Novel LRP1-binding peptide

Abstract

Disclosed is a linear peptide or cyclic peptide having an amino acid sequence represented by formula (1): XN-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15 (1), or a pharmacologically acceptable salt thereof. Binding this peptide to LRP1 makes it possible to deliver any drug to the brain tissue through LRP1-mediated transcytosis.

Description

Cross References

[0001] This application claims priority based on Patent Application No. 2022-125238 filed in Japan on August 5, 2022, and all contents of that application are incorporated herein by reference in their entirety. [Technical field]

[0002] The present invention relates to peptides that bind to LRP1, etc. The present invention relates to peptides that can pass through barrier tissues that separate peripheral tissues and brain tissues, such as the blood-brain barrier (BBB), via binding to LRP1, etc. In particular, the present invention relates to linear or cyclic peptides having a specific amino acid sequence, etc. [Background technology]

[0003] Central nervous system diseases (mental diseases), such as dementia, schizophrenia, and autism spectrum disorders, are estimated to affect 1 billion people worldwide, accounting for the largest number of disability-adjusted life years, and therefore impose a huge social burden. For example, it has been estimated that the combined economic burden in the United States and Europe in 2030 will be $6 trillion (Non-Patent Document 1). Given this background, it is expected that success in drug discovery for central nervous system diseases will lead to a huge market, but it is also known to be the drug discovery field with the highest failure rate (Non-Patent Document 1). One of the reasons for this is the presence of barrier tissues that separate peripheral tissues and brain tissues in the body: the blood-brain barrier, blood-cerebrospinal fluid barrier, blood-spinal cord barrier, and blood-arachnoid barrier. Among these, the blood-brain barrier (BBB) ​​in particular is a hurdle in drug discovery for central nervous system diseases. The blood-brain barrier is a physical barrier formed by pericytes and astrocytes surrounding brain capillary endothelial cells connected by tight junctions, and strictly controls the inflow and outflow of substances between peripheral tissues and brain tissues (Non-Patent Document 2). For example, it is known that substances that migrate from peripheral tissues to brain tissues by passive diffusion should preferably have a molecular weight of 400 g / mol or less and be highly lipid-soluble. For this reason, many attempts at drug discovery for central nervous system diseases have been made with low-molecular-weight compounds, but there are concerns about side effects due to off-target effects, and drug discovery targets are limited.

[0004] In particular, for drug discovery targets such as protein-protein interactions, which have attracted attention in recent years, there are few clear pockets to which conventional low molecular weight compounds can bind, and medium molecules such as peptides and polymers such as antibodies are better able to control them. In addition, because medium molecules and polymers have high specificity for drug discovery targets, it is expected that side effects due to off-target effects will be reduced. However, as mentioned above, due to the presence of barrier tissues, it is extremely difficult for medium molecules and polymers to migrate from peripheral tissues to brain tissues by passive diffusion, which is an issue.

[0005] To date, receptor-mediated transcytosis (RMT) has been studied as a method for transferring medium-sized molecules and polymers from peripheral tissues to brain tissues in order to transfer specific nutrients and essential components to the brain, using receptors expressed on the blood-brain barrier. Examples include transporters that use glucose or amino acids as substrates, and receptors for insulin, transferrin, and lipoproteins (Non-Patent Document 2). There are already drugs on the market that attempt to conjugate an antibody that binds to the human transferrin receptor with a medicinal molecule and transport the medicinal molecule to central tissues, and the concept of RMT has been demonstrated in human clinical trials (Non-Patent Document 3).

[0006] Low density lipoprotein receptor-related protein 1 (LRP1) is also one of the receptors expected to be involved in RMT. The expression levels of LRP1 at the blood-brain barrier are well correlated between humans, monkeys, and mice. In addition, the amino acid sequences of cluster 2 (CL2) and cluster 4 (CL4), which are ligand-binding domains that interact with various endogenous ligands, are highly conserved among species. Furthermore, a molecule conjugated to the anticancer drug paclitaxel and Angiopep-2 (ANG2), known as an LRP1-binding peptide, has completed phase II of human clinical trials, demonstrating the safety of RMT by LRP1 in humans (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] L Ereshefsky,R Evans,R Sood,Dwilliamson And BA English.“VENTURING INTO A NEW ERA OF CNS DRUG DEVELOPMENT TO IMPROVE SUCCESS”2015. [Non-Patent Document 2] VM Pulgar.Front Neurosci.2019,12:1019. [Non-licensed document 3] R Yamamoto and S Kawashima.Nihon Yakugaku Zasshi.2022,157(1):62-75.

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0008] The invention described herein aims to provide a novel peptide that binds to LRP1, and further aims to provide a method for using a "certain molecule" as a pharmaceutical, diagnostic, and / or research reagent by combining the peptide with the "certain molecule" and delivering the "certain molecule" to brain tissue via RMT by LRP1. [Means for solving the problem]

[0009] The present inventors have searched for molecules that bind to LRP1, and have completed the present invention. That is, one aspect of the present invention is a compound represented by the following formula (1): X N -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 (1) is a linear or cyclic peptide having an amino acid sequence represented by the formula (1), or a pharmacologically acceptable salt thereof.

[0010] Here, in formula (1), X N is any 1 to 4 amino acid residues, and X 5 and X 15 each independently represents serine, threonine, cysteine, D-cysteine, or proline; X 6 represents serine, homoserine, threonine, allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, lysine, arginine, proline, cis-4-hydroxy-proline, or trans-4-hydroxy-proline; X7 represents histidine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 8 represents ornithine, lysine, homolysine, arginine, or homoarginine; X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 10 represents methionine, leucine, norleucine, isoleucine, valine, lysine, or arginine; X 11 and X 14 each independently represents methionine, leucine, norleucine, isoleucine, valine, 2-aminoheptanoic acid, or 2-aminooctanoic acid; X 12 represents alanine, D-alanine, or 2-aminoisobutyric acid, and X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine, or glutamic acid, the N-terminal amino group and the C-terminal carboxy group may be modified or deleted, and X 5 and X 15 is cysteine ​​or D-cysteine ​​when involved in cyclization, and forms a covalent bond between the -SH groups in the side chains via a disulfide bond, or a linker of a methylene group, an acetylmethylene group, an ethylene group, or a propylene group, so that the peptide of formula (1) may have one cyclic structure within the molecule.

[0011] In the peptide of the above formula (1), X 1 From X 15 Preferred aspects of each amino acid residue will be described in detail in the following embodiments, but each preferred embodiment can be independently combined in any combination.

[0012] More specifically, the present invention includes the following embodiments. [1] The above formula (1): XN -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 (1) (In the formula, X N is any 1 to 4 amino acid residues, and X 5 and X 15 each independently represents serine, threonine, cysteine, D-cysteine, or proline; X 6 represents serine, homoserine, threonine, allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, lysine, arginine, proline, cis-4-hydroxy-proline, or trans-4-hydroxy-proline; X 7 represents histidine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 8 represents ornithine, lysine, homolysine, arginine, or homoarginine; X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 10 represents methionine, leucine, norleucine, isoleucine, valine, lysine, or arginine; X 11 and X 14 each independently represents methionine, leucine, norleucine, isoleucine, valine, 2-aminoheptanoic acid, or 2-aminooctanoic acid; X 12 represents alanine, D-alanine, or 2-aminoisobutyric acid, and X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine, or glutamic acid, the N-terminal amino group and the C-terminal carboxy group may be modified or deleted, and X 5and X 15 is cysteine ​​or D-cysteine ​​when involved in cyclization, and forms a covalent bond between the -SH groups in their side chains via a linker such as a disulfide bond, a methylene group, an acetylmethylene group, an ethylene group, or a propylene group, so that the peptide of formula (1) may have one cyclic structure in the molecule. [2] The following formula (3): X N -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -Pro-X 16 (3) (In the formula, X N is any 1 to 4 amino acid residues, and X 5 and X 16 each independently represents serine, threonine, cysteine, D-cysteine, or proline; X 6 represents serine, homoserine, threonine, allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, lysine, arginine, proline, cis-4-hydroxy-proline, or trans-4-hydroxy-proline; X 7 represents histidine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 8 represents ornithine, lysine, homolysine, arginine, or homoarginine; X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 10represents methionine, leucine, norleucine, isoleucine, valine, lysine, or arginine; X 11 and X 14 each independently represents methionine, leucine, norleucine, isoleucine, valine, 2-aminoheptanoic acid, or 2-aminooctanoic acid; X 12 represents alanine, D-alanine, or 2-aminoisobutyric acid, and X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine, or glutamic acid, the N-terminal amino group and the C-terminal carboxy group may be modified or deleted, and X 5 and X 16 is cysteine ​​or D-cysteine ​​when involved in cyclization, and forms a covalent bond between the -SH groups in their side chains via a linker such as a disulfide bond, a methylene group, an acetylmethylene group, an ethylene group, or a propylene group, thereby allowing the peptide of formula (3) to have one cyclic structure in the molecule. [3] The following formula (3): X N -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -Pro-X 16 (3) (In the formula, X N is any 1 to 4 amino acid residues, and X 6 and X 16 each independently represents serine, threonine, cysteine, D-cysteine, or proline; X 5 represents serine, homoserine, threonine, allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, lysine, arginine, proline, cis-4-hydroxy-proline, or trans-4-hydroxy-proline; X 7represents histidine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 8 represents ornithine, lysine, homolysine, arginine, or homoarginine; X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 10 represents methionine, leucine, norleucine, isoleucine, valine, lysine, or arginine; X 11 and X 14 each independently represents methionine, leucine, norleucine, isoleucine, valine, 2-aminoheptanoic acid, or 2-aminooctanoic acid; X 12 represents alanine, D-alanine, or 2-aminoisobutyric acid, and X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine, or glutamic acid, the N-terminal amino group and the C-terminal carboxy group may be modified or deleted, and X 6 and X 16 is cysteine ​​or D-cysteine ​​when involved in cyclization, and forms a covalent bond between the -SH groups in their side chains via a linker such as a disulfide bond, a methylene group, an acetylmethylene group, an ethylene group, or a propylene group, thereby allowing the peptide of formula (3) to have one cyclic structure in the molecule. [4] X N is Lys-Gly-Thr-Pro or Gly-Thr-Pro, or a pharmacologically acceptable salt thereof. [5] A linear or cyclic peptide having LRP1 binding activity, comprising an amino acid sequence in which one or more amino acids are deleted, added, and / or substituted in the amino acid sequence according to any one of [1] to [4], or a pharmacologically acceptable salt thereof. [6] A derivative and / or modified form of the peptide according to any one of [1] to [5] and

[14] to

[21] below. [7] The peptide derivative or modified product according to [6], wherein the derivative or modified product has an azide group, an alkyne group, a cyclooctyne group, or a tetrazine group at the N-terminus, the C-terminus, or the side chain of an amino acid, either directly or via a linker. [8] [1] to [7], or a pharmaceutical, diagnostic agent, and / or reagent comprising the peptide or its derivative or modified form according to any one of

[14] to

[21] below. [9] A peptide-drug conjugate in which the derivative or modified form of the peptide according to [6] is linked to a drug directly or via a linker.

