CN117642177A

CN117642177A - SCO-spondin derivative polypeptides for enhancing synaptic transmission

Info

Publication number: CN117642177A
Application number: CN202280048609.9A
Authority: CN
Inventors: S·B·A·勒玛查恩特; Y·戈德弗瑞恩; M·C·索里奥克斯
Original assignee: Axoritis Pharmaceuticals
Current assignee: Axoritis Pharmaceuticals
Priority date: 2021-07-09
Filing date: 2022-06-17
Publication date: 2024-03-01

Abstract

The present invention relates to polypeptides derived from SCO-spondin for increasing or enhancing basal excitatory synaptic transmission, in particular glutamatergic neurotransmission. More particularly, the invention relates to the use of said polypeptides in the treatment of disorders including psychotic disorders; drug addiction; viral infections (e.g., coronavirus, such as SARS CoV 2) associated neurological symptoms; NMDA receptor (NMDAr) and/or AMPA receptor (AMPAr) deficiency associated diseases, in particular anti-NMDAr encephalitis; the plant human state increases or enhances glutamatergic neurotransmission in diseases or conditions of hypoxic brain injury. The invention also relates to a method of treatment.

Description

SCO-spondin derivative polypeptides for enhancing synaptic transmission

Background

Disturbance of synaptic physiology may lead to major brain homeostasis defects, compromising execution of neural circuits and advanced brain functions (e.g., cognition and consciousness). Glutamate is the most abundant excitatory neurotransmitter in the human brain and plays a key role in, for example, long-term enhanced synaptic plasticity (Hansen et al, 2017). Synaptic targeted therapies that selectively enhance NMDA and/or AMPA receptor mediated glutamatergic activity in the critical brain circuit may improve brain activity in diseases or conditions in which cognition or consciousness is altered (e.g., mental disorders, drug addiction, viral infections, particularly coronavirus infections and their associated neurological symptoms, NMDAr and/or AMPAr deficiency related diseases, and plant human states).

Impaired glutamatergic neurotransmission is a common feature of many mental diseases (Tang et al 2020). Schizophrenia is a chronic debilitating mental disease that affects about 1% of the world's population. NMDA receptor dysfunction in schizophrenic patients is supported by clinical observations that indicate that administration of NMDA receptor antagonists (phencyclidine (PCP), ketamine) to normal healthy people induces a range of psychotic symptoms and cognitive impairment, similar to that exhibited by schizophrenic patients (Pratt et al, 2017; hashimoto, 2014).

There was also an alteration in hippocampal glutamate function in subjects with bipolar disorder, and a decrease in the number of NMDA receptors with open ion channels in certain areas of the hippocampus was observed (Scarr et al, 2003; chitty et al, 2015).

SARS CoV2 long new patients suffer from adverse neurological effects, including long-term cognitive impairment (Kumar et al, 2021; alnefeesi et al, 2020). Autopsy analysis of brain tissue of the patient with covd-19 revealed the presence of synaptic defects in upper excitatory neurons that are known to play a critical role in cognitive function (Yang et al, 2021). Thus, the neuronal population may be particularly susceptible to defects in neurotransmission by astrocytes and neurons affected by covd-19.

Many drugs, such as cocaine, amphetamines, opioids, alcohol, and cannabis, alter synaptic transmission and induce cognition-related symptoms (Gould, 2010). For example, prolonged abuse of cocaine alters long-term potentiation (Keralapurath et al, 2017) and produces cognitive deficit (jeedema et al, 2021). Similarly, NMDA receptor antagonists (PCP and ketamine) inhibit synaptic transmission and long-term enhancement in the hippocampus when used as drugs or anesthetics, which induce cognitive impairment, particularly working memory deficits (Roussy et al, 2021; medina-Kirchner and Evans,2021; luo et al, 2002; pratt et al, 2017; hashimoto,2014; stringer et al, 1983). Withdrawal of opioids (morphine and heroin) alters synaptic function and long-term enhancement, resulting in cognitive inflexibility (Gould, 2010; pu et al, 2002).

anti-NMDA receptor encephalitis is an autoimmune disease characterized by autoantibodies that target NMDAr present in the brain that significantly alter glutamatergic synaptic transmission (Wagnon et al 2020; tang et al 2020; finke et al 2012). anti-NMDAr encephalitis may occur in coronavirus infection-related diseases (Sarigecili et al, 2021;Alvarez Bravo and Ramio Torrenta,2020) and autoimmune autism (Tzang et al, 2019). This involves the detection of diseases and conditions that are anti-NMDAr autoantibodies, such as stroke (Stanca et al, 2015), schizophrenia (Pearlman and Najjar, 2014), major depression, dementia (Doss et al, 2014), age-related cognitive decline (Busse et al, 2014).

Botanic status is defined as a strong decrease in neural circuit activity involving consciousness due to traumatic and non-traumatic conditions. Thalamus plays a central role in the integration and transmission of neural information between subcortical and cortical areas. Changes in synaptic strength and plasticity of thalamocortical projections represent a major limitation of conscious restoration in plant human state patients (Bagnat et al, 2013; pistonia et al, 2010).

Hypoxia is a common condition in which some tissues of the body are hypoxic. Such lack of adequate oxygenation may have a significant impact on the entire affected tissue. Notably, insufficient brain oxygenation can lead to inhibition of synaptic transmission (commonly referred to as "hypoxia-induced synaptic transmission inhibition"), whereas prolonged exposure to hypoxia leads to neuronal cell loss and death. Cerebral hypoxia may thus lead to hypoxic brain damage if left untreated or untreated.

Disruption of synaptic function represents a major determinant of most neurodegenerative diseases, CNS injury and mental disease. Disturbances in synaptic physiology may imbalance brain homeostasis, compromising the functional integrity of neural circuits and the performance of advanced brain functions such as cognition and consciousness. Glutamate plays a key role in synaptic plasticity such as long-term potentiation (LTP) [ Hansen 2017 ]]. Selective enhancement of alpha-amino-3-hydroxy-5-methyl-4-iso in critical neural networksSynaptic targeted therapies for the transmission of glutamatergic mediated by the oxazolopropionate receptor (AMPAR) and/or the N-methyl-D-aspartate receptor (NMDAR) may thus improve diseases or conditions in which cognition or consciousness is altered (e.g. psychotic disorders [ Hashimoto 2014,Tang2020,Balu 2016,Nazakawa 2020)]Drug addiction [ Luo 2021 ]]Neurodegenerative diseases [ Benitez 2021; conway 2020; m is Milnerwood 2010；Lepeta 2016]Viral infections (especially coronavirus infections and associated neurological symptoms) [ Kumar2021 ]]anti-NMDAR encephalitis [ Finke 2012 ]]Plant human status [ Bagnato 2013]Or aging [ Kumar 2019, shi 2007 ]]) Is a brain activity in the middle (a) and (b).

Methods capable of increasing, enhancing or restoring glutamatergic neurotransmission would be highly beneficial to patients suffering from conditions associated with the above-described therapeutic settings.

SCO-spondin derived peptides have been described for their neuroprotective and neuroregenerative properties. Treatment of spinal cord injury and tauopathies with SCO-spondin-derived peptides has been studied in animal models. However, to date, their ability to improve glutamatergic synaptic transmission has not been demonstrated.

Summary of The Invention

The present invention relates to the effect of peptides on synaptic function/dysfunction. By using electrophysiology on mouse brain slices, we describe that NX218 enhances excitatory postsynaptic currents and increases basal synaptic transmission through AMPAR and GluN 2A-containing NMDAR (GluN 2A-NMDAR). Thus, a single acute systemic administration of NX218 improved spatial working memory in a pharmacological model of mice with NMDAR-induced synaptic dysfunction blocked by phencyclidine (PCP). In addition, repeated daily administrations of this peptide increased GluN2A-NMDAR protein levels and reversed the reduction in PCP-induced NMDAR drive signaling (phosphorylating cAMP response element binding protein; pCREB), which also restored memory.

Thus, the present inventors have unexpectedly identified novel properties of SCO-spondin-derived peptides. The present invention relates to SCO-spondin derived peptides or pharmaceutical compositions containing at least one such peptide for increasing or enhancing synaptic transmission, in particular basal excitatory synaptic transmission, in particular glutamatergic neurotransmission. As known and explained herein, impaired synaptic transmission, e.g. glutamatergic transmission, is a common feature of conditions or diseases such as schizophrenia, drug addiction (in particular drug addiction resulting from PCP, ketamine or scopolamine), NMDAr and/or AMPAr deficiency related diseases (in particular anti-NMDAr encephalitis), plant human status, inhibition of synaptic transmission caused by hypoxia, hypoxic brain damage. These conditions or diseases are pathological conditions encompassed by the therapeutic uses and corresponding methods of treatment.

Such effects on synaptic transmission or neurotransmission may be obtained on existing, fully functional synapses and/or on existing synapses whose function is impaired or inhibited. Peptides may augment, reconstitute or protect basal excitatory synaptic transmission, in particular glutamatergic neurotransmission. As explained below, this is more specifically NMDAr-related glutamatergic neurotransmission, in particular with the GluN2A subunit (GluN 2A-NMDAr neurotransmission) and/or with AMPAR (AMPAR neurotransmission). More precisely, it acts to increase or potentiate the intensity of neurotransmission in different neural circuits (i.e. hippocampal or thalamocortical synapses) by increasing the GluN2A-NMDAR and AMPAR excitatory postsynaptic currents.

In one aspect, the peptide induces an increase or enhancement in basal excitatory synaptic transmission (i.e., propagation of a signal from a presynaptic neuron to a postsynaptic neuron). Thus, when the peptide is administered, the postsynaptic response caused by the electrical stimulation of the presynaptic terminal increases.

In one aspect, the peptide more specifically induces an increase, enhancement or restoration of glutamatergic neurotransmission. Thus, the glutamatergic response recorded in postsynaptic neurons is increased by peptide application relative to the response recorded in the same neurons prior to peptide application. In particular, the magnitude of these postsynaptic currents mediated by glutamate receptors increases as shown in the examples of NMDAr postsynaptic current (EPSC) magnitude and AMPAr EPSC magnitude: NMDAr post-synaptic current (EPSC) amplitude: the peptide was 101pA when present, and 53pA before exposure; AMPAr EPSC amplitude: the peptide was 163pA in the presence of the peptide, and 142pA before exposure of the peptide. Such increased synaptic inputs may allow for recovery of the generation of action potentials in post-synaptic neurons. Such increased synaptic inputs will make the post-synaptic neurons more likely to trigger action potentials.

In one aspect, the increase or enhancement of NMDAr-related neurotransmission by the peptide is at least specifically induced by GluN2A subunits. When GluN2A current was isolated using a GluN2B subunit antagonist, it was shown that the addition of peptide significantly increased the magnitude of NMDAr current (GluN 2A EPSC magnitude (% of control): 79% GluN2B antagonist, 91% GluN2B antagonist and peptide, see examples). Thus, the GluN2A subunit is involved in peptide-mediated increase in NMDAr current magnitude. In contrast, when GluN2B current was isolated using a GluN2A subunit antagonist, it was shown that the addition of peptide had no significant effect on NMDAr current. Thus, the GluN2B subunit is not involved in peptide-mediated increase in NMDAr current magnitude. In addition, the computer docking method demonstrated that the peptide specifically binds to the GluN2A subunit of NMDAr and AMPA receptor.

Neurotransmission or synaptic transmission is the process by which one neuron communicates chemically with another neuron, which encodes information in the form of electrical pulses called action potentials. Once the action potential reaches the end of the neuron axon, it needs to be transferred to another neuron or tissue. To achieve this, it must span the synaptic cleft between the presynaptic neuron and the postsynaptic neuron. At the end of the presynaptic neuron axon terminal is a synaptic vesicle that contains chemical messengers, known as neurotransmitters. When presynaptic action potentials reach these synaptic vesicles, they release the contents of their neurotransmitters. Neurotransmitters then carry signals across the synaptic cleft. They bind to receptor sites on postsynaptic neurons, thereby completing the synaptic transmission process, resulting in excitatory postsynaptic potentials (EPSPs) (i.e., depolarization of the postsynaptic membrane) and possibly in the emission of postsynaptic action potentials. Among them, glutamate is the most abundant excitatory neurotransmitter in the human brain, and therefore glutamatergic neurotransmission plays a key role in brain activity.

Any alteration or enhancement of synaptic transmission, in particular glutamatergic neurotransmission, can be highlighted using electrophysiological methods. This can be done by recording any changes in the synaptic strength at the glutamatergic synapse measured in response to post-synaptic currents induced by pre-synaptic electrical stimuli (pos-Bennacoeur and Lozovaya, 2017).

By "increasing or enhancing glutamatergic neurotransmission" is meant that when a neuron or synapse is present with a peptide of the invention, the glutamatergic postsynaptic current will have a higher magnitude than expected (or than recorded under control conditions).

The magnitude of the NMDAr postsynaptic current in picoamperes (pA) can be increased, in particular, by at least about 10% to 300% after peptide administration relative to the pre-treatment state or control condition. Preferably, the increase is about 20% to about 40% for AMPA receptor mediated current and about 60% to about 230% for NMDA receptor mediated current.

In fact, the current amplitude (pA) can be measured using electrophysiology from brain slices of adult mice based on known methods, such as those disclosed in examples 4 and 5.

In one aspect of the invention, the peptide or pharmaceutical composition is used for preventing or treating diseases or conditions including psychotic disorders, drug addiction, diseases associated with NMDAr and/or AMPAr deficiency, in particular anti-NMDAr encephalitis, plant human status and hypoxic brain injury. In particular, peptides are beneficial to subjects suffering from or at risk of suffering from one of these diseases by increasing, enhancing or restoring glutamatergic neurotransmission, as disclosed herein. This is also beneficial for normal subjects in which an increase in the neurotransmission is desired. Thus, the peptide allows for improved (e.g., increased, enhanced or restored) glutamatergic neurotransmission, and thus improved function or mental capacity related to synapses in which the peptide increases, reconstructs or protects glutamatergic neurotransmission.

In one aspect, the invention is the use of an SCO-spondin derived peptide or a pharmaceutical composition comprising at least one such peptide for preventing or treating the detrimental effects of hypoxia on synaptic transmission (inhibition of synaptic transmission which manifests as a significant reduction in the slope of fEPSP), particularly in the hippocampus. Cerebral hypoxia may be hypoxia occurring during or at the time of a disease such as ischemic stroke, transient ischemic attacks or any other condition leading to cerebral ischemia, traumatic brain injury, cardiac arrest or other cardiac problems, pulmonary diseases (e.g. chronic obstructive pulmonary disease, emphysema, bronchitis, pneumonia and pulmonary oedema), perinatal Hypoxic Ischemic Encephalopathy (HIE), severe asthma attacks, obstructive sleep apnea, obesity Hypoventilation Syndrome (OHS), anaemia, infectious respiratory diseases (e.g. covd-19 syndrome) and more commonly any acute or chronic respiratory failure leading to chronic or recurrent hypoventilation, and any condition leading to insufficient cerebral oxygenation.

Peptides may allow, inter alia, maintenance of normal synaptic function or near normal, and/or restoration of normal synaptic function or increased synaptic transmission.

