WO2019006691A1 - 用于治疗、改善或预防脑出血的肽及其用途 - Google Patents

用于治疗、改善或预防脑出血的肽及其用途 Download PDF

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WO2019006691A1
WO2019006691A1 PCT/CN2017/091793 CN2017091793W WO2019006691A1 WO 2019006691 A1 WO2019006691 A1 WO 2019006691A1 CN 2017091793 W CN2017091793 W CN 2017091793W WO 2019006691 A1 WO2019006691 A1 WO 2019006691A1
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peptide
pharmaceutical composition
hemorrhage
seq
cerebral hemorrhage
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PCT/CN2017/091793
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English (en)
French (fr)
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韩化敏
芦颖
田雨佳
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拜西欧斯（北京）生物技术有限公司
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Priority to CN201780092737.2A priority Critical patent/CN110799522B/zh
Priority to PCT/CN2017/091793 priority patent/WO2019006691A1/zh
Publication of WO2019006691A1 publication Critical patent/WO2019006691A1/zh

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    • 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
    • A61K47/30—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
    • 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]
    • 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

Definitions

  • the present application relates generally to the field of medicine.
  • the present application provides peptides for treating, ameliorating or preventing cerebral hemorrhage and uses thereof.
  • Cerebral hemorrhage generally refers to bleeding caused by rupture of blood vessels in the parenchyma.
  • Non-traumatic cerebral hemorrhage accounts for 20% to 30% of all strokes, and the acute mortality rate is 30% to 40%.
  • the Src family is a class of non-receptor tyrosine kinases. Src is not only a cytoplasmic effector of the G protein-coupled receptor PAR1, but also a functional enzyme that regulates the ion channel NMDAR, which may be an important bridge connecting the G protein-coupled receptor (PAR1) and NMDAR.
  • the N-methyl-D-aspartate receptor (NMDAR) is a ligand-gated ion channel receptor composed of two subunits, GluN1 and GluN2. NMDA receptors are key molecules in many pathological processes of brain damage or diseases of the nervous system.
  • PSD95 Post-synaptic compact protein (PSD), receptor protein, cytoskeletal protein, and various signaling molecules (including protein kinases, phosphatases) can be directly or indirectly bound to NMDA after neuronal excitatory glutamate injury.
  • the receptor forms a complex and the process is reversible.
  • PSD95 includes three N-terminal PDZ domains (PDZ1, PDZ2, PDZ3), one SH3 domain and one C-terminal GK domain.
  • PSD95 binds to the NMDA receptor subunits GluN2 (GluN2A and GluN2B) via the PDZ2 domain, and its PDZ3 domain binds to the SH2 domain of Src PTK.
  • the GluN2A subunit of Src PTK and NMDA receptor forms a SRC-PSD95-GluN2A signal complex with PSD.
  • This signaling complex is very stable, resulting in sufficient contact of Src with GluN2A and promoting GluN2A tyrosine phosphorylation.
  • Activated NMDA receptors accelerate calcium flux, which exacerbates neuronal damage.
  • PAR contributes to the formation of the Src-PSD95-GluN2A signaling complex, and thus thrombin-PAR-SRC-PSD95-GluN2A is an important molecule that causes neuronal apoptosis in the ICH model.
  • the present application provides a pharmaceutical composition for treating cerebral hemorrhage, the pharmaceutical composition comprising a peptide or a pharmaceutically acceptable salt thereof, the peptide comprising the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a function thereof Sexual variants.
  • the peptide is a chimeric peptide comprising the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof, and an internalization peptide capable of promoting the chimeric peptide by the cell Ingestion.
  • the internalization peptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2).
  • the chimeric peptide comprises the amino acid sequence YGRKKRRQRRR YEKLLDTEI (SEQ ID NO: 3).
  • 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 pharmaceutically acceptable salt is selected from the group consisting of trifluoroacetate, acetate, hydrochloride, and phosphate.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, and/or excipient.
  • 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 present application provides a method of treating, ameliorating or preventing cerebral hemorrhage in an individual, the method comprising administering to the individual in need thereof the pharmaceutical composition of the first aspect.
  • the application provides the use of a peptide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating, ameliorating or preventing cerebral hemorrhage in an individual, the peptide comprising 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 pharmaceutically acceptable salt is selected from the group consisting of trifluoroacetate, acetate, hydrochloride, and phosphate.
  • the cerebral hemorrhage is selected from the group consisting of traumatic cerebral hemorrhage and non-traumatic cerebral hemorrhage.
  • the cerebral hemorrhage is selected from the group consisting of basal ganglia hemorrhage, cerebrospinal hemorrhage, thalamic hemorrhage, caudate nucleus hemorrhage, intraventricular hemorrhage, cerebral hemorrhage, cerebral hemorrhage, cerebellar hemorrhage Subarachnoid hemorrhage.
  • cerebral hemorrhage is caused by any one of the following factors or a combination thereof: microaneurysm or microangioma, cerebral arteriovenous malformation, amyloid cerebral vascular disease, cystic hemangioma, intracranial vein Thrombosis, dural arterial spasm, specific arteritis, fungal arteritis, moyamoya disease, arterial anatomical variation, carotid arteriovenous fistula, hypertension, migraine, anticoagulation, antiplatelet or thrombolytic therapy, Haemophilus infection , leukemia, thrombotic thrombocytopenia, intracranial tumors, alcohol, amphetamine, cocaine, sympathetic stimulants.
  • microaneurysm or microangioma cerebral arteriovenous malformation, amyloid cerebral vascular disease, cystic hemangioma, intracranial vein Thrombosis, dural arterial spasm, specific arteritis, fungal arteritis, moyamoya
  • 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 the scores of the balance beam test for each group of rats.
  • Figure 3 shows the scores of the Berderson test for each group of rats.
  • Figure 4 shows a comparison of brain hematoma volume in each group of rats.
