WO2014104227A1 - Peptides pour le transfert de molécules cibles à l'intérieur de cellules - Google Patents

Peptides pour le transfert de molécules cibles à l'intérieur de cellules Download PDF

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WO2014104227A1
WO2014104227A1 PCT/JP2013/084963 JP2013084963W WO2014104227A1 WO 2014104227 A1 WO2014104227 A1 WO 2014104227A1 JP 2013084963 W JP2013084963 W JP 2013084963W WO 2014104227 A1 WO2014104227 A1 WO 2014104227A1
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peptide
target molecule
introduction
cell
seq
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Japanese (ja)
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鈴木　康弘
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Tohoku University NUC
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Tohoku University NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • the present invention relates to a target molecule intracellular introduction peptide used for introducing a desired molecule into a cell, a target molecule complex obtained by binding the target molecule intracellular introduction peptide and a target molecule, and the target molecule cell.
  • the present invention relates to a vector comprising a polynucleotide encoding a peptide for internal transfer.
  • Non-patent Documents 1, 2 When HIV-1 -derived trans-activator of transcription protein (TAT protein) is added to the culture solution, it has been transferred to the cell through the cell membrane (Non-patent Documents 1, 2). Later, it was revealed that the protein derived from flies and the structural protein of HSV-1 also have the property of passing through the cell membrane. The amino acid sequence necessary for passage through the membrane has been determined, and the domain consisting of 10 to 20 amino acids has come to be called Protein-transduction domain (PTD) / Cell-penetrating peptide (CPP).
  • PTD Protein-transduction domain
  • CPP Cell-penetrating peptide
  • PTD intracellular introduction efficiency is greatly influenced by the properties of the target protein itself and the polymer compound to be introduced, and in most cases, sufficient introduction efficiency cannot be obtained even in vitro, and cell introduction efficiency There is a problem that is unstable.
  • the problem to be solved by the present invention is to provide a target molecule cell-introducing peptide capable of introducing a target polymer compound and nanocarrier into cells with higher efficiency than conventional peptides.
  • the present inventor has found that the pH-dependent membrane fusion peptide portion of the peptide for introduction into a target molecule into which a pH-dependent membrane fusion peptide and a protein introduction domain (PTD) are fused is described. It has been found that the above-mentioned problems can be solved by setting the number of constituent amino acids to 21 amino acids or longer, which is longer than those conventionally used (20 amino acids at the longest). The present inventors further studied the amino acid sequence of the peptide, the number of peptides for introduction into cells to be bound to one target molecule, and the like based on the above-mentioned novel findings, and completed the present invention.
  • PTD protein introduction domain
  • Item 1 A target molecule intracellular introduction peptide having a pH-dependent membrane fusion peptide and a protein introduction domain (PTD), wherein the membrane fusion peptide has 21 or more constituent amino acids.
  • PTD protein introduction domain
  • the membrane fusion peptide has the sequence X 1 X 2 X 3 [Wherein X 1 , X 2 and X 3 are the same or different and represent W, H, G, F, Y, C, L, or T] Item 2.
  • Item 3 The target molecule intracellular introduction peptide according to Item 1, which is any of the following: (1) The amino acid sequence of the membrane fusion peptide is GLFGAIAGFIENGWEGMIDGWYGF (SEQ ID NO: 1), GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 2), GFFGAIAGFLEGGWEGMIAGWHGY (SEQ ID NO: 3), GFFGAIAGFLEGGWEGMIAGWHG (SEQ ID NO: 4), LAGVIMAGVAIGIGTIGTAGCTG 6), GTFTWTLSDSSGVENPGGYCLT (SEQ ID NO: 7), AFFSWSLTDSSGKDTPGGYCL (SEQ ID NO: 8), AFFSWSLTDSSGKDMPGGYCL (SEQ ID NO: 9), AFFSWSLSDPKGNDMPGGYCL (SEQ ID NO: 10) or GIFSWTITDAVGNDMPGGYCL (SEQ ID NO: 11) peptide for introduction into the target molecule cell
  • Item 4. The peptide for intracellular introduction of a target molecule according to any one of Items 1 to 3, which is any of the following: (1) The peptide for introduction into a target molecule, wherein the amino acid sequence of the PTD is YGRKKRRQRRR (SEQ ID NO: 12), RRRRRRRRRRR (SEQ ID NO: 13), or RQIKIWFQNRRMKWKK (SEQ ID NO: 14) (2) The amino acid sequence of the PTD is YGRKKRRQRRR (SEQ ID NO: 12), RRRRRRRRRRR (SEQ ID NO: 13) or RQIKIWFQNRRMKWKK (SEQ ID NO: 14) is an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and permeability through the cell membrane A peptide for introduction into the target molecule into the cell.
