Neuroprotective polypeptide compounds and uses thereof

Abstract

The present invention provides a neuroprotective polypeptide compound and its use, which belongs to the field of pharmaceutical technology, including a polypeptide having the following chemical formula and its salt: (M)m-(Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu)-(N)n; m and n are integers from 0 to 3, but m and n are not 0 at the same time. M is beta-Ala-His, beta-Ala-1-Methyl-His, or beta-Ala-3-Methyl-His, and N is beta-Ala-His, beta-Ala-1-Methyl-His, or beta-Ala-3-Methyl-His. Unexpectedly, a significant therapeutic effect is achieved by intravenous administration at a dose of only 3 mg / kg, and the effect is similar to that of NA1. This polypeptide compound can be used in combination with thrombolytic drugs, providing new possibilities for the treatment of stroke.

Description

[Technical Field]

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to neuroprotective polypeptide compounds and their uses. [Background technology]

[0002] Nerinetide (NA-1), a neuroprotective agent, can inhibit postsynaptic density protein 95 (PSD-95) by halting intracellular NO radical production. It can reduce infarct size and improve functional outcomes in preclinical ischemic stroke models after cerebral ischemia and reperfusion. Adult patients with acute ischemic stroke due to large vessel occlusion within a 12-hour therapeutic window were randomly assigned to receive nerinetide at a single dose of 2.6 mg / kg (maximum dose of 270 mg) or saline placebo. The primary endpoint of the study was favorable functional outcome 90 days after randomization, defined as a modified Rankin Scale (mRS) score of 0 to 2. Secondary endpoints included neurological dysfunction, functional independence in activities of daily living, favorable functional outcome (mRS 0-1), and mortality. This study enrolled 1,105 patients, including 549 in the nerinetide group and 556 in the placebo group. The proportion of patients with an mRS score of 0-2 within 90 days was 337 (61.4%) in the nerinetide group and 329 (59.2%) in the placebo group. Secondary outcomes were similar between the two groups. At the same time, this study found that nerinetide treatment reduced the efficacy of alteplase in patients receiving alteplase. The incidence of serious adverse events was similar between the two groups. See Michael D Hill et al. Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke (ESCAPE-NA1): a multicenter, double-blind, randomized controlled trial. Lancet. 2020,395(10227),pp. 878-887.

[0003] Nerinetide is a fusion peptide consisting of the C-terminal nine residues of the NMDAR GluN2B subunit and the membrane-permeable peptide TAT derived from nuclear transport activator protein (NMTPS). It can bind to the PDZ-1 or PDZ-2 structural domain of PSD-95, thereby inhibiting the production of NO by nNOS. Therefore, it is also called Tat-NR2B9c. Its specific amino acid sequence is Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val. The latter partial sequence, Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val, is the NR2B9c sequence, specifically inhibiting the production of NO by nNOS. The preceding subsequence, Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, improves the bioavailability of NR2B9c. The amino acid sequence of active peptides such as NR2B9c consists of 3-25 amino acids derived from the C-terminus of the NMDA receptor or PDZ structural domains 1 and / or 2 derived from the PSD-95 receptor, which bind to an internalization peptide (A. Tasker, T. Doucette, M. Tymianski, K. Mendoza, MP Belmares, D. Garman, and PS Lu, 2013, United States Patent 8,536,129). Such active peptides have amino acid sequences including [E / D / N / Q]-[S / T]-[D / E / Q / N]-[V / L], e.g., KLSSIETDV and KLSSIESDV.

[0004] Carnosine (L-carnosine) is a dipeptide composed of two amino acids, β-alanine and L-histidine. Carnosine possesses excellent antioxidant properties and is beneficial to the human body. Carnosine has been shown to scavenge reactive oxygen radicals (ROS) and α,β-unsaturated aldehydes, which are generated by the peroxidation of fatty acids in cell membranes during oxidative stress. Carnosine's anti-inflammatory, anti-glycation, antioxidant, and chelating properties make it a promising non-prescription dietary supplement for the prevention and adjunctive treatment of chronic diseases such as cardiovascular disease and neurodegenerative disorders. Animal studies have confirmed that carnosine's neuroprotective mechanism protects against chronic cerebral ischemia. Carnosine is also an important intracellular antioxidant. Due to its nontoxicity and potent antioxidant properties, carnosine is gaining widespread attention as a novel food additive and pharmaceutical. Carnosine is involved in intracellular peroxidation, inhibiting not only the peroxidation process of cell membranes but also related intracellular peroxidation. Since the Russian scholar Gulewitsch first discovered carnosine in 1900, scientists around the world have extracted and isolated other histidine dipeptide derivatives from different muscle tissues. For example, anserine is a dipeptide composed of two amino acids, β-alanine and 1-methyl-L-histidine, while balenine (also known as ophidine) is a dipeptide composed of β-alanine and 3-methyl-L-histidine. The content and ratio of these histidine dipeptides vary depending on the species, with some degree of specificity. In addition to the presence of histidine dipeptides in muscle tissue, these dipeptides are also present in other tissues, such as brain tissue. These carnosine derivatives are highly water-soluble and have significant antioxidant, anti-aging, and uric acid-lowering properties. They are used in the food industry as natural antioxidants and uric acid-lowering drugs, and also have some neuroprotective properties.

[0005] Although carnosine and carnosine derivatives all have some degree of neuroprotective function, relatively large doses are often required, and the use of carnosine, anserine, ophidine, etc. alone is not effective, so there is still a need for new neuroprotective methods in this field. Summary of the Invention

[0006] The inventors have combined carnosine and carnosine derivatives with active peptides such as NR2B9c to produce new polypeptides, and have unexpectedly discovered that these novel combined polypeptides have excellent neuroprotective effects that are completely different from nerintide, and that the neuroprotective effects are not affected by thrombolytic products such as alteplase, providing a new method for neuroprotection, etc.

[0007] The present invention provides neuroprotective polypeptide compounds, including carnosine, anserine, ophidine, and other active peptides, such as NR2B9c, which are composed of beta-alanine and histidine, 1-methylhistidine, or 3-methylhistidine. The amino acid sequence of the active peptide is Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu, particularly NR2B9c Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val, thereby forming a series of combined polypeptide compounds that combine the characteristics of carnosine, anserine, or ophidine with Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu. These peptides unexpectedly penetrate the blood-brain barrier and exhibit excellent biological activity upon intravenous administration, making them highly promising for the treatment of neurological disorders, particularly brain injury and stroke.

