WO2023104052A1 - Blood-brain barrier crossing polypeptide as well as derivative and application thereof - Google Patents

Blood-brain barrier crossing polypeptide as well as derivative and application thereof Download PDF

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WO2023104052A1
WO2023104052A1 PCT/CN2022/137012 CN2022137012W WO2023104052A1 WO 2023104052 A1 WO2023104052 A1 WO 2023104052A1 CN 2022137012 W CN2022137012 W CN 2022137012W WO 2023104052 A1 WO2023104052 A1 WO 2023104052A1
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polypeptide
seq
modified
blood
brain barrier
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PCT/CN2022/137012
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French (fr)
Chinese (zh)
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赵华山
朱雯
张键
任培根
雷晓华
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深圳先进技术研究院
中国科学院深圳理工大学(筹)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides

Definitions

  • the invention belongs to the field of medical biology, and relates to a blood-brain barrier crossing polypeptide, derivatives and applications thereof.
  • the blood-brain barrier is an important structure to ensure the development and normal function of the nervous system. It not only protects the brain from external interference, but also restricts the delivery and transport of a large number of valuable drugs. It is known that except for some small molecules that can cross the blood-brain barrier, the transport of most macromolecules needs to be mediated by receptors. With the help of endogenous receptors, macromolecular drugs can be delivered to the brain Internally, drug delivery is achieved. However, so far, the research on mediating ligands is still very limited, and there are very few mediating ligands available. The discovery of such ligands is also something that can be encountered but not sought after, which is also what has been sought and explored in terms of drug delivery across the blood-brain barrier.
  • the purpose of the present invention is to provide a blood-brain barrier-crossing polypeptide and its derivatives and applications.
  • One aspect of the present invention provides a use of a polypeptide in the preparation of drugs or detection reagents that cross the blood-brain barrier;
  • the polypeptide is selected from Metabolitin or Osteocalcin.
  • metabolin is selected from the polypeptides shown in SEQ ID NO.1 or SEQ ID NO.3,
  • osteocalcin is selected from the polypeptides shown in SEQ ID NO.2 or SEQ ID NO.4,
  • Another aspect of the present invention provides a derivative of a polypeptide, and the derivative is a product obtained by modifying the terminal or side chain of the polypeptide or a product obtained by isotope labeling modification;
  • the modified product is selected from a product modified by a fluorescent group, or a product modified by phosphorylation, or a product modified by disulfide bond-based cyclization, or a product modified by biotin labeling, or a product modified by Products modified by photosensitizers, products modified by azide, products modified by PEG, products modified by methylation, products modified by fluorescent quenching groups, or products obtained by protein coupling Modified products, or products modified by small molecule compounds;
  • the peptide hormone is selected from Metabolitin or Osteocalcin.
  • metabolin is selected from the polypeptides shown in SEQ ID NO.1 or SEQ ID NO.3,
  • osteocalcin is selected from the polypeptides shown in SEQ ID NO.2 or SEQ ID NO.4,
  • the modification of the terminal is selected from the group consisting of N-terminal acetylation modification and C-terminal amination modification of the polypeptide.
  • the modification of the side chain is selected from the modification on the R group of the amino acid side chain in the polypeptide.
  • the fluorescent dye used in the modification of the fluorescent group is selected from FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, ICG, and the modification can be used for fluorescence detection.
  • the phosphorylation modification is selected from one or a combination of p-Ser, p-Thr, p-Tyr.
  • the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin and biotin-dUTP.
  • the modification of the photosensitizer can be used to prepare photosensitizers.
  • azide modification can be used in secondary connection reactions.
  • the PEG modification can be used to prepare drug carriers.
  • the isotopes used in the isotopic labeling are selected from one or more combinations of 13 C, 14 C, 15 N, 2 H, 3 H, 18 O, 34 S, and 36 S.
  • Another aspect of the present invention provides the use of the derivatives of the above polypeptides in the preparation of drugs or reagents capable of crossing the blood-brain barrier.
  • Another aspect of the present invention provides a carrier across the blood-brain barrier, wherein the surface of the carrier is modified with the above-mentioned polypeptide of the present invention.
  • the carrier is selected from liposomes and nanoparticles.
  • the carrier is loaded with active ingredients.
