JP2018165253A - Medicine transporter that penetrates blood brain barrier, peptide and use therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel peptide that can penetrate the blood brain barrier, and a medicine transporter containing the peptide, a blood brain barrier penetrating agent, and a vascular endothelial cell penetrating agent.SOLUTION: The present invention provides a peptide that contains one of (i) an amino acid sequence represented by MMATGKLLVSYAPPGAD (sequence number: 1), and (ii) an amino acid sequence having at least 80% of sequence identity to the amino acid sequence of the sequence number: 1, with the length of at least 16 amino acid residues; or a peptide that contains one of (iii) an amino acid sequence represented by RIVVPLSTPREMDILGFVSACNDFSVRLLRDTTH (sequence number: 2), and (iv) an amino acid sequence having at least 80% of sequence identity to the amino acid sequence of the sequence number: 2, with the length of at least 33 amino acid residues.SELECTED DRAWING: None

Description

  The present invention relates to a peptide that can permeate the blood brain barrier, a drug transporter containing the peptide, a blood brain barrier permeation agent, and a vascular endothelial cell permeation agent.

  The blood-brain barrier (BBB) in the central nervous system enters the central nervous system due to the fact that vascular endothelial cells are densely packed and physically narrowed, and the physiological functions of these cells. Incorporation of these substances is very limited. The mechanism of this blood-brain barrier permeation has not yet been elucidated, and even if a disorder occurs in the central nervous system, the drug cannot reach the target region of the central nervous system.

Examples of disorders of the central nervous system include infections caused by poliovirus, Japanese encephalitis virus, and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's chorea. For infections in the central nervous system, prevention by vaccine administration and symptomatic treatment after infection are mainstream, and no effective treatment method has yet been found.
For example, as a therapeutic agent for poliovirus, an antiviral composition containing citric acid and / or zinc and L-arginine together with a possible carrier (see Patent Document 1), or an antiviral agent containing selegiline as an active ingredient (patented) Therapeutic drugs such as literature 2) have also been proposed, but it is problematic in that these antiviral agents cannot penetrate the blood-brain barrier.

In recent years, in order to achieve safer and more effective use of drugs, not only dosage forms suitable for drugs but also methods for efficiently delivering drugs to target in vivo sites have been attracting attention. . In particular, there is a demand for practical use of a drug delivery system (DDS) as a transport system that efficiently transports a necessary amount of a drug to a required place when necessary.
In the central nervous system, a method using a viral vector has been proposed (see, for example, Patent Document 3). However, this method is a problem in terms of safety and has not been put into practical use.

So far, it has been found that poliovirus permeates the blood-brain barrier through the transferrin receptor, and from this finding, a peptide that permeates the blood-brain barrier has been proposed (see, for example, Patent Document 4).
However, since the blood-brain barrier permeation is not limited to the pathway through the transferrin receptor, development of a new technique different from the proposed peptide is strongly demanded at present.

JP 2009-298800 A JP 2004-292399 A JP 2009-159988 A International Publication No. 2012/105595

  An object of the present invention is to solve the above-described problems and achieve the following objects. That is, an object of the present invention is to provide a novel peptide that can permeate the blood brain barrier, a drug transporter containing the peptide, a blood brain barrier permeation agent, and a vascular endothelial cell permeation agent.

  In order to solve the above-mentioned problems, the present inventors have intensively studied. As a result, they found that VP3, which is a poliovirus outer shell protein, efficiently penetrates vascular endothelial cells, and thus completed the present invention.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> (i) an amino acid sequence represented by the following SEQ ID NO: 1, and (ii) at least 80% sequence identity to the amino acid sequence represented by the following SEQ ID NO: 1, and a long A peptide comprising any one of the amino acid sequences having a length of at least 16 amino acid residues.
MMATGKLLVSYAPPGAD (SEQ ID NO: 1)
<2> (iii) the amino acid sequence represented by SEQ ID NO: 2 below, and (iv) at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 below, and long A peptide comprising any one of the amino acid sequences having a length of at least 33 amino acid residues.
RIVVPLSTPREMDILGFVSACNDFSVRRLRDTH (SEQ ID NO: 2)
<3> A drug transporter comprising the peptide according to any one of <1> to <2>.
<4> A blood brain barrier permeation agent comprising the peptide according to any one of <1> to <2>.
<5> A vascular endothelial cell permeation agent comprising the peptide according to any one of <1> to <2>.

  ADVANTAGE OF THE INVENTION According to this invention, the said conventional problems can be solved, the said objective can be achieved, the novel peptide which can permeate | transmit a blood brain barrier, the drug transporter containing the said peptide, and a blood brain barrier permeation agent And vascular endothelial cell penetrants can be provided.

FIG. 1A is a diagram showing the results of confirming the protein size by SDS-PAGE in Test Example 1. FIG. In FIG. 1A, “Con.” Indicates the result of confirming the control fusion protein, “VP1” indicates the result of confirming the fusion protein containing VP1 which is the poliovirus outer shell protein, and “VP2” indicates the result of outside the poliovirus. The result of confirming the fusion protein containing the shell protein VP2 is shown, and “VP3” shows the result of confirming the fusion protein containing the poliovirus outer shell protein VP3. FIG. 1B is a diagram showing the results of immunostaining in Test Example 1. FIG. 2A is a diagram showing the results of confirming the protein size by SDS-PAGE in Test Example 2-1. In FIG. 2A, “M” indicates a marker, “Con.” Indicates the result of confirming the control fusion protein, and “N1” to “N6” indicate the results of confirming the fusion protein containing N1 to N6, respectively. Show. FIG. 2B is a diagram showing the results of fluorescence quantification in Test Example 2-1. FIG. 2C is a diagram showing the results of immunostaining in Test Example 2-2. FIG. 3 is a diagram showing the results of flow cytometry in Test Example 3. FIG. 4 is a diagram showing the results of flow cytometry in Test Example 4. FIG. 5A is a diagram showing the results when CPZ is used as an inhibitor in Test Example 5. FIG. 5B is a diagram showing the results when Dynasore was used as an inhibitor in Test Example 5. FIG. 5C is a diagram showing results when EIPA was used as an inhibitor in Test Example 5. FIG. 6A is a diagram showing the results of flow cytometry in Test Example 6-1. FIG. 6B is a diagram showing the results of flow cytometry in Test Example 6-2. FIG. 7A is a diagram showing the results of flow cytometry in Test Example 7-1. FIG. 7B is a diagram showing the results of flow cytometry in Test Example 7-2. FIG. 8 is a diagram showing the results of flow cytometry in Test Example 8. FIG. 9A is a diagram showing the results of measuring the protein permeability to vascular endothelial cells in Test Example 9. FIG. 9B is a diagram showing the results of measuring the protein permeability to the brain side in Test Example 9.

(peptide)
The peptide of the present invention includes at least one of the amino acid sequences represented by the following (i) to (iv), and further includes other amino acid sequences as necessary.
The peptide has a function of penetrating the blood brain barrier and vascular endothelial cells.
(I) The amino acid sequence represented by SEQ ID NO: 1 (ii) The sequence has at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and has a length of at least 16 amino acids An amino acid sequence which is a residue (iii) an amino acid sequence represented by SEQ ID NO: 2 below (iv) has at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 below, and Amino acid sequence MMATGKLLVSYAPPGAD (SEQ ID NO: 1) that is at least 33 amino acid residues in length
RIVVPLSTPREMDILGFVSACNDFSVRRLRDTH (SEQ ID NO: 2)

The peptide includes at least one of the amino acid sequences (i) to (iv), and is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose.
The peptide may consist of only one of the amino acid sequences (i) to (iv).

  The upper limit of the length of the peptide containing the amino acid sequence (i) is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Preferably, 50 amino acid residues or less are more preferable, and 25 amino acid residues or less are particularly preferable.

Specific examples of the peptide containing the amino acid sequence (i) include a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 (hereinafter referred to as “N4”). A peptide consisting of the amino acid sequence represented by SEQ ID NO: 12 (hereinafter sometimes referred to as “N4-2”), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 13 (hereinafter, referred to as “N4-2”). A peptide consisting of an amino acid sequence represented by SEQ ID NO: 14 (hereinafter sometimes referred to as “N4-4”), an amino acid sequence represented by SEQ ID NO: 15. A peptide comprising the amino acid sequence represented by SEQ ID NO: 16 (hereinafter sometimes referred to as “N4-6”), Peptide consisting of amino acid sequence represented by column number: 17 (hereinafter sometimes referred to as “N4-7”), Peptide consisting of amino acid sequence represented by SEQ ID NO: 18 (hereinafter referred to as “N4-8”) For example).
The peptide consisting of the amino acid sequence (i) may be referred to as “N4-9” hereinafter.

  The amino acid sequence (ii) is at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and has a length of at least 16 amino acid residues. There is no limitation and can be appropriately selected depending on the purpose. For example, in the amino acid sequence represented by SEQ ID NO: 1, one or several amino acids are substituted, deleted, added, or inserted. Examples include amino acid sequences.

  The sequence identity with respect to the amino acid sequence represented by SEQ ID NO: 1 in the amino acid sequence (ii) is not particularly limited as long as it is at least 80%, and can be appropriately selected depending on the purpose. 81% or more is preferable, 87% or more is more preferable, and 93% or more is particularly preferable.

  The length of the peptide containing the amino acid sequence (ii) is not particularly limited as long as it is at least 16 amino acid residues, and can be appropriately selected according to the purpose. The group is preferably 16 amino acid residues or more and 50 amino acid residues or less, more preferably 16 amino acid residues or more and 25 amino acid residues or less.

  Specific examples of the peptide containing the amino acid sequence of (ii) above include a peptide comprising the amino acid sequence represented by SEQ ID NO: 22 (hereinafter sometimes referred to as “N4-9-m1”), SEQ ID NO: 23 A peptide comprising the amino acid sequence represented by (hereinafter sometimes referred to as “N4-9-m2”), a peptide comprising the amino acid sequence represented by SEQ ID NO: 24 (hereinafter referred to as “N4-9-m3”) A peptide consisting of an amino acid sequence represented by SEQ ID NO: 25 (hereinafter sometimes referred to as “N4-9-m4”), a peptide consisting of an amino acid sequence represented by SEQ ID NO: 26 ( Hereinafter, it may be referred to as “N4-9-m5”), a peptide comprising the amino acid sequence represented by SEQ ID NO: 27 (hereinafter, sometimes referred to as “N4-9-m6”), SEQ ID NO: A peptide comprising the amino acid sequence represented by 28 (hereinafter sometimes referred to as “N4-9-m7”), a peptide comprising the amino acid sequence represented by SEQ ID NO: 29 (hereinafter referred to as “N4-9-m8”) And a peptide consisting of the amino acid sequence represented by SEQ ID NO: 30 (hereinafter sometimes referred to as “N4-9-m9”).