[0008] In one aspect, neuroprotective polypeptide compounds are provided, including polypeptides and salts thereof having the following formula: (M)m-(Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu)-(N)n In the formula, m is an integer of 0 to 3, and n is an integer of 0 to 3, but m and n are not simultaneously 0. M is beta-Ala-His, beta-Ala-1-Methyl-His, or beta-Ala-3-Methyl-His, and N is beta-Ala-His, beta-Ala-1-Methyl-His, or beta-Ala-3-Methyl-His. Glu / Asp indicates that the amino acid at that position may be either Glu or Asp, Ser / Thr indicates that the amino acid at that position may be either Ser or Thr, Asp / Glu indicates that the amino acid at that position may be either Asp or Glu, and Val / Leu indicates that the amino acid at that position may be either Val or Leu.

[0009] Preferably, Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu is Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val.

[0010] In some embodiments, neuroprotective polypeptide compounds of the present invention include polypeptides and salts thereof having the following formula: beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu; Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu-beta-Ala-His; beta-Ala-His-beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp-Val / Leu; Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu-beta-Ala-His-beta-Ala-His; beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu-beta-Ala-His; beta-Ala-His-beta-Ala-His-beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp-Val / Leu; beta-Ala-His-beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp-Val / Leu-beta-Ala-His; beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu-beta-Ala-His-beta-Ala-His; Lys-Leu-Ser-Ser-Ile-Glu / Asp-Ser / Thr-Asp / Glu-Val / Leu-beta-Ala-His-beta-Ala-His-beta-Ala-His. In the formula, His is His, 1-Methyl-His or 3-Methyl-His, i.e., the previously abbreviated M or N, which correspond to carnosine, anserine and ophidine, respectively.

[0011] Specifically, the neuroprotective polypeptide compounds of the present invention include polypeptides having the following chemical formula and salts thereof: beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Asp-Ser-Asp-Val; beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Thr-Asp-Val; beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Thr-Glu-Val; beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Glu-Val; beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Asp-Thr-Asp-Val; beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Asp-Ser-Glu-Val;

[0012] beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Asp-Thr-Glu-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His; Lys-Leu-Ser-Ser-Ile-Asp-Ser-Asp-Val-beta-Ala-His; Lys-Leu-Ser-Ser-Ile-Glu-Thr-Asp-Val-beta-Ala-His; Lys-Leu-Ser-Ser-Ile-Glu-Thr-Glu-Val-beta-Ala-His; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Glu-Val-beta-Ala-His; Lys-Leu-Ser-Ser-Ile-Asp-Thr-Asp-Val-beta-Ala-His; Lys-Leu-Ser-Ser-Ile-Asp-Ser-Glu-Val-beta-Ala-His; Lys-Leu-Ser-Ser-Ile-Asp-Thr-Glu-Val-beta-Ala-His;

[0013] beta-Ala-(1-methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; beta-Ala-(1-methyl-His)-Lys-Leu-Ser-Ser-Ile-Asp-Ser-Asp-Val; beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Thr-Asp-Val; beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Thr-Glu-Val; beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Glu-Val; beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Asp-Thr-Asp-Val; beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Asp-Ser-Glu-Val; beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Asp-Thr-Glu-Val;

[0014] Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His); Lys-Leu-Ser-Ser-Ile-Asp-Ser-Asp-Val-beta-Ala-(1-Methyl-His); Lys-Leu-Ser-Ser-Ile-Glu-Thr-Asp-Val-beta-Ala-(1-Methyl-His); Lys-Leu-Ser-Ser-Ile-Glu-Thr-Glu-Val-beta-Ala-(1-Methyl-His); Lys-Leu-Ser-Ser-Ile-Glu-Ser-Glu-Val-beta-Ala-(1-Methyl-His);

[0015] Lys-Leu-Ser-Ser-Ile-Asp-Thr-Asp-Val-beta-Ala-(1-Methyl-His); Lys-Leu-Ser-Ser-Ile-Asp-Ser-Glu-Val-beta-Ala-(1-Methyl-His); Lys-Leu-Ser-Ser-Ile-Asp-Thr-Glu-Val-beta-Ala-(1-Methyl-His); beta-Ala-(3-methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; beta-Ala-(3-methyl-His)-Lys-Leu-Ser-Ser-Ile-Asp-Ser-Asp-Val; beta-Ala-(3-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Thr-Asp-Val; beta-Ala-(3-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Thr-Glu-Val;

[0016] beta-Ala-(3-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Glu-Val; beta-Ala-(3-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Asp-Thr-Asp-Val; beta-Ala-(3-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Asp-Ser-Glu-Val; beta-Ala-(3-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Asp-Thr-Glu-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(3-Methyl-His); Lys-Leu-Ser-Ser-Ile-Asp-Ser-Asp-Val-beta-Ala-(3-Methyl-His); Lys-Leu-Ser-Ser-Ile-Glu-Thr-Asp-Val-beta-Ala-(3-Methyl-His);

[0017] Lys-Leu-Ser-Ser-Ile-Glu-Thr-Glu-Val-beta-Ala-(3-Methyl-His); Lys-Leu-Ser-Ser-Ile-Glu-Ser-Glu-Val-beta-Ala-(3-Methyl-His); Lys-Leu-Ser-Ser-Ile-Asp-Thr-Asp-Val-beta-Ala-(3-Methyl-His); Lys-Leu-Ser-Ser-Ile-Asp-Ser-Glu-Val-beta-Ala-(3-Methyl-His); Lys-Leu-Ser-Ser-Ile-Asp-Thr-Glu-Val-beta-Ala-(3-Methyl-His).

[0018] Preferably, the neuroprotective polypeptide compounds of the present invention include polypeptides and salts thereof having the following chemical formula: beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His; beta-Ala-(1-methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His); beta-Ala-(3-methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(3-Methyl-His).

[0019] More preferably, the neuroprotective polypeptide compounds of the present invention include polypeptides and salts thereof having the following chemical formula: beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His.

[0020] Most preferably, the neuroprotective polypeptide compounds of the present invention include polypeptides and salts thereof having the following formula: beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val.

