Polypeptides passing through blood-brain barrier and uses thereof
By developing a method for binding of blood-brain barrier penetrating peptides with specific amino acid sequences to target substances, the problem of drugs being unable to pass through the blood-brain barrier is solved, effective treatment and diagnosis of brain diseases are achieved, and treatment efficiency is significantly improved.
Patent Information
- Application Number
- CN202380040091.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2022-05-16
- Filing Date
- 2023-05-16
- Publication Date
- 2024-12-24
AI Technical Summary
Existing drugs for treating brain and central nervous system diseases are inefficient in treatment due to hydrophilicity.
A blood-brain barrier penetrating peptide containing a specific amino acid sequence is developed and bound to the target substance, passing through the blood-brain barrier through the form of a fusion protein, introducing drugs or diagnostic agents into brain tissue. This peptide can be fused with other cell membrane penetrating peptides and is connected to the target substance through covalent bonds, chemical bonds, etc. to achieve effective delivery of drugs.
Successfully delivers substances that cannot naturally pass through the blood-brain barrier to brain tissues for the treatment or diagnosis of brain and central nervous system diseases such as Alzheimer's disease and Parkinson's disease.
Smart Images
Figure CN119183455A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a polypeptide that crosses the blood-brain barrier or the cerebrovascular barrier and a substance delivery use of the polypeptide. Background Art
[0002] The blood-brain barrier or cerebrovascular barrier (BBB) is a barrier that separates cerebrospinal fluid from blood. Due to its high selective permeability, it plays a role in isolating the main regulatory centers of the human body from pathogens such as bacteria that can be transported through the blood and potential dangerous substances in the blood.
[0003] The blood-brain barrier allows glucose, essential amino acids, electrolytes, etc. to pass passively through endothelial cells, but prevents metabolites, toxins, and drugs in the blood from entering brain cells. However, fat-soluble substances such as water, air, and carbon dioxide can freely pass through the barrier, so alcohol, nicotine, anesthetics, etc. can affect the brain.
[0004] This selective permeability of the blood-brain barrier has become a major obstacle to drug treatment of brain and central nervous system (CNS) diseases such as Alzheimer's disease, Parkinson's disease, bacterial and viral infections, and cancer. This is because many therapeutic agents used to treat brain and central nervous system (CNS) diseases and disorders are hydrophilic and therefore cannot directly cross the blood-brain barrier. Summary of the invention
[0005] Technical issues
[0006] In view of the above situation, the present inventors conducted research to discover novel blood-brain barrier penetrating peptides and confirmed through in vivo experimental results that fusion proteins of five novel peptides and GST can cross the blood-brain barrier.
[0007] Therefore, an object of the present invention is to provide a novel blood-brain barrier penetrating peptide and use of the peptide for delivering a target substance to the brain.
[0008] Solution to the problem
[0009] In order to achieve the above object, one embodiment of the present invention provides a blood-brain barrier penetrating peptide comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.
[0010] According to a specific example of the present invention, the blood-brain barrier penetrating peptide comprising the amino acid sequence of sequence number 3 can be selected from the group consisting of sequence numbers 1 to 3, and the blood-brain barrier penetrating peptide comprising the amino acid sequence of sequence number 5 can be composed of the amino acid sequence of sequence number 4 or sequence number 5.
[0011] In the present invention, the blood-brain barrier penetrating peptide comprising the amino acid sequence of sequence number 3 may comprise an amino acid sequence having a sequence homology of more than 70%, preferably more than 80%, more preferably more than 90%, further preferably more than 95%, and most preferably more than 98% with the amino acid sequence selected from the group consisting of sequence numbers 1 to 3.
[0012] In the present invention, the blood-brain barrier penetrating peptide comprising the amino acid sequence of sequence number 5 may comprise an amino acid sequence having a sequence homology of more than 70%, preferably more than 80%, more preferably more than 90%, further preferably more than 95%, and most preferably more than 98% to the amino acid sequence of sequence number 5.
[0013] In addition, the above-mentioned blood-brain barrier penetrating peptide can be fused with other cell membrane penetrating peptides for use. Examples of cell membrane penetrating peptides include penetratin, TAT peptide, Pep-1 peptide, S413-PV, Magainin2 (bombesin 2) or Buforin 2 (buforin antimicrobial peptide).
[0014] The inventors of the present invention have confirmed through specific experiments that the fusion protein of the discovered blood-brain barrier penetrating peptide and GST can pass through the blood-brain barrier of mice ( Figures 1 to 5 ).
