Peptides for blood-brain barrier permeability and their applications

Abstract

This invention provides a blood-brain barrier (BBB) ​​permeable peptide for effectively delivering drugs used to treat brain diseases or brain tumors to the brain parenchyma. [Solution] A blood-brain barrier-penetrating peptide is provided, which is linked to a cerebral vascular endothelial cell surface protein-binding peptide and a cell-permeable peptide.

Description

[Technical Field]

[0001] The present invention relates to a blood-brain barrier-penetrating peptide and its applications, and more particularly to a blood-brain barrier-penetrating peptide and a complex to which an antibody, protein, or therapeutic nucleic acid drug is conjugated, and its applications in the treatment or prevention of brain diseases or brain tumors. [Background technology]

[0002] The blood-brain barrier (BBB), as the most powerful biological barrier, is a defense system that helps maintain the complex physiology of the brain by selectively allowing only blood substances essential for maintaining brain function to pass through. The BBB consists of capillaries formed from brain endothelial cells and the surrounding astrocytes and pericytes, and each cell contributes to strengthening the BBB's unique biological barrier. Unlike endothelial cells of other organs, brain endothelial cells have well-developed tight junctions that strongly inhibit the intercellular passage of hydrophilic substances with a density of 300 Da or more; this is called the physical barrier function. In addition, polarized endothelial cells have well-developed multidrug efflux pumps such as P-glycoprotein, MRP1, MRP4, and BCRP, which, by binding to hydrophobic drugs, hinder the permeation of drugs into brain tissue; this is called the metabolic barrier function. Astrocytes extend projections toward the capillaries that form in the brain, creating astrocyte endfeet that surround most of the surface of the blood vessels. Recently, it has been discovered that these endfeet play a central role in protecting the blood-brain barrier (BBB) ​​from harmful factors and maintaining the blood-brain barrier.

[0003] Therefore, neurological drugs such as large molecular weight drugs that should act inside the brain, or small molecular weight drugs with low brain permeability, cannot cross the blood-brain barrier (BBB).

[0004] On the other hand, conventional techniques utilize the transcytosis pathway, mediated by endogenous receptors expressed on the capillary endothelium of the brain, to cross the blood-brain barrier. Focusing on the fact that many transferrin receptors are present on the surface of brain vascular endothelial cells, antibodies or peptides against transferrin receptors were devised. However, antibodies bound to transferrin receptors cannot directly pass through the cell membrane and can only enter brain vascular endothelial cells via endocytosis. Once inside brain vascular cells via endocytosis, they form endosomes, and if they cannot escape from the endosomes, they are limited to translocating to lysosomes and being degraded.

[0005] On the other hand, in addition to transferrin receptors, brain vascular endothelial cells also contain low-density lipoprotein receptor (LDLR)-related protein (LRP) receptors. These LRP receptors endocytose LDL particles, allowing them to pass through the blood-brain barrier (BBB). These LRP receptors are advantageous for delivering drugs used to treat neurodegenerative diseases because they do not have to be translocated to lysosomes and degraded.

[0006] Previous studies have shown that IgG injected into the bloodstream permeates into the CNS compartment at a very low rate (approximately 0.1%) [Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)], resulting in a limitation in its pharmacokinetic effects within the CNS. Therefore, there is a need for a drug delivery system for neurological disorders that can effectively transport drugs across the blood-brain barrier to the brain.

[0007] Therefore, the inventors manufactured a blood-brain barrier (BBB) ​​permeable peptide by linking a cell-permeable peptide to a transferrin receptor-binding peptide or a low-density lipoprotein receptor-associated protein receptor-binding peptide. They confirmed that the manufactured peptide can directly pass through the cell membrane of brain endothelial cells without relying on endocytosis. They also confirmed that a blood-brain barrier permeable peptide-drug conjugate, to which an antibody, protein, or therapeutic nucleic acid is bound, is effectively delivered to the brain parenchyma and has a therapeutic effect on brain tumors, thus completing the present invention.

[0008] The information described in this background section is solely for the purpose of improving understanding of the background of the present invention, and therefore may not include information that constitutes prior art already known to a person with ordinary skill in the art to which the present invention belongs. [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a blood-brain barrier (BBB) ​​permeable peptide for effectively delivering drugs for the treatment of brain diseases or brain tumors to the brain parenchyma.

[0010] Another object of the present invention is to provide a blood-brain barrier-penetrating peptide-drug complex in which a drug is bound to the blood-brain barrier-penetrating peptide.

[0011] Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of brain diseases or brain tumors, comprising the blood-brain barrier-penetrating peptide-drug complex.

[0012] Another object of the present invention is to provide a method for preventing or treating a brain disease or brain tumor, comprising the step of administering the complex; uses of the complex for the prevention or treatment of a brain disease or brain tumor; and uses of the complex for the manufacture of a drug for the prevention or treatment of a brain disease or brain tumor. [Means for solving the problem]

[0013] To achieve the above objective, the present invention provides a blood-brain barrier (BBB) ​​permeable peptide in which a brain vascular endothelial cell surface protein-binding peptide and a cell-permeable peptide are linked.

[0014] The present invention also provides a blood-brain barrier-penetrating peptide-drug complex in which a drug is bound to the blood-brain barrier-penetrating peptide.

[0015] The present invention also provides nucleic acids that encode the complex.

[0016] The present invention also provides a recombinant vector into which the nucleic acid is introduced.

[0017] The present invention also provides recombinant cells into which the nucleic acid or the recombinant vector has been introduced.

[0018] The present invention also provides a method for producing a blood-brain barrier-penetrating peptide-drug complex, comprising the steps of (a) culturing the recombinant cells to express the blood-brain barrier-penetrating peptide-drug complex; and (b) recovering the expressed blood-brain barrier-penetrating peptide-drug complex.

[0019] The present invention also provides a pharmaceutical composition for the treatment or prevention of brain diseases or brain tumors, comprising the aforementioned blood-brain barrier-penetrating peptide-drug complex.

[0020] The present invention also provides a method for preventing or treating a brain disease or brain tumor, comprising the step of administering the complex, use of the complex for preventing or treating a brain disease or brain tumor, and use of the complex for producing a medicament for preventing or treating a brain disease or brain tumor. [BRIEF DESCRIPTION OF THE DRAWINGS]

[0021] [Figure 1] FIG. 1 is a schematic diagram of a complex of an antibody and a blood-brain barrier permeable peptide. [Figure 2] FIG. 2 is a schematic diagram of a complex of siRNA and a blood-brain barrier permeable peptide. [Figure 3] FIG. 3 shows the results of SDS-PAGE and Western blotting of the blood-brain barrier permeable peptide and antibody expressed and purified in pcDNA3.4-TOPO. [Figure 4] FIG. 4a shows the results of IVIS observation of complexes of blood-brain barrier permeable peptides and antibodies distributed in the brain, liver, lung, spleen, kidney and heart. FIG. 4b shows the results of three-dimensional observation of complexes of blood-brain barrier permeable peptides and antibodies distributed inside the brain. [Figure 5] FIG. 5 shows the concentrations of complexes of blood-brain barrier permeable peptides and antibodies present in blood and brain. [Figure 6] FIG. 6a is an image obtained by measuring bioluminescence of U87MG-Luc cells on day 16. FIG. 6b shows the results of measuring bioluminescence of U87MG-Luc cells over 16 days. FIG. 6c shows the results of observing the distribution of the complex of blood-brain barrier permeable peptide and antibody at the brain tumor site. [Figure 7] FIG. 7 shows the results of confirming the complex of blood-brain barrier permeable peptide and siRNA by electrophoresis. [Figure 8] FIG. 8 shows the results of confirming the complex of blood-brain barrier permeable peptide and siRNA with a transmission electron microscope. [Figure 9] FIG. 9 shows the permeability results of the blood-brain barrier permeable peptide and siRNA complex permeated in an in vitro BBB model. [Figure 10] FIG. 10 shows the results of observing the blood-brain barrier permeable peptide and siRNA complex permeated into the brain parenchyma. [Figure 11] This shows the results of measuring β-catenin mRNA levels, which were reduced in the cerebral cortex, cerebellum, and midbrain by a blood-brain barrier-penetrating peptide and siRNA complex, using qRT-PCR. [Modes for carrying out the invention]

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by skilled experts in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0023] In this invention, in order to prevent substances that have permeated brain vascular endothelial cells from moving from endosomes to lysosomes, a peptide capable of binding to the transferrin receptor was devised using a phage display technique instead of antibodies. This transferrin receptor-binding peptide is characterized by binding to a site different from the site where transferrin binds to the transferrin receptor. Furthermore, a low-density lipoprotein receptor (LDLR)-related protein (LRP) receptor-binding peptide was devised.

