ANG-2b9c peptide for stroke treatment with an enhanced therapeutic index

Abstract

The invention is based on linking PSD-95 inhibitors to an internalization peptide angiopep-2 (Ang) and variants thereof. The present examples show that such linkage results in reduced drop in blood pressure, if any, and reduced histamine release following intravenous administration compared with nerinetide, which incorporates a tat internalization peptide. The reduction in blood pressure drop and histamine release means that active agents incorporating Ang can be administered at higher doses than nerinetide and / or without an anti-inflammatory agent when a comparable dose of nerinetide would require co-administration of an anti-inflammatory agent.

Description

ANG-2B9c PEPTIDE FOR STROKE TREATMENT WITH AN ENHANCED THERAPEUTIC INDEX

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of US 63 / 902,492, filed October 20, 20025, and 63 / 789,431 filed April 16, 2025, each incorporated by reference in its entirety for all purposes.

[0003] SEQUENCE LISING

[0004] The application includes sequences in an electronic sequence listing named 650123SQLST. XML of 29,300 bytes created April 6, 2026.

[0005] BACKGROUND

[0006] Tat-NR2B9c (also known as NA-1 and nerinetide) is an agent that inhibits PSD-95, thus disrupting binding to N-methyl-D-aspartate receptors (NMDARs) and neuronal nitric oxide synthases (nNOS) and reducing excitotoxicity induced by cerebral ischemia. Treatment reduces infarction size and functional deficits in models of cerebral injury and neurodegenerative diseases. Tat-NR2B9c has undergone a successful phase II trial (see WO 2010144721 and Aarts et al., Science 298, 846-850 (2002), Hill et al., Lancet Neurol. 11:942-950 (2012)) and a successful Phase 3 trial (Hill et al, Lancet 395:878-887 (2020)).

[0007] WO2021140485 and WO 2022 / 150655 describe D-amino acid variants of Tat- NR2B9c conferring resistance to plasmin thereby facilitating combination treatment with a thrombolytic agent that induces plasmin.

[0008] SUMMARY OF THE CLAIMED INVENTION

[0009] The invention provides an active agent comprising an internalization peptide comprising the sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1), or having up to five substitutions, deletions or insertions, or a variant of any of these substituted with one or more D-, beta, N-methyl or alpha-methyl amino acids, linked to a PSD-95 inhibitor. Optionally, the internalization peptide has 3-7 D-amino acids. Optionally, the internalization peptide has 3-5 D-amino acids at non-adjacent positions. Optionally, the internalization peptide has 2-5 D-amino acids at non-adjacent positions. Optionally, the non-adjacent positions are R or K amino acids or amino acids C-terminal thereto. Optionally, the internalization peptide has 2 or 3 D-amino acid substitutions of R and K residues at non-adjacent positions Optionally, the PSD-95 inhibitor comprises IX1-[T / S]-X2V (SEQ ID NO:2) or [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ IDNO: 12) at the C-terminus. Optionally, the PSD-95 inhibitor comprises IESDV (SEQ ID NO:3) or IETDV (SEQ ID NO:4) at the C-terminus. Optionally, the PSD-95 inhibitor comprises KLSSIESDV (SEQ ID NO:5) or KLSSIETDV (SEQ ID NO:6). Optionally, the PSD-95 inhibitor comprises kLSSIESDV (SEQ ID NO:7) or KlSSIESDV (SEQ ID NO:8) or klSSIESDV (SEQ ID NO:9). Optionally, the active agent has an amino acid sequence comprising, consisting of or consisting essentially of TFFYGGSRGkRNNFkTEEYkLSSIESDV (SEQ ID NO: 10) or TFFYGGSrGKrNNFkTEEYkLSSIESDV (SEQ ID NO: 11)). Optionally, the C-terminus of the internalization peptide is linked to the N-terminus of the PSD-95 inhibitor. Optionally, the active agent is Ang-2B9c A6 or Ang-2B9c A7. Optionally, the active agent is in dimeric form.Optionally, the active agent is characterized by reduced drop in blood pressure and / or histamine release relative to nerinetide.

[0010] The invention further provides a pharmaceutical composition comprising an active agent as described herein and a pharmaceutically acceptable carrier.

[0011] The invention further provides a method of treating a subject having or at risk of a condition selected from stroke, cerebral ischemia, traumatic injury to the CNS, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy, or a neurodegenerative disease comprising administering an effective regime of an active agent described herein.Optionally, the condition is stroke. Optionally, the method further comprises effecting reperfusion in the subject, optionally by administering a thrombolytic agent, by mechanical reperfusion, or endovascular thrombectomy. Optionally, the active agent is administered at a dose of at least 10 mg / kg without co-administration of an anti-inflammatory. Optionally, the active agent is administered at a dose of at least 25 mg / kg without co-administration of an antiinflammatory.

[0012] The invention further provides an internalization peptide comprising the sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO:1), or having up to five substitutions, deletions or insertions, substituted with one or more D-, beta, N-methyl or alpha-methyl amino acids.Optionally, the internalization peptide comprises the sequence of SEQ ID NO: 1 substituted with 2 or 3 D-, beta, N-methyl or alpha-methyl amino acids. Optionally, The internalization peptide is linked to a heterologous molecule.

[0013] BRIEF DESCRIPTIONS OF THE FIGURES

[0014] Figs. 1, 2 and 3 show blood pressure, CMax and histamine release following intravenous injection of Ang-2B9c compared to nerinetide.

[0015] Figs. 4A and 4B show Ang-2B9c competes with nerinetide for binding to PSD-95 PDZ domain 2.

[0016] Fig. 5 shows Ang-2B9c is cleaved by plasmin similarly to nerinetide.

[0017] Fig 6. shows the deconvoluted mass spectra of ANG-2B9c digestion products after incubation with plasmin.

[0018] Fig. 7 shows that Ang-2B9c dissociates pre-formed GluN2B: PSD95 PDZ2 complexes in rat brain lysates.

[0019] Fig. 8 shows Ang-2B9c reduces infarct size.

[0020] Figs. 9A and 9B show that Ang-2B9c improves neurological outcome at 4 hr and 24 hr after induction of ischemia.

[0021] Fig. 10 shows Ang-2B9c A6 and A7 D-amino acid variants are resistant to cleavage by plasmin (TFFYGGSRGkRNNFkTEEYkLSSIESDV (SEQ ID NO: 10)) TFFYGGSrGKrNNFkTEEYkLSSIESDV (SEQ ID NO: 11)).

[0022] Fig. 11 shows Ang-2B9c A6 and A7 D-amino acid variants compete effectively with biotinylated nerinetide (Bio-NA1) for binding to PSD95-PDZ domain 2.

[0023] Fig. 12 shows Ang-2B9c A6 and A7 D-amino acid variants bind to LRP-1 similarly to Ang-2B9c.

[0024] Fig. 13 shows that both ANG2B9c A7 and ANG2B9c significantly reduced infarct volume.

[0025] DEFINITIONS

[0026] A "pharmaceutical formulation" or composition is a preparation that permits an active agent to be effective and lacks additional components which are toxic to the subjects to which the formulation would be administered.

[0027] For an amino acid sequence presented entirely in upper case single-letter codes, the upper case letters can refer to L-amino acids as well as D-amino acids unless the context indicates otherwise. For an amino acid sequence including both upper and lower case letters (e g., TFFYGGSRGkRNNFkTEEYkLSSIESDV (SEQ ID NO: 10)), the upper case letters are L-amino acids and the lower case letters are D-amino acids. Lower case single letter codes areused to indicate D-amino acids. Glycine does not have D- and L-forms and thus can be represented in either upper or lower case interchangeably. When a peptide is said to have a defined number of D-amino acids, glycine is not included in such count of D-amino acids. When a peptide is said to have a stretch of contiguous L-amino acids what is meant is the stretch is uninterrupted by a D-amino acid, but the stretch of amino acids may include glycine unless explicitly indicated otherwise. Such a stretch is also referred to as a contiguous stretch of amino acids selected independently from the group consisting of standard L-amino acids and glycine.

[0028] Numeric values such as concentrations or pH’s are given within a tolerance reflecting the accuracy with which the value can be measured. Unless the context requires otherwise, fractional values are rounded to the nearest integer. Unless the context requires otherwise, recitation of a range of values means that any integer or subrange within the range can be used.

[0029] The terms “disease” and “condition” are used synonymously to indicate any disruption or interruption of normal structure or function in a subject.

[0030] Indicated dosages should be understood as including the margin of error inherent in the accuracy with which dosages can be measured in a typical hospital setting.

[0031] The terms "isolated" or "purified" means that the object species (e.g., a peptide) has been purified from contaminants that are present in a sample, such as a sample obtained from natural sources that contain the object species. If an object species is isolated or purified it is the predominant macromolecular (e.g., polypeptide) species present in a sample (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, an isolated, purified or substantially pure composition comprises more than 80 to 90 percent of all macromolecular species present in a composition. Most preferably, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. The term isolated or purified does not necessarily exclude the presence of other components intended to act in combination with an isolated species. For example, an internalization peptide can be described as isolated notwithstanding that it is linked to an active peptide.

[0032] A "peptidomimetic" refers to a synthetic chemical compound which has substantially the same structural and / or functional characteristics of a peptide consisting of natural aminoacids. The peptidomimetic can contain entirely synthetic, non-natural analogues of amino acids, or can be a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The peptidomimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and / or inhibitory or binding activity. Polypeptide mimetic compositions can contain any combination of nonnatural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural amino acids in place of naturally occurring amino acids; or c) amino acids which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. In a peptidomimetic of a chimeric peptide comprising an active peptide and an internalization peptide, either the active moiety or the internalization moiety or both can be a peptidomimetic.

[0033] The term "specific binding" refers to binding between two molecules, for example, a ligand and a receptor, characterized by the ability of a molecule (ligand) to associate with another specific molecule (receptor) even in the presence of many other diverse molecules, i.e., to show preferential binding of one molecule for another in a heterogeneous mixture of molecules. Specific binding of a ligand to a receptor is also evidenced by reduced binding of a detectably labeled ligand to the receptor in the presence of excess unlabeled ligand (i.e., a binding competition assay).

[0034] Excitotoxicity is the pathological process by which neurons and surrounding cells are damaged and killed by the overactivation of receptors for the excitatory neurotransmitter glutamate, such as the NMDA receptors, e.g., NMDA receptors bearing the NMDAR2B subunit.

[0035] The term "subject" or “patient” includes humans and veterinary animals, such as mammals, as well as laboratory animal models, such as mice or rats used in preclinical studies.

[0036] Co-administration of a pharmacological agents means that the agents are administered sufficiently close in time for detectable amounts of the agents to present in the plasma simultaneously and / or the agents exert a treatment effect on the same episode of a condition or the agents act co-operatively, or synergistically in treating the same episode of a condition. For example, an anti-inflammatory agent acts cooperatively with an agent including a tat peptide when the two agents are administered sufficiently proximately in time that the anti-inflammatory agent can inhibit an anti-inflammatory response inducible by the internalization peptide.

[0037] Statistically significant refers to a p-value that is <0.05, preferably <0.01 and most preferably <0.001.

[0038] An episode of a condition means a period when signs and / or symptoms of the condition are present interspersed by flanked by longer periods in which the signs and / or symptoms or absent or present to a lesser extent.

[0039] The term " NMDA receptor," or " NMD AR," refers to a membrane associated protein that is known to interact with NMDA including the various subunit forms described below. Such receptors can be human or non-human (e.g., mouse, rat, rabbit, monkey).

[0040] Therapeutic index is used in accordance with convention as LD (lethal dose)50 / ED (effective dose)50 in animal studies or TD (toxic dose)50 / ED (effective dose)50 in humans.

[0041] “Comprising,” “consisting of’ and “consisting essentially of’ are each used in accordance with convention. Thus, “comprising” does not exclude additional elements or steps, and “consisting essentially of’ refers to the basic and novel features of an invention. Reference to feature(s) of an object or method in the specification should be understood, unless the context requires otherwise, as support for claiming any of the following alternatives: the object or method comprising, the object or method consisting of, or the object or method consisting essentially of the specified feature(s).