  • the blood-brain barrier-mediated polypeptide can be synthesized independently under general chemical laboratory conditions, or it can be synthesized industrially by a commercial reagent company.
  • the polypeptide is synthesized by a solid-phase method, and different amino acids on the resin are oriented to synthesize amino acid chains through condensation reactions. .
  • Derivatives of polypeptides are labeled with modifying groups after the amino acids are connected.
  • Another aspect of the present invention provides a drug that crosses the blood-brain barrier, and the drug is a conjugate of the above-mentioned polypeptide or its derivative and an active ingredient.
  • the polypeptide and its derivatives can be combined with diagnostic and/or therapeutic carriers through organic chemical methods to exert their application value on the basis of mediating the function of crossing the blood-brain barrier.
  • polypeptide probe that crosses the blood-brain barrier.
  • the polypeptide probe is the above-mentioned polypeptide or a derivative thereof, which is prepared by coupling with a fluorescent dye through an organic chemical reaction.
  • the fluorescent dye is a dye with a fluorescent group in the near-infrared region, preferably, the dye with a fluorescent group in the near-infrared region is selected from Cy5, Cy7, and ICG.
  • the mediator polypeptide provided by the present invention can be used to mediate trans-blood-brain barrier transport.
  • the mediator polypeptide provided by the present invention can be used as a modified polypeptide to enhance the targeting and therapeutic properties of drugs or drug carriers.
  • the mediating polypeptide provided by the present invention is easy to synthesize and modify, and the cost is low, so it has broad application prospects.
  • the multiple derivatives and candidate polypeptides provided by the present invention provide a wide range of preparation strategies for diagnostic and therapeutic compounds or drugs for their practical applications.
  • the present invention is suitable for drug-mediated applications in the human brain, and is also suitable as a research tool for brain disease modeling experimental animals.
  • Fig. 1 is an effect diagram of crossing the blood-brain screen in Example 3 of the present invention.
  • the figure shows the distribution and results of the fluorescent signal in the brains of the three groups of mice in Example 3.
  • Example 1 Preparation of polypeptide sequences across the blood-brain barrier
  • polypeptide sequence is as follows:
  • polypeptides are synthesized by conventional solid-phase synthesis or liquid-phase synthesis methods.
  • the solid-phase synthetic peptide method is used to react from the amino acid at the C-terminal to the N-terminal.
  • the amino acid linkage is completed one by one, and then precipitated and centrifuged with excess ether, and the crude peptide is purified by HPLC. Afterwards, mass spectrometry was performed, and freeze-dried in liquid nitrogen for further use.
  • SEQ ID NO.1 is a mouse metabolite
  • SEQ ID NO. ID NO.2 is mouse osteocalcin
  • SEQ ID NO.3 is human metabolin
  • SEQ ID NO.3 is human metabolite ID NO.4 is human osteocalcin.
  • This embodiment provides a polypeptide probe, using the polypeptide prepared in Example 1, and combining it with ICG through an organic chemical reaction.
  • the probe is prepared by linking the polypeptide with ICG by the click chemistry method, and the linker is the universal DBCO.
  • ICG with activating functional groups include amino NH 2 , carboxyl COOH, activated lipid NHS, maleimide MAL, mercapto SH, azide N 3 , alkyne ALK, and then prepared with Example 1 Peptide coupling to obtain cross-blood-brain barrier peptide ICG probes.
  • Example 3 Detection of the ability of mice to cross the blood-brain barrier
  • mice were used to inject the cross-blood-brain barrier polypeptide ICG probe (containing SEQ ID NO.1), ICG solution (2mM) and PBS prepared in Example 2 into the tail vein, each mouse was injected with 100 microliters, and after 24 hours , detected under a small animal imager.
  • ICG probe containing SEQ ID NO.1
  • ICG solution 2mM
  • PBS prepared in Example 2
  • the mediating polypeptide provided by this application can be used for the purpose of transporting across the blood-brain barrier.

Abstract

The present invention belongs to the field of bio-medicines, and a blood-brain barrier crossing polypeptide as well as a derivative and application thereof are disclosed. Specifically, a use of the polypeptide in preparation of blood-brain barrier crossing drugs or detection reagents is disclosed. The polypeptide is selected from a metabolitin or osteocalcin. The derivative of the polypeptide is further disclosed. The derivative is a product obtained after the polypeptide is modified by a tail end or a side chain or a product obtained after the polypeptide is modified by means of isotope labeling. This type of polypeptide molecules can enter brain tissue across a blood-brain barrier, thereby providing a specific blood-brain barrier crossing tool for diagnosis and treatment of brain diseases. Therefore, the polypeptide can be used for preparing detection reagents and blood-brain barrier crossing drugs.

