WO2023006083A1 - 一种神经保护多肽化合物及其应用 - Google Patents

一种神经保护多肽化合物及其应用 Download PDF

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WO2023006083A1
WO2023006083A1 PCT/CN2022/109048 CN2022109048W WO2023006083A1 WO 2023006083 A1 WO2023006083 A1 WO 2023006083A1 CN 2022109048 W CN2022109048 W CN 2022109048W WO 2023006083 A1 WO2023006083 A1 WO 2023006083A1
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李健雄
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Innerse Zhuhai Pharmaceutical Co Ltd
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Innerse Zhuhai Pharmaceutical Co Ltd
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Priority to EP22848687.4A priority patent/EP4378949A4/en
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21068—Tissue plasminogen activator (3.4.21.68), i.e. tPA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids

Definitions

  • the invention belongs to the technical field of medicines, in particular to a neuroprotective polypeptide compound and its application.
  • Nerinetide (NA-1) is a neuroprotective agent that interferes with postsynaptic density protein 95 (PSD-95) by stopping the production of intracellular NO free radicals. It reduces the infarct size of cerebral ischemia-reperfusion and improves its functional outcome in a preclinical ischemic stroke model. For adult patients with acute ischemic stroke due to large vessel occlusion within the 12-hour treatment window, randomized to receive a single dose of 2.6 mg/kg of Nerinetide with a maximum dose of 270 mg, or placebo in normal saline.
  • the primary endpoint of the study was good functional outcome 90 days after randomization, defined as a Modified Rankin Scale (mRS) score of 0-2; secondary endpoints were neurological disability, functional independence in activities of daily living, good Functional outcome (mRS0-1) and mortality.
  • mRS Modified Rankin Scale
  • the proportion of patients with mRS score 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.
  • this study found that in patients receiving alteplase, treatment with nerinetide resulted in suppression of the effect of alteplase.
  • Nerinetide is a fusion peptide composed of the C-terminal 9 residues of the NMDAR GluN2B subunit and the transmembrane peptide TAT derived from nuclear translocation activator, which can bind to the PDZ-1 or PDZ-2 domain of PSD-95 to inhibit nNOS
  • the production of NO so it is also called Tat-NR2B9c, and 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 following partial sequence Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val is the sequence of NR2B9c, which specifically inhibits the production of NO in nNOS.
  • the preceding partial sequence Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg increases the bioavailability of NR2B9c.
  • the amino acid sequences of active peptides like NR2B9c consist of 3–25 amino acids from the C-terminus of the NMDA receptor or from PDZ domains 1 and/or 2 of the PSD-95 receptor linked to internalization peptides (A. Tasker, T. Doucette, M. Tymianski, K. Mendoza, M. P.
  • Such active peptides have an amino acid sequence comprising [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L], such as KLSSIETDV and KLSSIESDV.
  • Carnosine is a dipeptide composed of ⁇ -alanine and L-histidine. Carnosine has a strong antioxidant capacity and is beneficial to the human body. Carnosine has been shown to scavenge reactive oxygen species (ROS) and ⁇ - ⁇ -unsaturated aldehydes formed by excessive oxidation of fatty acids in cell membranes during oxidative stress. Carnosine has anti-inflammatory, anti-glycation, anti-oxidation and chelating effects. It can be used as an over-the-counter food supplement and has good prospects in the prevention and auxiliary treatment of chronic diseases such as cardiovascular disease and neurodegenerative diseases. In animal experiments, the neuroprotective mechanism of carnosine can prevent permanent cerebral ischemia.
  • ROS reactive oxygen species
  • ⁇ - ⁇ -unsaturated aldehydes formed by excessive oxidation of fatty acids in cell membranes during oxidative stress.
  • Carnosine has anti-inflammatory, anti-glycation, anti-oxidation and chelating effects. It can be used as an
  • Carnosine is also an important intracellular antioxidant. Carnosine is not only non-toxic, but also has strong antioxidant properties, so it has attracted widespread attention as a new type of food additive and pharmaceutical reagent. Carnosine participates in the peroxidation reaction in the cell. In addition to inhibiting the peroxidation process of the cell membrane, it can also inhibit the related peroxidation reaction in the cell.
  • these dipeptides are also present in other tissues, such as brain tissue.
  • These carnosine derivatives are water-soluble and strong, and have significant anti-oxidation, anti-aging, and uric acid-lowering functions. They have been used as natural antioxidants and uric acid-lowering diet therapy in the food industry, and they also have certain neuroprotective functions. .
  • carnosine and carnosine derivatives have certain brain protective effects, they often require relatively large doses, and the effects of using carnosine, anserine, and snake carnosine alone are not good, and new neuroprotective methods are still needed in this field.
  • the inventors combined carnosine and carnosine derivatives with active peptides like NR2B9c to produce a new type of polypeptide, unexpectedly found that this new combination of polypeptides is completely different from Nerinetide, has good neuroprotective effect, and neuroprotective effect It is not affected by thrombolytic products such as alteplase, thus providing a new method for neuroprotection.
  • the present invention provides a neuroprotective polypeptide compound, which contains carnosine, anserine, snake carnosine, etc. composed of beta-alanine and histidine, 1-methylhistidine or 3-methylhistidine, etc. , and the active peptide represented by NR2B9c, the amino acid sequence of the active peptide is Lys-Leu-Ser-Ser-Ile-Glu/Asp-Ser/Thr-Asp/Glu-Val/Leu, especially NR2B9c Lys-Leu-Ser -Ser-Ile-Glu-Ser-Asp-Val, thus forming a series of carnosine, anserine or snake carnosine and Lys-Leu-Ser-Ser-Ile-Glu/Asp-Ser/Thr-Asp/Glu- Combination polypeptide compounds with Val/Leu characteristics.
  • These peptides penetrate the blood-brain barrier unexpectedly, and exhibit good biological activity through intravenous administration, thus showing broad
  • a neuroprotective polypeptide compound including a polypeptide having the following chemical formula and a salt thereof:
  • m is an integer of 0-3, n is an integer of 0-3, but m and n are not zero at the same time;
  • M is beta-Ala-His, beta-Ala-1-Methyl-His or beta-Ala- 3-Methyl-His,
  • N is beta-Ala-His, beta-Ala-1-Methyl-His or beta-Ala-3-Methyl-His.
  • Glu/Asp means that the amino acid at this position can be any one of Glu or Asp
  • Ser/Thr means that the amino acid at this position can be any one of Ser or Thr
  • Asp/Glu means that the amino acid at this position can be any of Asp or Glu
  • Any one of Val/Leu means that the amino acid at this position can be any one of Val or Leu.
  • Lys-Leu-Ser-Ser-Ile-Glu/Asp-Ser/Thr-Asp/Glu-Val/Leu is Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val.
  • neuroprotective polypeptide compounds of the present invention include polypeptides having the following chemical formula and salts thereof:
  • His is His, 1-Methyl-His or 3-Methyl-His, that is, M or N for short, corresponding to carnosine, anserine and snake carnosine respectively.
  • neuroprotective polypeptide compounds of the present invention include polypeptides having the following chemical formula and salts thereof:
  • the neuroprotective polypeptide compounds of the present invention include polypeptides having the following chemical formula and salts thereof:
  • neuroprotective polypeptide compounds of the present invention include polypeptides having the following chemical formula and salts thereof:
  • neuroprotective polypeptide compounds of the present invention include polypeptides having the following chemical formula and salts thereof:
  • the application of the aforementioned neuroprotective polypeptide compound in the preparation of medicines is also provided. Also provided is the aforementioned neuroprotective polypeptide compound for use in treating nervous system diseases. Also provided is a method for treating nervous system diseases using the aforementioned neuroprotective polypeptide compound.
  • the nervous system disease may be ischemic stroke, hemorrhagic stroke, brain trauma, Alzheimer's disease, Parkinson's disease or other neurodegenerative diseases.
  • the neurological disease is ischemic stroke.
  • the neuroprotective polypeptide compound provided by the invention can be prepared as medicine.
  • the aforementioned medicines are injections, oral medications, sublingual medications, spray medications or anal medications, etc., preferably injections.
  • the injection is powder injection or injection.
  • the aforementioned drugs are administered intravenously.
  • the aforementioned drugs can also be used as active ingredients to prepare other dosage forms, so as to facilitate corresponding medical applications.
  • the developed preparations can include oral administration and sublingual administration preparations.
  • the developed preparations can include spray administration, anal administration and other preparations, so that patients with no mobility can be treated. .