In one aspect, the peptides are used to promote excitatory postsynaptic potential (EPSP), in particular to promote recovery of EPSP from inhibition. As presented in examples 7 and 8, this effect has been measured on hippocampal slices and by measuring the field EPSP slope according to a well established method.

In one aspect, the peptide is used to protect and/or rescue synaptic transmission (as indicated by the restoration of the slope of the potential after a field excitatory synapse) when/if synaptic transmission is impaired.

In one aspect, peptides are used to promote better and faster recovery of synaptic transmission when/if synaptic transmission is impaired.

In one aspect, the peptides may be beneficial in preventing the detrimental effects of hypoxia in the brain, particularly those that may cause synaptic transmission (e.g., in the hippocampus), and/or allowing normal synaptic transmission to resume during or after hypoxia.

It was demonstrated herein that peptides as disclosed herein, such as NX218, trigger synaptic transmission via GluN2A-NMDAR and AMPAR and even NMDAR driven signaling, as shown by an increase in pCREB brain content following repeated administration of the peptide in vivo.

In one aspect, the peptide is used to treat or prevent a synaptic disorder.

Peptides such as NX218 as disclosed herein promote AMPAR and GluN2A-NMDAR mediated neurotransmission in brain regions associated with advanced functions (i.e., cortex and hippocampus). Treatment with this peptide resulted in favorable changes in NMDAR-dependent signaling and short term memory as demonstrated by pharmacological mouse models of synaptic dysfunction. Overall, modulation of GluN2A-NMDAR and AMPAR function by the peptides represents a therapeutic opportunity to improve outcome in the elderly and patients with CNS diseases or synaptic diseases with disabling synaptic defects.

Detailed description:

synaptic dysfunction detection:

today, there are several techniques available for assessing human synaptic dysfunction. Some electrophysiological measurements, imaging techniques, and liquid biomarker tests may allow for, among other things, diagnosis of synaptic dysfunction in a patient in a non-invasive manner. The oldest and most well known method is electroencephalography (EEG), which is an electrophysiological monitoring method that records the electrical activity produced by cerebral neurons (Cook et al, 1996). This electrical activity is represented by different EEG bands (called alpha, beta, gamma, theta and delta waves). Human EEG waves are well characterized by different parameter values (primary frequency, voltage and morphology). This enables the medical professional to quickly and easily detect any abnormal brain activity.

Evoked Potentials (EP) may be more useful in clinical medicine in order to more specifically study activity within a particular central nervous system pathway. EP is used to measure electrical activity in certain areas of the brain in response to external stimuli (whether visual, auditory, sensitive or motor). The delay between the stimulus and the recorded electrical response and its magnitude are compared to values typically obtained in healthy subjects. Thus, EP is useful for understanding whether a particular nervous system pathway is not functioning properly.

In addition to Magnetic Resonance Imaging (MRI), EP may be used as such: MRI will detect possible lesions, while EP will provide information about the functional impact of these lesions. More importantly, EP can diagnose dysfunction without any radiological abnormalities.

Recently, some imaging techniques have also been tailored to study synaptic function. This is the case with Positron Emission Tomography (PET) imaging, a quantitative imaging technique that is suitable for providing functional and physiological information throughout a living being. With respect to the brain, novel tracers have been developed to quantify synaptic function in the human brain. The most common to date is [11C ] UCB-J PET, a radiotracer that binds to presynaptic vesicles, allowing detection of loss of connectivity and/or tracking of changes in synaptic function in the brain (Finnmma et al 2016).

Some CSF biomarkers may also be used in dosages to assess synaptic function. In the last decade, significant advances in protein detection methods have enabled accurate quantification of presynaptic and postsynaptic proteins in biological fluids. Up to now, the major synaptic biomarkers used to study synaptic function are growth-related protein 43 (GAP-43), synaptotagmin 25 (SNAP-25), synaptotagmin-1 and neuropeptide (Camporesi et al 2020). Interestingly, many other biomarkers are currently emerging.

SCO-spondin-derived peptides for use and methods disclosed herein:

"SCO-Spondin" is a glycoprotein specific to the central nervous system and is found in all vertebrates, from prechordal animals to humans. It is called an extracellular matrix molecule, secreted by a specific organ (sub-connective organ) located at the top of the third ventricle. It is a large-size molecule. It consists of 4500 amino acids and has a multi-modular organization, including various retained protein modes, including in particular 26 TR or TSR modes. Certain peptides derived from SCO-Spondin starting from TSR mode are known to have biological activity in nerves or nerve cells (described in particular in WO-99/03890).

The "TSR or TR pattern" is a protein domain of about 55-60 residues based on an alignment of the remaining amino acids cysteine, tryptophan and arginine. These patterns were first isolated in TSP-1 (thrombospondin 1), a molecule that interferes with clotting. They are then described in many other molecules, such as SCO-Spondin. In fact, in all the proteins studied so far and previously mentioned, this thrombospondin type 1 unit (TSR) comprises about 55-60 Amino Acids (AA), some of which, such as cysteine (C), tryptophan (W), serine (S), glycine (G), arginine (R) and proline (P), are highly conserved.

SCO-Spondin peptides or peptide compounds are used to carry out the invention (different objects of the invention, e.g. peptides or compositions for use, methods of treatment, use of peptides for manufacturing a medicament, etc.). Pharmaceutical compositions comprising at least one peptide according to the invention and a pharmaceutically acceptable excipient, carrier or diluent are also used.

In particular, the invention uses peptides of the sequence X1-W-S-A1-W-S-A2-C-S-A3-A4-C-G-X2 (SEQ ID NO: 1),

wherein:

a1, A2, A3 and A4 consist of an amino acid sequence of 1 to 5 amino acids,

two cysteines form or do not form disulfide bonds,

x1 and X2 consist of an amino acid sequence of 1 to 6 amino acids; or X1 and X2 are absent;

the N-terminal amino acid may be acetylated (e.g., carrying H ₃ CCOHN-), the C-terminal amino acid may be amidated (e.g., carrying-CONH) ₂ ) Or the N-terminal amino acid may be acetylated and the C-terminal amino acid may be amidated.

In one embodiment, the amino acid sequence of formula SEQ ID NO: in 1, X1 or X2 or both X1 and X2 are absent. In one embodiment, in the absence of X1 and/or X2, the N-terminal W is acetylated and/or the C-terminal G is amidated. Preferably, both X1 and X2 are absent, and the N-terminal W is acetylated, and the C-terminal G is amidated.

In particular, the invention uses peptides of the sequence W-S-A1-W-S-A2-C-S-A3-A4-C-G (SEQ ID NO: 2),

wherein:

a1, A2, A3 and A4 consist of an amino acid sequence of 1 to 5 amino acids,

two cysteines form or do not form disulfide bonds.

In structural formula SEQ ID NO: in one embodiment of 1 and 2, the peptide is a linear peptide, or the cysteines present on peptide structures SEQ ID NO 1 and 2 do not form disulfide bonds (reduced forms).

In a preferred embodiment, the polypeptide appears in the peptide of formula SEQ ID NO: the two cysteines on 1 and 2 form disulfide bonds (oxidized forms).

Preferably, in structural formula SEQ ID NO: in 1 and 2, A1, A2, A3 and/or (preferably, and) A4 preferably consist of 1 or 2 amino acids, more preferably of 1 amino acid.

Preferably, A1 is selected from G, V, S, P and a, more preferably G, S.

Preferably, A2 is selected from G, V, S, P and a, more preferably G, S.

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

Preferably, A4 is selected from S, T, P and a, more preferably S, T.

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

Preferably, A3-A4 is selected from R-S or V-T or R-T.

Preferably, X1, X2, A1, A2, A3 and A4 do not comprise cysteine.

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

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

In one embodiment, SEQ ID NO:1 or 2, A1 and A2 in the peptide of 1 or 2 are independently selected from G and S, and A3-A4 are selected from R-S or V-T or R-T.

In a particular mode, such peptides are further acetylated and/or amidated. In one embodiment, the peptide is a linear peptide, or the cysteine does not form a disulfide bond. In another embodiment, the peptide has two cysteines forming a disulfide bond (C-ring glycosylation). In another embodiment, the peptides used in the present invention or administered to a patient include both oxidized and linear forms of peptides.

For the purposes of the present invention, the term "amino acid" refers to both natural and unnatural amino acids, and the skilled artisan can routinely make amino acid changes, including natural to unnatural changes, while maintaining the function or efficacy of the propeptide. "Natural amino acid" refers to L-amino acids that may be present in a natural protein, i.e., alanine, arginine, asparagine, aspartic acid, cysteine; glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. "unnatural amino acid" refers to the aforementioned D-form amino acids, as well as certain amino acids in homologous form, such as arginine, lysine, phenylalanine, and serine, or leucine or valine in canonical form. The definition also includes other amino acids such as alpha-aminobutyric acid, agmatine, alpha-aminoisobutyric acid, sarcosine, statins, ornithine, deaminated tyrosine. The nomenclature used to describe peptide sequences is the international nomenclature using the one-letter code, with the amino terminus shown on the left and the carboxy terminus shown on the right. Dashes "-" denote common peptide bonds of amino acids of the linker sequence.

In one embodiment, the peptide according to the invention, for example the sequence SEQ ID NO:1-63, including N-terminal acetylation, C-terminal amidation, or both N-terminal acetylation and C-terminal amidation.

In various embodiments, the invention relates to the use of a polypeptide consisting essentially of or consisting of the following amino acid sequence (table a):

in one embodiment, the sequence disclosed in table a SEQ ID NO: the peptides of 3-34 are linear peptides, or the cysteines do not form disulfide bonds (reducing peptides). In another embodiment, the sequence disclosed in table a, supra, SEQ ID NO:3-34 has two cysteines oxidized to form disulfide bonds (oxidized peptides). In another embodiment, the peptides used in the present invention or administered to a patient include both oxidized and linear peptides of the same peptide sequence. In yet another embodiment, the peptide used in the present invention or the peptide administered to a patient comprises a peptide selected from the group consisting of the sequences SEQ ID NO:3-34, wherein the mixture may be a mixture of at least two linear peptides, or a mixture of at least two oxidized peptides, or a mixture of at least one linear peptide and at least one oxidized peptide, e.g., having the same amino acid sequence.

In a preferred embodiment, the peptide consists of the amino acid sequence W-S-G-W-S-S-C-S-R-S-C-G (SEQ ID NO: 3). In one embodiment, the peptide is a linear peptide, or the cysteine does not form a disulfide bond (reduced form, referred to as NX 210). In another embodiment, the peptide has two cysteines oxidized to form a disulfide bond (oxidized form), referred to as NX218. In another embodiment, the peptides used in the present invention or administered to a patient include both oxidized and reduced forms.

In SEQ ID NO: in one embodiment of the peptide of 1,

x1 represents a hydrogen atom or P or A-P or L-A-P or V-L-A-P, and/or

X2 represents a hydrogen atom or L-G-L-I-F.

In various embodiments, the invention thus relates to the use of a polypeptide consisting of or consisting essentially of the following amino acid sequence (table B):

in one embodiment, the sequence disclosed in table B SEQ ID NO:35-63 or the sequence SEQ ID NO: the peptides of 3-63 are linear peptides, or the cysteines do not form disulfide bonds (reducing peptides). In another embodiment, the peptide has two cysteines oxidized to form a disulfide bond (oxidized peptide). In another embodiment, the peptides used in the present invention or administered to a patient by systemic route include both oxidized and linear peptides of the same peptide sequence. In another embodiment, the peptide used in the present invention or the peptide administered to a patient comprises two forms of the same peptide sequence: oxidized peptides and linear peptides. In another embodiment, the peptide used in the present invention or the peptide administered to a patient comprises a mixture of at least two peptides selected from these different peptides of sequence SEQ ID NO:35-63 or 3-63, wherein the mixture may be a mixture of at least two linear peptides, or a mixture of at least two oxidized peptides, or a mixture of at least one linear peptide and at least one oxidized peptide, e.g. having the same amino acid sequence.

The sequence SEQ ID NO: each of the peptides 3-63 may be acetylated, amidated, or both.

The peptides used in the present invention or peptides administered to a patient are defined by their amino acid sequence. The peptide used may be one of the peptides disclosed herein, or a mixture of at least two of the peptides disclosed herein. The mixture also comprises a mixture of linear peptides and oxidized peptides of the same or different amino acid sequences. If 100% pure peptide can be used, the invention may comprise, according to the invention: the peptide has a purity of greater than 80%, preferably 85%, more preferably 90%, even more preferably equal to or greater than 95%, 96%, 97%, 98% or 99%. Conventional purification methods, such as chromatography, may be used to purify the desired peptide compounds.

In one embodiment, the peptide used in the present invention or the peptide administered to the patient does include both oxidized peptide (Op) and linear peptide (Lp), e.g., in similar or dissimilar amounts, e.g., (in digital form%), op:10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, the remainder to 100% being Lp. The combined oxidized peptide and linear peptide may have the same sequence or different sequences. For example, the sequence SEQ ID NO:3 and the linear form of the peptide are so combined (NX 210 and NX 218), for example in the proportions disclosed above. The same applies to any of the peptides of sequences SEQ ID NO. 4-34 and 35-63.

In a general aspect, the peptides and pharmaceutical compositions of the invention are useful for enhancing or restoring excitatory synaptic transmission.

In one aspect, the peptides and pharmaceutical compositions are useful for enhancing basal excitatory synaptic transmission, in particular glutamatergic neurotransmission, more in particular NMDAr-related glutamatergic neurotransmission, in particular with respect to the GluN2A subunit.

In another aspect, the peptides and pharmaceutical compositions are used to enhance or restore excitatory synaptic transmission when/if it is impaired, in particular during or after hypoxia.

In particular aspects, peptides and pharmaceutical compositions:

for the treatment or prevention of schizophrenia,

for the treatment or prevention of bipolar disorders,

for the treatment or prophylaxis of drug addiction, in particular of drug addiction by PCP, ketamine or scopolamine,

for the prophylaxis or treatment of diseases associated with NMDAr and/or AMPAr deficiency, in particular against NMDAr encephalitis,

for the treatment of plant human states,

for preventing and/or treating synaptic transmission inhibition caused by hypoxia,

for preventing and/or treating hypoxic brain injury,

for the treatment or prophylaxis of synaptic defects caused by viral infections, in particular in SARS CoV2 and COVID-19, in particular in patients with long new coronaries,

For increasing, enhancing or restoring excitatory synaptic transmission

For preventing or treating the detrimental effects of hypoxia on excitatory synaptic transmission,

for increasing, enhancing or restoring excitatory synaptic transmission, in particular in the hippocampus, or for preventing or treating the detrimental effects of hypoxia on excitatory synaptic transmission, in particular at the hippocampus,

for increasing the NMDAr and/or AMPAr postsynaptic current (EPSC), in particular the EPSC amplitude,

-and combinations thereof.

Schizophrenia and drug addiction:

in one aspect, the peptide or pharmaceutical composition is for use in preventing or treating schizophrenia. In particular, the peptides are beneficial by increasing or enhancing glutamatergic neurotransmission, as disclosed herein. The peptide increases, reconstructs or protects glutamatergic neurotransmission. As disclosed herein, PCP and scopolamine animal models are useful models related to schizophrenia.

