WO2019085366A1 - 一种促红细胞生成素来源肽及其制备方法和用途 - Google Patents

一种促红细胞生成素来源肽及其制备方法和用途 Download PDF

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WO2019085366A1
WO2019085366A1 PCT/CN2018/078253 CN2018078253W WO2019085366A1 WO 2019085366 A1 WO2019085366 A1 WO 2019085366A1 CN 2018078253 W CN2018078253 W CN 2018078253W WO 2019085366 A1 WO2019085366 A1 WO 2019085366A1
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derived peptide
erythropoietin
epo
peptide according
amino acid
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PCT/CN2018/078253
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English (en)
French (fr)
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汪昕
丁晶
吴婷婷
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复旦大学附属中山医院
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Priority to JP2019542465A priority Critical patent/JP6929949B2/ja
Priority to KR1020217043158A priority patent/KR102414649B1/ko
Priority to SG11201906820WA priority patent/SG11201906820WA/en
Priority to EP18873437.0A priority patent/EP3569613A4/en
Priority to KR1020197023338A priority patent/KR20190098268A/ko
Priority to US16/320,493 priority patent/US11479592B2/en
Publication of WO2019085366A1 publication Critical patent/WO2019085366A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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
    • A61P25/08—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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biological genes, and relates to an erythropoietin, in particular to an erythropoietin-derived peptide, a preparation method thereof and use thereof.
  • neurological diseases represented by cerebrovascular diseases have caused a certain economic burden on the country and society.
  • the WHO calculated that a 10% reduction in stroke and myocardial infarction mortality is expected to be reduced annually. About $25 billion in economic expenditures for interventions. In a sense, neurological diseases are not only medical but also social problems.
  • Nervous system diseases are complicated by the pathogenesis and many symptoms, and many still lack effective drug treatment.
  • hypoxic encephalopathy hyperbaric oxygen chamber can improve brain tissue oxygen supply, but it is contraindicated in poorly controlled hypertensive patients and critically ill patients, and may cause adverse effects caused by secondary contraction of cerebrovascular vessels, and clinical application is limited.
  • Another example is ischemic stroke, a series of pathophysiological changes such as energy metabolism disorder and excitatory amino acid toxicity after cerebral ischemia. Thrombolysis is currently the only effective method, but the time window of thrombolytic therapy is short, only a few patients benefit .
  • the other direction is neuroprotective treatment, which blocks the death of nerve cells and saves nerve cells in the ischemic penumbra.
  • the main neuroprotective agents that may be protective at present are: glutamate antagonists, anti-inflammatory factors, calcium channel blockers, sodium channel blockers, potassium channel activators, free radical scavengers, GABA receptors. Antagonists, serotonin antagonists, NMDA receptor antagonists, low-temperature phenobarbital-induced anesthesia, and the like.
  • glutamate antagonists anti-inflammatory factors
  • calcium channel blockers sodium channel blockers
  • potassium channel activators free radical scavengers
  • GABA receptors GABA receptors.
  • Antagonists, serotonin antagonists, NMDA receptor antagonists, low-temperature phenobarbital-induced anesthesia, and the like There are currently more than 1,000 neuroprotective agents, which are effective in animal experiments. More than 100 drugs have been clinically tested, but they have had little clinical benefit. Therefore, the development and development of new effective neuroprotective agents is of great significance.
  • EPO Erythropoietin
  • EPO and EPO receptors have been widely distributed in various non-hematopoietic tissues, including nerves, heart, kidney, etc. Molecules with a wide range of tissue protective activities. In particular, research in the past 20 years has found that EPO can play a neuroprotective and neurotrophic role whether in vivo or in vitro.
  • EPO/EPOR is expressed in neurons, astrocytes, vascular endothelial cells, etc. EPO/EPOR is expressed in normal adult brain tissue, but when subjected to injury or ischemia, hypoxia and other stress Its expression increased significantly. However, the activated EPO/EPOR system is not sufficient to protect damaged nerve cells, and if exogenously supplemented with a large amount of recombinant erythropoietin (rhEPO), it is transmitted through receptor-mediated endocytosis or pinocytosis on the cell membrane.
  • rhEPO erythropoietin
  • the blood-brain barrier enters the nervous system, and EPO binds to overexpressed EPOR to induce Jak2 phosphorylation, activates signal transducer and activator STAT5 pathway, phosphatidylinositol 3-kinase (P13K)/AKT pathway, nuclear factor NF- ⁇ B Multiple pathways, through anti-inflammatory, anti-oxidative stress, anti-apoptosis, inhibition of excitotoxicity, promotion of nerve re-generation, protection of the blood-brain barrier and other mechanisms, reduce neuronal apoptosis, reduce neuronal Injury, play a neuroprotective role.
  • EPO autoimmune encephalomyelitis
  • EPO has been shown to have neuroprotective effects in vitro and in vivo, in recent decades, a series of clinical trials have further investigated whether EPO can exert neuroprotective and regenerative functions in humans, from cerebral ischemia to nerve damage. Patients with inflammation and neurodegeneration are more gratified that most of these studies have confirmed that EPO treatment is effective.
  • EPO erythropoiesis
  • CEPO carbamylated EPO
  • a derivative of erythropoietin protein modified by an acid EPO (Neuro-EPO) or the like is not combined with EPOR, but it can also preserve the neuroprotection and other tissue protection of EPO.
  • the red-promoting effect different from EPO is mediated by the dimer formed by two molecules of EPOR, and the tissue protective effect of EPO including neuroprotection is by EPOR and CD131, ⁇ cR ( ⁇ -community).
  • the composition of the heteropolymer is mediated.
  • Another type of derivative is a small molecule polypeptide that mimics the EPO spatial structure, including HBP, HBSP (ARA290), pHBSP, Epopeptide AB, MKX-2, JM4, Epotris, and the like. This kind of polypeptide derivative has the advantages of small molecular weight and easy to penetrate the blood-brain barrier, and has a promising clinical application, but further research and experiments are needed to clinically verify its role in the human body.
  • the present invention provides an erythropoietin-derived peptide, a preparation method thereof and a use thereof, wherein the erythropoietin-derived peptide has no erythropoiesis-producing effect, but has a nerve
  • the protective effect can avoid a series of serious medical problems such as polycythemia, hypertension and thrombosis caused by the promotion of erythropoiesis.
  • the present invention first refers to the published human EPO amino acid sequence (Proteomic databases, P01588), according to the results of the research published in the 1998 Nature Journal (Nature, 1998. 395 (6701): p. 511-6), one of which contains EPO and The amino acid sequence of the EPOR binding site and the EPO protein helix structure, and the deletion of some amino acids (for example, deletion of the 86th glutamine, 88th tryptophan, 89th glutamic acid, 90th ⁇ in the amino acid sequence of the EPO molecule) Or one or more of the 97-position lysine, preferably all of the above-mentioned deletions, and the original arginine at position 103 is replaced by alanine, and the erythropoietin-derived peptide is not promoted.
  • the role of erythropoiesis but with neuroprotective effects.