[0024] By linking a transferrin receptor-binding peptide or a low-density lipoprotein receptor-related protein receptor-binding peptide with a peptide possessing cell permeability, we developed a blood-brain barrier (BBB) ​​permeable peptide that can directly pass through the cell membrane of cerebral vascular endothelial cells without relying on endocytosis. This peptide was named the NIPEP-TPP-BBB shuttle (NIBEC Peptide-Target Tissue Penetrating Property-BBB shuttle).

[0025] Furthermore, we confirmed that when a drug such as an antibody, protein, or therapeutic nucleic acid was attached to the N-terminus of a blood-brain barrier-penetrating peptide using gene expression or chemical binding methods, and then intravenously injected into mice, it was transmitted to the brain parenchyma, and that when administered to animals in which glioblastoma had been induced, tumor cells were reduced.

[0026] Accordingly, in one aspect, the present invention relates to a blood-brain barrier (BBB) ​​permeable peptide to which a brain vascular endothelial cell surface protein-binding peptide and a cell-permeable peptide are linked.

[0027] In the present invention, the cerebral vascular endothelial cell surface protein can be characterized as a transferrin receptor or a low-density lipoprotein receptor-related protein (LRP) receptor. Accordingly, the blood-brain barrier permeable peptide can be characterized as a peptide having binding ability to transferrin receptors or low-density lipoprotein receptor-related protein (LRP) receptors linked to a cell-permeable peptide.

[0028] In the present invention, the transferrin receptor-binding peptide can be characterized by being represented by one of the amino acid sequences from SEQ ID NO: 1 to SEQ ID NO: 4. Sequence ID 1: VPALR Sequence ID 2: LRRERQSRLRRERQSR Sequence ID 3:VPALRRERQSRLRRERQSR Sequence ID 4: HAIYPRH

[0029] In this invention, the term "transferrin receptor" refers to a specific receptor for transferrin that allows iron to be trapped inside cells. It is composed of a homologous dimer of a polypeptide chain with a molecular weight of 95,000 Da, with one molecule of transferrin, each containing two iron ions, bound to each chain.

[0030] In this invention, the term "transferrin" refers to a type of glycoprotein, an iron-carrying protein that, when β-globulin is bound to two molecules of trivalent iron ions absorbed into the serum, supplies iron necessary for cell proliferation and hemoglobin production into the cell via the transferrin receptor. More than 99% of the iron in the serum binds to transferrin.

[0031] In the present invention, the low-density lipoprotein receptor-associated protein receptor-binding peptide can be characterized by being represented by the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. Sequence ID 5: YHFNGCEDPLCR Sequence ID 6: HPWCCGLRLDLR

[0032] In this invention, the term "low-density lipoprotein receptor-associated protein receptor (LRP receptor)" refers to a cell membrane protein known to be expressed in large numbers in cerebral vascular endothelial cells, small intestinal epithelium, etc., and functions to endocytose LDL particles and allow them to pass through the blood-brain barrier (BBB).

[0033] In this invention, the term "low-density lipoprotein receptor (LDLR)-related proteins (LRPs)" refers to 600 kDa glycoproteins belonging to the LDL receptor gene family that are expressed in various tissues, including hepatocytes, adipocytes, fibroblasts, macrophages, and neurons of the central nervous system (Herz J., et al., EMBO Journal, 20, 4119-27 (1988)), and are mainly distributed in large quantities in the liver and brain. The function of LRPs in the central nervous system is considered particularly important in the cerebellum, cerebral cortex, hippocampus, and brainstem (Qian Z., et al., Nature, 361, 453-457 (1993)).

[0034] In the present invention, the cell-permeable peptide can be characterized by being represented by one of the amino acid sequences of SEQ ID NOs: 7 to 10. Sequence ID 7:HRRCNKNNKKR Sequence ID 8:HRRCNPNNKKR Sequence ID 9: VSRRRRRRRGGRRRR Sequence ID 10: GKCSTRGRKCCRRKK

[0035] In the present invention, the cell-permeable peptide may be, but is not limited to, being directly linked to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the transferrin receptor-binding peptide or the low-density lipoprotein receptor-associated protein receptor-binding peptide.

[0036] In the present invention, the blood-brain barrier permeable peptide can be characterized by being represented by one amino acid sequence from SEQ ID NO: 11 to SEQ ID NO: 58 (Table 1).

[0037] [Table 1]

[0038] In the present invention, the brain vascular endothelial cell surface protein-binding peptide and the cell-permeable peptide are linked via spacers consisting of 2 to 10 amino acids G and 1 to 5 amino acids S.

[0039] In other aspects, the present invention relates to a blood-brain barrier-penetrating peptide-drug conjugate in which a drug is bound to the blood-brain barrier-penetrating peptide.

[0040] In the present invention, the blood-brain barrier-penetrating peptide and the drug may be linked by a linker, but is not limited thereto.

[0041] In the present invention, the drug may be an antibody, a protein, or a therapeutic nucleic acid, and the antibody may be an antibody that suppresses a protein that induces brain disease, and the protein may be a growth factor related to nerve cells, but is not limited thereto.

[0042] In the present invention, the protein can be characterized by being selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotropin-4 / 5, fibroblast growth factor (FGF)-2 and other FGFs, neurotropin (NT)-3, erythropoietin (EPO), epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF), neuruturin, platelet-derived growth factor (PDGF), heregulin, neuregulin, artemin, percephin, interleukin, GDNF family receptor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, and stem cell factor (SCF).

[0043] In the present invention, the antibody can be characterized by being represented by the amino acid sequence of SEQ ID NO: 59, and has efficacy in treating glioblastoma. Sequence ID 59: MAWVWTLLFLMAAAQSIQA EWIKQRPGQGLEWIGVIHPGNGGTNYNENFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCASGNDGSYWGQGTTVTVSSGGGGSGKGGSGGGGSGGGGSDIEL TQSPSSLTVTAGEKVTMSCKSSQSLLNSGDQKIYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSVSGTDFTLTISSVQAEDLAVYYCQNDYNYPYTFGGGTKC LEIKRGSENLYFQGGSGKPIPNPLLGLDSTGGSGGSHHHHHHH

[0044] In the present invention, the blood-brain barrier-penetrating peptide-drug conjugate, to which an antibody is bound, can be produced by gene expression or chemical conjugation, but the method of production is not limited thereto.

[0045] When producing a blood-brain barrier-penetrating peptide-drug conjugate using gene expression, a vector can be used that expresses both (i) an antibody capable of suppressing glioblastoma and (ii) a blood-brain barrier-penetrating peptide. In this invention, a pET vector was used for expression in E. coli, and pcDNA3.1-TOPO and pcDNA3.4-TOPO vectors were used for expression in mammalian cells.

[0046] In this case, a linker sequence can be further included between the antibody and the blood-brain barrier-penetrating peptide during expression. Preferably, the linker is CGGGG, but is not limited thereto. In the present invention, therapeutic antibodies can be expressed from genes, purified, and then chemically conjugated to a blood-brain barrier-penetrating peptide.

[0047] In the present invention, the blood-brain barrier-penetrating peptide-drug conjugate of a therapeutic antibody and a blood-brain barrier-penetrating peptide can be characterized in that a transferrin receptor-binding peptide selected from the group consisting of SEQ ID NOs. 1 to 4, or a low-density lipoprotein receptor-associated protein receptor-binding peptide of SEQ ID NOs. 5 or 6, and a cell-permeable peptide selected from the group consisting of SEQ ID NOs. 7 to 10 are chemically linked to the cysteine ​​at the C-terminus of the antibody of SEQ ID NO. 59 or a sugar in the Fc region. This is shown in the schematic diagram of the antibody-blood-brain barrier-penetrating peptide conjugate in Figure 1.