In one aspect, the peptide or pharmaceutical composition is used for preventing or treating drug addiction, for example, drug addiction resulting from PCP or ketamine or scopolamine. In particular, the peptides are beneficial by increasing or enhancing glutamatergic neurotransmission, as disclosed herein. The peptide increases, reconstructs or protects glutamatergic neurotransmission.

It is known that drug substances that significantly alter synaptic transmission by antagonizing NMDA and/or AMPA and acetylcholine (ACh) receptors may benefit from treatment with the peptides disclosed herein. Increasing or enhancing glutamatergic neurotransmission may play a key role in counteracting the cognitive deficit caused by these drugs. These include psychoactive substances such as PCP and ketamine, which are NMDAr antagonists, and anticholinergic agents, also known as tropane alkaloids, such as scopolamine.

Pharmacological blockade of glutamatergic or cholinergic synaptic transmission by PCP or scopolamine, respectively, is also the standard method for testing putative cognitive enhancers and psychoactive compounds.

PCP models are associated with schizophrenia and drug addiction. Administration of the NMDA receptor antagonist phencyclidine (PCP) produces symptoms resembling schizophrenia in healthy volunteers (Domino and Luby, 2012) and is therefore often used to mimic schizophrenia in rodents (Jones et al, 2011). Symptoms of acute and sub-chronic administration of PCP in rodents include positive and negative symptoms (hyperkinesias and social withdrawal, respectively), deficiency in prepulse inhibition, and cognitive impairment (Young et al 2012; jones et al 2011). Blocking NMDA receptor-dependent current-impaired synaptic transmission on postsynaptic neurons using antagonists such as PCP and ketamine (=pcp-like compounds) includes (1) long-term potentiation, especially in the hippocampus, which alters memory (Neill et al, 2010; ingram et al, 2018;Stringer and Guyenet,1983), and (2) altering the mismatch negative wave of automatic auditory change detection (Garrido et al, 2009).

The scopolamine acute model is associated with schizophrenia. Administration of scopolamine, a muscarinic ACh receptor antagonist, resulted in deficits in attention, working memory and learning in healthy volunteers, similar to those exhibited by schizophrenic and demented patients (Tang, 2019;Gilles and Luthringer,2007). The reduction of ACh release from presynaptic neurons at the synapses impairs synaptic transmission, including long-term enhancement, especially in the hippocampus, which alters memory (More et al 2016; hirotsu et al, 1989).

Other applications:

in one aspect, the peptide or pharmaceutical composition is for use in the prevention or treatment of a disorder associated with NMDAr and/or AMPAr deficiency, in particular against NMDAr encephalitis. In particular, peptides are beneficial by increasing or enhancing glutamatergic neurotransmission, as disclosed herein and supported by examples 4 and 5. Peptides increase, reconstitute or protect glutamatergic neurotransmission.

In one aspect, the peptide or pharmaceutical composition is for use in treating a plant human condition. In particular, peptides are beneficial by increasing or enhancing glutamatergic neurotransmission, as disclosed herein and supported in example 3. Peptides increase, reconstitute or protect glutamatergic neurotransmission.

In one aspect, the peptide or pharmaceutical composition is for use in treating hypoxia-induced synaptic transmission inhibition and/or hypoxia-induced brain damage. In particular, these peptides are beneficial by better and faster restoration of excitatory synaptic transmission, as disclosed herein and supported by examples 7 and 8. Peptides increase, reestablish or protect excitatory synaptic transmission.

In one aspect, the peptide or pharmaceutical composition is for use in the treatment of bipolar disorder.

In one aspect, the peptide or pharmaceutical composition is used to treat synaptic defects caused by viral infection, particularly in SARS CoV2 and COVID-19 (particularly in patients with long new crowns).

In one aspect, the peptide or pharmaceutical composition is for use in the treatment or prevention of a synaptic disorder, more particularly a synaptic dysfunction in the synaptic disorder. More precisely, the synaptic disease is a synaptic disease with impaired glutamatergic neurotransmission, in particular in relation to NMDAr and/or AMPAr, as disclosed herein.

In one aspect, the peptide or pharmaceutical composition is used for the treatment or prevention of psychotic disorders such as autism, schizophrenia, bipolar disorder, and depression.

CREB：

The transcription factor c-AMP response element binding protein (CREB) is critical for activity-induced gene expression that mediates memory formation (Silva et al, 1998). The CREB pathway responds to calcium increases caused by neuronal activity.

Abnormal CREB expression was observed in the brains of schizophrenic patients (Wang 2018). Autopsy pathology studies of patients provide evidence. Protein and gene levels of CREB and binding activity of CREB to CRE in the brain of schizophrenic patients are significantly reduced in the cingulate gyrus (Yuan et al, 2010; ren et al, 2014), a complete structure of the limbic system of the brain that is associated with emotion, learning and memory, and is found to be smaller and to have lower neural activity in schizophrenic and bipolar affective patients. Thus, the CREB pathway may represent a promising target for developing innovative interventions for schizophrenia and bipolar disorder.

An increase in phosphorylated CREB (pCREB) in T lymphocytes is significantly correlated with clinical improvement in patients receiving antidepressant drug treatment. In one study, authors focused on patients who received only psychotherapy to rule out direct pharmacological effects. After 6 weeks of psychotherapy, 17 patients responded to the treatment; the pCREB increased significantly after 1 week compared to the no-reaction group.

(Koch，2009)

The serum levels of CREB were lower in post-traumatic stress disorder (PTSD) patients compared to control psychologically healthy subjects. CREB levels in PTSD patients do not vary with wound type. CREB may be involved in the pathophysiology of PTSD (Olam 2019).

Impaired CREB signaling has been well documented in addiction, parkinson's disease, schizophrenia, huntington's disease, hypoxia, preconditioning effects, ischemia, alcoholism, anxiety and depression (Sharma 2020).

The positive effect of the peptides of the invention on CREB is of interest in current therapeutic indications (e.g. schizophrenia, hypoxia, bipolar disorder).

Pharmaceutical composition:

as used herein, a pharmaceutical composition comprises as an active ingredient a peptide or mixture of peptides as described previously, e.g., a peptide of different amino acid composition or a peptide of the same amino acid composition in oxidized and linear form, together with one or more pharmaceutically acceptable carriers, carriers or excipients.

The peptide compounds according to the invention may be used in pharmaceutical compositions or for the manufacture of a medicament for the prevention or treatment of basal excitatory synaptic transmission, in particular glutamatergic neurotransmission, as disclosed herein.

In these compositions or medicaments, the active ingredient may be incorporated into the composition in a variety of forms, i.e. in solution, typically in aqueous solution, or in lyophilized form, or in emulsion or in any other pharmaceutically and physiologically acceptable form suitable for the route of administration.

The route of administration may be a systemic route. Mention may in particular be made of the following injection or route of administration: intravenous, intrathecal, intraperitoneal, intranasal, subcutaneous, intramuscular, sublingual, oral, and combinations thereof.

Administration may also be topical, particularly using an intra-brain route, particularly intra-brain administration.

Compositions containing one or more of the peptides disclosed herein are sterile. These compositions are suitable for administration to deliver the peptide into a patient (e.g., in the blood circulation). Delivery to the patient is to deliver a sufficient amount of peptide, and the sufficient amount is associated with a beneficial effect. As disclosed herein, "pharmacodynamic" may include increasing or enhancing glutamatergic transmission.

In some embodiments, the active ingredient in the pharmaceutical composition comprises (1) a linear peptide as disclosed herein, (2) an oxidized peptide as disclosed herein, (3) NX210, (4) NX218, or (5) a mixture of linear peptide and oxidized peptide, such as in particular NX210 and NX218, in an amount similar or dissimilar to the amount disclosed above.

The active ingredient may be administered to animals and humans in unit administration form as a mixture with conventional pharmaceutical carriers, excipients or vehicles. Suitable unit administration forms include oral access forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols; an implant; subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal and intranasal forms of administration and forms of rectal administration.

Preferably, the pharmaceutical composition comprises a carrier, excipient or vehicle which is pharmaceutically acceptable for liquid formulations capable of being administered, e.g. injected, to deliver the active ingredient in a patient, e.g. in the blood stream. These solutions may in particular be ready-to-use solutions, such as isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts), or dry, in particular lyophilized, compositions, which, as the case may be, allow for the formulation of an applicable solution after addition of sterile water or physiological saline.

Pharmaceutical forms include sterile aqueous solutions or dispersions; the formula comprises sesame oil, peanut oil or water-based propylene glycol; sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy injection is possible. It must remain stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the active polypeptide in the required amount in an appropriate solvent and then filter sterilizing.

After formulation is complete, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. These formulations are readily administered in a variety of dosage forms, such as injectable solutions of the type described above, but drug delivery capsules and the like may also be used.

For example, for proper administration in aqueous solutions, the solution should be buffered appropriately if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for administration such as intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, one of ordinary skill in the art will recognize in light of this disclosure that sterile aqueous media may be used. In addition to the compounds of the invention formulated for administration by injection, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other oral solids; a liposome formulation; a slow-release capsule; and any other form currently in use, and delivers the active ingredient.

One dose of peptide is expressed as the weight of peptide per patient's body weight (kg) and may be in the range of about 1 μg/kg to about 1g/kg, especially in the range of about 10 μg/kg to about 100mg/kg, for example from about 50 μg/kg to about 50mg/kg.

The dosing regimen may include a single administration or repeated administration. According to one embodiment, repeated administration may include administration of a dose of therapy per day, for example, one dose per day or every 2 or 3 days during a treatment period. According to another embodiment, repeated administration may include at least two doses of treatment administered daily, for example 2, 3 or more doses per day over a treatment period. In these embodiments, the treatment period may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 days or more (e.g., up to 6 months). The purpose of the treatment is to "benefit" the patient from such treatment over a "period of time". The "benefit" may include the "efficacy" described above, i.e., the peptide is beneficial to a subject suffering from or at risk of suffering from one of these diseases by increasing, enhancing or restoring glutamatergic neurotransmission. Thus, the peptides allow for improved (increased, re-established or protected) glutamatergic neurotransmission and thus improve the function or mental capacity or cognition of the synapses involved in which the peptides increase, re-establish or protect glutamatergic neurotransmission. This "period of time" may depend on the dosing regimen and the patient itself, e.g., the severity of the disease and the patient's response to the therapeutic dose. "improvement" includes "partial improvement" and "total improvement". When partial recovery of glutamatergic neurotransmission and physiological function is observed in a subject, it is referred to as "partial"; there was a significant improvement in these with respect to the initial state of the subject prior to treatment, but it was still significantly lower than in healthy subjects. "complete recovery" refers to the subject's restoration of glutamatergic neurotransmission and physiological function; or they were not significantly different from healthy subjects.

The treatment method comprises the following steps:

in one aspect, the invention relates to a method of treating a subject in need thereof to enhance basal excitatory synaptic transmission, in particular glutamatergic neurotransmission, the method comprising administering to the subject a therapeutic amount of an SCO-Spondin-derived peptide and a pharmaceutically acceptable vehicle or excipient.

The subject in need thereof may be a subject with reduced basal excitatory synaptic transmission, in particular reduced glutamatergic neurotransmission, in particular NMDAr-related glutamatergic neurotransmission, in particular related to GluN2A subunits. In particular, such reduced transmission or neurotransmission is characterized by a sub-normal postsynaptic current. As explained previously, the enhanced transmission or neurotransmission may be characterized by an increased post-synaptic current magnitude, at least an increased NMDAr post-synaptic current magnitude.

The subject in need thereof may be a subject having normal basal excitatory synaptic transmission, in particular glutamatergic neurotransmission. Enhanced delivery or neurotransmission may be characterized by an increased post-synaptic current magnitude above a basal value, at least an increased post-synaptic current magnitude of NMDAr.

Preferably, the SCO-spondin-derived peptide is selected from the group consisting of the sequences SEQ ID NO:1 or 2. More specifically, the peptide is selected from the group consisting of the sequences SEQ ID NO: 3-63. Preferably, the peptide is NX218.

In one aspect of the invention, the method treats a disease or condition selected from the group consisting of: mental disorders, drug addiction, viral, in particular coronavirus infections and their associated neurological symptoms, diseases associated with NMDAr and/or AMPAr deficiency, plant human status and hypoxic brain injury. In particular, as disclosed herein, peptides are beneficial by increasing, enhancing, or restoring glutamatergic neurotransmission.

In some aspects, the methods treat schizophrenia, drug addiction (e.g., PCP, ketamine, and scopolamine), NMDAr and/or AMPAr deficiency-related diseases, plant human states, as disclosed herein.

In some aspects, the methods prevent and/or treat hypoxia-induced synaptic transmission inhibition or hypoxic brain damage, as disclosed herein.

In one aspect, the invention relates to a method of treating a subject in need thereof to increase, enhance or restore synaptic transmission, in particular synaptic transmission in the hippocampus, the method comprising administering to the subject a therapeutic amount of an SCO-Spondin-derived peptide and a pharmaceutically acceptable vehicle or excipient.

In one aspect, the invention relates to a method of treating a subject in need thereof to prevent or treat the effects of hypoxia on excitatory postsynaptic potentials, particularly in the hippocampus, comprising administering to the subject a therapeutic amount of an SCO-Spondin-derived peptide and a pharmaceutically acceptable vehicle or excipient.

Peptides may allow, inter alia, maintenance of normal synaptic transmission or near normal, and/or restoration of normal synaptic transmission or increase synaptic transmission.

Peptides may allow, inter alia, maintenance of normal or near normal EPSP, and/or restoration of normal EPSP or increase EPSP.

Other features disclosed in this specification are applicable to these methods of treatment. In particular, the description "for use" or "use for" should be regarded as the basis of "method of use".

Further definition:

the administration or use of "a peptide" or "peptides" or "one or more peptides" is a generic term and the present invention encompasses administration or use of one single peptide or more than one single peptide, i.e., administration or use of at least two peptides according to the present disclosure. Thus, in the present disclosure, singular or plural is not limited unless indicated to the contrary, and may include one single peptide or at least two peptides at a time. The same applies to the equivalent word "peptide compound", which may be used interchangeably with "peptide".

"treatment", "treating" or "treatment" refers to the delivery of some peptide compounds according to the invention to a subject. These terms as used herein refer to therapies that aim to slow down (reduce) an undesired physiological condition, disorder or disease, or to achieve a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, modulating, stabilizing, preferably increasing or enhancing glutamatergic neurotransmission; alleviation of symptoms caused by impaired glutamatergic neurotransmission; stabilization (i.e., not worsening) of the state of symptoms, conditions, disorders, or diseases; delaying the onset or slowing the progression of symptoms, conditions, disorders or diseases; improvement in symptoms, conditions, disorders, or disease states; and remission (whether partial or complete), with significant remodeling of glutamatergic neurotransmission, or enhancement or amelioration of a condition, disorder or disease. The terms "treat," "treating" or "treatment" may include preventing, inhibiting, suppressing, ameliorating or completely eliminating symptoms of a disease associated with glutamatergic neurotransmission. Preventing a disease may involve administering a composition of the invention to a subject prior to the onset of symptoms of the disease associated with glutamatergic neurotransmission. Inhibiting symptoms of a disease associated with glutamatergic neurotransmission may involve administering the composition of the invention to a subject after induction of the disease but before its clinical appearance. Inhibiting or ameliorating a disease symptom associated with glutamatergic neurotransmission may involve administering the composition of the invention to a subject after clinical occurrence of the disease symptom.