Description

一种跨血脑屏障的多肽及其衍生物和应用A polypeptide crossing the blood-brain barrier and its derivatives and applications 技术领域technical field
本发明属于医学生物学领域，涉及一种跨血脑屏障多肽及其衍生物和应用。The invention belongs to the field of medical biology, and relates to a blood-brain barrier crossing polypeptide, derivatives and applications thereof.
背景技术Background technique
血脑屏障是保障神经系统发育及正常发挥功能的重要结构，它在保护大脑不被外界侵扰的同时，也限制了大量有价值的药物的递送转运。已知除了一些小分子的物质可以穿过血脑屏障外，绝大多数大分子物质的转运是需要受体介导的，借助内源性受体的介导，可以将大分子药物递送至大脑内部，实现药物投递。然而，截至目前，在介导配体的研究方面，还非常有限，可用的介导配体，还很少。这类配体发现也是可遇而不可求的，这也是跨血脑屏障给药方面的一直在寻觅和探索的。The blood-brain barrier is an important structure to ensure the development and normal function of the nervous system. It not only protects the brain from external interference, but also restricts the delivery and transport of a large number of valuable drugs. It is known that except for some small molecules that can cross the blood-brain barrier, the transport of most macromolecules needs to be mediated by receptors. With the help of endogenous receptors, macromolecular drugs can be delivered to the brain Internally, drug delivery is achieved. However, so far, the research on mediating ligands is still very limited, and there are very few mediating ligands available. The discovery of such ligands is also something that can be encountered but not sought after, which is also what has been sought and explored in terms of drug delivery across the blood-brain barrier.
技术问题technical problem
截至目前，在介导配体的研究方面，还非常有限，可用的介导配体，还很少。这类配体发现也是可遇而不可求的，这也是跨血脑屏障给药方面的一直在寻觅和探索的。So far, the research on mediating ligands is still very limited, and there are very few available mediating ligands. The discovery of such ligands is also something that can be encountered but not sought after, which is also what has been sought and explored in terms of drug delivery across the blood-brain barrier.
技术解决方案technical solution
就上述背景技术中所提出的瓶颈问题，本发明的目的在于提供一种跨血脑屏障多肽及其衍生物和应用。With regard to the bottleneck problem raised in the above-mentioned background technology, the purpose of the present invention is to provide a blood-brain barrier-crossing polypeptide and its derivatives and applications.
本发明一方面提供了一种多肽在制备跨血脑屏障的药物或检测试剂中的用途；One aspect of the present invention provides a use of a polypeptide in the preparation of drugs or detection reagents that cross the blood-brain barrier;
所述多肽选自代谢素（Metabolitin）或骨钙素（Osteocalcin）。The polypeptide is selected from Metabolitin or Osteocalcin.
进一步地，代谢素选自如SEQ ID NO.1或SEQ ID NO.3所示的多肽，Further, the metabolin is selected from the polypeptides shown in SEQ ID NO.1 or SEQ ID NO.3,
YLGASVPSPDPLEPT                         SEQ ID No.1；YLGASVPSPDPLEPT SEQ ID No.1;
YLYQWLGAPVPYPDPLEPR              SEQ ID No.3。YLYQWLGAPVPYPDPLEPR SEQ ID No. 3.
进一步地，骨钙素选自如SEQ ID NO.2或SEQ ID NO.4所示的多肽，Further, osteocalcin is selected from the polypeptides shown in SEQ ID NO.2 or SEQ ID NO.4,
YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI                  SEQ ID No.2；YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI SEQ ID No.2;
YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No.4。YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No.4.