  • Figure 5 shows the results of hematoxylin-eosin staining of brain tissue sections of each group of rats, including A. normal group; B. sham operation group; C. model group; D.NA-1 administration group; E.GM Treatment group; F. P5 administration group (5 mg/kg) 1 hour after autologous blood injection; G. P5 administration group (20 mg/kg) 1 hour after autologous blood injection; H. P5 administration 1 hour after autologous blood injection Group (10 mg/kg); I. P5 administration group (10 mg/kg) 2 hours after autologous blood injection; J. P5 administration group (10 mg/kg) 3 hours after autologous blood injection.
  • Figure 6 shows the immunohistochemical analysis of brain tissue sections of each group of rats.
  • the antigen used in immunohistochemical analysis was Bax-2, of which A. normal group; B. sham operation group; C. model group; D.NA -1 administration group; E.GM treatment group; F. P5 administration group (5 mg/kg) 1 hour after autologous blood injection; G. P5 administration group (20 mg/kg) 1 hour after autologous blood injection; H. P5 administration group (10 mg/kg) 1 hour after autologous blood injection; I. P5 administration group (10 mg/kg) 2 hours after autologous blood injection; J. P5 administration group 3 hours after autologous blood injection (10 mg/kg) ).
  • Figure 7 shows the immunohistochemical analysis of brain tissue sections of each group of rats.
  • the antigen used in immunohistochemical analysis was Caspase-3, of which A. normal group; B. sham operation group; C. model group; D.NA -1 administration group; E.GM treatment group; F. P5 administration group (5 mg/kg) 1 hour after autologous blood injection; G. P5 administration group (20 mg/kg) 1 hour after autologous blood injection; H. P5 administration group (10 mg/kg) 1 hour after autologous blood injection; I. P5 administration group (10 mg/kg) 2 hours after autologous blood injection; J. P5 administration group 3 hours after autologous blood injection (10 mg/kg) ).
  • Figure 8 is a graph showing the levels of CK in the serum of each group of rats.
  • 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 targets of cerebral hemorrhage treatment, and designed and screened polypeptide neuroprotective agents through pharmacological and pharmacological experiments in vitro and in vivo, and screened them.
  • a peptide with desirable properties are desirable properties.
  • PDZ domain refers to a modular protein domain of approximately 90 amino acids characterized by a synaptic protein PSD-95, a Drosophila-separating connexin, a Discs-Large (DLG), and an epithelial tight junction protein Z01. (Z01) has significant (eg, at least 60%) sequence identity.
  • the PDZ domain is also known as Discs-Large homology repeats ("DHRs") and GLGF repeats.
  • DHRs Discs-Large homology repeats
  • the PDZ domain typically displays a retained core consensus sequence (Doyle, D.A., 1996, Cell 85: 1067-76).
  • Exemplary PDZ domain-containing proteins and PDZ domain sequences are disclosed in U.S. Patent Application Serial No. 10/714,537.
  • the term "specifically binds” refers to a bond between two molecules (eg, a ligand and a receptor) characterized by one molecule (ligand) and another specific molecule in the presence of many other different molecules ( The ability of a receptor to bind, ie, the ability to display a preferential binding of one molecule to another in a heterogeneous mixture of molecules. Specific binding of the ligand to the receptor is also demonstrated as follows: When an excess of unlabeled ligand is present, the binding of the detectably labeled ligand to the receptor is reduced (i.e., binding competition assay).
  • Statistically significant means a p value ⁇ 0.05, preferably p ⁇ 0.01, most preferably ⁇ 0.001.
  • a “functional variant” refers to a variant having the same or similar biological function and properties as the parent.
  • a “functional variant” can be obtained by performing one or more conservative substitutions in the parent.
  • lyophilization relates to a process by which the material to be dried is first frozen and then biochemically removed under vacuum to remove ice or frozen solvent.
  • the present application provides a pharmaceutical composition for treating cerebral hemorrhage, the pharmaceutical composition comprising a peptide or a pharmaceutically acceptable salt thereof, the peptide comprising the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a function thereof Sexual variants.
  • cerebral hemorrhage is related to a variety of factors.
  • non-traumatic cerebral hemorrhage it is mainly related to cerebrovascular disease, and may include hypertension, hyperlipemia, diabetes, vascular aging, smoking, amyloid cerebral vascular disease, cerebrovascular malformation, tumor stroke, leukemia and the like.
  • the present application demonstrates by direct cerebral hemorrhage models (see, for example, the Examples below) that the peptides of the present application have potent therapeutic and palliative effects for cerebral hemorrhage and its complications (eg, behavioral changes or cerebral hematoma formation).
  • the cerebral hemorrhage can be traumatic cerebral hemorrhage or non-traumatic cerebral hemorrhage.
  • cerebral hemorrhage may be brain basal ganglia hemorrhage, cerebrospinal hemorrhage, thalamic hemorrhage, caudate nucleus hemorrhage, ventricular hemorrhage, cerebral hemorrhage, cerebral hemorrhage, cerebellar hemorrhage Dry hemorrhage, subarachnoid hemorrhage or multiple sites of bleeding.
  • cerebral hemorrhage can be caused by microaneurysms or microangiomas, cerebral arteriovenous malformations, amyloid cerebral vascular disease, cystic hemangioma, intracranial venous thrombosis, dural arterial spasm, specific arteritis, and Fungal arteritis, moyamoya disease, arterial anatomical variation, cerebral hemorrhage caused by carotid artery venous fistula.
  • cerebral hemorrhage can be cerebral hemorrhage caused by migraine.
  • cerebral hemorrhage can be cerebral hemorrhage caused by anticoagulation, antiplatelet or thrombolytic therapy, Haemophilus infection, leukemia, thrombotic thrombocytopenia, and the like.
  • cerebral hemorrhage can be cerebral hemorrhage caused by an intracranial tumor.