  • One aspect of the present invention provides an erythropoietin-derived peptide having no erythropoiesis-producing action but having neuroprotective effects, which comprises the following (a) or (b):
  • SEQ ID NO. 1 is:
  • the amino acid sequence of the erythropoietin-derived peptide is the sequence shown in SEQ ID NO.
  • Another aspect of the invention provides a medicament comprising the erythropoietin-derived peptide, or a composition comprising the erythropoietin-derived peptide.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the erythropoietin-derived peptide, and an excipient or carrier.
  • the pharmaceutical compositions of the invention are administered in a pharmaceutically acceptable formulation.
  • Such formulations may conventionally contain a pharmaceutically acceptable salt, buffer, preservative, compatible carrier, and optionally other therapeutic agents.
  • Another aspect of the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding any one of the above-described erythropoietin-derived peptides.
  • the present invention also provides an expression vector of the above nucleic acid molecule.
  • the present invention also provides a host cell or virus of the above expression vector.
  • kits comprising any one of the following:
  • the invention also provides a preparation method of the above erythropoietin-derived peptide, comprising the following steps:
  • the starting resin to which leucine is attached is immersed in DMF (N,N-dimethylformamide) for 20 to 50 minutes, and then immersed in a capping solution to remove FMOC (fluorenylmethoxycarbonyl). ), after 20 to 50 minutes, rinse with dimethylformamide;
  • DMF N,N-dimethylformamide
  • FMOC fluorenylmethoxycarbonyl
  • amino acid linkage sequence in the preparation method of the present invention is leucine ⁇ alanine ⁇ arginine ⁇ leucine ⁇ leucine ⁇ threonine ⁇ threonine ⁇ leucine ⁇ serine ⁇ alanine Acid ⁇ leucine ⁇ glycine ⁇ serine ⁇ valine ⁇ alanine ⁇ aspartate ⁇ valine ⁇ histidine ⁇ leucine ⁇ glutamine ⁇ leucine ⁇ valine ⁇ serine ⁇ serine ⁇ Asparagine ⁇ Proline ⁇ Leucine ⁇ Leucine ⁇ Alanine ⁇ Glutamine ⁇ Glycine ⁇ Arginine ⁇ Leucine ⁇ Proline ⁇ Alanine ⁇ Glutamate ⁇ Serine.
  • the condensing agent described in step 2) is TBTU, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate.
  • the base described in step 2) is morpholine.
  • the capping solution described in the step 2) is a mixture of hexahydropyridine and N,N-dimethylformamide, wherein the volume ratio of the hexahydropyridine to the N,N-dimethylformamide is preferably 1:3 to 5, the best is 1:4.
  • the invention also provides the use of the above erythropoietin-derived peptide for treating nerve cell damage.
  • the invention also provides the use of the above erythropoietin-derived peptide in the treatment of hypoxic brain damage.
  • the invention also provides the use of the above erythropoietin-derived peptide for treating epilepsy.
  • the present invention also provides a medicament for treating nerve cell damage comprising the erythropoietin-derived peptide of the present invention.
  • the present invention also provides a medicament for treating hypoxic brain damage comprising the erythropoietin-derived peptide of the present invention.
  • the present invention also provides a medicament for treating epilepsy comprising the erythropoietin-derived peptide of the present invention.
  • the invention successfully prepares and synthesizes a novel EPO source peptide, and the preparation method of the invention has a purity of up to 95%.
  • the inventors designed an animal model of epilepsy. After the EPO-derived peptide was used in the animal model, the incubation period of seizure was prolonged, indicating that it may have the effect of alleviating nerve cell damage, and then designing experiments. It is confirmed that after using the novel EPO-derived peptide in nerve-damaged animals, the novel EPO-derived peptide can protect neurons from the blood-brain barrier during acute injury, exert neuroprotective effects, prolong the seizure latency, and reduce hippocampus nerves. Loss of the element, thus indicating that the novel EPO-derived peptide has a medicinal effect for treating nerve damage.
  • novel EPO-derived peptide of the present invention has been shown to have an anti-neuronal apoptosis effect in an in vitro experiment. After the cultured primary neurons were given glucose-free and anaerobic injury and NMDA injury, the neuronal damage of the new EPO-derived peptide intervention group was alleviated, and the cell survival rate was higher than that of the control group. In in vivo experiments, it was verified that novel EPO-derived peptides can reduce cerebral infarction volume, improve cerebral ischemia, and reduce the severity of neurobehavioral scores.
  • the novel EPO-derived peptide can alleviate nerve damage, has neuroprotective effect and reduce apoptosis; the mice are administered for a long time without significant side effects of erythropoiesis.
  • the novel EPO-derived peptide has a neuroprotective effect on acute hypoxic injury and is superior to EPO.
  • the technical progress of the present invention is remarkable compared to the prior art.
  • the invention provides a novel EPO source peptide with neuroprotective activity and no erythropoiesis-producing activity, the novel drug has small molecular weight and can penetrate the blood-brain barrier, and is a small molecule polypeptide derived from functional proteins in human body, compared with other In terms of the development of synthetic drugs, the side effects on humans are extremely small, and the clinical application prospects are considerable. If the invention is applicable to clinical practice, it will become a novel neuroprotective drug for preventing and treating central nervous system diseases, and providing a new therapeutic direction for patients with nerve damage.
  • Figure 1 is a high performance liquid chromatography HPLC report (A) and mass spectrometry Mass Spectrometry report (B).
  • Figure 2 is a comparison of red blood cell count histograms (3 days, 7 days, 13 days), wherein the abscissa: the number of days of detection, and the ordinate: the count of red blood cells (M/uL).
  • Figure 3 shows the MAP-2 and DAPI fluorescence double staining of cortical neurons, in which the neuronal cell bodies are stained green and the nucleus is blue.
  • Figure 4A shows cell viability of different drug-interfering glucose-deprived injured neurons; among them, abscissa: grouping, ordinate: cell viability; B shows lactate dehydrogenase release from different drugs in interventional glucose deprivation injured neurons Rate, where, abscissa: grouping, ordinate: lactate dehydrogenase release rate.
  • Figure 5A shows the cell viability of NMDA-injured neurons treated with different drugs; wherein, the abscissa: grouping, ordinate: cell viability; B shows the release rate of lactate dehydrogenase from different drugs in NMDA-injured neurons, Abscissa: grouping, ordinate: lactate dehydrogenase release rate.
  • Figure 6 shows cerebrospinal fluid biotin concentrations in mice of different treatment groups, wherein the abscissa: grouping, ordinate: biotin concentration.
  • Figure 7A shows the neurobehavioral scores of mice in different treatment groups, wherein the abscissa: grouping, ordinate: neurobehavioral score score; B shows the transient cerebral infarction volume of the middle cerebral artery in different treatment groups, wherein Abscissa: grouping, ordinate: cerebral infarct volume.
  • the step of synthesizing the EPO-derived peptide of the present invention the starting resin is leucine, the resin is emulsified with dimethylformamide for 30 minutes, and then the methoxycarbonyl group is removed with a capping solution, and after 30 minutes, it is washed with dimethylformamide. Then add the next amino acid (alanine), condensing agent and base, react for 30 minutes, then wash it with dimethylformamide. After the test, remove the methoxycarbonyl group with capping solution, and then use dimethyl group after 30 minutes. Formamide cleaning. Then, according to the designed sequence, the next amino acid (arginine) is taken until the last amino acid is obtained, and on the basis of ensuring the linear peptide pair, the purity is finally cut and purified to 95% or more.
