Novel LRP1 binding peptides

Abstract

A linear peptide or a cyclic peptide, or a pharmacologically acceptable salt thereof, having an amino acid sequence represented by formula (1). XN-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15 (1) The peptide, by binding to LRP1, is capable of delivering an arbitrary drug to brain tissue by endocytosis transport via LRP1.

Description

[0001] Cross Reference

[0002] This application claims priority based on Japanese Patent Application No. 2022-125238 filed in Japan on August 5, 2022, and all the contents described in the application are directly incorporated into this specification by reference. Technical Field

[0003] 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 from brain tissues, such as the blood-brain barrier (BBB), etc. by binding to LRP1. Specifically, the present invention relates to linear peptides or cyclic peptides having a specific amino acid sequence, etc. Background Art

[0004] Central nervous system diseases (mental illnesses) such as dementia, schizophrenia, and autism spectrum disorders are believed to have a patient population of 1 billion worldwide, accounting for the largest disability-adjusted life years, so their total social burden is very large. For example, the total economic burden in the United States and Europe in 2030 is expected to be 6 trillion US dollars (non-patent document 1). From these backgrounds, the development of drugs for central nervous system diseases is expected to be successful in a huge market, but on the other hand, it is also known to be the field of drug development with the highest failure rate (non-patent document 1). One of the reasons is that there are barrier tissues separating peripheral tissues and brain tissues in organisms: blood-brain barrier, blood-cerebrospinal fluid barrier, blood-spinal cord barrier, and blood-spinal barrier. Among them, the blood-brain barrier (Blood-Brain Barieer: BBB) is an obstacle to the development of drugs for central nervous system diseases. The blood-brain barrier is a physical barrier formed by pericytes and further astrocytes covering the periphery of brain capillary endothelial cells connected in a tightly bound manner, strictly controlling the inflow and outflow of substances between peripheral tissues and brain tissues (non-patent document 2). For example, it is known that substances that transfer from peripheral tissues to brain tissues by passive diffusion preferably have a molecular weight of 400 g / mol or less and a high lipid solubility. Therefore, drug development for central nervous system diseases is mostly attempted with low molecular weight compounds, but there are concerns about side effects caused by off-target effects, and drug development targets are also limited.

[0005] In particular, in drug development targets such as protein-protein interactions that have received attention in recent years, there are few clear pockets that low molecular weight compounds can bind to in the past, and the control of middle molecules such as peptides and macromolecules such as antibodies is excellent. In addition, since middle molecules and macromolecules have high specificity relative to drug development targets, it is also expected that side effects caused by off-target effects will be reduced. However, as mentioned above, due to the presence of barrier tissue, the technical problem is that it is extremely difficult for middle molecules and macromolecules to transfer from peripheral tissues to brain tissues by passive diffusion.

[0006] So far, as one of the methods for transferring middle molecules and macromolecules from peripheral tissues to brain tissues, the endocytic transport (receptor-mediated transcytosis RMT) of receptors expressed in the blood-brain barrier in order to transfer specific nutrients and essential components to the brain has been studied. For example, receptors for transporters, insulin, transferrin, and lipoproteins with glucose and amino acids as substrates can be cited (non-patent document 2). Regarding the attempt to couple antibodies and pharmacodynamic molecules that bind to human transferrin receptors and transport pharmacodynamic molecules to central tissues, there are already marketed drugs, and the concept verification of RMT has been carried out in human clinical trials (non-patent document 3).

[0007] Low density lipoprotein receptor-related protein 1 (LRP1) is also one of the receptors expected for RMT. The expression level of LRP1 in the blood-brain barrier is highly 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 between species. Moreover, a molecule in which paclitaxel, an anticancer agent, is coupled to Angiopep-2 (ANG2), known as an LRP1 binding peptide, has completed the second phase of human clinical trials, confirming the safety of LRP1-based RMT in humans (Non-patent Document 4).

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent document 1: L Ereshefsky, R Evans, R Sood, Dwilliamson and B.A English. “VENTURING INTO ANEW ERA OF CNS DRUG DEVELOPMENT TO IMPROVE SUCCESS” 2015.

[0011] Non-patent literature 2: VM Pulgar. Front Neurosci. 2019, 12: 1019.

[0012] Non-patent literature 3: R Yamamoto and S Kawashima. Nihon Yakugaku Zasshi (Japanese Journal of Pharmaceutical Sciences). 2022, 157(1): 62-75.

[0013] Non-patent document 4: P Kumthekar, Sc Tang, AJ Brenner, S Kesari, D. V Piccioni, C Anders, J Carrillo, P Chalasani, P Kabos, S Puhalla, K Tkaczuk, A. A Garcia, M. S Ahluwalia, JS Wefel, N Lakhani and N Lbrahm. Clin Cancer Res. (Clinical Cancer Research) 2020, 26(12): 2789-2799.

[0014] Non-patent document 5: K Sakamoto, T Shinohara, Y Adachi, T Asami and T Ohtaki. Biochem Biophys Rep. (Biochemistry and Biophysics Reports) 2017, 12: 135-139.

[0015] Non-patent document 6: F Tanaka, S Ohgu, A Yamauchi, J Matsumoto, T Machida, K Fujishita, K Shibata, Y Shinozaki, K Sato, Y Kataoka and S Koizumi. PLoS One. 2013, 8(1): e55166.

[0016] Non-patent literature 7: D Watanabe, S Nakagawa, Y Morofuji, A Toth, M Vastag, J Aruga, M Niwa and M Deli. Pharmaceutics. 2021, 13(9): 1484.

[0017] Non-patent document 8: DP Fairlie and A Dantas. Biopolymers 2016, 106(6): 843-852.

[0018] Non-patent document 9: YH Lau, P Andrade, Y Wu, and DR Spring. Chem Soc Rev. 2015, 44(1) 91-102. Summary of the invention

[0019] Technical problem to be solved by the invention

[0020] The invention described in this specification aims to provide a novel peptide that binds to LRP1. The invention also aims to provide a method for using "a certain molecule" as a pharmaceutical, diagnostic drug and / or research reagent by combining the peptide with "a certain molecule" and delivering "a certain molecule" to brain tissue via RMT based on LRP1.

[0021] Technical solutions for solving technical problems

[0022] The present inventors have searched for molecules that bind to LRP1 and have completed the present invention. Specifically, one embodiment of the present invention is a linear peptide or cyclic peptide having an amino acid sequence represented by the following formula (1), or a pharmacologically acceptable salt thereof.

[0023] 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)

[0024] Among them, in formula (1), X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7 represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8 represents ornithine, lysine, homolysine, arginine or homoarginine, X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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 12represents alanine, D-alanine or 2-aminoisobutyric acid, X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid. The N-terminal amino group and the C-terminal carboxyl group can be modified or deleted. X 5 and X 15 When cysteine ​​or D-cysteine ​​participates in the cyclization, covalent bonds are formed between the side chain -SH groups via disulfide bonds, methylene, acetylmethylene, ethylene or propylene linking groups, so that the peptide of formula (1) can have a cyclic structure in the molecule.

[0025] X in the peptide of the above formula (1) 1 ~X 15 Preferred embodiments of the amino acid residues are described in detail in the following embodiments, and the respective preferred embodiments can be arbitrarily combined independently of each other.

[0026] More specifically, the present invention includes the following embodiments.

[0027] [1]

[0028] A linear peptide or cyclic peptide having an amino acid sequence represented by the following formula (1), or a pharmacologically acceptable salt thereof.

[0029] 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)

[0030] (Where X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7 represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8 represents ornithine, lysine, homolysine, arginine or homoarginine, X 9represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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, X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid. The N-terminal amino group and the C-terminal carboxyl group can be modified or deleted. X 5 and X 15 In the case of participating in the cyclization, cysteine ​​or D-cysteine ​​is used, and a covalent bond is formed between each side chain -SH group via a disulfide bond, a methylene group, an acetyl methylene group, an ethylene group or a propylene group as a linking group, so that the peptide of formula (1) can have a cyclic structure in the molecule.

[0031] [2]

[0032] A linear peptide or cyclic peptide having an amino acid sequence represented by the following formula (3), or a pharmacologically acceptable salt thereof.

[0033] 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)

[0034] (Where X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7 represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8represents ornithine, lysine, homolysine, arginine or homoarginine, X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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, X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid. The N-terminal amino group and the C-terminal carboxyl group can be modified or deleted. X 5 and X 16 In the case of participating in the cyclization, cysteine ​​or D-cysteine ​​is used, and a covalent bond is formed between each side chain -SH group via a disulfide bond, a methylene group, an acetyl methylene group, an ethylene group or a propylene group as a linking group, so that the peptide of formula (3) can have a cyclic structure in the molecule.

[0035] 〔3〕

[0036] A linear peptide or cyclic peptide having an amino acid sequence represented by the following formula (3), or a pharmacologically acceptable salt thereof.

[0037] 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)

[0038] (Where X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8 represents ornithine, lysine, homolysine, arginine or homoarginine, X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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, X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid. The N-terminal amino group and the C-terminal carboxyl group can be modified or deleted. X 6 and X 16 In the case of participating in the cyclization, cysteine ​​or D-cysteine ​​is used, and a covalent bond is formed between each side chain -SH group via a disulfide bond, a methylene group, an acetyl methylene group, an ethylene group or a propylene group as a linking group, so that the peptide of formula (3) can have a cyclic structure in the molecule.

[0039] [4]

[0040] The linear peptide or cyclic peptide according to any one of [1] to [3], or a pharmacologically acceptable salt thereof, wherein X N It is Lys-Gly-Thr-Pro or Gly-Thr-Pro.

[0041] 〔5〕

[0042] A linear peptide or cyclic peptide having LRP1 binding activity, or a pharmacologically acceptable salt thereof, comprising an amino acid sequence in which one or more amino acids are deleted, added and / or substituted in the amino acid sequence described in any one of [1] to [4].

[0043] [6]

[0044] [1] to [5], and a derivative and / or modified form of the peptide described in any one of

[14] to

[21] below.

[0045] 〔7〕

[0046] The derivative or modified form of the peptide according to [6], wherein the derivative or modified form 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 directly or via a linking group.

[0047] 〔8〕

[0048] A medicine, diagnostic drug and / or reagent comprising the peptide or its derivative or modified form as described in any one of [1] to [7] and

[14] to

[21] below.

[0049] 〔9〕

[0050] A peptide-drug complex, which is a peptide derivative or modified form of the peptide described in [6] directly linked to a drug or linked via a linking group.

[0051] 〔10〕

[0052] The peptide drug complex according to [9], wherein the drug is an antibody, a nucleic acid or a bioactive peptide.

[0053]

[11]

[0054] 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 having a base sequence with a sequence identity of 70% or more with the polynucleotide and encoding a peptide having LRP1 binding activity.