  • Item 5. A target molecule complex obtained by binding the target molecule intracellular introduction peptide according to any one of items 1 to 4 and a target molecule.
  • Item 6. The complex according to Item 5, wherein two or more target molecule intracellular introduction peptides are bound to one target molecule.
  • Item 7. A vector comprising a polynucleotide encoding the target molecule intracellular introduction peptide according to any one of items 1 to 4.
  • Item 8. A pharmaceutical composition comprising the complex according to item 6 or 7 as an active ingredient.
  • Item 9 A method for introducing a target molecule into a cell, comprising the step of adding the complex according to Item 6 or 7 to the cell.
  • Item 10 A target molecule intracellular introduction agent comprising the target molecule intracellular introduction peptide according to any one of Items 1 to 4.
  • Item 11 Use of the peptide for intracellular introduction of a target molecule according to any one of Items 1 to 4 for producing an agent for intracellularly introducing a target molecule.
  • a peptide for introducing a target molecule cell that can introduce a target substance into cells with higher efficiency than conventional peptides for introducing a target molecule cell.
  • FIG. 1 shows a schematic diagram of cell membrane permeation by the complex of the present invention.
  • the target molecule intracellular introduction peptide is simply indicated as PTD.
  • A Shows the number of quantum dots taken up per cell 45 minutes after adding 30 pM of a peptide loaded with a target molecule into the cell at various valences on the quantum dot in the present invention.
  • B Quantum taken up per cell 45 minutes after adding 30 pM of the known bivalent PTD and the target molecule intracellular introduction peptide (20 valence) of the present invention loaded on the quantum dot onto the cell Indicates the number of dots.
  • the amino acid sequence of the peptide of the present invention includes both the N-terminal on the left side and the C-terminal on the left side.
  • the sequence “GLFGAIAGFIENGWEGMIDGWYGF” represents N-GLFGAIAGFIENGWEGMIDGWYGF-C or N-FGYWGDIMGEWGNEIFGAIAGFLG-C
  • the sequence “YGRKKRRQRRR” represents N-YGRKKRRQRRR-C or N-RRRQRRKKRGY-C
  • the sequence “GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR” represents N-GLFGAIAGFIENGWEGMIDGWYGFYGRKKRRQRRR-C or N-YGRKKRRQRRRFGYWGDIMGEWGNEIFGAIAGFLG-C.
  • the present invention relates to a peptide for introduction into a target molecule cell having a pH-dependent membrane fusion peptide and a protein introduction domain (PTD), and the membrane fusion peptide has 21 or more constituent amino acids.
  • PTD protein introduction domain
  • the protein transduction domain is a peptide having a function of causing local stimulation after adsorption to a cell membrane surface receptor and causing the target molecule intracellular transduction peptide to be taken into the cell by endocytosis and an equivalent function
  • the amino acid sequence is not particularly limited, and examples thereof include YGRKKRRQRRR, RRRRRRRRR, RQIKIWFQNRRMKWKK, QWTLNSAGYLLGKINLKALAALAKKIL, KLALKLALKALKAALKLA, MVTVLFRRLRIRRACGPPRVRV, and the like.
  • PTD includes YGRKKRRQRRR, RRRRRRRRRRR, RQIKIWFQNRRMKWKK, QWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 15), KLALKLALKALKAALKLA (SEQ ID NO: 16), or the number of amino acids represented by MVTVLFRRLRIRRACGPPRVRV (SEQ ID NO: 17).
  • amino acid sequence having an amino acid deleted, substituted or added for example, YGRKKRRQRRRPPQ (SEQ ID NO: 18) in which PPQ is added to the end of YGRKKRRQRRR), and GRKKRRQRRRPPQ (SEQ ID NO: 19 in which Y is deleted from the opposite end) ), RRRRRRRRR (SEQ ID NO: 20) in which RR is deleted from RRRRRRRRR, etc.) are also included.
