WO2016059277A1 - Peptide derived from trkb-fl and use thereof as a neuroprotector - Google Patents

Abstract

The invention relates to a neuroprotective peptide characterised in that it consists of 15 amino acids of the human TrkB-FL sequence corresponding to positions 458 to 472 of the canonical sequence, and can exert a neuroprotective effect when the pro-survival response time is increased. The invention also relates to the use of said peptide for preventing and/or treating neuronal damage in mammals caused by a pathology of the central nervous system associated with excitotoxicity. The invention further relates to a pharmaceutical composition characterised in that it comprises said neuroprotective peptide, and to the use of same in the treatment of cerebrovascular diseases and other pathologies of the CNS.

Description

 PEPTIDE DERIVED FROM TRKB-FL AND ITS USE AS A NEUROPROTECTOR

5

 SECTOR DESCRIPTION AND OBJECT OF THE INVENTION

 The present invention is encompassed in the pharmaceutical sector, and in the public, social and collective services sector. Specifically, the present invention is directed to pharmaceutical companies working in the field of neuroprotection, both referred to acute (ischemia, acute trauma) and chronic (neurodegenerative diseases). 5 STATE OF THE TECHNIQUE

 The pharmaceutical industry has great interest in human diseases of high incidence and increasing prevalence due to demographic changes and higher life expectancy. Cerebrovascular diseases (CVD) represent the second leading cause of death worldwide, with 6.7 million deaths and 0 11.9% of the total (according to recent WHO data from 2012). Likewise, cerebral ischemia is the second cause of dementia after Alzheimer's disease, and the first reason for disability in adults.

Cerebral ischemia or stroke is a phenomenon that characterizes CVDs and is defined5 as the reduction by different causes of blood supply to insufficient levels to maintain the normal metabolism and functioning of brain cells. There are two types of stroke according to their origin: hemorrhagic stroke, generated by the rupture of a cerebral vessel, vascular spasm associated with subarachnoid hemorrhage and intracranial hypertension, and ischemic stroke, caused by the lack of blood supply in a region of the brain due to the presence of a thrombus or atherosclerotic plaque. In the affected tissue we can differentiate two areas: the heart of the infarct, which suffers the most severe reduction of blood flow and experiences irreversible damage, and the area of ischemic penumbra, characterized by being functionally silent but structurally intact. However, if the blood flow does not recover in a period of time called a window Therapeutically, this region may undergo secondary neuronal degeneration processes that cause the heart attack to expand to the area of ischemic penumbra. The only treatment approved in clinical practice against ischemic stroke is thrombolysis with the tissue plasminogen activator (tPA), which contributes to the restoration of cerebral blood flow by dissolving the clot that obstructs the artery. However, thrombolytic therapy results in relatively frequent symptomatic intracerebral hemorrhages and, in addition, tPA has an associated toxicity that increases neuronal degeneration in experimental models. These adverse effects entail the existence of numerous contraindications for this type of therapy, which causes an additional problem: the therapeutic window in which the benefits of the treatment are greater than the risks narrows considerably, being within 3-4.5 hours after the onset of symptoms. This means that in Spain only 5-10% of patients with an ischemic stroke can benefit from the use of thrombolytic compounds. Therefore, even improving the evolution of some patients, the use of tPA is not risk-free, has a very limited use and is only effective in a narrow therapeutic window.

Among the therapeutic alternatives that are being tried to develop is the interference of the biochemical signaling cascades induced by CVD that lead to ischemic damage and secondary neuronal death in the twilight zone. Reducing this secondary neuronal death would be extremely beneficial because it would mitigate the neurological damage and the degree of disability of the patients. The fundamental mechanism of secondary neuronal death is excitotoxicity, a process induced by the increase in the concentration of the excitatory neurotransmitter glutamate in the extracellular space and the over-activation of its specific receptors, mainly those of the N-methyl-D-aspartate type (NMDARs) ) (Olney, 1986). Therefore, excitotoxicity is a fundamental target in the search for neuroprotection strategies. In the past, as a first approximation, drugs were used that by directly binding to NMDARs (antagonists) or indirectly (for example, glutamate release inhibitors, voltage-dependent calcium channel antagonists) decreased over-activation of the NMDAR However, these drugs showed encouraging results in models. Experiments of ischemia but were ineffective in the treatment of cerebral ischemia in humans and, therefore, did not pass clinical trials (Ikonomidou and Turski, 2002). A common defect to most of these drugs is their lack of selectivity, mainly due to the lack of knowledge of the dual nature of NMDARs, receptors that are critical at the same time in processes of survival and cell death (Hardingham et al., 2002 ). In addition, NMDARs also play a preponderant role in synaptic transmission and neuronal communication of the central nervous system (CNS), both during development and in adult life, and are critical in vital processes such as learning, memory, plasticity synaptic and synaptogenesis. Because of this, previously developed drugs in general block both the physiological activation of NMDAR and the pathological one and, therefore, have side effects on learning and produce drowsiness, hallucinations or even coma.

The interest in developing new drugs capable of reducing the excitotoxic process goes well beyond CVDs, since the functional alterations of NMDAR are also causes of neuronal degeneration or death in a large number of neurological pathologies, such as hypoglycemia, epilepsy and acute trauma (Choi, 1988). In addition, excitotoxicity also appears associated with certain neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's diseases and amyotrophic lateral sclerosis (ALS). In general, all these diseases lack etiological treatments and the therapies used are symptomatic or palliative. Thus, for example, in more moderate or severe cases of Alzheimer's disease, a more recent non-competitive NMDAR antagonist, memantine, is being used, which has some efficacy in reducing the excitotoxicity associated with said disease (Chen and Lipton, 2006). Finally, excitotoxicity is also critical in neurological disorders characterized by hyperexcitability or neuronal hypersensitization (some types of dyskinesias, neuropathic pain), and ocular pathologies (glaucoma, diabetic retinopathy, ischemic optic neuropathy and optic nerve trauma).

In recent years, small peptides permeable to the plasma membrane and the blood-brain barrier (BHE) have begun to develop, which do not alter the activation of NMDARs per se, but the formation of complexes with scaffolding proteins and postsynaptic density signaling (PSD) that are necessary for the pathological functions of NMDAR (Aarts et al., 2002). It has been shown that peptide NA-1 (Tat-NR2B9c, US patent 2010/0137224 A1) is capable of reducing ischemic damage in murine models (Aarts et al., 2002) and primates (Cook et al., 2012b) and, more recently, in patients undergoing endovascular surgery for the repair of an aneurysm participants in a small phase 2 clinical study (Hill et al., 2012; reviewed in Dolgin, 2012). Although promising, this therapeutic approach is not exempt from possible complications since the subunits of the target NMDAR of this peptide participate in processes of synaptic plasticity and memory formation in adult individuals (Brigman et al., 2010).

In neurons there are other mechanisms for controlling neuronal survival, functionally related to those mediated by NMDARs, although different from them, which are also altered in pathological conditions. Examples of these mechanisms are those regulated by neurotrophins such as BDNF (brain-derived neurotrophic factor), which by binding to its high affinity receptor TrkB-FL induces its dimerization and transphosphorylation, and the activation of various procellular intracellular signaling cascades survival. Neurotrophins further regulate other fundamental CNS processes such as neurotransmitter release, gene expression or synaptic transmission. The TrkB gene mRNA (Ntrk2) encodes for the complete TrkB-FL isoform, which is the catalytically active TrkB receptor, and for several truncated isoforms (TrkB-T1, TrkB-T2 and TrkB-T-Shc) lacking the tyrosine domain kinase involved in the modulation of the active receptor. Particularly, the TrkB-T1 isoform blocks the function of the TrkB-FL receptor by competition for BDNF binding or the formation of inactive TrkB-FL / TrkB-T1 heterodimers (Carim-Todd et al., 2009).

The reduction of neurotrophic support is an important component in the pathogenesis of numerous CNS pathologies. In neurodegenerative diseases such as Alzheimer's disease, Parkinson's, Huntington or ALS, altered levels of BDNF and TrkB have been observed, and a decrease in signaling (Dawbarn and Alien, 2003); BDNF / TrkB pathway alterations have also been observed in schizophrenia, Down syndrome models or depressive and stress states (Dawbarn and Alien, 2003). Recently, it has been shown that the BDNF / TrkB pathway is inhibited in situations of excitotoxicity and transient cerebral ischemia (Vidaurre et al., 2012). Specifically, the modification in the opposite direction of the levels has been observed neurons of the TrkB-FL and TrkB-T1 isoforms through two mechanisms: the inversion of the balance between the mRNAs of the isoforms, which leads to an increase in the expression of TrkB-T1 to the detriment of that of TrkB-FL, and the Calpain proteolysis of TrkB-FL, which reduces the levels of the active receptor and produces a truncated protein similar in size to TrkB-T1 and which, like it, could act as a negative dominant (Vidaurre et al., 2012). The increase in TrkB-T1 has been observed in cellular and animal models of excitotoxicity, and also in necropsies of patients who died as a result of CVD, supporting the relevance of this mechanism for ischemic pathology in humans. Through the use of lentiviral vectors that reverse the imbalance between isoforms, it has been shown that changes in TrkB expression are fundamental in this process of neuronal death (Vidaurre et al., 2012). These results suggest that the maintenance of TrkB-FL active neurotrophin receptor levels could be a useful therapeutic strategy in numerous CNS pathologies with an excitotoxic component. Specifically, the development of neuroprotective peptides with TrkB-FL as a target would allow the treatment and prevention of neuronal damage caused by pathologies of the SN in which the BDNF / TrkB survival pathway is altered, as CNS pathologies associated with the excitotoxicity process , CVDs such as stroke or cerebral ischemia, or in neuronal damage caused by lack of neurotrophic support, hypoxia, disconnection or mechanical damage due to brain and / or spinal trauma. In the case of stroke or cerebral ischemia, these therapeutic methods could constitute a complement and / or alternative to the current thrombolytic therapies, the only ones existing today. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a neuroprotective peptide, hereafter referred to as "neuroprotective peptide of the invention", characterized in that it consists of 15 amino acids of the human TrkB-FL sequence (SEQ ID NO: 1) corresponding to positions 458 to 472 of the canonical sequence (NCBI Gene ID: 4915, UniProt Q16620). The TrkB-FL protein is highly conserved in mammals and the neuroprotective peptide sequence of the invention is identical in rat (NCBI Gene ID: 25054, UniProt Q63604) and mouse (NCBI Gene ID: 18212, UniProt P15209) proteins, where occupies residues 457 to 471. The neuroprotective peptide of the invention has been designed to interfere in the processing of TrkB-FL by proteases and, in particular, by calpain. The reduction of TrkB-FL processing increases its levels in the neuronal plasma membrane and, in this way, increases the responsiveness of these cells to the BDNF neurotrophin, which activates various neuronal cascades of pro-survival signaling. The reduction of neurotrophic support is a common common factor in pathological situations of neuronal damage associated with the excitotoxicity process. The stimulants that cause this form of cell death can be: cerebrovascular accidents (CVAs, such as ischemic stroke or hemorrhagic stroke), traumatic brain injuries (including neurosurgery injuries), CNS tumors, side effects of cancer treatment , or chronic pathologies of the central nervous system associated with an excitotoxicity process (neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington and amyotrophic lateral sclerosis or ALS).