[0055]

[12]

[0056] An expression vector comprising the polynucleotide described in

[11] .

[0057] 〔13〕

[0058] A cell, yeast, bacterium, virus, liposome or nanoparticle presenting the amino acid sequence described in any one of [1] to [5], or

[14] to

[21] below.

[0059]

[14]

[0060] A linear peptide or cyclic peptide comprising an amino acid sequence represented by the following formula (6), or a pharmacologically acceptable salt thereof.

[0061] X 1 -X 2 -X 3 -X 4 -X L -X 7 -X 8 -X 9 -X 10 -X11 -X 12 -X 13 -X 14 -X M (6)

[0062] (X L For X 5 -X 6 , X M For X 15 -X 16 , when participating in the cyclization within the peptide molecule, each independently represents an amino acid residue having an amino group, a carboxyl group, a thiol group, an allyl group, an alkynyl group, an azide group or a halogen atom, or a derivative thereof; when not participating 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, its derivative or deletion, X 7 represents an amino acid residue having a hydrocarbon group which may have a substituent, an amino acid residue having an aromatic carbocyclic group which may have a substituent, an amino acid residue having an aromatic heterocyclic group which may have a substituent, or a derivative thereof, and X 8 represents amino acid residues or their derivatives with positive charge, X 11 , X 12 and X 14 Each independently represents an amino acid residue having a hydrocarbon group which may have a substituent, or a derivative thereof, X 13 represents an amino acid residue having a hydrocarbon group which may have a substituent, an amino acid residue having a negative charge, or a derivative thereof, and 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, its derivative or deletion, X L With X M A direct covalent bond or an indirect covalent bond via a linking group is formed between the two, so that the peptide of formula (6) can have a cyclic structure in the molecule. In this case, X L With X M The covalent bonds between them can be any bonds between their main chains, main chains and side chains, side chains and main chains, or side chains and side chains. The N-terminal amino group and the C-terminal carboxyl group can be modified or missing).

[0063] 〔15〕

[0064] The linear peptide or cyclic peptide according to

[14] , or a pharmacologically acceptable salt thereof, wherein X 5 With X 15 The bonds between Cα carbon atoms, X 6 With X 15 The bonds between Cα carbon atoms, X 5 With X 16 The bond between Cα carbon atoms or X 6 With X 16 The bonds between the Cα carbon atoms of each independently include the structure represented by the following formula (7).

[0065] Cα L -(CH2) A -Z-(CH2) B -Cα M (7)

[0066] (wherein, L is 4 or 5, M is 14 or 15, A and B each independently represent an integer from 0 to 2, the sum of A and B is an integer from 0 to 4, and Z represents 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-CH 2. 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 on the phenylene ring is any one of the ortho position, meta position and para position), S-CH2-C(=O)-CH2-S, or S-CH2-C(=CH2)-CH2-S.)

[0067]

[16]

[0068] The linear peptide or cyclic peptide according to

[14] or

[15] , or a pharmacologically acceptable salt thereof, wherein X 82,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, lysine, homolysine, arginine, homoarginine, D-amino acids thereof, N-methylated amino acids thereof, α-methylated amino acids thereof, or derivatives thereof.

[0069]

[17]

[0070] The linear peptide or cyclic peptide according to any one of

[14] to

[16] , or a pharmacologically acceptable salt thereof, wherein X 12 Alanine, D-alanine, 2-aminoisobutyric acid, 2-aminobutyric acid, D-2-aminobutyric acid, isovaline, D-isovaline, valine, D-valine, isoleucine, D-isoleucine, leucine, D-leucine, their N-methylated amino acids, their α-methylated amino acids, or their derivatives.

[0071]

[18]

[0072] The linear peptide or cyclic peptide according to any one of

[14] to

[17] , or a pharmacologically acceptable salt thereof, wherein X 11 and X 14 Each is independently methionine, an amino acid residue represented by the following formula (8), the following formula (9), or the following formula (10), or a derivative thereof.

[0073] [Chemistry 1]

[0074]

[0075] (wherein the wavy line represents the point of attachment to the carbonyl or nitrogen atom forming the amide bond of the main chain, 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 from 0 to 10)

[0076] [Chemistry 2]

[0077]

[0078] (where Z represents C or N, R 6 represents a hydrogen atom or a methyl group, and n represents any integer from 1 to 6)

[0079] [Chemistry 3]

[0080]

[0081] (In the formula, the wavy line represents the bonding site with the carbonyl group or nitrogen atom forming the amide bond of the main chain, Z represents C or N, and R 6 represents a hydrogen atom or a methyl group, and n represents any integer from 1 to 6)

[0082]

[19]

[0083] The linear peptide or cyclic peptide according to any one of

[14] to

[18] , or a pharmacologically acceptable salt thereof, wherein X 7 It is an amino acid residue represented by the following formula (8), the following formula (9), the following formula (10), the following formula (11), the following formula (12) or the following formula (13), or a derivative thereof.

[0084] [Chemistry 4]

[0085]

[0086] (wherein the wavy line represents the point of attachment to the carbonyl or nitrogen atom forming the amide bond of the main chain, 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 from 0 to 10)

[0087] [Chemistry 5]

[0088]

[0089] (where Z represents C or N, R 6 represents a hydrogen atom or a methyl group, and n represents any integer from 1 to 6)

[0090] [Chemistry 6]

[0091]

[0092] (In the formula, the wavy line represents the bonding site with the carbonyl group or nitrogen atom forming the amide bond of the main chain, Z represents C or N, and R 6 represents a hydrogen atom or a methyl group, and n represents any integer from 1 to 6)

[0093] [Chemistry 7]

[0094]

[0095] (Where R 12represents 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thiazolyl, 4-thiazolyl, 2-oxazolyl, 4-oxazolyl, 1-pyrazolyl, 1H-imidazol-4-yl, 1-imidazolyl, 1,2,3-triazol-1-yl or 1,2,4-triazol-1-yl, R 6 represents a hydrogen atom or a methyl group)

[0096] [Chemistry 8]

[0097]

[0098] (wherein the wavy line represents the point of attachment to the carbonyl or nitrogen atom forming the amide bond of the main chain, 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. )

[0099] [Chemistry 9]

[0100]

[0101] (Where 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. )

[0102] 〔20〕

[0103] The linear peptide or cyclic peptide according to any one of

[14] to

[19] , or a pharmacologically acceptable salt thereof, wherein X 13 It is glycine, alanine, aspartic acid, glutamic acid, asparagine, glutamine, D-alanine, D-aspartic acid, D-glutamic acid, D-asparagine, D-glutamine, their N-methylated amino acids, their α-methylated amino acids, or their derivatives.

[0104] 〔twenty one〕

[0105] The linear peptide or cyclic peptide or a pharmacologically acceptable salt thereof according to any one of

[14] to

[20] , wherein X which does not participate in cyclization in the peptide molecule L and X Meach independently represents alanine, isovaline, norvaline, serine, homoserine, threonine, allothreonine, O-methylated serine, O-methylated homoserine, O-methylated threonine, O-methylated allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline, trans-4-hydroxyproline, azetidine-2-carboxylic acid, pipecolic acid, D-serine, D-homoserine, D-threonine, D-allothreonine, D-methylated amino acids, D-O-methylated serine, D-methylated homoserine, D-methylated threonine, D-methylated allothreonine, D-2,3-diaminopropionic acid, D-2,4-diaminobutyric acid, D-ornithine, D-lysine, D-arginine, D-proline, D-cis-4-hydroxyproline, D-trans-4-hydroxyproline, D-azetidine-2-carboxylic acid, D-pipecolic acid, their N-methylated amino acids, their α-methylated amino acids, or their derivatives.

[0106] Effects of the Invention

[0107] According to the present invention, there is provided a novel linear peptide and cyclic peptide useful as a pharmaceutical, diagnostic drug and / or research reagent for transferring "a certain molecule" combined with the present peptide to brain tissue via RMT via binding to LRP1. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 The results are obtained by evaluating the concentration-dependent binding activity of sequences 45 and 46, which are representative examples of biotin-labeled peptides of the present invention, to LRP1 cluster 2 (CL2)-Fc chimeric protein, LRP1 cluster 3 (CL3)-Fc chimeric protein, and LRP1 cluster 4 (CL4)-Fc chimeric protein. The concentrations of the biotin-labeled peptides used in the experiment are, from left to right, (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, and the columns are shown with shades.

[0109] Figure 2 The following is an overview of a competitive binding test using the ELISA method used to evaluate both the binding activity of the peptide of the present invention to LRP1 cluster 4 and the resistance to protease degradation. In the figure, SA represents streptavidin, B represents biotin, and HRP represents horseradish peroxidase.

[0110] Figure 3The results are as follows: 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 was evaluated in the presence of sequence 2 (2000-63 nM) immediately after mixing with mouse plasma or 24 hours after mixing. The concentrations of the peptide 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 the columns are shown with shades.

[0111] Figure 4 The results show the results of evaluating the inhibitory activity (Inhibition (%)) of Sequence 46 (500 nM), a representative example of the biotin-labeled peptide of the present invention, against the 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 after mixing.

[0112] Figure 5 The outline of the in vitro blood-brain barrier model for evaluating the ability to cross the blood-brain barrier is presented.

[0113] Figure 6 The figures show 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 cross the blood-brain barrier using an in vitro blood-brain barrier model (rat type and monkey type).

[0114] Figure 7 The structural formulas of Sequence 2 and Sequence 46 are shown as representative examples of the peptides of the present invention used in Examples.

[0115] Figure 8 These are the results of RP-HPLC analysis of a solution in which the peptide-modified DBCO-KS-487 of the present invention (before reaction) and 6-azidohexanoic acid were reacted at room temperature (after reaction). DETAILED DESCRIPTION

[0116] Next, various embodiments of the present invention will be described with reference to the accompanying drawings. It should be noted that the various embodiments described below do not limit the invention involved in the claims, and all of the various elements and their combinations described in the various embodiments are not necessarily required for the solution of the present invention.

[0117] (definition)

[0118] In this specification, a peptide refers to a substance obtained by bonding two or more amino acids through an amide bond (peptide bond), for example, a substance obtained by bonding 2 to 20 amino acids through an amide bond. 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 (carboxyl terminus). The first carbon atom adjacent to the carbonyl group forming the peptide bond is called the Cα carbon.