[0015] Therefore, another embodiment of the present invention provides a target substance brain delivery system comprising a blood-brain barrier permeable peptide and a target substance bound to one end of the blood-brain barrier permeable peptide.
[0016] The target substance can be selected from the group consisting of compounds, proteins, glycoproteins, peptides, nucleotides, nucleic acids, carbohydrates, lipids, glycolipids, nanoparticles, liposomes, drugs, lipid-based formulations, viruses, quantum dots and fluorochromes.
[0017] In the present invention, the blood-brain barrier penetrating protein and the target substance can be linked by methods known in the art for binding proteins to target substances, for example, they can be linked by covalent bonds, chemical bonds, peptide bonds, and the like.
[0018] As described above, the present inventors have confirmed that a fusion protein of a blood-brain barrier penetrating peptide and GST can cross the blood-brain barrier. Therefore, when a protein (target protein) is used as a target substance, it can be introduced into brain tissue in the form of a fusion protein of a blood-brain barrier penetrating protein and the target protein.
[0019] In addition, if a therapeutic or diagnostic agent for a brain or central nervous system disease is used as a target substance, the target substance brain delivery system can be used as a pharmaceutical composition for treating or preventing a brain or central nervous system disease, or a composition for diagnosing a brain or central nervous system disease.
[0020] For example, in the present invention, if a substance that inhibits aggregation by selectively binding to various target substances whose aggregation increases in degenerative brain diseases, such as alpha-synuclein, amyloid beta, tau, superoxide dismutase 1 (SOD1), and TDP-43, is linked to a blood-brain barrier-penetrating peptide and then injected into the body, the distribution of the above-mentioned target substances in the brain parenchyma can be imaged early or their aggregation can be inhibited by PET.
[0021] The above-mentioned brain or central nervous system diseases include so-called degenerative neurological diseases, and include Alzheimer's dementia, Parkinson's disease, frontotemporal dementia, Lewy dementia, Huntington's disease, and the like.
[0022] The above-mentioned composition for diagnosing brain or central nervous system diseases can be provided in the form of a risk prediction or diagnosis kit for brain or central nervous system diseases. The kit contains not only a preparation capable of detecting blood-brain barrier penetrating peptides, but also one or more other constituent compositions, solutions or devices suitable for the analysis method.
[0023] The pharmaceutical composition for treating or preventing the above-mentioned brain or central nervous system diseases can be administered orally or parenterally (for example, administered intravenously, subcutaneously, intraperitoneally or topically) according to the target method, and when forming a preparation, it is manufactured using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.
[0024] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, lozenges, etc. Such solid preparations are manufactured by mixing at least one or more excipients such as starch, calcium carbonate, sucrose, lactose or gelatin with active ingredients. In addition, in addition to simple excipients, lubricants such as magnesium stearate talc are also used. As liquid preparations for oral administration, there are suspensions, oral solutions, emulsions or syrups, etc., in addition to water and liquid paraffin commonly used as simple diluents, various excipients such as wetting agents, sweeteners, spices, preservatives, etc. can also be included.
[0025] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, lyophilized preparations, suppositories, etc. As non-aqueous solvents and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As bases for suppositories, witepsol (semi-synthetic fatty acid esters), polyethylene glycol (macrogol), tween 61 (Tween 61), cocoa butter (cacao butter), laurin butter (laurin butter), glycerin, gelatin, etc. can be used.
[0026] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio that can be applied to medical treatment, and the effective dose level can be determined based on factors including the patient's disease type, severity, activity of the drug, sensitivity to the drug, administration time, route of administration and excretion ratio, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. For example, the pharmaceutical composition of the present invention can be administered once a day or three times a day at a dosage of 0.1 mg / kg to 200 mg / kg, preferably 1 mg / kg to 50 mg / kg. Regarding the above dosage, the scope of the present invention will not be limited in any way. However, it can be increased or decreased according to the route of administration, the severity of obesity, gender, weight, age, etc., so the above dosage does not limit the scope of the present invention in any way.
[0027] Still another aspect of the present invention provides a polynucleotide encoding the above-mentioned blood-brain barrier penetrating peptide.
[0028] The term "polynucleotide" used in this specification is a polymer of deoxyribonucleotides or ribonucleotides in a single-stranded or double-stranded form. It covers RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specifically mentioned.
