Therapeutic peptides

Abstract

The invention relates generally to peptides comprising an amino acid sequence as set forth in SEQ ID NO: 1: CPRILX1, wherein X1 is a chain of 3 or more R residues, and to peptides comprising an amino acid sequence as set forth in SEQ ID NO:2: KLX2X3IESX4X5 wherein: X2, X3 and X4 are any amino acid residue; and X5 is selected from V and I, provided that SEQ ID NO:2 is not KLSSIESDV. The invention further relates to fusion peptides thereof, methods of delivering an active agent across a cell membrane and / or across the blood brain barrier, and methods of treating or preventing a condition associated with neuronal hyperexcitation-associated neurotoxicity.

Description

Therapeutic peptidesRelated Applications

[0001] This application claims priority to Australian Provisional Application No. 2024900908 entitled “Therapeutic Peptides” filed 3 April 2024, the contents of which are incorporated herein by reference in their entirety.Field of the Art

[0002] The present disclosure relates to cell membrane and blood brain barrier penetrating peptides, peptides for use in the treatment of neurological disorders such as Alzheimer's disease, and fusion peptides comprising said peptides.Background

[0003] Alzheimer’s disease (AD) is the most common neurodegenerative condition, causing progressive cognitive decline and memory loss. The disease presents with pathological deposition of the amyloid-P (AP) and tau proteins. There is no cure, and therapeutic options to date are limited.

[0004] As such, new therapeutic approaches based on a deep understanding of underlying molecular disease mechanisms are needed. In recent years, the present inventors have defined a molecular disease pathway that involves neuronal hyperexcitation-associated neurotoxicity (exci totoxi city) mediated by a post-synaptic excitotoxic signalling complex of proteins, and reported the critical role of the tai protein in excitotoxicity in AD. This work highlighted the potential of targeting the excitotoxic signalling complex for the development of novel treatments for AD (Ittner LM, et al. Dendritic Function of Tau Mediates Amyloid-beta Toxicity in Alzheimer's Disease Mouse Models. Cell (2010) 142: 387-397; Ittner A, et al. Site-specific phosphorylation of tau inhibits amyloid-beta toxicity in Alzheimer's mice. Science (2016) 354: 904-908; Ittner A and Ittner LM. Dendritic Tau in Alzheimer's Disease. Neuron (2018) 99: 13- 27).

[0005] Protein-protein interactions (PPIs) are highly specific associations between two (ormore) proteins that govern pivotal biological functions and aberrant PPIs contribute to various diseases, making them an attractive target for drug development. PPI interfaces are often large, flat and lack suitable pockets, making them difficult targets for small molecule drugs. In fact, PPIs were regarded as ‘undruggable’ for a long time. One specific way of targeting protein complexes is by using short amino acid sequences (peptides), but their therapeutic potential is limited due to inherent instability in the organism, being highly susceptible to hydrolysis and enzymatic digestion in the organism, resulting in short half-life. Peptides also bring the potential for immune reactions in the organism. Poor crossing of the blood brain barrier also limits the therapeutic use of peptides, in particular for CNS conditions.

[0006] There remains a need for further avenues for targeting disease-relevant PPIs, including for treating neurodegeneration.Summary of the Disclosure

[0007] As described and exemplified herein, the inventors have designed novel peptides which aid in cell penetration and penetration of the blood brain barrier, as well as peptides which find use in the treatment of diseases associated with a molecular disease pathway involving neuronal hyperexcitation-associated neurotoxicity, in particular Alzheimer's disease, as well as fusion peptides thereof.

[0008] A first aspect of the invention provides a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 :CPRILXi [SEQ ID NO:!] wherein Xi is a chain of 3 or more R residues.

[0009] In an embodiment, Xi is a chain of at least 4 R residues, optionally a chain of 4 R residues.

[0010] The peptide may act as a cell membrane permeability agent and / or a blood-brain barrier permeability agent, optionally, both as a cell membrane permeability agent and a bloodbrain barrier permeability agent.

[0011] The peptide may further comprise an additional active agent selected from atherapeutic agent, a diagnostic agent, a detection agent or an agent for imaging biological tissue. In an exemplary embodiment the additional active agent is a therapeutic agent. In particular embodiments, the active agent comprises a peptide sequence.

[0012] In a particular exemplary embodiment, the therapeutic agent is therapeutic for a condition associated with neuronal hyperexcitation-associated neurotoxicity. The condition may be a neurological condition such as, for example, Alzheimer's disease.

[0013] In an exemplary embodiment the therapeutic agent comprises a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2:KLX2X3IESX4X5 [SEQ ID NO:2] whereinX2, X3 and X4 are any amino acid residue; andX5 is selected from V and I.

[0014] In a particular embodiment of the first aspect, the peptide comprises a sequence set forth in SEQ ID NO: 1 linked to a sequence set forth in SEQ ID NO:2.

[0015] The peptide may comprise a linker sequence L between the sequence of SEQ ID NO: 1 and the additional active agent, optionally between the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO:2. In particular embodiments, the additional active agent, optionally comprising a sequence of SEQ ID NO:2 is located at or adjacent to the C-terminal of the sequence of SEQ ID NO: 1, optionally separated from the sequence of SEQ ID NO: 1 by a linker sequence L. The linker sequence L may comprise a sequence of from 15 to 25 amino acid residues in length, optionally a sequence of 18, 19 or 20 amino acids in length.

[0016] In particular embodiments, the linker sequence L may comprise an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11, or a sequence having at least or about 90% sequence identity thereto:CRRDSDCPGACICRGNGYC [SEQ ID NO:6] CRRDSDCPGACICRGGYC [SEQ ID NO: 10] CRRDSDCPGACICRGNSGYC [SEQ ID NO: 11],

[0017] In particular embodiments, a peptide of the first aspect, comprising a sequence ofSEQ ID NO: 1 and an additional active agent comprising a peptide sequence, optionally separated by a linker sequence L, together comprise a cyclic peptide.

[0018] Another aspect of the invention provides a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2:KLX2X3IESX4X5 [SEQ ID NO:2] whereinX2, X3 and X4 are any amino acid residue; andX5 is selected from V and I, provided that SEQ ID NO: 2 is not KLSSIESDV [SEQ ID NO:3].

[0019] In an exemplary embodiment of the second aspect, X2 is S or T. Optionally, X2 is T.

[0020] In an exemplary embodiment of the second aspect, X3 is S or T. Optionally, X3 is T.

[0021] In an exemplary embodiment of the second aspect, X4 is D or E. Optionally, X4 is E.

[0022] In an exemplary embodiment of the second aspect, X5 is V or I. Optionally, X5 is I.

[0023] In an embodiment of the second aspect, the peptide further comprises an additional active agent selected from a therapeutic agent, a diagnostic agent, a detection agent or an agent for imaging biological tissue, a cell membrane permeability agent and a blood-brain barrier permeability agent. In a particular embodiment the additional active agent facilitates both cell membrane permeability and blood-brain barrier permeability. In particular embodiments, the additional active agent comprises a peptide sequence, optionally a sequence set forth in SEQ ID NO: 1.

[0024] In a particular embodiment of the second aspect, the peptide comprises a sequence set forth in SEQ ID NO:2 linked to a sequence set forth in SEQ ID NO: 1.