[0021] According to other aspects, there is further provided the use of said neuroprotective polypeptide compounds in the preparation of a medicament, particularly a medicament for treating a nervous system disorder. There is further provided the use of said neuroprotective polypeptide compounds in the treatment of a nervous system disorder. There is further provided a method of treating a nervous system disorder with said neuroprotective polypeptide compounds.

[0022] In particular, the nervous system disease may be ischemic stroke, hemorrhagic stroke, brain injury, Alzheimer's disease, Parkinson's disease or other neurodegenerative diseases. Preferably, the nervous system disease is ischemic stroke.

[0023] The neuroprotective polypeptide compound provided by the present invention can be manufactured as a pharmaceutical. The pharmaceutical may be an injection, oral preparation, sublingual preparation, spray, or rectal preparation, preferably an injection. Specifically, the injection may be a powder injection or an injection solution. Furthermore, the pharmaceutical may be administered intravenously. The active ingredient of the pharmaceutical may be in other dosage forms depending on the corresponding medical application. Formulations developed as neuroprotective agents may include formulations for oral administration and sublingual administration. Formulations developed as pharmaceuticals for emergency use at the scene of stroke, for emergency care for incapacitated patients, may include formulations for spray, rectal administration, etc.

[0024] Furthermore, the pharmaceutical product contains a pharmaceutically acceptable diluent and / or carrier. In yet another aspect, the present invention further provides a method for preparing various neuroprotective polypeptide compounds of the present invention by solid-phase synthesis, although for some peptides, solution-phase synthesis or fragment synthesis may be more convenient. Salt formation of polypeptide pharmaceuticals is a common means for improving the physicochemical properties of drug molecules and increasing drug formability, and the pharmaceuticals may be in any form of salt.

[0025] According to another aspect, there is further provided a combination of a neuroprotective polypeptide compound described herein with a thrombolytic agent.

[0026] The thrombolytic drug may be a first-generation thrombolytic drug represented by streptokinase and urokinase, a second-generation thrombolytic drug represented by tissue-type plasminogen activator (tPA), alteplase, and pro-urokinase, or a third-generation thrombolytic drug based on recombinant human tissue-type plasminogen activator (rtPA). Many commercially available products are known in the art, such as injectable urokinase (Tianjin Biochemical Pharmaceutical Co., Ltd.), injectable recombinant streptokinase (e.g., Sikaitong), injectable alteplase (e.g., Actilyse), and injectable recombinant human TNK tissue-type plasminogen activator (e.g., Mingfule).

[0027] The neuroprotective polypeptide compounds and thrombolytic drugs described herein may be administered separately, before, after, or simultaneously, or may be mixed in any suitable ratio and administered as a combined pharmaceutical.

[0028] The combination may be used in the preparation of a medicament, in particular for the preparation of a medicament for the treatment of a nervous system disorder.

[0029] The nervous system disease may be ischemic stroke, hemorrhagic stroke, brain injury, Alzheimer's disease, Parkinson's disease or other neurodegenerative diseases. Preferably, the nervous system disease is ischemic stroke.

[0030] To confirm the use of the synthetic peptides provided herein for neurological disorders, SD rats were used as experimental subjects, and a cerebral ischemia model rat was created by middle cerebral artery occlusion (MCAO). Medication was intravenously injected 1-2 hours after ischemia, and behavioral observation and scoring of each animal was performed 22-24 hours later. After behavioral observation, the experimental rats were euthanized, and their brains were removed. Brain tissue was excised and stained with TTC for quantitative analysis, and the cerebral infarction volume (%) was calculated.

[0031] The inventors have creatively combined neuroprotective polypeptide compounds with active peptides such as NR2B9c, carnosine, anserine, or ophidine, and the resulting combined polypeptides unexpectedly penetrate the blood-brain barrier and enter the brain via intravenous injection, effectively treating nervous system disorders. Such synthetic peptides are preferably used in the treatment of ischemic stroke, hemorrhagic stroke, brain injury, Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases. In the present invention, intravenous administration of the synthetic polypeptide at a dose of just 3 mg / kg unexpectedly achieved significant therapeutic effects, comparable to those achieved by the clinically active standard polypeptide NA1.

[0032] Furthermore, the synthetic peptides of the present invention can be easily synthesized, and during the synthesis process, carnosine, anserine, or ophidine derivatives can be used as synthetic fragments instead of the corresponding two amino acids, further simplifying the synthesis of the target peptide. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a diagram of brain tissue sections from the model group. [Figure 2] FIG. 1 is a diagram of a brain tissue section from the S1 group. [Figure 3]FIG. 10 is a diagram of a brain tissue section from the S3 group. [Figure 4] FIG. 10 is a photograph of a brain tissue section from a sham-operated group. [Figure 5] The results show the area of ​​cerebral infarction and the inhibition rate of cerebral infarction, where A: area of ​​cerebral infarction (Infarction Area%), B: inhibition rate of cerebral infarction (Inhibition Ratio%), ** indicates P<0.01 compared to the model control group, and *** indicates P<0.005 compared to the model control group. [Figure 6] These are the results of NSS scoring. All data in the figure are expressed as mean ± standard deviation (Mean ± SD). A: Results of NSS scoring. B: Results of the reduction rate of NSS score for each treatment group compared to the model control group one day after surgery. * indicates P≦0.05 compared to the model control group animals. [Figure 7] FIG. 10 shows the results of TTC staining of animals in the model control group. [Figure 8] FIG. 10 shows the results of TTC staining of animals in the S3 group. [Figure 9] FIG. 10 shows the results of TTC staining of animals in the t-PA group. [Figure 10] FIG. 10 shows the results of TTC staining of animals in the S3+tPA group. DETAILED DESCRIPTION OF THE INVENTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0035] Example 1: Synthesis of beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val 1. Fmoc-Val-CTC resin was obtained by coupling the solid-supported 2-CTC resin with Fmoc-Val-OH in the presence of an activator system (HoBT, DIC). The Fmoc protecting group on Fmoc-Val-CTC was removed with 2.20% piperidine, and after removal, it was washed with DMF. A 3.3-fold excess of Fmoc-Asp(0tBu)-OH and a 3-fold excess of the activator were weighed out and completely dissolved in a small amount of DMF. After dissolution, the resin was added to the washed resin and reacted for 1 hour, followed by washing with DMF.