[0048] The blood-brain barrier-penetrating peptide can be chemically linked to the antibody by a linker and a crosslinking agent. Any linker can be used as long as it can provide space to form a functional structure. For example, the linker may be a peptidolytic linker of natural and / or synthetic origin. A peptidolytic linker of natural and / or synthetic origin may consist of an amino acid chain of 1 to 50 amino acids and may include a repeating amino acid sequence of a naturally occurring polypeptide, such as a polypeptide with hinge function. In another embodiment, the peptidolytic linker amino acid sequence may be a synthetic linker amino acid sequence specified to be rich in glycine, glutamine, and / or serine residues. These residues may be arranged, for example, in small repeating units of 5 or fewer amino acids, and these small repeating units may be repeated to form a multimer unit. Up to 6 additional arbitrary naturally occurring amino acids may be added at the amino-terminus and / or carboxy-terminus of the multimer unit. Other synthetic peptide linkers may be single amino acid compositions repeated 10 to 20 times, or they may have up to six additional any naturally occurring amino acids at the amino-terminus and / or carboxy-terminus. On the other hand, the linker may be in a chemically modified form of amino acid, and can be used, for example, in the form of Fmoc-6-aminohexanoic acid to which Fmoc-(9-Fluorenylmethoxycarbonyl) is attached as a blocking group, but is not limited thereto.

[0049] In some aspects of the present invention, the sulfhydryl group of cysteine ​​located at the N-terminus of the blood-brain barrier-penetrating peptide can be linked to the amine group of lysine in the exposed portion of the stereostructure of SEQ ID NO: 59.

[0050] In this case, the crosslinking agents that can be used are 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethyleneglycol (BM[PEO]4), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), succinimidyl-4-[N-maleimidomethylcyclohexane-1-carboxy-[6-amidocaproate]] (SMCC) and its sulfonated salt (sulfo-SMCC), succinimidyl 6-[3-(2-pyridyldithio)-lopionamide]hexanoate (succimidyl Examples include, but are not limited to, 6-[3-(2-pyridyldithio)-ropionamido]hexanoate (SPDP) and its sulfonated salt (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and its sulfonated salt (sulfo-MBS), succimidyl[4-(p-maleimidophenyl)butyrate] (SMPB) and its sulfonated salt (sulfo-SMPB), etc.

[0051] In the present invention, a method for producing a blood-brain barrier-penetrating peptide-drug conjugate using the chemical bonding method includes the following steps: (a) A step of activating the amine group of lysine present in the cysteine ​​or exposed portion at the C-terminus of the therapeutic antibody with a crosslinking agent; (b) The step of linking the blood-brain barrier-penetrating peptide to the antibody activated with a crosslinking agent; and (c) A step of purifying the blood-brain barrier-penetrating peptide-drug conjugate, on which the antibody and the blood-brain barrier-penetrating peptide are bound.

[0052] In the present invention, the therapeutic nucleic acid may be selected from the group consisting of DNA, siRNA, miRNA, and mRNA, and preferably the therapeutic nucleic acid has the function of suppressing or increasing the expression of a target gene. The siRNA may have a nucleotide sequence complementary to the nucleotide sequence encoding a tumor-inducing protein or a protein that induces brain disease.

[0053] In one embodiment of the present invention, the expression of mutant KRAS is suppressed using siRNA against mutant KRAS, wherein the siRNA is represented by SEQ ID NO: 60 and SEQ ID NO: 61. KRAS siRNA sequence: Sense-GUGCAAUGAAGGGACCAGUA(Sequence ID 60); Anti-sense-UACUGGUCCCUCAUUGCAC(Sequence ID 61)

[0054] In the present invention, the siRNA may be in its natural form or a chemically modified form.

[0055] In the present invention, the therapeutic nucleic acid is reacted with a cationic peptide, a cationic polymer, or an RNA-binding peptide to produce a primary complex, and then the primary complex is reacted with an anionic amino acid or anionic polymer and a blood-brain barrier-penetrating peptide to which a spacer is linked to form nanoparticles.

[0056] In the present invention, the cationic peptide is represented by the amino acid sequence of SEQ ID NO: 9, the cationic polymer is poly-L-lysine, polyethyleneimine, or chitosan, the RNA-binding peptide is represented by one of the amino acid sequences of SEQ ID NOs. 62 to 67, the anionic amino acid is glutamic acid or aspartic acid, the anionic polymer is heparin or hyaluronic acid, and the spacer is 5 to 10 glycine molecules, but is not limited to these characteristics.

[0057] To allow therapeutic nucleic acids to pass through the blood-brain barrier, an RNA-binding peptide can be introduced into the blood-brain barrier permeability peptide, characterized in that the RNA-binding peptide is represented by one of the amino acid sequences from SEQ ID NO: 62 to SEQ ID NO: 67. Sequence ID 62:CPISQVHEIGIKRNMTVHFKVLREEGPAHMKNF Sequence ID 63: CPISQVHEIGIKR Sequence ID 64: CNMTVHFKVLREEG Sequence ID 65: CPAHMKNFITA Sequence ID 66:CIVTEGEGNGKKVSKKRAAEKMLVEL Sequence ID 67: CEGNGKKVSKKRAA

[0058] The peptides of SEQ ID NOs. 62 to 67 can be used by linking them to the N-terminus of the complex of SEQ ID NOs. 11 to 58, but are not limited to this.

[0059] In the present invention, the complex of siRNA and blood-brain barrier-penetrating peptide can be characterized by the self-assembly of a transferrin receptor-binding peptide selected from the group consisting of SEQ ID NOs. 1 to 4, or a low-density lipoprotein receptor-associated protein receptor-binding peptide of SEQ ID NOs. 5 or 6; a cell-permeable peptide selected from the group consisting of SEQ ID NOs. 7 to 10; and an RNA-binding peptide of SEQ ID NOs. 62 to 67, and the siRNA of SEQ ID NOs. 60 and 61. This complex forms nano-sized particles, characterized by a size of 10 to 200 nm.

[0060] In the present invention, the complex of siRNA and the blood-brain barrier-penetrating peptide can be produced in two steps. 1) The siRNA is first reacted with a cationic peptide or cationic polymer. The cationic peptide may be the peptide of SEQ ID NO: 9, and the cationic polymer may be poly-L-lysine, polyethyleneimine, or chitosan. 2) In the next step, the complex can be bound with the blood-brain barrier-penetrating peptide linked to an anionic amino acid or an anionic polymer. At this time, the complex of the blood-brain barrier-penetrating peptide, spacer, and anionic amino acid may be characterized by being represented by one of the amino acid sequences of SEQ ID NOs: 68 to 85.

[0061] Figure 2 is a schematic diagram of a complex of siRNA and a blood-brain barrier-penetrating peptide. Figure 2 is an illustrative diagram showing the case where the therapeutic nucleic acid is siRNA. When the siRNA is DNA, miRNA, or mRNA, a primary complex can be produced by reacting it with a DNA, miRNA, or mRNA-binding peptide. The blood-brain barrier-penetrating peptide-therapeutic nucleic acid complex may be characterized by containing a peptide to which an anionic amino acid consisting of 5 to 10 glutamic acid or aspartic acid molecules, a spacer consisting of 5 to 10 glycine molecules, one cell-penetrating functional peptide selected from the group consisting of SEQ ID NOs: 7 to SEQ ID NOs: 10, and one brain vascular endothelial cell surface protein-binding peptide selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NOs: 6 is linked. Sequence ID 68: EEEGEEEGE-GGGGG-HRRCNKNNKKR-VPALR Sequence ID 69: EEEGEEEGE-GGGGG-HRRCNPNNKKR-GGGGG-VPALR Sequence ID 70: EEEGEEEGE-GGGGG-VSRRRRRRGGRRRR-GGGGG-VPALR Sequence ID 71: EEEGEEEGE-GGGGG-GKCSTRGRKCCRRKK-GGGGG-VPALR Sequence ID 72: EEEGEEEGE-GGGGG-HRRCNKNNKKR-LRRERQSRLRRERQSR Sequence ID 73: EEEGEEEGE-GGGGG-HRRCNPNNKKR-LRRERQSRLRRERQSR Sequence ID 74: EEEGEEEGE-GGGGG-VSRRRRRRGGRRRR-GGGGG-LRRERQSRLRRERQSR Sequence ID 75: EEEGEEEGE-GGGGG-GKCSTRGRKCCRRKK-LRRERQSRLRRERQSR Sequence ID 76: EEEGEEEGE-GGGGG-HRRCNKNNKKR-VPALRRERQSRLRRERQSR Sequence ID 77: EEEGEEEGE-GGGGG-HRRCNPNNKKR-VPALRRERQSRLRRERQSR Sequence ID 78: EEEGEEEGE-GGGGG-VSRRRRRRGGRRRR-VPALRRERQSRLRRERQSR Sequence ID 79: EEEGEEEGE-GGGGG-GKCSTRGRKCCRRKK-VPALRRERQSRLRRERQSR Sequence ID 80: EEEGEEEGE-GGGGG-HRRCNKNNKKR-HAIYPRH Sequence ID 81: EEEGEEEGE-GGGGG-HRRCNPNNKKR-HAIYPRH Sequence ID 82: EEEGEEEGE-GGGGG-VSRRRRRRGGRRRR-HAIYPRH Sequence ID 83: EEEGEEEGE-GGGGG-GKCSTRGRKCCRRKK-HAIYPRH Sequence ID 84: EEEGEEEGE-GGGGG-VSRRRRRRGGRRRR-GGGGG-YHFNGCEDPLCR Sequence ID 85: EEEGEEEGE-GGGGG-VSRRRRRRGGRRRR-GGGGG-HPWCCGLRLDLR

[0062] Furthermore, instead of the anionic amino acids glutamic acid or aspartic acid, anionic polymers such as heparin or hyaluronic acid can be used.