[0119] In this specification, "any amino acid residue or its derivative" is used in its broadest sense and includes, in addition to natural amino acids, artificial amino acids with non-natural structures, chemically synthesized compounds with properties known in the art that are characteristic of amino acids, and carboxylic acids with functional groups. Examples of non-natural amino acids include D-type amino acids, α / α-disubstituted amino acids whose main chain structures are different from those of natural ones (α-methylated amino acids such as 2-aminoisobutyric acid), N-alkyl amino acids (N-methylated amino acids), N-substituted glycine (peptides), amino acids whose main chains are extended (β-homoamino acids, γ-homoamino acids), amino acids whose side chain structures are different from those of natural ones (cyclohexylalanine, allylglycine, 2-(2-pyridyl)-glycine, 3-(1H-benzimidazol-2-yl)-alanine, etc.), amino acids whose side chains are partially substituted (norleucine, diaminopropionic acid, 3-(2-pyridyl)-alanine, etc.), amino acids whose side chains have extra functional groups; amino acids whose side chains have extra C, alkyl, or methyl groups (homonorleucine, γ-methylleucine, etc.), and amino acids whose side chains have halogen atoms (F, Cl, Br, I). The present invention also includes amino acids having a halogen atom (F, Cl, Br, I) on the side chain (3-chloropropionic acid, etc.), carboxylic acids having a functional group on the side chain (3-butenoic acid, etc.), amino acids having an extra N or amino group on the side chain (β-azidoalanine, ornithine, etc.), amino acids having an extra O or methoxy group on the side chain (O-methylserine, O-methylthreonine, etc.), amino acids having an extra hydroxyl group on the side chain (3-hydroxyphenylalanine, etc.), amino acids having an extra carboxyl group (-COOH) on the side chain (3-carboxyphenylalanine, etc.), amino acids having an extra S on the side chain (ethionine, etc.), amino acids having an ester-protected carboxylic acid functional group on the side chain (4-methyl aspartate, etc.), amino acids having a sulfenyl group (-S-) on the side chain oxidized to be converted into a sulfinyl group (-S(=O)-) or a sulfonyl group (-S(=O)2-) (methionine sulfoxide), etc., but is not limited to these.

[0120] In the present specification, "LRP1" refers to LRP1 of mammals such as mouse, rat, dog, monkey, and human.

[0121] In this specification, "having LRP1 binding activity" means that in an in vitro test, the peptide of the present invention or the combination of the peptide of the present invention and "a certain molecule" binds to the full-length protein of LRP1, partial proteins such as cluster 4, or chimeras of proteins other than these in a concentration-dependent manner. The presence or absence of LRP1 binding activity can be confirmed by a known method with reference to Non-Patent Document 5, etc., but the present invention is not limited to this.

[0122] In the present specification, "barrier tissue" refers to the barrier tissue of mammals such as mice, rats, dogs, monkeys, and humans.

[0123] In the present specification, "penetrating the barrier tissue" means: (1) in an experiment using an in vitro blood-brain barrier model, the peptide of the present invention or the combination of the peptide of the present invention and "a certain molecule" is transferred from the pores on the blood vessel side to the pores on the brain side; (2) in a pharmacokinetic experiment using LC-MS / MS, etc., the peptide of the present invention or the combination of the peptide of the present invention and "a certain molecule" administered peripherally is detected from the brain tissue; (3) in a pharmacokinetic experiment using an in vivo imaging system, etc., the peptide of the present invention or the combination of the peptide of the present invention and "a certain molecule" administered peripherally is detected in the brain, etc. In the case of any of these effects, it is called "penetrating the barrier tissue". Regarding the presence or absence of the ability to pass through the barrier tissue, those skilled in the art can refer to non-patent documents 6 and 7, etc., and confirm it according to a known method, but it is not limited to this.

[0124] (Peptide of the present invention)

[0125] [1] The linear peptide and cyclic peptide in one embodiment of the present invention have an amino acid sequence represented by the following formula (1).

[0126] 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)

[0127] In formula (1), X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7 represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8 represents ornithine, lysine, homolysine, arginine or homoarginine, X 9represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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, X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid. The N-terminal amino group and the C-terminal carboxyl group may be modified or deleted. 5 and X 15 When cysteine ​​or D-cysteine ​​participates in the cyclization, covalent bonds are formed between the side chain -SH groups via disulfide bonds, methylene, acetylmethylene, ethylene or propylene linking groups, so that the peptide of formula (1) can have a cyclic structure in the molecule.

[0128] In the peptide of the above formula (1), X N Preferably, Lys-Gly-Thr-Pro or Gly-Thr-Pro. When the N-terminus is lysine, it can be bonded to a molecule via the epsilon amino group of its side chain. When the N-terminus is glycine, it can be bonded to a molecule via the alpha amino group of its side chain. Preferably, X 5 and X 15 It is cysteine ​​or D-cysteine, and the side chain -SH groups thereof form a covalent bond via a disulfide bond or a methylene linking group, thereby forming a ring structure in the molecule.

[0129] As a further preferred embodiment of the peptide of the above formula (1), for example, an amino acid sequence represented by the following formula (2) can be cited. A covalent bond is formed between each side chain -SH group via a disulfide bond or a methylene linking group, thereby having a ring structure in the molecule, or bonding to a certain molecule via the ε amino group of Lys1.

[0130] Ac-Lys-Gly-Thr-Pro-Cys-X 6 -X 7 -Lys-X 9 -X 10 -Leu-Ala-Glu-X 14 -Cys-OH(2)

[0131] In formula (2), X 6 represents serine, threonine or 2,3-diaminopropionic acid, X 7represents 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.

[0132] [2] The linear peptide and cyclic peptide in another embodiment of the present invention have an amino acid sequence represented by the following formula (3).

[0133] 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)

[0134] In the above formula (3), X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7 represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8 represents ornithine, lysine, homolysine, arginine or homoarginine, X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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, X 13represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid. The N-terminal amino group and the C-terminal carboxyl group may be modified or deleted. 5 and X 16 When cysteine ​​or D-cysteine ​​participates in the cyclization, covalent bonds are formed between the side chain -SH groups via disulfide bonds, methylene, acetyl methylene, ethylene or propylene linking groups, so that the peptide of formula (3) can have a cyclic structure in the molecule.

[0135] In the peptide of the above formula (3), X N Preferably, Lys-Gly-Thr-Pro or Gly-Thr-Pro. When the N-terminus is lysine, it can be bonded to a molecule via the epsilon amino group of its side chain. When the N-terminus is glycine, it can be bonded to a molecule via the alpha amino group of its side chain. Preferably, X 5 and X 16 It is cysteine ​​or D-cysteine, and the side chain -SH groups thereof form a covalent bond via a disulfide bond or a methylene linking group, thereby forming a ring structure in the molecule.

[0136] As a further preferred embodiment of the peptide of the above formula (3), for example, an amino acid sequence represented by the following formula (4) can be cited. A covalent bond is formed between each side chain -SH group via a disulfide bond or a methylene linking group, thereby having a ring structure in the molecule, or bonding to a certain molecule via the ε amino group of Lys1.

[0137] Ac-Lys-Gly-Thr-Pro-Cys-X 6 -X 7 -Lys-X 9 -X 10 -Leu-Ala-Glu-X 14 -Pro-Cys-OH(4)

[0138] 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.

[0139] [3] In another embodiment of the present invention, the linear peptide and the cyclic peptide have an amino acid sequence represented by the following formula (3).

[0140] XN -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -Pro-X 16 (3)

[0141] In the above formula (3), X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7 represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8 represents ornithine, lysine, homolysine, arginine or homoarginine, X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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, X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid. The N-terminal amino group and the C-terminal carboxyl group can be modified or deleted. X 6 and X 16 When cysteine ​​or D-cysteine ​​participates in the cyclization, covalent bonds are formed between the side chain -SH groups via disulfide bonds, methylene, acetyl methylene, ethylene or propylene linking groups, so that the peptide of formula (3) can have a cyclic structure in the molecule.

[0142] In the peptide of the above formula (3), X NPreferably, Lys-Gly-Thr-Pro or Gly-Thr-Pro. When the N-terminus is lysine, it can be bonded to a molecule via the epsilon amino group of its side chain. When the N-terminus is glycine, it can be bonded to a molecule via the alpha amino group of its side chain. Preferably, X 6 and X 16 It is cysteine ​​or D-cysteine, and the side chain -SH groups thereof form a covalent bond via a disulfide bond or a methylene linking group, thereby forming a ring structure in the molecule.

[0143] As a further preferred embodiment of the peptide of the above formula (3), for example, an amino acid sequence represented by the following formula (5) can be cited. A covalent bond is formed between each side chain -SH group via a disulfide bond or a methylene linking group, thereby having a cyclic structure in the molecule, or bonding to a certain molecule via the ε amino group of Lys1.

[0144] Ac-Lys-Gly-Thr-Pro-X 5 -Cys-X 7 -Lys-X 9 -X 10 -Leu-Ala-Glu-X 14 -Pro-Cys-OH(5)

[0145] 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.

[0146] [4] The peptides involved in this embodiment include peptides having binding activity to LRP1 even if they are homologous peptides in which 1 to several amino acids are deleted, added and / or substituted in the amino acid sequences shown in [1] to [3] above. In this specification, when referred to as "a peptide in which 1 to several amino acids are deleted, added and / or substituted", the number of these amino acids is not particularly limited as long as the peptide has LRP1 binding activity, and is preferably 1 to 5, and more preferably 1 or 2. The site of deletion, addition and / or substitution may be at the end of the peptide or in the middle, and may be 1 place or 2 or more places.

[0147] Examples of such amino acid sequences in which one to several amino acids are deleted, added and / or substituted in the above amino acid sequence include amino acid sequences that have an identity of at least 50% or more, preferably 70% or more, further preferably 80% or more, and particularly preferably 90% or more with the above 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., initially set parameters).

[0148] In addition, it is known that there are peptides and protein domains that have highly similar three-dimensional structures even if the homology in the primary structure is low. Therefore, peptides having a three-dimensional structural homology of at least 50%, preferably more than 70%, more preferably more than 80%, and particularly preferably more than 90% with the above-mentioned amino acid sequence, and having Ras binding activity are also included in the present embodiment. The homology of the three-dimensional structures of such peptides can be carried out by predicting the three-dimensional structure of the amino acid sequence of the peptide with unknown three-dimensional structure using homology modeling methods, etc. For example, when an arbitrary amino acid sequence (target sequence) having a sequence similar to the cyclic peptide (reference peptide) of the present embodiment is given, an alignment between the target sequence and the reference sequence is given (the sequences are placed side by side). If an alignment calculated by FASTA, PSI-BLAST, LIBRA, etc. is used, the corresponding relationship between each amino acid between the target sequence and the reference sequence is determined, so based on this relationship, the 3-dimensional coordinates of each amino acid on the target sequence are made by the 3-dimensional coordinates of the reference peptide. In the construction of 3D coordinates, sometimes inappropriate gaps, conflicts, and deformations are generated between amino acid residues, so these structural deformations are eliminated by energy minimization calculations. According to the modeling software, in order to smoothly eliminate the deformation of the structure, sometimes all the atoms of the peptide are not carried out at the same time, but are carried out in stages. That is, first the α carbon atom that forms the skeleton of the peptide is carried out, then the main chain atoms containing the α carbon atom are carried out, and finally the entire peptide containing the side chain atoms is carried out. If the comparison for the target sequence is obtained in this way, its three-dimensional structure can be predicted and constructed. About the index of three-dimensional structural homology, the difference of the XYZ coordinates when the best overlap can be used, i.e. RMSD (Root Mean Square Deviation) etc. for comparison.