A summary of the polynucleotides and peptides described in the present invention is described in the following table:

Table 1. List of sequences mentioned in the invention.

 SEQ ID NO SEQ ID NO

 Name

 nucleotide amino acid

 TrkB-FL 458-472 1 26

 TrkB-FL 483-496 2

 TrkB-FL 542-556 3

 TrkB-FL 640-653 4

 c-Myc 408-421 5

 TFL457 6 27

TFL ₄₈₂ 7

 TFL541 8

 TFL639 9

 TMyc 10

 MAP 11

 pAntp43-68 / Penetratin 12

 Transport / TP10 13

SBP 14 FBP 15

 TAT48-60 16

 TAT47-57 17 28

 SynB1 18

 SynB3 19

 pVEC 20

 Pep-1 21

 OctaArginine 22

 NonaArginine 23

 CADY 24

 RVG 25

Thus a first object of the invention is a neuroprotective peptide, hereafter referred to as the "neuroprotective peptide of the invention", characterized in that it consists of 15 amino acids of the TrkB-FL sequence corresponding to positions 458-472 of the human canonical sequence or positions 457-471 of the rat (NCBI Gene ID: 25054, UniProt Q63604) and mouse sequences (NCBI Gene ID: 18212, UniProt P15209) (SEQ ID NO: 1).

The expression: "neuroprotective peptide", as used in the present invention, refers to an amino acid sequence capable of preventing, mitigating or delaying the biochemical processes that occur in the nervous system and cause neurodegeration or neuronal death of apoptotic type or necrotic.

The term "cell death induced by excitotoxicity" or "excitotoxicity" refers to a pathological process by which neurons are damaged and destroyed by over-activation mediated by binding to their membrane receptors of excitatory neurotransmitters, primarily glutamate or its analogues. Pharmacological: NMDA (in English, N-methyl-D-aspartate receptor), AMPA (in English, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) and kainic acid. The presence in the nervous system of pathologically elevated levels of glutamate, or its NMDA or kainate analogues, can cause excitotoxicity by allowing elevated levels of calcium ions to enter neurons. The entry of Ca ⁺² into cells activates a series of enzymes, including phospholipases, endonucleases, and proteases such as calpain that damage cell structures, such as those that make up the cytoskeleton, membrane and DNA. Other important contributions to the excitotoxicity process are the generation of free radicals and mitochondrial dysfunction.

Preferably, said neuroprotective peptide comprises an "internalization agent"; with the term "internalization agent" refers to a molecule capable of facilitating the passage of a specific peptide through the blood brain barrier and the plasma membrane to allow access to the interior of neurons and / or glial cells. Said molecule may be covalently bound or not to the neuroprotective peptide sequence. Non-limiting examples can be cited: cyclodextrin, poloxamers, acetyl choline receptor binding peptides, viruses and VLPs (Virus Like Particles), Cereport (RMP-7), nanocarriers (including polyethylene glycols: PEG-PLA, PEG-PCL, PEG-PHDCA, etc.), or cellular penetrating peptides.

In a preferred embodiment of this invention, the neuroprotective peptide of the invention is covalently linked to a cellular penetrating peptide or CPP (Cell Penetrating Peptide) (Milletti, 2012; Regberg et al., 2013). Some examples of CPPs can be seen in Table 2.

Table 2: Examples of CPPs and their sequences. Abbreviations: MAP: model amphipathic peptide; Antp: Antennapedia; SBP: sequence signal-based peptide; FBP, sequence-based peptide fusion; TAT: HIV-1 trans-activating transcriptor, pVEC: vascular endothelial-cadherin; RVG: Rabies Virus Glycoprotein fragment.

SEQ ID NO

 Peptide Name

 amino acid

 MAP 11

 pAntp43-68 / Penetratin 12

 Transport / TP10 13

 SBP 14

 FBP 15

TAT48-60 16 TAT47-57 17

 SynB1 18

 SynB3 19

 pVEC 20

 Pep-1 21

 OctaArginine 22

 NonaArginine 23

 CADY 24

 RVG 25

In the present invention "CPPs" or "cell penetrating peptides" refers to a group of short peptides of less than 30 amino acids that are capable of penetrating the membrane along with the charge they carry to the cell interior without producing cytolytic effects; they are positively charged peptides, amphipathic characteristics, theoretical hydrophobicity, helical moment or able to interact with lipid membranes and adopt a distinctive secondary structure after their association with lipids. CPPs penetrate the cell primarily by endocytosis or macropinocytosis mediated by lipid raffis. CPPs are used as a means of transport to introduce bioactive molecules into cells; usually by a covalent bond, although non-covalent bonds are also possible.

Thus, in an even more preferred embodiment, the neuroprotective peptide of the invention is linked to the basic domain of the Tat immunodeficiency virus (HIV) transactivating protein, TAT47-57 (SEQ ID NO: 17) or TAT48-60 (SEQ ID NO: 16).

In a particular object of the invention the sequence of the neuroprotective peptide object of the present invention is a chimeric peptide with sequence SEQ ID NO: 6 and is characterized by comprising: i. the 11 amino acid sequence of CPP TAT47-57 (SEQ ID NO: 17), fused with

I. the 15 amino acid sequence of TrkB-FL corresponding to positions 458-472 of the human protein (SEQ ID NO: 1), with the particularity that the C-terminal arginine of the Tat sequence is also the first amino acid of the TrkB-FL sequence. As "chimeric peptide" or "fusion protein" is understood in the present invention, a peptide created from the binding in a fusion gene or by chemical synthesis of two or more polynucleotides with different known activities. When a "fusion gene" is used we refer to genes or gene fragments, which originally code for separate peptides, but whose translation results in an individual peptide with functional properties derived from each of the original peptides. Peptides or polypeptides whose amino acid sequence is identical or homologous to the sequences described in the present invention are also included within the scope of the present invention; preferably, the percentage of identity is between 70-95%, even more preferably the percentage of identity is between 90-95%, and even more preferably the percentage of identity is between 95-99%.

Protein or peptide fusion is a technique that is frequently used in molecular biology. It is usually related to the production of proteins or peptides in living systems, such as, but not limited to, bacteria, yeasts, or mammalian cells. The inclusion of the coding sequence of interest in phase, respecting the reading pattern, allows the production of a chimeric protein or peptide.

A fusion or chimeric peptide, such as that of the present invention, is easily purified, using the characteristics of the proteins and peptides to bind to a chromatographic matrix, or to precipitate under certain conditions. Purification of a fusion peptide can be accomplished by, for example, but not limited to, sepharose-glutathione columns. The chemical synthesis of small-sized polypeptides is known to those skilled in the art and can be carried out according to conventional methods known in the state of the art, such as by solid phase peptide synthesis methods (Stewart and Young, 1984; Bodanzsky and Bodanzsky, 1994; Lloyd-Williams et al., 1997), solution synthesis, a combination of solid phase and solution synthesis methods or enzymatic synthesis (Kullmann, 1980). The term "homology", as used herein, refers to the similarity between two structures due to a common evolutionary ancestry, and more specifically to the similarity or identity between nucleotides of equivalent positions in two or more polynucleotides.

The term "identity", as used herein, refers to the proportion of identical amino acids between two polypeptides that are compared. Sequence comparison methods are known in the state of the art, and include, but are not limited to, the BLASTP or BLASTN, ClustalW and FASTA program. Since two proteins are considered homologous if they have the same evolutionary origin, in general, it is assumed that similarity or identity values greater than 60% indicate homologous structures. We can consider, therefore, that identity percentages of at least 80% will maintain the same properties of said polypeptide.

In addition, with the information provided, one skilled in the art is able to combine the mutations to generate new variants of the neuroprotective peptide with similar or improved activity. One possibility is the conservative substitution of amino acids; Thus, for example, conservative substitution is understood as one that maintains the polarity and charge characteristics of the substituted amino acid. For example, lysine and arginine are amino acids whose side chains are positively charged at neutral pH, so it is accepted that changes of lysine by arginine or vice versa represent conservative changes. The 20 amino acids that constitute the basis of all natural proteins have been classified according to their conservativity into groups: (i) aromatic amino acids (phenylalanine, tyrosine, tryptophan); (ii) aliphatic amino acids (glycine, alanine, valine, leucine, isoleucine and methionine); (iii) basic ionizable amino acids (histidine, lysine and arginine); (iv) ionizable amino acids acids (aspartic and glutamic acids); (v) acid amino acid amides (asparagine and glutamine); and (vi) hydroxylated amino acids (serine and threonine). Some authors would include cysteine in this last group.