Unless otherwise defined, "effective amount," "sufficient amount," and the like as "therapeutically effective amount" are used interchangeably herein and refer to the amount of one or more peptides of the invention that is effective over the period of time required to achieve the desired therapeutic result. The effective dosage can be determined by one of skill in the art and can vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the drug to elicit a desired response in the individual. The term as used herein may also refer to an amount effective to cause a desired in vivo effect in a subject. It is within the scope of the present disclosure to administer the peptide multiple times during the course of treatment of the subject. The time at which the dose is administered and used will depend on several factors, such as the therapeutic goal (therapeutic versus prophylactic), the condition of the subject, etc., and can be readily determined by one of skill in the art. A therapeutically effective amount also refers to an amount that therapeutically beneficial effects exceed any toxic or detrimental effects of the substance. "prophylactically effective amount" refers to an effective amount of dosage and period of time required to achieve the desired prophylactic result. Typically, since a prophylactic dose is administered to a subject prior to or during the early stages of the disease, a prophylactically effective amount may be less than a therapeutically effective amount. An "effective amount", "sufficient amount" may also take into account combinations of different peptides, if the amount of peptide is taken alone, and/or in combination with another effective ingredient, for example, the dosage of one or both drugs in the combination may be reduced due to a combined or synergistic effect.

The terms "comprising," "including," "having," "possibly," "containing," and variations thereof as used herein are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of other acts or products (e.g., peptides, compounds, or drugs). The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising" embodiments or elements shown herein, "consisting of" and "consisting essentially of, whether or not explicitly stated.

"patient or subject" refers to an animal, particularly a mammal, including a human. In one embodiment, the subject is a human. In other embodiments, the subject is a large animal or livestock, a pet (e.g., cat, dog), or a sports animal (e.g., horse).

In embodiments of the use or method of treatment of the invention, one or more of the following diseases may be excluded: parkinson's Disease (PD), multiple Sclerosis (MS), muscle diseases, non-brain nervous system injuries such as Spinal Cord Injury (SCI) or Optic Nerve Injury (ONI), tauopathies (i.e. Tau protein positive neurodegenerative diseases including any of Alzheimer's Disease (AD), progressive Supranuclear Palsy (PSP), tau protein positive frontotemporal dementia such as Pick's disease, dementia with lewy bodies, corticobasal degeneration, niemann-Pick type C disease, chronic traumatic encephalopathy including dementia pugilistica, postencephalitis parkinson's disease); alzheimer's Disease (AD) can be excluded alone; progressive Supranuclear Palsy (PSP); tau-positive frontotemporal dementia, such as pick's disease; dementia with lewy bodies; degeneration of cortical basal ganglia; niemann-Pick C disease; chronic traumatic encephalopathy, including dementia of boxers; postencephalitis Parkinson's disease; cerebral ischemia; CNS neuron trauma (i.e., spinal cord or brain injury) pathology; viral neurodegeneration; amyotrophic lateral sclerosis; spinal muscular atrophy; huntington's disease; prion diseases; PSP; multiple system atrophy; adrenoleukodystrophy; down syndrome.

The invention will now be described in more detail using non-limiting examples with reference to the accompanying drawings:

fig. 1: normalized mean slope of fEPSP induced during input-output (I/O) responses in somatosensory cortex following stimulation of thalamoventral basal nuclei in the presence of vehicle and NX218 peptide. Data are expressed as mean ± SEM and analyzed using repeated two-way ANOVA followed byAnd (5) comparing and checking afterwards. Using two-way ANOVA, a statistical increase in the slope of fEPSP was observed following application of NX218 peptide compared to vehicle alone (p=0.0027 between treatment groups), especially for stimulation intensities of 150 (, p=0.049), 200 (, p=0.034), 250 (, p=0.044), 300 (, p=0.016), 350 (, p=0.009), 400 (, p=0.037) and 450 (, p=0.036).

Fig. 2: examples of isolated NMDA excitatory postsynaptic currents induced in the absence and presence of NX 218.

Fig. 3: examples of isolated AMPA excitatory postsynaptic currents induced in the absence and presence of NX 218.

Fig. 4: amplitude of isolated NMDA excitatory postsynaptic current induced in the absence and presence of NX 218.

Fig. 5: amplitude of isolated AMPA excitatory postsynaptic current induced in the absence and presence of NX 218.

Fig. 6: normalized amplitude of isolated NMDA excitatory postsynaptic currents induced before (baseline) and after continuous addition of ifenprodil (3 μm) (GluN 2B antagonist) and NX218 peptide (250 μg/mL). After ifenprodil (3 μm) was applied, NMDA EPSC was significantly reduced in magnitude (absolute:, p <0.0001; normalized:, p <0.0001, rm one-way ANOVA). Addition of NX218 (250 μg/mL) significantly increased NMDA excitatory postsynaptic current (baseline versus ifenprodil+nx218: absolute ns; normalized ns; ifenprodil versus ifenprodil+nx218: absolute: #, p= 0.0529; normalized:, p=0.0263, rm one-way ANOVA).

Fig. 7: normalized amplitude of isolated NMDA excitatory postsynaptic currents induced before (baseline) and after continuous addition of NVP-AAM077 (0.4 μm) (GluN 2B antagonist) and NX218 peptide (250 μg/mL). After application of NVP-AAM077 (0.4 μm), NMDA EPSC was significantly reduced in magnitude (absolute:, p <0.0001; normalized:, p <0.0001, rm one-way ANOVA). The addition of NX218 (250 μg/mL) had no significant effect on EPSC amplitude (baseline versus NVP-AAM077+NX218: absolute: p=0.0002; normalized: p <0.0001; absolute: ns; normalized: ns; RM one-way ANOVA).

Fig. 8: effect of intraperitoneal administration of NX210 or NX218 on scopolamine-induced spatial short-term working memory deficit in mice: y maze motion. NX210 or NX2183 doses (10, 15 and 20 mg/kg) were administered 24 hours prior to the trial. Donepezil (DPZ) (positive control) was administered 1 hour prior to testing. Doses are expressed in mg/kg, n being 6 for each group; * P <0.001, # with respect to Veh/chop group, p <0.001, one-way ANOVA with respect to Veh/Veh group, followed by Dunnett test.

Fig. 9: time course of mean normalized f-EPSP slope caused by Schaffer collateral stimulation during and after hypoxia/glucose deprivation (OGD) in mouse hippocampal slices of perfusion vehicle (n=9) or 250 μg/mL NX218 (n=9). Black circles represent responses in vehicle treated sections (control sections, n=9) before, during and after OGD induction. Gray triangles represent responses in the NX218 treated sections (treated sections, n=9) before, during and after OGD induction.

Fig. 10: bar graph of mean normalized f-EPSP slope caused by Schaffer collateral stimulation during and after hypoxia/glucose deprivation (OGD) in mouse hippocampal slices of perfusion vehicle (n=9) or 250 μg/mL NX218 (n=9). The white bars represent the slope of f-EPSP in vehicle-treated sections (control sections, n=9) before the start of OGD induction (baseline) and after various time points. The shaded bars represent the slope of f-EPSP in the sections treated with NX218 (treated sections, n=9) before the start of OGD induction (baseline) and after various time points. Summary of P values: * p <0.05, < p <0.01, < p <0.001.

Fig. 11: time course of mean normalized f-EPSP slope during and after hypoxia/glucose deprivation (OGD) caused by Schaffer collateral stimulation in mouse hippocampal slices starting with perfused vehicle (n=10) or 250 μg/mL NX218 (n=10) from the end of OGD. Black circles represent responses in vehicle treated sections (control sections, n=10) before, during and after OGD induction. Gray triangles represent responses in the NX218 treated sections (treated sections, n=10) before, during and after OGD induction.

Fig. 12: bar graph of mean normalized f-EPSP slope caused by Schaffer collateral stimulation during and after hypoxia/glucose deprivation (OGD) in mouse hippocampal slices of perfused vehicle (n=10) or 250 μg/mL NX218 (n=10) at the end of OGD. The white bars represent the slope of f-EPSP in vehicle-treated sections (control sections, n=10) before OGD induction starts (baseline) and after various time points. The shaded bars represent the slope of f-EPSP in the sections treated with NX218 (treated sections, n=10) before the start of OGD induction (baseline) and after various time points. Summary of P values: * p <0.05, < p <0.01, < p <0.001.

Fig. 13: time course of mean normalized f-EPSP slope caused by Schaffer collateral stimulation during and after hypoxia/glucose deprivation (OGD) in mouse hippocampal slices perfused with 250 μg/mL NX218 during recovery plateau at t=60 min and t=70 min (each time point n=1) after OGD onset.

Fig. 14: normalized slope of fEPSP induced during I/O recordings in the CA1 region following Schaffer side branch stimulation in hippocampal slices of continuous perfusion vehicle and 250 μg/mL NX 218. Input-output (I/O) curves were obtained by plotting the fEPSP slope for different intensities of presynaptic stimulus (from 0 to 850 μa, step size 50 μa). All data were normalized to the maximum slope of fEPSP recorded under control conditions (vehicle infusion). Data are presented as mean ± SEM and analyzed using two-way ANOVA and subsequent Sidak multiple comparison test. N=10 slices/group. * P <0.01 relative to control. * P <0.05 relative to control.

Fig. 15: spatial memory in vehicle, nicotine (0.4 mg/kg 30 min), NX218 mg/kg 2 hours (D14), NX218 mg/kg 24 hours (D13), NX218 mg/kg48h+24h+2h (D12, D13 and D14 respectively) treated vehicle and sub-chronic PCP mice was assessed as spontaneous alternation of the T maze. Data for all groups are expressed as mean ± s.e.m of n=10, except for NX218 5mg/kg 24h group (n=9), where significant outliers were identified by QuickCalcs. The least significant difference between one-way ANOVA and subsequent Fisher protected was used for pairwise comparison: * P <0.001 relative to sub-chronic PCP. # # relative to control, p <0.0001.* P <0.0001 relative to PCP.

Fig. 16: protein levels of pCREB in cortical samples in mice models of cognitive deficits induced by sub-chronic administration of PCP. Results are expressed as mean +/-SEM (n=4-5) as a percentage of control conditions. One-way ANOVA and subsequent Tukey test (×p <0.05 was considered significant compared to PCP group). # relative to control, p <0.05.* P <0.05 relative to PCP.

Fig. 17: gluN2A levels in cortical samples in a mouse model of cognitive deficit induced by sub-chronic administration of PCP. Results are expressed as mean +/-SEM (n=4-5) as a percentage of control conditions. One-way ANOVA and subsequent Tukey test (×p <0.05 was considered significant compared to PCP group). # relative to control, p <0.05.* P <0.01 relative to PCP.

Example 1: synthesis of NX peptides

The sequence SEQ ID NO: 1. 2 or sequences 3-63, in particular those peptides used in part of the examples, such as NX210 (SEQ ID NO: 3), is based on solid phase peptide synthesis, which uses N-alpha-Fmoc (side chain) protected amino acids as building blocks in peptide assembly. The protocol used involved coupling the C-terminal glycine N- α -Fmoc protected amino acid to the MPPA linker peptide on MBHA resin, followed by Fmoc/deprotection sequence coupling. After assembling the peptide onto the resin, a step of simultaneously cleaving the peptide from the resin and deprotecting the amino acid side chain is performed.

The crude peptide was precipitated, filtered and dried. The peptides were dissolved in an aqueous solution containing acetonitrile prior to purification by preparative reverse phase chromatography. The purified peptide in solution is concentrated prior to the ion exchange step to obtain the peptide in acetate form.

Further details of the synthesis can be obtained by the person skilled in the art with reference to US 6,995,140 and WO2018146283, and the oxidized forms of the peptides disclosed herein with reference to WO 2017/051135, all of which are incorporated herein by reference.

The skilled artisan can also use standard methods to produce any of the disclosed peptides of the invention, including N-terminal and C-terminal modified or protected peptides. Regarding acetylation and/or amidation of peptides at the N-and C-terminus, respectively, the person skilled in the art can refer to standard techniques, for example those described in Biophysical Journal, volume 95, month 11 2008, 4879-4889, which are also incorporated by reference.

Example 2: synthesis of cyclic NX peptides

The polypeptide of sequence W-S-G-W-S-S-C-S-R-S-C-G was added to Human Serum Albumin (HSA) in a ratio of 1:1 and incubated with stirring in air at room temperature for 1 to 3 hours. By using HPLC we observed the formation of peaks corresponding to the polypeptide sequence W-S-G-W-S-S-C-S-R-S-C-G, wherein 2 cysteines are linked by disulfide bonds. After removal of albumin by precipitation, the product was purified and analyzed by HPLC. The use of different proportions of albumin and polypeptide corresponding to the sequence W-S-G-W-S-S-C-S-R-S-C-G may affect the rate of cyclization and the final yield of cyclization, while knowing that smaller amounts of albumin are more readily eliminated. The cyclized compound is NX218.

Further details of the synthesis can be obtained by the person skilled in the art with reference to WO 2017051135. This document is incorporated herein by reference.

Example 3: measurement of the effect of NX218 on synaptic Transmission on mouse brain sections in the thalamus cortical region

The effect of the NX218 peptide on thalamocortical synaptic responses was determined. The cortico field excitatory postsynaptic potential (fEPSP) was recorded in response to ventral basal thalamus stimulation during focal application of NX218 peptide on thalamus radiation on mouse brain sections. Comparison of postsynaptic responses under control versus NX218 peptide conditions was evaluated.

Preparation of mouse brain sections:

5 male mice (C57 Bl 6/J) of 4-5 weeks of age were obtained from Charles River, france and kept in animal facilities. Animal care is in compliance with the recommendations of the french national and local ethics committee. Mice were deeply anesthetized by inhalation of isoflurane and then decapitated. The dissected brain was rapidly placed in a solution containing 214mM sucrose, 2.5mM KCl, 1.25mM NaH ₂ PO ₄ 、26mM NaHCO ₃ 、2mM MgSO ₄ 、2mM CaCl ₂ And 10mM D-glucose (95% O) ₂ /5％ CO ₂ ) In solution.

10 thalamocortical sections (2 sections per mouse) were prepared as described by Agmon and Connors (1991) and Varela et al (2013). After recovery, the brain is placed on a support to raise the tail of the brain until the back makes an angle of 10 degrees with the horizontal plane. Then, a slice was taken at 55 degrees with respect to the midline and the beak was removed. In the slicing chamber, the brain is stuck to the slice surface. Then, the brain was sliced at a thickness of 400 μm. The sections were immediately transferred to a holding chamber containing artificial cerebrospinal fluid (aCSF) consisting of 124mM NaCl, 2.5mM KCl, 1.25mM NaH ₂ PO ₄ 、26mM NaHCO ₃ 、2mM MgSO ₄ 、2mM CaCl ₂ And 10mM D-glucose. The holding chamber was continuously oxygenated and maintained at 35 ℃. After a recovery period of 30 minutes, the sections were incubated at room temperature for at least 30 minutes.

Electrophysiological recording:

for electrophysiological recordings, a single slice is placed in immersion and continuously perfused with aeration (95% O) at a constant rate (2 mL/min) ₂ ，5％CO ₂ ) In the recording chamber (room temperature) of aCSF of (A)And (5) proofing experiments.