[0042] When the specification refers to a numbered position in the sequence of TFFYGGSRGKRNNFKTEEY (SEQ ID NO:1) or TFFYGGSRGKRNNFKTEEYKLSSIESDV (SEQ ID NO: 12), or other defined sequence, the specification should be understood as also disclosing a corresponding position in a variant of such sequence when the variant is maximally aligned with such sequence.

[0043] Standard amino acids refer to the 20 L-amino acids found in nature. Non-standard amino acid are analogs of one of the standard amino acids, typically conferring improved resistance to protease digestion, such as a D-amino acid, beta amino acid, N-methyl amino acid or alpha-methyl amino acid. Cognate standard and non-standard amino acids (e.g., L-lysine, D-lysine, beta lysine, N-methyl lysine and alpha-methyl lysine) are referred to as amino acids of the same type.

[0044] Bioavailability can be assessed by any pharmacokinetic parameter, such as plasma AUC, CMax, half-life, concentration at a defined time point, such as 1 or 3 hr, or time for plasmaconcentration to decay to zero following administration. Bioavailability can be assessed in a human or animal model, such a non-human primate, dog, rat or mouse.

[0045] Sequence identity is determined by maximally aligning a query sequence with a reference sequence (e.g., TFFYGGSRGKRNNFKTEEY (SEQ ID NO:1) or TFFYGGSRGKRNNFKTEEYKLSSIESDV (SEQ ID NO: 12)) and dividing the number of matched amino acids over the total number of amino acids of the reference. Alignment can be by BLAST (basic local alignment search tool) using default parameters as defined in the national center for biotechnology information web site (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), or by inspection. A standard L- and D-amino acid of the same type are considered a match for this purpose. Likewise, a standard L-amino acid and beta, N-methyl or alpha-methyl variants of the same amino acids are considered as match for this purpose. Flanking amino acids in the variant outside the sequence aligned with the reference are not scored.

[0046] DETAILED DESCRIPTION

[0047] I. General

[0048] The invention is based on linking PSD-95 inhibitors to an internalization peptide angiopep-2 (Ang or ANG) derived from the Kunitz domain of human aprotinin, and variants thereof. The present examples show that such linkage result in reduced drop in blood pressure, if any, and reduced histamine release following intravenous administration compared with nerinetide, which incorporates a tat internalization peptide. The reduced drop in blood pressure and histamine release means that active agents incorporating Ang can be administered at higher doses than nerinetide and / or without an anti-inflammatory agent when a comparable dose of nerinetide would require co-admini strati on of an anti-inflammatory agent.

[0049] II. Active Agents

[0050] Active agents of the invention include an inhibitor peptide specifically binding to PSD-95 (e.g.., Stathakism, Genomics 44(l):71-82 (1997)) so as to inhibit its binding to NMDA Receptor 2 subunits including NMDAR2B (e.g., GenBank ID 4099612) and / or NOS (e.g., neuronal or nNOS Swiss-Prot P29475), and an internalization peptide to facilitate passage of the inhibitor peptide across cell membranes and the blood brain barrier. The inhibitor peptide and / or internalization peptide can each be present in one or multiple copies per molecule of active agent.

[0051] Internalization peptides include angiopep-2 TFFYGGSRGKRNNFKTEEY (SEQ ID NO:1) (di Polidoro et al., Molecules. 2022 Oct 8;27(19):6696. doi:10.3390 / molecules27196696). Internalization peptide also include variants of this sequence having up to five substitutions, deletions or insertions. Variants also include peptides having at least 75%, 80%, 85%, 90%, or 95% sequence identity to this sequence. Variants are preferably conservative substitutions and / or such as to maintain the net charge of the parent sequence (+1), (e.g., 0, +1 or +2), and / or maintain binding to LRP-1 (e.g., within at least 50, 75 or 90% of that of Ang or within a factor of 2 of that of Ang). Any of these variants can have any or all standard L-amino acids (i.e., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19) substituted with D-amino acids or other non-standard amino acids of the same type as the replaced standard L-amino acid. The angiopep-2 internalization peptide and variants thereof can facilitate passage of a linked inhibitor peptide across cell membranes and / or the blood brain barrier. Although an understanding of mechanism is not required for practice of the invention, it is believed the angiopep-2 internalization peptide facilitates such passage by binding to an LRP-1 receptor, which is expressed on endothelial cells of the blood brain barrier. The human form of LRP-1 receptor is assigned UniProtKB accession number Q07954. Variants of ang-2 internalization peptide can be screened for retention of binding to an LRP-1 receptor or extracellular domain thereof, and for retention of facilitating passage of linked peptides across cells expressing LRP-1. Such variants can also be screened for binding to PSD-95, or a PDZ domain 2 thereof, optionally in competition with nerinetide. Such variants can also be screened for plasmin resistance.Preferably variants have at least 50, 75 or 90%, and more preferably the same (within experimental error) or greater relative to Ang of each of the following: binding to an LRP-1 receptor, binding to PSD-95 PDZ domain 2, ability to facilitate passage of linked peptides across cells expressing LRP-1 and plasmin resistance.

[0052] Preferred PSD-95 inhibitor peptides inhibit the human forms of PSD-95 NMD AR 2B and NOS for use in a human subject. However, inhibition can also be shown from species variants of the proteins. Some inhibitor peptides have a C-terminal motif amino acid sequence comprising [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO: 12). Exemplary peptides comprise: ESDV (SEQ ID NO: 14), ESEV (SEQ ID NO: 15), ETDV (SEQ ID NO: 16), ETAV (SEQ ID NO: 17), ETEV (SEQ ID NO: 18), DTDV (SEQ ID NO: 19), and DTEV (SEQ ID NO:20) as the C-terminal amino acids. Some peptides have an amino acid sequence comprising [I]-[E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO:21) at their C-terminus. Exemplary peptides comprise: IESDV (SEQ ID NO:3), IESEV (SEQ ID NO:22), IETDV (SEQ ID NO:4), IETAV (SEQ ID NO:23), IETEV (SEQ ID NO:24), IDTDV (SEQ ID NO:25), and IDTEV (SEQ ID NO:26) as the C-terminal amino acids. Some inhibitor peptides having an amino acid sequence comprising X1-[T / S]-X2V (SEQ ID NO:27) at the C-terminus, wherein [T / S] are alternative amino acids, XI is selected from among E, Q, A, or D or an analogue thereof, X2 is selected from among A, Q, D, E, N, N-Me-A, N-Me-Q, N-Me-D, and N-Me-N or an analog thereof (see Bach, J. Med. Chem. 51, 6450-6459 (2008) and WO 2010 / 004003). Optionally the peptide is N-alkylated in the P3 position (third amino acid from C-terminus, i.e., position occupied by [T / S]). The peptide can be N-alkylated with a cyclohexane or aromatic substituent and further comprises a spacer group between the substituent and the terminal amino group of the peptide or peptide analogue, wherein the spacer is an alkyl group, preferably selected from among methylene, ethylene, propylene and butylene. The aromatic substituent can be a naphthalen-2-yl moiety or an aromatic ring substituted with one or two halogen and / or alkyl group. Some inhibitor peptides having an amino acid sequence comprising IX1-[T / S]-X2V (SEQ ID NO:2) at the C-terminus. Exemplary inhibitor peptides have sequences IESDV (SEQ ID NO:3), IETDV (SEQ ID NO:4), KLSSIESDV (SEQ ID NO:5), and KLSSIETDV (SEQ ID NO:6). Inhibitor peptides usually have 3-25 amino acids (without an internalization peptide), peptide lengths of 5-10 amino acids, and particularly 9 amino acids (also without an internalization peptide) are preferred.

[0053] Active agents of the invention typically include an inhibitor peptide and an internalization peptide configured such that inhibitor peptide has a free C-terminus and an N-terminus linked to the C-terminus of the internalization peptide. In some such agents, at least the four C-terminal amino acids of the inhibitor peptide and sometimes at least the five C-terminal amino acids of the inhibitor peptide are standard L-amino acids. The remainder of the inhibitor peptide is sometimes referred to as central segment or spacer because it lies between an internalization peptide and a C-terminal motif, which is primarily responsible for binding to PSD-95. The central or spacer segment can contribute to PSD-95 binding by various mechanisms, which may include influencing the spatial relationship of the C-terminal motif to the highly charged internalization peptide and direct interactions with PSD-95.

[0054] In NR2B9c, the central segment includes the sequence LSSI (SEQ ID NO:28). These amino acids are responsible for loss of active agent following subcutaneous administration andconsequently reduced bioavailability. The loss of active agent is presumptively due to proteases proximate to the skin acting on the active agent after subcutaneous administration.

[0055] Loss of active agent can be avoided by substituting 1, 2, 3, 4 or all 5 of the standard L-amino acids present in the KLSSI (SEQ ID NO:29) segment of NR2B9c. Some variants include a substitution in either K or SSI (SEQ ID NO:30) of KLSSI (SEQ ID NO:29). Some variants include a substitution in one amino acid in SSI (SEQ ID NO:30). Substitution can be by standard L-amino acids of a different type, non-standard amino acids of the same type (e.g., D-amino acids, beta amino acids, N-methyl or alpha-methyl amino acids) or non-standard amino acids of a different type. If multiple standard L-amino acids are substituted the replacement nonstandard amino acids can all be the same type of non-standard amino acids (e.g., all D-amino acids) or can be different types (e.g., one D-amino acid and one beta amino acid). Some variants have substitutions of both L and I. Some variants have substitution of one or both S’s. A substitution can also be by a different type of amino acid. For example, the first serine in LSSI (SEQ ID NO:28) can be substituted with D, E or P and the second serine can be substituted with V, Q or T.

[0056] Active agents can also include substitutions with D-amino acids or other non-standard amino acids to confer resistance to plasmin cleavage.

[0057] Although inclusion of D-amino acids or other nonstandard amino acids in at least one of the two amino acids bridging a proteolytic cleavage site is advantageous for increasing plasmin resistance and plasma half-life, it has been found that D-amino acids can also give rise to nephrotoxicity. The extent of the nephrotoxicity depends on the dose of active agent, content of D-amino acids of the active peptide and use of an inhibitor of D-amino acid oxidase to reduce the toxicity. Surprisingly, it has been found that most and sometimes all (e.g., over 50, 75, 90, 99 or 100%) of the benefit of increased plasmin resistance and plasma half-live can be achieved by including D-amino acids at fewer positions than there are plasmin cleavage sites (i.e., not every plasmin cleavage site has a bordering D-amino acid) with the additional benefit of reduced nephrotoxicity (e.g., reduced by 10, 25, 50, 75 or 90%) for the same dosage. In other words, the benefits and nephrotoxicity associated with D-amino acids do not show the same correlation with content of D-amino acids.

[0058] Although understanding of mechanism is not required for practice of the invention, the above results can be explained in part by a D-amino acid providing protection against plasmincleavage and sometimes other proteases not only when the D-amino acid is one of the two amino acids across a cleavage site but at a greater distance from the cleavage site, such as two, three or four amino acids distant (with amino acid one being an amino acid immediately next to a cleavage site). Additionally, nephrotoxicity from D-amino acids appears to increase more than proportionally with the number of D-amino acids in a peptide, as well as with peptide dose. In consequence using a minimal number of D-amino acids can confer most if not all the benefit of stability of a peptide completely made of D-amino acids without significant nephrotoxicity.

[0059] For example, some active agents have only 1, 2, 3, 4, 5, 6 or 7 amino acids of the internalization peptide as D-amino acids. Some active agents have 2-3, 2-4, 2-5 or 3-5 positions as D-amino acids in the internalization peptide.

[0060] Plasmin-resistance and plasma half-life can be increased by replacing select standard L-amino acids with D- or other non-standard amino acids in an active agent, and / or deleting standard L-amino acids not essential to the active agent’s activity.