[0036] 4. Steps 2 and 3 were repeated to couple amino acids with N-Fmoc protection and side chain protection in the order of the main chain, from Ser to β-Ala. 5. After the synthesis was completed, the peptide resin was decomposed using a decomposition reagent ratio of TFA:EDT:Tis:TA:anisole:HO=80:5:1:5:5:4 (volume ratio). 10 mL of the decomposition reagent was used for 1 g of peptide resin. The decomposition was carried out at room temperature for approximately 2 hours (120 r / min), followed by precipitation with ice-cold methyl tert-butyl ether. The lower precipitate was the crude product.

[0037] 6. The crude peptide obtained in the previous steps was taken out, dissolved, and purified by preparative HPLC with 0.1% TFA / acetonitrile. 7. After purification, lyophilization was carried out, after which the powder was removed and dispensed, and quality control was carried out to obtain the polypeptide beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val.

[0038] Example 2: Synthesis of Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His 1. Fmoc-His(Trt)-CTC resin was obtained by coupling the solid-phase supported 2-CTC resin with Fmoc-His(Trt)-OH in the presence of an activating agent system (HoBT, DIC). The Fmoc protecting group on Fmoc-His(Trt)-CTC was removed with 2.20% piperidine, and after removal, it was washed with DMF. A 3.3-fold excess of Fmoc-beta-Ala-OH and a 3-fold excess of the activator were weighed out and completely dissolved in a small amount of DMF. After dissolution, the resin was added to the washed resin and reacted for 1 hour, followed by washing with DMF.

[0039] 4. Steps 2 and 3 were repeated to couple amino acids with N-Fmoc protection and side chain protection in the order of the main chain from Val to Lys. 5. After the synthesis was completed, the peptide resin was decomposed using a decomposition reagent ratio of TFA:EDT:Tis:TA:anisole:HO=80:5:1:5:5:4 (volume ratio). 10 mL of the decomposition reagent was used for 1 g of peptide resin. The decomposition was carried out at room temperature for approximately 2 hours (120 r / min), followed by precipitation with ice-cold methyl tert-butyl ether. The lower precipitate was the crude product.

[0040] 6. The crude peptide obtained in the previous steps was taken out, dissolved, and purified by preparative HPLC with 0.1% TFA / acetonitrile. 7. After purification, lyophilization was carried out, after which the powder was removed and dispensed, and quality control was carried out to obtain the polypeptide Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His.

[0041] Example 3: Synthesis of beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His 1. Fmoc-His(Trt)-CTC resin was obtained by coupling the solid-phase supported 2-CTC resin with Fmoc-His(Trt)-OH in the presence of an activating agent system (HoBT, DIC). The Fmoc protecting group on Fmoc-His(Trt)-CTC was removed with 2.20% piperidine, and after removal, it was washed with DMF. A 3.3-fold excess of Fmoc-beta-Ala-OH and a 3-fold excess of the activator were weighed out and completely dissolved in a small amount of DMF. After dissolution, the resin was added to the washed resin and reacted for 1 hour, followed by washing with DMF.

[0042] 4. Steps 2 and 3 were repeated to couple amino acids with N-Fmoc protection and side chain protection in the order of the main chain, from Val to beta-Ala. 5. After the synthesis was completed, the peptide resin was decomposed using a decomposition reagent ratio of TFA:EDT:Tis:TA:anisole:HO=80:5:1:5:5:4 (volume ratio). 10 mL of the decomposition reagent was used for 1 g of peptide resin. The decomposition was carried out at room temperature for approximately 2 hours (120 r / min), followed by precipitation with ice-cold methyl tert-butyl ether. The lower precipitate was the crude product.

[0043] 6. The crude peptide obtained in the previous steps was taken out, dissolved, and purified by preparative HPLC with 0.1% TFA / acetonitrile. 7. After purification, lyophilization was carried out, after which the powder was removed and dispensed, and quality control was carried out to obtain the polypeptide beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His.

[0044] Example 4: The following polypeptides can be synthesized in a similar manner. beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His); beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His); beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val;

[0045] Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His); beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His).

[0046] Example 5: Animal Experiment Method Monitor for small animals: Shenzhen Rongxianda Technology Co., Ltd., VT200. SD rats: National Institutes for Food and Drug Control, 180 - 200 g. Suture emboli: Maiyue Biotechnology (M8507). TTC staining solution: Yuanye Biotechnology (R24053).

[0047] Operating procedure for animal model: Measure the weight of the SD rats used in the experiment and anesthetize them with chloral hydrate. After anesthesia, fix the limbs of the rats, lay them on their backs, connect them to the monitor for small animals, and monitor important physiological indicators such as the body temperature, blood pressure, and heart rate of the rats. Depilate the necks of the rats, disinfect them with 75% alcohol cotton, and then make an incision about 2 cm in the center of the necks of the experimental rats. During the separation process, avoid the submandibular glands of the rats as much as possible and separate them bluntly. Next, bluntly separate the left common carotid artery (CCA) of the experimental rats, and carefully separate the internal carotid artery (ICA) and external carotid artery (ECA) upward along the CCA. Make sure not to damage the vagus nerve during the separation process. Use two arterial clips to clamp the CCA and ICA, cut off the distal end of the ECA with scissors, insert a silicon suture embolus from the ECA "port", insert it into the ICA arterial clip, briefly release the arterial clip, quickly insert the head end of the suture embolus from there, clamp the ICA again, then slowly release the arterial clip of the ICA, and continue to insert the suture embolus until the black mark of the suture embolus passes through the bifurcation of the ECA and ICA. After the head end of the suture embolus blocks the middle cerebral artery, tightly tie the ECA "port" and the suture embolus with a suture to prevent the suture embolus from "falling out" or bleeding when the rat wakes up. Loosen and remove the arterial clips of the CCA and ICA, return the tissue to its original position, and add an appropriate amount of penicillin to prevent wound infection. Suture the wound with a medical suture needle with thread, seal it, and sterilize it again with iodoform. Observe whether the indicators of the experimental rats are within the normal range with the monitor for small animals. Then, place the rats on an electric blanket for small animals to keep their body temperature until they wake up, and put them in an animal cage.

