Brain-targeting peptide, transport carrier, and method for screening peptide binding to target cell

Abstract

The present invention is a brain-targeting peptide containing an amino acid sequence represented by SEQ ID NO: 1 and a method for screening a peptide that binds to a target cell in a non-human organism, the screening method comprising: bringing a peptide library comprising a 7-amino acid peptide into contact with the target cell; recovering a peptide group bound to the target cell to form a peptide population binding to the target cell; and selecting, from the peptide population, a peptide having the same amino acid sequence as a portion of a protein expressed in the non-human organism and having the same amino acid sequence as a portion of a protein constituting a virus as a peptide that binds to the target cell.

Description

Cerebral targeting peptides, transport carriers, and screening methods for peptides that bind to target cells

[0001] The present invention relates to a peptide that targets the cerebrum, a transport carrier and a pharmaceutical composition using the peptide. The present invention also relates to a method for screening peptides that bind to target cells. This application claims priority to Japanese Patent Application No. 2023-204750, filed on December 4, 2023, the contents of which are incorporated herein by reference.

[0002] Techniques for targeting specific organs are expected to be applied to, for example, drug delivery systems (DDS) for delivering drugs. For example, transport efficiency can be improved by modifying a transport carrier targeted to a specific tissue with a peptide that specifically binds to cells constituting the tissue. Examples of peptides targeting specific tissues or cells include a neovascularization-specific peptide described in Patent Document 1, a cardiac-specific peptide described in Patent Document 2, and a spinal cord tissue-specific peptide described in Patent Document 3. Furthermore, Patent Document 4 describes a peptide that can penetrate the blood-brain barrier and is used to modify a transport carrier targeted to the brain.

[0003] International Publication No. 2000 / 023476 U.S. Patent No. 6,303,573 Japanese Patent No. 6479331 Japanese Patent No. 7378046

[0004] The present invention aims to provide a peptide that specifically binds to cells that constitute the cerebrum and is suitable for modifying transport carriers that target the cerebrum, a transport carrier that targets the cerebrum containing the peptide, and a method for screening peptides that bind to target cells.

[0005] The present invention includes the following aspects: [1] A cerebrum-targeting peptide comprising the amino acid sequence represented by SEQ ID NO: 1 (EVGTARY). [2] A transport carrier for specifically transporting to the cerebrum, which is a complex in which a cerebrum-targeting peptide comprising the amino acid sequence represented by SEQ ID NO: 1 is linked to a cationic polymer. [3] The transport carrier of [2] above, wherein the cerebrum is a cerebral nerve cell. [4] A pharmaceutical composition comprising the cerebrum-targeting peptide of [1] above, the transport carrier of [2] above, or the transport carrier of [3] above. [5] The pharmaceutical composition of [4] above, further comprising a therapeutic agent or a detection agent. [6] A method for screening for peptides that bind to target cells in a non-human living organism, comprising: contacting a peptide library consisting of 7-amino acid peptides with the target cells, recovering a group of peptides that bind to the target cells to form a peptide population that binds to the target cells, and selecting from the peptide population, as peptides that bind to the target cells, peptides that have the same amino acid sequence as a portion of a protein expressed in the non-human living organism and the same amino acid sequence as a portion of a protein that constitutes a virus. [7] The method for screening for peptides that bind to target cells according to [6], comprising recovering from the peptide population, a group of peptides that specifically bind to the target cells, and selecting from the peptide population, as peptides that have the same amino acid sequence as a portion of a protein expressed in the non-human living organism and the same amino acid sequence as a portion of a ligand recognized by a virus during infection.

[0006] The present invention provides peptides that target the cerebrum and transport carriers that target the cerebrum and contain the peptides. Furthermore, the present invention enables efficient screening of peptides that specifically bind to specific tissues or cells in the body and are suitable for modifying transport carriers used in DDS.