  • cerebral hemorrhage can be cerebral hemorrhage caused by alcohol, amphetamine, cocaine, sympathetic stimulants, and the like.
  • the cerebral hemorrhage can be hypertensive cerebral hemorrhage.
  • the peptide is a chimeric peptide comprising the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) or a functional variant thereof, and an internalization peptide capable of promoting the chimeric peptide by the cell Ingestion.
  • Internalized peptides also known as transmembrane peptides, are widely used in the field of protein drugs, and their function is to promote the uptake and absorption of active peptides bound thereto by cells. It will be understood by those skilled in the art that the purpose of chimerizing the active peptide and the internalization peptide is mainly to make the active peptide better reach the target of the action. Therefore, 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.
  • the internalization peptide which is specifically adapted to the neuronal cell.
  • 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 is specific Phenol-1,4-disulfonyl chloride (which reacts mainly with an amino group); 1,6-hexamethylene diisocyanate or diisothiocyanate, or phenylazo-p-diisocyanate (mainly with an amino group) Reaction); glutaraldehyde (which reacts with several different side chains) and double nitrogen benzidine (which reacts primarily 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 is specific Ph
  • the peptides described above can optionally be derivatized (eg, acetylated, phosphorylated, and/or glycosylated) to promote affinity with the inhibitor, promote the ability of the inhibitor to be transported across the cell membrane, or promote stabilization. Sex.
  • derivatized eg, acetylated, phosphorylated, and/or glycosylated
  • 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; Ostergaard (1997) Mol. Divers. 3: 17-27; Ostresh (1996) Methods Enzymol. 267: 220-234.
  • the functional variant is a variant produced following one or more conservative substitutions of the LDTEI moiety of SEQ ID NO: 1.
  • NMDAR2B has GenBank ID 4099612 with a C-terminal 20 amino acids of FNGSSNGHVYEKLSSLESDV and a 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, LDTV, 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 the same at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or even higher than the peptides mentioned above.
  • Sexual 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 synthetically produced, for example, by modification of one or more of the above-described peptide sequences disclosed herein and as assessed herein using any of a variety of techniques well known in the art. Its biological activity.
  • a pharmaceutically acceptable salt is a salt prepared by substantially retaining the biological activity of the free base and reacting with a mineral acid. Pharmaceutical salts tend to be more soluble in water and other protic solvents than the corresponding free base forms.
  • the pharmaceutically acceptable salt can be in any suitable pharmaceutically acceptable salt form.
  • the pharmaceutically acceptable salt is a trifluoroacetate salt.
  • the pharmaceutically acceptable salt is an acetate.
  • the pharmaceutically acceptable salt is the hydrochloride salt.
  • the pharmaceutically acceptable salt is a phosphate.
  • the pharmaceutical compositions disclosed herein 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 active peptide or chimeric peptide into a pharmaceutically acceptable formulation. Proper formulation depends on the route of administration chosen.
  • administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. It is preferably administered intravenously.
  • the pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic.
  • the active peptide or chimeric peptide or a pharmaceutically acceptable salt thereof can be used.
  • Formulated into an aqueous solution preferably formulated into a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to alleviate discomfort at the site of injection).
  • the solution may contain formulas such as suspending, stabilizing and/or dispersing agents.
  • 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.
  • the active peptide or chimeric peptide or a pharmaceutically acceptable salt thereof can be formulated with a pharmaceutically acceptable carrier into tablets, pills, lozenges, capsules, liquids, condensates Gum, syrup, slurry, 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. Further, a flavoring agent, a preservative, a coloring agent, or the like may be added.
  • the pharmaceutical compositions can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the pharmaceutical composition may 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 Slightly soluble salt.
  • Chimeric peptides can be delivered using liposomes and emulsions. Certain organic solvents such as dimethyl sulfoxide can also be used. Additionally, the pharmaceutical composition 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 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.
  • the compounds described herein, or pharmaceutically acceptable salts thereof 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.
  • the pharmaceutical compositions provided herein are used in an amount effective to achieve the intended purpose (e.g., to alleviate or alleviate cerebral hemorrhage).
  • a therapeutically effective amount means: in a patient (or animal model population) treated with a pharmaceutical composition disclosed herein, relative to cerebral hemorrhage in a control population of a patient (or animal model) not treated with the pharmaceutical composition disclosed herein.
  • An amount of a pharmaceutical composition sufficient to significantly reduce damage caused by cerebral hemorrhage. If 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 measured by cerebral hematoma volume or disability index), then the amount is also considered 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. Usually a single application is sufficient.
  • a preferred dosage range for the pharmaceutical compositions provided herein comprises from 0.001 to 20 [mu]mol per kg patient body weight, optionally between 0.03 and 3 [mu]mol per kg patient body weight, including any value therebetween or between any two values.
  • 0.1-20 [mu]mol of the pharmaceutical composition of the present application is administered per kg of patient body weight over 6 hours.
  • 0.1 to 10 ⁇ mol of the pharmaceutical composition of the present application is administered per kg of patient body weight within 6 hours, more preferably about 0.3 ⁇ mol of the pharmaceutical composition of the present application per kg of patient body weight within 6 hours.
  • the dosage range is from 0.005 to 0.5 [mu]mol of the pharmaceutical composition of the present application per kg patient body weight.
  • a suitable dose of the pharmaceutical composition of the present application for humans in grams may be 0.01 to 100 mg/kg of patient body weight, or more preferably 0.01 to 30 mg/kg of patient body weight or 0.01 to 10 mg/kg of patient body weight, or 0.01 to 1 mg. /kg Patient weight, including any value in between or a range between any two values.
  • the amount of pharmaceutical composition administered will depend 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 or even undetectable. Treatment can be provided alone or in combination with other drugs.