[0061] The inhibitor peptide linked to the internalization peptides just discussed can comprise four C-terminal amino acids as a C-terminal motif, according to the formula [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO: 13). Preferably the inhibitor peptide comprises ESDV (SEQ ID NO: 14), IESDV (SEQ ID NO:3), ETDV (SEQ ID NO: 16) or IETDV (SEQ ID NO:4) at the C-terminus. At least the four and sometimes five terminal amino acids, such as ESDV (SEQ ID NO: 14), IESDV (SEQ ID NO:3), ETDV (SEQ ID NO: 16) or IETDV(SEQ ID NO:4) of the inhibitor peptide are standard L-amino acids.

[0062] Other substitutions, deletions or additions (besides replacement of standard L-amino acids with D-amino acids or other non-standard amino acids) can optionally made to disclosed sequences (e.g., up to 1, 2, 3, 4, or 5 substitutions, deletions or additions, preferably while retaining, at least substantially, binding, inhibition, plasmin-resistance and plasma half-life and without increasing nephrotoxicity as discussed further below. For example, some variants include 1, 2, 3, 4 or 5 substitution(s) relative to a disclosed sequence. Some variants include a combination of 1, 2, 3, 4, or 5 substitution(s) and deletion(s) relative to a disclosed sequence. Some variants include a combination of 1, 2, 3, 4 or 5 substitution(s), deletion(s) or addition(s) relative to a disclosed sequence.

[0063] The D- or other non-standard amino acids present in the active agents and their internalization peptide and inhibitor peptide components disclosed above may or may not becontiguous with one another. For example, in some active agents at least 2 D- or other nonstandard amino acids are separated from one another by one or more standard L-amino acids or glycine. In some internalization peptides at least 3 D- or other non-standard amino acids are each separated from one another by one or more standard L-amino acids or glycine.

[0064] In some active agents, at least one or all the standard L-amino acids replaced by D- or other non-standard amino acids are K and R or amino acids immediately on the C-terminal side of a K or R amino acid because these amino acids flank potential sites of plasmin cleavage. In some active agents, an amino acid immediately C-terminal to a stretch of two or more consecutive K or R amino acids is replaced by a D- or other non-standard amino acid. Other standard L-amino acids besides K, R and immediately C-terminal adjacent amino acids can also be replaced with D-or other non-standard amino acids.

[0065] Some active agents having an amino acid sequence having at least 75, 80, 85, 90 or 95% amino acid sequence identity to the sequence TFFYGGSRGKRNNFKTEEYKLSSIESDV (SEQ ID NO: 12) wherein 1-9, 2-9, 3-8, 3-7, 4-7, 5-6, 2-5, 3-5, or 3-4 amino acids at positions other than the five C-terminal amino acids are D- or other non-standard amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 such D- or other non-standard amino acids). In such active agents at least one of amino acids LSSI (SEQ ID NO:28) can be substituted with a standard L-amino acid of a different type or is a non-standard amino acid of the same or different type. In some such active agents, none of the D-or other non-standard amino acids occupies adjacent positions in the active agent. In some such active agents, each of the D- or other non-standard amino is separated by at least two or at least three standard amino acids selected from the group consisting of L-amino acids and glycine. Some active agents have 2, 3, 4 or 5 D- or other non-standard amino acids. Some active agents lack a stretch of more than three contiguous acids selected from the group consisting of standard L-amino acids and glycine, which stretch includes two adjacent amino acids selected independently from R and K (i.e., a plasmin cleavage site). In other words, one standard L-form R or K does not occur adjacent to another standard L-form R or K as part of a stretch of more than three contiguous amino acids that are standard L-amino acids or glycine. Some active agents lack a stretch of more than three contiguous standard L-amino acids or glycine including an R or K or both. In other words, a standard L-form R or K does not occur as part of a stretch of more than three contiguous amino acids that are standard L-amino acids or glycine. Some active agents lack a stretch of more than four contiguous standard L-amino acidsor glycine including two adjacent amino acids selected independently from R and K (i.e., a plasmin cleavage site). In other words, a standard L-form R or K does not occur adjacent to another standard L-form R or K as part of a stretch of more than four contiguous amino acids that are L-amino acids or glycine. If a stretch of four such contiguous amino acids is present, there is preferably only one such stretch, and it is located at the N-terminus of the active agent (i.e., first four amino acids), and elsewhere there are stretches of no more than two or three such contiguous amino acids Some active agents do not contain a stretch of more than four contiguous amino acids selected independently from the group consisting of standard L-amino acids and glycine, which stretch includes either an R or a K (or both). In other words, a standard L-form R or K does not occur as part of a stretch of more than four contiguous amino acids that are standard L-amino acids or glycine. If a stretch of four such contiguous amino acids is present, there is preferably only one such stretch, and it is located at the N-terminus of the active agent (i.e., first four amino acids) and elsewhere there are stretches of no more than two or three such contiguous amino acids.

[0066] Some active agents include 2-5, 3-4, or 3-5 D- or other non-standard amino acids in total each replacing a corresponding standard L amino acid in the sequence TFFYGGSRGKRNNFKTEEYKLSSIESDV (SEQ ID NO: 12), at least one of which is an amino acid in the sequence KLSSI SEQ ID NO:29), LSSI (SEQ ID NO:28), SSI (SEQ ID NO:30). Some active agents include 2 or 3 D-substitutions of K or R residues in the sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1) and one D-substitution of K or L in the sequence KLSSIESDV (SEQ ID NO:5). Some active agents have 2-5 D-amino acid substitutions of the K and R residues, or residues C-terminal thereto in the sequence TFFYGGSRGKRNNFKTEEYKLSSIESDV (SEQ ID NO: 12), where each D-amino acid substitution is separated by at least two amino acids from another D-amino acid substitution. Some active agents have 2-4 D-amino acid substitutions of the K and R residues of the sequence TFFYGGSRGKRNNFKTEEYKLSSIESDV (SEQ ID NO: 12) provided no two immediately adjacent positions are both substituted.

[0067] Some active agents include 2-3, 2-5, 3-4, 3-5 D- or other non-standard amino acids in total each replacing a corresponding standard L-amino acid in the TFFYGGSRGKRNNFKTEEYKLSSIESDV (SEQ ID NO: 12) sequence and at least one standard amino acid in KLSSI SEQ ID NO:29), LSSI (SEQ ID NO:28) or SSI (SEQ ID NO:30) replacedwith a standard amino acid of a different type. Two exemplary D-substituted Ang-2B9c peptides are as follows:

[0068] Table 1

[0069] Some active agents have enhanced stability in rat or human plasma compared with Ang-2B9c or an otherwise identical all-L active agent. Stability can be measured as in the examples. Some active agents have enhanced plasmin resistance and / or plasma half-life compared with Ang-2B9c or an otherwise identical all-L active agent. Plasmin resistance and plasma half-life can be measured as in the examples. Some active agents have increased plasma area under the curve and / or CMax compared with any of Ang-2B9c and / or an otherwise identical all-L active agent, particularly following subcutaneous administration. Some active agents have increased plasma bioavailability following subcutaneous administration relative to any of Ang-2B9c, and / or an otherwise identical all-L active agent. Bioavailability can be measured by any suitable pharmacokinetic parameter, such as AUC, CMax, concentration after a defined time period, such as 1 or 3 hr, half-life or time to decay to undetectable.

[0070] Active agents preferably bind to PSD-95 within 1.5-fold, 2-fold, 3-fold or 5-fold of Ang-2B9c (all standard L) or an otherwise identical all standard L peptide or have indistinguishable binding within experimental error. Preferred active agents compete for binding with Ang-2B9c or a peptide containing the last 15-20 amino acids of a NMDA Receptor subunit 2 sequence that contains the PDZ binding domain for binding to PSD-95 (e.g., a ten-fold excess of active agent reduces Tat-NR2B9c binding) by at least 10%, 25% or 50%. Competition provides an indication that the active agent binds to the same or overlapping binding site as Ang-2B9c. Possession of the same or overlapping binding sites can also be shown by alanine mutagenesis of PSD-95. If mutagenesis of the same or overlapping set of amino acids reduces binding of an active agent and Ang-2B9c, then the active agent and Ang-2B9c bind to the same or overlapping sites on PSD-95. Some active agents show reduced kidney toxicity and / or greatertherapeutic index compared with an active agent comprising an internalization peptide of sequence TFFYGGSRGKRNNFKTEEYKLSSIESDV (SEQ ID NO: 12). Enhancement of a desired activity, such as half-life, or reduction of an undesired activity, such as nephrotoxicity, refers to enhancement or reduction beyond experimental error, in other words statistically significant at 95% probability. Optionally, the enhancement is by a factor of at least 10%, 25%, 50%, 100%, 200%, 500% or 1,000% and reduction is by a factor of at last 10%, 25%, 50%, 75%, 95 or 99%.

[0071] Much of the above disclosure concerns replacement of select L-amino acids with D-amino acids to confer plasmin resistance or resistance to degradation following subcutaneous administration. Such disclosure also applies to replacing L-amino acids with other non-standard amino acids conferring protease resistance, such as beta amino acids, N-methyl and alpha-methyl amino acids.

[0072] Active agents of the invention can contain modified amino acids for example, amino acids that are N-alkylated. N-terminal alkyl modifications can include e.g., N-Methyl, N-Ethyl, N-Propyl, N-Butyl, N-Cyclohexylmethyl, N-Cyclyhexylethyl, N-Benzyl, N-Phenylethyl, N-phenylpropyl, N-(3, 4-Dichlorophenyl)propyl, N-(3,4-Difluorophenyl)propyl, and N-(Naphthalene-2-yl)ethyl). Active agents can also include retro peptides. A retro peptide has a reverse amino acid sequence. Peptidomimetics also include retro inverse peptides in which the order of amino acids is reversed so the originally C-terminal amino acid appears at the N-terminus and D-amino acids are used in place of L-amino.

[0073] Appropriate pharmacological activity of peptides, peptidomimetics or other agent can be confirmed if desired, using previously described rat models of stroke before testing in the primate and clinical trials described in the present application. Peptides or peptidomimetics can also be screened for capacity to inhibit interactions between PSD-95 and NMD AR 2B using assays described in e.g., US 20050059597, which is incorporated by reference. Useful peptides typically have IC50 values of less than 50 μM, 25 μM, 10 μM, 0.1 μM or 0.01 μM in such an assay. Preferred peptides typically have an IC50 value of between 0.001-1 μM, and more preferably 0.001-0.05, 0.05-0.5 or 0.05 to 0.1 μM. When a peptide or other agent is characterized as inhibiting binding of one interaction, e.g., PSD-95 interaction to NMDAR2B, such description does not exclude that the peptide or agent also inhibits another interaction, for example, inhibition of PSD-95 binding to nNOS.

[0074] Peptides such as those just described can optionally be derivatized (e.g., acetylated, phosphorylated, myristoylated, geranylated, pegylated and / or glycosylated) to improve the binding affinity of the inhibitor, to improve the ability of the inhibitor to be transported across a cell membrane or to improve stability. As a specific example, for inhibitors in which the third amino acid from the C-terminus is S or T, this amino acid can be phosphorylated before use of the peptide.

[0075] Internalization peptides can be attached to inhibitor peptides by conventional methods. For example, the inhibitor peptides can be joined to internalization peptides by chemical linkage, for instance via a coupling or conjugating agent. Numerous such agents are commercially available and are reviewed by S. S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991). Some examples of cross-linking reagents include J-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or N, N'-(l,3-phenylene) bismal eimide; N, N'-ethylene-bis-(iodoacetamide) or other such reagent having 6 to 11 carbon methylene bridges (which relatively specific for sulfhydryl groups); and l,5-difluoro-2,4-dinitrobenzene (which forms irreversible linkages with amino and tyrosine groups). Other cross-linking reagents include p, p'-difluoro-m, m'-dinitrodiphenylsulfone (which forms irreversible cross-linkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4-disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts principally with amino groups); glutaraldehyde (which reacts with several different side chains) and disdiazobenzidine (which reacts primarily with tyrosine and histidine).