[0007] In Example 2, nucleic acid-PEI-P ＣＮ1 shows a nuclear staining image and a dsRed fluorescence image of the cerebrum of a mouse administered with a nucleic acid-PEI-Benzyl complex in Example 2. 1 shows a nuclear staining image and a dsRed fluorescence image of the cerebrum of a mouse administered with dsRed in Example 2. 1 shows a nuclear staining image and a dsRed fluorescence image of the cerebrum of a mouse administered with a nucleic acid-PEI-P in Example 2. ＣＮ 1 shows a nuclear staining image and a dsRed fluorescence image of the heart of a mouse administered with a nucleic acid-PEI-Benzyl complex in Example 2. 1 shows a nuclear staining image and a dsRed fluorescence image of the heart of a mouse administered with dsRed in Example 2. 1 shows a nuclear staining image and a dsRed fluorescence image of the heart of a mouse administered with a nucleic acid-PEI-P in Example 2. ＣＮ 1 shows a nuclear staining image and a dsRed fluorescence image of the small intestine of a mouse administered with a nucleic acid-PEI-Benzyl complex in Example 2. 1 shows a nuclear staining image and a dsRed fluorescence image of the small intestine of a mouse administered with a nucleic acid-PEI-Benzyl complex in Example 2. 1 shows a nuclear staining image and a dsRed fluorescence image of the small intestine of a mouse administered with dsRed in Example 2. 1 shows a nucleic acid-PEI-P ＣＮ 1 shows a nuclear staining image and a dsRed fluorescence image of the liver of a mouse administered with the complex in Example 2. 1 shows a nuclear staining image and a dsRed fluorescence image of the liver of a mouse administered with the nucleic acid-PEI-Benzyl complex in Example 2. 1 shows a nuclear staining image and a dsRed fluorescence image of the liver of a mouse administered with dsRed in Example 2.

[0008] Hereinafter, embodiments of the present invention will be specifically described.

[0009] In the present invention and this specification, "X1 to X2 (X1 and X2 are real numbers satisfying X1<X2)" means "greater than or equal to X1 and less than or equal to X2."

[0010] In the present invention and this specification, the term "polypeptide" refers to a polymer of 25 or more amino acids bonded together by peptide bonds, and the term "peptide" refers to a polymer of 2 to 24 amino acids bonded together by peptide bonds.

[0011] In the present invention and this specification, the term "comprise" means that it may contain components other than the target component. The term "consist of" means that it does not contain components other than the target component. The term "consist essentially of" means that it does not contain components other than the target component in a manner that exerts a special function (such as a manner that completely loses the effects of the invention). In the present invention and this specification, when it is written "comprise", it includes an embodiment that "consists of" and an embodiment that "consists essentially of".

[0012] In the present invention and this specification, "targeting (specific cells or tissues)" means targeting specific cells or tissues as the binding target. For example, a "(specific cell or tissue) targeting peptide" means a peptide that binds to a specific cell or tissue. A "(specific cell or tissue) targeted transport carrier" means a transport carrier for transporting a substance to a specific cell or tissue.

[0013] In the present invention and this specification, the term "specifically bind (to a specific cell or tissue)" means that the affinity (strength of binding) for a specific cell or tissue is higher than the affinity for the majority of other cells or tissues.

[0014] <Cerebrum-targeting peptide> The cerebrum-targeting peptide of this embodiment is a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 (EVGTARY). A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is a peptide that specifically binds to cells that constitute cerebral tissue, particularly cerebral neurons. Examples of cerebral neurons include cortical neurons, striatal neurons, hippocampal neurons, hypothalamic neurons, PVN neurons, and substantia nigra neurons. By comprising the amino acid sequence represented by SEQ ID NO: 1, the cerebrum-targeting peptide of this embodiment can specifically bind to cells that constitute cerebral tissue and functions as a peptide that targets the cerebrum.

[0015] The cerebrum-targeting peptide of this embodiment may be a peptide consisting solely of the amino acid sequence represented by SEQ ID NO: 1, or may contain other amino acid sequence portions. The other amino acid sequence portions are not particularly limited, as long as they do not impair the specific binding ability of the amino acid sequence portion represented by SEQ ID NO: 1 to the cerebrum. Examples of the other amino acid sequence portions include various signal peptides and tag peptides. These signal peptides and tag peptides can be appropriately selected from various signal peptides and tag peptides commonly used in the production of recombinant proteins. Examples of the tag peptides include His tags, HA (hemagglutinin) tags, Myc tags, and Flag tags. The other amino acid sequence portions preferably include linkers for binding to other substances or amino acid residues that contribute to binding to other substances. Examples of amino acid residues that contribute to binding to other substances include cysteine ​​residues.

[0016] The cerebrum-targeting peptide of this embodiment may be a linear or cyclic peptide. Cyclization of the peptide is expected to improve hydrolysis resistance and in vivo stability.

