Plasmin-resistant peptides for improving the therapeutic index

Abstract

According to the present invention, variants of the aforementioned active agent Tat-NR2B9c for treating stroke are provided. Target binding properties are retained by including an L-amino acid at the C-terminus, and plasmin resistance is conferred by including a D-amino acid. Nephrotoxicity associated with D-amino acids can be reduced by including D-amino acids at minimal positions and / or by co-administration with an inhibitor of D-amino acid oxidase. The resulting active agent has several advantages, including administration simultaneously with thrombolytic agents without significant loss of activity due to plasmin digestion. The resulting agent is also more suitable for administration by alternative routes of intravenous infusion, such as subcutaneous, intranasal and intramuscular, and for multiple dose regimens for the treatment of chronic diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 221,874, filed July 14, 2021, U.S. Patent No. 63 / 147,711, filed February 9, 2021, and U.S. Patent No. 63 / 135,498, filed January 8, 2021, each of which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains sequences in a text file (42 kilobytes) with the file name 572703-SQLIST, created on January 6, 2022, which is incorporated herein by reference. [Background technology]

[0003] Tat-NR2B9c (also known as NA-1 and nerinetide) is a drug that inhibits PSD-95, interfering with its binding to N-methyl-D-aspartate receptors (NMDARs) and neuronal nitric oxide synthase (nNOS), reducing excitotoxicity caused by cerebral ischemia. Treatment reduces infarct size and functional impairment in models of brain injury and neurodegenerative disease. Tat-NR2B9c has been successfully tested in Phase II trials (WO2010144721; Aarts et al., Science 298, 846-850 (2002), Hill et al., Lancet Neurol. 11: 942-950 (2012)) and Phase III trials (Hill et al., Lancet 395: 878-887 (2020)).

[0004] With the exception of glycine, all standard α-amino acids can exist in either of two optical isomers, which are mirror images of each other, called L- and D-amino acids. Proteins and most natural peptides are formed entirely of amino acids in the L configuration. D-amino acids have been detected in only a few natural peptides. These D-amino acids are formed when L-amino acids undergo post-translational modification. Because D-amino acids are rare in nature, they are generally not recognized by L-proteins at least to the same extent as L-amino acids. Simply replacing a D-amino acid with an L is not effective in creating a mimic of the parent molecule, as it changes the orientation of the side chain relative to the target site. Substituting an L or D-amino acid and reversing the order of the amino acids results in a side chain topology similar to the parent molecule, but with an inverted amide peptide bond that adopts a left-handed helix, whereas L-peptides adopt a right-handed helix. Thus, target binding can still be lost or altered.

[0005] U.S. Patent No. 62 / 959,091, filed January 9, 2020, and WO 2021140485 are each incorporated by reference in their entirety for all purposes. Summary of the Invention

[0006] The present invention provides an active agent, comprising an internalization peptide or a variant thereof linked to an inhibitor peptide, the inhibitor peptide inhibiting binding of PSD-95 to NOS and / or NMDAR2B, the internalization peptide having an amino acid sequence comprising RKKRRQRRR (SEQ ID NO: 13), the inhibitor peptide having a sequence comprising ESDV (SEQ ID NO: 14) at the C-terminus, the variant having up to a total of 5 substitutions or deletions in the internalization peptide and the inhibitor peptide, at least 4 C-terminal amino acids of the inhibitor peptide are L-amino acids, and at least one, but not all, residues of the internalization peptide are D-amino acids.

[0007] Optionally, the C-terminus of the internalization peptide is linked to the N-terminus of the inhibitor peptide to form a fusion peptide. Optionally, the internalization peptide has an amino acid sequence comprising RKKRRQRRR (SEQ ID NO: 13) and the inhibitor peptide has a sequence comprising IE[S / T]DV (SEQ ID NO: 4). Optionally, the 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acid residues of RKKRRQRRR (SEQ ID NO: 13) are D-amino acids. Optionally, the 2-8, 2-7, 2-6, 2-5, 2-4, 2-3 residues of RKKRRQRRR (SEQ ID NO: 13) are D-amino acids. Optionally, residues 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8 of RKKRRQRRR (SEQ ID NO: 13) are D-amino acids. Optionally, the internalization peptide comprises GRKKRRQRRR (SEQ ID NO: 11) of which residues 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 are D-amino acids. Optionally, residues 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 of the internalization peptide are D-amino acids. Optionally, residues 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8 of the internalization peptide are D-amino acids. Optionally, the internalization peptide comprises YGRKKRRQRRR (SEQ ID NO:1), of which residues 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 are D-amino acids. Optionally, residues 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 of the internalization peptide are D-amino acids. Optionally, residues 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-9, 4-8, 4-7, 4-6, 4-5, 5-9, 5-8, 5-7, 5-6, 6-9, 6-8, 6-7, 7-9, 7-8, or 8-9 of the internalization peptide are D-amino acids. Optionally, the inhibitor peptide comprises [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO:3) at the C-terminus. Optionally, the inhibitor peptide comprises IE-[S / T]-DV (SEQ ID NO:4) at the C-terminus. Optionally, the inhibitor peptide comprises IESDV (SEQ ID NO:5) at the C-terminus.Optionally, each of the five C-terminal amino acids of the inhibitor peptide is an L-amino acid. Optionally, the inhibitor peptide comprises KLSSIESDV (SEQ ID NO:2), where each residue of the inhibitor peptide is an L-amino acid except K, which can be an L or D amino acid. Optionally, the internalization peptide comprises at least two D-amino acids separated from each other by one or more amino acids independently selected from L-amino acids and glycine. Optionally, the internalization peptide comprises at least three D-amino acids, each separated from each other by one or more amino acids independently selected from L-amino acids and glycine. Optionally, at least one D-amino acid is R or K, or is located immediately C-terminal to an R or K residue. Optionally, all D-amino acids are R or K, or are located immediately C-terminal to an R or K residue. Optionally, at least one D-amino acid is not R or K and is not located immediately C-terminal to an R or K residue. Optionally, the internalization peptide has an amino acid sequence consisting of or including one of the following sequences: yGrkkrrqrrr (SEQ ID NO: 88) YGrkkrrQrrr (SEQ ID NO:89) YGrkkrrQrRr (SEQ ID NO: 90) YGrKKRrQrRr (SEQ ID NO:91) YGrKKRrQrRR (SEQ ID NO:92) YGrKKRrQRrR (SEQ ID NO:93) YGRKkrrQrrr (SEQ ID NO:94) YGRKkRrQrrRV (SEQ ID NO: 95) ygrkkrrqrrr (SEQ ID NO:96) rkkrrqrrr (SEQ ID NO:97) RkkrrQrRr (SEQ ID NO:98) RkkrrQrrR (SEQ ID NO: 99) RKkrrQrrR (SEQ ID NO: 100) rKKRrQrRr (SEQ ID NO: 101) RKkRrQrrR (SEQ ID NO: 102) rKKRrQrRR (SEQ ID NO: 103) rKKRrQRRR (SEQ ID NO: 104) rKKRRQrRR (SEQ ID NO: 105) RKKRrQrRR (SEQ ID NO: 106) RKKRRQrRR (SEQ ID NO: 107) rKKRRQRRR (SEQ ID NO: 108) RKKRrQRRR (SEQ ID NO: 109)

[0008] Here, lower case letters indicate D-amino acids and upper case letters indicate L-amino acids.

[0009] Optionally, the active agent has an amino acid sequence consisting of or including RkkrrQrRrIESDV (SEQ ID NO: 110, NoNO411), RKkRrQrrRIESDV (SEQ ID NO: 111) or rKKRrArRRIESDV (SEQ ID NO: 112).

[0010] According to the present invention, there is provided an active agent comprising an internalization peptide or a variant thereof linked to an inhibitor peptide. The inhibitor peptide inhibits binding of PSD-95 to NOS and / or NMDAR2B. The internalization peptide has an amino acid sequence comprising RKKRRQRRR (SEQ ID NO: 13). The inhibitor peptide has a sequence comprising ESDV (SEQ ID NO: 14) at the C-terminus, the variant having up to 5 substitutions or deletions in the internalization peptide and the inhibitor peptide, wherein at least 4 C-terminal amino acids of the inhibitor peptide are L-amino acids and 3-5 residues of the internalization peptide are D-amino acids. Optionally, the inhibitor peptide comprises IE[S / T]DV (SEQ ID NO: 4) at the C-terminus, wherein IE[S / T]DV (SEQ ID NO: 4) is an L-amino acid. Optionally, each D residue is K or R, or C-terminal to a K or R residue. Alternatively, the active agent lacks a stretch of four or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising contiguous amino acids independently selected from R or K. Alternatively, the active agent lacks a stretch of four or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising contiguous amino acids independently selected from R or K. Alternatively, the active agent lacks a stretch of five or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising contiguous amino acids independently selected from R or K, and if there is a stretch of four consecutive amino acids, said stretch is located at the N-terminus of the internalization peptide. Alternatively, the active agent lacks a stretch of five or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising contiguous amino acids independently selected from R or K, and if there is a stretch of four consecutive amino acids, said stretch is located at the N-terminus of the internalization peptide. Optionally, the internalization peptide has an amino acid sequence comprising YGRKKRRQRRR (SEQ ID NO: 1) having three D residues, a D residue between positions 3 and 5, a D residue between positions 7 or 8, and a D residue between positions 9 and 11, numbered from the N-terminus. Optionally, the inhibitor peptide is linked to the internalization peptide via a spacer.Optionally, the spacer is a peptide. Optionally, the spacer is a peptide of 2-4 residues. Optionally, the spacer lacks lysine or arginine residues. Optionally, the spacer comprises one or more residues independently selected from glycine, alanine, serine, and leucine (each in the L-form). Optionally, the spacer is KLSS (SEQ ID NO: 113), where K and / or L are in the D-form.

[0011] According to the present invention, there is provided an active agent having an amino acid sequence that comprises or includes YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 58), or a variant thereof having up to 5 deletions or additions thereto, wherein the 3-5 amino acids at positions other than the 5 C-terminal amino acids are D-amino acids; or an amino acid sequence having at least 75% amino acid sequence identity with the sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 58), wherein the 3-5 amino acids at positions other than the 5 C-terminal amino acids are D-amino acids. Optionally, there are no D-amino acids occupying adjacent positions in the active agent. Optionally, each D-amino acid is separated from another D-amino acid by at least two amino acids selected from L-amino acids and glycine. Optionally, each D-amino acid is separated from another D-amino acid by at least three amino acids independently selected from L-amino acids and glycine. Optionally, the active agent comprises four D-amino acids. Optionally, the active agent comprises three D-amino acids. Alternatively, the active agent lacks a stretch of four or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising two adjacent amino acids independently selected from R and K. Alternatively, the active agent lacks a stretch of four or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising amino acids selected from R or K. Alternatively, the active agent lacks a stretch of five or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising two adjacent amino acids independently selected from R and K, and if said stretch of four consecutive amino acids is present, said stretch is located at the N-terminus of the active agent. Alternatively, the active agent lacks a stretch of five or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising amino acids independently selected from R or K, and if said stretch of four consecutive amino acids is present, said stretch is located at the N-terminus of the internalization peptide.Optionally, the active agent comprises three or four D-amino acids, a first D-amino acid between positions 3-5, a second D-amino acid between positions 7 or 8, a third D-amino acid between positions 9-11, and optionally a fourth D-amino acid at position 12 or 13. Optionally, the active agent comprises at least two copies of an inhibitor peptide. Optionally, the two copies of the inhibitor peptide are linked to an internalization peptide via a branched linker. Optionally, the active agent comprises an amino acid sequence comprising or consisting of YGrKKRrQrRRkLSSIESDV (SEQ ID NO: 114), YGRkKRrQRrRKLSSIESDV (SEQ ID NO: 115), or YGrKKRrQrRRKlSSIESDV (SEQ ID NO: 116).

[0012] Optionally, the active agent has improved plasmin resistance compared to nerinetide. Optionally, the active agent has improved plasma half-life compared to nerinetide. Optionally, the active agent competes with nerinetide for binding to PSD-95. Optionally, the active agent has and / or an active agent comprising an internalization peptide of sequence ygrkkrrqrrr (SEQ ID NO:96) linked to an inhibitor peptide of sequence KLSSIESDV (SEQ ID NO:2), where each amino acid (except glycine) of said internalization peptide is a D-amino acid and each amino acid of said inhibitor peptide is an L-amino acid; and / or an active agent comprising an internalization peptide of sequence ygrkkrrqrrr (SEQ ID NO:96) linked to klssIESDV (SEQ ID NO:117) (lower case letters indicate D-amino acids and upper case letters indicate L-amino acids), where each amino acid (except glycine) of said internalization peptide is a D-amino acid; and / or an active agent having the amino acid sequence vdseisslkrrrqrrkkrgy (SEQ ID NO:118) (lower case letters indicate D-amino acids). Optionally, said active agent has a binding affinity for PSD-95 that is within 2-fold of nerinetide. Optionally, the active agent has an IC50 for inhibiting PSD-95 binding to NMDAR2B that is within 2-fold of Tat-NR2B9c. Optionally, the active agent has a higher area under the curve (AUC) or CMax than nerinetide (e.g., when administered subcutaneously).

[0013] Optionally, the active agent is a chloride salt.

[0014] Further provided in accordance with the present invention is a formulation of an active agent having histidine, trehalose, and optionally sodium benzoate. Further provided in accordance with the present invention is a formulation of an active agent having phosphate buffer and optionally sodium benzoate.

[0015] The present invention further provides a co-formulation comprising an active agent and an anti-inflammatory agent. Optionally, the anti-inflammatory agent is a mast cell degranulation inhibitor or an antihistamine.

[0016] The present invention further provides a co-formulation comprising an active agent and a thrombolytic agent.

[0017] The present invention further provides a co-formulation comprising the above active agent and an inhibitor of D-amino acid oxidase, optionally wherein the inhibitor of D-amino acid oxidase is risperidone, sodium benzoate or 5-chloro-benzo[d]isoxazol-3-ol.

[0018] The present invention further provides a method for treating a subject suffering from or at risk of suffering from a condition selected from stroke, cerebral ischemia, CNS trauma, concussion, reperfusion injury, subarachnoid hemorrhage, pain, anxiety, epilepsy or a neurodegenerative disease, comprising administering to said subject an effective dosage regimen of an active agent or a formulation as described above. Optionally, said method further comprises administering an inhibitor of D-amino acid oxidase activity.

[0019] According to the present invention, there is further provided a method of treating ischemic stroke in a subject suffering from or at risk of suffering from stroke, comprising administering to said subject an effective dosing regimen of the above-mentioned active agent or formulation, wherein said active agent and thrombolytic agent are administered at a sufficiently short interval such that cleavage of the active agent induced by the thrombolytic agent is reduced by the inclusion of a D-amino acid in said active agent. Optionally, said method further comprises administering an inhibitor of D-amino acid oxidase activity. Optionally, said thrombolytic agent is administered within a 60, 30 or 15 minute window before said active agent is administered. Optionally, said active agent and thrombolytic agent are administered simultaneously.

[0020] The present invention further provides a method of delivering an active agent or formulation to a subject, comprising administering the active agent via a non-intravenous route, wherein the active agent is delivered to plasma at therapeutic levels. Optionally, the active agent is administered subcutaneously. Optionally, the active agent is administered intramuscularly. Optionally, the active agent is administered intranasally or intrapulmonary. Optionally, the dose is greater than 3 mg / kg, 10 mg / kg, 20 mg / kg. Optionally, the dose is less than 10 mg / kg and the active agent is administered without co-administration of a mast cell degranulation inhibitor or an antihistamine. Optionally, the dose is greater than 10 mg / kg and the active agent is administered with a mast cell degranulation inhibitor or an antihistamine. Optionally, the subject is suffering from or at risk of suffering from a condition selected from stroke, cerebral ischemia, CNS traumatic injury, concussion, reperfusion injury, pain, anxiety, epilepsy, subarachnoid hemorrhage or a neurodegenerative disease (e.g., Alzheimer's disease or Parkinson's disease).

[0021] The present invention further provides a method for treating a subject suffering from or at risk of suffering from a condition selected from stroke, cerebral ischemia, traumatic injury of the CNS, subarachnoid hemorrhage, pain, anxiety, epilepsy, which comprises administering to the subject an effective dosing regimen of an active agent and an inhibitor of D-amino acid oxidase activity, the active agent comprising an internalization peptide or a variant thereof linked to an inhibitor peptide, the inhibitor peptide inhibiting binding of PSD-95 to NOS and / or NMDAR2B, the internalization peptide having an amino acid sequence comprising RKKRRQRRR (SEQ ID NO: 13), the inhibitor peptide having a sequence at its C-terminus comprising ESDV (SEQ ID NO: 14), the variant comprising up to five substitutions or deletions in the internalization peptide and the inhibitor peptide, at least four C-terminal amino acids of the inhibitor peptide being L-amino acids, and at least one residue of the internalization peptide being a D-amino acid. According to the present invention, there is further provided a method of treating ischemic stroke in a subject suffering from or at risk of suffering from stroke, comprising administering to the subject an effective dosing regimen of an active agent and an inhibitor of D-amino acid oxidase activity, wherein a thrombolytic agent is co-administered to the subject, wherein the active agent comprises an internalization peptide or a variant thereof linked to an inhibitor peptide, wherein the inhibitor peptide inhibits binding of PSD-95 to NOS, the internalization peptide having an amino acid sequence comprising RKKRRQRRR (SEQ ID NO: 13), the inhibitor peptide having a sequence at its C-terminus comprising ESDV (SEQ ID NO: 14), the variant comprising up to five substitutions or deletions in the internalization peptide and the inhibitor peptide, wherein at least the four C-terminal amino acids of the inhibitor peptide are L-amino acids, and at least one residue of the internalization peptide is a D-amino acid. The active agent and thrombolytic agent are administered with an interval sufficiently short that cleavage of the active agent induced by the thrombolytic agent is reduced by the inclusion of a D-amino acid in the active agent.Optionally, in such methods, the active agent comprises an internalization peptide or a variant thereof linked to an inhibitor peptide, wherein the inhibitor peptide inhibits binding of PSD-95 to NOS, wherein the internalization peptide has an amino acid sequence comprising YGRKKRRQRRR (SEQ ID NO:1), and wherein the inhibitor peptide has a sequence comprising KLSSIESDV (SEQ ID NO:2), and wherein the variant has up to five substitutions or deletions in the internalization peptide and the inhibitor peptide, wherein at least the four C-terminal amino acids of the inhibitor peptide are L-amino acids, and wherein a contiguous segment of amino acids comprising all of the R and K residues are D-amino acids.

[0022] The active agent comprises an internalization peptide or variant thereof linked to an inhibitor peptide. The inhibitor peptide inhibits binding of PSD-95 to NOS and / or NMDAR2B. The internalization peptide has an amino acid sequence comprising YGRKKRRQRRR (SEQ ID NO:1), and the inhibitor peptide has a sequence comprising KLSSIESDV (SEQ ID NO:2). The variant has up to a total of five substitutions or deletions in the internalization peptide and the inhibitor peptide. At least four C-terminal amino acids of the inhibitor peptide are L-amino acids, and a contiguous segment of amino acids comprising R and K residues are D-amino acids. Optionally, the C-terminal residue immediately adjacent to the most C-terminal R or K residue is also a D-residue. Optionally, the C-terminus of the internalization peptide is linked to the N-terminus of the inhibitor peptide to form a fusion peptide. Optionally, the inhibitor peptide comprises [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO:3) at the C-terminus. Alternatively, the inhibitor peptide comprises IE-[S / T]-DV (SEQ ID NO: 4) at the C-terminus. Alternatively, the inhibitor peptide comprises IESDV (SEQ ID NO: 5) at the C-terminus.