[0076] A linker, e.g., a polyethylene glycol linker, can be used to dimerize an inhibitor peptide the active moiety of the peptide or the peptidomimetic to enhance its affinity and selectivity towards proteins containing tandem PDZ domains. See e.g., Bach et al., (2009) Angew. Chem. Int. Ed. 48:9685-9689 and WO 2010 / 004003. A PL motif-containing peptide is preferably dimerized via joining the N-termini of two such molecules, leaving the C-termini free. Bach further reports that a pentamer peptide IESDV (SEQ ID NO:3) from the C-terminus of NMDAR 2B was effective in inhibiting binding of NMDAR 2B to PSD-95. IETDV (SEQ ID NO:4) can also be used instead of IESDV (SEQ ID NO:3). Optionally, about 2-10 copies of a PEG can be joined in tandem as a linker. Optionally, the linker can also be attached to aninternalization peptide or lipidated to enhance cellular uptake. An example of a dimeric inhibitor is shown below (IETDV, SEQ ID NO:4) (see Bach et al., PNAS 109 (2012) 3317-3322).

[0077] R is any of the internalization peptides disclosed herein N can be replaced by O. Any of the PSD-95 inhibitors disclosed herein can be used instead of IETDV (SEQ ID NO:4), particularly IESDV (SEQ ID NO:3). Other linkers to that shown can also be used.

[0078] Internalization peptides can also be linked to inhibitor peptide as fusion peptides, preferably with the C-terminus of the internalization peptide linked to the N-terminus of the inhibitor peptide leaving the inhibitor peptide with a free C-terminus.

[0079] Instead of or as well as linking a peptide to an internalization peptide, such a peptide can be linked to a lipid (lipidation) to increase hydrophobicity of the conjugate relative to the peptide alone and thereby facilitate passage of the linked peptide across cell membranes and / or across the brain barrier. Lipidation is preferably performed on the N-terminal amino acid but can also be performed on internal amino acids, provided the ability of the peptide to inhibit interaction between PSD-95 and NMD AR 2B is not reduced by more than 50%. Preferably, lipidation is performed on an amino acid other than one of the five most C-terminal amino acids. Lipids are organic molecules more soluble in ether than water and include fatty acids, glycerides and sterols. Suitable forms of lipidation include myristoylation, palmitoylation or attachment of other fatty acids preferably with a chain length of 10-20 carbons, such as lauric acid and stearicacid, as well as geranylation, geranylgeranylation, and isoprenylation. Lipidations of a type occurring in posttranslational modification of natural proteins are preferred. Lipidation with a fatty acid via formation of an amide bond to the alpha-amino group of the N-terminal amino acid of the peptide is also preferred. Lipidation can be by peptide synthesis including a prelipidated amino acid, can be performed enzymatically in vitro or by recombinant expression, by chemical crosslinking or chemical derivatization of the peptide. Amino acids modified by myristoylation and other lipid modifications are commercially available.

[0080] Inhibitor peptides, optionally fused to internalization peptides, can be synthesized by solid phase synthesis or recombinant methods. Peptidomimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.

[0081] III. Salts

[0082] Peptides of the type described above are typically made by solid state synthesis. Because solid state synthesis uses trifluoroacetate (TFA) to remove protecting groups or remove peptides from a resin, peptides are typically initially produced as trifluoroacetate salts. The tri fluoroacetate can be replaced with another anion by for example, binding the peptide to a solid support, such as a column, washing the column to remove the existing counterion, equilibrating the column with a solution containing the new counterion and then eluting the peptide, e g., by introducing a hydrophobic solvent such as acetonitrile into the column. Replacement of tri fluoroacetate with acetate can be done with an acetate wash as the last step before peptide is eluted in an otherwise conventional solid state synthesis. Replacing trifluoroacetate or acetate with chloride can be done with a wash with ammonium chloride followed by elution. Use of a hydrophobic support is preferred and preparative reverse phase HPLC is particularly preferred for the ion exchange. Trifluoroacetate can be replaced with chloride directly or can first be replaced by acetate and then the acetate replaced by chloride.

[0083] Counterions, whether tri fluoroacetate, acetate or chloride, bind to positively charged atoms on Ang-2B9c and D-variants thereof, particularly the N-terminal amino group and amino side chains arginine and lysine amino acids.

[0084] Although replacement of one counterion by another takes place efficiently, the purity of the final counterion may be less than 100%. Thus, reference to a chloride salt of Ang-2B9c or its described herein means that in a preparation of the salt, chloride is the predominant anion by weight (or moles) over all other anions present in the aggregate in the salt. In other words, chloride constitutes greater than 50% and preferably greater than 75%, 95%, 99%, 99.5% or 99.9% by weight or moles of all anions present in the salt. In such a salt or formulation prepared from the salt, acetate and trifluoroacetate in combination and individually constitutes less than 50%, 25%, 5%, 0.5% or 0.1 of the anions in the salt or formulation by weight or moles.

[0085] IV. Formulations

[0086] Active agents can be incorporated into liquid formulation or lyophilized formulations. A liquid formulation can include a buffer, salt and water. A preferred buffer is sodium phosphate. A preferred salt is sodium chloride. Sodium benzoate can also be present as a dual purposes preservative and inhibitor of D-amino acid oxidases. Insulin or other excipient can be added to facilitate subcutaneous administration. The pH can be e.g., pH 7.0 or about physiological. The volume of a unit dose for subcutaneous administration to a human is preferably, no more than 5 ml, 3 ml or 1.5 ml.

[0087] Lyophilized formulations can be prepared from a prelyophilized formulation comprising an active agent, a buffer, a bulking agent and water. Other components, such as cryo or lyopreservatives, a tonicity agent pharmaceutically acceptable carriers and the like may or may be present. A preferred buffer is histidine. Again, sodium benzoate can also be present as a dual purpose preservative and inhibitor of D-amino acid oxidases. A preferred bulking agent is trehalose. Trehalose also serves as a cryo and lyo-preservative. An exemplary prelyophilized formulation comprises the active agent, histidine (10-100 mM, 15-100 mM 15-80 mM, 40-60 mM or 15-60 mM, for example, 20 mM or optionally 50 mM, or 20-50 mM)) and trehalose (50-200 mM, preferably 80-160 mM, 100-140 mM, more preferably 120 mM). The pH is 5.5 to 7.5, more preferably, 6-7, more preferably 6.5. The concentration of active agent is 20-200 mg / ml, preferably 50-150 mg / ml, more preferably 70-120 mg / ml or 90 mg / ml. Thus, an exemplary prelyophilized formulation is 20 mM histidine, 120 mM trehalose, and 90 mg / ml chloride salt of active agent. Optionally an acetylation scavenger, such as lysine can be included, as described in US 10,206,878, to further reduce any residual acetate or trifluoroacetate in the formulation

[0088] After lyophilization, lyophilized formulations have a low-water content, preferably from about 0%-5% water, more preferably below 2.5% water by weight. Lyophilized formulations can be stored in a freezer (e.g., -20 or -70°C), in a refrigerator (0-40°C) or at room temperature (20-25 °C).

[0089] Active agents can be reconstituted in an aqueous solution, preferably water for injection or optionally normal saline (0.8-1.0% saline and preferably 0.9% saline).Reconstitution can be to the same or a smaller or larger volume than the prelyophilized formulation. Preferably, the volume is larger post-reconstitution than before (e g., 3-6 times larger). For example, a prelyophilization volume of 3-5 ml can be reconstituted as a volume of 10 mL, 12 mL, 13.5 ml, 15 mL or 20 mL or 10-20 mL among others. After reconstitution, the concentration of histidine is preferably 2-20 mM, e.g., 2-7 mM, 4.0-6.5 mM, 4.5 mM or 6 mM; the concentration of trehalose is preferably 15-45 mM or 20-40 mM or 25-27 mM or 35-37 mM. The concentration of lysine is preferably 100-300 mM, e.g., 150-250 mM, 150-170 mM or 210-220 mM. The active agent is preferably at a concentration of 10-30 mg / ml, for example 15-30, 18-20, 20 mg / ml of active agent or 25-30, 26-28 or 27 mg / mL active agent. An exemplary formulation after reconstitution has 4-5 mM histidine, 26-27 mM trehalose, 150-170 mM lysine and 20 mg / ml active agent (with concentrations rounded to the nearest integer). A second exemplary formulation after reconstitution has 5-7 mM histidine, 35-37 mM trehalose, 210-220 mM lysine and 26-28 mg / ml active agent (with concentrations rounded to the nearest integer). The reconstituted formulation can be further diluted before administration such as by adding into a fluid bag containing normal saline.

[0090] V. Conditions

[0091] The active agents are useful in treating a variety of conditions, particularly neurological conditions, and especially conditions mediated in part by excitotoxity. Such conditions include stroke, epilepsy, hypoxia, subarachnoid hemorrhage, concussion, traumatic injury to the CNS not associated with stroke such as traumatic brain injury and spinal cord injury, other cerebral ischemia, reperfusion injury, neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Such conditions can also include disorders or diseases of the eye or ear, including retinopathies, retinal ischemia associated other oculardisorders, or tinnitus. Other neurological conditions treatable by active agents of the invention not known to be associated with excitotoxicity include anxiety and pain (either neuropathic or inflammatory).

[0092] A stroke is a condition resulting from impaired blood flow in the CNS regardless of cause. Potential causes include embolism, hemorrhage and thrombosis. Some neuronal cells die immediately as a result of impaired blood flow. These cells release their component molecules including glutamate, which in turn activates NMDA receptors, which raise intracellular calcium levels, and intracellular enzyme levels leading to further neuronal cell death (the excitotoxicity cascade). The death of CNS tissue is referred to as infarction. Infarction Volume (i.e., the volume of dead neuronal cells resulting from stroke in the brain) can be used as an indicator of the extent of pathological damage resulting from stroke. The symptomatic effect depends both on the volume of an infarction and where in the brain it is located. Disability index can be used as a measure of symptomatic damage, such as the Rankin Stroke Outcome Scale (Rankin, Scott Med J;2:200-15 (1957)) and the Barthel Index. The Rankin Scale is based on directly assessing the global conditions of a subject as follows.

[0093] 0: No symptoms at all1: No significant disability despite symptoms; able to carry out all usual duties and activities.2: Slight disability; unable to cany out all previous activities but able to look after own affairs without assistance.3: Moderate disability requiring some help, but able to walk without assistance 4: Moderate to severe disability; unable to walk without assistance and unable to attend to own bodily needs without assistance.5: Severe disability; bedridden, incontinent, and requiring constant nursing care and attention.

[0094] The Barthel Index is based on a series of questions about the subject's ability to carry out 10 basic activities of daily living resulting in a score between 0 and 100, a lower score indicating more disability (Mahoney et al, Maryland State Medical Journal 14:56-61 (1965)).

[0095] Alternatively stroke severity / outcomes can be measured using the NIH stroke scale, available at world wide web ninds.nih.gov / doctors / NIH Stroke ScaleJBooklet.pdf.

[0096] The scale is based on the ability of a subject to carry out 11 groups of functions that include assessments of the subject's level of consciousness, motor, sensory and language functions.

[0097] An ischemic stroke refers more specifically to a type of stroke that caused by blockage of blood flow to the brain. The underlying condition for this type of blockage is most commonly the development of fatty deposits lining the vessel walls. This condition is called atherosclerosis. These fatty deposits can cause two types of obstruction. Cerebral thrombosis refers to a thrombus (blood clot) that develops at the clogged part of the vessel " Cerebral embolism" refers generally to a blood clot that forms at another location in the circulatory system, usually the heart and large arteries of the upper chest and neck. A portion of the blood clot then breaks loose, enters the bloodstream and travels through the brain's blood vessels until it reaches vessels too small to let it pass. A second important cause of embolism is an irregular heartbeat, known as arterial fibrillation. It creates conditions in which clots can form in the heart, dislodge and travel to the brain. Additional potential causes of ischemic stroke are hemorrhage, thrombosis, dissection of an artery or vein, a cardiac arrest, shock of any cause including hemorrhage, and iatrogenic causes such as direct surgical injury to brain blood vessels or vessels leading to the brain or cardiac surgery. Ischemic stroke accounts for about 83 percent of all cases of stroke.

[0098] Transient ischemic attacks (TIAs) are minor or warning strokes. In a TIA, conditions indicative of an ischemic stroke are present and the typical stroke warning signs develop.However, the obstruction (blood clot) occurs for a short time and tends to resolve itself through normal mechanisms. Subjects undergoing heart surgery are at particular risk of transient cerebral ischemic attack.