[0149] The peptide of this embodiment also includes a salt of the peptide. As a salt of the peptide, a salt with a physiologically acceptable base or acid can be used, for example, an addition salt of an inorganic acid (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), an addition salt of an organic acid (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), an addition salt of an inorganic base (ammonium hydroxide, or alkali, or alkaline earth metal hydroxide, carbonate, bicarbonate, etc.), an amino acid, etc.

[0150] (Peptide derivatives and modifications)

[0151] The peptide of the present embodiment also includes various derivatives and / or modified forms thereof as long as they can solve the technical problems of the present invention. Examples of the derivatives include: derivatives in which a saturated fatty chain of a peptide is replaced by an unsaturated fatty chain, derivatives in which a part of the atoms of a peptide is replaced by other atoms containing radioactive or non-radioactive isotope atoms, derivatives in which an amide bond of a peptide is replaced by a thioamide bond (-NH-C(=S)-), derivatives in which an amide bond of a peptide is replaced by an olefin (-C=C-), derivatives in which an amide bond of a peptide is replaced by an alkyl (-CC-), derivatives in which an amide bond of a peptide is replaced by a hydroxyethylene (-C(-OH)-C-), derivatives in which an amide bond of a peptide is replaced by an ester (-OC(=O)-), derivatives in which an amide bond of a peptide is replaced by an olefin (-C=C-), derivatives in which an amide bond of a peptide is replaced by (-C-NH-), or derivatives in which an amide bond of a peptide is replaced by (-C(=O)-C-); examples of the modified form include: a modified form in which the α-carbon of a peptide is disubstituted, a modified form of a peptide The invention also includes a modified form in which an amide bond is N-alkylated, a modified form in which a part of the functional groups of the peptide is modified by halogenation, cyanation, nitration, oxidation, hydroxylation, amination, deamination, dehydrogenation, amidation, acetylation, methoxylation, prenylation, alkylation, or the like (for example, a modified form in which a part of the amino groups of the peptide is acetylated, alkylated, or deaminated, a modified form in which a part of the carboxyl groups of the peptide is amide or ester, etc.), a modified form in which S of the peptide is sulfoxide S(=O) or sulfone S(=O)2, a modified form in which the peptide is polymerized via a chemical linking group, a modified form in which the peptide is labeled with biotin, a modified form in which the peptide is fluorescently labeled, a modified form in which the peptide is luminescently labeled, and a modified form in which an alkyl chain, polyethylene glycol, antibody, lectin, sugar chain, enzyme, peptide, peptoid, membrane permeable peptide, low molecular weight compound, nucleic acid, or molecule that induces ubiquitination of protein is fused or coupled to the peptide directly or via a linking group, etc., but the invention is not limited to these.

[0152] According to some embodiments of the present invention, the peptide of the present invention comprises an amino acid residue having an azido group at its N-terminus or C-terminus. Therefore, a "certain molecule" having an alkyne group can be covalently bonded to the N-terminus or C-terminus of the peptide of the present invention by a "monovalent copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction" (or simply referred to as a "click" reaction). According to another embodiment of the present invention, an azido group is introduced by reacting a linking group having an azido group and an N-hydroxysuccinimide (NHS) ester group with the α-amino group or ε-amino group of the peptide of the present invention. Therefore, a "certain molecule" having an alkyne group can be covalently bonded to the peptide of the present invention by a CuAAC reaction. According to another embodiment of the present invention, 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 azido group can be covalently bonded to the N-terminus or C-terminus of the peptide of the present invention by a CuAAC reaction. According to another embodiment of the present invention, an alkyne group is introduced by reacting a linking group having an alkyne group and an NHS ester group with the α-amino group or ε-amino group of the peptide of the present invention. Therefore, the "certain molecule" having an azide group can be covalently bonded to the peptide of the present invention through a CuAAC reaction. The CuAAC reaction is as follows: Scheme 1:

[0153] [Chemistry 10]

[0154] Solution 1

[0155]

[0156] The CuAAC reaction yields 1,5-disubstituted-1,2,3-triazoles. The reaction between alkynes and azides is very selective, and alkyne and azide groups do not exist in natural biological molecules. Furthermore, the reaction is rapid and pH-insensitive.

[0157] The above-mentioned alkyne group can be replaced by a cyclooctyne group. According to some embodiments of the present invention, the peptide of the present invention comprises 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 a "strain-promoted azide-alkyne click chemistry (SPAAC) reaction". According to another embodiment of the present invention, an azide group is introduced by reacting a linking group 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 a SPAAC reaction. According to another embodiment of the present invention, the peptide of the present invention comprises 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 a SPAAC reaction. According to another embodiment of the present invention, a cyclooctyne group is introduced by reacting a linking group having a cyclooctyne group and an NHS ester group with an α-amino group or an ε-amino group of the peptide of the present invention. Therefore, the "certain molecule" having an azide group can be covalently bonded to the peptide of the present invention through a SPAAC reaction. The SPAAC reaction is as follows:

[0158] [Chemistry 11]

[0159] Solution 2

[0160]

[0161] Alternatively, in a specific embodiment, a "molecule" having a tetrazine group or a cyclooctenyl group is bonded to the cyclooctenyl group or the tetrazine group corresponding to the peptide or the linker group through an "inverse electron demand Diels-Alder (iEDDA) reaction" as shown in the following Scheme 3.

[0162] [Chemistry 12]

[0163] Solution 3

[0164]

[0165] Examples of the amino acid residue 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 the amino acid residue having an alkynyl group include L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-HPG), and β-homopropargylglycine (β-HPG).

[0166] The deformed alkyne group present at the free end of the linking group may be a cyclooctenyl group, such as a trans-cyclooctenyl (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 present at the free end of the linking group may include 1,2,3,4-tetrazine group, 1,2,3,5-tetrazine group, and 1,2,4,5-tetrazine group, and derivatives thereof, such as 6-methyltetrazine group, but is not limited thereto.

[0167] In a preferred embodiment of the present invention, the derivatives and modified forms of the peptide have an azido group, an alkyne group or a tetrazine group directly or via a linking group at the N-terminus, C-terminus or amino acid side chain. As an example of such a derivative, the following examples can be cited. For example, if 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 the peptide, a peptide (SEQ ID NOs. 49 and 50) to which DBCO or BCN is added via a PEG linking group can be obtained.

[0168] [Chemistry 13]

[0169]

[0170] Alternatively, as shown in the following Scheme 4, by reacting DBCO-PEG5-DBCO (BP-22450) with an N-terminal lysine into which an azide group has been previously introduced using Fmoc-Lys(εN3)-OH, a peptide (SEQ ID NOs. 51 and 52) having a DBCO group introduced into the side chain of the amino acid can be obtained.

[0171] [Chemistry 14]

[0172] Solution 4

[0173]

[0174] Furthermore, in the following scheme 5, N3-PEG4-NHS (N-Succinimidyl 15-azido-4,7,10,13-tetraoxapentadecanoate) can be reacted with the α-amino group of glycine at the N-terminus of the peptide shown in sequence number 53 to synthesize a peptide introduced with an azido group (sequence number 54), and then DBCO-PEG5-DBCO can be used to obtain a peptide introduced with a DBCO group at the N-terminus (sequence number 55).

[0175] [Chemistry 15]

[0176] Solution 5

[0177]

[0178] (Peptide Drug Complex)

[0179] The peptide of the present invention may be a conjugate (complex). The complex is a complex comprising the peptide of the present invention, a linking group bound to the peptide, and a "certain molecule" bound to the linking group. The complex is preferably a complex in which at least the "certain molecule" can pass through the barrier tissue. It may also be a complex as a whole that can pass through the barrier tissue.

[0180] Examples of the linking group include, but are not limited to, a linking group consisting of amino acids of about 1 to 15 residues, a linking group consisting of fatty acids, a linking group consisting of polyethylene glycol, or derivatives thereof. The linking group may be a linking group that deviates or separates under specific environments and conditions, or a linking group that maintains a stable structure.

[0181] The "certain molecule" described in this specification can be any molecule desired by a person skilled in the art as long as it is a molecule desired to be delivered to the brain. However, since the mechanism of passing through the barrier tissue is via RMT bound to LRP1, molecules that are too large to be transported by the same mechanism are not preferred. Examples of "certain molecules" include fluorescent pigments, low molecular weight compounds, peptides, peptoids, nucleic acids, sugar chains, lipids, antibodies, proteins, enzymes, liposomes, micelles, nanoparticles, carbon nanotubes, carbon nanohorns, carbon nanosheets, and radioactive isotope-labeled bodies thereof, but are not limited to these. In a preferred embodiment, "certain molecule" refers to a drug, such as an antibody, nucleic acid, or a bioactive peptide.

[0182] The complex of the peptide of the present invention and "a certain molecule" may be a fusion or a graft. For example, the peptide of the present invention may be grafted into a loop region fused to the N-terminus and / or C-terminus of an antibody or protein.

[0183] The above-mentioned fusions and transplants may be expressed as a part of the extracellular domain of cells, fungi, viruses, etc., or may be coupled to the extracellular domain of cells, fungi, viruses, etc.

[0184] The complex of the peptide of the present invention and "a certain molecule" may be via "a molecule having a binding activity with a certain molecule". The peptide of the present invention and "a molecule having a binding activity with a certain molecule" may be any of a conjugate, a fusion and / or a transplant. The binding between "a certain molecule" and "a molecule having a binding activity with a certain molecule" may be reversible or irreversible.

[0185] The complex of the peptide of the present invention and "a certain molecule" may be a crystal, and the crystal form may be a single crystal form or a mixture of crystal forms, all of which are included in the peptide of the present invention. The crystal can be produced by crystallization using a known crystallization method.

[0186] The complex of the peptide of the present invention and "a certain molecule" may be a pharmaceutically acceptable cocrystal or cocrystallized salt. Here, cocrystal or cocrystallized salt refers to a crystalline substance having different physical properties (e.g., structure, melting point, heat of fusion, hygroscopicity, solubility, and stability, etc.) and composed of two or more unique solids at room temperature. Cocrystal or cocrystallized salt can be produced according to a cocrystallization method known per se.