[0048] Administration Instructions: A 3 mg / mL solution of the test drug was prepared and administered via the tail vein 1 to 2 hours after ligation. When administering the drug, the rat was fixed with a restrainer, and the test drug was drawn up into a 1 mL syringe at a dose of 3 mg / kg, and then slowly injected into the tail vein of the rat to reduce the cardiopulmonary load of the experimental rat. The animals were divided into different experimental groups of six animals each. Model group: After ligation, an equal volume of saline was administered into the tail vein. Drug groups S1 (NA1) and S3 (beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val): After ligation, drugs were administered into the tail vein. Sham-operated group: The internal carotid artery (ICA) and external carotid artery (ECA) were isolated, but not ligated, and an equal volume of saline was administered into the tail vein.

[0049] Behavioral observation: Behavioral observations and scoring were performed on each animal 22-24 hours after ligation. A blinded evaluation was performed by a technician who was not involved in the study according to the following evaluation criteria: 0 points: Walks normally in a straight line. 1 point: Weakness of the front legs. 2 points: Weakness in hind legs. 3 points: Mild rotation. 4 points: Severe rotation. 5 points: Hemiplegia.

[0050] TTC staining and quantitative analysis: After behavioral observation, the experimental rats were euthanized and their brains were removed. The brain tissue was cut into six 2-mm-thick slices transversely, transferred to TTC staining solution, and incubated in a 37°C incubator for 10 minutes in the dark. Photographs were then taken (the results are shown in Figures 1-4). The TTC-stained brain tissue and the remaining small amount of unstained brain tissue were then stored at -20°C.

[0051] Photographs after TTC staining were quantitatively analyzed using ImageJ software. Cerebral infarct volume % = (total infarct area * section thickness) / (total brain section area * section thickness) * 100%. Test Results: 1. The TTC quantitative results are shown in Table 1.

[0052] [Table 1]

[0053] As shown in Table 1, the infarct volume of rats injected with saline was 0.390 ± 0.028 mm, and that of rats injected with 3 mg / kg Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val (S3) was 0.150 ± 0.047 mm. The infarct volume of the control group was 0.044 ± 0.005 mm. Compared with the model group, S3 showed significant biological activity, with no statistical difference between S1 and S2. Among them, NA1 (Nerinetide), a neuroprotective agent (Michael Tymianski and Jonathan D. Garman. Model systems and treatment regimes for treatment of neurological disease. 2015, US Patent, US8940699B2), inhibits postsynaptic density protein 95 (PSD-95) by terminating intracellular NO radical production, reducing infarct size and improving functional outcomes in experimental rhesus monkey cerebral ischemia-reperfusion preclinical ischemic stroke models, making it a good positive control. Dr. Hill investigated the efficacy and safety of intravenously administered NA-1 (2.6 mg / kg) in patients with acute ischemic stroke (AIS) who underwent endovascular thrombectomy, and found that NA1 administration improved patient outcomes. (Michael D Hill et al. Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke (ESCAPE-NA1): a multicentre, double-blind, randomized controlled trial. Lancet. Published online February 20,2020).

[0054] 2. The behavioral evaluation results are shown in Table 2.

[0055] [Table 2]

[0056] As can be seen from the results in Table 2, the synthetic peptides provided by the present invention were physiologically active. The behavioral score of cerebral ischemia rats injected with saline was 3.8±0.8, while the score of cerebral ischemia rats injected with 3 mg / kg of beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val (S3) was 2.5±1.0. In comparison, the score of cerebral ischemia rats intravenously administered with 3 mg / kg of NA1 was 2.0±0.6. The result for the control sham-operated group was 0.0±0.0. Compared with the model group, S3 showed significant biological activity, and there was no statistical difference between S3 and S1.

[0057] Example 6: Combined administration test of S3 (beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val) and the stroke drug t-PA 1. Preparation of test sample: t-PA was prepared using recombinant human TNK tissue-type plasminogen activator (Mingfule) for injection from Guangzhou Mingkang Bio-Engineering Co., Ltd., at 1.0x10E7IU / 16mg / animal. For example, when the animal weighed 300g, the preparation method was as shown in Table 3 below.

[0058] [Table 3]

[0059] All preparations for administration must be performed aseptically in a biological safety cabinet, and all consumable materials used must be sterilized. The prepared S3 was stored in an icebox in a dark place and allowed to return to room temperature before injection. After preparing t-PA, it was temporarily stored at room temperature.

[0060] 2. Animal-based / grade Animal system: Sprague-Dawley (SD) rats. Grade: SPF grade. Number and sex of animals at the start of the adaptation period: 40, males. Number and sex of animals used: 36 males. Age and weight of the animals

[0061] Body weight: 151.78-186.9g at the start of the adaptation period, 242.95-289.31g at the time of group allocation. Age: Approximately 5-7 weeks old at the start of the adaptation period, 6-8 weeks old at the time of group allocation. Source: Sibaifu (Beijing) Biotechnology Co., Ltd. Adaptation of animals to the environment: After receiving the animals, they were allowed to adapt to the environment for 5 days. The main inspection items during the adaptation period included whether the quality indicators required at the time of ordering were met, their general condition, and whether their body weight reached the weight range required for the study. Unsuitable animals were not allowed to participate in the study.

[0062] Animal housing: Plastic rat cages (L x W x H: 46.6 cm x 30 cm x 21.5 cm); 5 rats / cage for adaptation period, 3-4 rats / cage for main test. Rearing environment conditions standard: National Standard of the People's Republic of China GB14925-2010. Animal environment control system: WINCC7.3 EMS series machine room environment monitoring system. Temperature: Room temperature 20~26°C (daily difference ≦4°C). Relative humidity: 40~70%. Lighting: Artificial lighting, 12 / 12 hour day / night alternating light and dark.

[0063] Ventilation rate: More than 15 ventilations per hour. Feed type: rat feed, feed lot number 21103213, purchased from Beijing Keao Xieli Feed Co., Ltd.