[0023] Optionally, each of the five C-terminal amino acids of the inhibitor peptide is an L-amino acid. Optionally, each other residue of the active agent is a D-amino acid. Optionally, the active agent has the amino acid sequence of ygrkkrrqrrrklssIESDV (SEQ ID NO:6), ygrkkrrqrrrkssIESDV (SEQ ID NO:7), ygrkkrrqrrrksIESDV (SEQ ID NO:8) or ygrkkrrqrrrkIESDV (SEQ ID NO:9). Optionally, the active agent has the amino acid sequence of ygrkkrrqrrrklssIESDV (SEQ ID NO:6), where lowercase letters are D-amino acids and uppercase letters are L-amino acids (except for glycine, which has no chiral form and can be represented as uppercase or lowercase).

[0024] Optionally, the active agent has a higher plasma stability than nerinetide.Optionally, the active agent has a higher plasmin resistance than nerinetide.Optionally, the active agent has a binding affinity for PSD-95 that is within 2-fold of nerinetide.Optionally, the active agent has an IC50 for inhibiting the binding of PSD-95 to NMDAR2B that is within 2-fold of nerinetide.

[0025] Optionally, the active agent is a chloride salt.

[0026] There is further provided in accordance with the invention a formulation of any one of the above active agents further comprising histidine and trehalose.

[0027] There is further provided in accordance with the invention a formulation of any one of the above active agents, further comprising a phosphate buffer.

[0028] The present invention further provides a co-formulation comprising any one of the active agents described above and an anti-inflammatory agent. Optionally, the anti-inflammatory agent is a mast cell degranulation inhibitor or an antihistamine.

[0029] There is further provided in accordance with the present invention a co-formulation comprising any one of the active agents described above and a thrombolytic agent.

[0030] Further provided in accordance with the present invention is a method of treating a subject suffering from or at risk of suffering from a condition selected from stroke, cerebral ischemia, traumatic injury of the CNS, subarachnoid hemorrhage, pain, anxiety, epilepsy, comprising administering to the subject an effective dosage regimen of any of the above-mentioned active agents.

[0031] The present invention further provides a method for treating ischemic stroke in a patient suffering from or at risk of suffering from stroke. The method comprises administering to the subject an effective dosing regimen of an active agent, wherein the subject is co-administered with a thrombolytic agent. The active agent comprises an internalization peptide linked to an inhibitor peptide, the inhibitor peptide inhibiting PSD-95 to NOS and / or NMDAR2B. At least four C-terminal amino acids of the inhibitor peptide are L-amino acids, and at least one of the remaining amino acids of the active agent is a D-amino acid. The active agent and the thrombolytic agent are administered at a sufficiently short dosing interval that cleavage of the active agent induced by the thrombolytic agent is reduced by the inclusion of at least one D-amino acid. Optionally, the internalization peptide is linked at its N-terminus to the C-terminus of the inhibitor peptide to form a fusion protein. Optionally, the inhibitor peptide comprises [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO:3) as the last four residues. Optionally, the inhibitor peptide comprises [I]-[E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO:10) as the last five residues, each of which is an L-amino acid. Optionally, the internalization peptide is a tat peptide. Optionally, at least eight residues of the tat peptide are D-amino acids. Optionally, each residue of the tat peptide is a D-amino acid. Optionally, the internalization peptide comprises GRKKRRQRRR (SEQ ID NO:11) which is linked at its N-terminus to KLSSIESDV (SEQ ID NO:2) or KLSSIETDV (SEQ ID NO:12) as the inhibitor peptide to form a fusion protein. Optionally, the active agent comprises a contiguous segment of D-residues, each of which comprises a K residue and an R residue. Optionally, the active agent comprises ygrkkrrqrrrklssIESDV (SEQ ID NO: 6), where lower case letters represent D-amino acids (other than glycine) and upper case letters represent L-amino acids. Optionally, the thrombolytic agent is administered within a 60 minute, 30 minute, or 15 minute window prior to the active agent. Optionally, the active agent and the thrombolytic agent are administered simultaneously.

[0032] According to the present invention, there is further provided a method of delivering an active agent to a subject. The method comprises administering the above-mentioned active agent via a non-intravenous route. The active agent is delivered to the plasma at therapeutic levels. Optionally, the active agent is administered subcutaneously. Optionally, the active agent is administered intramuscularly. Optionally, the active agent is administered intranasally or intrapulmonary. Optionally, the dose is greater than 3 mg / kg. Optionally, the dose is greater than 10 mg / kg. Optionally, the dose is greater than 20 mg / kg. Optionally, the dose is less than 10 mg / kg and variants are administered without co-administration of a mast cell degranulation inhibitor or an antihistamine. Optionally, the dose is greater than 10 mg / kg and variants are administered. Optionally, the subject suffers from or is at risk of suffering from a disease selected from stroke, cerebral ischemia, traumatic injury of the CNS, pain, anxiety, epilepsy, subarachnoid hemorrhage, Alzheimer's disease and Parkinson's disease.

[0033] According to the present invention there is further provided a chimeric agent comprising an internalization peptide or a variant thereof linked to an agent useful for the treatment of a disorder, said internalization peptide having an amino acid sequence comprising RKKRRQRRR (SEQ ID NO: 13), said variant having one, two or up to three substitutions or deletions (not including L to D substitutions of the same amino acid), and wherein residues 3-5 of said internalization peptide are D-amino acids. [Brief description of the drawings]

[0034] [Figure 1] Plasmin cleavage site on NA-1 (SEQ ID NO:58).

[0035] [Diagram 2] When administered simultaneously with rt-PA, the NA-1 content in rat plasma was significantly decreased.

[0036] [Diagram 3] When administered simultaneously with rt-PA, the NA-1 content in human plasma is significantly reduced.

[0037] [Figure 4] When administered concomitantly with rt-PA (5.4 mg / kg), NA-1 Cmax and AUC were significantly decreased.

[0038] [Diagram 5] D-Tat-L-NR2B9c exhibits superior stability in rat plasma in the presence of rt-PA compared to NA-1.

[0039] [Figure 6] D-Tat-L-NR2B9c is resistant to proteolysis upon infusion of rt-PA in human plasma.

[0040] [Figure 7] When administered concomitantly with TNK, NA-1 content in human plasma is decreased, whereas D-Tat-L-NR2B9c content is maintained.

[0041] [Figure 8] When administered simultaneously with TNK, NA-1 content in rat plasma is decreased, whereas D-Tat-L-NR2B9c content is maintained.

[0042] [Figure 9] D-Tat-L-NR2B9c is resistant to plasmin cleavage in PBS medium.

[0043] [Figure 10] As a result, D-Tat-L-NR2B9c dissociates the NR2B:PSD95 complex formed in rat brain lysates.

[0044] [Figure 11] D-Tat-L-NR2B9c and D-Tat-L-IESDV (SEQ ID NO: 6) effectively bind to the target protein PSD95-PDZ2.

[0045] [Figure 12]As a result, NA-1 and D-Tat-L-NR2B9c have high binding affinity to the PSD95-PDZ2 domain.

[0046] [Figure 13] Subcutaneous NA-1 achieved similar plasma exposure as IV NA-1.

[0047] [Figure 14] Subcutaneous NA-3 (D-Tat-L-IESDV SEQ ID NO:6) achieved higher plasma concentrations and greater plasma exposure compared to subcutaneous NA-1.

[0048] [Figure 15] FIG. 15A (table) and FIG. 15B (graph) show that subcutaneous NA-3 (D-Tat-L-IESDV SEQ ID NO:6) achieved greater plasma exposure compared to SQ NA-1.

[0049] [Figure 16] Intrapulmonary instillation of D-NA-1 and NA-3 (D-Tat-L-IESDV SEQ ID NO:6) achieved higher plasma concentrations and greater plasma exposure compared to intrapulmonary NA-1.

[0050] [Figure 17] Following subcutaneous administration of NA-3 (D-Tat-L-IESDV SEQ ID NO: 6) at a dose of 8.3 mg / kg or 2.8 mg / kg, histamine release was significantly reduced.

[0051] [Figure 18] There was no significant histamine release after intravenous administration of a coformulation of D-Tat-L-NR2B9c (7.6 mg / kg) and lodoxamide (0.6 mg / kg).

[0052] [Figure 19] Intravenous administration of D-Tat-L-NR2B9c and lodoxamide 1 hour after stroke onset reduced infarct volume and hemispheric swelling in eMCAo model animals.

[0053] [Figure 20] Administration of D-Tat-L-NR2B9c and lodoxamide improved neurological outcome 24 hours after stroke onset.

[0054] [Figure 21] Effect of subcutaneous NA-3 and nerinetide on infarct volume.

[0055] [Figure 22] Plasma concentrations of nerinetide and NA-3 at 15 minutes after subcutaneous administration.

[0056] [Figure 23] Compared to nerinetide IV infusion, subcutaneous NA-3 at 25 mg / kg resulted in higher Cmax and AUC.

[0057] [Figure 24] Pharmacokinetic profile of NA-3 after subcutaneous administration.

[0058] [Diagram 25] Physiological parameters of experimental animals in the NHP NA-3 pharmacokinetic study.

[0059] [Figure 26] 26A and 26B show that following administration of NA-3 SQ (25 mg / kg), (A) BUN and creatine and (B) other renal function biomarker levels were increased.

[0060] [Figure 27] Dose reduction, decreased D-amino acid content, and DAAO inhibition have all been shown to lower creatinine levels.

[0061] [Figure 28] Reduction in dose, reduction in D-amino acid content, and DAAO inhibition have all been shown to lower BUN levels.

[0062] [Figure 29] PSD-95 inhibitors containing small amounts of D-amino acids may confer resistance to plasmin. NoNO411: RkkrrQrRrIESDV (SEQ ID NO: 110), NoNO414: RKkRrQrrRIESDV (SEQ ID NO: 111), NoNO415: rKKRrQrRRIESDV (SEQ ID NO: 134).

[0063] [Diagram 30] Figures 30A and 30B show that nerinetide is effective in reducing (A) infarct size and (B) neurological deficits in a rat tMCAo model over at least the dose range of 0.25-25 mg / kg.

[0064] [Diagram 31] 1 shows Nerinetide and other peptides containing D-amino acids that compete with biotinylated Nerinetide for binding to PSD-95 domain 2.

[0065] [Diagram 32] 1 shows plasma concentrations in rats given various doses of NoNO42 IV compared to nerinetide.

[0066] [Diagram 33] 1 shows blood pressure versus time in rats treated with various doses of NoNO42 compared to nerinetide.

[0067] [Diagram 34] 1 shows the change in body weight of rats treated with various doses of NoNO42 compared to nerinetide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] definition A "pharmaceutical formulation" or composition is a preparation in which an active agent is effective and contains no additional components that are toxic to the subject to which the formulation is administered.

[0069] The use of capitalized one-letter amino acid codes can refer to either D- or L-amino acids unless the context dictates otherwise. Lowercase one-letter codes are used to indicate D-amino acids. Glycine does not have a D- or L-form and can therefore be represented interchangeably by uppercase and lowercase letters. When a peptide is said to contain a defined number of D-amino acids, glycine is not included in such number of D-amino acids. When a peptide is said to have a stretch of contiguous L-amino acids, it is meant that the stretch is not interrupted by D-amino acids, although the stretch of amino acids may include glycine unless otherwise stated. Such a stretch is also referred to as a contiguous stretch of amino acids independently selected from L-amino acids and glycine.

[0070] Numerical values ​​such as concentration or pH are given within tolerances that reflect the precision with which the values ​​can be measured. Small values ​​are rounded to the nearest integer unless the context requires otherwise. Recitations of ranges of values ​​refer to any integer or subrange within the available range, unless the context requires otherwise.

[0071] The terms "disease" and "condition" are used interchangeably to refer to any disruption or arrest of the normal structure or function of a subject.

[0072] The stated doses should be understood to include the error inherent in the precision with which doses can be measured using typical hospital equipment.

[0073] The terms "isolated" or "purified" mean that the subject species (e.g., peptide) has been purified from contaminants present in a sample, such as a sample from a natural source that contains the subject species. When a subject species is isolated or purified, it is the predominant macromolecular (e.g., polypeptide) species in the sample (i.e., on a molar basis, it is more abundant than any other individual species in the composition), and preferably the subject species comprises at least 50% (on a molar basis) of all macromolecular species present. Generally, an isolated, purified, or substantially pure composition comprises greater than 80-90% of all macromolecular species present in the composition. More preferably, the subject species is purified to essential homogeneity (i.e., conventional detection methods cannot detect contaminating species in the composition), such that the composition is essentially composed of a single macromolecular species. The terms isolated or purified do not necessarily exclude the presence of other components that are intended to act in combination with the isolated species. For example, an internalizing peptide is described as isolated even though it is linked to an active peptide.

[0074] A "peptide mimetic" refers to a synthetic chemical entity that has substantially the same structure and / or functional properties as a peptide composed of natural amino acids. A peptidomimetic can include fully synthetic non-natural amino acid analogs, or can be a chimeric molecule of partly natural peptide amino acids and partly non-natural amino acid analogs. A peptidomimetic can also incorporate any number of conservative substitutions of natural amino acids, so long as the substitutions do not substantially alter the mimetic's structure and / or inhibitory or binding activity. A polypeptide mimetic composition can contain any combination of non-natural structural components that are generally derived from three structural groups: a) residue linkages other than the natural amide bond ("peptide bond") linkage; b) non-natural residues replacing naturally occurring amino acid residues; or c) residues that induce secondary structure mimicry, i.e., residues that induce or stabilize secondary structures such as, for example, beta-turn, gamma-turn, beta-sheet, alpha-helical conformations. In a peptidomimetic of a chimeric peptide that includes an active peptide and an internalizing peptide, one or both of the active portion and the internalizing portion can be a peptidomimetic.

[0075] The term "specific binding" refers to binding between two molecules, e.g., a ligand and a receptor, that is characterized by the ability of one molecule (the ligand) to associate with another specific molecule (the receptor) even in the presence of many other molecules, i.e., exhibiting preferential binding of one molecule over another in a heterogeneous mixture of molecules. Specific binding of a ligand to a receptor can also be demonstrated by the binding of a detectably labeled ligand to the receptor being reduced in the presence of an excess of unlabeled ligand (i.e., binding competition assays).

[0076] Excitotoxicity is a pathological process in which neurons and surrounding cells are damaged and killed by overactivation of receptors for the excitatory neurotransmitter glutamate, such as NMDA receptors, e.g., NMDA receptors with the NMDAR2B subunit.

[0077] The term "subject" or "patient" includes humans, as well as veterinary animals, such as mammals, and laboratory animals, such as mice or rats, used in preclinical trials.

[0078] By tat peptide is meant a peptide consisting of or including RKKRRQRRR (SEQ ID NO: 13) in which no more than five residues have been deleted, substituted or inserted, and which maintains the ability to promote cellular uptake of a linked peptide or other agent. Preferably, any amino acid changes are conservative substitutions. Preferably, any substitutions, deletions or internal insertions in the assembly leave the peptide with a net positive charge, preferably similar to that of the sequence above. Such can be achieved, for example, by not substituting any R or K residues, or by keeping the sum of R and K residues the same. The amino acids of the tat peptide can be derivatized with biotin or a similar molecule to reduce inflammatory responses.

[0079] Co-administration of pharmacologically active agents means that the agents are administered close enough in time that detectable amounts of the agents are present in the plasma at the same time and / or that the agents exert a therapeutic effect on the same episode of a disease state or that the agents act synergistically or synergistically on the same episode of a disease state. For example, an anti-inflammatory agent acts synergistically when both agents are administered close enough in time to an agent that includes a tat peptide that the anti-inflammatory agent can inhibit an anti-inflammatory response that may be induced by the internalized peptide.

[0080] Statistically significant refers to a p-value of <0.05, preferably <0.01, and most preferably <0.001.

[0081] An episode of a medical condition means a period during which a sign or symptom of a medical condition is present that is interspersed with longer periods during which the sign and / or symptom is absent or present to a lesser extent.

[0082] The term "NMDA receptor" or "NMDAR" refers to a membrane-bound protein known to interact with NMDA, including the various subunit types described below. Such receptors can be human or non-human (e.g., rat, rabbit, monkey).

[0083] The therapeutic index is conventionally used as LD50 (lethal dose) / ED50 (effective dose) in animal studies and TD50 (toxic dose) / ED50 (effective dose) in humans.

[0084] The terms "comprising," "consisting," and "consisting essentially of" are each used according to convention. Thus, "comprising" does not exclude additional elements or steps, whereas "consisting essentially of" refers to a basic and novel feature of the invention. Reference to product or method features in this specification should be understood, unless the context requires otherwise, as support for claiming any of the following alternatives: a product or method that comprises, consists of, or consists essentially of the specified features.

[0085] When the specification refers to a numbered position in the sequence of Nerinetide (e.g., the third amino acid) or the tat internalization peptide of Nerinetide, it should be understood that the specification also discloses a variant of Nerinetide or the corresponding position in the internalization peptide of Nerinetide, if the variant maximally aligns with Nerinetide or the internalization peptide of Nerinetide. Detailed Description

[0086] I. Overview

[0087] According to the present invention, there is provided a variant of the aforementioned active agent (Tat-NR2B9c) for treating stroke, in which the four or five amino acids at the C-terminus are L-amino acids and one or more of the remaining amino acids are D-amino acids. The inclusion of D-amino acids inhibits proteolysis of the agent, particularly by plasmin, which is naturally present in plasma and is induced by administration of thrombolytic agents. D-amino acids can also inhibit other proteases encountered by Tat-NR2B9c, such as in plasma and subcutaneous tissue. Retention of L-amino acids at the C-terminus is sufficient to retain the binding and inhibitory properties of Tat-NR2B9c, regardless of the presence or absence of D-amino acids in some or all of the remaining molecule. Nephrotoxicity associated with D-amino acids can be mitigated by including D-amino acids at minimal positions and / or by co-administration with an inhibitor of D-amino acid oxidase. The resulting active agent has several advantages, including extended half-life and resistance to plasmin induced by co-administered or co-formulated thrombolytic agents. The resulting drug is also suitable for administration by alternative routes to intravenous infusion (e.g., subcutaneous, intranasal, and intramuscular administration), since the drug's longer half-life can compensate for the longer time required by these routes to reach therapeutic plasma concentrations. Administration by such routes allows for higher doses to be administered without significant histamine release and is more suitable for field rather than medical facility implementation. The longer half-life of the active drug of the present invention makes it more suitable for maintaining therapeutic concentrations over an extended period of time in a multiple dose regimen. Such a regimen is useful for promoting recovery from pathological and cognitive impairments resulting from stroke and for reducing initial impairment. Multiple dose regimens are also useful for treating chronic diseases such as Alzheimer's disease and Parkinson's disease.