  • a therapeutically effective dose of a pharmaceutical composition 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. Pharmaceutical compositions which exhibit a high therapeutic index are preferred (see, for example, Fingl et al, 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1).
  • the application provides a method of treating, ameliorating or preventing cerebral hemorrhage in an individual, the method comprising administering to the individual the pharmaceutical composition of the first aspect.
  • the application provides the use of a peptide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating, ameliorating or preventing cerebral hemorrhage in an individual, the peptide comprising the amino acid sequence YEKLLDTEI (SEQ ID NO: 1) Or a functional variant thereof.
  • “Individuals” as used herein include birds, reptiles, and mammals.
  • the animal 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 is 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; electrophoresis buffer was PBS; concentration gradient was 6.25n, 12.5n, 25n, 50n, 100n, 200n, 400nM using Kinetic Analysis Wizard mode; injection time It was 1 minute; the dissociation time was 2 min; the 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
  • the chimeric peptide P5 was further tested in the following experiments, and in some experiments, NA-1 and YE-NA-1 were used as controls.
  • 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).
  • both the P5 and PDZ1/2 domains were contained in the elution band of the chimeric peptide P5, thereby confirming that the chimeric peptide P5 was able to bind to the PDZ1/2 domain.
  • Example 3 Therapeutic effect of P5 polypeptide on rat cerebral hemorrhage model
  • Animals Adult SD rats, SPF grade, weighing 400 ⁇ 30 g.
  • the cerebral hemorrhage model used in this experiment is a brain infusion based on autologous blood.
  • the specific steps are as follows.
  • the tail of the rat was soaked in warm water at 50 °C, and the blood vessels in the tail of the rat were filled.
  • the tip of the tail was about 5 mm, the tail was lightly tapped, 120 ⁇ l of blood was squeezed out, and 100 ⁇ l of blood was collected by a 100 ⁇ l syringe.
  • the collagenase was injected into the same site, the needle was 6 mm, and 110 ⁇ l of the tail tip derived autologous blood was injected within 12 minutes. After coagulation for 20 minutes, the needle was evenly removed within 3 minutes to close the cranial hole.
  • the total bolus time of collagenase + autologous blood is about 35 to 40 minutes, and the subsequent administration time is started at the time when all the autologous blood is injected.
  • the experiment was divided into normal control group, sham operation group (injection of collagenase with 1 ⁇ l saline instead of autologous blood), model group (untreated group, labeled as ICH group in the drawing), positive drug treatment group, P5 peptide Drug group, NA-1 administration group.
  • the P5 polypeptide administration group included the following subgroups: 1 hour after the whole blood injection, the tail vein was injected with the P5 administration groups of 20 mg/kg, 10 mg/kg, and 5 mg/kg, respectively; A dose group of 10 mg/kg of P5 was administered 2 hours and 3 hours after the injection.
  • the positive drug treatment group was intramuscularly injected with 0.4 ml/kg GM (ganglioside) treatment group 1 hour after the whole blood injection.
  • the NA-1 administration group was administered with a 10 mg/kg NA-1 administration group 1 hour after the autologous blood was injected. 6 to 8 rats per group.
  • the behavioral behavior was evaluated 24 hours after the modeling of each group (0 o'clock when all the autologous blood was injected).
  • Balance beam score balance beam length 80cm, width 2.5cm, 10cm from the ground, a total of 6 grades. 0 points: jump on the balance beam, walk does not fall; 1 point: jump on the balance beam, the probability of falling is less than 50%; 2 points: jump on the balance beam, the probability of falling is greater than 50%; 3 points: jump on the balance beam, the affected side can not help move; 4 points: Can not walk, but can sit on it; 5 points: fell from the balance beam.
  • Berderson scoring method lightly grasp the tail, lift 10cm above the table, and straighten the front paws. 0 points: no neurological impairment; 1 point: lesions of the contralateral wrist joint, elbow flexion, shoulder flexion; 2 points: the above signs + paralysis thrust force decreased; 3 points: active, circle and rear-end.
  • the rats were decapitated. Brain tissue was taken, frozen sectioned, and cut into 2 mm thick sections. Take the middle 6 pieces, calculate the area of each piece with software ImageJ and calculate the total volume. For the brain slices with similar bleeding areas on both sides, the posterior area was multiplied by the thickness of 2 mm to record the hematoma volume; for the brain slices with large difference in the bleeding area between the front and the back, the larger hemorrhage area was multiplied by 1 mm to record the hematoma volume. Finally, the hematoma of each brain slice accumulates and adds the total volume.
  • Fig. 4 The results of measuring the volume of cerebral hematoma are shown in Fig. 4. The results show that 1 hour -5mg / kg P5 give There was significant statistical difference (p ⁇ 0.01) between the drug group, 1 hour-10 mg/kg P5 administration group and 1 hour-20 mg/kg P5 administration group compared with the model group; 2 hours-10 mg/kg P5 administration group There was a statistical difference (P ⁇ 0.05) relative to the model group.
  • the brain was perfused with formaldehyde, and the brain tissue was dehydrated, transparent, dipped in wax, embedded, and sliced by conventional gradient. The brain tissue sections were then stained with hematoxylin-eosin and observed under a light microscope.
  • Fig. 5 The results of brain histopathological observation are shown in Fig. 5.
  • Immunohistochemical studies were performed on the obtained pathological sections of brain tissue. Immunohistochemical antigens were selected for Bax-2 (Fig. 6) and Caspase-3 (Fig. 7).
  • Creatine kinase is present in the mitochondria of brain cells, which rarely enter the bloodstream under normal conditions. When the brain cells are damaged, the disintegrated brain cells release CK into the blood. Therefore, the severity of damage to brain tissue can be assessed by measuring the CK content in serum.