  • the aforementioned medicines include pharmaceutically acceptable diluents or/and carriers and the like.
  • polypeptide drugs in another aspect, it also provides a method for preparing various neuroprotective polypeptide compounds of the present invention by solid-phase synthesis, but it is more convenient to use liquid-phase synthesis or fragment synthesis for some of the peptides.
  • Salt formation of polypeptide drugs is one of the common means to improve the physical and chemical properties of drug molecules and enhance their druggability.
  • the aforementioned drugs can be salts in any form.
  • a combination of the neuroprotective polypeptide compound described herein and a thrombolytic drug is also provided.
  • the thrombolytic drug can be a first-generation thrombolytic drug represented by streptokinase and urokinase, and represented by tissue-type plasminogen activator (tissue-type plasminogen activator, tPA) alteplase and pro-urokinase
  • tissue-type plasminogen activator tissue-type plasminogen activator
  • rtPA recombinant human tissue plasminogen activator
  • urokinase for injection (Tianjin Biochemical Pharmaceutical Co., Ltd.), recombinant streptokinase for injection (such as Sikaitong),reteplase for injection (such as Aitongli), Recombinant human TNK tissue-type plasminogen activator for injection (such as Mefole).
  • the neuroprotective polypeptide compound described herein and the thrombolytic drug can be administered separately or simultaneously, or mixed in any suitable ratio and administered as a combination drug.
  • the combination can be used to prepare medicines, especially medicines for treating nervous system diseases.
  • the nervous system disease may be ischemic stroke, hemorrhagic stroke, brain trauma, Alzheimer's disease, Parkinson's disease or other neurodegenerative diseases.
  • the neurological disease is ischemic stroke.
  • the inventors creatively combined the neuroprotective polypeptide compound with active peptides like NR2B9c, carnosine, anserine or snake carnosine, and the developed combination polypeptide unexpectedly penetrated the blood-brain barrier through intravenous injection and entered the brain, effectively It plays a role in the treatment of neurological diseases.
  • Such synthetic peptides are preferably used in the treatment of ischemic stroke, hemorrhagic stroke, brain trauma, Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases.
  • the present invention only needs 3 mg/kg intravenous injection dose of the synthetic polypeptide, which unexpectedly can achieve significant therapeutic effect, and achieves the same effect as the clinically active standard polypeptide NA1.
  • the synthetic peptide of the present invention is easy to synthesize.
  • derivatives of carnosine, anserine or snake carnosine can also be used as synthetic fragments to replace the corresponding two amino acids to further simplify the synthesis of the target peptide.
  • Figure 1 Slices of the brain tissue of the model group.
  • Figure 2 Slices of the brain tissue of the S1 group.
  • Figure 3 Slicing diagram of brain tissue of S3 group.
  • Figure 4 Slices of brain tissue from the sham group.
  • Figure 5 The results of cerebral infarction area and cerebral infarction inhibition rate, where A: cerebral infarction area (Infarction Area%), B: cerebral infarction inhibition rate (Inhibition Ratio%); ** indicates that compared with the model control group, P ⁇ 0.01, *** indicates P ⁇ 0.005 compared with the model control group.
  • NSS score results the data in the figure are expressed as mean ⁇ standard deviation (Mean ⁇ SD), where A: NSS score results, B: the reduction rate of NSS scores in each administration group relative to the model control group 1 day after operation ; * means P ⁇ 0.05 compared with model control animals.
  • Figure 7 TTC staining results of animals in the model control group.
  • FIG. 8 TTC staining results of animals in group S3.
  • FIG. 10 TTC staining results of animals in the S3+tPA group.
  • the Fmoc-Val-CTC resin is obtained by coupling the solid-phase support 2-CTC resin and Fmoc-Val-OH.
  • the Fmoc-His(Trt)-CTC resin was obtained by coupling the solid-phase support 2-CTC resin and Fmoc-His(Trt)-OH.
  • the Fmoc-His(Trt)-CTC resin was obtained by coupling the solid-phase support 2-CTC resin and Fmoc-His(Trt)-OH.
  • Example 4 The following polypeptides can also be synthesized in the same way
  • Thread bolt Maiyue Bio (M8507).
  • TTC staining solution source leaf organisms (R24053).
  • the experimental SD rats were weighed and anesthetized with chloral hydrate. After anesthesia, the limbs of the rats were fixed, placed on their backs, connected to a small animal monitor, and important physiological indicators such as body temperature, blood pressure and heart rate of the rats were monitored.
  • the neck of the rat was depilated, and after disinfection with 75% alcohol cotton ball, a median incision of about 2 cm in length was made on the neck of the experimental rat, and the submandibular gland of the rat was bluntly separated, and the damage to the gland was avoided as far as possible during the separation process, and then
  • the left common carotid artery (CCA) of the experimental rat was bluntly separated, and the internal carotid artery (ICA) and external carotid artery (ECA) were carefully separated upward along the CCA. Avoid damaging the vagus nerve during the separation.
  • the drug to be tested was made into a 3mg/mL solution. 1-2h after the ligation of the tail vein administration.
  • the rats When administering the drug, the rats were fixed with a fixer, and a 1mL syringe was used to inhale the drug to be tested at a dose of 3 mg/kg, and then the rats were injected into the tail vein, and the injection was injected slowly to reduce the cardiopulmonary load of the experimental rats.
  • Drug groups S1 (NA1) and S3 (beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val), administered through the tail vein after ligation;
  • ICA internal carotid artery
  • ECA external carotid artery
  • mice After behavioral observations, experimental rats were euthanized and their brains were removed. The brain tissue was cut transversely into 6 slices with a thickness of 2 mm, then transferred to TTC staining solution, incubated in a 37°C incubator in the dark for 10 minutes, and photographed (results shown in Figures 1-4). Then the TTC-stained brain tissue and the remaining small amount of non-TTC-stained brain tissue were stored at -20°C.
  • Cerebral infarct volume % (total infarct area*slice thickness)/(total brain slice area*slice thickness)*100%.
  • Model systems and treatment regimens for treatment of neurological disease can interfere with postsynaptic density protein 95 (PSD-95) by terminating the production of intracellular NO free radicals, and can reduce animal experimental ( Macaque) infarct size of cerebral ischemia reperfusion, and improve its functional prognosis, so it is a good positive control product.
  • Dr. Hill studied the efficacy and safety of intravenous administration of a novel neuropeptide NA-1 (2.6 mg/kg) in patients with acute ischemic stroke (AIS) undergoing endovascular thrombectomy and showed that treatment with NA1 improved patient outcomes prognosis.
  • AIS acute ischemic stroke
  • ESCAPE-NA1 a multicentre, double-blind, randomised controlled trial. Lancet. Published online February 20, 2020).
  • Example 6 Combined administration test of S3 (beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val) and stroke drug t-PA
  • t-PA chooses recombinant human TNK tissue plasminogen activator for injection (Mingfule) from Guangzhou Mingkang Bioengineering Co., Ltd., 1.0x10E7IU/16mg/piece. Taking an animal with a body weight of 300 g as an example, the preparation method is shown in Table 3 below.
  • the drug preparations are prepared in a biosafety cabinet for aseptic operation, and the consumables used need to be sterilized.
  • the preparation of S3 was stored in an ice box away from light, and the temperature was returned to room temperature before injection, and the t-PA was prepared and placed in room temperature for temporary storage.
  • Body weight 151.78-186.9g when entering the adaptation period, and 242.95-289.31g when grouping;
  • Age about 5-7 weeks old in the adaptation period, 6-8 weeks old in the test group;
  • Animal environment adaptation Animals adapt to the environment for 5 days after receiving them.
  • the main inspection items during the adaptation period include whether they are consistent with the quality indicators required at the time of ordering, their general status, and whether their weight reaches the weight range required by the test. Unqualified animals are not included in this test;
  • Animal feeding plastic rat cage (L ⁇ W ⁇ H: 46.6cm ⁇ 30cm ⁇ 21.5cm); 5 rats/cage during the environmental adaptation period, 3-4 rats/cage for the formal test;
  • Breeding environment control system WINCC7.3EMS series machine room environment monitoring system
  • Relative humidity 40-70%
  • Lighting artificial lighting, 12/12 hours day and night alternating light and dark;
  • Number of air changes no less than 15 air changes per hour
  • Feed type Rat and mouse breeding feed, feed batch number 21103213, purchased from Beijing Keaoxieli Feed Co., Ltd.