  • the range of “one or more” is not particularly limited as long as the peptide for introducing into a target molecule, which is a combination of the PTD and a pH-dependent membrane fusion peptide, has cell membrane permeability.
  • the number is 15, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 4, particularly preferably 1 to 3, and still more preferably 1 or 2.
  • Techniques for deleting, substituting and / or adding one or more amino acids in a specific amino acid sequence are known.
  • the peptide thus deleted, substituted and / or added is composed of an amino acid sequence having 50% or more identity to the amino acid sequence before deletion, substitution and addition, and PTD.
  • the peptide having a cell membrane permeability as a peptide for introduction into a target molecule into which a pH-dependent membrane fusion peptide is combined.
  • the identity of amino acids is usually 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 97% or more, and still more preferably 98%. That's it.
  • cell membrane permeability refers to the property of not only being taken into endosomes by endocytosis but also having a small hole in the endosomal membrane to reach the cytoplasm.
  • the pH-dependent membrane fusion peptide refers to a peptide that is inserted into the endosome and then inserted into the endosome membrane, and whose secondary structure changes with pH. More specifically, a peptide whose steric structure changes under neutral conditions and an acidic region such as pH 5 or lower (for example, pH 4 or lower or pH 3 or lower) is shown.
  • pH-dependent membrane fusion peptides include the sequences X 1 X 2 X 3 [wherein X 1 , X 2 and X 3 are the same or different, and W, H, G, F, Y, A, C] , L, or T (preferably W, H, G, F, or Y)] The thing which has is mentioned.
  • the position of the sequence X 1 X 2 X 3 is not particularly limited, but the terminal on the side that binds to the PTD is preferable.
  • Examples of the array X 1 X 2 X 3 include YGF, WYG, HGY, WHG, ALY, CLT, and YCL.
  • pH-dependent membrane fusion peptides examples include all amino acid sequences of membrane fusion peptides derived from various viruses (eg, influenza A, influenza B, influenza C, etc.) and having pH dependency. Although a part can be used, it is important that the pH-dependent membrane fusion peptide has 21 or more amino acids, preferably 23 or more.
  • the upper limit of the constituent amino acids is not particularly limited, but is preferably 26 or less, and more preferably 24 or less.
  • the amino acid sequence of such pH dependent membrane fusion peptide but not particularly limited as long as it has a number of the constituent amino acids, for example, GLFGAIAGFIENGWEGMIDGWYGF, GLFGAIAGFIENGWEGMIDGWYG, GFFGAIAGFLEGGWEGMIAGWHGY, GFFGAIAGFLEGGWEGMIAGWHG, LAGVIMAGVAIGIATAAQITAGVALY, GTFTWTLSDSEGKDTPGGYCLT, GTFTWTLSDSSGVENPGGYCLT, AFFSWSLTDSSGKDTPGGYCL, AFFSWSLTDSSGKDMPGGYCL, AFFSWSLSDPKGNDMPGGYCL, GIFSWTITDAVGNDMPGGYCL, etc.
  • the pH dependent membrane fusion peptide the GLFGAIAGFIENGWEGMIDGWYGF, GLFGAIAGFIENGWEGMIDGWYG, GFFGAIAGFLEGGWEGMIAGWHGY, GFFGAIAGFLEGGWEGMIAGWHG, AGVIMAGVAIGIATAAQITAGVALY, GTFTWTLSDSEGKDTPGGYCLT, GTFTWTLSDSSGVENPGGYCLT, AFFSWSLTDSSGKDTPGGYCL, AFFSWSLTDSSGKDMPGGYCL, 1 or several amino acids in the amino acid sequence shown in AFFSWSLSDPKGNDMPGGYCL or GIFSWTITDAVGNDMPGGYCL In which amino acid sequences are deleted, substituted or added.
  • the range of “one or more” is not particularly limited as long as the peptide for introduction into a target molecule cell in which PTD is combined with the pH-dependent membrane fusion peptide has cell membrane permeability. Is, for example, 1 to 15, preferably 1 to 10, more preferably 1 to 7, and still more preferably 1 to 5. Techniques for deleting, substituting and / or adding one or more amino acids in a specific amino acid sequence are known.