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Abstract

本申请提供了用于治疗脑出血的药物组合物，所述药物组合物包含含有氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体的肽或者所述肽的药学可接受的盐。本申请还提供了所述药物组合物的医学应用。

Description

用于治疗、改善或预防脑出血的肽及其用途 技术领域
本申请大体上涉及医学领域。具体而言，本申请提供了用于治疗、改善或预防脑出血的肽及其用途。
发明背景
脑出血一般指脑实质内血管破裂引起的出血。非外伤性脑出血占全部脑卒中的20％～30％，急性期病死率为30％～40％。
有研究报道，脑出血发病后，活化的凝血酶受体PAR1诱导Src激活，其以Src激酶为媒介，增强Src-PSD95-GluN2A信号通路，上调GluN2A磷酸化，进而调节NMDA受体的活性。并且有研究报道，PAR1与Src-PSD95-GluN2A1信号通路在ICH中参与神经元细胞的凋亡。
Src家族是一类非受体酪氨酸激酶。Src不仅是G蛋白偶联受体PAR1的细胞质效应酶，也是调节离子通道NMDAR的功能性酶，其可能是连接G蛋白偶联受体(PAR1)及NMDAR的重要桥梁。N-甲基-D-天冬氨酸受体(NMDAR)是一种配体门控离子通道受体，由GluN1和GluN2两个亚基构成。NMDA受体是许多脑损伤病理过程或者神经系统疾病的关键分子。
在神经元发生兴奋性谷氨酸损伤后，突触后致密蛋白(PSD)，受体蛋白，细胞骨架蛋白，以及各种信号分子(包括蛋白激酶，磷酸酶)可以直接或间接地结合于NMDA受体形成复合物，该过程可逆。PSD95包括三个N-末端PDZ结构域(PDZ1、PDZ2、PDZ3)，一个SH3结构域和一个C末端GK结构域。PSD95可以通过PDZ2结构域结合到NMDA受体亚基GluN2(GluN2A和GluN2B)，其PDZ3结构域结合到Src PTK的SH2结构域上。Src PTK和NMDA受体的GluN2A亚基与PSD形成SRC-PSD95-GluN2A信号复合物。这种信号复合物很稳定，导致Src与GluN2A充分接触并促进GluN2A酪氨酸磷酸化。活化的NMDA受体加速钙离子流动，从而加剧了神经元损伤。这一结论证明了PAR对Src-PSD95-GluN2A信号复合物的形成有贡献，因此凝血酶-PAR-SRC-PSD95-GluN2A均为ICH模型中引起神经元凋亡的重要分子。这些结果表明阻断Src-PSD95-GluN2A复合物形成，或者寻找一种安全有效的抑制PSD95-GluN2A相互作用的分子可开发为用于脑出血的治疗的药物。
鉴于脑出血在临床的高发病率以及可能对机体健康带来的严重影 响，开发有效治疗方案具有重要意义。
发明概述
第一方面，本申请提供了用于治疗脑出血的药物组合物，所述药物组合物包含肽或者其药学可接受的盐，所述肽包含氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体。
在一些实施方案中，所述肽为含有氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体以及内化肽的嵌合肽，所述内化肽能促进所述嵌合肽被细胞摄取。
在一些实施方案中，内化肽包含氨基酸序列YGRKKRRQRRR(SEQ ID NO:2)。
在一些实施方案中，嵌合肽包含氨基酸序列YGRKKRRQRRR YEKLLDTEI(SEQ ID NO:3)。
在一些实施方案中，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，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，药学可接受的盐选自三氟乙酸盐、醋酸盐、盐酸盐和磷酸盐。
在一些实施方案中，药物组合物还包含药学可接受的载体、稀释剂和/或赋形剂。
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。
第二方面，本申请提供了治疗、改善或预防个体中的脑出血的方法，所述方法包括向有需要的个体施用第一方面所述的药物组合物。
第三方面，本申请提供了肽或者其药学可接受的盐在制备用于治疗、改善或预防个体中的脑出血的药物中的用途，所述肽包含氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体。
在一些实施方案中，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，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，药学可接受的盐选自三氟乙酸盐、醋酸盐、盐酸盐和磷酸盐。
在上述任一方面的一些实施方案中，脑出血选自外伤性脑出血和非外伤性脑出血。
在上述任一方面的一些实施方案中，脑出血选自脑基底节区出血、壳脑出血、丘脑出血、尾状核出血、脑室出血、脑叶出血、大脑出血、小脑出血、脑干出血和蛛网膜下腔出血。
在上述任一方面的一些实施方案中，脑出血由下述任一因素或其组合导致：微动脉瘤或者微血管瘤、脑动静脉畸形、淀粉样脑血管病、囊性血管瘤、颅内静脉血栓形成、硬膜动脉瘘、特异性动脉炎、真菌性动脉炎、烟雾病、动脉解剖变异、颈动静脉瘘、高血压、偏头痛、抗凝、抗血小板或溶栓治疗、嗜血杆菌感染、白血病、血栓性血小板减少症、颅内肿瘤、酒精、苯异丙胺、可卡因、交感神经兴奋药。
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显示了各组大鼠平衡木测试的评分。
图3显示了各组大鼠Berderson测试的评分。
图4显示了各组大鼠的脑部血肿体积比较。
图5显示了各组大鼠的脑组织切片苏木精-伊红染色结果，其中A.正常组；B.假手术组；C.模型组；D.NA-1给药组；E.GM治疗组；F.自体血注入后1小时P5给药组(5mg/kg)；G.自体血注入后1小时P5给药组(20mg/kg)；H.自体血注入后1小时P5给药组(10mg/kg)；I.自体血注入后2小时P5给药组(10mg/kg)；J.自体血注入后3小时P5给药组(10mg/kg)。
图6显示了各组大鼠的脑组织切片免疫组化分析图，免疫组化分析中所用抗原为Bax-2，其中A.正常组；B.假手术组；C.模型组；D.NA-1给药组；E.GM治疗组；F.自体血注入后1小时P5给药组(5mg/kg)；G.自体血注入后1小时P5给药组(20mg/kg)；H.自体血注入后1小时P5给药组(10mg/kg)；I.自体血注入后2小时P5给药组(10mg/kg)；J.自体血注入后3小时P5给药组(10mg/kg)。
图7显示了各组大鼠的脑组织切片免疫组化分析图，免疫组化分析中所用抗原为Caspase-3，其中A.正常组；B.假手术组；C.模型组；D.NA-1给药组；E.GM治疗组；F.自体血注入后1小时P5给药组(5mg/kg)；G.自体血注入后1小时P5给药组(20mg/kg)；H.自体血注入后1小时P5给药组(10mg/kg)；I.自体血注入后2小时P5给药组(10mg/kg)；J.自体血注入后3小时P5给药组(10mg/kg)。