Bipolar tungsten stimulation electrodes were placed in the ventral basal nuclei of the thalamus (which contain predominantly glutamatergic neurons and a small number of gabaergic interneurons) and extracellular field potentials in the somatosensory cortex were recorded using glass microelectrodes.

A synaptic transmission input/output (I/O) curve was constructed to evaluate changes in synaptic transmission using a series of stimulus intensities from 0 to 850 ua at intervals of 50 ua. Increasing the stimulation intensity resulted in a linear increase until the maximum plateau under vehicle conditions was reached.

The signal was amplified using an Axopatch 200B amplifier (Molecular Devices, union City, calif.) digitized through a Digidata 1322A interface (Axon Instruments, molecular Devices, USA) and sampled at 10 kHz. Records were obtained using Clampex (Molecular Devices) and analyzed using Clampfit (Molecular Devices). The experimenters of all experiments were blinded to the treatment group.

NX218 drug delivery system:

NX218 or vehicle is delivered to thalamus radiation areas by rapid focal infusion. The system consists of a syringe containing the corresponding solution and tubing fused to a low dead volume manifold mounted on a micromanipulator. The tip mounted on the manifold (300 μm diameter) was located within the required distance of less than 1mm from the target area, with a flow rate of about 0.2mL/min. The tubing line is controlled by an electrically powered pinch valve that opens and closes by transistor-transistor logic signals sent from the DigiData system and parameters set in the priming and recording protocol in clamtex 10.3.

Results:

the data were analyzed by measuring the slope of individual fEPSP using a Clampfit linear fit. The slope of fEPSP was plotted for different stimulus intensities (from 0 to 850. Mu.A). The effect of NX218 was assessed by the change in the slope of fEPSP, expressed as a percentage of the maximum of the I/O curve during vehicle infusion. Table 1 and fig. 1 show the results of 10 validated slices.

Table 1: normalized mean slope of fEPSP induced during input/output (I/O) reactions in somatosensory cortex following stimulation of thalamo ventral basal nuclei in the presence of vehicle and NX218 peptide

In this study, somatosensory cortico fEPSP in response to ventral basal thalamus stimulation was recorded during the application of vehicle and NX218 peptide in ex vivo brain sections of C57Bl6/J mice. A significant increase in the slope of fEPSP was observed after application of NX218 compared to vehicle alone, especially for stimulus intensities of 150 to 450 μΑ (fig. 1). The effect of NX218 on thalamocortical basal synaptic transmission is independent of stimulus intensity.

The results indicate that the NX218 peptide enhances basal synaptic transmission between thalamoventral basal nuclei and somatosensory cortex.

Example 4: influence of NX218 recorded on mouse brain sections in mouse hippocampal CA1 neurons on NMDA-and AMPA-receptor currents

To determine the effect of the NX218 peptide on NMDA or AMPA excitatory postsynaptic current (EPSC), electrophysiological recordings were performed under control conditions or during application of NX218 using whole cell patch clamp methods.

Preparation of mouse brain sections:

12 (C57 Bl 6/J) 4-5 week old male mice were obtained from Charles River, france and kept in an animal facility. Animal care is in compliance with the recommendations of the french national and local ethics committee. Sagittal hippocampal brain sections (about 6 sections per mouse) were obtained using standard brain section methods (Knobloch et al, 2007). Mice were anesthetized with isoflurane and then decapitated. The brain was dissected out of the skull and immediately immersed in ice-cold freshly prepared aCSF containing: 124mM NaCl, 3.75mM KCl, 2mM MgSO ₄ 、2mM CaCl ₂ 、26.5mM NaHCO ₃ 、1.25mM NaH ₂ PO ₄ 10mM glucose, continuously oxygenated (ph=7.4), for a total duration of 3-4 minutes. Acute sections (350 μm thick) were prepared using a vibrating microtome (VT 1000S;Leica Microsystems,Bannockburn,IL). The sections were incubated in standard aCSF for at least 1 hour at room temperature prior to recording.

Electrophysiological recording:

for electrophysiological recording, a single slice is placed in immersion and at a constant rate (2 ml. Min ^-1 ) Continuous infusion aerated (95% O) ₂ ，5％CO ₂ ) In the recording room (room temperature) of the aCSF to alert the experiment.

In the first series of experiments, the GABA receptor antagonist dicranostine (20. Mu.M), α -amino-3-hydroxy-5-methylisoxygen, was addedAzole-4-propionic acid and rhodopsin (AMPA/KA) receptor antagonists 1,2,3, 4-tetrahydro-6-nitro-2, 3-dioxo-benzo [ f]Quinoxaline-7-sulfonamide (NBQX; 10. Mu.M) the NMDAr component of EPSC was isolated. For this series of experiments, low Mg was used ²⁺ (0.1 mM) solution. In order to maintain the same extracellular divalent cation concentration, the perfusion medium contains CaCl ₂ ：3.7mM。

In a second series of experiments, the combination of NMDAr competitive antagonist, phosphoramidate (APV; 20. Mu.M), S) -1- (2-amino-2-carboxyethyl) -3- (2-carboxy) pyrimidine-2, 4-dione rhodophycin receptor antagonist (UBP-302; 10. Mu.M) and GABA receptor antagonists dicentraine (20. Mu.M) separated the AMPAr component of EPSC. All chemicals were from aloone or Tocris Bioscience.

For whole cell recordings of somatic cells, a patch pipette was loaded with a solution containing the following components: 140mM K-gluconate, 5mM NaCl, 2mM MgCl ₂ 10mM HEPES, 0.5mM EGTA, 2mM MgATP, 0.4mM NaGTP, osmotic pressure 305Osm/L, and pH was adjusted to 7.25 with KOH. As previously described (Jaffe and Brown 1994b;Stuart and Sakmann 1994;Stuart et al 1993), using a combination of infrared light and differential interference contrast optics, after recording the visual proximity of the pipette, the cell bodies of large CA1 pyramidal neurons are identified and patch clamped. The resistance of the diaphragm electrode at the time of filling was about 5mΩ. When the series resistance exceeds 40mΩ, recording is terminated. The signal is digitized and low pass filtered at 10 kHz.

EPSC was induced in response to Schaffer collateral stimulation. For the recording of EPSC, the recording was carried out at a specified holding potential (-60 mV) in a voltage clamp. The stimulus intensity was adjusted to elicit EPSCs with acceptable amplitude.

The signal was amplified using an Axopatch 200B amplifier (Molecular Devices, union City, calif.), digitized via a Digidata 1550 interface (Molecular Devices) and sampled with Clampex 10 (Molecular Devices). Records were obtained using Clampex (Molecular Devices) and analyzed using Clampfit (Molecular Devices). One or more cells from each mouse were used and the data averaged as described below. The experimenters of all experiments were blinded to the treatment group.

Experimental timeline and recorded set:

series 1: effect of NX218 peptide on isolated NMDAr currents in hippocampal CA1 pyramidal neurons

A total of 10 validated neurons were contained in series 1.

Under each condition (stable T10 (10 min), NX218 peptide condition T20 (20 min) and flush T30 (30 min)), 10 EPSCs were induced and averaged in response to Schaffer collateral stimulation.

Series 2: effect of NX218 peptide on isolated AMPAr currents in hippocampal CA1 pyramidal neurons

A total of 10 validated neurons were contained in series 2.

Under each condition (stable T10, NX218 peptide condition T20 and flush T30), 10 EPSCs were induced in response to Schaffer collateral stimulation. At T10, T20 and T30, 10 EPSCs were induced and averaged in response to Schaffer side branch stimulation.

Data were analyzed by measuring the amplitude (average of 10 EPSCs) under each condition using Clampfit (Molecular Devices):

stabilization (control)

-NX218 peptide

Flushing out

Data are expressed as mean ± SEM. Statistical comparisons of group averages were performed using Prism8 (Graph Pad) to evaluate significant differences using RM one-way ANOVA followed by Tukey post-hoc testing. Summary of P values: * p <0.05, < p <0.01, < p <0.001. Data charts were drawn using Prism8 (Graph Pad).

Results:

the results are shown in fig. 2, 3, 4 and 5 and in tables 2 and 3 below.

Examples of traces of isolated NMDA and AMPA excitatory postsynaptic currents induced in the absence and presence of NX218 are shown in figures 2 and 3, respectively.

NX218 treatment electrophysiological assessment of induced isolated NMDA excitatory postsynaptic currents (series 1)

Table 2: amplitude of isolated NMDA excitatory postsynaptic currents induced in the absence and presence of NX 218.

	Control	NX218 250μg/mL	Flushing
				Average value of	53.1145899	101.4928607	90.1073967
SEM	5.20352103	24.42031133	14.9938705
				N	10	10	10

NX218 significantly increased the magnitude of induced isolated NMDA excitatory postsynaptic currents (x, p=0.0347, rm one-way ANOVA followed by Tukey post-hoc test). After 10 minutes of flushing, this effect is not reversed (fig. 4).

NX218 treatment electrophysiological assessment of induced isolated AMPA excitatory postsynaptic currents (series 2)

Table 3: amplitude of isolated AMPA excitatory postsynaptic currents induced in the absence and presence of NX 218.

	Control	NX218 250μg/mL	Flushing
				Average value of	141.690904	163.0687985	152.965846
SEM	8.16972957	7.392187323	9.2606305
				N	10	10	10

NX218 significantly increased the magnitude of induced isolated AMPA excitatory postsynaptic currents (×p=0.0226, rm one-way ANOVA followed by Tukey post-hoc test). After 10 minutes of flushing, this effect is not reversed (fig. 5).

Conclusion:

in this study, induced NMDA and AMPA excitatory postsynaptic currents (EPSCs) of mouse hippocampal CA1 neurons in response to Schaffer collateral stimulation were recorded under control conditions and during application of NX218 peptide.

The use of the NX218 peptide significantly increased NMDA-and AMPA-EPSC. After 10 minutes of flushing, this increase was not reversed.

Example 5: gluN2 subunit involved in NX218 determination of the effect on NMDA receptor currents recorded in mouse hippocampal CA1 neurons

To determine which GluN2 subunit was involved in the increasing effect of NX218 of NMDAR excitatory postsynaptic current (EPSC), a whole cell patch clamp method was used. The experiments were performed in the absence or presence of 250 μg/mL NX218 and after 10 minutes of flushing. Selective antagonists of GluN2A and GluN2B subunits are also used.

Preparation of mouse brain sections:

12 (C57 Bl 6/J) 4-5 week old male mice were obtained from Charles River, france and kept in an animal facility. Animal care is in compliance with the recommendations of the french national and local ethics committee. Sagittal hippocampal brain sections (about 6 sections per mouse) were obtained using standard brain section methods (Knobloch et al, 2007). Mice were anesthetized with isoflurane and then decapitated. The brain was dissected out of the skull and immediately immersed in ice-cold freshly prepared aCSF containing: 124mM NaCl, 3.75mM KCl, 2mM MgSO ₄ 、2mM CaCl ₂ 、26.5mM NaHCO ₃ 、1.25mM NaH ₂ PO ₄ 10mM glucose, continuously oxygenated (ph=7.4), for a total duration of 3-4 minutes. Preparation of acute cuts using a vibrating microtome (VT 1000S;Leica Microsystems,Bannockburn,IL)Sheet (350 μm thick). The sections were incubated in standard aCSF for at least 1 hour at room temperature prior to recording.

Electrophysiological recording:

for electrophysiological recordings, a single slice is placed in immersion and continuously perfused with aeration (95% O) at a constant rate (2 mL/min) ₂ ，5％CO ₂ ) In the recording room (room temperature) of the aCSF to alert the experiment.

The NMDAR component of EPSC is prepared by adding GABA receptor antagonist dicranostine (20 μm), a-amino-3-hydroxy-5-methylisoxymethylAzole-4-propionic acid and rhodopsin (AMPA/KA) receptor antagonists 1,2,3, 4-tetrahydro-6-nitro-2, 3-dioxo-benzo [ f]Quinoxaline-7-sulfonamide (NBQX; 10. Mu.M) was isolated. Using low Mg ²⁺ (0.1 mM) solution. To maintain the same extracellular divalent cation concentration, the perfusion medium contained 3.7mM CaCl ₂ 。

The experiment was performed in 3 stages as follows:

stage 0 of the study was intended to show the effect of selective continuous blocking of GluN2A and GluN2B subunits on NMDA EPSC. First, the GluN2A subunit was blocked by adding the GluN2A antagonist NVP-AAM077 (PEAQX tetrasodium hydrate) (0.4. Mu.M) (Li et al, 2007). NVP-AAM077 is a relatively selective GluN1/GluN2A antagonist that exhibits a 100-fold preferential blocking of GluN1/GluN2A compared to GluN1/GluN2B (Auberson et al, 2002). Concomitant blockade of the GluN2B subunit was then achieved using the putative GluN2B antagonist ifenprodil tartrate (3 μm) (Li et al, 2007). Ifenprodil is one of the most selective GluN2B antagonists, with a preference for GluN1/GluN2B over 200-fold compared to GluN1/GluN2A (Williams 1993).

For phase 1 of the study (evaluation of GluN2A subunit participation), gluN 2A-mediated EPSCs were isolated by addition of GluN2B antagonist ifenprodil (3. Mu.M) prior to (T10-T20) and during (T20-T30) infusion of the NX218 peptide (250. Mu.g/mL).

For phase 2 of the study (evaluation of GluN2B subunit participation), gluN 2B-mediated EPSCs were isolated by addition of GluN2A antagonist NVP-AAM077 (0.4. Mu.M) before (T10-T20) and during (T20-T30) perfusion of the NX218 peptide (250. Mu.g/mL).

All chemicals were obtained from aloone Labs or Tocris Bioscience (see appendix 5).

For whole cell recordings of somatic cells, a patch pipette was loaded with a solution containing the following components: 140mM K-gluconate, 5mM NaCl, 2mM MgCl ₂ 10mM HEPES, 0.5mM EGTA, 2mM MgATP, 0.4mM NaGTP, osmotic pressure 305mOsm/L, pH was adjusted to 7.25 with KOH. As previously described (Jaffe and Brown 1994b;Stuart and Sakmann 1994;Stuart et al, 1993), a combination of infrared light and Differential Interference Contrast (DIC) optics was used to identify and patch clamp the cell bodies of large CA1 pyramidal neurons after visual access to the pipette was recorded. The resistance of the diaphragm electrode at the time of filling was about 5mΩ. When the series resistance exceeds 40mΩ, recording is terminated. The signal is digitized and low pass filtered at 10 kHz.

Induced post-synaptic current (EPSC) is induced in response to Schaffer collateral stimulation using bipolar electrodes. To record EPSC, experiments were performed at the indicated holding potential (-60 mV) in a voltage clamp. The liquid junction potential is corrected before recording is performed.

The stimulation duration was 0.1ms and the stimulation intensity was adjusted to induce EPSC with acceptable amplitude (amplitude range-40 pA).

The signal was amplified using an Axopatch 200B amplifier (Molecular Devices, unionCity, calif.) and digitized via a Digidata 1550 interface (Molecular Devices). Records were obtained using clampox 10 (Molecular Devices) and analyzed using Clampfit (Molecular Devices). Two cells from each mouse were used and the data averaged. The experimenters of all experiments were blinded to the treatment group.

