Neuroprotective PSD-95 polypeptide inhibitor and use thereof

Abstract

Provided are a neuroprotective PSD-95 polypeptide inhibitor and the use thereof. Specifically, provided are a series of active peptides that can bind to a PDZ1 domain or PDZ2 domain of PSD-95, and a pharmaceutically acceptable salt and pharmaceutical composition thereof. The active peptide has a structure as represented by formula (I), (I), and can be used as an inhibitor of protein-protein interaction mediated by PSD-95 for the treatment of stroke and nervous system related diseases.

Description

A neuroprotective PSD-95 polypeptide inhibitor and its application

[0001] This application claims priority to Chinese patent application CN202310027484.8 filed on January 9, 2023 and Chinese patent application CN202311125942.8 filed on September 1, 2023. Technical Field

[0002] The present disclosure belongs to the field of biomedicine, and specifically relates to an active peptide capable of binding to the PDZ1 domain or PDZ2 domain of PSD-95, which can be used as an inhibitor of PSD-95-mediated protein-protein interaction for the treatment of stroke and nervous system-related diseases. Background Art

[0003] Postsynaptic density protein-95 (PSD-95) is an important scaffold protein located in the postsynaptic membrane of the central nervous system. It consists of three repeating PDZ domains at the N-terminus: PDZ1, PDZ2, and PDZ3; an intermediate SH3 domain; and a C-terminal guanylate kinase domain. PDZ domains are common protein-protein interaction domains. PSD-95 binds to the tSXV-COOH of NMDARs (N-methyl-D-aspartate receptors) through PDZ1 and PDZ2, and to the PDZ of nNOS (neuronal nitric oxide synthase) through PDZ2 (Acta Pharmacol. Sin., 2018, 39:661–668).

[0004] After ischemic stroke, local cerebral blood flow obstruction causes glutamate to accumulate in the extracellular space. Excessive release of excitatory glutamate continuously acts on glutamate receptors, leading to neuronal depolarization and calcium influx. Overloaded Ca 2+ Activation of nNOS by calmodulin leads to abnormal increase in nitric oxide (NO) production, ultimately causing neuronal damage.

[0005] PSD-95 inhibitors effectively interfere with the intracellular interaction between NMDA receptors and PSD-95. Disrupting the GluN2B-PSD95-nNOS complex can inhibit NMDA-mediated NO production and protect neurons. Nerinetide (also known as TAT-NR2B9c or NA-1) is a PSD-95 inhibitor developed by NoNO Inc. of Canada (Science, 2002, 298:846-50). It is a neuroexcitotoxicity inhibitor. Nerinetide is composed of 20 amino acid residues. TAT is derived from the human (HIV-1) transcriptional transactivator and can cross multiple cell membranes. NR2B9c is selected from the nine amino acids at the C-terminus of the NR2B subunit.

[0006] In preclinical animal models (rats and cynomolgus monkeys), administration of nerinetide after ischemic stroke significantly reduced infarct size and improved neurobehavioral function (Sci. Transl. Med., 2021, 13, eabb1498). Results from a Phase II clinical study in patients undergoing endovascular repair of cerebral aneurysms (ENACT) and a Phase III clinical study in patients with severe acute ischemic stroke (AIS) undergoing endovascular thrombectomy (EVT) showed no serious drug-related adverse events were observed, and the safety profile of nerinetide was comparable to that of placebo.

[0007] Current research on PSD-95 inhibitors includes WO2015078477A, ​​WO2022150655A, etc. In order to meet clinical needs, it is still necessary to develop PSD-95 inhibitors with high stability and good pharmacokinetic properties to meet the treatment of diseases such as ischemic stroke.

[0008] Summary of the Invention

[0009] The present disclosure provides a compound represented by formula (G) or a pharmaceutically acceptable salt thereof,

[0010] Said X1 is selected from Gly or is missing;

[0011] X2 is selected from Aib, Ala, Ile, Cha, Phe, Trp, 1-Nal, Ser, Lys, Arg, Nle, Nva, Orn, cyclopropylAla, 4-thiazolylAla, homoLeu or missing;

[0012] X3 is selected from Dap, Dab, Thr, Aib, Ala, Cha, Phe, Trp, 1-Nal, Asn, Glu, Lys, Arg, 1Me-Trp, 2-Nal, Gln, Thr, 4-thiazolylAla or missing;

[0013] Said X4 is selected from Thr, homoArg, Ser, Phe or deletion;

[0014] X5 is selected from Tle, homoArg, Arg, Lys, 4-thiazolylAla, Chg, Cha, homoPhe, 4F-Phe, 3F-Phe, 2F-Phe, 4-Pal, 3-Pal, 2-Pal, Cit, Orn or Phe;

[0015] X6 is selected from Tle, Ile, Phe, Trp, Leu, I-Nal, Nle, D-Nle, D-Trp or D-Phe;

[0016] Said X7 is selected from Thr or Ser;

[0017] X8 is Asp, Cha, Leu, Trp, 1-Nal, Phe, Val, Ala, Abu, Ser or Thr;

[0018] The X9 is Val.

[0019] Another aspect of the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0020] Said X1 is selected from Gly or is missing;

[0021] Said X1 is selected from Gly or is missing;

[0022] X2 is selected from Aib, Ala, Ile, Cha, Phe, Trp, 1-Nal, Ser, Lys, Arg, Nle, Nva, Orn, cyclopropylAla, 4-thiazolylAla, homoLeu or missing;

[0023] X3 is selected from Dap, Dab, Thr, Aib, Ala, Cha, Phe, Trp, 1-Nal, Asn, Glu, Lys, Arg, 1Me-Trp, 2-Nal, Gln, Thr, 4-thiazolylAla or missing;

[0024] Said X4 is selected from Thr, homoArg, Ser, Phe or deletion;

[0025] X5 is selected from Tle, homoArg, Arg, Lys, 4-thiazolylAla, Chg, Cha, homoPhe, 4F-Phe, 3F-Phe, 2F-Phe, 4-Pal, 3-Pal, 2-Pal, Cit, Orn or Phe;

[0026] X6 is selected from Tle, Ile, Phe, Trp, Leu, I-Nal, Nle, D-Nle, D-Trp or D-Phe;

[0027] Said X7 is selected from Thr or Ser;

[0028] X8 is Asp, Cha, Leu, Trp, 1-Nal, Phe, Val, Ala, Abu, Ser or Thr;

[0029] X9 is Val;

[0030] The L1 is a chemical bond or contains polyethylene glycol, one or two oxygen atoms of the polyethylene glycol are optionally replaced by nitrogen atoms, and the CPP is an internalization peptide.

[0031] In an optional embodiment, the present disclosure provides a compound represented by formula (G), formula (I) or a pharmaceutically acceptable salt thereof,

[0032] Said X1 is selected from Gly or is missing;

[0033] X2 is selected from Aib, Ala, Ile, Cha, Phe, Trp, 1-Nal, Ser, Lys, Arg, Nle, Nva, Orn, cyclopropylAla, 4-thiazolylAla, homoLeu or missing;

[0034] X3 is selected from Dap, Dab, Thr, Aib, Ala, Cha, Phe, Trp, 1-Nal, Asn, Glu, Lys, Arg, 1Me-Trp, 2-Nal, Gln, Thr, 4-thiazolylalanine or deletion;

[0035] Said X4 is selected from Thr, homoArg, Ser or deletion;

[0036] Said X5 is selected from Tle, homoArg, Chg;

[0037] Said X6 is selected from Tle or Ile;

[0038] Said X7 is selected from Thr or Ser;

[0039] X8 is Asp;

[0040] The X9 is Val.

[0041] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X7 is Thr.

[0042] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X6 is Tle.

[0043] In some embodiments, the present disclosure provides a compound represented by Formula (G) or Formula (I) or a pharmaceutically acceptable salt thereof, wherein X5 is Tle.

[0044] In some embodiments, the present disclosure provides a compound represented by formula (G), formula (I) or a pharmaceutically acceptable salt thereof, wherein X6-X7 is selected from Tle-Thr, Ile-Thr or Tle-Ser.

[0045] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X6-X7 is selected from Tle-Thr or Ile-Thr.

[0046] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X6-X7 is Tle-Thr.

[0047] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X5-X6 is selected from Tle-Tle, homoArg-Ile or Chg-Tle.

[0048] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X5-X6 is selected from Tle-Tle and Chg-Tle.

[0049] In some embodiments, the present disclosure provides a compound represented by Formula (G) or Formula (I) or a pharmaceutically acceptable salt thereof, wherein X5-X6 is Tle-Tle.

[0050] In some embodiments, the present disclosure provides a compound represented by formula (G), formula (I) or a pharmaceutically acceptable salt thereof, wherein X5-X6-X7 is Tle-Tle-Thr.

[0051] In some embodiments, the present disclosure provides compounds of formula (G) or formula (I) or pharmaceutically acceptable salts thereof, wherein one or more of X1, X2, X3, and X4 are optionally missing.

[0052] In some embodiments, the present disclosure provides a compound represented by Formula (G) or Formula (I) or a pharmaceutically acceptable salt thereof, wherein X1 is missing.

[0053] In some embodiments, the present disclosure provides a compound represented by Formula (G) or Formula (I) or a pharmaceutically acceptable salt thereof, wherein X2 and / or X3 are missing.

[0054] In some embodiments, the present disclosure provides compounds of formula (G) or formula (I) or pharmaceutically acceptable salts thereof, wherein X1, X2, X3, and X4 are all missing.

[0055] In some embodiments, the present disclosure provides a compound represented by formula (G), formula (I) or a pharmaceutically acceptable salt thereof, wherein X4 is selected from Thr, homoArg or Ser.

[0056] In some embodiments, the present disclosure provides a compound represented by formula (G), formula (I) or a pharmaceutically acceptable salt thereof, wherein X4 is selected from Thr or Ser.