[0099] Hemorrhagic stroke accounts for about 17 percent of stroke cases. It results from a weakened vessel that ruptures and bleeds into the surrounding brain. The blood accumulates and compresses the surrounding brain tissue. The two general types of hemorrhagic strokes are intracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic stroke result from rupture of a weakened blood vessel ruptures. Potential causes of rupture from a weakened blood vessel include a hypertensive hemorrhage, in which high blood pressure causes a rupture of a blood vessel, or another underlying cause of weakened blood vessels such as a ruptured brain vascular malformation including a brain aneurysm, arteriovenous malformation (AVM) or cavernous malformation. Hemorrhagic strokes can also arise from a hemorrhagic transformation of anischemic stroke which weakens the blood vessels in the infarct, or a hemorrhage from primary or metastatic tumors in the CNS which contain abnormally weak blood vessels. Hemorrhagic stroke can also arise from iatrogenic causes such as direct surgical injury to a brain blood vessel. An aneurysm is a ballooning of a weakened region of a blood vessel. If left untreated, the aneurysm continues to weaken until it ruptures and bleeds into the brain. An arteriovenous malformation (AVM) is a cluster of abnormally formed blood vessels. A cavernous malformation is a venous abnormality that can cause a hemorrhage from weakened venous structures. Any one of these vessels can rupture, also causing bleeding into the brain. Hemorrhagic stroke can also result from physical trauma. Hemorrhagic stroke in one part of the brain can lead to ischemic stroke in another through shortage of blood lost in the hemorrhagic stroke.

[0100] One subject class amenable to treatments are subjects undergoing a surgical procedure that involves or may involve a blood vessel supplying the brain, or otherwise on the brain or CNS. Some examples are subjects undergoing cardiopulmonary bypass, carotid stenting, diagnostic angiography of the brain or coronary arteries of the aortic arch, vascular surgical procedures and neurosurgical procedures. Additional examples of such subjects are discussed in section IV above. Subjects with a brain aneurysm are particularly suitable. Such subjects can be treated by a variety of surgical procedures including clipping the aneurysm to shut off blood, or performing endovascular surgery to block the aneurysm with small coils or introduce a stent into a blood vessel from which an aneurysm emerges, or inserting a microcatheter. Endovascular procedures are less invasive than clipping an aneurysm and are associated with a better subject outcome but the outcome still includes a high incidence of small infarctions. Such subjects can be treated with an inhibitor of PSD95 interaction with NMD AR 2B and particularly the agents described above. The timing of administration relative to performing surgery can be as described above for the clinical trial.

[0101] Another class of subjects amenable to treatment are subjects having a subarachnoid hemorrhage with or without an aneurysm (see US 61 / 570,264). Another class of subjects is those with ischemic strokes who are candidates for endovascular thrombectomy to remove the clot, such as the ESCAPE-NA1 trial (NCT02930018). Drug can be administered before or after the surgery to remove the clot and is expected to improve outcome from both the stroke itself and any potential iatrogenic strokes associated with the procedures as discussed supra. Another example is those who have been diagnosed with a potential stroke without the use of imagingcriteria and receive treatment within hours of the stroke, preferably within the first 3 hours but optionally the first 6, 9 or 12 hour after stroke onset (similar to NCT02315443).

[0102] VI. Effective Regimes of Administration

[0103] An active agent is administered in an amount, frequency and route of administration effective to cure, reduce or inhibit further deterioration of at least one sign or symptom of a condition in a subject having the condition being treated. A therapeutically effective amount (before administration) or therapeutically effective plasma concentration after administration means an amount or level of active agent sufficient significantly to cure, reduce or inhibit further deterioration of at least one sign or symptom of the condition to be treated in a population of subjects (or animal models) suffering from the condition treated with an agent of the invention relative to the damage in a control population of subjects (or animal models) suffering from that condition who are not treated with the agent. The amount or level is also considered therapeutically effective if an individual treated subject achieves an outcome more favorable than the mean outcome in a control population of comparable subjects not treated by methods of the invention. A therapeutically effective regime involves the administration of a therapeutically effective dose at a frequency and route of administration needed to achieve the intended purpose.

[0104] For a subject suffering from stroke or other ischemic condition, the active agent is administered in a regime comprising an amount frequency and route of administration effective to reduce the damaging effects of stroke or other ischemic condition. When the condition requiring treatment is stroke, the outcome can be determined by infarction volume or disability index, and a dosage is considered therapeutically effective if an individual treated subject shows a disability of two or less on the Rankin scale and 75 or more on the Barthel scale, or if a population of treated subjects shows a significantly improved (i.e., less disability) distribution of scores on a disability scale than a comparable untreated population, see Lees et at L, N Engl J Med 2006;354:588-600. A single dose of agent can be sufficient for treatment of stroke.

[0105] The invention also provides methods and formulations for the prophylaxis of a condition in a subject at risk of that condition. Usually such a subject has an increased likelihood of developing the condition (e.g., illness, disorder or disease) relative to a control population. The control population for instance can comprise one or more individuals selected at random from the general population (e.g., matched by age, gender, race and / or ethnicity) who have not been diagnosed or have a family history of the condition. A subject can be considered at risk fora disorder if a "risk factor" associated with that condition is found to be associated with that subject. A risk factor can include any activity, trait, event or property associated with a given disorder, for example, through statistical or epidemiological studies on a population of subjects. A subject can thus be classified as being at risk for a disorder even if studies identifying the underlying risk factors did not include the subject specifically. For example, a subject undergoing heart surgery is at risk of transient cerebral ischemic attack because the frequency of transient cerebral ischemic attack is increased in a population of subjects who have undergone heart surgery as compared to a population of subjects who have not.

[0106] Other common risk factors for stroke include age, family history, gender, prior incidence of stroke, transient ischemic attack or heart attack, high blood pressure, smoking, diabetes, carotid or other artery disease, atrial fibrillation, other heart diseases such as heart disease, heart failure, dilated cardiomyopathy, heart valve disease and / or congenital heart defects; high blood cholesterol, and diets high in saturated fat, trans fat or cholesterol.

[0107] In prophylaxis, an active agent is administered to a subject at risk of a condition but not yet having the condition in an amount, frequency and route sufficient to prevent, delay or inhibit development of at least one sign or symptom of the condition. A prophylactically effective amount before administration or plasma level after administration means an amount or level of agent sufficient significantly to prevent, inhibit or delay at least one sign or symptom of the condition in a population of human subjects (or animal models) at risk of the condition relative treated with the agent compared to a control population of human subjects (or animal models) at risk of the condition not treated with an active agent of the invention. The amount or level is also considered prophylactically effective if an individual treated subject achieves an outcome more favorable than the mean outcome in a control population of comparable subjects not treated by methods of the invention. A prophylactically effective regime involves the administration of a prophylactically effective dose at a frequency and route of administration needed to achieve the intended purpose. For prophylaxis of stroke in a subject at imminent risk of stroke (e.g., a subject undergoing heart surgery), a single dose of agent is usually sufficient.

[0108] Depending on the agent, administration can be parenteral, intravenous, intrapulmonary, nasal, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular.

[0109] Tat-NR2B9c has previously been administered to humans by single dose intravenous infusion at 2.6 mg / kg. Higher doses are associated with mast cell degranulation and histamine release in some patients. The present active agents can be administered at the same or higher doses as Tat-NR2B9c with little if any mast cell degranulation and histamine release. Depending on the route of administration and whether an anti-inflammatory is co-administered, doses of up to about 10 mg / kg, 25 mg / kg or even 50 mg / kg can be administered. In some methods doses, of at least 10 mg / kg, 25 mg / kg or 50 mg / kg are administered with or without an anti-inflammatory agent.

[0110] The need for an-inflammatory at higher doses can alternatively be reduced or eliminated by administration of the active agent over a longer time period (e.g., administration in less than 1 minute, 1-10 minutes, and greater than ten minutes constitute alternative regimes in which for constant dosage histamine release and need for an anti-inflammatory is reduced or eliminated with increased time period).

[0111] The active agents can be administered as a single dose or as a multi-dose regime. A single dose regime can be used for treatment of an acute condition, such as acute ischemic stroke, to reduce infarction and cognitive deficits. Such a dose can be administered before onset of the condition if the timing of the condition is predictable such as with a subject undergoing neurovascular surgery, or within a window after the condition has developed (e.g., up to 1, 3, 6 or 12 hours later).

[0112] A multi -dose regime can be designed to maintain the active agent at a detectable level in the plasma over a prolonged period, such as at least 1, 3, 5 or 10 days, or at least a month, three months, six months or indefinitely. For example, the active agents can be administered every hour, 2, 3, 4, 6, or 12 times per day, daily, every other day, weekly and so forth. Such a regime can reduce initial deficits from an acute condition as for single dose administration and thereafter promote recovery from such deficits as still develop. Such a regime can also be used for treating chronic conditions, such as Alzheimer’s and Parkinson’s disease. Active agents are sometimes incorporated into a controlled release formulation for use in a multi-dose regime.

[0113] Active agents can be prepared with carriers that protect the compound against rapid elimination from the body, such as controlled formulations or coatings. Such carriers (also known as modified, delayed, extended or sustained release or gastric retention dosage forms, such as the Depomed GR™ system in which agents are encapsulated by polymers that swell inthe stomach and are retained for about eight hours, sufficient for daily dosing of many drugs). Controlled release systems include microencapsulated delivery systems, implants and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, matrix controlled release devices, osmotic controlled release devices, multiparticulate controlled release devices, ion-exchange resins, enteric coatings, multilayered coatings, microspheres, nanoparticles, liposomes, and combinations thereof. The release rate of an active agent can also be modified by varying the particle size of the active agent: Examples of modified release include, e.g., those described in U. S. Pat. Nos: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5, 120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739,108;5,891,474; 5,922,356; 5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6, 113,943; 6, 197,350; 6,248,363; 6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; and 6,699,500.

[0114] Active agents including D-amino acids are subject to nephrotoxicity, which increases with D-amino acid content of the agent and the dose. Nephrotoxicity can be reduced by coadministration of the active agent with an inhibitor of D-amino acid oxidase. Such inhibitors have previously been proposed for treatment of e.g., schizophrenia. Numerous examples of such inhibitors have been described (see, e.g., Sacchi et al., Curr. Phar. Ds. 19:2499-511 (2013); Szilagyi et al., Molecules 24, 290 (2019); Lorenzo et al., Biosci Rep. 34, e00133 (2014); Katane et al., J. Med. Chem. 56, 1894–1907 (2013); Sacchi et al., Current Pharmaceutical Design 19, 2499-2511 (2013)). Examples of such inhibitors include, risperidone, sodium benzoate or 5-chloro-benzo[d]isoxazol-3-ol. Such an inhibitor can be administered before, after or at the same time as the active agent. If the latter, it can be administered as a co-formulation or separately. Sodium benzoate is also used as a preservative in pharmaceutical compositions so can serve a dual purpose in a co-formulation.

[0115] VII. Co-administration with anti-inflammatories

[0116] Depending on the dose and route of administration the active agents of the invention can induce an inflammatory response characterized by mast cell degranulation and release of histamine and its sequelae.

[0117] A wide variety of anti-inflammatory agents are readily available to inhibit one or more aspects of the of the inflammatory response. A preferred class of anti-inflammatory agent is mastcell degranulation inhibitors. This class of compounds includes cromolyn (5,5'-(2-hydroxypropane-l,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylic acid) (also known as cromoglycate), and 2-carboxylatochromon-5'-yl-2 -hydroxypropane derivatives such as bis(acetoxymethyl), disodium cromoglycate, nedocromil (9-ethyl-4,6-dioxo-10-propyl-6,9-dihydro-4H-pyrano[3,2-g]quinoline-2,8-di- carboxylic acid) and tranilast (2-{[(2E)-3-(3,4-dimethoxyphenyl)prop-2-enoyl]amino}), and lodoxamide (2-[2-chloro-5-cyano-3-(oxaloamino)anilino]-2-oxoacetic acid). Reference to a specific compound includes pharmaceutically acceptable salts of the compound Cromolyn is readily available in formulations for nasal, oral, inhaled or intravenous administration. Although practice of the invention is not dependent on an understanding of mechanism, it is believed that these agents act at an early stage of inflammatory response induced by an internalization peptide and are thus most effective at inhibiting development of its sequelae including a transient reduction in blood pressure. Other classes of anti-inflammatory agent discussed below serve to inhibit one or more downstream events resulting from mast cell degranulation, such as inhibiting histamine from binding to an Hl or H2 receptor but may not inhibit all sequelae of mast cell degranulation or may require higher dosages or use in combinations to do so. Table 2 below summarizes the names, chemical formulate and FDA status of several mast cell degranulation inhibitors that can be used with the invention.