[0088] II. Active Agents

[0089] The active agents of the present invention include peptide inhibitors that specifically bind to PSD-95 (e.g., Stathakism, Genomics 44(1):71-82(1997)) and thereby inhibit the binding of PSD-95 to NMDA receptor 2 subunits, including NMDAR2B (e.g., GenBank ID 4099612) and / or NOS (e.g., neuronal or nNOS Swiss-Prot P29475), and internalization peptides that facilitate the peptide inhibitors crossing cell membranes and the blood-brain barrier. Preferred peptides inhibit the human form of PSD-95 NMDAR 2B and NOS used in human subjects. However, inhibition may be exhibited from species variants of the protein. Some peptide inhibitors have an amino acid sequence at their C-terminus that includes [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO:3). Exemplary peptides include 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 at their C-terminus that includes [I]-[E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO:10). Exemplary peptides include IESDV (SEQ ID NO:5), IESEV (SEQ ID NO:21), IETDV (SEQ ID NO:22), 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 have an amino acid sequence at their C-terminus that includes X1-[T / S]-X2V (SEQ ID NO:27). where [T / S] is an alternative amino acid, X1 is selected from E, Q, A, D or analogs thereof, and X2 is selected from A, Q, E, D, N, N-Me-A, N-Me-Q, N-Me-D, N-Me-N and analogs thereof (Bach, J. Med. Chem. 51, 6450-6459 (2008); and WO 2010 / 004003). Optionally, the peptide is N-alkylated at the P3 position (the third amino acid from the C-terminus, i.e., the position occupied by [T / S]).The peptides may be N-alkylated with cyclohexane or aromatic substituents and may further comprise a spacer group between the substituent and the terminal amino group of the peptide or peptide analog. The spacer group is an alkyl group, preferably selected from methylene, ethylene, propylene, butylene. The aromatic substituent may be a naphthalene-2-yl moiety or an aromatic ring substituted with one or two halogens and / or alkyl groups. Some inhibitor peptides have an amino acid sequence at the C-terminus comprising IX1-[T / S]-X2V (SEQ ID NO:28). Exemplary inhibitor peptides have the sequences IESDV (SEQ ID NO:5), IETDV (SEQ ID NO:22), KLSSIESDV (SEQ ID NO:2) and KLSSIETDV (SEQ ID NO:12). Inhibitor peptides usually comprise 3-25 amino acids (without internalization peptide), with peptide lengths of 5-10 amino acids, particularly 9 amino acids (without internalization peptide) being preferred.

[0090] Internalizing peptides are a well-known class of relatively short peptides that allow many cellular or viral proteins to cross membranes. They can also facilitate the passage of a linking peptide through a cell membrane or the blood-brain barrier. Internalizing peptides are also known as cell membrane transduction peptides, protein transduction domains, brain shuttle or cell penetrating peptides and may have, for example, 5 to 30 amino acids. Such peptides generally have a positive charge (compared to general proteins) from the above general notation of arginine and / or lysine residues, which is believed to facilitate membrane penetration. Some such peptides have at least 5, 6, 7 or 8 arginine and / or lysine residues. Examples include the antennapedia protein (Bonfanti, Cancer Res. 57, 1442-6 (1997)) (and variants thereof), the tat protein of the human immunodeficiency virus, protein VP22, the product of the UL49 gene of herpes simplex virus type 1, penetratin, SynB1 and 3, Transportan, Amphipathic, gp41NLS, polyArg, and several plant and bacterial protein toxins such as ricin, abrin, modeccin, diphtheria toxin, cholera toxin, anthrax toxin, heat-labile toxin, and Pseudomonas aeruginosa exotoxin A (ETA).Other examples are described in the following publications: Temsamani, Drug Discovery Today, 9(23):1012-1019, 2004; De Coupade, Biochem J., 390:407-418, 2005; Saalik Bioconjugate Chem. 15:1246-1253, 2004; Zhao, Medicinal Research Reviews 24(1):1-12, 2004; Deshayes, Cellular and Molecular Life Sciences 62:1839-49, 2005; Gao, ACS Chem. Biol. 2011, 6, 484-491, SG3(RLSGMNEVLSFRWL) (SEQ ID NO: 29)), Stalmans PLoS ONE 2013, 8(8)e71752, 1-11 and supplement; Figueiredo et al., IUBMB Life 66, 182-194(2014); Copolovici et al., ACS Nano, 8, 1972-94(2014); Lukanowski, Biotech J. 8, 918-930(2013); Stockwell, Chem. Biol. Drug Des. 83, 507-520(2014); Stanzl et al., Accounts. Chem. Res / 46, 2944-2954(2013); Oller-Salvia et al., Chemical Society Reviews 45:10.1039 / c6cs00076b(2016); Behzad Jafari et al.,(2019) Expert Opinion on Drug Delivery, 16:6, 583-605(2019) (all incorporated by reference). Yet another strategy involves using additional methods or compositions to enhance delivery of cargo molecules, such as PSD-95 inhibitors, to the brain (Dong, Theranostics 8(6):1481-1493 (2018)).

[0091] A preferred internalization peptide is tat from the HIV virus. The tat peptide reported in previous studies comprises or consists of the standard amino acid sequence YGRKKRRQRRR (SEQ ID NO: 1) found in the HIV tat protein. RKKRRQRRR (SEQ ID NO: 13) and GRKKRRQRRR (SEQ ID NO: 11) can also be used. When additional residues flanking such a tat motif are present (besides the pharmacologically active agent), they can be, for example, naturally occurring amino acids adjacent to this segment from the tat protein, or spacer or linker amino acids of the type commonly used to connect two peptide domains, such as gly(ser)4 (SEQ ID NO:30), TGEKP (SEQ ID NO:31), GGRRGGGS (SEQ ID NO:32), or LQRDGERP (SEQ ID NO:33) (see, e.g., Tang et al. (1996), J. Biol. Chem. 271, 15682-15686; Hennecke et al. (1998), Protein Eng. 11, 405-410), or any other amino acid that does not significantly diminish the ability to effect incorporation of the variant without flanking residues. Preferably, the number of flanking amino acids other than the active peptide does not exceed 10 on either side of YGRKKRRQRRR (SEQ ID NO:1), although preferably there are no flanking amino acids. One suitable tat peptide or other inhibitor peptide that includes additional amino acid residues adjacent to the C-terminus of YGRKKRRQRRR (SEQ ID NO:1) is YGRKKRRQRRRPQ (SEQ ID NO:34). Other tat peptides that can be used include GRKKRRQRRRPQ (SEQ ID NO:35) and GRKKRRQRRRP (SEQ ID NO:36).

[0092] Variants of the above tat peptides having reduced ability to bind to N-type calcium channels are described in International Patent Application Publication No. WO / 2008 / 109010. Such variants may comprise or consist of the amino acid sequence XGRKKRRQRRR (SEQ ID NO:37), where X is an amino acid other than Y, or may comprise or consist of the amino acid sequence GRKKRRQRRR (SEQ ID NO:11). An exemplary tat peptide has the N-terminal Y residue substituted with F. Thus, an exemplary tat peptide comprises or consists of FGRKKRRQRRR (SEQ ID NO:3). Another exemplary mutant tat peptide comprises or consists of GRKKRRQRRR (SEQ ID NO:1). Another exemplary tat peptide comprises or consists of RRRQRRKKRG or RRRQRRKKRGY (amino acids 1-10 or 1-11 of SEQ ID NO:9). Other tat-derived peptides that promote uptake of pharmacologically active agents without inhibiting N-type calcium channels include those shown in Table 1A below.

[0093] [Table 1A]

[0094] X can be a free amino terminus, one or more amino acids, or a linking moiety.

[0095] The active agents of the present invention typically include an inhibitor peptide and an internalization peptide. The inhibitor peptide has a free C-terminus and an N-terminus bound to the C-terminus of the internalization peptide. In such agents, at least four C-terminal residues of the inhibitor peptide, preferably five C-terminal residues of the inhibitor peptide, are L-amino acids, and at least one of the remaining residues in the inhibitor peptide and the internalization peptide is a D-residue. The position for including the D-residue can be selected so that the D-residue appears immediately (i.e., C-terminal) to any basic residue (i.e., arginine or lysine). Plasmin acts by cleaving peptide bonds C-terminal to such basic residues. By including D-residues at sites adjacent to the cleavage site, particularly C-terminal to the basic residue, peptide cleavage is reduced or eliminated. Some or all of the residues C-terminal to the basic residue can be D-residues. Any basic residue can also be a D-amino acid.

[0096] As an example, FIG. 1 shows a diagram of actual and potential plasmin cleavage sites in Tat-NR2B9c. There are seven actual sites (sites where cleavage has been detected) and two potential sites (sites where plasmin cleavage may occur). Some active agents contain at least one D-amino acid in both the internalization peptide and the inhibitor peptide. Some active agents have an inhibitor peptide that contains a D-amino acid at each position of the internalization peptide. Some active agents contain a D-amino acid at each position of the inhibitor peptide except for the four or five C-terminal residues, which are L-amino acids. Some active agents contain a D-amino acid at each position of the internalization peptide and at each position of the inhibitor peptide except for the last four or five C-terminal amino acid residues, which are L-amino acids.

[0097] Tat-NR2B9c, also known as NA-1 or nerinetide, has the amino acid sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO:58). Preferred active agents of the invention are variants of the above sequences in which ESDV (SEQ ID NO:14) or IESDV (SEQ ID NO:5) is an L-amino acid and at least one of the remaining amino acids is a D-amino acid. In some active agents, at least the L and / or K residues at positions 8 and 9 from the C-terminus are D-residues. In some active agents, at least one of the R, R, Q, R, R residues occupying positions 6, 7, 8, 10 and 11 from the N-terminus is a D-residue. In some active agents, these residues are all D-residues. In some active agents, residues 4-8 and residues 10-13 are each D-amino acids. In some active agents, residues 4-13 or 3-13 are each D-amino acids. In some active agents, each of the 11 residues of the internalization peptide (except for the chiral glycine) is a D-amino acid. Some exemplary active agents include ygrkkrrqrrrklssIESDV (SEQ ID NO:6) (also called NA-3), ygrkkrrqrrrklssiESDV (SEQ ID NO:59), ygrkkrrqrrrklsSIESDV (SEQ ID NO:60), ygrkkrrqrrrklSSIESDV (SEQ ID NO:61), ygrkkrrqrrrkssIESDV (SEQ ID NO:7), ygrkkrrqrrrksIESDV (SEQ ID NO:8), or ygrkkrrqrrrkIESDV (SEQ ID NO:9). Other active agents include variants of the above sequence in which the S at the third position from the C-terminus is replaced with a T (ygrkkrrqrrrklssIETDV (SEQ ID NO:62), ygrkkrrqrrrklssiETDV (SEQ ID NO:63), ygrkkrrqrrrklsSIETDV (SEQ ID NO:64), ygrkkrrqrrrkssIETDV (SEQ ID NO:65), ygrkkrrqrrrksIETDV (SEQ ID NO:66) and ygrkkrrqrrrkIETDV (SEQ ID NO:67)). Active agents include ygrkkrrqrrrIESDV (SEQ ID NO:68) (D-Tat-L-2B5c) and ygrkkrrqrrrIETDV (SEQ ID NO:69).

[0098] The invention further includes an active agent comprising, for example, an internalization peptide bound to an inhibitor peptide to form a fusion peptide, and an inhibitor peptide, or a variant of said active agent having up to 1, 2, 3, 4 or 5 substitutions or deletions throughout the internalization peptide and the inhibitor peptide. The inhibitor peptide inhibits the binding of PSD-95 to NOS and / or NMDAR2B. The internalization peptide has an amino acid sequence comprising YGRKKRRQRRR (SEQ ID NO: 1), GRKKRRQRRR (SEQ ID NO: 11) or RKKRRQRRR (SEQ ID NO: 13). The inhibitor peptide has a sequence comprising KLSSIESDV (SEQ ID NO: 2). In such an active agent, at least the four or five C-terminal amino acids of the inhibitor peptide are L-amino acids, and the contiguous segment of amino acids comprising all R and K residues and the C-terminal residue immediately adjacent to the most C-terminal R or K residue are D-amino acids. Thus, in a peptide containing the sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO:58), the contiguous segment from the first R to the L-residue is a D-amino acid (except for glycine, which has no chiral form).

[0099] One example of tolerated substitutions is provided by the motif [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO:3) at the C-terminus of the inhibitor peptide. For example, the third amino acid from the C-terminus can be S or T. Preferably, each of the five C-terminal amino acids of the inhibitor peptide are L-amino acids. Optionally, each of the other amino acids are D-amino acids, similar to the active agent ygrkkrrqrrrklssIESDV (SEQ ID NO:6), where lower case letters are D-amino acids and upper case letters are L-amino acids (except for glycine, which has no chiral form).

[0100] It has been found that the inclusion of D-amino acids in the above nerinetide variants is advantageous in increasing plasmin resistance and plasma half-life, but D-amino acids can also cause nephrotoxicity. The degree of nephrotoxicity depends on the dose of the active agent, the D-amino acid content of the active peptide, and the use of D-amino acid oxidase inhibitors to reduce toxicity. Surprisingly, it has been found that most, and in some cases all (e.g., 50, 75, 90, 99% or 100%) of the benefits of increased plasmin resistance and plasma half-life can be achieved by including D-amino acids at fewer positions than the above active agent, with the additional benefit of reduced nephrotoxicity (e.g., reduced by 10, 25, 50, 75 or 90%) at the same dose. In other words, the benefits and nephrotoxicity associated with D-amino acids do not show the same correlation with the content of D-amino acids.

[0101] Although an understanding of the mechanism is not necessary to practice the invention, the above results can be explained in part by the fact that D-amino acids provide protection against plasmin cleavage and possibly other proteases not only when they are one of the two amino acids spanning the cleavage site, but also when they are more distant from the cleavage site, e.g., 2, 3 or 4 amino acids away (with the amino acid immediately adjacent to the cleavage site). Furthermore, nephrotoxicity from D-amino acids appears to increase proportionally with the number of D-amino acids in the peptide and the dose of the peptide. As a result, the use of a minimal number of D-amino acids provides most, if not all, of the stability benefits of peptides made entirely of D-amino acids without significant nephrotoxicity.

[0102] For example, some active agents have only 1, 2, 3, 4, 5 or 6 amino acids (not including glycine, if present) of the internalization peptide as D-amino acids. The inhibitor peptide is formed with all L residues except the first K and / or L residue. Some active agents have 2-6, 2-5, 2-4, 3-6, 3-5 or 4-5 amino acids (not including glycine, if present) of the internalization peptide as D-amino acids and the remaining amino acids of the internalization peptide and the amino acids except the first K and / or L residue of the inhibitor peptide as L-amino acids or glycine. Some active agents have 2-6, 2-5, 2-4, 3-6, 3-5 or 4-5 amino acids (not including glycine, if present) of the internalization peptide as D-amino acids and the remaining amino acids of the internalization peptide and the inhibitor peptide as L-amino acids or glycine.

[0103] Plasmin resistance and plasma half-life can be improved by substituting selected L-amino acids in the active agent with D-amino acids and / or deleting L-amino acids that are not essential for the activity of the active agent. Exemplary amino acids that can be deleted from nerinetide without significantly impairing pharmaceutical activity include the first two amino acids Y and G of the internalization peptide YGRKKRRQRRR (SEQ ID NO: 1), and the first four or five amino acids from the N-terminus of the inhibitor peptide KLSSIESDV (SEQ ID NO: 2). Thus, some active agents are based on an internalization peptide that includes RKKRRQRRR (SEQ ID NO: 13). At least one, but not all, amino acids of the internalization peptide are D-amino acids. For example, 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in RKKRRQRRR (SEQ ID NO: 13) can be D-amino acids. In some active agents, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues of RKKRRQRRR are D-amino acids. In some active agents, residues 2-8, 2-7, 2-6, 2-5, 2-4, 2-3 of RKKRRQRRR (SEQ ID NO: 13) are D-amino acids. In some active agents, residues 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8 of RKKRRQRRR (SEQ ID NO: 13) are D-amino acids.

[0104] Some active agents are based on an internalization peptide containing GRKKRRQRRR (SEQ ID NO:11). In some active agents, 1, 2, 3, 4, 5, 6, 7, or 8 residues of GRKKRRQRRR (SEQ ID NO:11) are D-amino acids (glycine, which does not have a chiral form, is not counted as a D residue in this or similar counts of D amino acids in a peptide). In some such active agents, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 residues of GRKKRRQRRR (SEQ ID NO:11) are D-amino acids. In some such active agents, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 residues of GRKKRRQRRR (SEQ ID NO:11) are D-amino acids. In some such active agents, residues 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8 of GRKKRRQRRR (SEQ ID NO:11) are D-amino acids.

[0105] Some active agents are based on an internalization peptide comprising YGRKKRRQRRR (SEQ ID NO: 1). In some active agents, residues 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 in the internalization peptide are D-amino acids. In some such active agents, residues 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 in the internalization peptide are D-amino acids. In some such active agents, residues 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-9, 4-8, 4-7, 4-6, 4-5, 5-9, 5-8, 5-7, 5-6, 6-9, 6-8, 6-7, 7-9, 7-8, or 8-9 in the internalization peptide are D-amino acids. In some such active agents, 1, 2, 3, 4, 5, 6, 7, 8, or 9 residues in the internalization peptide are D-amino acids.

[0106] The inhibitor peptide attached to the internalization peptide comprises four C-terminal amino acids according to the formula [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L] (SEQ ID NO:3). Preferably, the inhibitor peptide comprises ESDV (SEQ ID NO:14), IESDV (SEQ ID NO:5), ETDV (SEQ ID NO:16) or IETDV (SEQ ID NO:22) at the C-terminus. The four or five terminal L-amino acids of the inhibitor peptide, e.g., ESDV (SEQ ID NO:14), IESDV (SEQ ID NO:5), ETDV (SEQ ID NO:16) or IETDV (SEQ ID NO:22), are L-amino acids. When additional amino acids are present in the inhibitor peptide, they are typically KLSS (SEQ ID NO:113), KLS, KL, K, S, SS or SL attached to the N-terminus of IE[S / T]DV (SEQ ID NO:4). Such additional amino acids of the inhibitor peptide are typically amino acids, but K may be an L or D amino acid. KLSSIESDV (SEQ ID NO:2) or KLSSIETDV (SEQ ID NO:12) are exemplary inhibitor peptides in which each residue of the inhibitor peptide is an L-amino acid (except that K and L may independently be an L or D-amino acid). In some inhibitor peptides, K and L are both L-amino acids. In some inhibitor peptides, K and L are both D-amino acids. In some inhibitor peptides, K is a D-amino acid and L is an L-amino acid, or vice versa.

[0107] Other substitutions, deletions, or additions (in addition to the replacement of L-amino acids with D-amino acids) can be optionally made to the disclosed sequences (e.g., up to 1, 2, 3, 4, or 5 substitutions, deletions, or additions) that at least substantially retain binding, inhibition, plasmin resistance, and plasma half-life, and do not increase nephrotoxicity, as discussed further below. For example, some variants include 1, 2, 3, 4, or 5 substitutions relative to the disclosed sequences. Some variants include combinations of 1, 2, 3, 4, or 5 substitutions and deletions compared to the disclosed sequences. Some variants include combinations of 1, 2, 3, 4, or 5 substitutions, deletions, or additions compared to the disclosed sequences.

[0108] The D residues present in the internalization peptides disclosed above may or may not be contiguous. For example, in some internalization peptides, at least two D-amino acids are separated from each other by one or more L-amino acids or glycines. In some internalization peptides, at least three D-amino acids are separated from each other by one or more L-amino acids or glycines.