Another object of the invention relates to an isolated nucleotide sequence, hereafter referred to as the "nucleotide sequence of the invention", which codes for the peptide of the invention, or to the nucleotide sequence complementary to said sequence. nucleotide, without limiting it to other nucleotide sequences that could be generated by the degeneracy of the genetic code and give rise to the same polypeptide sequence. The sequences SEQ ID NO: 26 and SEQ ID NO: 27 describe the nucleotide sequences from which the amino acid sequences SEQ ID NO: 1, and SEQ ID NO: 6, respectively, are derived.

The terms "nucleotide sequence", "nucleotide sequence", "nucleic acid", "oligonucleotide" and "polynucleotide" interchangeably refer to a polymer composed of a multiplicity of nucleotide units (deoxyribonucleotides or ribonucleotides or related structural variants or synthetic analogs thereof) linked through phosphodiester bonds (or related structural variants or synthetic analogs thereof). The term polynucleotide includes genomic DNA or double or single stranded coding DNA, RNA, any synthetic and genetically manipulated polynucleotide and both both the coding chain and the antisense (although only the coding chain is highlighted herein). This includes single and double stranded molecules, such as DNA-DNA, DNA-RNA and RNA-RNA hybrids.

The nucleotide sequence of the invention can be obtained artificially by conventional cloning and selection methods widely known in the state of the art.

The nucleotide sequence of the invention, in addition to the coding sequence, can carry other elements, such as, but not limited to, introns, non-coding sequences at the 5 'or 3' ends, ribosome binding sites, stabilizing sequences, sequences of cell detection, etc. These polynucleotides can additionally also include coding sequences for additional amino acids that may be useful, for example, but not limited, to increase the stability of the peptide generated from it or to allow a better purification thereof.

The polynucleotide sequences of the invention can be found as part of vectors that allow their multiplication or cloning as well as their expression. Said vector may be, for example a cloning vector or an expression vector or a recombinant vector. The cloning of the nucleotide sequence of the invention can be perform using an expression or recombinant vector, or a plasmid. These vectors comprise the nucleotide sequence of the invention or, where appropriate, the polynucleotide sequence encoding a carrier protein and the nucleotide sequence of the invention, and a series of amino acid coding sequences that are protease cleavage targets. The advantage of this structure is that, once the fusion protein or peptide is produced, the expression system is lysed and purified by, for example, although without limitation, affinity chromatography, the carrier protein of the peptide of the invention can be cleaved by digestion with a protease and repurifying the peptide of the invention by the same chromatographic system.

The term "vector", as used herein, refers to a nucleic acid molecule that is capable of transferring nucleic acid sequences contained therein to the cell that transduces and is produced by means of techniques. of molecular biology. Some examples of recombinant vectors are linear DNA, plasmid DNA, modified viruses, adenoviruses / adeno-associated viruses, retroviral and viral vectors, etc .; all of them widely described in the literature and that can be used following standard molecular biology techniques or purchased from suppliers.

Vectors or plasmids can be introduced, for example, but not limited to, by transfection, transformation or infection of host cells, such as, but not limited to, plant, mammalian, bacterial, yeast or insect cells. The introduction of the expression vector into the host cell can be carried out by any of the physical or biological methods to give rise to transformed or transfected cells. Such biological methods include, but are not limited to, the use of viral DNA and RNA vectors. The main advantage of physical methods is that they are not associated with oncogenic or pathological virus processes. However, physical methods are less accurate and often result in insertions of multiple copies, random integrations, disruption of own and foreign genetic sequences, as well as unpredictable expression. Among the most used viral vectors for the introduction of genes into mammalian cells, there are vectors of poxvirus, herpes simplex, adenovirus, vectors associated with adenovirus, etc. Therefore, another embodiment of the invention relates to an expression vector or a recombinant vector, hereinafter "vector of the invention", comprising the nucleotide sequences of the invention SEQ ID NO: 26 and / or SEQ ID NO : 27. The term "cloning vector", as used in the present description, refers to a DNA molecule in which another DNA fragment can be integrated, without losing the ability to replicate. Examples of expression vectors are, but are not limited to, plasmids, cosmids, DNA phages or artificial yeast chromosomes.

The term "expression vector", as used herein, refers to a cloning vector suitable for expressing a nucleic acid that has been cloned therein after being introduced into a cell, called a host cell. Said nucleic acid is generally operatively linked to control sequences.

The term "recombinant vector", as used herein, refers to a vector suitable for expressing a nucleic acid that has been cloned therein, so that the expression of the neuroprotective peptide is performed directly in the tissue. or target cell. Generally said vector is a virus, and is produced by the union of different fragments of nucleic acids from different sources and whose expression gives rise to a viral particle with infective capacity characteristically composed of protein capsid, viral genome and proteins associated with the viral genome. ; This virus is designed in such a way that it allows the expression in the target tissue or cell of the nucleotide or peptide sequences of interest. Expression in a tissue or cell of interest is carried out by the operative binding of the polynucleotide to control sequences, preferably specific control sequences of the tissue or cell where it is to be expressed; preferably said control sequences are promoters or enhancers of the CNS. A recombinant vector according to the invention can therefore be used both as a biotechnological tool to multiply the virus and be used in pharmaceutical compositions as a pharmacological treatment per se. A typical recombinant vector is selected from the group consisting of a lentiviral vector, an adenoviral vector and / or an adeno-associated virus vector. The term "expression" refers to the process by which a polypeptide is synthesized from a polynucleotide. It includes transcription of the polynucleotide into a messenger RNA (mRNA) and the translation of said mRNA into a protein or a polypeptide. Expression can take place in a host cell, but also by any process of protein expression in vivo.

The term "host cell" or "host cell", as used herein, refers to any prokaryotic or eukaryotic organism that is the recipient of an expression vector, cloning or any other DNA molecule.

As used herein, a "host cell" or "host cell" includes any cultivable cell that can be modified by introducing non-naturally occurring DNA into the cell, hereinafter "host cell of the invention".

Preferably, a host cell is one in which the polynucleotide of the invention can be expressed, giving rise to a stable polypeptide, post-translationally modified and located in the appropriate subcellular compartment. The choice of a suitable host cell may also be influenced by the choice of the detection signal. For example, the use of constructs with reporter genes (eg, lacZ, luciferase, thymidine kinase or the green fluorescent protein "GFP") may provide a signal to be selected by activating or inhibiting transcription of the gene of interest in response to a transcription regulatory protein. In order to achieve optimal screening or screening, the host cell phenotype should be considered.

A host cell of the present invention includes prokaryotic and eukaryotic cells. Prokaryotes include Gram negative (for example, Escherichia coli) or Gram positive organisms (for example, bacteria of the genus Bacillus). The prokaryotic cells will preferably be used for the propagation of the transcription control sequence of the vector containing the polynucleotide (s) object (s) of the invention, which will allow a greater number of copies of the vector to be achieved. containing the polynucleotide (s) object (s) of the invention. Among suitable prokaryotic host cells for the transformation of this vector are found, for example, but not limited to E. coli, Bacillus subtilis, Salmonella typhimurium, and other species within the genera Pseudomonas, Streptomyces and Staphylococcus. Eukaryotic cells include, among others, yeast cells, plant cells, fungal cells, insect cells, mammalian cells, and parasitic organism cells (eg, Trypanosomas). As used herein, the term "yeast" does not only include yeast in the strict taxonomic sense, that is, unicellular organisms, but also multicellular fungi similar to yeasts or filamentous fungi. Examples of species are Kluyveromyces lactis, Schizosaccharomyces pombe, and Ustilago maydis, with Saccharomyces cerevisiae and Pichia pastoris as preferred organisms. Other yeasts that can be used in the production of the polypeptide sequence (s) of the present invention are Neurospora crassa, Aspergillus niger, Aspergillus nidulans, Candida tropicalis, and Hansenula polymorpha. Culture systems with mammalian host cells include established cell lines such as COS cells, L cells, 3T3 cells, Chinese hamster ovary (CHO) cells, embryonic stem cells, with BHK, HeK or HeLa cells as preferred cells. Eukaryotic cells are preferably used for the expression of the recombinant gene by the application of the transcriptional regulation sequence or the vector of the present invention.

A culture of host cells refers to the process of maintaining and growing host cells. Cell cultures need controlled conditions of temperature, pH, percentages of gases (oxygen and carbon dioxide), as well as the presence of adequate nutrients to allow viability and cell division. Cell cultures can be grown on solid substrates such as agar, or in a liquid medium, allowing large numbers of suspended cells to be cultured. The choice of one cell type and another for the propagation of the vector of the invention depends on its characteristics, as is known in the state of the art. In addition, when the vector of the invention is a virus, viral cultures also require host cells that provide the cellular and metabolic machinery they lack. This allows the virus not only to be maintained but also to multiply so that the expression of the polynucleotide of the invention in said cell culture can be used to propagate the vector of the invention. Thus, another object of the invention is the cell that contains at least one of the polynucleotides of the invention, hereafter referred to as the "host cell of the invention". A particular embodiment of the invention relates to the host cell of the invention containing at least one of the polynucleotides of the invention SEQ ID NO: 26 or SEQ ID NO: 27.

Thus, another embodiment of the invention relates to the use of the host cell of the invention to obtain the neuroprotective peptide of the invention, to reproduce and maintain the polynucleotide of the invention and / or to obtain the vector of the invention.