[0063] Through self-assembly, the siRNA can be reacted first with the peptide of Sequence ID No. 9, and then bound to the blood-brain barrier permeability peptide. This complex can form nano-sized particles, characterized by a size of 10 to 200 nm.

[0064] In the present invention, the method for forming a complex of therapeutic nucleic acids, including siRNA, by the self-assembly described above includes the following steps: (a) A step in which a cationic peptide, cationic polymer, or RNA-binding peptide is reacted with a therapeutic nucleic acid; (b) A step in which the anionic peptide is reacted with a blood-brain barrier-penetrating peptide; (c) Self-assembly process for nanoparticle formation.

[0065] In another aspect, the present invention relates to nucleic acids encoding the blood-brain barrier-penetrating peptide-drug complex.

[0066] In another aspect, the present invention also relates to a recombinant vector into which the nucleic acid is introduced.

[0067] In the present invention, the vector may be a pET vector, pcDNA3.4, pcDNA3.1, pcDNA3.1-TOPO, pcDNA3.4-TOPO, or a pSecTag vector, but is not limited to these. Preferably, the vector is a pET vector or a pcDNA3.1-TOPO or pcDNA3.4-TOPO vector.

[0068] In another aspect, the present invention also relates to recombinant cells into which the nucleic acid or the recombinant vector has been introduced.

[0069] In the present invention, the recombinant cells may be, but are not limited to, Escherichia coli or mammalian cells.

[0070] In the present invention, the mammalian cell line may be, but is not limited to, the Chinese hamster ovarian cell line (CHO) or the human embryonic kidney cell line (HEK293).

[0071] In another aspect, the present invention also relates to a method for producing the blood-brain barrier-penetrating peptide-drug conjugate, comprising the following steps: (a) The step of culturing the recombinant cells and expressing the blood-brain barrier-penetrating peptide-drug complex; and (b) A step of recovering the expressed blood-brain barrier-penetrating peptide-drug complex.

[0072] Among antibodies that suppress brain diseases, antibodies that suppress glioblastoma, a representative disease, or siRNAs that suppress KRAS mutations, cannot cross the blood-brain barrier and therefore do not have a significant therapeutic effect. On the other hand, in other embodiments of the present invention, when the neurotransmission effect or tumor growth inhibitory effect of antibodies or siRNAs bound to blood-brain barrier-penetrating peptides was confirmed, both in vitro and animal experiments confirmed that they had a significant effect in suppressing the proliferation of the tumor cells.

[0073] Accordingly, the present invention also relates in another aspect to a pharmaceutical composition for the treatment or prevention of brain diseases or brain tumors comprising the blood-brain barrier-penetrating peptide-drug complex.

[0074] In another aspect, the present invention relates to a method for preventing or treating a brain disease or brain tumor, comprising the step of administering the blood-brain barrier-penetrating peptide-drug conjugate.

[0075] In another aspect, the present invention relates to the use of the blood-brain barrier-penetrating peptide-drug conjugate for the prevention or treatment of brain diseases or brain tumors.

[0076] In another aspect, the present invention also relates to the use of the blood-brain barrier-penetrating peptide-drug conjugate for the manufacture of agents for the prevention or treatment of brain diseases or brain tumors.

[0077] In the present invention, an antibody or therapeutic nucleic acid substance that can be used as a therapeutic agent for brain diseases other than brain tumor suppression can be used by conjugating it with a blood-brain barrier permeable peptide.

[0078] In the present invention, the brain disease can be characterized by being selected from the group consisting of encephalitis, Parkinson's disease, epilepsy, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, spinocerebellar degeneration, cerebellar atrophy, multiple sclerosis, senile dementia resulting from the loss of nerve cells, stroke, amnesia syndrome, depression, and post-traumatic stress disorder.

[0079] In the present invention, the brain tumor can be characterized by being selected from the group consisting of glioma, glioblastoma multiforme, meningioma, astrocytoma, acoustic neuroma, chondroma, oligodendroglioma, medulloblastoma, ganglioglioma, schwannoma, neurofibroma, neuroblastoma, and epidural, intramedullary, or intradural tumors.

[0080] In the present invention, the single dose of the blood-brain barrier-penetrating peptide-drug complex may be 1 μg / kg to 100 mg / kg, preferably 5 μg / kg to 50 mg / kg, and may be administered once a day or once to three times a week, but the dose and administration interval are not limited thereto.

[0081] In the present invention, the pharmaceutical composition may be formulated into any one dosage form selected from the group consisting of injections, oral preparations, liquid preparations (e.g., for injection) such as aqueous solutions, suspensions, and emulsions, capsules, granules, tablets, and mucosal administration preparations, but is not limited thereto. These preparations can be manufactured by conventional methods used in formulation in the art or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, and can be formulated into various formulations depending on the disease or component.

[0082] In the present invention, the pharmaceutical composition of the present invention may further contain one or more pharmaceutically acceptable carriers in addition to a complex of an antibody or siRNA conjugated with a blood-brain barrier-penetrating peptide. The pharmaceutically acceptable carriers may be one or more selected from the group consisting of physiological saline, sterile water, Ringer's solution, buffered physiological saline, dextrose solution, maltodextrin solution, glycerol, and ethanol, but are not limited to these.

[0083] The pharmaceutical composition of the present invention may optionally further contain pharmaceutically acceptable adjuvants. The adjuvants may be one or more selected from the group consisting of excipients, diluents, dispersants, buffers, antimicrobial preservatives, bacteriostatic agents, surfactants, binders, lubricants, antioxidants, thickeners, and viscosity modifiers, but are not limited to these.

[0084] The pharmaceutical composition according to the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) by the intended method, and the dosage can be varied and used in a range that is determined by expert opinion, depending on the patient's weight, age, sex, health condition, diet, administration time, method of administration, excretion rate, and severity of the disease.

[0085] The present invention will be described in more detail below through examples. It will be obvious to those of ordinary skill in the art that these examples are solely for illustrative purposes and that the scope of the present invention should not be construed as being limited by these examples.

