting the central nervous system, said traumas or diseases being characterized by stable or progressive lesions of the central nervous system which are generally irreversible. Such lesions may be due to vascular accidents, traumatic lesions, surgical lesions or degenerative diseases of the central nervous system such as lesions of the optic nerve or the retina, posttraumatic brain or spinal cord injuries, accidents ischemic or hemorrhagic cerebrovascular diseases, intracranial aneurysms, spinal cord injuries resulting in monoplegias, diplegia, paraplegia, hemiplegia or quadriplegia, injuries related to diving accidents, neurodegenerative diseases such as Alzheimer's disease, Pick's disease, Parkinson's disease, and diffuse Lewy body disease, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy or Creutzfeldt-Jakob disease.

Preferably, the pharmaceutical composition according to the invention is in a form suitable for parenteral, oral, rectal, nasal, transdermal, pulmonary, systemic, or topical administration. Preferably, the pharmaceutical composition of the invention is injectable, the injection being carried out intravenously, intraperitoneally, intramuscularly, subcutaneously or transdermally. The injection is preferably carried out intravenously.

According to one embodiment of the invention, a therapeutically effective amount of the pharmaceutical composition of the invention is administered to a subject suffering from trauma or disease affecting the central nervous system.

A therapeutically effective amount is sufficient to increase axonal regeneration. This amount may vary with the age, sex of the subject and the stage of the disease and will be determined by those skilled in the art.

The fusion peptides of the present invention overcome many of the disadvantages of the methods in use and present a decidedly new approach for the treatment of diseases of the central nervous system, such as brain and spinal cord injuries.

The treatment of many diseases affecting the central nervous system is made difficult because of limitations in the transport of therapeutic molecules beyond the blood-brain and cerebrospinal barriers. Effective treatment requires that the therapeutic molecules reach their target in sufficient concentration and within a reasonable time. One of the main advantages of the method is that the fusion peptides which are the subject of the invention can by themselves overcome the transport limitations due to the haematoencephalic and cerebrospinal barriers. Conjugation of neuroactive peptides to SynB-type vector peptides significantly improves their penetration into the central nervous system following intravenous administration. Concurrent pharmacological strategies to use lipid vectors such as liposomes, have multiple drawbacks essentially related to their slow penetration of the haematoencephalic and cerebrospinal membranes. Another advantage of the invention is that the fusion peptides which are the subject of the invention can themselves directly reach the glial scars bordering the lesion sites and attach to them by means of a peptide targeting the glial scar. This advantage overcomes the many disadvantages of invasive neurosurgical methods to use biomimetic matrices integrating grafted peptides, reverse microdialysis techniques, or to implant stem cells. Another advantage of the present invention is that the fusion peptides which are the subject of the invention provide signals promoting the growth of neurons and can greatly improve axonal regeneration in situ. Another advantage of the present invention is that the subject fusion peptides of the invention provide neuron guidance signals and can promote guided axonal regeneration in situ. Consequently, a major advantage of the present invention is that the fusion peptides produced according to the methods described have a much higher efficiency than the methods in use known to those skilled in the art. Of course, various modifications may be made by those skilled in the art to the processes described, presented solely by way of non-limiting examples, without departing from the scope of protection defined by the appended claims.

DESCRIPTION OF THE FIGURES

Figure 1: Structure of human tenascin-C. Tenascin-C has a hexameric structure consisting of two trimeric structures associated with a disulfide bridge (central nodule). Each of the six arms of tenascin-C is composed of 14 EGF (epidermal growth factor) domains, 6 to 17 FN-III domains (fibronectin type III) and one fbg domain (fibrinogen). The FN-III domains 1 to 8 (fnl-8) are present in each of the arms (constitutive splicing). The longest arm differs from the shorter one by the inclusion of the nine FN-III domains A1, A2, A3, A4, B, AD2, ADI, C and D (fnA-D) between domains FN-III 5 (fn5 ) and 6 (fn6) (alternative splicing).

Figure 2: Motif of the fnD domain of human tenascin-C (TNC) promoting the growth of neurites and similar motifs of other species. The amino acids FD and FV required for receptor binding and activation are underlined. Abbreviations: bTNC: Bos taurus (cow); CTN: Gallus gallus (chicken); dTNC: Canis familiaris (dog); hTNC: Homo sapiens (man); mTNC: Mus musculus (mouse); pTNC: Sus scrofa (pork); TCN: Rattus norvegicus (rat).