[0025] The peptide may comprise a linker sequence L between the sequence of SEQ ID NO:2 and the additional active agent, optionally between the sequence of SEQ ID NO:2 and the sequence of SEQ ID NO: 1. In particular embodiments, the additional active agent, optionallycomprising a sequence of SEQ ID NO: 1 is located at or adjacent to the N-terminal of the sequence of SEQ ID NO:2, optionally separated from the sequence of SEQ ID NO:2 by a linker sequence L. The linker sequence L may comprise a sequence of from 15 to 25 amino acid residues in length, optionally a sequence of 18, 19 or 20 amino acids in length.

[0026] In particular embodiments, the linker sequence L may comprise an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11, or a sequence having at least or about 90% sequence identity thereto:CRRDSDCPGACICRGNGYC [SEQ ID NO:6] CRRDSDCPGACICRGGYC [SEQ ID NO: 10] CRRDSDCPGACICRGNSGYC [SEQ ID NO: 11],

[0027] In some particular embodiments a peptide of the first aspect, comprising a sequence of SEQ ID NO: 1 and an additional active agent comprising a peptide sequence, optionally separated by a linker sequence L, together comprise a cyclic peptide.

[0028] In accordance with the above aspects and embodiments, a peptide of the present invention may comprise an amino acid sequence as set forth in SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 13:CPRILRRRRCRRDSDCPGACICRGNGYCKLSSIESDV [SEQ ID NO:7] CPRILRRRRCRRDSDCPGACICRGNGYCKLSSIESDI [SEQ ID NO:8] CPRILRRRRCRRDSDCPGACICRGNSGYCKLSSIESDV [SEQ ID NO: 12] GYCKLSSIESDVCPRILRRRRCRRDSDCPGACICRG [SEQ ID NO: 13],

[0029] In some particular embodiments, a peptide of the present invention is a cyclic peptide. Thus, a peptide comprising the sequence of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO: 12 may be provided as a cyclic peptide.

[0030] In accordance with the above aspects and embodiments, a peptide of the present invention may be an isolated peptide.

[0031] Another aspect of the present invention provides a method of delivering an active agent across a cell membrane and / or across the blood brain barrier, comprising contacting the active agent with or delivering the active agent to a cell or the blood brain barrier, wherein the active agent is linked to a peptide comprising a cell membrane or blood-brain barrier penetratingpeptide sequence according to SEQ ID NO: 1.

[0032] Another aspect of the present invention provides a method of treating or preventing a condition associated with neuronal hyperexcitation-associated neurotoxicity, comprising administering to a subject in need thereof an effective amount of a peptide as herein defined, wherein the peptide comprises a therapeutic agent.

[0033] In exemplary embodiments, the peptide comprises a peptide comprising a sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 12 or SEQ ID NO: 13.

[0034] In exemplary embodiments the condition is a neurological condition such as, for example, Alzheimer's disease.

[0035] Also provided herein is the use of a peptide as herein defined, wherein the peptide comprises a therapeutic agent, in the manufacture of a medicament for the treatment or prevention of a condition associated with neuronal hyperexcitation-associated neurotoxicity.Brief Description of the Drawings

[0036] Figure 1. Structure of exemplary peptide according to the invention. (A) Amino acid sequence of exemplary cyclic peptide c5R-NR2B9c (SEQ ID NO: 7), with positions of disulphide bridges between cysteine (C) residues shown. (B) 3D structure of the peptide of (A), with locations of disulphide bridges indicated by arrows.

[0037] Figure 2. Cell internalisation of TAMRA labelled c5R-RN2B9c peptide in neurons. Peptides are instantly taken up by the cells and reach saturation at 40 min.

[0038] Figure 3. Treatment of neurons with c5R-NR2B9c resulting in 80% and 70% transduced neuronal cells in 0.5 pM and 0.1 pM respectively.

[0039] Figure 4. Comparison of stability in blood plasma of peptide c5R-NR2B9c with linear Tat-NR2B9c peptide, showing enhanced stability of peptide c5R-NR2B9c, as measured by liquid chromatography and mass spectrometry.

[0040] Figure 5. Effect on excitotoxic cell death induced by N-methyl D-aspartate (NMD A) in cultured neurons, in the presence of c5R-NR2B9c, Ac5R-NR2B9c and c5R-NR2B9c intein peptides compared to linear Tat-NR2B9c peptide, vehicle and inactive double alaninesubstituted controls (c5R-NR2B9AA, Tat-NR2BAA and c5R-NR2BAA intein). (A) c5R- NR2B9c efficiently prevented excitotoxic neuronal death induced by N-methyl D-aspartate (NMD A) in cultured neurons, as determined by propidium iodide staining of dead cells (bottom row), relative to DAPI staining of all cells (top row). (B) and (C) Percent dying cells (as determined according to (A)) in the presence of c5R-NR2B9c, c5R-NR2B9aa, Ac5R-NR2B9c, c5R-NR2B9c intein, c5R-NR2BAA intein, Tat-NR2B9c and Tat-NR2BAA.

[0041] Figure 6. Seizure occurrence with administration of c5R-NR2B9c, c5R-NR2B9aa and Tat-NR2B9c. Both (A) intravenous (i.v.) and (B) oral treatment of mice with c5R-NR2B9c significantly reduced severe forms of induced seizures (status epilepticus). ** P < 0.005; * P < 0.05.

[0042] Figure 7. Enhanced prevention of excitotoxic cell death in cultured neurons by an amino acid exchange. ** P < 0.01.

[0043] Figure 8. Modelling of the PSD95 tau and Fyn interacting domains (circled) based on chemically stabilized complexes.Detailed DescriptionDefinitions

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[0045] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0046] In the context of this specification, the term "about," is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0047] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0048] The term "optionally" is used herein to mean that the subsequently described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiments in which the event or circumstance occurs as well as embodiments in which it does not.

[0049] The term “peptide” means a polymer made up of amino acids linked together by peptide bonds. The term “polypeptide” may also be used to refer to such a polymer although in some instances a polypeptide may be longer (i.e. composed of more amino acid residues) than a peptide. Typically, the term peptide is used to define a sequence of amino acids of up to about 70 amino acids, although the terms "peptide" and "polypeptide" may be used interchangeably herein.

[0050] As used herein, "isolated" with reference to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is substantially free of cellular material or other contaminating proteins from the cells from which the polynucleotide or polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.

[0051] The term “fusion peptide” as used herein relates to a peptide comprising two or more heterologous regions or domains not found operably linked in nature. Optionally a linker sequence may connect or join the two or more heterologous regions or domains.

[0052] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant peptide. Whether an amino acid change results in a functional peptide can readily be determined by assaying its activity.

[0053] As used herein the terms "treating", "treatment", “preventing”, “prevention” and grammatical equivalents refer to any and all uses which remedy, prevent, retard or delay the establishment of a disorder, such as a neurological disorder, such as Alzheimer's disease, or otherwise prevent, hinder, retard, or reverse the progression of such a disorder. Thus the terms "treating" and “preventing” and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery. Where the disorder displays or a characterized by multiple symptoms, the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms, but may prevent, hinder, retard, or reverse one or more of said symptoms.