[0057] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X4 is Thr.

[0058] In some embodiments, the present disclosure provides a compound represented by Formula (G) or Formula (I) or a pharmaceutically acceptable salt thereof, wherein X3 is selected from Dap or Dab.

[0059] In some embodiments, the present disclosure provides a compound represented by Formula (G) or Formula (I) or a pharmaceutically acceptable salt thereof, wherein X3 is Dab.

[0060] In some embodiments, the present disclosure provides a compound represented by Formula (G) or Formula (I) or a pharmaceutically acceptable salt thereof, wherein X2 is selected from Aib, Ala or Ile.

[0061] In some embodiments, the present disclosure provides a compound represented by Formula (G) or Formula (I) or a pharmaceutically acceptable salt thereof, wherein X2 is Aib.

[0062] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X1 is selected from Nle, Gly or deletion.

[0063] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X1 is Gly.

[0064] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein L1 is a chemical bond.

[0065] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein L1 comprises polyethylene glycol, and one or two oxygen atoms of the polyethylene glycol are optionally replaced by a nitrogen atom.

[0066] In an optional embodiment, L1 comprises polyethylene glycol, and the number of polyethylene glycols is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28.

[0067] In some embodiments, the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein L1 is AEEA.

[0068] In some embodiments, the present disclosure provides a compound represented by formula (G) or formula (I) or a pharmaceutically acceptable salt thereof, wherein X1-X2-X3-X4-X5-X6-X7-X8-X9 is selected from SEQ ID NO.1 to SEQ ID NO.8:

[0069] In some embodiments, the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide is derived from HIV virus.

[0070] In some embodiments, the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide has a structure as described in WO2022150655A, WO2010072406A, WO2010072405A, or WO2021140485A.

[0071] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide has an amino acid sequence comprising or selected from SEQ ID NO: 9 to SEQ ID NO.11, and the amino acid residues in SEQ ID NO: 9 to SEQ ID NO.11 are optionally D amino acids: SEQ ID NO.9 RKKRRQRRR SEQ ID NO.10 GRKKRRQRRR SEQ ID NO.11 YGRKKRRQRRR.

[0072] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide has an amino acid sequence comprising or selected from SEQ ID NO.11, and the amino acid residues in SEQ ID NO.11 are optionally D amino acids.

[0073] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide has an amino acid sequence comprising or selected from SEQ ID NO.9, and the amino acid residues in SEQ ID NO.9 are optionally D amino acids.

[0074] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residues of the internalization peptide are D-amino acids.

[0075] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residues of the internalization peptide are D-amino acids.

[0076] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 9, 10 or 11 of the amino acid residues of the internalization peptide are D-amino acids.

[0077] In an optional embodiment, the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the amino acid residue R of the internalization peptide is a D-amino acid.

[0078] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 1, 2, 3, 4, 5 or 6 of the amino acid residues R are D-amino acids.

[0079] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 3, 4, 5 or 6 of the amino acid residues R are D-amino acids.

[0080] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 5 or 6 of the amino acid residues R are D-amino acids.

[0081] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the first amino acid residue R of the internalization peptide starting from the C-terminus is a D-amino acid, and the amino acid residues optionally separated by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are D-amino acids.

[0082] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the first amino acid residue R of the internalization peptide starting from the C-terminus is a D-amino acid, and the amino acid residues optionally separated by 0, 1, 2, 3, 4, 5, 6, 7 or 8 amino acids are D-amino acids.

[0083] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the first amino acid residue R of the internalization peptide starting from the C-terminus is a D-amino acid, and the amino acid residues optionally separated by 0, 1, 2 or 3 amino acids are D-amino acids.

[0084] In an optional embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide has an amino acid sequence comprising or selected from SEQ ID NO: 12 or SEQ ID NO: 13: SEQ ID NO.12 yGrkkrrqrrr SEQ ID NO.13 rKKRrQRRr.

[0085] Note: Lowercase single letters represent D-amino acids.

[0086] In an optional embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof provided by the present disclosure is selected from SEQ ID NO.14 to SEQ ID NO.22: Note: Lowercase single letters represent D-amino acids.

[0087] Another aspect of the present disclosure provides an active peptide or a pharmaceutically acceptable salt thereof, wherein the active peptide comprises or consists of formula (G),

[0088] Said X1, X2, X3, X4, X5, X6, X7, X8 and X9 are respectively defined as the compound shown in formula (I).

[0089] In some embodiments, the number of amino acid residues in the active peptide is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0090] In some embodiments, the active peptide comprises or is selected from the amino acid sequence shown in SEQ ID NO.1 to SEQ ID NO.8.

[0091] Another aspect of the present disclosure provides a peptide comprising an active peptide and an internalization peptide, wherein the active peptide comprises or is selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 8, and the internalization peptide is as defined herein. In some embodiments, the internalization peptide comprises or is selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 13.

[0092] In some embodiments, the peptide comprises or is selected from the group consisting of the amino acid sequences shown in SEQ ID NO. 14 to SEQ ID NO. 22.

[0093] Another aspect of the present disclosure provides an active peptide comprising or selected from the group consisting of the following SEQ ID NO.23 to SEQ ID NO.39:

[0094] In some embodiments, the active peptide provided by the present disclosure is selected from SEQ ID NO. 23 to SEQ ID NO. 39 and is linked to an internalization peptide.

[0095] In an optional embodiment, the internalization peptide has a structure as described in WO2022150655A, WO2010072406A, WO2010072405A, or WO2021140485A.

[0096] In an optional embodiment, the internalization peptide has an amino acid sequence comprising or selected from any one of SEQ ID NO: 9 to SEQ ID NO. 11, and the amino acid residues in SEQ ID NO: 9 to SEQ ID NO. 11 are optionally D amino acids, SEQ ID NO. 9RKKRRQRRR SEQ ID NO. 10GRKKRRQRRR SEQ ID NO. 11YGRKKRRQRRR.

[0097] In an optional embodiment, the active peptides shown in SEQ ID NO. 23 to SEQ ID NO. 39 and the internalization peptide are optionally linked via L2, wherein L2 comprises polyethylene glycol, and one or two oxygen atoms of the polyethylene glycol are optionally replaced by nitrogen atoms.

[0098] In an optional embodiment, L2 comprises polyethylene glycol, and the number of polyethylene glycols is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28.

[0099] In some embodiments, the L2 is AEEA.

[0100] In an optional embodiment, the active peptides shown in SEQ ID NO.23 to SEQ ID NO.39 and the internalization peptide form the peptides shown in SEQ ID NO.40 to SEQ ID NO.61 as follows: Note: Lowercase single letters represent D-amino acids.

[0101] In some embodiments, the peptide disclosed herein comprises or is selected from the group consisting of the amino acid sequences shown in SEQ ID NO. 40 to SEQ ID NO. 61.

[0102] The active peptides of the present disclosure (in the absence of an internalization peptide) have a length of 3-25 amino acids, 4-15 amino acids, 4-10 amino acids, such as 9 amino acids, such as 3, 4, 5, 6, 7, 8, 9 amino acids.

[0103] The compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof provided by the present disclosure is an active peptide capable of binding to the PDZ1 domain or PDZ2 domain of PSD-95.

[0104] The compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof provided by the present disclosure can be used as an inhibitor of protein-protein interactions mediated by PSD-95.

[0105] On the other hand, the present disclosure also provides a pharmaceutical composition containing a therapeutically effective amount of the aforementioned compound or a pharmaceutically acceptable salt thereof, and / or the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0106] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0107] In certain embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01-99.99% of the aforementioned compound or its pharmaceutically acceptable salt or the aforementioned active peptide or peptide or its pharmaceutically acceptable salt. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned compound or its pharmaceutically acceptable salt or the aforementioned active peptide or peptide or its pharmaceutically acceptable salt. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound or its pharmaceutically acceptable salt or the aforementioned active peptide or peptide or its pharmaceutically acceptable salt. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound or its pharmaceutically acceptable salt or the aforementioned active peptide or peptide or its pharmaceutically acceptable salt. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound or its pharmaceutically acceptable salt or the aforementioned active peptide or peptide or its pharmaceutically acceptable salt.

[0108] In certain embodiments, the pharmaceutical composition comprises 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition comprises 0.1% to 99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 1% to 99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 2% to 98% of a pharmaceutically acceptable excipient.

[0109] The present disclosure provides a compound represented by the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof as a drug.

[0110] The present disclosure provides a compound represented by the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition, which can improve brain neural function and reduce cerebral infarction area.

[0111] The present disclosure provides a compound represented by the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition, which can reduce the ability to release histamine.

[0112] The present disclosure provides a compound represented by the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition, which can increase plasma exposure.

[0113] The present invention provides a method for extending T 1 / 2 The compound represented by the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof or the aforementioned pharmaceutical composition.

[0114] The present disclosure provides a compound represented by formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition with high plasma stability.

[0115] On the other hand, the present disclosure provides the use of the compound represented by the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition in the preparation of a medicament, wherein the medicament is used to treat and / or prevent stroke, cerebral ischemia, traumatic injury to the central nervous system, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy or neurodegenerative diseases (Alzheimer's disease or Parkinson's disease) or diseases with the above-mentioned risks.

[0116] On the other hand, the present disclosure provides a method for treating and / or preventing stroke, cerebral ischemia, traumatic injury to the central nervous system, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy or neurodegenerative diseases (Alzheimer's disease or Parkinson's disease) or diseases with the above-mentioned risks, by administering to the patient a therapeutically effective amount or a prophylactically effective amount of the compound represented by the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

[0117] On the other hand, the present disclosure provides a compound shown in the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, a compound shown in formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition combined with a thrombolytic drug for the preparation of a medicament for treating and / or preventing acute ischemic stroke (AIS). On the other hand, the present disclosure provides a method for treating and / or preventing acute ischemic stroke (AIS), administering to a patient a therapeutically effective amount or a prophylactic effective amount of a compound shown in the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, a compound shown in formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition and thrombolytic drug.