Figure 3: Motif of the fnC domain of human tenascin-C (TNC) promoting the attraction / retention of neurites and similar motifs of other species. Abbreviations: bTNC: Bos taurus (cow); CTN: Gallus gallus (chicken); dTNC: Canis familiaris (dog); hTNC: Homo sapiens (man); mTNC: Mus musculus (mouse).

FIG. 4: SG1 motif of the human integrin α4 (ITA4) subunit ensuring the binding of the α4131 integrin to the MCSP proteoglycan and analogous motifs of other species. Abbreviations: bITA4: Bos taurus (cow); chITA4: Pan troglodytes (chimpanzee); dITA4: Canis familiaris (dog); bITA4: Homo sapiens (man); mITA4: Mus musculus (mouse); oITA4: Monodelphis domestica (opossum); rITA4: Rattus norvegicus (rat); rmITA4: Macaca mulatta (rhesus macaque). Figure 5: Translocation vector peptides. (A) Peptides of the family of protegrins (peptides PG-1 to PG-5) and linear peptides derived (peptides SynB1, SynB3 and SynB5). (B) Peptides of the tachyplesin family (peptides T-1 to T-3) and linear peptide derivatives (SynB4 peptide). Basic residues are shown in bold type. The cysteine residues are underlined.

FIG. 6: Fusion peptides associating a neuroactive peptide and a SynB-type vector peptide. The SynB peptide is bound to the neuroactive peptide by a specifically cleaved disulfide bridge when the fusion peptide reaches the central nervous system. The disulfide bond binds the SynB peptide either to the peptide comprising a neurite attraction pattern or a pattern for neurite growth, or to the glial scar targeting peptide.

Figure 7: Translocation of fusion peptides beyond the blood-brain barrier.

Injected intravenously, the fusion peptides pass through the blood-brain membrane via an endocytosis / exocytosis mechanism. Cleavage of the disulfide bridge in the central nervous system (CNS) releases the neuroactive peptide.

Figure 8: Conjugation of a neuroactive peptide and a SynB-type vector peptide by a disulfide bond. Both peptides are treated such that a succinimidyl-3- (2-pyridyl-dithio) propionate linker (SPDP) is coupled at their N-terminus to form a 2-pyridyl disulfide structure. The modified neuroactive peptide is converted to an aliphatic thiol by reaction with dithiothreitol (DTT). The formed thiol peptide is coupled to the SynB-2-pyridyl disulfide derivative by thiol disulfide exchange.

Figure 9: Growth of the filopods induced by NGF (Verve Growth Factor). (A) In the quiescent state, the cytoskeleton (dashed lines) is static and the f31 integrins are randomly distributed at the level of the filopodia. (B) NGF induces the formation of filopodia and stimulates the transport and accumulation of integrins 131 not bound to a substrate at their ends. (C) NGF activates the retrograde transport of R1 integrins bound to a substrate. The coupling of extracellular matrix-bound (MEC) X31 integrins (hatched surface) to the retrograde transport mechanism (oval structures) creates a traction favoring the growth of the filopods.

Figure 10: DEKQ motif of mouse NGF loop 4 involved in TrkA receptor binding and similar motifs of other species. The amino acids required for binding to the TrkA receptor and its activation are underlined. Abbreviations: bNGF: Bos taurus (cow); cNGF: Gallus gallus (chicken); hNGF: Homo sapiens (man); mNGF: Mus musculus (mouse); pNGF: Sus scrofa (pork); rNGF: Rattus norvegicus (rat).

11: Peptides fusion associating an NGF peptidomimetic and a SynB-type vector peptide. A disulfide bond binds the SynB peptide, either to the NGF peptidomimetic, or to the glial scar targeting peptide when present.

FIG. 12: Peptides fusion associating a peptidomimetic of PDGF and a vector peptide of SynB type. A disulfide bond binds the SynB peptide, either to the peptidomimetic PDGF, or to the glial scar targeting peptide when present.