[0187] (Effects of cyclic peptides)

[0188] As shown in the examples described below, sequences 1 to 46, which are representative examples of the amino acid sequence group shown in this embodiment, have LRP1 binding activity. Moreover, sequence 2 is transferred from the blood side hole to the brain side hole in the in vitro blood-brain barrier model, and therefore has the ability to cross the blood-brain barrier. The amino acid sequence group shown in this embodiment has amino acid sequences and three-dimensional structural characteristics similar to these representative examples, so it is believed that they are very likely to also have LRP1 binding activity and the ability to cross the blood-brain barrier. Moreover, it is believed that the conjugates, fusions, and transplants with the peptides of the present invention are also very likely to have LRP1 binding activity and the ability to cross the blood-brain barrier.

[0189] As shown in the Examples described below, sequences 1 to 46, which are representative examples of the amino acid sequence group described in this embodiment, have LRP1 binding activity. This indicates the following. 5 With X 15 The bonds between Cα carbon atoms, X 6 With X 15 The bonds between Cα carbon atoms, X 5 With X 16 The bond between Cα carbon atoms or X 6 With X 16 The bonds between Cα carbon atoms include structures of a wide range of shapes, such as amide bonds, disulfide bonds, sulfide bonds, C=C bonds, CC bonds, bonds via triazole, bonds via dithiotetrafluorobenzene, bonds via dimethylphenylene (the bonding position of the methylene group to the phenylene ring may be any of the ortho, meta, and para positions).

[0190] The amino acid sequence of LRP1 cluster 4 is highly conserved in mammals, and sequence 2, a representative example of the peptide of the present invention, shows the ability to cross the blood-brain barrier in rat and monkey in vitro blood-brain barrier models. Therefore, it is believed that the peptide group containing the amino acid sequence shown in this embodiment, and the complex of the peptide group and "a certain molecule" are very likely to bind to LRP1 of mammals other than mice, rats, dogs, monkeys, etc., and show the ability to cross the barrier tissue.

[0191] (Method for producing cyclic peptide)

[0192] The peptide of the present embodiment can be produced by a known peptide production method or the like, such as a chemical synthesis method such as a liquid phase method, a solid phase method, or a hybrid method combining the liquid phase method and the solid phase method.

[0193] The solid phase method can use a commercially available automatic synthesizer. For example, the hydroxyl group of a resin having a hydroxyl group is subjected to an esterification reaction with the carboxyl group of a first amino acid (usually the C-terminal amino acid of the target peptide) whose α-amino group is protected by a protecting group such as an Fmoc group. As an esterification catalyst, a well-known dehydration condensation agent such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used. Next, the protecting group of the α-amino group of the first amino acid is removed, and a second amino acid whose functional groups other than the carboxyl group of the main chain are protected is added to activate the carboxyl group, so that the first amino acid and the second amino acid are bonded. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid whose functional groups other than the carboxyl group of the main chain are protected is added to activate the carboxyl group, so that the second amino acid and the third amino acid are bonded. Repeat this operation to synthesize a peptide of the target length. The linear peptide is cleaved from the resin and purified, and then the functional group used for cyclization of the peptide is deprotected and the peptide is cyclized according to a conventional method.

[0194] As resins for solid phase synthesis, Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin, Merck, HMPA-PEGA resin, etc. can be cited. These resins can be used after washing with a solvent (dimethylformamide (DMF), 2-propanol, dichloromethane, etc.). As protective groups for α-amino groups, benzyloxycarbonyl (Cbz) group, tert-butyloxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, allyloxycarbonyl (Alloc) group, etc. can be cited. 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-carboxyl group can be protected by methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, etc. As other functional groups of amino acids, the hydroxyl groups of serine, threonine, etc. can be protected by benzyl and tert-butyl groups, and the hydroxyl groups of tyrosine, etc. can be protected by 2-bromobenzyloxycarbonyl and tert-butyl groups. The amino groups of the side chains of lysine, etc., and the carboxyl groups of glutamic acid, aspartic acid, etc. can be protected in the same way as the α-amino group and the α-carboxyl group.

[0195] The activation of the carboxyl group can be carried out using a condensing agent. As a condensing agent, for example, 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) can be mentioned. The peptide chain can be cut from the resin by treating with an acid such as TFA or hydrogen fluoride (HF).

[0196] Regarding the peptide of the present invention, as one mode, it can be cyclized. In this specification, cyclization refers to the direct covalent bonding or indirect covalent bonding via a linking group of two or more amino acids separated by one or more amino acids in one peptide, to produce one or more cyclic structures in the molecule. Cyclization can be implemented according to the methods described in non-patent literature 8 and non-patent literature 9. For example, it can be cyclized by an amide bond between an amino group and a carboxyl 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 between a thiol group and an allyl group based on a thiol-ene reaction, a C=C bond between an allyl group and an allyl group based on an olefin metathesis reaction (the C=C bond can be converted into a CC bond by a reduction reaction), a triazole bond between an alkynyl group and an azide group via a click reaction, a thioether bond between a linking group having a halogen group and two thiol groups, etc., but is not limited to these. The direct covalent bond or the indirect covalent bond via a linking group used for cyclization may be any of main chain-main chain, main chain-side chain, side chain-main chain, and side chain-side chain.

[0197] In the cyclization of the peptide of the present invention, for example, the following can be used: (1) using cysteine, D-cysteine, homocysteine, or D-homocysteine ​​each independently as amino acid 1 and amino acid 2 to form a disulfide bond between their thiol groups; (2) using an amino acid having a halogen atom (chlorine, bromine, or iodine) as a nucleophilic group (e.g., 3-chloroalanine) or a carboxylic acid having a halogen atom (chlorine, bromine, or iodine) (e.g., 3-chloropropionic acid) as amino acid 1 to form a thioether bond between amino acid 1 and amino acid 2 having a thiol group. ; (3) a thioether bond formed between an amino acid 1 and an amino acid 2 having a thiol group and a linking group having a halogen atom (chlorine, bromine or iodine) 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); (4) using β-azidoalanine having an azido group as an amino acid 1 and a 2-amino5-hydroxy-1-nitropropane having an alkynyl group as a linking group; (5) using an amino acid having an allyl group (e.g., allylglycine, D-allylglycine, homoallylglycine, D-homallylglycine, etc.) or a carboxylic acid having an allyl group (e.g., 3-butenoic acid) as amino acid 1, and forming a thioether bond through a thiol-ene reaction with an amino acid 2 having a thiol group; (6) using an amino acid having an allyl group or a carboxylic acid having an allyl group as amino acid 1 and amino acid 2, respectively, and forming a thioether bond through this reaction. (7) C-C bond formed by reducing a C-C bond formed by olefin metathesis reaction; (8) an amide bond between an amino acid 1 having an amino group (e.g., 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, lysine, their D-type amino acids, β-alanine, etc.) and an amino acid 2 having a carboxyl group (e.g., aspartic acid, glutamic acid, their D-type amino acids, etc.) or a C-terminal carboxyl group; (9) an amide bond between an N-terminal amino group and an amino acid having a carboxyl group or a C-terminal carboxyl group. Either of amino acid 1 and amino acid 2 may be located on the N-terminal side.

[0198] (Medicines, diagnostic drugs, and research reagents including cyclic peptides)

[0199] The pharmaceutical composition of the present invention comprises a peptide containing the above-mentioned amino acid sequence as an active ingredient, and the peptide binds to LRP1 and can be transferred to the central nervous system tissue via the RMT of LRP1. The administration method of the above-mentioned pharmaceutical composition is not particularly limited, and it can be oral administration or parenteral administration. As parenteral administration, for example, injection administration such as intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, transmucosal administration (nasal, oral cavity, eye, lung, vaginal or rectal administration) and the like can be cited. The peptide in the pharmaceutical composition can be variously modified in view of the properties of easy metabolism and excretion. For example, by adding alkyl chains, polyethylene glycol or sugar chains to the peptide, the retention time in the blood can be prolonged and the antigenicity can be reduced. In addition, biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared from unsaturated fatty acids, nanoparticles, microparticles, nanospheres, etc. can also be used as sustained-release bases to enclose the peptide therein. In the case of transdermal administration, a weak electric current may be passed through the skin surface and penetrate the stratum corneum (iontophoresis).

[0200] The above-mentioned pharmaceutical composition can be used directly as an active ingredient, or can be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. As dosage forms, for example, liquids (injections, etc.), dispersants, suspensions, tablets, pills, powders, suppositories, powders, fine particles, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nose drops, ear drops, ointments, etc. These preparations can be controlled release preparations such as immediate release preparations or sustained release preparations (sustained release microcapsules, etc.). For example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH regulators, preservatives, antioxidants, etc. can be appropriately used for formulation, and it is carried out by conventional methods. Examples of ingredients used for formulation include purified water, saline, phosphate buffer, pharmaceutically acceptable organic solvents such as dextrose, glycerol, and ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, gum tragacanth, casein, agar, polyethylene glycol, diglycerol, glycerol, propylene glycol, vaseline, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, and human serum albumin, but are not limited to these. In the case where the peptide is difficult to absorb through the mucosa, surfactants such as polyoxyethylene lauryl ether, sodium lauryl sulfate, and saponin can be used as absorption enhancers to improve the absorption of poorly absorbed drugs; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylates; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, nitric oxide donors, etc.

[0201] Pills or tablets may also be coated with sugar coating, gastric soluble substances, or enteric soluble substances. Injections may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, cosolvents, preservatives, etc. may be added. Conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents, and other additives may also be used appropriately and in appropriate amounts as needed.