Methods for obtaining the neuroprotective polypeptide of the invention are known to the person skilled in the art. These comprise both the culture methods of the host cell of the invention and subsequent purification of the peptide, as well as chemical synthesis methods described above.

The term "purify" as used in the description refers to the isolation and concentration of the polypeptide of the invention with respect to the rest of polypeptides present in the culture medium and the host cell of the invention. The isolation of the polypeptide of the invention can be carried out by differential solubility, chromatography, electrophoresis or isoelectric focusing techniques. Chromatography techniques can be based on molecular weight, ionic charge (based on the ionization state of amino acids in working conditions), protein affinity for certain chromatographic matrices or columns, or by purification labels, and can be done in column, on paper or on plate. Protein isolation can be carried out, for example, by precipitation with ammonium sulfate, fast liquid chromatography (FPLC) or "High Performance Liquid Chromatography" (HPLC). , using automated systems that significantly reduce the purification time and increase the purification performance.

The term "purification tag" or "affinity tag", as used herein, refers to an amino acid sequence that has been incorporated (generally, by genetic engineering) into a protein to facilitate its purification. The tag, which can be another protein or a short amino acid sequence, allows the protein to be purified, for example, by affinity chromatography. Purification tags known in the state of the art are, for example, but not limited to, calmodulin-binding peptide (CBP), glutathione-S-transferase enzyme (GST) or a tail of histidine residues.

Another embodiment of the invention relates to the use of the first host cell of the invention for obtaining the polypeptide of the invention. Preferably, the host cell of the invention is a bacterium, more preferably Escherichia coli.

 Another object of the invention relates to a pharmaceutical composition or medicament, hereinafter "pharmaceutical composition of the invention", comprising at least one of the following: i. the neuroprotective peptide of the invention,

 I. the polynucleotide of the invention,

 Neither. The vector of the invention. In a more preferred embodiment, the pharmaceutical composition comprises the neuroprotective peptide of the invention, and more preferably comprises peptides with sequence SEQ ID NO: 1 and / or SEQ ID NO: 6.

Another embodiment is the use of the neuroprotective peptide of the invention in the manufacture of a pharmaceutical composition or medicament.

In another more preferred embodiment, the pharmaceutical composition comprises the vector of the invention, such that the peptide of the invention can be expressed directly in the desired tissues and / or target cells. In an even more preferred embodiment, the vector of the invention is a lentiviral vector, an adenoviral vector and / or an adeno-associated virus vector.

Another embodiment is the use of the vector of the invention in the manufacture of a pharmaceutical composition or medicament. The present invention also refers to the neuroprotective peptide of the invention, as well as the polynucleotide that encodes it and the vector that expresses it, for use in medicine. Likewise, the present invention also refers to the use of the neuroprotective peptide object of the invention, as well as to the polynucleotide of the invention, the vector of the invention and / or the host cell of the invention for the preparation or manufacture of a pharmaceutical composition. or medication

In the present invention, the term: "medicament or pharmaceutical composition", as used refers to any substance used for prevention, relief, treatment and / or cure of diseases in man and / or animals. In the context of the present invention it refers to a pharmaceutical composition or a medicament characterized by comprising the peptide of the invention or the polynucleotides and vectors that allow its expression in the organism to be treated, so that the neuroprotective peptide exerts its function in the target tissue / cell.

In a preferred embodiment, the pharmaceutical composition or medicament of the invention further comprises a pharmaceutically acceptable carrier or excipient. In a more preferred embodiment, the pharmaceutical composition or medicament of the invention further comprises an adjuvant. In an even more preferred embodiment, the pharmaceutical composition or medicament of the invention further comprises another active ingredient (additional active ingredient).

In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the agent or compound capable of increasing neuronal survival in situations of excitotoxicity, calculated to produce the desired effect and, in general, will be determined, to the case of a therapeutic composition, due to the characteristics of the compounds, the route, form and frequency of administration thereof, and other factors, including the age, condition of the patient, as well as the severity of the alteration or disorder.

The term "excipient" refers to a substance that helps the absorption of the elements of the composition of the invention, stabilizes said elements and activates or aids the preparation of the composition in the sense of giving it consistency or providing flavors that make it nicer. Thus, the excipients could have the function of keeping the ingredients together, such as in the case of starches, sugars or cellulose, the function of sweetening, the function as a dye, the function of protection of the composition, for example, to isolate it from air and / or moisture, the filling function of a tablet, capsule or any other Form of presentation, as for example, is the case of dibasic calcium phosphate, the disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.

The term "vehicle", like the excipient, refers to a substance that is used in the pharmaceutical composition or medicament to dilute any of the components of the present invention comprised therein to a certain volume or weight. The "pharmacologically acceptable carrier" is an inert substance or action analogous to any of the elements of the present invention. The function of the vehicle is to facilitate the incorporation of other elements, allow a better dosage and administration or give consistency and form to the composition. When the form of presentation is liquid, the pharmacologically acceptable carrier is the diluent.

Here, the term "adjuvant" refers to an agent that enhances the neuroprotective effect of the peptide of the invention when it is delivered jointly to it or as part of the same treatment protocol.

In another particular embodiment, said pharmaceutical composition is prepared in the form of a solid form or aqueous suspension, in a pharmaceutically acceptable diluent. The therapeutic composition provided by this invention may be administered by any appropriate route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen. In a particular embodiment, the administration of the therapeutic composition provided by this invention is carried out parenterally, orally, intraperitoneally, subcutaneously, etc. A review of the different pharmaceutical forms of drug administration and of the excipients necessary to obtain them can be found, for example, in the "Galician Pharmacy Treaty", C. Faulí i Trillo, 1993, Luzán 5, SA Ediciones , Madrid. Another embodiment of the present invention is the use of the pharmaceutical composition or medicament of the invention for the prevention and / or treatment of cell death induced by excitotoxicity. "Cell death induced by excitotoxicity" or "excitotoxicity", as described earlier in this invention, is a pathological process by which neurons are damaged and destroyed by overactivations of glutamate excitatory neurotransmitter receptors, such as the NMDA receptor ( in English, N-methyl-D-aspartate receptor) and AMPA receptors (in English, a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptor) and kainate. Excitotoxins such as NMDA and kainic acid that bind to these receptors, as well as high pathological levels of glutamate, can cause excitotoxicity by allowing elevated levels of calcium ions to enter the cell. The entry of Ca ²⁺ into cells activates a series of enzymes, including phospholipases, endonucleases, and proteases such as calpain. These enzymes continue to damage cellular structures such as those that make up the cytoskeleton, membrane and DNA.

A preferred embodiment of the present invention refers to the use of the pharmaceutical composition or medicament of the invention for the prevention and / or treatment of neuronal damage caused by a CNS pathology associated with excitotoxicity, such as cerebrovascular accidents (CVAs). such as ischemic stroke or hemorrhagic stroke), traumatic brain injuries (including neurosurgery injuries), hypoxia, hypoglycemia, epilepsy, CNS tumors and their cancer treatment, chronic central nervous system pathologies associated with an excitotoxicity process (diseases neurodegeneratives such as Alzheimer's, Parkinson's, Huntington and amyotrophic lateral sclerosis or ALS), etc. Excitotoxicity is also critical in eye diseases such as glaucoma, diabetic retinopathy, ischemic optic neuropathy and optic nerve trauma. Preferably, the use of the pharmaceutical composition is to prevent or treat a stroke.

The expression: "cerebrovascular accidents" or "ACVs", as used in the present invention, refers to an event that causes blood flow to a part of the brain to stop or limit causing cell death. LCAs are classified according to the mechanism of blood flow block, so we speak of ischemic stroke when the blood vessel is blocked by the formation of a thrombus (thrombotic stroke) or the thrombus comes from another part of the body (cerebral embolism or embolic stroke). On the other hand, there is talk of hemorrhagic stroke when the interruption of blood flow is due to hemorrhage, caused by the rupture of a blood vessel due to trauma or congenital defects; Congenital defects causing a hemorrhagic stroke can be, among others, the aneurysm (a weak area in the wall of a blood vessel that causes it to bulge or bulge) and arteriovenous malformation or AVM (an abnormal connection between the arteries and veins in the brain) Preferably, stroke is selected from the following: stroke or stroke, transient ischemic attack, intracerebral hemorrhages, including subarachnoid and vascular dementia.

The term "traumatic brain injuries", as used in the present invention, refers to damage caused to the brain caused by a blow or a violent head shake. Lesions are classified as penetrating injuries and closed head injuries. Penetrating injuries are caused by the introduction of a foreign object in the brain (such as a bullet or the scalpel of a neurosurgeon) that causes damage to specific regions of the brain along the path of penetration of the object. Closed head injuries include those resulting from blows to the head, such as a car accident or a fall.