[0109] In some active agents, the L-amino acids substituted with at least one or all D-amino acids are K and R, or the residues immediately C-terminal to a K or R residue, since these residues are adjacent to potential sites of plasmin cleavage. In some active agents, the residue immediately C-terminal to a stretch of two or more consecutive K or R residues is substituted with a D-amino acid. Other L residues besides K, R, and the residue immediately adjacent to the C-terminus can also be substituted with a D residue.

[0110] Some exemplary internalization peptides have the sequences shown in Table 1B.

[0111] [Table 1B]

[0112] Some exemplary active agents have an amino acid sequence that includes or consists of any of RkkrrQrRrIESDV (SEQ ID NO:110, NoNO411), RKkRrQrrRIESDV (SEQ ID NO:111), or rKKRrArRRIESDV (SEQ ID NO:112).

[0113] Some preferred active agents have any of the above internalization peptides linked to kLSSIESDV (SEQ ID NO: 122), KlSSIESDV (SEQ ID NO: 123) or klSSIESDV (SEQ ID NO: 70).

[0114] Some active agents include an internalization peptide or a variant thereof linked to an inhibitor peptide, the inhibitor peptide inhibiting the binding of PSD-95 to NOS and / or NMDAR2B, the internalization peptide has an amino acid sequence including RKKRRQRRR (SEQ ID NO: 13), and the inhibitor peptide has a sequence including ESDV (SEQ ID NO: 4) at the C-terminus. The variant has up to 1, 2, 3, 4 or 5 substitutions, additions or deletions in the internalization peptide and the inhibitor peptide (not including L to D substitutions of the same amino acid). Some active agents have a sequence including RKKRRQRRR (SEQ ID NO: 13), GRKKRRQRRR (SEQ ID NO: 11) or YGRKKRRQRRR (SEQ ID NO: 1), and the inhibitor peptide has ESDV (SEQ ID NO: 14) at the C-terminus, and the variant has up to 1, 2, 3, 4 or 5 substitutions, additions or deletions in the internalization peptide and the inhibitor peptide (not including L to D substitutions of the same amino acid). At least the four C-terminal amino acids of the inhibitor peptide are L-amino acids. 2-6, preferably 3-5, e.g., 3 or 4 residues (excluding glycine) of the internalization peptide are D-amino acids. The C-terminal amino acids are preferably IE[S / T]DV (SEQ ID NO: 4), all of which are L-amino acids. The attachment position of the inhibitor peptide or the spacer peptide between the internalization peptide and the inhibitor peptide can be L or D. In some such agents, each D residue is located at a K or R or C-terminal to a K or R residue. The D residues in such active agents are preferably spaced for maximum effectiveness in reducing cleavage by plasmin and other proteases that the active agent may encounter. Some such active agents do not include a stretch of four or more consecutive amino acids independently selected from L-amino acids and glycine. The stretch includes adjacent amino acids independently selected from R and K (i.e., the plasmin cleavage site). In other words, one L-form R or K is not adjacent to another L-form R or K as part of a stretch of consecutive amino acids that are four or more L-amino acids or glycine. Some active agents do not include a stretch of four or more consecutive amino acids independently selected from L-amino acids and glycine.The stretch comprises R and / or K. In other words, an L-form R or K does not occur as part of a stretch of four or more consecutive amino acids that are L-amino acids or glycine. Some such active agents do not comprise a stretch of five or more consecutive amino acids independently selected from L-amino acids and glycine. The stretch does not comprise adjacent amino acids independently selected from R and K (i.e., a plasmin cleavage site). In other words, an L-form R or K does not occur adjacent to another L-form R or K as a stretch of five or more consecutive amino acids that are L-amino acids or glycine. If a stretch of four such consecutive amino acids is present, preferably, only one such stretch is present and is located at the N-terminus of the internalization peptide (i.e., the first four residues), with no more than two or three such stretches of consecutive amino acids at other positions. Some active agents do not comprise a stretch of five or more consecutive amino acids selected from L-amino acids or glycine that comprise R and / or K. In other words, an L-form R or K does not occur as part of a stretch of five or more consecutive amino acids that are glycine, L-amino acids. If a stretch of four such consecutive amino acids is present, preferably there is only one such stretch and it is located at the N-terminus of the internalization peptide (i.e. the first four residues), with no more than two or three such stretches of consecutive amino acids in other positions.

[0115] Thus, for example, some active agents have three D-residues (excluding glycine) in the internalization peptide having YGRKKRRQRRR (SEQ ID NO: 1), with a D-amino acid between positions 3-5, 7 or 8, and 9-11, numbered from the N-terminus. Some active agents have a spacer peptide between the inhibitor peptide and the internalization peptide. The spacer peptide is not necessary for the inhibitor peptide to bind to PSD-95, but is so called because it facilitates spacing between the inhibitor peptide and the internalization peptide so that the internalization peptide can increase the binding of the inhibitor peptide to PSD-95. The spacer is usually a peptide of 2-4 amino acids, but may be any form of linker with similar spacing. Some spacers include some or all amino acids independently selected from glycine, alanine, serine, or leucine amino acids. Such amino acids are often included in the spacer to provide flexibility between the molecules being linked. In some spacers, lysine and / or arginine are not used to avoid the introduction of plasmin or other cleavage sites. The spacer may be KLSS (SEQ ID NO: 113), the amino acids between IESDV (SEQ ID NO: 5) of nerinetide and the internalization peptide, where K and / or L can be D-type to reduce cleavage by plasmin and other proteases. Such agents may optionally contain two or more copies of an inhibitor peptide linked to the internalization peptide by a branched linker.

[0116] Some active agents have an amino acid sequence comprising or consisting of YGRKKRRQRRRKLSSIESDV (SEQ ID NO:58), or a variant thereof, said variant having up to 1, 2, 3, 4 or 5 substitutions, deletions or additions (excluding L-to-D substitutions of the same amino acid) in said sequence, and 2-7, 2-6, 3-6 or preferably 3-5 amino acids (excluding glycine) at positions other than the 5 C-terminal amino acids are D-amino acids.Some active agents comprise an amino acid sequence having at least 75, 80, 85, 90 or 95% amino acid sequence identity to the sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO:58), and 2-7, 2-6, 3-6 or preferably 3-5 amino acids (excluding glycine) at positions other than the 5 C-terminal amino acids are D-amino acids. Herein, identity is determined by maximally aligning the variant sequence with the reference sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 58) and dividing the number of matching amino acids by the total number of amino acids in the reference. L-amino acids and D-amino acids of the same type are considered suitable for this purpose. Adjacent amino acids in the variant outside the sequence aligned with the reference are not scored. In some such active agents, there are no D-amino acids occupying adjacent positions in the active agent. In some such active agents, each D-amino acid is separated from each other by at least two or at least three amino acids selected from L-amino acids and glycine. Some active agents contain three, four or five D-amino acids (glycine is not counted as a D-amino acid). Some active agents lack a stretch of four or more adjacent amino acids selected from L-amino acids and glycine, the stretch containing two adjacent amino acids independently selected from R and K (i.e., a plasmin cleavage site). In other words, an L-form R or K does not occur adjacent to another L-form R or K as part of a stretch of four or more L-amino acids or glycines. Some active agents lack a stretch of four or more L-amino acids or glycines that includes an R and / or K. In other words, an L-form R or K does not occur as a position in a stretch of four or more L-amino acids or glycines.NoNO42 is an example of an active agent that contains a stretch of three (KKR) consecutive amino acids, rather than four or more of the above consecutive amino acids. Some active agents lack a stretch of five or more consecutive L-amino acids or glycines that contains two adjacent amino acids independently selected from R and K (i.e., plasmin cleavage sites). In other words, one L-form R or K is not present adjacent to another L-form R or K as part of a stretch of consecutive amino acids that is five or more L-amino acids or glycines. If there is a stretch of four such consecutive amino acids, preferably there is only one such stretch and it is located at the N-terminus of the active agent (i.e., the first four residues), with no more than two or three such stretches of consecutive amino acids at other positions. NA-411.4 is an example of such an active agent, with a stretch of four such consecutive amino acids (YGRK SEQ ID NO: 124) at the N-terminus, with no more than two such stretches of consecutive amino acids at other positions. Some active agents do not include a stretch of 5 or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch including R and / or K. In other words, one L-form R or K is not adjacent to another L-form R or K as part of a stretch of consecutive amino acids that is 5 or more L-amino acids or glycine. If there is a stretch of 4 such consecutive amino acids, preferably there is only one such stretch and it is located at the N-terminus of the active agent (i.e., the first 4 residues), with no more than 2 or 3 such stretches of consecutive amino acids at other positions. Some active agents include 3 or 4 D-amino acids (glycine does not count as a D-amino acid), with the first D-amino acid being between positions 3-5, the second D-amino acid being between positions 7 or 8, the third D-amino acid being between positions 9-11, and optionally the fourth D-amino acid being at positions 12 or 13.

[0117] Preferred active agents have an amino acid sequence comprising, consisting of, or consisting essentially of YGrKKRrQrRRkLSSIESDV (NA4.2, SEQ ID NO: 114), YGRkKRrQRrRKLSSIESDV (NA4.4.3, SEQ ID NO: 115) or YGrKKRrQrRRKlSSIESDV (NA4.4.2, SEQ ID NO: 116), where the D-amino acids are shown in lower case. These agents have three or four D-amino acids and have a favorable balance of increased plasma half-life and plasmin resistance compared to nerinetide and reduced or absent nephrotoxicity compared to ygrkkrrqrrrrklssIESDV (SEQ ID NO: 6). Other preferred active agents are the plasmin resistant peptides shown in Example 10, Table 6.

[0118] Preferred active agents have improved stability and / or plasma half-life in rat or human plasma compared to Tat-NR2B9c or an otherwise identical all-L-active agent. Stability can be measured as described in the Examples. Preferred active agents have improved plasmin resistance compared to Tat-NR2B9c or an otherwise identical all-L-active agent. Plasmin resistance and plasma half-life can be measured as described in the Examples. Some active agents have increased area under the curve and / or CMax compared to Tat-NR2B9c or an otherwise identical all-L active agent, especially after subcutaneous administration. Active agents preferably bind to PSD-95 within 1.5-fold, 2-fold, 3-fold or 5-fold compared to Tat-NR2B9c (all-L) or an otherwise identical all-L-peptide, or have binding that is indistinguishable within experimental error. Preferred active agents compete for binding by at least 10%, 25% or 50% with Tat-NR2B9c or a peptide comprising the last 15-20 amino acids of the NMDA receptor subunit 2 sequence that contains the PDZ binding domain for binding to PSD-95 (e.g., a 10-fold excess of active agent reduces Tat-NR2B9c binding). This competition suggests that the active agent binds to the same or overlapping binding site as Tat-NR2B9c. Having the same or overlapping binding site can also be demonstrated by alanine mutagenesis of PSD-95. If mutagenesis of the same or overlapping set of residues reduces binding of the active agent and Tat-NR2B9c, then the active agent and TAT-NR2B9c bind to the same or overlapping site on PSD-95.Some active agents exhibit reduced nephrotoxicity and / or a higher therapeutic index compared to an active agent comprising an internalization peptide of sequence ygrkkrrqrrr (SEQ ID NO:96) linked to an inhibitor peptide of sequence KLSSIESDV (SEQ ID NO:2), where each amino acid of the internalization peptide is a D-amino acid and each amino acid of the inhibitor peptide is an L-amino acid; and / or an active agent comprising an internalization peptide of sequence ygrkkrrqrrr (SEQ ID NO:96) linked to klssIESDV (SEQ ID NO:117) (lower case letters indicate D-amino acids and upper case letters indicate L-amino acids), where each amino acid of the internalization peptide is a D-amino acid; and / or an active agent having the amino acid sequence vdseisslkrrrqrrkkrgy (SEQ ID NO:118) (lower case letters indicate D-amino acids). Nephrotoxicity or therapeutic index can be measured as described in the Examples or herein. An enhancement of a desired activity, such as half-life, or a reduction in an undesired activity, such as nephrotoxicity, refers to an enhancement or reduction beyond experimental error, in other words, statistically significant with a probability of 95%. Optionally, enhancement refers to an improvement of at least 10%, 25%, 50%, 100%, 200%, 500% or 1,000%, and reduction refers to a decrease of at least 10%, 25%, 50%, 75%, 95 or 99%.

[0119] The active agents of the present invention can include modified amino acid residues (e.g., N-alkylated residues). N-terminal alkyl modifications can include, for example, N-methyl, N-ethyl, N-propyl, N-butyl, N-cyclohexylmethyl, N-cyclohexylethyl, N-benzyl, N-phenylethyl, N-phenylpropyl, N-(3,4-dichlorophenyl)propyl, N-(3,4-difluorophenyl)propyl, and N-(naphthalen-2-yl)ethyl). The active agent can include a retropeptide. A retropeptide has a reverse amino acid sequence. Peptide mimetics also include retro-inverso peptides, in which the order of amino acids is reversed, so that the original C-terminal amino acid appears at the N-terminus and D-amino acids are used instead of L-amino acids (e.g., amino acid vdseisslkrrrqrrkkrgy (SEQ ID NO: 118), also known as RI-NA-1).

[0120] The appropriate pharmacological activity of peptides, peptidomimetics or other agents can be confirmed, if desired, using the rat model of stroke described above prior to testing in primates and clinical trials as described herein. Peptides or peptidomimetics can also be screened for their ability to inhibit the interaction of PSD-95 with NMDAR2B using the assays described in U.S. Patent Application Publication No. US20050059597, which is incorporated by reference. Useful peptides generally have IC50 values ​​in such assays of 50 μM, 25 μM, 10 μM, 0.1 μM or 0.01 μM. Preferred peptides generally have IC50 values ​​of 0.001-1 μM, more preferably IC50 values ​​of 0.001-0.05, 0.05-0.5 μM or 0.05-0.1 μM. When a peptide or other agent is characterized as inhibiting the binding of one interaction, e.g., the interaction of PSD-95 to NMDAR2B, such a statement does not exclude that the peptide or agent also inhibits other interactions, e.g., the binding of PSD-95 to nNOS.

[0121] Such peptides can be optionally derivatized (e.g., acetylated, phosphorylated, myristoylated, geranylated, pegylated, and / or glycosylated) to increase binding affinity to the inhibitor, increase the inhibitor's ability to penetrate cell membranes, or increase stability. As a specific example, for inhibitors in which the third residue from the C-terminus is S or T, this residue can be phosphorylated prior to use of the peptide.

[0122] The internalization peptide can be attached to the inhibitor peptide by conventional methods. For example, the inhibitor peptide can be attached to the internalization peptide by chemical bonds (e.g., via a binding or conjugation agent). Many such agents are commercially available and are reviewed by S SWong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991). Some examples of cross-linking agents include J-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N,N'-(1,3-phenylene)bismaleimide; N,N'-ethylenebis-(iodoacetamide) or other such agents with 6-11 carbon methylene bridges (relatively specific for sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which forms an irreversible bond between the amino and tyrosine groups). Other cross-linking reagents include p,p'-difluoro-m,m'-dinitrodiphenylsulfone (reacts primarily with amino and phenolic groups); dimethyladipimidic acid (specific for amino groups); phenol-1,4-disulfonyl chloride (reacts primarily with amino groups); hexamethylene diisocyanate or diisothiocyanate or azophenyl-p-diisocyanate (reacts primarily with amino groups); glutaraldehyde (reacts with several different side chains) and disdiazobenzidine (reacts primarily with tyrosine and histidine).

[0123] Linkers (e.g., polyethylene glycol linkers) can be used to dimerize the active portion of inhibitor peptides, peptides, or peptidomimetics to improve their affinity and selectivity for proteins containing tandem PDZ domains. See Bach et al., (2009) Angew. Chem. Int. Ed. 48:9685-9689 and International Patent Application Publication No. WO2010 / 004003. PL motif-containing peptides are preferably dimerized by linking the N-terminus of two molecules, leaving the C-terminus free. Bach further reports that the pentameric peptide IESDV (SEQ ID NO: 5), derived from the C-terminus of NMDAR2B, is effective in inhibiting the binding of NMDAR2B to PSD-95. IETDV (SEQ ID NO: 22) can also be used in place of IESDV (SEQ ID NO: 5). Alternatively, 2-10 copies of PEG can be linked in series to form a linker. Optionally, the linker can be linked to an internalization peptide or lipidated to enhance cellular uptake. An example of a dimeric inhibitor is shown below (IETDV, SEQ ID NO: 22) (Bach et al., PNAS 109 (2012) 3317-3322). [ka]

[0124] R is any of the internalization peptides disclosed herein that contain one or more D-amino acids. N can be replaced with O. Any of the PSD-95 inhibitors disclosed herein can be used in place of IETDV (SEQ ID NO: 22), particularly IESDV (SEQ ID NO: 5), and any internalization peptide or lipidation moiety can be used in place of tat. Other linkers as shown can also be used.

[0125] The internalization peptide can also be linked to the inhibitor peptide to form a fusion peptide, preferably with the C-terminus of the internalization peptide linked to the N-terminus of the inhibitor peptide, with the C-terminus of the inhibitor peptide being a free terminus.

[0126] Alternatively or in addition to linking peptides to internalization peptides, such peptides can be linked to lipids (lipidation) to increase the hydrophobicity of the complex relative to the peptide alone, thereby facilitating the penetration of the linked peptide through cell membranes and / or the brain barrier. Lipidation is preferably performed at the N-terminal amino acid, but can also be performed at internal amino acids, provided that the ability of the peptide to inhibit the interaction of PSD-95 with NMDAR2B is not reduced by more than 50%. Preferably, lipidation is performed at an amino acid other than one of the four most C-terminal amino acids. Lipids are organic molecules that are more soluble in ether than in water, and include fatty acids, glycerides and steroids. Suitable forms of lipidation are myristoylation, palmitoylation, or the addition of other fatty acids, such as lauric acid and stearic acid, preferably with a chain length of 10 to 20 carbons, as well as geranylation, geranylgeranylation and isoprenylation. Lipidation of the kind that occurs in post-translational modifications of natural proteins is preferred. Lipidation with fatty acids is also preferred, via the formation of an amide bond to the alpha amino group of the N-terminal amino acid of the peptide. Lipidation can be performed by peptide synthesis containing pre-lipidated amino acids, enzymatically in vitro, or by recombinant expression, by chemical cross-linking, or by chemical derivatization of the peptide. Amino acids modified by myristoylation and other lipid modifications are commercially available. The use of lipids instead of internalizing peptides reduces the number of K and R residues that provide plasmin cleavage sites. Some exemplary lipidated molecules include KLSSIESDV (SEQ ID NO:2), klSSIESDV (SEQ ID NO:70), lSSIESDV (SEQ ID NO:71), LSSIESDV (SEQ ID NO:72), SSIESDV (SEQ ID NO:73), SIESDV (SEQ ID NO:74), IESDV (SEQ ID NO:5), KLSSIETDV (SEQ ID NO:12), klSSIETDV (SEQ ID NO:75), lSSIETDV (SEQ ID NO:76), LSSIETDV (SEQ ID NO:77), SSIETDV (SEQ ID NO:78), SIETDV (SEQ ID NO:79), IETDV (SEQ ID NO:22), preferably with N-terminus lipidation.

[0127] Inhibitor peptides, optionally fused to internalization peptides, can be synthesized by solid-phase synthesis or recombinant gene synthesis. Peptide mimetics can be synthesized using a variety of procedures and techniques described in the scientific and patent literature, for example, 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.