EXAMPLES

Different fusion peptides of the invention were made using the following sequences: These peptide fusions are constructed in the sense: Peptide favoring the attraction or the growth of neurites or peptidomimetic of NGF or PDGF - flexible linker - targeting peptide the glial-ligable scar capable of forming a disulfide bridge such as (N-hydroxysuccinimidyl 3- (2-pyridyldithio) -propionate (SPDP) or succinimidyl 6- [3- (2-pyridyldithio) -propionamido] hexanoate); translocation vector peptide, or in the sense: targeting peptide of flexible glial hyposisin - Peptide promoting the attraction or growth of neurites or peptidomimetics of NGF or PDGF to a linker capable of forming a disulfide bridge such as ( N-hydroxysuccinimidyl 3- (2-pyridyl-dithio) -propionate (SPDP) or succinimidyl 6- [3- (2-pyridyldithio) -propionamido] -hexanoate) peptide translocation vector.

1 / Peptides that promote the guidance and / or growth of neurites in glial scars and thus axonal regeneration in the central nervous system Peptide favoring pattern of attraction of DINPYGFTVSWMASE attraction or neurites (SEQ ID NO: 23) Neurite Growth Pattern for ADEGVFDNFVLKIRDTKKQ Neurite Growth (SEQ ID NO: 1) Glial Scar Targeting Peptide TAASGVRSMH (SEQ ID NO: 31) LTLRWVGLMS (SEQ ID NO: 32) KKEKDIMKKTI (SEQ ID NO: 33) Peptide Vector of Translocation Through RGGRLSYSRRRFSTSTGR (SEQ ID NO: 56) RRLSYSRRRF (SEQ ID NO: 57) Blood and Cerebrospinal Barriers 2 / Peptides Fusion Comprising NGF Peptidomimetic or Peptidomimetic Peptidomimetic Peptidomimetic of NGF Glu E) 'd Lysine OH (Lys, K) O ~ O NH2 Peptide favoring L-serins the attraction or the H (Hse) HN O HN NH growth of ~ ° Glycine neurites OH (GIy, G) EKHseG-OH (03) Peptidomimetic N -Ac-ISRRLIDRTNANFLC (N-Ac- PDGF SEQ ID NO: 68) Optionally, glial targeting peptide TAASGVRSMH (SEQ ID NO: 31) glial LTLRWVGLMS (SEQ ID NO: 32) KKEKDIMKKTI (SEQ ID NO: 33) Peptide vector translocation through RGGRLSYSRRRFSTSTGR ( SEQ blood-brain barrier and ID NO: 56) cerebrospinal RRLSYSRRRF (SEQ ID NO: 57) REFERENCES 1. Beglova N., LeSauteur L., Ekiel I., Saragovi HU and Gehring K., 1998. Solution structure and internai motion of a bioactive peptide derived from nerve growth factor. J. Biol. Chem. 273 (37), 23652-23658. 2. Burg M.A., Pasqualini R., Arap W., Ruoslahti E. and Stallcup W.B., 1999. NG2 proteoglycan-binding peptides target tumor neovasculature. Cancer Res. 59, 2869-2874. 3. Chao J.T., Martinez-Lemus L.A., Kaufman S.J., Meininger G.A., Ramos K.S. and Wilson E., 2006. Modulation of a7-integrin-mediated adhesion and expression by platelet-derived growth factor in vascular smooth muscle cells. Am. J. Physiol. Cell Physiol. 290, 972-980. 4. Drin G., Rousselle C., Schermann J.M., Rees A.R. and Temsamani J., 2002. Peptide delivery to the brain via absorptive-mediated endocytosis: advances with SynB vectors. AAPS Pharm. Sci. 4 (4), article 26. 5. Iida J., Meijne AML, Oegema TR, Yednock TA, Kovach Ni., Furcht LT and McCarthy JB, 1998. A role of chondroitin sulfate glycosaminoglycan binding site in a * integrin-mediated melanoma cell adhesion. J. Biol. Chem. 273 (10), 5955-5962. 6. Jehanli A.M., Patel G., Olabiran Y., Brennand D.M. and Kakkar V.V., 1999. Platelet-derived growth factor analogues. U.S. Patent 6001802. 7. Liu H.Y., Nur-E-Kamal A., Schachner M. and Meiners S. 2005. Neurite guidance by the FnC Tenascin-C: neurite attraction vs. neurite retention. Eur. J. Neurosci., 22, 1863-1872. 8. Maliartchuk S., Feng Y., Ivanisevic L., Debeir T., Cuello A.C., Burgess K. and Saragovi H.U., 2000. A designed peptidomimetic agonistic ligand of TrkA NGF receptors. Mol. Pharmacol. 57 (2), 385-391. 9. Meiners S., Nur-E-Kamal M.S.A. and Mercado M.L.T., 2001. Identification of a neurite outgrowth-promoting motif in the alternatively spliced region of human tenascin-C. J. Neurosci. 21 (18), 7215-7225. 10. Mercado M.L.T., Nur-E-Kamal A., Liu H.Y., Gross S., Movahed R. and Meiners S., 2004. Neurite outgrowth by the alternatively spliced region of tenascin-C is mediated by neuronal a7131 integrin. J. Neurosci. 24 (1), 238-247.