A total of 12 mice were used for this study. In total 23 neurons were recorded as described below (10 per group (stage 1 and 2), 3 neurons were recorded for the control stage (stage 0). During each recording, 10 EPSCs were induced in response to Schaffer side branch stimulation. Measurements of amplitude (average of 10 EPCS) were evaluated. Stage 0 represents the validation stage, aiming to confirm that GluN2A and GluN2B are the main NR2 subunits of EPSCs involved in Schaffer side branch stimulation. We expect that the residual current at the end of stage 0 is minimal.

Results:

the results are shown in tables 4 and 5 below and in fig. 6 and 7.

Continuous application of NVP-AAM077 and ifenprodil electrophysiological assessment of isolated NMDA excitatory postsynaptic currents induced in hippocampal CA1 pyramidal neurons

The use of NVP-AAM077 (0.4. Mu.M) and NVP-AAM077 (0.4. Mu.M) +ifenprodil (3. Mu.M) significantly reduced the NMDA EPSC amplitude (data not shown).

Evaluation of the effect of GluN2A subunit on isolated induced NMDAR currents in CA1 hippocampal neurons by involvement of the NX218 peptide observed (GluN 2B subunit blocked with ifenprodil)

Table 4: normalized amplitude of isolated NMDA excitatory postsynaptic currents induced before (control) and after continuous addition of ifenprodil (3 μm) (GluN 2B antagonist) and NX218 peptide (250 μg/mL).

	Control	Ifenprodil	ifenprodil+NX 218 250 μg/mL
				Average value of	100	78.74	90.55
SEM		2.78	4.20
				N	10	10	10

Evaluation of GluN2B subunit involvement observed influence of NX218 peptide on isolated induced NMDAR currents in CA1 hippocampal neurons (GluN 2A subunit blocked with NVP-AAM 077)

Table 5: normalized amplitude of isolated NMDA excitatory postsynaptic currents induced before (control) and after serial addition of NVP-AAM077 (0.4 μm) (GluN 2B antagonist) and NX218 peptide (250 μg/mL).

	Control	NVP-AAM077	NVP-AAM077+NX218 250μg/mL
				Average value of	100	56.35	63.69
SEM		2.94	4.39
				N	10	10	10

Conclusion:

in this study, pharmacological tools were used to isolate postsynaptic currents mediated by NMDAR containing GluN2A and GluN 2B. Isolated induced NMDAR currents in response to Schaffer collateral stimulation in mouse hippocampal CA1 neurons under control conditions and during the application of 250 μg/mL of NX218 peptide with selective antagonists of GluN2A and GluN2B subunits were recorded using the whole cell patch clamp method. The objective was to determine which GluN2 subunit was involved in the increasing role of NX218 for NMDAR EPSC current.

In this study, we observed that:

after continued application of NVP-AAM077 (0.4 μm) and ifenprodil (3 μm), NMDA EPSC amplitude was significantly reduced, indicating that excitatory postsynaptic currents are mediated primarily by ionic glutamate receptors.

Blocking GluN2B subunit with ifenprodil (3 μm) followed by addition of NX218 (250 μg/mL) significantly increased NMDA excitatory postsynaptic current. Thus, the GluN2A subunit is involved in the increasing role of NX218 for NMDAR EPSC current.

Adding NX218 (250 μg/mL) after blocking GluN2A subunit with NVP-AAM077 (0.4 μΜ) had no significant effect on NMDAR EPSC current. Thus, gluN2B subunit is not involved in the increasing role of NX218 for NMDAR EPSC current.

Example 6: evaluation of the role of NX210 and NX218 in scopolamine mouse amnesia model

To evaluate the effect of NX210 and its circulating form NX218 on scopolamine-induced learning and memory impairment, a Y maze cognitive test was used in a scopolamine mouse amnesia model.

The method comprises the following steps:

male Swiss mice (weighing 30-35 g) from JANVIER (Saint Berchemin, france) were housed in groups, and food and water were available ad libitum except during behavioral experiments. They were kept in temperature and humidity controlled animal facilities with a light/dark cycle of 12 hours/12 hours (07:00 lights off at night). The mice were numbered by marking their tails with permanent markers. All animal procedures strictly adhere to the EU directive (2010/63/UE) at 9 and 22 days of 2010. The treatment protocol was the same in each cage (n=6 per cage). Animals were tested in a randomized and blind fashion.

The NX210 and NX218 compounds were dissolved in water for injection (vehicle) and administered intraperitoneally 1, 2, 24, or 48 hours prior to Y Maze (YM) testing.

Donepezil (DPZ) used as a positive control was orally administered at a dose of 1mg/kg 1 hour prior to the YM test.

Scopolamine was subcutaneously injected 30 minutes before YM onset at a dose of 0.5mg/kg.

After administration of the compounds, all animals were tested for spontaneous alternating manifestations in YM (which is an index of spatial working memory) at different time points. YM was designed according to Itoh and its co-workers (1993) and Hiramatsu and Inoue (1999) and was made of grey polyvinyl chloride. Each arm is 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at the top, and converging at equal angles. Each mouse was placed at the end of one arm and allowed to move freely in the maze over a period of 8 minutes. A series of arm entries were visually inspected, including the case of a possible return to the same arm. Alternation is defined as the continuous entry into all three arms. Thus, the maximum number of alternations is the total number of arm entries minus two, and the percentage of alternations is calculated as (actual alternations/maximum alternations) x100. Parameters include alternating percentages (called memory index) and total number of arm entries (called exploration index) (Maurice et al, 1996,1988; meunier et al, 2006; villard et al, 2009,2011).

Animals exhibiting extreme behavior (alternating percentage <20% or >90% or number of arm entries < 10) will be excluded from the calculation. Accordingly, no animals were discarded.

Results:

results of administration of either NX210 or NX218 24 hours prior to YM testing (doses of 10, 15 or 20 mg/kg) are shown in Table 6 and FIG. 8. The value is spontaneous alternation%.

Table 6: the effect of intraperitoneal administration of NX210 or NX218 on scopolamine-induced spatial short-term working memory deficit in mice was studied using YM cognitive testing. 3 doses (10, 15 and 20 mg/kg) of NX210 or NX218 were administered 24 hours prior to testing. Donepezil (DPZ) (positive control) was administered 1 hour prior to testing.

Conclusion:

scopolamine is a cholinergic blocker that induces a amnestic effect on spatial short-term working memory as highlighted in YM.

Donepezil as a positive control significantly reversed the defect in alternation in YM when administered 1 hour prior to behavioural testing.

When administered once at different doses 24 hours (24 hour pretreatment) prior to YM testing, NX210 and NX218 peptides exhibited dose-response effects in preventing spatial short-term working memory deficits caused by acute scopolamine administration.

The beneficial effects of test compound NX210 were demonstrated starting at a dose of 10mg/kg, showing the greatest reversal of the spatial short-term working memory impairment at a dose of 15mg/kg, followed by a decrease but still significant at a dose of 20mg/kg (bell curve). No effect was shown when the dose of NX210 was below 10mg/kg or administered 2 hours or 1 hour prior to YM testing (data not shown). The positive effect of NX210 was maintained, but less pronounced (data not shown) when administered once 48 hours prior to YM testing, and only at the highest dose (15 mg/kg).

When administered 24 hours prior to YM testing, 5mg/kg of test compound NX218 showed a complete reversal of spatial short-term working memory impairment (data not shown). This recovery was only partial when applied 2 hours prior to testing, but not when applied 1 hour prior to YM testing (data not shown). At 24 hours prior to this cognitive test, the lowest test dose (i.e., 2.5 mg/kg) has shown efficacy, with the greatest effect observed at 10 mg/kg. As demonstrated by the YM test (bell-shaped dose response curve), the two highest doses of 15 and 20mg/kg only partially blocked scopolamine induced memory defects. At 48 hours prior to testing, NX218 at 5 and 15mg/kg partially blocked scopolamine effects in YM tests, while the 10mg/kg dose completely offset scopolamine-induced short-term memory defects (data not shown).

Example 7: assessment of the influence of NX218 on functional recovery in an in vitro hypoxia model

Acute hypoxia may lead to inhibition of synaptic activity in many brain regions. In the previous examples, we show that NX218 (250 μg/mL) acts through the GluN2A subunit to increase NMDA-mediated current amplitude in CA1 hippocampal neurons following Schaffer collateral stimulation. The aim of this study was to determine whether the NX218 peptide could improve functional recovery in an in vitro hypoxia model (H dou et al, 2008; farrinelli et al, 2012). To achieve this goal, functional recovery was assessed by recording evoked field excitatory postsynaptic potentials (fepsps) in mouse hippocampal neurons before and after oxygen-glucose deprivation (OGD). To evaluate the effect of NX218, experiments were performed on different sections of either NX218 or vehicle of interest.

Experiment design:

the method comprises the following steps:

animals

Experiments were performed using 4-5 week old male C57Bl6/J mice (about 20 grams) obtained from Charles River, france. A total of 9 mice were used in this study. Two sections were recorded from each mouse (one for control conditions and the other for NX218 conditions). Animals were acclimatized to laboratory feeding conditions for 13 days prior to experimental use. Standard foods (type a04, SAFE, france) are available at will. The filtered main reference water (0.22. Mu.M) was available ad libitum.

Preparation of mouse brain slices

Sagittal hippocampal brain sections (approximately 6 sections per mouse) were obtained using standard brain slice methods (Knobloch et al, 2007). Mice were anesthetized with 5% isoflurane and then decapitated. The brain was dissected out of the skull and immediately immersed in ice-cold freshly prepared artificial cerebrospinal fluid (aCSF), which contains: 124mM NaCl, 3.75mM KCl, 2mM MgSO ₄ 、2mM CaCl ₂ 、26.5mM NaHCO ₃ 、1.25mM NaH ₂ PO ₄ 10mM glucose, continuously oxygenated (95% O) ₂ ，5％CO ₂ ) (ph=7.4) for a total duration of 3-4 minutes. Acute sections (350 μm thick) were prepared using a vibrating microtome (VT 1000S;Leica Microsystems,Bannockburn,IL). Prior to recording, the sections were incubated with standard aCSF (124 mM NaCl, 3.75mM KCl, 2mM MgSO) ₄ 、2mM CaCl ₂ 、26.5mM NaHCO ₃ 、1.25mM NaH ₂ PO ₄ 10mM glucose) at room temperature for at least 1 hour.

Electrophysiological recording

For electrophysiological recording, a single slice is placed in immersion and at a constant rate (2 mL min ^-1 ) Continuous infusion of aerated aCSF (95% O) ₂ 、5％CO ₂ The method comprises the steps of carrying out a first treatment on the surface of the ph=7.4) to alert the experiment. Extracellular field excitatory postsynaptic potential (fEPSP) in the CA1 radiological layer was recorded using a glass micropipette filled with aCSF. fEPSP is prepared by electrically stimulating the Schaffer collateral ligation pathway at 0.1Hz (i.e., one pulse per 10 seconds) with a glass stimulation electrode (borosilicate capillary glass with filaments, standard wall; OD:1.5mm; ID:0.86mm; length: 75mm; ref: W3 30-0060, from Harvard Apparatus) placed in the radiation layerInduced.

At the beginning of each experiment (when a stable fEPSP response is reached), an input/output (I/O) curve is obtained by gradually increasing the stimulus intensity (at intervals from 0 to 100. Mu.A, 10. Mu.A) to determine the intensity of the stimulus that is retained to alert the experiment. A stable baseline fEPSP was then recorded over 10 minutes by stimulation at 80% of the maximum field amplitude (one pulse every 10 seconds, i.e. 0.1 Hz).

Recordings were made in hippocampal slices of mice perfused with vehicle (WFI) or NX218 (250. Mu.g/mL) before and after OGD (10-15 minutes). The field excitatory postsynaptic potential (fEPSP) induced after oxygen-glucose deprivation (OGD) was measured continuously for 90 minutes from the start of OGD induction.

The signal was amplified using an Axopatch 200B amplifier (Molecular Devices, unionCity, CA), digitized via Digidata 1322A interface (Axon Instruments, molecular Devices, USA), and sampled at 10 kHz. Records were obtained using Clampex (Molecular Devices) and analyzed using Clampfit (Molecular Devices).

The experimenter was blinded to the treatment of all experiments. To ensure strict comparability of the two sets of data, two sections were used per mouse: one served as a control slice and the other served as a treatment slice.

Oxygen-glucose deprivation

After 10 minutes of stabilization of baseline, glucose was depleted by perfusion and with 95% N ₂ 、5％CO ₂ Aerated sucrose aCSF (0 mM glucose, replaced with equimolar sucrose) acute sections were exposed to 10-15 min hypoxic/glycemic conditions. OGD was applied in the sections until the f-EPSP slope reached a value representing at least less than 10% of the average baseline value. At this point we believe that the effects of OGD have been induced. In this study, the average OGD duration was 9.6±2.8 minutes.

Data processing and statistical analysis

The data were analyzed by measuring the magnitude of f-EPSP under each condition using Clampfit (Molecular Devices). Two-way ANOVA statistical test was performed using Prism 8 (Graph Pad), followed by Multiple comparison tests to assess significant differences. Summary of P values: * P is p<0.05，**p<0.01，***p<0.001. All data are expressed as mean ± SEM (standard error of mean) except for OGD mean duration, which is expressed as mean ± SD (standard deviation).

Results

Electrophysiological evaluation of the effect of NX218 on functional recovery in an in vitro hypoxic mouse model:

the results are shown in FIG. 9, table 7 and FIG. 10.

Table 7: effect of NX218 perfusion on mean normalized f-EPSP slope before (baseline) and at different time points after the start of OGD induction.

Conclusion(s)

In this study, we observed that NX218 treatment improved functional recovery after acute hypoxia in vitro. Indeed, significant synaptic inhibition (i.e., inhibition of the fEPSP slope) occurred at the Schaffer collateral CA1 synapses following OGD induction in hippocampal slices. Unexpectedly, treatment with NX218 (250 μg/mL) at various time points after the start of OGD induction statistically increased recovery after OGD in hippocampal slices: as early as 30-60 minutes after OGD induction, the fEPSP slope in the NX218 treated sections was significantly higher than in vehicle treated sections (fEPSP slope expressed as% of baseline: NX218 61.7±5.0 compared to vehicle 47.1±4.5; p=0.045). This benefit was more pronounced at 60-90 minutes (88.0±2.6 for NX218 versus 69.6±3.5 for vehicle; p < 0.001) and continued until the last 10 minutes after OGD induction (92.6±2.4 for NX218 versus 72.4±2.9 for vehicle; p < 0.001).

Example 8: assessment of the influence of NX218 on functional recovery in an in vitro hypoxia model

In an in vitro model of hypoxia (H dou et al, 2008; farrinelli et al, 2012), we have previously shown in example 7 that NX218 (250 μg/mL) promotes recovery of synaptic transmission when added from the onset of oxygen/glucose deprivation (OGD). The purpose of this study was to determine if this beneficial effect on post-hypoxia recovery could be maintained even with the later addition of NX218 (after OGD has ended). Two additional sections were also recorded to assess the potential therapeutic capacity of NX218 for hypoxia. For this purpose, NX218 is added during the recovery plateau after OGD (more precisely t=60 min and t=70 min after the start of OGD).

Design of experiment

Method

Animals: see example 7. A total of 10 mice were used in this study. Two additional sections were obtained using additional mice for preliminary recording ("test section").