[10] The peptide-drug conjugate according to [9], wherein the drug is an antibody, a nucleic acid, or a physiologically active peptide.

[11] A polynucleotide encoding an amino acid sequence consisting only of natural amino acids among the amino acid sequences described in any one of [1] to [5] and

[14] to

[21] below, or a polynucleotide encoding a peptide having a base sequence with 70% or more sequence identity to the polynucleotide and having LRP1 binding activity.

[12] An expression vector comprising the polynucleotide according to

[11] .

[13] A cell, yeast, bacterium, virus, liposome, or nanoparticle presenting an amino acid sequence according to any one of [1] to [5] and

[14] to

[21] below.

[14] The following formula (6): X 1 -X 2 -X 3 -X 4-X L -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X M (6) (X L X 5 -X 6 and X M X 15 -X 16 When involved in the cyclization within the peptide molecule, each independently represents an amino acid residue having an amino group, a carboxy group, a thiol group, an allyl group, an alkynyl group, an azide group, or a halogen atom, or a derivative thereof. When not involved in the cyclization within the peptide molecule, X 5 , X 6 , or X 15 each independently represents any amino acid residue or a derivative thereof; X 16 represents any amino acid residue, derivative or deletion thereof, and X 7 represents an amino acid residue having a hydrocarbon group which may be substituted, an amino acid residue having an aromatic carbocyclic group which may be substituted, an amino acid residue having an aromatic heterocyclic group which may be substituted, or a derivative thereof; X 8 represents a positively charged amino acid residue or a derivative thereof, and X 11 , X 12 and X 14 each independently represents an amino acid residue having a hydrocarbon group which may be substituted, or a derivative thereof; X 13 represents an amino acid residue having a hydrocarbon group which may be substituted, an amino acid residue having a negative charge, or a derivative thereof; X 9 , X 10 each independently represents any amino acid residue or a derivative thereof; X 1 , X 2 , X 3 , X 4 each independently represents any amino acid residue, derivative, or deletion thereof; X L and XM The peptide of formula (6) may have one cyclic structure in the molecule by forming a direct covalent bond between X or an indirect covalent bond via a linker. In this case, L and X M The covalent bond between may be any of main chain-to-main chain, main chain-to-side chain, side chain-to-main chain, or side chain-to-side chain, and the N-terminal amino group and the C-terminal carboxy group may be modified or deleted. A linear peptide or cyclic peptide having an amino acid sequence represented by the formula:

[15] X 5 and X 15 The bond between the Cα carbon atoms of X 6 and X 15 The bond between the Cα carbon atoms of X 5 and X 16 The bond between the Cα carbon atoms of 6 and X 16 The bonds between the Cα carbon atoms are each independently represented by the following formula (7): Cα L -(CH2) A -Z-(CH2) B -Cα M (7) (In the formula, L is 4 or 5, M is 14 or 15, A and B each independently represent an integer of 0 to 2, the sum of A and B is an integer of 0 to 4, Z is SS, CH2-CH2, S-CH2, CH2-S, O-CH2, CH2-O, CH=CH, NH-C(=O), N(CH3)-C(=O), NH-C(=S), N(CH3)-C(=S), OC(=O), C(=O)-NH, C(=O)-N(CH3), C(=S)-NH, C(=S)-N(CH3), C(=O)-O, CH2-CH2-CH2, S-CH2-CH2, CH2-CH2-S, CH2-S- CH2, O-CH2-CH2, CH2-CH2-O, CH2-O-CH2, S-CH2-S, SC(=CH2)-S, S-(C(=O)-CH3)-S, S-(CH2)2-S, S-(CH2)3-S, S-CH2-C(=O)-CH2-S, S-CH2-C(=CH2)-CH2-S, S-(CH2)4-S, S-CH2-CH=CH-CH2-S, S-CH2-C6H4-CH2-S (the bonding position of the methylene group to the phenylene ring may be any of ortho, meta and para), S-CH2-C(=O)-CH2-S, or S-CH2-C(=CH2)-CH2-S.) The linear peptide or cyclic peptide according to

[14] , comprising a structure represented by the following formula:

[16] X 8 The linear peptide and cyclic peptide according to claim 14 or 15, wherein is 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, lysine, homolysine, arginine, homoarginine, D-amino acids thereof, N-methylated amino acids thereof, α-methylated amino acids thereof, or derivatives thereof, or a pharmacologically acceptable salt thereof.

[17] X 12is alanine, D-alanine, 2-aminoisobutyric acid, 2-aminobutanoic acid, D-2-aminobutanoic acid, isovaline, D-isovaline, valine, D-valine, isoleucine, D-isoleucine, leucine, D-leucine, N-methylated amino acids thereof, α-methylated amino acids thereof, or derivatives thereof. The linear peptide and cyclic peptide according to any one of

[14] to

[16] , or a pharmacologically acceptable salt thereof.

[18] X 11 and X 14 are independently methionine, the following formula (8): [ka] (In the formula, the wavy line represents the attachment point with a carbonyl group or nitrogen atom that forms an amide bond in the main chain, and R 1 , R 2 and R 6 each independently represents a hydrogen atom or a methyl group; R 3 , R 4 , R 5 each independently represents a hydrogen atom, a methyl group, or a halogen atom (F, Cl, Br, I), and n represents an integer of 0 to 10; or The following formula (9) [ka] (Wherein, Z represents C or N, R 6 represents a hydrogen atom or a methyl group, and n represents an integer of 1 to 6; or The following formula (10) [ka] (In the formula, the wavy line represents a bonding site with a carbonyl group or a nitrogen atom that forms an amide bond in the main chain, Z represents C or N, R 6 represents a hydrogen atom or a methyl group, and n represents an integer of 1 to 6. or a derivative thereof, or a pharmacologically acceptable salt thereof.

[19] X 7 However, the following formula (8): [ka] (In the formula, the wavy line represents the attachment point with a carbonyl group or nitrogen atom that forms an amide bond in the main chain, and R 1 , R 2 and R 6 each independently represents a hydrogen atom or a methyl group; R 3 , R 4 , R 5 each independently represents a hydrogen atom, a methyl group, or a halogen atom (F, Cl, Br, I), and n represents an integer of 0 to 10; or The following formula (9) [ka] (Wherein, Z represents C or N, R 6 represents a hydrogen atom or a methyl group, and n represents an integer of 1 to 6; or The following formula (10) [ka] (In the formula, the wavy line represents a bonding site with a carbonyl group or a nitrogen atom that forms an amide bond in the main chain, Z represents C or N, R 6 represents a hydrogen atom or a methyl group, and n represents an integer of 1 to 6; or The following formula (11) [ka] (In the formula, R 12represents a 2-thienyl group, a 3-thienyl group, a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 2-thiazolyl group, a 4-thiazolyl group, a 2-oxazolyl group, a 4-oxazolyl group, a 1-pyrazolyl group, a 1H-imidazol-4-yl group, a 1-imidazolyl group, a 1,2,3-triazol-1-yl group, or a 1,2,4-triazol-1-yl group; R 6 represents a hydrogen atom or a methyl group); or The following formula (12) [ka] (In the formula, the wavy line represents the attachment point with a carbonyl group or nitrogen atom that forms an amide bond in the main chain, and R 7 , R 8 , R 9 , R 10 , R 11 each independently represents a hydrogen atom, a hydroxyl group, a methoxy group, a methyl group, a tert-butyl group, a halogenated methyl group, or a halogen atom (F, Cl, Br, I); R 6 represents a hydrogen atom or a methyl group; or The following formula (13) [ka] (In the formula, R 13 , R 14 , R 15 , R 16 each independently represents a hydrogen atom, a hydroxyl group, a methoxy group, a methyl group, a tert-butyl group, a halogenated methyl group, or a halogen atom (F, Cl, Br, I); R 6 represents a hydrogen atom or a methyl group. The linear peptide or cyclic peptide according to any one of

[14] to

[18] , which is an amino acid residue represented by the following formula:

[20] X 13is glycine, alanine, aspartic acid, glutamic acid, asparagine, glutamine, D-alanine, D-aspartic acid, D-glutamic acid, D-asparagine, D-glutamine, N-methylated amino acids thereof, α-methylated amino acids thereof, or derivatives thereof, or a linear or cyclic peptide according to any one of

[14] to

[19] , or a pharmacologically acceptable salt thereof. 〔twenty one〕 X not involved in intramolecular cyclization L and X M are independently classified as alanine, isovaline, norvaline, serine, homoserine, threonine, allothreonine, O-methyl-serine, O-methyl-homoserine, O-methyl-threonine, O-methyl-allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, lysine, arginine, proline, cis-4-hydroxy-proline, trans-4-hydroxy-proline, azetidine-2-carboxylic acid, pipecolic acid, D-serine, D-homoserine, D-threonine, D-allothreonine, D-O-methyl-serine, D-O-methyl The linear peptide and cyclic peptide according to any one of

[14] to

[20] , which is N-homoserine, D-O-methylated-threonine, D-O-methylated-allothreonine, D-2,3-diaminopropionic acid, D-2,4-diaminobutanoic acid, D-ornithine, D-lysine, D-arginine, D-proline, D-cis-4-hydroxy-proline, D-trans-4-hydroxy-proline, D-azetidine-2-carboxylic acid, D-pipecolic acid, N-methylated amino acids thereof, α-methylated amino acids thereof, or derivatives thereof, or a pharmacologically acceptable salt thereof. Effect of the Invention

[0013] According to the present invention, novel linear and cyclic peptides are provided that are useful for transporting a "certain molecule" combined with the peptide into brain tissue by RMT via binding to LRP1 and for use as a pharmaceutical, diagnostic agent, and / or research reagent. [Brief description of the drawings]