  • the peptide thus deleted, substituted and / or added consists of an amino acid sequence having 50% or more identity to the amino acid sequence before deletion, substitution and addition, and Examples are peptides in which a peptide for introduction into a target molecule into which a PTD is combined with a pH-dependent membrane fusion peptide has cell membrane permeability.
  • the identity of amino acids is usually 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 97% or more, and still more preferably 98%. That's it.
  • GLFX 4 AIAX 5 FIEX 6 GWEGX 7 IX 8 GWYG SEQ ID NO: 21
  • X 4 represents D, E, G or N.
  • X 5 represents D, E, G or N.
  • X 6 represents D, E, G or N.
  • X 7 represents M or L.
  • X 8 represents D, E, G or N.
  • the combination of (X 4 , X 5 , X 6 , X 7 , X 8 ) (G, G, N, M, D) is excluded.
  • the like the like.
  • a sequence in which four of the sequence GLFGAIAGFIENGWEGMIDGWYG are substituted for example, a peptide consisting of GLF E AIA E FIE G GWEG L I E GWYG (underlined is a substituted portion, SEQ ID NO: 22) and the like can be mentioned.
  • the pH-dependent membrane fusion peptide and the PTD may be bonded directly or via a linker.
  • a linker what is used in the said field
  • the peptide for introduction into a target molecule of the present invention has a domain for binding a target substance, specifically, for example, a DNA binding domain (zinc finger domain, HMG-box, etc.), a double-stranded RNA binding domain ( Histone, RDE-4 protein, double-stranded RNA binding domain derived from protamine, etc., various labeled tag protein recognition compounds (anti-Myc binding protein (for Myc-tag)), Halotag ligand (Halotag protein binding molecule), nickel molecule (His-tag protein binding molecule) etc.) may be present at the end of the PTD side among the end of the fusion peptide and the end of the PTD.
  • a DNA binding domain zinc finger domain, HMG-box, etc.
  • Histone, RDE-4 protein double-stranded RNA binding domain derived from protamine, etc.
  • various labeled tag protein recognition compounds anti-Myc binding protein (for Myc-tag)
  • Halotag ligand Halotag protein
  • examples of the peptide for introduction into a target molecule cell of the present invention include the following: N-GLFGAIAGFIENGWEGMIDGWYGF YGRKKRRQRRR- C (Peptide 1, SEQ ID NO: 23) N-GLFGAIAGFIENGWEGMIDGWYGF RRRQRRKKRGY- C (Peptide 2, SEQ ID NO: 24) N- YGRKKRRQRRR FGYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 3, SEQ ID NO: 25) N- RRRQRRKKRGY FGYWGDIMGEWGNEIFGAIAGFLG-C (Peptide 4, SEQ ID NO: 26) N-GFFGAIAGFLEGGWEGMIAGWHGY YGRKKRRQRRR- C (peptide 5, SEQ ID NO: 27) N-GFFGAIAGFLEGGWEGMIAGWHGY RRRQRRKKRGY- C (Peptide 6, SEQ ID NO: 28) N- YGRKKRRQRRQRR
  • the peptide for introduction into a target molecule cell of the present invention can be prepared by a conventionally known genetic engineering method, chemical synthesis method or the like.
  • a desired peptide may be obtained after inserting a polynucleotide encoding the aforementioned peptide for introduction into a target molecule into a vector and culturing a transformant in which the vector is incorporated.
  • the peptide for introduction into a target molecule of the present invention may be obtained by isolating and purifying from a microorganism transformed to have the ability to produce the peptide.
  • the target molecule intracellular introduction peptide of the present invention may be synthesized by a conventionally known chemical synthesis method in accordance with the information of the amino acid sequence of the target molecule intracellular introduction peptide or the nucleotide sequence encoding the peptide.
  • the chemical synthesis method includes a peptide synthesis method using a liquid phase method or a solid phase method.
  • the peptide for introducing a target molecule into the cell of the present invention is produced by the above method by binding a pH-dependent membrane fusion peptide and PTD (or further binding a domain for binding a target substance).
  • the pH-dependent membrane fusion peptide and PTD may be synthesized by the above method and then bound to each other.
  • the present invention provides a target molecule complex formed by binding the above-described target molecule intracellular introduction peptide and a target molecule.
