WO2016148213A1 - Blood-brain barrier permeable peptide - Google Patents

Abstract

Disclosed are: any one polypeptide selected from (a) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO: 1, (b) a polypeptide which comprises an amino acid sequence produced by deleting, substituting, inserting and/or adding 1 to 6 amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 and which has a brain-penetrating activity and (c) a polypeptide which comprises an amino acid sequence produced by adding 1 to 5 arbitrary amino acid residues to the C-terminal side and/or the N-terminal side of the polypeptide (a) or (b) and which has a brain-penetrating activity; a brain delivery carrier molecule comprising the polypeptide; a complex comprising the polypeptide and a protein, polypeptide, oligopeptide, low-molecular-weight compound or nucleic acid bonded to the polypeptide; a pharmaceutical composition containing the complex; and a method for preventing and/or treating a brain disease and a method for diagnosing a brain disease, in each of which the complex is used.

Description

Blood brain barrier permeability peptide

The present invention relates to a polypeptide having blood-brain barrier (BBB) permeability and brain translocation activity, and a carrier molecule for delivery in the brain containing the polypeptide. Furthermore, the present invention relates to a complex containing the above polypeptide and a pharmaceutical composition comprising the complex. The present invention also relates to a method for preventing and / or treating a brain disease using the complex, and a method for diagnosing a brain disease.

It is known that water-soluble drugs and proteins administered to peripheral tissues hardly migrate into the brain. This is because the BBB functions as a physical and physiological barrier that separates the brain from peripheral tissues and strictly restricts the passive diffusion of substances into the brain. In fact, almost all substances except small molecule compounds with high fat solubility are very difficult to move into the brain via the bloodstream. Therefore, in order to deliver a target substance administered to peripheral tissues into the brain, it is essential to develop a carrier molecule that efficiently permeates the BBB. With the advent of an aging society, the number of patients with central nervous system diseases such as Alzheimer's disease and Parkinson's disease is expected to increase significantly, but many of these diseases are intractable diseases that still lack effective treatment. From the viewpoint of developing therapeutic agents for central nervous system diseases, there is a need for the development of carrier molecules for intracerebral delivery that penetrate the BBB.

Tat peptide has been reported in the past as a peptide carrier molecule for delivering substances that do not migrate into the brain such as proteins and water-soluble drugs into the brain. Tat peptide is a peptide that is widely used as an intracellular delivery molecule such as protein, as a kind of cell translocation peptide (Protein Transduction Domain, PTDs; Cell-Penetrating Peptides, CPPs). It has been reported that this also moves into the brain. For example, Schwarze et al. Reported that β-Gal activity was confirmed in many organs including the brain when a mouse was injected subcutaneously with a protein fused with Tat peptide and β-galactosidase (Tat-β-galactosidase). (Science 285, 1569-1572 (1999)). Also, Cao et al. Reported that neuronal cell death in the brain was suppressed when the apoptosis inhibitor Bcl-xL was fused with a Tat peptide and injected subcutaneously into cerebral ischemia model mice (J. Neurosci. 22, 5423-5431 (2002).).

In recent years, several groups have been re-evaluated regarding the ability of the Tat peptide to enter the brain. In the first place, almost all cases of introducing proteins using the Tat peptide into the brain were performed using a model in which partial damage to the cerebral vascular endothelial cells constituting the BBB cannot be ruled out, which is a model animal of cerebral ischemia or stroke. It does not show BBB permeability in a strict sense. In connection with this, Simon et al. Examined the vascular endothelial cell layer to evaluate the BBB permeability of the Tat peptide, and found that it did not penetrate at all. It is pointed out that the delivery method is not generally effective (Ann. Biomed. Eng. 39, 394-401 (2010)).

In addition, Cai et al. Conducted a follow-up experiment using Tat-β-galactosidase and found that β-Gal activity in the brain was hardly detectable in any of subcutaneous injection, intravenous injection and oral administration. Reported with the author responsible for the article published in the above Science journal (Eur. J. Pharm. Sci. 27, 311-319 (2006)). At present, no BBB-penetrating peptide has been reported that has been demonstrated to reliably enter the brain, including Tat peptide, in model animals with normal BBB.