[0128] III. Salt

[0129] Peptides of the above types are generally made by solid-phase synthesis. In solid-phase synthesis, the peptides are generally initially produced as trifluoroacetate salts, since trifluoroacetate (TFA) is used to remove protecting groups or to remove the peptide from the resin. The trifluoroacetate can be replaced by another anion, for example, by binding the peptide to a solid support (e.g., a column), washing the column to remove existing counterions, equilibrating the column with a solution containing the new counterion, and eluting the peptide, for example, by introducing a hydrophobic solvent (e.g., acetonitrile) into the column. Replacement of trifluoroacetate with acetate can be achieved with an acetate wash as a final step before eluting the peptide with another conventional solid-phase synthesis method. Replacement of trifluoroacetate or acetate with chloride can be achieved by washing with ammonium chloride followed by elution. The use of a hydrophobic support is preferred, and preparative reversed-phase HPLC is preferred, especially ion-exchange type. The trifluoroacetate can be directly displaced with chloride or can be first displaced with acetate and then the acetate can be displaced with chloride.

[0130] The counterion, whether trifluoroacetate, acetate or chloride, is attached to the positively charged atoms of Tat-NR2B9c and its D variants, particularly to the N-terminal amino group and the amino side chains of arginine and lysine residues. Although an understanding of the exact stoichiometry of peptide to anion in salts of Tat-NR2B9c and its D variants is not necessary to the practice of the invention, it is believed that up to about nine counterion molecules are present per molecule of salt.

[0131] Although the substitution of an ion for its counterion occurs efficiently, the final counterion purity may be less than 100%. Thus, reference to a chloride salt of TAT-NR2B9c or its D variant means that in the salt preparation, chloride is the predominant anion by weight (or mole) relative to all other anions present in the salt aggregate. In other words, chloride constitutes more than 50% by weight or mole, preferably more than 75%, more than 95%, more than 99% by weight or mole, or more than 99.9% by weight or mole, of all anions present in the salt. In such salts or formulations prepared from this salt, acetate and trifluoroacetate alone and in combination constitute less than 50%, 25%, 5%, 0.5% or 0.1% by weight or mole of the anions in the salt or formulation.

[0132] IV. Formulation

[0133] The active agent can be formulated into a liquid formulation or a lyophilized formulation. The liquid formulation can include a buffer, a salt, and water. A preferred buffer is sodium phosphate. A preferred salt is sodium chloride. Sodium benzoate can be present as a preservative and an inhibitor of D-amino acid oxidase (dual purpose). The pH can be, for example, pH 7.0 or at physiological levels.

[0134] Lyophilized formulations can be prepared from non-lyophilized formulations that include active agent, buffer, bulking agent, and water. Other components, such as frozen or lyophilized preservatives, pharma- ceutically acceptable carriers, etc., may or may not be present. A preferred buffer is histidine. Sodium benzoate can be present as a preservative and inhibitor of D-amino acid oxidase (dual purpose). A preferred bulking agent is trehalose. Trehalose also serves as a frozen or lyophilized preservative. An exemplary non-lyophilized formulation comprises an active agent, histidine (e.g., 10-100 mM, 15-100 mM, 15-80 mM, 40-60 mM or 15-60 mM, e.g., 20 mM or alternatively 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 5.5-7.5, more preferably 6-7, more preferably 6.5. The concentration of the 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 non-lyophilized formulation is 20 mM histidine, 120 mM trehalose and 90 mg / ml of the chloride salt of the active agent. Optionally, as described in US 10,206,878, an acetylated scavenger (eg, lysine) can be included to further reduce any residual acetate or trifluoroacetate in the formulation.

[0135] After lyophilization, the lyophilized formulation has a low moisture content by weight, preferably about 0%-5% water, more preferably less than 2.5%. The lyophilized formulation can be stored in a freezer (e.g., -20 or -70°C), a refrigerator (0-40°C), or at room temperature (20-25°C).

[0136] The active agent may be reconstituted in an aqueous solution, preferably water for injection or alternatively normal saline (0.8-1.0% saline and preferably 0.9% saline). The reconstituted product may be in the same or similar volume as the unlyophilized formulation or in a larger volume. Preferably, the volume is greater (e.g., 3-6 times greater) in the reconstituted product than before reconstitution. For example, an unlyophilized volume of 3-5 ml may be reconstituted in a volume of 10 ml, 12 ml, 13.5 ml, 15 ml or 20 ml, or in particular 10-20 ml. 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) and 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, e.g., 15-30, 18-20, 20 mg / ml 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 (concentrations rounded to the nearest whole number). 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 (concentrations rounded to the nearest whole number). The reconstituted formulation can be further diluted prior to administration, for example, by addition to a fluid bag containing normal saline.

[0137] IV. Symptoms

[0138] The active agent is effective for treating various conditions, especially neurological conditions and especially conditions that are partially mediated by excitotoxicity. Such conditions include stroke, epilepsy, hypoxia, subarachnoid hemorrhage, concussion, trauma to the CNS that is not associated with stroke (e.g. traumatic brain injury and spinal cord injury), other cerebral ischemia, reperfusion injury, neurodegenerative diseases (e.g. Alzheimer's disease and Parkinson's disease). Such conditions can also include retinopathy, other ocular disorders associated with retinal ischemia, or eye or ear disorders or diseases, including tinnitus. Other neurological conditions that are not known to be associated with excitotoxicity and can be treated by the active agent of the present invention include anxiety and pain (neuropathic or inflammatory).

[0139] Stroke is a disease that is the result of impaired blood flow in the CNS, regardless of the cause. Possible causes include embolism, hemorrhage, and thrombosis. As a result of impaired blood flow, some neuronal cells die immediately. These cells release their component molecules, including glutamate, which activates NMDA receptors, which increase intracellular calcium levels and intracellular enzyme levels, leading to the death of additional neuronal cells (excitotoxicity cascade). The death of CNS tissue is called infarction. Infarction volume (i.e., the volume of neuronal cells that die in the brain as a result of stroke) can be used as an indicator of the extent of pathological damage caused by stroke. Symptomatic effects depend on both the volume of the infarction and where it is located in the brain. Disability indices such as the Rankin Stroke Outcome Scale (Rankin, Scott Med J;2:200-15 (1957)) and the Barthel Index can be used to measure symptomatic damage. The Rankin Scale is based on a direct assessment of the subject's overall condition, as follows:

[0140] 0: Completely asymptomatic. 1: No significant impairment despite symptoms; able to perform usual duties and activities. 2: Mild impairment; unable to perform all previous activities, but able to care for self without assistance. 3: Moderate disability; requires some assistance but can walk unaided. 4: Moderate to severe disability; unable to walk without assistance and unable to care for themselves without assistance. 5: Severely disabled; bedridden, incontinent and requiring constant care and attention.

[0141] The Barthel Index is based on a series of questions about a subject's ability to perform 10 basic activities of daily living, and the results are expressed as a score from 0 to 100, with lower scores indicating greater disability (Mahoney et al., Maryland State Medical Journal 14:56-61 (1965)).

[0142] Alternative stroke severity / outcomes can be measured using the NIH Stroke Scale, available on the World Wide Web at ninds.nih.gov / doctors / NIH Stroke Scale Booklet.pdf.

[0143] The scale is based on the subject's ability to perform 11 groups of functions, including assessment of the subject's level of consciousness, motor, sensory and language function.

[0144] Ischemic stroke more specifically refers to the type of stroke caused by an obstruction in blood flow to the brain. The underlying condition for this type of obstruction is most commonly the growth of fatty deposits lining the walls of blood vessels. This condition is called atherosclerosis. These fatty deposits can cause two types of obstruction. Cerebral thrombosis refers to a blood clot (thrombus) that grows in a blocked section of a blood vessel. A "cerebral embolism" generally refers to a blood clot that forms elsewhere in the circulatory system, usually in the heart or the large arteries in the upper thorax and neck. Part of the blood clot then breaks free and enters the bloodstream, travelling through the blood vessels of the brain and eventually reaching vessels that are too small to pass through. A second important cause of embolism is an irregular heartbeat known as atrial fibrillation. This creates a situation in which blood clots can form in the heart and break free and reach the brain. Further possible causes of ischemic stroke include hemorrhage, clot formation, dissection of an artery or vein, cardiac arrest, shock of any cause including hemorrhage, and iatrogenic causes such as surgery on the cerebral blood vessels or blood vessels supplying the brain or cardiac surgery. Ischemic stroke accounts for approximately 83% of all stroke cases.

[0145] A transient ischemic attack (TIA) is a mini-stroke or warning stroke. In a TIA, conditions indicative of an ischemic stroke are present and warning signs of a typical stroke are present; however, the blockage (blood clot) tends to occur for a short period of time and resolve naturally by normal mechanisms. Subjects undergoing cardiac surgery are at particular risk for a transient ischemic attack.

[0146] Hemorrhagic stroke accounts for approximately 17% of stroke cases. It is the result of a weakened blood vessel bursting and bleeding into the surrounding brain. Blood backs up and presses on the surrounding brain tissue. Two common types of hemorrhagic stroke are intracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic stroke is the result of the rupture of a weakened blood vessel. Possible causes of the rupture of a weakened blood vessel include hypertensive bleeding, where high blood pressure causes the blood vessel to burst, or other underlying causes of a weakened blood vessel, such as a ruptured cerebral vascular malformation, including cerebral aneurysm, arteriovenous malformation (AVM), or cavernous malformation. Hemorrhagic stroke can also result from hemorrhagic transformation of an ischemic stroke that weakens the blood vessel at the infarct site, or bleeding from a primary or metastatic tumor in the CNS that contains an abnormally weak blood vessel. Hemorrhagic stroke can also result from iatrogenic causes, such as direct surgical injury to a cerebral blood vessel. An aneurysm is a dilation of a weakened area of ​​a blood vessel. If left untreated, the aneurysm will continue to weaken and eventually rupture, bleeding into the brain. Arteriovenous malformations (AVMs) are abnormally formed clusters of blood vessels. Cavernous malformations are venous abnormalities that can cause bleeding from weakened venous structures. Any of these blood vessels can rupture, causing bleeding into the brain. Hemorrhagic strokes can also occur due to physical trauma. A hemorrhagic stroke in one part of the brain can lead to an ischemic stroke in another part through lack of blood lost in the hemorrhagic stroke.

[0147] One class of subjects suitable for treatment is those undergoing surgical procedures involving or potentially involving blood vessels supplying the brain, or otherwise involving or potentially involving the brain or CNS. In some examples, subjects undergoing cardiopulmonary bypass, carotid artery stenting, diagnostic angiograms of the coronary arteries of the brain or aortic arch, vascular surgery, and neurosurgery. Further examples of such subjects are described in Section IV above. Cerebral aneurysm subjects are particularly suitable. Such subjects can be treated by various surgical procedures, including clamping the aneurysm and closing off the blood, or performing an endovascular procedure to block the aneurysm using small coils or by introducing a stent or inserting a microcatheter into the blood vessel in which the aneurysm appears. Endovascular procedures are less invasive than clamping the aneurysm and are associated with better subject outcomes, although this outcome still involves a high incidence of small infarcts. Such subjects can be treated with the inhibitors of PSD95 interaction with NMDAR 2B described above, and in particular with the above agents. The timing of administration relative to performing the surgery may be subject to clinical trials, as described above.

[0148] Another class of subjects amenable to treatment are those with subarachnoid hemorrhage with or without aneurysm. (See US61 / 570,264). Another class of subjects are those with ischemic stroke suitable for endovascular thrombectomy to remove clots, such as the ESCAPE-NA1 trial (NCT02930018). Drugs can be administered before or after surgery to remove clots and are expected to improve the outcome of both the stroke itself and potential iatrogenic strokes associated with the above procedures. Another example is subjects diagnosed with possible stroke without the use of imaging criteria and treated within hours after stroke onset, preferably within the first 3 hours after stroke onset, and optionally within the first 6, 9 or 12 hours (similar to NCT02315443).

[0149] IV. Effective Dosage Regimens

[0150] The active agent is administered such that it 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 the condition being treated in a subject suffering from the condition. A therapeutically effective amount (before administration) or therapeutically effective plasma concentration after administration refers to an amount or level of the active agent sufficient to cure, reduce or inhibit further deterioration of at least one sign or symptom of the condition or condition being treated in a group of subjects (or animal models) suffering from the condition being treated with the agent of the present invention, significantly compared to the damage in a control group of subjects (or animal models) suffering from the condition not treated with the agent of the present invention. The amount or level is also considered to be therapeutically effective if the individual treated subjects achieve an outcome that is better than the average outcome in a comparable control group not treated by the method of the present invention. A therapeutically effective dosing regimen includes administering a therapeutically effective amount at a frequency and route of administration necessary to achieve the objectives of the present invention.

[0151] For subjects suffering from stroke or other ischemic conditions, the active agent is administered in a dosage regimen that includes a dosage, frequency and route that is effective for reducing the damaging effects of stroke or other ischemic conditions.When the condition requiring treatment is stroke, outcome can be determined by infarct volume or disability index, and a dosage is considered to be therapeutically effective if the treated subjects individually show a disability score of 2 or less and less than 2 and a Barthel scale score of 75 or more than 75, or if the treated subjects show a significantly improved disability score distribution (i.e., less disability) than the comparison untreated group (see Lees et al. at L, N Engl J Med 2006;354:588-600).A single dosage of the agent may be sufficient for the treatment of stroke.

[0152] The present invention also provides methods and formulations for the prevention of a condition in a subject at risk for the condition. Typically, such subjects are more likely to develop a condition (e.g., a disease, disorder, or disorder) compared to a control group. The control group can include, for example, one or more individuals selected from a population (e.g., an age-, sex-, race-, and / or ethnicity-matched population) who have neither been diagnosed nor have a family history of the condition. A subject can be considered at risk for a disorder if a "risk factor" associated with the condition is found to be associated with the subject. Risk factors can include any activity, trait, event, or characteristic associated with a given disorder through statistical or epidemiological studies of a group of subjects. Thus, a subject can be classified as at risk for a disorder even if the subject was not specifically included in a study identifying the underlying risk factor. For example, a subject undergoing cardiac surgery is at risk for transient ischemic attacks because of an increased frequency of transient ischemic attacks compared to subjects who have undergone cardiac surgery compared to subjects who have not.

[0153] Other common risk factors for stroke include age, family history, sex, prior incidence of stroke, transient ischemic attack or heart attack, high blood pressure, smoking, diabetes, carotid or other arterial disease, atrial fibrillation, other heart diseases (e.g., heart disease, heart failure, dilated cardiomyopathy, valvular heart disease and / or congenital heart defects), high blood cholesterol and a diet high in saturated fat, trans fat or cholesterol.

[0154] In prophylaxis, the active agent is administered to a subject at risk for a condition but not yet suffering from the condition in an amount, frequency and route sufficient to prevent, delay or inhibit the progression of at least one sign or symptom of the condition. A prophylactically effective amount before administration or plasma concentration after administration refers to an amount or level of the agent sufficient to prevent, inhibit or delay at least one sign or symptom of the condition in a group of human subjects (or animal models) at risk for the condition treated with the agent, compared to a control group of human subjects (or animal models) at risk for the condition not treated with the active agent of the invention. The amount or level is also considered to be prophylactically effective if each individual treated subject achieves an outcome that is better than the average outcome in a comparable control group not treated by the method of the invention. A prophylactically effective dosing regimen includes administering a prophylactically effective amount at a dosing frequency and route necessary to achieve the objectives of the invention. For the prevention of stroke in subjects at imminent risk of stroke (e.g., subjects undergoing cardiac surgery), a single dose of the agent is usually sufficient.

[0155] Depending on the agent, administration may be parenteral, intravenous, intrapulmonary, nasal, oral, subcutaneous, intraarterial, intracerebral, intrathecal, intraperitoneal, topical, intranasal, or intramuscular.

[0156] Traditionally, Tat-NR2B9c has been administered to humans by a single intravenous injection at 2.6 mg / kg. The active agent of the present invention can obtain a higher CMax and AUC when administered by a non-intravenous route, such as subcutaneously, intranasally or intramuscularly, compared to Tat-NR2B9c. The longer half-life is to compensate for the additional time required for the active agent to reach the plasma. Administration by such a non-intravenous route also allows for administration at higher doses without significant amounts of histamine release due to mast cell degranulation. For example, doses up to about 10 mg / kg can be used without significant histamine release, and doses up to 25 mg / kg release detectable histamine, but much less than the same dose administered intravenously.

[0157] Various doses can be administered depending on the route of administration and whether or not it is co-administered with an anti-inflammatory drug to reduce histamine release or its downstream effects. In the case of intravenous administration, the agent of the present invention can be administered at a dose similar to Tat-NR2B9c (e.g., 3 mg / kg or less, 0.1-3 mg / kg, 2-3 mg / kg or 2.6 mg / kg) when not co-administered with an anti-inflammatory drug, or at a higher dose (e.g., 5 or more, 10, 15, 20 or 25 mg / kg) when co-administered with an anti-inflammatory drug (see Figures 30A and 30B, which show efficacy in the range of at least 0.25 mg / kg to 25 mg / kg). In the case of routes such as subcutaneous, intranasal, intrapulmonary or intramuscular, the dose can be up to 10, 15 or 20 mg / kg when not co-administered with an anti-inflammatory drug, and more than 10, 15, 20, 25 or 50 mg / kg when co-administered with an anti-inflammatory drug. The need to administer higher doses of anti-inflammatory drugs can be reduced or eliminated by administration of the active agent over a longer period of time (e.g., administration for less than 1 minute, 1-10 minutes, and greater than 10 minutes constitute alternative dosing regimens in which the need for constant histamine release and anti-inflammatory drug is reduced or eliminated over time).

[0158] The active agent can be administered as a single dose or a multiple dose schedule. A single dose schedule can be used to treat acute conditions, such as acute ischemic stroke, to reduce infarction and cognitive impairment. Such doses can be administered before the onset of conditions, such as subjects undergoing neurovascular surgery, when the timing of the condition is predictable, or can be administered within a window (e.g., up to 1, 3, 6 or 12 hours) after the onset of conditions.

[0159] A multiple dose regimen can be designed to maintain detectable levels of the active agent in plasma for an extended period of time (e.g., at least 1, 3, 5, or 10 days, or at least 1, 3, 6, or indefinitely). For example, the active agent can be administered hourly, 2, 3, 4, 6, or 12 times a day, daily, every other day, weekly, etc. Such a regimen can provide initial relief from acute disease conditions, as in the case of a single dose, and then promote recovery from disease conditions that are still progressing. Such a regimen can also be used to treat chronic diseases, such as Alzheimer's disease and Parkinson's disease. The active agent may be incorporated into a controlled release formulation for use in a multiple dose regimen.

[0160] The active agent may be prepared with carriers that protect the compound against rapid elimination from the body, such as controlled release formulations or coatings. Such carriers are also known as modified, delayed, sustained or extended release or gastric retention dosage forms, and are, for example, Depomed GR TMIn the system, the drug is encapsulated by a polymer that swells in the stomach and remains there for approximately 8 hours, sufficient for daily dosing of many drugs. Controlled release systems include microencapsulated delivery systems, implants, biodegradable and biocompatible polymers (e.g., collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid), matrix-type controlled release devices, osmotic-type controlled release devices, multiparticulate controlled release devices, ion exchange resins, enteric coatings, multilayer coatings, microspheres, nanoparticles, liposomes, and combinations thereof. The release rate of the active agent can also be altered by varying the particle size of the active agent. Examples of modified release include those disclosed in U.S. Pat. Nos. 3,845,770, 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, and 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.