  • the binding mode of the target molecule and the peptide for introduction into the target molecule into the cell is not particularly limited.
  • the target molecule may be bound through the above-described binding domain, may be cross-linked by —SS—bonding, etc.
  • the avidin system may be used, may be electrostatically bonded, or may be chemically modified and bonded.
  • the number of target molecule intracellular introduction peptides to be bound to one target molecule is not particularly limited, but 2 or more (eg, 3 or more, more preferably 8 or more) target molecule intracellular introduction peptides are target molecules. It is preferable from the viewpoint of increasing the efficiency of cell surface receptor binding and activating the cell surface receptor uptake mechanism.
  • the upper limit of the number of the target molecule-introducing peptide to be bound to one target molecule is not particularly limited, but is preferably 30 or less, and more preferably 20 or less.
  • the target substance is not particularly limited and may be either a polymer or a low molecule.
  • polymer compounds include nucleic acid molecules such as DNA and RNA (siRNA, shRNA, etc.); peptides such as oligopeptides and proteins (antibodies, antibody fragments, enzymes, cytokines, chemokines, receptor polypeptides, etc.), sugars, etc. Examples include chains.
  • small molecules include antibiotics, anticancer agents, anti-inflammatory agents, liposomes, micelles, dendrimers, nanotubes, nanocarriers such as amino acid nanoparticles, quantum dots, fluorescent dyes, intracellular molecular visualization reagents, nanomagnetic materials, Examples thereof include compounds that can visualize target cell dynamics in vivo, such as nanogold.
  • a complex of the present invention containing the transcription factor as a target molecule and administer this to a subject to treat the cancer.
  • an antigen when introduced into an antigen-presenting cell using the method of the present invention, the antigen is presented to MHC-class I and cytotoxic T cells are activated. Therefore, application to vaccines against many viral infections and cancers that require the induction of cytotoxic T cells is conceivable.
  • a substance for example, antibody, antibody fragment (scFv (Single-chain variable fragment), etc.) that specifically binds to a specific antigen is bound to the peptide and complex for introduction into the target molecule of the present invention.
  • a target molecule can be specifically introduced into a specific cell in a sample or subject containing various cells.
  • the complex of the present invention can be applied to a conditional knockout animal. Specifically, for example, in a flox mouse having a gene locus in which a target gene region is sandwiched between Cre recombinase target sequences loxP, the target molecule intracellular introduction peptide of the present invention and Cre recombinase or a gene encoding the same are added. A method of administering the bound complex is exemplified.
  • Cre recombinase or a gene encoding it can be introduced into somatic cells of flox mice, and the target gene can be deleted by the recombinase. Therefore, this embodiment is useful because a target gene region can be deleted at a desired timing.
  • deletion of a target gene region can be caused at a desired timing and at a desired site.
  • the complex of the present invention can also be used to produce iPS cells.
  • the OCT3 / 4 / SOX2 / NANOG / LIN28 4 gene expression vector or 4 protein or the like is used as a target molecule, and the complex of the present invention is added to cells in vitro, or By directly administering to a subject animal, a gene as a target molecule is introduced into the cell, and an iPS cell can be established.
  • a reaction substrate that reacts to changes in the environment outside the cell inside the cell, for example, a decrease in pH after incorporation into the endosome changes its structure in response to pH reactivity, and the target substance is introduced from the introduced peptide.
  • the method includes separation from the target substance in the cell using, for example, pH-reactive para-nitrophenyl (pNP) -PEG-PE, SS bond between cysteine and cysteine, and the like.
  • the present invention also provides a vector comprising a polynucleotide encoding a peptide for introduction into a target molecule into a cell.
  • a vector comprising a polynucleotide encoding a peptide for introduction into a target molecule into a cell.
  • any vector such as a plasmid vector, an adenovirus vector, or a retrovirus vector can be used.
  • a method for cloning a nucleotide sequence containing a polynucleotide encoding a peptide for introduction into a target molecule into a cell a method known per se can be used.
  • expression control signals transcription initiation and translation initiation signals
  • Etc. can be designed so that the gene can be self-expressed in microbial cells depending on the host microorganism.
  • the vector of the present invention includes not only those encoding the target molecule intracellular introduction peptide but also those encoding a complex in which the target molecule intracellular introduction peptide and the target substance are bound.