Non-Patent Documents 1-3 report an MPG peptide (Ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-Cya (Ac: acetyl, Cya: cysteamide)), which is a peptide carrier molecule for efficiently delivering oligonucleotides to cultured cells. . Since the MPG and the oligonucleotide are bound by electrostatic interaction, and since a plurality of MPG molecules are bound to coat the oligonucleotide, the oligonucleotide is stabilized and protected from degradation.

Non-Patent Document 4 reports on MPG-8 with improved MPG (βAla-FLGWLGAWGTMGWSPKKKRK-Cya), which forms nanoparticles with siRNA and efficiently delivers siRNA in cultured cells and in vivo. It is described to promote.

However, Non-Patent Documents 1-4 do not mention the brain transferability of MPG and MPG-8, and there is no clear data indicating the brain transferability.

Therefore, an object of the present invention is to provide a polypeptide having high BBB permeability and brain translocation activity, and a carrier molecule for intracerebral delivery containing the polypeptide. Furthermore, an object of this invention is to provide the composite_body | complex containing the said polypeptide, and the pharmaceutical composition containing this composite_body | complex. Another object of the present invention is to provide a method for preventing and / or treating a brain disease using the complex, and a method for diagnosing a brain disease.

As a result of intensive studies to achieve the above object, the present inventors have found that mMPG8 ((βAla) -FLGWLGAWGTMGWSPKKKRK-CONH ₂ ) has high BBB permeability and brain migration, and mMPG8 is shared The present inventors have found that fluorescein, which is a small molecule water-soluble drug model bound by binding, can be rapidly and efficiently transferred into the brain.

The present invention has been completed based on these findings, and has been completed. The following polypeptide, carrier molecule for delivery in the brain, complex, pharmaceutical composition, prevention and / or treatment method, and diagnosis method are as follows. Is to provide.

Item 1. The following polypeptide (a), (b) or (c):
(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1
(b) a polypeptide comprising an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1 and having brain translocation activity
(c) It consists of an amino acid sequence in which any one to 5 amino acids are added to the C-terminal side and / or N-terminal side of the polypeptide shown in (a) or (b), and has a brain transition activity Polypeptide.
Item 2. The following polypeptide (d), (e) or (f):
(d) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2
(e) a polypeptide comprising an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having a brain transition activity
(f) It consists of an amino acid sequence in which 1 to 5 arbitrary amino acids are added to the C-terminal side and / or the N-terminal side of the polypeptide shown in (d) or (e), and has brain translocation activity Polypeptide.
Item 3. Item 3. A carrier molecule for intracerebral delivery comprising the polypeptide according to Item 1 or 2.
Item 4. Item 3. A complex comprising the polypeptide according to item 1 or 2, and a protein, polypeptide, oligopeptide, low molecular compound, or nucleic acid bound thereto.
Item 5. Item 5. A pharmaceutical composition comprising the complex according to Item 4.
Item 6. Item 6. The pharmaceutical composition according to Item 5, which is used for prevention and / or treatment of brain disease.
Item 7. Item 6. The pharmaceutical composition according to Item 5, which is used for diagnosis of a brain disease.
Item 8. Item 5. A method for preventing and / or treating a brain disease, comprising a step of administering an effective amount of the complex according to Item 4 to a patient.
Item 9. A method for diagnosing a brain disease, comprising a step of administering an effective amount of the complex according to Item 4 to a patient.
Item 10. Item 5. Use of the complex according to Item 4 in the manufacture of a pharmaceutical composition for prevention and / or treatment of brain disease.
Item 11. Item 5. Use of the complex of Item 4 in the manufacture of a pharmaceutical composition for diagnosis of brain disease.

The polypeptide of the present invention has high BBB permeability and brain transportability, and efficiently binds proteins, peptides, low-molecular compounds, etc. that do not inherently enter the brain by binding them to the polypeptide of the present invention. It can be transferred into the brain.

