WO2019006690A1 - 多肽的药学可接受的盐及其应用 - Google Patents

多肽的药学可接受的盐及其应用 Download PDF

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Publication number
WO2019006690A1
WO2019006690A1 PCT/CN2017/091792 CN2017091792W WO2019006690A1 WO 2019006690 A1 WO2019006690 A1 WO 2019006690A1 CN 2017091792 W CN2017091792 W CN 2017091792W WO 2019006690 A1 WO2019006690 A1 WO 2019006690A1
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Prior art keywords
pharmaceutically acceptable
polypeptide
pharmaceutical composition
stroke
acceptable salt
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PCT/CN2017/091792
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English (en)
French (fr)
Inventor
韩化敏
田雨佳
贾红军
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拜西欧斯（北京）生物技术有限公司
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Priority to JP2020522762A priority Critical patent/JP7002788B2/ja
Priority to AU2017422458A priority patent/AU2017422458B2/en
Priority to KR1020207003472A priority patent/KR102403089B1/ko
Priority to US16/628,083 priority patent/US11767344B2/en
Priority to BR112020000115-5A priority patent/BR112020000115A2/pt
Priority to CN201780092744.2A priority patent/CN110809579B/zh
Priority to PCT/CN2017/091792 priority patent/WO2019006690A1/zh
Priority to EA202090143A priority patent/EA039314B1/ru
Priority to EP17916979.2A priority patent/EP3650460A4/en
Publication of WO2019006690A1 publication Critical patent/WO2019006690A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • A61P25/08—Antiepileptics; Anticonvulsants
    • 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
    • A61P25/22—Anxiolytics
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • This application relates generally to the field of biomedicine.
  • the application provides pharmaceutically acceptable salts, compositions, and methods for treating, ameliorating, or preventing a polypeptide associated with a nervous system.
  • Stroke is a common acute cerebrovascular disease in middle-aged and elderly people, and tends to be younger. It is one of the three diseases (cancer, cardiovascular disease, diabetes) that are the most harmful to humans in the world today. According to statistics, China has nearly 3 million deaths from cerebrovascular diseases each year, 4 to 5 times higher than that of European and American countries, 3.5 times that of Japan, and even higher than that of developing countries such as Thailand and India; the incidence rate rises at an annual rate of 8.7%. The recurrence rate was over 30%, and the recurrence rate was 54% within 5 years; 75% of stroke survivors lost their ability to work and 40% were disabled.
  • Stroke can be broadly divided into two categories, namely ischemic stroke and hemorrhagic stroke, of which ischemic stroke accounts for 85% of the total number of stroke patients.
  • the therapeutic drugs for ischemic stroke are mainly divided into the following categories: vasodilators (such as dipyridamole), drugs that improve microcirculation, expand blood volume (such as low molecular dextran, etc.), drugs that dissolve thrombus (such as urine).
  • Kinase, etc. anticoagulant therapy, drugs that prevent platelet aggregation (such as aspirin), traditional Chinese medicine, neuroprotective agents, etc., but because most of these drugs have side effects, potential risks, or ineffective effects, so research
  • drugs that prevent platelet aggregation such as aspirin
  • traditional Chinese medicine such as aspirin
  • neuroprotective agents such as aspirin
  • the pathogenesis of stroke and drug development for its mechanism have important social significance for the prevention and treatment of cerebrovascular disease.
  • Stroke is characterized by neuronal cell death in areas of ischemia, cerebral hemorrhage, and/or traumatic areas.
  • Neuronal death or injury caused by cerebral ischemia is a process of injury cascade. After cerebral ischemia, blood perfusion decreases, excitatory neurotransmitters increase, and NMDA and AMPA receptors are activated, causing ion channel opening, calcium ion Inflow, activation of a large number of enzymes triggers a signal cascade that causes multiple pathways of neuronal damage.
  • Its downstream post-synaptic density 95 protein (PSD-95) triggers a series of ischemic injuries through interaction with various proteins, is a key site for cerebral ischemic injury, and is also a potential target for drug therapy. Therefore, the development of PSD-95 inhibitors has great medicinal significance for neurological damage caused by various excitatory neurotoxicity including stroke.
  • NMDA neurodegenerative diseases
  • ALS amyotrophic lateral sclerosis
  • Huntington's disease various neurodegenerative diseases
  • NMDA receptor NMDA receptor
  • the onset of epilepsy includes three different and continuous pathophysiological processes, including initiation, maintenance and expansion of episodic discharge, and inhibition of episodes. During this process, excitatory neurotransmitters such as glutamate and aspartate play an important role. .
  • PSD-95 In Alzheimer's disease, PSD-95 is involved in the neurotoxic mechanism that leads to it through the GluR6-PSD-95-MLK3 pathway. In addition, in Huntington's disease, PSD-95 is a mediator of neurotoxicity of NMDA receptors and huntingtin mutants. Therefore, the development of PSD-95 inhibitors is also important for the treatment, improvement and prevention of the above diseases.
  • the application provides a pharmaceutically acceptable salt of a polypeptide, wherein the polypeptide comprises the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof.
  • the functional variant is a variant produced following one or more conservative substitutions of the LDTEI moiety of SEQ ID NO:1.
  • the conservative substitution is selected from the group consisting of a substitution between D and E, a substitution between L, V and I, and a substitution between T and S.
  • the functional variant is a variant produced by replacing the LDTEI portion of SEQ ID NO: 1 with any of the following sequences: LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL, LDSEV, LDSDI, LDSDL, LDSDV, LETEI, LETEL, LETEV, LETDI, LETDL, LETDV, VDTEI, VDTEL, VDTEV, VDTDI, VDTDL, VDTDV, IDTEI, IDTEL, IDTEV, IDTDI, IDTDL, IDTDV, IETEI, IETEL, IETEV, IETDI, IETDL, IETDVD.
  • the polypeptide is a chimeric peptide comprising an internalization peptide moiety and an active peptide moiety, the active peptide moiety being the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof,
  • the peptide moiety can facilitate uptake of the chimeric peptide by the cell.
  • the internalized peptide portion comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2).
  • the chimeric peptide comprises the amino acid sequence YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 3).
  • the pharmaceutically acceptable salt is selected from the group consisting of trifluoroacetate, acetate, hydrochloride, and phosphate.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable salt of the polypeptide of the first aspect, and a pharmaceutically acceptable carrier, excipient and/or diluent.
  • the pharmaceutical composition is a pre-lyophilized formulation, preferably comprising histidine and trehalose.
  • the pharmaceutical composition is a lyophilized formulation, preferably prepared by lyophilizing the pre-lyophilized formulation described above.
  • the pharmaceutical composition is a reconstituted formulation, preferably prepared by combining the lyophilized formulation described above with an aqueous solution.
  • the pharmaceutical composition is for treating, ameliorating or preventing a nervous system injury, a disease or pain associated with nervous system damage, a neurodegenerative disease, anxiety or epilepsy in an individual.
  • the pharmaceutical composition is used as a neuroprotective agent.
  • the present application provides a method of treating, ameliorating or preventing a nervous system injury, a neurological damage-related disease or pain, a neurodegenerative disease, anxiety or epilepsy in an individual, the method comprising administering a first to the individual A pharmaceutically acceptable salt of the polypeptide of aspect or a pharmaceutical composition of the second aspect.
  • the present application provides the pharmaceutically acceptable salt of the polypeptide of the first aspect or the pharmaceutical composition of the second aspect for the preparation of a nervous system for the treatment, amelioration or prevention of damage to the nervous system A medicament for a related disease or pain, a neurodegenerative disease, anxiety or epilepsy or in the preparation of a neuroprotective agent.
  • the nervous system damage is a nervous system damage caused by excitatory neurotoxicity.
  • the neurological damage caused by the excitatory neurotoxicity comprises an episode selected from stroke, spinal cord injury, ischemic or traumatic injury of the brain or spinal cord, damage to central nervous system (CNS) neurons, including Acute CNS injury, ischemic stroke or spinal cord injury, as well as hypoxia, ischemia, mechanical injury and neurodegenerative diseases, anxiety, epilepsy, stroke-induced damage.
  • CNS central nervous system
  • the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease or Huntington's disease .
  • the nervous system injury or pain is located in the peripheral nervous system or the central nervous system.
  • the neurological damage-related disease is stroke.
  • the stroke comprises ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke.
  • the stroke is an ischemic stroke.
  • the individual is a mammal, such as a non-primate or primate, such as a human.
  • Figure 1 shows the Pull-down assay to detect the interaction of P5 with the PDZ1/2 domain.
  • M represents the protein molecular weight marker; Lane 1 is His+PDZ1/2+P5; Lane 2 is P5 alone; Lane 3 is His+P5; Lane 4 is His+PDZ1/2.
  • the elution band shown in lane 1 contains both P5 and PDZ1/2, confirming that P5 is capable of binding to the PDZ1/2 domain.
  • Figure 2 shows a comparison of experimental data on the pharmacodynamics of different polypeptide salts in rats.
  • Figure 3 shows the results of cytotoxicity assays for different polypeptide salts.
  • Figure 4 shows the stability of different polypeptide salts, wherein the A and B graphs show the content and impurity species of different solid forms of the polypeptide salt after exposure to light + ultraviolet, high temperature and high humidity; C and D, respectively; The figure shows the content of the aqueous solution form of different polypeptide salts and the number of impurity species after being placed under light and high temperature conditions.
  • the inventors of the present application conducted intensive studies on peptides that are capable of reducing the damaging effects of at least a portion of NMDAR-excitatory neurotoxicity-mediated neurological disorders. Without wishing to be bound by any theory, it is believed that such peptides function, at least in part, by inhibiting the interaction between NMDAR and postsynaptic density 95 protein (PSD-95) (i.e., PSD-95 inhibitors).
  • PSD-95 postsynaptic density 95 protein
  • the inventors of the present application have intensively considered various therapeutic targets of diseases related to nervous system, and carried out the design and screening of polypeptide neuroprotective agents through pharmacological and pharmacological experiments in vitro and in vivo.
  • the peptide obtained by screening is further modified to obtain a pharmaceutically acceptable salt of a polypeptide having desirable properties.
  • the application provides a pharmaceutically acceptable salt of a polypeptide, wherein the polypeptide comprises the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof.
  • the term "functional variant” refers to a variant having the same or similar biological function and properties as the parent. As a non-limiting example, a “functional variant” can be obtained by performing one or more conservative substitutions in the parent.
  • the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof is also referred to herein as the "active peptide moiety", which is used herein as an active part of the treatment of central nervous system damage or as a neuroprotective agent. .
  • NMDAR-based structures some active peptides that inhibit the interaction between NMDAR and PSD-95 are NMDAR-based structures.
  • NMDAR2B has GenBank ID 4099612 and the C-terminal 20 amino acids are FNGSSNGHVYEKLSSLESDV and PL motif ESDV.
  • Some of the existing active peptides have selected a partial amino acid sequence at the C-terminus of NMDAR2B, thereby producing competitive inhibition of PSD-95 with NMDAR2B.
  • Studies have suggested that the ESDV or LESDV segments in the above peptides play an important role in inhibiting the interaction between NMDAR and PSD-95 proteins.