  • the target molecule can be produced by covalently binding the target molecule-introducing peptide and the target substance by gene expression.
  • compositions comprising the complex as an active ingredient.
  • the complex as the active ingredient can be used alone, but depending on the route of administration, it should be formulated into a suitable dosage form using a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be administered to a subject in various administration routes, specifically oral or parenteral administration (eg, intravenous injection, intramuscular injection, nasal mucosal administration, oral mucosal administration, transdermal Administration, subcutaneous administration, intradermal administration, rectal administration, etc.).
  • parenteral administration such as intravenous injection, intramuscular injection, or intranasal mucosal administration is used.
  • the subject include mammals (human or non-human mammal), and examples of the non-human mammal include mice, rats, rabbits, dogs, monkeys, and the like.
  • Preferred examples of the dosage form include parenteral preparations such as injections (including drops), ointments, eye drops, eye ointments, nasal drops, ear drops, poultices, lotions and the like.
  • parenteral preparations such as injections (including drops), ointments, eye drops, eye ointments, nasal drops, ear drops, poultices, lotions and the like.
  • oral preparations include tablets, powders, fine granules, granules, coated tablets, capsules, syrups, and lozenges.
  • Carriers that can be used to formulate these preparations include, for example, excipients, binders, disintegrants, lubricants, colorants, and flavoring agents that are commonly used in the pharmaceutical field, and, if necessary, Stabilizer, emulsifier, absorption promoter, surfactant, pH adjuster, preservative, antioxidant, extender, wetting agent, surface active agent, dispersant, buffer, preservative, solubilizer, Examples include soothing agents.
  • the present invention provides a method for introducing a target molecule into a cell, comprising the step of adding the complex to a cell.
  • the complex may be added to cells either in vitro or in vivo.
  • PTD binds to heparan sulfate proteoglycan etc. on the membrane surface near the cell membrane surface to cause local stimulation, and the target molecule intracellular introduction peptide is taken into the cell by endocytosis (FIG. 1). ). Then, a pH-dependent membrane fusion peptide is inserted into the endosome membrane. Thereafter, the pH in the endosome rapidly changes to acidic with time. Along with the change in pH, the three-dimensional structure of the pH-dependent membrane fusion peptide is changed while it is inserted into the endosomal membrane, and is bent to form small pores in the endosomal membrane. As a result, the peptide for introduction into the target molecule into the cell is released into the cytoplasm through the endosome membrane.
  • Target molecule intracellular introduction agent comprising the aforementioned target molecule intracellular introduction peptide.
  • the target molecule intracellular introduction peptide itself may be used as the target molecule intracellular introduction agent, or the target molecule intracellular introduction peptide is mixed with the above-described carrier or the like to prepare a formulation. May be.
  • Example 1 A peptide consisting of the following amino acid sequence was synthesized by Fmoc (N- (9-fluorenyl) methoxycarbonyl) solid phase synthesis method by requesting Toray Industries, Inc .: N-GLFGAIAGFIENGWEGMIDGWYGF YGRKKRRQRRR -C Peptide 1 The synthesized peptide was purified to 95% or more by reverse phase HPLC with an acetonitrile / H 2 O / trifluoroacetic acid gradient (molecular weight 4149.7).
  • Examples 2 to 6 A peptide was synthesized in the same manner as in Example 1 except that a peptide consisting of the following amino acid sequence was synthesized: N- YGRKKRRQRRR FGYWGDIMGEWGNEIFGAIAGFLG-C (peptide 3, molecular weight 4149.7) N- RRRQRRKKRGY YGHWGAIMGEWGGELFGAIAGFFG-C (peptide 8, molecular weight 4072.6) N- YGRKKRRQRRR GYWGDIMGEWGNEIFGAIAGFLG-C (peptide 11, molecular weight 4002.6) N- RRRRRRRRRRRR GYWGEILGEWGGEIFEAIAEFLG-C (peptide 26, molecular weight 4261.9) N- YGRKKRRQRRR GYWGEILGEWGGEIFEAIAEFLG-C (peptide 29, molecular weight 4085.6) In the above sequence, the underlined portion indicates PTD, and the other portion is a pH-dependent membrane
  • Example 1 Further, in the above Fmoc solid phase synthesis method, N N was biotinylated in the same manner as in Example 1 except that the PTD side was biotinylated using a biotinylated amino acid for the extension of the last amino acid (N-terminal G in peptide 1). A peptide biotinylated at the end was synthesized. The obtained compound is defined as Compound 1 (molecular weight 4394.1).