The term "hypoxia", as used in the present invention, refers to that situation in which the organism or a part thereof, such as the CNS, does not receive a sufficient supply of oxygen. It can occur as a result of various circumstances such as ischemia; hypoxemic hypoxia that can be caused by respiratory alkalosis, by the short-circuit ("shunting") of the physiological or pathological blood circulation, and / or by pulmonary ventilation problems due to lung lesions, alteration of the perfusion ventilation ratio ( V / Q ratio) and / or physical circumstances such as exposure to high altitudes (mountaineering) or depths (diving); the alteration of the dissociation rate of 0 ₂ of hemoglobin; anemia carbon monoxide poisoning; cyanide poisoning; and / or the intake of some chemical compounds (such as sodium nitrite) or drugs. The term "hypoglycemia", as used in the present invention, refers to an abnormally low blood glucose concentration, generally associated with alterations and / or loss of consciousness. The normal functioning of the CNS and neurons is dependent on a continuous supply of glucose; If the amount of blood glucose falls the CNS is one of the first organs affected. The set of effects on the brain and the CNS are known as neuroglycopenia and include: reduced mental efficiency (values below 65mg / dl of blood glucose concentration), impaired motor and reasoning abilities ( values below 40mg / dl), seizures, and even coma (values below 10mg / dl). Severe or prolonged hypoglycemia can cause permanent damage to the CNS, which includes impaired cognitive function, motor control, and even consciousness. The causes of hypoglycemia can be multiple, and include, but are not limited to: prolonged voluntary or secondary fasting to a disease (such as diarrhea), hyperinsulinemia (due to a failure in the administration of insulin of for example an individual with diabetes , congenital hyperinsulinemia, insulin-secreting pancreatic tumors, reactive hypoglycemia, idiopathic postprandial syndrome), sepsis, Addison's disease, congenital hypopituitarism, metabolic diseases (glycogen storage disease, fatty acid oxidation diseases, etc.) , the ingestion of certain chemicals (alcohol, propanolol, sulfonylureas, some medications), acquired adrenal insufficiency, etc.

The term "epilepsy", as used in the present invention, refers to a disorder caused by an imbalance in the electrical activity of neurons in some area of the brain. It is characterized by one or several neurological disorders that leave a predisposition in the brain to suffer from recurrent seizures, which usually lead to neurobiological, cognitive and psychological consequences. The role of excitotoxicity-induced death in epilepsy is associated with persistent activation of the glutamate, NMDA and kainate pathways in different epileptic seizures.

The expression "tumors of the central nervous system", as used in the present invention, refers to a mass of transformed cells, with abnormal growth and multiplication located in the brain and / or spinal cord. Its origin may be in the brain cells, in the membranes around the brain (meninges), or in the nerves or glands of the head. The treatment of tumors through the usual treatments (cancer treatment) usually leads to cell death of healthy surrounding tissues, whether it is the use of chemotherapeutics or radiation (by-stander effect).

The expression "chronic pathologies of the central nervous system", as used in the present invention refers to a set of diseases that mainly affect the brain and / or spinal cord in its pathology and whose development over time is prolonged. Among these diseases are neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington or amyotrophic lateral sclerosis (ALS). In all of them, the involvement of the cell survival mechanism based on the BDNF / TrkB signaling pathway, in which the neuroprotective peptide of the invention interferes with increasing the pro-survival response time thereof, has been seen.

The term "glaucoma", as used in the present invention, refers to an ocular pathology that is characterized by the pathological increase in infraocular pressure, for lack of drainage of aqueous humor, and which ultimately results in optic neuropathy. It is characterized by the progressive loss of nerve fibers of the optic nerve and changes in their appearance.

The term "diabetic retinopathy", as used in the present invention, refers to an ocular complication of diabetes that is caused by the deterioration of blood vessels that supply the retina; Damage to the blood vessels can be accompanied by proliferation of fibrous tissue in the retina, which leads to impaired vision.

 The term "ischemic optic neuropathy", as used in the present invention, refers to a sudden loss of central vision, lateral vision or both due to a decrease or interruption of blood flow to the optic nerve.

Another preferred embodiment of the present invention refers to the use of the pharmaceutical composition or medicament of the invention for the prevention and / or treatment of neuronal damage caused by a stroke. In an even more preferred embodiment the LCA is an ischemic stroke. Another preferred embodiment of the present invention refers to the use of the pharmaceutical composition or medicament of the invention for the prevention and / or treatment of neuronal damage caused by a traumatic brain injury.

Another preferred embodiment of the present invention refers to the use of the pharmaceutical composition or medicament of the invention, characterized in that it is used alone or in combination with other medicaments to prevent and / or treat CNS damage caused by exocytotoxicity, and more preferably, to prevent and / or treat ischemic stroke. For example, and in a non-limiting way it can be found in combination with: anticoagulant medications (such as heparin, warfarin, acetylsalicylic acid, clopidogrel, acenocoumarol, dabigatran, rivaroxaban, apixaban, fondaparinux, etc.), antihypertensive medications (such as captopril, enalapril, lisinpril, lisinprin, , ramipril, valsartan, telmisartan, losartan, irbesartan, olmesartan, acetazolamide, vorzolamide, ketoconazole, amiloride, triamterene, spironolactone, canrenoate, eplerenone, mannitol, alprenolol, bucindolol, carteolol, carvedilol, pyrololol, pyrololol, pyrololol, pyrololol , sotalol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, nebivolol, butaxaminam, dihydropyridines (bepridyl, mibefradil, amlodipine, felodipine, nicardipine) and non-dihydropyridisbolic thrombolyroid drugs, etc.) (tPA), lanoteplase, reteplasa, staphylokinase, streptokinase (SK), tenecteplase, urokinase, etc.) Other known peptides with neuroprotective properties, such as Tat-NR2B9c or NA-1, are also included here. In a preferred embodiment, the pharmaceutical composition or medicament of the invention is used in medicine. Preferably, it is used in the prevention and / or treatment of neuronal damage. Even more preferably, said neuronal damage is caused by a CNS pathology associated with excitotoxicity. And more preferably, the excitotoxicity is caused by a stroke, and more preferably even by an ischemic stroke.

Another embodiment of the present invention refers to a method for the prevention and / or treatment of neuronal damage characterized by comprising the administration of a therapeutically effective amount of the pharmaceutical composition of the invention. Even more preferably, said neuronal damage is caused by a CNS pathology associated with excitotoxicity. Preferably, said neuronal damage is caused by a stroke.

The present invention also refers to a method for the prevention and / or treatment of ACVs and CNS diseases associated with excitotoxicity, characterized by comprising the administration to the patient of a therapeutically effective amount of the pharmaceutical composition of the invention.

The present invention also refers to a method for the prevention and / or treatment of a stroke, preferably cerebral ischemia, and / or a traumatic brain or spinal lesion, characterized by understanding the administration to the patient of a therapeutically effective amount of the pharmaceutical composition of the invention. The term "prevention", as understood in the present invention, is to prevent or reduce the occurrence of neuronal damage caused by excitotoxicity. The term "treatment" means combating neuronal damage caused by excitotoxicity, to stabilize the condition of individuals or prevent further damage.

Another embodiment of the present invention refers to a method for preventing and / or treating ischemic damage characterized by comprising the administration of a therapeutically effective amount of the pharmaceutical composition of the invention used alone or in combination with other medicaments to prevent and / or treat damage caused by excitotoxicity, preferably a stroke and more preferably an ischemic stroke; for example, and in a non-limiting way, it can be found in combination with: anticoagulant medications (such as heparin, warfarin, acetylsalicylic acid, clopidogrel, acenocoumarol, dabigatran, rivaroxaban, apixaban, fondaparinux, etc.), antihypertensive medications (such as captopril, enalapril, lislapril, lislapril, lislapril, lislapril, lislapril, lislapril , ramipril, valsartan, telmisartan, losartan, irbesartan, olmesartan, acetazolamide, vorzolamide, ketoconazole, amiloride, triamterene, spironolactone, canrenoate, eplerenone, mannitol, alprenolol, bucindolol, carteolol, carvedilol, pyrololol, pyrololol, pyrololol, pyrololol , sotalol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, nebivolol, butaxaminam, dihydropyridines (bepridyl, mibefradil, amlodipine, felodipine, nicardipine) and no dihydropyridines, etc.), thrombolytic medications (such as tissue plasminogen activator (tPA), lanoteplase, reteplase, staphylokinase, streptokinase (SK), tenecteplase, urokinase, etc.), other peptides with neuroprotective properties known as Tat are also included here. NR2B9c or NA-1.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration and are not intended to be limiting of the present invention.

MODE OF CARRYING OUT THE INVENTION Description of the figures

Fig. 1. Designed Tat peptides. A. Partial sequence of the rat TrkB-FL protein (amino acids 401-821) indicating the position of the residues included in the different designed Tat peptides (gray boxes), as well as their transmembrane region (amino acids 430-453, italic) and the tyrosine kinase domain (amino acids 537-806, bold). This sequence has an identity of 97.6% with respect to the corresponding human sequence (amino acids 402-822) and 100% for residues included in Tat peptides. B. Sequence of designed Tat peptides. The 11 amino acids corresponding to the HIV Tat protein occupy N-terminal positions and are common to all peptides (italic). The sequences corresponding to TrkB-FL occupy C-terminal positions, indicating in each case the positions of the first and last amino acid contained according to the numbering of the rat protein. In the case of TFL ₄₅₇ peptides (with SEQ ID NO 6, corresponding to the binding of HIV TAT47-57 CPP, SEQ ID NO 17, and the human TrkB-FL 458-472 fragment, SEQ ID NO 1) and TFL ₅₄ i (with SEQ ID NO 8, corresponding to the binding of HIV TAT47-57 CPP, SEQ ID NO 17, and the human TrkB-FL 542-556 fragment, SEQ ID NO 3) _, the C-terminal arginine of the Tat sequence is also the first amino acid of the 15 amino acid sequence of TrkB-FL contained therein. TFL ₄₈ 2 peptides (with SEQ ID NO 7, corresponding to the binding of HIV TAT47-57 CPP, SEQ ID NO 17, and the TrkB-FL 483-496 fragment human, SEQ ID NO 2) and TFL ₆ 39 (with SEQ ID NO 9, corresponding to the junction of HIV TAT47-57 CPP, SEQ ID NO 17, and the human TrkB-FL 640-653 fragment, SEQ ID NO 4 ) _, like the previous 25 amino acids, only contain 14 amino acids of TrkB-FL. The amino acids of TFL ₄₅₇ also present in the TrkB-T1 isoform are shown in bold. In the TMyc control peptide (with SEQ ID NO 10, corresponding to the binding of HIV TAT47-57 CPP, SEQ ID NO 17, and the human c-Myc 408-421 fragment, SEQ ID NO 5), the Tat sequences are 408-421 amino acid-bound transcription factor c-Myc. Fig. 2. Permeability of the TMyc peptide in neurons. Primary cultures of rat embryonic cortical neurons of 13 DIVs were incubated for 1 h with the TMyc peptide (15 μΜ) labeled with FITC (fluorescein isothiocyanate) (C and D) and compared with control cultures that did not receive the peptide (A and B). The cells were fixed, permeabilized and analyzed by immunofluorescence with antibodies to the neuronal protein NeuN. The images shown are individual sections of 0.5-1 μηι thickness of confocal microscopy. Scale bar: 10 μΜ.