[0118] Table 2

[0119] Another class of anti-inflammatory agent is anti-histamine compounds. Such agents inhibit the interaction of histamine with its receptors thereby inhibiting the resulting sequelae of inflammation noted above. Many anti-histamines are commercially available, some over the counter. Examples of anti-histamines are azatadine, azelastine, burfroline, cetirizine, cyproheptadine, doxantrozole, etodroxizine, forskolin, hydroxyzine, ketotifen, oxatomide, pizotifen, proxicromil, N, N'-substituted piperazines or terfenadine. Anti-histamines vary in their capacity to block anti-histamine in the CNS as well as peripheral receptors, with second and third generation anti-histamines having selectivity for peripheral receptors. Acrivastine, Astemizole, Cetirizine, Loratadine, Mizolastine, Levocetirizine, Desloratadine, and Fexofenadine are examples of second and third generation anti-histamines. Anti-histamines are widely available in oral and topical formulations. Some other anti-histamines that can be used are summarized in Table 3 below.

[0120] Table 3

[0121] Another class of anti-inflammatory agent useful in inhibiting the inflammatory response is corticosteroids. These compounds are transcriptional regulators and are powerful inhibitors of the inflammatory symptoms set in motion by release of histamine and other compounds resulting from mast cell degranulation. Examples of corticosteroids are Cortisone, Hydrocortisone (Cortef), Prednisone (Deltasone, Meticorten, Orasone), Prednisolone (Delta-Cortef, Pediapred, Prelone), Triamcinolone (Aristocort, Kenacort), Methylprednisolone (Medrol), Dexamethasone (Decadron, Dexone, Hexadrol), and Betamethasone (Celestone). Corticosteriods are widely available in oral, intravenous and topical formulations.

[0122] Nonsteroidal anti-inflammatory drugs (NSAIDs) can also be used. Such drugs include aspirin compounds (acetylsalicylates), non-aspirin salicylates, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, naproxen, naproxen sodium, phenylbutazone, sulindac, and tometin. However, the anti-inflammatoryeffects of such drugs are less effective than those of anti-histamines or corticosteroids. Stronger anti-inflammatory drugs such as azathioprine, cyclophosphamide, leukeran, and cyclosporine can also be used but are not preferred because they are slower acting and / or associated with side effects. Biologic anti-inflammatory agents, such as Tysabri® or Humira® can also be used but are not preferred for the same reasons.

[0123] Different classes of drugs can be used in combinations in inhibiting an inflammatory response. A preferred combination is a mast cell degranulation inhibitor and an anti-histamine.

[0124] In methods in which a pharmacological agent linked to an internalization peptide is administered with an anti-inflammatory agent, the two entities are administered sufficiently proximal in time that the anti-inflammatory agent can inhibit an inflammatory response inducible by the internalization peptide. The anti-inflammatory agent can be administered before, at the same time as or after the pharmacologic agent. The preferred time depends in part on the pharmacokinetics and pharmacodynamics of the anti-inflammatory agent. The anti-inflammatory agent can be administered at an interval before the pharmacologic agent such that the antiinflammatory agent is near maximum serum concentration at the time the pharmacologic agent is administered. Typically, the anti-inflammatory agent is administered between 6 hours before the pharmacological agent and one hour after. For example, the anti-inflammatory agent can be administered between 1 hour before and 30 min after the pharmacological agent. Preferably the anti-inflammatory agent is administered between 30 minutes before and 15 minutes after the pharmacologic agent, and more preferably within 15 minutes before and the same time as the pharmacological agent. In some methods, the anti-inflammatory agent is administered before the pharmacological agent within a period of 15, 10 or 5 minutes before the pharmacological agent is administered. In some methods, the agent is administered 1-15, 1-10 or 1-5 minutes before the pharmacological agent.

[0125] When administration of an agent is not instantaneous, such as with intravenous infusion, the anti-inflammatory agent and pharmacological agent are considered to be administered at the same time if their periods of administration are co-extensive or overlap. Time periods of administration before administration start from the beginning of its administration. Time periods after administration start from the end of its administration. Time periods referring to the administration of the anti-inflammatory agent refer to the beginning of its administration.

[0126] When an anti-inflammatory agent is said to be able to inhibit the inflammatory response of a pharmacological agent linked to an internalization peptide what is meant is that the two are administered sufficiently proximate in time that the anti-inflammatory agent would inhibit an inflammatory response inducible by the pharmacological agent linked to the internalization peptide if such a response occurs in a particular subject, and does not necessarily imply that such a response occurs in that subject. Some subjects are treated with a dose of pharmacological agent linked to an internalization peptide that is associated with an inflammatory response in a statistically significant number of subjects in a controlled clinical or nonclinical trial. It can reasonably be assumed that a significant proportion of such subjects although not necessarily all develop an anti-inflammatory response to the pharmacological agent linked to the internalization peptide. In some subjects, signs or symptoms of an inflammatory response to the pharmacological agent linked to the internalization peptide are detected or detectable.

[0127] In clinical treatment of an individual subject, it is not usually possible to compare the inflammatory response from a pharmacological agent linked to an internalization peptide in the presence and absence of an anti-inflammatory agent. However, it can reasonably be concluded that the anti-inflammatory agent inhibits an anti-inflammatory response inducible by the peptide if significant inhibition is seen under the same or similar conditions of co-administration in a controlled clinical or pre-clinical trial. The results in the subject (e.g., blood pressure, heart rate, hives) can also be compared with the typical results of a control group in a clinical trial as an indicator of whether inhibition occurred in the individual subject. Usually, the anti-inflammatory agent is present at a detectable serum concentration at some point within the time period of one hour after administration of the pharmacologic agent. The pharmacokinetics of many antiinflammatory agents is widely known and the relative timing of administration of the antiinflammatory agent can be adjusted accordingly. The anti-inflammatory agent is usually administered peripherally, i.e., segregated by the blood brain barrier from the brain. For example, the anti-inflammatory agent can be administered orally, nasally, intravenously or topically depending on the agent in question. If the anti-inflammatory agent is administered at the same time as the pharmacologic agent, the two can be administered as a combined formulation or separately.

[0128] In some methods, the anti-inflammatory agent is one that does not cross the blood brain barrier when administered orally or intravenously at least in sufficient amounts to exert a detectable pharmacological activity in the brain. Such an agent can inhibit mast cell degranulation and its sequelae resulting from administration of the active agent in the periphery without itself exerting any detectable therapeutic effects in the brain. In some methods, the antiinflammatory agent is administered without any co-treatment to increase permeability of the blood brain barrier or to derivatize or formulate the anti-inflammatory agent so as to increase its ability to cross the blood brain barrier. However, in other methods, the anti-inflammatory agent, by its nature, derivatization, formulation or route of administration, may by entering the brain or otherwise influencing inflammation in the brain, exert a dual effect in suppressing mast-cell degranulation and / or its sequelae in the periphery due to an internalization peptide and inhibiting inflammation in the brain. Strbian et al., WO 04 / 071531 have reported that a mast cell degranulation inhibitor, cromoglycate, administered i.c.v. but not intravenously has direct activity in inhibiting infarctions in an animal model.

[0129] In some methods, the subject is not also treated with the same anti-inflammatory agent co-administered with the active agent in the day, week or month preceding and / or following coadministration with the active agent. In some methods, if the subject is otherwise being treated with the same anti-inflammatory agent co-administered with the active agent in a recurring regime (e.g., same amount, route of delivery, frequency of dosing, timing of day of dosing), the co-administration of the anti-inflammatory agent with the active agent does not comport with the recurring regime in any or all of amount, route of delivery, frequency of dosing or time of day of dosing. In some methods, the subject is not known to be suffering from an inflammatory disease or condition requiring administration of the anti-inflammatory agent co-administered with the active agent in the present methods. In some methods, the subject is not suffering from asthma or allergic disease treatable with a mast cell degranulation inhibitor. In some methods, the antiinflammatory agent and active agent are each administered once and only once within a window as defined above, per episode of a condition, an episode being a relatively short period in which symptoms of the condition are present flanked by longer periods in which symptoms are absent or reduced.

[0130] The anti-inflammatory agent is administered in a regime of an amount, frequency and route effective to inhibit an inflammatory response to an internalization peptide under conditionsin which such an inflammatory response is known to occur in the absence of the antiinflammatory. An inflammatory response is inhibited if there is any reduction in signs or symptoms of inflammation as a result of the anti-inflammatory agent. Symptoms of the inflammatory response can include redness, rash such as hives, heat, swelling, pain, tingling sensation, itchiness, nausea, rash, dry mouth, numbness, airway congestion. The inflammatory response can also be monitored by measuring signs such as blood pressure, or heart rate.Alternatively, the inflammatory response can be assessed by measuring plasma concentration of histamine or other compounds released by mast cell degranulation. The presence of elevated levels of histamine or other compounds released by mast cell degranulation, reduced blood pressure, skin rash such as hives, or reduced heart rate are indicators of mass cell degranulation. As a practical matter, the doses, regimes and routes of administration of most of the antiinflammatory agents discussed above are available in the Physicians' Desk Reference and / or from the manufacturers, and such anti-inflammatories can be used in the present methods consistent with such general guidance.

[0131] VII. Co-administration with thrombolytic agents or mechanical reperfusion

[0132] Plaques and blood clots (also known as emboli) causing ischemia can be dissolved, removed or bypassed by both pharmacological and physical means. The dissolving, removal of plaques and blood clots and consequent restoration of blood flow is referred to as reperfusion. One class of agents acts by thrombolysis. Thrombolytic agents work by promoting production of plasmin. Plasmin clears cross-linked fibrin mesh (the backbone of a clot), making the clot soluble and subject to further proteolysis by other enzymes, and restores blood flow in occluded blood vessels. Examples of thrombolytic agents include tissue plasminogen activator t-PA, alteplase (Activase®), reteplase (Retavase®), tenecteplase (TNKase®), anistreplase (Eminase®), streptokinase (Kabikinase®, Streptase®), and urokinase (Abbokinase®).

[0133] Another class of drugs that can be used for reperfusion is vasodilators. These drugs act by relaxing and opening up blood vessels thus allowing blood to flow around an obstruction. Some examples of types of vasodilators alpha-adrenoceptor antagonists (alpha-blockers), Angiotensin receptor blockers (ARBs), β2-adrenoceptor agonists (β2-agonists), calcium-channel blockers (CCBs), centrally acting sympatholytics, direct acting vasodilators, endothelin receptor antagonists, ganglionic blockers, nitrodilators, phosphodiesterase inhibitors, potassium-channel openers, and renin inhibitors.

[0134] Another class of drugs that can be used for reperfusion is hypertensive drugs (i.e., drugs raising blood pressure), such as epinephrine, phenylephrine, pseudoephedrine, norepinephrine; norephedrine; terbutaline; salbutamol; and methylephedrine. Increased perfusion pressure can increase flow of blood around an obstruction.

[0135] Mechanical methods of reperfusion include angioplasty, catheterization, and artery bypass graft surgery, stenting, embolectomy, or endarterectomy. Such procedures restore plaque flow by mechanical removal of a plaque, holding a blood vessel open, so blood can flow around a plaque or bypassing a plaque.