[0202] The pharmaceutical composition of the present invention delivers a molecule to the central nervous system tissue by binding to LRP1, and the molecule is effective for the treatment or prevention of various diseases as a target. For example, when a molecule is an anticancer agent, brain tumors (e.g., pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, degenerative astrocytoma, etc.), schwannoma, etc., can be cited. When a molecule targets a molecule related to a central nervous system disease, mental disorders (schizophrenia, schizoaffective disorder, schizophrenia-like disorder, delusional disorder, etc.), childhood mental disorders (attention deficit disorder, attention deficit / hyperactivity disorder, behavioral disorders, autism, etc.), neurodegenerative disorders, neural stem cell disorders, neural precursor disorders, ischemic disorders, neurodegenerative ... Traumatic disorders, affective disorders, psychomotor disorders, sleep disorders (hypersomnia, circadian rhythm sleep disorders, insomnia, abnormal behavior during sleep, sleep blockage, etc.), anxiety-related mental disorders (acute stress disorder, generalized anxiety disorder, social anxiety disorder, panic disorder, post-traumatic stress disorder, agoraphobia, obsessive-compulsive disorder, etc.), pseudopsychotic disorders (acute hallucinogenic mania, etc.), impulse control disorders (compulsive gambling, intermittent explosive disorder, etc.), mood disorders (bipolar I disorder, bipolar II disorder, mania, mixed mood states, etc.), major depressive disorder, chronic depression , seasonal depression, psychotic depression, seasonal depression, cognitive impairment (amnesia, Alzheimer's disease, HIV-related dementia, Alzheimer's disease, Huntington's disease, Lewy body dementia, vascular dementia, drug-related dementia, tardive dyskinesia, myoclonus, dystonia, delirium, Pick's disease, Creutzfeldt-Jakob disease, AIDS, Tourette syndrome, epilepsy, muscle spasm, mild cognitive impairment, etc.), mental retardation (spasticity, Down syndrome, fragile X syndrome, etc.); premenstrual syndrome (PMS), premenstrual dysthymia (PDD), postpartum depression, neuronal damage Disorders (eye damage, retinal disease or macular degeneration of the eye, tinnitus, hearing impairment, cerebral edema, etc.), Parkinson's disease, Parkinson-like disorders, migraine, epilepsy, Alzheimer's disease, brain damage, stroke, cerebrovascular disease (cerebral arteriosclerosis, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, cerebral hypoxia-ischemia, etc.), drug dependence (narcotics dependence, alcoholism, amphetamine dependence, cocaine addiction, nicotine dependence, drug withdrawal syndrome, etc.), eating disorders (anaesthesia, overeating, binge eating, polyphagia, obesity, compulsive eating disorder, pagophagia, etc.), etc., but not limited to these.

[0203] The pharmaceutical composition of the present invention can also be administered in combination with other medicines and treatment methods useful for the above-mentioned diseases. For example, in the case of malignant tumors, it can be combined with various chemotherapy, surgical treatment, and radiotherapy.

[0204] The dosage of the pharmaceutical composition of the present invention when administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), especially humans, varies depending on the symptoms, age, sex, body weight, sensitivity of the patient, administration method, administration interval, type of active ingredient, and type of preparation, and is not particularly limited. For example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg can be administered once or in divided doses.

[0205] (Polynucleotide encoding the peptide of the present invention)

[0206] In one embodiment, a polynucleotide encoding a peptide of the present invention is provided. The polynucleotide may be a polynucleotide encoding an amino acid sequence consisting only of natural amino acids in the amino acid sequence of the peptide of the present invention or a polynucleotide having a base sequence having a specified sequence identity with the polynucleotide. The degree of consistency of the base 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 base sequence identity can be determined by a method known per se. For example, the base sequence identity (%) can be determined by the same method as the above-mentioned amino acid sequence identity (%).

[0207] In another embodiment, the polynucleotide may be a base sequence in which one or more nucleotides in the base sequence of a polynucleotide encoding an amino acid sequence consisting only of natural amino acids in the amino acid sequence of the peptide of the present invention are subjected to one or more modifications selected from substitution, addition, deletion and insertion. 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).

[0208] (Expression vector)

[0209] The expression vector of one embodiment of the present invention may include a polynucleotide encoding a target polypeptide to be expressed or a target polynucleotide to be expressed, and a promoter functionally linked to the polynucleotide. "Promoter functionally linked to polynucleotide" means that the promoter is combined with the polynucleotide encoding the gene to enable the expression of the polynucleotide itself or the expression of the polypeptide encoded by the polynucleotide under its control.

[0210] The backbone of the expression vector of the present embodiment is not particularly limited as long as it can produce the target substance in a predetermined cell, and examples thereof include plasmid vectors and viral vectors. When the expression vector is used as a medicine, adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus and other viral vectors can be cited as vectors suitable for administration to mammals.

[0211] When using prokaryotic cells as host cells, expression vectors that can use prokaryotic cells as host cells can be used. Such expression vectors can include elements such as promoter-operator region, initiation codon, polynucleotides encoding the polypeptide of the present embodiment or its partial peptides, termination codon, terminator region, replication origin, etc. The promoter-operator region for expressing the polypeptide of the present invention in bacteria includes promoter, operator and Shine-Dalgarno (SD) sequence. About these elements, self known elements can be used.

[0212] In addition, when using eukaryotic cells as host cells, expression vectors that can use eukaryotic cells as host cells can be used. At this time, the promoter used is not particularly limited as long as it can function in eukaryotic organisms such as mammals. In the case of the expression of polypeptides as the purpose, as such a promoter, for example, viral promoters such as the initial promoter from SV40, cytomegalovirus LTR, Rous sarcoma virus LTR, LTR from MoMuLV, initial promoter from adenovirus, and β-actin gene promoter, PGK gene promoter, transferrin gene promoter, etc. can be cited. In the case of the expression of polynucleotides as the purpose, the promoter can be a polIII promoter (e.g., tRNA promoter, U6 promoter, H1 promoter).

[0213] The expression vector of the present invention may also contain sites for transcription initiation and transcription termination, as well as a ribosome binding site required for translation in the transcribed region, a replication origin, and a selective 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 (e.g., refer to the above-mentioned Molecular Cloning, etc.).

[0214] (Cells, liposomes or nanoparticles presenting the peptide of the present invention)

[0215] In another embodiment of the present invention, a cell, liposome or nanoparticle presenting the peptide of the present invention is provided. The cell can be made as follows: the desired cell is separated from the organism, the peptide of the present invention is coupled to the cell in vitro, or the vector expressing the peptide of the present invention is introduced into the cell and presented on the cell surface. The liposome or nanoparticle can be made in the following manner: coupling the peptide of the present invention, or preparing a coupling body of the peptide of the present invention and a fatty chain, and inserting the fatty chain into the lipid membrane, so that it is presented on the surface of the liposome or nanoparticle.

[0216] The following embodiments are merely illustrative and are intended to explain the present invention in detail together with the above-mentioned embodiments, but are not intended to limit the present invention. A person skilled in the art can modify the present invention in various ways without departing from the meaning of the present invention, and the modifications are also included in the scope of the present invention.

[0217] Example

[0218] The abbreviations used in this specification have the following meanings.

[0219] BSA: bovine serum albumin

[0220] RP-HPLC: Reversed-Phase High-Performance Liquid Chromatography

[0221] HRP: Horseradish peroxidase

[0222] SA: Streptavidin

[0223] D-PBS: Dulbecco's phosphate buffered saline

[0224] ELISA: Enzyme-linked immunosorbent assay

[0225] Ac:Acetyl

[0226] Cys: L-cysteine

[0227] Gly: Glycine

[0228] Ala: L-Alanine

[0229] Ser: L-serine

[0230] Thr:L-Threonine

[0231] Pro: L-Proline

[0232] cHyp: cis-4-hydroxy-L-proline

[0233] tHyp: trans-4-hydroxy-L-proline

[0234] Tyr: L-Tyrosine

[0235] Trp: L-Tryptophan

[0236] Phe: L-phenylalanine

[0237] 4fF: 4-Fluoro-L-phenylalanine

[0238] 4cF: 4-Chloro-L-phenylalanine

[0239] His:L-histidine

[0240] Lys: L-lysine

[0241] Orn: L-Ornithine

[0242] Arg: L-arginine

[0243] Val: L-Valine

[0244] Leu: L-Leucine

[0245] Ile: L-isoleucine

[0246] Nle: L-norleucine

[0247] Ahep: (S)-2-aminoheptanoic acid

[0248] Aoc: (S)-2-aminooctanoic acid

[0249] Aib: 2-aminoisobutyric acid

[0250] Asp: L-Aspartic acid

[0251] Glu: L-glutamic acid

[0252] 5-FAM: 5-carboxyfluorescein

[0253] DX: D-amino acid X

[0254] c(XY): Cyclization between amino acids X and Y

[0255] (Peptide Synthesis)

[0256] The chemical synthesis of all peptides used in this example was entrusted to SCRUM Co., Ltd. (Tokyo, Japan), and a standard solid phase synthesis method using 9-fluorenylmethoxycarbonyl (Fmoc group) as a protecting group for the α-amino group was carried out using an automatic synthesizer SyroII (manufactured by Biotage). The side chain protected amino acid-resin located at the C-terminus was placed in a synthesis column and the device was set up. Next, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) / diisopropylethylamine (DIEA) was added to the secondary amino acid protected by the Fmoc group for activation, and the column was placed for reaction. After the reaction was completed, the Fmoc group was deprotected using 20% ​​piperidine after washing. By repeating this step, the peptide chain was extended, and after the Fmoc group of the final amino acid was deprotected, the peptide resin was removed from the device. 30% hexafluoro-2-propanol (HFIP) / dichloromethane (DCM) was added to the peptide resin, and the linear side chain protected peptide was cut out 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) (manufactured by Waters), and then freeze-dried. The cyclization of the peptide was carried out by referring to the methods described in non-patent literature 8 and non-patent literature 9. The theoretical molecular weight, measured molecular weight, purity, cyclization type, and amino acid sequence of the peptide synthesized in this example are shown in Tables 1 and 2. In addition, the structural formulas of sequence 2 and sequence 46 are shown in Figure 7 It should be noted that in Tables 1 and 2, amino acid residues that are not expressed in D-form represent L-form.

[0257] [Table 1]

[0258]

[0259] [Table 2]

[0260]

[0261] (Construction of binding test using ELISA method)

[0262] In order to evaluate the binding activity of the peptide to LRP1, a binding test using the ELISA method was constructed. The following is a brief description of the 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. After coating overnight at 4°C, 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, LRP1 cluster 2-Fc chimeric protein (Catalog No. 2368-L2-050, manufactured by R&D Systems), LRP1 cluster 3-Fc chimeric protein (Catalog No. 48248-L3-050, manufactured by R&D Systems), or LRP1 cluster 4-Fc chimeric protein (Catalog No. 5395-L4-050, manufactured by R&D Systems) as recombinant proteins of LRP1 were added at 100 ng / 50 μL / well using 0.5% BSA / D-PBS, and reacted with the coated antibody at room temperature for 30 minutes. After washing the plate with 0.1% Tween20 / PBS, Sequence 45 and Sequence 46 as biotinylated peptides were prepared at an arbitrary concentration using 0.5% BSA / D-PBS and added at 50 μL / well. After reacting at room temperature for 30 minutes, the plate was washed with 0.1% Tween20 / D-PBS. Sequence 45 and sequence 46 bound to the recombinant protein of LRP1 captured on the plate were detected with SA-HRP (Catalog No. ab7403, manufactured by Abcam). For quantification of HRP, absorbance at 450 nm was measured using TMB-ELISA Substrate Solution (Catalog No. 34028, manufactured by Thermo Fisher).