[0086] Example 1: Synthesis of a blood-brain barrier-penetrating peptide The amino acids and reagents necessary for synthesis were purchased from GL biochem and Sigma-Aldirich. The peptide was synthesized using a peptide synthesizer via solid-phase chemical synthesis of F-moc from the C-terminus. Specifically, it was synthesized using a rink resin (0.075 mmol / g, 100-200 mesh, 1% DVB crosslinking) to which Fmoc-(9-Fluorenylmethoxycarbonyl) was conjugated as a blocking group. After adding 50 mg of the rink resin to the synthesizer, the resin was swelled with DMF, and then a 20% piperidine / DMF solution was used to remove the Fmoc- group. The amino acids were reacted in sequence from the C-terminus with 5, 10, and 5 equivalents each of 0.5 M amino acid solution (solvent: dimethylformamide, DMF), 1.0 M DIPEA (solvent: dimethylformamide & N-methylpyrrolidone, DMF & NMP), and 0.5 M HBTU (solvent: dimethylformamide, DMF), and the reaction was carried out under a nitrogen stream for 1-2 hours. After each deprotection and coupling step, the molecule was washed twice with DMF and isopropanol. Deprotection was performed again after coupling the last amino acid to remove the Fmoc- group. After removing the F-moc protecting group from the last amino acid at the N-terminus, the synthesis was continued by adding DIPEA (10 equivalents), HBTU (5 equivalents), and palmitic acid (5 equivalents) in the presence of DMF. The reaction time was allowed to proceed until a negative result was obtained using Kaiser test solution. After the reaction was complete, the resin was washed with DMF and MeOH and dried in a vacuum oven. Trifluoroacetic acid (TFA) cleavage cocktail was added at a ratio of 20 ml per gram of resin and shaken for 3 hours, after which the resin and the cocktail containing the dissolved peptide were separated by filtration.After removing the filtered solution using a rotary evaporator, cold ether was added, or an excess of cold ether was directly added to the TFA cocktail solution containing the peptides to crystallize the peptides into a solid phase. This was then separated by centrifugation. During this process, the TFA cocktail was completely removed by repeated washing with ether and centrifugation. The resulting peptides were dissolved in distilled water and freeze-dried. After freeze-drying, they were separated and purified by high-performance liquid chromatography (Shimadzu, Japan). (C. 4.6 mm in diameter.) 18 Analysis was performed using a column by passing 0.1% TFA / H2O and 0.092% TFA / acetonitrile at a flow rate of 1 ml / min for 30 minutes, varying the concentration from 0 to 60%, with a UV detector wavelength of 220 nm. Purification was performed using a 2.2 cm diameter column at a flow rate of 20 ml / min under the same conditions for solvent and detection wavelength. The molecular weight of the purified peptide was confirmed by mass spectrometry.

[0087] Alexandrite TM 1 mg of 680 NHS ester (=Cy5.5-NHS ester, Invitrogen, A37567) and 2 mg of the synthesized peptide were dissolved in 0.1 ml of DMF. After adding 0.05 ml of pyridine, the mixture was reacted in a 50°C heat block for 15 minutes. After adding 0.7 ml of ethyl acetate to form a precipitate, the mixture was centrifuged at 10,000 rpm for 2 minutes. After removing the upper layer, the mixture was washed twice in the same manner with 0.7 ml of ethyl acetate and dried in air.

[0088] Example 2: Production of blood-brain barrier permeable peptide and antibody complex A linker CGGGG was introduced to the N-terminus of a blood-brain barrier-penetrating peptide (SEQ ID NO: 41) prepared using the method of Example 1, and the antibody of SEQ ID NO: 59 was chemically conjugated to it. 1.02 mg of antibody SEQ ID NO: 59 was dissolved in 1 mL of PBS buffer (pH 8.3). 2.12 mg of SPDP (succinimidyl 3-(2-pyridyldithio)propionate, ThermoFisher, 21857) was dissolved in 120 μL of DMSO (Dimethyl sulfoxide). 40 μL of SMCC solution was added to this solution, and the reaction was allowed to proceed for 1 hour in the dark, repeating this process three times. After the reaction was complete, desalting was performed using a PD-10 desalting column (GE Healthcare) to obtain 3.5 mL of pyridyldithiol-activated antibody. 2.5 mg of SEQ ID NO: 42 solution was mixed with 300 μL of tertiary purified water, and the pH was adjusted to 8.3 using 1 M Tris buffer (pH=9). After shielding from light, the reaction was carried out overnight at 4°C with mixing. Antibodies conjugated with the blood-brain barrier-penetrating peptide were purified using an FPLC (Akta Pure, GE Healthcare) and a heparin column (HiTrap, GE Healthcare).

[0089] For expression in animal cells, a gene sequence was synthesized in which the C term of the antibody of SEQ ID NO: 59 was linked to the blood-brain barrier permeability peptides of SEQ ID NOs: 15 to 18. Sequence ID 59 - Sequence ID 15 MAWVWTLLFLMAAAQSIQA EWIKQRPGQGLEWIGVIHPGNGGTNYNENFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCASGNDGSYWGQGTTVTVSSGGGGSGKGGSGGGGSGGGGSDIELTQSPSSLTV TAGEKVTMSCKSSQSLLNSGDQKIYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSVSGTDFTLTISSVQAEDLAVYYCQNDYNYPYTFGGGTKCHRRCNKNNKKR-VPALR LEIKRGSENLYFQGGSGKPIPNPLLGLDSTGGSGGSHHHHHHH Sequence ID 59 - Sequence ID 16 MAWVWTLLFLMAAAQSIQA EWIKQRPGQGLEWIGVIHPGNGGTNYNENFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCASGNDGSYWGQGTTVTVSSGGGGSGKGGSGGGGSGGGGSDIELTQSPSSLTV TAGEKVTMSCKSSQSLLNSGDQKIYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSVSGTDFTLTISSVQAEDLAVYYCQNDYNYPYTFGGGTKCHRRCNPNNKKR-VPALR LEIKRGSENLYFQGGSGKPIPNPLLGLDSTGGSGGSHHHHHHH Sequence ID 59 - Sequence ID 17 MAWVWTLLFLMAAAQSIQA EWIKQRPGQGLEWIGVIHPGNGGTNYNENFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCASGNDGSYWGQGTTVTVSSGGGGSGKGGSGGGGSGGGGSDIELTQSPSSLTVT AGEKVTMSCKSSQSLLNSGDQKIYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSVSGTDFTLTISSVQAEDLAVYYCQNDYNYPYTFGGGTKCVSRRRRRRGGRRRR-VPALR LEIKRGSENLYFQGGSGKPIPNPLLGLDSTGGSGGSHHHHHHH Sequence ID 59 - Sequence ID 18 MAWVWTLLFLMAAAQSIQA EWIKQRPGQGLEWIGVIHPGNGGTNYNENFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCASGNDGSYWGQGTTVTVSSGGGGSGKGGSGGGGSGGGGSDIELTQSPSSLTVTA GEKVTMSCKSSQSLLNSGDQKIYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSVSGTDFTLTISSVQAEDLAVYYCQNDYNYPYTFGGGTKCGKCSTRGRKCCRRKK-VPALR LEIKRGSENLYFQGGSGKPIPNPLLGLDSTGGSGGSHHHHHHH

[0090] pcDNA3.1-TOPO or pcDNA3.4-TOPO was cleaved with EcoR I and BamH I, and 900-960 bp KRAS mutant scFv gene fragments were recovered by agarose electroelution. These fragments were amplified by PCR to insert into pcDNA3.1-TOPO and pcDNA3.4-TOPO vectors, and BamH I and EcoR I restriction enzyme sites were synthesized simultaneously. The cleaved vectors and inserts were mixed in a 4 μl-4 μl ratio and reacted at 4°C for 16-18 hours using T4 DNA ligase in a ligation buffer solution consisting of Tris-HCl 500 mM, MgCl2 100 mM, DTT 200 mM, and ATP 10 mM, using ligation buffer solution.

[0091] The manufactured vector was transformed into the Chinese hamster ovary cell line Expi-CHO-S. The Chinese hamster ovary cell line Expi-CHO-S was supplied by Thermo Fisher Scientific (USA) and cultured in ExpiCHO Expression Medium (GIBCO, USA) medium. Subsequently, the ExpiCHO-S cell line (3X10) was transformed. 8 Cells were placed in a 250 mL disposable Erlenmeyer flask with 50 mL of culture medium. 160 μL of expifectamine CHO reagent (Gibco, Cat # A20130), diluted in 1.84 mL of Opti-MEM, was mixed with 2 mL of Opti-MEM containing 50 μg of plasmid. The mixture was allowed to stand at room temperature for 5 minutes, then uniformly infused into prepared Chinese hamster ovary cells. After 18 hours of incubation at 37°C in an 8% CO2 incubator, 300 μL of ExpiCHO enhancer and 12 mL of ExpiCHO feed were added, and the cells were incubated for 5 days. Six days after transfection, the culture medium was collected, sterilized, and purified using FPLC. Nickel chromatography (HisTrap) was used for the column. TMThe proteins were separated and purified using Excel (GE Healthcare). A nickel column was pre-moistened and homogenized with a buffer under the conditions [20 mM sodium phosphate, 0.5 M NaCl, pH 7.4], and then the culture medium was applied. After washing with the aforementioned buffer containing 25 mM imidazole, the imidazole concentration was increased to 125 mM to separate the proteins. The separated proteins were collected, desolding to remove the imidazole, and stored.