  The upper limit of the length of the peptide containing the amino acid sequence (iii) is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Preferably, 75 amino acid residues or less are more preferable, and 40 amino acid residues or less are particularly preferable.

Specific examples of the peptide containing the amino acid sequence (iii) include a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 11 (hereinafter referred to as “N6”). A peptide consisting of an amino acid sequence represented by SEQ ID NO: 31 (hereinafter sometimes referred to as “N6-2”), a peptide consisting of an amino acid sequence represented by SEQ ID NO: 33 (hereinafter, referred to as “N6-2”). A peptide consisting of the amino acid sequence represented by SEQ ID NO: 37 (hereinafter sometimes referred to as “N6-8”), an amino acid sequence represented by SEQ ID NO: 38 And the like (hereinafter sometimes referred to as “N6-9”).
The peptide having the amino acid sequence (iii) may be referred to as “N6-10” hereinafter.

  The amino acid sequence (iv) is at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and has a length of at least 33 amino acid residues. There is no limitation and can be appropriately selected according to the purpose. For example, in the amino acid sequence represented by SEQ ID NO: 2, one or several amino acids are substituted, deleted, added, or inserted. Examples include amino acid sequences.

  The sequence identity with respect to the amino acid sequence represented by SEQ ID NO: 2 in the amino acid sequence (iv) is not particularly limited as long as it is at least 80%, and can be appropriately selected depending on the purpose. 90% or more is preferable, 93% or more is more preferable, and 96% or more is particularly preferable.

  The length of the peptide containing the amino acid sequence (iv) is not particularly limited as long as it is at least 33 amino acid residues, and can be appropriately selected according to the purpose. The group is preferably not more than 33, more preferably not less than 33 amino acid residues and not more than 75 amino acid residues, particularly preferably not less than 33 amino acid residues and not more than 40 amino acid residues.

  Specific examples of the peptide containing the amino acid sequence (iv) include a peptide consisting of the amino acid sequence represented by SEQ ID NO: 32 (hereinafter sometimes referred to as “N6-3”), represented by SEQ ID NO: 34. A peptide comprising the amino acid sequence (hereinafter sometimes referred to as “N6-5”), a peptide comprising the amino acid sequence represented by SEQ ID NO: 35 (hereinafter sometimes referred to as “N6-6”), A peptide comprising the amino acid sequence represented by SEQ ID NO: 40 (hereinafter sometimes referred to as “N6-10-m1”), a peptide comprising the amino acid sequence represented by SEQ ID NO: 41 (hereinafter referred to as “N6-10”) -M2 "), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 42 (hereinafter sometimes referred to as" N6-10-m3 "), and the like.

  Among the peptides including the amino acid sequences (i) to (iv), the peptide including the amino acid sequence (i) or (ii) is preferable in that the permeability of the blood brain barrier is excellent.

  In addition, the peptide may be one having a chemical modification at the N-terminus or C-terminus. There is no restriction | limiting in particular as said chemical modification, According to the objective, it can select suitably, For example, acetylation, myristoylation, amidation, cystinylation etc. are mentioned.

  There is no restriction | limiting in particular as the preparation method of the said peptide, According to the objective, it can select suitably, For example, it obtains by the molecular biological method based on the method of chemical synthesis, the gene arrangement | sequence of a poliovirus (PV; Poliovirus) The method etc. are mentioned.

  There is no restriction | limiting in particular as the method of the chemical synthesis of the said peptide, According to the objective, it can select suitably, For example, the method of chemically synthesizing using a peptide synthesizer (for example, Shimadzu Corporation make) etc. are mentioned. It is done.

The method obtained by molecular biological techniques based on the poliovirus gene sequence is not particularly limited and can be appropriately selected according to the sequence of the peptide. For example, genomic RNA prepared from poliovirus is reversed. Examples include a method in which a DNA synthesized by a transcription enzyme is used as a template, amplified by PCR, and cloned.
The conditions of the PCR method and the primers used are not particularly limited and can be appropriately selected depending on the sequence of the peptide.

-Other sequences-
The other sequences are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples thereof include linkers, proteins, and tags.
The linker can be provided, for example, between the peptide and the protein, and the sequence thereof is not particularly limited and can be appropriately selected according to the purpose. For example, the linker is represented by SEQ ID NO: 44. The amino acid sequence etc. which are made are mentioned.
There is no restriction | limiting in particular as said protein, According to the objective, it can select suitably, For example, GFP analogs, such as GST protein, GFP, and Venus protein, etc. are mentioned.
There is no restriction | limiting in particular as said tag, According to the objective, it can select suitably, For example, a FLAG tag, a His tag, etc. are mentioned.
The said other arrangement | sequence may be used individually by 1 type, may use 2 or more types together, and does not need to use it.

<Application>
The use of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose. However, the peptide is preferably used for the drug transporter, blood brain barrier permeation agent, vascular endothelial cell permeation agent and the like of the present invention described later. Can do.

(Drug transporter)
The drug transporter of the present invention contains at least the peptide of the present invention, and further contains other components as necessary.

<Peptide>
The peptide is the peptide of the present invention described above, and the preferred embodiment is also the same.
The content of the peptide in the drug transporter is not particularly limited as long as the drug can be transported, and can be appropriately selected according to the purpose.

  In the peptide, the site to which other components such as a carrier and a drug to be described later coordinate is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include the N-terminus and C-terminus of the peptide. .

<Other ingredients>
The other components are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Examples thereof include carriers, drugs, additives, adjuvants, and water. These may be used alone or in combination of two or more.

-Carrier-
The form of the carrier is not particularly limited and can be appropriately selected depending on the purpose. For example, the form in which the drug is enclosed in a closed space formed by a film, the form in which the drug is enclosed, or a film The form etc. which were included in the inside are mentioned, The form by these combination may be sufficient. As described above, various forms of the carrier are conceivable. Therefore, “supporting” widely means inclusion, encapsulation, interaction, and the like depending on the form.
Specific examples of the form of the carrier include macromolecules, microassemblies, microparticles, microspheres, nanospheres, liposomes, and emulsions.
There is no restriction | limiting in particular as said carrier, According to the objective, it can select suitably from what is known in the field of a pharmaceutical formulation.

  The amount of the carrier in the drug transporter is not particularly limited and can be appropriately selected depending on the purpose.

-Drug-
The drug is not particularly limited and may be appropriately selected depending on the purpose. For example, a drug for performing at least one of prevention, treatment, and diagnosis, and other pharmacologically acceptable pharmacology Active substance, physiologically active substance, diagnostic substance and the like.
There is no restriction | limiting in particular as a property of the said drug, According to the objective, it can select suitably, A hydrophilic drug may be sufficient and a hydrophobic drug may be sufficient.
The drug may be transported by being carried on the peptide, may be carried by being carried by the carrier, or may be carried by being carried by both.

There is no restriction | limiting in particular as a kind of said drug, According to the objective, it can select suitably, For example, a nucleic acid, a polynucleotide, a gene, and these analogs; Glycosaminoglycan and its derivative; Oligosaccharide, polysaccharide , And derivatives thereof; physiologically active substances such as proteins and peptides; anti-neurologic agents; antiviral agents; anticancer agents; antibiotics; enzyme agents; antioxidant agents; anti-inflammatory agents; steroid agents; Inhibitors; Vasodilators; Smooth muscle cell proliferation and / or migration inhibitors; Platelet aggregation inhibitors; Anticoagulants; Chemical mediator release inhibitors; Immunosuppressants; Lipid uptake inhibitors; Hormonal agents; Angiotensin receptors Antagonist; Vascular endothelial cell proliferation or inhibitor; Aldose reductase inhibitor; Mesangial cell proliferation inhibitor; Lipoxygenase inhibitor; Activator; Maillard reaction inhibitor; Amyloidosis inhibitor; Nitric oxide synthase (NOS): Inhibitor of advanced glycation end products (AGEs); Pharmacologically active substances such as radical scavenger Is mentioned. These may be used alone or in combination of two or more.
Since the drug transporter can suitably pass through the blood brain barrier, among these drugs, a drug that can be used for treatment and / or diagnosis of the central nervous system is preferable.

  Specific examples of the anti-nerve agent include anxiolytic agents such as Constant, Sepazone, Celsin, Serenal, Solanax, Depas, Balance, Meirax, Liese, Ribotolyl, Lexotan, Wybax, Sedir, Grandaxin, Erispan; Anafuranil, Tofranil, Antidepressants such as tryptanol, amoxan, amplito, prothiaden, tethypur, tetramide, ludiomir, dezirell, reslin, abilit, dogmatil, miradol, ritalin, depromail, paxil, lubox, toledomine; amoban, halcione, evermile, myslee, rismy, Lendrmin, Lolamet, Cyrace, Doral, Benzalin, Eurozine, Lohypnol, Insmin, Somerin, Dalmate, Fenovar, Iso Anti-insomnia drugs such as tar; tranquilizers such as winteramine, contamine, neuleptil, hirunamin, PZC, merylyl, impromene, serenace, aurap, cremin, cromecton, defecton, holit, rhodopine, attalux; Antiepileptics such as ethotoin, phenytoin, acetylphenetride, primidone, sultiam, ethosuximide, clonazepam, carbamazepine, sodium valproate, zonisamide; levodopa, pergolide mesylate, amandadine hydrochloride, trihexyphenidyl hydrochloride, pyroheptin hydrochloride, hydrochloric acid Examples include Parkinson's disease therapeutic agents such as mazaticol, methixene hydrochloride, biperidene, prophenamine, and droxidopa.

  Specific examples of the antiviral agent include acyclovir, ganciclovir, didanosine, zidovudine, sorivudine, vidarabine and the like.