Claims (11)

REVENDICATIONS1. A fusion peptide comprising: (a) a neuroactive peptide comprising: a peptide comprising a neurite attraction pattern or a pattern for neurite growth, and - a glial scar targeting peptide, (b) a translocation vector peptide through the haematoencephalic and cerebrospinal barriers, wherein the peptide comprising a neurite attraction pattern is derived from the fnC region of tenascin-C, the peptide comprising a pattern for neurite outgrowth derived from the fnD region of the tenascin-C, the targeting peptide of the glial scar specifically binds to the proteoglycan NG2 / HM, and the translocation vector peptide across the blood brain and cerebrospinal barriers is a linear peptide derived from peptides of the family Protégrines or family Tachyplesins. A fusion peptide according to claim 1, wherein said peptide comprising a pattern for neurite growth is a peptide of not more than 30 amino acids comprising one of the following: VFDNFVLK (SEQ ID NO: 2), VFDSFVLK (SEQ ID NO: 4) or IFDSFVIR (SEQ ID NO: 5). A fusion peptide according to claim 2, wherein said peptide comprising a pattern for neurite growth is a peptide of up to 30 amino acids comprising one of the following sequences: VFDNFVLKIRDTKKQ (SEQ ID NO: 6), VFDSFVLKIRDTKKQ (SEQ ID NO: 7), IFDSFVIRIRDTKKQ (SEQ ID NO: 8), VFDSFVLKIRDTKRK (SEQ ID NO: 9), VFDNFVLKIRDTKRK (SEQ ID NO: 10), or IFDSFVIRIRDTKRK (SEQ ID NO: 11), ADEGVFDNFVLKIRDTKKQ (SEQ ID NO. : 1), ADEGVFDSFVLKIRDTKKQ (SEQ ID NO: 12), ADEGIFDSFVIRIRDTKKQ (SEQ ID NO: 13), ADDGVFDSFVLKIRDTKRK (SEQ ID NO: 14), ADEGVFDNFVLKIRDTKRK (SEQ ID NO: 15), ADEGVFDSFVLKIRDTKRK (SEQ ID NO: 16), ADEGIFDSFVIRIRDTKRK (SEQ ID NO: 17), ADDGVFDNFVLKIRDTKKQ (SEQ ID NO: 18), ADDGVFDSFVLKIRDTKKQ (SEQ ID NO: 19), ADDGIFDSFVIRIRDTKKQ (SEQ ID NO: 20), ADDGVFDNFVLKIRDTKRK (SEQ ID NO: 21), ADDGIFDSFVIRIRDTKRK (SEQ ID NO: 22). 4. A fusion peptide according to claim 1, wherein said peptide comprising a neurite attraction pattern is a peptide of at most 30 amino acids comprising one of the following motifs: DINPYGFTVSWM (SEQ ID NO: 24), DTNPYGFTVSWT (SEQ ID NO: 25), NPYGFTVSWM (SEQ ID NO: 26), NPYGFTVSWT (SEQ ID NO: 27). A fusion peptide according to claim 4, wherein said peptide comprising a neurite attraction pattern is a peptide of at most 30 amino acids comprising one of the following sequences: DINPYGFTVSWMASE (SEQ ID NO: 23), DTNPYGFTVSWTASE (SEQ ID NO: 28), NPYGFTVSWMASE (SEQ ID NO: 29), NPYGFTVSWTASE (SEQ ID NO: 30). A fusion peptide according to claim 1, wherein said glial scar targeting peptide is a peptide of at most 30 amino acids comprising one of the following: TAASGVRSMH (SEQ ID NO: 31), LTLRWVGLMS (SEQ ID NO: 31), ID NO: 32), KKEKDIMKKTI (SEQ ID NO: 33), KKEKDIIKKTI (SEQ ID NO: 48), KKEKDIIEKTI (SEQ ID NO: 49), KKERDIIEKTI (SEQ ID NO: 50), KKEKDIIRKTI (SEQ ID NO: 51) , KKEKDVIRKMI (SEQ ID NO: 52), KKEKDIIREKI (SEQ ID NO: 53), GGGTRAGMKY (SEQ ID NO: 34), WGKIEDPLRA (SEQ ID NO: 35), AGQTLTASGD (SEQ ID NO: 36), DLLAVSWLRA (SEQ ID NO: 37), SAERGVVAMS (SEQ ID NO: 38), AIHSELMWVS (SEQ ID NO: 39), FWTERAGWAY (SEQ ID NO: 40), MVWSKGPLFL (SEQ ID NO: 41), AGTRMSWEVL (SEQ ID NO: 42), VSRSSRWGSI (SEQ ID NO: 43), DAHVLVPRTP (SEQ ID NO: 44), AQGIVLQLAL (SEQ ID NO: 45), LSPLLSPATA (SEQ ID NO: 46), CLSGSLSC (SEQ ID NO: 47). 