[0118] On the other hand, the present disclosure provides a compound shown in the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, a compound shown in formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition for treating and / or preventing acute ischemic stroke (AIS), the compound shown in the aforementioned formula (G) or a pharmaceutically acceptable salt thereof, the compound shown in formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition in combination with a thrombolytic drug. Two different active ingredients are in the same or different containers.

[0119] Another aspect of the present disclosure provides a thrombolytic drug for treating and / or preventing acute ischemic stroke (AIS), wherein the thrombolytic drug is used in combination with the compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition. The two different active ingredients are in the same or different containers.

[0120] The thrombolytic drug described in the present disclosure is selected from urokinase, streptokinase, anistreplase, staphylokinase, recombinant staphylokinase, prourokinase, vampire bat salivary plasminogen activator, ranteplase, pamiplase, monteplase, alteplase, reteplase, tenecteplase, etc.

[0121] In an optional embodiment, the thrombolytic drug disclosed herein is alteplase (rt-PA).

[0122] Another aspect of the present disclosure provides a method for treating central nervous system damage caused by ischemia, comprising administering a therapeutically effective amount of a compound represented by formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the aforementioned active peptide or peptide or a pharmaceutically acceptable salt thereof, or the aforementioned combination to a patient suffering from or at risk of suffering from ischemia, and performing reperfusion therapy on the patient. For explanations of "ischemia," "reperfusion," and "reperfusion therapy" in this disclosure, reference may be made to WO2012176172A.

[0123] In the polypeptide compound sequences provided herein, lowercase single letters represent D-amino acids. Unless otherwise specified, uppercase single letters represent L-amino acids (wherein Gly and Aib have no stereo configuration), for example, RKKRRQRRR (SEQ ID NO. 9), when it is specified that the amino acid residues in SEQ ID NO: 9 are optionally D amino acids, then the amino acids are optionally D-amino acids.

[0124] The compounds and derivatives provided by the present invention are synthesized by a solid-phase method, and the synthesis support is Fmoc-Val-Wang resin. The α-amino group of the amino acid derivatives used in the synthesis process is protected by an Fmoc group (fluorenylcarbonyl), and the side chains of the amino acids are selected from the following protecting groups according to different functional groups: the side chain amino group of glutamine (L / D) is protected by Trt (trityl), the side chain guanidine group of homoarginine (homoArg) and arginine (L / D) is protected by Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), the side chain indole group of tryptophan, the side chain amino group of lysine (L / D), Dab and Orn are protected by Boc (tert-butyloxycarbonyl), and the side chain hydroxyl group of threonine, the side chain phenol group of tyrosine (L / D), the side chain hydroxyl group of serine, the side chain carboxyl group of aspartic acid and the side chain carboxyl group of glutamic acid are protected by t-Bu (tert-butyl). The synthesis process involves first fully swelling Fmoc-Val-Wang resin in N,N-dimethylformamide (DMF). The Fmoc protecting group on the α-amino group is removed using a DMF solution containing 20% ​​4-methylpiperidine. The carboxyl group of the C-terminal amino acid residue is then condensed to the high-molecular-weight, insoluble resin via an amide bond. The Fmoc protecting group on the α-amino group is then removed using a DMF solution containing 20% ​​4-methylpiperidine. The solid support is then condensed with the next amino acid derivative in the sequence in excess to form an amide bond, extending the peptide chain. The washing, deprotection, washing, next amino acid condensation, and washing steps are repeated to achieve the desired peptide chain length. Finally, the peptide is cleaved from the solid support by reacting with a mixture of trifluoroacetic acid, water, and triisopropylsilane (90:5:5, v:v:v). The solid is then precipitated with chilled methyl tert-butyl ether, centrifuged, and the supernatant removed to yield the crude peptide, which is then dried overnight. The crude polypeptide solid product is dissolved in deionized water and then purified and separated using a C-18 reverse phase preparative chromatography column to obtain pure polypeptide and its derivatives.

[0125] Detailed Description of the Invention

[0126] "Internalization peptides" are a class of relatively short peptides known to enable many cellular or viral proteins to cross the cell membrane. Internalization peptides, also known as cell membrane transduction peptides or cell penetrating peptides, have, for example, 5-30 amino acids. These peptides typically have a cationic charge derived from arginine and / or lysine residues, which are believed to facilitate their transmembrane transport (usually relative to proteins). Some such polypeptides have at least 5, 6, 7, or 8 arginine and / or lysine residues.

[0127] “Natural amino acids” refer to the 20 common amino acids (i.e., alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine (Y).

[0128] "Non-natural amino acids" refer to amino acids that are not naturally encoded or found in the genetic code of any organism. They can be purely synthetic compounds. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamate, O-phosphoserine, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid (Dap), tert-leucine (Tle), desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid (Dab), N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline. The non-natural amino acids include amino acids, such as d-aminobutyric acid, d-hydroxylysine, d-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine (Orn), D-ornithine, D-arginine, homoarginine (homoArg), D-tyrosine, D-lysine, D-glutamine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline. In addition, the non-natural amino acids include natural or non-natural amino acids wherein the C-terminal carboxyl group, the N-terminal amino group, and / or their side chain functional groups are chemically modified.

[0129] The corresponding relationships between some amino acid abbreviations and structural abbreviations in this disclosure are as follows:

[0130] The term "AEEA" is structured as follows:

[0131] "Optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "C1-6 alkyl optionally substituted with halogen or cyano" means that halogen or cyano may but need not be present, and the description includes instances where the alkyl is substituted with halogen or cyano and instances where the alkyl is not substituted with halogen or cyano.

[0132] In the chemical structures of the compounds disclosed herein, unless otherwise indicated, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or or include both and Although all of the above structural formulae are drawn in certain isomeric forms for simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0133] The term "subject" or "patient" includes humans and veterinary animals, such as mammals, as well as laboratory animal models, such as mice or rats used in preclinical studies.

[0134] "Ac-y" in the present disclosure means that the amino group of the amino acid y is acetylated. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1A. Rat whole blood hemolysis risk test;

[0136] Figure 1B. Human whole blood hemolysis risk test;

[0137] Figure 1C. Rabbit whole blood hemolysis risk test;

[0138] Figure 2A. Human plasma stability test of peptide compounds;

[0139] Figure 2B. Rat plasma stability test of polypeptide compounds;

[0140] Figure 2C. Human plasma stability test of polypeptide compounds;

[0141] Figure 2D. Rat plasma stability test of polypeptide compounds;

[0142] Figure 3. Pharmacokinetic results of peptide compounds in rats;

[0143] Figure 4. Pharmacokinetic results of peptide compound 14 at different doses;

[0144] Figure 5A. Nephrotoxicity results of peptide compounds in rats - serum urea nitrogen level, ns indicates no statistical difference, * indicates P < 0.05, ** indicates P < 0.01;

[0145] Figure 5B. Nephrotoxicity results of peptide compounds in rats - serum creatinine levels, ns indicates no statistical difference;

[0146] Figure 6. Pharmacokinetic results of peptide compounds in beagle dogs;

[0147] Figure 7. Ability of polypeptide compounds to induce histamine release in beagle dogs;

[0148] Figure 8A. Pharmacological efficacy of polypeptide compound 14 in rat tMCAO model - cerebral infarction area, ** indicates P < 0.01, *** indicates P < 0.001;

[0149] Figure 8B. Pharmacological efficacy of polypeptide compound 14 in rat tMCAO model - neurological function injury score, * indicates P < 0.05, ** indicates P < 0.01;

[0150] Figure 8C. Pharmacological efficacy of polypeptide compound 22 in rat tMCAO model - cerebral infarction area, * indicates P < 0.05, ** indicates P < 0.01;

[0151] Figure 8D. Pharmacological efficacy of polypeptide compound 22 in rat tMCAO model - neurological function injury score, * indicates P < 0.05, ** indicates P < 0.01. DETAILED DESCRIPTION

[0152] In order to illustrate the present disclosure in more detail, this specification provides the following specific embodiments, but the embodiments of the present disclosure are not limited thereto.

[0153] 1. Experimental Reagents

[0154] Table 1-1

[0155] 2. Experimental Instruments

[0156] Table 1-2

[0157] 3. Specific experimental plan

[0158] 3.1 Chemical synthesis of polypeptide compound 14

[0159] yGrkkrrqrrrG-Aib-Dab-T-Tle-Tle-TDV(SEQ ID NO.14)

[0160] 3.1.1 Resin swelling and removal of Fmoc protecting groups

[0161] Weigh the solid-phase synthesis support Fmoc-Val-Wang resin (286 mg, 0.1 mmol, degree of substitution: 0.349 mmol / g) into a disposable polypropylene peptide synthesis solid-phase reaction tube. Add DMF (10 mL) to swell the resin for 10 minutes. Vacuum the DMF and wash the resin with DMF (5 mL). Repeat washing twice before draining. Add 4-methylpiperidine / DMF (20% v / v, 5 mL) to the resin, shake at room temperature for 8 minutes, then drain. Add 4-methylpiperidine / DMF (20% v / v, 5 mL), shake at room temperature for 8 minutes, then remove the solution. After deprotection, wash the resin four times with DMF (5 mL).