  Specific examples of the anticancer agent include cyclophosphamide, ifosfamide, nitrogen mustard-N-oxide hydrochloride, thiotepa, brusphan, carbocon, nimustine hydrochloride, ranimustine, melphalan, improsulfan tosylate, dacarbazine, Procarbazine hydrochloride, cytarabine, cytarabine oxfert, inositabine, mercaptopurine, thioinosine, fluorouracil, doxyfluridine, tegafur, methotrexate, carmofur, hydroxycarbamide, vincristine sulfate, vinblastine sulfate, vindesine sulfate, etoposide, dammorubicin hydrochloride, dauporubicin hydrochloride hydrochloride Alaclubicin hydrochloride, pirarubicin, epirubicin hydrochloride, dactinomycin, mitoxantrone hydrochloride, hydrochloric acid Leomycin, pepromycin sulfate, mitomycin C, neocarnostatin, L-asparaginase, acegraton mitopronitol, sodium dextran sulfate, octreotide acetate, cisplatin, carboplatin, tamoxifen citrate, medroxyprogesterone acetate, estramustine sodium phosphate, Examples include goserelin acetate and leuprorelin acetate.

  Specific examples of the antibiotics include benzylpenicillin potassium, benzylpenicillin benzathine, phenoxymethyl penicillin potassium, pheneticillin potassium, cloxacillin sodium, flucloxacillin sodium, ampicillin, sultamicillin tosylate, bacampicillin hydrochloride, tarampicillin hydrochloride , Lenampicillin, hetacillin potassium, cyclacillin, amoxicillin, pibmesililin hydrochloride, aspoxicillin, carbenicillin sodium, calindacillin sodium, sulbenicillin sodium, ticarcillin sodium, piperacillin sodium, cephaloridine, cephalothin sodium, cefazolin sodium, cefapirin sodium, cefradine sodium, cefradine sodium Cephalexin, cephatridin propylene glycol, cephalexin Loxazine, cefaclor, cefadroxyl, cefotiam hydrochloride, cefotiam hexetyl, cefuroxime sodium, cefuroxime axetyl, cefamandol sodium, cefdinir, cefetametpipoxil hydrochloride, ceftipten, cefmetazole sodium, cefoxitin sodium, Cefotetan sodium, cefminox sodium, cefpuperazone sodium, cefpyramide sodium, cefthrodine sodium, cefotaxime sodium, cefoperazone sodium, ceftizoxime sodium, cefmenoxime hydrochloride, ceftriaxone sodium, ceftazidime, cefpimi Zole sodium, cefixime, cefterampipoxil, cefzonam sodium, cefpodoxyproxetil, cefodizime, Pyrom, latamoxef sodium, flomoxef sodium, imipenem, silastatin sodium, aztreonam, carmonam sodium, strepeptomycin sulfate, kanamycin sulfate, fradiomycin sulfate, amikacin sulfate, gentamicin sulfate, paromomycin sulfate, pecanamic acid sulfate, sulfate Lipostamycin, dibekacin sulfate, tobramycin, sisomycin sulfate, myromycin sulfate, astronomycin sulfate, netilmycin sulfate, isepamicin sulfate, arbekacin sulfate, erythromycin, kitasamycin, acetyl kitasamycin, oleandomycin phosphate, josamycin, acetyl spiramycin , Midecamycin, midecamycin acetate, rokitamycin, roxithromycin, clarithromycin, Tracycline, oxytetracycline hydrochloride, tetracycline metaphosphate, demethylchlortetracycline hydrochloride, lolitetracycline, doxycycline hydrochloride, minocycline hydrochloride, chloramphenicol, chloramphenicol sodium succinate, chloramphenicol palmitate, thianphenicol, Aminoacetic acid thianphenicol hydrochloride, colistin sulfate, colistin sodium methanesulfonate, polymyxin B sulfate, bacitracin, vancomycin hydrochloride, lincomycin hydrochloride, clindamycin, spectinomycin hydrochloride, fosfomycin sodium, phosphomycin calcium, etc. It is done.

  Specific examples of the enzyme agent include chymotrypsin, crystalline trypsin, streptokinase / streptodolase, hyaluronidase, urokinase, nasarplase, alteplase, lysozyme chloride, semi-alkaline proteinase, serrapeptase, tisokinase, duteprase, batroxobin, pronase, promeline, etc. Can be mentioned.

  Specific examples of the antioxidant include tocopherol, ascorbic acid, uric acid and the like.

  Specific examples of the anti-inflammatory agent include choline salicylate, sazapyrine, sodium salicylate, aspirin, diflunisal, flufenamic acid, mefenamic acid, fructophenine, tolfenamic acid, diclofenacnatrim, tolmetin sodium, sulindac, fenbufen, ferbinacethyl, indomethacin, indomethacin Farnesyl, acemetacin, progourmetacin maleate, ampenac sodium, nabumetone, ibuprofen, flurbiprofen, flurbiprofen axetil, ketoprofen, naproxen, protidic acid, pranoprofen, fenoprofen calcium, thiaprofenic acid, oxaprozin , Loxoprofen sodium, aluminoprofen, zaltoprofen, phenylbutazone, black Ezon, keto phenylbutazone, piroxicam, tenoxicam, ampiroxicam, tiaramide hydrochloride, hydrochloric tinoridine, benzydamine hydrochloride, epirizole, Erumofazon the like.

  Specific examples of the steroidal agent include cortisone acetate, hydrocortisone (phosphate ester, acetate), hydrocortisone butyrate, hydrocortisone sodium succinate, prednisolone (acetate, succinate, tertiary butyl acetate, phosphate ester), methylprednisolone (Acetate), methylprednisolone sodium succinate, triamcinolone, triamcinolone acetonide (triamcinolone acetate), dexamethasone (phosphate ester, acetate salt, sodium phosphate salt, sulfate ester), dexamethasone palmitate, betamethasone (phosphate, disodium) Salt), acetic acid parameterzone, fludrocortisone acetate, halopredone acetate, clobetasol propionate, halcinonide, beclomethasone propionate, valeric acid Tametazon, betamethasone acetate, acetic acid cortisone and the like.

  Specific examples of the angiotensin converting enzyme inhibitor include alacepril, imidapril hydrochloride, temocapril hydrochloride, delapril hydrochloride, benazepril hydrochloride, captopril, cilazapril, enalapril maleate, lisinopril and the like.

  Specific examples of the vasodilator include theophylline, diprofylline, proxyphylline, aminophylline, choline theophylline, prostaglandin, prostaglandin derivatives, alprostadil alphadex, alprostadil, limaprosto alphadex, papaverine, cyclandelate, cinnarizine , Bencyclane fumarate, cinepazide maleate, dirazep hydrochloride, trapidil, diphenidol hydrochloride, nicotinic acid, inositol hexanicotinate, nicotinate citrate, nicotinic alcohol tartrate, tocopherol nicotinate, hepronicart, isoxpurine hydrochloride, bamethane sulfate, tralizone hydrochloride, mesyl Dihydroergotoxin acid, ifenprodil tartrate, moxysilito hydrochloride, nicergoline, nicardipine hydrochloride Nilvadipine, nifedipine, benidipine hydrochloride, diltiazem hydrochloride, nisoldipine, nitrendipine, manidipine hydrochloride, varnidipine hydrochloride, efonidipine hydrochloride, verapamil hydrochloride, trimetazidine hydrochloride, captopril, enalapril maleate, alacepril, delapril hydrochloride, cilazapril, hydrazopril hydrochloride, ricinopril hydrochloride, Examples include todralazine hydrochloride, budralazine, cadralazine, indapamide, carbochromene hydrochloride, efloxate, etaphenone hydrochloride, oxyfedrine hydrochloride, nicorandil, amyl nitrite, and isosorbide nitrate.

  Specific examples of the smooth muscle cell proliferation and / or migration inhibitor include heparin sodium, dalteparin sodium (low molecular heparin), heparin calcium, dextran sulfate and the like.

  Specific examples of the platelet aggregation inhibitor include ticlopidine hydrochloride, cilostazol, ethyl icosapentate, beraprost sodium, salpgrelate hydrochloride, batroxobin, dipyridamole and the like.

  Specific examples of the anticoagulant include heparin sodium, dalteparin sodium (low molecular heparin), heparin calcium, dextran sulfate, warfarin potassium, argatroban and the like.

  Specific examples of the chemical mediator release inhibitor include tranilast, ketofetin fumarate, azelastine hydrochloride, oxatomide, amlexanox, and repirinast.

  Examples of the immunosuppressive agent include cyclosporine.

  Moreover, there is no restriction | limiting in particular as a chemical | medical agent used for a diagnosis, According to the objective, it can select suitably, For example, an X-ray contrast agent, a radioisotope labeling nuclear medicine diagnostic agent, a diagnostic agent for nuclear magnetic resonance diagnosis, etc. In-vivo diagnostic agents.

  Specific examples of the X-ray contrast agent include meglumine amidotrizoate, sodium iotaramate, meglumine iotaramate, gastrografin, iodamid meglumine, lipiodol ultrafluid, adipiodon meglumine, oxagrate, meglumine iotroxate, iotrolan, iopanoic acid Iopamidol, iohexol, ioversol, iopodate sodium, iomeprol, isopaque, iodoxamic acid and the like.

  The amount of the drug carried on the drug transporter is not particularly limited, and can be appropriately selected according to the type of drug.

-Additives, adjuvants-
The additive or the adjuvant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include bactericides, preservatives, binders, thickeners, fixing agents, binders, and coloring agents. , Stabilizers, pH adjusters, buffers, isotonic agents, solvents, antioxidants, UV inhibitors, crystal precipitation inhibitors, antifoaming agents, physical property improvers, preservatives, and the like. These may be used individually by 1 type and may use 2 or more types together.

Other components such as the additive, adjuvant, and water may be blended together with the drug transporter, or may be blended in the carrier.
There is no restriction | limiting in particular as content in the drug transporter of other components, such as the said additive, adjuvant, and water, According to the objective, it can select suitably.

  There is no restriction | limiting in particular as said bactericidal agent, According to the objective, it can select suitably, For example, cationic surfactants, such as benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, etc. are mentioned.

  There is no restriction | limiting in particular as said preservative, According to the objective, it can select suitably, For example, paraoxybenzoic acid esters, chlorobutanol, cresol, etc. are mentioned.

  The binder, thickener and fixing agent are not particularly limited and may be appropriately selected depending on the intended purpose. For example, starch, dextrin, cellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropyl Cellulose, hydroxypropylmethylcellulose, carboxymethyl starch, pullulan, sodium alginate, ammonium alginate, propylene glycol alginate, guar gum, locust bean gum, gum arabic, xanthan gum, gelatin, casein, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, ethylene propylene A block polymer, sodium polyacrylate, polyvinylpyrrolidone, etc. are mentioned.

  The binder is not particularly limited and may be appropriately selected depending on the intended purpose. For example, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxy Examples include propyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinyl pyrrolidone.