本发明另一方面提供了一种多肽的衍生物，所述的衍生物为多肽经过末端或侧链的修饰后所得产物或经过同位素标记修饰得到的产物；Another aspect of the present invention provides a derivative of a polypeptide, and the derivative is a product obtained by modifying the terminal or side chain of the polypeptide or a product obtained by isotope labeling modification;
所述修饰所得产物选自经过荧光基团修饰得到的产物，或经过磷酸化修饰得到的产物，或基于二硫键的环化修饰得到的产物，或经过生物素标记修饰得到的产物，或经过光敏剂修饰得到的产物，或叠氮修饰得到的产物，或经过PEG修饰得到的产物，或经过甲基化修饰得到的产物，或经过荧光淬灭基团修饰得到的产物，或经过蛋白偶联修饰得到的产物，或经过小分子化合物修饰得到的产物；The modified product is selected from a product modified by a fluorescent group, or a product modified by phosphorylation, or a product modified by disulfide bond-based cyclization, or a product modified by biotin labeling, or a product modified by Products modified by photosensitizers, products modified by azide, products modified by PEG, products modified by methylation, products modified by fluorescent quenching groups, or products obtained by protein coupling Modified products, or products modified by small molecule compounds;
所述肽类激素选自代谢素（Metabolitin）或骨钙素（Osteocalcin）。The peptide hormone is selected from Metabolitin or Osteocalcin.
进一步地，代谢素选自如SEQ ID NO.1或SEQ ID NO.3所示的多肽，Further, the metabolin is selected from the polypeptides shown in SEQ ID NO.1 or SEQ ID NO.3,
YLGASVPSPDPLEPT                         SEQ ID No.1；YLGASVPSPDPLEPT SEQ ID No.1;
YLYQWLGAPVPYPDPLEPR              SEQ ID No.3。YLYQWLGAPVPYPDPLEPR SEQ ID No. 3.
进一步地，骨钙素选自如SEQ ID NO.2或SEQ ID NO.4所示的多肽，Further, osteocalcin is selected from the polypeptides shown in SEQ ID NO.2 or SEQ ID NO.4,
YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI                  SEQ ID No.2；YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI SEQ ID No.2;
YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No.4。YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No.4.
进一步地，所述末端的修饰选自多肽N端乙酰化修饰和C端的胺化修饰。Further, the modification of the terminal is selected from the group consisting of N-terminal acetylation modification and C-terminal amination modification of the polypeptide.
进一步地，所述侧链的修饰选自多肽中氨基酸侧链R基上的修饰。Further, the modification of the side chain is selected from the modification on the R group of the amino acid side chain in the polypeptide.
进一步地，所述荧光基团的修饰中所用的荧光染料选自FITC、Rhodamine、Cy3、Cy5、Cy5.5、Cy7、ICG，该修饰可用于荧光检测。Further, the fluorescent dye used in the modification of the fluorescent group is selected from FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, ICG, and the modification can be used for fluorescence detection.
进一步地，所述磷酸化修饰选自p-Ser、p-Thr、p-Tyr中的一种或多种的组合。Further, the phosphorylation modification is selected from one or a combination of p-Ser, p-Thr, p-Tyr.
进一步地，所述生物素的标记，所述生物素选自D-生物素、生物素酰肼、光敏生物素和生物素-dUTP。Further, for the labeling of biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin and biotin-dUTP.
进一步地，所述光敏剂修饰，可用于制备光敏感制剂。Furthermore, the modification of the photosensitizer can be used to prepare photosensitizers.
进一步地，所述叠氮修饰，可用于次级的连接反应。Furthermore, the azide modification can be used in secondary connection reactions.
进一步地，所述PEG修饰，可用于制备药物载体。Furthermore, the PEG modification can be used to prepare drug carriers.
进一步地，所述同位素标记中所用的同位素选自 13C、 14C、 15N、 2H、 3H、 18O、 34S、 36S中的一种或多种的组合。 Further, the isotopes used in the isotopic labeling are selected from one or more combinations of 13 C, 14 C, 15 N, 2 H, 3 H, 18 O, 34 S, and 36 S.
本发明另一个方面提供了上述多肽的衍生物在制备能够跨血脑屏障的药物或试剂中的用途。Another aspect of the present invention provides the use of the derivatives of the above polypeptides in the preparation of drugs or reagents capable of crossing the blood-brain barrier.
本发明另一个方面提供了一种跨血脑屏障的载体，所述载体表面修饰有本发明上述多肽。Another aspect of the present invention provides a carrier across the blood-brain barrier, wherein the surface of the carrier is modified with the above-mentioned polypeptide of the present invention.