[0017] The cerebrum-targeting peptide of this embodiment may be modified in various ways as long as the specific binding ability to the cerebrum due to the amino acid sequence portion represented by SEQ ID NO: 1 is not impaired. Examples of such modifications include acetylation of the N-terminus and amidation of the C-terminus.

[0018] The cerebrum-targeting peptide of this embodiment may be a peptide consisting only of L-amino acids, a peptide containing both L-amino acids and D-amino acids, or a peptide consisting only of D-amino acids. Furthermore, some or all of the amino acids constituting the cerebrum-targeting peptide of this embodiment may be replaced with artificial amino acids.

[0019] The cerebral targeting peptide of this embodiment can be easily synthesized by commonly used peptide synthesis techniques, and can also be easily produced by using commonly used expression systems such as Escherichia coli.

[0020] The cerebrum-targeting peptide of this embodiment may be bound to a substance other than a peptide or polypeptide. The substance can be appropriately selected from various labeling substances used to label peptides. Examples of the labeling substance include low-molecular-weight compounds such as biotin, fluorescent substances, etc.

[0021] When the cerebrum-targeting peptide of this embodiment is a substance in which a peptide or polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 is linked to a labeling substance, the two substances may be linked directly or indirectly via a linker. The linker is not particularly limited as long as it is a divalent or higher linking group that does not impair the specific binding ability of the amino acid sequence portion represented by SEQ ID NO: 1 to the cerebrum. Examples of the linking group include a hydrocarbon group, -NH-, -O-, -CO-, -S-, -SO-, a divalent or higher aromatic ring group, a maleimide group, and combinations of two or more of these. The peptide or polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 can be linked to a labeling substance using chemical reactions commonly used in the synthesis of chemical substances.

[0022] The cerebrum-targeting peptide of this embodiment can be bound to the cerebrum or cells constituting the cerebrum by contacting the cerebrum-targeting peptide of this embodiment with the cerebrum or cells constituting the cerebrum. Contact of the cerebrum-targeting peptide of this embodiment with the cerebrum or cells constituting the cerebrum may be performed in vivo, in vitro, or ex vivo. The method for contacting the cerebrum-targeting peptide of this embodiment with the cerebrum or cells constituting the cerebrum is not particularly limited. For example, when contacting the cerebrum-targeting peptide of this embodiment in vitro or ex vivo, an appropriate amount of the peptide may be contacted with the cerebrum or a subject containing the cerebrum, and then incubated as necessary. Furthermore, when targeting the cerebrum-targeting peptide of this embodiment in vivo, it can be performed by direct injection into the cerebrum, intravenous, subcutaneous, intramuscular, or peritoneal injection, as well as oral administration, inhalation administration, transmucosal administration, etc.

[0023] The cerebrum-targeting peptide of this embodiment specifically binds to the cerebrum and the cells that make up the cerebrum, and is therefore useful for the observation and diagnosis of cerebral tissue. The peptide can be used for various applications in the pharmaceutical field, such as molecular imaging of cerebral tissue, prevention, alleviation, or treatment of diseases in cerebral tissue, and as a tool for evaluating the therapeutic effects of diseases. Diseases in cerebral tissue include, for example, cerebral neurological diseases such as Alzheimer's disease and Parkinson's disease, and brain tumors. Furthermore, when the cerebrum-targeting peptide of this embodiment is a peptide linked to a labeling substance such as a fluorescent substance, the cerebrum-targeting peptide can be used to label the cerebrum and the cells that make up the cerebrum, and is useful for the observation and diagnosis of the cerebrum.

[0024] The biological species of the cerebrum and cells constituting the cerebrum targeted by the cerebrum-targeting peptide of this embodiment are not particularly limited. Examples of animals include vertebrates such as mammals, birds, reptiles, amphibians, and fish. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, and humans.

[0025] <Transport carrier targeting the cerebrum> The transport carrier of this embodiment is a transport carrier for transporting specifically to the cerebrum (cerebrum-targeted transport carrier), and is a complex of a cerebrum-targeting peptide containing the amino acid sequence represented by SEQ ID NO: 1 and a cationic polymer linked together. The transport carrier of this embodiment utilizes the specific binding ability to the cerebrum due to the amino acid sequence portion represented by SEQ ID NO: 1, and is therefore suitable for use in delivering a substance to be delivered to cerebral tissue. The transport carrier of this embodiment can transport a substance to cerebral tissue more efficiently, thereby allowing the beneficial effects of the substance to be delivered to be efficiently exerted in spinal cord tissue. Furthermore, the transport carrier of this embodiment can reduce or avoid side effects on tissues and cells other than cerebral tissue.