[0064] During the adaptation period, the animals' general condition was observed and no abnormalities were found. After the adaptation period, the animals' weights were 185.46-230.82g, which was below the weight required for constructing an animal model, so the adaptation period was extended to 12 days. After the adaptation period, animals weighing 240-290g were selected and divided into groups to construct the model. 24 hours after administration, animal 2M006 in group S3 died, and an autopsy revealed that animal 1M005 in the model control group had lost brain tissue, which was confirmed to be a brain development abnormality. Furthermore, during autopsy, subarachnoid hemorrhage was found in animal numbered 2M001 in the S3 group and animal numbered 4M002 in the S3 + t-PA group. The cerebral infarction area of ​​animal numbered 4M009 in the S3 + t-PA group was 25.19%, which was greater than the mean value ±3 standard deviations of the remaining animals in this group and was determined to be an outlier. The data of these animals were not included in the final statistical analysis, while the data of the remaining animals in each group were all included in the final statistical analysis. The number of animals finally included in the statistical analysis was 8 in the model control group, 7 in the S3 group, 9 in the t-PA group, and 7 in the S3 + t-PA group, respectively.

[0065] 3. Construction of the Rat Model 1) Induction of anesthesia: The rat was placed in an anesthesia induction box filled with 3.0% isoflurane, and anesthesia was induced. 2) Fixation: After anesthesia induction, the animal was moved to the operating table and anesthesia was continued by administering 2.0% to 2.5% isoflurane at 200 mL / min using a small animal gas anesthesia machine. The rat's eyelid reflex and nociceptive response were observed, and surgery began once the eyelid reflex and nociceptive response in the limbs and tail had disappeared. 3) Cerebral ischemia-reperfusion surgery steps:

[0066] A. Isolation and exposure of blood vessels: The skin at the surgical site was shaved, and the rat was placed under a surgical microscope. The skin was cut along the midline with ophthalmic scissors to a length of approximately 2 cm. The right cervical musculature was bluntly separated and retracted with microscope tweezers to expose the right common carotid artery (CCA). The common carotid artery was then cut upward along its length, exposing the external carotid artery (ECA) and internal carotid artery (ICA). B. The proximal end of the CCA was ligated, and the CCA at the Y-branch was temporarily clamped with an arterial clip. A thread was passed from the CCA ligation site to the provisional occlusion site of the ICA, loosely tied for backup (pre-ligation), and a small opening was cut at the proximal end of the pre-ligation. C. Pushing the suture: A No. 4-0 suture was inserted through the CCA incision and slowly and gently pushed into the internal carotid artery. When it reached the ICA arterial clip, the suture was temporarily tightened (to prevent excessive bleeding during the push). The clip was then removed, preventing blood flow from the ICA. The suture was then pushed into the ICA until it was inside the skull. Care was taken to avoid the suture entering the pterygopalatine artery, a branch of the internal carotid artery. (The pterygopalatine artery is classified as an extracranial branch of the ICA, and once the suture penetrated approximately 10 mm deep, it could no longer be inserted further. Therefore, the suture was withdrawn slightly, adjusted, and then resumed.)

[0067] D. Fixation of the suture embolus and closure of the incision: The suture embolus was inserted approximately 18 mm deep from the bifurcation of the common carotid artery. If slight resistance was felt, this indicated that the tip of the suture embolus had entered the anterior cerebral artery (ACA) and that the side wall of the suture embolus had occluded the opening of the middle cerebral artery. Therefore, insertion was stopped, the time was recorded, the arterial clip of the CCA was removed, and the incision was closed after confirming that there was no active bleeding. E. Cerebral reperfusion: The ischemic rat was placed at room temperature, and anesthesia was induced 120 minutes later. While maintaining the anesthetized state, the suture embolus was slowly and gently pulled, causing the head end to return to the common carotid artery, thereby achieving reperfusion of the middle cerebral artery. F. The incision was disinfected with iodoform.

[0068] 4. Grouping of animals Group design: Four groups were established: model control group, S3 group, t-PA group, and S3+t-PA group. Number of animals: 9 animals per group, 36 in total. Gender ratio: According to reports by JW Simpkins and RL Roof, estrogen and progesterone have neuroprotective effects against ischemic stroke in adult rats. However, to exclude the influence of estrogen and progesterone on the results of this study, all test animals were male.

[0069] Grouping method: The rats were randomly assigned to groups based on their most recent body weight before grouping. Specific grouping information is shown in Table 4 below.

[0070] [Table 4]

[0071] Note: The first digit of the animal number indicates the group (1, 2, 3, and 4 represent the model control group, S3 group, t-PA group, and S3 + t-PA group, respectively), the second letter indicates the sex (M means male), the last three digits indicate the animal's sequence number, a indicates administration of 3 mL of S3 and 3 mL of sterile water for injection, b indicates administration of 3 mL of t-PA and 3 mL of sterile water for injection, and c indicates administration of 3 mL of S3 and 3 mL of t-PA. Administration route: The drug was administered by injection into the tail vein. Administration time: immediately after ischemia-reperfusion (within 5 minutes). Dosage frequency and interval: Administered once. The day of model construction was defined as D0, and the day before as D-1.

[0072] 5. Observation and measurement details General condition observation Observed animals: All surviving experimental animals were observed. Observation time: Once a day. If any abnormalities are observed in the animals, the number of observations can be increased. Observations include, but are not limited to, general appearance, behavior, and signs of toxicity in the eyes, mouth, nose, ears, hair, skin, feces, urine, and genitals. Any abnormalities must be detailed.

[0073] body weight Animal measurements: All living experimental animals were included in the measurements. Measurement time: Body weight was measured at least twice during the adaptation period, once 24 hours before surgery for grouping, and once 24 hours after surgery.

[0074] Behavioral assessment Bederson scoring Detection time: 50-60 min after ischemia on the day of model construction was used to evaluate whether animal ischemia was successful or not. Animals studied: All animals that survived surgery. Detection method: Animals were scored according to the Bederson scoring criteria.

[0075] [Table 5]

[0076] Rats with a post-ischemic score of 1 or more are considered to have successfully constructed a model, and rats that fail to construct a model are culled.

[0077] Rat neurological function scoring Detection time: Scoring was performed once 24 hours before model construction, once 24 hours after model construction, and once 72 hours after model construction. Animals measured: All living experimental animals were included in the measurements. Detection methods: Animal motor function tests, sensory tests, balance tests, reflex and abnormal movement tests, specifically refer to the rat neurological scoring scale (NSS).

[0078] [Table 6]

[0079] Autopsy and pathological examination Dissection time: D1 (corresponding to 24 h after model construction) (the day of model construction is defined as D0); Dissected animals: all living animals of each group; Anesthesia and euthanasia method: The animals were anesthetized by intraperitoneal injection of 3% sodium pentobarbital at a dose of 60 mg / kg. After anesthesia, the animals were euthanized by exsanguination from the abdominal aorta.