[0092] Figure 3 shows the results of SDS-PAGE and Western blotting of blood-brain barrier-penetrating peptides and antibodies expressed and purified using pcDNA3.4-TOPO. The arrows indicate the expressed complex.

[0093] Experimental Example 1: Confirmation of the neurotransmission effect of blood-brain barrier permeable peptide and antibody complex in normal animals. Hairless mice (SKH-1, female, 6 weeks old, Orient Bio) were intravenously injected at a dose of 4 mg / kg each with the Cy5.5-labeled blood-brain barrier permeable peptide (SEQ ID NO: 17), the Cy5.5-labeled antibody (SEQ ID NO: 59), and a complex of the Cy5.5-labeled blood-brain barrier permeable peptide (SEQ ID NO: 17) and the SEQ ID NO: 59 antibody. Fluorescence in each organ (brain, liver, lungs, spleen, kidneys, heart) was measured using IVIS (Perkin Elmer) at 685 nm (excitation) and 706 nm (emission). Additionally, a 3D image of the brain was separately taken using Optix MX3 (ART Advanced Research Technologies Inc.). Figure 4 shows the results of observing the delivery of the blood-brain barrier permeable peptide and antibody complex to the animal brain.

[0094] As shown in Figure 4(a), when the Cy5.5-labeled blood-brain barrier permeable peptide of SEQ ID NO: 17 and the Cy5.5-labeled blood-brain barrier permeable peptide of SEQ ID NO: 17 and the antibody complex of SEQ ID NO: 59 were injected, fluorescence could be observed in the brain. However, the Cy5.5-labeled antibody of SEQ ID NO: 59 did not produce fluorescence in the brain. Furthermore, as can be seen from the 3D image in Figure 4(b), when the Cy5.5-labeled blood-brain barrier permeable peptide of SEQ ID NO: 17 and the antibody complex of the Cy5.5-labeled blood-brain barrier permeable peptide of SEQ ID NO: 17 and the antibody complex of SEQ ID NO: 59 were injected, a large degree of penetration into the brain was observed. However, the antibody of SEQ ID NO: 59 was observed to a low extent inside the brain.

[0095] Experimental Example 2: Confirmation of the concentrations of blood-brain barrier permeable peptide and antibody complexes in blood and brain tissue in normal animals. A control cell permeabilization peptide (TAT) and the antibody of SEQ ID NO: 59 were prepared using the same method as in the expression method of Example 2, and labeled with cy5.5. A complex of the blood-brain barrier permeabilization peptide of SEQ ID NO: 41 (in which CGGGG was introduced into the N term prepared in Example 2) and the antibody of SEQ ID NO: 59 was labeled with Cy5.5. The antibody of SEQ ID NO: 59 was labeled with Cy5.5 and intravenously injected at 10 mg / kg into hairless mice (SKH-1, female, 6 weeks old, Orient Bio). Brain and blood concentrations were measured using IVIS (Perkin Elmer) at excitation: 685 nm and emission: 706 nm, and the results are shown in Figure 5.

[0096] When the antibody was administered alone, it was completely eliminated from the blood within 12 days. Both the antibody linked to TAT and the antibody linked to the blood-brain barrier-penetrating peptide of SEQ ID NO: 41 were maintained until 24 days. However, the blood concentrations of the complex of the blood-brain barrier-penetrating peptide and antibody of SEQ ID NO: 41 were higher.

[0097] Furthermore, no fluorescence was detected in brain tissue when the antibody was administered alone. Antibodies linked to TAT showed fluorescence at 2.4% of the initial dose in brain tissue 4 days after intravenous injection. The complex of the blood-brain barrier-penetrating peptide (SEQ ID NO: 41) and the antibody (SEQ ID NO: 59) showed fluorescence at approximately 7% of the initial dose in brain tissue 4 days after intravenous injection. This result demonstrates that the complex of the blood-brain barrier-penetrating peptide (SEQ ID NO: 41) and the antibody (SEQ ID NO: 59) exhibits higher brain transmission efficiency.

[0098] Experimental Example 3: Confirmation of the efficacy of brain tumor treatment using an animal model of brain tumors. Using an animal model of brain tumor, we measured the penetration of a blood-brain barrier permeable peptide and antibody complex into the brain, its ability to suppress brain tumors, and its distribution into the brain parenchyma. Specifically, we used U87MG-Luc2 (ATCC, HTB-14-LUC2), a glioblastoma of U87MG that expresses luciferase in DMEM medium supplemented with 10% FBS and 1% antibiotic. TM Cells were cultured. 5-7 week old nude mice (Orient Bio) were anesthetized with isoflurane and fixed using an ear bar in a stereotactic device. After incising the scalp at the surgical site, a hole was drilled using a sterile drill 2 mm to the right and 2 mm below the bregma. Then, 1.0 × 10⁻¹⁶ cells were injected through the hole. 5 10 μL of U87MG-Luc-2 cells were injected using a 26G Hamilton syringe (1 μl / min). After injection, the holes were sealed with bone wax and the scalp was sutured closed. To assess the proliferation of the transplanted glioblastomas, mice were injected with 150 mg / kg of D-luciferin potassium (15 mg / mL dissolved in PBS, sigma), a substrate of luciferase, and sacrificed 10 minutes later. Bioluminescence was measured using IVIS (Perkin Elmer), and the results are shown in Figure 6.

[0099] Four days after glioblastoma transplantation, a complex of the blood-brain barrier-penetrating peptide CGGGG-SEQ ID NO: 41 and the antibody SEQ ID NO: 59, at a dose of 1 mg / kg, and the antibody SEQ ID NO: 59 were administered intravenously. The administration interval was every 3 days for a total of 4 doses, and observation continued until day 16. Figure 6 shows the results of observing that the blood-brain barrier-penetrating peptide-antibody complex penetrated the brain, suppressed brain tumors, and was distributed to the brain parenchyma in an animal model of brain tumor. Figure 6(a) is a photograph of the bioluminescence of U87MG-Luc cells observed with IVIS on day 16. Figure 6(b) shows the results of measuring the bioluminescence of U87MG-Luc cells with IVIS on days 4, 10, and 16. After 16 days of bioluminescence observation, the group that received glioblastoma transplantation but no treatment continued to show increased bioluminescence. This indicates that a large number of transplanted brain tumor cells survived. While many brain tumor cells survived when only antibodies were administered, the bioluminescence of brain tumor cells decreased when a complex of the blood-brain barrier-penetrating peptide CGGGG-SEQ ID NO: 41 and the antibody SEQ ID NO: 59 was injected.

[0100] The same animal model was injected with a complex of the CGGGG-SEQ ID NO: 41 blood-brain barrier-penetrating peptide labeled with cy5.5 and the antibody labeled with SEQ ID NO: 59, 8 hours before sacrificing, and the brain was removed. After freezing, the fluorescence distribution was observed using a confocal microscope. Figure 6(c) confirms that the complex of the CGGGG-SEQ ID NO: 41 blood-brain barrier-penetrating peptide labeled with cy5.5 and the antibody labeled with SEQ ID NO: 59 was distributed in the area where the brain tumor developed.

[0101] Example 3: Preparation of a blood-brain barrier permeable peptide and a complex of RNA-binding peptide and siRNA. Nanoparticles were produced by self-assembly of siRNAs of SEQ ID NO: 60 and 61, RNA-binding peptide of SEQ ID NO: 62, and blood-brain barrier-penetrating peptide of SEQ ID NO: 40, mixed in a fixed ratio. The RNA-binding peptide and blood-brain barrier-penetrating peptide were dissolved at a concentration of 5 μg / μL in RNase, DNase-free, PCR-certified water (Tech&Innovation, BWA-8000). siRNA was dissolved at a concentration of 1 μg / μL in RNase, DNase-free, PCR-certified water (Tech&Innovation, BWA-8000). The mixing ratio of siRNA solution to blood-brain barrier-penetrating peptide was 1:10 to 1:20. 10 μL of siRNA was mixed with 12 μL of blood-brain barrier-penetrating peptide solution (1:10) in 2 μL portions, and left to stand for 30 minutes or more. The blood-brain barrier-penetrating peptides SEQ ID NOs. 63-40, 64-40, 65-40, 66-40, and 67-40 were also complexed with the siRNA of SEQ ID NOs. 60 and 61 using the same method as described above.