It is a confocal microscope picture of mouse neuroblastoma Neuro2A cells to which each labeled peptide was administered. Upper: Fluorescein, Lower: Merged with Hoechst staining 2 is a graph showing the amount of intracellular migration of each labeled peptide when administered to rat-derived cerebral vascular endothelial cells (showing an average value of results obtained three times; hereinafter referred to as n = 3). It is a graph which shows the evaluation result of BBB permeability | transmittance of each labeled peptide by an in-vitro BBB reconstruction system (n = 3). It is a graph which shows the transendothelial electrical resistance value (TEER) of each labeled peptide in an in vitro BBB reconstruction system (n = 3). It is a graph which shows the time-dependent change of the concentration ratio in brain / perfusate concentration of mMPG8-Fl, Na-F and pVEC-Fl (n = 3-5). It is a graph which shows the density | concentration ratio in brain / perfusate concentration (after 1 minute of perfusion start) of mMPG8-Fl and FITC-Alb (n = 4-6). It is a graph showing the concentration ratio in the brain / perfusate (1 minute after the start of perfusion) when the administration concentration of mMPG8-Fl is 10 μg / mL, 50 μg / mL, 100 μg / mL (n = 4-5) . It is a graph showing the concentration ratio of mMPG8-Fl in the brain / perfusate (1 minute after the start of perfusion) when unlabeled mMPG8 is not administered simultaneously and unlabeled mMPG8 200 μg / mL and 500 μg / mL are administered simultaneously (n = 5-6). It is a graph showing cell viability after 1 hour, 6 hours, and 24 hours after administration of mMPG8 and pVEC (0 μM, 5 μM, 10 μM, 30 μM) to mouse brain vascular endothelial cell line MBEC4 (n = 3) .

Hereinafter, the present invention will be described in detail.

In the present specification, “comprise” includes the meaning of “essentially consist of” and the meaning of “consistently”.

Polypeptide The polypeptide of the present invention is any of the following (a), (b) or (c).
(a) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 (FLGWLGAWGTMGWSPKKKRK)
(b) a polypeptide comprising an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1 and having brain translocation activity
(c) It consists of an amino acid sequence in which any one to 5 amino acids are added to the C-terminal side and / or N-terminal side of the polypeptide shown in (a) or (b), and has a brain transition activity Polypeptide.

Hereinafter, in the present specification, the term “polypeptide of the present invention” means the above-mentioned polypeptide (a), (b) or (c).

In the present invention, “brain transfer activity” means an activity of transferring a molecule such as a polypeptide into the brain tissue when the molecule is administered into the body by intravenous administration or the like.

The polypeptide of the present invention has high BBB permeability and brain migration activity. While not wishing to be bound by any theory, as shown in the examples below, BBB permeation by the polypeptides of the present invention involves an uptake mechanism via some transport carrier present in BBB. Presumed not to do. In addition, the polypeptide of the present invention can transfer other molecules into the brain by binding to the other molecules.

The animal that can be a target for which the polypeptide of the present invention exhibits brain translocation activity is not particularly limited as long as it is an animal having BBB. Among the animals, mammals are preferred, and examples of mammals include rats, mice, rabbits, dogs, cats, goats, cows, monkeys, humans, and the like.

The polypeptide of the present invention includes a salt thereof. Here, the “salt” is any pharmacologically acceptable salt of a polypeptide, for example, sodium salt, potassium salt, calcium salt, hydrochloride, sulfate, nitrate, magnesium salt, ammonium of the polypeptide. Salt, phosphate, organic acid salt (acetate, trifluoroacetate, citrate, maleate, oxalate, malate, lactate, succinate, propionate, fumarate, formic acid Salt, picrate, benzoate, benzenesulfonate, etc.).

The polypeptide of the present invention also includes derivatives thereof. Here, the “derivative” refers to a derivative obtained by modifying the functional group of the polypeptide of the present invention by modification, addition, mutation, substitution, deletion or the like by a known method. For example, the N-terminal, C-terminal, or amino acid side chain of the polypeptide of the present invention is modified with a protecting group. Examples of the derivatives include acetylation, palmitoylation, amidation, myristylation, dansylation, acrylation, biotinylation, phosphorylation, anilide, succinylation, benzyloxycarbonylation, formylation, nitration, sulfonation, Aldehydation, glycosylation, cyclization, monomethylation, dimethylation, trimethylation, guanidylation, maleylation, trifluoroacetylation, trinitrophenylation, carbamylation, polyethylene glycolation, acetoacetylation, labeling (e.g. PET radionuclides, fluorescent dyes, etc.). Of these, acetylation at the N-terminus and amidation at the C-terminus are preferred because they impart resistance to exopeptidases that degrade the polypeptide from the terminus.