图8为显示各组大鼠血清中的CK水平的图。
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，本申请的发明人对脑出血治疗的多个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，并筛选得到了具有理想性质的肽。
定义
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。
术语“PDZ结构域”是指约90个氨基酸的模块蛋白质结构域，其特征是对脑突触蛋白PSD-95、果蝇(Drosophila)分隔连接蛋白Discs-Large(DLG)和上皮紧密连接蛋白Z01(Z01)具有显著(例如至少60％)的序列同一性。PDZ结构域也称作Discs-Large同源性重复(“DHRs”)和GLGF重复。PDZ结构域通常显示保留核心共有序列(Doyle,D.A.,1996,Cell 85：1067-76)。示例性的含PDZ结构域的蛋白质和PDZ结构域序列在美国申请10/714,537号中公开。
术语“特异性结合”是指两个分子(例如配体和受体)之间的结合，其特征是甚至在存在许多其他不同分子时，一种分子(配体)与另一种特异分子(受体)结合的能力，即在分子的异质混合物中显示一种分子对另一分子的优先结合的能力。配体与受体的特异性结合也如下被证明：存在过量未标记的配体时，经可检测标记的配体与受体的结合降低(即结合竞争实验)。
统计学显著的是指p值<0.05，优选地p<0.01，最优选地<0.001。
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。
术语“冻干”涉及一种工艺，通过该工艺，待干燥的原料先被冷冻，然后在真空环境下生化而去除冰或冻结的溶剂。
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，本申请提供了用于治疗脑出血的药物组合物，所述药物组合物包含肽或者其药学可接受的盐，所述肽包含氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体。
有研究表明，脑出血的发生与多种因素有关。对于非外伤性脑出血而言，其主要与脑血管的病变相关，可能诱因包括高血压、高血脂、糖尿病、血管老化、吸烟、淀粉样脑血管病、脑血管畸形、瘤卒中、白血病等。
本申请通过直接的脑出血模型证明(例如，参见下文实施例)，本申请的肽对于脑出血及其并发症(例如，行为学改变或脑血肿形成)具有有效的治疗和缓解效果。
在一些实施方案中，脑出血可以为外伤性脑出血或非外伤性脑出血。
在一些实施方案中，脑出血可以为脑基底节区出血、壳脑出血、丘脑出血、尾状核出血、脑室出血、脑叶出血、大脑出血、小脑出血、脑 干出血、蛛网膜下腔出血或以上多个部位出血。
在一些实施方案中，脑出血可以是由微动脉瘤或者微血管瘤、脑动静脉畸形、淀粉样脑血管病、囊性血管瘤、颅内静脉血栓形成、硬膜动脉瘘、特异性动脉炎和真菌性动脉炎、烟雾病、动脉解剖变异、颈动静脉瘘导致的脑出血。
在一些实施方案中，脑出血可以是由偏头痛导致的脑出血。
在一些实施方案中，脑出血可以是由抗凝、抗血小板或溶栓治疗、嗜血杆菌感染、白血病、血栓性血小板减少症等导致的脑出血。
在一些实施方案中，脑出血可以是由颅内肿瘤导致的脑出血。
在一些实施方案中，脑出血可以是由酒精、苯异丙胺、可卡因、交感神经兴奋药等导致的脑出血。
在一些实施方案中，脑出血可以为高血压性脑出血。
在一些实施方案中，所述肽为含有氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体以及内化肽的嵌合肽，所述内化肽能促进所述嵌合肽被细胞摄取。
“内化肽”也可称为穿膜肽，在蛋白质药物领域被广泛使用，其功能是促进与其结合的活性肽被细胞摄取和吸收。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)；以及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)。
此外，前文所述的肽能够任选地被衍生化(例如乙酰化、磷酸化和/或糖基化)以促进与抑制剂的亲合力，促进抑制剂跨越细胞膜被转运的能力，或促进稳定性。
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；Ostergaard(1997)Mol.Divers.3:17-27；Ostresh(1996)Methods Enzymol.267:220-234。
在一些实施方案中，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。
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，其C末端20个氨基酸为FNGSSNGHVYEKLSSLESDV，并含有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。在不受任何理论束缚的情况下，本申请的发明人出人意料地发现，本文公开的活性肽YEKLLDTEI(SEQ ID NO:1)中，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，本申请证实这样的改变能够增强活性肽与PDZ1/2结构域的相互作用。同时相对于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％、70％、75％、80％、85％、90％、95％，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，也可以是合成产生的，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。
药学可接受的盐是基本保留游离碱的生物活性并通过与无机酸反应而制备的盐。药物盐倾向于比相应的游离碱形式更易溶于水和其它质子溶剂。
在一些实施方案中，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，药学可接受的盐为三氟乙酸盐。在一些实施方案中，药学可接受的盐为醋酸盐。在一些实施方案中，药学可接受的盐为盐酸盐。在一些实施方案中，药学可接受的盐是磷酸盐。
在一些实施方案中，本文公开的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活性肽或嵌合肽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地是无菌和基本等渗的。对注射而言，可以将活性肽或嵌合肽或其药学可接受的盐 配制进水溶液中，优选地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。