进一步地，所述载体选自脂质体、纳米粒。Further, the carrier is selected from liposomes and nanoparticles.
进一步地，所述载体中负载活性成分。Further, the carrier is loaded with active ingredients.
所述的跨血脑屏障介导多肽可通过一般化学实验室条件自主合成，也可以由商业化试剂公司工业合成，采用固相法合成多肽，在树脂上不同氨基酸，经过缩合反应定向合成氨基酸链。多肽的衍生物，是在氨基酸完成连接后，再标记上修饰基团。The blood-brain barrier-mediated polypeptide can be synthesized independently under general chemical laboratory conditions, or it can be synthesized industrially by a commercial reagent company. The polypeptide is synthesized by a solid-phase method, and different amino acids on the resin are oriented to synthesize amino acid chains through condensation reactions. . Derivatives of polypeptides are labeled with modifying groups after the amino acids are connected.
本发明再一方面提供了一种跨血脑屏障的药物，所述药物为上述多肽或其衍生物与活性成分的偶联物。所述的多肽及其衍生物可通过有机化学方法结合于诊断和/或治疗载体上发挥介导跨血脑屏障的功能基础上的应用价值。Another aspect of the present invention provides a drug that crosses the blood-brain barrier, and the drug is a conjugate of the above-mentioned polypeptide or its derivative and an active ingredient. The polypeptide and its derivatives can be combined with diagnostic and/or therapeutic carriers through organic chemical methods to exert their application value on the basis of mediating the function of crossing the blood-brain barrier.
本发明再一方面提供了一种跨血脑屏障的多肽探针，所述多肽探针为上述多肽或其衍生物，与荧光染料通过有机化学反应偶联制得。Another aspect of the present invention provides a polypeptide probe that crosses the blood-brain barrier. The polypeptide probe is the above-mentioned polypeptide or a derivative thereof, which is prepared by coupling with a fluorescent dye through an organic chemical reaction.
进一步地，所述荧光染料选择具有近红外区荧光基团的染料，优选地，所述具有近红外区荧光基团的染料选自Cy5、Cy7、ICG。Further, the fluorescent dye is a dye with a fluorescent group in the near-infrared region, preferably, the dye with a fluorescent group in the near-infrared region is selected from Cy5, Cy7, and ICG.
有益效果Beneficial effect
本发明的有益效果：Beneficial effects of the present invention:
（1）本发明提供的介导多肽，可用于介导跨血脑屏障转运。(1) The mediator polypeptide provided by the present invention can be used to mediate trans-blood-brain barrier transport.
（2）本发明提供的介导多肽，可作为修饰多肽以增强药物或药物载体的靶向性和治疗性。(2) The mediator polypeptide provided by the present invention can be used as a modified polypeptide to enhance the targeting and therapeutic properties of drugs or drug carriers.
（3）本发明提供的介导多肽，其易于合成修饰，成本低廉，因此应用前景广泛。(3) The mediating polypeptide provided by the present invention is easy to synthesize and modify, and the cost is low, so it has broad application prospects.
（4）本发明提供的多元的衍生物备选多肽，为其实际应用提供了广泛的诊疗化合物或药物的制备策略。(4) The multiple derivatives and candidate polypeptides provided by the present invention provide a wide range of preparation strategies for diagnostic and therapeutic compounds or drugs for their practical applications.
（5）本发明提供的多肽分子由于序列的特异性，本发明适用于人类脑部药物介导的应用，也适用于作为脑疾病造模实验动物中的研究工具。(5) Due to the sequence specificity of the polypeptide molecules provided by the present invention, the present invention is suitable for drug-mediated applications in the human brain, and is also suitable as a research tool for brain disease modeling experimental animals.
附图说明Description of drawings
图1为本发明实施例3中跨血脑屏效果图。图中显示实施例3中3组小鼠脑部荧光信号分布和结果。Fig. 1 is an effect diagram of crossing the blood-brain screen in Example 3 of the present invention. The figure shows the distribution and results of the fluorescent signal in the brains of the three groups of mice in Example 3.
本发明的实施方式Embodiments of the present invention
为了更好地理解本发明的内容，下面结合具体实施案例，对本发明内容作进一步说明，但本发明的保护内容不局限以下实施例。In order to better understand the content of the present invention, the content of the present invention will be further described below in conjunction with specific implementation examples, but the protection content of the present invention is not limited to the following examples.