However, the polypeptide of the present invention does not include those in which the C-terminus is modified with cysteamide. From this point, the polypeptide of the present invention is different from MPG-8 described in Non-Patent Document 4 in which the C-terminus is modified with cysteamide.

The amino acid constituting the polypeptide of the present invention may be either L-form or D-form. Moreover, the amino acid which comprises the polypeptide of this invention is not limited to a natural amino acid, A non-natural amino acid may be sufficient.

In the polypeptide (b), the number of amino acids to be deleted, substituted, inserted and / or added is preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, particularly preferably Preferably 1 or 2, more particularly preferably 1. When an amino acid is substituted, it is considered that the activity of the original polypeptide is easily maintained if the amino acid is substituted with an amino acid having similar properties.

In the polypeptide (c), the number of arbitrary amino acids added to the C-terminal side and / or the N-terminal side is preferably 1 to 4, more preferably 1 to 3, and still more preferably 1 or 2. One, particularly preferably one.

Arbitrary amino acids are not particularly limited. Of the amino acids, cysteine is preferred. The addition of a cysteine residue is desirable because the compound can be bound to the polypeptide of the present invention using the SH group of cysteine. When cysteine is added, cysteine can be added in a state where other amino acids (for example, glycine) are interposed, instead of directly adding cysteine.

A technique for deleting, substituting, inserting and / or adding one or more amino acids in a specific amino acid sequence is known.

The polypeptide of the present invention can be produced by using a known synthesis method such as solid phase synthesis or liquid phase synthesis, or by culturing a transformant introduced with a gene encoding the polypeptide. . Examples of the host for producing the transformant include Escherichia coli, yeast, mammalian cells, plant cells, and insect cells.

The purified polypeptide can be purified by affinity chromatography, ion exchange chromatography, hydroxyapatite column chromatography, ammonium sulfate salting-out method or the like.

As shown in Examples described later, in addition to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (RQIKIWFQNRRMKWKK) is also highly BBB permeable. Indicates. Therefore, in the above description of the polypeptide of the present invention, SEQ ID NO: 1 can be replaced with SEQ ID NO: 2.

Carrier molecule for intracerebral delivery The carrier molecule for intracerebral delivery of the present invention is characterized by comprising the polypeptide of (a), (b) or (c) above.

Since the polypeptide of (a), (b) or (c) has high BBB permeability and brain translocation activity, the polypeptide of the present invention delivers other molecules and substances into the brain. Can be used as a carrier molecule. For example, by binding the carrier molecule of the present invention to other molecules and substances, it is possible to transfer other molecules and substances into the brain.

It can also be used as a carrier molecule for delivery in the brain by binding liposomes, micelles or microcapsules to the polypeptide of the present invention. In this case, these can be transferred into the brain by encapsulating target molecules and substances in liposomes, micelles or microcapsules.

Complex The complex of the present invention comprises the above-mentioned polypeptide (a), (b) or (c), and a protein, polypeptide, oligopeptide, low molecular compound, or nucleic acid (hereinafter referred to as `` compound A '') bound thereto. It is characterized by containing.

Since the polypeptide of the present invention has brain migration activity, the compound A to be bound to the polypeptide of the present invention is imparted with brain migration activity. The polypeptide of the present invention has high BBB permeability and brain transportability, and it is possible to efficiently transport Compound A, which does not naturally travel into the brain, into the brain.

Examples of compound A include compounds that are desirably transferred to brain tissue for the prevention or treatment of brain diseases. By binding Compound A to the polypeptide of the present invention, it is expected to efficiently migrate to brain tissue and exert a therapeutic effect.

Examples of the protein include protein drugs such as antibodies, antibody fragments, antagonists and agonists, and anticancer agents.