[0026] The cerebrum-targeting peptide in the transport carrier of this embodiment can be the cerebrum-targeting peptide of this embodiment. Furthermore, the transport carrier of this embodiment may be a complex in which the cerebrum-targeting peptide and the cationic polymer are directly linked, or in which the cerebrum-targeting peptide and the cationic polymer are indirectly linked via a linker. The linker is not particularly limited, as long as it is a divalent or higher linking group that does not impair the specific binding ability of the amino acid sequence portion represented by SEQ ID NO: 1 to the cerebrum. Examples of such linking groups include hydrocarbon groups, —NH—, —O—, —CO—, —S—, —SO—, divalent or higher aromatic ring groups, maleimide groups, and combinations of two or more of these. Linking the cerebrum-targeting peptide to the cationic polymer, or linking the cerebrum-targeting peptide, the cationic polymer, and the linker can be performed using chemical reactions commonly used in the synthesis of chemical substances.

[0027] Cationic polymers in the transport carrier of this embodiment are relatively easy to chemically modify and are widely used, and therefore include DEAE-dextran, Dendrimer, Polybrene (registered trademark) (CAS: 28728-55-4), Polyethyleneimine (PEI), and various derivatives thereof. Instead of the cationic polymer, a complex in which calcium phosphate or a cationic lipid is linked to a cerebral targeting peptide can also be used as a cerebral targeting transport carrier.

[0028]

[0029] The transport carrier of this embodiment may be directly or indirectly linked to the substance to be transported, or may not be linked. When the surface of the substance to be transported is anionic, the transport carrier of this embodiment and the substance to be transported can be mixed in an appropriate solvent to form a complex through electrostatic interaction with the cationic polymer moiety in the transport carrier. The complex formed between the transport carrier and the substance to be transported can be used to specifically transport the substance to the cerebrum.

[0030] Examples of substances that can be transported by the transport carrier of this embodiment include nucleic acids, peptides, proteins (e.g., protein-based drugs such as antibodies, antibody fragments, antagonists, and agonists), lipids, peptide lipids, sugars, low-molecular-weight compounds, and other synthetic or natural compounds. These substances can be used alone or in combination of two or more. The size of the substance to be transported is not particularly limited, as long as it is large enough to be introduced into cells that make up the cerebrum using the transport carrier of this embodiment.

[0031] Among the substances to be transported by the transport carrier of this embodiment, nucleic acids are particularly preferred. Examples of nucleic acids include DNA such as plasmid DNA, cDNA, and antisense DNA, and RNA such as siRNA, shRNA, miRNA, antisense RNA, mRNA, tRNA, and rRNA. Examples of such nucleic acids include nucleic acids that serve as active ingredients in gene therapy and mRNA vaccine therapy.

[0032] <Pharmaceutical Composition> The pharmaceutical composition of this embodiment includes the cerebrum-targeting peptide of this embodiment or the transport carrier of this embodiment. For example, a transport carrier of this embodiment containing a therapeutic agent or detection agent targeted to the cerebrum as a transported substance is suitable as an active ingredient of a pharmaceutical composition targeting the cerebrum. For example, by using a drug that acts as a therapeutic agent among substances targeted to the cerebrum as a transported substance and the transport carrier of this embodiment, the therapeutic agent can be efficiently transported to the cerebrum, and improved therapeutic effects can be expected. Furthermore, by using a detection agent for detecting the cerebrum as a transported substance and the transport carrier of this embodiment, the cerebrum can be efficiently detected. Similarly, among the cerebrum-targeting peptides of this embodiment, peptides in which a therapeutic agent or detection agent targeted to the brain as a target tissue is linked directly or indirectly to a peptide moiety consisting of the amino acid sequence represented by SEQ ID NO: 1 are also suitable as active ingredients of pharmaceutical compositions targeting the cerebrum.

[0033] The content of the substance to be delivered in the pharmaceutical composition of this embodiment is not limited as long as the useful effect of the substance to be delivered is exerted in cerebral tissue, and can be appropriately determined by a person skilled in the art depending on the form of the pharmaceutical composition, the manner of use, the weight, type, and condition of the cerebral tissue, etc.