[0054] As used herein the term "effective amount" includes within its meaning a non-toxic but sufficient amount or dose of an agent or compound to provide the desired effect. The exact amount or dose required will vary from subject to subject depending on factors such as the species being treated, the age, size, weight and general condition of the subject, the severity of the disease or condition being treated, the particular agent being administered and the mode of administrationand so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

[0055] The term "subject" as used herein refers to mammals and includes humans, primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs), performance and show animals (e.g. horses, livestock, dogs, cats), companion animals (e.g. dogs, cats) and captive wild animals. Preferably, the mammal is human or a laboratory test animal. Even more preferably, the mammal is a human.

[0056] It will be appreciated that the above described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting.Therapeutic peptides, blood-brain barrier and cell membrane permeability peptides, and fusion peptides

[0057] The present inventors have identified new blood-brain barrier and cell membrane permeability peptide sequences which allow delivery of additional active agents, such as therapeutic peptide sequences, across the blood brain barrier. The present inventors have also developed new therapeutic peptide sequences effective against diseases such as Alzheimer's disease with exci totoxi city as an underlying disease mechanism. The present inventors have also developed new peptide sequences comprising blood-brain barrier and cell membrane permeability peptide sequences as well as therapeutic peptide sequences joined by linker peptide sequences, which allow delivery of said therapeutic peptides across the blood-brain barrier and which are particularly stable. The inventors have further developed a synthesis process which cyclizes peptides including such sequences, to provide brain active cyclic fusion peptides with a long half-life and low immunogenicity, for use in the treatment of conditions such as Alzheimer's disease. When the aforementioned blood-brain barrier and cell membrane permeability peptide sequences and therapeutic peptide sequences were incorporated into a fusion peptide as described herein, and, according to some embodiments, when incorporated into a cyclic peptide, peptides which exhibited increased resistance to in vivo degradation compared to some linear alternatives were obtained. Thus, as described and exemplified herein, the inventors have designed and synthesised novel therapeutic peptides, including cyclic therapeutic peptides, comprising blood-brain barrier and cell membrane permeability peptide sequences and therapeutic peptide sequences which find use in the treatment of neurological disorders such as Alzheimer's disease.

[0058] Previous work by the present inventors identified a molecular disease pathway underlying Alzheimer's Disease which involves neuronal hyperexcitation-associated neurotoxicity ('excitotoxicity') mediated by a post-synaptic excitotoxic signalling complex constituting N-methyl-D-aspartate (NMDA) receptors, the postsynaptic density protein 95 (PSD95), the microtubule-associated protein tau and the Src kinase Fyn (=NMDAR-PSD95-tau- Fyn; Ittner LM, el al. Dendritic Function of Tau Mediates Amyloid-beta Toxicity in Alzheimer's Disease Mouse Models. Cell (2010) 142: 387-397). The present invention looks to the excitotoxic signalling complex as a target for the treatment of Alzheimer's Disease and other conditions, especially neurological conditions, with underlying excitotoxicity. Advantageously, peptides according to embodiments of the present invention are resistant to degradation in vivo and thus have a long half-life, are brain active, immune privileged and tissue directed to target the excitotoxic signalling complex therapeutically. Peptides including cell membrane penetrating peptides according to the present invention exhibit high cell membrane and blood-brain barrier penetration.Blood-brain barrier and cell membrane permeability sequences

[0059] According to one aspect, the present disclosure provides a peptide comprising a sequence according to SEQ ID NO: 1 :CPRILXi [SEQ ID NO:!] wherein Xi is a chain of 3 or more R residues. In some embodiments, Xi is a chain of 4 or more R residues. In some embodiments, Xi is a chain of from 3 to 8 R residues , for example from 4 to 8 R residues. In some embodiments, Xi is a chain of exactly 4 R residues.

[0060] A cell membrane permeability peptide / peptide sequence is understood as a peptide / peptide sequence which facilitates efficient penetration of cellular membranes. In some examples, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the cell membrane permeability peptide / peptide sequence and / or active agent attached thereto that is contact with a cell penetrates the cell membrane. A blood-brainbarrier penetrating peptide / peptide sequence is understood as a peptide / peptide sequence which facilitates delivery of active agents across the blood-brain barrier. The blood-brain barrier is a semi-permeable barrier between the circulatory system and the central nervous system (CNS), by which the microvasculature of the CNS tightly regulates the movement of molecules, ions, and cells between the blood and the CNS. In some examples, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the cell membrane permeability peptide / peptide sequence and / or active agent attached thereto that is delivered to (or contacted with) a blood brain barrier crosses the blood brain barrier the cell membrane. The peptide according to SEQ ID NO: 1 acts as a blood-brain barrier and cell membrane permeability sequence. The peptide has been demonstrated by the present inventors to impart effective bloodbrain barrier penetration to fusion peptides in which it is integrated, including cyclic fusion peptides, allowing brain activity of therapeutic peptides linked to or comprising the peptide according to SEQ ID NO: 1 to be achieved. When incorporated into a fusion peptide as described below, for example a cyclic peptide, the sequence demonstrates stability and resistance to in-vivo degradation, providing a longer half-life than alternative linear peptides incorporating alternative cell penetration sequences tested. The permeability sequences of the present invention may advantageously be incorporated into other peptides for which blood brain barrier permeability is required.

[0061] A peptide comprising the cell membrane penetrating peptide according to SEQ ID NO: 1 as described herein may further comprise one or more additional active agents attached thereto, for example at either terminus or via a linker or spacer as described below. The active agent may be, for example, a therapeutic agent, a diagnostic agent, a detection agent or an agent for imaging biological tissue. Suitable active agents include nucleic acid-based constructs such as antisense oligonucleotides, peptide nucleic acids and protein-, polypeptide- or peptide-based moieties, including antibodies and fragments thereof. Other active agents may include small molecules. Conjugation of active agents to the peptides can be achieved using a variety of conjugation chemistry techniques that will be well known to those skilled in the art. By way of example, suitable techniques are described in: Karas et al., 2018, Methods Mol Biol 1828:355- 363; Patil etal., 2019, Bioconjug Chem 30:793-799; Shabanpoor and Gait, 2013, Chem Commun (Camb) 49: 10260-10262; and Shabanpoor et al., 2015, Nucleic Acids Res 43:29-39. In the context of fusion peptides described herein, the terms "linked", "attached" and "conjugated" are used interchangeably and relate to any type of interaction that conjugates (joins) two or more peptides and include covalent bonds or non-covalent bonds, such as, for example,hydrophobic / hydrophilic interactions, van der Waals forces, ionic bonds, disulphide bonds or hydrogen bonds. In an exemplary embodiment, two or more peptide components are joined via a linker or spacer sequence or moiety.Therapeutic peptides

[0062] In particular embodiments, the additional active agent is a therapeutic agent suitable for use in the treatment or prevention of any disease or disorder. In particular embodiments, the site of action of the agent is within the central nervous system, requiring that the agent crosses the blood-brain barrier. The skilled person will appreciate that the scope of the present disclosure is not limited by reference to any specific type or identity of therapeutic agent. In some exemplary embodiments, the therapeutic agent is therapeutic for a neurological condition, such as a CNS disorder or condition. In some embodiments, the condition is associated with neuronal hyperexcitation-associated neurotoxicity. In some embodiments, the condition is Alzheimer's disease.