  • a functional variant provided herein is a variant produced following one or more conservative substitutions of the LDTEI moiety of SEQ ID NO: 1.
  • the conservative substitution is selected from the group consisting of a substitution between D and E, a substitution between L, V and I, and a substitution between T and S.
  • the functional variant is a variant produced by replacing the LDTEI portion of SEQ ID NO: 1 with any of the following sequences: LDTEL, LDTEV, LDTDI, LDTDL, LDTDV, LDSEI, LDSEL , LDSEV, LDSDI, LDSDL, LDSDV, LETEI, LETEL, LETEV, LETDI, LETDL, LETDV, VDTEI, VDTEL, VDTEV, VDTDI, VDTDL, VDTDV, IDTEI, IDTEL, IDTEV, IDTDI, IDTDL, IDTDV, IETEI, IETEL, IETEV, IETDI, IETDL, IETDVD.
  • the functional variants disclosed herein further comprise at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% with the peptides mentioned above. , 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity amino acid sequence. It is known in the art that "identity" between two proteins is determined by aligning the sequence of a second protein substituted with the amino acid sequence of one protein and its conserved amino acid. The degree of identity between the two proteins is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm.
  • the functional variants disclosed herein include substitutions, deletions, additions and/or amino acid residues at 1, 2, 3, 4, 5 or more compared to the peptides mentioned above. Or insert a specific peptide that differs from the above disclosure.
  • a functional variant can be distinguished from a particular peptide disclosed above by one or more substitutions, deletions, additions, and/or insertions. These variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the above-described peptide sequences disclosed herein and evaluating them according to any of a variety of techniques well known in the art as described herein. Biological activity.
  • the polypeptide is a chimeric peptide comprising an internalization peptide moiety and an active peptide moiety, the active peptide moiety being the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof,
  • the peptide moiety can facilitate uptake of the chimeric peptide by the cell.
  • the internalization peptide suitable for the present application is not limited to a specific species, as long as The purpose of transmembrane and internalization can be achieved. It will also be understood by those skilled in the art that since the target of action of the active peptide is mainly located inside the neuronal cell, it is preferred that the internalization peptide which is specifically adapted to the neuronal cell. In some embodiments, the internalization peptide can be a Tat peptide.
  • the amino acid sequence of the Tat peptide is YGRKKRRQRRR (SEQ ID NO: 2).
  • the chimeric peptide comprises the amino acid sequence YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 3).
  • the internalization peptide may be linked to the active peptide by an amide bond as a fusion peptide, but may also be joined by other suitable means, such as chemical bond ligation. Coupling of the two components can be achieved by a coupling agent or a conjugating agent.
  • a coupling agent or a conjugating agent A large number of such reagents are commercially available and can be found in S. S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991).
  • Some examples of cross-linking reagents include J-succinimide-3-(2-pyridyldithio)propionate (SPOP) or N,N'-(1,3-phenylene)dimale.
  • crosslinking reagents include P,P'-difluoro-m,m'-dinitrodiphenyl sulfone (which forms irreversible crosslinks with amino and phenolic groups); dimethyl diethylamine hexanoate (for Amino group is specific); phenol-1,4-disulfonyl chloride (which is mainly reacted with an amino group); 1,6-hexamethylene diisocyanate or diisothiocyanate, or phenylazo-p-diisocyanate (which Main Reaction with an amino group; glutaraldehyde (which reacts with several different side chains) and double nitrogen benzidine (which reacts mainly with tyrosine and histidine).
  • P,P'-difluoro-m,m'-dinitrodiphenyl sulfone which forms irreversible crosslinks with amino and phenolic groups
  • dimethyl diethylamine hexanoate for Amino group
  • the active peptide of the present application and the fusion peptide fused to the internalization peptide can be synthesized by solid phase synthesis or recombinant methods.
  • Peptidomimetics can be synthesized using a variety of protocols and methods described in the scientific literature and patent literature, for example, Organic Syntheses Collective Volumes, Gilman et al. (ed.) John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9: 205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1: 14-119; 0 stergaard (1997) Mol. Divers. 3: 17-27; Ostresh (1996) Methods Enzymol. 267: 220-234.
  • the drug may be desirable to improve certain undesirable physicochemical or biopharmaceutical properties of the drug, such as altering the solubility or dissolution of the drug, by combining a molecule or ion that is oppositely charged with the drug to form a salt with the drug. , reduce hygroscopicity, improve stability, change the melting point, and the like.
  • the final determination of the ideal salt form requires a balance between physicochemical properties and biopharmaceutical properties.
  • the choice of the pharmaceutically acceptable salt form of the drug should give priority to the following requirements: solubility, wettability, stability to environmental factors under different conditions.
  • the pharmaceutically acceptable salt of the polypeptide of the present application may be in any suitable pharmaceutically acceptable salt form.
  • the pharmaceutically acceptable salt of the polypeptide is a trifluoroacetate salt. In some embodiments, the pharmaceutically acceptable salt of the polypeptide is acetate. In some embodiments, the pharmaceutically acceptable salt of the polypeptide is the hydrochloride salt. In some embodiments, the pharmaceutically acceptable salt of the polypeptide is a phosphate. In some embodiments, the pharmaceutically acceptable salt of the polypeptide is an acetate or hydrochloride salt.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable salt of the polypeptide of the first aspect, and a pharmaceutically acceptable carrier, excipient and/or diluent.
  • the compounds described herein can be prepared in the form of a lyophilized formulation.
  • the application provides a lyophilized formulation.
  • the lyophilized preparation is prepared from a pre-lyophilized preparation by lyophilization comprising at least an active ingredient, a buffer, a filler and water, wherein the active ingredient is a compound of the present application or a pharmaceutically acceptable salt thereof.
  • a preferred buffer is histidine.
  • Other buffers are selected from the group consisting of succinate, citrate, gluconate, acetate, phosphate, and Tris. Fillers provide structure for the lyophilized compound.
  • the filler is selected from the group consisting of mannitol, trehalose, dextran-40, glycine, lactose, sorbitol, and sucrose, and the like, with trehalose being preferred.
  • the lyophilized formulation of the present application comprises a compound described above, or a pharmaceutically acceptable salt thereof, and histidine and trehalose.
  • the lyophilized formulation can be reconstituted by rehydrating the lyophilized formulation with a solution to a solution of microparticles that are invisible to the naked eye.
  • the application provides a reconstituted formulation prepared by combining a lyophilized formulation with an aqueous solution.
  • the aqueous solution is water for injection.
  • the aqueous solution is physiological saline.
  • lyophilization relates to a process in which the material to be dried is first frozen and then sublimed in a vacuum to remove ice or frozen solvent.
  • a pharmaceutically acceptable salt of a polypeptide disclosed herein can be in the form of a pharmaceutical composition It is administered.
  • the pharmaceutical compositions can be made by conventional methods of mixing, dissolving, granulating, tableting, milling, emulsifying, encapsulating, capturing or lyophilizing.
  • the pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or excipients which facilitate processing the pharmaceutically acceptable salt of the polypeptide into a pharmaceutically acceptable formulation. Proper formulation depends on the route of administration chosen.
  • the administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular, preferably intravenous.
  • the pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic.
  • a pharmaceutically acceptable salt of the polypeptide can be formulated into an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce the site of injection). Discomfort).
  • the solution may contain formulas such as suspending, stabilizing and/or dispersing agents.
  • the pharmaceutically acceptable salt of the polypeptide may be in the form of a powder for constitution with a suitable vehicle, such as sterile non-pyrogenic water, prior to use.
  • penetrants appropriate to the barrier to be penetrated are used in the formulation. This route of administration can be used to deliver a compound to the nasal cavity or for sublingual administration.
  • a pharmaceutically acceptable salt of a polypeptide can be formulated into a tablet, pill, lozenge, capsule, liquid, gel, syrup, slurry with a pharmaceutically acceptable carrier. , suspension, etc., for oral ingestion by the patient being treated.
  • suitable excipients include fillers such as sugars such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, Potato starch, gelatin, tragacanth, methylcellulose, carboxypropylmethylcellulose, sodium carboxymethylcellulose and/or povidone (PVP); granulating agents and binders.
  • a disintegrating agent such as crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate may be added.
  • the solid dosage form can be sugar coated or enteric coated using standard techniques.
  • suitable carriers, excipients or diluents include water, glycerol, oil, alcohol.
  • a flavoring agent, a preservative, a coloring agent, or the like may be added.
  • the pharmaceutically acceptable salts of the polypeptides can also be formulated into a depot preparation.
  • Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compound can be formulated with a suitable polymeric or hydrophobic material (for example, as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, for example, formulated as a slightly soluble solution. Salt.
  • Chimeric peptides can be delivered using liposomes and emulsions. Certain organic solvents such as dimethyl sulfoxide can also be used. Additionally, the compound can be delivered using a sustained release system, such as a semipermeable matrix of a solid polymer containing a therapeutic agent.
  • sustained release capsules release the chimeric peptide for several weeks up to over 100 days.
  • Other strategies for protein stabilization can be used depending on the chemical nature and biostability of the therapeutic agent.
  • a pharmaceutically acceptable salt of a polypeptide effective to achieve the intended purpose eg, mitigating traumatic stroke and related conditions
  • the amount of damage effect used.
  • a therapeutically effective amount means that a pharmaceutically acceptable salt of a polypeptide disclosed herein is used in relation to a central nervous system injury in a patient (or animal model) control population that has not been treated with a pharmaceutically acceptable salt of a polypeptide disclosed herein.
  • the amount of pharmaceutically acceptable salt of the polypeptide in the treated patient (or animal model population) that is sufficient to significantly reduce the damage caused by stroke.
  • an individual treated patient achieves a better output than a mean output in a comparable patient control population that is not treated by the methods disclosed herein (as determined by infarct volume or disability index), then the amount is also considered to be It is therapeutically effective.
  • the amount is also considered to be a therapeutically effective amount if the individual being treated shows 2 or fewer disability in the Rankin scale and 75 or more in the Barthel scale.
  • the dose is also considered therapeutically effective if the population of treated patients shows a significant improvement (ie less disability) score distribution on the disability scale compared to comparable untreated populations, see Lees et al. N Engl J Med 2006; 354: 588-600.
  • a therapeutically effective regimen represents a combination of a therapeutically effective dose and the frequency of administration required to achieve the above intended purpose.
  • a preferred dosage range for a pharmaceutically acceptable salt of a polypeptide comprises administering from 0.001 to 20 [mu]mol of the salt of the present application per kg patient body weight, optionally 0.03 to 3 [mu]mol per kg patient body weight, including any value therebetween. Or a range between any two values.
  • 0.1-20 [mu]mol of the salt of the present application per kg patient body weight is administered within 6 hours.
  • 0.1 to 10 ⁇ mol of the salt of the present application per kg patient body weight is administered within 6 hours, more preferably about 0.3 ⁇ mol of the salt of the present application per kg patient body weight within 6 hours.
  • the dosage range is from 0.005 to 0.5 [mu]mol of the salt of the present application per kg patient body weight.
  • the different surface area: mass ratio can be compensated by dividing by 6.2, while the dose per kg of body weight is converted from rat to human.