[0263] The selective and peptide concentration-dependent binding of Sequence 45 and Sequence 46 used in this binding assay to the LRP1 cluster 4-Fc chimeric protein 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 the wells of the recombinant protein that did not capture LRP1 from the absorbance obtained when the biotinylated peptide was added to the wells of the recombinant protein that captured LRP1. As a result, both sequence 45 and sequence 46 bound selectively to the LRP1 cluster 4-Fc chimeric protein and dependent on the added concentration of the peptide. There was little or no binding to the LRP1 cluster 2-Fc chimeric protein and the LRP1 cluster 3-Fc chimeric protein. The binding EC50 values ​​of sequence 45 and sequence 46 were calculated to be 10.5nM and 11.0nM, showing equivalent binding activity regardless of the presence or absence of a ring structure.

[0264] (Construction of competitive binding assay using ELISA method)

[0265] In order to compare the binding activity to LRP1 and the resistance to protease degradation of the peptide introduced with amino acid substitutions in sequence 1 with those of sequence 2, a peptide was constructed. Figure 2 The competitive binding test using the ELISA method shown. Immediately after mixing sequence 2 or the amino acid replacement peptide with mouse plasma (Plasma incubation) (0 hours) or after 24 hours at 37°C, it is further diluted to an arbitrary final concentration in a manner that the plasma concentration is sufficiently reduced, mixed with sequence 46 (500nM), and subjected to the above-mentioned competitive binding test. The binding of sequence 46 to the LRP1 cluster 4-Fc chimeric protein is inhibited depending on the concentration of sequence 2 or the amino acid replacement peptide due to competition for the binding of sequence 2 or the amino acid replacement peptide to the LRP1 cluster 4-Fc chimeric protein coexisting in the solution. That is, the binding activity of sequence 2 or the amino acid replacement peptide to the LRP1 cluster 4-Fc chimeric protein is detected as a competitive inhibitory activity relative to the binding of sequence 46. The binding value of sequence 46 to the wells without captured LRP1 cluster 4-Fc chimeric protein was set as 100% inhibitory activity, and the binding value of sequence 46 to the wells without added sequence 2 and amino acid replacement peptide was set as 0% inhibitory activity. The inhibitory activity values ​​of sequence 2 and amino acid replacement peptide were calculated in %.

[0266] (Evaluation of the binding activity of amino acid substituted peptides to LRP1)

[0267] The results of the competition binding assay with LRP1 cluster 4-Fc chimeric protein at n=4 (±SEM) are shown in Figure 3. Plasma inhibited the binding of sequence 46 to sequence 2 cultured for 0 hours depending on the added concentration. The binding of sequence 46 (500 nM) in the wells to which 500 nM of sequence 2 was added was 52.4% on average, indicating that sequence 2 and sequence 46 competed 1:1 for binding to LRP1 cluster 4. Plasma also inhibited the binding of sequence 46 to sequence 2 cultured for 24 hours depending on the added concentration. However, the binding of sequence 46 (500 nM) in the wells to which 2000 nM of sequence 2 was added was 50.6% on average. That is, although 2000 nM was added to the wells, only an inhibitory activity equivalent to 500 nM was shown, indicating that about 75% of sequence 2 was decomposed during the 24-hour culture with mouse plasma. DMSO was mixed with mouse plasma and immediately subjected to the same test, and the results showed that the binding of sequence 46 (500 nM) was not inhibited at all, and no systemic inhibition occurred when plasma was mixed into the binding system.

[0268] Immediately after mixing with mouse plasma or after 24 hours at 37°C, sequences 1 to 44 were further diluted to a final concentration of 2000 nM in a manner that sufficiently reduced the plasma concentration, mixed with sequence 46 (500 nM), and subjected to the above-mentioned competitive binding assay. Figure 4 The inhibitory activities of sequence 1 and sequence 2 were estimated to be 70.7% and 79.4%, respectively. The inhibitory activities of sequence 6, sequence 7, sequence 9, sequence 19, sequence 24, sequence 34, and sequence 43 were 91.9%, 87.6%, 85.8%, 79.1%, 77.4%, 82.0%, and 83.5%, respectively, showing inhibitory activities equal to or higher than those of sequence 1 and sequence 2. The inhibitory activities of other sequences tended to be weakened.

[0269] Similar to Sequence 2, many of the amino acid substituted peptides showed reduced inhibitory activity 24 hours after mixing with mouse plasma compared to the case where they were immediately mixed with mouse plasma and subjected to competition assay, indicating that they were degraded in mouse plasma.

[0270] (Evaluation using an in vitro blood-brain barrier model)

[0271] Figure 5The summary of the in vitro blood-brain barrier model for evaluating the ability to pass through the blood-brain barrier is shown. The rat BBB kit (catalog No. RBT-24H, manufactured by Pharmacocell Co., Ltd.) and the monkey BBB kit (catalog No. MBT-24H, manufactured by Pharmacocell Co., Ltd.) were used for the test. These kits maintain the BBB characteristics in vivo (the presence or absence of expression of various receptors and transporters, the ability to form tight junctions, etc.), and are widely used as an evaluation system for estimating the brain metastasis of drugs (non-patent literature 6 and 7). The embedding hole is used as the vascular side, and the bottom hole is used as the brain side, and the amount of the peptide added in the embedding hole through the bottom surface of the three layers composed of endothelial cells / filter membrane / pericytes is evaluated. In addition, the TEER value (transepithelial electrical resistance) representing the BBB function is measured before and after the test, and it is confirmed that the addition of the peptide does not affect the BBB function.

[0272] The results of evaluating the BBB permeability of sequence 2 are shown in Table 1, using ANG2 (Non-Patent Document 4) and L57 (Non-Patent Document 5) for which LRP1 binding activity and BBB permeability have been reported as comparison objects. Figure 6 . 5-FAM-labeled ANG (sequence 47), 5-FAM-labeled L57 (sequence 48), and sequence 2 were added to the embedding wells at final concentrations of 10 μM, 3 μM, and 1 μM, respectively. After culturing at 37°C for 24 hours, the culture supernatant of the bottom well was recovered. Each 5-FAM-labeled peptide was stepwise diluted with a culture medium to an arbitrary concentration, thereby preparing a standard curve, and calculating the concentration of the 5-FAM-labeled peptide contained in each recovered culture supernatant. The amount of peptide added to the embedding well (blood vessel side) (number of moles) was set to 100%, and the amount of peptide transferred to the bottom well (brain side) (number of moles) was calculated as %. Regarding the BBB permeability of sequence 2, both the rat BBB shown in the upper figure and the monkey BBB shown in the lower figure were significantly higher than those of 5-FAM-labeled ANG and L57 at any concentration (n=4, ±SD, *p<0.01, Dunnett's test for sequence 2). After 24 hours of culture, the TEER values ​​(transepithelial electrical resistance) of all wells were 150Ω×cm 2 The above results show that the BBB function is maintained. These results show that the peptide of the present invention can pass through barrier tissues, especially the blood-brain barrier. In addition, it was confirmed that the BBB permeability of sequence 2, which is a representative example of the peptide of the present invention, is superior to that of the existing ANG2 and L57.

[0273] (Synthesis and evaluation of modified peptide DBCO-KS-487)

[0274] <Synthesis method>

[0275] The synthesis was outsourced to SCRUM Co., Ltd. (Tokyo, Japan). The following linear peptide precursor was first synthesized by a standard solid phase synthesis method using an automatic synthesizer SyruII (manufactured by Biotage) using a 9-fluorenylmethoxycarbonyl group (Fmoc group) as a protecting group for the α-amino group.

[0276] 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

[0277] Next, the resin was immersed in dimethylformamide (DMF) and diisopropylethylamine (DIEA) was added to adjust the pH to 8. At pH 8, N3-PEG4-NHS (Cas#944251-24-5, catalog #J64834, manufactured by AlfaAesar) dissolved in DMF was added to react, thereby introducing an azide group (N3-) into the N-terminal α-amino group to obtain the following substance.

[0278] 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

[0279] Next, trifluoroacetic acid (TFA) / ultrapure water (Water) / thioanisole (Thioanisole) / 1,2-ethanedithiol (EDT) / triisopropylsilyl chloride (Tips) (83 / 5 / 5 / 5 / 2) were added to deprotect the side chains of each amino acid residue and to cut 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 cooled ether to precipitate it. The precipitate was dissolved in a small amount of ultrapure water and freeze-dried to obtain the following substance.

[0280] N3-PEG4-Gly-Thr-Pro-Cys-Thr-Tyr-Lys-Tyr-Nle-Leu-Ala-Glu-Nle-Cys-OH (SEQ ID NO: 56)

[0281] The lyophilized product was dissolved in DMSO, and an aqueous solution of 0.1 M NH4CO3 was added little by little. The oxidation reaction was carried out with stirring at room temperature for 1 - 2 days, thereby forming S - S bonds between the side chains of Cys residues. Separation and purification were performed by RP - HPLC, followed by lyophilization, thus obtaining the following cyclic peptide.

[0282] N3 - PEG4 - Gly - Thr - Pro - c(Cys - Thr - Tyr - Lys - Tyr - Nle - Leu - Ala - Glu - Nle - Cys) - OH (Sequence number 54)

[0283] The purified cyclic peptide and DBCO - PEG5 - DBCO (Cas#2363130 - 04 - 3, Catalog#BP - 22450, manufactured by BROADPHARM) were respectively dissolved in dimethyl sulfoxide (DMSO). After mixing, 50 mM phosphate buffer was added. After this addition, the reaction was carried out at room temperature for 2 hours. Separation and purification were performed by RP - HPLC, followed by lyophilization, thus obtaining the following DBCO - KS - 487 (theoretical molecular weight 2701.3, measured molecular weight 2702.0, purity 95.0%). Here, the cyclic peptide part with the N - terminal modification part (DBCO - PEG5 - Taz - PEG4) removed was called KS - 487.

[0284] DBCO - PEG5 - Taz - PEG4 - Gly - Thr - Pro - c(Cys - Thr - Tyr - Lys - Tyr - Nle - Leu - Ala - Glu - Nle - Cys) - OH (Sequence number 55)

[0285] <Confirmation of the reactivity of DBCO>

[0286] An appropriate amount of DBCO - KS - 487 and 6 - azidohexanoic acid were mixed in DMSO / PBS (50 / 50) (pH 7.5), and after reacting at room temperature, the shift of the peak position of DBCO - KS - 487 was evaluated by RP - HPLC. The results are shown in Figure 8 . In the Figure 8 chart before the reaction, a peak from DBCO - KS - 487 (10.200 min) was shown, but in the Figure 8 chart after the reaction, the peak from DBCO - KS - 487 (10.248 min) almost disappeared. As shown in the Figure 8 chart after the reaction, new peaks (7.904 min and 9.285 min) appeared. The results shown in Figure 8 (before and after the reaction) indicate that the DBCO group attached to the cyclic peptide retains the activity for click reaction.