  • the animal numbered 2M006 in the S3 group was found dead, and the brain tissue of the animal numbered 1M005 in the model control group was found to be missing during autopsy, which was determined to be Abnormal brain development, in addition, subarachnoid hemorrhage was found in the animals of S3 group numbered 2M001 and S3+t-PA group numbered 4M002, and the range of cerebral infarction in S3+t-PA group numbered 4M009 was 25.19%, which was larger than that of this group The mean ⁇ 3 standard deviation of the remaining animals was judged as an outlier. The data of the above animals were not included in the final statistical analysis, and the data of the remaining animals in each group were included in the final statistical analysis. The number of animals included in the final statistical analysis was 8 in the model control group. , 7 rats in S3 group, 9 rats in t-PA group and 7 rats in S3+t-PA group.
  • Anesthesia induction put the rat into an anesthesia induction box filled with 3.0% isoflurane for anesthesia induction;
  • CCA right common carotid artery
  • ECA external carotid artery
  • ICA internal carotid artery
  • E. Cerebral reperfusion The ischemic rats are placed at room temperature, and anesthesia can be induced after 120 minutes. While maintaining anesthesia, the thread plug is slowly pulled gently to make the head end return to the common carotid artery, and the middle cerebral artery is reperfused. perfusion;
  • Group design 4 groups were set up, namely model control group, S3 group, t-PA group and S3+t-PA group;
  • Grouping method random grouping according to the latest body weight of rats before grouping
  • the first digit of the animal number represents the group (1, 2, 3 and 4 represent the model control group, S3 group, t-PA group and S3+t-PA group respectively), and the second letter represents the gender (M is male ), the last 3 digits represent the serial number of the animal, a represents the administration of 3mL S3 and 3mL sterile water for injection, b represents the administration of 3mL t-PA and 3mL sterile water for injection, and C represents the administration of 3mL S3 and 3mL t-PA;
  • the day of modeling was defined as D0, and the day before was defined as D-1.
  • Observation time Observe once a day, if the animal is abnormal, the frequency of observation can be increased;
  • Observation content including but not limited to general manifestations, behavioral status, eyes, mouth, nose and mouth, ears, hair and skin, feces, urine, genitals and other toxic symptoms. If there is any abnormality, a detailed description is required.
  • Measuring animals all surviving experimental animals that are planned to be measured;
  • 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.
  • Detection time 50-60 minutes after ischemia on the day of modeling, it is used to evaluate whether the animal ischemia is successful;
  • Detection method Refer to the Bederson scoring standard to score the animals;
  • Detection time 24h before modeling, 24h and 72h after modeling
  • Measuring animals all surviving experimental animals that are planned to be measured;
  • Detection method Carry out motor function test, sensory test, balance test, reflex and abnormal movement test on animals, refer to rat neurological function score scale (NSS) for details.
  • NSS neurological function score scale
  • D1 (equivalent to 24 hours after modeling) (the day of modeling is defined as D0);
  • Dissected animals all surviving animals included in the group;
  • Anesthesia and euthanasia method 3% pentobarbital sodium was used for intraperitoneal injection anesthesia, the injection dose was 60 mg/kg, and the abdominal aorta was bled for euthanasia after anesthesia.
  • Test animals all live animals
  • Inspection method euthanize the animal and quickly remove the brain, put the mouse brain in a -20°C refrigerator and freeze it until the brain tissue hardens, and then take it out. 8 slices were placed in six-well plates filled with 0.5% TTC solution (prepared in PBS), then placed in a 37°C incubator and incubated in the dark for 20 minutes, taken out and stored in 10% formaldehyde solution in the dark.
  • Measurement indicators are expressed as mean ⁇ standard deviation. When the number of samples is less than 3, the data of this group will not be included in the statistical comparison.
  • the data were input and statistically analyzed by Excel 2010, GraphPad Prism 7, SPSS 22.0 and Stata 15.0 software.
  • the LEVENE variance homogeneity test was first used for the measurement indicators. When the variances were homogeneous (p>0.05), the results of the variance analysis could be directly quoted to determine whether the overall difference was statistically significant. When the overall difference was statistically significant (p ⁇ 0.05), use The Dunnett-t test was used to compare the differences between groups.
  • the central link in the treatment of acute ischemic stroke is to minimize the degree and scope of neurocyte lesions caused by ischemia.
  • drug intervention was performed immediately after ischemia-reperfusion, and TTC staining was performed on the brains of model animals 24 hours later.
  • the anti-ischemic stroke pharmacodynamics of S3 and S3 combined with t-PA were evaluated by measuring the infarct size of animals in each group and calculating the inhibition rate.
  • the range of cerebral infarction and the inhibition rate of cerebral infarction in animals are shown in Table 7 and Figures 5, 7-10.
  • the range of cerebral infarction in the model control group was 21.493 ⁇ 2.734% 24 hours after operation.
  • the range of cerebral infarction 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, which were significantly lower than those in the model control group (P ⁇ 0.05).
  • the inhibition rates of cerebral infarction were 32.279 ⁇ 7.291%, 22.802 ⁇ 5.719% and 28.301 ⁇ 8.746% in the group, t-PA group and S3+t-PA group respectively.
  • Table 7 The range of cerebral infarction and the inhibition rate of cerebral infarction in each group of animals
  • Q value is the average cerebral infarction inhibition rate of S3+t-PA group/(average cerebral infarction inhibition rate of S3 group+average cerebral infarction inhibition rate of t-PA-
  • the average cerebral infarction inhibition rate of S3 group ⁇ t-PA average cerebral infarction inhibition rate) is that Q ⁇ 0.85 is an antagonistic effect, 0.85 ⁇ Q ⁇ 1.15 is an additive effect, and Q ⁇ 1.15 is a synergistic effect.
  • the main purpose of cerebral infarction treatment is to restore the patient's neurological function as much as possible and improve the quality of life after stroke.
  • the Bederson score was performed on the animals 1 hour after ischemia to judge the ischemia situation of the animals, and the neurobehavioral function of the surviving animals was evaluated with reference to the NSS score table 24 hours after the model was established, so as to evaluate the neurological function of S3 and S3+t-PA nerves. Evaluate the effect of functional improvement. See Tables 8 and 10 for the Bederson score results of the animals, and Tables 9 and 10 and Figure 6 for the NSS score results.
  • the data in the table are expressed as mean ⁇ standard deviation (Mean ⁇ SD); "N” represents the number of animals in each group for statistical analysis; * indicates P ⁇ 0.05 compared with model control group animals; Q value is S3+t -PA group average NSS reduction rate/(S3 group average NSS reduction rate+t-PA average cerebral infarction reduction rate-S3 group average cerebral infarction reduction rate ⁇ t-PA average cerebral infarction reduction rate), the judgment standard of Q value is Q ⁇ 0.85 is antagonistic effect, 0.85 ⁇ Q ⁇ 1.15 is additive effect, Q ⁇ 1.15 is synergistic effect.
  • Cerebral ischemia-reperfusion is an acute injury model, and the weight of experimental animals will drop sharply after MCAO modeling.
  • the weight of experimental animals is one of the most basic sensitive indicators to comprehensively reflect the health status of animals.
  • the body weight of the surviving animals was monitored for 1 day, and the changes in the body weight of the animals were observed and recorded.
  • the experiment showed that there was no significant difference in the body weight of animals in each group before modeling and 24 hours after modeling. The above results indicated that each group had no significant effect on the body weight of animals under the experimental conditions.
  • the animals in the model control group and each drug treatment group showed symptoms such as unsteady gait, salivation and circling, and no difference was found between the groups; 24 hours after operation, most of the surviving animals showed unsteady gait, circling, etc. , piloerection, salivation, nasal, eye, and mouth filth and other stroke-related abnormal symptoms, there was no significant difference in the type and severity of abnormal symptoms among the groups.
  • S3 and S3 combined with t-PA have a significant inhibitory effect on cerebral infarction in stroke rats.
  • the size of cerebral infarction in the S3 group, t-PA group and S3+t-PA group was significantly reduced on the first day after operation (P ⁇ 0.05), and the S3 group, t-PA group and S3+t-PA group
  • the inhibition rates of cerebral infarction in animals were 32.279 ⁇ 7.291%, 22.802 ⁇ 5.719% and 28.301 ⁇ 8.746%. Cerebral infarction has a significant inhibitory effect.
  • S3 and t-PA have a synergistic effect on the improvement of neurological impairment in stroke rats.