[0063] In some embodiments, the therapeutic agent is a therapeutic peptide sequence according to SEQ ID NO:2:KLX2X3IESX4X5 [SEQ ID NO:2] wherein X2, X3 and X4 are any amino acid residue, and X5 is selected from V and I. The present inventors have found such therapeutic peptide sequences to be stable in blood plasma and efficient in preventing or reducing excitotoxic death of neurons as well as preventing or reducing excitotoxic seizures, when incorporated into a peptide comprising the cell membrane penetrating sequence of SEQ ID NO: 1, such as cyclic peptides comprising the cell membrane penetrating sequence of SEQ ID NO: 1.

[0064] Accordingly, according to a further aspect, the present disclosure provides a peptide comprising a sequence according to SEQ ID NO:2:KLX2X3IESX4X5 [SEQ ID NO:2] wherein: X2, X3 and X4 are any amino acid residue; and X5 is selected from V and I. In particular embodiments, SEQ ID NO:2 is not KLSSIESDV [SEQ ID NO:3].

[0065] According to some embodiments, therapeutic peptides of the above aspect may further comprise one or more additional active agents attached thereto. Additional active agents include those described above (e.g. a therapeutic agent, a diagnostic agent, a detection agent or an agent for imaging biological tissue) and agents that facilitate transport of the peptide across cell membranes and / or the blood brain barrier. In some particular embodiments, the one or more additional active agents may be a cell membrane permeability sequence, a blood-brain barrier permeability sequence, or a sequence which is both a cell membrane permeability sequence and a blood-brain barrier permeability sequence. Such active agents allow delivery of the therapeutic peptide across the blood-brain barrier to the central nervous system, as described herein. Cellpenetrating peptides that are effective in enhancing transport and cellular penetration and uptake include, for example, an arginine-rich peptide transporter, Penetratin or the TAT peptide, which are disclosed, for example, in US Publication No. 20100016215. In particular examples, the therapeutic peptide comprising a sequence according to SEQ ID NO:2 is attached to a peptide comprising a sequence according to SEQ ID NO: 1.

[0066] In some embodiments of the above aspects of a therapeutic peptide: X2 is S or T, for example X2 is T; X3 is S or T, for example X3 is T; X4 is D or E, for example X4 is E; and / or X5 is V or I, for example X5 is I. In some embodiments, X2 is S, X3 is S, X4 is E and X5 is V. In some embodiments, X2 is S, X3 is T, X4 is D and X5 is V. In some embodiments, X2 is T, X3 is S, X4 is D and X5 is V. In particularly preferred embodiments, X5 is I. For example, in some embodiments, X2 is S, X3 is S, X4 is D and X5 is I. Particular embodiments wherein X5 is I have been found to be particularly effective in preventing excitotoxic cell death in cultured neurons. Exemplary therapeutic peptides of the present disclosure include those comprising the sequence KLSSIESDV [SEQ ID NO:3], KLSSIESDI [SEQ ID NO:4],

[0067] The potential therapeutic activity of peptides against conditions associated with exci totoxi city may be measured, for example, by assessing excitotoxic neuronal cell death induced by N-methyl D-asparate (NMDA) in cultured neurons, as determined by propidium iodide staining of dead cells (as described in Example 3). Assessment of the activity of the therapeutic peptide (e.g. a therapeutic peptide comprising the sequence set forth in SEQ ID NO:2) may be performed with a therapeutic peptide that is either attached or not attached to a cell membrane permeability peptide (or comprises or does not comprise a cell membrane permeability sequence). Moreover, assessment of the activity of the therapeutic peptide may be performed with a therapeutic peptide that is either linear or cyclic.

[0068] A peptide of the present disclosure may further comprise one or more additional active agent and moieties. For example, a lipid moiety, such as a myristoyl group, may be attached to the N-terminal end of the peptide. The peptide may comprise a moiety to facilitate or enhance uptake by brain cells, such as a folate moiety (for example 5-methyltetrahydrofolate) to bind to the folate receptor or sugar moiety (for example glucose or N-acetyl-galactosamine) to bind to class I hexose (glucose) transporters.Linkers

[0069] Different functional moieties of peptides disclosed herein, such as cell-penetrating peptides disclosed herein and therapeutic peptides, may be joined by linkers, e.g. to form fusion peptides. As used herein, the terms "linker" and “spacer” refer to any molecule or group of molecules that binds or joins two components and may be used interchangeably. Linkers may comprise functional groups enabling the conjugation of active agents to the peptide. Linkers or spacers may provide for optimal spacing of components of the fusion peptide, for example providing flexibility to the construct and enabling each component of the construct, including any active agent conjugated to the fusion peptide, to interact with its respective target, and optimise or increase cell uptake and activity. Linkers may also provide for optimal conformation of the fusion peptide, for example by providing flexibility and optimal spacing of each component of the peptide and by facilitating and / or participating in intramolecular bonds, such as intramolecular disulphide bonds formed between cysteine residues which support the peptide conformation. Without wishing to be bound by theory, it is thought that these secondary structural features make peptides according to embodiments of the present invention exceptionally resistant to enzymatic degradation, hydrolysis, thermal denaturation and chemical modification, providing them with high stability and a long half-life.

[0070] The linkers of the present disclosure may, in preferred embodiments, be in the form of peptide sequences. In some particular embodiments, the linker is a sequence of from 5 to 50, 10 to 30, or 15 to 25 amino acid residues in length, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid residues in length. In some particularly preferred embodiments, the linker ("L") is a sequence according to SEQ ID NO:6, SEQ ID NO: 10 or SEQ ID NO: 11, or a sequence having at least or about 70%, 80% or 90% sequence identity thereto (e.g. having 1, 2, 3 or 4 amino acid substitutions, deletions or additions):CRRDSDCPGACICRGNGYC [SEQ ID NO:6]CRRDSDCPGACICRGGYC [SEQ ID NO: 10]CRRDSDCPGACICRGNSGYC [SEQ ID NO: 11]Cyclic peptides

[0071] The peptides of the present disclosure may be linear or cyclic. In particular embodiments, peptides of the present disclosure are cyclic peptides. Cyclic peptides, also known as cyclotides, are typically formed through a head to tail cyclisation of a peptide backbone, which may be further stabilized by intramolecular bonds, such as intramolecular disulphide bonds formed between cysteine residues which support its cyclic structure. Without wishing to be bound by theory, it is thought that these secondary structural features make cyclotides exceptionally resistant to enzymatic degradation, hydrolysis, thermal denaturation and chemical modification. The present inventors have found that cyclic peptides according to certain embodiments of the present invention exhibit a longer half-life and low immunogenicity in comparison to some linear peptides also investigated, thought to be due to the stability provided by their secondary structure (as described above) and the fact that cyclic peptides are not typically substrates for proteases.

[0072] Various means of providing cyclised peptides will be known and available to a skilled person. In particular embodiments, the peptide may be cyclized through N-to-C cyclization (head to tail cyclization), preferably through an amide bond (i.e. an amide bond between the N- and C- termini of the linear peptide). Such peptides do not possess N- or C-terminal amino acid residues. In particular embodiments, the peptides of the present disclosure have an amide-cyclized peptide backbone. In other embodiments, the peptides of the present disclosure are cyclized using sidechain to side-chain cyclization, such as through a disulfide bond or a lactam bridge.