[0014] [Figure 1] 1 shows the results of evaluating the concentration-dependent binding activity of the representative biotin-labeled peptides of the present invention, i.e., sequence 45 and sequence 46, to the LRP1 cluster 2 (CL2)-Fc chimeric protein, the LRP1 cluster 3 (CL3)-Fc chimeric protein, and the LRP1 cluster 4 (CL4)-Fc chimeric protein. The concentrations of the biotin-labeled peptides used in the experiment were (1) 1000 nM, (2) 330 nM, (3) 110 nM, (4) 37 nM, (5) 12 nM, (6) 4.1 nM, (7) 1.4 nM, and 0 nM for each protein, from the left, and are indicated by shading on the bars. [Diagram 2] 2 shows an outline of a competitive binding test by ELISA used to evaluate both the binding activity of the peptides of the present invention to LRP1 cluster 4 and their resistance to protease degradation. In the figure, SA represents streptavidin, B represents biotin, and HRP represents horseradish peroxidase. [Diagram 3] 3 shows the results of evaluating the binding activity (binding (%)) of sequence 46 (500 nM), a representative example of the biotin-labeled peptide of the present invention, to the LRP1 cluster 4-Fc chimeric protein in the presence of sequence 2 (2000 to 63 nM) immediately after mixing with mouse plasma or 24 hours later. The peptide concentrations of sequence 2 are (1) 2000 nM, (2) 1000 nM, (3) 500 nM, (4) 250 nM, (5) 130 nM, and (6) 63 nM, respectively, from the left, and are indicated by shading on the bars. [Figure 4] Figure 4 shows the results of evaluating the inhibitory activity (Inhibition (%)) of sequence 46 (500 nM), a representative example of a biotin-labeled peptide of the present invention, against LRP1 cluster 4-Fc chimeric protein in the presence of sequences 1 to 44 (2000 nM) immediately after mixing with mouse plasma or 24 hours later. [Diagram 5] FIG. 5 shows an overview of an in vitro blood-brain barrier model for assessing the ability to cross the blood-brain barrier. [Figure 6]FIG. 6 shows the results of evaluating the ability of Sequence 2, a representative example of the peptide of the present invention labeled with 5-FAM (fluorescent dye), to pass through the blood-brain barrier using in vitro blood-brain barrier models (rat and monkey types). [Figure 7] FIG. 7 shows the structural formulae of Sequence 2 and Sequence 46, which are representative examples of the peptides of the present invention used in the Examples. [Figure 8] FIG. 8 shows the results of RP-HPLC analysis of the modified peptide DBCO-KS-487 of the present invention (before reaction) and a solution reacted with 6-azido-hexanoic acid at room temperature (after reaction). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Next, each embodiment of the present invention will be described with reference to the drawings. Note that each embodiment described below does not limit the invention according to the claims, and all of the elements and combinations thereof described in each embodiment are not necessarily essential to the solution of the present invention.

[0016] (definition) In this specification, a peptide refers to a compound in which two or more amino acids are bonded by amide bonds (peptide bonds), and can be, for example, a compound in which 2 to 20 amino acids are bonded by amide bonds. In addition, according to the convention of peptide notation, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxy terminus). The first carbon atom adjacent to the carbonyl group that forms the peptide bond is called the Cα carbon.

[0017] As used herein, "any amino acid residue, or derivative thereof" is used in its broadest sense and includes, in addition to natural amino acids, artificial amino acids having unnatural structures, chemically synthesized compounds having properties known in the art that are characteristic of amino acids, and also carboxylic acids having functional groups. Examples of unnatural amino acids include D-amino acids, α / α-disubstituted amino acids with a backbone structure different from that of natural amino acids (such as α-methylated amino acids such as 2-aminoisobutyric acid), N-alkyl-amino acids (such as N-methylated amino acids), N-substituted glycines (peptoids), amino acids with extended backbones (such as β homoamino acids and γ homoamino acids), amino acids with a side chain structure different from that of natural amino acids (such as cyclohexylalanine, allylglycine, 2-(2-pyridyl)-glycine, and 3-(1H-benzimidazol-2-yl)-alanine), amino acids with partially substituted side chains (such as norleucine, diaminopropanoic acid, and 3-(2-pyridyl)-alanine), amino acids with extra functional groups in the side chain; amino acids with extra C, alkyl groups, or methyl groups in the side chain (such as homonorleucine and γ-methylleucine), amino acids with halogen atoms (F, Cl, Br, I) in the side chain (such as 3-chloro-alanine), and amino acids with halogen atoms (F, Examples of such amino acids include, but are not limited to, carboxylic acids having an additional N or amino group in the side chain (such as 3-chloropropanoic acid), carboxylic acids having a functional group in the side chain (such as 3-butenoic acid), amino acids having an extra N or amino group in the side chain (such as β-azidoalanine and ornithine), amino acids having an extra O or methoxy group in the side chain (such as O-methyl-serine and O-methyl-threonine), amino acids having an extra hydroxyl group in the side chain (such as 3-hydroxy-phenylalanine), amino acids having an extra carboxyl group (-COOH) in the side chain (such as 3-carboxy-phenylalanine), amino acids having an extra S in the side chain (such as ethionine), amino acids in which the carboxylic acid functional group in the side chain is protected with an ester (such as aspartic acid-4-methyl ester), and amino acids in which the thio group (-S-) in the side chain is oxidized to a sulfinyl group (-S(=O)-) or a sulfonyl group (-S(=O)2-) (methionine sulfoxide).

[0018] As used herein, "LRP1" refers to LRP1 from mammals such as mice, rats, dogs, monkeys, and humans.

[0019] As used herein, the term "having LRP1 binding activity" means that in an in vitro test, the peptide of the present invention or a combination of the peptide of the present invention and "a certain molecule" binds to the full-length LRP1 protein, partial proteins such as cluster 4, or chimeras of these and other proteins in a concentration-dependent manner. The presence or absence of LRP1 binding activity can be confirmed by those skilled in the art according to known methods, with reference to Non-Patent Document 5, etc., but is not limited thereto.

[0020] As used herein, the term "barrier tissue" refers to barrier tissue of mammals such as mice, rats, dogs, monkeys, and humans.

[0021] In the present specification, the term "passing through the barrier tissue" means (1) that in a test using an in vitro blood-brain barrier model, the peptide of the present invention or a combination of the peptide of the present invention and "a certain molecule" migrates from a well on the blood vessel side to a well on the brain side, (2) that in a pharmacokinetic test using LC-MS / MS or the like, the peptide of the present invention or a combination of the peptide of the present invention and "a certain molecule" administered peripherally is detected in brain tissue, and (3) that in a pharmacokinetic test using an in vivo imaging system or the like, the peptide of the present invention or a combination of the peptide of the present invention and "a certain molecule" administered peripherally is detected in the brain, etc., and if any one of these effects is exhibited, it is said to "pass through the barrier tissue". The presence or absence of the ability to pass through the barrier tissue can be confirmed by a person skilled in the art according to a known method with reference to Non-Patent Documents 6 and 7, etc., but is not limited thereto.

[0022] (Peptides of the Invention) [1] In one embodiment of the present disclosure, the linear and cyclic peptides have the following formula (1): X N -X 5 -X 6 -X 7 -X8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -X 15 (1) It has an amino acid sequence represented by the following formula: In formula (1), X N is any 1 to 4 amino acid residues, and X 5 and X 15 each independently represents serine, threonine, cysteine, D-cysteine, or proline; X 6 represents serine, homoserine, threonine, allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, lysine, arginine, proline, cis-4-hydroxy-proline, or trans-4-hydroxy-proline; X 7 represents histidine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 8 represents ornithine, lysine, homolysine, arginine, or homoarginine; X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 10 represents methionine, leucine, norleucine, isoleucine, valine, lysine, or arginine; X 11 and X 14 each independently represents methionine, leucine, norleucine, isoleucine, valine, 2-aminoheptanoic acid, or 2-aminooctanoic acid; X 12 represents alanine, D-alanine, or 2-aminoisobutyric acid, and X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine, or glutamic acid. The N-terminal amino group and the C-terminal carboxy group may be modified or deleted. X 5 and X 15is cysteine ​​or D-cysteine ​​when involved in cyclization, and forms a covalent bond between the -SH groups in the side chains via a disulfide bond, or a linker of a methylene group, an acetylmethylene group, an ethylene group, or a propylene group, so that the peptide of formula (1) may have one cyclic structure within the molecule.

[0023] In the peptide of the above formula (1), X N is preferably Lys-Gly-Thr-Pro or Gly-Thr-Pro. When the N-terminus is lysine, it may be bonded to a molecule via the ε-amino group of the side chain. When the N-terminus is glycine, it may be bonded to a molecule via the α-amino group. 5 and X 15 is preferably cysteine ​​or D-cysteine, and a disulfide bond or a covalent bond is formed between the -SH groups of each side chain via a linker of a methylene group, thereby forming a ring structure within the molecule.

[0024] A more preferred embodiment of the peptide of formula (1) above is, for example, an amino acid sequence represented by the following formula (2): A disulfide bond or a covalent bond may be formed between the -SH groups of each side chain via a methylene group linker, thereby forming a cyclic structure within the molecule, or the peptide may be bound to a certain molecule via the ε-amino group of Lys1.

[0025] Ac-Lys-Gly-Thr-Pro-Cys-X 6 -X 7 -Lys-X 9 -X 10 -Leu-Ala-Glu-X 14 -Cys-OH (2) In formula (2), X 6 represents serine, threonine, or 2,3-diaminopropionic acid; X 7 represents histidine, tyrosine, phenylalanine, or tryptophan; X 9 represents tyrosine, methionine, norleucine, or arginine; X10 represents methionine, norleucine, arginine, or lysine; X 14 represents leucine, isoleucine, methionine, or norleucine.

[0026] [2] In another embodiment of the present disclosure, the linear peptide and the cyclic peptide have the following formula (3): X N -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -Pro-X 16 (3) It has an amino acid sequence represented by the following formula: In the above formula (3), X N is any 1 to 4 amino acid residues, and X 5 and X 16 each independently represents serine, threonine, cysteine, D-cysteine, or proline; X 6 represents serine, homoserine, threonine, allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, lysine, arginine, proline, cis-4-hydroxy-proline, or trans-4-hydroxy-proline; X 7 represents histidine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 8 represents ornithine, lysine, homolysine, arginine, or homoarginine; X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 10 represents methionine, leucine, norleucine, isoleucine, valine, lysine, or arginine; X 11 and X14 each independently represents methionine, leucine, norleucine, isoleucine, valine, 2-aminoheptanoic acid, or 2-aminooctanoic acid; X 12 represents alanine, D-alanine, or 2-aminoisobutyric acid, and X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine, or glutamic acid. The N-terminal amino group and the C-terminal carboxy group may be modified or deleted. X 5 and X 16 is cysteine ​​or D-cysteine ​​when involved in cyclization, and forms a covalent bond between the -SH groups in the side chains via a disulfide bond, or a linker of a methylene group, an acetylmethylene group, an ethylene group, or a propylene group, so that the peptide of formula (3) may have one cyclic structure within the molecule.