  • the Q values of S3 combined with t-PA in terms of NSS score and cerebral infarction inhibition rate were calculated according to the neurological function reduction rate and cerebral infarction inhibition rate, and the Q values of S3 combined with t-PA in NSS score and cerebral infarction inhibition were calculated respectively. It is 1.308 and 0.593, and the Q value in the NSS score is greater than 1.15, suggesting that S3 and t-PA have a synergistic effect in improving the neurological damage of stroke rats, but there is no obvious effect in the inhibition of cerebral infarction under the experimental conditions. synergistic effect.
  • Table 11 Summary of individual data for the extent of cerebral infarction

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Abstract

本发明公开了一种神经保护多肽化合物及其应用，属于药物技术领域。包括具有如下化学式的多肽及其盐：(M)m-(Lys-Leu-Ser-Ser-Ile-Glu/Asp-Ser/Thr-Asp/Glu-Val/Leu)-(N)n； m和n为0-3的整数，但m和n不同时为零；M为beta-Ala-His、beta-Ala-1-Methyl-His或beta-Ala-3-Methyl-His，N为beta-Ala-His、beta-Ala-1-Methyl-His或beta-Ala-3-Methyl-His。只需要3mg/kg的静脉注射剂量，出乎意料地可达到显著的治疗效果，与NA1达到了同样的效果。该多肽化合物可以与溶血药物组合使用，为脑卒中治疗提供了新的可能。

Description

一种神经保护多肽化合物及其应用 技术领域
本发明属于药物技术领域，特别涉及一种神经保护多肽化合物及其应用。
背景技术
Nerinetide(NA-1)是一种神经保护剂，可通过终止细胞内NO自由基的产生，来干扰突触后密度蛋白95(PSD-95)。它在临床前缺血性脑卒中模型中可减少脑缺血再灌注的梗死面积，并改善其功能预后。针对成年的12小时治疗窗口内的大血管闭塞急性缺血性脑卒中患者，随机接受单次剂量为2.6mg/kg最大剂量为270mg的Nerinetide，或者接受生理盐水安慰剂。研究的主要终点是随机化90天后的功能预后良好，定义为修正的Rankin量表(mRS)评分为0-2分；次要终点是神经功能残疾、日常生活活动中的功能独立性、良好的功能结果(mRS0-1)和死亡率。该实验共有1105例患者参与，其中Nerinetide组549人，安慰剂组556人。90天内mRS评分为0-2的患者比例为：Nerinetide组337人(61.4％)，安慰剂组329人(59.2％)。两组间的次要结果相似。同时本研究发现在接受阿替普酶的患者中，Nerinetide治疗导致阿替普酶效果受抑制。两组间严重不良事件发生率相同。参见Michael D Hill et al.Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke(ESCAPE-NA1)：a multicentre，double-blind，randomised controlled trial.Lancet.2020，395(10227)，P878-887。
Nerinetide是一条由NMDAR GluN2B亚单位的C端9个残基和核转运激活蛋白衍生的穿膜肽TAT组成的融合肽，可以结合PSD-95的PDZ-1或PDZ-2结构域，从而抑制nNOS的NO的产生,所以也叫Tat-NR2B9c，其具体的氨基酸序列为：Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val。后面的部分序列Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val为NR2B9c的序列，具体抑制nNOS的NO的产生。前面的部分序列Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg则是提升NR2B9c的生物利用度。像NR2B9c这样的活性肽的氨基酸序列由来自NMDA受体C末端的3-25 个氨基酸或来自连接到内化肽的PSD-95受体的PDZ结构域1和/或2组成(A.Tasker,T.Doucette,M.Tymianski,K.Mendoza,M.P.Belmares,D.Garman,和P.S.Lu,2013，United States Patent 8,536,129)。这类活性肽具有包含[E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L]的氨基酸序列，比如KLSSIETDV和KLSSIESDV。
肌肽(L-Carnosine)，是一种由β-丙氨酸和L-组氨酸两种氨基酸组成的二肽。肌肽具有很强的抗氧化能力，对人体有益。肌肽已被证实可清除在氧化应激过程中使细胞膜的脂肪酸过度氧化而形成的活性氧自由基(ROS)以及α-β不饱和醛。肌肽具有抗炎、抗糖化、抗氧化和螯合作用，可作为一种非处方食品补充剂，在预防和辅助治疗心血管疾病和神经退行性等慢性疾病方面具有良好的前景。在动物实验里，肌肽的神经保护机制可以防止永久性脑部缺血。肌肽还是一种重要的细胞内抗氧化剂。肌肽不仅无毒，而且具备强的抗氧化性，因此以它作为一种新型的食品添加剂与药用试剂已经引起广泛关注。肌肽参与细胞内的过氧化反应，除了具有抑制细胞膜的过氧化过程，还能抑制细胞内的相关的过氧化反应。1900年，俄国学者Gulewitsch最早发现了肌肽，之后各国学者又在不同的肌肉组织中还提取分离了其它组氨酸二肽衍生物，如鹅肌肽(Anserine)，其是一种由β-丙氨酸和1-甲基-L-组氨酸两种氨基酸组成的二肽，以及鲸肌肽或蛇肌肽(Balenine，又称Ophidine,)，蛇肌肽则是由β-丙氨酸和3-甲基-L-组氨酸组成的二肽。不同物种之间这几种组氨酸二肽的含量及比例也各不相同，具有一定的特异性。除了在肌肉组织中存在着组氨酸二肽以外，这些二肽还存在于别的组织中，如脑组织。这些肌肽衍生物具有水溶性和较强的而且具有显著的抗氧化、抗衰老、降尿酸等功能，在食品工业中已用作天然的抗氧化剂和降尿酸食疗，也同样具有一定的神经保护功能。
尽管肌肽以及肌肽衍生物都有一定的脑保护作用，但是它们往往需要较大的剂量，单独使用肌肽、鹅肌肽和蛇肌肽等效果并不好，本领域仍然需要新的神经保护方法。
发明内容
本发明人将肌肽以及肌肽衍生物与像NR2B9c这样的活性肽组合起来产 生了新型多肽，出乎意外地发现该类全新组合的多肽完全不同于Nerinetide，具有良好的神经保护效果，且神经保护效果不受阿替普酶等溶血栓产品的影响，从而为神经保护等提供了新的办法。
本发明提供了一种神经保护多肽化合物，它包含由beta-丙氨酸与组氨酸、1-甲基组氨酸或3-甲基组氨酸等组成的肌肽、鹅肌肽、蛇肌肽等，以及以NR2B9c为代表的活性肽，活性肽的氨基酸序列为Lys-Leu-Ser-Ser-Ile-Glu/Asp-Ser/Thr-Asp/Glu-Val/Leu，尤其是NR2B9c Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val，从而形成了一系列兼具肌肽、鹅肌肽或蛇肌肽与Lys-Leu-Ser-Ser-Ile-Glu/Asp-Ser/Thr-Asp/Glu-Val/Leu特征的组合多肽化合物。这些肽类出乎意料之外地透过血脑屏障，通过静脉途径给药展示了很好的生物活性，从而在治疗神经系统疾病，尤其是脑损伤，脑卒中方面显示了广阔的前景。
一方面，提供了一种神经保护多肽化合物，包括具有如下化学式的多肽及其盐：
(M)m-(Lys-Leu-Ser-Ser-Ile-Glu/Asp-Ser/Thr-Asp/Glu-Val/Leu)-(N)n
式中，m为0-3的整数，n为0-3的整数，但m和n不同时为零；M为beta-Ala-His、beta-Ala-1-Methyl-His或beta-Ala-3-Methyl-His，N为beta-Ala-His、beta-Ala-1-Methyl-His或beta-Ala-3-Methyl-His。Glu/Asp表示该位置的氨基酸可以是Glu或Asp中的任何一个，Ser/Thr表示该位置的氨基酸可以是Ser或Thr中的任何一个，Asp/Glu表示该位置的氨基酸可以是Asp或Glu中的任何一个，Val/Leu表示该位置的氨基酸可以是Val或Leu中的任何一个。