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，可以将活性肽或嵌合肽或其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、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，也可以将药物组合物配制成储存制剂。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，例如可将药物组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)递送药物组合物。
根据其化学性质，持续释放胶囊可释放肽数周直至超过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。
可将本申请所述的化合物或其药学可接受的盐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。
本文提供的药物组合物以有效达到预期目的(例如减轻或缓解脑出血)的量使用。治疗有效量表示：相对于未用本文公开的药物组合物治疗的患者(或动物模型)对照群体中的脑出血而言，在用本文公开的药物组合物治疗的患者(或动物模型群体)中，足以显著降低脑出血引起的损伤的药物组合物的量。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(通过脑血肿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，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。通常单一施用就足够了。
在一些实施方案中，本文提供的药物组合物的优选的剂量范围包括每kg患者体重施用0.001到20μmol，任选地每kg患者体重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小时内每kg患者体重施用0.1-20μmol本申请的药物组合物。在一些方法中，在6小时内每kg患者体重施用0.1-10μmol本申请的药物组合物，更优选在6小时内每kg患者体重施用约0.3μmol本申请的药物组合物。在其他情况下，剂量范围是每kg患者体重施用0.005到0.5μmol本申请的药物组合物。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，用于人的本申请的药物组合物的合适剂量可以是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，施用的药物组合物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节。在症状可检测时或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，本文公开的药物组合物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药物组合物(见例如Fingl等，1975,In:The Pharmacological Basis of Therapeutics,第1章，第1页)。
第二方面，本申请提供了治疗、改善或预防个体中的脑出血的方法，所述方法包括向所述个体施用第一方面所述的药物组合物。
第三方面，本申请提供了肽或者其药学可接受的盐在制备用于治疗、改善或预防个体中的脑出血的药物中的用途，所述肽包含氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体。
本申请所述的“个体”包括鸟类、爬行类和哺乳类动物。在一些实施方案中，所述动物是哺乳动物，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6.25n、12.5n、25n、50n、100n、200n、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(SEQ ID NO:3)
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，据发明人的推测，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。
以下实验中对嵌合肽P5进行进一步地测试，并在部分实验中将NA-1和YE-NA-1作为对照。
实施例2：Pμll-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Pμll-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:P5多肽对大鼠脑出血模型的治疗效果
实验用动物及材料：
动物：采用成年SD大鼠，SPF级，体重400±30g。
器械和药品：P5多肽、NA-1多肽由金斯瑞生物科技公司合成；神经节苷脂(GM)购自齐鲁制药有限公司，国药准字H20046213，脑定位仪、石蜡切片机、涡轮牙钻机、牙科水泥由北京众实迪创科技发展有限责任公司提供。
脑出血模型的建立：
本实验采用的脑出血模型是基于自体血液的脑部输注，具体步骤如下。
剪开大鼠颅顶处头皮伤口5～10mm，轻轻割去颅骨膜，圆心落于前颅右3mm后2mm处，直径约1mm的圆。圆心处打孔，10μl注射器进针6mm，5分钟内注入1μl 0.6CDU胶原酶Ⅳ，间隔15分钟后缓慢退针。全过程约3分钟。
50℃温水浸泡大鼠尾巴，待大鼠尾部血管充盈，断尾尖约5mm，轻揉尾部，挤压出120μl以上的血液，100μl注射器收集血液110μl。随后胶原酶注射同一位点，进针6mm，12分钟内注射110μl尾尖来源自体血。凝固20分钟后，3分钟内均匀退针封闭颅孔。胶原酶+自体血全部推注时间约35～40分钟，后续的给药时间以自体血全部注射完毕时刻开始计时。
实验分组：
实验分正常对照组、假手术组(用1μl生理盐水代替胶原酶注入，并且不注入自体血)、模型组(未治疗组，附图中标记为ICH组)、阳性药治疗组、P5多肽给药组、NA-1给药组。P5多肽给药组包括下述亚组：在自体血全部注入后1小时，尾静脉分别注射20mg/kg、10mg/kg、5mg/kg三个剂量的P5的给药组；分别在自体血全部注入后2小时、3小时给予10mg/kg剂量的P5的给药组。阳性药治疗组为自体血全部注入后1小时，肌肉注射0.4ml/kg GM(神经节苷脂)的治疗组。NA-1给药组为自体血全部注入后1小时，尾静脉注射10mg/kg NA-1的给药组。每组6～8只大鼠。
1.行为学观察结果
在各组造模后24小时(自体血全部注射完毕时为时间0点)，对其行为学进行评价。