[0161] Active drugs containing D-amino acids are susceptible to nephrotoxicity, which increases with the D-amino acid content and dose of the drug. Nephrotoxicity can be reduced by coadministration of the active drug and a D-amino acid oxidase inhibitor. Such inhibitors have been previously proposed, for example, for the treatment of schizophrenia. Many examples of such inhibitors have been described (e.g., Sacchi et al., Curr. Phar. Ds. 19: 2499-511 (2013); Szilagyi et al., Molecules 24, 290 (2019); Terry 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 inhibitors can be administered before, after, or simultaneously with the active agent. In the latter case, they can be administered as a co-formulation or separately. Sodium benzoate is also used as a preservative in pharmaceutical compositions, so it can serve a dual purpose in the co-formulation.

[0162] V. Coadministration with Anti-Inflammatory Drugs

[0163] Depending on the dose and route of administration, the active agents of the invention can induce an inflammatory response characterized by degranulation of mast cells and the release of histamine and its sequelae. For example, a dose of at least 3 mg / kg by IV administration and at least 10 mg / kg by other routes causes the release of histamine.

[0164] Anti-inflammatory drugs for inhibiting one or more aspects of the inflammatory response are numerous and readily available. A preferred class of anti-inflammatory drugs are mast cell degranulation inhibitors. Compounds in this class include cromolyn (5,5'-(2-hydroxypropane-1,3-diyl)bis(oxy)bis(4-oxy-4H-chromene-2-carboxylic acid) (also known as cromoglycate), 2-carboxylate chromone-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-dicarboxylic acid), tranilast (2-{[(2E)-3-(3,4-dimethoxyphenyl)prop-2-enoyl]amino}), and lodoxamide (2-[2-chloro-5-cyano-3-(oxaloamino)anilino]-2-oxoacetic acid). The compounds include pharma- ceutical acceptable salts of the compounds. Cromolyn is readily available in formulations for nasal, oral, inhaled, or intravenous administration. Although the practice of the present invention does not depend on understanding the mechanism, it is believed that these agents act at the early stage of the inflammatory response induced by internalized peptides and are therefore most effective in inhibiting the development of sequelae, including a temporary drop in blood pressure. Other classes of anti-inflammatory drugs, described below, inhibit one or more downstream events resulting from mast cell degranulation, such as inhibiting histamine to H1 or H2 receptors, but are unable to inhibit all sequelae of mast cell degranulation or require higher doses or combinations. Table 2 below shows the names, chemical formulas, and FDA status of some mast cell degranulation inhibitors that can be used in combination with the present invention.

[0165] [Table 2] JPEG2024502463000006.jpg204162

[0166] Another class of anti-inflammatory drugs are antihistamine compounds. Such drugs inhibit the sequelae of the above inflammation by inhibiting the interaction of histamine with its receptors. Many antihistamines are available over the counter, some are OTC. Examples of antihistamines include azatadine, azelastine, verflorin, cetirizine, cyproheptadine, doxantrozole, etodroxyzine, forskolin, hydroxyzine, ketotifen, oxatomide, pizotifen, proxicromil, N,N'-substituted piperazines or terfenadine. Antihistamines differ in their ability to block CNS and peripheral receptors, with second and third generation antihistamines having selectivity for peripheral receptors. Acrivastine, astemizole, cetirizine, loratadine, mizolastine, levocetirizine, desloratadine and fexofenadine are examples of second and third generation antihistamines. Antihistamines are widely available as oral and topical preparations. Some other antihistamines that can be used are listed in Table 3 below.

[0167] [Table 3] JPEG2024502463000008.jpg144157

[0168] Another class of anti-inflammatory drugs useful in inhibiting inflammatory responses are the corticosteroids. These compounds are transcription regulators and potent inhibitors of inflammatory conditions caused by the release of histamine and other compounds resulting from the degranulation of mast cells. Examples of corticosteroids include cortisone, hydrocortisone (Cortef), prednisone (Deltasone, Meticorten, Orasone), prednisolone (Delta-Cortef, Pediapred, Prelone), triamcinolone (Aristocort, Kenacort), methylprednisolone (Medrol), dexamethasone (Decadron, Dexone, Hexadrol) and betamethasone (Celestone). Corticosteroids are widely available in oral, intravenous and topical formulations.

[0169] Nonsteroidal anti-inflammatory drugs (NSAIDs) can also be used. Such drugs include aspirin compounds (acetylsalicylate), non-aspirin salicylates, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, naproxen, naproxen sodium, phenylbutazone, sulindac, and tometin. However, the anti-inflammatory effect of such drugs is less than that of antihistamines and corticosteroids. Strong anti-inflammatory drugs such as azathioprine, cyclophosphamide, leukeran, and cyclosporine can also be used, but are undesirable because they act slowly and / or cause side effects. Biological anti-inflammatory drugs such as Tysabri® and Humira® can also be used, but are undesirable for the same reasons.

[0170] In inhibiting the inflammatory response, drugs from different classes can be used in combination, with a preferred combination being a mast cell degranulation inhibitor and an antihistamine.

[0171] In the method in which the pharmacological agent bound to the internalization peptide is administered together with an anti-inflammatory agent, the two are administered with a sufficiently short administration interval so that the anti-inflammatory agent inhibits the inflammatory response induced by the internalization peptide. The anti-inflammatory agent can be administered before, simultaneously with, or after the administration of the pharmacological agent. The preferred administration interval 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 administration of the pharmacological agent so that the anti-inflammatory agent is close to its maximum serum concentration at the time the pharmacological agent is administered. Typically, the anti-inflammatory agent is administered 6 hours before to 1 hour after the administration of the pharmacological agent. For example, the anti-inflammatory agent can be administered 1 hour before to 30 minutes after the administration of the pharmacological agent. Preferably, the anti-inflammatory agent is administered 30 minutes before to 15 minutes after the administration of the pharmacological agent, and more preferably, the anti-inflammatory agent is administered 15 minutes before to at the time of administration of the pharmacological agent. In some methods, the anti-inflammatory agent is administered within 15, 10, or 5 minutes before the pharmacological agent is administered. In some methods, the anti-inflammatory agent is administered 1-15, 1-10, or 1-5 minutes before administration of the pharmacological agent.

[0172] When administration of the drugs is not instantaneous, such as with intravenous infusion, the anti-inflammatory drug and the pharmacological agent are considered to be administered simultaneously if their administration periods are comparable or overlap. A pre-administration administration period begins at the start of administration. A post-administration period begins at the end of administration. A period with respect to administration of an anti-inflammatory drug refers to the start of its administration.

[0173] When an anti-inflammatory drug is said to be capable of inhibiting an inflammatory response to a pharmacological agent bound to an internalization peptide, it means that both are administered with a sufficiently short interval between doses that the anti-inflammatory drug inhibits an inflammatory response inducible by the pharmacological agent bound to the internalization peptide, if such a response may occur in a particular subject (not necessarily meaning that such a response will occur in the subject). Some subjects are treated with a dose of the agent bound to the internalization peptide that is associated with an inflammatory response in a statistically significant number of subjects in a controlled clinical trial or non-clinical trial. It can be reasonably assumed that a significant proportion of such subjects, but not necessarily all, will have an anti-inflammatory response to the pharmacological agent bound to the internalization peptide. In some subjects, signs or symptoms of an inflammatory response to the agent bound to the internalization peptide are detected or are detectable.

[0174] In clinical treatment of individual subjects, it is usually not possible to compare the inflammatory response from a pharmacological agent bound to an internalized peptide in the presence and absence of an anti-inflammatory drug. However, if significant inhibition is seen under the same or similar co-administration conditions in a controlled clinical trial or preclinical trial, it can be reasonably concluded that the anti-inflammatory drug inhibits the inflammatory response induced by the peptide. The subject's results (blood pressure, heart rate, urticaria, etc.) can also be compared to typical results from a control group in a clinical trial as an indication of whether inhibition has occurred in an individual subject. Typically, the anti-inflammatory drug is present in detectable serum concentrations at some point within one hour after administration of the pharmacological agent. The pharmacokinetics of many anti-inflammatory drugs are widely known, and the relative timing of administration of the anti-inflammatory drug can be adjusted accordingly. Anti-inflammatory drugs are usually administered peripherally, i.e., separated from the brain by the blood-brain barrier. For example, anti-inflammatory drugs can be administered orally, nasally, intravenously, or topically, depending on the drug under discussion. When an anti-inflammatory drug is administered simultaneously with a pharmacological agent, the two can be administered as a combined formulation or they can be administered separately.

[0175] In some methods, the anti-inflammatory drug does not cross the blood-brain barrier when administered orally or intravenously at least in an amount sufficient to exert detectable pharmacological activity in the brain. Such drugs can inhibit mast cell degranulation and its sequelae resulting from administration of an active drug in the periphery without exerting a detectable therapeutic effect in the brain itself. In some methods, the anti-inflammatory drug is administered without co-treatment to increase the permeability of the blood-brain barrier, or is derivatized or formulated to increase its ability to cross the blood-brain barrier. However, in other methods, the anti-inflammatory drug, depending on its nature, derivatization, formulation, or route of administration, can exert a dual effect of suppressing mast cell degranulation and / or its sequelae in the periphery due to internalized peptides and inhibiting inflammation in the brain by entering the brain or affecting inflammation in the brain. WO04 / 071531 (Strbian et al.) reports that icv administration (non-intravenous administration) of cromoglycate, a mast cell degranulation inhibitor, has a direct inhibitory effect on infarction in animal models.

[0176] In some methods, the subject has not been treated with the same anti-inflammatory drug co-administered with the active agent within days, weeks or months before and / or after co-administration with the active agent. In some methods, if the subject is treated with the same anti-inflammatory drug co-administered with the active agent in a repeated dosing regimen (e.g., the same amount, route of administration, frequency of administration, timing of days of administration), the co-administration of the anti-inflammatory drug and the active agent does not match the repeated dosing regimen in any or all of the amount, route of administration, frequency of administration and timing of days of administration. In some methods, the subject is not known to suffer from an inflammatory disease or condition that requires administration of the anti-inflammatory drug co-administered with the active agent in the method. In some methods, the subject does not suffer from asthma or allergy disease treatable with a mast cell degranulation inhibitor. In some methods, the anti-inflammatory drug and the active agent are each administered only once within the window defined above per episode of the disease. An episode is a relatively short period during which symptoms of the disease are present, adjacent to a longer period during which symptoms are absent or reduced.

[0177] The anti-inflammatory drug is administered in an amount, frequency and route of administration regimen effective to inhibit an inflammatory response to an internalized peptide under conditions known to occur in the absence of the anti-inflammatory drug. An inflammatory response is suppressed if the signs or symptoms of inflammation are reduced as a result of the anti-inflammatory drug. Symptoms of an inflammatory response include redness, rash such as hives, heat, swelling, pain, tingling, itching, nausea, rash, dry mouth, numbness, airway congestion, and the like. An inflammatory response can also be monitored by measuring signs such as blood pressure and heart rate. Alternatively, an inflammatory response can be assessed by measuring the plasma concentration of histamine or other compounds released by mast cell degranulation. Increased levels of histamine or other compounds released by mast cell degranulation, decreased blood pressure, skin rash such as hives, or decreased heart rate are indicators of mast cell degranulation. For practical purposes, dosages, dosing schedules, and routes of administration for most of the above anti-inflammatory drugs are available from the Physicians' Desk Reference and / or from the manufacturers, and such anti-inflammatory drugs can be used in the methods of the present invention consistent with such general guidance.

[0178] VI. Coadministration of Thrombolytic Agents or Mechanical Reperfusion

[0179] Plaques and blood clots (also known as emboli) that cause ischemia can be dissolved, removed, or bypassed by both pharmacological and physical means. The dissolution and removal of plaques and blood clots and the resulting restoration of blood flow is called reperfusion. One class of drugs acts by thrombolysis. Thrombolytic agents function by promoting the production of plasmin, which clears the cross-linked fibrin meshwork (the framework of the blood clot), solubilizing the clot and subjecting it to further proteolysis by other enzymes, restoring blood flow in the occluded vessel. Examples of thrombolytic agents include tissue plasminogen activator t-PA, alteplase (Activase), reteplase (Retavase), tenecteplase (TNKase), anistreplase (Eminase), streptokinase (Kabikinase, Streptase), and urokinase (Abbokinase).

[0180] Another class of pharmacological agents that can be used for reperfusion are vasodilators. These pharmacological agents work by relaxing and widening blood vessels, allowing blood to flow around the occlusion. Some examples of types of vasodilators include alpha-adrenergic receptor antagonists (alpha-blockers), angiotensin receptor blockers (ARBs), beta2-adrenergic receptor antagonists (beta2-blockers), calcium channel blockers (CCBs), centrally acting sympatholytic agents, direct acting vasodilators, endothelin receptor antagonists, ganglion blockers, nitrodilators, phosphodiesterase inhibitors, potassium channel openers, and renin inhibitors.

[0181] Another class of pharmacological agents that can be used for reperfusion are vasopressors (i.e., agents that increase blood pressure), such as epinephrine, phenylephrine, pseudoephedrine, norepinephrine, norephedrine, terbutaline, salbutamol, and methylephedrine. Increased perfusion pressure can increase blood flow around the obstruction.

[0182] Mechanical means for reperfusion include angioplasty, catheter insertion, and arterial bypass graft surgery, stent placement, embolectomy, or endarterectomy. These procedures restore flow through the mechanical removal of plaque, keeping the blood vessel open so blood can flow around or around the plaque.

[0183] Other methods of reperfusion include the use of devices that divert blood flow from other areas of the body to the brain. One example is a catheter that partially occludes the aorta, such as the CoAxia NeuroFlo™ catheter device, which has recently been subjected to randomized trials and may be FDA approved for stroke treatment. This device has been used in subjects presenting with stroke up to 14 hours after the onset of ischemia.

[0184] The active agent of the present invention comprising a D-amino acid can be administered to a subject susceptible to treatment in any form of reperfusion therapy. However, the active agent of the present invention is particularly advantageous for administration with a thrombolytic agent, since the inclusion of one or more D-amino acids in the active agent reduces the susceptibility of the active agent to cleavage by plasmin induced by the thrombolytic agent. Thus, the active agent comprising one or more D-amino acids can be co-administered with the thrombolytic agent in a dosing regimen that results in cleavage of the active agent induced by the thrombolytic agent. For example, the thrombolytic agent can be administered within a 60 minute, 30 minute, or 15 minute window before the active agent is administered. In some methods, the active agent can be administered simultaneously with the thrombolytic agent. The active agent and the thrombolytic agent can be co-formulated or administered separately. In some methods, the thrombolytic agent is administered before the active agent is administered and persists at detectable levels in serum when the active agent is administered.

[0185] For the treatment of ischemia that cannot be predicted in advance, the active agent can be administered as soon as possible or as soon as practical after the onset of ischemia. For example, the active agent can be administered within 0.5, 1, 2, 3, 4, 5, 6, 9, 12, or 24 hours after the onset of ischemia. For ischemia that can be predicted in advance, the active agent can be administered before, at the same time as, or after the onset of ischemia. For example, for ischemia caused by surgery, the PSD-95 inhibitor can be administered periodically, regardless of whether ischemia has occurred or is likely to occur, for a period starting 30 minutes before the start of surgery and ending 1, 2, 3, 4, 5, 6, 9, 12, or 24 hours after surgery. Since the active agent has no significant side effects, it can be administered when stroke or other conditions of ischemia are suspected, even if a diagnosis has not been made according to standards accepted in the art. For example, the active agent can be administered at the site where the stroke occurred (e.g., at the subject's home) or in an ambulance transporting the subject to a hospital. The active agents may also be safely administered to subjects who are at risk for pre-symptomatic stroke or other conditions of ischemia, and who may or may not actually develop the condition.

[0186] Following administration of the active agent, or sometimes prior to administration, subjects who exhibit signs and / or symptoms of ischemia may be further subjected to diagnostic evaluation to determine whether the subject has ischemia in or otherwise affecting the CNS and whether the subject has or is suspected of having a hemorrhage. In subjects who exhibit symptoms of stroke, particularly tests to determine whether the stroke is the result of hemorrhage or ischemia, with hemorrhage accounting for 17% of strokes. Diagnostic tests may include scans of one or more organs, such as CAT scans, MRI or PET imaging scans, or blood tests for biomarkers that suggest a stroke is occurring. Several biomarkers related to stroke are known, including nerve growth factor type B, von Willebrand factor, matrix metalloproteinase-9, and monocyte chemotactic protein-1 (see Reynolds et al., Clinical Chemistry 49:1733-1739 (2003)). Organs scanned include any organs suspected to be the site of ischemia (e.g., brain, heart, limbs, spine, lungs, kidneys, retina), as well as any other organs suspected to be the source of hemorrhage instead. Brain scanning is a routine procedure for distinguishing between ischemic and hemorrhagic strokes. Diagnostic evaluation may also include obtaining or reviewing the subject's medical history and performing other tests.The presence of any of the following factors, alone or in combination, can be used to assess whether reperfusion therapy poses an unacceptable risk: the subject's symptoms are not severe or improve rapidly; the subject had a seizure at the onset of the stroke; the subject had another stroke or severe brain trauma within the past 3 months; the subject had major surgery within the last 14 days; the subject has a known history of intracranial hemorrhage; the subject has a sustained systolic blood pressure of >185 mmHg; the subject has a sustained diastolic blood pressure of >110 mmHg; vs. Aggressive treatment is required to lower the elephant's blood pressure; the subject has symptoms suggestive of subarachnoid hemorrhage; the subject has had gastrointestinal or urinary bleeding within the last 21 days; the subject has had an arterial puncture at a non-compressible site within the last 7 days; the subject has received heparin within the last 48 days and has an elevated PTT; the subject has a prothrombin time (PT) >15 seconds; the subject has a platelet count <100,000 μL; the subject has a serum glucose <50 mg / dL or >400 mg / dL; the subject is hemophilic or has other coagulation deficiencies.

[0187] Further diagnostic investigations determine, according to accepted criteria, or at least to a greater extent, whether the subject has an ischemic disease prior to the investigation, and whether the subject has a hemorrhage, has an unacceptable risk of hemorrhage, or is otherwise excluded from undergoing reperfusion therapy due to an unacceptable risk of side effects. Subjects with a confirmed diagnosis of ischemic disease in or otherwise affecting the CNS and who do not have an unacceptable risk of side effects may be subjected to reperfusion therapy. Reperfusion therapy is performed as soon as possible after completion of any diagnostic procedures.

[0188] Both treatment with active agent and treatment with reperfusion therapy have the ability to reduce infarct size and functional impairment due to ischemia individually. When used in combination, the reduction in infarct size and / or functional impairment is preferably greater than the reduction resulting from the use of either agent alone administered under the equivalent dosing schedule but not for the combination. More preferably, the reduction in infarct size and / or functional impairment is at least in addition to, and preferably greater than, the reduction achieved with a single agent administered under the equivalent dosing schedule but excluding the combination. In some dosing schedules, reperfusion therapy is effective at reducing infarct size and / or functional impairment at a time point (e.g., more than 4.5 hours) after the onset of ischemia when it is not effective without co-administration or prior administration of a PSD-95 inhibitor. In other words, when a subject is administered with active agent and reperfusion therapy, reperfusion therapy is preferably at least as effective as when it is administered at an earlier time point without active agent. Thus, the active agent effectively improves the effect of reperfusion therapy by reducing one or more damaging effects of ischemia before or when reperfusion therapy takes effect. The active agent can therefore compensate for a delay in performing reperfusion, whether that delay is due to a delay in the subject recognizing his or her early risk of symptoms while transporting the subject to a hospital or other medical facility, or a delay in performing diagnostic procedures to confirm the presence of ischemia and / or the absence or unacceptable risk of hemorrhage. A statistically significant combined effect, including an additive or synergistic effect, of the active agent and reperfusion therapy can be demonstrated between populations in clinical trials or between populations in animal models in preclinical studies.