[0073] In some embodiments, the N- and C-termini are linked using a linking moiety. The linking moiety may be a peptide linker such that cyclization produces an amide-cyclized peptide backbone. Variation within the peptide sequence of the linking moiety is possible, such that the linking moiety may be modified to alter the physicochemical properties of the peptides and potentially reduce side effects of the peptides of the present disclosure or otherwise improve the therapeutic use of the peptides, for example, by improving stability. The linking moiety will be of suitable length to span the distance between the N- and C-termini of the peptide without substantially altering the structural conformation of the peptide, for example, a peptidic linkingmoiety may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues in length. In some embodiments, longer or shorter peptidic linking moieties may be required. In alternative embodiments, the peptide is an acyclic molecule.

[0074] As described above, intramolecular bonds may form cross-links within a cyclic, or acyclic, structure. While any covalent cross-links, including chemical linkers, are envisaged, suitable covalent cross-links include, but are not limited to, a disulfide bond, a diselenide bond, a lanthionine bridge, an amide bond, an ester linkage, a thioester linkage, a carbamoyl linkage, an alkyl linkage, an alkenyl linkage, an ether linkage, a thioether linkage, an amine linkage or a thioamide linkage.

[0075] A skilled person will be well aware of suitable amino acids which will allow the formation of the covalent cross-links. For example, in some embodiments, amino acids in positions suitable for participating in cross-linking are independently selected from the group consisting of C, K, R, D, E, penicillamine, selenocysteine, diaminopropionic acid, mercaptoproline and modified forms thereof; especially C, K, R, D, E, penicillamine, selenocysteine, diaminopropionic acid or mercaptoproline; more especially C, K, R, D, E, penicillamine, selenocysteine or di aminopropionic acid; most especially C, K, R, D or E.

[0076] For example, cysteine residues may be included for formation of a disulfide bond, acidic (e.g. D or E) and basic residues (e.g. R or K) may be included for formation of a lanthionine bridge, selenocysteine residues may be included for formation of a diselenide bond, penicillamine residues may be included for formation of a disulfide bond or for formation of a thioether linkage with 2-methylbenzoyl, for example, and diaminopropionic acid may be included to participate in a lanthionine bridge.

[0077] In some particular embodiments, the linker(s) present in the peptide facilitate the formation of a cyclic peptide, by use of a linker which includes amino acids, such as cysteine residues, in positions which allow the formation of intramolecular interactions such as cross links, in particular such as disulphide bridges across the cycle. The exemplary linker of SEQ ID NO:6 facilitates the formation of such cross-linking disulphide bridges in the cyclic peptide, for example as depicted in Figure 1 with respect to exemplary peptide c5R-NR2B9c. Other linkers that facilitate the formation of a cyclic peptide that comprises, for example, the peptide of SEQID NO: 1 and the peptide of SEQ ID NO:2, include the linker of SEQ ID NO: 11, and further examples are known or can be developed by those skilled in the art.

[0078] Similarly, according to some alternative embodiments, the linker(s) present in the peptide facilitate the formation of an acyclic peptide (i.e. not head-to-tail cyclised) which has a secondary structure and spatial conformation similar to the cyclic peptides described herein, by use of a linker which includes amino acids, such as cysteine residues, in positions which allow the formation of intramolecular interactions such as cross links, in particular such as disulphide bridges across the peptide secondary structure. The exemplary linker of SEQ ID NO: 10 may facilitate the formation of such cross-linking disulphide bridges in an acyclic peptide, such as a peptide comprising the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO:2, in the form of exemplary peptide Ac5R-NR2B9c (SEQ ID NO: 13).

[0079] Accordingly, in some embodiments, linear peptides according to the invention (i.e. peptides which are not head-to-tail cyclised) have a cyclic-like conformation (i.e. wherein the N- and C- termini are held in proximity to one another). Typically said conformation is supported by intramolecular bonds, for example intramolecular disulphide bonds, as described above and as depicted in Figure 1 for the cyclic peptide c5R-NR2B9c. Peptides of the present disclosure may, in some embodiments, comprise one, two, or three intramolecular bonds such as disulphide bonds, for example three intramolecular bonds, for example three disulphide bonds.

[0080] It will be appreciated that, when a peptide has a cyclic structure, functional moieties of a fusion peptide may be joined by a linker at one respective end, and may be directly joined (such as by a direct N-C amide bond) at their other respective end within the cyclic structure. Alternatively, more than one linker may be presentjoining the functional moieties of the peptide at both ends within a cyclic structure.

[0081] In particular embodiments, the peptide is a peptide according to SEQ ID NO:7:CPRILRRRRCRRDSDCPGACICRGNGYCKLSSIESDV [SEQ ID NO:7] or SEQ ID NO:8:CPRILRRRRCRRDSDCPGACICRGNGYCKLSSIESDI [SEQ ID NO:8];or SEQ ID NO: 12:CPRILRRRRCRRDSDCPGACICRGNSGYCKLSSIESDV [SEQ ID NO: 12].

[0082] In such embodiments, the peptides are typically cyclic peptides. In said cyclic peptides according to SEQ ID NO:7 and SEQ ID NO:8, cross-linking disulphide bridges are present between cysteine resides at positions 1 and 20, 10 and 22, 16 and 28. One such embodiment with a cyclic structure is shown in Figure 1. A cyclic peptide having the amino acid sequence shown in SEQ ID NO:7 is also referred to herein as c5R-NR2B9c. A cyclic peptide having the amino acid sequence shown in SEQ ID NO: 12 is also referred to herein as “c5R- NR2B9c with intein” or “c5R-NR2B9c intein”.

[0083] In some particular embodiments, the peptide is a peptide according to SEQ ID NO: 13:GYCKLSSIESDVCPRILRRRRCRRDSDCPGACICRG [SEQ ID NO: 13],

[0084] A peptide according to such an embodiment is typically a linear (i.e. non-cyclised) peptide. A linear peptide having the amino acid sequence shown in SEQ ID NO: 13 is also referred to herein as “Ac5R-NR2B9c”.

[0085] Embodiments of the disclosure contemplate derivatives of peptide sequences disclosed herein. As used herein the term "derivative" is intended to encompass chemical modification to a peptide or one or more amino acid residues of a peptide, including chemical modification in vitro, for example by introducing a group in a side chain in one or more positions of a peptide, such as a nitro group in a tyrosine residue or iodine in a tyrosine residue, by conversion of a free carboxylic group to an ester group or to an amide group, by converting an amino group to an amide by acylation, by acylating a hydroxy group rendering an ester, by alkylation of a primary amine rendering a secondary amine, or linkage of a hydrophilic moiety to an amino acid side chain. Other derivatives may be obtained by oxidation or reduction of the side-chains of the amino acid residues in the peptide. Modification of an amino acid may also include derivation of an amino acid by the addition and / or removal of chemical groups to / from the amino acid, and may include substitution of an amino acid with an amino acid analog (such as a phosphorylated or glycosylated amino acid) or a non-naturally occurring amino acid such asa N-alkylated amino acid (e.g. N-methyl amino acid), D-amino acid, 0-amino acid or y-amino acid.