[0027] In the peptide of formula (3), X N is preferably Lys-Gly-Thr-Pro or Gly-Thr-Pro. When the N-terminus is lysine, it may be bonded to a molecule via the ε-amino group of the side chain. When the N-terminus is glycine, it may be bonded to a molecule via the α-amino group. 5 and X 16 is preferably cysteine ​​or D-cysteine, and a disulfide bond or a covalent bond is formed between the -SH groups of each side chain via a linker of a methylene group, thereby forming a ring structure within the molecule.

[0028] A more preferred embodiment of the peptide of formula (3) above is, for example, an amino acid sequence represented by the following formula (4): A disulfide bond or a covalent bond via a methylene group linker may be formed between the -SH groups of each side chain, thereby forming a cyclic structure within the molecule, or the peptide may be bonded to a certain molecule via the ε-amino group of Lys1.

[0029] Ac-Lys-Gly-Thr-Pro-Cys-X 6 -X 7-Lys-X 9 -X 10 -Leu-Ala-Glu-X 14 -Pro-Cys-OH (4) In formula (4), X 6 represents serine, threonine, or 2,3-diaminopropionic acid; X 7 represents histidine, tyrosine, phenylalanine, or tryptophan; X 9 represents tyrosine, methionine, norleucine, or arginine; X 10 represents methionine, norleucine, arginine, or lysine; X 14 represents leucine, isoleucine, methionine, or norleucine.

[0030] [3] In another embodiment of the present disclosure, the linear peptide and the cyclic peptide are represented by the following formula (3): X N -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -Pro-X 16 (3) It has an amino acid sequence represented by the following formula: In the above formula (3), X N is any 1 to 4 amino acid residues, and X 6 and X 16 each independently represents serine, threonine, cysteine, D-cysteine, or proline; X 5 represents serine, homoserine, threonine, allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, lysine, arginine, proline, cis-4-hydroxy-proline, or trans-4-hydroxy-proline; X 7 represents histidine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X8 represents ornithine, lysine, homolysine, arginine, or homoarginine; X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyl-tyrosine, phenylalanine, 4-amino-phenylalanine, 4-fluoro-phenylalanine, or 4-chloro-phenylalanine; X 10 represents methionine, leucine, norleucine, isoleucine, valine, lysine, or arginine; X 11 and X 14 each independently represents methionine, leucine, norleucine, isoleucine, valine, 2-aminoheptanoic acid, or 2-aminooctanoic acid; X 12 represents alanine, D-alanine, or 2-aminoisobutyric acid, and X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine, or glutamic acid, the N-terminal amino group and the C-terminal carboxy group may be modified or deleted, and X 6 and X 16 is cysteine ​​or D-cysteine ​​when involved in cyclization, and forms a covalent bond between the -SH groups in the side chains via a disulfide bond, or a linker of a methylene group, an acetylmethylene group, an ethylene group, or a propylene group, so that the peptide of formula (3) may have one cyclic structure within the molecule.

[0031] In the peptide of formula (3), X N is preferably Lys-Gly-Thr-Pro or Gly-Thr-Pro. When the N-terminus is lysine, it may be bonded to a molecule via the ε-amino group of the side chain. When the N-terminus is glycine, it may be bonded to a molecule via the α-amino group. 6 and X 16 is preferably cysteine ​​or D-cysteine, and a disulfide bond or a covalent bond is formed between the -SH groups of each side chain via a linker of a methylene group, thereby forming a ring structure within the molecule.

[0032] A more preferred embodiment of the peptide of formula (3) above is, for example, an amino acid sequence represented by formula (5) below. A disulfide bond or a covalent bond via a methylene group linker may be formed between the -SH groups of each side chain, thereby forming a cyclic structure within the molecule, or the peptide may be bonded to a certain molecule via the ε-amino group of Lys1.

[0033] Ac-Lys-Gly-Thr-Pro-X 5 -Cys-X 7 -Lys-X 9 -X 10 -Leu-Ala-Glu-X 14 -Pro-Cys-OH (5) In formula (5), X 5 represents serine, threonine, or 2,3-diaminopropionic acid; X 7 represents histidine, tyrosine, phenylalanine, or tryptophan; X 9 represents tyrosine, methionine, norleucine, or arginine; X 10 represents methionine, norleucine, arginine, or lysine; X 14 represents leucine, isoleucine, methionine, or norleucine.

[0034] [4] The peptide according to this embodiment includes peptides having homology to the amino acid sequences shown in [1] to [3] above, in which one to several amino acids have been deleted, added, and / or substituted, as long as they have binding activity to LRP1. In the present specification, when referring to a "peptide in which one to several amino acids have been deleted, added, and / or substituted," the number of amino acids is not particularly limited as long as the peptide has LRP1 binding activity, but is preferably 1 to 5, more preferably 1 or 2. The deletion, addition, and / or substitution may be at the end or middle of the peptide, and may be at one or more locations.

[0035] Such amino acid sequences in which one to several amino acids have been deleted, added, and / or substituted in the above-mentioned amino acid sequences include those that have an identity of at least 50% or more, preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more, with the above-mentioned amino acid sequence when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, using default, i.e., initial setting parameters).

[0036] It is also known that there are peptides or protein domains that have a high degree of similarity in three-dimensional structure even if the homology in the primary structure is low. Therefore, peptides having at least 50% or more, preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more of three-dimensional structure homology with the above amino acid sequence and having Ras binding activity are also included in this embodiment. The homology of the three-dimensional structure between such peptides can be predicted as follows from the amino acid sequence of a peptide with an unknown three-dimensional structure using a homology modeling method or the like. For example, when an arbitrary amino acid sequence (target sequence) having a similar sequence to the cyclic peptide (reference peptide) of this embodiment is given, an alignment (a sequence juxtaposed) between the target sequence and the reference sequence is given. If an alignment calculated by FASTA, PSI-BLAST, LIBRA, or the like is used, the correspondence relationship between each amino acid between the target sequence and the reference sequence is determined, and based on this relationship, three-dimensional coordinates for each amino acid in the target sequence are created from the three-dimensional coordinates of the reference peptide. In constructing the three-dimensional coordinates, structurally inappropriate gaps, collisions, and distortions may occur between amino acid residues, so these structural distortions are eliminated by energy minimization calculations. Some modeling software performs this structural distortion elimination stepwise rather than simultaneously for all atoms of the peptide in order to smoothly eliminate it. That is, first, the α carbon atoms that form the peptide backbone are performed, then the main chain atoms including the α carbon atoms, and finally the entire peptide including the side chain atoms. If an alignment for the target sequence is obtained in this way, its three-dimensional structure can be predicted and constructed. The index of three-dimensional structural homology can be compared using RMSD (Root Mean Square Deviation), which is the difference between the XYZ coordinates when optimally superimposed.

[0037] The peptide of the present embodiment also includes salts of peptides. As the salts of peptides, salts with physiologically acceptable bases or acids are used, and examples thereof include addition salts with inorganic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), addition salts with organic acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), addition salts with inorganic bases (ammonium hydroxide, or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), and addition salts of amino acids.

[0038] (Peptide derivatives and modifications) The peptide of this embodiment also includes various derivatives and / or modifications thereof, so long as they solve the problems of the present invention. Such derivatives include those in which the saturated fatty chain of the peptide is replaced with an unsaturated fatty chain, those in which some of the atoms of the peptide are replaced with other atoms including radioactive or non-radioactive isotope atoms, those in which the amide bond of the peptide is replaced with a thioamide bond (-NH-C(=S)-), those in which the amide bond of the peptide is replaced with an alkene (-C=C-), those in which the amide bond of the peptide is replaced with an alkyl (-CC-), those in which the amide bond of the peptide is replaced with a hydroxyethylene (-C(-OH)-C-), those in which the amide bond of the peptide is replaced with an ester (-OC(=O)-), those in which the amide bond of the peptide is replaced with an alkene (-C=C-), those in which the amide bond of the peptide is replaced with (-C-NH-), or those in which the amide bond of the peptide is replaced with (-C(=O)-C-).Such modified forms include those in which the α-carbon of the peptide is disubstituted, those in which the amide bond of the peptide is N-alkylated, those in which the functional groups of the peptide are replaced with a hydroxyethylene (-C(-OH)-C-), those in which the amide bond of the peptide is replaced with an ester (-OC(=O)-), Examples of the peptide include, but are not limited to, peptides in which some of the functional groups have been modified by halogenation, cyanation, nitration, oxo-ation, hydroxylation, ammination, deamination, dehydrogenation, amidation, acetylation, methoxylation, prenylation, alkylation, or the like (for example, peptides in which some of the amino groups of the peptide have been acetylated, alkylated, or deaminated, and peptides in which some of the carboxyl groups of the peptide have been amides or esters), peptides in which S is a sulfoxide S(=O) or a sulfone S(=O)2, peptides in which the peptide is polymerized via a chemical linker, peptides in which the peptide is labeled with biotin, peptides in which the peptide is fluorescently labeled, peptides in which the peptide is luminescently labeled, and peptides in which the peptide is fused or conjugated, directly or via a linker, with alkyl chains, polyethylene glycol, antibodies, lectins, sugar chains, enzymes, peptides, peptoids, membrane-permeable peptides, low molecular weight compounds, nucleic acids, or molecules that induce ubiquitination of proteins.

[0039] According to some embodiments of the present disclosure, the peptide of the present invention comprises an amino acid residue having an azide group at its N-terminus or C-terminus. Thus, a "certain molecule" having an alkyne group can be covalently bound to the N-terminus or C-terminus of the peptide of the present invention by "copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction" (or abbreviated as "click" reaction). According to other embodiments of the present disclosure, an azide group is introduced by reacting a linker having an azide group and an N-hydroxysuccinimide (NHS) ester group with an α-amino group or an ε-amino group of the peptide of the present invention. Thus, a "certain molecule" having an alkyne group can be covalently bound to the peptide of the present invention by the CuAAC reaction. According to other embodiments of the present disclosure, the peptide of the present invention comprises an amino acid residue having an alkyne group at its N-terminus or C-terminus. Thus, a "certain molecule" having an azide group can be covalently bound to the N-terminus or C-terminus of the peptide of the present invention by the CuAAC reaction. According to another embodiment of the present disclosure, an alkyne group is introduced by reacting a linker having an alkyne group and an NHS ester group with an α-amino group or an ε-amino group of the peptide of the present invention. Thus, a "certain molecule" having an azide group can be covalently bonded to the peptide of the present invention by CuAAC reaction. The CuAAC reaction is shown in Scheme 1 below.