  • a suitable dose of the salt of the present application for human administration in grams may be from 0.01 to 100 mg/kg of patient body weight, or more preferably from 0.01 to 30 mg/kg of patient body weight or from 0.01 to 10 mg/kg of patient body weight, or from 0.01 to 1 mg/ The weight of a kg patient, including any value in between or a range between any two values.
  • the amount of pharmaceutically acceptable salt of the administered polypeptide depends on the subject being treated, the weight of the subject, the severity of the pain, the mode of administration, and the adjustment of the prescribing physician.
  • the treatment can be repeated when the symptoms are detectable, even when it is not detectable. Treatment can be provided alone or in combination with other drugs.
  • a therapeutically effective dose of a pharmaceutically acceptable salt of a polypeptide disclosed herein is capable of providing a therapeutic benefit without causing significant toxicity.
  • the toxicity of the chimeric peptide can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, for example by measuring LD50 (a dose that kills 50% of the population) or LD100 (a dose that kills 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Preference is given to pharmaceutically acceptable salts of polypeptides which exhibit a high therapeutic index (see for example Fingl et al, 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1).
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable salt of the polypeptide of the first aspect, and a pharmaceutically acceptable carrier, excipient and/or diluent.
  • the pharmaceutical composition is for treating, ameliorating or preventing a nervous system injury, a disease or pain associated with nervous system damage, a neurodegenerative disease, anxiety or epilepsy in an individual.
  • the pharmaceutical composition is used as a neuroprotective agent.
  • the nervous system damage is a nervous system damage caused by excitotoxicity.
  • neurological damage caused by excitotoxicity comprises an episode of stroke, spinal cord injury, ischemic or traumatic injury of the brain or spinal cord, damage to central nervous system (CNS) neurons, including acute CNS Injury, ischemic stroke or spinal cord injury, as well as hypoxia, ischemia, mechanical injury and neurodegenerative diseases, anxiety, epilepsy, stroke-induced damage.
  • CNS central nervous system
  • the pharmaceutical composition is for treating, ameliorating or preventing neurological damage caused by ischemic stroke or ischemic stroke. In some embodiments, the pharmaceutical composition is for treating, ameliorating or preventing neurological damage caused by hemorrhagic stroke or hemorrhagic stroke. In some embodiments, the pharmaceutical composition is for treating, ameliorating or preventing a neurological injury caused by a hemorrhagic stroke converted from an ischemic stroke or a hemorrhagic stroke converted from an ischemic stroke.
  • Stroke is a condition caused by impaired blood flow in the CNS. Possible causes include embolism, bleeding, and thrombosis. Some neuronal cells die immediately due to impaired blood flow. These cells release their component molecules (including glutamate), which then activate the NMDA receptor, which increases intracellular calcium levels and intracellular enzyme levels, resulting in more neuronal cell death ( Excitatory neurotoxicity cascade amplification). The death of the CNS organization is called infarction.
  • the infarct volume i.e., the volume of neuronal cells in the brain caused by stroke
  • Symptomatic effects depend both on the infarct volume and on where the infarct is located in the brain.
  • Rankin Stroke Outcome Scale Rankin, Scott MedJ; 2:200-15 (1957)
  • Barthel Index Barthel Index
  • the Barthel Index is based on a series of questions about the patient's ability to perform 10 basic activities of daily living, which scores between 0 and 100, with lower scores indicating more disability (Mahoney et al., Maryland State Medical Journal) 14:56-61 (1965).
  • stroke severity/output can be measured using the NIH Stroke Scale, available on the World Wide Web at ninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf.
  • the scale is based on the patient's ability to perform 11 sets of functions, including assessing the patient's level of consciousness, movement, feel, and language function.
  • Ischemic stroke is more clearly expressed as a type of stroke caused by blockage of blood flow to the brain.
  • the potential for such blockages is most commonly the occurrence of fatty deposits along the walls of blood vessels. This condition is called atherosclerosis.
  • These fat deposits can cause two types of obstruction.
  • Cerebral thrombosis refers to a thrombus (blood clot) that is produced in the obstructed portion of a blood vessel.
  • Cerebral embolism usually refers to various emboli in the blood (such as a wall thrombus in the heart, atherosclerotic plaque, fat, tumor cells, fibrocartilage or air, etc.) blocked by blood flow into the cerebral artery.
  • Blood vessels when the collateral circulation can not be compensated, cause ischemic necrosis of brain tissue in the blood supply area of the artery, and focal neurological deficit occurs.
  • the second important cause of embolism is an irregular heartbeat called arterial fibrillation. It causes a condition in which a blood clot can form in the heart, move and transfer to the brain.
  • Other potential causes of ischemic stroke are hemorrhage, thrombosis, arterial or venous severing, cardiac arrest, shock from any cause (including bleeding), and iatrogenic causes, such as cerebral blood vessels or blood vessels leading to the brain. Direct surgical injury or cardiac surgery. Ischemic stroke constitutes approximately 83% of all stroke cases.
  • the pharmaceutical composition is for treating, ameliorating or preventing a neurodegenerative disease, anxiety or epilepsy, wherein the neurodegenerative disease comprises Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease or Huntington's disease.
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease Huntington's disease.
  • the pharmaceutical composition is used as a neuroprotective agent.
  • the present application provides a method of treating, ameliorating or preventing a nervous system injury, a neurological damage-related disease or pain, a neurodegenerative disease, anxiety or epilepsy in an individual, the method comprising administering a first to the individual A pharmaceutically acceptable salt of the polypeptide of aspect or a pharmaceutical composition of the second aspect.
  • the nervous system injury is a neurological damage caused by excitatory neurotoxicity, wherein the injury or pain is located in the peripheral nervous system or the central nervous system.
  • neurological damage caused by excitatory neurotoxicity comprises an episode of stroke or spinal cord injury, ischemic or traumatic injury of the brain or spinal cord, and damage to central nervous system (CNS) neurons, including acute CNS.
  • CNS central nervous system
  • the neurodegenerative disease includes Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease or Huntington's disease.
  • the disease is a nervous system injury caused by ischemic stroke or ischemic stroke. In some embodiments, the disease is a neurological injury caused by a hemorrhagic stroke or a hemorrhagic stroke. In some embodiments, the disease is a neurological injury caused by a hemorrhagic stroke converted from ischemic stroke or a hemorrhagic stroke converted from ischemic stroke.
  • the present application provides the pharmaceutically acceptable salt of the polypeptide of the first aspect or the pharmaceutical composition of the second method for preparing a nervous system damage or nervous system damage for treating, ameliorating or preventing the individual A medicament for a related disease or pain, a neurodegenerative disease, anxiety or epilepsy or in the preparation of a neuroprotective agent.
  • the nervous system injury is a neurological damage caused by excitatory neurotoxicity, wherein the injury or pain is located in the peripheral nervous system or the central nervous system.
  • neurological damage caused by excitatory neurotoxicity comprises an episode of stroke or spinal cord injury, ischemic or traumatic injury of the brain or spinal cord, and damage to central nervous system (CNS) neurons, including acute CNS.
  • CNS central nervous system
  • the neurodegenerative disease includes Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease or Huntington's disease.
  • the disease is a nervous system injury caused by ischemic stroke or ischemic stroke. In some embodiments, the disease is a neurological injury caused by a hemorrhagic stroke or a hemorrhagic stroke. In some embodiments, the disease is a neurological injury caused by a hemorrhagic stroke converted from ischemic stroke or a hemorrhagic stroke converted from ischemic stroke.
  • individual refers to an animal comprising birds, reptiles, and mammals.
  • the individual is a mammal, including primates and non-primates, such as humans, chimpanzees, cows, horses, pigs, sheep, goats, dogs, cats, and such as rats and mice. Rodents.
  • the Tat transmembrane peptide YGRKKRRQRRR (SEQ ID NO: 2) was selected and ligated to a different number of amino acids to form a peptide library.
  • the chimeric peptide molecules in the peptide library were respectively interacted with the PDZ1/2 domain expressed and purified in vitro, and the polypeptide was initially screened according to the strength of the interaction force.
  • the immobilized molecule is PDZ1/2 protein, molecular weight: ⁇ 20kD, concentration: 2mg/ml; molecular phase of mobile phase (analyte): polypeptide to be screened, molecular weight: ⁇ 2kD, concentration: 10mg/ml.
  • the CM5 chip was used for fixation using a Biacore 3000 instrument.
  • the running buffer was PBS + 0.005% Tween 20. Fixation was carried out using an amino coupling method.
  • the concentration of the ligand was 10 ⁇ g/ml.
  • the fixing buffer was 10 mM sodium acetate, pH 4.0. Fixed amount: 1400 RU, fixed to flow cells 2.
  • the flow rate used was 10 ⁇ l/ml and the ligand was injected for 1 minute.
  • 10 mM Gly at pH 2.0 + 2.5 was used as a regenerant, and regeneration was carried out at a flow rate of 30 ⁇ l/min.
  • the injection time is 30s.
  • Kinetic analysis was performed using the following conditions: control channel: flow cell 1; running buffer was PBS; using Kinetic Analysis Wizard mode, concentration gradient was 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM; injection time was 1 minute; dissociation time was 2 min; flow rate was 30 ⁇ l/min.
  • the data was fitted using the Biaevaluation 4.1 software.
  • the quasi-sum model is a 1:1 binding model.
  • the dissociation constant KD value is inversely proportional to the force.
  • control chimeric peptide NA-1 was introduced with the following sequence:
  • NA-1 YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 4)
  • YE-NA-1 YGRKKRRQRRRYEKLSSIESDV (SEQ ID NO: 5)
  • the chimeric peptides YE-NA-1 and P5 interacted more strongly with the PDZ1/2 domain than the control chimeric peptide NA-1, and the action properties of P5 were better. Therefore, according to the inventors' hypothesis, the additional YE two amino acid residues at the N-terminus of the active peptide have a certain potentiating effect on the interaction of the polypeptide with the PDZ1/2 domain. Furthermore, P5 reduced two less hydrophobic serines (SS) relative to the carboxy terminus of YE-NA-1, which, according to the inventors' hypothesis, may thus further increase the interaction of the polypeptide with the PDZ1/2 domain.
  • SS hydrophobic serines
  • Example 2 Pull-down assay to detect the interaction between P5 and PDZ1/2 domain
  • the column was equilibrated with 100 ⁇ l of His beads and 1 ml of MCAC-0 buffer for 5 min. Concussion at 4 °C. The mixture was centrifuged at 5000 g for 1 minute at 4 ° C, and the supernatant was discarded. 1 mg of PDZ1/2 protein was added to the mixture and made up to 1 ml with buffer. The mixture was spun for 1 hour at 4 °C. The mixture was centrifuged at 5000 g for 1 minute at 4 ° C, and the supernatant was discarded. Wash 3 times with 1 ml of MCAC-0 buffer for 5 minutes each time (at 4 ° C, shake wash).
  • the eluted band of the chimeric peptide P5 contains both the P5 and PDZ1/2 domains, This confirmed that the chimeric peptide P5 is capable of binding to the PDZ1/2 domain.
  • Example 3 Therapeutic effect of different P5 salts on rat MCAO model
  • P5-trifluoroacetate P5-TFA
  • P5-acetate P5-Ac
  • P5-hydrochloride P5-Cl
  • Animals Adult SD rats (Vitronius), SPF grade, body weight 220-250 g, male.