  • the next amino acid arginine
  • the histogram of the above data is shown in Fig. 2. As shown in the graph, as the number of administrations increases, the number of administrations increases, and the EPO group is significantly higher than the other groups, while the EPO-derived peptide group (T3) of the present invention There was no significant increase in red blood cells in the saline group (Saline) and the solvent group (ACN+Milliq), and the difference between the red blood cell count and the low dose group in the high-dose EPO-derived peptide group was also not statistically significant.
  • Digestion Move the shredded tissue block into a clean 50ml centrifuge tube, add 5ml of pre-warmed 0.125% trypsin-EDTA at 37 °C, and digest it in a 37 °C water bath for about 5-10 minutes;
  • Termination of digestion The digestion was terminated by adding 5 ml of 37 ° C preheated FBS-DMEM medium. Aspirate the flocculent cell mass in a clean 15 ml centrifuge tube, and add 10% FBS-DMEM culture solution to wash away the residual digestive juice;
  • the dropper is repeatedly blown to disperse into single cells, and the cells are filtered through a 200 mesh filter to prepare a single cell suspension;
  • the 7-14-day cultured neurons were placed on a glass slide and washed with pre-warmed PBS for 5 min;
  • Blocking add blocking solution (5% sheep serum), and block at room temperature for 30-60min;
  • the culture plate was placed in an anoxic incubator at 37 ° C, and the oxygen concentration in the chamber was set to 1%.
  • the results are shown in Fig. 4.
  • the release rate of neuronal lactate dehydrogenase in the EPO-derived peptide group (T3) group was lower than that in the control group, and the cell survival rate was higher than that in the control group, indicating that the EPO-derived peptide of the present invention can be alleviated.
  • Acute nerve damage reduces neuronal apoptosis.
  • NMDA N-methyl D-aspartate
  • the results are shown in Fig. 5.
  • the release rate of lactate dehydrogenase in the EPO-derived peptide group (T3) group was lower than that in the control group, and the cell survival rate was higher than that in the control group, indicating that the EPO-derived peptide of the present invention can be alleviated.
  • Acute nerve damage reduces neuronal apoptosis.
  • Example 4 EPO-derived peptide of the present invention passed the blood-brain barrier test
  • biotin-labeled new EPO-derived peptide was administered, and 5-15 ul of mouse cerebrospinal fluid was taken under general anesthesia 1 hour after administration.
  • the biotin detection kit was used to detect whether the biotin-labeled new EPO-derived peptide passed through the blood brain.
  • the barrier enters the cerebrospinal fluid and the results are shown in Figure 6.
  • mice 1. 4% chloral hydrate anesthetized mice;
  • the mouse is lying prone, placing the head at a 135° angle to the head and the head on the stereotactic instrument. Under the stereo microscope, the subcutaneous tissue and muscle are bluntly separated along the midline. At this angle, the incision under the occipital bone The dura mater and spinal cord can be seen more clearly (the main features are white clear appearance, cyclic pulsation of spinal cord blood vessels and adjacent cerebrospinal fluid areas);
  • the needle puller sets the heat value to 300, the pressure value is 330, the capillary glass tube is pulled into a pointed end, and the scissors trims the tip to make the blunt head diameter 0.5 mm;
  • the capillary tube is connected to the negative pressure device to maintain the negative pressure state of the collection system in advance;
  • control group the biotinylated EPO-derived peptide group of the present invention, epilepsy, and biotinylated cerebrospinal fluid of the EPO-derived peptide group of the present invention were respectively taken, and the biotin assay kit was used to detect the biotin concentration.
  • Example 5 Neuroprotective effect of EPO-derived peptide of the present invention on a transient mouse model of middle cerebral artery occlusion
  • ICR mice male, 25-30g, 18, purchased from Shanghai Slack Laboratory Animal Co., Ltd., divided into control group (ACN+Millq), 0.2ml/only), EPO group (50ug/kg), new type
  • EPO-derived peptide group T3, 500 ug/kg
  • 6/group The mouse middle cerebral artery occlusion model was performed by mouse thread suture method. The mice were anesthetized with ketamine hydrochloride anesthesia and fixed on the operating table. After 60 min of ischemia, the plug was removed and reperfused, and the solvent (ACN+Millq), EPO, and T3 were administered separately. Neurobehavioral scores were performed on the third day after surgery, and then the mice were sacrificed and brain slices were taken and stained with tar purple. The result is shown in Figure 7.
  • the results are shown in Fig. 7.
  • the neurobehavioral scores and cerebral infarction volume of the EPO group and the EPO-derived peptide group of the present invention were lower than those of the control group, indicating that EPO and the EPO-derived peptide of the present invention have cerebral ischemia in vivo and have neuroprotective effects.
  • Example 6 Neuroprotective effect of EPO-derived peptides of the present invention on animal models of epilepsy
  • C57/BL6 mice male, 20-25g, 24, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.
  • normal saline group Saline, 0.2ml / only
  • solvent group ACN + Milliq, 0.2ml
  • EPO group 50ug/kg
  • new EPO source peptide group T3, 500ug/kg
  • 6/group 6/group.
  • Pilocarpine was administered 1 day, 4 hours, and 2 hours before epilepsy, and the seizure latency and severity of seizures were recorded. The result is shown in Figure 8.
  • Level I gaze, oral and facial activity
  • Grade III unilateral or bilateral forelimb twitching
  • Class V Lost posture, repeated jumps, fall to the ground, tonic clonic seizures
  • the EPO-derived peptide of the present invention When the EPO-derived peptide of the present invention is prepared into a medicament, an effective amount of the polypeptide, and at least one pharmaceutically acceptable carrier, diluent or excipient can be combined.
  • the active ingredient is usually mixed with excipients, or diluted with excipients, or enclosed in a carrier in the form of capsules or sachets.
  • the excipient acts as a diluent, it can be a solid, semi-solid or liquid material as a vehicle for the vehicle, carrier or active ingredient. Therefore, the drug may be a tablet, a pill, a powder, a solution, a syrup, a sterile injectable solution or the like.
  • excipients examples include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like.
  • the preparation may also include a wetting agent, an emulsifier, a preservative (such as methyl and propyl hydroxybenzoate), a sweetener, and the like.
  • the novel EPO-derived peptide of the present invention can protect neuronal cells and reduce apoptosis in acute injury; long-term administration of mice has no significant side effects of erythropoiesis; it can penetrate the blood-brain barrier, Administration after ischemic injury in vivo can reduce the volume of cerebral infarction and improve cerebral ischemia. Pre-dosing can prolong the incubation period of seizures, and it can be confirmed that drugs for treating nerve damage can be prepared. In vivo hematopoietic experiments were carried out on the EPO-derived peptide of the present invention, and it was confirmed that it had no effect of promoting red blood cells.
  • the inventors confirmed that the novel EPO-derived peptide has a protective effect on nerve cells by in vitro anti-cortical neuronal apoptosis experiments.