实施例1：跨血脑屏障多肽序列的制备Example 1: Preparation of polypeptide sequences across the blood-brain barrier
采用人工合成的方法合成跨血脑屏障多肽序列，Synthesize the polypeptide sequence across the blood-brain barrier by artificial synthesis method,
所述多肽序列如下所述：The polypeptide sequence is as follows:
YLGASVPSPDPLEPT                         SEQ ID No.1；YLGASVPSPDPLEPT SEQ ID No.1;
YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI                  SEQ ID No.2；YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI SEQ ID No.2;
YLYQWLGAPVPYPDPLEPR              SEQ ID No.3；YLYQWLGAPVPYPDPLEPR SEQ ID No.3;
YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No.4。YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No.4.
采用常规的固相合成或液相合成的方法合成上述多肽。其中利用固相合成多肽法为从C端的氨基酸向N端反应，经过树脂活化、氨基酸链接、洗脱保护、检测等步骤，逐个完成氨基酸链接，然后用过量乙醚沉淀离心，对粗肽经过HPLC纯化后，质谱分析，在液氮中速冷冻干备用。The above-mentioned polypeptides are synthesized by conventional solid-phase synthesis or liquid-phase synthesis methods. Among them, the solid-phase synthetic peptide method is used to react from the amino acid at the C-terminal to the N-terminal. After the steps of resin activation, amino acid linkage, elution protection, and detection, the amino acid linkage is completed one by one, and then precipitated and centrifuged with excess ether, and the crude peptide is purified by HPLC. Afterwards, mass spectrometry was performed, and freeze-dried in liquid nitrogen for further use.
其中SEQ ID NO.1为鼠源代谢素，SEQ ID NO.2为鼠源骨钙素，SEQ ID NO.3为人源代谢素，SEQ ID NO.4为人源骨钙素。Wherein SEQ ID NO.1 is a mouse metabolite, and SEQ ID NO. ID NO.2 is mouse osteocalcin, SEQ ID NO.3 is human metabolin, SEQ ID NO.3 is human metabolite ID NO.4 is human osteocalcin.
实施例2：跨血脑屏障多肽探针的制备Example 2: Preparation of polypeptide probes across the blood-brain barrier
本实施例提供一种多肽探针，采用实施例1制备的多肽，将其与ICG通过有机化学反应结合。This embodiment provides a polypeptide probe, using the polypeptide prepared in Example 1, and combining it with ICG through an organic chemical reaction.
具体方法为购买或合成，本实施例中，通过点击化学方法将多肽与ICG连接完成探针制备，其中的连接子为通用的DBCO。具有活化功能基团的ICG，活化功能基团包含氨基NH 2、羧基COOH、活化脂NHS、马来酰亚胺MAL、巯基SH、叠氮N 3、炔烃ALK，然后与实施例1制备的多肽偶联，获得跨血脑屏障多肽ICG探针。 The specific method is purchase or synthesis. In this example, the probe is prepared by linking the polypeptide with ICG by the click chemistry method, and the linker is the universal DBCO. ICG with activating functional groups, the activating functional groups include amino NH 2 , carboxyl COOH, activated lipid NHS, maleimide MAL, mercapto SH, azide N 3 , alkyne ALK, and then prepared with Example 1 Peptide coupling to obtain cross-blood-brain barrier peptide ICG probes.
实施例3：小鼠跨血脑屏障能力检测Example 3: Detection of the ability of mice to cross the blood-brain barrier
使用成年小鼠，尾静脉注射实施例2制备的跨血脑屏障多肽ICG探针（含SEQ ID NO.1）、ICG溶液（2mM）以及PBS，每只小鼠注射100微升，24小时后，在小动物成像仪下检测。Adult mice were used to inject the cross-blood-brain barrier polypeptide ICG probe (containing SEQ ID NO.1), ICG solution (2mM) and PBS prepared in Example 2 into the tail vein, each mouse was injected with 100 microliters, and after 24 hours , detected under a small animal imager.