  • the preparation of the focal cerebral ischemia-reperfusion model was based on the reversible middle cerebral artery occlusion (MCAO) suture method proposed by Longa and improved according to the anatomical structure of the rat brain to prepare a focal cerebral ischemia-reperfusion model.
  • MCAO middle cerebral artery occlusion
  • CCA common carotid artery
  • ECA external carotid artery
  • pterygopalatine 0.26mm monofilament nylon fishing line head 0.5cm with paraffin
  • the length of the suture was about 18-20 mm from the CCA bifurcation, depending on the animal's weight, embolization right
  • the middle cerebral artery is then sutured, and the end of the suture is fixed to the skin.
  • the suture was carefully taken out to form a reperfusion.
  • the body temperature was maintained at (37 ⁇ 0.5) °C during ischemia and 2 h after reperfusion.
  • the success of the model is that the left limb is paralyzed after the patient is anesthetized, and the standing is unstable. When the tail is lifted, it turns to one side.
  • control group normal group
  • model group saline group
  • NBP positive drug group
  • P5 salt administration group One hour after the ischemic modeling, physiological saline, the positive drug Enb (2.5 mg/kg), and each P5 salt (10 mg/kg) were administered to each group via tail vein injection.
  • the rats were decapitated and the brain tissue was quickly placed in a -20 ° C refrigerator. After 10 minutes, the brain was placed in a room temperature environment. The brain was placed in a rat brain slice mold to remove the olfactory bulb, cerebellum and low brain. After drying, cut the five-knife in a 2 mm interval as shown in the map, and cut into six consecutive continuous coronal slices. Then, the brain slices were quickly placed in a 5 ml solution containing 2% TTC, and incubated at 37 ° C for 30 minutes in the dark, and the brain slices were flipped once every 5 minutes. After TTC staining, the normal tissue was rose red, and the infarcted tissue was unstained and white.
  • Each group of brain slices was arranged neatly, photographed and saved, and processed by image analysis system software and counted.
  • the infarct area of each brain slice was calculated, multiplied by the thickness of each slice of brain 2 mm, and the infarct area of each brain slice of each animal was multiplied by The thickness is added, which is the volume of cerebral infarction. Volume is expressed as a percentage of the brain's hemisphere to eliminate the effects of cerebral edema.
  • Test cell line PC12 cells vertical laminar flow clean bench, steam sterilizer, centrifuge, microscope, microplate reader, coverslip, blood cell counting plate, manual counter, alcohol lamp, pipette, pipette , tips, centrifuge tubes, 96-well plates, PBS/salt, high glucose DMEM medium (containing 10% FBS and 1% double antibody).
  • CCK8 was purchased from Soleil.
  • Viable cell rate (%) (experimental group - corresponding blank control group) / (0 ⁇ M experimental group - 0 ⁇ M blank control group) ⁇ 100%
  • This example tested the stability of the three P5 salts of Example 3 under illumination, high temperature and high humidity conditions, respectively.
  • the powders of the three P5 salts prepared in Example 3 were treated with light (3000 Lx) + ultraviolet, high temperature (60 ° C) and high humidity (75% RH) for 10 days. After the treatment, the powder is dissolved in water to prepare a solution having a concentration of 2 mg/ml, After precision measurement, 10 ⁇ l was injected into the liquid chromatograph, and the chromatogram was recorded. The relevant materials were calculated according to the area normalization method to analyze the content and the number of impurity species.
  • High Performance Liquid Chromatograph (Agilent, 1260 EZChrom); Column (Agilent, ZORBAX300SB-C18 (4.6*250mm, 5 ⁇ m) SN: USHH008416); Analytical Balance (Sedolis, BT25S); Filter Membrane (Millipore, 0.45 ⁇ m PTFE) ); acetonitrile (MREDA); water (love); TFA (MREDA); comprehensive drug stability test chamber three-box type (Shanghai Zuocheng Experimental Instrument Co., Ltd., model: SHH-3SDT)
  • the percentage of the main peak area of the three salts is above 99.5%, there is no significant difference, and the acetate is the most stable in comparison with the number of impurities;
  • Example 3 The three P5 salts of Example 3 were dissolved in water to prepare a solution of 2 mg/ml. It was treated with light (3000 Lx) and high temperature (60 ° C) for 10 days. After the treatment, a precise amount of 10 ⁇ l was injected into the liquid chromatograph, and the chromatogram was recorded. The relevant substances were calculated according to the area normalization method to analyze the content and the number of impurity species.