  • the biotin-labeled polypeptide in the cerebrospinal fluid was measured by animal administration, and it was confirmed that the novel EPO-derived peptide can function through the blood-brain barrier.

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Abstract

本发明提供了一种促红细胞生成素来源肽及其制备方法和用途，所述促红细胞生成素来源肽的氨基酸序列如SEQ ID NO.1所示。本发明的促红细胞生成素来源肽是来源于人体内功能蛋白的小分子多肽，能透过血脑屏障，可用于预防和治疗中枢神经系统疾病，如缺氧性脑损伤、癫痫。

Description

一种促红细胞生成素来源肽及其制备方法和用途 技术领域
本发明属于生物基因领域，涉及一种促红细胞生成素，具体来说是一种促红细胞生成素来源肽及其制备方法和用途。
背景技术
随着人类寿命的延长，神经系统疾病正越来越成为患者、家人和社会的沉重负担。2016年《柳叶刀》杂志发表了国际疾病负担组(GBD)新的全球死因分析报告，2015年脑血管病死因顺位再次上升，跃升为人类死因的第二位。而在我国，脑血管疾病是我国人口首位的死亡原因和致残原因，其患病率逐年上升，并有年轻化的趋势。而阿尔兹海默病、其他类型痴呆、癫痫、帕金森病等其它神经系统疾病的发病率也是逐年上升，其死亡人数从1990年到2015年增长率超过20％。除了影响健康，以脑血管疾病为代表的神经系统疾病给国家和社会造成了一定的经济负担，WHO计算得出，减少10％的卒中和心肌梗死所致的死亡率，预计每年会减少用于干预措施的约250亿美元的经济支出。从某种意义上说，神经系统疾病不仅是医学问题，也是社会问题。
神经系统疾病由于发病机制复杂，症状繁多，仍有不少尚缺乏有效的药物治疗。比如缺氧性脑病，高压氧舱可以改善脑组织供氧，但禁用于控制不佳的高血压患者和危重患者，且可能产生脑血管继发性收缩而引起的不良作用，临床应用受到限制。再比如缺血性卒中，脑缺血后发生能量代谢障碍、兴奋性氨基酸毒性作用等一系列病理生理改变，溶栓是目前唯一有效的方法，但溶栓治疗时间窗较短，只有少数患者受益。另一方向则是神经保护治疗，阻断神经细胞的死亡，挽救缺血半暗带的神经细胞。目前可能具有保护作用的主要神经保护剂有：谷氨酸拮抗剂、抗炎因子、钙离子通道阻断剂、钠离子通道阻断剂、钾离子通道激活剂、自由基清除剂、GABA受体拮抗剂、5-羟色胺拮抗体、NMDA受体拮抗剂、低温苯巴比妥类药物诱导麻醉等。当前有超过1000多个神经保护剂，在动物实验中有效，超过100种药物进行了临床试验，然而在临床上却收效甚微。因此研制和开发在新的有效的神经保护剂有重要意义。
促红细胞生成素(EPO)是主要由肾脏产生的由165个氨基酸组成的34kDa的糖蛋白激素，是最早应用于临床的细胞因子。以往促红细胞生成素在临床被广泛应用于治疗贫血，近年来研究发现，EPO及EPO受体(EPOR)还广泛分布于各种非造血组织，包括神经、心脏、肾脏等，是一种多功能分子，具有广泛的组织保护活性。特别地，近20年来的研究发现，无论在体内或体外实验，EPO均可起到神经保护和神经营养作用。
在神经系统，EPO/EPOR表达在神经元、星形胶质细胞、血管内皮细胞等，在正常成人脑组织内EPO/EPOR呈低表达状态，但当受到损伤或缺血、缺氧等应激时其表达显著增加。但激活的EPO/EPOR系统尚不足以保护受损的神经细胞，而若外源性补充大量重组促红细胞生成素(rhEPO)，通过细胞膜上的受体介导的胞吞或胞饮作用透过血脑屏障进入神经系统，EPO与过度表达的EPOR结合后诱导Jak2磷酸化，激活信号转导子及转录激活子STAT5通路、磷脂酰肌醇3激酶(P13K)/AKT通路、核因子NF-κB等多个通路，通过抗炎、抗氧化应激、抗凋亡、抑制兴奋性毒性、促进神经再成、保护血脑屏障等多种机制联合作用，减少神经细胞的凋亡，减轻神经元的损伤，发挥神经保护作用。并且，EPO的神经保护作用已在多种动物疾病模型中证实，包括：脑缺血、脑缺血缺氧性损伤、脑缺血再灌注损伤、脊髓损伤、实验性自身免疫性脑脊髓膜炎(EAE)、蛛网膜下腔出血、癫痫、帕金森、多系统萎缩、坐骨神经压迫、视网膜神经节损伤等。
随着EPO在体内外实验均被证实具有神经保护作用后，最近十几年，一系列的临床试验进一步研究EPO是否在人体内可以发挥神经保护及再生的功能，从脑缺血、神经损伤到炎症及神经退行性变患者，比较欣慰的是，这些研究大多证实了EPO的治疗是有效的。但大剂量或长期使用EPO，由于其具有促进红细胞生成的作用，会直接导致红细胞增多症、高血压、血栓形成等一系列严重的医学问题，因此，研发无造血功能但具有神经保护功效的EPO衍生物、变构体等制剂，成为目前神经科学家研究的热点。
近年来，研究者相继开发了一些具有神经保护作用但无促红细胞生成作用的EPO衍生物，一类是以氨甲酰化EPO(CEPO)为代表，包括去唾液酸EPO(asialoEPO)和低唾液酸EPO(Neuro-EPO)等的对促红细胞生成素蛋白修饰的 衍生物。且随着对EPO及CEPO研究的深入，发现CEPO不与EPOR结合，但还能保留EPO的神经保护和其它组织保护作用。因而进一步提出，不同于EPO的促红作用是由2分子的EPOR所形成的二聚体所介导，EPO所发挥的组织保护作用包括神经保护作用则是由EPOR与CD131即βcR(β共同体)组成的异聚体所介导。另一类衍生物是模拟EPO空间结构的小分子多肽，包括HBP、HBSP(ARA290)、pHBSP、Epopeptide AB、MKX-2、JM4、Epotris等。这一类多肽衍生物具有分子量小，易透过血脑屏障的优点，临床应用前景可观，但仍需进一步深入研究及试验于临床验证其在人体内的作用。
发明内容
针对现有技术中的上述技术问题，本发明提供了一种促红细胞生成素来源肽及其制备方法和用途，所述的这种促红细胞生成素来源肽没有促红细胞生成的作用，但具备神经保护作用，能够避免因促进红细胞生成的作用，导致的红细胞增多症、高血压、血栓形成等一系列严重的医学问题。