[0102] Figure 7 shows the results of electrophoresis confirming the complex of the blood-brain barrier-penetrating peptide and siRNA. It was confirmed that siRNA alone moves downward, but the complex does not.

[0103] Example 4: Preparation of blood-brain barrier permeable peptides and complexes of cationic peptides and siRNAs Example 4-1: Preparation of a complex of a blood-brain barrier permeable peptide containing a transferrin receptor-binding peptide and KRAS siRNA. The cationic peptide of SEQ ID NO: 9 was dissolved at a concentration of 5 μg / μL in RNase, DNase-free, PCR-certified water (Tech&Innovation, BWA-8000). The siRNAs of SEQ ID NO: 60 and 61 were dissolved at a concentration of 1 μg / μL in RNase, DNase-free, PCR-certified water (Tech&Innovation, BWA-8000). The mixing ratio of the siRNA solution to the peptide of SEQ ID NO: 9 was set to 1:10. 10 μL of siRNA was mixed with 12 μL of the peptide solution of SEQ ID NO: 9 (1:10), divided into 2 μL portions, and left to stand for at least 30 minutes.

[0104] The blood-brain barrier-penetrating peptide of SEQ ID NO: 70 was dissolved at a concentration of 1 μg / μL in RNase, DNase-free, PCR-certified water (Tech&Innovation, BWA-8000). 12 μL of the blood-brain barrier-penetrating peptide solution of SEQ ID NO: 70 was added to a mixture of siRNA and the peptide of SEQ ID NO: 9, in 2 μL portions, and the mixture was left to stand for at least 30 minutes. The formed complex was confirmed using a transmission electron microscope (TEM, JEOL, JEM 1010) (Figure 8(a)).

[0105] Example 4-2: Preparation of a complex of a blood-brain barrier permeable peptide containing a transferrin receptor-binding peptide and β-catenin siRNA. RNA-binding peptide of sequence number 64 was dissolved at a concentration of 5 μg / μL in RNase, DNase-free, PCR-certified water (Tech&Innovation, BWA-8000). β-catenin siRNA (Dhamacon, #J-040628-05) was dissolved at a concentration of 1 μg / μL in RNase, DNase-free, PCR-certified water (Tech&Innovation, BWA-8000). The mixing ratio of siRNA solution to RNA-binding peptide was set to 1:10. 10 μL of siRNA was mixed with 12 μL of RNA-binding peptide solution (1:10), divided into 1 μL portions, and left to stand for at least 30 minutes.

[0106] The blood-brain barrier-penetrating peptide of SEQ ID NO: 70 was dissolved at a concentration of 1 μg / μL in RNase, DNase-free, PCR-certified water (Tech&Innovation, BWA-8000). 12 μL of the blood-brain barrier-penetrating peptide solution of SEQ ID NO: 70 was added to a mixture of β-catenin siRNA and RNA-binding peptide of SEQ ID NO: 64, in 1 μL increments, and the mixture was allowed to stand for at least 30 minutes. The formed complex was confirmed using a transmission electron microscope (TEM, JEOL, JEM 1010) (Figure 8(b)).

[0107] Example 4-3: Preparation of a complex of blood-brain barrier-penetrating peptides containing β-catenin siRNA and LRP-binding peptide. The peptide of SEQ ID NO: 64 was dissolved in RNase, DNase-free & PCR-certified water (Tech&Innovation, BWA-8000) at a concentration of 5 μg / μL. The siRNAs of SEQ ID NO: 60 and SEQ ID NO: 61 were dissolved in RNase, DNase-free & PCR-certified water (Tech&Innovation, BWA-8000) at a concentration of 1 μg / μL. The mixing ratio of the siRNA solution to the peptide carrier was set to 1:10. 1 μL each of the peptide solution of SEQ ID NO: 64 (1:10) totaling 12 μL was mixed into 10 μL of the siRNA, and the mixture was left to stand for 30 minutes or more. The blood-brain barrier permeation peptide of SEQ ID NO: 84 was dissolved in RNase, DNase-free & PCR-certified water (Tech&Innovation, BWA-8000) at a concentration of 1 μg / μL. 2 μL each of the blood-brain barrier permeation peptide solution of SEQ ID NO: 84 totaling 12 μL was mixed into the mixture of siRNA and the peptide of SEQ ID NO: 64, and the mixture was left to stand for 30 minutes or more. The formed complex was confirmed using a transmission electron microscope (TEM, JEOL, JEM 1010) (Figure 8(c)).

[0108] Figure 8 shows the results of confirming the complex of the blood-brain barrier permeation peptide and siRNA with a transmission electron microscope, and it was confirmed that each prepared complex forms particles with a size of 100 nm.

[0109] Experimental Example 4: Confirmation of permeability in an in vitro BBB model Alexa Fluor TM 488 (Invitrogen)-labeled siRNAs of SEQ ID NO: 60 and SEQ ID NO: 61 were used to prepare a complex with the blood-brain barrier permeation peptide by the same method as in Example 4-1, and the permeability was evaluated using an in vitro BBB model. As a control group, Lipofectamine TM 2000 (Invitrogen) was reacted with the Alexa Fluor TM 488 (Invitrogen)-labeled siRNAs of SEQ ID NO: 60 and SEQ ID NO: 61 according to the manufacturer's protocol. Immortalized mouse brain endothelial cells (bEnd.3, CRL-2299, ATCC) 5×10 4The cells were seeded and placed in 24 wells. 800 μL of Dulbecco's modified Eagle medium (DMEM) was placed in the lower chamber. The medium was changed every two days. Cell layer density was monitored using a transendothelial electrical resistance (TEER) analyzer (World Precision Instruments, Inc., Sarasota), and the TEER value was 200 Ω cm. 2 The experiment was conducted under the above conditions. The culture medium was replaced with DMEM that did not contain FBS, and a complex of the blood-brain barrier permeable peptide of SEQ ID NO: 70 labeled with Cy5.5 and the siRNAs of SEQ ID NO: 60 and SEQ ID NO: 61, and lipofectamine were added. TM siRNA complexes of 2000, SEQ ID NO: 60, and SEQ ID NO: 61 were added to the upper chamber at a concentration of 200 nM each. The assembled transwell was left in a shaking incubator at 37°C and 50 rpm for 1, 4, and 8 hours. 500 μL was taken from the lower chamber (basolateral compartment) and refilled with the same volume of fresh medium. The TEER value was checked again at the end of the experiment. The transport ratio (%) of the transmitted complexes was measured by fluorescence sensitivity using a spectrofluorometer (Thermo Scientific, USA). Examples 4-2 and 4-3 also had their permeability measured in an in vitro BBB model using the same method as described above.

[0110] Figure 9 shows the results of the permeability of blood-brain barrier permeable peptide and siRNA complexes permeated in an in vitro BBB model. The permeability of the complex from Example 4-1 increased with increasing time (Figure 9(a)). Furthermore, 8 hours after treatment of cells, the permeability was 6 times higher than that of the complex of lipofectamine and siRNA of SEQ ID NO: 60 and SEQ ID NO: 61. The complexes from Examples 4-2 and 4-3 also showed higher permeability up to 4 hours than lipofectamine. TM It was found that the degree of transmission was 8 to 10 times higher (Figures 9(b) and 9(c)).

[0111] This means that the complex in Example 4 effectively allows siRNA to pass through the blood-brain barrier (BBB).

[0112] Experimental Example 5: Confirmation of the neurotransmission effect of blood-brain barrier-penetrating peptides and siRNA complexes in normal animals. Alexandrite TM Using siRNAs of SEQ ID NO: 60 and SEQ ID NO: 61, which are labeled with 680 (Invitrogen), complexes were prepared with blood-brain barrier permeability peptides in the same manner as in Example 4-1.

[0113] Hairless mice (SKH-1, female, 6 weeks old, Orient Bio) were intravenously injected with the complex from Example 4-1 and siRNAs of SEQ ID NO: 60 and SEQ ID NO: 61 at a dose of 150 μg / kg each. After 5 hours, the brains were removed and fluorescence was observed using a confocal microscope (Carl Zeiss LSM700).

[0114] Alexandrite TM Using β-catenin siRNA labeled with 488 (Invitrogen), a complex was prepared with a blood-brain barrier-penetrating peptide in the same manner as in Examples 4-2 and 4-3.