优选地，Lys-Leu-Ser-Ser-Ile-Glu/Asp-Ser/Thr-Asp/Glu-Val/Leu为Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val。
在一些实施方案中，本发明的神经保护多肽化合物包括具有如下化学式的多肽及其盐：
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；
式中，His为His、1-Methyl-His或3-Methyl-His，即前面简称的M或N，分别对应肌肽，鹅肌肽和蛇肌肽。
具体地，本发明的神经保护多肽化合物包括具有如下化学式的多肽及其盐：
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；
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；
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；
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-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；
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)；
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)。
优选地，本发明的神经保护多肽化合物包括具有如下化学式的多肽及其盐：
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)。
更优选地，本发明的神经保护多肽化合物包括具有如下化学式的多肽及其盐：
beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val；
Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His。
最优选地，本发明的神经保护多肽化合物包括具有如下化学式的多肽及其盐：
beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val。
另一方面，还提供了前述神经保护多肽化合物在制备药物(尤其是治疗神经系统疾病药物)中的应用。还提供了前述神经保护多肽化合物用于治疗神经系统疾病。还提供了利用前述神经保护多肽化合物治疗神经系统疾病的方法。
具体地，神经系统疾病可以是缺血性脑卒中、出血性脑卒中、脑创伤、阿尔茨海默病、帕金森病或其它神经退行性疾病。优选地，神经系统疾病是缺血性脑卒中。
本发明提供的神经保护多肽化合物可以制备为药物。前述药物为注射剂、口服用药、舌下用药、喷雾用药或肛门用药等，优选为注射剂。具体地，注射剂为粉针剂或者注射液。进一步地，前述药物采用静脉的方式给药。前述药物也可以作为活性成分制备其它剂型，以便于相应的医疗应用。作为神经 保护用药，开发的制剂可以包括口服给药和舌下给药制剂，作为脑卒中现场急救用药，开发的制剂可以包括喷雾给药，肛门给药等制剂，便于没有行动能力的患者得到救治。
进一步地，前述药物中包括药学上可接受的稀释剂或/和载体等。
又一方面，还提供了通过固相合成制备本发明各类神经保护多肽化合物的方法，但其中的一些肽用液相合成或片段合成更加方便。多肽药物成盐是改善药物分子理化性质、提高其成药性的常见手段之一，前述药物可以是任何形式的盐。
再一方面，还提供了本文所述的神经保护多肽化合物与溶栓药物的组合。
所述溶栓药物可以为以链激酶和尿激酶为代表的第一代溶栓药，以组织纤溶酶原激活剂(tissue—type plasminogen activator，tPA)阿替普酶和尿激酶原为代表的第二代溶栓药物，或者基于重组人组织型纤溶酶原激活剂(rtPA)的第三代溶栓药物。本领域已知很多这样的市售产品，比如注射用尿激酶(天津生物化学制药有限公司)，注射用重组链激酶(例如思凯通)，注射用阿替普酶(例如爱通立)，注射用重组人TNK组织型纤溶酶原激活剂(例如铭复乐)。
本文所述神经保护多肽化合物与溶栓药物可以先后或同时单独施用，或者以任何合适的比例混合作为组合药物施用。
所述组合可用于制备药物，尤其是治疗神经系统疾病药物。
所述神经系统疾病可以是缺血性脑卒中、出血性脑卒中、脑创伤、阿尔茨海默病、帕金森病或其它神经退行性疾病。优选地，神经系统疾病是缺血性脑卒中。
为了确定本文提供的合成肽在神经系统疾病方面的应用，用SD大鼠作为实验对象，采用脑中动脉阻断法(MCAO)制备脑缺血大鼠模型，缺血1-2小时后静脉注射药物，22-24小时对每只动物做行为学观察和评分。行为学观察之后，将实验大鼠进行安乐死并取出其大脑，脑组织切片，TTC染色后进行量化分析，计算脑梗死体积％。
本发明人创造性地把神经保护多肽化合物与像NR2B9c这样的活性肽、肌肽、鹅肌肽或蛇肌肽等组合起来，开发的组合多肽出乎意料地通过静脉注射透过血脑屏障，进入大脑，有效地在治疗神经系统疾病中发挥了作用。该 类合成肽优选地应用于治疗缺血性脑卒中、出血性脑卒中、脑创伤、阿尔茨海默病、帕金森病及其他神经退行性疾病。本发明只需要3mg/kg的合成多肽静脉注射剂量，出乎意料地可达到显著的治疗效果，与已有临床活性的标准多肽NA1达到了同样的效果。
同时本发明的合成肽便于合成，合成过程中还可以使用肌肽，鹅肌肽或蛇肌肽的衍生物作为合成片段，代替对应的两个氨基酸来进一步简化目标肽的合成。
附图说明
图1：模型组的脑组织的切片图。
图2：S1组的脑组织的切片图。
图3：S3组的脑组织的切片图。
图4：假手术组的脑组织的切片图。
图5：脑梗死范围和脑梗死抑制率结果，其中A：脑梗死范围(Infarction Area％)，B：脑梗死抑制率(Inhibition Ratio％)；**表示与模型对照组相比P＜0.01，***表示与模型对照组相比P＜0.005。
图6：NSS评分结果，图中数据均以均值±标准差(Mean±SD)表示，其中A：NSS评分结果，B：术后1天各给药组NSS评分相对于模型对照组降低率结果；*表示与模型对照组动物相比P≤0.05。
图7：模型对照组动物TTC染色结果图。
图8：S3组动物TTC染色结果图。
图9：t-PA组动物TTC染色结果图。
图10：S3+tPA组动物TTC染色结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。
实例1：beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val的合成
1.在活化剂系统(HoBT,DIC)的存在下，由固相载体2-CTC树脂和Fmoc-Val-OH偶联得到Fmoc-Val-CTC树脂。
2.用20％哌啶脱除Fmoc-Val-CTC上的Fmoc保护基，脱除干净后用DMF洗涤干净。
3.称取3倍过量的Fmoc-Asp(0tBu)-OH，与三倍过量的活化剂，加入少量DMF充分溶解，溶解完之后加入至洗涤干净的树脂中，反应1h后用DMF洗涤干净。
4.重复第二步骤与第三步骤，按照主链顺序从Ser到β-Ala依次偶联具有N端Fmoc保护且侧链带保护的氨基酸。
5.合成完毕之后进行裂解，裂解试剂配比为TFA：EDT：Tis：TA：苯甲醚：H 2O＝80：5：1：5：5：4(体积比)，按1g肽树脂需10ml裂解试剂配比，室温下裂解约2h(120r/min)，裂解之后用冰甲基叔丁基醚沉淀，下层沉淀即为粗品。
6.取上步的粗肽溶解，在制备型HPLC上用0.1％TFA/乙腈纯化。
7.纯化完之后进行冻干，冻干结束后取出粉末进行分装,质检得多肽beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val。
实例2：Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His的合成
1.在活化剂系统(HoBT,DIC)的存在下，由固相载体2-CTC树脂和Fmoc-His(Trt)-OH偶联得到Fmoc-His(Trt)-CTC树脂。
2.用20％哌啶脱除Fmoc-His(Trt)-CTC上的Fmoc保护基，脱除干净后用DMF洗涤干净。
3.称取3倍过量的Fmoc-beta-Ala-OH，与三倍过量的活化剂，加入少量DMF充分溶解，溶解完之后加入至洗涤干净的树脂中，反应1h后用DMF洗涤干净。
4.重复第二步骤与第三步骤，按照主链顺序从Val到Lys依次偶联具有N端Fmoc保护且侧链带保护的氨基酸。
5.合成完毕之后进行裂解，裂解试剂配比为TFA：EDT：Tis：TA：苯甲醚：H 2O＝80：5：1：5：5：4(体积比)，按1g肽树脂需10ml裂解试剂配比，室温下裂解约2h(120r/min)，裂解之后用冰甲基叔丁基醚沉淀，下层沉淀即为粗品。
6.取上步的粗肽溶解，在制备型HPLC上用0.1％TFA/乙腈纯化。
7.纯化完之后进行冻干，冻干结束后取出粉末进行分装,质检得多肽Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His。