本发明首先参考公开的人类EPO氨基酸序列(Proteomic databases，P01588)，根据1998年Nature杂志(Nature,1998.395(6701):p.511-6)上公开的研究结果，选取了其中一段包含有EPO与EPOR结合位点及EPO蛋白螺旋结构的氨基酸序列，并对部分氨基酸进行缺失(例如，缺失EPO分子氨基酸序列中第86位谷氨酰胺、88位色氨酸、89位谷氨酸、90位脯氨酸、97位赖基酸中的一位或者多位，优选为前述各位全部缺失)，将原来位于第103位的精氨酸，替换为丙氨酸，该促红细胞生成素来源肽没有促红细胞生成的作用，但具备神经保护作用。
本发明一个方面提供了一种没有促红细胞生成作用，但具备神经保护作用的促红细胞生成素来源肽，其包括以下(a)或(b)：
(a)氨基酸序列如SEQ ID NO.1所示的多肽；
(b)在(a)中氨基酸序列基础上经过取代、缺失或添加一个或几个氨基酸衍生的多肽。
具体地，SEQ ID NO.1为：
SEAVLRGQAL LVNSSP LQLHVDAVSGLASLTTLLRAL。
在本发明的一种优选实施方式中，所述促红细胞生成素来源肽的氨基酸序列即为如SEQ ID NO.1所示的序列。
进一步地，上述如SEQ ID NO.1所示氨基酸序列的所述促红细胞生成素来源肽的结构式如下：
Figure PCTCN2018078253-appb-000001
本发明另一方面提供了包含所述促红细胞生成素来源肽的药物，或提供了包含所述促红细胞生成素来源肽的组合物。
进一步，本发明提供了一种药物组合物，包含所述促红细胞生成素来源肽，以及赋形剂或载体。当被施用时，本发明的药物组合物以药学上可接受的制剂被施用。这种制剂可常规地含有药学上可接受的浓度的盐、缓冲剂、防腐剂、相容的载体和任选的其它治疗剂。
本发明另一方面提供了一种核酸分子，包含编码上述任意一种促红细胞生成素来源肽的核苷酸序列。
进一步，本发明还提供了上述核酸分子的表达载体。
进一步，本发明还提供了上述表达载体的宿主细胞或病毒。
更进一步，本发明还提供了一种包含以下各项中任意一项的试剂盒：
a)本发明所述的促红细胞生成素来源肽；
b)本发明所述的组合物；
c)本发明所述的核酸分子；
d)本发明所述的表达载体；
e)本发明所述的宿主细胞；
f)本发明所述的病毒。
本发明还提供了上述促红细胞生成素来源肽的制备方法，包括如下步骤：
1)采用固相合成法，将连接有亮氨酸的起始树脂用DMF(N,N-二甲基甲酰胺)浸泡20～50分钟，然后用脱帽液浸泡，去除FMOC(芴甲氧羰基)，20～50分钟后用二甲基甲酰胺清洗；
2)加入下一个氨基酸、缩合剂和碱，反应20～50分钟，再用二甲基甲酰胺清洗；
3)用脱帽液去除笏甲氧羰基，30分钟后再用二甲基甲酰胺清洗；
4)重复步骤2)和步骤3)，依次连接氨基酸。
进一步地，本发明的制备方法中的氨基酸连接顺序为亮氨酸→丙氨酸→精氨酸→亮氨酸→亮氨酸→苏氨酸→苏氨酸→亮氨酸→丝氨酸→丙氨酸→亮氨酸→甘氨酸→丝氨酸→缬氨酸→丙氨酸→天冬氨酸→缬氨酸→组氨酸→亮氨酸→谷氨酰胺→亮氨酸→脯氨酸→丝氨酸→丝氨酸→天冬酰胺→缬氨酸→亮氨酸→亮氨酸→丙氨酸→谷氨酰胺→甘氨酸→精氨酸→亮氨酸→缬氨酸→丙氨酸→谷氨酸→丝氨酸。
优选地，步骤2)中所述的缩合剂为TBTU即O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯。
优选地，步骤2)中所述的碱为吗啡啉。
优选地，步骤2)中所述的脱帽液为六氢吡啶和N,N-二甲基甲酰胺组成的混合液，其中六氢吡啶和N,N-二甲基甲酰胺的体积比优选为1:3～5，最佳为1:4。
本发明还提供了上述促红细胞生成素来源肽在治疗神经细胞损伤中的应用。
本发明还提供了上述促红细胞生成素来源肽在治疗缺氧性脑损伤中的应用。
本发明还提供了上述促红细胞生成素来源肽在治疗癫痫中的应用。
本发明还提供了一种用于治疗神经细胞损伤的药物，其包含本发明的促红细胞生成素来源肽。
本发明还提供了一种用于治疗缺氧性脑损伤的药物，其包含本发明的促红细胞生成素来源肽。
本发明还提供了一种用于治疗癫痫的药物，其包含本发明的促红细胞生成素 来源肽。
本发明成功制备合成了新型EPO来源肽，应用本发明的制备方法其纯度可达95％。
为了进一步研究新型EPO来源肽的功能，发明人设计了癫痫动物模型，在对该动物模型使用了EPO来源肽后，癫痫发作潜伏期延长，说明其可能有减轻神经细胞损伤的作用，进而设计实验，证实了对神经损伤动物使用新型EPO来源肽后，所述的新型EPO来源肽能够在急性损伤时保护神经元能够透过血脑屏障，发挥神经保护作用，延长癫痫发作潜伏期，可以减少海马区神经元的丢失，因此说明新型EPO来源肽具有治疗神经损伤的药用效果。
本发明的新型EPO来源肽在体外实验中显示具有抗神经元凋亡的作用。培养的原代神经元予以无糖无氧损伤及NMDA损伤后，新型EPO来源肽干预组的神经元损伤减轻，细胞存活率较对照组提高。在体内实验中，验证了新型EPO来源肽可以减小脑梗死体积改善脑缺血，降低神经行为学评分严重程度。提示新型EPO来源肽可以减轻神经损伤，具有神经保护作用，减少细胞凋亡；小鼠长期给药，没有显著促红细胞生成的副作用。因而新型EPO来源肽对于急性缺氧损伤具有神经保护的作用，并优于EPO。
本发明和已有技术相比，其技术进步是显著的。本发明提供了一种具有神经保护作用而无促红细胞生成活性的新型EPO来源肽，该新药分子量小、能透过血脑屏障，且其是来源于人体内功能蛋白的小分子多肽，较其它研制合成的药物而言，对人体副作用极小，临床应用前景可观。该发明若可应用于临床，将成为预防和治疗中枢神经系统疾病新型的神经保护药物，为神经损伤患者提供新的治疗方向。
附图说明
图1为高效液相色谱法HPLC报告(A)和质谱法Mass Spectrometry报告(B)。
图2为红细胞数比较直方图(3天、7天、13天)，其中，横坐标：检测天数，纵坐标：血红细胞计数(M/uL)。
图3显示了皮层神经元MAP－2及DAPI荧光双染图，其中，神经元胞体染为绿色，细胞核为蓝色。
图4A显示了不同药物干预糖氧剥夺损伤神经元的细胞存活率；其中，横坐 标：分组，纵坐标：细胞存活率；B显示了不同药物干预糖氧剥夺损伤神经元的乳酸脱氢酶释放率，其中，横坐标：分组，纵坐标：乳酸脱氢酶释放率。
图5A显示了不同药物干预NMDA损伤神经元的细胞存活率；其中，横坐标：分组，纵坐标：细胞存活率；B显示了不同药物干预NMDA损伤神经元的乳酸脱氢酶释放率，其中，横坐标：分组，纵坐标：乳酸脱氢酶释放率。