[0086] The peptide sequences comprising the peptides of the present disclosure may be produced using any method known in the art, including synthetically or by recombinant techniques such as expression of nucleic acid constructs encoding the components in host cells (e.g. bacteria, yeast, plant or mammalian cells). For example a peptide may be synthesized using the Fmoc-polyamide mode of solid-phase peptide synthesis. Other synthesis methods include solid phase t-Boc synthesis and liquid phase synthesis. Purification can be performed by any one, or a combination of, techniques such as re-crystallization, size exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography and reverse-phase high performance liquid chromatography using, for example, acetonitrile / water gradient separation. Fusion peptides may be synthesised as a single peptide sequence, including where appropriate one or more amino acid or peptide linkers. Alternatively, components of fusion peptides may be synthesised individually and may be conjugated or linked post-synthesis by suitable techniques known to those skilled in the art.

[0087] A fusion peptide of the present disclosure may be produced when two or more heterologous nucleotide sequences encoding each component of the fusion peptide, optionally including nucleotides sequences encoding a linker or spacer amino acid(s), are fused together in the correct translational reading frame and are expressed. Accordingly, the present disclosure also provides isolated nucleic acid molecules encoding peptides and fusion peptides and components thereof as described herein.

[0088] The present disclosure also provides vectors comprising a nucleotide sequence(s) encoding peptide sequences and fusion peptides described herein. Typically the nucleotide sequence(s) is operably linked to a promoter to allow for expression of the peptide or fusion peptide. The vectors can be episomal vectors (z.e., that do not integrate into the genome of a host cell), or can be vectors that integrate into a host cell genome. Vectors may be replication competent or replication-deficient. Exemplary vectors include, but are not limited to, plasmids, cosmids, and viral vectors, such as adeno-associated virus (AAV) vectors, lentiviral, retroviral, adenoviral, herpesviral, parvoviral and hepatitis viral vectors. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

[0089] In some embodiments, the peptides of the invention may be cyclized. Cyclization may be performed using several techniques, for example, as described in Davies (2003) J P ept Sci, 9: 471-501; or Thongyoo etal. (2006) Chem Commun (Camb), 27: 2848-2850. For example, N-to-C cyclization may be conducted in the solution phase, using a dilute solution of the linear peptide in the presence of a coupling agent such as BOP (1-benzotriazole-tris-dimethyl aminophosphonium hexafluorophosphate), PyBOP (1-benzotriazolyloxy-tris-pyrrolidino phosphonium hexafluorophosphate), PyAOP (7-azabenzotriazol-l-yloxy tris pyrrolidino phosphonium hexafluorophosphate), AOP (7-azabenzotriazol-l-yloxy-tris-dimethyl aminophosphonium hexafluorophosphate), HBTU (O-(benzotriazol-l-yl)-l,l,3,3-tetramethyl uronium hexafluorophosphate), TBTU (O-(benzotriazol-l-yl)-l,l,3,3-tetramethyl uronium tetrafluoroborate), HATU (O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyl uronium hexafluorophosphate), HAPyU (O-(7-azabenzotriazol-l-yl)-l,l,3,3-tetramethylene uronium hexafluorophosphate), HAPipU (O-(7-azabenzotriazol-l-yl)-l,l,3,3-pentamethylene uranium hexafluorophosphate), DCC (A,A-dicyclohexylcarbodiimide), DIC (7V,7V- diisopropylcarbodiimide), and / or EDC [l-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride]. The cyclized peptide may then be deprotected (i.e. the side chain protecting groups may then be removed) using standard techniques, followed by purification using suitable methods, such as preparative chromatography. Alternatively, N-to-C cyclization may be achieved on resin using a suitable coupling agent, such as those described above, and a suitable resin, such as a Kaiser oxime resin, and / or linker (e.g. a safety catch linker), or via native chemical ligation as described in Thongyoo et al. (2006) Chem Commun (Camb), 27: 2848-2850.Methods and uses

[0090] As exemplified herein, the inventors have developed cell membrane and blood-brain barrier penetrating peptide sequences. Accordingly, provided herein are methods for effecting transport of an agent across a cell membrane and / or across the blood brain barrier, comprising delivering a peptide comprising a cell membrane or blood-brain barrier penetrating peptide sequence described herein (e.g. one comprising a sequence set forth in SEQ ID NO: 1) to the cell or blood brain barrier (or contacting a cell membrane or blood-brain barrier penetrating peptide sequence with a cell or blood brain barrier). In some examples, as described above, the peptide is linked to an additional active agent. Thus, provided are methods for delivering an active agent across a cell membrane and / or across the blood brain barrier, by contacting the agent with (or delivering the agent to) a cell or a blood brain barrier, wherein the agent is linked to a peptidecomprising a cell membrane and / or blood-brain barrier penetrating peptide sequence described herein. Optionally, the method includes first linking the active agent to the peptide comprising a cell membrane or blood-brain barrier penetrating peptide sequence. In some particular embodiments, the peptide and the additional active agent are in the form of a cyclic peptide, as described above. In alternative embodiments, the peptide and the additional active agent are in the form of a linear (non-cyclised) peptide, as described above.

[0091] The inventors have also developed therapeutic sequences for conditions associated with excitotoxicity, such as Alzheimer's disease. The present inventors have incorporated such peptide sequences into linear and cyclic peptides, including linear and cyclic peptides also comprising a cell membrane and blood-brain barrier penetrating peptide sequence, such as the cell membrane and blood-brain barrier penetrating peptide sequences of the present disclosure. In particular embodiments, the present inventors have incorporated such therapeutic peptide sequences into fusion peptides to provide stable therapeutic peptides with long half-lives, low immunogenicity and which are effective capable of crossing the blood-brain barrier for use in therapy. In some exemplary embodiments, such stable peptide sequences are cyclic peptides (such as cyclic peptides also comprising a cell membrane and blood-brain barrier penetrating peptide sequence of the present disclosure). In some alternative embodiments, such peptides are linear (i.e. non-cyclised).

[0092] Accordingly, provided herein are methods for the treatment or prevention of a disorder or condition, for example a disease or disorder associated with excitotoxicity and / or a neurological condition. Peptides according to embodiments of the invention find particular use in the treatment and / or prevention of conditions associated with neuronal hyperexcitation- associated neurotoxicity. In particular embodiments, the condition is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Parkinson's disease (PD), epilepsy, seizure or a condition associated with or characterised by seizures, or stroke (including neuronal damage due to stroke). Said methods comprise administering to a subject in need thereof an effective amount of a peptide according to the present invention. The subject may be suffering from, having, predisposed to, or at risk of developing a disease or condition, such as those described above.

[0093] For in vivo methods contemplated herein, the route of the administration of the peptides (including fusion peptides) is not limited. For example, an effective amount of a peptide 1can be administered to a subject via, for example, intravitreal, intrathecal, intracerebroventricular, intravenous, intraperitoneal, subcutaneous, epicutaneous, intradermal, intramuscular, pulmonary, oral, buccal, or nasal routes. In exemplary embodiments, the peptide may be administered by intravenous administration. The peptide can be administrated as a single dose or multiple doses, and at varying intervals. In particular embodiments administration may comprise multiple doses be for a period of days, weeks or months, depending on a variety of the peptide administered, the route of administration, the severity of the condition to be treated and the general health and wellbeing of the subject.