[0136] Other mechanical methods of reperfusion include use of a device that diverts blood flow from other areas of the body to the brain. An example is a catheter partially occluding the aorta, such as the CoAxia NeuroFlo™ catheter device, which has recently been subjected to a randomized trial and may get FDA approval for stroke treatment. This device has been used on subjects presenting with stroke up to 14 hours after onset of ischemia.

[0137] Active agents of the invention including D-amino acid(s) can be administered with any of the forms of reperfusion therapy to a subject amenable to treatment. However, the active agents of the invention are particularly advantageous for administration with thrombolytic agents because the inclusion of one or more D-amino acids in the active agent reduces the susceptibility of the active agent to cleavage by plasmin, which is induced by thrombolytic agents. Thus, the active agents including one or more D-amino acids can be co-administered with thrombolytic agents in which regimes, which would otherwise result in cleavage of the active agent induced by the thrombolytic agent. For example, the thrombolytic agent can be administered within a window of 60, 30, or 15 minutes before the active agent. In some methods, the active agent is administered at the same time as the thrombolytic agent. The active agent and thrombolytic agent can be co-formulated or administered separately. In some methods, the thrombolytic agent is administered before the active agent and persists at a detectable level in the serum when the active agent is administered.

[0138] For treatment of ischemia that cannot be predicted in advance, an active agent can be administered as soon as possible or practical after onset of ischemia. For example, an active agent can be administered within a period of 0.5, 1, 2, 3, 4, 5, 6, 9, 12 or 24 hours after the onset of ischemia. For ischemia’s that can be predicted in advance, an active agent can be administered before, concurrent with or after onset of ischemia. For example, for an ischemia resulting fromsurgery, the PDS-95 inhibitor is sometimes routinely administered in a period starting 30 minutes before beginning surgery and ending 1, 2, 3, 4, 5, 6, 9, 12 or 24 hours after surgery without regard to whether ischemia has or will develop. Because the active agents are free of serious side effects, they can be administered when stroke or other ischemic conditions are suspected without a diagnosis according to art-recognized criteria having been made. For example, an active agent can be administered at the location where the stroke has occurred (e.g., in the subjects' home) or in an ambulance transporting a subject to a hospital. An active agent can also be safely administered to a subject at risk of stroke or other ischemic conditions before onset who may or may not actually develop the condition.

[0139] Following, or sometimes before, administration of an active agent, a subject presenting with sign(s) and / or symptom(s) of ischemia can be subject to further diagnostic assessment to determine whether the subject has ischemia within or otherwise affecting the CNS and determine whether the subject has or is susceptible to hemorrhage. Most particularly in subjects presenting with symptoms of stroke, testing attempts to distinguish whether the stroke is the result of hemorrhage or ischemia, hemorrhage accounting for about 17% of strokes. Diagnostic tests can include a scan of one or more organs, such as a CAT scan, MRI or PET imaging scan or a blood test for a biomarker that suggests that a stroke has occurred. Several biomarkers associated with stroke are known including B-type neurotrophic growth factor, von Willebrand factor, matrix metalloproteinase-9, and monocyte chemotactic protein-1 (see Reynolds et al., Clinical Chemistry 49: 1733-1739 (2003)). The organ(s) scanned include any suspected as being the site of ischemia (e.g., brain, heart, limbs, spine, lungs, kidney, retina) as well as any otherwise suspect of being the source of a hemorrhage. A scan of the brain is the usual procedure for distinguishing between ischemic and hemorrhagic stroke. Diagnostic assessment can also include taking or reviewing a subject's medical history and performing other tests. Presence of any of the following factors alone or in combination can be used in assessing whether reperfusion therapy presents an unacceptable risk: subject's symptoms are minor or rapidly improving, subject had seizure at onset of stroke, subject has had another stroke or serious head trauma within the past 3 months, subject had major surgery within the last 14 days, subject has known history of intracranial hemorrhage, subject has sustained systolic blood pressure >185 mmHg, subject has sustained diastolic blood pressure >110 mmHg, aggressive treatment is necessary to lower the subject's blood pressure, subject has symptoms suggestive of subarachnoid hemorrhage, subjecthas had gastrointestinal or urinary tract hemorrhage within the last 21 days, subject has had arterial puncture at noncompressible site within the last 7 days, subject has received heparin with the last 48 hours and has elevated PTT, subject's prothrombin time (PT) is >15 seconds, subject's platelet count is <100,000 / μL. subject's serum glucose is <50 mg / dL or >400 mg / dL, subject is a hemophiliac or has other clotting deficiencies.

[0140] The further diagnostic investigation determines according to recognized criteria or at least with greater probability that before the investigation whether the subject has an ischemic condition, and whether the subject has a hemorrhage, has an unacceptable risk of hemorrhage or is otherwise excluded from receiving reperfusion therapy due to unacceptable risk of side effects. Subjects in which a diagnosis of an ischemic condition within or otherwise likely to affect the CNS is confirmed who are without unacceptable risk of side effects can then be subject to reperfusion therapy. Reperfusion therapy can be performed as soon as practical after completion of any diagnostic procedures.

[0141] Both treatment with an active agent and reperfusion therapy independently have ability to reduce infarction size and functional deficits due to ischemia. When used in combination according to the present methods, the reduction in infarction size and / or functional deficits is preferably greater than that from use of either agent alone administered under a comparable regime other than for the combination (i.e., co-operative). More preferably, the reduction in infarction size and / or functional deficits is at least additive or preferably more than additive (i.e., synergistic) of reductions achieved by the agents alone under a comparable regime except for the combination. In some regimes, the reperfusion therapy is effective in reducing infarction size and / or functional deficits at a time post onset of ischemia (e.g., more than 4.5 hr) when it would be ineffective but for the concurrent or prior administration of the PSD-95 inhibitor. Put another way, when a subject is administered an active agent and reperfusion therapy, the reperfusion therapy is preferably at least as effective as it would be if administered at an earlier time without the active agent. Thus, the active agent effectively increases the efficacy of the reperfusion therapy by reducing one or more damaging effects of ischemia before or as reperfusion therapy takes effects. The active agent can thus compensate for delay in administering the reperfusion therapy whether the delay be from delay in the subject recognizing the danger of his or her initial symptoms delays in transporting a subject to a hospital or other medical institution or delays in performing diagnostic procedures to establish presence of ischemia and / or absence ofhemorrhage or unacceptable risk thereof. Statistically significant combined effects of an active agent and reperfusion therapy including additive or synergistic effects can be demonstrated between populations in a clinical trial or between populations of animal models in preclinical work.

[0142] EXAMPLES

[0143] Example 1: Effects of ANG-2B9c on MABP and histamine concentration in plasmin SD rats

[0144] This example evaluated the effect of ANG-2B9c on mean arterial blood pressure in male Sprague-Dawley rats when given by a single intravenous 10 min infusion. The following parameters and end points were evaluated during the study:

[0145] Mean arterial blood pressure was monitored continuously with a left femoral artery catheter from 5 min before treatment and over 60 minutes after drug injection.

[0146] ANG-2B9c and nerinetide concentration in rat plasma was quantified.

[0147] Histamine in rat plasma was quantified.

[0148] No hypotension or histamine was observed during the 10-minute infusion of ANG-2B9c(7.6mg / kg, 4.5mg / kg, or 3.8mg / kg) (Figs. 1 and 3).

[0149] Coax of Ang2-B9c was lower than for nerinetide (Fig. 2).

[0150] Example 2: ANG-2B9c Binding Affinity to the target protein PSD-95 PDZ2

[0151] Binding was evaluated with a competitive ELISA assay. A plate was coated with 1 ug / ml PSD95PDZ2 in 50 mM bicarbonate buffer overnight at 4C. The plate was blocked in 2% BSA in PBST (0.05%) for 2 h at room temperature. Then, the plate was incubated with the mixture of 150 ng / ml of biotinylated-NA-1 and the different test compounds at concentrations starting from 120 ug / ml in a 3 -fold dilution overnight at 4C, after proper washing with PBS-T, the plate was incubated with (1:3000) SA-HRP for 30 min. The wells were washed again, and then incubated with TMB solution for 10 min. The reaction was stopped with 100 μL H2SO4. Absorbance was determined at 450 nm with the synergy Hl reader.

[0152] Figs. 4A and 4B show that Ang-2B9c effectively competes the binding of biotin labelled nerinetide to PSD-95 domain 2 showing that the two entities have the same or overlapping binding sites.

[0153] Example 3: ANG-2B9c susceptibility to plasmin cleavage

[0154] The stability of ANG-2B9c compared to nerinetide and NoNO-42 (YGrKKRrQrRRkLSSIESDV SEQ ID NO:31) in the presence of plasmin in phosphate buffered saline (PBS) was evaluated to test whether ANG-2B9c was plasmin resistant. Test peptides were added at 65 pg / ml (NoNO-42 and nerinetide) or 85 ug / mL (ANG-2B9c, to produce an equimolar solution due to the increased molecular weight) to 600 pL of PBS and incubated at 37°C for 2 min to obtain baseline samples. Plasmin (10 pg / ml) was then added (spiked) after the first baseline sample acquisition. After the addition of plasmin, samples were collected at 5 min, 15 min, 30 min, and 45 min post-spiking. Experiments were performed in triplicate. At each time point, including baseline, lOOpL of the reaction mixture was collected using a fresh syringe from each vial. Samples were stored at -80°C until analysis. Samples were analyzed by a reversephase high-performance liquid chromatography method (RP-HPLC) to determine peptide stability, allowing for the relative quantitative determination of the peptide absorbance peak. This method is based on the polarity of the compound tested and the interactions with the column material. Peptide content % was defined as the peak absorbance for every time point divided by the peak absorbance acquired from the baseline sample multiplied by 100.

[0155] Fig. 5 shows that Ang-2B9c is cleaved by plasmin on a similar timescale as is nerinetide. NoNO-42 is not cleaved by plasmin.

[0156] Determination of ANG-2B9c main fragments in the presence of Plasmin by LC / ESI / MS. ANG-2B9c (85 ug / ml) was added to 500 pL of PBS and incubated at 37°C for 2 min to obtain baseline sample. Plasmin (10 pg / ml) was then added (spiked) after baseline sample acquisition. After the addition of plasmin, sample was collected at 2 min and the reaction was then stopped by cooling to -80°C until further testing. The various peptide fragments of ANG-2B9c in the mixture were then separated by means of liquid chromatography and identified by means of mass spectrometry (LC-MS / MS).

[0157] Fig. 6 shows the deconvoluted mass spectra of ANG-2B9c digestion products after incubation with plasmin. (Left) Deconvoluted mass spectra of the ANG-2B9c in PBS prior digestion with plasmin. (Right) Deconvoluted mass spectra of the ANG-2B9c 2 min after digestion with plasmin.

[0158] Example 4: Ability of ANG-2B9c to Disrupt PSD-95: GluN2B pre-formed Complexes Ischemic cerebral cortex was harvested from male 280-320g Sprague-Dawley rat 2 hours after 3 pial vessel occlusion (3PVO). In brief, rats were anesthetized with 2% isoflurane withouttracheal intubation. After midline skin incision, the skull was exposed. Over the left somatosensory cortex, a 6mm cranial window was opened using a dental drill so that the pial arteries were visualized. Dura was left to intact. To ensure blockage, the three to four pial arteriolar middle cerebral artery branches within this cranial window were coagulated using a bipolar forceps through the dura. Nerinetide (7.6mg / kg), Tat-ADA (7.4 mg / kg), ANG2B9c (9.8 mg / kg), or placebo was given 1 hour after 3PV0 induction. Harvested samples were immediately frozen on dry ice and stored at -80°C until used. Animals were excluded when subarachnoid hemorrhage were observed during the surgery and when they died before sample collection.

[0159] Samples were homogenized in RIPA buffer, centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatants were collected. Protein concentrations were measured using the BioRad Protein Assay. Protein G beads were incubated with the anti-GluN2B antibody and then mixed with 1 mg of brain lysate to immunoprecipitated the PSD-95 / NR2B complex. Then the proteins were precipitated and washing to remove unbound proteins. The captured complexes were then eluted, separated by 8% SDS-PAGE, and analyzed by Western blot to identify interacting proteins, these were detected via HRP-conjugated secondary antibodies and chemiluminescence. The optical density (OD) of the protein bands was quantified using the profile function of MultiGauge software (Fujifilm). The OD ratio of PSD-95 to NR2B was calculated and compared between the ANG-2B9c and placebo groups.