[0162] 3.1.2 Coupling of peptide chain sequences

[0163] According to the peptide chain sequence of polypeptide compound 14, the synthesis was carried out in the order from the carboxyl end to the amino end. First, Fmoc-Asp(OtBu)-OH (1 mmol) was weighed and dissolved in DMF to prepare a 0.34 M solution. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1 mmol) was weighed and dissolved in DMF to prepare a 0.34 M solution. 4-Methylmorpholine (NMM, 2 mmol) was measured and dissolved in DMF to prepare a 1 M solution. 3 mL of Fmoc-Asp(OtBu)-OH solution, 3 mL of HATU solution and 1.5 mL of 4-methylmorpholine solution were mixed and added to the resin obtained in step 3.1.1. The mixture was shaken at room temperature for 35 minutes. After the reaction, it was washed with DMF 3 times.

[0164] Add 4-methylpiperidine / DMF (20% v / v, 5 mL) to the resin to remove the Fmoc protecting group from the amino acid N-terminus. Shake the mixture at room temperature for 8 minutes, remove the solvent, and then add 4-methylpiperidine / DMF (20% v / v, 5 mL). Shake the mixture at room temperature for 8 minutes, then remove the solvent. After the reaction, wash the resin three times with DMF (5 mL).

[0165] Repeat the above condensation process of amino acid derivatives, and condense in sequence: Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Aib-OH, Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pb f)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH, and finally a complete resin peptide containing polypeptide compound 14 was obtained.

[0166] 3.1.3 Cleavage of on-resin peptides

[0167] The resin peptide containing polypeptide compound 14 obtained in the above steps was washed three times with DMF and DCM, followed by vacuum drying. 8 mL of freshly prepared lysis buffer (trifluoroacetic acid: triisopropylsilane: water = 90:5:5, v:v:v) was then added and shaken for 6 hours at room temperature. After completion of the reaction, the resin was filtered and washed twice with trifluoroacetic acid. The filtrates were combined and a large amount of chilled methyl tert-butyl ether was added to precipitate the solid. After centrifugation, the supernatant was removed to obtain the crude polypeptide, which was then dried overnight.

[0168] 3.1.4 Reverse-phase liquid chromatography purification of crude peptide

[0169] The crude peptide was purified twice: first in a TFA system and then in a 25mM ammonium bicarbonate (pH 8.0) system. TFA system: The presence of multiple basic amino acids in the target molecule makes it more polar. The crude peptide was dissolved in water and sonicated until completely dissolved. It was then filtered through a 0.22μm membrane and separated using a WATERS Prep150 HPLC system with mobile phases A (0.1% trifluoroacetic acid, 10% acetonitrile / water, v / v) and B (0.1% trifluoroacetic acid, 90% acetonitrile / water, v / v). The chromatographic column was an X-SELECT OBD C-18 (WATERS, 10μm, 19×250mm) reversed-phase column. During the purification process, the chromatograph detection wavelength was set at 220nm and the flow rate was 15mL / min. The product-related fractions were collected, lyophilized, and then purified again using a 25mM ammonium bicarbonate (pH 8.0) system. The product obtained after the first purification step was dissolved in 25mM ammonium bicarbonate buffer and loaded onto the column; the ammonium bicarbonate system: mobile phase A (25mM ammonium bicarbonate aqueous solution) and B (100% acetonitrile). The chromatographic column was an X-SELECT OBD C-18 (WATERS, 5μm, 19×250mm) reversed-phase column. During the purification process, the chromatograph detection wavelength was set at 220nm and the flow rate was 10mL / min. The relevant fractions were collected and lyophilized to obtain the pure product of polypeptide compound 14, with a yield of approximately 20%. The purity of the peptide was determined by WATERS H-CLASS analytical ultra-high performance liquid chromatography (HPLC) system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The purity was 97.92%. The molecular weight of the compound was confirmed by Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The ion current shown in the mass spectrum was 815.4899 [M+3H] 3+ / 3.

[0170] 3.2 Chemical synthesis of polypeptide compound 15

[0171] yGrkkrrqrrr-AEEA-Tle-Tle-TDV(SEQ ID NO.15)

[0172] The synthesis of polypeptide compound 15 was carried out according to the synthesis steps of polypeptide compound 14, except that the condensation order of amino acid derivatives was: Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-AEEA-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc c-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH. The purity of the peptide was determined by WATERS H-CLASS analytical ultra-high performance liquid chromatography (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6×150 mm)) and was 98.73%. The molecular weight of the compound was confirmed by Agilent Q-TOF 6530 system (chromatographic column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6×150 mm). The ion current shown in the mass spectrum is: 749.4533 [M+3H] 3+ / 3.

[0173] 3.3 Chemical synthesis of polypeptide compound 16

[0174] yGrkkrrqrrr-Nle-TT-Tle-Tle-TDV (SEQ ID NO.16)

[0175] The synthesis of polypeptide compound 16 was carried out according to the synthesis steps of the above polypeptide compound 14, except that the condensation order of the amino acid derivatives was: Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Nle-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D -Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH , Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH. The purity of the peptide was determined by WATERS H-CLASS analytical ultra-high performance liquid chromatography (HPLC) system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The purity was 97.93%. The molecular weight of the compound was confirmed by Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The ion current shown in the mass spectrum was 806.1549 [M+3H] 3+ / 3.

[0176] 3.4 Chemical synthesis of polypeptide compound 17

[0177] yGrkkrrqrrrT-homoArg-homoArg-ITDV (SEQ ID NO.17)

[0178] The synthesis of polypeptide compound 17 was carried out according to the synthesis steps of polypeptide compound 14, except that the condensation order of amino acid derivatives was: Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-homoArg(Pbf)-OH, Fmoc-homoArg(Pbf)-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc- D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH , Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH. The purity of the peptide was determined by WATERS H-CLASS analytical ultra-high performance liquid chromatography (HPLC) system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The purity was 98.77%. The molecular weight of the compound was confirmed by Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The ion current shown in the mass spectrum was 810.4980 [M+3H]. 3+ / 3.

[0179] 3.5 Chemical synthesis of polypeptide compound 18

[0180] yGrkkrrqrrrG-Aib-Dab-homoArg-Tle-Tle-TDV (SEQ ID NO.18)

[0181] The synthesis of polypeptide compound 18 was carried out according to the synthesis steps of polypeptide compound 14, except that the condensation order of amino acid derivatives was: Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-homoArg(Pbf)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Aib-OH, Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-O H, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf )-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH. The purity of the peptide was determined by WATERS H-CLASS analytical ultra-high performance liquid chromatography (HPLC) system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The purity was 98.68%. The molecular weight of the compound was confirmed by Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The ion current shown in the mass spectrum was 838.5120 [M+3H] 3+ / 3.

[0182] 3.6 Chemical synthesis of polypeptide compound 19

[0183] yGrkkrrqrrrG-Aib-Dab-T-Chg-Tle-SDV (SEQ ID NO.19)

[0184] The synthesis of polypeptide compound 19 was carried out according to the synthesis steps of polypeptide compound 14, except that the condensation order of amino acid derivatives was: Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tle-OH, Fmoc-Chg-OH, Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Aib-OH, Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf) -OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH. The purity of the peptide was determined by a WATERS H-CLASS analytical ultra-high performance liquid chromatography system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6×150 mm) and the purity was 98.95%. The molecular weight of the compound was confirmed by an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6×150 mm). The ion current shown in the mass spectrum was 819.4897 [M+3H] 3+ / 3.

[0185] 3.7 Chemical Synthesis of Peptide Compound 20

[0186] yGrkkrrqrrrGT-Tle-Tle-TDV (SEQ ID NO.20)

[0187] The synthesis of polypeptide compound 20 was carried out according to the synthesis steps of polypeptide compound 14, except that the condensation order of amino acid derivatives was: Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf )-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmo c-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH. The purity of the peptide was determined by WATERS H-CLASS analytical ultra-high performance liquid chromatography (HPLC) system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The purity was 97.06%. The molecular weight of the compound was confirmed by Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The ion current shown in the mass spectrum was 753.7843 [M+3H]. 3+ / 3.

[0188] 3.8 Chemical Synthesis of Peptide Compound 21

[0189] yGrkkrrqrrrGT-Chg-Tle-SDV (SEQ ID NO.21)

[0190] The synthesis of polypeptide compound 21 was carried out according to the synthesis steps of polypeptide compound 14, except that the condensation order of amino acid derivatives was: Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tle-OH, Fmoc-Chg-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf )-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmo c-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH. The purity of the peptide was determined by WATERS H-CLASS analytical ultra-high performance liquid chromatography (HPLC) system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The purity was 98.54%. The molecular weight of the compound was confirmed by Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The ion current shown in the mass spectrum was 757.7843 [M+3H]. 3+ / 3.

[0191] 3.9 Chemical Synthesis of Peptide Compound 22

[0192] rKKRrQRRrG-Aib-Dab-T-Tle-Tle-TDV (SEQ ID NO.22)

[0193] The synthesis of polypeptide compound 22 was carried out according to the synthesis steps of polypeptide compound 14, except that the condensation order of amino acid derivatives was: Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Aib-OH, Fmoc-Gly- OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-A rg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH. The purity of the peptide was determined by WATERS H-CLASS analytical ultra-high performance liquid chromatography (HPLC) system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The purity was 97.82%. The molecular weight of the compound was confirmed by Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm). The ion current shown in the mass spectrum was 742.1292 [M+3H]. 3+ / 3.

[0194] Biological test evaluation

[0195] The present disclosure is further described and explained below in conjunction with test examples, but these embodiments are not intended to limit the scope of the present disclosure.

[0196] 1. Experimental Reagents

[0197] Table 1-3

[0198] 2. Experimental instruments

[0199] Table 1-4

[0200] 3. Test Case

[0201] 3.1. Evaluation of the affinity of peptide compounds for human PSD-95

[0202] 3.1.1 Experimental Purpose

[0203] The purpose of this test example is to determine the affinity of a peptide compound for human PSD95-PDZ2 using a competitive ELISA method.