  The colorant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include titanium oxide and iron oxide.

  There is no restriction | limiting in particular as said stabilizer, According to the objective, it can select suitably, For example, tragacanth, gum arabic, gelatin, sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid etc. are mentioned.

  There is no restriction | limiting in particular as said pH adjuster and said buffer, According to the objective, it can select suitably, For example, sodium citrate, sodium acetate, sodium phosphate etc. are mentioned.

  The isotonizing agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include sodium chloride and glucose.

<Average particle size>
There is no restriction | limiting in particular as long as the average particle diameter of the said drug transporter does not impair the effect of this invention, According to the objective, it can select suitably.
The method for incorporating the drug transporter into the cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include endocytosis.
Many cells are known to be due to endocytosis. The size that can be taken up by endocytosis is about 300 nm at the maximum, and the amount taken up may be extremely reduced.
Therefore, the average particle diameter of the drug transporter is preferably smaller than 300 nm. The average particle diameter can be measured with a particle size distribution measuring apparatus.

<Transport of drug transporter>
The method for transporting the drug transporter into the cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method performed using the function and characteristics of the cell to be transported. .
Examples of cell functions and characteristics that can be used for transport of the drug transporter include cell phagocytosis and endocytosis.
Prior to administration of the drug transporter, cells may be activated in advance to facilitate phagocytosis and endocytosis. In addition, when cells are highly activated and adhesion is excessively increased or aggregation occurs, the cells may be inactivated by an appropriately selected method.

  The cells to which the drug transporter can be applied are not particularly limited as long as the above-described peptide of the present invention can be recognized, and can be appropriately selected according to the purpose. Examples thereof include vascular endothelial cells.

<Administration>
There is no restriction | limiting in particular as an administration method, dosage amount, administration time, and administration subject of the said drug transporter, According to the objective, it can select suitably.
There is no restriction | limiting in particular as said administration method, According to the objective, it can select suitably, For example, a local administration method, an enteral administration method, a parenteral administration method etc. are mentioned.
The topical administration method is not particularly limited and may be appropriately selected depending on the intended purpose. For example, dermal administration method, inhalation administration method, injection administration method, instillation on the conjunctiva, nasal administration method, vagina An internal administration method etc. are mentioned.
There is no restriction | limiting in particular as said enteral administration method, According to the objective, it can select suitably, For example, an oral administration method, a tube feeding method, an enema administration method etc. are mentioned.
The parenteral administration method is not particularly limited and may be appropriately selected depending on the purpose. For example, intravenous administration method, transarterial administration method, intramuscular administration method, intracardiac administration method, subcutaneous administration method, Intraosseous administration, intradermal administration, subarachnoid administration, intraperitoneal administration, intravesical administration, transdermal administration, transmucosal administration, inhalation administration, epidural administration, intravitreal administration, etc. Is mentioned.

  The dosage is not particularly limited and may be appropriately selected in consideration of various factors such as the age, weight, constitution, symptom, and presence / absence of administration of a drug containing other ingredients as active ingredients. it can.

  The animal species to be administered is not particularly limited and can be appropriately selected according to the purpose. For example, human, monkey, pig, cow, sheep, goat, dog, cat, mouse, rat, bird, etc. Is mentioned. Among these, it is preferably used for humans.

<Use>
The drug transporter may be used alone or in combination with a drug or drug transporter that has other components as effective components. Moreover, you may use in the state mix | blended with the pharmaceutical which uses another component as an active ingredient.

<Application>
Since the drug transporter of the present invention can permeate vascular endothelial cells and permeate the blood-brain barrier, it can be used for prevention, treatment or diagnosis of disorders in the central nervous system, such as diabetes and cancer. It can be suitably used as a drug transporter for diseases in which blood vessels are a problem.

(Brain-brain barrier permeation agent, vascular endothelial cell permeation agent)
<Blood-brain barrier permeation agent>
The blood brain barrier permeation agent of the present invention contains at least the peptide of the present invention, and further contains other components as necessary.
The peptide is the peptide of the present invention described above, and the preferred embodiment is also the same.
The content of the peptide in the blood brain barrier permeation agent is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. The blood brain barrier permeating agent may be the peptide itself.

<Vascular endothelial cell penetrant>
The vascular endothelial cell permeation agent of the present invention contains at least the peptide of the present invention, and further contains other components as necessary.
The peptide is the peptide of the present invention described above, and the preferred embodiment is also the same.
The content of the peptide in the vascular endothelial cell permeation agent is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. The vascular endothelial cell penetrating agent may be the peptide itself.

-Other ingredients-
The other components in the blood brain barrier permeation agent and the vascular endothelial cell permeation agent are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. And an acceptable carrier.
There is no restriction | limiting in particular as said support | carrier, According to a dosage form etc., it can select suitably, For example, what was described in the other item of the above-mentioned drug transporter etc. are mentioned.
Moreover, there is no restriction | limiting in particular as content of the said other component in the said blood brain barrier permeation agent and the said vascular endothelial cell permeation agent, According to the objective, it can select suitably.

  The blood brain barrier permeation agent and the vascular endothelial cell permeation agent may be used singly or in combination of two or more, and used in combination with a pharmaceutical comprising other ingredients as active ingredients. May be. Further, the blood brain barrier permeating agent and the vascular endothelial cell permeating agent may be used in a state of being blended in a medicine containing other components as active ingredients.

<Dosage form>
The dosage form of the blood-brain barrier permeating agent and the vascular endothelial cell permeating agent is not particularly limited and can be appropriately selected according to a desired administration method. Examples thereof include solid agents, semi-solid agents, and liquid agents. Can be mentioned.
There is no restriction | limiting in particular as a manufacturing method of the said blood brain barrier permeation agent and the said vascular endothelial cell permeation | transduction agent, According to dosage form, it can select suitably from a conventional method.

-Solid agent-
The solid preparation is not particularly limited and may be appropriately selected depending on the intended purpose. However, when used as an internal preparation, for example, a tablet, chewable tablet, effervescent tablet, orally disintegrating tablet, troche, drop Agents, hard capsules, soft capsules, granules, powders, pills, dry syrups, soaking agents and the like.
When the said solid agent is used as an external preparation, a suppository, a poultice, a plaster agent etc. are mentioned, for example.

-Semi-solid agent-
There is no restriction | limiting in particular as said semi-solid preparation, Although it can select suitably according to the objective, When using as an internal preparation, a licking agent, a chewing gum agent, a whipping agent, a jelly agent etc. are mentioned, for example.
When the semi-solid preparation is used as an external preparation, examples thereof include an ointment, a cream, a mousse, an inhaler, and a nazar gel.

-Liquid-
There is no restriction | limiting in particular as said liquid agent, Although it can select suitably according to the objective, When used as an internal preparation, a syrup agent, a drink agent, a suspension agent, an alcoholic agent etc. are mentioned, for example.
When the said liquid agent is used as an external preparation, a liquid agent, eye drops, an aerosol agent, a spray agent etc. are mentioned, for example.

<Administration>
There are no particular limitations on the administration method, dose, administration timing, and administration target of the blood brain barrier permeation agent and the vascular endothelial cell permeation agent, and it can be appropriately selected according to the purpose.
There is no restriction | limiting in particular as said administration method, According to the objective, it can select suitably, For example, a local administration method, an enteral administration method, a parenteral administration method etc. are mentioned.
The dosage is not particularly limited and may be appropriately selected in consideration of various factors such as the age, weight, constitution, symptom, and presence / absence of administration of a drug containing other ingredients as active ingredients. it can.
The animal species to be administered is not particularly limited and can be appropriately selected according to the purpose. For example, human, monkey, pig, cow, sheep, goat, dog, cat, mouse, rat, bird, etc. Among these, it is preferably used for humans.

  The present invention will be specifically described below with reference to test examples, but the present invention is not limited to these test examples.

(Test Example 1: Cell permeability to vascular endothelial cells)
<Plasmid construction>
A base sequence encoding a poliovirus outer shell protein VP1 (SEQ ID NO: 3), VP2 (SEQ ID NO: 4), or VP3 (SEQ ID NO: 5) from a poliovirus (Mahoney strain) by PCR. Cloned.

  Oligo DNA encoding a FLAG tag (DYKDDDDK (SEQ ID NO: 43)) was obtained by oligo synthesis.

The cloned PCR product and oligo DNA were cloned in-frame at the multicloning site of pGEX6T-1 vector (GE Healthcare) to construct a plasmid having sequences encoding the following proteins. As a control, a plasmid not containing a sequence encoding a poliovirus outer coat protein was also constructed.
The base sequence encoding the following linker (SRGSGGGGSGGGGSSG (SEQ ID NO: 44)) was inserted by PCR.
"GST protein"-"FLAG tag"-"Linker"-"VP1"-"6 x His"
"GST protein"-"FLAG tag"-"linker"-"VP2"-"6 x His"
"GST protein"-"FLAG tag"-"Linker"-"VP3"-"6 × His"
"GST protein"-"FLAG tag"-"linker"-"6 x His"

<Production of protein>
The Rosseta2 strain (Novagen) was transformed with the above plasmid.
The obtained Escherichia coli was cultured at 37 ° C. until the OD value at 600 nm reached 0.4, and then induced with 0.3 mM IPTG at 20 ° C. for 16 hours.
Thereafter, E. coli was recovered and dissolved, and then His tag purification was performed using Ni column resin to obtain the target protein.
The size of the obtained protein was confirmed by electrophoresing the protein by SDS-PAGE and coomassie staining (see FIG. 1A).

<Intracellular permeability test>
The day before the test, mouse vascular endothelial cells (MBEC4) were seeded in a 96-well plate at 1 × 10 ⁴ per well.
On the day of the test, each protein obtained above was added to the medium so as to be 1 μM.
After the addition, the cells were cultured for 24 hours, and then immunostained.

-Immunostaining-
The cells to which the protein had been added were fixed with 4% PFA, the cell membrane was disrupted with 0.2% Triton X100, and a primary antibody reaction was performed with FLAG antibody (SIGMA).
Next, a secondary antibody reaction was performed with an anti-mouse secondary antibody (Alexa Fluor 488 (Abcam)).
Thereafter, nuclear staining was performed with Hoechst (Invitrogen) and observed with an inverted microscope. The results are shown in FIG. 1B.