[0189] Binding of X.Tat variants to other drugs The tat variants described above can be conjugated with any other drug to facilitate drug uptake through cell membranes and / or the blood-brain barrier. The use of a chimeric drug comprising or consisting of a tat variant and a drug in a therapeutic treatment improves bioavailability at the intended site, increases plasma half-life and / or therapeutic index, and / or improves pharmacokinetic values ​​such as CMax and AUC at the same dose compared to the use of the drug alone. The tat variants are particularly useful for drugs with intracellular targets and / or neuroactive drugs that must cross the blood-brain barrier to exert their activity. Some, but not all, of the drugs suitable for conjugation of tat variants are peptides. The use of tat variants is particularly useful for existing pharmaceutical drugs that have low bioavailability, high doses, or short half-lives.

[0190] Some guidance regarding the selection of agents, methods of attachment, and their use has been provided by previous scientific and patent literature regarding tat peptides (e.g., US 6,316,003 and US 5,804,604).All of the above discussion regarding chimeric peptides comprising inhibitor peptides attached to tat variants for the treatment of stroke and related diseases applies to chimeric agents comprising a tat variant attached to another agent.

[0191] The present invention includes a chimeric agent. The chimeric agent includes an internalization peptide or a variant thereof linked to an agent for treating a disorder. The internalization peptide has an amino acid sequence including RKKRRQRRR (SEQ ID NO: 13). The variant includes up to 1, 2 or 3 substitutions or deletions (not including L to D substitutions of the same amino acid). Residues 3-5 of the internalization peptide are D-amino acids. In some chimeric agents, the internalization peptide has an amino acid sequence including RKKRRQRRR (SEQ ID NO: 13), GRKKRRQRRR (SEQ ID NO: 11) or YGKKRRQRRR (SEQ ID NO: 125) in which residues 3-5 are D-amino acids. In some chimeric agents, the internalization peptide includes an amino acid sequence having at least 75, 80, 85 or 90% identity to any of the reference RKKRRQRRR (SEQ ID NO: 13), GRKKRRQRRR (SEQ ID NO: 11) or YGKKRRQRRR (SEQ ID NO: 125). In some chimeric agents, each D residue is located at a K or R, or at the C-terminal residue to a K or R. In some active agents, the internalization peptide lacks a stretch of four or more contiguous amino acids independently selected from L-amino acids and glycine, said stretch comprising adjacent amino acids independently selected from R or K. In some active agents, the internalization peptide lacks a stretch of four or more contiguous amino acids independently selected from L-amino acids and glycine, said stretch comprising amino acids selected from R or K. In some active agents, the internalization peptide lacks a stretch of five or more contiguous amino acids independently selected from L-amino acids and glycine, said stretch comprising adjacent amino acids independently selected from R or K, and if a stretch of four contiguous amino acids is present, this stretch is located at the N-terminus of the internalization peptide. In some active agents, the internalization peptide lacks a stretch of five or more consecutive amino acids independently selected from L-amino acids and glycine, the stretch comprising an amino acid selected from R or K, and if a stretch of four consecutive amino acids is present, the stretch is located at the N-terminus of the internalization peptide.In some active agents, the internalization peptide has an amino acid sequence comprising YGRKKRRQRRR (SEQ ID NO:1) and has three D residues, a D residue between positions 3 and 5, a D residue between positions 7 or 8, and a D residue between positions 9 and 11, numbered from the N-terminus. In some active agents, the agent is attached to the internalization peptide via a spacer.

[0192] The table below provides the names of the drugs (some approved), the diseases they help treat (acute and chronic), the route of drug administration (established range), and a comment on problems with existing drugs that may be partially overcome by the improved transport across membranes afforded by tat variant peptides.

[0193] Chimeric drugs comprising a tat variant peptide conjugated to a drug can be used at the same or lower doses on a molar basis as the drug alone, can be administered by the same routes, and can be administered to treat the same diseases as the drug alone. Preferred methods of administration of the disclosed peptide:active conjugates are intravenous, intraarterial, intranasal / inhalation, intramuscular, intraperitoneal, sublingual, intrarectal, and topical (for lesions proximal to the dermis or epithelial cells).

[0194] [Table 4] JPEG2024502463000010.jpg198134JPEG2024502463000011.jpg201136JPEG2024502463000012.jpg196135

[0195] Although the present invention has been described in detail for clarity of understanding, certain modifications may be made within the scope of the appended claims. All documents, accession numbers, and patent documents cited in this application are incorporated herein by reference in their entirety, exactly as if each were individually described. If one or more sequences are associated with an accession number at different times, the sequence associated with that accession number is intended to be as of the effective filing date of this application. 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, function, or feature of the present invention can be practiced in combination with others.

[0196] Working Example

[0197] The examples relate to peptides with the following names and sequences, where lower case letters indicate D-amino acids and upper case letters indicate L-amino acids: NA-1 (aka Tat-NR2B9c or Nerinetide): YGRKKRRQRRRKLSSIESDV (SEQ ID NO:58) D-TAT-L-2B9c: ygrkkrrqrrrKLSSIESDV (SEQ ID NO: 80) NA-3: ygrkkrrqrrrklssIESDV (SEQ ID NO: 6) D-NA-1: ygrkkrrqrrrklssiesdv (SEQ ID NO: 81)

[0198] 1. Plasmin cleavage site in NA-1

[0199] Plasmin is a serum protease that is induced by thrombolytic agents such as tPA. Plasmin cleavage sites may occur C-terminal to basic amino acid residues in peptides composed of L-amino acids.

[0200] NA-1 was digested with plasmin and the products were analyzed by mass spectrometry. The following cleavage products were detected: YGRKKRRQRRRKLSSIESDV (SEQ ID NO:58) (full length NA-1, undigested) RRQRRRKLSSIESDV (SEQ ID NO:82) RQRRRKLSSIESDV (SEQ ID NO: 83) QRRRKLSSIESDV (SEQ ID NO:84) RRKLSSIESDV (SEQ ID NO: 85) RKLSSIESDV (SEQ ID NO:86) KLSSIESDV (SEQ ID NO:2) LSSIESDV (SEQ ID NO: 87)

[0201] These cleavage products indicate that NA-1 is cleaved at seven of the nine potential sites, as shown in Figure 1. However, cleavage at the other two sites may occur to a lesser extent.

[0202] 2. Degradation of NA-1 administered simultaneously with tPA in rat or human plasma Rat or human plasma was treated with NA-1 alone or in combination with tPA at the following concentrations: NA-1 alone [65ug / mL] (N=4) NA-1[65ug / mL]+rt-PA[22.5ug / mL](N=4) NA-1[65ug / mL]+rt-PA[67.5ug / mL](N=4) NA-1[65ug / mL]+rt-PA[135ug / mL](N=4)

[0203] Samples were collected at six different time points.

[0204] Figures 2 and 3 show that NA-1 content in rat plasma (in vitro) or human plasma (in vitro) was rapidly decreased when co-administered with tPA, compared to administration of NA-1 alone. Figure 4 shows that CMax and AUC were similarly decreased after administration of NA-1 and tPA to rats, and plasma was collected at various time points to determine the level of NA-1. Thus, tPA induces cleavage of NA-1 in rat or human plasma when administered together with tPA in vitro or in vivo. Neither tPA nor TNK directly cleaves NA-1 in phosphate buffered saline alone (data not shown). Thus, cleavage of NA-1 is the result of activation of plasminogen in animal plasma or blood.

[0205] 3. Decomposition of peptides containing D-amino acids

[0206] In Figure 5, NA-1 and D-Tat-L-2B9C (also called D-Tat-L-NA-1) administered alone or co-administered with tPA in rat plasma in vitro. NA-1 treated with tPA decreased to zero within about 15 minutes, while D-Tat-L-2B9C showed little degradation after co-administration with tPA. Figure 6 shows the results for human plasma similar to those for rat plasma. Such degradation increased with the dose of tPA.

[0207] The experiment was repeated using TNK-tissue plasminogen activator instead of tPA. TNK-tissue plasminogen activator is a bioengineered variant of tPA with a longer half-life. Similar results were obtained using TNK instead of tPA. NA-1 showed rapid degradation upon coadministration of TNK, whereas D-Tat-L-2B9C was stable (Figure 7, Figure 8).

[0208] Figure 9 shows similar results for treatment of NA-1 or D-Tat-L-2B9C with plasmin in PBS. NA-1 was rapidly degraded, while D-Tat-L-2B9C showed similar stability with or without plasmin. Control treatment with tPA in PBS buffer (no plasma) showed no degradation of NA-1 or D-Tat-L-2B9C, since tPA does not generate plasmin without plasma supply.

[0209] 4. D-Tat-L-NR2B9c disrupts the PSD-95:NR2B9c complex.

[0210] Three pial vessel models (3PVo) were constructed using Sprague-Dawley rats. One hour after stroke onset, placebo, NA-1 or D-Tat-L-2B9C were administered to the rats at 7.6 mg / kg, respectively. Two hours after stroke onset, brains were harvested. Cortical stroke areas were collected for analysis. Immunoprecipitation was performed using anti-PSD-95 or anti-NMDAR2B. The amount of PSD-95 and NMDAR2B in the samples was analyzed by Western blotting. The reduction in PSD-95-NMDAR2B complex formation was evaluated by the fold reduction of placebo versus treatment. Figure 10 shows that both NA-1 and D-Tat-L-2B9C can dissociate preformed NMDAR2B:PSD-95 complexes and act effectively in vivo.

[0211] 5. Binding affinity to PSD-95

[0212] Binding was assessed by competitive ELISA assay. Plates were coated with 1 μg / ml PSD95PDZ2 in 50 mM bicarbonate buffer and left overnight at 4°C. Plates were blocked with 2% BSA (0.05%) in PBST for 2 hours at room temperature. Plates were then incubated overnight at 4°C with a mixture of 150 ng / ml biotinylated NA-1 and various test compounds (initial concentration 120 ug / ml, 3-fold dilutions) and after appropriate washing with PBS-T, plates were incubated with (1:3000) SA-HRP for 30 minutes. Wells were washed again and incubated with TMB solution for 10 minutes. The reaction was stopped with 100 ul H2SO4. Absorbance was measured at 450 nm using a Synergy H1 reader.

[0213] FIG. 12 shows that biotinylated NA-1, D-Tat-L-2B9C and D-Tat-L-IESDV (SEQ ID NO: 6) each bound to PSD-95 domain 2, and also shows the EC50 of NA-1, D-Tat-L-2B9C and D-NA-1. The EC50 of NA-1 and D-Tat-L-2B9C were almost the same within experimental error, while the EC50 of D-NA-1 was about 10-fold lower. This result provides evidence that converting the C-terminal residue of NA-1, which is most involved in binding to PSD-95, to a D-amino acid reduces the binding affinity. D-Tat-L-2B9C and D-Tat-L-IESDV (SEQ ID NO: 6) bind to the target protein PS95. PDZ2 Both test peptides bind to IC 50 achieving values ​​<5 μM (Figure 11). Figure 11 shows that the IC50s were within a factor of 2 of each other, which was within the error range of the experiment.

[0214] 6. Pharmacokinetic Analysis

[0215] Rats were anesthetized (isoflurane 1.5%) in the supine position and spontaneously breathed at 0.5 L / min O2. The left femoral artery was cannulated for blood sampling.

[0216] Test drugs were prepared at a stable concentration in the total volume of vehicle. Intrapulmonary instillation was performed by intubation with a 14G catheter connected to a 1 cc syringe through which test drug was delivered. Injections were given subcutaneously (SQ or SC) in the left flank area with a total volume not exceeding 2 ml per site.

[0217] Compounds NA-1, D-Tat-L-NA1, D-Tat-L-IESDV (SEQ ID NO: 6) and D-NA-1 were tested. For each dosing strategy, each dose was evaluated in 3 rats. The planned dose levels and routes are shown in Table 5 below. In the first experiment, two different routes of administration (SQ and PI) were evaluated and blood samples were taken at 8 different time points (7 time points pre-dose and post-dose: 1, 2.5, 5, 10, 15, 30, 60 min (250ul / sample)). In the 24-hour PK curve experiment, blood samples were taken at 11 time points (pre-dose, 2.5, 5, 10, 15, 30, 60 min, 3 hr, 6 hr, 12 hr and 24 hr post-dose).

[0218] [Table 5]

[0219] HPLC quantitative analysis Plasma was separated from blood and stored at -80°C until use. Each sample was precipitated by adding 1M HCl (10ul / 100ul sample) at >80°C, centrifuged (12,000rpm x 15min) and collected. A 25cm C-18 RP-HPLC column was equilibrated with 10% acetonitrile and 0.1% TFA at 40°C, samples were injected and analyzed on an Agilent 1260 Infinity Quaternary LC System (1.5mL / min for 30min; gradient from 10% to 35% acetonitrile in 0.1% TFA; absorbance detection at 220nm). HPLC standard curves were constructed using samples spiked with known amounts of test reagent.

[0220] As shown in Figure 13, subcutaneous NA-1 had a much lower CMax, a slightly lower AUC, and a longer half-life than intravenous NA-1 at the same dose. Intramuscular NA-1 had a lower CMax, a slightly higher AUC, and a longer half-life than intravenous NA-1.

[0221] As shown in Figure 14, subcutaneous D-Tat-L-IESDV (SEQ ID NO: 6) (NA-3) increased the Cmax and AUC compound compared to subcutaneous NA-1. Subcutaneous D-Tat-L-2B9C and D-NA also increased the Cmax and AUC compared to subcutaneous NA-1, but not to the same extent as D-Tat-L-IESDV (SEQ ID NO: 6). As shown in Figures 15A-B, the Cmax and AUC of subcutaneous D-Tat-L-IESDV (SEQ ID NO: 6) are dose-dependent and increase linearly with dose.

[0222] As shown in FIG. 16, intrapulmonary instillation of D-Tat-L-IESDV (SEQ ID NO: 6) resulted in a higher CMax compared to NA-1 or D-Tat-L-2B9C.

[0223] 7. Effect of peptides on histamine release

[0224] The effect of D-Tat-L-IESDV (SEQ ID NO: 6) injection on histamine release was tested using plasma samples from rats administered NA-3[SQ] at three different doses. Plasma samples were collected at 11 time points (pre-dose, 2.5, 5, 10, 15, 30, 60 min, 3 hr, 6 hr, 12 hr and 24 hr post-dose). Histamine levels were quantified using a commercially available histamine ELISA assay kit (Histamine ELISA-H1531-K01, Eagle Bioscience). Plates were coated with plasma samples (50 μl / well) and incubated at room temperature for 60 min on an orbital shaker at medium frequency. Then, 100 μl of enzyme conjugate was added to the wells and incubated at room temperature for 20 min. Samples were washed again and incubated with TMB solution for 25 min at room temperature. The reaction was stopped with 100 μl of H2SO4. Absorbance was measured at 450 nm by an ELISA plate reader.

[0225] Blood samples were taken pre-injection and at 0, 1, 2.5, 5, 10, 15, 30, and 60 min post-dose for quantification of histamine levels using a commercial kit. This sampling period covers the period of histamine elevation observed with NA-1 after IV injection in rat samples (N=3 animals / group).

[0226] As shown in Figure 17, subcutaneous administration of D-Tat-L-IESDV (SEQ ID NO: 6) at a dose of 8.3 mg / kg or 2.8 mg / kg did not result in significant histamine release. D-Tat-L-NA1 at 7.6 mg / kg (IV) resulted in significant histamine release. D-Tat-L-IESDV (SEQ ID NO: 6) at 25 mg / kg (SQ) resulted in histamine release, but much less than at 7.6 mg / kg (IV). Histamine induced by D-Tat-L-2B9C at 7.6 mg / kg (IV) was abrogated by co-administration of lodoxamide (Figure 18).

[0227] Thus, subcutaneous administration of active agents, including D-amino acids, reduces histamine release at higher doses compared to intravenous administration.

[0228] 8. Efficacy of D-Tat-L-2B9C as a neuroprotectant in an embolic MCA occlusion model

[0229] Animals were anesthetized with isoflurane (5% for induction, 2% for surgery, 1.5% for maintenance). The femoral vein and artery were cannulated with PE-50 tubing for drug administration, blood pressure monitoring and blood sampling. Full monitoring (cerebral blood flow, arterial blood gases, plasma glucose, temperature) was performed before and during surgery. All physiological parameters were maintained within normal ranges. Relative cerebral blood flow was continuously measured using a PR407-1 straight-needle LDF probe (Perimed, Jarfalla, Stockholm, Sweden) connected to a standard laser Doppler monitor (PF5010 LDPM unit and PF5001 main unit, Perimed, Jarfalla, Stockholm, Sweden). In case of middle cerebral artery embolic stroke, a PE-50 tube with a PE-10 5 cm tip was inserted from the internal carotid artery via the external carotid artery to the base of the skull and a previously prepared single red blood clot was manually injected. After 7 min, the common carotid artery (CCA) catheter and clips were removed. Animals were kept anesthetized during the entire procedure and injections. Therapeutic drugs were administered simultaneously 1 h after stroke onset. Neuroprotectants were injected by intravenous bolus (<30 s), thrombolytics were administered by bolus injection with the first 10% administered within 1 min, and the remaining 90% of the total dose infused over 1 h. After administration, animals were collected in clean cages with heat lamps. Due to the acute nature of the stroke model, only neurological score tests (postural reflex and forelimb placing test (grade: 0-12)) were performed as behavioral evaluation. Immediately after neuroscore test (24 h after stroke onset), animals were euthanized. Brains were removed and 8 slices with a thickness of 1.5 mm were cut coronally and placed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) at 37 °C for staining. Sections were scanned and infarct volumes were measured with ImageJ software. Brain swelling was also measured.

[0230] The study included the following groups: Sham (no surgery) (N=10) Placebo (negative control) (N=12) NA-1 alone [7.6 mg / kg] (positive control) (N=11) D-TAT-L-NA1 Lodo [7.6mg / kg](N=12) rt-PA alone [5.4mg / kg] (N=10) NA-1 [7.6 mg / kg] + rt-PA [5.4 mg / kg] (negative control) (N = 12) D-TAT-L-NA1 Lodo [7.6mg / kg]+rt-PA[5.4mg / kg](N=17)

[0231] As shown in Figure 19, in the absence of tPA treatment, the combination of lodoxamide with either NA-1 or D-Tat-L-2B9C significantly reduced infarct volume and right hemisphere swelling. When tPA was co-administered, only the combination of D-Tat-L-NA1 with lodoxamide significantly protected against infarction and right hemisphere swelling. This result may be explained by tPA-induced proteolysis of NA-1 reducing its efficacy. D-Tat-L-NA1 remains effective because it is protected from such proteolysis by including D-residues. Figure 20 shows a similar effect on neurological outcome. Thus, D-Tat-L-2B9C shows improved plasma stability (reduced stroke volume and improved behavioral outcome) even when administered simultaneously with a thrombolytic agent such as rt-PA.