[0204] 3.1.2 Expression and purification of human PSD95-PDZ2 protein

[0205] Using human PSD95 (Uniprot Entry: P78352) as a PDZ template, the amino acid sequence of the PDZ2 protein was designed: PDZ2 (155-249, the italicized portion is the Flag-His-Avitag-TEVsite tag, which is subsequently used for purification and biotin labeling): The PDZ2 gene was constructed into a pET expression vector and induced for expression in BL21-DE3 Escherichia coli at 37°C with 1mM IPTG for 4h. The pellet was then collected by centrifugation at 10,000g for 10min at 4°C, resuspended in 1×PBS, and disrupted with a homogenizer. The supernatant was collected by high-speed centrifugation and filtered through 0.45µM. A Ni-Sepharose affinity column was equilibrated with 20mM phosphate buffer (pH 8.0) for 5 column volumes. The sample was centrifuged at high speed to remove impurities and then loaded onto the column for binding. The column was rinsed with 20mM phosphate buffer until the A280 reading dropped to baseline. Elution was then performed with a 20mM phosphate buffer gradient containing 0-500mM imidazole to collect the protein and identify the target protein. The purified sample was exchanged with 1×PBS and concentrated to 2mL. It was further purified using a Superdex200 (GE) gel chromatography system equilibrated in 1×PBS, and the target peak was collected for subsequent use.

[0206] 3.1.3 Experimental methods

[0207] Site-directed biotinylated Biotin-NA-1 (sequence: Biotin–YGRKKRRQRRRKLSSIESDV; SEQ ID NO. 63) was used in competitive ELISA binding assays. The positive controls for this test example were NA-1 (sequence: YGRKKRRQR RRKLSSIESDV; SEQ ID NO. 64) and NoNO42 (sequence: YGrKKRrQrRRkLSSIESDV; SEQ ID NO. 65), and the negative control was NA-1, an Ala mutant at positions 0 and -2 of NA-1. (ADA) (Sequence: YGRKKRRQRRRKLSSIEADA; SEQ ID NO. 66), which has been shown to have no PDZ2 binding ability (Science, 2002, 298: 846-50).

[0208] PSD95-PDZ2 (tag removed by TEV enzyme) was diluted to 1 μg / mL in 1× PBS buffer and added to a 96-well ELISA plate (Corning, 9018, 25 / box 96-well clear flat bottom plate) at a volume of 100 μL / well. The plate was incubated at 4°C overnight for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4, 0.05% Tween-20) buffer. 4% BSA blocking solution diluted in PBST buffer was added (300 μL / well) and blocked by incubation at 37°C for 1 hour. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer. Biotin-NA-1 was then added at a constant concentration of 0.3 μM and the test compound (100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0 μM) was diluted tenfold in PBS buffer at an initial concentration of 100 μM. The plate was incubated at 37°C for 1 hour. After incubation, the reaction solution in the ELISA plate was discarded and the plate was washed three times with PBST. 100 μL of HRP-SA secondary antibody (1:2000 dilution) was added to each well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL of TMB chromogenic substrate was added and incubated at room temperature for 1-3 minutes. The reaction was terminated by adding 100 μL of 1 M sulfuric acid.

[0209] 3.1.4 Sample analysis and data processing

[0210] The absorbance was read at 450 nm using a SpectraMax M5 microplate reader, and the IC values ​​of the test compounds for PSD95-PDZ2 protein binding were calculated using nonlinear fitting using GraphPad Prism 9. 50 The specific data are shown in Table 1.

[0211] Table 1-5. Binding ability of peptide compounds to human PSD95-PDZ2 *The test error is within 3 times; **rIC 50 Represents the affinity ratio of the peptide compound to NA-1.

[0212] 3.1.5 Experimental Conclusions:

[0213] The results showed that the compounds disclosed herein can effectively bind to the target protein human PSD95-PDZ2. Given the high conservation of the PSD95-PDZ domain across different species, this facilitates subsequent animal evaluation.

[0214] 3.2. Evaluation of the specificity of peptide compounds binding to human PSD95-PDZ2

[0215] 3.2.1 Experimental Purpose

[0216] The three PDZ domains in the PSD95 protein have similar structures. Binding to the PDZ1 and PDZ2 domains can effectively block PSD95-mediated neurotoxicity, but the biological function of binding to the PDZ3 domain is unclear. Therefore, to avoid potential safety issues, this test case examines the selectivity of peptide compounds for the PDZ2 and PDZ3 domains to select peptide compounds that specifically bind to the PDZ2 domain.

[0217] 3.2.2 Expression and purification of human PSD95-PDZ3 protein

[0218] Refer to the steps in 3.1.2 for expression and purification of PSD95-PDZ3 protein. The amino acid sequence of PDZ3 protein is: PDZ3 (309-401, the italicized part is the Flag-His-Avitag-TEVsite tag, which is used for subsequent purification and biotin labeling):

[0219] 3.2.3 Experimental methods

[0220] Site-directed biotinylated Biotin-PDZ2 and Biotin-PDZ3 proteins were used for binding ELISA detection. The positive controls for this test example were NA-1 and NoNO42 (WO2022150655), and the negative control was NA-1 (ADA) The test compound was diluted to 2 μM in 1× PBS buffer and added to a 96-well ELISA plate (Corning, 9018 25 / box 96well clear flat bottom plate) at a volume of 100 μL / well. The plate was incubated at 4°C overnight for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4, 0.05% Tween-20) buffer. 4% BSA blocking solution diluted in PBST buffer was added (300 μL / well) and the plate was incubated at 37°C for 1 hour for blocking. After blocking, discard the blocking solution and wash the plate three times with PBST buffer. Then, add Biotin-PDZ2 or Biotin-PDZ3 with an initial concentration of 10 μM and a ten-fold dilution gradient of 7 (10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0 μM) in 1× PBS buffer and incubate at 37°C for 1 hour. After incubation, discard the reaction solution in the ELISA plate, wash the plate three times with PBST, add 100 μL of HRP-SA secondary antibody (1:2000 dilution) to each well, and incubate at 37°C for 1 hour. After washing the plate three times with PBST, add 100 μL of TMB chromogenic substrate, incubate at room temperature for 1-3 minutes, and add 100 μL of 1M sulfuric acid to terminate the reaction.

[0221] 3.2.4 Sample analysis and data processing

[0222] The absorbance was read at 450 nm using a SpectraMax M5 microplate reader, and the EC50 values ​​of the binding of Biotin-PDZ2 or Biotin-PDZ3 to the test compounds were calculated using GraphPad Prism 9. Specific data are shown in Tables 2-1 and 2-2.

[0223] Table 2-1. Binding ability of peptide compounds to human PSD95-PDZ2 and human PSD95-PDZ3

[0224] Table 2-2. Binding ability of peptide compounds to human PSD95-PDZ2 and human PSD95-PDZ3

[0225] 3.2.5 Experimental Conclusion

[0226] The results showed that the compounds disclosed herein all selectively bind to the PSD95-PDZ2 domain, among which the selectivity of polypeptide compounds 14, 15, 16, 19, 21 and 22 is comparable to that of the positive drugs NA-1 and NoNO42.

[0227] 3.3 Hemolytic risk assessment of peptide compounds in rat, rabbit, and human whole blood

[0228] Hemolysis refers to the destruction of the red blood cell membrane, resulting in an increased transparency and a deep red color. Certain pharmaceutical ingredients and excipients contain hemolytic components, which can cause hemolysis in the human body, leading to adverse reactions such as local swelling and circulatory dysfunction. Based on the principle that hemoglobin released by ruptured red blood cells absorbs visible light, a solution of the test compound was added to a rat red blood cell suspension. After incubation, the degree of hemolysis was measured using a microplate reader.

[0229] 3.3.1 Experimental Purpose

[0230] This test example examines whether a polypeptide compound induces hemolytic reactions in rat, rabbit, and human whole blood.

[0231] 3.3.2 Experimental methods

[0232] Preparation of red blood cell suspension: Take 100 μL of fresh whole blood, then add 900 μL of 1× PBS solution, place on a plate shaker, shake at 30 rpm for 5 minutes, then centrifuge at 1000g for 5 minutes, discard the supernatant; repeat the above washing steps until the supernatant no longer appears red, and use it for testing.

[0233] Preparation of test peptide solution: Add an appropriate amount of 1× PBS solution to the peptide powder to dissolve to obtain a standard stock solution. Then dilute with 1× PBS to obtain test sample solutions at concentrations of 1, 3, 10, 30, 100, and 300 μg / mL. Set up two replicate wells for each concentration. Simultaneously, use a blank 1× PBS solution as a negative control, and a 1× PBS solution containing 0.1% Triton X-100 as a positive control.

[0234] Incubation process: Add 500 μL of the test solution to the red blood cell suspension, then shake on a plate shaker at 30 rpm for 5 minutes to mix it thoroughly. Then incubate the suspension at 37°C for 1 hour and then centrifuge at 1000g for 5 minutes.

[0235] 3.3.3 Sample analysis and data processing

[0236] 100 μL of the supernatant was transferred to a single well of a microplate. The absorbance at 540 nm was measured using a SpectraMax M5 microplate reader, and data were analyzed using GraphPad Prism 9. The hemolysis rate (%) was calculated as (test sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%. A result less than 5% was considered non-hemolytic; a result greater than 5% was considered hemolytic. The results are shown in Table 3 and Figures 1A and 1B.

[0237] Table 3. Hemolytic risk test of peptide compounds

[0238] 3.3.4 Experimental Conclusions:

[0239] The results showed that in rat and human whole blood, polypeptide compound 14 and positive drugs NA-1 and NoNO42 met the requirement of hemolysis rate less than 5% in the concentration range of 1 to 50 μM, and there was no hemolysis risk.