Preparation of mouse brain sections: see example 7

Electrophysiological recording:

the conditions of example 7 were used except that:

from the end of the OGD and until the end of the experiment, each slice was perfused with vehicle (WFI) or with NX218 at 250 μg/mL.

Measurement of post-OGD induced field excitatory postsynaptic potential (fEPSP) recovery continues for 120 minutes starting from T0 (OGD onset). Two "test slices" were also performed according to the experimental design described above.

Oxygen-glucose deprivation: see example 7. In this study, the average OGD duration was 10.6±1.0 minutes.

Data processing and statistical analysis: see example 7.

Results

the results are shown in FIG. 11, table 8 and FIG. 12.

Table 8: effect of more delayed NX218 perfusion on average normalized f-EPSP slope before (baseline) and at different time points after the start of OGD induction.

Evaluation of efficacy of NX218 on neuronal activity after OGD (preliminary sample study recorded using single slice; n=2):

the results are shown in FIG. 13.

Conclusion(s)

Here we observed that NX218 improved functional recovery after acute hypoxia in vitro, even with more delayed administration of perfusion (hypoxic/non-glycemic condition, average duration = 10.6 minutes; NX218 perfusion starting from the end of OGD). In fact, treatment with NX218 (250 μg/mL) at the end of OGD statistically increased recovery of hippocampal neuronal activity from t=90 minutes (NX 218 88.0±1.9 compared to 77.1±2.7 for vehicle; p=0.004) and continued until the end of the experiment 120 minutes after the start of OGD (NX 218 90.3±2.7 compared to 79.3±2.7 for vehicle; p=0.01).

Two additional sections were made to evaluate the therapeutic ability of NX218 to treat hypoxia. The shape of the recovery phase was changed by further rising toward baseline during the recovery plateau after OGD (t=60 min or t=70 min) with 250 μg/mL NX 218. This preliminary study reveals the potential efficacy of NX218 after hypoxia.

Example 9: effect of NX218 on basal hippocampal synaptic transmission measured on mouse brain slices

To determine the effect of NX218 peptide on basal excitatory synaptic transmission in the CA1 region of the mouse hippocampus, extracellular field excitatory postsynaptic potential (fEPSP) in response to Schaffer collateral stimulation was recorded on a mouse hippocampal slice of infusion vehicle followed by 250 μg/mL NX 218. Comparison of postsynaptic responses under control versus NX218 peptide conditions was evaluated.

Preparation of mouse brain sections:

5 male mice (C57 Bl 6/J) of 4-5 weeks of age were obtained from Charles River, france and kept in animal facilities. Animal care is in compliance with the recommendations of the national and local ethics committee of france. Ten sagittal hippocampal brain sections (2 per mouse) were obtained using standard brain slice methods (Knobloch et al, 2007). Mice were anesthetized with 5% isoflurane and then decapitated. The brain was dissected out of the skull and immediately immersed in ice-cold freshly prepared artificial cerebrospinal fluid (aCSF), which contains: 124mM NaCl, 3.75mM KCl, 2mM MgSO ₄ 、2mM CaCl ₂ 、26.5mM NaHCO ₃ 、1.25mM NaH ₂ PO ₄ 10mM glucose, continuously oxygenated (95% O) ₂ ，5％CO ₂ ) (ph=7.4) for a total duration of 3-4 minutes. Acute sections (350 μm thick) were prepared using a vibrating microtome (VT 1000S;Leica Microsystems,Bannockburn,IL). Prior to recording, the sections were incubated with standard aCSF (124 mM NaCl, 3.75mM KCl, 2mM MgSO) ₄ 、2mM CaCl ₂ 、26.5mM NaHCO ₃ 、1.25mM NaH ₂ PO ₄ 10mM glucose) at room temperature for at least 1 hour.

Electrophysiological recording:

for electrophysiological recording, a single slice is placed in immersion and at a constant rate (2 mL min ^-1 ) Continuous infusion of aerated aCSF (95% O) ₂ 、5％CO ₂ The method comprises the steps of carrying out a first treatment on the surface of the ph=7.4) to alert the experiment. Extracellular field excitatory postsynaptic potential (fEPSP) in the CA1 radiological layer was recorded using a glass micropipette filled with aCSF. fEPSP is a Schaffe stimulated electrically at 0.1Hz (i.e., one pulse per 10 seconds) by stimulating the electrode (borosilicate capillary glass with filaments, standard wall; OD:1.5mm; ID:0.86mm; length: 75mm; ref: W3 30-0060, from Harvard Apparatus) with glass placed in the radiation layer The r-branch commissure pathway.

After stabilization for 10 minutes, basal synaptic transmission is assessed. To this end, the stimulation intensity was gradually increased from 0.1Hz (i.e., one pulse every 10 seconds) from 0 to 850 μa (in 50 μa increments) from the stimulation electrode, and the fEPSP slope response from the recording electrode was quantified. This procedure was repeated 3 times, each time 30 seconds apart. A single final I/O curve was obtained by plotting the average rational slope of fEPSP as a function of stimulus intensity. Each time respectively occurs in the following steps:

-a: after 10 minutes of vehicle perfusion, the sections were still perfused with vehicle during the I/O recording.

-B: after 10 minutes of NX218 perfusion, the sections were still perfused with NX218 during I/O recording.

Results:

all data were normalized to the maximum slope of fEPSP recorded under control conditions (during vehicle bath application). Thus, the results obtained for the NX218 condition were normalized to the control condition (vehicle) in this way. The following criteria were used to identify potential outliers for exclusion: the group mean was deviated by >3 standard deviations in either direction. The results from 10 validated slices are listed in table 9 and fig. 14.

Table 9: after sequential infusion of vehicle and NX218 (250 μg/mL) in hippocampal slices, normalized mean slope of fEPSP was induced during input-output (I/O) recordings of CA1 region.

In this study, a statistical increase in basal excitatory synaptic transmission of CA3-CA1 synapses in NX218 infused mouse hippocampal slices was observed compared to control conditions.

The results indicate that the NX218 peptide enhances basal hippocampal synaptic transmission.

Example 10: effect of NX218 on sub-chronic PCP-induced cognitive deficit in mice

Phencyclidine (PCP) is an antagonist of NMDA receptors whose administration in healthy humans produces symptoms resembling schizophrenia, and thus PCP is commonly used to simulate schizophrenia in rodents. To evaluate the effect of NX218 on sub-chronic PCP-induced cognitive deficits in mice, the T maze was used for consecutive alternating tasks. In addition, brain samples were collected shortly after T maze test to measure the levels of brain biomarkers associated with synaptic plasticity.

The method comprises the following steps:

animals:

sixty (60) male Swiss CD-1 mice were purchased from Janvier Labs (Le Genest-Saint-Isle, france) for environmental adaptation one week prior to study initiation. The mice were housed in groups (6-8 mice per European Standard III cage) and maintained in temperature-controlled (21-22 ℃) rooms at 12 hours/12 hours with inverted light/dark cycles (lights on: 5:30pm; lights off: 05:30 am) with food and water ad libitum. The mice had a body weight in the range of 25.9 to 36.4 grams.

Animals were checked daily for health. Animals were checked daily for general condition and activity, while body weight was monitored prior to PCP and peptide administration. All animal procedures were carried out in accordance with the current laws and regulations in France (European directive 2010/63/EU incorporating French laws, revised by the act of 2013-118 on month 18 of 2013).

Compound administration and treatment group description:

PCP (or physiological saline for control group) was administered at a dose of 0.2mg/kg twice daily for 12 days (day 0 to day 11) by subcutaneous injection.

NX218 was dissolved in water for injection (vehicle) and administered intraperitoneally at a dose of 5mg/kg 48 hours, 24 hours and/or 2 hours prior to the T maze test.

Nicotine used as a positive control was administered intraperitoneally at a dose of 0.4mg/kg 30 minutes prior to the T maze test

Experimental procedure:

after administration of the compounds at different time points, all animals were tested for consecutive alternating tasks in the T maze. The T maze was made of gray Plexiglas, with a trunk (55 cm long by 10 cm wide by 20 cm high) and two arms positioned at 90 degrees to the trunk (30 cm long by 10 cm wide by 20 cm high). The start box (15 cm long by 10 cm wide) is separated from the trunk by a gate. A horizontal gate is also provided to close a particular arm during forced selection of alternate tasks. The protocol included a single session starting with 1 "forced selection" trial followed by 14 "free selection" trials (Gerlai, 1998). In the first "forced selection" trial, the mice were restrained in the starting arm for 5 seconds and then released while blocking the left or right target arm by closing the sliding door. It then passes through the maze, eventually into an open target arm, and then back to the starting position. Immediately after the mouse returns to the home position, the left or right target door is opened (i.e., both target doors are open), and the mouse is free to choose between the left and right target door arms (the first "free choice test"). When a mouse placed its four paws into the arm, the mouse was considered to have entered the arm. The opposing target door is then closed until the animal returns to the starting arm. Then, by opening the closed target doors (i.e., opening both target doors), the mice were allowed to choose to enter either the right or left arm, and a second "free choice test" was initiated. After completing 14 free-selection trials in less than 10 minutes, the session was considered to be complete and the mice were removed from the maze. The time required to complete these 14 "free choices" was recorded. None of the mice took more than 10 minutes to perform 14 selections, which would be an exclusion criterion.

The percentage of spontaneous alternation is calculated as the number of spontaneous alternations divided by the number of free-choice trials, which is an index of the spatial short-term working memory. The device was cleaned between each animal with alcohol (70%). Urine and faeces have been removed from the maze. During the trial, animal handling and operator visibility was reduced as much as possible. Animals were tested in a randomized and blind fashion.

Brain sampling:

about 5 minutes after testing, mice were anesthetized with a 5% isoflurane oxygen mixture. The brains were immediately extracted and the cortex of each hemisphere was then collected. The left and right cortex were transferred to clean pre-labeled microtubes and the exact weight of each sample was recorded. Finally, the samples were stored at-80 ℃ for further analysis.

Protein extraction and automatic analysis:

brain samples were lysed using defined lysis buffer consisting of cellytmt reagent and 1% protease and phosphatase inhibitor cocktail (60 μl per well). At the end of the experiment, the lysate was treated at +4℃and stored at-80 ℃. For each sample, the amount of protein was determined using the mini kit BCA (Pierce). Briefly, lysates were centrifuged and diluted 1/20 in PBS and mixed with a micro BCA equivalent volume. After incubation at 60 ℃ for 1 hour, protein amounts were measured at 562nm using spectrophotometer Nanovue (GE Healthcare) and compared to bovine serum albumin standard curve (BSA, pierce).

Using WES ^TM System and method for controlling a systemAutomated protein analysis was performed:

according to the manufacturer with respect to WES ^TM The reagents were prepared and used according to the use advice (ProteinSimple, https:// www.Proteinsimple.com/wes. Html).

The operation is performed according to manufacturer's recommendations. The capillary, sample (3. Mu.L solution of 0.2mg/mL for GluN2A and 2mg/mL for phosphorylated CREB), antibody and matrix were then all loaded into the instrument. Simple western blotting is performed using capillaries filled with separation matrix, stacking matrix and protein sample. Then, the capillaries were incubated with primary antibody at room temperature (23 ℃,.+ -. 3 ℃) for 2 hours:

anti-pCREB antibody (Cell signaling,9198S,43 kDa)

Total anti-GluN ₂ Antibody A (Millipore Sigma, M264-10UG,. About.180 kDa)

Each protein was evaluated independently. The capillaries were washed and incubated with highly sensitive horseradish peroxidase (HRP) -conjugated secondary antibodies for 1 hour at room temperature (23 ℃, ±3 ℃). After removal of unbound secondary antibody, the capillary was incubated with luminol-S/peroxide substrate at room temperature (23 ℃, + -3 ℃) and WES was used ^TM The Charge Coupled Device (CCD) camera collects chemiluminescent signals at six different exposure times (30, 60, 120, 240, 480 and 960 seconds). Using WES ^TM Compass software (ProteinSimple) above. 5 samples were analyzed for each condition (biological replicates).

Results:

t maze assay:

the results of NX218 administration on sub-chronic PCP-induced cognitive deficits are shown in table 10 and fig. 15.

Table 10: using the T maze cognition test, intraperitoneal administration of NX218 had an effect on subclinical PCP-induced cognitive deficits in mice.

The spontaneous and continuous alternating manifestations of sub-chronic PCP treated mice in the T maze were divided into 31%. This behavior is significantly different from saline-treated mice (66%), reflecting PCP-induced sub-chronic defects in this task.

Pretreatment with NX218 for 2 hours on day 14 significantly reversed sub-chronic PCP-induced defects in mouse T maze alternation, as shown by a spontaneous alternation rate of 53%. The reversal effect of NX218 was even greater (62% spontaneous alternation) when PCP mice received three consecutive pretreatments (48 hours, 24 hours, and 2 hours) at D12, D13, and D14, respectively. In contrast, a single 24 hour NX218 pretreatment was ineffective against defects, as shown by a 35% spontaneous alternation rate. Taken together, these results indicate that NX218 reverses sub-chronic PCP-induced cognitive deficits in mice, but the presence of 2 hours pretreatment appears to be critical.

Nicotine (0.4 mg/kg) tested in parallel produced 62% spontaneous alternation, which was significantly different from sub-chronic PCP treated mice.

Brain biomarker level (protein expression measured by automated WB):

the effect of NX218 on relevant brain biomarker levels in cortical samples of mice chronically treated with PCP is shown in tables 11 and 12 and fig. 16 and 17.

Table 11: pCREB protein levels in cortical samples in mice models of cognitive deficits induced by sub-chronic administration of PCP (results expressed as a percentage of control conditions)

Table 12: gluN2A levels in cortical samples in mice models of cognitive deficits induced by sub-chronic administration of PCP (results expressed as a percentage of control conditions)

There was no change in GluN2A levels in the cortex of PCP treated mice compared to control mice. However, a significant increase in the total amount of GluN2A was observed in mice chronically treated with NX218 compared to control mice (double increase) and PCP treated mice. A significant decrease in nuclear transcription factor pCREB was observed in the cortex of PCP treated mice, which recovered after chronic administration of NX 218.

Overall, the results indicate that NX218 can regulate levels and/or phosphorylation of proteins involved in synaptic plasticity in the cortex.

Conclusion:

finally, NX218 can reverse PCP-induced cognitive deficits in mice. Repeated administration of the peptide 2 hours prior to cognitive tasks or single acute administration of NX218 significantly improved cognitive performance in mice. The effect of repeated administration of NX218 at synapses is translated into an increase in GluN2A subunit cortex content and a restoration of CREB phosphorylation, two associated mechanisms that may explain the restoration of cognitive function.

It is shown herein that NX218 may enhance the intensity of neurotransmission in different neural circuits (i.e., hippocampal or thalamocortical synapses) by increasing GluN2A-NMDAR and AMPAR excitatory postsynaptic currents. In a mouse model that exhibited defects in synaptic transmission induced by chronic administration of NMDAR antagonist phencyclidine, a single acute systemic injection of NX218 significantly improved spatial working memory. Furthermore, daily repeat treatments with NX218 increased GluN2A-NMDAR cortical content while restoring NMDAR-dependent phosphorylation of CREB, supporting a complete restoration of memory function.