[0040] [ka]

[0041] The CuAAC reaction affords 1,5-disubstituted-1,2,3-triazoles. The reaction between alkynes and azides is highly selective, and no alkyne or azide groups are present in natural biomolecules. Furthermore, the reaction is rapid and pH insensitive.

[0042] The alkyne group may be replaced by a cyclooctyne group. According to some embodiments of the present disclosure, the peptide of the present invention contains an amino acid residue having an azide group at its N-terminus or C-terminus. Therefore, a "certain molecule" having a cyclooctyne group can be covalently bonded to the N-terminus or C-terminus of the peptide of the present invention by "strain-promoted azide-alkyne click chemistry (SPAAC) reaction". According to other embodiments of the present disclosure, an azide group is introduced by reacting a linker having an azide group and an NHS ester group with an α-amino group or an ε-amino group of the peptide of the present invention. Therefore, a "certain molecule" having a cyclooctyne group can be covalently bonded to the peptide of the present invention by the SPAAC reaction. According to other embodiments of the present disclosure, the peptide of the present invention contains an amino acid residue having a cyclooctyne group at its N-terminus or C-terminus. Thus, a "certain molecule" having an azide group can be covalently bonded to the N-terminus or C-terminus of the peptide of the present invention by the SPAAC reaction. According to another embodiment of the present disclosure, a linker having a cyclooctyne group and an NHS ester group is reacted with an α-amino group or an ε-amino group of the peptide of the present invention to introduce a cyclooctyne group. Thus, a "certain molecule" having an azide group can be covalently bonded to the peptide of the present invention by the SPAAC reaction. The SPAAC reaction is shown in Scheme 2 below.

[0043] [ka]

[0044] Alternatively, in certain embodiments, a molecule having a tetrazine or cyclooctene group is bonded to the corresponding cyclooctene or tetrazine group of a peptide or linker by an inverse electron demand Diels-Alder (iEDDA) reaction, as shown in Scheme 3 below.

[0045] [ka]

[0046] Examples of amino acid residues having an azido group include L-azidohomoalanine (AHA), 4-azido-L-phenylalanine, 4-azido-D-phenylalanine, 3-azido-L-alanine, 3-azido-D-alanine, 4-azido-L-homoalanine, 4-azido-D-homoalanine, 5-azido-L-ornithine, 5-azido-d-ornithine, 6-azido-L-lysine, and 6-azido-D-lysine. Examples of amino acid residues having an alkyne group include L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-HPG), and β-homopropargylglycine (β-HPG).

[0047] The strained alkyne group at the free end of the linker can be a cyclooctene group, such as a trans-cyclooctene (TCO) group; or a cyclooctyne group, such as a dibenzocyclooctyne (DBCO) group, a difluorocyclooctyne (DIFO) group, a bicyclononyne (BCN) group, and a dibenzocyclooctyne (DICO) group. Alternatively, the tetrazine group at the free end of the linker can be, but is not limited to, a 1,2,3,4-tetrazine group, a 1,2,3,5-tetrazine group, and a 1,2,4,5-tetrazine group, and derivatives thereof, such as a 6-methyltetrazine group.

[0048] In a preferred embodiment of the present disclosure, the peptide derivatives and modifications have an azide group, an alkyne group, or a tetrazine group at the N-terminus, C-terminus, or the side chain of an amino acid, either directly or via a linker. Examples of such derivatives include the following. For example, when DBCO-PEG4-NHS ester (D5922) or BCN-PEG4-NHS ester (BP-22851) is reacted with the epsilon amino group of the N-terminal lysine of a peptide, a peptide (SEQ ID NOs: 49 and 50) to which DBCO or BCN is added via a PEG linker is obtained.

[0049] [ka]

[0050] Alternatively, as shown in Scheme 4 below, a peptide having a DBCO group introduced into the side chain of an amino acid (SEQ ID NO: 51 and SEQ ID NO: 52) can be obtained by reacting DBCO-PEG5-DBCO (BP-22450) with an N-terminal lysine to which an azide group has been introduced using Fmoc-Lys(εN3)-OH.

[0051] [ka]

[0052] Furthermore, in the following Scheme 5, N3-PEG4-NHS (N-succinimidyl 15-azido-4,7,10,13-tetraoxapentadecanoate) is reacted with the α-amino group of the N-terminal glycine of the peptide shown in SEQ ID NO: 53 to synthesize a peptide having an azide group introduced therein (SEQ ID NO: 54), and further, DBCO-PEG5-DBCO can be used to obtain a peptide having a DBCO group introduced therein at the N-terminus (SEQ ID NO: 55).

[0053] [ka]

[0054] (Peptide drug conjugates) The peptide of the present disclosure may be a conjugate (complex). This complex is a complex including the peptide of the present disclosure, a linker bound to the peptide, and a "certain molecule" bound to the linker. This complex is preferably a complex in which at least the "certain molecule" can pass through a barrier tissue. The entire complex may be capable of passing through a barrier tissue.

[0055] Examples of linkers include, but are not limited to, those composed of about 1 to 15 amino acid residues, those composed of fatty acids, those composed of polyethylene glycol, and derivatives thereof, etc. The linker may be one that dissociates or separates under specific environments or conditions, or one that maintains a stable structure.

[0056] The "certain molecule" described herein may be any molecule desired by a person skilled in the art, so long as it is a molecule that is desired to be delivered to the brain. However, since the mechanism of passage through the barrier tissue is RMT via binding to LRP1, molecules that are too large to be transported by this mechanism are not preferred. Examples of the "certain molecule" include, but are not limited to, fluorescent dyes, low molecular weight compounds, peptides, peptoids, nucleic acids, sugar chains, lipids, antibodies, proteins, enzymes, liposomes, micelles, nanoparticles, carbon nanotubes, carbon nanohorns, carbon nanosheets, and radioisotope-labeled versions thereof. In a preferred embodiment, the "certain molecule" is a drug, and may be, for example, an antibody, a nucleic acid, or a physiologically active peptide.

[0057] The complex of the peptide of the present disclosure and a "certain molecule" may be a fusion or graft. For example, the peptide of the present invention may be fused to the N-terminus and / or C-terminus of an antibody or protein, or grafted into a loop region.

[0058] The above fusions and transplants may be expressed as part of an ectodomain of a cell, fungus, virus, etc., or may be conjugated to an ectodomain of a cell, fungus, virus, etc.

[0059] A complex between the peptide of the present disclosure and a "certain molecule" may be formed via a "molecule having binding activity to a certain molecule". The peptide of the present disclosure and the "molecule having binding activity to a certain molecule" may be a conjugate, a fusion, and / or a graft. The bond between the "certain molecule" and the "molecule having binding activity to a certain molecule" may be reversible or irreversible.

[0060] The complex of the peptide of the present disclosure and a "certain molecule" may be in the form of a crystal, and the peptide of the present invention includes both a single crystal form and a mixture of crystal forms. The crystal can be produced by crystallization using a crystallization method known per se.

[0061] The complex of the peptide of the present disclosure and a "certain molecule" may be a pharma- ceutically acceptable cocrystal or cocrystal salt. Here, a cocrystal or a cocrystal salt refers to a crystalline substance composed of two or more distinct solids at room temperature, each of which has different physical properties (e.g., structure, melting point, heat of fusion, hygroscopicity, solubility, and stability). A cocrystal or a cocrystal salt can be prepared according to a cocrystallization method known per se.

[0062] (Actions and Effects of Cyclic Peptides) As shown in the examples described below, sequences 1 to 46, which are representative examples of the amino acid sequence group represented by this embodiment, have LRP1 binding activity. Sequence 2 migrates from the blood side well to the brain side well in an in vitro blood-brain barrier model, and therefore has the ability to pass through the blood-brain barrier. The amino acid sequence group represented by this embodiment has similar amino acid sequences and three-dimensional structural characteristics to these representative examples, and therefore it is highly likely that they also have LRP1 binding activity and the ability to pass through the blood-brain barrier. It is also highly likely that conjugates, fusions, and transplants with the peptide of the present invention also have LRP1 binding activity and the ability to pass through the blood-brain barrier.

[0063] As shown in the Examples below, sequences 1 to 46, which are representative examples of the amino acid sequence group represented by this embodiment, have LRP1 binding activity. This indicates the following. 5 and X 15 The bond between the Cα carbon atoms of X 6 and X 15 The bond between the Cα carbon atoms of X 5 and X 16 The bond between the Cα carbon atoms of 6 and X 16 The bond between the Cα carbon atoms can have a wide variety of structures, such as an amide bond, a disulfide bond, a thioether bond, a C=C bond, a CC bond, a bond via a triazole, a bond via a dithiotetrafluorobenzene, or a bond via a dimethylphenylene group (the bonding site of the methylene group to the phenylene ring may be any of ortho, meta, and para).

[0064] The amino acid sequence of LRP1 cluster 4 is highly conserved among mammals, and sequence 2, which is a representative example of the peptide of the present invention, exhibits the ability to pass through the blood-brain barrier in rat and monkey in vitro blood-brain barrier models. Therefore, it is considered highly likely that the peptide group consisting of the amino acid sequence represented in this embodiment, and the complex of this peptide group with "a certain molecule", will also bind to LRP1 in mammals other than humans, such as mice, rats, dogs, and monkeys, and exhibit the ability to pass through the barrier tissue.

[0065] (Method of Producing Cyclic Peptides) The peptide of this embodiment can be produced by known peptide production methods, such as chemical synthesis methods including a liquid phase method, a solid phase method, or a hybrid method that combines a liquid phase method and a solid phase method.

[0066] For the solid phase method, a commercially available automatic synthesizer can be used. For example, the hydroxyl group of a resin having a hydroxyl group and the carboxyl group of a first amino acid (usually the C-terminal amino acid of the target peptide) whose α-position amino group is protected with a protecting group such as an Fmoc group are subjected to an esterification reaction. As an esterification catalyst, a known dehydration condensation agent such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), etc. can be used. Next, the protecting group of the α-position amino group of the first amino acid is removed, and a second amino acid in which all functional groups except the carboxyl group of the main chain are protected is added, the carboxyl group is activated, and the first and second amino acids are bonded. Furthermore, the α-position amino group of the second amino acid is deprotected, and a third amino acid in which all functional groups except the carboxyl group of the main chain are protected is added, the carboxyl group is activated, and the second and third amino acids are bonded. This is repeated to synthesize a peptide of the desired length. The linear peptide is cleaved from the resin and purified, after which the functional groups for cyclizing the peptide are deprotected and the peptide is cyclized according to a standard method.