Examples of the nucleic acid include plasmids, siRNA related to disease-related genes, and antisense DNA.

The size of the compound A is not particularly limited, and usually the upper limit is a size that can physically pass through the BBB.

The polypeptide of the present invention and compound A can be appropriately combined using a known method.

When the polypeptide of the present invention has a cysteine residue, the cysteine residue of the polypeptide of the present invention and compound A can be bound via an -SS- bond, and bound via an appropriate crosslinking agent. It can also be made. In addition, by a conventional method such as introducing a DNA ligated with the DNA of the present invention and a compound A (protein, polypeptide or oligopeptide) into a vector and expressing it in a host cell such as E. coli. A complex can also be obtained.

The cross-linking agent is not particularly limited as long as it is an at least divalent cross-linking agent capable of binding the polypeptide of the present invention and compound A. For example, N- (6-maleimidocaproyloxy) succinimide ester (EMCS) and the like Is mentioned.

Pharmaceutical composition The pharmaceutical composition of the present invention is characterized by comprising the above complex.

When preparing as a pharmaceutical composition, the above complex is used as it is, or together with a non-toxic carrier, diluent or excipient that is acceptable in pharmaceuticals, tablets (plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets). , Capsules, pills, powders (powder), fine granules, granules, solutions, suspensions, emulsions, syrups, pastes, injections (when used, It is possible to prepare a pharmaceutical preparation by preparing it in a form such as distilled water, amino acid infusion, electrolyte infusion, etc.

The content of the above complex in the pharmaceutical composition of the present invention may be appropriately selected from the range of 0.0001 to 100% by weight, preferably 0.001 to 99.9% by weight, more preferably 0.01 to 99% by weight, based on the total amount of the pharmaceutical composition. Is possible.

The administration method of the pharmaceutical composition of the present invention is not particularly limited, and can be performed by, for example, intraarterial administration, intravenous administration, buccal administration, rectal administration, enteral administration, transdermal administration, oral administration, and the like.

The pharmaceutical composition of the present invention is administered to mammals including humans.

The dosage of the pharmaceutical composition of the present invention can be appropriately determined according to various conditions such as the patient's weight, age, sex, and symptoms.

The pharmaceutical composition of the present invention is useful for the prevention and / or treatment of brain diseases. The pharmaceutical composition of the present invention is also useful for diagnosing brain diseases.

The brain diseases in the present invention include, for example, brain tumors, metastatic brain tumors, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, cerebrovascular disorders such as stroke, brain infections such as moyamoya disease, encephalitis and brain abscess, Multiple sclerosis, Down's syndrome, frontotemporal dementia, Pick's disease, progressive supranuclear palsy (PSP), prion disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, multisystem atrophy, metabolic Examples include brain disorders, headaches, brain development disorders such as autism, and manic depression.

Since the polypeptide of the present invention has a brain transition activity, the compound having a preventive and / or therapeutic effect on brain disease bound to the polypeptide is efficiently transferred to brain tissue, and thus brain disease can be prevented and / or Expected to exert therapeutic effects.

In addition, by labeling the polypeptide of the present invention with a PET radionuclide, a fluorescent dye, etc., and then administering a complex bound with an antibody that specifically binds to a causative agent of brain disease, into the body, It is considered that the complex can be efficiently transferred to the brain tissue and the diagnosis of the brain disease becomes possible.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Production Examples Table 1 shows the amino acid sequences of the peptides used in the experiments in this study. Each peptide has a Gly-Cys sequence for introducing fluorescein on the carboxy terminus (C terminus) side. The amino terminal (N terminal) is acetylated and the C terminal is an amide. However, as an exception, C6R8 and mMPG8 each have hexanoic acid or β-alanine at the N-terminal instead of an acetyl group.