  FIG. 1B shows the result at a field magnification of 200 times. “GST-FLAG-Con.” Is “GST protein”-“FLAG tag”-“linker”-“6 × His” (control) ”. The result when added is shown, “GST-FLAG-VP1” shows the result when “GST protein”-“FLAG tag”-“linker”-“VP1”-“6 × His” is added, “GST-FLAG-VP2” indicates the result when “GST protein” — “FLAG tag” — “linker” — “VP2” — “6 × His” ”is added, and“ GST-FLAG-VP3 ” The results when “GST protein”-“FLAG tag”-“linker”-“VP3”-“6 × His” ”are added are shown.

From the result of FIG. 1B, it was confirmed that the fusion protein containing VP3 was strongly stained. Moreover, although the fusion protein containing VP1 and the fusion protein containing VP2 were weak in this order, staining was confirmed. In the control, staining was not confirmed.
Therefore, it was confirmed that VP3 has the property of intracellular permeability to vascular endothelial cells.

(Test Example 2: Identification of VP3 responsible region for cell permeation)
<Test Example 2-1>
-Plasmid construction-
By PCR, a base sequence encoding an amino acid sequence obtained by dividing the VP3 sequence into six as follows was cloned.
GLPVMNTPGSNQYLTADNFQSPCALPEFDVTPPIDIPGEVKNMM (SEQ ID NO: 6, hereinafter sometimes referred to as “N1”)
VKNMMELAEIDTM IPFDLSATKKNTMEMYRVRLSDKPHTDDPILC (SEQ ID NO: 7, hereinafter sometimes referred to as “N2”)
LCLSLSPASDPRLSHTMLGEILNYYTHWAG (SEQ ID NO: 8, hereinafter sometimes referred to as “N3”)
SLKFTFLFCGSMMATGKLLVSYAPPGADPPPKKRKKEAML (SEQ ID NO: 9, hereinafter sometimes referred to as “N4”)
GTHVIWDIGLQSSSTMVVPWISNTTYRQTIDDSFTEGGYISVFY (SEQ ID NO: 10, hereinafter sometimes referred to as “N5”)
YISVFYQTRIVVPLSTPREMDILGFVSACNDFSVRRLRDTHIEQKALAQ (SEQ ID NO: 11, hereinafter may be referred to as “N6”)

  The nucleotide sequence encoding the GFP analog Venus protein (SEQ ID NO: 47) was cloned by an artificial gene synthesis method (Fusmac).

The cloned PCR product was cloned in frame into the multi-cloning site of pGEX6T-1 vector (GE Healthcare) to construct each plasmid having sequences encoding the following proteins. As a control, a plasmid not containing a sequence encoding VP3 was also constructed.
The base sequence encoding the following linker (SRGSGGGGSGGGGSSG (SEQ ID NO: 44)) was inserted by PCR.
"GST protein"-"Venus protein"-"Linker"-"N1"-"6 x His"
"GST protein"-"Venus protein"-"Linker"-"N2"-"6 x His"
"GST protein"-"Venus protein"-"Linker"-"N3"-"6 × His"
"GST protein"-"Venus protein"-"Linker"-"N4"-"6 x His"
"GST protein"-"Venus protein"-"Linker"-"N5"-"6 x His"
"GST protein"-"Venus protein"-"Linker"-"N6"-"6 × His"
"GST protein"-"Venus protein"-"Linker"-"6 x His"

-Production of protein-
Each protein was prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.
The size of the obtained protein was confirmed by electrophoresing the protein by SDS-PAGE and coomassie staining (see FIG. 2A).

-Intracellular permeability test-
The day before the test, mouse vascular endothelial cells (MBEC4) were seeded in a 96-well plate at 1 × 10 ⁴ per well.
On the day of the test, each protein obtained above was added to the medium so as to be 1 μM.
After the addition, the cells were cultured for 24 hours, and then the amount of the protein adhered to or taken into the cells was detected based on the fluorescence of the Venus protein as follows.

--Fluorescence determination--
The cells to which the protein had been added were washed twice with PBS, and then the cells were lysed with Passive Lysis buffer (Promega).
Thereafter, the amount of protein adhering to the cell or incorporated into the cell was calculated from the amount of fluorescence using a Glomax multiluminometer (Promega). The result is shown in FIG. 2B.

  In FIG. 2B, “Con.” Indicates the result when “GST protein”-“Venus protein”-“linker”-“6 × His” (control) ”is added, and“ N1 ”indicates“ GST protein ” "-" Venus protein "-" linker "-" N1 "-" 6 × His "" shows the results when "NST" indicates "GST protein"-"Venus protein"-"linker"-" The results when N2 "-" 6xHis "are added are shown, and" N3 "is" GST protein "-" Venus protein "-" Linker "-" N3 "-" 6xHis "" “N4” indicates the result when “GST protein” − “Venus protein” − “linker” − “N4” − “6 × His” ”is added, and“ N5 ”indicates “GST protein” — “Venus protein” — “linker” — “N5” — “6 × His” ”shows the result when“ N6 ”is“ GST protein ”—“ Venus protein ”—“ The result when adding “linker”-“N6”-“6 × His” ”is shown.

  From the results of FIG. 2B, it was confirmed that the responsible sites for intracellular permeability of VP3 protein to vascular endothelial cells are the N4 site and the N6 site.

<Test Example 2-2>
-Plasmid construction-
In the same manner as in Test Example 1, a plasmid having a sequence encoding the following protein was constructed.
"GST protein"-"FLAG tag"-"linker"-"6 x His"
"GST protein"-"FLAG tag"-"Linker"-"VP3"-"6 × His"

In the same manner as in Test Example 1, except that “VP3” in the above plasmid was replaced with “N4” (SEQ ID NO: 9) or “N6” (SEQ ID NO: 11), sequences encoding the following proteins were obtained. A plasmid was constructed.
"GST protein"-"FLAG tag"-"Linker"-"N4"-"6 x His"
"GST protein"-"FLAG tag"-"linker"-"N6"-"6 x His"

-Production of protein-
Each protein was prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.

-Intracellular permeability test-
The day before the test, mouse vascular endothelial cells (MBEC4) were seeded in a 96-well plate at 1 × 10 ⁴ per well.
On the day of the test, each protein obtained above was added to the medium so as to be 1 μM.
After the addition, the cells were cultured for 24 hours, and then immunostained in the same manner as in Test Example 1. Results are shown in FIG. 2C.

FIG. 2C shows the result at a field magnification of 200 times. “GST-FLAG-Con.” Is “GST protein” — “FLAG tag” — “linker” — “6 × His” (control) ”. The result when added is shown, “GST-FLAG-VP3” shows the result when “GST protein”-“FLAG tag”-“linker”-“VP3”-“6 × His” is added, “GST-FLAG-VP3-N4” indicates the result when “GST protein”-“FLAG tag”-“linker”-“N4”-“6 × His” is added, and “GST-FLAG-VP3” “-N6” indicates the result when “GST protein”-“FLAG tag”-“linker”-“N6”-“6 × His” ”is added.
From the results of FIG. 2C, it was confirmed that the responsible sites for intracellular permeability of VP3 protein to vascular endothelial cells were the N4 site and the N6 site.

(Test Example 3: Transmission directivity to normal cells)
<Plasmid construction>
In the same manner as in Test Example 2-1, a plasmid having a sequence encoding the following protein was constructed.
"GST protein"-"Venus protein"-"Linker"-"6 x His"
"GST protein"-"Venus protein"-"Linker"-"N4"-"6 x His"
"GST protein"-"Venus protein"-"Linker"-"N6"-"6 × His"

In the same manner as in Test Example 2-1, except that “N4” in the plasmid was replaced with a base sequence encoding the following amino acid sequence (hereinafter sometimes referred to as “Tat”), ““ GST protein ”— A plasmid having a sequence encoding “Venus protein”-“linker”-“Tat”-“6 × His” was constructed.
The Tat is a human immunodeficiency virus (HIV-1) protein known to have cell permeability and was used as a positive control.
MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCMFKALGISYSYGRKKRRQRRRAHQNSQTHQASLSKQPTSQSRGDPGTGPKE (SEQ ID NO: 45, Tat)

<Production of protein>
Each protein was prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.

<Intracellular permeability test>
On the day before the test, the following cells were seeded in a 96-well plate at 1 × 10 ⁴ cells per well.
・ Normal human fibroblasts (NHDF)
・ Human peripheral blood mononuclear cells (PBMCs)
・ Human cord blood-derived vascular endothelial cells (HUVEC)

On the day of the test, each protein obtained above was added to the medium so as to be 1 μM.
After the addition, the cells were cultured for 24 hours, and then an experiment by flow cytometry was performed.

-Flow cytometry-
Cells to which protein has been added are washed twice with PBS, suspended by trypsin treatment, and dead cells are stained with propidium iodide (SIGMA). The ratio of the expression level of fluorescent protein in living cells alone, fluorescence intensity Was quantified and evaluated with a flow cytometer (Sony icite 800). Moreover, the experiment by flow cytometry was similarly performed about the cell which did not add protein.
The results are shown in FIG.

  In FIG. 3, “Non-treated” indicates the results for cells to which no protein was added, and “Empty” added ““ GST protein ”—“ Venus protein ”—“ linker ”—“ 6 × His ” “VP3-N4” shows the results for cells added with “GST protein”-“Venus protein”-“linker”-“N4”-“6 × His”, “VP3-N6” shows the result for cells added with “GST protein”-“Venus protein”-“linker”-“N6”-“6 × His” ”, and“ Tat (positive control) ”is“ GST ”. The result about the cell which added "protein"-"Venus protein"-"linker"-"Tat"-"6xHis" "is shown.

  From the results shown in FIG. 3, the fusion protein containing “N4” and the fusion protein containing “N6” exhibited efficient intracellular entry into human normal fibroblasts (NHDF) and human peripheral blood mononuclear cells (PBMCs). Not shown, showed the property of intracellular entry effective only on human umbilical cord blood-derived vascular endothelial cells (HUVEC).

(Test Example 4: Comparison of cell permeability in various cell lines)
<Plasmid construction>
In the same manner as in Test Example 2-1, a plasmid having a sequence encoding the following protein was constructed.
"GST protein"-"Venus protein"-"Linker"-"N4"-"6 x His"
"GST protein"-"Venus protein"-"Linker"-"N6"-"6 × His"

<Production of protein>
Each protein was prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.