[0160] Fig. 7 shows that Ang-2B9c dissociates pre-formed GluN2B: PSD95 PDZ domain 2 complexes in rat brain lysates.

[0161] Example 5: Efficacy of ANG-2B9c as a Neuroprotectant in a transient MCA Occlusion Model (taco)

[0162] taco was induced in Sprague Dawley rats at time zero and maintained for 1 hour before injecting the stud drug or placebo. Reperfusion was effected 30 minutes after injection. The dose of Ang-2B9c was 9.8 mg / kg and that of nerinetide was 7.6 mg / kg. Neurological score was determined 4 and 24 hr after MCAO induction.

[0163] Figs. 8, 9A and 9B show that Ang-2B9c effectively reduced infarct volume an improved neurological outcome.

[0164] Example 6: D-amino acid substitution variants of Ang-2B9c.

[0165] Two D-variants of Ang-2B9c designated variants A6 and A7 (sequences shown above) were tested for plasmin-resistance, inhibition of PSD-95 and binding to LRP-1.

[0166] For plasmin-resistance, he stability of ANG-2B9c Analogs (A6 & A7) compared to nerinetide, NoNO-42 and ANG-2B9c in the presence of plasmin in phosphate buffered saline (PBS) was evaluated to test whether the D-amino acid substitutions within both analogs will render the peptide plasmin resistant. Test peptides were added at 65 pg / ml (NoNO-42 and nerinetide) or 85 ug / mL (ANG-2B9c, ANG-2B9C A6 and ANG-2B9C A7 to produce an equimolar solution due to the increased molecular weight) to 600 uL of PBS and incubated at 37°C for 2 min to obtain baseline samples. Plasmin (10 pg / ml) was then added (spiked) after the first baseline sample acquisition. After the addition of plasmin, samples were collected at 5 min, 15 min, 30 min, and 45 min post-spiking. Experiments were performed in triplicate. At each time point, including baseline, lOOpL of the reaction mixture was collected using a fresh syringe from each vial. Samples were stored at -80°C until analysis. Samples were analyzed by a reversephase high-performance liquid chromatography method (RP-HPLC) to determine peptide stability, allowing for the relative quantitative determination of the peptide absorbance peak. This method is based on the polarity of the compound tested and the interactions with the column material. Peptide content% was defined as the peak absorbance for every time point divided by the peak absorbance acquired from the baseline sample multiplied by 100. Fig. 10 shows that both ANG-2B9c A6 and ANG-2B9c A7 are resistant to plasmin cleavage; however ANG-2B9c is cleaved by plasmin on a similar timescale as is nerinetide.

[0167] Inhibition of PSD-95 was assessed by measuring competition between Ang-NR2B9c variants and nerinetide. Binding was evaluated with a competitive ELISA assay. A plate was coated with I ug / ml PSD95poz2 in 50 mM bicarbonate buffer overnight at 4C. The plate was blocked in 2% BSA in PBST (0.05%) for 2 hat room temperature. Then, the plate was incubated with the mixture of 0.07uM of biotinylated-NA-1 and the different test compounds at concentrations starting from 40 ump in a 5-fold dilution overnight at 4C, after proper washing with PBS-T, the plate was incubated with (1:2000) SA-HRP for 30 min. The wells were washed again, and then incubated with TMB solution for 10 min. The reaction was stopped with 100 ul H2SO4. Absorbance was determined at 450 nm with the CLAIRO reader. Fig. 11 shows that ANG-2B9c A6 and ANG-2B9c A7 effectively competes the binding of biotin labelled nerinetide to PSD-95 domain 2.

[0168] Binding of Ang2-NR2B9c variants to LRP-1 was evaluated with an ELISA assay (Human LRP-1 ELISA). A plate was coated with an affinity tag label antibody for LRP-1 and a reporter conjugated detector antibody which immunocaptures the sample analyte in solution. Recombinant LRP-1 extracellular domain fragment was used. The different test compounds at concentrations starting from 5uM in a 3-fold dilution were incubated with the antibody cocktail (LRP-1 Capture and Detector antibody) for 1 hour. After incubation, the wells were washed to removed unbound material. lOOul of TMB solution was added to each well and incubated for 10 min. The reaction was stopped with 100 ul H2SO4. Absorbance was determined at 450 nm with the CLAIRO reader.

[0169] Fig. 12 show that ANG-2B9c A6 and ANG-2B9c A7 are comparable to ANG-2B9c at competing for LRP-1 binding.

[0170] Example 7

[0171] This example compares nerinetide, ANG-2B9c, ANG-2B9c A7 and saline placebo for effect on infarct volume in a rat stroke model.

[0172] MATERIALS AND METHODS

[0173] Study Design

[0174] Acute cerebral ischemia was induced in young Sprague-Dawley rats using the permanent three-pial vessel occlusion (3PVO) model. Sample size calculations were performed using G Power version 3.1. Based on these parameters and prior pilot studies, estimated group sizes ranged from 10-12 animals per group.

[0175] Animals

[0176] Experiments were performed using male Sprague-Dawley rats aged 10-12 weeks and weighing 270-310 g (Charles River Laboratories, Montreal, QC, Canada). Animals were housed in standard laboratory cages with ad libitum access to food and water under controlled environmental conditions throughout the study period.

[0177] Permanent Distal Middle Cerebral Artery (3PVO) Model

[0178] Neuroprotective efficacy was evaluated using the permanent distal middle cerebral artery pial vessel occlusion (3PVO) model. Male Sprague-Dawley rats were anesthetized with 5% isoflurane for induction and maintained at 2% isoflurane throughout the surgical procedure. Core body temperature was continuously monitored and maintained within physiological limits.

[0179] A left-sided rostro caudal cranial window (6-8 mm in length) was created over the somatosensory cortex without disrupting the dura mater. Ischemia was induced by permanent bipolar coagulation of three distal arteriolar branches of the left middle cerebral artery.

[0180] Mean arterial blood pressure (MABP) was continuously monitored using a calibrated physiological pressure transducer (MLT844; AD Instruments, Colorado Springs, CO, USA) connected to a PowerLab 8 / 35 data acquisition system with LabChart Pro v8 software (AD Instruments). MABP was recorded during a 5-minute baseline period, throughout drug infusion, and for 5 minutes following treatment administration.

[0181] Treatment Administration

[0182] Animals were randomly assigned to receive placebo sterile 0.9% saline, nerinetide (7.6mg / kg), ANG-2B9c (9.8 mg / kg), or ANG-2B9c A7 (9.8mg / kg) (also referred to as D-ANG2B9c A7). Treatments were initiated 1 hour after stroke induction and administered as a 10-minute intravenous infusion.

[0183] Infarct Volume Assessment

[0184] At the experimental endpoint (24hrs), brains were harvested and sectioned into 1.5-mm coronal slices using a rat brain matrix. Sections were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) to delineate infarcted tissue. Digital images of brain slices were analyzed using NIH ImageJ software by an investigator blinded to treatment allocation.

[0185] Infarct volume and hemispheric swelling were calculated simultaneously. Edema-corrected hemispheric infarct volumes were expressed as a percentage of the contralateral hemisphere using the McBride formula (McBride et al., 2015).

[0186] Statistical Analysis

[0187] Statistical analyses were performed using GraphPad Prism version 10. Data are presented for animals that received assigned treatment and met predefined inclusion criteria. Animals were excluded if death occurred prior to drug administration or if frank subarachnoid hemorrhage was identified intraoperatively or postmortem by investigators blinded to treatment allocation.

[0188] Group differences in infarct volume were analyzed by t-test.

[0189] RESULTS

[0190] Fig. 13 shows that both ANG2B9c A7 and ANG2B9c significantly reduced infarct volume. Median volume is indicated by a line and mean with an x. P values are relative to the saline group.

[0191] All publications, patents and patent applications, accession numbers, websites and the like mentioned in this specification are incorporated by reference to the same extent as if each individual publication, patent or patent application was so individually denoted. To the extent more different content is associated with an accession number or other reference at different times, the content in effect as of the effective filing date of this application is meant. The effective filing date is the date of the earliest priority application disclosing the accession number in question. Unless otherwise apparent from the context any element, embodiment, step, feature or aspect of the invention can be performed in combination with any other, and steps can be performed in various orders beyond those exemplified.

Claims

What is claimed is:

1. An active agent comprising an internalization peptide comprising the sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1), or having up to five substitutions, deletions or insertions, or a variant of any of these substituted with one or more D-, beta, N-methyl or alpha-methyl amino acids, linked to a PSD-95 inhibitor.

2. The active agent of claim 1, wherein the internalization peptide has 3-7 D-amino acids.

3. The active agent of claim 1, wherein the internalization peptide has 2-5 D-amino acids at non-adjacent positions.

4. The active agent of claim 3, wherein the non-adjacent positions are R or K amino acids or amino acids C-terminal thereto.

5. The active agent of claim 1, wherein the internalization peptide has 2 or 3 D-amino acid substitutions of R and K residues at non-adjacent positions.

6. The active agent of any preceding claim, wherein the PSD-95 inhibitor comprises (a) IXi- [T / S]-X2V (SEQ ID NO:2) at the C-terminus, wherein [T / S] are alternative amino acids, XI is selected from among E, Q, A, or D or an analogue thereof, X2 is selected from among A, Q, D, E, N, N-Me-A, N-Me-Q, N-Me-D, and N-Me-N or an analog thereof, or (b) [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO: 12) at the C-terminus.

7. The active agent of any preceding claim, wherein the PSD-95 inhibitor comprises IESDV (SEQ ID NO:3) or IETDV (SEQ ID NO:4) at the C-terminus.

8. The active agent of any preceding claim, wherein the PSD-95 inhibitor comprises KLSSIESDV (SEQ ID NO:5) or KLSSIETDV (SEQ ID NO:6).

9. The active agent of any preceding claim, wherein the PSD-95 inhibitor comprises kLSSIESDV (SEQ ID NOY) or KlSSIESDV (SEQ ID NO:8) or klSSIESDV (SEQ ID NO: 9).

10. The active agent of any preceding claim, wherein the C-terminus of the internalization peptide is linked to the N-terminus of the PSD-95 inhibitor.

11. The active agent of claim 1, which has an amino acid sequence consisting of TFFYGGSRGkRNNFkTEEYkLSSIESDV (SEQ ID NO: 10) or TFFYGGSrGKrNNFkTEEYkLSSIESDV (SEQ ID NO: 11)).

12. The active agent of any preceding claim in dimeric form.

13. The active agent of any preceding claim characterized by reduced drop in blood pressure and / or histamine release relative to nerinetide on administration to a subject.

14. A pharmaceutical composition comprising the active agent of claim 1 and a pharmaceutically acceptable carrier.

15. A method of treating a subject having or at risk of a condition selected from stroke, cerebral ischemia, traumatic injury to the CNS, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy, or a neurodegenerative disease comprising administering an effective regime of the active agent of any one of claims 1- 13.

16. The method of claim 14, wherein the condition is stroke.

17. The method of claim 15 or 16, further comprising effecting reperfusion in the subject, optionally by administering a thrombolytic agent or by mechanical reperfusion.

18. The method of claim 17, wherein the reperfusion is effected by endovascular thrombectomy.

19. The method of any one of claims 15-18, wherein the active agent is administered at a dose of at least 10 mg / kg without co-admini strati on of an anti-inflammatory.

20. The method of any one of claims 15-18, wherein the active agent is administered at a dose of at least 25 mg / kg without co-administration of an anti-inflammatory.

21. An internalization peptide comprising the sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1), or having up to five substitutions, deletions or insertions, substituted with one or more D-, beta, N-methyl or alpha-methyl amino acids.

22. The internalization peptide of claim 21 comprising the sequence of SEQ ID NO: 1 substituted with 2 or 3 D-, beta, N-methyl or alpha-methyl amino acids.

23. The internalization peptide of claim 21 linked to a heterologous molecule, optionally a peptide.

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