实例3：beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His的合成
1.在活化剂系统(HoBT,DIC)的存在下，由固相载体2-CTC树脂和Fmoc-His(Trt)-OH偶联得到Fmoc-His(Trt)-CTC树脂。
2.用20％哌啶脱除Fmoc-His(Trt)-CTC上的Fmoc保护基，脱除干净后用DMF洗涤干净。
3.称取3倍过量的Fmoc-beta-Ala-OH，与三倍过量的活化剂，加入少量DMF充分溶解，溶解完之后加入至洗涤干净的树脂中，反应1h后用DMF洗涤干净。
4.重复第二步骤与第三步骤，按照主链顺序从Val到beta-Ala依次偶联具有N端Fmoc保护且侧链带保护的氨基酸。
5.合成完毕之后进行裂解，裂解试剂配比为TFA：EDT：Tis：TA：苯甲醚：H 2O＝80：5：1：5：5：4(体积比)，按1g肽树脂需10ml裂解试剂配比，室温下裂解约2h(120r/min)，裂解之后用冰甲基叔丁基醚沉淀，下层沉淀即为粗品。
6.取上步的粗肽溶解，在制备型HPLC上用0.1％TFA/乙腈纯化。
7.纯化完之后进行冻干，冻干结束后取出粉末进行分装,质检得多肽beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His。
实例4：同样的办法也可以合成如下多肽
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)。
实例5：动物实验方法
小动物监护仪：深圳市荣显达科技有限公司，VT200。
SD大鼠：中国食品药品鉴定研究院，180-200g。
线栓：迈越生物(M8507)。
TTC染色液：源叶生物(R24053)。
动物模型操作步骤：
对实验用SD大鼠进行称重，使用水合氯醛麻醉。麻醉后对大鼠四肢进行固定，仰卧，连接小动物监护仪，对大鼠体温、血压和心率等重要生理指标进行监护。对大鼠颈部进行脱毛，用75％酒精棉球消毒后对实验大鼠颈部进行长度2cm左右的正中切口，通过钝性分开大鼠颌下腺，分离过程中尽量避免对腺体的破坏，然后钝性分离出实验大鼠左侧的颈总动脉(CCA)，沿着CCA向上小心分离出颈内动脉(ICA)和颈外动脉(ECA)。分离过程中避免损伤迷走神经。使用两个动脉夹分别将CCA和ICA夹闭，剪断ECA远端并将硅胶线栓从ECA“端口”插入，插到ICA动脉夹处短暂松一下动脉夹并迅速将线栓头端插过动脉夹后再次夹闭ICA，然后轻轻松开ICA上的动脉夹并不断使线栓插入，直到线栓黑标插过ECA和ICA的分叉，线栓头端堵住大脑中动脉后用缝合线将ECA“端口”和线栓紧紧系紧，以防止大鼠醒后线栓“退出”或出血。松开并拿掉CCA和ICA上的动脉夹，使组织恢复原位，并滴加适量的青霉素以防伤口感染。使用医用带线缝合针进行缝合，封好之后再次用碘伏消毒。观察小动物监护仪判断实验大鼠指标是否正常，然后将大鼠放在小动物电热毯上使其保持体温直至苏醒放置动物笼中。
给药流程：
将待测药物配成3mg/mL的溶液。在结扎后1-2h尾静脉给药。
给药时将大鼠用固定器固定，利用1mL注射器按照3mg/kg剂量吸取待测药物，然后对大鼠进行尾静脉推注，注射时缓缓推注以降低实验大鼠的心肺负荷。
动物分为不同的试验小组，每组六只。
模型组：结扎后尾静脉给等量的生理盐水；
药物组S1(NA1)和S3(beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val)，结扎后尾静脉给药；
假手术组：分离颈内动脉(ICA)和颈外动脉(ECA)后不做结扎处理，尾静脉给等量的生理盐水。
行为学观察：
结扎后22-24h对每只动物做行为学观察和评分。
按照如下评分表，让未参与该实验的技术员盲评。
0分：正常直线行走；
1分：前肢无力；
2分：后肢无力；
3分：轻度转圈；
4分：严重转圈；
5分：偏瘫。
TTC染色和量化分析：
行为学观察之后，将实验大鼠进行安乐死并取出其大脑。将脑组织横切成6片2mm厚的切片，再移至TTC染色液中，37℃孵箱中避光孵育10min，拍照(结果见图1-4)。然后将TTC染色后的脑组织和剩余的少量未进行TTC染色的脑组织-20℃保存。
使用Image J软件对TTC染色后的照片做量化分析。
脑梗死体积％＝(总梗死面积*切片厚度)/(总脑切片面积*切片厚度)*100％。
实验结果：
1、TTC量化结果如表1所示：
表1
编号 1 2 3 4 5 6 平均值
模型组 0.407 0.402 0.392 0.420 0.377 0.340 0.390
S1(NA1) 0.187 0.218 0.153 0.145 0.107 0.134 0.157
S3 0.128 0.186 0.075 0.158 0.210 0.146 0.128
假手术组 0.048 0.038 0.042 0.051 0.039 0.044 0.044
从表1的结果可见,注射生理盐水的脑缺血大鼠，梗死体积0.390±0.028；注射3mg/kg Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val(S3)的脑缺血大鼠，梗死体积0.150±0.047。作为对照的假手术组的结果则为梗死体积0.044±0.005。与模型组比较，S3有明显的生物活性且S3与S1没有统计学区别，其中，NA1(Nerinetide)是一种神经保护剂(Michael Tymianski and Jonathan D.Garman.Model systems and treatment regimes for treatment of neurological disease.2015,US Patent,US8940699B2)，可通过终止细胞内NO自由基的产生来干扰突触后密度蛋白95(PSD-95)，在临床前缺血性脑卒中模型中可减少动物实验性(猕猴)脑缺血再灌注的梗死面积，并改善其功能预后，因此是一个很好的阳性对照产品。Hill博士研究了静脉注射新型神经肽NA-1(2.6mg/kg)用于血管内血栓切除术的急性缺血性脑卒中(AIS)患者的疗效和安全性，结果表明使用NA1治疗可改善患者预后。(Michael D Hill et al.Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke(ESCAPE-NA1)：a multicentre,double-blind,randomised controlled trial.Lancet.Published online February 20,2020)。
2、行为学测评结果如表2所示：
表2
Figure PCTCN2022109048-appb-000001
从表2可以看出本发明提供的合成肽具有生理活性。注射生理盐水的脑缺血大鼠，行为学评分为3.8±0.8；注射3mg/kg beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val(S3)的脑缺血大鼠，评分为2.5±1.0；作为比较，静脉给于3mg/kg NA1的脑缺血大鼠，评分为2.0±0.6。作为对照的假手术组的结果则为0.0±0.0。与模型组比较，S3有明显的生物活性，且S3和S1没有统计学区别。
实例6：S3(beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val)与脑卒中药物t-PA的联合给药试验
1.供试品配制：t-PA选择广州铭康生物工程有限公司的注射用重组人TNK组织型纤溶酶原激活剂(铭复乐)，1.0x10E7IU/16mg/只。以动物体重为300g举例，配制方法如下表3。
表3
Figure PCTCN2022109048-appb-000002
给药制剂配制均在生物安全柜内进行无菌操作，所使用的耗材等需经过灭菌处理。
S3配制避光保存于冰盒，注射前使其温度恢复至常温，t-PA配制后放置于常温暂存。
2.动物品系/级别
品系：Sprague-Dawley(SD)大鼠；
等级：SPF级；
进入适应期动物数量和性别：40只，雄性；
使用动物数量和性别：36只，雄性；
动物年龄及体重
体重：进入适应期时151.78-186.9g，分组时体重242.95-289.31g；
年龄：适应期时约5-7周龄，试验分组时6-8周龄；
来源：斯贝福(北京)生物科技有限公司；
动物环境适应：动物接收后适应环境5天，适应期的主要检查内容包括是否与订购时要求的质量指标一致、一般状态、体重是否达到试验要求的体重范围，不合格的动物不纳入本试验；
动物饲养：塑料大鼠笼具(L×W×H：46.6cm×30cm×21.5cm)；环境适应期5只/笼，正式试验3-4只/笼；
饲养环境条件标准：中华人民共和国国家标准GB14925-2010；
饲养环境控制系统：WINCC7.3EMS系列机房环境监控系统；
温度：室温20-26℃(日温差≤4℃)；
相对湿度：40-70％；
光照：人工照明，12/12小时昼夜明暗交替；
换气次数：每小时空气更换不少于15次；
饲料种类：大小鼠育成饲料，饲料批号21103213，购自北京科澳协力饲料有限公司。