[0080] Pathological (TTC staining) evaluation Detected animals: all living animals; Testing method: The brains of euthanized animals were quickly removed, and the rat brains were placed in a -20°C refrigerator and frozen until the brain tissue hardened. They were then removed and cut into 2mm-thick slices, positioned from the olfactory bulb toward the back, for a total of 6-8 slices. Each slice was placed in a 6-well plate containing 0.5% TTC solution (prepared with PBS), and then placed in a 37°C incubator and incubated away from light for 20 minutes. After that, the slices were removed and placed in 10% formaldehyde solution and stored away from light.

[0081] Normal tissue was stained pink, and infarcted tissue was stained white. Each brain plane was placed on filter paper in turn and photographed with a digital camera. The white tissue was carefully removed and weighed. The infarct area (%) was calculated as the ratio of the infarcted tissue weight to the total brain weight, or the cerebral infarction area was calculated using image processing. The inhibition rate (%) of each drug treatment group was calculated based on the infarct area, and the calculation formula is as follows: JPEG0007770535000007.jpg20167

[0082] statistical analysis Measurements are expressed as mean ± standard deviation. Groups with fewer than three samples were not included in statistical comparisons.

[0083] Data were entered and statistically analyzed using Excel 2010, GraphPad Prism 7, SPSS 22.0, and Stata 15.0 software. Measurement indices were first subjected to the Levene test for equality of variance. If the variances were consistent (p>0.05), the results of the analysis of variance were used to determine whether the overall difference was statistically significant. If the overall difference was statistically significant (p<0.05), the Dunnett t-test was used to compare the differences between groups. If the overall difference was not statistically significant (p≥0.05), the statistical analysis was terminated. If the Levene test for equality of variances showed that the variances were not equal (p≦0.05), a nonparametric test (Kruskal-Wallis H test) was used; if the Kruskal-Wallis H test showed that the overall difference was statistically significant (p<0.05), the Mann-Whitney U test was used to compare the differences between groups; statistical analysis was terminated if the Kruskal-Wallis H test showed that the overall difference was not statistically significant (p≧0.05).

[0084] 6. Test Results Effects of S3 and S3+t-PA on the extent of cerebral infarction in animal models: A central part of acute ischemic stroke treatment is to minimize the extent and range of neuronal damage caused by ischemia. In this study, drug intervention was performed immediately after ischemia-reperfusion, and 24 hours later, TTC staining was performed on the brains of model animals. The infarct size of each group was measured and the inhibition rate was calculated to evaluate the pharmacological efficacy of S3 and the combination of S3 and t-PA on ischemic stroke. The cerebral infarction size and inhibition rate of cerebral infarction in the animals are shown in Table 7 and Figures 5, 7-10.

[0085] The cerebral infarction area in the model control group 24 hours after surgery was 21.493 ± 2.734%. The cerebral infarction areas in the S3 group, t-PA group, and S3 + t-PA group were 16.248 ± 1.749%, 18.522 ± 1.372%, and 17.203 ± 2.098%, respectively, all of which were significantly lower than those in the model control group (P ≤ 0.05). The cerebral infarction suppression rates in the S3 group, t-PA group, and S3 + t-PA group were 32.279 ± 7.291%, 22.802 ± 5.719%, and 28.301 ± 8.746%, respectively. These results suggest that under the experimental conditions, the S3 group, t-PA group, and S3 + t-PA group have significant suppressive effects on cerebral infarction in rats with ischemic stroke. The Q value for the combined use of S3 and t-PA was estimated based on the average cerebral infarction suppression rate for each drug group, and was found to be 0.593, suggesting that under the experimental conditions, 3 mg / kg of S3 and 3 mg / kg of t-PA did not have a significant synergistic effect on the suppression of cerebral infarction.

[0086] [Table 7]

[0087] Note: All data in the table are expressed as mean ± standard deviation (Mean ± SD). "N" is the number of animals in each group statistically analyzed. "-" indicates no data for that item. ** indicates P<0.01 compared to the model control group. *** indicates P<0.005 compared to the model control group. The Q value is calculated as follows: mean cerebral infarction inhibition rate of S3 + t-PA group / (mean cerebral infarction inhibition rate of S3 group + mean cerebral infarction inhibition rate of t-PA - mean cerebral infarction inhibition rate of S3 group × mean cerebral infarction inhibition rate of t-PA). The Q value criteria are: Q<0.85 indicates antagonism, 0.85≦Q<1.15 indicates additive effect, and Q≧1.15 indicates synergism.

[0088] Effects of S3 and S3+t-PA on neurobehavioral scoring in animal models: The primary goal of clinical stroke treatment is to maximize neurological recovery and improve the patient's ability to function. In this study, animals were scored by Bederson 1 hour after ischemia to determine their ischemic state. Neurobehavioral function of surviving animals 24 hours after model construction was assessed using the NSS scoring system to evaluate the neurological function improvement effects of S3 and S3 + t-PA. The Bederson scoring results for the animals are shown in Tables 8 and 10, and the NSS scoring results are shown in Tables 9 and 10 and Figure 6.

[0089] One hour after ischemia, the Bederson score for all model animals was 3, indicating that all model animals were ischemic. One day after surgery, the NSS score for the ischemia-reperfusion model control group was 7.75±1.71, while the NSS scores for the S3 group, t-PA group, and S3+t-PA group were 6.71±1.79, 6.83±0.75, and 5.36±1.30, respectively. The NSS score for the S3+t-PA group was significantly lower than that of the model control group (P=0.0385). The reduction rate of NSS scores for each treatment group compared to the model control group was calculated; the reduction rates for the S3 group, t-PA group, and S3+t-PA group were 13.3% and 13.4%, respectively. The Q value for the combination of S3 and t-PA was calculated based on the rate of reduction in NSS, and the resulting Q value was 1.308, suggesting that S3 and t-PA have a synergistic effect on improving NSS scores. These results suggest that under the experimental conditions of this study, the combination of S3 and t-PA has a significant improving effect on neurobehavior in stroke rats, and that S3 and t-PA have a synergistic effect on improving neurological function damage in ischemia-reperfusion rats.