Fig. 3. Specificity of interference mediated by the TFL ₄₅₇ peptide on the processing of TrkB-FL induced under conditions of excitotoxicity. TO.

Primary cultures of rat cortical neurons of 13 DIVs were pre-incubated for 30 min with the TMyc peptides or the different TFL peptides (25 μΜ) and then treated with NMDA (100 μΜ) and glycine (10 μΜ) for 2 h as indicated . Cultures in which pretreatment with Tat peptides was omitted were used as control. TrkB-FL levels (Mr 145 kDa) were analyzed by immunoblot with the TrkB-ECD antibody, which recognizes an extracellular region common to all TrkB isoforms including truncated forms (trkB; Mr 95 kDa). As a control, we also analyze neuronal specific enolasa (NSE), a neuronal protein that is not processed by calpain. B. Quantification of interference by TFL ₄₅₇ from the processing of TrkB-FL by calpain induced under conditions of excitotoxicity. TrkB-FL levels were determined by densitometric analysis of the bands present in the immunoblots and normalized with respect to those of the NSE protein. The results are represented as a percentage of the value obtained in neurons not preincubated with peptide or treated with NMDA, to which 100% was arbitrarily assigned. Mean values are shown ± sem of 4 Independent experiments and statistical calculations were performed using the ANOVA test followed by a post-hoc HSD Tukey test, comparing the levels obtained in pre-incubated cultures with the different peptides with respect to the corresponding ones not pre-incubated, whether or not treated with NMDA. Only significant differences were found for TFL ₄₅₇ (* p <0.05) but not for the rest of cultures treated with NMDA and pre-incubated with TMyc, TFL ₄₈ 2, TFL ₅₄₁ or TFL ₆ 39 or for those not subjected to excitotoxicity.

Fig. 4. Neuroprotective effect of preincubation with the TFL ₄₅₇ peptide on induced neuronal death under conditions of chronic NMDA treatment.

Primary neuronal cultures of 13 DIVs were pre-incubated for 30 min with the TMyc peptides and: TFL ₄₅₇ (A) _, TFL ₄₈ 2 (B) TFL ₅₄₁ (C) or TFL ₆ 39 (D), (all at concentration: 25 μΜ) and treated with NMDA as before for 0, 2, 4 or 6 h. Neural viability was established by the MTT tetrazolium salt reduction test and the results are presented as relative values with respect to those obtained in control cultures not treated with NMDA, to which a value of 100% was arbitrarily assigned. To establish the contribution of glial cells present in mixed cultures to total cell viability, sister cultures were treated in parallel with NMDA 400 μΜ and glycine 10 μΜ for 24 h, conditions that induce neuronal death in its entirety without affecting the viability of glial cells. Neural viability was calculated by subtracting this absorbance value from those obtained for each of the conditions studied. Mean values ± sem are shown (n = 1 1, TMyc and TFL _457; n = 7, TFL ₅₄₁ and FL ₆ 3g _; n = 3, TFL ₄₈ 2). Statistical calculations were performed using the Kruskal-Wallis test followed by a U-Mann Whitney test, comparing for each of the treatment times with NMDA the neuronal viability of cultures pre-incubated with TMyc or the different TFLs (* p <0.05; ** p <0.01, *** p <0.001).

Fig. 5. TFL ₄₅₇ peptide dose dependence for the effects exerted on TrkB-FL processing and excitotoxicity-induced neuronal death. Rat cortical neurons were pre-incubated as before with TMyc or TFL ₄₅₇ (5, 15 or 25 μΜ) peptides and then treated with NMDA (100 μΜ) and glycine (10 μΜ) for 2 (A) or 4 h (B) . A. Analysis by immunoblot of the TrkB-FL levels with the TrkB-ECD antibody, of the active form of the pro-survival transcription factor CREB by means of a phospho-antibody specific for its Ser 133 (pCREB) and NSE. A culture not treated with peptide or NMDA is shown as control. B. Quantification by MTT assay of neuronal viability in cultures pre-incubated with different concentrations of peptide and treated with NMDA. The results are presented as relative values with respect to those obtained in pre-treated control cultures with an equal concentration of the same peptide but not treated with NMDA, to which a value of 100% was arbitrarily assigned. In all cases, mean values ± sem (n = 6) are shown. Statistical calculations were performed using the Kruskal-Wallis test followed by a Mann Whitney U test, comparing for each of the peptide concentrations the neuronal viability of cultures treated with NMDA and pre-incubated with TMyc or TFL ₄₅₇ (* p <0.05; ** p <0.01, *** p <0.001).

Fig. 6. Kinetics of interference mediated by TFL ₄₅₇ peptide from TrkB-FL processing induced under conditions of excitotoxicity. TO.

Primary neuronal cultures were pre-incubated as before for 30 min with the TMyc or TFL ₄₅₇ peptides (25 μΜ) and then treated with NMDA (100 μΜ) and glycine (10 μΜ) for 0, 2, 4 or 6 h. Protein extracts were analyzed by immunoblot with the TrkB-ECD antibody, or antibodies specific for the TrkB-T1 isoform, pCREB, spectrin and NSE. Quantification of TFL ₄₅₇ interference from TrkB-FL (B) calpain processing and CREB inactivation (C) induced under conditions of excitotoxicity. TrkB-FL and pCREB levels were determined by densitometric analysis of the bands present in the immunoblots and normalized with respect to those of the NSE protein. The results are represented as a percentage of the value obtained in neurons without dealing with NMDA, to which we assign an arbitrary value of 100%. Mean values are shown ± sem of 5 independent experiments and statistical calculations were performed using the Kruskal-Wallis test followed by a Mann Whitney U test, comparing the levels obtained in cultures treated with NMDA and pre-incubated with TMyc or TFL ₄₅₇ (* p <0.05; ** p <0.01, *** p <0.001). Fig. 7. Neuroprotective effect of TFL ₄₅₇ peptide transduction subsequently added to the induction of neuronal death by acute treatment with NMDA. Primary neuronal cultures of 13 DIVs were treated with NMDA (50 μΜ) and glycine (10 μΜ) for 1 h. This medium was then replaced by agonist-free conditioned medium containing the DL-AP-5 NMDAR antagonist (200 μΜ) and the TFL ₄₅₇ or TMyc (15 μΜ) peptides. Neural viability was established as previously by the MTT test 20 h after the change of medium and the results are presented as relative values with respect to those obtained in cultures incubated with TMyc and not treated with NMDA, to which a value of 100% was arbitrarily assigned. Mean values are shown ± sem of 5 independent experiments. Statistical calculations were performed using the unpaired Student's t-test, comparing the viability of cultures treated with NMDA and incubated with TMyc or TFL ₄₅₇ (* p <0.05).

Fig. 8. Comparison of the neuroprotective effect of the TFL ₄₅₇ peptide and calpain inhibitors. Cultures of rat cortical neurons were pre-incubated with calpain inhibitors, calpeptin (Calp) and inhibitor III (Cilll; both at 10 μΜ) as indicated and, 30 min later with TMyc or TFL ₄₅₇ (25 μΜ) peptides. After an additional 30 min, the cultures were treated with NMDA as before for 4 h. Neural viability was established by the MTT test and the results are presented as relative values with respect to those obtained in control cultures pre-incubated with TMyc and treated in the same way but not with NMDA, to which a value of 100% was arbitrarily assigned. . Mean values ± sem (n = 10) are shown. Statistical calculations were performed using the Kruskal-Wallis test followed by a Mann Whitney U test, comparing the neuronal viability of cultures pre-incubated with TMyc and treated with NMDA, pre-incubated or not with calpain inhibitors (* p <0, 05), or that of those pre-incubated with TMyc or TFL ₄₅₇ for each of the conditions (** p <0.01, *** p <0.001).

Examples:

Example 1. Design of a TrkB-FL receptor processing inhibitor peptide

First, a detailed biochemical analysis of the N-terminal fragments of TrkB-FL generated by calpain was carried out in primary cultures of rat cortical neurons subjected to excitotoxicity, both for the endogenous receptor and for the expressed recombinant HA-TrkB-FL protein by neuro-specific lentiviral vectors (Vidaurre et al., 2012). These experiments allowed us to establish that a majority processing sequence in TrkB-FL must be found in the 100 intracellular amino acids closest to its transmembrane region (amino acids 430-453 of the rat sequence; Fig. 1A), overlapping with the onset of the tyrosine kinase domain. The precise location of putative calpain processing sites in their substrates is a complex process since a consensus sequence of recognition for this protease has not been established, which seems to recognize higher order structural elements and has a tendency to process separation zones between different functional domains of their substrates (Tompa et al., 2004). Therefore, the TrkB-FL region mentioned above was then analyzed in silico using different network databases (www.calpain.org, www.dmbr.ugent.be) and the GPS-CDD application (http: // gps.biocuckoo.org/), tools that allow theoretical predictions about possible calpain processing sites in their substrates. Several of the predictive results of greater statistical value were concentrated around amino acids 463, 489-494, 541-546 and 549-554 of TrkB-FL, which suggested that in these sequences a major target of calpain processing could be found . On the other hand, the analysis of the C-terminal fragments of TrkB-FL generated by calpain by in vitro digestions of recombinant forms of proteins expressed in heterologous systems suggested the possible existence of a second processing site in its tyrosine kinase region. In silico analysis of these sequences using the above algorithms indicated amino acids around 638 and 645-650 of TrkB-FL as possible additional targets of calpain processing.