[0029] In the present invention, the polynucleotide comprises not only a nucleotide sequence encoding a cell membrane permeable peptide, but also a sequence complementary to the sequence. The complementary sequence comprises not only a completely complementary sequence, but also a substantially complementary sequence. In addition, the polynucleotide sequence may be modified, and the modification includes the addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.
[0030] Still another aspect of the present invention provides a recombinant vector comprising a polynucleotide sequence encoding the above-mentioned blood-brain barrier penetrating protein.
[0031] The term "vector" used in this specification refers to a tool for expressing a target gene in a host cell. For example, a plasmid vector, a cosmid vector, a phage vector, an adenovirus vector, a retrovirus vector, and adeno-associated virus vectors may be included, but is not limited thereto. The vector that can be used as the above-mentioned recombinant vector can be made by operating a plasmid commonly used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), a phage (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.) or a virus (e.g., CMV, SV40, etc.).
[0032] The above-mentioned recombinant vector may comprise the above-mentioned polynucleotide sequence and a promoter operatively linked to the polynucleotide sequence.
[0033] The term "operably linked" used in this specification refers to the functional combination between a nucleotide expression regulatory sequence (eg, a promoter sequence) and other nucleotide sequences, whereby the regulatory sequence regulates the transcription and / or translation of the other nucleotide sequences.
[0034] According to a specific example of the present invention, when a protein is used as a target substance, polynucleotide sequences encoding the blood-brain barrier penetrating protein and the target protein can be introduced into a vector together to express them in the form of a fusion protein.
[0035] Therefore, the above-mentioned recombinant vector may contain a tag sequence that makes the fusion protein easy to purify, for example, continuous histidine codons, maltose binding protein codons and Myc codons, etc. In addition, the above-mentioned recombinant vector may additionally contain a fusion partner for improving the solubility of the fusion protein, etc. In addition, it may contain a sequence that is specifically cut by an enzyme to remove unnecessary parts when the fusion protein is expressed, an expression regulatory sequence, and a marker or reporter gene sequence for confirming intracellular delivery.
[0036] Effects of the Invention
[0037] By using the blood-brain barrier penetrating polypeptide of the present invention, substances that cannot naturally cross the blood-brain barrier can be effectively delivered to brain tissue. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1The results are as follows: a protein fused with GST and the peptide of SEQ ID NO: 1 was labeled with a fluorescent substance and administered to mice, and the blood-brain barrier penetration of the peptide in brain tissue at different times was confirmed (red: blood vessels, green: peptide).
[0039] Figure 2 The results are as follows: a protein fused with GST and the polypeptide of SEQ ID NO: 2 was labeled with a fluorescent substance and administered to mice, and the blood-brain barrier penetration of the polypeptide in brain tissue at different times was confirmed (red: blood vessels, green: polypeptide).
[0040] Figure 3 The results are as follows: the polypeptide of SEQ ID NO: 3 was labeled with a fluorescent substance and administered to mice, and the blood-brain barrier penetration of the polypeptide in brain tissue at different times was confirmed (red: blood vessels, green: polypeptide).
[0041] Figure 4 The results are as follows: the polypeptide of SEQ ID NO: 4 was labeled with a fluorescent substance and administered to mice, and the blood-brain barrier penetration of the polypeptide in brain tissue at different times was confirmed (red: blood vessels, green: polypeptide).
[0042] Figure 5 The results are as follows: the polypeptide of SEQ ID NO: 5 was labeled with a fluorescent substance and administered to mice, and the blood-brain barrier penetration of the polypeptide in brain tissue at different times was confirmed (red: blood vessels, green: polypeptide). DETAILED DESCRIPTION
[0043] Hereinafter, one or more specific examples will be described in more detail by way of examples. However, these examples are for the purpose of illustrating one or more specific examples, and the scope of the present invention is not limited to these examples.
[0044] Example 1: Peptide preparation
[0045] The peptides were prepared as follows. The peptide sequences are shown in Table 1.
[0046] [Table 1]
[0047]
[0048] In order to evaluate whether the penetrating peptide can be used as a drug delivery body, GST, which cannot naturally penetrate the blood-brain barrier, was coupled to the N-terminus of the peptide corresponding to SEQ ID NO: 1 or 2.
[0049] Specifically, the peptide sequence of sequence number 1 or 2 and the nucleotide sequence encoding GST were inserted into the pGEX vector system to create a recombinant vector, and the recombinant vector was introduced into the Escherichia coli strain BL21 (DE3). The above strain was cultured in LB medium at 26°C for 3 hours, treated with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG), and then cultured again at 18°C for 12 hours.