[0090] [Table 8]

[0091] Note: All data in the table are expressed as mean ± standard deviation (Mean ± SD), and "N" is the number of animals in each group that were statistically analyzed.

[0092] [Table 9]

[0093] Note: All data in the table are expressed as mean ± standard deviation (Mean ± SD), "N" is the number of animals in each group statistically analyzed, * indicates P<0.05 compared to animals in the model control group, and Q value is calculated as follows: mean NSS reduction rate of S3 + t-PA group / (mean NSS reduction rate of S3 group + mean t-PA reduction rate of cerebral infarction - mean S3 group reduction rate of cerebral infarction × mean t-PA reduction rate of cerebral infarction), with Q value criteria being Q<0.85 indicating antagonism, 0.85≦Q<1.15 indicating additive effect, and Q≧1.15 indicating synergism.

[0094] Effects of S3 and S3+tPA on body weight in model animals: Cerebral ischemia-reperfusion was used as an acute injury model. After the MCAO model was established, the experimental animals' body weight significantly decreased. Animal weight is one of the most basic and sensitive indicators for comprehensively reflecting an animal's health. In this experiment, the body weight of surviving animals was monitored one day before and one day after surgery, and changes in body weight were observed and recorded. The results showed that there was no significant difference in the body weight of animals in each group before and 24 hours after model establishment. These results suggest that under the experimental conditions, there is no significant effect on the weight changes of animals in each group.

[0095] General condition observations: After constructing the animal behavioral MCAO models in the control and treatment groups, all animals exhibited symptoms such as unsteady gait, salivation, and rolling, with no significant differences between groups. 24 hours after surgery, surviving animals frequently exhibited abnormal stroke symptoms, such as unsteady gait, rolling, piloerection, salivation, and stains around the nose, eyes, and mouth. There were no significant differences in the type or severity of abnormal symptoms between groups.

[0096] As described above, this study used the classical suture embolization method to establish a MCAO ischemia-reperfusion model. We evaluated the effects of S3 and the combination of S3 and t-PA on stroke and explored whether there was a synergistic effect between S3 and t-PA. Both the control and treatment groups received a single dose of S3 immediately (within 5 minutes) after ischemia-reperfusion. One day after surgery, neurological damage was assessed using NSS scores. Brain tissue was dissected and TTC stained to measure the extent of cerebral infarction. The neuroprotective effects of S3 and the combination of S3 and t-PA on ischemic stroke in rats were evaluated based on the extent of cerebral infarction and improvement in neurological function. The results are as follows:

[0097] A) S3 and the combination of S3 and t-PA significantly suppressed cerebral infarction in rats with stroke. One day after surgery, the cerebral infarction areas of animals in the S3 group, t-PA group, and S3 + t-PA group were significantly reduced compared to the model control group (P ≤ 0.05). The cerebral infarction suppression rates of animals in the S3 group, t-PA group, and S3 + t-PA group were 32.279 ± 7.291%, 22.802 ± 5.719%, and 28.301 ± 8.746%, respectively. These results suggest that under the experimental conditions, S3 group, t-PA group, and S3 + t-PA have a significant suppressive effect on cerebral infarction in rats with ischemic stroke. B) The combination of S3 and t-PA significantly reduced neurological damage in rats with stroke. Twenty-four hours after surgery, the NSS scores of animals in the S3, t-PA, and S3 + t-PA groups were reduced to different degrees compared with the model control group. The NSS scores of animals in the S3 + t-PA group were significantly reduced compared with the model control group (P = 0.0385). The reduction rates of NSS scores of animals in each treatment group compared with the model control group were 13.36 ± 23.1%, 11.83 ± 9.62%, and 30.88 ± 16.79%, respectively. These results suggest that, under the experimental conditions, the combination of S3 and t-PA significantly improved neurological damage in rats with ischemic stroke.

[0098] C) S3 and t-PA had a synergistic effect on improving neurological impairment in stroke-affected rats. In this study, the NSS score and Q value of the cerebral infarction inhibition rate were calculated based on the rate of neurological decline and the rate of cerebral infarction inhibition when S3 was combined with t-PA. The NSS score and Q value of the cerebral infarction inhibition rate when S3 was combined with t-PA were 1.308 and 0.593, respectively. The Q value for NSS score was greater than 1.15, suggesting that S3 and t-PA have a synergistic effect on improving neurological impairment in stroke-affected rats, but that there was no clear synergistic effect in terms of cerebral infarction inhibition under the experimental conditions.

[0099] In short, the results of this study demonstrated that S3, t-PA, and the combination of S3 and t-PA had significant therapeutic effects on acute ischemic stroke, and that S3 and t-PA had a synergistic effect on improving neurobehavior in rats with acute ischemic stroke.

[0100] [Table 10]

[0101] [Table 11]

[0102] [Table 12]

[0103] The above content is only a preferred embodiment of the present invention and is not intended to restrict the present invention, and any modifications, equivalent replacements, improvements, etc. made within the spirit and rules of the present invention shall be included in the protection scope of the present invention.

Claims

1. A neuroprotective polypeptide compound characterized by being a polypeptide having the following chemical formula or a salt thereof: beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His; beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His; beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His); beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His); beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His); beta-Ala-(1-Methyl-His)-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-(1-Methyl-His).

2. The neuroprotective polypeptide compound of claim 1, which is a polypeptide having the following chemical formula or a salt thereof: beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val; Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His.

3. The neuroprotective polypeptide compound of claim 1, which is a polypeptide having the following chemical formula or a salt thereof: beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val.

4. 4. Use of a neuroprotective polypeptide compound according to any one of claims 1 to 3 in medicine.

5. The neuroprotective polypeptide compound according to any one of claims 1 to 3, for use in the treatment of a nervous system disease selected from ischemic stroke, hemorrhagic stroke, brain injury, Alzheimer's disease and Parkinson's disease.

6. A pharmaceutical comprising the neuroprotective polypeptide compound according to any one of claims 1 to 3, the pharmaceutical being in the form of an injection, oral preparation, sublingual preparation, spray, or anal preparation.

7. A composition comprising a neuroprotective polypeptide compound according to any one of claims 1 to 3 and a thrombolytic agent.

8. 8. The composition of claim 7, which is used to treat a nervous system disease selected from ischemic stroke, hemorrhagic stroke, brain injury, Alzheimer's disease, and Parkinson's disease.

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