[0287] Industrial Applicability

[0288] The peptides of the present invention have LRP1 binding activity, and by binding to LRP1, the peptides can pass through barrier tissues separating peripheral tissues and brain tissues, such as the blood-brain barrier (BBB), via the RMT of LRP1. Therefore, it is believed that the peptides of the present invention are useful as a means for transporting any molecule to the brain tissue by combining with any molecule.

Claims

1. A linear peptide or cyclic peptide, or a pharmacologically acceptable salt thereof, characterized in that: It has the amino acid sequence shown in 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) In formula (1), X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7 represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8 represents ornithine, lysine, homolysine, arginine or homoarginine, X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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, X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid, the N-terminal amino group and the C-terminal carboxyl group are modified or unmodified, or, are missing or not missing, X 5 and X 15 When cysteine ​​or D-cysteine ​​participates in the cyclization, covalent bonds are formed between the side chain -SH groups via disulfide bonds, methylene, acetyl methylene, ethylene or propylene linking groups, so that the peptide of formula (1) may or may not have a cyclic structure in the molecule.

2. A linear peptide or cyclic peptide, or a pharmacologically acceptable salt thereof, characterized in that: It has the amino acid sequence shown in 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 formula (3), X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7 represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8 represents ornithine, lysine, homolysine, arginine or homoarginine, X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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, X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid, the N-terminal amino group and the C-terminal carboxyl group are modified or unmodified, or, are missing or not missing, X 5 and X 16 When cysteine ​​or D-cysteine ​​participates in the cyclization, covalent bonds are formed between the side chain -SH groups via disulfide bonds, methylene, acetyl methylene, ethylene or propylene linking groups, so that the peptide of formula (3) may or may not have a cyclic structure in the molecule.

3. A linear peptide or cyclic peptide, or a pharmacologically acceptable salt thereof, characterized in that: It has the amino acid sequence shown in 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 formula (3), X N is 1 to 4 arbitrary amino acid residues, 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-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline or trans-4-hydroxyproline, X 7 represents histidine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, X 8 represents ornithine, lysine, homolysine, arginine or homoarginine, X 9 represents methionine, norleucine, lysine, arginine, tyrosine, O-methyltyrosine, phenylalanine, 4-aminophenylalanine, 4-fluorophenylalanine or 4-chlorophenylalanine, 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, X 13 represents glycine, alanine, asparagine, aspartic acid, glutamine or glutamic acid, the N-terminal amino group and the C-terminal carboxyl group are modified or unmodified, or, are missing or not missing, X 6 and X 16 When cysteine ​​or D-cysteine ​​participates in the cyclization, covalent bonds are formed between the side chain -SH groups via disulfide bonds, methylene, acetyl methylene, ethylene or propylene linking groups, so that the peptide of formula (3) may or may not have a cyclic structure in the molecule.

4. The linear peptide or cyclic peptide according to any one of claims 1 to 3, or a pharmacologically acceptable salt thereof, wherein: X N It is Lys-Gly-Thr-Pro or Gly-Thr-Pro.

5. A linear peptide or cyclic peptide having LRP1 binding activity, or a pharmacologically acceptable salt thereof, characterized in that: An amino acid sequence comprising one or more amino acids deleted, added and / or substituted in the amino acid sequence according to any one of claims 1 to 3.

6. A derivative and / or modified form of the peptide according to any one of claims 1 to 3.

7. The peptide derivative or modified form according to claim 6, wherein The derivative or modified form has an azide group, an alkynyl group, a cyclooctyne group or a tetrazine group at the N-terminus, the C-terminus or the side chain of an amino acid directly or via a linking group.

8. A medicine, diagnostic drug and / or reagent, characterized in that The invention comprises the peptide according to any one of claims 1 to 3, or a derivative or a modified form thereof.

9. A peptide drug complex, characterized in that The peptide derivative or modified form of claim 6 is directly linked to the drug or linked via a linker group.

10. The peptide-drug complex according to claim 9, wherein The drug is an antibody, a nucleic acid or a bioactive peptide.

11. A polynucleotide, characterized in that A polynucleotide encoding an amino acid sequence consisting only of natural amino acids among the amino acid sequences described in any one of claims 1 to 3, or a polynucleotide having a base sequence having a sequence identity of 70% or more with the polynucleotide and encoding a peptide having LRP1 binding activity.

12. An expression vector, characterized in that: Comprising the polynucleotide of claim 11.

13. A cell, yeast, bacterium, virus, liposome or nanoparticle, characterized in that: The amino acid sequence according to any one of claims 1 to 3 is presented.

14. A linear peptide or cyclic peptide, or a pharmacologically acceptable salt thereof, characterized in that: Contains the amino acid sequence represented by 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) In formula (6), X L For X 5 -X 6 , X M For X 15 -X 16 , when participating in the cyclization within the peptide molecule, each independently represents an amino acid residue having an amino group, a carboxyl group, a thiol group, an allyl group, an alkynyl group, an azide group or a halogen atom, or a derivative thereof; when not participating 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, its derivative or deletion, 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, and X 8 represents an amino acid residue with a positive charge, or a derivative thereof, 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, its derivative or deletion, X L With X M A direct covalent bond or an indirect covalent bond via a linking group is formed between the two, so that the peptide of formula (6) may or may not have a cyclic structure in the molecule. In this case, X L With X M The covalent bonds between them are any bonds between their main chains, main chains and side chains, side chains and main chains, or side chains and side chains, and the N-terminal amino group and the C-terminal carboxyl group are modified or unmodified, or are missing or not missing.

15. The linear peptide and cyclic peptide according to claim 14, or a pharmacologically acceptable salt thereof, wherein: X 5 With X 15 The bonds between Cα carbon atoms, X 6 With X 15 The bonds between Cα carbon atoms, X 5 With X 16 The bonds between Cα carbon atoms or X 6 With X 16 The bonds between the Cα carbon atoms of each independently comprise the structure represented by the following formula (7): Cα N -(CH2) A -Z-(CH2) B -Cα M (7) In formula (7), N is 4 or 5, M is 14 or 15, A and B each independently represent an integer from 0 to 2, the sum of A and B is an integer from 0 to 4, and Z represents 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-C H2, 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, S-CH2-C(=O)-CH2-S or S-CH2-C(=CH2)-CH2-S, wherein in S-CH2-C6H4-CH2-S, the bonding position of the methylene group on the phenylene ring is any one of the ortho position, meta position and para position.

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

17. The linear peptide or cyclic peptide according to claim 14 or 15, or a pharmacologically acceptable salt thereof, wherein: X 12 Alanine, D-alanine, 2-aminoisobutyric acid, 2-aminobutyric acid, D-2-aminobutyric acid, isovaline, D-isovaline, valine, D-valine, isoleucine, D-isoleucine, leucine, D-leucine, their N-methylated amino acids, their α-methylated amino acids, or their derivatives.

18. The linear peptide or cyclic peptide according to claim 14 or 15, or a pharmacologically acceptable salt thereof, wherein: X 11 and X 14 Each is independently methionine, an amino acid residue represented by the following formula (8), the following formula (9), or the following formula (10), or a derivative thereof: In formula (8), the wavy line represents the point of attachment to the carbonyl group or nitrogen atom forming the amide bond of 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, wherein the halogen atom is F, Cl, Br, or I, and n represents any integer from 0 to 10; In formula (9), Z represents C or N, R 6 represents a hydrogen atom or a methyl group, and n represents any integer from 1 to 6; In formula (10), the wavy line represents the bonding site to the carbonyl group or nitrogen atom forming the amide bond of the main chain, Z represents C or N, and R 6 represents a hydrogen atom or a methyl group, and n represents any integer of 1 to 6.

19. The linear peptide or cyclic peptide according to claim 14 or 15, or a pharmacologically acceptable salt thereof, wherein: X 7 It is an amino acid residue represented by the following formula (8), the following formula (9), the following formula (10), the following formula (11), the following formula (12) or the following formula (13), or a derivative thereof: In formula (8), the wavy line represents the point of attachment to the carbonyl group or nitrogen atom forming the amide bond of 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, wherein the halogen atom is F, Cl, Br, or I, and n represents any integer from 0 to 10; In formula (9), Z represents C or N, R 6 represents a hydrogen atom or a methyl group, and n represents any integer from 1 to 6; In formula (10), the wavy line represents the bonding site to the carbonyl group or nitrogen atom forming the amide bond of the main chain, Z represents C or N, and R 6 represents a hydrogen atom or a methyl group, and n represents any integer from 1 to 6; In formula (11), R 12 represents 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thiazolyl, 4-thiazolyl, 2-oxazolyl, 4-oxazolyl, 1-pyrazolyl, 1H-imidazol-4-yl, 1-imidazolyl, 1,2,3-triazol-1-yl or 1,2,4-triazol-1-yl, R 6 represents a hydrogen atom or a methyl group; In formula (12), the wavy line represents the point of attachment to the carbonyl group or nitrogen atom forming the amide bond of 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 halomethyl group or a halogen atom, wherein the halogen atom is F, Cl, Br, I, R 6 represents a hydrogen atom or a methyl group; In formula (13), 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 halomethyl group or a halogen atom, wherein the halogen atom is F, Cl, Br, I, R 6 represents a hydrogen atom or a methyl group.

20. The linear peptide or cyclic peptide according to claim 14 or 15, or a pharmacologically acceptable salt thereof, wherein: X 13 It is glycine, alanine, aspartic acid, glutamic acid, asparagine, glutamine, D-alanine, D-aspartic acid, D-glutamic acid, D-asparagine, D-glutamine, their N-methylated amino acids, their α-methylated amino acids, or their derivatives.

21. The linear peptide or cyclic peptide according to claim 14 or 15, or a pharmacologically acceptable salt thereof, wherein: X does not participate in the cyclization within the peptide molecule L and X M each independently represents alanine, isovaline, norvaline, serine, homoserine, threonine, allothreonine, O-methylated serine, O-methylated homoserine, O-methylated threonine, O-methylated allothreonine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, lysine, arginine, proline, cis-4-hydroxyproline, trans-4-hydroxyproline, azetidine-2-carboxylic acid, pipecolic acid, D-serine, D-homoserine, D-threonine, D-allothreonine, D-methylated amino acids, D-O-methylated serine, D-methylated homoserine, D-methylated threonine, D-methylated allothreonine, D-2,3-diaminopropionic acid, D-2,4-diaminobutyric acid, D-ornithine, D-lysine, D-arginine, D-proline, D-cis-4-hydroxyproline, D-trans-4-hydroxyproline, D-azetidine-2-carboxylic acid, D-pipecolic acid, their N-methylated amino acids, their α-methylated amino acids, or their derivatives.

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