<Intracellular permeability test>
On the day before the test, the following cells were seeded in a 96-well plate at 1 × 10 ⁴ cells per well.
-Melanoma-
Mewo, A2058, GAK, HMY-1, VMRC-MELG.
-Glioma-
WERI-Rb-1, HS-PSS, GI-1, T98G, TM-31, A172, NH-12, NB16, NB69.
-Oral cancer cells-
HSQ-89, EC-GI-10, HSC-4, 8305C.
-Lung cancer cells-
STC1, SBC-5, ABC-1, NCI-H1975, HCC827, A549, NCI-H23, H1299, NCI-H1650, Lu99B, EBC-1.
-Stomach-derived cancer cells-
NUGC-4, AZ521, HGC-27, GSS, GSU, H-111-TC.
-Solid organ cancer cells-
LoVo, CACO-2, CW-2, COLO201, HCT-116, MIA Paca2, PK-1, KP4, Li-7, TKK, OS-RC-2, MDA-MB-453, ME-180, HeLa, OVK18, JMSSU1, 5637.
-Normal cells-
Human peripheral blood mononuclear cells (PBMCs), human umbilical cord blood-derived vascular endothelial cells (HUVEC), human normal fibroblasts (NHDF).

On the day of the test, each protein obtained above was added to the medium so as to be 1 μM.
After the addition, the cells were cultured for 24 hours, and then an experiment by flow cytometry was performed.

-Flow cytometry-
In the same manner as in Test Example 3, an experiment by flow cytometry was performed. The results are shown in FIG.

  FIG. 4 is a graph showing Geo-mean which is fluorescence intensity. The upper row shows the results with and without the addition of “VP3-N4 (“ GST protein ”—“ Venus protein ”—“ linker ”—“ N4 ”—“ 6 × His ”)”, and the lower row shows “VP3-N6 (“ GST The result by the presence or absence of addition of "protein"-"Venus protein"-"linker"-"N6"-"6 * His") is shown. The horizontal axis shows each cell, the left graph in each cell item shows the results in the non-protein-added cells, and the right graph shows the results in the protein-added cells.

  From the results of FIG. 4, the fusion protein containing “N4” and the fusion protein containing “N6” do not show efficient intracellular invasion in most cells, and are strongly resistant to human umbilical cord blood-derived vascular endothelial cells (HUVEC). It showed a specific intracellular invasion property.

(Test Example 5: Examination of permeation route into cells)
<Plasmid construction>
In the same manner as in Test Example 2-1, a plasmid having a sequence encoding the following protein was constructed.
"GST protein"-"Venus protein"-"Linker"-"N4"-"6 x His"
"GST protein"-"Venus protein"-"Linker"-"N6"-"6 × His"

<Production of protein>
Each protein was prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.

<Intracellular permeability test>
The day before the test, mouse vascular endothelial cells (MBEC4) were seeded in a 96-well plate at 1 × 10 ⁴ per well.

On the day of the test, each protein obtained above was added to the medium so as to be 1 μM.
Simultaneously with the addition of the protein, any one of the following three inhibitors that inhibit each of the endocytosis pathways was added at the following concentration.
Chloropromazine (hereinafter sometimes referred to as “CPZ”, Wako Pure Chemical Industries, Ltd.)
Addition concentration: 0 μM, 0.2 μM, 1 μM, 2 μM, or 10 μM
CPZ inhibits clathrin-dependent intracellular indexing.
・ Dynasore (SIGMA)
Addition concentration: 0 μM, 50 μM, 100 μM, or 200 μM
Dynasore is an inhibitor of dynamin, an enzyme that cleaves endosomes that have been interrupted by clathrin-dependent intracellular interruption, caveolin-dependent intracellular interruption, and lipid raft-dependent intracellular interruption.
・ EIPA (Wako Pure Chemical Industries, Ltd.)
Addition concentration: 0 μM, 2 μM, 5 μM, or 10 μM
EIPA inhibits macropinocytosis-dependent intracellular indexing.

  After the addition, the cells were cultured for 24 hours, and then an experiment by flow cytometry was performed.

-Flow cytometry-
The cells to which the protein has been added are washed twice with PBS, suspended by trypsin treatment, and dead cells are stained with propidium iodide (SIGMA). Quantification and evaluation were performed with a meter (Sony icite 800).
The results are shown in FIGS. The ratio (%) of fluorescence-stained cells in cells not added with an inhibitor was expressed as a ratio of 100.

FIG. 5A shows the results when CPZ, a clathrin inhibitor, is added, FIG. 5B shows the results when Dynasore, a dynamin inhibitor, is added, and FIG. 5C shows EIPA, a macropinocytosis inhibitor. The result when added is shown.
The horizontal axis indicates the additive concentration of the inhibitor, and the graph on the left side of each additive concentration item is a fusion protein containing “N4” (“GST protein”-“Venus protein”-“linker”-“N4”-“6 XHis ") is shown, and the graph on the right shows the fusion protein containing" N6 "(" GST protein "-" Venus protein "-" Linker "-" N6 "-" 6xHis "). The result when added is shown.

From the results of FIGS. 5A to 5C, the fusion protein containing “N4” showed a decrease in permeation efficiency dependent on the dynamin inhibitor. On the other hand, the fusion protein containing “N6” was sensitive to any inhibitor.
Thus, it was shown that the permeation pathway of the “N4” peptide into cells is dynamin-dependent endocytosis and is intracellular entry via caveolin and lipid rafts. On the other hand, it was shown that the permeation pathway of the “N6” peptide into the cell may utilize all endocytosis pathways of clathrin, caveolin, lipid raft, and macropinocytosis.

(Test Example 6: Identification of cell permeation responsible region in “N4” site of VP3)
<Test Example 6-1>
-Plasmid construction-
In the same manner as in Test Example 2-1, a plasmid having a sequence encoding the following protein was constructed.
“GST protein” — “Venus protein” — “linker” — “N4” — “6 × His” (hereinafter sometimes referred to as “VP3-N4”)

By PCR, a nucleotide sequence encoding an amino acid sequence in which the sequence of the N4 site of VP3 was changed as follows was cloned.
SLKFTFLFCGSMMATGKLLVSYAPPGADPPPKKR (SEQ ID NO: 12, hereinafter sometimes referred to as “N4-2”)
SLKFTFLFCGSMMATGKLVLVYAPPGAD (SEQ ID NO: 13, hereinafter sometimes referred to as “N4-3”)
FLFCGSMMMATGKLLVSYAPPGADPPKKRKKEAML (SEQ ID NO: 14, hereinafter sometimes referred to as “N4-4”)
FLFCGSMMMATGKLLVSYAPPGADPPKKR (SEQ ID NO: 15, hereinafter sometimes referred to as “N4-5”)
MMATGKLLVSYAPPGADPKKRKKEAML (SEQ ID NO: 16, hereinafter sometimes referred to as “N4-6”)
FLFCGSMMMATGKLLVSYAPPGAD (SEQ ID NO: 17, hereinafter sometimes referred to as “N4-7”)
MMATGKLLVSYAPPGADPKKR (SEQ ID NO: 18, hereinafter sometimes referred to as “N4-8”)
MMATGKLLVSYAPPGAD (SEQ ID NO: 1, hereinafter sometimes referred to as “N4-9”)
MMATGKLLVSYAPPG (SEQ ID NO: 19, hereinafter sometimes referred to as “N4-10”)
ATGKLLVSYAPPGAD (SEQ ID NO: 20, hereinafter sometimes referred to as “N4-11”)
ATGKLLVSYAPPG (SEQ ID NO: 21, hereinafter sometimes referred to as “N4-12”)

“N4” in the plasmid having a sequence encoding the “GST protein”-“Venus protein”-“linker”-“N4”-“6 × His” protein is referred to as “N4-2” to “N4-12”. A plasmid having a sequence encoding the following protein was constructed instead of the base sequence encoding the amino acid sequence of
“GST protein” — “Venus protein” — “linker” — “N4-2” — “6 × His” (hereinafter sometimes referred to as “VP3-N4-2”)
"GST protein"-"Venus protein"-"linker"-"N4-3"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-3")
"GST protein"-"Venus protein"-"linker"-"N4-4"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-4")
"GST protein"-"Venus protein"-"linker"-"N4-5"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-5")
"GST protein"-"Venus protein"-"linker"-"N4-6"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-6")
"GST protein"-"Venus protein"-"linker"-"N4-7"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-7")
"GST protein"-"Venus protein"-"linker"-"N4-8"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-8")
"GST protein"-"Venus protein"-"linker"-"N4-9"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-9")
"GST protein"-"Venus protein"-"linker"-"N4-10"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-10")
"GST protein"-"Venus protein"-"linker"-"N4-11"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-11")
"GST protein"-"Venus protein"-"linker"-"N4-12"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-12")

-Production of protein-
Each protein was prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.

-Intracellular permeability test-
The day before the test, mouse vascular endothelial cells (MBEC4) were seeded in a 96-well plate at 1 × 10 ⁴ per well.
On the day of the test, each protein obtained above was added to the medium so as to be 1 μM.
After the addition, the cells were cultured for 24 hours, and then an experiment by flow cytometry was performed.

-Flow cytometry-
The cells to which the protein has been added are washed twice with PBS, suspended by trypsin treatment, dead cells are stained with propidium iodide (SIGMA), and the fluorescence intensity of the fluorescent protein of only living cells is measured by a flow cytometer ( Sony icite 800) was quantified and evaluated.

The results are shown in FIG. 6A. The ratio (%) of the fluorescence-stained cells in the cells to which the fusion protein “VP3-N4” was added is shown as a ratio of 100.
In FIG. 6A, the vertical axis indicates the added protein, and the horizontal axis indicates the fluorescence intensity.

  From the result of FIG. 6A, it was confirmed that even when “N4-9” in which the N4 site of VP3 was reduced to 17 amino acids was used, the transmission efficiency was almost equivalent to that of “N4” before reduction.