All peptides were manually synthesized by the Fmoc solid phase synthesis method. For peptide synthesis, solid phase carrier (Rink Amide Resin) manufactured by Novabiochem, Fmoc amino acid and condensing agent (1-hydroxybenzotriazole) manufactured by Peptide Laboratories were used, and diisopropylethylamine, acetic anhydride, and dimethylformamide were combined. A product made by Kojun Pharmaceutical Co., Ltd. was used. The protected peptide synthesized on the solid support was deprotected with trifluoroacetic acid (Watanabe Chemical Co., Ltd.) containing 5% (v / v) of ethanedithiol (Wako Pure Chemical Industries, Ltd.). The peptide after deprotection was purified as a main product by reversed-phase HPLC (JASCO HPLC was connected with Nacalai Tesque column 5C4AR-300). The purified peptide was confirmed for molecular weight by electrospray ionization mass spectrometry (ESI-MS, LCQ Deca XP from Thermo-Fischer Scientific) (this is referred to as unlabeled peptide).

Next, a 1: 1 mixed solvent containing unlabeled peptide and 5- (iodoacetamido) fluorescein (Sigma Aldrich), dimethylformamide / methanol (0.1% (v / v) N-methylmorpholine), both The peptide in which fluorescein was introduced into the C-terminal Cys side chain was synthesized by reacting for 1.5 hours in Kojunkaku Kogyo Co., Ltd. (this is referred to as a labeled peptide). All of the labeled peptides were purified as main products by reverse phase HPLC, and the molecular weight was confirmed by ESI-MS. Table 2 shows the measured molecular weights and the theoretical values as physical properties by ESI-MS.

Test Example 1: Primary screening It was examined how efficiently the peptide (1)-(13) shown in Table 1 can deliver a substance that does not originally migrate into the cell. Fluorescein, a fluorescent dye, was used as a water-soluble model substance that does not inherently migrate into cells. Two days before the experiment, Dulbecco's modified Eagle's medium (DMEM, Gibco) was used for mouse neuroblastoma Neuro2A cells seeded in a 4-well chamber slide (Lab-Tek II chamber slide, Nunc) at 50,000 cells / well. After the labeled peptide (1)-(13) diluted to 10 μM was administered and cultured for 1 hour, the cell nucleus was stained with Hoechst33342 (Molecular Probes) and then observed with a confocal microscope (Olympus FV1000). Performed (FIG. 1).

As a result, when a labeled peptide other than SAP (8) was administered, remarkable fluorescein fluorescence was observed in the cells. On the other hand, almost no fluorescence signal was observed in the cells administered with SAP (8). This suggested that at least peptides other than SAP (8) have the ability to deliver fluorescein into cells.

Next, 10 μM labeled peptide (1)-(13) was administered to rat cerebral vascular endothelial cells for 1 hour, and the fluorescence intensity of fluorescein in the cells was measured. From this measured value, the intracellular migration amount of the labeled peptide was calculated (FIG. 2). In this test example, fluorescein sodium salt (Na-F), which is a compound that hardly shows intracellular migration, was used as a negative control.

Three days before the experiment, primary cultured rat brain capillary endothelial cells were seeded at 15,000 cells / well in 96 で well culture plate 、 (Iwaki), and 10% Plasma derived serum (Animal) in DMEM / F12 (Wako Pure Chemical Industries, Ltd.) technology), bFGF® (1.5 ng / mL; Wako Pure Chemical Industries, Ltd.), heparin® (100 μg / mL; Sigma Aldrich), and ITS supplement® (Sigma Aldrich). Labeled peptides (1)-(13) and Na-F diluted to 10 μM in the culture solution were administered and cultured for 1 hour. The culture solution was removed, and the cells were lysed with 0.2N NaOH after washing 3 times with phosphate buffer. Fluorescence intensity of the cell lysate was measured with a fluorescence plate reader (Wallac 1420 ARVO Multilabel Counter, Perkin Elmer) (Ex: 485 nm, Em: 535 nm). Concentration was calculated.

As a result of the experiment, high intracellular migration was confirmed in Penetratin (2), pVEC (4), mMPG (8) and the like. On the other hand, almost no fluorescein fluorescence was detected in the cells to which SAP (8) was administered, and the intracellular transfer amount was almost the same as that of Na-F. From this, it was suggested that the intracellular delivery capability of each peptide is greatly different.

Based on the above studies, six types of peptides (Tat (1), Penetratin (2), pVEC (4), Rev (7), C6R8 (10), mMPG8 (13)), which are suggested to have high intracellular delivery The following examination was advanced.