7. A fusion peptide according to claim 1, wherein said peptide vector for translocation across the blood brain and cerebrospinal barriers is a linear peptide derived from peptides of the Protégrine family or of the Tachyplesin family corresponding to one of the following formulas: or to a fragment of one of these formulas: RXIXIRX1X2X1X2RRRX1X2X1X2X1X1R (SEQ ID NO: 54) RX1X2X1RX1X2X1RX1X1X2X1RRX2R (SEQ ID NO: 55) in which R represents arginine, the groups X1 identical or different represent a natural amino acid or not aliphatic or aromatic, selected from glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteineA penicillamine, methionine, serine, threonine, asparagine, glutamine , phenylalanine, histidine, tryptophan, tyrosine, proline, amino-butyric acid, amino-1-cyclohexane carboxylic acid, amino-isobutyric acid 2-aminotetralin carboxylic acid, 4-bromophenylalanine, tert-leucine, 4-chlorophenylalanine, (3-cyclohexylalanine, 3,4-dichlorophenylalanine, 4-fluorophenylalanine, homoleucine, 3-homoleucine, homophenylalanine, 4-methylphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 4-nitrophenylalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridylalanine, [2-thienyl] alanine, and X2 represents serine or threonine. A fusion peptide according to claim 7, wherein said translocating vector peptide across the hematoencephalic and cerebrospinal barriers is a peptide having one of the following sequences: RGGRLSYSRRRFSTSTGR (SEQ ID NO: 56), RRLSYSRRRF (SEQ ID NO: 57), RGGRLAYLRRRWAVLGR (SEQ ID NO: 58), AWSFRVSYRGISYRRSR (SEQ ID NO: 59) or their retroinversed sequences: RGTSTSFRRRSYSLRGGR (SEQ ID NO: 60), FRRRSYSLRR (SEQ ID NO: 61), RGLVAWRRRLYALRGGR (SEQ ID NO: 62) ), RSRRYSIGRYSVRFSWA (SEQ ID NO: 63). 9. A pharmaceutical composition comprising at least one fusion peptide according to any one of claims 1 to 8 and at least one pharmaceutically acceptable carrier. 10. Pharmaceutical composition according to claim 9 for treating trauma or diseases affecting the central nervous system. 11. Pharmaceutical composition according to claim 10, for treating lesions due to vascular accidents, traumatic lesions, surgical lesions or degenerative diseases of the central nervous system such as lesions of the optic nerve or the retina, brain lesions or post-traumatic medullaries, ischemic or hemorrhagic strokes, intracranial aneurysms, spinal cord injuries resulting in monoplegias, diplegia, paraplegia, hemiplegia or quadriplegia, injuries related to diving accidents, neurodegenerative diseases such as Alzheimer's disease, Pick's disease, Parkinson's disease, and diffuse Lewy body disease, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy or Creutzfeldt-Jakob disease .