图6显示了不同处理组小鼠脑脊液生物素浓度，其中，横坐标：分组，纵坐标：生物素浓度。
图7A显示了不同处理组小鼠神经行为学评分，其中，横坐标：分组，纵坐标：神经行为学评分分数；B显示了不同处理组小鼠短暂性大脑中动脉阻塞脑梗死体积，其中，横坐标：分组，纵坐标：脑梗死体积。
图8不同药物干预匹鲁卡品致痫小鼠发作潜伏期，其中，横坐标：分组，纵坐标：发作潜伏期。
具体实施方式
实施例1 本发明的EPO来源肽的制备
合成本发明的EPO来源肽的步骤：起始树脂是亮氨酸，用二甲基甲酰胺泡树脂30分钟，然后用脱帽液去除笏甲氧羰基，30分钟后用二甲基甲酰胺清洗，然后加入下一个氨基酸(丙氨酸)、缩合剂和碱，反应30分钟，再用二甲基甲酰胺清洗检测，检测通过后用脱帽液去除笏甲氧羰基，30分钟后再用二甲基甲酰胺清洗。再按设计好的序列接下一个氨基酸(精氨酸)，直到接至最后一个氨基酸，在保证线性肽对的基础上，最后切割、纯化至纯度达95％以上。
鉴定新型EPO来源肽纯度方法：见图1
将粗肽溶于按体积计ACN(乙腈)：H 2O＝1:2溶液中，经HPLC纯化，HPLC条件：流动相A相：0.1％TFA三氟乙酸/100％ACN乙腈；流动相B相：0.1％TFA三氟乙酸/100％水；色谱柱：Kromasil C18,4.6×250mm,5μm；
梯度：A B
0.0min 38％ 62％
25.0min 63％ 37％
25.1min 100％ 0％
30.0min Stop
流速：1.0ml/min
柱温：25℃
仪器平衡设定好后，启动仪器走10min左右，将粗肽注入仪器，采集基线，停止，最终获取的样品纯度为95.25％。
实施例2 本发明的EPO来源肽造血实验
18-22g雄性C57/BL6小鼠(购自上海西普尔-必凯动物有限公司，共50只，每组10只)，腹腔注射EPO(50ug/kg)、本发明的EPO来源肽(50ug/kg)、本发明的EPO来源肽(250ug/kg)、生理盐水(0.1ml/mouse)和溶剂(ACN+Milliq，0.1ml/mouse)，每天1次。第3天、7天、13天分别通过尾静脉取血法，用ProCyte Dx全自动血细胞分析仪测定血红细胞数。
试验结果如下表：
表1 RBC生成比较表(3天、7天、13天)单位：M/uL
Saline ACN+MiNiQ T3 50ug/kg T3 250ug/kg Epo 50ug/kg
3天 7.03±0.59 8.10±0.44 9.29±0.13 8.16±0.28 9.14±0.56
7天 7.36±0.47 8.63±0.52 8.75±0.36 9.56±0.54 12.79±0.24
13天 6.28±0.17 5.81±0.14 6.77±0.18 6.52±0.12 11.15±0.23
上述数据作直方图如图2，由图表看出，随给药次数增多，随给药次数增多，EPO组较其它组红细胞计数相比明显增高，而本发明的EPO来源肽组(T3)与生理盐水组(Saline)、溶剂组(ACN+Milliq)相比红细胞没有明显增高，而且高剂量EPO来源肽组的红细胞计数同低剂量组之间的差异也无统计学意义。
实施例3 本发明的EPO来源肽神经元保护实验
采用文献方法进行胎鼠皮层神经元培养，采用
Figure PCTCN2018078253-appb-000002
Medium(市购)无血清培养基，5％CO2培养箱7-14天后，进行神经元特异性标记：微管相关蛋白-2(MAP-2)染色鉴定，同时予4，6-联脒-2-苯基吲哚(DAPI)荧光染料标记细胞核。荧光显微镜下观测结果，见图3。
具体操作过程如下：
胎鼠皮层神经元原代培养
1.孕18天SD孕鼠(上海西普尔-必凯动物有限公司)10％水合氯醛麻醉；
2. 75％酒精消毒腹部皮肤，打开腹腔，分离子宫，剥出胎鼠；
3.分离组织：剪下胎鼠头部，立即放入预冷的PBS缓冲液中，冰上操作，仔细分离出脑组织，小心分离皮层，仔细剥离脑膜及血管，PBS液洗2次；
4.剪碎：用眼科剪将皮层剪碎，约1mm3即可；
5.消化：将剪碎的组织块移入一干净50ml离心管中，加入5ml 37℃预热的0.125％胰酶-EDTA，放入37℃水浴箱中消化5-10min左右；
6.终止消化：加5ml 37℃预热的FBS-DMEM培养液终止消化。吸出絮状细胞团块于一干净15ml离心管中，加入10％FBS-DMEM培养液洗掉残留的消化液；
7.滴管反复吹打，使其分散成单细胞，用200目滤网过滤细胞制成单细胞悬液；
8. 4℃，2000rpm离心5分钟，弃上清，加入含10％胎牛血清的种植培养基，重悬细胞；
9.显微镜下计数，以1×10 6个/cm 2的密度种植在多聚赖氨酸包被过夜的玻片或培养板上，置37℃、5％CO2、95％湿度培养箱内培养；
10.培养8-12h后，全部更换为NeurobasalA+B27+谷氨酰胺无血清培养基维持培养，每隔3天半量换液；
11. 7-14天后进行神经元鉴定。
神经元MAP-2/DAPI鉴定
1.取种植在载玻片上的培养7-14天的神经元，用预热PBS洗涤5min；
2.加4％多聚甲醛室温固定30min；
3.PBS洗涤5min×3次；
4.加0.2％TritonX-100PBS室温作用通透胞膜20min；
5.PBS洗涤5min×3次；
6.封闭：滴加封闭液(5％羊血清)，室温下封闭30-60min；
7.吸掉液体后，滴加1:100一抗(兔抗MAP-2多克隆抗体，Abcam)，4℃过夜，阴性对照用PBS替代；
8.弃去液体，PBS洗涤5min×3次；
9.吸干水分后，滴加1：200二抗(Alexa Fluor488驴抗兔荧光二抗，Invitrogen)， 室温避光，孵育60min；
10.弃去液体，PBS洗涤5min×3次；
11.弃去液体，DAP(sigma-Aldrich)0I染色，吸干水分，室温10min；
12.弃去液体，PBS洗5min x 3次；
13.滴加抗荧光淬灭封片液(50％甘油封片)
14.荧光显微镜下观察、拍照。
结果见图3，绿色为MAP-2阳性神经元，蓝色为细胞核，结果表明胎鼠皮层神经元原代培养纯度可达90％以上，培养成功。
糖氧剥夺(oxygen glucose deprivation/OGD)对神经元损伤及本发明的EPO来源肽保护