[0034] The pharmaceutical composition of this embodiment may consist solely of the cerebrum-targeting peptide of this embodiment or the transport carrier of this embodiment, or may contain a pharmaceutically acceptable carrier, if necessary. The carrier is not limited as long as it achieves the effects of the present invention, and examples thereof include purified water, buffer solution, physiological saline, RNase-free water, DNase-free water, protease-free water, aqueous glucose solution, isotonicity agents, excipients, binders, lubricants, disintegrants, fluidizing agents, diluents, thickeners, stabilizers, buffers, preservatives, antioxidants, flavoring agents, coloring agents, etc., and these can be appropriately selected by those skilled in the art depending on the intended use.

[0035] The dosage form of the pharmaceutical composition of this embodiment is not particularly limited and can be appropriately selected from commonly used dosage forms, taking into consideration the administration method, etc. Examples of such dosage forms include tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (pulverized drugs), granules, fine granules, liquids, suspensions, emulsions, pastes, syrups, and injections (including those prepared as liquids by mixing with distilled water or infusions such as amino acid infusions or electrolyte infusions at the time of use).

[0036] The method of administration of the pharmaceutical composition of this embodiment is not particularly limited, and can be, for example, intra-arterial administration, intravenous administration, oral administration, rectal administration, enteral administration, transdermal administration, oral administration, etc. The pharmaceutical composition of this embodiment is administered to vertebrates having a cerebrum, and is preferably administered to mammals, and particularly preferably administered to primates including humans.

[0037] The amount of the cerebrum-targeting peptide of this embodiment or the transport carrier of this embodiment contained in the pharmaceutical composition of this embodiment is not particularly limited. The pharmaceutical composition of this embodiment is preferably adjusted so that a sufficient amount of the target substance can be delivered to the cerebrum by the cerebrum-targeting peptide and the transport carrier. The dosage of the pharmaceutical composition of this embodiment can be appropriately determined depending on various conditions such as the patient's weight, age, sex, symptoms, administration route, and dosage form.

[0038] <Method for screening peptides that bind to target cells> The screening method of this embodiment is a method for screening peptides that bind to target cells in a non-human living organism. Specifically, first, (A) a peptide library consisting of 7-amino acid peptides is contacted with the target cells, and a group of peptides that bind to the target cells is recovered, and the recovered group of peptides is used as a peptide population that binds to the target cells. Next, (B) peptides that have the same amino acid sequence as a portion of a protein expressed in the non-human living organism and the same amino acid sequence as a portion of a protein that constitutes a virus are selected from the peptide population as peptides that bind to the target cells.

[0039] In (A), the method for contacting target cells with a peptide library consisting of 7-amino acid peptides is not particularly limited. For example, a phage population displaying the peptide library may be administered to a non-human organism, followed by recovery of tissue containing target cells from the non-human organism, and, if necessary, isolation of the target tissue from the recovered tissue, followed by recovery of peptides derived from the administered peptide library. Alternatively, a phage population displaying the peptide library may be introduced into a culture medium for target cells and incubated, followed by recovery of peptides bound to the target cells. Recovery of tissues or cells, and recovery of peptides from the recovered tissue, etc., can be performed by conventional methods.

[0040] The phage may be any phage typically used in a phage display library, but for example, M13 phage may be used. As a phage population displaying a peptide library, a phage display library may be used in which a gene encoding a peptide library is inserted into a gene encoding the pIII coat protein of M13 phage. As a peptide library, a library of random peptides consisting of 7 amino acids is preferred.

[0041] Non-human living organisms are not particularly limited, and include animals, plants, etc. Examples of animals include vertebrates and invertebrates. Examples of vertebrates include mammals, birds, reptiles, amphibians, fish, etc. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, and humans. Examples of invertebrates include arthropods and mollusks.

[0042] The phage population can be administered to a non-human organism using a method appropriate for the non-human organism and the target cells. The target cells may be a cell population of all cells constituting an organ or tissue, or a single cell. For example, a phage population displaying a peptide library is administered to a non-human organism, and then the cerebrum of the non-human cell is collected. Peptides derived from the administered peptide library are then recovered from the cerebrum, thereby obtaining a peptide population that binds to cerebral cells (including all the various types of cells constituting the cerebrum). Specific cells, such as cortical neurons, striatal neurons, hippocampal neurons, hypothalamic neurons, PVN neurons, and substantia nigra neurons, are isolated from the collected cerebrum, and peptides derived from the peptide library are recovered from each isolated cell, thereby obtaining a peptide population that binds to a specific single cell.