<Test Example 6-2>
-Plasmid construction-
In the same manner as in Test Example 6-1, a plasmid having a sequence encoding the following protein was constructed.
"GST protein"-"Venus protein"-"linker"-"N4-9"-"6 × His" (hereinafter sometimes referred to as "VP3-N4-9")

A plasmid having a sequence encoding a mutant peptide in which the sequence of the “N4-9” site was changed as follows was constructed by PCR. In the following sequences, the underlined portion indicates a portion mutated from the amino acid sequence “N4-9”.
· M A ATGKLLVSYAPPGAD (SEQ ID NO: 22, hereinafter sometimes referred to as "N4-9-m1")
MMA A GKLLVSYAPPGAD (SEQ ID NO: 23, hereinafter may be referred to as “N4-9-m2”)
MMAT A KLLVSYAPPGAD (SEQ ID NO: 24, hereinafter may be referred to as “N4-9-m3”)
MMATG A LLVSYAPPGAD (SEQ ID NO: 25, hereinafter may be referred to as “N4-9-m4”)
MMATGKLLVSYAPP A AD (SEQ ID NO: 26, hereinafter may be referred to as “N4-9-m5”)
MMATGKLLVSYAP A GAD (SEQ ID NO: 27, hereinafter may be referred to as “N4-9-m6”)
· M A ATGKLLVSYAPP A AD (SEQ ID NO: 28, hereinafter sometimes referred to as "N4-9-m7")
· MMATGKLLVSYAPPGA A D (SEQ ID NO: 29, hereinafter sometimes referred to as "N4-9-m8")
・ MMATGKLLVSYAPPG D (SEQ ID NO: 30, hereinafter may be referred to as “N4-9-m9”)

Oligo DNA encoding a control peptide (EFYGTAPLE (SEQ ID NO: 46)) was obtained by oligo synthesis.
A plasmid was constructed in the same manner as in Test Example 1 except that “N4-9” in the plasmid was replaced with an oligo DNA encoding the control peptide.

-Production of protein-
The following proteins were prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.
“GST protein” — “Venus protein” — “linker” — “N4-9” — “6 × His” (hereinafter sometimes referred to as “N4-9”)
“GST protein” — “Venus protein” — “linker” — “N4-9-m1” — “6 × His” (hereinafter sometimes referred to as “M1”)
“GST protein” — “Venus protein” — “linker” — “N4-9-m2” — “6 × His” (hereinafter sometimes referred to as “M2”)
“GST protein” — “Venus protein” — “linker” — “N4-9-m3” — “6 × His” (hereinafter sometimes referred to as “M3”)
“GST protein” — “Venus protein” — “linker” — “N4-9-m4” — “6 × His” (hereinafter sometimes referred to as “M4”)
“GST protein” — “Venus protein” — “linker” — “N4-9-m5” — “6 × His” (hereinafter sometimes referred to as “M5”)
“GST protein” — “Venus protein” — “linker” — “N4-9-m6” — “6 × His” (hereinafter sometimes referred to as “M6”)
“GST protein” — “Venus protein” — “linker” — “N4-9-m7” — “6 × His” (hereinafter sometimes referred to as “M7”)
“GST protein” — “Venus protein” — “linker” — “N4-9-m8” — “6 × His” (hereinafter sometimes referred to as “M8”)
“GST protein” — “Venus protein” — “linker” — “N4-9-m9” — “6 × His” (hereinafter sometimes referred to as “M9”)
“GST protein” — “Venus protein” — “linker” — “control peptide” — “6 × His” (hereinafter sometimes referred to as “E”)

-Intracellular permeability test-
The day before the test, umbilical cord blood-derived vascular endothelial cells (HUVEC) were seeded in a 96-well plate at 1 × 10 ⁴ cells per well.
On the day of the test, each protein obtained above was added to the medium so as to be 1 μM.
After the addition, the cells were cultured for 24 hours, and then an experiment by flow cytometry was performed.

-Flow cytometry-
The cells to which the protein has been added are washed twice with PBS, suspended by trypsin treatment, dead cells are stained with propidium iodide (SIGMA), and the fluorescence intensity of the fluorescent protein of only living cells is measured by a flow cytometer ( Sony icite 800) was quantified and evaluated.

The result is shown in FIG. 6B. In addition, the ratio (%) of the fluorescence-stained cells in the cells to which the fusion protein “VP3-N4-9” was added is shown as a ratio of 1.
In FIG. 6B, the vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the added protein.

  From the result of FIG. 6B, even when a mutation such as a substitution mutation, an insertion mutation, or a deletion mutation is added to the amino acid sequence of “N4-9”, the same permeability as that of the fusion protein containing “N4-9” It was confirmed to show efficiency.

(Test Example 7: Identification of responsible region of cell permeation at “N6” site of VP3)
<Test Example 7-1>
-Plasmid construction-
In the same manner as in Test Example 2-1, a plasmid having a sequence encoding the following protein was constructed.
“GST protein” — “Venus protein” — “linker” — “N6” — “6 × His” (hereinafter sometimes referred to as “VP3-N6”)

A nucleotide sequence encoding an amino acid sequence obtained by changing the sequence of the “N6” site of VP3 as follows was cloned by PCR.
YQTRIVVPLSPREMDILGFVSACNDFSVRRLRDTHIEQKALAQ (SEQ ID NO: 31, hereinafter sometimes referred to as “N6-2”)
VVPLSPREMDILGFVSACNDFSVRLLRDTHIEQKALAQ (SEQ ID NO: 32, hereinafter may be referred to as “N6-3”)
YQTRIVVPLSTPREMDILGFVSACNDFSVRRLRDTHTHIEQ (SEQ ID NO: 33, hereinafter may be referred to as “N6-4”)
YQTRIVVPLSTPREMDILGFVSACNDFSVRLLRDT (SEQ ID NO: 34, hereinafter may be referred to as “N6-5”)
VVPLSPREMDILGFVSACNDFSVRRLRDTHTHIEQ (SEQ ID NO: 35, hereinafter may be referred to as “N6-6”)
VVPLSPREMDILGFVSACNDFSVRRLRDT (SEQ ID NO: 36, hereinafter may be referred to as “N6-7”)

“N6” in the plasmid having the sequence encoding the “GST protein”-“Venus protein”-“linker”-“N6”-“6 × His” protein is referred to as “N6-2” to “N6-7” above. A plasmid having a sequence encoding the following protein was constructed instead of the base sequence encoding the amino acid sequence of
“GST protein” — “Venus protein” — “linker” — “N6-2” — “6 × His” (hereinafter sometimes referred to as “VP3-N6-2”)
"GST protein"-"Venus protein"-"linker"-"N6-3"-"6 × His" (hereinafter sometimes referred to as "VP3-N6-3")
"GST protein"-"Venus protein"-"linker"-"N6-4"-"6 × His" (hereinafter sometimes referred to as "VP3-N6-4")
"GST protein"-"Venus protein"-"linker"-"N6-5"-"6 × His" (hereinafter sometimes referred to as "VP3-N6-5")
“GST protein” — “Venus protein” — “linker” — “N6-6” — “6 × His” (hereinafter sometimes referred to as “VP3-N6-6”)
“GST protein” — “Venus protein” — “linker” — “N6-7” — “6 × His” (hereinafter sometimes referred to as “VP3-N6-7”)

-Production of protein-
Each protein was prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.

-Intracellular permeability test-
An experiment by flow cytometry was performed in the same manner as in Test Example 6-1 except that the protein in Test Example 6-1 was replaced with the above protein.
The results are shown in FIG. 7A. In addition, the ratio (%) of the fluorescence-stained cells in the cells to which the fusion protein “VP3-N6” was added is shown as a ratio of 100.
In FIG. 7A, the vertical axis represents the added protein, and the horizontal axis represents the fluorescence intensity.

  From the result of FIG. 7A, it was confirmed that even when the N6 site of VP3 was reduced to 35 amino acids, the transmission efficiency was almost the same as that of N6 before the reduction.

<Test Example 7-2>
-Plasmid construction-
In the same manner as in Test Example 2-1, a plasmid having a sequence encoding the following protein was constructed.
“GST protein” — “Venus protein” — “linker” — “N6” — “6 × His” (hereinafter sometimes referred to as “N6”)

A nucleotide sequence encoding an amino acid sequence obtained by changing the sequence of the “N6” site of VP3 as follows was cloned by PCR.
QTRIVVPLSTPREMDILGFVSACNDFSVRRLRDTHIE (SEQ ID NO: 37, hereinafter may be referred to as “N6-8”)
TRIVVPLSPREMDILGFVSACNDFSVRRLRDTTHI (SEQ ID NO: 38, hereinafter may be referred to as “N6-9”)
RIVVPLSTPREMDILGFVSACNDFSVRLLRDDTTH (SEQ ID NO: 2, hereinafter sometimes referred to as “N6-10”)
IVVPLSPREMDILGFVSACNDFSVRLLRDTT (SEQ ID NO: 39, hereinafter may be referred to as “N6-11”)

  “N6” in a plasmid having a sequence encoding the “GST protein”-“Venus protein”-“linker”-“N6”-“6 × His” protein is referred to as “N6-8” to “N6-11” above. A plasmid was constructed in which the nucleotide sequence encoding the amino acid sequence was replaced.

A plasmid having a sequence encoding a mutant peptide in which the sequence of the “N6-10” site was changed as follows was constructed by PCR. In the following sequences, the underlined portion indicates a portion mutated from the amino acid sequence “N6-10”.
RA VVPLSPREMDILGFVSACNDFSVRRLRDTH (SEQ ID NO: 40, hereinafter may be referred to as “N6-10-m1”)
RA A IVVPLSPREMDILGFVSACNDFSVRRLRDTH (SEQ ID NO: 41, hereinafter may be referred to as “N6-10-m2”)
・ R VVPLSPREMDILGFVSACNDFSVRRLRDTTH (SEQ ID NO: 42, hereinafter may be referred to as “N6-10-m3”)

  In the same manner as in Test Example 6-2, a plasmid having a sequence encoding “GST protein” — “Venus protein” — “linker” — “control peptide” — “6 × His” was constructed.

-Production of protein-
The following proteins were prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.
“GST protein” — “Venus protein” — “linker” — “N6” — “6 × His” (hereinafter sometimes referred to as “N6”)
"GST protein"-"Venus protein"-"linker"-"N4-6-8"-"6 × His" (hereinafter sometimes referred to as "N6-8")
“GST protein” — “Venus protein” — “linker” — “N4-6-9” — “6 × His” (hereinafter sometimes referred to as “N6-9”)
“GST protein” — “Venus protein” — “linker” — “N4-6-10” — “6 × His” (hereinafter sometimes referred to as “N6-10”)
"GST protein"-"Venus protein"-"linker"-"N4-6-11"-"6 × His" (hereinafter sometimes referred to as "N6-11")
"GST protein"-"Venus protein"-"linker"-"N4-6-10-m1"-"6 × His" (hereinafter sometimes referred to as "N6-10-m1")
“GST protein” — “Venus protein” — “linker” — “N4-6-10-m2” — “6 × His” (hereinafter sometimes referred to as “N6-10-m2”)
"GST protein"-"Venus protein"-"linker"-"N4-6-10-m3"-"6 × His" (hereinafter sometimes referred to as "N6-10-m3")
“GST protein” — “Venus protein” — “linker” — “control peptide” — “6 × His” (hereinafter sometimes referred to as “E”)

-Intracellular permeability test-
An experiment by flow cytometry was performed in the same manner as in Test Example 6-2 except that the protein in Test Example 6-2 was replaced with the above protein.
The result is shown in FIG. 7B. In addition, the ratio (%) of the fluorescence staining cell in the cell to which the fusion protein “N6” was added is shown as a ratio of 1.
In FIG. 7B, the vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the added protein.