[0050] Then, the culture medium was recovered, the cells were recovered by centrifugation, and the cells were sonicated. A GST+peptide fusion protein was obtained by affinity chromatography. The salt concentration of the obtained fusion protein was adjusted with PBS (pH 7.4) buffer for 2 to 3 days to improve the purity.
[0051] Peptides of sequence numbers 3, 4 and 5, excluding sequence numbers 1 and 2, were synthesized and used in subsequent experiments after confirming that the purity was 95% or more.
[0052] Example 2: Evaluation of BBB penetration ability of peptides
[0053] Whether or not the peptide of the present invention can penetrate the blood-brain barrier in a living system was confirmed as follows.
[0054] In order to track the peptide in vivo, Dylight488, which exhibits a fluorescent signal, was bound to the GST+peptide fusion protein prepared in Example 1 or the peptides of sequence numbers 3 to 5. Each fusion protein or peptide sample bound to a fluorescent substance was injected into mice (strain name: C57BL / 6N, 8 weeks old) known to exhibit metabolic effects similar to those of humans, and the presence or absence of the sample was observed in brain tissue and blood vessels.
[0055] Mice were anesthetized with respiratory anesthesia (isoflurane) to limit movement and prevent improper interpretation of the results. In order to distinguish brain tissue from blood vessels, blood vessels were stained with anti-CD31 and FSD fluor 647 compounds. For tissue imaging, the skin tissue on the skull was cut, and a cranial window was attached to the incision site to obtain a fixed image. Then, the tissue image before the sample was injected (0 hr) was taken using a fluorescence microscope (in vivo confocal two-photon convertible microscope, IVM-CM Confocal & Two-photon convertible microscopy), and the sample (10 mg / kg) was injected intravenously to observe the fluorescence signal appearance that changes over time.
[0056] As a result, the fluorescence signal of peptide 1 and 2 can be observed in the blood vessels in the brain tissue from 0.16 hours (10 minutes) after injection, and it can be seen that the fluorescence signal in the brain tissue is strongest at 1 hour after injection of peptide 1 and 4 hours after injection of peptide 2 ( Figure 1 and Figure 2 ).
[0057] For all peptides 3 to 5, fluorescence signals were observed in the blood vessels in the brain tissue starting from 0.5 hours (30 minutes) after injection, and the fluorescence signals were strongest at 1 hour after injection ( Figures 1 to 5 ).
[0058] pass Figures 1 to 5 The results of this study demonstrate that the peptide of the present invention can penetrate the blood-brain barrier and can effectively deliver large substances such as GST to brain tissue.
Claims
1. A blood-brain barrier penetrating peptide comprising the amino acid sequence of sequence number 3 or sequence number 5.
2. The blood-brain barrier penetrating peptide according to claim 1, wherein The blood-brain barrier penetrating peptide comprising the amino acid sequence of SEQ ID NO: 3 is selected from the group consisting of SEQ ID NOs: 1 to 3.
3. The blood-brain barrier penetrating peptide according to claim 1, wherein The blood-brain barrier penetrating peptide comprising the amino acid sequence of SEQ ID NO: 5 consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:
5.
4. A target substance brain delivery system comprising: the blood-brain barrier penetrating peptide according to any one of claims 1 to 3; and a target substance bound to one end of the blood-brain barrier penetrating peptide.
5. The target substance brain delivery system according to claim 4, wherein: The target substance is selected from the group consisting of compounds, proteins, glycoproteins, peptides, nucleotides, nucleic acids, carbohydrates, lipids, glycolipids, nanoparticles, liposomes, drugs, lipid-based preparations, viruses, quantum dots and fluorescent pigments.
6. The target substance brain delivery system according to claim 4, wherein: The blood-brain barrier penetrating peptide is linked to the target substance via a covalent bond. 7 . A polynucleotide encoding the blood-brain barrier penetrating peptide according to claim 1 . A recombinant vector comprising the polynucleotide according to claim 7.
9. A pharmaceutical composition for treating or preventing brain or central nervous system diseases, comprising the target substance brain delivery system according to any one of claims 4 to 6 as an active ingredient. 10 . A composition for diagnosing a brain or central nervous system disease, comprising the target substance brain delivery system according to any one of claims 4 to 6 as an active ingredient.