[0094] Those skilled in the art will appreciate that in accordance with the methods of the present disclosure peptides described herein may be administered alone or in conjunction with one or more additional compounds or agents for treating the disease or condition that is to be treated using the peptide of the disclosure. For such combination therapies, each component of the combination therapy may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect. Alternatively, the components may be formulated together in a single dosage unit as a combination product. When administered separately, it may be preferred for the components to be administered by the same route of administration, although it is not necessary for this to be so.

[0095] The peptides are typically administered to subjects in the form of pharmaceutical compositions. Suitable compositions for use with the methods of the invention may be prepared according to methods and procedures that are known to those of ordinary skill in the art and accordingly may include pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants. The diluents, adjuvants and excipients should be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.

[0096] Examples of pharmaceutically acceptable carriers, diluents excipients and / or adjuvants include demineralised or distilled water; saline solution; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween™, Pluronics™ or polyethylene glycol (PEG); silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones;mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3 -butylene glycol or glycerin; and fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate.

[0097] One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of a pharmaceutical composition which would be required to achieve the desired therapeutic effect. For example, an effective dosage may be in the range of about O.OOOlmg to about lOOOmg per kg body weight per 24 hours; typically, about O.OOlmg to about 750mg per kg body weight per 24 hours; about O.Olmg to about 500mg per kg body weight per 24 hours; about O. lmg to about 500mg per kg body weight per 24 hours; about O. lmg to about 250mg per kg body weight per 24 hours; about 1.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about 1.Omg to about 200mg per kg body weight per 24 hours; about 1.Omg to about lOOmg per kg body weight per 24 hours; about l.Omg to about 50mg per kg body weight per 24 hours; about l.Omg to about 25mg per kg body weight per 24 hours; about 5. Omg to about 50mg per kg body weight per 24 hours; about 5. Omg to about 20mg per kg body weight per 24 hours; about 5. Omg to about 15mg per kg body weight per 24 hours.

[0098] The present disclosure further relates to use of the peptides according to the present disclosure in the manufacture of a medicament for the treatment or prevention of conditions as described above. The present disclosure further relates to peptides according to the present disclosure for use in the treatment or prevention of conditions as described above.

[0099] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0100] The present disclosure will now be described with reference to the following specific examples, which should not be construed as in any way limiting the scope of the disclosure.Examples

[0101] The following examples are illustrative of the disclosure and should not be construed as limiting in any way the general nature of the disclosure of the description throughout this specification.Example 1 — Peptide synthesis

[0102] Linear peptides were synthesised using an automated solid phase peptide synthesiser (Liberty Blue, CEM) based on Fmoc-chemistry using diisopropylcarbodiimide (DIC) and Oxyma as coupling reagent. Peptides to be cyclised were cleaved with 20% HFIP in DCM for 1.5 h to yield protected linear peptides. Thioesterification of the C-terminus was performed with PyBOP (5 eq.), p-acetamidothiophenol (15 eq.), and DIPEA (0.2 mL) in DCM overnight. Peptides were then subjected to global deprotection with TFA / TIPS / H2O (95 / 2.5 / 2.5) for 3 hours and precipitated to cold diethyl ether. The resulting residue was lyophilised.

[0103] The linear Ac5R peptides were cleaved with 92.5% TFA, 2.5% TIPS, 2.5% water, 2.5% thioanisole for 3.5h to yield linear peptides. Cleaved linear peptides were precipitated in diethyl ether to yield crude peptides.

[0104] Cyclisation and oxidation of the cyclic peptides was achieved by incubating the linear peptides in ammonium bicarbonate (0.1M, pH 8.0) in isopropanol for 24 hours. Crude peptides were purified by semi-preparative HPLC using an acetonitrile / water gradient.

[0105] Oxidation of linear Ac5R peptides was achieved by incubating the linear Ac5R peptides in 90% morpholine / 10% DMSO solution with 100 equivalents of cysteamine and 10 equivalents of cystamine for 4 hours. Crude peptides were purified by semi-preparative HPLC using an acetonitrile / water gradient.Example 2 — Peptide uptake and stability

[0106] To determine the ability of neurons to internalise the peptides, primary neurons at DIV5 (days in vitro.5) were first transferred to microscope incubation chambers at 37 °C, 5% CO2 and left to stabilise for 30 minutes. Peptides were then added directly into the primary neuron media. Peptide intake was imaged every minute at 550 nM wavelengths (Zeiss LSM 880). Asshown in Figure 2, fluorescent (TAMRA)-labelled cyclic peptide c5R-NR2B9c is immediately taken up and internalised by neurons. Contacting neurons with 0.5 pM and 0.1 pM c5R-NR2B9c resulted in transduction levels of 80% and 70%, respectively (Figure 3).

[0107] To determine peptide stability, TAMRA-labelled peptides c5R-NR2B9c and Tat- NR2B9c (100 pM) were spiked in mice plasma and incubated at 37 °C. Plasma was collected at 0, 15, 30, 60, 120 and 300 min time points. At each time point, 100 pL of plasma was collected. Plasma samples were stored at -80°C until analysed. Peptide contents were analysed with HPLC at 550 nM (for TAMRA detection). Enhanced stability of c5R-NR2B9c relative to linear Tat- NR2B9c over at least 5 hours is clearly shown in Figure 4.Example 3 — Excitoxicity and in vivo studies

[0108] Primary cortical and hippocampal neurons were cultured from El 6.5 embryonic brains at a density of 220,000 cells / cm2as described previously (Fath et al., 2009 Nature Protocols 4:78-85). At DIV11, primary neurons were pre-treated with Tat-NR2B9c, c5R- NR2B9c, Ac5R-NR2B9c or c5R-NR2B9c intein, or the double alanine-substituted controls Tat- NR2Baa, c5R-NR2Baa or c5R-NR2Baa intein, 1 hour prior to N-methyl D-aspartate (NMDA) exposure. After 1 hour, media were replaced with fresh media. Cells were then incubated for 24 hours. Dead and dying cells were visualised using propidium iodide (PI) uptake that was added to the medium 5 min prior to fixation with PFA. DAPI was used to stain all cells. As shown in Figure 5, cells exposed to c5R-NR2B9c, Ac5R-NR2B9c, c5R-NR2B9c intein and Tat-NR2B9c peptides resulted in significantly reduced dead and dying cells relative to vehicle (DMSO) and cells exposed to the control peptides c5R-NR2B9aa, c5R-NR2Baa intein and Tat-NR2Baa.

[0109] C57BL6 mice were pre-treated with peptide (either c5R-NR2B9c, Tat-NR2B9c or c5R-NR2B9aa) via tail-vein injection (i.v., 3 nmol / g) or oral gavage (300 nmol / g). After 1 hour, mice were administered pilocarpine (100 mg / g, i.v.) to induce seizure (status elipticus). Seizure severity and mouse survival were monitored for 2 hours following pilocarpine administration. Both intravenous and oral administration of c5R-NR2B9c significantly reduced seizures (Figure 6).