Test Example 2: BBB permeability was evaluated for the six peptides selected in Secondary Screening Test Example 1. BBB permeability was evaluated using an in vitro BBB reconstitution system (BBB kit, Cell. Mol. Neurobiol. 27, 687-694 (2007).) Developed by Nakagawa et al. In this test example, fluorescein-labeled dextran (FD4), which is a compound having almost the same molecular weight as that of the labeled peptide (approximately 4000) and hardly showing BBB permeability, and a peptide that showed almost no intracellular delivery in Test Example 1 were used. A SAP (8) was used as a negative control. 1 μM labeled peptide was administered to the blood vessel side of the BBB kit, and the fluorescein fluorescence intensity in the brain side culture solution was measured over time. The fluorescence intensity was measured with a fluorescence plate reader, and the concentration of the labeled peptide in the brain side culture solution was calculated from the measured value (FIG. 3).

As a result, 5 types of peptides (Tat (1), Penetratin (2), Rev (7), C6R8 (10), mMPG8 (13)) except for pVEC (4) were compared with FD4 and SAP (8). And was detected at a significantly higher concentration in the brain-side culture medium. On the other hand, pVEC (4) was hardly detected in the brain side culture solution, and its concentration was similar to FD4 and SAP (8).

At the same time, transendothelial electrical resistance (TEER), which is an index of cell layer tightness, was measured (FIG. 4). TEER was measured using an electrical resistance measuring instrument (EVOM, World Precision Instruments) and a cup-type electrode (ENDOHM, WPI). The electric resistance value obtained from the BBB kit was subtracted from the electric resistance value of only the insert membrane on which cells were not seeded, and was calculated by multiplying the culture area of the insert membrane. As a result, there was no difference in the change of TEER value in any peptide and FD4, and no significant decrease was confirmed.

From these results, the above five peptides have the ability to permeate the BBB and deliver fluorescein from the blood vessel side to the brain side, and this delivery may involve a decrease in the barrier function of the BBB cell layer Was suggested to be low. Of these, three peptides (Penetratin (2), C6R8 (10), and mMPG8 (13)) are higher than Tat (1), which some researchers have suggested in the brain BBB permeability was shown.

Test Example 3: In Vivo Experiment In Test Example 2, mMPG8 (13), which showed the highest BBB permeability, was evaluated for its ability to enter the brain using an animal model. In this test example, Na-F, which is a compound that hardly shows the ability to enter the brain, and pVEC (4), which is a peptide that hardly shows BBB permeability in Test example 2, were used as negative controls.

A labeled peptide (100 μg / ml) was perfused from the left ventricle of a male ICR mouse (8 weeks old) at a flow rate of 2 ml / min, and the brain was collected 0.5, 1, 1.5, and 2 minutes later. The collected brain was weighed and then homogenized by adding 2 volumes of 0.5M aqueous boric acid (pH 10.0). After centrifugation at 1000 g for 15 minutes, 2 volumes of 99.5% ethanol was further added and stirred. After centrifugation at 15,000 g for 15 minutes, the fluorescence intensity in the supernatant was measured with a fluorescence plate reader (CytoFluor, Perspective Biosystems), and the brain concentration was determined from the calibration curve. The labeled peptide concentration in the perfusate was also determined in the same manner, and the amount of transferred brain was calculated as the brain concentration / perfusate concentration ratio over time. The brain concentration / perfusate concentration ratio was plotted on the vertical axis and the perfusion time on the horizontal axis (FIG. 5), and the slope was calculated as the permeation clearance from the blood side to the brain parenchyma side (Table 3).

As a result, mMPG8 (13) was detected in the brain at a significantly higher concentration than Na-F and pVEC (4), and the permeation clearance also showed a significantly higher value. Compared with the result of perfusion of albumin fluorescein-labeled protein (FITC-Alb, Sigma-Aldrich), which is a protein that hardly passes through the normal BBB, under the same conditions, the amount of brain migration was approximately 18 times. (FIG. 6). From these results, it was suggested that mMPG8 (13) has a high ability to migrate into the brain.