平衡木评分法：平衡木长80cm，宽2.5cm，距地面10cm，评分共6级。0分：跳上平衡木，行走不跌倒；1分：跳上平衡木，跌倒几率小于50％；2分：跳上平衡木，跌倒几率大于50％；3分：跳上平衡木，受累侧不能帮助移动；4分：不能行走，但可坐在上面；5分：从平衡木上摔落。
平衡木测试的行为学观察的结果显示于图2中。结果显示，GM治疗组、1小时-5mg/kg P5给药组、1小时-10mg/kg P5给药组、1小时-20mg/kg P5给药组、以及2小时-10mg/kg P5给药组相对于模型组均存在显著的统计学差异；自体血全部注入后1小时P5-10mg/kg给药组相对于模型组存在统计学显著差异。
Berderson评分法：轻抓尾巴，提起高于桌面10cm，前爪伸直。0分：无神经功能损伤；1分：病变对侧腕关节、肘关节屈曲，肩内收屈曲；2分：上述体征+麻痹侧推组力下降；3分：活动时，打圈追尾。
采用Berderson评分法进行行为学评估的结果显示于图3中。结果显示1小时-10mg/kg P5给药组与模型组存在统计学显著差异。
2.脑血肿体积的测定
在行为学评价后，立即将大鼠断头处死。取脑组织，进行冷冻切片，切成2mm厚度的切片。取中间6片，用软件ImageJ计算各片面积再计算总体积。对于前后两面出血面积相近的脑片，以后面面积乘以厚度2mm记为血肿体积；对于前后两面出血面积相差较大的脑片，以较大的出血面积乘以1mm记为血肿体积。最后，各脑片血肿体积累加得到总体积。
脑血肿体积的测定结果如图4所示。结果显示，1小时-5mg/kg P5给 药组、1小时-10mg/kg P5给药组、1小时-20mg/kg P5给药组相对于模型组存在显著的统计学差异(p<0.01)；2小时-10mg/kg P5给药组相对于模型组存在统计学差异(P<0.05)。
3.脑组织病理形态学观察
用甲醛灌注取脑，将脑组织经常规梯度脱水、透明、浸蜡、包埋、切片。随后将脑组织切片进行苏木精-伊红染色，并在光镜下观察。
脑组织病理学形态观察的结果如图5所示。另外，对得到的脑组织病理切片进行免疫组化研究。免疫组化的抗原分别选择Bax-2(图6)和Caspase-3(图7)。
结果显示，正常脑组织神经细胞核仁清晰、核圆形，核膜完整；而脑出血模型组大鼠的脑组织出现严重的神经细胞坏死，细胞肿胀，胞核浓缩，胞浆疏松淡染及空泡化；1小时-10mg/kg P5给药组、1小时-20mg/kg P5给药组、2小时-10mg/kg P5给药组的显微图片显示的结果均优于GM治疗组和NA-1给药组，1小时-5mg/kg P5给药组的结果优于模型组。这些结果充分说明P5肽给药能够有效治疗或改善脑出血症状。
4.血清生化检测指标的测定
肌酸激酶(CK)存在于脑细胞的线粒体中，正常情况下，其很少进入血液中。当脑细胞受损时，崩解的脑细胞将CK释放至血液中。因此可以通过测定血清中的CK含量来评估脑组织受损伤破坏的严重程度。
各组血清中CK水平的测定结果如图8所示。结果显示，相对于模型组，1小时-20mg/kg P5给药组中血清CK水平降低。
以上结果表明本申请公开的P5肽能够显著治疗脑出血，且在某些指标的评价中，治疗效果优于市售治疗脑出血的阳性药物神经节苷脂。
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 (20)

  1. 用于治疗脑出血的药物组合物，所述药物组合物包含肽或者其药学可接受的盐，所述肽包含氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体。
  2. 如权利要求1所述的药物组合物，其中所述肽为含有氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体以及内化肽的嵌合肽，所述内化肽能促进所述嵌合肽被细胞摄取。
  3. 如权利要求2所述的药物组合物，其中所述内化肽包含氨基酸序列YGRKKRRQRRR(SEQ ID NO:2)。
  4. 如权利要求2所述的药物组合物，其中所述嵌合肽包含氨基酸序列YGRKKRRQRRRYEKLLDTEI(SEQ ID NO:3)。
  5. 如前述权利要求中任一项所述的药物组合物，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。
  6. 如权利要求5所述的药物组合物，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。
  7. 如权利要求1所述的药物组合物，还包含药学可接受的载体、稀释剂和/或赋形剂。
  8. 如前述权利要求中任一项所述的药物组合物，其为预冻干制剂，优选包含组氨酸和海藻糖。
  9. 如权利要求1-7中任一项所述的药物组合物，其为冻干制剂，优选通过将权利要求8所述的预冻干制剂冻干而制备。
  10. 如权利要求1-7中任一项所述的药物组合物，其为复原制剂，优选通过将权利要求9所述的冻干制剂与水溶液结合而制备。
  11. 治疗、改善或预防个体中的脑出血的方法，所述方法包括向有需要的个体施用权利要求1-10中任一项所述的药物组合物。
  12. 肽或者其药学可接受的盐在制备用于治疗、改善或预防个体中的脑出血的药物中的用途，所述肽包含氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体。
  13. 如权利要求12所述的用途，其中所述肽为含有氨基酸序列YEKLLDTEI(SEQ ID NO:1)或其功能性变体以及内化肽的嵌合肽，所述内化肽能促进所述嵌合肽被细胞摄取。
  14. 如权利要求13所述的用途，其中所述内化肽包含氨基酸序列YGRKKRRQRRR(SEQ ID NO:2)。
  15. 如权利要求13所述的用途，其中所述嵌合肽包含氨基酸序列YGRKKRRQRRRYEKLLDTEI(SEQ ID NO:3)。
  16. 如权利要求12-15中任一项所述的用途，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。
  17. 如权利要求16所述的用途，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。
  18. 如前述权利要求中任一项所述的药物组合物、方法或用途，其中所述脑出血选自外伤性脑出血和非外伤性脑出血。
  19. 如前述权利要求中任一项所述的药物组合物、方法或用途，其中所述脑出血选自脑基底节区出血、壳脑出血、丘脑出血、尾状核出血、脑室出血、脑叶出血、大脑出血、小脑出血、脑干出血和蛛网膜下腔出血。
  20. 如前述权利要求中任一项所述的药物组合物、方法或用途，其中所述脑出血由下述任一因素或其组合导致：微动脉瘤或者微血管瘤、脑动静脉畸形、淀粉样脑血管病、囊性血管瘤、颅内静脉血栓形成、硬膜动脉瘘、特异性动脉炎、真菌性动脉炎、烟雾病、动脉解剖变异、颈动静脉瘘、高血压、偏头痛、抗凝、抗血小板或溶栓治疗、嗜血杆菌感染、白血病、血栓性血小板减少症、颅内肿瘤、酒精、苯异丙胺、可卡因、交感神经兴奋药。
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