In order to explore these hypotheses, a series of peptides were designed that, by containing the previously established TrkB-FL sequences (Fig. 1A), could specifically interfere with the processing of this protein by calpain. As a method to facilitate the passage of interfering peptides through BHE and the plasma membrane, it was chosen to use peptide transduction for several reasons (Dietz and Bahr, 2004). First, this methodology takes advantage of the fact that the permeability properties of certain proteins, such as the Tat transactivator protein of the human immunodeficiency virus (HIV), are mediated by short sequences generally of a basic nature, such as amino acids 47- 57 (YGRKKRRQRRR, SEQ ID NO 17) in the case of Tat. In addition, as mentioned, these sequences have recently been used successfully as a vehicle to introduce various sequences into neurons, including the C-terminal end of the GluN2B subunit of the NMDAR. These sequences were able to interfere with the interaction of GluN2B with the PSD-95 postsynaptic density scaffolding protein (Tat-NR2B9c or NA-1 peptide) (Aarts et al., 2002); (Cook et al., 2012a, b). The designed Tat peptides, therefore, contain amino acids 457-471 (TFL ₄₅₇ of SEQ ID NO 6, corresponding to the binding of HIV TAT47-57 CPP, SEQ ID NO 17, and the human TrkB-FL 458-472 fragment , SEQ ID NO 1), 482-495 (TFL ₄₈₂ of SEQ ID NO 7, corresponding to the junction of the CPP TAT47-57 of HIV, SEQ ID NO 17, and the human TrkB-FL 483-496 fragment, SEQ ID NO 2), 541-555 (TFL ₅₄ i of SEQ ID NO 8, corresponding to the junction of the CPP TATt47-57 of HIV, SEQ ID NO 17, and the human TrkB-FL 542-556 fragment, SEQ ID NO 3) and 639-652 (TFL ₆₃₉ of SEQ ID NO 9, corresponding to the junction of the HIV TAT47-57 CPP, SEQ ID NO 17, and the human TrkB-FL 640-653 fragment, SEQ ID NO 4) of the TrkB- sequence Rat FL fused with the Tat sequence mentioned above (Fig. 1 B). In the case of the TFL ₄₅₇ peptide, the first 9 amino acids of the TrkB-FL sequence are also present in the truncated isoform of the TrkB-T1 receptor while the 5 C-terminal residues are exclusive to the complete isoform. A peptide of the same size as the previous ones containing the Tat sequence linked to amino acids 408-421 of the transcription factor c-Myc (TMyc with SEQ ID NO 10, corresponding to the binding of the CPP TAT47-57 of HIV, SEQ ID NO 17 , and the human c-Myc 408-421 fragment, SEQ ID NO 5) was synthesized and used as a negative control in the experiments performed.

All TrkB-FL sequences used in the above peptides are completely conserved in the human protein sequence; therefore, the neuroprotective effects of the peptides designed in murine excitotoxicity models would be extrapolar for human cells.

Example 2. The TMycFITC peptide is capable of crossing the neuronal plasma membrane.

Next, the neuronal plasma membrane permeability of those peptides containing the transduction sequences of the Tat protein was confirmed (Dietz and Bahr, 2004). For this, primary cultures of mature 13-day rat cortical neurons in vitro (IVDs) were incubated for 1 h with TMyc (15 μΜ) labeled with FITC (Fig. 2, panels C and D) and compared with untreated control cultures (Fig. 2, panels A and B). Since these cultures consist of a mixed population of neurons and glial cells, we employ immunoflurescence with a specific neuronal antibody (NeuN) to identify this cellular subpopulation. The cells, grown on coverslips, were fixed in a 4% paraformaldehyde solution prepared in 0.1 M PBS for 30 min at room temperature. After blocking for 30 min with a solution of 1% BSA with 0.1% Triton X-100 in PBS, the cells were incubated for 2 h with anti-NeuN antibody diluted in blocking solution. The immunoreactivity was detected with secondary antibodies conjugated with fluorophores, incubated for 1 h at room temperature. The coverslips were mounted with Prolong medium (Invitrogen, Carlsbad, CA, USA) on the slides. By confocal microscopy it was observed that, under these conditions, the TMycFITC peptide was able to cross the membrane of the neurons present in these primary cultures. Example 3. The effects of the TFL ₄₅₇ peptide on the processing of TrkB-FL and neuronal death under conditions of excitotoxicity are specific, depend on the dose of peptide used and follow parallel kinetics.

The effect of the different TFL peptides generated on the processing of TrkB-FL induced by NMDAR overactivation was then analyzed (Fig. 3). Primary cultures of 13 IVDs were incubated with the TMyc, TFL _457, TFL ₄₈ 2, TFL ₅₄₁ or TFL ₆ 39 (25 μΜ) peptides for 30 min prior to their treatment for 2 h with NMDAR agonists, NMDA (100 μΜ) and glycine (10 μΜ). We used as a control cultures in which pretreatment with the peptides was omitted. Using immunoblot, the TrkB-FL levels (Mr 145 kDa) (Fig 3A) were studied using the TrkB-ECD antibody, which recognizes an extracellular region common to all TrkB isoforms including truncated forms (trkB; Mr 95 kDa). As a control, neuronal specific enolasa (NSE), a neuronal protein that is not processed by calpain, was also analyzed. TrkB-FL processing interference was quantified for each of the peptides, showing the results of 4 independent experiments (Fig. 3B). In cultures that did not receive peptide, there was a notable reduction in TrkB-FL levels and an increase in truncated forms (tTrkB) that were induced by excitotoxicity conditions, as previously demonstrated (Vidaurre et al., 2012 ). A similar result was obtained with the TMyc control peptide and with the different TFL peptides, with the exception of TFL ₄₅₇ which greatly reduced Remarkable TrkB-FL processing induced by NMDA treatment. For example, in cells preincubated with TFL ₄₅₇ and subjected to excitotoxicity, TrkB-FL levels only underwent a moderate reduction, reaching values of 80 ± 7% with respect to the same cultures without treating with NMDA, a value significantly higher than that obtained in pre-incubated cells with TMyc (36 ± 1 1%, p <0.5). The treatment with the different peptides did not have a significant effect on the basal levels of TrkB-FL found in the cells not subjected to excitotoxicity conditions. The effect of the generated TFL peptides on neuronal death induced by the excitotoxicity process was then investigated (Fig. 4). For this, the primary cultures were pre-incubated with the TMyc, TFL ₄₅₇ , TFL ₄₈ 2, TFL ₅₄₁ or TFL ₆ 3g peptides and treated chronically with NMDA and glycine as above for 0, 2, 4 or 6 h. Neural viability was established by the MTT tetrazolium salt reduction test and the results of 3-1 independent experiments were represented as relative values compared to those obtained in control cultures treated with the same peptide but not NMDA. As can be seen, cultures pretreated with TFL ₄₅₇ presented a significantly greater neuronal viability compared to those incubated with the control peptide at all times evaluated for treatment with NMDA (Fig. 4A). Thus, for example, the viability of cultures incubated for 2 hours with NMDA in the presence of TMyc was 49 ± 5% compared to untreated cells, while these values reached 80 ± 8% in cultures treated with TFL ₄₅₇ (p <0.01). For the rest of the TFL peptides no significant differences were observed with respect to TMyc (Figs. 4C and D), with the exception of TFL ₄₈ 2 that had a very transient neuroprotective effect (Fig. 4B) that did not correlate with greater stability of TrkB-FL in excitotoxic neurons (Fig. 3). Together, the previous data demonstrated that only the TFL ₄₅₇ peptide had a sustained and significant neuroprotective effect on the excitotoxicity induced by chronic treatment with NMDA, which corresponded with its ability to interfere with the processing of the TrkB-FL protein.

In order to establish the most appropriate treatment conditions with the TFL ₄₅₇ peptide, the primary cultures were then incubated with different concentrations of TMyc or TFL ₄₅₇ (5, 15 or 25 μΜ) for 30 min prior to their treatment with the NMDAR agonists for 2 (Fig. 5A) or 4 (Fig. 5B) h. He Analysis of TrkB-FL levels in cultures pre-incubated with TMyc showed results similar to those above at all peptide concentrations used. In contrast, TFL ₄₅₇ was able to interfere with the processing of TrkB-FL induced by NMDA, modestly at the lowest concentration used and very noticeably for 15 and 25 μΜ (Fig. 5A). A very important additional result was that the stabilization of the active isoform of TrkB mediated by pretreatment with TFL ₄₅₇ resulted in the maintenance under conditions of excitotoxicity of the activity of the pro-survival transcription factor CREB, established by analysis with a phosphorus. specific antibody for its Ser 133 (Fig. 5A). In contrast, NMDA treatment of cultures pre-incubated with the TMyc control peptide resulted in the inactivation of pCREB, as described above (Hardingham et al., 2002). The activation of CREB by phosphorylation in this residue occurs as a consequence of the physiological activation of TrkB receptors and NMDARs in neurons and is an important survival mechanism since pCREB induces the expression, among others, of BDNF (Tao et al., 1998) and TrkB (Deogracias et al., 2004). In contrast, under conditions of excitotoxicity, inactivation of the transcriptional activity of pCREB is a critical factor in the neuronal death process (Hardingham et al., 2002). The effects of TFL ₄₅₇ observed on the activation status of CREB are relevant to its clinical utility since it has previously been shown that the increase in pCREB is a factor that stimulates neurogenesis in the dentate gyrus of adult brain, a repair mechanism. brain that is activated after ischemic damage (Zhu et al., 2004).