适应期动物一般状态观察未见任何异常，适应期结束后动物体重为185.46-230.82g，未达到动物造模需要的体重，因此动物适应期延长到12天。适应期结束后选择体重在240-290g内的动物进行分组造模，给药24h后S3组编号为2M006的动物被发现死亡，解剖发现模型对照组编号为1M005的动物脑部组织缺失，确定为脑部发育异常，另外S3组编号为2M001和S3+t-PA组编号为4M002的动物解剖发现蛛网膜下腔出血，S3+t-PA组编号为4M009脑梗死范围为25.19％，大于本组其余动物的平均值±3标准差判定为离群值，以上动物的数据不纳入最终统计分析，各组其余动物数据均纳入最终统计分析，最终纳入统计分析的动物数量分别为模型对照组8只、S3组7只、t-PA组9只和S3+t-PA组7只。
3.造模
1)麻醉诱导：将大鼠放入充满3.0％异氟烷的麻醉诱导盒进行麻醉诱导；
2)固定：经过麻醉诱导的动物转移至操作台上，使用小动物气体麻醉机以2.0％-2.5％异氟烷按200mL/min进行麻醉维持，观察大鼠眼睑反射和痛觉反应，在眼睑反射反应及四肢和尾部痛觉反应消失后方可开始手术；
3)大脑缺血再灌注手术步骤：
A.分离暴露血管：手术区域备皮后，将大鼠置于手术显微镜下，沿正中线用眼科剪剪开大鼠皮肤，长度约2cm，经右侧颈旁入路，显微镊钝性分离并牵开右侧颈部的肌肉组织，显露右侧颈总动脉(common carotid artery，CCA)，然后沿颈总动脉向上分离，继续显露颈外动脉(external carotid artery，ECA)、颈内动脉(internal carotid artery，ICA)；
B.结扎CCA近心端，用动脉夹暂时夹闭Y形分支处CCA，在CCA结扎部位至ICA暂闭部位穿线，系一松结(预结扎)备用，并在预结扎近心端剪一小口；
C.线栓推入：将4-0号线栓经CCA的切口插入，再缓慢轻轻地推入颈内动脉，到达ICA的动脉夹处暂停，进一步套紧预结扎线(避免推线栓时出血过多)，然后将阻断ICA血流的动脉夹取出，立即将线栓推入ICA直到进入颅内，此时注意不要将线栓插入颈内动脉的另一分支翼腭动脉(翼腭动脉属于ICA在颅外的分支，当线栓进入该动脉深度为10mm左右时即不能继续插入，此时，稍微退出线栓，调整方向，重新操作)；
D.固定线栓、缝合切口：当线栓插入深度距离颈总动脉分叉约18mm左右时，如果有轻微阻力感，说明线栓的头端已经进入大脑前动脉(cerebral anterior artery，ACA)，线栓侧壁已经堵塞了大脑中动脉开口，此时停止插入，并记录时间，移去CCA上的动脉夹，观察无活动性出血后关闭切口；
E.大脑再灌注：缺血大鼠置于室温，120min后即可进行诱导麻醉，在维持麻醉状态下缓慢地轻拉线栓，使其头端回到颈总动脉内，即实现大脑中动脉再灌注；
F.碘伏消毒切口。
4.动物分组
组别设计：共设置4个组，分别为模型对照组、S3组、t-PA组和S3+t-PA组；
动物数量：各组9只动物，合计36只；
性别比例：根据JW Simpkins和RL Roof等的报道，在成年大鼠中雌性激素和黄体酮等对缺血性脑梗死具有神经保护作用，为排除雌激素和黄体酮等对试验结果的影响，本试验全部动物均为雄性；
分组方法：根据分组前最近一次大鼠体重进行随机分组；
具体分组信息见下表4。
表4
Figure PCTCN2022109048-appb-000003
注：动物编号的首位数字代表组别(1、2、3和4分别代表模型对照组、S3组、t-PA组和S3+t-PA组)，第二位字母代表性别(M为雄性)，后3位数字代表动物序列号，a表示给予3mL S3和3mL无菌注射用水，b表示给予3mL t-PA和3mL无菌注射用水，C表示给予3mL S3和3mL t-PA；
给药途径：尾静脉注射给药；
给药时间：缺血再灌注后即刻(5min内)；
给药频率及周期：给药1次；
造模当天定义为D0，前一天定义为D-1。
5.观察和测定内容
一般状态观察
观察动物：所有计划观察的存活实验动物；
观察时间：每天观察一次，如果动物出现异常，可增加观察频率；
观察内容：包括但不限于一般表现、行为状态、眼睛、口腔、鼻口部、耳、毛发及皮肤、粪便、尿、生殖器等毒性症状，若出现异常，需进行详细描述。
体重
测定动物：所有计划测定的存活实验动物；
测定时间：适应期至少测量体重2次，手术前24h称量1次用于分组，术后24h称量1次。
行为学评价
Bederson评分
检测时间：造模当天缺血50-60min后，用于评估动物缺血是否成功；
测定动物：所有手术存活动物；
检测方法：参考Bederson评分标准对动物进行评分；
表5：大鼠Bederson评分标准
Figure PCTCN2022109048-appb-000004
缺血后评分大于等于1分的大鼠为造模成功，造模未成功大鼠则淘汰。
大鼠神经功能评分
检测时间：造模前24h、造模后24h和72h各评分一次；
测定动物：所有计划测定的存活实验动物；
检测方法：对动物进行运动功能测试、感觉测试、平衡测试和反射和异常动作测试，具体参考大鼠神经功能评分表(NSS)。
表6：大鼠神经功能评分表(NSS)
Figure PCTCN2022109048-appb-000005
解剖及病理学检查
解剖时间：D1(相当于造模后24h)(造模当天定义为D0)；
解剖动物：所有入组存活动物；
麻醉及安乐死方法：采用3％戊巴比妥钠进行腹腔注射麻醉，注射剂量为60mg/kg，麻醉后腹主动脉放血安乐死。
病理学(TTC染色)评价
检测动物：所有活体动物；
检查方法：安乐死动物快速取脑，并将鼠脑置于-20℃冰箱内冷冻至脑组织变硬后取出，将大鼠大脑切成2mm厚的切片，切片位置从嗅球向后，总共6-8片，分别放置于装有0.5％TTC溶液(PBS配制)的六孔板格中，然后置于37℃恒温箱中避光温孵20min，取出后置于10％甲醛溶液中避光保存。
正常组织经染色后呈玫瑰红色，梗死组织呈白色。将每个脑平面按序摆放在滤纸上，数码相机拍照，小心取下白色组织并称重，以梗死组织重量占全脑重量的百分比作为梗塞范围(％)或者以图像处理计算脑梗死范围。并以梗塞范围计算各药物治疗组的抑制率(％)，抑制率计算公式如下：
Figure PCTCN2022109048-appb-000006
统计分析
计量指标采用均数±标准差表示。样本数小于3时，该组数据不纳入统计比较。
数据经Excel 2010、GraphPad Prism 7、SPSS 22.0和Stata 15.0软件进行录入与统计分析。计量指标先采用LEVENE方差齐性检验，当方差齐时(p＞0.05)，可直接引用方差分析的结果判断总体差异是否有统计学意义，总体差异有统计学意义时(p＜0.05)，用Dunnett-t检验对组间差异进行比较，总体差异无统计学意义时(p≥0.05)，统计分析结束；当LEVENE方差齐性检验显示方差不齐时(p≤0.05)，则采用非参数检验(Kruskal-Wallis H检验)，Kruskal-Wallis H检验显示总体差异有统计学意义时(p＜0.05)，用 Mann-Whitney U检验进行组间差异的比较，当Kruskal-Wallis H检验显示总体差异无统计学意义时(p≥0.05)，统计分析结束。
6.试验结果
S3和S3+t-PA对模型动物脑梗死范围的影响：
急性缺血性脑卒中治疗的中心环节是尽量减少缺血所致的神经细胞病变程度及范围，本试验采用缺血再灌注后即刻进行药物干预，24h后对造模动物脑部进行TTC染色，通过测定各组动物梗死范围并计算抑制率，对S3和S3联合t-PA的抗缺血性脑卒中药效学进行评价。动物脑梗死范围及脑梗死抑制率见表7及图5、7-10。
术后24h模型对照组脑梗死范围为21.493±2.734％。S3组、t-PA组和S3+t-PA组动物脑梗死范围分别为16.248±1.749％、18.522±1.372％和17.203±2.098％，相对于模型对照组均显著降低(P≤0.05)，S3组、t-PA组和S3+t-PA组动物脑梗死抑制率分别为32.279±7.291％、22.802±5.719％和28.301±8.746％，以上结果提示本试验条件下S3组、t-PA组和S3+t-PA对缺血性脑卒中大鼠脑梗死具有显著的抑制作用；根据各药物组的平均脑梗死抑制率计算预估S3和t-PA的联合用药的Q值，得Q值为0.593，提示本试验条件下3mg/kg S3和3mg/kg t-PA对脑梗死的抑制无显著的协同效应。
表7：各组动物脑梗死范围和脑梗死抑制率
Figure PCTCN2022109048-appb-000007
备注：表中数据均以均值±标准差(Mean±SD)表示；“N”代表各组进行统计分析的动物数量，“-”表示该项没有数据，**表示与模型对照组相比P＜0.01，***表示与模型对照组相比P＜0.005；Q值为S3+t-PA组平均脑梗死抑制率/(S3组平均脑梗死抑制率+t-PA平均脑梗死抑制率-S3组平均脑梗死抑制率×t-PA平均脑梗死抑制率)，Q值的判断标准为Q＜0.85为拮抗作用，0.85≤Q＜1.15为相加作用，Q≥1.15为协同作用。
S3和S3+t-PA对模型动物神经行为学评分的影响：
临床上脑梗死治疗的主要目的是尽量恢复患者的神经功能，提高卒中后生活质量。本试验于缺血后1h对动物进行Bederson评分，对动物缺血情况进行判断，于造模后24h参照NSS评分表对存活动物的神经行为功能进行评价，从而对S3和S3+t-PA神经功能改善效果进行评估。动物Bederson评分结果见表8和10，NSS评分结果见表9、10和图6。