[0240] 3.3.5 Risk Assessment of Peptide Compounds on Rabbit Whole Blood Hemolysis

[0241] A similar method to that used in Section 3.3.2 was used, except that fresh rabbit whole blood was used in this test. The experimental results are shown in Figure 1C. The results demonstrate that peptide compounds 14 and 22, along with the positive agents NA-1 and NoNO42, all met the requirement of less than 5% hemolysis in rabbit whole blood at concentrations ranging from 1 to 50 μM, demonstrating no hemolytic risk.

[0242] 3.4 In vitro stability study of peptide compounds in rat and human plasma

[0243] There are a variety of hydrolases in plasma that can decompose and metabolize drug molecules, resulting in a rapid decrease in the concentration of drug molecules in plasma, which cannot reach an effective concentration, causing a higher clearance rate and a shorter half-life, and poor pharmacokinetic and pharmacodynamic properties. Therefore, plasma stability is an important indicator affecting drugability.

[0244] 3.4.1 Experimental Purpose

[0245] This test case investigates the in vitro stability of a peptide compound in rat and human plasma and is divided into two parts: 1) the stability of the peptide compound alone in rat and human plasma; 2) the stability of the peptide compound in rat and human plasma when administered simultaneously with alteplase (rt-PA).

[0246] 3.4.2 Experimental methods

[0247] Solution Preparation: Weigh an appropriate amount of test peptide compound and dissolve in 1× PBS to obtain a 1 mM test solution. NA-1 and NoNO42 are positive agents, propantheline bromide is used as the control in human plasma, and lovastatin is used as the control in rat plasma.

[0248] Plasma stability test: Take 12.5μL of 1mM test solution and add it to 495μL of pre-incubated plasma sample. Divide the prepared plasma sample into 50μL each centrifuge tube with different incubation times (0min, 10min, 20min, 30min, 60min, 120min), and then incubate in a 37°C water bath with shaking at 60rpm. Two samples are parallel for each incubation time. Then add 200μL of stop solution to stop the incubation and vortex for 5min. Then centrifuge at 10000rpm and 4°C for 10min to remove the protein in the sample, and take 70μL of supernatant to a new 96-well plate (70μL of water was added to each well before) and mix well.

[0249] Plasma stability test with alteplase: Weigh an appropriate amount of solid alteplase, add an appropriate volume of water, and vortex to dissolve to obtain a 1 mg / mL alteplase solution. Add 250 μL of this 1 mg / mL alteplase solution to 4750 μL of plasma to obtain plasma containing 50 μg / mL alteplase. All other procedures are the same as those in the "Plasma Stability Test" above, except that plasma containing 50 μg / mL alteplase is used instead of plasma.

[0250] 3.4.3 Sample analysis and data processing:

[0251] The content of the remaining compound at each time point was detected by LC-MS / MS, and the relative content of the remaining compound at other time points was calculated with 0 min as the standard (100%).

[0252] Sample pretreatment method: Take 30 μL of plasma sample, add 120 μL of 5% formic acid methanol solution containing 1 ng / mL internal standard (verapamil), vortex for 5 minutes, centrifuge at 10000 rpm for 10 minutes, take 70 μL of supernatant, add 70 μL of 0.1% formic acid aqueous solution, mix well, and analyze by LC-MS instrument.

[0253] The LC-MS analysis method was as follows: (1) Chromatographic conditions: Mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile; flow rate was 0.5 mL / min; injection volume was 10 μL; chromatographic column was a nanomicro Unisil C18aq (4.6 mm × 150 mm, 5 μm); column temperature was 40°C. (2) Mass spectrometry conditions: Mass spectrometry was performed using an electrospray ionization source (ESI) in positive ion analysis mode and a multiple reaction monitoring (MRM) scan. The experimental results are shown in Tables 4-1 and 4-2, and Figures 2A, 2B, 2C, and 2D.

[0254] Table 4-1. Plasma stability of polypeptide compounds

[0255] Table 4-2. Plasma stability of polypeptide compounds

[0256] 3.4.4 Experimental Conclusion

[0257] Results showed that within 2 hours, NA-1 underwent significant degradation in both human and rat plasma, while neither NoNO42 nor peptide compound 14 could be degraded. Co-incubation with alteplase accelerated the degradation rate of NA-1, while peptide compound 14 remained stable in both species' plasma. However, NoNO42 showed reduced stability in rat plasma but remained stable in human plasma. These results are consistent with the Phase III clinical trial results of NA-1, which showed that the addition of alteplase rapidly disrupted the molecular structure of NA-1, rendering it inactive. Peptide compound 14's resistance to alteplase in plasma makes it a promising candidate to address NA-1's clinical limitations.

[0258] The polypeptide compound 22 is stable in human and rat plasma. After the addition of alteplase, the polypeptide compound 22 remains stable in human plasma, and its degradation rate in rat plasma is significantly slower than that of NA-1.

[0259] The resistance of peptide compounds 14 and 22 to alteplase in plasma enables them to compensate for the clinical disadvantages of NA-1.

[0260] 3.5 Pharmacokinetic study of peptide compounds in rats

[0261] 3.5.1 Experimental Purpose

[0262] Male SD rats were used as test animals to study the pharmacokinetic behavior of the polypeptide compound in rats (plasma) after a single intravenous injection.

[0263] 3.5.2 Experimental methods

[0264] Male SD rats weighing 170 to 200 grams and aged 4-6 weeks were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. After dissolving and preparing the polypeptide compound solution in 1×PBS, the drug was administered by intravenous injection at a dose of 3 nmol / g over 10 minutes, with 3 animals in each group. 0.2 mL of blood was collected at 2 minutes, 8 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 120 minutes after the infusion, and transferred to a centrifuge tube containing EDTA-K2 anticoagulant. The whole blood sample was centrifuged at 4000g for 5 minutes at 4°C to separate the plasma, which was stored frozen at -80°C.

[0265] A similar method was used to investigate the pharmacokinetic parameters of the peptide compound 22 after intravenous injection in rats. The difference was that the enzyme inhibitor was added immediately after blood was drawn.

[0266] 3.5.3 Sample analysis and data processing

[0267] The blood drug concentration of each analyte in plasma was determined using the method of Test Example 3.4.3. The blood drug concentration-time curve was plotted, and the pharmacokinetic parameters were calculated using PKSolver software. The experimental results are shown in Table 5-1, Table 5-2 and Figure 3.

[0268] 3.5.4 Experimental Conclusion

[0269] The results in Table 5-1 and Figure 3 demonstrate that the pharmacokinetic properties of the active drugs NA-1 and NoNO42 in rats are similar, with half-lives of less than 3 minutes. The pharmacokinetic properties of the tested peptide compounds 14, 19, and 21 are significantly superior to those of the two active drugs. Compared to NA-1, the half-life is significantly prolonged by 18-23 times, the area under the curve (AUC) increases by 18-33 times, and the maximum plasma concentration (Cmax) increases by 4-5 times. At the same concentration, peptide compounds 14, 19, and 21 exhibit a longer duration of action and greater drug exposure.

[0270] The results in Table 5-2 demonstrate that the addition of the enzyme inhibitor reduced the degradation of NA-1 after blood collection, resulting in improved AUC and Cmax values. Compared to NA-1, peptide compound 22 significantly prolonged its half-life by 29-fold, increased its area under the curve (AUC) by 19.4-fold, and increased its maximum plasma concentration (Cmax) by 2.3-fold.

[0271] Table 5-1. Pharmacokinetic parameters of intravenous injection in rats (n=3)

[0272] Table 5-2. Pharmacokinetic parameters of intravenous injection in rats (n=3)

[0273] 3.6 Pharmacokinetic study of different doses of peptide compounds in rats

[0274] 3.6.1 Experimental Purpose

[0275] The neuroprotectant NA-1 has been associated with dose-related safety issues in both preclinical and clinical settings. For example, at high doses, excessive histamine release can lead to symptoms such as tachycardia and decreased blood pressure. Therefore, the pharmacokinetic properties of peptide compound 14 were re-examined at three doses: high (3 nmol / g), medium (1 nmol / g), and low (0.3 nmol / g). These doses were then compared with a high dose of NA-1 (3 nmol / g) to provide a dosage reference for subsequent efficacy studies.

[0276] 3.6.2 Experimental methods

[0277] The experimental method of Test Example 3.5.2 was used, with 3 animals in each group, and the blood collection points were set as: 2 minutes, 8 minutes, 15 minutes, 60 minutes, 120 minutes, and 180 minutes after the end of the intravenous push injection.

[0278] 3.6.3 Sample analysis and data processing

[0279] The blood drug concentration of each analyte in plasma was determined using the method of Test Example 3.4.3. The blood drug concentration-time curve was plotted, and the pharmacokinetic parameters were calculated using WinNonlin software. The experimental results are shown in Table 6 and Figure 4.

[0280] Table 6. Pharmacokinetic parameters of intravenous injection in rats (different doses, n=3)

[0281] 3.6.4 Experimental Conclusion

[0282] As shown in Table 6, the plasma exposure of peptide compound 14 at low doses was comparable to that of NA-1 at high doses. Since plasma exposure is correlated with efficacy, it can be inferred that peptide compound 14 may still be effective at a 10-fold dose reduction. The Cmax value is believed to be related to histamine release. The Cmax values ​​of peptide compound 14 at both medium and low doses were lower than those of NA-1 at high doses, indicating a better safety profile, which will be verified in subsequent testing.

[0283] 3.7 Test of the ability of polypeptide compounds to induce histamine release

[0284] Histamine is an important endogenous component that mediates allergic and inflammatory responses. Drugs can induce mast cell degranulation and release large amounts of histamine, thereby causing inflammatory symptoms such as redness, rash, decreased blood pressure, and decreased heart rate. The histamine-releasing effect of drugs is often evaluated using plasma histamine levels, and the commonly used detection method is liquid chromatography-mass spectrometry.