[0232] 9. Subcutaneous administration of PSD-95 inhibitors

[0233] A series of animal studies were performed to demonstrate that a PSD-95 inhibitor containing a D-amino acid in addition to the five amino acids at the C-terminus is effective in a model of stroke and can be administered by subcutaneous injection. Figure 21 compares subcutaneous administration of three dose levels (2.6, 7.6 or 25 mg / kg) of nerinetide or NA-3 in a rat three-leptomeningeal vessel occlusion model of stroke. Treatment was administered subcutaneously by bolus injection 60 minutes after stroke onset. Rats administered NA-3 and nerinetide at a concentration of 25 mg / kg showed a significant reduction in infarct volume compared to placebo. NA-3 at 7.6 mg / kg also significantly reduced infarct volume, while nerinetide at the same concentration failed to achieve a reduction in infarct volume. Data are presented as mean ± SD (N=10 / group). Asterisks (*) represent statistical significance when compared to placebo (ANOVA with Tukey's post-hoc analysis, * P<0.0332, ** P<0.0021, *** P<0.0002 and **** P<0.0001). NA-3 was effective in this model, demonstrating that changing all amino acids except the C-terminal five amino acids (IESDV, SEQ ID NO:5) to D-amino acids was effective in stroke and PSD-95 inhibition. Furthermore, the increased stability may contribute to improved efficacy over subcutaneously administered Nerinetide. Equivalent neuroprotection (reduction in infarct volume) was observed between the 25 mg / kg dose of Nerinetide and the 7.6 mg / kg dose of NA-3, suggesting that a 3-fold lower dose of NA-3 is similarly effective.

[0234] Unlike the transient model of stroke in rats, where much lower doses were required for neuroprotection, plasma concentrations of nerinetide of 2 μg / mL (or molar equivalents) or greater appear to be required for neuroprotection in the three pial vessel occlusion model of stroke. Figure 22 shows plasma concentrations of NA-3 and nerinetide 15 minutes after dosing from animals from the previous model (Figure 21). Plasma concentrations continue to rise for approximately 3 hours and then decline, but for emergency indications such as stroke, rapid accumulation in the blood and brain is important. This indicates that subcutaneous administration of PSD-95 inhibitors with the structures described herein can achieve therapeutic concentrations in a rapid time frame. To analyze pharmacokinetic samples, calibration standard samples were prepared with concentrations of 0, 2.5, 5, 10, 15, 20, and 40 μg / mL of nerinetide by adding 1 μL of the appropriate stock to 100 μL of plasma. To generate an NA-3 standard curve for pharmacokinetic sample analysis, calibration standard samples were prepared at concentrations of 0, 2.5, 5, 10, 15, 20, and 40 ug / mL of NA-3 by spiking 1 μL of the appropriate stock into 100 μL of plasma. Blood samples were collected 15 min after subcutaneous administration of Nerinetide (25 mg / kg (N=6)), Nerinetide (7.6 mg / kg (N=8)), Nerinetide (2.5 mg / kg (N=4)), or NA-3 (25 mg / kg (N=7)), NA-3 (7.6 mg / kg (N=9)), NA-3 (2.5 mg / kg (N=9)), and placebo (N=8). Data are presented as mean ± SD. Figure 22 shows the dose proportionality between therapeutic dose and Cmax following a single subcutaneous administration of Nerinetide or NA-3. NA-3 exhibits greater plasma stability and higher concentrations 15 minutes after administration compared to the same dose of nerinetide.

[0235] To ensure that the plasma concentrations were comparable or greater than the effective concentrations known from human studies (plasma concentrations of approximately 10ug / mL at a dose of 2.6mg / kg), 25mg / kg or 7.6mg / Kg of NA-3 were administered by subcutaneous injection to non-human primates (cynomolgus monkeys) and plasma samples were tested at different time points (Figure 23). Both concentrations were also higher than those proven effective in humans and were able to achieve higher plasma concentrations than an intravenous dose of 2.6mg / kg of NA-1 (Hill, Lancet 2020). Figure 24 shows the pharmacokinetic profile of the injected levels tested. All values ​​are expressed as mean ± SD. Statistical significance compared to nerinetide alone is indicated by * (one-way ANOVA with post-hoc turkey's correction, *P<0.01) (C max : maximum plasma concentration based on the extrapolated time zero value; t 1 / 2 : Terminal half-life;t max : Time to reach Cmax; AUC 0-t : Area under the concentration-time curve from 0 to the last measurement; AUC 0-inf : area under the concentration-time curve extrapolated from 0 to infinity; Cl: total clearance). Data are expressed as mean ± SD of 3–4 animals per group. (-) indicates AUC 0-inf represents data not reported because AUC is greater than 20% and R2 is below 0.9. 0-inf The extrapolated value of exceeds 20%, and R 2 represents data not reported because σ is less than 0.9.

[0236] Physiological parameters of NHP animals following subcutaneous administration of NA-3 were examined and no significant safety issues were identified compared to intravenous nerinetide (Figure 25). No significant reduction in blood pressure was observed compared to NA-1, as observed at doses of NA-1 above 3.75 mg / kg in humans or at 7.6 mg / kg intravenous in rats or dogs, indicating an improvement.

[0237] Next, an MTD study was performed in rats administered NA-3 to assess whether there were any dose-limiting adverse events. Subcutaneous doses of 25 mg / kg or 50 mg / kg in rats showed dose-dependent increases in creatinine and BUN (blood urea nitrogen), indicating adverse effects on the kidney (Figure 26A,B). These increases were observed 1 day after administration in the 25 mg / kg dose group of NA-3 (left panel) and persisted until day 7. No out-of-range values ​​were observed in the 7.6 mg / kg or 2.6 mg / kg subcutaneous doses of NA-3. Thus, although NA-3 can be administered effectively to treat stroke, the therapeutic index (a measure of the difference between the minimum effective dose and the maximum level of side effects not observed) is low. It is advantageous for a therapeutic drug to have a large therapeutic index.

[0238] To increase the therapeutic index of plasmin-resistant PSD-95 inhibitors, nephrotoxicity was characterized and the impact of administering large amounts of D-amino acids was determined. Three strategies were identified to increase the therapeutic window: reducing the dose, reducing the number of D-amino acids in the inhibitor, and providing an inhibitor of D-amino acid oxidase with activity associated with nephrotoxicity at high concentrations. Figures 27 and 28 show the results of these treatment strategies on creatinine and BUN, comparing subcutaneous doses before and after 1 day. For the first strategy, the second and third sets of pre / post bars can be compared. Reducing the dose of NA-3 by a factor of two significantly reduces the level of NA-3 creatinine induced by 25 mg / kg NA-3. When reducing the number of D-amino acids in the second, the first set of bars (25 mg / kg NA-3) can be compared to the fourth set (25 mg / kg D-Tat-L-2B9c). When a peptide inhibitor containing a D residue at the 11 / 20 position is administered at the same dose level, much less creatinine is observed than with NA-3, which contains a D-amino acid at the 14 / 20 position. The figure shows that the addition of sodium benzoate, an inhibitor of D-amino acid oxidase, also reduces the creatinine and BUN levels induced by 25 mg / kg NA-3 (compare the pre / post pair of the first and second groups - 25 mg / kg NA-3 vs 25 mg / kg NA-3+SB). Thus, the therapeutic index of PSD-95 inhibitors can be enhanced by multiple strategies.

[0239] Since D-Tat-L-2B9c has been shown to be stable in the presence of plasmin, variants of the Tat internalization peptide containing 1-10 D-amino acids were generated. Representative internalization peptides are shown in the table above, and FIG. 29 shows the plasmin resistance of exemplary PSD-95 inhibitors containing the last 5 amino acids of L-type nerinetide and D-amino acid-containing variants of the tat transduction domain. Nerinetide itself is degraded within 5 minutes in the presence of 10 μg / mL plasmin, while the other variants are stable, with no degradation observed within 45 minutes. These variants have improved plasmin stability over nerinetide and improved therapeutic index compared to NA-3.

[0240] 9. Capacity range

[0241] Nerinetide and lodoxamide were administered intravenously to rats as a bolus injection 60 min after tMCAo. Figure 30A shows the measurement of hemispheric infarct volume 24 h after tMCAo. Bars in A represent the mean ± SD, and all separate data points are plotted. Asterisks in A indicate P<0.01 when compared to the vehicle group (one-way ANOVA post hoc Tukey correction for multiple comparison tests) (N=12-14 animals / group). Figure 30B shows the neurological scores 24 h after tMCAo. Significant differences are indicated by asterisks when compared to the vehicle group (Kruskal-Wallis analysis of variance on ranks with post hoc Dunn correction for multiple comparison tests, *P<0.01). Vehicle: PBS alone. Scramble: ADA peptide unable to bind to PSD-95. Doses as low as 0.25 mg / kg produced significant reductions in infarct volume (P=0.01) and improved neurological function. Doses up to at least 25 mg / kg were also effective, with the highest efficacy observed at approximately 15 mg / kg. The wide therapeutic range observed was due to nerinetide, but not to the mast cell degranulation inhibitor lodoxamide, which was present in all solutions to avoid potential hypotension due to histamine release.

[0242] 10. Plasmin Stability of Nerinetide Variants

[0243] The following peptides were synthesized and tested for plasmin sensitivity. Approximately 65 μg / ml of peptide in PBS was challenged with 10 μg / ml of plasmin at 37°C. Nerinetide was completely degraded in approximately 5 minutes as assessed by HPLC. Peptides that retained at least 85% intact peptide over 30 minutes were classified as plasmin resistant, while the other peptides were classified as plasmin sensitive. NoNO42, NA4.4.1, and NA411.4 showed nearly 100% intact peptide after 30 minutes, and NA4.4.3 showed 85% or greater intact peptide retention (Table 6 below). Despite literature reports that tryptophan inhibits plasmin cleavage when it is within 3-4 amino acids of a potential cleavage site, the R to W substitution at position 8 of nerinetide did not improve plasmin sensitivity compared to nerinetide.

[0244] [Table 6]

[0245] 11. Further testing of NoNO42

[0246] NoNO42, NA5.10, NA5.6, and NA5.1 were tested for their ability to compete with biotinylated nerinetide for binding to PSD-95 PDZ domain 2. ADA was a negative control peptide similar in sequence to nerinetide but unable to bind to PSD-95 domain 2. Each peptide, except the negative control, competed with biotinylated nerinetide in a dose-dependent manner as shown in FIG. 31. Symbols indicate the mean ± SD of duplicates. Absorbance data were normalized by subtracting A650nm from A450nm values, and then absorbance data were averaged for each time point in duplicate wells. Thus, the introduction of D-amino acids into the internalization peptide of NoNO42 does not affect binding to PSD-95 domain 2.

[0247] NoNO42 was compared to Nerinetide for Cmax administered intravenously. Rats were given a 10 minute IV infusion at doses of 7.6 mg / kg, 5.7 mg / kg, and 3.8 mg / kg. Plasma samples were collected pre-injection and 10, 15, 20, 30, 45, and 60 minutes after dosing. Each point represents the mean (±SD) of three animals per group. The C-max of NoNO42 was higher than Nerinetide (Figure 32).

[0248] Various doses of NoNO42 were compared with Nerinetide for the fall in blood pressure (due to mast cell degranulation) after administration to rats. The peptide was administered by intravenous infusion for 10 min. A fall in blood pressure was observed in rats treated with NoNO42 (7.6 mg / kg) (N=3) (Figure 33). Reducing the dose of NoNO42 to 5.7 mg / kg results in a similar fall in blood pressure as seen with Nerinetide 7.6 mg / kg. These doses of NONO42 and Nerinetide result in the same CMax. Thus, the hypotension of NoNO42 is less severe than that of Nerinetide at a dose that expresses a comparable CMax. Further reduction of the dose of NoNO42 to 3.8 mg / kg largely prevents the hypotension. Control: Nerinetide injection (N=3). Results are presented as % of baseline values ​​(mean ± SD).

[0249] NoNO42 was tested in comparison to nerinetide for its effect on various renal biomarkers or electrolyte imbalance after administration to rats, as shown in Table 7. No significant differences were observed despite the presence of four D-amino acids in NoNO42.

[0250] [Table 7]

[0251] NoNO42 was also compared to nerinetide for its effect on weight gain in rats. No significant differences were found (Figure 34). Normal weight gain indicates normal function and no toxicity due to the D-amino acid in NoNO42.

Claims

1. 1. An active agent comprising an internalization peptide or a variant thereof linked to an inhibitor peptide, The inhibitor peptide inhibits binding of PSD-95 to NOS and / or NMDAR2B; 1. An active agent wherein the internalization peptide has an amino acid sequence comprising RKKRRQRRR (SEQ ID NO: 13) and the inhibitor peptide has a sequence comprising ESDV (SEQ ID NO: 14) at its C-terminus, and the variant has up to a total of five substitutions or deletions in the sequences of the internalization peptide and the inhibitor peptide, wherein at least four C-terminal amino acids of the inhibitor peptide are L-amino acids and 3 to 5 residues of the internalization peptide are D-amino acids.

2. The active agent of claim 1, wherein the inhibitor peptide comprises an IE[S / T]DV (SEQ ID NO: 4) at the C-terminus, the IE[S / T]DV (SEQ ID NO: 4) being an L-amino acid.

3. 3. The active agent of claim 1 or 2, wherein each D residue is at a K or R or is C-terminal to a K or R residue.

4. 4. An active agent according to any one of claims 1 to 3, comprising at least two copies of the inhibitor peptide.

5. The internalization peptide is YGrKKRrQrRr (SEQ ID NO:91), YGrKKRrQrRR (SEQ ID NO:92), YGrKKRrQRrR (SEQ ID NO: 93), YGRKkRrQrrRV (SEQ ID NO:95), RKkrrQrrR (SEQ ID NO: 100), rKKRrQrRr (SEQ ID NO: 101), RKkRrQrrR (SEQ ID NO: 102), or rKKRrQrRR (SEQ ID NO: 103) having an amino acid sequence consisting of or including one of the following:

5. The active agent of any one of claims 1 to 4, wherein lower case letters indicate D-amino acids and upper case letters indicate L-amino acids.

6. or having an amino acid sequence consisting of or comprising YGRKKRRQRRRKLSSIESDV (SEQ ID NO:58), or a variant of said amino acid sequence, said variant having up to 5 substitutions, deletions or additions in said amino acid sequence, wherein 3-5 amino acids at positions other than the 5 C-terminal amino acids are D-amino acids; or 2. The active agent of claim 1, comprising an amino acid sequence having at least 75, 80, 85, 90 or 95% sequence identity to the sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO:58), wherein 3-5 amino acids at positions other than the 5 C-terminal amino acids are D-amino acids.

7. There are no D-amino acids occupying adjacent positions in the active agent; or each D-amino acid is separated from another D-amino acid by at least two amino acids independently selected from L-amino acids and glycine; or each D-amino acid is separated from another D-amino acid by at least three amino acids independently selected from L-amino acids and glycine; or The active agent of claim 6, comprising three or four D-amino acids.

8. lacking a stretch of four or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising two adjacent amino acids independently selected from R and K; or lacking a stretch of four or more consecutive amino acids selected from L-amino acids and glycine, said stretch comprising amino acids selected from R or K; or lacking a stretch of five or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising two adjacent amino acids independently selected from R and K, and if said stretch of four consecutive amino acids is present, said stretch is located at the N-terminus of said active agent; or 8. The active agent of claim 6 or 7, lacking a stretch of five or more consecutive amino acids independently selected from L-amino acids and glycine, said stretch comprising amino acids selected from R or K, and wherein said stretch of four consecutive amino acids, if present, is located at the N-terminus of the internalization peptide.

9. 9. The active agent of any one of claims 6 to 8, comprising three or four D-amino acids: a D-amino acid between positions 3 and 5, a D-amino acid at positions 7 or 8, a D-amino acid between positions 9 and 11, and optionally a D-amino acid at positions 12 or 13.

10. 10. The active agent of any one of claims 6 to 9, wherein the amino acid sequence has zero substitutions, deletions or additions, except for the D to L substitution, relative to the amino acid sequence: YGRKKRRQRRRKLSSIESDV (SEQ ID NO:58).

11. 11. The active agent of any one of claims 1 to 10, having an amino acid sequence consisting of or including: YGrKKRrQrRRkLSSIESDV (SEQ ID NO: 114), YGRkKRrQRrRKLSSIESDV (SEQ ID NO: 115), YGrKKRrQrRRKlSSIESDV (SEQ ID NO: 116), RKkRrQrrRIESDV (SEQ ID NO: 111), or rKKRrArRRIESDV (SEQ ID NO: 112).

12. has improved plasmin resistance and / or improved plasma half-life compared to nerinetide, or competes with nerinetide for binding to PSD-95; or and / or an active agent comprising an internalization peptide of sequence ygrkkrrqrrr (SEQ ID NO:96) linked to an inhibitor peptide of sequence KLSSIESDV (SEQ ID NO:2), wherein each amino acid of the internalization peptide is a D-amino acid and each amino acid of the inhibitor peptide is an L-amino acid; and / or and / or an internalization peptide linked to klssIESDV (SEQ ID NO: 117) (lower case letters indicate D-amino acids and upper case letters indicate L-amino acids); and / or An active agent having the amino acid sequence vdseisslkrrrqrrkkrgy (SEQ ID NO:118) (lower case letters indicate D-amino acids); or exhibiting reduced nephrotoxicity and / or a higher therapeutic index compared to a binding affinity for PSD-95 that is within 2-fold of nerinetide and / or an IC50 for inhibiting the binding of PSD-95 to NMDAR2B that is within 2-fold of nerinetide; or 12. The active agent of any one of claims 1 to 11, wherein the AUC or CMax is higher than that of nerinetide, and optionally, the AUC or CMax is for subcutaneous administration.

13. 13. An active agent according to any one of claims 1 to 12, which is a chloride salt.

14. 14. The active agent formulation of any one of claims 1 to 13, further comprising histidine, trehalose, and optionally sodium benzoate.

15. 14. The active agent formulation of any one of claims 1 to 13, further comprising a phosphate buffer, and optionally sodium benzoate.

16. 14. A co-formulation comprising a formulation of an active agent according to any one of claims 1 to 13, an anti-inflammatory drug, and optionally a mast cell degranulation inhibitor or an antihistamine.

17. 14. A co-formulation comprising a formulation of an active agent according to any one of claims 1 to 13 and a thrombolytic agent.

18. A co-formulation comprising a formulation of an active agent according to any one of claims 1 to 13 and an inhibitor of D-amino acid oxidase, optionally risperidone, sodium benzoate or 5-chloro-benzo[d]isoxazol-3-ol.

19. 1. A pharmaceutical composition for treating stroke, cerebral ischemia, CNS traumatic injury, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy, or a neurodegenerative disease, comprising:

19. A pharmaceutical composition comprising an active agent according to any one of claims 1 to 13 or a formulation according to any one of claims 14 to 18.

20. 20. The pharmaceutical composition for treating stroke, cerebral ischemia, CNS traumatic injury, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy, or neurodegenerative disorders according to claim 19, optionally in combination with reperfusion by thrombolytic agents, mechanical reperfusion, or endovascular thrombectomy.

21. The pharmaceutical composition for treating stroke, cerebral ischemia, CNS traumatic injury, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy, or neurodegenerative disease according to claim 19, in combination with a D-amino acid oxidase activity inhibitor.

Images