[0067] Examples of resins for solid-phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), and HMPA-PEGA resin (Merck). These resins may be washed with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.) before use. Examples of protecting groups for the α-position amino group include benzyloxycarbonyl (Cbz) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, and allyloxycarbonyl (Alloc) group. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine. The α-position carboxyl group can be protected with a methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, etc. As for other functional groups of amino acids, the hydroxyl group of serine, threonine, etc. can be protected with a benzyl group or a tert-butyl group, and the hydroxyl group of tyrosine, etc. can be protected with a 2-bromobenzyloxycarbonyl group or a tert-butyl group. The amino group of the side chain of lysine, etc., and the carboxyl group of glutamic acid, aspartic acid, etc. can be protected in the same manner as the α-position amino group and the α-position carboxyl group.

[0068] The activation of the carboxyl group can be carried out using a condensation agent. Examples of the condensation agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), etc. The cleavage of the peptide chain from the resin can be carried out by treating with an acid such as TFA or hydrogen fluoride (HF).

[0069] The peptide of the present disclosure may be cyclized as one embodiment. In this specification, cyclization means that two or more amino acids separated by one or more amino acids in one peptide are covalently bonded directly or indirectly via a linker to form one or more ring structures in the molecule. The cyclization can be performed according to the methods described in Non-Patent Document 8 and Non-Patent Document 9. For example, cyclization can be performed by an amide bond between an amino group and a carboxy group, a disulfide bond between a thiol group and a thiol group, a thioether bond between a thiol group and a halogen group, a thioether bond by a thiol-ene reaction between a thiol group and an allyl group, a C=C bond by an olefin metathesis reaction between allyl groups (the C=C bond may be converted to a CC bond by a reduction reaction), a bond via a triazole by a click reaction between an alkynyl group and an azide group, a thioether bond between a linker having a halogen group and two thiol groups, etc., but is not limited to these. The covalent bond for cyclization, which may be direct or indirect via a linker, may be between the main chain and the main chain, between the main chain and the side chain, between the side chain and the main chain, or between the side chain and the side chain.

[0070] For example, the cyclization of the peptide of the present disclosure can be carried out by using (1) cysteine ​​having a thiol group, D-cysteine, homocysteine, or D-homocysteine ​​as the amino acid 1 and the amino acid 2, respectively, independently, and using a disulfide bond formed between the thiol groups of these amino acids; (2) using an amino acid having a halogen atom (chloro, bromo, or iodo) that is a nucleophilic group (e.g., 3-chloroalanine) or a carboxylic acid having a halogen atom (chloro, bromo, or iodo) (e.g., 3-chloropropanoic acid) as the amino acid 1 and using a thiol group (3) a thioether bond formed between amino acid 1 and amino acid 2 having a thiol group and a linker having a halogen atom (chloro, bromo, or iodo) as a nucleophilic group (e.g., 1,1-diiodomethane, 1,1-dichloroacetone, 1,2-diiodoethane, 1,3-diiodopropane, 1,4-diiodobutane, α,α'-dibromo-o-xylene, α,α'-dibromo-m-xylene, α,α'-dibromo-p-xylene, hexafluorobenzene, etc.) (4) A covalent bond via a triazole formed by a click reaction between β-azidoalanine having an azide group as amino acid 1 and 2-amino-5-hexynoic acid having an alkynyl group as amino acid 2 can be used. (5) A thioether bond formed by a thiol-ene reaction between an amino acid having an allyl group (e.g., allylglycine, D-allylglycine, homoallylglycine, D-homoallylglycine, etc.) or a carboxylic acid having an allyl group (e.g., 3-butenoic acid) as amino acid 1 and an amino acid having a thiol group as amino acid 2 can be used. (6) An amino acid having an allyl group or a carboxylic acid having an allyl group can be used as amino acid 1 and amino acid 2, respectively, independently, and a C=C bond formed by an olefin metathesis reaction between the allyl groups can be used. (7) A CC bond formed by reducing a C=C bond formed by an olefin metathesis reaction can be used. (8) An amino acid having an amino group (e.g., 2,3-diaminopropanoic acid, 2,An amide bond can be used between an amino acid 1 having a carboxy group (4-diaminobutanoic acid, ornithine, lysine, D-amino acids thereof, β-alanine, etc.) and an amino acid 2 having a carboxy group (aspartic acid, glutamic acid, D-amino acids thereof, etc.) or a C-terminal carboxy group, (9) an amide bond can be used between an N-terminal amino group and an amino acid having a carboxy group or a C-terminal carboxy group. Either amino acid 1 or amino acid 2 may be on the N-terminal side.

[0071] (Medicines, diagnostic agents, and research reagents containing cyclic peptides) The pharmaceutical composition according to the present disclosure contains a peptide having the above-mentioned amino acid sequence as an active ingredient, and the peptide binds to LRP1 and can be transferred to central nervous system tissues via the RMT of LRP1. The administration form of the pharmaceutical composition is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injection administration such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (transnasal, oral, ocular, pulmonary, vaginal, or rectal administration). The peptide in the pharmaceutical composition may be modified in various ways in consideration of its tendency to be metabolized and excreted. For example, the peptide may be added with an alkyl chain, polyethylene glycol, or sugar chain to increase the blood residence time and reduce antigenicity. In addition, biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, microparticles, nanospheres, etc. may be used as sustained-release bases, and peptides may be encapsulated in them. For transdermal administration, a weak electric current can be passed through the skin surface to allow penetration through the stratum corneum (iontophoresis method).

[0072] The above pharmaceutical composition may be prepared by adding an active ingredient as it is, or by adding a pharma- ceutically acceptable carrier, excipient, additive, etc. The dosage form may be, for example, a liquid (such as an injection), a dispersion, a suspension, a tablet, a pill, a powder, a suppository, a powder, a fine granule, a granule, a capsule, a syrup, a lozenge, an inhalant, an ointment, an eye drop, a nasal drop, an ear drop, a cataplasm, etc. These preparations may be a release-controlled preparation such as an immediate-release preparation or a sustained-release preparation (such as sustained-release microcapsules). The preparation may be carried out by a conventional method using, for example, an excipient, a binder, a disintegrant, a lubricant, a dissolving agent, a dissolving aid, a colorant, a flavoring agent, a stabilizer, an emulsifier, an absorption promoter, a surfactant, a pH adjuster, a preservative, an antioxidant, etc., as appropriate. Examples of ingredients used in formulations include purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharma- ceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, anhydrous silicic acid, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin and the like, but are not limited thereto.When a peptide is poorly absorbed via the mucosa, absorption enhancers that improve the absorption of poorly absorbed drugs may be used, such as surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, saponin, etc.; bile salts such as glycocholic acid, deoxycholic acid, taurocholic acid, etc.; chelating agents such as EDTA, salicylic acids, etc.; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, mixed micelles, etc.; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, nitric oxide donors, etc.

[0073] Pills or tablets can be coated with sugar, gastric or enteric substances. Injections can contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizing agents, preservatives, etc. can be added. If necessary, additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents, etc. can be used appropriately and in appropriate amounts.

[0074] The pharmaceutical composition of the present disclosure delivers a molecule to central nervous system tissues through binding to LRP1, and is effective in treating or preventing various diseases targeted by the molecule. For example, when the molecule is an anticancer agent, examples of the agent include brain tumors (e.g., pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, etc.) and schwannoma. When the molecule targets a molecule involved in a central nervous system disease, examples of the agent include psychiatric disorders (schizophrenia, schizoaffective disorder, schizophreniform, delusional disorder, etc.), childhood psychiatric disorders (attention deficit disorder, attention deficit / hyperactivity disorder, conduct disorder, autism, etc.), neurodegenerative disorders, neural stem cell disorders, neural precursor disorders, ischemic disorders, neurotraumatic disorders, emotional disorders, psychomotor disorders, Sleep disorders (hypersomnia, circadian rhythm sleep disorder, insomnia, parasomnia, sleep deprivation, etc.), mental disorders such as anxiety (acute stress disorder, generalized anxiety disorder, social anxiety disorder, panic disorder, post-traumatic stress disorder, agoraphobia, obsessive-compulsive disorder, etc.), factitious disorder (acute hallucinatory mania, etc.), impulse control disorders (compulsive gambling, intermittent explosive disorder, etc.), mood disorders (bipolar I disorder, bipolar II disorder, mania, mixed affective states, etc.), major depression, chronic depression, seasonal depression, psychotic depression, seasonal depression, cognitive disorders (amnesia, geriatric cognition, etc.) Dementia, HIV-associated dementia, Alzheimer's disease, Huntington's disease, Lewy body dementia, vascular dementia, drug-associated dementia, tardive dyskinesia, myoclonus spasms, dystonia, delirium, Pick's disease, Creutzfeldt-Jakob disease, HIV disease, Gilles de la Tourette syndrome, epilepsy, muscle spasms, mild cognitive impairment, etc.), mental retardation (spasticity, Down's syndrome, Fragile X syndrome, etc.); Premenstrual syndrome (PMS), Premenstrual dysphoric disorder (PDD), Postpartum depression, Neuronal damage disorders (eye damage, ocular retinopathy or macular degeneration) , tinnitus, hearing impairment, cerebral edema, etc.), Parkinson's disease, Parkinson-like disorders, migraine, epilepsy, Alzheimer's disease, brain injury, stroke, cerebrovascular disease (cerebral arteriosclerosis, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, cerebral hypoxia-ischemia, etc.), drug addiction (narcotic addiction, alcoholism, amphetamine addiction, cocaine addiction, nicotine addiction, drug withdrawal syndrome, etc.), eating disorders (anorexia, bulimia, binge eating disorder, bulimia, obesity, compulsive eating disorder, pagophagia, etc.).

[0075] The pharmaceutical composition of the present disclosure may be administered in combination with other medicines or treatments useful for the above diseases, for example, in the case of malignant tumors, it may be combined with various chemotherapy, surgical treatment, or radiation therapy.

[0076] When the pharmaceutical composition of the present disclosure is administered to a mammal (e.g., a human, a mouse, a rat, a guinea pig, a rabbit, a dog, a horse, a monkey, a pig, etc.), particularly a human, the dosage varies depending on the symptoms, the patient's age, sex, weight, difference in sensitivity, administration method, administration interval, type of active ingredient, and type of formulation, and is not particularly limited, but for example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg can be administered once or in several divided doses.