[0110] Primary cortical and hippocampal neurons were cultured from E16.5 embryonic brains at a density of 220,000 cells / cm2(Fath et al., 2009 Nature Protocols 4:78-85). At DIV11,primary neurons were pre-treated with Tat-NR2B9c, or a variant of Tat-NR2B9c containing a single amino acid substitution. Tat-NR2B9c variants were generated with an amino acid substitution at either position 7 of the NR2B9 component (SEQ ID NO:3), to generate variant peptides Tat-NR2B9c S7T and Tat-NR2B9c S7Y, or at position 9 of the NR2B9 component (SEQ ID NO:3), to generate variant peptides Tat-NR2B9c V9I and Tat-NR2B9c V9L. The primary neurons were pre-treated with the Tat-NR2B9c, or variant peptide NR2B9c S7T, Tat- NR2B9c S7Y, Tat-NR2B9c V9I or Tat-NR2B9c V9L, 1 hour prior to N-methyl D-aspartate (NMDA) exposure. After 1 hour, media were replaced with fresh media. Cells were then incubated for 24 hours. Dead and dying cells were visualised using propidium iodide (PI) uptake that was added to the medium 5 min prior to fixation with PF A. DAPI was used to stain all cells. As shown in Figure 7, cells exposed to Tat-NR2B9c V9I peptide resulted in significantly reduced dead and dying cells relative to the original Tat-NR2B9c peptide.

[0111] Tau (AplhaFold PDB: AF-P10636-F1) protein-protein interaction with PSD-95 (PDB: 5YPR) and Fyn (PDB: 1G83) was predicted using Schrodinger Bioluminate with parameter of 70000 ligands rotation and no restraints. The interaction region of tau-PSD95 and tau-Fyn (circled in Figure 8) indicate a likely binding region of the cyclotides.Table 1. Peptide sequences disclosed herein

Claims

Claims1. A peptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 :CPRILXi [SEQ ID NO:!] wherein Xi is a chain of 3 or more R residues.

2. The peptide according to claim 1, wherein Xi is a chain of at least 4 R residues.

3. The peptide according to claim 2, wherein Xi is a chain of 4 R residues.

4. The peptide according to any one of claims 1 to 3, wherein the peptide acts as a cell membrane permeability agent, a blood-brain barrier permeability agent, or both as a cell membrane permeability agent and a blood-brain barrier permeability agent.

5. The peptide according to any one of claims 1 to 4, further comprising an additional active agent selected from a therapeutic agent, a diagnostic agent, a detection agent or an agent for imaging biological tissue.

6. The peptide according to claim 5, wherein the additional active agent is a therapeutic agent.

7. The peptide according to claim 6, wherein the therapeutic agent is therapeutic for a condition associated with neuronal hyperexcitation-associated neurotoxicity.

8. The peptide according to claim 7, wherein the condition is a neurological condition.

9. The peptide according to claim 8, wherein the condition is Alzheimer's disease.

10. The peptide according to any one of claims 6 to 9, wherein the therapeutic agent comprises a therapeutic peptide comprising an amino acid sequence as set forth in SEQ ID NO:2:KLX2X3IESX4X5 [SEQ ID NO:2] wherein:X2, X3 and X4 are any amino acid residue; andX5 is selected from V and I.

11. A peptide comprising an amino acid sequence as set forth in SEQ ID NO:2:KLX2X3IESX4X5 [SEQ ID NO:2] wherein:X2, X3 and X4 are any amino acid residue; andX5 is selected from V and I, provided that SEQ ID NO:2 is not KLSSIESDV [SEQ ID NO:3].

12. The peptide according to claim 11, further comprising an additional active agent selected from a therapeutic agent, a diagnostic agent, a detection agent or an agent for imaging biological tissue, a cell membrane permeability agent and a blood-brain barrier permeability agent.

13. The peptide according to claim 12, wherein the additional active agent facilitates both cell membrane permeability and blood-brain barrier permeability.

14. The peptide according to any one of claims 10 to 13, wherein X2 is S or T.

15. The peptide according to claim 14, wherein X2 is T.

16. The peptide according to any one of claims 10 to 15, wherein X3 is S or T.

17. The peptide according to claim 16, wherein X3 is T.

18. The peptide according to any one of claims 10 to 17, wherein X4 is D or E.

19. The peptide according to claim 18, wherein X4 is E.

20. The peptide according to any one of claims 10 to 19, wherein X5 is V or I.

21. The peptide according to claim 20, wherein X5 is I.

22. The peptide according to any one of claims 5 to 10, further comprising a linker sequence L connecting SEQ ID NO: 1 and the additional active agent.

23. The peptide according to claim 22, wherein the additional active agent is located at or adjacent to the C-terminal of the sequence of SEQ ID NO: 1 separated from the sequence of SEQ ID NO: 1 by the linker sequence L.

24. The peptide according to claim 12, further comprising a linker sequence L connecting SEQ ID NO:2 and the additional active agent.

25. The peptide according to claim 24, wherein the additional active agent is located at or adjacent to the N-terminal of the sequence of SEQ ID NO:2 separated from the sequence of SEQ ID NO:2 by the linker sequence L.

26. The peptide according to any one of claims 22 to 25, wherein the linker sequence L comprises a sequence of from 15 to 25 amino acid residues in length.

27. The peptide according to claim 26, wherein the linker sequence L comprises a sequence of 18, 19 or 20 amino acid residues in length.

28. The peptide according to any one of claims 23 to 27, wherein the linker sequence L comprises an amino acid sequence as set forth in SEQ ID NO:6, SEQ ID NO: 10 or SEQ ID NO: 11 or a sequence having at least or about 90% sequence identity thereto:CRRDSDCPGACICRGNGYC [SEQ ID NO:6] CRRDSDCPGACICRGGYC [SEQ ID NO: 10] CRRDSDCPGACICRGNSGYC [SEQ ID NO: 11],29. The peptide according to any one of claims 23 to 28, comprising an amino acid sequence as set forth in SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 13:CPRILRRRRCRRDSDCPGACICRGNGYCKLSSIESDV [SEQ ID NO:7] CPRILRRRRCRRDSDCPGACICRGNGYCKLSSIESDI [SEQ ID NO:8]. CPRILRRRRCRRDSDCPGACICRGNSGYCKLSSIESDV [SEQ ID NO: 12] GYCKLSSIESDVCPRILRRRRCRRDSDCPGACICRG [SEQ ID NO: 13],30. The peptide according to any one of claims 1 to 29, which is a cyclic peptide.

31. The peptide according to any one of claims 1 to 30, which is an isolated peptide.

32. A method of delivering an active agent across a cell membrane and / or across the blood brain barrier, comprising contacting the active agent with or delivering the active agent to a cell or the blood brain barrier, wherein the active agent is linked to a peptide comprising a cell membrane or blood-brain barrier penetrating peptide sequence according to SEQ ID NO: 1.

33. A method of treating or preventing a condition associated with neuronal hyperexcitation- associated neurotoxicity, comprising administering to a subject in need thereof an effective amount of a peptide according to any one of claims 6 to 29, or a peptide according to claim 30 or 31 when dependent on any one of claims 6 to 29.

34. The method according to claim 33, wherein the condition is a neurological condition.

35. The method according to claim 34, wherein the condition is Alzheimer's disease.

36. Use of a peptide according to any one of claims 6 to 29, or a peptide according to claim 30 or 31 when dependent on any one of claims 6 to 29, in the manufacture of a medicament for the treatment or prevention of a condition associated with neuronal hyperexcitation-associated neurotoxicity.

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