  • High Performance Liquid Chromatograph (Agilent, 1260 EZChrom); Column (Agilent, ZORBAX300SB-C18 (4.6*250mm, 5 ⁇ m) SN: USHH008416); Analytical Balance (Sedolis, BT25S); Filter Membrane (Millipore, 0.45 ⁇ m PTFE) ); acetonitrile (MREDA); water (love); TFA (MREDA); comprehensive drug stability test chamber three-box type (Shanghai Zuocheng Experimental Instrument Co., Ltd., model: SHH-3SDT)
  • solutions of the three salts also showed better stability under different treatments.

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Abstract

提供了多肽的药学可接受的盐及其药物组合物，所述多肽包含氨基酸序列YEKLLDTEI或其功能性变体。

Description

多肽的药学可接受的盐及其应用 技术领域
本申请大体上涉及生物医学领域。具体而言，本申请提供了用于治疗、改善或预防神经系统相关病症的多肽的药学可接受的盐、组合物和方法。
发明背景
神经系统相关疾病的表现形式多种多样，并且严重危害人们的健康状态以及生活质量。
脑卒中是中老年常见的急性脑血管病，并出现年轻化的趋势。它是当今世界对人类危害最大的三种疾病(癌症，心脑血管疾病，糖尿病)之一。据统计，中国每年死于脑血管疾病近300万人，高于欧美国家4到5倍，是日本的3.5倍，甚至高于泰国、印度等发展中国家；发病率以每年8.7％的速率上升，复发率超过30％，5年内再次发生率达54％；脑卒中患者幸存者中75％不同程度丧失劳动能力，40％重残。
脑卒中大致可以分为两大类，即缺血性脑卒中和出血性脑卒中，其中缺血性脑卒中占脑卒中病人总数的85％。目前，缺血性脑卒中的治疗药物主要分为如下几类：血管扩张药(如潘生丁等)、改善微循环、扩充血容量的药物(如低分子右旋糖酐等)、溶解血栓的药物(如尿激酶等)、抗凝治疗、防止血小板凝聚的药(如阿司匹林等)、中药、神经元保护剂等，但是由于这些药物大多存在副作用大、具有潜在的危险性或疗效不显著等问题，因此研究脑卒中的发病机制并针对其机制进行药物研发，对防治脑血管病发生和发展具有重要的社会意义。
脑卒中的特征是局部缺血区域、脑出血区域和/或创伤区域的神经元细胞死亡。而由于脑缺血引起的神经元死亡或损伤是一个损伤级联反应过程，脑缺血后组织血液灌注下降，兴奋性神经递质增加，激活NMDA和AMPA受体，引起离子通道开放，钙离子内流，激活大量的酶引发信号级联反应，造成多途径的神经细胞损伤。其下游的突触后密度95蛋白(PSD-95)通过与多种蛋白的相互作用，引发一系列缺血性损伤，是脑缺血损伤的关键性位点，同时也是药物治疗的潜在靶点，因此PSD-95抑制剂的研发对于包括脑卒中在内的多种兴奋性神经毒性引起的神经系统损伤具有很大的药用意义。
此外，研究显示兴奋性神经递质NMDA在焦虑，癫痫和多种神经退行性疾病如阿尔茨海默氏病、肌萎缩性侧索硬化症(ALS)、帕金森氏病或亨廷顿氏病等中都发挥重要作用。例如，研究显示中枢的谷氨酸能系统过度兴奋可引发焦虑，而NMDA受体(NMDAR)负责谷氨酸兴奋性神经毒性的主要部分。癫痫的发作包含起动、发作性放电的维持与扩展以及发作性放电的抑制3个不同而连续的病理生理过程，在该过程中兴奋性神经递质如谷氨酸、天门冬氨酸起重要作用。在阿尔兹海默症中，PSD-95通过GluR6-PSD-95-MLK3通路参与导致其的神经毒性机制。此外，在亨廷顿氏病中， PSD-95是NMDA受体和huntingtin突变体神经毒性的介体。因此PSD-95抑制剂的研发对于上述疾病的治疗、改善和预防也具有重要意义。
发明概述
第一方面，本申请提供了多肽的药学可接受的盐，其中所述多肽包含氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体。
在一些实施方案中，所述功能性变体为SEQ ID NO:1中的LDTEI部分发生一处或多处保守型取代后产生的变体。
在一些实施方案中，保守型取代选自D和E之间的取代，L、V和I之间的取代以及T和S之间的取代。
在一些实施方案中，所述功能性变体为SEQ ID NO:1中的LDTEI部分被替换为下述任一序列后产生的变体：LDTEL、LDTEV、LDTDI、LDTDL、LDTDV、LDSEI、LDSEL、LDSEV、LDSDI、LDSDL、LDSDV、LETEI、LETEL、LETEV、LETDI、LETDL、LETDV、VDTEI、VDTEL、VDTEV、VDTDI、VDTDL、VDTDV、IDTEI、IDTEL、IDTEV、IDTDI、IDTDL、IDTDV、IETEI、IETEL、IETEV、IETDI、IETDL、IETDV。
在一些实施方案中，所述多肽是包含内化肽部分和活性肽部分的嵌合肽，所述活性肽部分为氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体，所述内化肽部分能促进所述嵌合肽被细胞摄取。
在一些实施方案中，所述内化肽部分包含氨基酸序列YGRKKRRQRRR(SEQ ID NO:2)。
在一些实施方案中，所述嵌合肽包含氨基酸序列YGRKKRRQRRRYEKLLDTEI(SEQ ID NO:3)。
在一些实施方案中，所述药学可接受的盐选自三氟乙酸盐、醋酸盐、盐酸盐和磷酸盐。
第二方面，本申请提供了药物组合物，其包含第一方面所述的多肽的药学可接受的盐，以及药学可接受的载体、赋形剂和/或稀释剂。
在一些实施方案中，所述药学组合物为预冻干制剂，优选包含组氨酸和海藻糖。
在一些实施方案中，药物组合物为冻干制剂，优选通过将以上所述预冻干制剂冻干而制备。
在一些实施方案中，药物组合物为复原制剂，优选通过将以上所述的冻干制剂与水溶液结合而制备。
在一些实施方案中，所述药物组合物用于治疗、改善或预防个体的神经系统损伤、神经系统损伤相关的疾病或疼痛、神经退行性疾病、焦虑或癫痫。
在一些实施方案中，所述药物组合物用作神经元保护剂。
第三方面，本申请提供了治疗、改善或预防个体的神经系统损伤、神经系统损伤相关的疾病或疼痛、神经退行性疾病、焦虑或癫痫的方法，所述方法包括向所述个体施用第一方面所述的多肽的药学可接受的盐或第二方面所述的药物组合物。
第四方面，本申请提供了第一方面所述的多肽的药学可接受的盐或第二方面所述的药物组合物在制备用于治疗、改善或预防个体中的神经系统损伤、神经系统损伤相关的疾病或疼痛、神经退行性疾病、焦虑或癫痫的药物或在制备神经元保护剂中的用途。
在第二、第三或第四方面的一些实施方案中，所述神经系统损伤为兴奋性神经毒性引起的神经系统损伤。
在一些实施方案中，所述兴奋性神经毒性引起的神经系统损伤包括选自脑卒中、脊髓损伤、脑或脊髓的缺血性或创伤性损伤、中枢神经系统(CNS)神经元的损伤，包括急性CNS损伤、缺血性脑卒中或脊髓损伤，以及缺氧、缺血、机械损伤和神经退行性疾病、焦虑、癫痫、脑卒中引起的损伤。
在第二、第三和第四方面的一些实施方案中，所述神经退行性疾病选自阿尔茨海默氏病、肌萎缩性侧索硬化症(ALS)、帕金森氏病或亨廷顿氏病。
在第二、第三或第四方面的一些实施方案中，所述神经系统损伤或疼痛位于外周神经系统或中枢神经系统。
在第二、第三或第四方面的一些实施方案中，所述神经系统损伤相关的疾病为脑卒中。在一些实施方案中，所述脑卒中包括缺血性脑卒中、出血性脑卒中和由缺血性脑卒中转化成的出血性脑卒中。在一些实施方案中，所述脑卒中是缺血性脑卒中。
在第二、第三或第四方面的一些实施方案中，所述个体是哺乳动物，例如非灵长类或灵长类动物，例如人。
附图简要描述
图1显示了Pull-down实验检测P5与PDZ1/2结构域的相互作用。M代表蛋白质分子量标识；泳道1为His+PDZ1/2+P5；泳道2为单独的P5；泳道3为His+P5；泳道4为His+PDZ1/2。泳道1所示的洗脱条带包含P5与PDZ1/2两者，证实P5能够结合PDZ1/2结构域。
图2显示了不同的多肽盐在大鼠体内药效学实验数据的对比图。
图3显示了不同的多肽盐的细胞毒性测定结果。
图4显示了不同的多肽盐的稳定性，其中A和B图分别显示了不同的多肽盐的固体形式在光照+紫外、高温和高湿条件下放置后的含量和杂质种类数；C和D图分别显示了不同多肽盐的水溶液形式在光照和高温条件下放置后的含量和杂质种类数。
发明详细描述
本申请的发明人对能降低至少部分由NMDAR兴奋性神经毒性介导的神经学病症的损伤效应的肽进行了深入研究。不希望受任何理论的束缚，据信这类肽至少部分通过抑制NMDAR与突触后密度95蛋白(PSD-95)之间的相互作用来发挥作用(即PSD-95抑制剂)。在此基础上，本申请的发明人对神经系统相关疾病的多个治疗靶点进行了深入思考，通过体内外的药理药效实验，进行了多肽类神经元保护剂的设计和筛选，并通过对筛选得到的肽进行进一步改进，得到了具有理想性质的多肽的药学可接受的盐。
除非另外指明，本申请中所用的术语具有本领域技术人员通常理解的含义。
本申请中对于氨基酸使用的单字母或三字母缩写遵循国际惯例。
本说明书和权利要求书中，词语“包括”、“包含”和“含有”意指“包括但不限于”，且并非意图排除其它部分、添加物、组分或步骤。
第一方面，本申请提供了多肽的药学可接受的盐，其中所述多肽包含氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体。
术语“功能性变体”是指与母体具有相同或相近的生物学功能和性质的变体。作为非限制性的实例，“功能性变体”可以通过在母体中进行一处或多处保守型取代获得。氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体在本申请中也被称为“活性肽部分”，其在本申请中作为治疗中枢神经系统损伤或作为神经元保护剂的活性部分。
根据已有的研究，一些抑制NMDAR与PSD-95之间的相互作用的活性肽是基于NMDAR的结构。例如，NMDAR2B具有GenBank ID4099612，C末端20个氨基酸为FNGSSNGHVYEKLSSLESDV和PL基序ESDV。已有的一些活性肽选取了NMDAR2B的C末端的部分氨基酸序列，从而与NMDAR2B产生对PSD-95的竞争性抑制。有研究认为上述肽中的ESDV或LESDV区段在抑制NMDAR与PSD-95蛋白之间的相互作用中发挥重要作用。本申请的发明人通过分析和验证，得到肽序列YEKLLDTEI，其相对于上述NMDAR2B的C末端氨基酸组成，不含有KL之后的SS两个残基，同时相对于PL基序增加了N端方向的YEKL氨基酸序列，本申请的发明人证实该序列能够增强活性肽与PDZ1/2结构域的相互作用。相对于YEKL基序，其C端的LDTEI可以进行变化，预期不影响活性肽的活性或有可能增加其活性。因此，在一些实施方案中，本申请提供的功能性变体为SEQ ID NO:1中的LDTEI部分发生一处或多处保守型取代后产生的变体。
在一些实施方案中，保守型取代选自D和E之间的取代，L、V和I之间的取代以及T和S之间的取代。
在更具体的一些实施方案中，功能性变体为SEQ ID NO:1中的LDTEI部分被替换为下述任一序列后产生的变体：LDTEL、LDTEV、LDTDI、LDTDL、LDTDV、LDSEI、LDSEL、LDSEV、LDSDI、LDSDL、LDSDV、LETEI、LETEL、LETEV、LETDI、LETDL、LETDV、VDTEI、VDTEL、VDTEV、VDTDI、VDTDL、VDTDV、 IDTEI、IDTEL、IDTEV、IDTDI、IDTDL、IDTDV、IETEI、IETEL、IETEV、IETDI、IETDL、IETDV。