实验结果见图1，通过对比3组结果可知，只有跨血脑屏障多肽探针组在脑部有特异信号，而其他两组均未显示在脑部有荧光信号。以上实验结果可知，本发明的跨血脑屏障多肽探针能够实现跨血脑屏障作用，由于本发明的多肽的介导转运的能力，进一步地，能够预期本发明的多肽可修饰与药物载体表面促进跨血脑屏障的转运效率，进而实现其他活性成分的转运。The experimental results are shown in Figure 1. By comparing the results of the three groups, it can be seen that only the cross-blood-brain barrier polypeptide probe group has specific signals in the brain, while the other two groups do not show fluorescent signals in the brain. From the above experimental results, it can be seen that the blood-brain barrier-crossing polypeptide probe of the present invention can realize the effect of crossing the blood-brain barrier. Promote the transport efficiency across the blood-brain barrier, and then realize the transport of other active ingredients.
综上，本申请提供的介导多肽可用于跨血脑屏障转运的目的。To sum up, the mediating polypeptide provided by this application can be used for the purpose of transporting across the blood-brain barrier.
以上所述仅为本发明的具体实施方式，不是全部的实施方式，本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。The above is only the specific implementation mode of the present invention, not all the implementation modes. Any equivalent transformation taken by those of ordinary skill in the art to the technical solution of the present invention by reading the description of the present invention is covered by the claims of the present invention. .

Claims (10)

  1. 一种多肽在制备跨血脑屏障的药物或检测试剂中的用途，其特征在于，所述多肽选自代谢素或骨钙素。 A use of a polypeptide in the preparation of drugs or detection reagents that cross the blood-brain barrier, characterized in that the polypeptide is selected from metabolin or osteocalcin.
  2. 根据权利要求1所述的用途，其特征在于，所述的代谢素选自如SEQ ID NO.1或SEQ ID NO.3所示的多肽， The use according to claim 1, characterized in that, the metabolin is selected from the polypeptides shown in SEQ ID NO.1 or SEQ ID NO.3,
    YLGASVPSPDPLEPT                         SEQ ID No.1；YLGASVPSPDPLEPT SEQ ID No.1;
    YLYQWLGAPVPYPDPLEPR              SEQ ID No.3；YLYQWLGAPVPYPDPLEPR SEQ ID No.3;
    所述的骨钙素选自如SEQ ID NO.2或SEQ ID NO.4所示的多肽，The osteocalcin is selected from the polypeptides shown in SEQ ID NO.2 or SEQ ID NO.4,
    YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI                  SEQ ID No.2；YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI SEQ ID No.2;
    YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No.4。YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No. 4.
  3. 一种多肽的衍生物，其特征在于，所述的衍生物为多肽经过末端或侧链的修饰后所得产物或经过同位素标记修饰得到的产物； A derivative of a polypeptide, characterized in that the derivative is a product obtained by modifying the terminal or side chain of the polypeptide or a product obtained by isotope labeling modification;
    所述修饰所得产物选自经过荧光基团修饰得到的产物，或经过磷酸化修饰得到的产物，或基于二硫键的环化修饰得到的产物，或经过生物素标记修饰得到的产物，或经过光敏剂修饰得到的产物，或叠氮修饰得到的产物，或经过PEG修饰得到的产物，或经过甲基化修饰得到的产物，或经过荧光淬灭基团修饰得到的产物，或经过蛋白偶联修饰得到的产物，或经过小分子化合物修饰得到的产物；The modified product is selected from a product modified by a fluorescent group, or a product modified by phosphorylation, or a product obtained by disulfide bond-based cyclization modification, or a product modified by biotin labeling, or a product modified by Products modified by photosensitizers, products modified by azide, products modified by PEG, products modified by methylation, products modified by fluorescent quenching groups, or products obtained by protein coupling Modified products, or products modified by small molecule compounds;
    所述多肽选自代谢素或骨钙素。The polypeptide is selected from metabolin or osteocalcin.
  4. 根据权利要求3所述的衍生物，其特征在于，所述的代谢素选自如SEQ ID NO.1或SEQ ID NO.3所示的多肽， The derivative according to claim 3, wherein the metabolin is selected from the polypeptides shown in SEQ ID NO.1 or SEQ ID NO.3,
    YLGASVPSPDPLEPT                         SEQ ID No.1；YLGASVPSPDPLEPT SEQ ID No.1;
    YLYQWLGAPVPYPDPLEPR              SEQ ID No.3。YLYQWLGAPVPYPDPLEPR SEQ ID No. 3.