[0077] Polynucleotides Encoding the Peptides of the Disclosure In one embodiment, a polynucleotide encoding the peptide of the present disclosure is provided. This polynucleotide may be a polynucleotide encoding an amino acid sequence consisting of only natural amino acids among the amino acid sequences of the peptide of the present disclosure, or a polynucleotide having a nucleotide sequence having a predetermined sequence identity with the polynucleotide. The degree of identity of the nucleotide sequence may be about 70% or more, preferably about 80% or more, more preferably about 90% or more, and even more preferably about 95% or more. The nucleotide sequence identity can be determined by a method known per se. For example, the nucleotide sequence identity (%) can be determined by the same method as the amino acid sequence identity (%) described above.

[0078] In another embodiment, the polynucleotide may be a base sequence in which one or more nucleotides have been subjected to one or more modifications selected from substitution, addition, deletion, and insertion in the base sequence of a polynucleotide encoding an amino acid sequence consisting only of natural amino acids among the amino acid sequences of the peptide of the present disclosure. The number of modified nucleotides is not particularly limited as long as it is one or more, and may be, for example, 1 to about 50, preferably 1 to about 30, more preferably 1 to about 10, and even more preferably 1 to about 5 (e.g., 1 or 2).

[0079] (Expression Vector) An expression vector according to an embodiment of the present disclosure may include a polynucleotide encoding a polypeptide of interest to be expressed or a polynucleotide of interest to be expressed, and a promoter operably linked to the polynucleotide. "A promoter operably linked to a polynucleotide" means that the promoter is linked to a polynucleotide encoding a gene so as to allow expression of the polynucleotide itself or the polypeptide encoded by the polynucleotide under its control.

[0080] The backbone of the expression vector of this embodiment is not particularly limited as long as it can produce a target substance in a predetermined cell, and examples thereof include a plasmid vector and a virus vector. When the expression vector is used as a medicine, examples of vectors suitable for administration to mammals include virus vectors such as adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, polio virus, Sindbis virus, and Sendai virus.

[0081] When a prokaryotic cell is used as a host cell, an expression vector that can be used as a host cell for a prokaryotic cell can be used. Such an expression vector can contain elements such as a promoter-operator region, a start codon, a polynucleotide encoding the polypeptide of this embodiment or a partial peptide thereof, a stop codon, a terminator region, and a replication origin. The promoter-operator region for expressing the polypeptide of the present invention in bacteria contains a promoter, an operator, and a Shine-Dalgarno (SD) sequence. These elements can be those that are known per se.

[0082] In addition, when eukaryotic cells are used as host cells, expression vectors that can be used as eukaryotic cells as host cells can be used. In this case, the promoter used is not particularly limited as long as it can function in eukaryotes such as mammals. When expressing a polypeptide, such promoters include, for example, virus promoters such as SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, and adenovirus-derived early promoter, as well as mammalian constitutive protein gene promoters such as β-actin gene promoter, PGK gene promoter, and transferrin gene promoter. When expressing a polynucleotide, the promoter can be a polIII promoter (e.g., tRNA promoter, U6 promoter, H1 promoter).

[0083] The expression vector of the present invention may further include sites for transcription initiation and termination, a ribosome binding site that may be required for translation in the transcribed region, a replication origin, and a selection marker gene (e.g., ampicillin, tetracycline, kanamycin, spectinomycin, erythromycin, chloramphenicol), etc. The expression vector of the present invention can be prepared by a method known per se (see, for example, Molecular Cloning, supra).

[0084] Cells, liposomes, or nanoparticles presenting the peptides of the present disclosure In another aspect of the present disclosure, a cell, liposome, or nanoparticle presenting the peptide of the present disclosure is provided. The cell can be prepared by isolating a desired cell from a living organism and conjugating the peptide of the present disclosure to the cell ex vivo, or by introducing a vector expressing the peptide of the present disclosure into the cell to present it on the cell surface. The liposome or nanoparticle can be prepared by conjugating the peptide of the present disclosure or by preparing a conjugate of the peptide of the present disclosure and a lipid chain, and inserting it into a lipid membrane via the lipid chain to present it on the surface of the liposome or nanoparticle.

[0085] The following examples are merely illustrative and are intended to describe the present invention in detail together with the above-mentioned embodiments, and are not intended to limit the present invention. Those skilled in the art can modify the present invention in various aspects without departing from the meaning of the present invention, and such modifications are also included in the scope of the present invention. EXAMPLES

[0086] The abbreviations used in this specification have the following meanings. BSA: Bovine serum albumin RP-HPLC: Reverse-phase high performance liquid chromatography HRP: Horseradish peroxidase SA: Streptavidin D-PBS:Dulbecco's Phosphate buffered saline ELISA:Enzyme-linked immunosorbent assay Ac: Acetyl Cys:L-Cysteine Gly:Glycine Ala: L-Alanine Ser:L-Serine Thr: L-Threonine Pro: L-Proline cHyp: cis-4-Hydroxy-L-Proline tHyp:trans-4-Hydroxy-L-Proline Tyr: L-Tyrosine Trp: L-Tryptophan Phe: L-Phenylalanine 4fF:4-fluoro-L-Phenylalanine 4cF:4―chloro―L-Phenylalanine His:L-Histidine Lys: L-Lysine Orn: L - Ornithine Arg: L - Arginine Val: L - Valine Leu: L - Leucine Ile: L - Isoleucine Nle: L - Norleucine Ahep: (S) - 2 - Aminoheptanonic acid Aoc: (S) - 2 - Aminooctanonic acid Aib: 2 - Aminoisobutyric acid Asp: L - Aspartic acid Glu: L - Glutamic acid 5 - FAM: 5 - Carboxyfluorescein DX: D - amino acid X c(XY): Cyclization between amino acid X to amino acid Y

[0087] (Peptide synthesis) Chemical synthesis of all peptides used in this example was entrusted to Scrum Co., Ltd. (Tokyo, Japan), and standard solid-phase synthesis using 9-fluorenylmethoxycarbonyl group (Fmoc group) as a protecting group for the α-amino group was performed with an automatic synthesizer SyroII (Biotage). The side chain-protected amino acid-resin at the C-terminus was placed in a synthesis column, and the device was set up. Next, the next amino acid protected with the Fmoc group was activated by adding 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) / diisopropylethylamine (DIEA), and reacted in the column. After the reaction was completed, the column was washed and the Fmoc group was deprotected using 20% ​​piperidine. By repeating this process, the peptide chain was extended, and the Fmoc group of the final amino acid was deprotected, after which the peptide resin was removed from the device. 30% hexafluoro-2-propanol (HFIP) / dichloromethane (DCM) was added to the peptide resin to cut out the linear side-chain protected peptide from the resin, and the side-chain protected peptide was recovered by ether precipitation. The side-chain protected peptide was purified by RP-HPLC using a SunFire C18 column (10×150 mm) (Waters) and then lyophilized. The peptide was cyclized with reference to the methods described in Non-Patent Documents 8 and 9. The theoretical molecular weight, actual molecular weight, purity, cyclization type, and amino acid sequence of the peptides synthesized in this example are shown in Tables 1 and 2. The structural formulas of Sequence 2 and Sequence 46 are shown in FIG. 7. In Tables 1 and 2, amino acid residues without D-form notation indicate L-form.

[0088] [Table 1]

[0089] [Table 2]

[0090] (Establishment of binding test by ELISA method) To evaluate the binding activity of peptides to LRP1, a binding test was established using the ELISA method. The following is a brief description of this ELISA method. First, goat anti-human IgG-Fcγ polyclonal antibody (catalog No. 109-0005-008, manufactured by Jackson ImmunoResearch) was added to a 96-well Maxisorp plate (catalog No. 439454, manufactured by Nunc) at 1 μg / 50 μL / well using D-PBS, and the plate was coated overnight at 4°C. After that, 0.5% BSA / D-PBS was further added at 300 μL / well and blocked at room temperature for 30 minutes. After washing the plate with 0.1% Tween20 / D-PBS, recombinant LRP1 proteins, LRP1 cluster 2-Fc chimeric protein (catalog No. 2368-L2-050, R&D system), LRP1 cluster 3-Fc chimeric protein (catalog No. 48248-L3-050, R&D system), or LRP1 cluster 4-Fc chimeric protein (catalog No. 5395-L4-050, R&D system), were added at 100ng / 50μL / well using 0.5% BSA / D-PBS and reacted with the coated antibody for 30 minutes at room temperature. After washing the plate with 0.1% Tween20 / PBS, biotinylated peptides, sequence 45 and sequence 46, were prepared at arbitrary concentrations using 0.5% BSA / D-PBS and added at 50μL / well. After 30 minutes of incubation at room temperature, the plate was washed with 0.1% Tween 20 / D-PBS. Sequences 45 and 46 bound to the LRP1 recombinant protein captured on the plate were detected with SA-HRP (catalog no. ab7403, Abcam). HRP was quantified using TMB-ELISA Substrate Solution (catalog no. 34028, Thermo Fisher Scientific), and the absorbance was measured at 450 nm.

[0091] The selective and peptide concentration-dependent binding of sequences 45 and 46 to the LRP1 cluster 4-Fc chimeric protein used in this binding study is shown in Figure 1 (n = 4, ± SEM). The specific binding activity was estimated by subtracting the absorbance obtained when the biotinylated peptide was added to wells in which the LRP1 recombinant protein was not captured from the absorbance obtained when the biotinylated peptide was added to wells in which the LRP1 recombinant protein was captured. As a result, both sequences 45 and 46 selectively bound to the LRP1 cluster 4-Fc chimeric protein in a peptide concentration-dependent manner. Almost no or only slight binding was observed to the LRP1 cluster 2-Fc chimeric protein and the LRP1 cluster 3-Fc chimeric protein. The binding EC50 values ​​of sequences 45 and 46 were calculated to be 10.5 nM and 11.0 nM, respectively, and showed equivalent binding activity regardless of the presence or absence of a cyclic structure.

[0092] (Establishment of competitive binding test by ELISA method) To compare the binding activity of peptides with amino acid substitutions introduced into sequence 1 to LRP1 and the resistance to protease degradation with those of sequence 2, a competitive binding test was established by ELISA as shown in Figure 2. Immediately after mixing sequence 2 or the amino acid-substituted peptide with mouse plasma (Plasma incubation) (0 hours) or after 24 hours at 37°C, the peptide was diluted to an arbitrary final concentration so that the plasma concentration was sufficiently low, and mixed with sequence 46 (500 nM) and subjected to the competitive binding test described above. The binding of sequence 46 to the LRP1 cluster 4-Fc chimeric protein competes with the binding of sequence 2 or the amino acid-substituted peptide coexisting in the solution to the LRP1 cluster 4-Fc chimeric protein, and is inhibited in a concentration-dependent manner by sequence 2 or the amino acid-substituted peptide. In other words, the binding activity of sequence 2 or the amino acid-substituted peptide to the LRP1 cluster 4-Fc chimeric protein is detected as a competitive inhibitory activity against the binding of sequence 46. The binding value of sequence 46 to wells that did not capture LRP1 cluster 4-Fc chimeric protein was defined as 100% inhibitory activity, and the binding value of sequence 46 to wells that did not contain sequence 2 or amino acid substituted peptide was defined as 0% inhibitory activity, and the inhibitory activity values ​​of sequence 2 and the amino acid substituted peptide were calculated as a percentage.