在一些实施方案中，本文所公开的功能性变体还包括与以上提到的肽具有至少60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％甚至更高的同一性的氨基酸序列。本领域已知，两种蛋白之间的“同一性”通过将一种蛋白的氨基酸序列和它的保守氨基酸取代的第二种蛋白的序列进行比对来确定。使用本领域技术人员公知的计算机算法和方法确定两种蛋白之间的同一性程度。两个氨基酸序列之间的同一性优选地通过利用BLASTP算法确定。
在一些实施方案中，本文所公开的功能性变体包括与以上提到的肽相比，具有1、2、3、4、5或更多处的氨基酸残基的取代、缺失、添加和/或插入区别于上述公开的具体的肽。
如上所述，功能性变体可以通过一个或多个取代、缺失、添加和/或插入区别于上述公开的具体的肽。这些变体可以是天然存在的或可以是合成产生的，例如，通过修饰一个或多个本文公开的上述肽序列并按照本文所述用本领域内公知的多种技术中的任何一种评估其生物活性。
在一些实施方案中，所述多肽是包含内化肽部分和活性肽部分的嵌合肽，所述活性肽部分为氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体，所述内化肽部分能促进所述嵌合肽被细胞摄取。
本领域技术人员应当理解，将活性肽和内化肽嵌合的目的主要在于使活性肽更好地到达作用靶点，因此，适用于本申请的内化肽并不局限于特定种类，只要能实现穿膜、内化的目的即可。本领域技术人员还应当理解，由于活性肽的作用靶点主要位于神经元细胞内部，因此能特异性地适合于神经元细胞的内化肽是优选的。在一些实施方案中，内化肽可以为Tat肽。在一些实施方案中，Tat肽的氨基酸序列为YGRKKRRQRRR(SEQ ID NO:2)。在一些实施方案中，嵌合肽包含氨基酸序列YGRKKRRQRRRYEKLLDTEI(SEQ ID NO:3)。
应当理解，内化肽可以与活性肽通过酰胺键连接而作为融合肽，但是也可以通过其他合适的方式进行接合，例如化学键接合。两种组分的偶联可以通过偶联剂或缀合剂实现。大量这类试剂是可商业获得的，并可以参见S.S.Wong，Chemistry of Protein Conjugation and Cross-Linking,CRC Press(1991)。交联试剂的一些例子包括J-琥珀酰亚胺-3-(2-吡啶二硫代)丙酸盐(SPOP)或N，N'-(1，3-亚苯基)双马来酰亚胺；N，N’-亚乙基-双-(碘乙酰胺)或具有6到11个碳亚甲基桥的其他这类试剂(其它巯基相对特异)；和1，5-二氟-2，4-二硝基苯(其与氨基和酪氨酸基形成不可逆的连接)。其他交联试剂包括P，P’-二氟-m，m’-二硝基二苯砜(其与氨基和酚基形成不可逆的交联)；二乙胺代己酸二甲酯(其对氨基是特异的)；苯酚-1，4-二磺酰氯(其主要与氨基反应)；1，6-己二异氰酸酯或二异硫氰酸酯，或苯基偶氮-对-二异氰酸酯(其主要 与氨基反应)；戊二醛(其与若干不同的侧链反应)和双重氮基联苯胺(其主要与酪氨酸和组氨酸反应)。
可通过固相合成或重组方法合成本申请的活性肽以及与内化肽融合的融合肽。可使用科学文献和专利文献中所述的多种方案和方法合成拟肽，所述科学文献和专利文献例如为Organic Syntheses Collective Volumes,Gilman等(编)John Wiley&Sons,Inc.,NY,al-Obeidi(1998)Mol.Biotechnol.9:205-223；Hruby(1997)Curr.Opin.Chem.Biol.1：114-119；0stergaard(1997)Mol.Divers.3:17-27；Ostresh(1996)Methods Enzymol.267:220-234。
不希望被任何理论所束缚，用一种与药物带相反电荷的分子或离子与药物结合成盐，预期能改善药物某些不理想的理化性质或生物药学性质，如改变药物的溶解度或溶出度、降低吸湿性、提高稳定性、改变熔点等。理想的盐形式的最终确定需要根据理化性质和生物药学性质之间寻找平衡。选择药物的药学可接受的盐型应优先考虑以下要求：溶解性、引湿性、不同状态下对环境因素的稳定性。本申请的多肽的药学可接受的盐可以为任何合适的药学可接受的盐形式。在一些实施方案中，多肽的药学可接受的盐为三氟乙酸盐。在一些实施方案中，多肽的药学可接受的盐为醋酸盐。在一些实施方案中，多肽的药学可接受的盐为盐酸盐。在一些实施方案中，多肽的药学可接受的盐是磷酸盐。在一些具体实施方案中，多肽的药学可接受的盐为醋酸盐或盐酸盐。
第二方面，本申请提供了药物组合物，其包含第一方面所述的多肽的药学可接受的盐，以及药学可接受的载体、赋形剂和/或稀释剂。
可将本申请所述的化合物制备为冻干制剂形式。在一些实施方案中，本申请提供冻干制剂。冻干制剂由预冻干制剂通过冻干而制备，其至少包含活性成分、缓冲液、填充剂和水，其中活性成分即为本申请的化合物或其药学可接受的盐。在一些实施方案中，优选的缓冲液是组氨酸。其它缓冲液选自琥珀酸盐、柠檬酸盐、葡萄酸盐、醋酸盐、磷酸盐以及Tris等。填充剂为冻干化合物提供结构。在一些实施方案中，填充剂选自甘露醇、海藻糖、右旋糖酐-40、甘氨酸、乳糖、山梨醇和蔗糖等，其中优选海藻糖。在一些实施方案中，本申请的冻干制剂包含以上所述的化合物或其药学可接受的盐以及组氨酸和海藻糖。
可将冻干制剂复原，即用溶液将冻干制剂再水化为肉眼看不见的微粒的溶液。在一些实施方案中，本申请提供复原制剂，其通过将冻干制剂与水溶液结合而制备。在一些实施方案中，所述水溶液为注射用水。在一些实施方案中，所述水溶液为生理盐水。
术语“冻干”涉及一种工艺，待干燥的原料先被冷冻，然后在真空环境下升华而去除冰或冻结的溶剂。
在一些实施方案中，本文公开的多肽的药学可接受的盐可以以药物组合物的形式 被施用。药物组合物可以通过常规的混合、溶解、制粒、制锭、研磨、乳化、包封、捕获或冻干方法制造。
可以使用一种或多种生理学可接受的便于将多肽的药学可接受的盐加工成可药用制剂的载体、稀释剂、赋形剂或辅料，以常规方式配制药物组合物。适当的配制依赖于选择的施用途径。
在一些实施方案中，施用可以是肠胃外、静脉内、经口、皮下、动脉内、颅内、鞘内、腹膜内、局部、鼻内或肌内的，优选静脉内施用。
在一些实施方案中，用于肠胃外施用的药物组合物优选是无菌和基本等渗的。对注射而言，可以将多肽的药学可接受的盐配制进水溶液中，优选配制进生理学兼容的缓冲液例如Hank’s溶液、Ringer’s溶液，或生理盐水或乙酸缓冲液中(以减轻注射位点处的不适)。溶液可以含有配制剂例如悬浮剂、稳定剂和/或分散剂。
或者，多肽的药学可接受的盐可以是用于在使用前用合适的运载体(例如无菌无热源水)构建的粉末形式。
对跨粘膜施用而言，在配制物中使用适合要穿透的屏障的穿透剂。该施用途径可被用于将化合物递送至鼻腔或用于舌下施用。
在一些实施方案中，对经口施用而言，可以将多肽的药学可接受的盐与可药用的载体一起配制为片剂、丸剂、锭剂、胶囊、液体、凝胶、糖浆、浆体、悬浮液等，用于由被治疗的患者经口摄入。对于口服固体配制物例如粉末、胶囊和片剂而言，合适的赋形剂包括填充剂例如糖，如乳糖、蔗糖、甘露醇和山梨糖醇；纤维素制剂例如玉米淀粉、小麦淀粉、水稻淀粉、马铃薯淀粉、明胶、西黄蓍胶、甲基纤维素、羧丙基甲基纤维素、羧甲基纤维素钠和/或聚维酮(PVP)；制粒剂和粘合剂。如果需要，可以添加崩解剂，例如交联的聚乙烯吡咯烷酮、琼脂，或海藻酸或其盐，例如海藻酸钠。如果需要，可以使用标准技术对固体剂型进行糖包裹或肠溶衣包裹。对于口服液体制剂例如悬浮液、酏剂和溶液而言，合适的载体、赋形剂或稀释剂包括水、甘油、油、醇。另外，可以添加调味剂、防腐剂、着色剂等。
除了先前所述的配制物以外，也可以将多肽的药学可接受的盐配制成储存制剂。可以通过植入(例如皮下或肌内)或通过肌内注射来施用这类长效配制物。因此，例如可将化合物与合适的多聚体材料或疏水材料(例如配制为可接受的油中的乳剂)或离子交换树脂配制在一起，或配制为略溶的衍生物，例如配制为略溶的盐。
或者，可以使用其他药物递送系统。可使用脂质体和乳剂递送嵌合肽。也可以使用某些有机溶剂例如二甲基亚砜。另外，可以使用持续释放的系统(例如含有治疗剂的固体聚合物的半渗透性基质)递送化合物。
根据其化学性质，持续释放胶囊可释放嵌合肽数周直至超过100天。根据治疗试剂的化学性质和生物稳定性，可以使用用于蛋白质稳定的其他策略。
多肽的药学可接受的盐以有效达到预期目的(例如减轻损伤性脑卒中和相关病症 的损伤效果)的量使用。治疗有效量表示：相对于未用本文公开的多肽的药学可接受的盐治疗的患者(或动物模型)对照群体中的中枢神经系统损伤而言，在用本文公开的多肽的药学可接受的盐治疗的患者(或动物模型群体)中，足以显著降低脑卒中引起的损伤的多肽的药学可接受的盐的量。如果与未通过本文公开的方法治疗的可比较的患者对照群体中的平均输出(通过梗死体积或残疾指数测定)相比，个体经治疗的患者达到更良好的输出，则该量也被认为是治疗上有效的。如果个体被治疗的患者在Rankin标度中显示2或更少的残疾以及在Barthel标度中显示75或更多，则所述量也被认为是治疗上有效的量。如果与可比较的未治疗群体相比，被治疗的患者群体在残疾标度上显示显著改进(即更少残疾)的分值分布，则剂量也被认为是治疗上有效的，参见Lees等，N Engl J Med 2006；354:588-600。治疗上有效的方案表示治疗上有效的剂量和达到上述预期目的所需的施用频率的组合。
在一些实施方案中，多肽的药学可接受的盐的优选的剂量范围包括每kg患者体重施用0.001到20μmol的本申请的盐，任选地每kg患者体重施用0.03到3μmol，包括其间的任意值或者任意两个数值之间的范围。在一些方法中，在6小时内施用每kg患者体重0.1-20μmol本申请的盐。在一些方法中，在6小时内施用每kg患者体重0.1-10μmol本申请的盐，更优选在6小时内施用每kg患者体重约0.3μmol本申请的盐。在其他情况下，剂量范围是每kg患者体重施用0.005到0.5μmol本申请的盐。可以通过除以6.2来补偿不同的表面积：质量比，而将每kg体重的剂量从大鼠转化为人。以克计，用于人的本申请的盐的合适剂量可以是施用0.01到100mg/kg患者体重，或更优选0.01到30mg/kg患者体重或0.01到10mg/kg患者体重，或0.01到1mg/kg患者体重，包括其间的任意值或者任意两个数值之间的范围。
在一些实施方案中，施用的多肽的药学可接受的盐的量取决于被治疗的受试者、受试者的体重、痛苦的严重性、施用方式和开处方的医师的调整。在症状可检测时，甚至不可检测时可重复治疗。治疗可单独提供或者与其他药物组合提供。
在一些实施方案中，本文公开的多肽的药学可接受的盐的治疗上有效的剂量能够提供治疗益处而不引起重大的毒性。可以通过标准药物步骤在细胞培养物或实验动物中测定嵌合肽的毒性，例如通过测定LD50(使50％群体致死的剂量)或LD100(使100％群体致死的剂量)来实现。毒性效应和治疗效应的剂量比例是治疗指数。优选显示高治疗指数的多肽的药学可接受的盐(参见例如Fingl等，1975,In:The Pharmacological Basis of Therapeutics,第1章，第1页)。
第二方面，本申请提供了药物组合物，其包含第一方面所述的多肽的药学可接受的盐，以及药学可接受的载体、赋形剂和/或稀释剂。
在一些实施方案中，药物组合物用于治疗、改善或预防个体的神经系统损伤、神经系统损伤相关的疾病或疼痛、神经退行性疾病、焦虑或癫痫。
在一些实施方案中，药物组合物被用作神经元保护剂。