[0285] 3.7.1 Experimental Purpose

[0286] Male SD rats were used as test animals to study the ability of a single intravenous injection of a polypeptide compound to induce histamine release in rats (plasma).

[0287] 3.7.2 Experimental methods

[0288] The same test was carried out as in Test Example 3.6, with 3 animals in each group. The blood collection points were set as: before drug administration, 2 minutes, 8 minutes, and 15 minutes after the end of intravenous injection.

[0289] 3.7.3 Sample analysis and data processing

[0290] The levels of histamine and 3-methylhistamine at each time point were determined by LC-MS / MS. The LC-MS detection method for compounds was as follows: (1) Chromatographic conditions: Mobile phase A was acetonitrile / water / 100 mM ammonium acetate, 50 / 45 / 5 (v / v / v), 2% FA; Mobile phase B was acetonitrile / 100 mM ammonium acetate, 95 / 5 (v / v), 2% FA; Flow rate was 0.4 mL / min; Chromatographic column was Waters BEH HILIC 1.7 μm, 2.1×150 mm; Column temperature was 50°C. Injection volume was 20 μL; (2) Mass spectrometry conditions: Mass spectrometry was performed using an electrospray ionization source (ESI) in positive ion analysis mode and multiple reaction monitoring (MRM) scanning.

[0291] Sample pretreatment: 30 μL of plasma sample was added to 200 μL of acetonitrile containing 100 ng / mL of tetradeuterated histamine (D4-histamine). The sample was vortexed for 1 minute and then centrifuged at 5800 rpm for 10 minutes. 100 μL of the supernatant was transferred to the sample tray for analysis. Table 7 shows the sum of the detected histamine and 3-methylhistamine levels.

[0292] Table 7. Histamine levels at different time points Note: Compared with 0 min: *P<0.05, **P<0.01.

[0293] 3.7.4 Experimental Conclusion

[0294] Table 7 shows that histamine release after drug administration is a transient response to external stimuli. At the same high dose and concentration, both NA-1 and polypeptide compound 14 showed the strongest histamine release stimulation at 2 minutes, a significant difference from pre-dose levels. This stimulation quickly weakened, with the peak level of polypeptide compound 14 being nearly twice that of NA-1. However, no significant increase in histamine levels was detected within 15 minutes at either the medium or low doses of polypeptide compound 14. This result corroborates the aforementioned pharmacokinetic results, indicating that the medium and low doses of polypeptide compound 14, due to their lower blood concentrations than the high dose of NA-1, are less potent than NA-1 in inducing histamine release and therefore offer a higher safety profile.

[0295] 3.8 Nephrotoxicity Assessment of Peptide Compounds in Rats

[0296] The kidneys are a key target organ for drug toxicity, and nephrotoxicity is a significant factor restricting new drug development. Therefore, potential nephrotoxicity assessment is essential during the new drug development process to mitigate drug development risks. Serum urea nitrogen (BUN) and creatinine are important indicators for evaluating kidney function. Abnormally elevated levels of these two indicators in the blood indicate possible renal impairment or damage.

[0297] 3.8.1 Experimental Purpose

[0298] Male SD rats were used as test animals to study the renal toxicity of a single intravenous injection of a polypeptide compound in rats (serum).

[0299] 3.8.2 Experimental methods

[0300] Conducted in parallel with Test Example 3.6, blood sampling was performed at the following times: before administration (day 0), and on days 2, 4, and 7 after administration. At each time point, 0.3 mL of blood was collected and transferred to a centrifuge tube. The tube was allowed to stand at room temperature for 1 hour. The whole blood sample was then centrifuged at 4000 g for 5 minutes at 4°C to separate the serum sample, which was then stored at -80°C.

[0301] 3.8.3 Sample analysis and data processing

[0302] Serum urea nitrogen (BUN) and creatinine levels at each time point were measured using commercially available kits. Urea nitrogen was measured using the urease continuous monitoring method, while creatinine was measured using the sarcosine oxidase method. After thawing the sample at 2 to 8°C, vortex-mix the sample, and pipette 100 μL into a sample cup. The sample was then placed in the sample holder of a biochemical analyzer and assayed according to the kit instructions. The results are shown in Tables 8-1 and 8-2 and Figures 5A and 5B.

[0303] 3.8.4 Experimental Conclusion

[0304] During the 7-day monitoring period, NA-1 blood urea nitrogen levels increased significantly on days 4 and 7 compared to pre-dose levels. However, the numerical increase was within a 2-fold range and was not clinically significant. Therefore, the test results indicate that within the tested concentration range, NA-1 and polypeptide compound 14 have no significant renal safety issues.

[0305] Table 8-1. Blood urea nitrogen (BUN) levels at different time points (in mg / dL) Note: Compared with before administration: *P<0.05, **P<0.01

[0306] Table 8-2. Serum creatinine (Crea) levels at different time points (in mg / dL)

[0307] 3.9 Pharmacokinetic study of peptide compounds in beagle dogs

[0308] 3.9.1 Experimental Purpose

[0309] Ordinary beagle dogs were used as test animals to study the pharmacokinetic behavior of a single intravenous infusion of a polypeptide compound in beagle dogs (plasma).

[0310] 3.9.2 Experimental methods

[0311] Peptide compound 22 was set up in two dose groups of 1.2 nmol / g and 0.4 nmol / g, and NA-1 was set up in one dose group of 1.2 nmol / g, with 4 animals in each group, half male and half female. After dissolving and preparing the peptide compound solution in 1×PBS, the drug was administered by intravenous infusion over 10 minutes. The blood collection points were: before infusion (0h), 5min after the start of the bolus injection, 0min, 2min, 5min, 10min, 15min, 30min, 1h, 2h, 4h, 8h, and 24h after the end of the bolus injection. 0.5mL of blood was collected at each time point and transferred to a centrifuge tube containing EDTA-K2 anticoagulant. 50μL of protease inhibitor was added at the same time. After mixing, the tube was centrifuged at 2000g for 10min (4°C). Plasma was separated within 1h and dispensed into cryovials. The tubes were stored at -60 to -90°C. The blood collection and centrifugation process were operated under ice bath conditions.

[0312] 3.9.3 Sample analysis and data processing

[0313] The blood drug concentration of each analyte in plasma was determined using the method of Test Example 3.4.3. The blood drug concentration-time curve was plotted, and the pharmacokinetic parameters were calculated using PKSolver software. The experimental results are shown in Table 9 and Figure 6.

[0314] 3.9.4 Experimental Conclusion

[0315] The results showed that the pharmacokinetic properties of the tested polypeptide compound 22 were significantly better than those of NA-1. 1 / 2 , NA-1 is 2.7min, polypeptide compound 22 is 5.1h; the average AUC, polypeptide compound 22 is 21.3 times that of NA-1; the average C max The drug exposure of peptide compound 22 was 1.3 times that of NA-1. The drug exposure of peptide compound 22 was positively correlated with the dosage.

[0316] Table 9. Pharmacokinetic parameters of intravenous infusion in beagle dogs

[0317] 3.10 Test of the ability of polypeptide compounds to induce histamine release

[0318] 3.10.1 Experimental Purpose

[0319] Beagle dogs were used as test animals to study the ability of a single intravenous infusion of a polypeptide compound to induce histamine release in dogs (plasma).

[0320] 3.10.2 Experimental methods

[0321] The same test was carried out as in Test Example 3.9, with 4 animals in each group. The blood collection points were set as: before administration (0 h), 2 min, 5 min and 15 min after the end of intravenous infusion.

[0322] 3.10.3 Sample analysis and data processing

[0323] Sample analysis and data processing were performed according to the method in 3.7.3. Table 10 and Figure 7 show the sum of the detected histamine and 3-methylhistamine.

[0324] Table 10. Histamine levels at different time points

[0325] 3.10.4 Experimental Conclusion

[0326] Compared with before administration, there was no significant change in histamine levels at both doses of polypeptide compound 22, indicating good safety.

[0327] 3.11 Study on the pharmacodynamic effects of peptide compounds in the rat tMCAO model

[0328] 3.11.1 Experimental Purpose

[0329] This study aims to investigate the pharmacodynamic effects of peptide compounds on the rat tMCAO (transient middle cerebral artery occlusion) model. High-dose NA-1 (3 nmol / g) was used as a positive drug. Peptide compounds 14 and 22 were set at high (3 nmol / g), medium (1 nmol / g), and low (0.3 nmol / g) doses. By comparing the cerebral infarction area and neurological function scores of rats, and combining with previous tests, a safe and effective dosage reference was provided for the in-depth evaluation of peptide compounds 14 and 22.

[0330] 3.11.2 Experimental methods

[0331] Modeling and Dosing: Male Sprague-Dawley rats weighing 240 to 260 grams and aged 6-8 weeks were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. A suture-bolus method was used to establish the MCAO model in rats. Dosing was performed 60 minutes after suture insertion and 90 minutes after suture removal for reperfusion. Baseline cerebral blood flow was monitored before suture insertion. After suture insertion, a decrease in cerebral blood flow of more than 50% from baseline and a return to more than 50% of pre-reperfusion blood flow immediately after suture removal were considered inclusion criteria. Animals that did not meet these criteria were excluded. The peptide compound was dissolved in saline to the desired concentration. One hour after suture insertion, the peptide compound was slowly injected into the tail vein over a period of 4 to 5 minutes. A control group received a 4-5 minute intravenous injection of saline. The experimental endpoint was 24 hours after dosing. During the dosing period, the animals were observed for their overall status, including voluntary activity, food and water intake, mortality, and other abnormalities.

[0332] Infarct size measurement: 24 hours after MCAO, surviving rats in each group were dissected and perfused with pre-chilled PBS. The brains were removed by decapitation and the sections were stained with 2% tetrazolium chloride (TTC) solution to calculate the infarct size and percentage of infarct area. Percentage of infarct area = infarct area / total brain area × 100%.