AMPAR and NMDAR are major participants in excitatory synaptic transmission, whose sustained changes trigger plasticity through molecular cascades in various CNS regions to ensure basic functions such as learning and memory or neuroendocrine functions. Mechanistically, activation of AMPAR results in rapid depolarization of postsynaptic membranes that accelerate electrical communication between neurons, while activation of NMDAR regulates neuronal gene expression to maintain long term changes induced by AMPAR. The diversity in AMPAR and NMDAR subunit composition and transport leads to many different forms of synaptic plasticity, such as LTP and long term inhibition (LTD), which are crucial for learning and memory processes, homeostatic plasticity, or synaptic re-plasticity. In this study, we provided the first evidence that NX218 enhances the strength of excitatory neurotransmission at CA3-CA1 hippocampus and thalamocortical synapses. Thus, NX218 represents a therapeutic opportunity to enhance excitatory neurotransmission in different diseases and conditions in which glutamatergic synaptic transmission is impaired (e.g., schizophrenia or even normal aging). Furthermore, a decrease in glutamatergic system activity is often associated with a decrease in gabaergic system activity to maintain a balance between excitatory and inhibitory transmission; thus, the activity of both systems can be enhanced by exogenously supplying NX 218. One major challenge in the treatment of excitatory synaptic dysfunction is achieving rapid rebalancing of excitation and inhibition within the CNS without promoting death of excitatory toxic neurons. Interestingly, we observed that GluN2A triggers an increase in EPSC in the presence of NX218, a subunit known to promote neurotransmission and neuronal survival.

As mentioned above, NMDAR plays a critical role in synaptic transmission and plasticity and cognitive processes. Thus, short-term or long-term reduction of glutamate activity resulting from acute or chronic exposure to NMDAR antagonist PCP impairs short-term spatial memory in rodents, primates and humans. In addition to its effect on excitatory currents and subsequent neurotransmission at synapses, NX218 also promotes NMDAR driven signaling and plasticity, as shown by increased levels of pCREB and GluN2A-NMDAR protein in the presence of peptides in vivo. Furthermore, we provide evidence that the effect of NX218 alleviates short term memory deficits in mice. We hypothesize that the effect of NX218 on synaptic plasticity and related function (i.e., memory in this study) may be associated with post-synaptic increase in GluN2A-NMDAR protein levels, rather than with an increase in presynaptic release in glutamate-containing vesicles.

Taken together, our studies indicate that NX218 promotes AMPAR and GluN2A-NMDAR mediated neurotransmission in brain regions associated with higher order functions (i.e., cortex and hippocampus). Consistent with these findings, we observed that NX218 treatment caused favorable changes in NMDAR-dependent signaling and short term memory in pharmacological mouse models of synaptic dysfunction. Overall, modulation of GluN2A-NMDAR and AMPAR functions by NX218 represents an innovative therapeutic opportunity to improve outcome in the elderly and in patients with CNS diseases with disabling synaptic defects.

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Tzang, R.F. et al (2019) Autism Associated With Anti-NMDAR Encephalitis: glamate-Related therapy, front Psychiary, 10,440.Doi:10.3389/fpsyt.2019.00440.

Varela,C.(2013).The gating of neocortical information by modulators.J Neurophysiol,109(5),1229-1232.doi:10.1152/jn.00701.2012.

Villard, V.et al (2009) Antiamnesic and neuroprotective effects of the aminotetrahydrofuran derivative ANAVEX1-41 against amyloid beta (25-35) -induced toxicity in mice.neuroopsychloropharmacology, 34 (6), 1552-1566.Doi:10.1038/npp.2008.212.

Villard, V.et al (2011), anti-amnesic and neuroprotective potentials of the mixed muscarinic receptor/sigma 1 (σ1) ligand ANAVEX2-73,a novel aminotetrahydrofuran derivative.J Psychopharmacol,25 (8), 1101-1117.Doi:10.1177/0269881110379286.

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Sequence listing

<110> Exx Olivitis pharmaceutical Co

<120> SCO-spondin-derived polypeptides for enhancing synaptic transmission

<130> BEX22L0068

<160> 63

<170> PatentIn version 3.5

<210> 1

<211> 40

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<220>

<221> MISC_FEATURE

<222> (1)..(6)

<223> Xaa is absent or 1 amino acid

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> Xaa is 1 amino acid

<220>

<221> MISC_FEATURE

<222> (10)..(13)

<223> Xaa is absent or 1 amino acid

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> Xaa is 1 amino acid

<220>

<221> MISC_FEATURE

<222> (17)..(20)

<223> Xaa is absent or 1 amino acid

<220>

<221> MISC_FEATURE

<222> (23)..(24)

<223> Xaa is 1 amino acid

<220>

<221> MISC_FEATURE

<222> (25)..(32)

<223> Xaa is absent or 1 amino acid

<220>

<221> MISC_FEATURE

<222> (35)..(40)

<223> Xaa is absent or 1 amino acid

<400> 1

Xaa Xaa Xaa Xaa Xaa Xaa Trp Ser Xaa Xaa Xaa Xaa Xaa Trp Ser Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Cys Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

Cys Gly Xaa Xaa Xaa Xaa Xaa Xaa

35 40

<210> 2

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Xaa is 1 amino acid

<220>

<221> MISC_FEATURE

<222> (4)..(7)

<223> Xaa is absent or 1 amino acid

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Xaa is 1 amino acid

<220>

<221> MISC_FEATURE

<222> (11)..(14)

<223> Xaa is absent or 1 amino acid

<220>

<221> MISC_FEATURE

<222> (17)..(18)

<223> Xaa is 1 amino acid

<220>

<221> MISC_FEATURE

<222> (19)..(26)

<223> Xaa is absent or 1 amino acid

<400> 2

Trp Ser Xaa Xaa Xaa Xaa Xaa Trp Ser Xaa Xaa Xaa Xaa Xaa Cys Ser

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Gly

20 25

<210> 3

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 3

Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10

<210> 4

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 4

Trp Ser Ser Trp Ser Gly Cys Ser Arg Ser Cys Gly

1 5 10

<210> 5

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 5

Trp Ser Ser Trp Gly Ser Cys Ser Arg Ser Cys Gly

1 5 10

<210> 6

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 6

Trp Ser Ser Trp Gly Gly Cys Ser Arg Ser Cys Gly

1 5 10

<210> 7

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 7

Trp Ser Ser Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10

<210> 8

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 8

Trp Ser Gly Trp Ser Gly Cys Ser Arg Ser Cys Gly

1 5 10

<210> 9

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 9

Trp Ser Gly Trp Gly Ser Cys Ser Arg Ser Cys Gly

1 5 10

<210> 10

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 10

Trp Ser Gly Trp Gly Gly Cys Ser Arg Ser Cys Gly

1 5 10

<210> 11

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 11

Trp Ser Ser Trp Ser Ser Cys Ser Val Ser Cys Gly

1 5 10

<210> 12

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 12

Trp Ser Ser Trp Ser Gly Cys Ser Val Ser Cys Gly

1 5 10

<210> 13

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 13

Trp Ser Ser Trp Gly Ser Cys Ser Val Ser Cys Gly

1 5 10

<210> 14

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 14

Trp Ser Ser Trp Gly Gly Cys Ser Val Ser Cys Gly

1 5 10

<210> 15

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 15

Trp Ser Gly Trp Ser Ser Cys Ser Val Ser Cys Gly

1 5 10

<210> 16

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 16

Trp Ser Gly Trp Ser Gly Cys Ser Val Ser Cys Gly

1 5 10

<210> 17

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 17

Trp Ser Gly Trp Gly Ser Cys Ser Val Ser Cys Gly

1 5 10

<210> 18

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 18

Trp Ser Gly Trp Gly Gly Cys Ser Val Ser Cys Gly

1 5 10

<210> 19

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 19

Trp Ser Ser Trp Ser Ser Cys Ser Val Thr Cys Gly

1 5 10

<210> 20

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 20

Trp Ser Ser Trp Ser Gly Cys Ser Val Thr Cys Gly

1 5 10

<210> 21

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 21

Trp Ser Ser Trp Gly Ser Cys Ser Val Thr Cys Gly

1 5 10

<210> 22

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 22

Trp Ser Ser Trp Gly Gly Cys Ser Val Thr Cys Gly

1 5 10

<210> 23

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 23

Trp Ser Gly Trp Ser Ser Cys Ser Val Thr Cys Gly

1 5 10

<210> 24

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 24

Trp Ser Gly Trp Ser Gly Cys Ser Val Thr Cys Gly

1 5 10

<210> 25

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 25

Trp Ser Gly Trp Gly Ser Cys Ser Val Thr Cys Gly

1 5 10

<210> 26

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 26

Trp Ser Gly Trp Gly Gly Cys Ser Val Thr Cys Gly

1 5 10

<210> 27

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 27

Trp Ser Ser Trp Ser Ser Cys Ser Arg Thr Cys Gly

1 5 10

<210> 28

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 28

Trp Ser Ser Trp Ser Gly Cys Ser Arg Thr Cys Gly

1 5 10

<210> 29

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 29

Trp Ser Ser Trp Gly Ser Cys Ser Arg Thr Cys Gly

1 5 10

<210> 30

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 30

Trp Ser Ser Trp Gly Gly Cys Ser Arg Thr Cys Gly

1 5 10

<210> 31

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 31

Trp Ser Gly Trp Ser Ser Cys Ser Arg Thr Cys Gly

1 5 10

<210> 32

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 32

Trp Ser Gly Trp Ser Gly Cys Ser Arg Thr Cys Gly

1 5 10

<210> 33

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 33

Trp Ser Gly Trp Gly Ser Cys Ser Arg Thr Cys Gly

1 5 10

<210> 34

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 34

Trp Ser Gly Trp Gly Gly Cys Ser Arg Thr Cys Gly

1 5 10

<210> 35

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 35

Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10

<210> 36

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 36

Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10

<210> 37

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 37

Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10 15

<210> 38

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 38

Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10 15

<210> 39

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 39

Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu

1 5 10

<210> 40

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 40

Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly

1 5 10

<210> 41

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 41

Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu

1 5 10 15

<210> 42

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 42

Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu Ile

1 5 10 15

<210> 43

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 43

Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu Ile

1 5 10 15

Phe

<210> 44

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 44

Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu

1 5 10

<210> 45

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 45

Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly

1 5 10 15

<210> 46

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 46

Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu

1 5 10 15

<210> 47

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 47

Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu

1 5 10 15

Ile

<210> 48

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 48

Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly Leu

1 5 10 15

Ile Phe

<210> 49

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 49

Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu

1 5 10 15

<210> 50

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 50

Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly

1 5 10 15

<210> 51

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 51

Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly

1 5 10 15

Leu

<210> 52

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 52

Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly

1 5 10 15

Leu Ile

<210> 53

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 53

Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu Gly

1 5 10 15

Leu Ile Phe

<210> 54

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 54

Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu

1 5 10 15

<210> 55

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 55

Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu

1 5 10 15

Gly

<210> 56

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 56

Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu

1 5 10 15

Gly Leu

<210> 57

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 57

Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu

1 5 10 15

Gly Leu Ile

<210> 58

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 58

Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly Leu

1 5 10 15

Gly Leu Ile Phe

20

<210> 59

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 59

Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10 15

Leu

<210> 60

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 60

Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10 15

Leu Gly

<210> 61

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 61

Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10 15

Leu Gly Leu

<210> 62

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 62

Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10 15

Leu Gly Leu Ile

20

<210> 63

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> peptide Compounds

<400> 63

Val Leu Ala Pro Trp Ser Gly Trp Ser Ser Cys Ser Arg Ser Cys Gly

1 5 10 15

Leu Gly Leu Ile Phe

20

Claims

1. Peptides of amino acid sequence X1-W-S-A1-W-S-A2-C-S-A3-A4-C-G-X2

Wherein:

a1, A2, A3 and A4 consist of an amino acid sequence of 1 to 5 amino acids,

-X1 and X2 consist of an amino acid sequence of 1 to 6 amino acids; or X1 and X2 are absent;

the N-terminal amino acid may be acetylated, the C-terminal amino acid may be amidated, or the N-terminal amino acid may be acetylated and the C-terminal amino acid may be amidated,

Which are useful for enhancing or restoring GluN2A-NMDAr mediated glutamatergic neurotransmission.

2. The peptide for use according to claim 1 for enhancing or restoring AMPAr and GluN2A-NMDAr mediated glutamatergic neurotransmission.

3. The peptide for use according to claim 1 for enhancing or restoring excitatory synaptic transmission when/if it is impaired, in particular during or after hypoxia.

4. Peptides of amino acid sequence X1-W-S-A1-W-S-A2-C-S-A3-A4-C-G-X2

Wherein:

a1, A2, A3 and A4 consist of an amino acid sequence of 1 to 5 amino acids,

which is used for the treatment or prevention of schizophrenia,

which are useful for the treatment or prophylaxis of drug addiction, in particular those resulting from PCP, ketamine or scopolamine,

which is used for treating the NMDAr encephalitis,

it is used for treating plant human state, or

For the treatment or prevention of hypoxia-induced synaptic transmission inhibition and/or hypoxia-induced brain damage,

Which is useful for the treatment of bipolar disorders,

which is useful for treating or preventing synaptic defects caused by viral infection, in particular in SARS CoV2 and COVID-19 (especially in patients with long new coronaries),

which is useful for treating or preventing synaptic dysfunction of a synaptic disorder.

5. The peptide for use according to any of the preceding claims, wherein the peptide has the amino acid sequence W-S-A1-W-S-A2-C-S-A3-A4-C-G, wherein A1, A2, A3 and A4 consist of an amino acid sequence of 1 to 5 amino acids.

6. The peptide for use according to any of the preceding claims, wherein

A1 is selected from G, V, S, P and A, preferably G, S,

a2 is selected from G, V, S, P and A, preferably G, S,

-A3 is selected from R, A and V, preferably R, V, and/or

-A4 is selected from S, T, P and a, preferably S, T.

7. The peptide for use according to any of the preceding claims, wherein A1 and A2 are independently selected from G and S, and/or A3-A4 are selected from R-S or V-T or R-T.

8. The peptide for use according to any of the preceding claims, wherein the peptide has a sequence selected from the group consisting of SEQ ID NOs: 3-63.

9. The peptide for use according to any one of the preceding claims, wherein the peptide is a linear peptide or a cyclized peptide in which two cysteines form a disulfide bond.

10. The peptide for use according to any one of claims 1 to 9, which is the sequence SEQ ID NO:3, wherein the peptide is a linear peptide or a cyclized peptide in which two cysteines form a disulfide bond, or a mixture of both.

11. The peptide for use according to any one of claims 1 to 9, which is the sequence SEQ ID NO:3, wherein the peptide is a cyclized peptide in which two cysteines form a disulfide bond.

12. The peptide for use according to any one of the preceding claims, wherein the peptide increases, enhances or restores excitatory synaptic transmission.

13. The peptide for use according to any one of claims 1 to 11, wherein the peptide prevents or treats the detrimental effect of hypoxia on excitatory synaptic transmission.

14. The peptide for use according to any one of claims 1 to 11 for increasing, enhancing or restoring excitatory synaptic transmission, in particular in the hippocampus, or for preventing or treating the detrimental effects of hypoxia on excitatory synaptic transmission, in particular at the hippocampus.