在一些实施方案中，神经系统损伤为兴奋性神经毒性引起的神经系统损伤。
在一些实施方案中，兴奋性神经毒性引起的神经系统损伤包括选自脑卒中、脊髓损伤、脑或脊髓的缺血性或创伤性损伤、中枢神经系统(CNS)神经元的损伤，包括急性CNS损伤、缺血性脑卒中或脊髓损伤，以及缺氧、缺血、机械损伤和神经退行性疾病、焦虑、癫痫、脑卒中引起的损伤。
在一些实施方案中，药物组合物用于治疗、改善或预防缺血性脑卒中或缺血性脑卒中导致的神经系统损伤。在一些实施方案中，药物组合物用于治疗、改善或预防出血性脑卒中或出血性脑卒中导致的神经系统损伤。在一些实施方案中，药物组合物用于治疗、改善或预防由缺血性脑卒中转化成的出血性脑卒中或者由缺血性脑卒中转化成的出血性脑卒中导致的神经系统损伤。
脑卒中是由CNS中受损的血流导致的病症。可能的原因包括栓塞、出血和血栓形成。一些神经元细胞由于受损的血流而立即死亡。这些细胞释放其组分分子(包括谷氨酸)，所述组分分子随后活化NMDA受体，所述NMDA受体提高细胞内钙水平和胞内酶水平，导致更多的神经元细胞死亡(兴奋性神经毒性级联放大)。CNS组织的死亡被称作梗死。梗死体积(即脑中由脑卒中导致的死亡的神经元细胞的体积)可以被用作脑卒中导致的病理学损伤程度的指标。症状性效果既取决于梗死体积，也取决于梗死在脑中位于何处。残疾指数可被用作症状性损伤的度量，例如Rankin中风输出标度(Rankin Stroke Outcome Scale,Rankin,Scott MedJ；2:200-15(1957))和Barthel指数(Barthel Index)。Rankin标度以如下直接评价患者的全局病症为基础：
0完全没有症状。
1尽管有症状但是没有显著的残疾；能够进行所有日常工作和活动。
2轻微残疾；不能进行所有先前的活性，但是能够照顾自己的事务而
不需要帮助。
3需要一些帮助的中度残疾，但是能够行走而不需要帮助。
4中度到严重的残疾，不受帮助时不能行走，并且不受帮助时不能照
顾自身的身体需要。
5严重残疾；卧床不起，失禁，并需要持久的护理和关注。
Barthel指数以关于患者进行10种基本日常生活活动的能力的一系列问题为基础，所述问题得到0和100之间的分数，较低的分数表示较多的残疾(Mahoney等,Maryland State Medical Journal 14:56-61(1965)。
或者可使用NIH脑卒中标度测量脑卒中严重性/输出，所述NIH脑卒中标度可在万维网ninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf上获得。该标度以患者实行11组功能的能力为基础，所述功能包括评价患者的意识、运动、感受和语言功能水平。
缺血性脑卒中更明确地表示为由于通向大脑的血流被堵塞而引起的一类脑卒中。 这类堵塞的潜在病症最常见地是沿血管壁的脂肪沉着物的发生。该病症被称作动脉粥样硬化。这些脂肪沉着物能够引起两种梗阻。脑血栓形成是指在血管的阻塞部分产生的血栓(血块)。“脑栓塞”通常是指血液中的各种栓子(如心脏内的附壁血栓、动脉粥样硬化的斑块、脂肪、肿瘤细胞、纤维软骨或空气等)随血流进入脑动脉而阻塞血管，当侧枝循环不能代偿时，引起该动脉供血区脑组织缺血性坏死，出现局灶性神经功能缺损。栓塞的第二个重要原因是不规则的心博，称作动脉肌纤维震颤。其引起下述病症，其中血块可在心中形成，移动并转移至脑。缺血性脑卒中的其他潜在原因是出血、血栓形成、动脉或静脉的切割、心脏停博、任何原因(包括出血)引起的休克，和医源性原因，如对脑血管或导向脑的血管的直接手术损伤或心脏手术。缺血性脑卒中构成所有脑卒中病例的约83％。
若干种其他神经病学病症也可以通过NDMAR介导的兴奋性神经毒性导致神经死亡。这些病症包括神经退行性疾病、焦虑、癫痫、缺氧、与脑卒中无关的对CNS的创伤如创伤性脑损伤和脊髓损伤。因此，在一些实施方案中，药物组合物用于治疗、改善或预防神经退行性疾病、焦虑或癫痫，其中所述神经退行性疾病包括阿尔茨海默氏病、肌萎缩性侧索硬化症(ALS)、帕金森氏病或亨廷顿氏病。
在一些实施方案中，药物组合物被用作神经元保护剂。
第三方面，本申请提供了治疗、改善或预防个体的神经系统损伤、神经系统损伤相关的疾病或疼痛、神经退行性疾病、焦虑或癫痫的方法，所述方法包括向所述个体施用第一方面所述的多肽的药学可接受的盐或第二方面所述的药物组合物。
在一些实施方案中在一些实施方案中，神经系统损伤为兴奋性神经毒性引起的神经系统损伤，其中所述损伤或疼痛位于外周神经系统或中枢神经系统。在一些实施方案中，兴奋性神经毒性引起的神经系统损伤包括选自脑卒中或脊髓损伤、脑或脊髓的缺血性或创伤性损伤以及中枢神经系统(CNS)神经元的损伤，包括急性CNS损伤、缺血性脑卒中或脊髓损伤，以及缺氧、缺血、机械损伤和神经退行性疾病、焦虑、癫痫、脑卒中引起的损伤。
在一些实施方案中，神经退行性疾病包括阿尔茨海默氏病、肌萎缩性侧索硬化症(ALS)、帕金森氏病或亨廷顿氏病。
在一些实施方案中，所述疾病为缺血性脑卒中或缺血性脑卒中导致的神经系统损伤。在一些实施方案中，所述疾病为出血性脑卒中或出血性脑卒中导致的神经系统损伤。在一些实施方案中，所述疾病为由缺血性脑卒中转化成的出血性脑卒中或者由缺血性脑卒中转化成的出血性脑卒中导致的神经系统损伤。
第四方面，本申请提供了第一方面所述的多肽的药学可接受的盐或第二方法所述的药物组合物在制备用于治疗、改善或预防个体中的神经系统损伤、神经系统损伤相关的疾病或疼痛、神经退行性疾病、焦虑或癫痫的药物或在制备神经元保护剂中的用途。
在一些实施方案中在一些实施方案中，神经系统损伤为兴奋性神经毒性引起的神经系统损伤，其中所述损伤或疼痛位于外周神经系统或中枢神经系统。在一些实施方案中，兴奋性神经毒性引起的神经系统损伤包括选自脑卒中或脊髓损伤、脑或脊髓的缺血性或创伤性损伤以及中枢神经系统(CNS)神经元的损伤，包括急性CNS损伤、缺血性脑卒中或脊髓损伤，以及缺氧、缺血、机械损伤和神经退行性疾病、焦虑、癫痫、脑卒中引起的损伤。
在一些实施方案中，神经退行性疾病包括阿尔茨海默氏病、肌萎缩性侧索硬化症(ALS)、帕金森氏病或亨廷顿氏病。
在一些实施方案中，所述疾病为缺血性脑卒中或缺血性脑卒中导致的神经系统损伤。在一些实施方案中，所述疾病为出血性脑卒中或出血性脑卒中导致的神经系统损伤。在一些实施方案中，所述疾病为由缺血性脑卒中转化成的出血性脑卒中或者由缺血性脑卒中转化成的出血性脑卒中导致的神经系统损伤。
本申请所述的“个体”，是指包含鸟类、爬行类和哺乳类的动物。在一些实施方案中，所述个体是哺乳动物，包括灵长类和非灵长类动物，例如人、黑猩猩、牛、马、猪、绵羊、山羊、狗、猫，以及诸如大鼠和小鼠的啮齿类动物。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
实施例
提供以下实施例仅仅是对本申请的一些实施方案进行举例说明，没有任何限制的目的或性质。
实施例1：活性肽分子的筛选
根据已报道的研究结果，选取Tat穿膜肽YGRKKRRQRRR(SEQ ID NO:2)，并将其与不同数目的氨基酸相连接，形成肽库。将肽库中的嵌合肽分子，分别与体外表达并纯化的PDZ1/2结构域相互作用，根据相互作用力的强弱，对多肽进行初步筛选。
固定的分子(配体)为PDZ1/2蛋白，分子量：～20kD，浓度：2mg/ml；流动相的分子(分析物)：待筛选多肽，分子量：～2kD，浓度：10mg/ml。使用Biacore 3000仪器，CM5芯片进行固定。电泳缓冲液为PBS+0.005％吐温20。使用氨基偶联方法进行固定。配体的浓度为10μg/ml。固定缓冲液为10mM醋酸钠，pH 4.0。固定量：1400RU，固定至流动细胞2。使用的流速为10μl/ml，配体进样1分钟。使用PH2.0+2.5的10mM Gly作为再生液，以30μl/分钟的流速进行再生。进样时间为30s。
使用下述条件进行动力学分析：对照通道：流动细胞1；电泳缓冲液为PBS；使用Kinetic Analysis Wizard模式，浓度梯度为6.25nM、12.5nM、25nM、50nM、100nM、 200nM、400nM；进样时间为1分钟；解离时间为2min；流速为30μl/分钟。
用拟和软件BIAevaluation 4.1软件对数据进行拟合。拟和模型为1：1结合模型。解离常数KD值与作用力呈反比。
通过筛选，获得了与PDZ1/2结构域具有较强相互作用能力的嵌合肽，将其命名为P5，序列如下：
P5:YGRKKRRQRRRYEKLLDTEI
为了直接与已报道的研究中的类似嵌合肽进行比较，引入了对照嵌合肽NA-1，序列如下：
NA-1：YGRKKRRQRRRKLSSIESDV(SEQ ID NO:4)
此外，通过比较P5与NA-1的结构差异，另外引入了在嵌合肽NA-1的活性肽的N端加入YE两个残基的嵌合肽YE-NA-1，序列如下：
YE-NA-1：YGRKKRRQRRRYEKLSSIESDV(SEQ ID NO:5)
将嵌合肽NA-1、YE-NA-1和P5同时进行上文所述的与PDZ1/2结构域相互作用的测试，结果如下文表1所示：
表1.三种嵌合肽与PDZ1/2结构域相互作用力检测
嵌合肽 NA-1 YE-NA-1 P5
KD(M) 7.53E-08 5.44E-08 2.99E-08
如表1所示，相比于对照嵌合肽NA-1，嵌合肽YE-NA-1及P5与PDZ1/2结构域相互作用力更强，并且P5的作用性质更佳。因此，据发明人推测，活性肽的N端额外的YE两个氨基酸残基对多肽与PDZ1/2结构域的相互作用有一定的增强作用。此外，P5相对于YE-NA-1的羧基端减少了两个疏水性较弱的丝氨酸(SS)，据发明人的推测，这可能因此进一步增加了多肽与PDZ1/2结构域的相互作用。
实施例2：Pull-down实验检测P5与PDZ1/2结构域的相互作用
为证明P5能与PDZ1/2结构域相互作用，进行Pull-down实验。
用100μl的His珠子和1ml的MCAC-0缓冲液将柱子平衡5min。在4℃震荡。将混合物在4℃，以5000g离心1分钟，弃上清。向混合物中加入1mg PDZ1/2蛋白，并用缓冲液补齐至1ml。在4℃，将所述混合物旋转结合1小时。将所述混合物在4℃，以5000g离心1分钟，弃上清。用1ml的MCAC-0缓冲液清洗3次，每次5分钟(在4℃，震荡洗涤)。向混合物中加入1mg P5蛋白，并用缓冲液补齐至1ml。在4℃，将所述混合物旋转结合2小时。将所述混合物在4℃，以5000g离心1分钟，弃上清。用1ml裂解液进行清洗3次，每次5分钟(在4℃，震荡洗涤)。清洗之后加入20μl MCAC-300。离心，取洗脱液进行SDS-PAGE检测。实验结果显示于图1。
如图1所证实，嵌合肽P5的洗脱条带中同时包含P5和PDZ1/2结构域两者，由 此证实嵌合肽P5能够结合PDZ1/2结构域。
实施例3：不同P5盐对大鼠MCAO模型的治疗效果
基于实施例1和2获得的嵌合肽P5，本申请的发明人设计了P5-三氟乙酸盐(P5-TFA)、P5-醋酸盐(P5-Ac)、P5-盐酸盐(P5-Cl)并委托杭州中肽生化有限公司合成。将制备得到的3种P5盐在大鼠MCAO模型上测试其治疗效果。
实验用动物及材料
动物：选用成年SD大鼠(维通利华)，SPF级，体重220-250g，雄性。
器械和药品：线剪1把、眼外科剪2把、弯镊4把、4#，5#手术缝线、6×17三角形缝针、0.26mm直径的栓线、持针钳1把。恩比普氯化钠注射液(NBP)(石药集团恩必普药业有限公司)，水合氯醛、速尿(20mg/支)、硫酸庆大霉素(80mg/支)，棉签，医用托盘等。
MCAO模型方法：