[0115] Hairless mice (SKH-1, female, 6 weeks old, Orient Bio) were intravenously injected with a complex of Examples 4-2 and 4-3 and β-catenin siRNA at 1 mg / kg each. One hour later, the brains were removed and fluorescence was observed using a confocal microscope (Carl Zeiss LSM700).

[0116] Figure 10 shows the results of observing the blood-brain barrier-penetrating peptide and siRNA complex that were permeated into the brain parenchyma. It was found that the complex from Example 4-1 effectively delivered siRNA of SEQ ID NO: 60 and SEQ ID NO: 61 into the brain (Figure 10(a)). Furthermore, it was found that the complexes from Examples 4-2 and 4-3 also effectively delivered β-catenin siRNA into the brain (Figure 10(b)).

[0117] Experimental Example 6: Confirmation of β-catenin gene suppression in brain tissue by a blood-brain barrier permeable peptide and β-catenin siRNA complex in normal animals. We observed whether β-catenin siRNA transmitted to brain tissue effectively reduces β-catenin mRNA.

[0118] Eight-week-old mice (C57BL / 6, Orient Bio) were purchased, and the complex was prepared using the same method as in Examples 4-2 and 4-3. The complex was then intravenously injected at doses of 2.5 mg / kg and 10 mg / kg (siRNA-based). At 24 and 96 hours after injection, the brain tissue was perfused with 20 ml of PBS three times to remove blood. The cerebral cortex, cerebellum, and midbrain were isolated, and β-catenin mRNA in the brain tissue was measured by qRT-PCR (Thermo Fisher - QuantStudio 3). The PCT conditions and primer sequences used are shown in Tables 2 and 3, respectively.

[0119] [Table 2]

[0120] [Table 3]

[0121] Figure 11 shows the results of measuring β-catenin mRNA in each brain region. Both the complexes in Examples 4-2 (Figure 11(a)) and 4-3 (Figure 11(b)) effectively reduced β-catenin mRNA. β-catenin mRNA decreased more significantly at 96 hours after injection than at 24 hours, and the levels of β-catenin mRNA decreased as the administered dose increased. Furthermore, the similar reduction in β-catenin mRNA levels across different brain regions confirms that the complex was transmitted to each brain region. [Industrial applicability]

[0122] The blood-brain barrier-penetrating peptide of the present invention, in which a cell-permeable peptide is linked to a cerebral vascular endothelial cell surface protein-binding peptide, passes through the cell membrane of cerebral vascular endothelial cells with excellent efficiency. Furthermore, the peptide-drug conjugate, in which a drug such as an antibody, protein, or therapeutic nucleic acid is linked to the blood-brain barrier-penetrating peptide, has the advantage of effectively delivering the drug to the brain parenchyma, thereby maximizing the therapeutic effect on brain diseases and brain tumors.

[0123] Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A blood-brain barrier (BBB) ​​permeable peptide in which a peptide that binds to cerebral vascular endothelial cell surface proteins and a cell-permeable peptide are linked.

2. The blood-brain barrier permeable peptide according to claim 1, characterized in that the cerebral vascular endothelial cell surface protein is a transferrin receptor or a low-density lipoprotein receptor-related protein (LRP) receptor.

3. The blood-brain barrier permeable peptide according to claim 2, characterized in that the transferrin receptor-binding peptide is represented by one amino acid sequence from SEQ ID NO: 1 to SEQ ID NO:

4.

4. The blood-brain barrier-penetrating peptide according to claim 2, characterized in that the low-density lipoprotein receptor-associated protein receptor-binding peptide is represented by the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

5. The cell-permeable peptide is represented by one amino acid sequence from SEQ ID NOs: 7 to SEQ ID NOs: 10, as described in claim 1, for use as a blood-brain barrier permeable peptide.

6. The blood-brain barrier-penetrating peptide according to claim 1, characterized in that the aforementioned blood-brain barrier-penetrating peptide is represented by one amino acid sequence from SEQ ID NO: 11 to SEQ ID NO:

58.

7. The blood-brain barrier-penetrating peptide according to claim 1, characterized in that the cerebral vascular endothelial cell surface protein-binding peptide and the cell-permeable peptide are linked via spacers consisting of 2 to 10 amino acids G and 1 to 5 amino acids S.

8. A blood-brain barrier-penetrating peptide-drug conjugate comprising a drug bound to a blood-brain barrier-penetrating peptide according to any one of claims 1 to 7.

9. The blood-brain barrier-penetrating peptide-drug conjugate according to claim 8, characterized in that the drug is an antibody, a protein, or a therapeutic nucleic acid.

10. The blood-brain barrier-penetrating peptide-drug complex according to claim 8, characterized in that the blood-brain barrier-penetrating peptide and the drug are linked by a linker.

11. The blood-brain barrier-penetrating peptide-drug conjugate according to claim 10, characterized in that, when the drug is an antibody, the linker is CGGGG.

12. The blood-brain barrier-penetrating peptide-drug conjugate according to claim 9, characterized in that the antibody is represented by the amino acid sequence of SEQ ID NO:

59.

13. The peptide-drug conjugate for blood-brain barrier penetration according to claim 9, characterized in that the protein is selected from the group consisting of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotropin-4 / 5, fibroblast growth factor (FGF)-2 and other FGFs, neurotropin (NT)-3, erythropoietin (EPO), epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF), neuruturin, platelet-derived growth factor (PDGF), heregulin, neuregulin, artemin, percefin, interleukin, GDNF family receptor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, and stem cell factor (SCF).

14. The peptide-drug conjugate for blood-brain barrier penetration according to claim 9, characterized in that the therapeutic nucleic acid is selected from the group consisting of DNA, siRNA, miRNA, and mRNA.

15. The peptide-drug conjugate for blood-brain barrier penetration according to claim 14, characterized in that the siRNA is represented by SEQ ID NO: 60 and SEQ ID NO:

61.

16. The blood-brain barrier-penetrating peptide-drug complex according to claim 9, characterized in that the therapeutic nucleic acid is reacted with a cationic peptide, a cationic polymer, or an RNA-binding peptide to produce a primary complex, and the primary complex is reacted with the blood-brain barrier-penetrating peptide according to claim 1, which is linked to an anionic amino acid or anionic polymer and a spacer, to form nanoparticles.

17. The blood-brain barrier-penetrating peptide-drug complex according to claim 16, characterized in that the cationic peptide is represented by the amino acid sequence of SEQ ID NO: 9, the cationic polymer is poly-L-lysine, polyethyleneimine, or chitosan, the RNA-binding peptide is represented by one amino acid sequence from SEQ ID NO: 62 to SEQ ID NO: 67, the anionic amino acid is glutamic acid or aspartic acid, the anionic polymer is heparin or hyaluronic acid, and the spacer is 5 to 10 glycine molecules.

18. A nucleic acid encoding a peptide-drug complex for blood-brain barrier penetration according to claim 8.

19. A recombinant vector into which the nucleic acid described in claim 18 has been introduced.

20. Recombinant cells into which the nucleic acid according to claim 18 or the recombinant vector according to claim 19 has been introduced.

21. A method for producing a blood-brain barrier-penetrating peptide-drug conjugate according to claim 8, comprising the following steps: (a) Culturing recombinant cells according to claim 20 and expressing a blood-brain barrier-penetrating peptide-drug complex according to claim 8; and (b) A step of recovering the expressed blood-brain barrier-penetrating peptide-drug complex.

22. A pharmaceutical composition for the treatment or prevention of brain diseases or brain tumors, comprising a blood-brain barrier-penetrating peptide-drug complex as described in claim 8.

23. The pharmaceutical composition according to claim 22, characterized in that the brain disease is selected from the group consisting of encephalitis, Parkinson's disease, epilepsy, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, spinocerebellar degeneration, cerebellar atrophy, multiple sclerosis, senile dementia resulting from loss of nerve cells, stroke, amnesia syndrome, depression, and post-traumatic stress disorder.

24. The pharmaceutical composition according to claim 22, characterized in that the brain tumor is selected from the group consisting of glioma, glioblastoma multiforme, meningioma, astrocytoma, acoustic neuroma, chondroma, oligodendroglioma, medulloblastoma, ganglioglioma, schwannoma, neurofibroma, neuroblastoma, and epidural, intramedullary, or intradural tumors.

25. The pharmaceutical composition according to claim 22, characterized in that the single dose of the blood-brain barrier permeable peptide-drug conjugate is 1 μg / kg to 100 mg / kg.

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