采用原代培养的胎鼠皮层神经元，于无糖DMED培养液，置于缺氧培养箱损伤神经元3h后，更换回无血清培养液，分别加入EPO、新型EPO来源肽组(T3)以及培养液，于5％CO2常氧培养箱培养4h，测定各组的LDH释放率及MTT细胞存活率。
依据上述结果，给予原代皮层神经元NMDA 250umol/L，60分钟后，结果见图5。
具体操作过程如下：
1.取培养7-14d左右，生长良好的神经元，吸除神经元的旧培养液，PBS缓冲液冲洗3遍，再加入无糖DMEM；
2.将培养板置于37℃缺氧培养箱内，设置箱内氧浓度为1％。
3.缺氧3小时后将培养板取出，吸掉无糖培养液，更换回原无血清维持培养基，分别加入EPO(5ug/ml、100ug/ml)、本发明的EPO来源肽(5ug/ml、50ug/ml、100ug/ml)、培养液，放置于37℃、5％CO2和95％空气的培养箱中继续培养4小时。
4. 4小时后，分别测定各组细胞的细胞存活率(MTT试剂盒，碧云天生物技术有限公司)、LDH乳酸脱氢酶释放率(LDH试剂盒，Promega公司试剂盒)。
结果见图4，加入EPO、本发明的EPO来源肽组(T3)组的神经元乳酸脱氢酶释放率较对照组减少，细胞存活率较对照组增高，说明本发明的EPO来源肽可以减轻急性神经损伤，减少神经元凋亡。
N甲基D天冬氨酸(NMDA)对神经元损伤及本发明的EPO来源肽保护
1.取培养7-14d左右，生长良好的神经元，分别加入EPO(5ug/ml)、本发明的EPO来源肽(100ug/ml)；
2.加入NMDA，最终浓度为250umol/L，37度培养箱培养60min；
3.弃去培养液，分别测定各组细胞的细胞存活率(MTT试剂盒，碧云天生物技术有限公司)、LDH乳酸脱氢酶释放率(LDH试剂盒，Promega公司试剂盒)。
结果见图5，加入EPO、本发明的EPO来源肽组(T3)组的神经元乳酸脱氢酶释放率较对照组减少，细胞存活率较对照组增高，说明本发明的EPO来源肽可以减轻急性神经损伤，减少神经元凋亡。
实施例4 本发明的EPO来源肽透过血脑屏障试验
腹腔注射，给予生物素标记的新型EPO来源肽，于给药后1小时于全麻下抽取小鼠脑脊液5-15ul，生物素检测试剂盒检测生物素标记的新型EPO来源肽是否透过血脑屏障进入脑脊液中，结果如图6。
具体操作过程如下：
1. 4％的水合氯醛麻醉小鼠；
2.用湿纱布擦试颈背部，剪去背毛暴露皮肤。在枕骨下方皮肤做一矢状位切口，将皮分离两侧，扩大视野；
3.小鼠俯卧，以身体与头部135°的角度放置并固定头部在立体定向仪上，在体视显微镜下，沿中线钝性分离皮下组织及肌肉，在此角度下，枕骨下方切口内可以较清楚看到硬脑膜和脊髓(主要特征是白色清晰的外观，脊髓血管的循环搏动以及邻近的脑脊液区域)；
4.拉针仪设置热量值为300，压力值为330，将毛细玻璃管拉成尖头，剪刀修剪尖端使其钝头直径为0.5mm；
5.毛细玻璃管连接负压装置，预先保持收集系统负压状态；
6.用棉棒擦拭血液，保持穿刺点周围清晰视野；
7.避开血管分布区，正对小脑延髓池，在体视显微镜下操作，用尖头毛细玻璃管缓慢穿刺硬脑膜，突破后可见脑脊液流进玻璃管，等待管内脑脊液缓慢上升至5-20ul，可适当再增加负压以收集更多脑脊液。
8.待管内脑脊液不再上升或足量后，缓慢抽去玻璃管，转移脑脊液至1.5ml ep管内，离心后-80℃冻存或立即检测。
分别抽取对照组、生物素化本发明的EPO来源肽组、癫痫及生物素化本发明的EPO来源肽组小鼠的脑脊液，生物素检测试剂盒检测生物素浓度。
表3 不同处理组小鼠脑脊液生物素浓度
Figure PCTCN2018078253-appb-000003
结果见图6，给予本发明的EPO来源肽组小鼠脑脊液中可以测得生物素，而对照组几乎无生物素存在，其差异具有统计学意义；癫痫后给药组小鼠脑脊液中生物素浓度高于单纯给药组，说明癫痫发作后血脑屏障破坏，通透性增高。综上说明，本发明的EPO来源肽可以透过血脑屏障进入脑脊液发挥作用。
实施例5 本发明的EPO来源肽对短暂性大脑中动脉阻塞小鼠模型的神经保护作用试验
ICR小鼠(雄性，25-30g，18只，购自上海斯莱克实验动物有限公司)，分为对照组(ACN+Millq)，0.2ml/只)，EPO组(50ug/kg)，新型
EPO来源肽组(T3，500ug/kg)，6只/组。分别行小鼠线栓法大脑中动脉阻塞模型手术，用盐酸氯胺酮麻醉剂腹腔注射麻醉小鼠，固定于手术台。缺血60min后拔出线栓再灌注，并分别给予溶剂(ACN+Millq)、EPO、T3。术后第3天行神经行为学评分，随后处死小鼠灌注取脑切片，行焦油紫染色。结果如图7。
1.消毒颈部，于颈正中切口，在体视显微镜下用显微镊暴露左侧颈总动脉，分离颈内动脉、颈外动脉和翼腭动脉，结扎颈总动脉、颈内动脉，用两根线在颈外动脉上打两个结，一根位于远端，为死结，一根在近端，为活扣；
2.用维纳斯剪将颈外动脉剪开一个小口，约为血管的一半大小；
3.剪断颈外动脉，将线栓小心地从颈外动脉反向插入至颈外动脉和颈内动脉的分叉处，然后松开颈内动脉上的结，再把线栓插入至颈内动脉，直至遇到轻微阻力，即位于大脑中动脉的起始处，放回至笼中；
4.脑缺血60分钟后，再次麻醉固定，在体视显微镜下重新暴露颈总动脉、颈内动脉和线栓部位；
5.用显微镊稍微松开结扎于颈外动脉近端用于固定线栓的线，慢慢抽出线栓，并结扎颈外动脉残端，松开结扎于颈总动脉上的线，实现再灌注；
6.缝合颈部切口，消毒，放回笼中观察。
表4 不同处理组小鼠神经行为学评分
Ctrl组 ctrl+T3组 致痫组+T3组
神经行为学评分 8.0±1.0 6.3±0.7 5.7±0.3
表5 不同处理组小鼠短暂性大脑中动脉阻塞脑梗死体积
Ctrl组 ctrl+T3组 致痫组+T3组
脑梗死体积(mm3) 49.65±10.29 40.54±5.78 37.72±9.20
结果见图7，EPO组、本发明的EPO来源肽组神经行为学评分及脑梗死体积较对照组减低，说明EPO及本发明的EPO来源肽在体内改善脑缺血，具有神经保护作用。
实施例6 本发明的EPO来源肽对癫痫动物模型的神经保护作用试验
C57/BL6小鼠(雄性，20-25g，24只，购自上海杰思捷实验动物有限公司)，分为生理盐水组(Saline，0.2ml/只)，溶剂组(ACN+Milliq，0.2ml/只)，EPO组(50ug/kg)，新型EPO来源肽组(T3，500ug/kg)，6只/组。匹罗卡品致痫前1天、4小时、2小时给药，记录癫痫发作潜伏期，发作严重程度。结果如图8。