局灶性脑缺血再灌注模型的制备根据longa提出的可逆性大脑中动脉闭塞(MCAO)线栓法并根据大鼠脑解剖结构图加以改进制作局灶性脑缺血再灌注模型，以10％水合氯醛0.3ml/kg腹腔麻醉，颈正中切口，暴露颈总动脉(CCA)、颈外动脉(ECA)及翼腭动脉，将0.26mm单丝尼龙鱼线头端0.5cm用石蜡包被，并于20mm长处标记，全部大鼠均通过右侧CCA切口处插入，翼腭动脉短暂夹闭以防误插，栓线长度自CCA分叉处约18～20mm，根据动物体重而定，栓塞右侧大脑中动脉，然后缝合皮肤，栓线尾端部分固定于皮肤上。缺血达到2h后小心抽出栓线，即形成再灌注。在缺血期间及再灌注后2h保持体温在(37±0.5)℃。模型成功的标志为大鼠手术麻醉清醒后出现左侧肢体瘫痪，站立不稳，提尾时向一侧转圈。
实验分组
实验分对照组(正常组)、模型组(生理盐水组)，阳性药物恩必普组(NBP)和P5盐给药组。在缺血造模后1h，分别将生理盐水、阳性药恩必普(2.5mg/kg)、每种P5盐(10mg/kg)经尾静脉注射给予各组大鼠。
梗死体积计算
给药24小时后，将大鼠断头处死，迅速将取出的脑组织置于-20℃冰箱，10min后置室温环境，将脑置于大鼠脑切片模具中，切除嗅球，小脑和低位脑干后按图谱所示间隔2mm冠状切五刀，切成6个大脑连续冠状粗切片。然后迅速将脑片置于5ml含2％TTC的溶液中，37℃恒温、避光孵育30min，期间每隔5min将脑片翻动一次。 经TTC染色后，正常组织呈玫瑰红色，梗死组织未被染色而呈白色。将每组脑片排列整齐，拍照保存，应用图像分析系统软件处理并作统计，计算每张脑片的梗塞面积，乘以每片脑片的厚度2mm，每只动物所有脑片梗塞面积乘以厚度相加，即为脑梗塞体积。体积以所占大脑半球的百分率表示，以消除脑水肿的影响。
实验结果如图2所示。结果显示与模型组相比，P5-TFA、P5-Ac、P5-Cl的给药显著降低了大鼠脑部的梗塞体积，其中p＜0.01；与阳性药NBP相比，P5-TFA、P5-Ac、P5-Cl的给药降低大鼠脑部梗塞体积的效果也显著优于阳性药NBP，其中p＜0.05。
实施例4：不同P5盐的细胞毒性测定
本实施例测试了实施例3中的P5-Ac和P5-Cl这两种多肽盐的细胞毒性。
实验材料
测试用细胞系PC12细胞；垂直层流超净工作台、蒸汽灭菌器、离心机、显微镜、酶标仪、盖玻片、血球计数板、手动计数器、酒精灯、移液器、移液管、枪头、离心管、96孔板、PBS/生理盐水、高糖DMEM培养基(含有10％FBS和1％双抗)。CCK8购自索莱宝。
实验步骤
消化对数期细胞，将其以4×104/孔的密度传入96孔板，每组设置3个复孔，并将细胞培养过夜；用培养基配制P5盐(例如5μM)；弃去96孔板内的上清，PBS/生理盐水清洗2-3次，随后每孔加入100μl P5盐，空白对照(无细胞)同样加入相同的P5盐，将细胞培养24h；向96孔板的每孔中加入10μl CCK8，孵育1h；取出96孔板，使用酶标仪测定450nm波长下的吸光度。根据测得的数值计算细胞活率。计算公式为：
活细胞率(％)＝(实验组-相应的空白对照组)/(0μM实验组-0μM空白对照组)×100％
实验结果如图3所示。结果显示，P5-Ac和P5-Cl这两种P5多肽的药学可接受的盐在5μM的浓度下无显著的细胞毒性。
实施例5：不同P5盐的稳定性
本实施例分别测试了实施例3中的3种P5盐在光照、高温和高湿条件下的稳定性。
P5盐粉末的稳定性分析
将实施例3中制备得到三种P5盐的粉末用光照(3000Lx)+紫外、高温(60℃)和高湿(75％RH)条件处理10天。处理后，将粉末溶于水配制成浓度为2mg/ml的溶液，然 后精密量取10μl注入液相色谱仪，记录色谱图，有关物质按照面积归一化法计算，以分析含量和杂质种类数。
仪器及试剂
高效液相色谱仪(Agilent,1260 EZChrom)；色谱柱(Agilent,ZORBAX300SB-C18(4.6*250mm，5μm)SN:USHH008416)；分析天平(赛多利斯,BT25S)；过滤膜(Millipore,0.45μm PTFE)；乙腈(MREDA)；水(爱夸)；TFA(MREDA)；综合药品稳定性试验箱三箱式(上海佐诚实验仪器有限公司，型号：SHH-3SDT)
色谱条件
流动相：A 0.065％TFA-水；B 0.05％TFA-ACN
检测波长λ＝220nm；流速V＝1.0ml/min；温度T＝36℃
进样量Inj＝10μl
梯度条件：0-30min，B％＝5-65％
结果分析
三种P5盐粉末稳定性分析的结果如下文表2以及图4中A和B图所示：
表2
Figure PCTCN2017091792-appb-000001
结果表明：
在光照+紫外条件下处理后，三种盐的主峰面积百分比、杂质种类数有明显差异，相对稳定性顺序为：三氟乙酸盐>醋酸盐>盐酸盐；
在高温条件下处理后，三种盐的主峰面积百分比均在99.5％以上，没有明显差异，以杂质种类数对比，醋酸盐最稳定；
在高湿条件下处理后，醋酸盐主峰面积百分比明显减小，相对稳定性顺序为：三氟乙酸盐>盐酸盐>醋酸盐。
总体来说，三种盐在不同处理下表现出较好的稳定性。
P5盐水溶液的稳定性分析
将实施例3的3种P5盐溶于水中配成2mg/ml的溶液。用光照(3000Lx)和高温(60℃)条件处理10天。处理后，精密量取10μl注入液相色谱仪，记录色谱图，有关物质按照面积归一化法计算，以分析含量和杂质种类数。
仪器及试剂
高效液相色谱仪(Agilent,1260 EZChrom)；色谱柱(Agilent,ZORBAX300SB-C18(4.6*250mm，5μm)SN:USHH008416)；分析天平(赛多利斯,BT25S)；过滤膜(Millipore,0.45μm PTFE)；乙腈(MREDA)；水(爱夸)；TFA(MREDA)；综合药品稳定性试验箱三箱式(上海佐诚实验仪器有限公司，型号：SHH-3SDT)
色谱条件
流动相：A 0.065％TFA-水；B 0.05％TFA-ACN
检测波长λ＝220nm；流速V＝1.0ml/min；温度T＝36℃
进样量Inj＝10μl
梯度条件：0-30min，B％＝5-65％
结果分析
三种P5盐的水溶液的稳定性分析的结果如表3以及图4中C和D图所示：
表3
Figure PCTCN2017091792-appb-000002
结果表明：
在光照条件下处理后，三种盐溶液的主峰面积百分比、杂质种类数有差异，相对稳定性顺序为：醋酸盐>三氟乙酸盐>盐酸盐；
在高温条件下处理后，盐酸盐和醋酸盐的主峰面积百分比、杂质种类数有差异，相对稳定性顺序：醋酸盐>盐酸盐。
总体来说，三种盐的溶液在不同处理下也表现出较好的稳定性。
将本说明书中引用的所有出版物和专利文献引入本文作为参考，如同每个出版物 或专利被分别明确指明引入本文作为参考。在不偏离本申请公开的真实思想和范围的情况下，可对本申请公开的各实施方案进行多种改变和用等同物替换。除非上下文中另有说明，否则本公开的实施方案的任何特征、步骤或实施方案都可以与任何其他特征、步骤或实施方案组合使用。

Claims (21)

  1. 多肽的药学可接受的盐，其中所述多肽包含氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体。
  2. 如权利要求1所述的多肽的药学可接受的盐，其中所述功能性变体为SEQ ID NO:1中的LDTEI部分发生一处或多处保守型取代后产生的变体，优选地，所述保守型取代选自D和E之间的取代，L、V和I之间的取代以及T和S之间的取代。
  3. 如权利要求2所述的多肽的药学可接受的盐，其中所述功能性变体为SEQ ID NO:1中的LDTEI部分被替换为下述任一序列后产生的变体：LDTEL、LDTEV、LDTDI、LDTDL、LDTDV、LDSEI、LDSEL、LDSEV、LDSDI、LDSDL、LDSDV、LETEI、LETEL、LETEV、LETDI、LETDL、LETDV、VDTEI、VDTEL、VDTEV、VDTDI、VDTDL、VDTDV、IDTEI、IDTEL、IDTEV、IDTDI、IDTDL、IDTDV、IETEI、IETEL、IETEV、IETDI、IETDL、IETDV。
  4. 如前述权利要求中任一项所述的多肽的药学可接受的盐，其中所述多肽是包含内化肽部分和活性肽部分的嵌合肽，所述活性肽部分为氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体，所述内化肽部分能促进所述嵌合肽被细胞摄取。
  5. 如权利要求4所述的多肽的可药用的盐，其中所述内化肽部分包含氨基酸序列YGRKKRRQRRR(SEQ ID NO:2)。
  6. 如权利要求5所述的多肽的可药用的盐，其中所述嵌合肽包含氨基酸序列YGRKKRRQRRRYEKLLDTEI(SEQ ID NO:3)。
  7. 如前述权利要求中任一项所述的多肽的药学可接受的盐，其中所述盐选自三氟乙酸盐、醋酸盐、盐酸盐和磷酸盐。
  8. 药物组合物，其包含权利要求1-7中任一项所述的多肽的药学可接受的盐，以及药学可接受的载体、赋形剂和/或稀释剂。
  9. 如权利要求8所述的药物组合物，其为预冻干制剂，优选包含组氨酸和海藻糖。
  10. 如权利要求8所述的药物组合物，其为冻干制剂，优选通过将权利要求9所述的预冻干制剂冻干而制备。
  11. 如权利要求8所述的药物组合物，其为复原制剂，优选通过将权利要求10所述的冻干制剂与水溶液结合而制备。
  12. 如权利要求8-11中任一项所述的药物组合物，其用于治疗、改善或预防个体的神经系统损伤、神经系统损伤相关的疾病或疼痛、神经退行性疾病、焦虑或癫痫，或者用作神经元保护剂。
  13. 治疗、改善或预防个体的神经系统损伤、神经系统损伤相关的疾病或疼痛、神经退行性疾病、焦虑或癫痫的方法，包括向所述个体施用权利要求1-7中任一项所 述的多肽的药学可接受的盐或权利要求8-12中任一项所述的药物组合物。
  14. 权利要求1-7中任一项所述的多肽的药学可接受的盐或权利要求8-12中任一项所述的药物组合物在制备用于治疗、改善或预防个体中的神经系统损伤、神经系统损伤相关的疾病或疼痛、神经退行性疾病、焦虑或癫痫的药物或在制备神经元保护剂中的用途。
  15. 如权利要求12所述的药物组合物、权利要求13所述的方法或权利要求14所述的用途，其中所述神经系统损伤为兴奋性神经毒性引起的神经系统损伤。
  16. 如权利要求15所述的药物组合物、方法或用途，其中所述兴奋性神经毒性引起的神经系统损伤包括选自脑卒中、脊髓损伤、脑或脊髓的缺血性或创伤性损伤、中枢神经系统(CNS)神经元的损伤，包括急性CNS损伤、缺血性脑卒中或脊髓损伤，以及缺氧、缺血、机械损伤和神经退行性疾病、焦虑、癫痫、脑卒中引起的损伤。
  17. 如权利要求12所述的药物组合物、权利要求13所述的方法或权利要求14所述的用途，其中所述神经退行性疾病选自阿尔茨海默氏病、肌萎缩性侧索硬化症(ALS)、帕金森氏病或亨廷顿氏病。
  18. 如权利要求12所述的药物组合物、权利要求13所述的方法或权利要求14所述的用途，其中所述神经系统损伤或疼痛位于外周神经系统或中枢神经系统。
  19. 如权利要求12所述的药物组合物、权利要求13所述的方法或权利要求14所述的用途，其中所述神经系统损伤相关的疾病为脑卒中。
  20. 如权利要求19所述的药物组合物、方法或用途，其中所述脑卒中选自缺血性卒中、出血性脑卒中和由缺血性脑卒中转化成的出血性脑卒中。
  21. 如权利要求20所述的药物组合物、方法或用途，其中所述脑卒中为缺血性脑卒中。
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