The effect of TFL ₄₅₇ on neuronal death induced by the excitotoxicity process at the different peptide concentrations used was then investigated (Fig. 5B). The results of 6 independent experiments of neuronal viability were represented as relative values with respect to those obtained in control cultures treated with peptide but without NMDA. As can be seen, the cultures pretreated with TFL ₄₅₇ presented a significantly greater neuronal viability with respect to those incubated with the control peptide at the three peptide concentrations analyzed, the most significant differences being found for the concentration of 25 μΜ. Thus, for example, the viability of cultures incubated with NMDA in the presence of TMyc was 17 ± 2% compared to untreated cells, while these values reached 50 ± 5% in cultures treated with TFL ₄₅₇ (p <0.001 ). Previous data showed that the TFL ₄₅₇ peptide had a neuroprotective effect significant on the excitotoxicity induced by chronic treatment with NMDA, this being dose dependent. This effect on viability corresponded with the ability of TFL ₄₅₇ to interfere with the processing of the TrkB-FL protein and with the maintenance of pCREB levels, an active form of the pro-survival CREB transcription factor.

In order to delve into the correlation found in the different effects of TFL _457, we analyzed the time course of TrkB-FL processing and CREB inactivation induced by NMDAR overactivation (Fig. 6) and compared them with those previously obtained for neuronal viability (Fig. 4A). For this, the primary cultures were incubated with TMyc or TFL ₄₅₇ (25 μΜ) for 30 min prior to their treatment with NMDA for 0, 2, 4 or 6 h. The analysis of TrkB levels showed, in both cases, the reduction of TrkB-FL and the progressive increase of truncated tTrkB forms from early times of treatment with NMDA (Fig. 6A) (Vidaurre et al., 2012). However, it was observed that in cultures pretreated with the TFL ₄₅₇ peptide, the reduction in TrkB-FL levels after treatment with NMDA was markedly lower than that obtained in the presence of TMyc. The quantification of the results corresponding to 5 independent experiments showed that the differences observed between cultures pretreated with TFL ₄₅₇ and TMyc were statistically significant at 2 and 4 h of treatment with NMDA (Fig. 6B). The effect of TFL ₄₅₇ was specific for the TrkB-FL protein and no differences were found in the processing of other calpain substrates such as spectrin, which resulted in the appearance of 150 and 145 kDa fragments in cultures pretreated with TFL ₄₅₇ or TMyc with similar kinetics. As an additional control, NSE was analyzed, a protein that we already saw is not a calpain substrate nor is it regulated under conditions of excitotoxicity. The stabilization of TrkB-FL in cultures pretreated with TFL ₄₅₇ had as a very important consequence the maintenance under conditions of excitotoxicity of the activity of pCREB (Fig. 6C), a protein below TrkB in the signaling cascades and fundamental for the BDNF / TrkB pro-survival pathway function. In cultures pre-incubated with the control peptide, treatment with NMDA, however, caused the inactivation of pCREB from very early times as described (Hardingham et al., 2002). The effects exerted by TFL ₄₅₇ on TrkB-FL and pCREB showed a kinetics very similar to that previously observed for neuronal viability (Fig. 4A), reinforcing the correlation between both phenomena. Together, the previous data demonstrated that the TFL ₄₅₇ peptide had a specific and significant neuroprotective effect on the excitotoxicity induced by chronic NMDA treatment that corresponded with its ability to interfere with the processing of the TrkB-FL protein and the maintenance of levels of activation of the CREB transcription factor.

Example 4. The TFL ₄₅₇ peptide is also capable of reducing neuronal death induced by acute neuronal damage.

The cellular model used in the previous experiments, in which neurons previously exposed to the neuroprotective peptide are faced with a chronic excitotoxic stimulus, tries to reproduce a clinical situation in which we try to reduce the secondary neuronal death of a patient who has suffered a stroke by Post-ischemia administration of a neuroprotective peptide to protect neurons from ischemic penumbra. However, in order to evaluate in a more global way the neuroprotective potential of the TFL ₄₅₇ peptide for clinical use, we also found it interesting to investigate its effectiveness when used immediately after induction of acute neuronal damage, conditions that would simulate some other processes of ischemia produced in vivo as a result of endovascular and cardiac surgeries.

Experiments were performed in which the neurons were subjected only temporarily to the excitotoxic conditions and the peptides were subsequently administered to the induction of acute neuronal damage (Fig. 7). For this, the primary cultures were treated with NMDA (50 μΜ) and glycine (10 μΜ) for 1 h, then replacing this medium with a medium free from the agonists and containing the generic NMDAR antagonist, DL-AP-5 ( 200 μΜ), which allows limiting the over-activation of NMDAR to a short period of time. TFL ₄₅₇ or TMyc (15 μ () peptides were added in the fresh medium together with the DL-AP-5. Neural viability was established as before 20 h after the change of medium and the average values of 5 experiments were represented as relative values to those obtained in the corresponding cultures not treated with NMDA. The relative neuronal viability for cultures treated with TMyc was 36 ± 9%, significantly lower than that obtained in neurons treated with TFL ₄₅₇ , which reached 85 ± 13% (p <0.05). Together, the previous results demonstrate that the TFL ₄₅₇ peptide exerts a significant neuroprotective effect both when it is present in neurons subsequently subjected to chronic excitotoxic damage and if it is administered immediately after acute damage.

Example 5. Inhibition of calpain does not block the neuroprotective effect of the TFL457 peptide in neurons subjected to excitotoxicity.

We have tested the hypothesis that the mechanism of action of the TFL ₄₅₇ peptide on TrkB-FL depends on its inhibition of calpain activity specifically on this substrate by neuroprotection tests performed in the presence of generic inhibitors of this protease (Fig. 8 ). To do this, the primary cultures were pre-incubated with the calpain inhibitors, calpeptin (Calp, 10 μΜ) and inhibitor III (Cilll; 10 μΜ) and, 30 min later, with the TMyc or TFL ₄₅₇ (25 μΜ) peptides. After an additional 30 min, the cells were treated chronically with NMDA (4 h). In control cultures pre-incubated with TMyc we observed a modest but significant effect of calpain inhibitors on neuronal viability (28 ± 3% versus 21 ± 2%, p <0.05). However, the neuroprotective effect of TFL ₄₅₇ was very similar regardless of whether or not treatment with protease inhibitors (48 ± 5% versus 47 ± 3%). Therefore, contrary to the initial hypothesis, the effect of TFL ₄₅₇ on the processing of TrkB-FL induced under conditions of excitotoxicity could be mainly due to the interference of an alternative proteolytic system to calpain that would also be activated in these situations and would participate in the regulation of the TrkB neurotrophin receptor in response to neuronal damage.

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9457.

Claims

1. Peptide with neuroprotective effect against death induced by excitotoxicity characterized by consisting of the sequence SEQ ID NO: 1.

2. Peptide according to claim 1 attached to an internalizing agent.

3. Peptide according to claim 2 characterized in that the internalizing agent is a cellular penetrating peptide, and wherein said cellular penetrating peptide is covalently bound to the peptide consisting of SEQ ID NO: 1.

4. Peptide according to claim 3 characterized in that the sequence of the cell penetrating peptide is TAT47-57 (SEQ ID NO: 17) or TAT48-60 (SEQ ID NO: 16).

5. Peptide according to claim 4 consisting of the sequence SEQ ID NO: 6, wherein the sequence SEQ ID NO: 6 consists of the penetrating peptide TAT47-57 (SEQ ID NO: 17) in which the C-terminal arginine of the sequence SEQ ID NO: 17 is also the first amino acid of the sequence SEQ ID NO: 1.

6. Polynucleotide encoding the peptide according to any one of claims 1 to 5.

7. Polynucleotide according to claim 6 characterized in that it consists of the sequence SEQ ID NO: 26 or SEQ ID NO: 27.

8. Vector comprising the polynucleotide according to any of claims 6 or 7.

9. Cell comprising the polynucleotide according to claims 6 or 7.

10. Use of the cell according to claim 9 for obtaining the peptide according to any of claims 1 to 5.

1 1. Pharmaceutical composition characterized by comprising the neuroprotective peptide according to any one of claims 1 to 5, or the polynucleotide according to any one of claims 6 to 7, or the vector according to claim 8.

12. Pharmaceutical composition according to claim 11 characterized in that it comprises the peptide consisting of the sequence SEQ ID NO: 1 and / or SEQ ID NO: 6.

13. Pharmaceutical composition according to claim 12 characterized in that it comprises the peptide consisting of the sequence SEQ ID NO: 1.

14. Pharmaceutical composition according to claim 12 characterized in that it comprises the peptide consisting of the sequence SEQ ID NO: 6.

15. Pharmaceutical composition according to claim 11 characterized by comprising the vector of the invention according to claim 8.

16. Use of the peptide described in claims 1 to 5 in the manufacture of a pharmaceutical composition or medicament according to claims 1 1 to 14.

17. Use of the vector described in claim 8 in the manufacture of a pharmaceutical composition or medicament according to claim 15.

18. Use according to any of claims 16 or 17 wherein the pharmaceutical composition or medicament is for the prevention and / or treatment of excitotoxicity induced cell death.

19. Use according to claim 18 wherein the pharmaceutical composition is for the prevention and / or treatment of a stroke.

20. Use according to claim 19 wherein the pharmaceutical composition is for the prevention and / or treatment of an ischemic stroke.

21. Use according to claim 18 wherein the pharmaceutical composition is for the prevention and / or treatment of a traumatic brain injury.