[0043] The amino acid sequence of each peptide in the collected peptide group can be identified by genetic analysis of the phage displaying the peptide. Specifically, the base sequences encoding the peptides displayed by the collected phage population are decoded using a next-generation sequencer to obtain a group of reads. The peptides encoded by the obtained group of reads are peptides that specifically bind to the target site in the body.

[0044] The peptide group prepared in (A) is again contacted with target cells, and the peptide group that binds to the target cells is recovered. By repeating this process, a peptide group with higher affinity for target cells can be obtained (panning). By repeating this process a sufficient number of times, for example, two or more times, preferably three or more times, more preferably four or more times, a peptide group consisting of peptides that specifically bind to target cells can be obtained.

[0045] Peptides that have binding affinity to target cells may also have binding affinity to sites other than the target cells. By using a next-generation sequencer, peptides that have binding affinity to cells other than the target cells can be removed from the data (in silico) (virtual subtraction). By performing virtual subtraction, the sensitivity and specificity of the binding affinity of the resulting peptide group to the target cells can be significantly improved, resulting in a peptide group consisting of peptides that specifically bind to the target cells.

[0046] From the peptide population prepared in (A), peptides that have the same amino acid sequence as a portion of a protein expressed in the non-human organism and the same amino acid sequence as a portion of a protein constituting a virus are selected as peptides that bind to the target cells (B). The peptides in the peptide population prepared in (A) consist of seven amino acids. A "peptide with the same amino acid sequence as a portion of a protein" refers to a peptide that consists of an amino acid sequence that is completely identical to the amino acid sequence of a portion (7 amino acids) of the full-length amino acid sequence of the protein. The full-length of the protein is not particularly limited as long as it is more than seven amino acids, and may be a peptide of about 10 amino acids or a protein consisting of several thousand amino acids.

[0047] Peptides with an amino acid sequence identical to a portion of a protein expressed in a non-human organism have low immunogenicity to the non-human organism and can be administered relatively safely. Furthermore, peptides with an amino acid sequence identical to a portion of a protein constituting a virus are expected to have an effect similar to that of the virus when administered in vivo. For example, a peptide with an amino acid sequence identical to a portion of a protein constituting a virus known to infect the same species of organism as the target cells is expected to infect the target cells as easily as the virus. In the screening method of this embodiment, it is particularly preferable to select, from the peptide population prepared in (A), a peptide with an amino acid sequence identical to a portion of a protein expressed in a non-human organism and having an amino acid sequence identical to a peptide or a portion thereof known as an infectious ligand for the virus, as a peptide that binds to target cells.

[0048] While preferred embodiments of the present invention have been described above, it should be understood that these are illustrative of the present invention and should not be construed as limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the present invention. Accordingly, the present invention is not to be deemed limited by the foregoing description, but is limited only by the scope of the appended claims.

[0049] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0050] [Experimental Example 1] A peptide that specifically binds to cortical neurons in the mouse cerebral cortex was identified.

[0051] (In vivo panning using mice) 1 × 10 １１ The phage library (cfu) was administered via the tail vein. ＴＭ The phage library was constructed using a "Peptide Library Kit" (New England Biolabs) that expressed random peptides consisting of seven amino acids on the phage surface.

[0052] Five minutes after administration of the phage library, mice were anesthetized, cardiac blood was collected, and then perfused and bled with phosphate-buffered saline (PBS). Subsequently, target tissues were collected from the mice. Thirty-seven organs and 152 types of cells were collected from the target tissues. Cells were collected by laser capture microdissection (LCM).

[0053] Subsequently, each of the collected target sites was homogenized to obtain a phage solution. The phage solution was infected to Escherichia coli, added to top agar, and plated on an LB plate. The plate was cultured overnight at 37°C. After confirming the phage plaques, the plate was cultured in SM buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 8 mM MgSO ４ The phages were collected and amplified using SM buffer (0.01% gelatin). PCR amplification was performed using SM buffer as a sample, and the PCR products were electrophoresed, gel extracted, and purified to prepare samples for next-generation sequencing for each target site.