    所述的骨钙素选自如SEQ ID NO.2或SEQ ID NO.4所示的多肽，The osteocalcin is selected from the polypeptides shown in SEQ ID NO.2 or SEQ ID NO.4,
    YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI                  SEQ ID No.2；YLGASVPSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI SEQ ID No.2;
    YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No.4。YLYQWLGAPVPYPDPLEPREVCELNPDCDELADHIGFQEAYRRFYGPV SEQ ID No.4.
  5. 根据权利要求3或4所述的多肽的衍生物，其特征在于，所述末端的修饰选自多肽N端乙酰化修饰和C端的胺化修饰； The derivative of the polypeptide according to claim 3 or 4, wherein the modification of the terminal is selected from the group consisting of N-terminal acetylation modification and C-terminal amination modification of the polypeptide;
    所述侧链的修饰选自肽类激素中氨基酸侧链R基上的修饰。The modification of the side chain is selected from the modification on the R group of the amino acid side chain in peptide hormones.
    所述荧光基团的修饰中所用的荧光染料选自FITC、Rhodamine、Cy3、Cy5、Cy5.5、Cy7、ICG；The fluorescent dye used in the modification of the fluorescent group is selected from FITC, Rhodamine, Cy3, Cy5, Cy5.5, Cy7, ICG;
    所述磷酸化修饰选自p-Ser、p-Thr、p-Tyr中的一种或多种的组合；The phosphorylation modification is selected from one or more combinations of p-Ser, p-Thr, p-Tyr;
    所述生物素的标记，所述生物素选自D-生物素、生物素酰肼、光敏生物素和生物素-dUTP；The labeling of the biotin, the biotin is selected from D-biotin, biotin hydrazide, photosensitive biotin and biotin-dUTP;
    所述同位素标记中所用的同位素选自 13C、 14C、 15N、 2H、 3H、 18O、 34S、 36S中的一种或多种的组合。 The isotopes used in the isotopic labeling are selected from one or more combinations of 13 C, 14 C, 15 N, 2 H, 3 H, 18 O, 34 S, and 36 S.
  6. 权利要求2或3所述的多肽的衍生物在制备能够跨血脑屏障的药物或试剂中的用途。 Use of the derivative of the polypeptide described in claim 2 or 3 in the preparation of drugs or reagents capable of crossing the blood-brain barrier.
  7. 一种跨血脑屏障的载体，其特征在于，所述载体表面修饰有权利要求1-2任一项中的多肽或权利要求3-5任一项所述的衍生物； A carrier across the blood-brain barrier, characterized in that the surface of the carrier is modified with the polypeptide according to any one of claims 1-2 or the derivative according to any one of claims 3-5;
    优选地，所述载体选自脂质体、纳米粒；Preferably, the carrier is selected from liposomes, nanoparticles;
    优选地，所述载体中负载活性成分。Preferably, the carrier is loaded with active ingredients.
  8. 一种跨血脑屏障的药物，其特征在于，所述药物为权利要求1-2任一项中的多肽或权利要求3-5任一项所述的衍生物与活性成分的偶联物。 A drug that crosses the blood-brain barrier, characterized in that the drug is a conjugate of the polypeptide according to any one of claims 1-2 or the derivative described in any one of claims 3-5 and an active ingredient.
  9. 一种跨血脑屏障的多肽探针，其特征在于，所述多肽探针为1-2任一项中的多肽或权利要求3-5任一项所述衍生物，与荧光染料通过有机化学反应偶联制得。 A polypeptide probe crossing the blood-brain barrier, characterized in that, the polypeptide probe is the polypeptide in any one of 1-2 or the derivative described in any one of claims 3-5, and is combined with a fluorescent dye by organic chemistry Prepared by reaction coupling.
  10. 根据权利要求9所述的多肽探针，其特征在于，所述荧光染料选择具有近红外区荧光基团的染料， The polypeptide probe according to claim 9, wherein the fluorescent dye is selected from a dye with a fluorescent group in the near-infrared region,
    优选地，所述具有近红外区荧光基团的染料选自Cy5、Cy7、ICG。Preferably, the dye with a fluorescent group in the near-infrared region is selected from Cy5, Cy7, and ICG.
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