1.在注射匹罗卡品前1天、4小时、2小时给药，腹腔注射；
2.提前30分钟腹腔注射东莨胆碱(1mg/kg)，以降低匹罗卡品所致的周围胆碱能反应；
3.腹腔注射匹罗卡品(350mg/kg)，记录给药时间、发作时间、发作严重程度。
4.四级以上发作后，出现自发活动，癫痫持续状态，造模成功，造模成功后30min安定(10mg/kg)终止发作。
Racine癫痫持续状态行为学分级标准
Ⅰ级：凝视，口面部活动；
Ⅱ级：点头，单次抽搐；
Ⅲ级：单侧或双侧前肢阵挛抽搐；
Ⅳ级：站立；
Ⅴ级：失去姿势，反复跳起，倒地，强直阵挛发作
表6 不同药物干预匹鲁卡品致痫小鼠发作潜伏期
epo T3 Saline ACN+miliq
seizure latency(min) 41.16±3.52 40.58±3.67 32.50±1.80 27.20±2.48
结果见图8，EPO组、本发明的EPO来源肽组癫痫发作潜伏期较生理盐水及溶剂组延长，差异有统计学意义，说明EPO及本发明的EPO来源肽在体内具有神经保护作用。
实施例7
将本发明EPO来源肽制备成药物时，可以将有效量的多肽，以及至少一种药学上可接受的载体，稀释剂或赋形剂组合进行配置。在制备这些组合物时，通常将活性成分与赋形剂混合，或用赋形剂稀释，或包在可以胶囊或药囊形式存在的载体中。当赋形剂起稀释剂作用时，它可以是固体、半固体或液体材料作为赋形剂、载体或活性成分的介质。因此，药物可以是片剂、丸剂、粉剂、溶液剂、糖浆剂、灭菌注射溶液等。合适的赋形剂的例子包括:乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、等。制剂还可包括:湿润剂、乳化剂、防腐剂(如羟基苯甲酸甲酯和丙酯)、甜味剂等。
综上所述，本发明的新型EPO来源肽能够在急性损伤时保护神经元细胞，减少细胞凋亡；小鼠长期给药，没有显著促红细胞生成的副作用；其能够透过血脑屏障，在体内缺血损伤后给药可以减小脑梗死体积，改善脑缺血，预给药可以延长癫痫发作潜伏期，证实可制备治疗神经损伤药物。通过对本发明的EPO来源肽进行了体内造血实验，证实其没有促红细胞的作用。进而发明人通过体外抗皮层神经元细胞凋亡的实验证实了新型EPO来源肽有保护神经细胞的作用。再通过动物给药后测得脑脊液中存在生物素标记的多肽，证明该新型EPO来源肽能透过血脑屏障发挥作用。

Claims (21)

  1. 一种促红细胞生成素来源肽，其包含EPO与EPOR结合位点及EPO蛋白螺旋结构的氨基酸序列，其没有促红细胞生成的作用，但具备神经保护作用。
  2. 根据权利要求1所述的促红细胞生成素来源肽，其中EPO第86位谷氨酰胺、88位色氨酸、89位谷氨酸、90位脯氨酸、97位赖基酸中的一位或者多位缺失。
  3. 根据权利要求1或2所述的促红细胞生成素来源肽，其中EPO第103位的精氨酸被丙氨酸所取代。
  4. 一种没有促红细胞生成作用，但具备神经保护作用的促红细胞生成素来源肽，其包括以下(a)或(b)：
    (a)氨基酸序列如SEQ ID NO.1所示的多肽；
    (b)在(a)中氨基酸序列基础上经过取代、缺失或添加一个或几个氨基酸衍生的多肽。
  5. 一种促红细胞生成素来源肽，其氨基酸序列如SEQ ID NO.1所示。
  6. 根据权利要求5所述的一种促红细胞生成素来源肽，其特征在于：其结构式如下：
    Figure PCTCN2018078253-appb-100001
  7. 一种药物，其包含如权利要求1-6中任一项所述的促红细胞生成素来源肽。
  8. 一种组合物，其包含如权利要求1-6中任一项所述的促红细胞生成素来源肽。
  9. 一种药物组合物，其包含如权利要求1-6中任一项所述的促红细胞生成素来源肽，以及赋形剂或载体。
  10. 一种核酸分子，其包含编码权利要求1-6中任一项所述的促红细胞生成素来源肽的核苷酸序列。
  11. 一种表达载体，其包含权利要求10所述的核酸分子。
  12. 一种宿主细胞，其包含权利要求10所述的核酸分子或权利要求11所述的表达载体。
  13. 一种病毒，其包含权利要求10所述的核酸分子或权利要求11所述的表达载体。
  14. 一种试剂盒，其包含以下各项中的任意一项：
    a)1-6中任一项所述的促红细胞生成素来源肽；
    b)权利要求8所述的组合物；
    c)权利要求10所述的核酸分子；
    d)权利要求11所述的表达载体；
    e)权利要求12所述的宿主细胞；
    f)权利要求13所述的病毒。
  15. 如权利要求5或6所述的促红细胞生成素来源肽的制备方法，其包括如下步骤：
    1)采用固相合成法，将连接有亮氨酸的起始树脂用N,N-二甲基甲酰胺浸泡，然后用脱帽液浸泡，去除芴甲氧羰基，用二甲基甲酰胺清洗；
    2)加入下一个氨基酸、缩合剂和碱，反应后用二甲基甲酰胺清洗，
    3)用脱帽液去除芴甲氧羰基，再用二甲基甲酰胺清洗；
    4)重复步骤2)和步骤3)，依次连接氨基酸。
  16. 根据权利要求15所述的促红细胞生成素来源肽的制备方法，其中步骤2)中所述的缩合剂为O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯。
  17. 根据权利要求15所述的促红细胞生成素来源肽的制备方法，其中步骤2)中所述的碱为吗啡啉。
  18. 根据权利要求15所述的促红细胞生成素来源肽的制备方法，其中步骤3)中 所述的脱帽液为六氢吡啶和N,N-二甲基甲酰胺组成的混合液。
  19. 如权利要求1-6中任一项所述的促红细胞生成素来源肽在治疗神经细胞损伤中的应用。
  20. 如权利要求1-6中任一项所述的促红细胞生成素来源肽在治疗缺氧性脑损伤中的应用。
  21. 如权利要求1-6中任一项所述的促红细胞生成素来源肽在治疗癫痫中的应用。
PCT/CN2018/078253 2017-11-01 2018-03-07 一种促红细胞生成素来源肽及其制备方法和用途 WO2019085366A1 (zh)

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