[0054] Next, the phages collected from each target site were mixed and injected into the mouse again at 1 × 10 １１ cfu was administered via the tail vein, and a second in vivo panning was performed in the same manner as above to prepare samples for next-generation sequencing for each target site.

[0055] Subsequently, the phages collected from each target site in the second in vivo panning were mixed and injected again into mice at 1 × 10 １１ cfu was administered via the tail vein, and a third round of in vivo panning was performed in the same manner as above to prepare samples for next-generation sequencing.

[0056] Next, the base sequences of the prepared samples for next-generation sequencing were analyzed using a next-generation sequencer.

[0057] (Analysis results by next-generation sequencing) Focusing on cerebral cortical neurons, the amino acid sequences of peptides with specific binding to cerebral cortical neurons were analyzed based on the results of next-generation sequencing after the third round of panning.

[0058] Of the 152 cells, a group of peptides that bound exclusively to cerebral cortical neurons was selected. From this group of peptides, five peptides were selected in descending order of the number of detected reads based on the amino acid sequences of peptides expressed on the surface of phages adsorbed to cerebral cortical neurons. The five selected peptides (P1) to (P5) are shown in Table 1. Proteins with the respective amino acid sequences of these five peptides (P1) to (P5) were searched using NCBI's protein BLAST search. As a result, only peptide (P3) was found to have an amino acid sequence identical to a portion of a mouse protein (TGF-β type 2 receptor) and a portion of a viral protein (serine / threonine kinase). These results suggest that peptide P3 is highly expressed throughout the cerebrum, does not produce antibodies when administered to mice, and is readily taken up into cells. Therefore, a peptide (CEVGTARYC: SEQ ID NO: 6) in which cysteine ​​residues were added to both ends of this peptide P3 was designated as the cerebral targeting peptide P ＣＮ This was used in the subsequent experiments.

[0059]

[0060] In this experiment, none of the blood-brain barrier-permeable peptides described in Patent Document 4 were obtained. The blood-brain barrier-permeable peptides described in Patent Document 4 were obtained by screening peptides that bind to hCMEC / D3 cells in vitro, and it is presumed that this is because, when administered in vivo, they were adsorbed to blood vessels and did not reach the brain.

[0061] [Experimental Example 2] Cerebrum-targeting peptide P obtained in Example 1 ＣＮ A cationic polymer was linked to (SEQ ID NO: 6) via a linker to prepare a transport carrier for specific transport to the cerebrum.

[0062] (Preparation of Cationic Polymer) Branched Polyethyleneimine (BPEI) having a molecular weight of 25,000 was used as the cationic polymer. ２５，０００Specifically, as shown in the following chemical reaction formula, the —OH of 5-hexynoic acid was chlorinated with thienyl chloride, and then BPEI ２５，０００ and reacting with BPEI ２５，０００ -Alkyne was synthesized. The obtained BPEI ２５，０００ The alkyne introduction rate of -Alkyne was 4%.

[0063]

[0064] (Formation of a complex between a cerebral targeting peptide and a cationic polymer) P ＣＮ The carboxy group of the C-terminal cysteine ​​residue of the compound is linked to a solid support, and the reactive groups of the side chains, i.e., -SH of the cysteine ​​residue, -OH of the tyrosine residue, -OH of the threonine residue, and -NH of the arginine residue, are ２ The —COOH of the glutamic acid residue was protected. Then, an azide linker, which connected the azide group and the carboxyl group with a polyethylene glycol chain, was attached to the protected P ＣＮ The amino acid sequence was then linked to the N-terminal amino group of P. ＣＮ All protecting groups of the side chains of ＣＮ obtained.

[0065]

[0066] Next, BPEI ２５，０００ -Azide-modified P to the alkynyl group of Alkyne ＣＮ The azide group of PEI-P is linked by a click reaction. ＣＮ was synthesized.

[0067]

[0068] (Formation of Linker-Attached Cationic Polymer) BPEI ２５，０００ The azide group of an azide linker, in which an azide group and N-benzyl acrylamide are linked via a polyethylene glycol chain, was linked to the alkynyl group of -Alkyne by a click reaction to synthesize PEI-Benzyl.