[0333] Neurological impairment scoring: Animals were scored for neurological impairment using a blinded method before modeling (animals with abnormal behavioral scores before modeling) and 24 hours after drug administration according to the scoring criteria in the attached table. The total score is 16 points. Higher scores indicate more severe impairment. Detailed scoring criteria are shown in Table 11.

[0334] Table 11. Scoring criteria for neurological impairment in MCAO rats

[0335] 3.11.3 Sample analysis and data processing

[0336] IBM SPSS Statistics 25.0 statistical software was used for data analysis. Data with homogeneous variance were compared using the LSD test, while data with unequal variance were compared using the Dunnett's T3 test. P < 0.05 was considered statistically significant. Specific experimental results are shown in Tables 12-1 and 12-2, and Figures 8A, 8B, 8C, and 8D.

[0337] Table 12-1. Summary of pharmacodynamic results in rat tMCAO model Note: Compared with the model group: *P<0.05, **P<0.01, ***P<0.001.

[0338] Table 12-2. Summary of pharmacodynamic results in rat tMCAO model

[0339] 3.11.4 Experimental Conclusion

[0340] The results showed that a single dose of peptide compounds 14 and 22 improved neurological function and cerebral infarction size in rats with tMCAO in a dose-dependent manner. A medium dose (1 nmol / g) of peptide compounds 14 and 22 was comparable in efficacy to a high dose (3 nmol / g) of the active drug NA-1.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, CPP-L1-X1-X2-X3-X4-X5-X6-X7-X8-X9 (I) The X1 is selected from Gly or missing; X2 is selected from Aib, Ala, Ile, Cha, Phe, Trp, 1-Nal, Ser, Lys, Arg, Nle, Nva, Orn, cyclopropylAla, 4-thiazolylAla, homoLeu or missing; X3 is selected from Dap, Dab, Thr, Aib, Ala, Cha, Phe, Trp, 1-Nal, Asn, Glu, Lys, Arg, 1Me-Trp, 2-Nal, Gln, Thr, 4-thiazolylalanine or missing; The X4 is selected from Thr, homoArg, Ser or deletion; The X5 is selected from Tle, homoArg, Chg; The X6 is selected from Tle or Ile; The X7 is selected from Thr or Ser; X8 is Asp; X9 is Val; L1 is a chemical bond or comprises polyethylene glycol, wherein one or two oxygen atoms of the polyethylene glycol are optionally replaced by nitrogen atoms; The CPP is an internalization peptide.

2. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein X7 is Thr.

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2, wherein X6 is Tle.

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein X5 is Tle.

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein X6-X7 is selected from Tle-Thr, Ile-Thr or Tle-Ser, preferably Tle-Thr or Ile-Thr, most preferably Tle-Thr.

6. The compound of formula (I) as claimed in claim 1 or a pharmaceutically acceptable salt thereof, wherein X5-X6 is selected from Tle-Tle, homoArg-Ile or Chg-Tle, preferably Tle-Tle or Chg-Tle, most preferably Tle-Tle.

7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein X5-X6-X7 is Tle-Tle-Thr.

8. The compound of formula (I) according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, wherein one or more of X1, X2, X3, and X4 are optionally missing, preferably, X2 and / or X3 are missing, or all of X1, X2, X3, and X4 are missing.

9. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein X4 is selected from Thr, homoArg or Ser, preferably Thr or Ser, most preferably Thr.

10. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein X3 is selected from Dap or Dab, preferably Dab.

11. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein X2 is selected from Aib, Ala or Ile, preferably Aib.

12. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein X1 is selected from Nle, Gly or missing, preferably Gly.

13. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein L1 is a chemical bond.

14. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein L1 comprises polyethylene glycol, one or two oxygen atoms of the polyethylene glycol are optionally replaced by nitrogen atoms, preferably AEEA.

15. The compound of formula (I) according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof, wherein X1-X2-X3-X4-X5-X6-X7-X8-X9 is selected from any one of SEQ ID NO.1 to SEQ ID NO.8: SEQ ID NO.1 G-Aib-Dab-T-Tle-Tle-TDV SEQ ID NO.2 Tle-Tle-TDV SEQ ID NO.3 Nle-TT-Tle-Tle-TDV SEQ ID NO.4 T-homoArg-homoArg-ITDV SEQ ID NO.5 G-Aib-Dab-homoArg-Tle-Tle-TDV SEQ ID NO.6 G-Aib-Dab-T-Chg-Tle-SDV SEQ ID NO.7 GT-Tle-Tle-TDV SEQ ID NO.8 GT-Chg-Tle-SDV.

16. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15, wherein the internalization peptide comprises an amino acid sequence as shown in any one of SEQ ID NO: 9 to SEQ ID NO. 11, preferably comprises an amino acid sequence as shown in SEQ ID NO. 11; Optionally, the amino acid residues in SEQ ID NO: 9 to SEQ ID NO. 11 are D amino acids, SEQ ID NO.9 RKKRRQRRR SEQ ID NO.10 GRKKRRQRRR SEQ ID NO. 11 YGRKKRRQRRR.

17. The compound of formula (I) according to claim 16 or a pharmaceutically acceptable salt thereof, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acid residues of the internalization peptide are D-amino acids, preferably, 4, 5, 6, 7, 8, 9, 10 or 11 of the amino acid residues of the internalization peptide are D-amino acids, and most preferably, 9, 10 or 11 of the amino acid residues of the internalization peptide are D-amino acids.

18. The compound of formula (I) according to claim 16 or a pharmaceutically acceptable salt thereof, wherein the amino acid residue R of the internalization peptide is a D-amino acid, preferably, 1, 2, 3, 4, 5 or 6 of the amino acid residues R are D-amino acids, preferably, 3, 4, 5 or 6 of the amino acid residues R are D-amino acids, most preferably, 5 or 6 of the amino acid residues R are D-amino acids.

19. The compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in claim 16, wherein the first amino acid residue R of the internalization peptide starting from the C-terminus is a D-amino acid, and the amino acid residues optionally spaced therefrom by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are D-amino acids, preferably the amino acid residues optionally spaced therefrom by 0, 1, 2, 3, 4, 5, 6, 7 or 8 amino acids are D-amino acids, and most preferably the amino acid residues optionally spaced therefrom by 0, 1, 2 or 3 amino acids are D-amino acids.

20. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 16 to 19, wherein the internalization peptide has an amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO. 13: SEQ ID NO.12 yGrkkrrqrrr SEQ ID NO.13 rKKRrQRRr.

21. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 16 to 20, selected from any one of SEQ ID NO.14 to SEQ ID NO.22: SEQ ID NO.14 yGrkkrrqrrrG-Aib-Dab-T-Tle-Tle-TDV SEQ ID NO.15 yGrkkrrqrrr-AEEA-Tle-Tle-TDV SEQ ID NO.16 yGrkkrrqrrr-Nle-TT-Tle-Tle-TDV SEQ ID NO.17 yGrkkrrqrrrT-homoArg-homoArg-ITDV SEQ ID NO.18 yGrkkrrqrrrG-Aib-Dab-homoArg-Tle-Tle-TDV SEQ ID NO.19 yGrkkrrqrrrG-Aib-Dab-T-Chg-Tle-SDV SEQ ID NO.20 yGrkkrrqrrrGT-Tle-Tle-TDV SEQ ID NO.21 yGrkkrrqrrrGT-Chg-Tle-SDV SEQ ID NO. 22 rKKRrQRRrG-Aib-Dab-T-Tle-Tle-TDV.

22. An active peptide or a pharmaceutically acceptable salt thereof, wherein the active peptide comprises formula (G), X1-X2-X3-X4-X5-X6-X7-X8-X9 (G), The X1, X2, X3, X4, X5, X6, X7, X8, and X9 are respectively defined as any one of claims 1 to 12 or 15.

23. The active peptide or a pharmaceutically acceptable salt thereof according to claim 22, wherein the active peptide comprises an amino acid sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.

8.

24. A peptide or a pharmaceutically acceptable salt thereof, comprising: The active peptide defined in claim 22 or 23, and An internalization peptide as defined in any one of claims 16 to 21. 25 . The peptide or a pharmaceutically acceptable salt thereof according to claim 24 , wherein the peptide comprises an amino acid sequence as shown in any one of SEQ ID NO. 14 to SEQ ID NO.

22.

26. A pharmaceutical composition comprising: The compound represented by formula (I) according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and / or the active peptide according to any one of claims 22 to 23 or a pharmaceutically acceptable salt thereof, and / or the peptide according to any one of claims 24 to 25 or a pharmaceutically acceptable salt thereof, and Pharmaceutically acceptable carriers and / or excipients.

27. Use of the compound of formula (I) according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and / or the active peptide according to any one of claims 22 to 23 or a pharmaceutically acceptable salt thereof, and / or the peptide according to any one of claims 24 to 25 or a pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition according to claim 26 in the preparation of a medicament; The drug is used for treating and / or preventing stroke, cerebral ischemia, traumatic injury of the central nervous system, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy, neurodegenerative diseases and / or diseases with the above risks.

28. Use of the compound of formula (I) according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and / or the active peptide according to any one of claims 22 to 23 or a pharmaceutically acceptable salt thereof, and / or the peptide according to any one of claims 24 to 25 or a pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition according to claim 26 in combination with a thrombolytic drug in the preparation of a medicament for treating and / or preventing acute ischemic stroke, wherein the thrombolytic drug is selected from: urokinase, streptokinase, anistreplase, staphylokinase, recombinant staphylokinase, prourokinase, vampire bat salivary plasminogen activator, ranteplase, pamiplase, monteplase, alteplase, reteplase, tenecteplase; preferably alteplase.