From the result of FIG. 7B, it was confirmed that even when “N6-10” in which the “N6” portion of VP3 was reduced to 34 amino acids was used, the transmission efficiency was almost the same as that of “N6” before reduction.
Further, even when a mutation such as a substitution mutation, an insertion mutation, or a deletion mutation was added to the amino acid sequence of “N6-10”, it was confirmed that the transmission efficiency was equivalent to that of “N6-10”. .

(Test Example 8: Examination of coordination of “N4” peptide and “N6” peptide)
<Plasmid construction>
In the same manner as in Test Example 2-1, a base sequence encoding the following amino acid sequence was cloned.
SLKFTFLFCGSMMATGKLLVSYAPPGADPPPKKRKKEAML (SEQ ID NO: 9, hereinafter sometimes referred to as “N4”)
YISVFYQTRIVVPLSTPREMDILGFVSACNDFSVRRLRDTHIEQKALAQ (SEQ ID NO: 11, hereinafter may be referred to as “N6”)

  In the same manner as in Test Example 2-1, the base sequence encoding the Venus protein, which is a GFP analog, was cloned.

  In the same manner as in Test Example 6-2, an oligo DNA encoding a control peptide (EFYGTAPLE (SEQ ID NO: 46)) was obtained.

Each plasmid having a sequence encoding the following protein was constructed in the same manner as in Test Example 2-1, except that the PCR product in Test Example 2-1 was replaced with the cloned or oligo DNA.
The base sequence encoding the following linker (SRGSGGGGSGGGGSSG (SEQ ID NO: 44)) was inserted by PCR.
“GST protein” — “control peptide” — “linker” — “Venus protein” — “6 × His” (hereinafter sometimes referred to as “GST-peptide-Ve-E”)
“GST protein” — “N4” — “linker” — “Venus protein” — “6 × His” (hereinafter sometimes referred to as “GST-peptide-Ve-N4”)
“GST protein” — “N6” — “linker” — “Venus protein” — “6 × His” (hereinafter sometimes referred to as “GST-peptide-Ve-N6”)
“GST protein” — “Venus protein” — “linker” — “control peptide” — “6 × His” (hereinafter sometimes referred to as “GST-Ve-peptide-E”)
“GST protein” — “Venus protein” — “linker” — “N4” — “6 × His” (hereinafter sometimes referred to as “GST-Ve-peptide-N4”)
“GST protein” — “Venus protein” — “linker” — “N6” — “6 × His” (hereinafter sometimes referred to as “GST-Ve-peptide-N6”)

<Production of protein>
Each protein was prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.

<Intracellular permeability test>
The day before the test, umbilical cord blood-derived vascular endothelial cells (HUVEC) were seeded in a 96-well plate at 1 × 10 ⁴ cells per well.
On the day of the test, each protein obtained above was added to the medium so as to be 1 μM.
After the addition, the cells were cultured for 24 hours, and then an experiment by flow cytometry was performed.

-Flow cytometry-
The cells to which the protein has been added are washed twice with PBS, suspended by trypsin treatment, and dead cells are stained with propidium iodide (SIGMA). Quantification and evaluation were performed with a meter (Sony icite 800).

The results are shown in FIG.
In FIG. 8, the vertical axis represents the fusion protein uptake ratio, and the horizontal axis represents the added fusion protein. In addition, the item of “-” on the horizontal axis indicates the result when the fusion protein is not added.

From the results shown in FIG. 8, it was confirmed that the “N4” peptide and the “N6” peptide showed equivalent cell permeability when inserted at the N-terminal side of the Venus protein and when inserted at the C-terminal side. It was.
Therefore, it was considered that the peptide of the present invention has no insertion site dependency.

(Test Example 9: Examination of blood-brain barrier permeability of “N4” peptide and “N6” peptide)
<Plasmid construction>
In the same manner as in Test Examples 2-1 and 6-2, a plasmid having a sequence encoding the following protein was constructed.
“GST protein” — “Venus protein” — “linker” — “N4” — “6 × His” (hereinafter sometimes referred to as “VP3-N4”)
“GST protein” — “Venus protein” — “linker” — “N6” — “6 × His” (hereinafter sometimes referred to as “VP3-N6”)
“GST protein” — “Venus protein” — “linker” — “control peptide” — “6 × His” (hereinafter sometimes referred to as “E”)

<Production of protein>
Each protein was prepared in the same manner as in Test Example 1 except that the plasmid in Test Example 1 was replaced with the above plasmid.

<Blood-brain barrier permeability test>
Each protein was added to a commercially available rat in vitro blood brain barrier (BBB) model kit (Pharmacol) at a concentration of 10 μg / well, and the permeability of the protein to rat vascular endothelial cells on the blood vessel side over time was added. And the permeability of the protein to the brain side.
The protein permeability to the blood vessel side rat vascular endothelial cells was measured with a flow cytometer.
The amount of the protein transferred to the brain side was measured by detecting the His protein connected to the C-terminal side of the added protein by the ELISA method (His Tag ELISA Detection Kit (GenScript Corp.)).

The result of measuring the protein permeability to the vascular endothelial cells is shown in FIG. 9A, and the result of measuring the protein permeability to the brain side is shown in FIG. 9B.
9A and 9B, “black circle” indicates the result when “VP3-N4” is added, “black square” indicates the result when “VP3-N6” is added, and “black triangle” indicates “E” ”Is added to the result.

  From the results of FIGS. 9A and 9B, “VP3-N4” and “VP3-N6” were observed to permeate vascular endothelial cells 3 hours after addition, and also permeated to the brain side. Among these, “VP3-N4” was confirmed to have higher permeability in blood-brain barrier permeation.

Examples of the aspect of the present invention include the following.
<1> (i) an amino acid sequence represented by the following SEQ ID NO: 1, and (ii) at least 80% sequence identity to the amino acid sequence represented by the following SEQ ID NO: 1, and a long A peptide comprising any one of the amino acid sequences having a length of at least 16 amino acid residues.
MMATGKLLVSYAPPGAD (SEQ ID NO: 1)
<2> (iii) the amino acid sequence represented by SEQ ID NO: 2 below, and (iv) at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 below, and long A peptide comprising any one of the amino acid sequences having a length of at least 33 amino acid residues.
RIVVPLSTPREMDILGFVSACNDFSVRRLRDTH (SEQ ID NO: 2)
<3> The peptide according to any one of <1> to <2>, which permeates the blood brain barrier.
<4> The peptide according to any one of <1> to <2>, which permeates vascular endothelial cells.
<5> A drug transporter comprising the peptide according to any one of <1> to <4>.
<6> The drug transporter according to <5>, further including a carrier.
<7> The drug transporter according to <6>, wherein the carrier is at least one selected from macromolecules, microassemblies, microparticles, microspheres, nanospheres, liposomes, and emulsions.
<8> The drug transporter according to any one of <6> to <7>, wherein a drug for performing at least one of diagnosis, prevention, and treatment is carried inside the carrier.
<9> Drugs for performing at least one of diagnosis, prevention, and treatment are nucleic acids, polynucleotides, genes, and analogs thereof; glycosaminoglycans and derivatives thereof; oligosaccharides, polysaccharides, and derivatives thereof Proteins and peptides; anti-neurologic agents; antiviral agents; anti-cancer agents; antibiotics; enzyme agents; antioxidant agents; anti-inflammatory agents; steroid agents; angiotensin converting enzyme inhibitors; vasodilators; Migration inhibitor; platelet aggregation inhibitor; anticoagulant; chemical mediator release inhibitor; immunosuppressant; lipid uptake inhibitor; hormone agent; angiotensin receptor antagonist; vascular endothelial cell proliferation or inhibitor; aldose reductase Inhibitor; mesangial cell growth inhibitor; lipoxygenase inhibitor; immunostimulant; Maillard reaction inhibitor; The drug transporter according to <8>, which is at least one selected from the group consisting of a dosis inhibitor; a nitric oxide synthase inhibitor; a terminal glycation product inhibitor; and a radical scavenger.
<10> The drug transporter according to any one of <5> to <9>, wherein the drug transporter is transported into a cell by endocytosis.
<11> The drug transporter according to <10>, wherein the cell is a vascular endothelial cell.
<12> The drug transporter according to any one of <5> to <11>, which is used for oral administration.
<13> A blood-brain barrier permeation agent comprising the peptide according to any one of <1> to <4>.
<14> A vascular endothelial cell permeation agent comprising the peptide according to any one of <1> to <4>.

  The peptide of the present invention can transport a drug in a cell-specific manner by being applied to an outer membrane such as a liposome. Therefore, for example, it can be suitably used as a drug transporter that transports a drug to cells of the central nervous system by permeating the blood brain barrier. Furthermore, since the peptide of the present invention can transport a drug specifically to vascular endothelial cells, it can be suitably used as a drug transporter for diseases in which blood vessels such as diabetes and cancer are problematic.

Claims (8)

(I) the amino acid sequence represented by SEQ ID NO: 1 below, and (ii) at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 1 below, and having a length of at least A peptide comprising any one of the amino acid sequences of 16 amino acid residues.
MMATGKLLVSYAPPGAD (SEQ ID NO: 1) (Iii) the amino acid sequence represented by SEQ ID NO: 2 below, and (iv) at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 below, and having a length of at least A peptide comprising any one of the amino acid sequences of 33 amino acid residues.
RIVVPLSTPREMDILGFVSACNDFSVRRLRDTH (SEQ ID NO: 2)   A drug transporter comprising the peptide according to claim 1.   The drug transporter according to claim 3, further comprising a carrier.   The drug transporter according to claim 4, wherein the carrier is at least one selected from macromolecules, microassemblies, microparticles, microspheres, nanospheres, liposomes, and emulsions.   The drug transporter according to any one of claims 4 to 5, wherein a drug for performing at least one of diagnosis, prevention, and treatment is carried inside the carrier.   A blood brain barrier permeation agent comprising the peptide according to claim 1.   A vascular endothelial cell permeation agent comprising the peptide according to claim 1.