缺血后1h，所有模型动物Bederson评分均为3分，提示所有模型动物缺血成功。
术后1天缺血再灌注模型对照组组动物NSS得分为7.75±1.71，S3组、t-PA组和S3+t-PA组动物NSS评分分别为6.71±1.79、6.83±0.75和5.36±1.30，其中S3+t-PA组动物NSS相对于模型对照组显著降低(P＝0.0385)，计算各给药组动物NSS评分相对于模型对照组降低率，得S3组、t-PA组和S3+t-PA组动物NSS得分降低率分别为13.36±23.1％、11.83±9.62％和30.88±16.79％，根据NSS降低率计算S3和t-PA的联合用药的Q值，得Q值为1.308提示对NSS评分的改善S3和t-PA具有协同作用，以上结果提示在本试验条件下S3联合t-PA对脑卒中大鼠神经行为具有显著改善效果，且S3和t-PA对缺血再灌注大鼠神经功能损伤的改善具有协同作用。
表8：动物Bederson评分结果
Figure PCTCN2022109048-appb-000008
Figure PCTCN2022109048-appb-000009
注：表中数据均以均值±标准差(Mean±SD)表示；“N”代表各组进行统计分析的动物数量。
表9.动物NSS评分结果
Figure PCTCN2022109048-appb-000010
注：表中数据均以均值±标准差(Mean±SD)表示；“N”代表各组进行统计分析的动物数量；*表示与模型对照组动物相比P<0.05；Q值为S3+t-PA组平均NSS降低率/(S3组平均NSS降低率+t-PA平均脑梗死降低率-S3组平均脑梗死降低率×t-PA平均脑梗死降低率)，Q值的判断标准为Q＜0.85为拮抗作用，0.85≤Q＜1.15为相加作用，Q≥1.15为协同作用。
S3和S3+tPA对模型动物体重的影响：
脑缺血再灌注作为一个急性损伤模型，实验动物在MCAO造模后体重会急剧下降，试验动物体重作为综合反映动物健康状况最基本的灵敏指标之一，本试验在术前1天、术后1天对存活动物进行体重监测，动物体重变化进行观察记录。实验表明造模前和造模24h后各组动物体重无显著差异，以上结果提示本试验条件下各组对动物体重变化无明显影响。
一般状态观察结果：
模型对照组和各给药组动物行MCAO造模后，均出现步态不稳、流涎和转圈等症状，组间未见任何差异；术后24小时，存活动物多出现步态不稳、转圈、竖毛、流涎、鼻周、眼周和口周污秽等脑卒中相关的异常症状，各组间异常症状种类和严重程度没有明显差异。
综上，本试验采用经典的线栓法建立MCAO缺血再灌注模型，对S3和S3联合t-PA给药的抗卒中效果进行评价，同时探索S3和t-PA是否存在协同 效应。模型对照组和各给药组均在缺血再灌注时即刻(5min内)给药1次。术后1天参照NSS评分对神经功能损伤进行评估并解剖动物对脑组织进行TTC染色测定脑梗死范围，通过脑梗死范围和神经功能改善，评价S3和S3联合t-PA对大鼠缺血性脑卒中的神经保护作用，结果表明：
A)S3和S3联合t-PA对卒中大鼠脑梗死具有显著的抑制效果。术后1天S3组、t-PA组和S3+t-PA组动物脑梗死范围相对于模型对照组均显著降低(P≤0.05)，S3组、t-PA组和S3+t-PA组动物脑梗死抑制率分别为32.279±7.291％、22.802±5.719％和28.301±8.746％，以上结果提示本试验条件下S3组、t-PA组和S3+t-PA对缺血性脑卒中大鼠脑梗死具有显著的抑制作用。
B)S3联合t-PA对卒中大鼠神经功能损伤具有显著的抑制效果。术后24h，S3组、t-PA组和S3+t-PA组动物NSS评分相对于模型对照组有不同程度降低，其中S3+t-PA组动物NSS相对于模型对照组显著降低(P＝0.0385)，各给药组动物NSS评分相对于模型对照组降低百分比分别为13.36±23.1％、11.83±9.62％和30.88±16.79％，以上结果提示在本试验条件下S3联合t-PA对缺血性脑卒中大鼠神经功能损伤均具显著的改善。
C)S3与t-PA对卒中大鼠神经功能损伤的改善具有协同效应。本试验中根据神经功能降低率和脑梗死抑制率分别计算S3联合t-PA在NSS评分和脑梗死抑制率方面的Q值，S3联合t-PA在NSS评分和脑梗死抑制方面的Q值分别为1.308和0.593，其中在NSS评分中的Q值大于1.15，提示S3和t-PA在改善脑卒中大鼠神经功能损伤方面具有协同效应，而在脑梗死抑制方面在本实验条件下无明显的协同效果。
概言之，本试验结果表明S3、t-PA和S3联合t-PA在急性缺血性脑卒中有明显治疗效果，且在对急性缺血性脑卒中大鼠神经行为改善方面S3和t-PA具有协同效应。
表10：神经行为学评分个体数据汇总
Figure PCTCN2022109048-appb-000011
Figure PCTCN2022109048-appb-000012
Figure PCTCN2022109048-appb-000013
表11：脑梗死范围个体数据汇总
Figure PCTCN2022109048-appb-000014
Figure PCTCN2022109048-appb-000015
表12：动物体重(g)数据汇总
Figure PCTCN2022109048-appb-000016
Figure PCTCN2022109048-appb-000017
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (11)

  1. 一种神经保护多肽化合物，其特征在于，包括具有如下化学式的多肽及其盐：
    (M)m-(Lys-Leu-Ser-Ser-Ile-Glu/Asp-Ser/Thr-Asp/Glu-Val/Leu)-(N)n；
    式中，m和n为0-3的整数，但m和n不同时为零；
    M为beta-Ala-His、beta-Ala-1-Methyl-His或beta-Ala-3-Methyl-His，N为beta-Ala-His、beta-Ala-1-Methyl-His或beta-Ala-3-Methyl-His。
  2. 根据权利要求1所述的神经保护多肽化合物，其特征在于，包括具有如下化学式的多肽及其盐：
    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；
    式中，His为His、1-Methyl-His或3-Methyl-His。
  3. 根据权利要求1所述的神经保护多肽化合物，其特征在于，包括具有 如下化学式的多肽及其盐：
    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；
    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；
    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；
    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-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；
    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)；
    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)。
  4. 根据权利要求1所述的神经保护多肽化合物，其特征在于，包括具有如下化学式的多肽及其盐：
    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)。
  5. 根据权利要求1所述的神经保护多肽化合物，其特征在于，包括具有 如下化学式的多肽及其盐：
    beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val；
    Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-beta-Ala-His。
  6. 根据权利要求1所述的神经保护多肽化合物，其特征在于，包括具有如下化学式的多肽及其盐：
    beta-Ala-His-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val。
  7. 权利要求1-6任一项所述的神经保护多肽化合物用在医药中。
  8. 权利要求1-6任一项所述的神经保护多肽化合物用于治疗神经系统疾病，例如缺血性脑卒中、出血性脑卒中、脑创伤、阿尔茨海默病、帕金森病或其它神经退行性疾病。
  9. 包含权利要求1-6任一项所述的神经保护多肽化合物的药物，例如注射剂、口服用药、舌下用药、喷雾用药或肛门用药。
  10. 权利要求1-6任一项所述的神经保护多肽化合物与溶栓药物的组合。
  11. 权利要求10任一项所述的组合，用于治疗神经系统疾病，例如缺血性脑卒中、出血性脑卒中、脑创伤、阿尔茨海默病、帕金森病或其它神经退行性疾病。
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