[0093] (Evaluation of the binding activity of amino acid substituted peptides to LRP1) The results of a competitive binding test for the LRP1 cluster 4-Fc chimeric protein, n = 4 (± SEM), are shown in Figure 3. Sequence 2, which was incubated with plasma for 0 hours, inhibited the binding of sequence 46 in a concentration-dependent manner. The binding of sequence 46 (500 nM) in wells to which 500 nM of sequence 2 was added was an average of 52.4%, indicating that sequence 2 and sequence 46 competed 1:1 for binding to LRP1 cluster 4. Sequence 2, which was incubated with plasma for 24 hours, also inhibited the binding of sequence 46 in a concentration-dependent manner. However, the binding of sequence 46 (500 nM) in wells to which 2000 nM equivalent of sequence 2 was added was an average of 50.6%. In other words, even though 2000 nM equivalent was added to the wells, only the inhibitory activity equivalent to 500 nM was shown, indicating that approximately 75% of sequence 2 was decomposed during 24 hours of incubation with mouse plasma. When DMSO was mixed with mouse plasma and then subjected to a similar test immediately thereafter, the binding of sequence 46 (500 nM) was not inhibited at all, indicating that the plasma contaminating the binding system did not inhibit the system.

[0094] Immediately after mixing sequences 1 to 44 with mouse plasma or after 24 hours at 37°C, the sequences were further diluted to a final concentration of 2000 nM so that the plasma concentration was sufficiently low, and mixed with sequence 46 (500 nM) and subjected to the above-mentioned competitive binding test. The results are shown in Figure 4. The inhibitory activities of sequences 1 and 2 were estimated to be 70.7% and 79.4%, respectively. The inhibitory activities of sequences 6, 7, 9, 19, 24, 34, and 43 were 91.9%, 87.6%, 85.8%, 79.1%, 77.4%, 82.0%, and 83.5%, respectively, showing inhibitory activities equal to or greater than those of sequences 1 and 2. The inhibitory activities of the other sequences tended to be weakened. As with sequence 2, many of the amino acid substituted peptides consistently showed weakened inhibitory activity 24 hours after mixing with mouse plasma compared to when they were subjected to the competitive test immediately after mixing with mouse plasma, indicating that they were degraded in mouse plasma.

[0095] (Evaluation using an in vitro blood-brain barrier model) FIG. 5 shows an outline of an in vitro blood-brain barrier model for evaluating the ability to pass through the blood-brain barrier. The test was performed using a rat-type BBB kit (catalog No. RBT-24H, Pharmacocell) and a monkey-type BBB kit (catalog No. MBT-24H, Pharmacocell). These kits retain in vivo BBB characteristics (such as the presence or absence of expression of various receptors and transporters and the ability to form tight junctions), and are widely used as evaluation systems for estimating the brain migration of drugs (Non-Patent Documents 6 and 7). The insert well was set on the blood vessel side, and the bottom well was set on the brain side, and the amount of peptide added to the insert well that permeated the bottom surface of the three layers consisting of endothelial cells / membrane filter / pericytes was evaluated. In addition, the TEER value (transepithelial electrical resistance value), which indicates BBB function, was measured before and after the test, and it was confirmed that the addition of the peptide did not affect the BBB function.

[0096] The results of evaluating the BBB permeability of sequence 2 using ANG2 (Non-Patent Document 4) and L57 (Non-Patent Document 5), which have been reported to have LRP1 binding activity and BBB permeability, as comparison subjects are shown in FIG. 6. 5-FAM-labeled ANG (Sequence 47), 5-FAM-labeled L57 (Sequence 48), and Sequence 2 were added to the insert wells at final concentrations of 10 μM, 3 μM, and 1 μM, respectively, and cultured at 37° C. for 24 hours, and the culture supernatant of the bottom well was collected. A calibration curve was created by serially diluting each 5-FAM-labeled peptide with medium to an arbitrary concentration, and the concentration of the 5-FAM-labeled peptide contained in each collected culture supernatant was calculated. The amount of peptide (molar number) added to the insert well (blood vessel side) was set to 100%, and the amount of peptide (molar number) transferred to the bottom well (brain side) was calculated as %. The BBB permeability of Array 2 was significantly higher than that of 5-FAM-labeled ANG and L57 at all concentrations, regardless of whether it was the rat BBB shown in the upper graph or the monkey BBB shown in the lower graph (n=4, ±SD, *p<0.01, Dunnett's test for Array 2). After 24 hours of culture, the TEER value (transepithelial electrical resistance) in all wells was 150 Ω×cm 2These results indicate that the peptide of the present invention can pass through barrier tissues, particularly the blood-brain barrier. It was also confirmed that the BBB permeability of sequence 2, a representative example of the peptide of the present invention, is superior to that of existing ANG2 and L57.

[0097] (Synthesis and evaluation of modified peptide DBCO-KS-487) <Synthesis method> The synthesis was outsourced to Scrum Co., Ltd. (Tokyo, Japan). First, the following linear peptide precursors were synthesized by standard solid-phase synthesis using 9-fluoromethoxycarbonyl (Fmoc) as a protecting group for the α-amino group on an automated synthesizer, SyruII (Biotage).

[0098] NH2-Gly-Thr(tBu)-Pro-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Lys(Boc)-Tyr(tBu)-Nle-Leu-Ala-Glu(OtBu)-Nle-Cys(Trt)-Trt(2-Cl)-Resin

[0099] Next, the resin was immersed in dimethylformamide (DMF) and diisopropylethylamine (DIEA) was added to adjust the pH to 8. At this pH of 8, N3-PEG4-NHS (Cas#944251-24-5, catalog #J64834, Alfa Aesar) dissolved in DMF was added and reacted to introduce an azide group (N3-) to the N-terminal α-amino group, obtaining the following:

[0100] N3-PEG4-Gly-Thr(tBu)-Pro-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Lys(Boc)-Tyr(tBu)-Nle-Leu-Ala-Glu(OtBu)-Nle-Cys(Trt)-Trt(2-Cl)-Resin

[0101] Next, trifluoroacetic acid (TFA) / ultrapure water (Water) / thioanisole / 1,2-ethanedithiol (EDT) / triisopropylsilyl chloride (Tips) (83 / 5 / 5 / 5 / 2) was added to deprotect the side chains of each amino acid residue and to cleave the peptide from the resin. After the addition, the reaction was carried out at room temperature for 2 hours. After the reaction, the resin was filtered, and the obtained filtrate was added to cold ether to cause precipitation. The precipitate was dissolved in a small amount of ultrapure water and lyophilized to obtain the following.

[0102] N3-PEG4-Gly-Thr-Pro-Cys-Thr-Tyr-Lys-Tyr-Nle-Leu-Ala-Glu-Nle-Cys-OH (SEQ ID NO: 56)

[0103] The lyophilized product was dissolved in DMSO, and 0.1M NH4CO3 aqueous solution was added in small amounts to carry out an oxidation reaction with stirring at room temperature for 1-2 days to form SS bonds between the side chains of Cys residues. The product was purified by RP-HPLC and lyophilized to obtain the following cyclic peptide.

[0104] N3-PEG4-Gly-Thr-Pro-c(Cys-Thr-Tyr-Lys-Tyr-Nle-Leu-Ala-Glu-Nle-Cys)-OH (SEQ ID NO: 54)

[0105] The purified cyclic peptide and DBCO-PEG5-DBCO (Cas#2363130-04-3, catalog#BP-22450, manufactured by BROADPHARM) were dissolved in dimethyl sulfoxide (DMSO), mixed, and then 50 mM phosphate buffer was added. After the addition, the mixture was reacted at room temperature for 2 hours. The product was purified by RP-HPLC and lyophilized to obtain DBCO-KS-487 (theoretical molecular weight 2701.3, actual molecular weight 2702.0, purity 95.0%). Here, the cyclic peptide portion excluding the N-terminal modification portion (DBCO-PEG5-Taz-PEG4) is referred to as KS-487.

[0106] DBCO-PEG5-Taz-PEG4-Gly-Thr-Pro-c(Cys-Thr-Tyr-Lys-Tyr-Nle-Leu-Ala-Glu-Nle-Cys)-OH (SEQ ID NO: 55)

[0107] <Confirmation of Reactivity of DBCO> DBCO-KS-487 and 6-azido-hexanoic acid were mixed in an appropriate amount in DMSO / PBS (50 / 50) (pH 7.5) and reacted at room temperature. Then, the shift of the peak position of DBCO-KS-487 was evaluated by RP-HPLC. The results are shown in Figure 8. A peak (10.200 min) derived from DBCO-KS-487 is shown in the graph before the reaction in Figure 8, but the peak (10.248 min) derived from DBCO-KS-487 almost disappeared in the graph after the reaction in Figure 8. As shown in the graph after the reaction in Figure 8, new peaks (7.904 min and 9.285 min) emerged. From the results shown in Figure 8 (before and after the reaction), it was shown that the DBCO group added to the cyclic peptide retains the activity for click reaction.

Industrial Applicability

[0108] The peptide according to the present invention has LRP1 binding activity, and when the peptide binds to LRP1, it can pass through a barrier tissue that separates peripheral tissue and brain tissue via the RMT of LRP1, for example, the blood-brain barrier (BBB). Therefore, the peptide according to the present invention is considered to be useful as a means for transporting any molecule to the brain tissue by combining it with any molecule.

Claims

1. A linear or cyclic peptide comprising an amino acid sequence represented by a sequence number selected from the group consisting of sequence numbers 1 to 46 and having LRP1 binding activity, or a pharmacologically acceptable salt thereof.

2. A pharmaceutical, diagnostic agent and / or reagent comprising the peptide of claim 1.

3. A polynucleotide encoding an amino acid sequence comprising only naturally occurring amino acids among the amino acid sequences described in claim 1.

4. An expression vector comprising the polynucleotide of claim 3.

5. A cell, yeast, bacterium, virus, liposome, or nanoparticle presenting the amino acid sequence of claim 1.

Images