Next, the possibility that some transport carrier existing in BBB is involved in the migration of mMPG8 (13) into the brain was examined. In general, when membrane transport of a substance depends on the transport carrier, if an excessive amount of the substance is administered, the transport efficiency decreases due to carrier saturation. Therefore, perfusion experiments similar to those described above were performed at different administration concentrations, and changes in the amount of brain transfer were examined (FIG. 7). As a result, the amount of mMPG8 (13) transferred to the brain increased depending on the administration concentration.

In addition, an excessive amount of unlabeled mMPG8 (13) was administered together with a certain amount of labeled mMPG8 (13), and the change in the amount of labeled body transferred to the brain was examined (FIG. 8). As a result, the amount of labeled substance transferred to the brain markedly increased as the concentration of unlabeled substance increased.

In all experiments, there was no decrease in transport efficiency with increasing dose concentration, so mMPG8 (13) translocation into the brain was not an uptake mechanism via any transport carrier present in the BBB. This suggests that other unknown mechanisms may be involved.

Test Example 4: Toxicity Test The cytotoxicity of mMPG8 (13) under the test conditions of Test Examples 1 to 3 was evaluated. In this test example, pVEC (4), which is a peptide that showed high intracellular transferability in Test Example 1 but hardly showed BBB permeability and brain transfer in Test Example 2 and Test Example 3, was negative. Used as a control. To the mouse brain vascular endothelial cell line MBEC4 seeded in a 96-well plate (Iwaki) at 5,000 cells / well two days before the experiment, an unlabeled peptide (5 μM, 10 μM, or 30 μM) diluted with DMEM was administered, Cultured for 1 hour, 6 hours, or 24 hours. After culture, cell proliferation reagent WST-1 (Roche) was added, and after 1 hour, the absorbance at 450 nm was measured with an absorption microplate reader (Multiskan FC, Thermo Fisher Scientific) to evaluate the cell viability (FIG. 9). ).

As a result, in mMPG8 (13), a slight decrease in cell viability was observed only when administered at a concentration of 30 μM (corresponding to approximately 100 μg / ml) for 24 hours (viability 87.0 ± 7.8%). No decrease in cell viability was observed under other conditions. On the other hand, when pVEC (4) was administered at a concentration of 10 μM or 30 μM for 24 hours, a decrease in cell viability was observed (94.9 ± 2.1% or 68.1 ± 3.2%, respectively). From these results, it is considered that pVEC (4) has higher cytotoxicity although cytotoxicity is extremely low in any peptide.

From the above, under the experimental conditions (administration concentration or administration time) in Test Examples 1 to 3, mMPG8 (13) has almost no cytotoxicity, and BMP permeability and translocation into the brain of mMPG8 (13). This suggests that it is unlikely that cytotoxicity is involved.

Claims (10)

1. The following polypeptide (a), (b) or (c):
   (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1
   (b) a polypeptide comprising an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1 and having brain translocation activity
   (c) It consists of an amino acid sequence in which any one to 5 amino acids are added to the C-terminal side and / or N-terminal side of the polypeptide shown in (a) or (b), and has a brain transition activity Polypeptide.
2. A carrier molecule for delivery in the brain comprising the polypeptide according to claim 1.
3. A complex comprising the polypeptide according to claim 1 and a protein, polypeptide, oligopeptide, low molecular compound or nucleic acid bound thereto.
4. A pharmaceutical composition comprising the complex according to claim 3.
5. The pharmaceutical composition according to claim 4, which is used for prevention and / or treatment of brain diseases.
6. The pharmaceutical composition according to claim 4, which is used for diagnosis of brain diseases.
7. A method for preventing and / or treating a brain disease comprising a step of administering an effective amount of the complex according to claim 3 to a patient.
8. A method for diagnosing brain disease, comprising a step of administering an effective amount of the complex according to claim 3 to a patient.
9. Use of the complex according to claim 3 in the manufacture of a pharmaceutical composition for prevention and / or treatment of brain diseases.
10. Use of the complex according to claim 3 in the manufacture of a pharmaceutical composition for diagnosis of brain disease.

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