[0069]

[0070] (Nucleic acid and PEI-P ＣＮUptake of PEI-P complex into mouse brain ＣＮ Nucleic acids were administered to mice using this as a transport carrier, and their uptake into the cerebrum and other tissues was examined. The nucleic acid to be delivered was the fluorescent protein vector dsRed plasmid (6155 bp, a modified version of a plasmid donated by Dr. Oka Kazuhiro, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA; the same type is available commercially from Takara).

[0071] 200 μg of dsRed plasmid was added to PEI-P in phosphate-buffered saline (PBS). ＣＮ When mixed at various concentrations, dsRed plasmid and PEI-P ＣＮ dsRed plasma:PEI-P ＣＮ = 1:200 (molar ratio) complex (nucleic acid-PEI-P ＣＮ Furthermore, when 200 μg of dsRed plasmid was mixed with PEI-Benzyl at various concentrations, it was confirmed that the dsRed plasmid and PEI-Benzyl formed a complex (nucleic acid-PEI-Benzyl complex) at a molar ratio of dsRed plasmid:PEI-Benzyl = 1:50.

[0072] Each of the formed complexes was administered to mice at a dose of 3 × 10 dsRed plasmid per mouse. １１ After intravenous injection of a particle-forming amount of dsRed plasmid, mice were maintained for four days. For comparison, an equal amount of dsRed plasmid alone was also administered to mice. No health problems were observed in any of the mice, and food intake remained normal, as before administration. Four days after administration, brains, hearts, small intestines, and livers were collected from the mice, and formalin-fixed, paraffin-embedded sections were prepared according to standard methods. Nuclei in each tissue section were stained with DAPI (4',6-diamidino-2-phenylindole), and blue fluorescent images (nuclear staining images) and red fluorescent images (dsRed fluorescent images) were taken to examine the expression status of dsRed in each tissue.

[0073] As a result, the expression of dsRed in the brain of mice administered with the nucleic acid-PEI-Benzyl complex was at the same level as that of mice administered with dsRed alone, but ＣＮ In mice administered with the complex, the fluorescence intensity was clearly stronger and the expression level was higher (Fig. 1A-C). On the other hand, in the heart (Fig. 2A-C), small intestine (Fig. 3A-C), and liver (Fig. 4A-C), the nucleic acid-PEI-P ＣＮ The mice administered with the complex showed similar levels of activity to the mice administered with the nucleic acid-PEI-Benzyl complex or dsRed alone. ＣＮ It was confirmed that modification with promotes specific uptake into cerebral tissue.

[0074] The cerebral targeting peptide and the transport carrier of this embodiment are very useful for molecular imaging of cerebral tissue, observation of the condition of cerebral diseases, disease treatment with delivered substances, etc. Furthermore, by further clarifying the state of cerebral tissue using the present invention, it will lead to further progress in methods for diagnosing, preventing, and treating diseases, as well as in personalized medicine, drug discovery, and elucidation of the causes of diseases.

Claims

1. A cerebral targeting peptide comprising the amino acid sequence represented by SEQ ID NO:1 (EVGTARY).

2. A transport carrier for specifically transporting a cerebrum to the cerebrum, which is a complex linking a cerebrum targeting peptide containing the amino acid sequence represented by sequence number 1 (EVGTARY) with a cationic polymer.

3. The transport carrier according to claim 2, wherein the cerebral cortex is a cerebral neuron.

4. A pharmaceutical composition comprising the cerebral targeting peptide of claim 1, the transport carrier of claim 2, or the transport carrier of claim 3.

5. The pharmaceutical composition of claim 4, further comprising a therapeutic or detection agent.

6. A method for screening for peptides that bind to target cells in a non-human living organism, comprising: contacting a peptide library consisting of 7-amino acid peptides with the target cells; recovering a group of peptides that bind to the target cells to create a peptide population that binds to the target cells; and selecting from the peptide population a peptide that has the same amino acid sequence as a portion of a protein expressed in the non-human living organism and the same amino acid sequence as a portion of a protein that constitutes a virus, as a peptide that binds to the target cells.

7. A method for screening peptides that bind to target cells as described in claim 6, comprising recovering a group of peptides that specifically bind to the target cells from the peptide population, and selecting from the group of peptides a peptide that has an amino acid sequence identical to a portion of a protein expressed in the non-human organism and an amino acid sequence identical to a portion of a ligand recognized by a virus during infection as a peptide that binds to the target cells.

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