  • Example 2'-6 ' In the Fmoc solid phase synthesis method, the PTD side was biotinylated by using a biotinylated amino acid for the extension of the last amino acid (N-terminal G in peptide 1) (compounds 3, 8, 11, 26).
  • the molecular weight of each compound is as follows: Compound 3, molecular weight 4394.1 Compound 8, molecular weight 4316.9 Compound 11, molecular weight 4246.9 Compound 26, molecular weight 4329.9
  • a peptide having the following amino acid sequence and biotinylated at the N-terminus was also synthesized in the same manner (Compound 33).
  • N-GYWGEILGEWGGEIFEAIAEFLGRRRRRRRRR-C (peptide 33, molecular weight 4506.2) Comparative Examples 1 and 2
  • a biotinylated peptide was synthesized in the same manner as Example 1 ′ except that a peptide consisting of the following amino acid sequence was synthesized (referred to as compounds 31 to 32, respectively): N-RRRQRRKKRGYGDIMGEWGNEIFGAIAGFLG-C (Peptide 31) N-YGRKKRRQRRR-C (peptide 32)
  • Examples 31 and 32 and Comparative Examples 3 and 4 Reaction of above-mentioned compounds 11 and 26 with various molar ratios up to 1,2,4,8,12,16,20 to 1 ⁇ mol of commercially available streptavidin fluorescent quantum dots (QD) (QD655, invitrogen; diameter 30 nm) Then, various numbers of peptides for introduction into the target molecule were polymerized on QD (this is expressed as valence, and
  • complexes 11-1 to 11-20 and 26-1 to 26-20 are referred to as complexes 11-1 to 11-20 and 26-1 to 26-20, respectively.
  • HeLa cells were used as living cells, and the HeLa cells were cultured in a cell culture medium X-VIVO buffer.
  • Complexes 11-1 to 11-20, 26-1 to 26-20, and 50 pM were added to the cells at the same time as endosome staining reagent 0.25% DiO. After 1 hour, the cells were washed with the culture medium, and then 2 hours. After culture, reference 1.Duan H, Nie S. J Am Chem Soc. (2007) Vol. 129,3333-8.
  • composites 31 and 32 were prepared in the same manner as in Example 31 except that compounds 31 and 32 synthesized in 1 and 2 were used as comparative examples, and the residual ratio of QD particles to endosomes was measured.
  • complex 32 derived from HIV-1 Tat
  • H-PTD complex 31
  • a control (20, which has the highest efficiency in the same study)
  • the peptide for introducing a target molecule into the cell of the present invention it is possible not only to introduce the target molecule in the form of being encapsulated in the endosome but also to introduce a small pore in the endosome into the cytoplasm. Therefore, it is very useful for studying the function of the target molecule in the cytoplasm.
  • the action point of many drugs since the action point of many drugs is considered to be in the cytoplasm, it is very important from the viewpoint of application to a drug delivery system.
  • Examples of the use of the peptide for intracellular introduction of a target molecule of the present invention include introduction of an anti-apoptotic peptide from a coronary artery against a myocardial ischemic damage caused by acute ischemic heart disease and directly introducing it into the cell. For use in protecting the myocardium.
  • Target molecule Peptide SEQ ID No. 48 into introduction into cells
  • Target molecule Peptide sequence number 49 for introduction into cell Peptide sequence number 50 for introduction into target molecule cell Peptide sequence number 51 for introduction into target molecule cell Peptide sequence number 52 for introduction into target molecule cell Peptide for introduction into target molecule cell Plastid

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Abstract

L'objectif de la présente invention est de fournir des peptides pour transférer des molécules cibles à l'intérieur de cellules, lesdits peptides permettant à des composés polymères cibles et des nanosupports d'être transférés à l'intérieur de cellules avec une efficacité supérieure aux peptides de l'état antérieur de la technique. Les peptides pour le transfert de molécules cibles à l'intérieur de cellules comprennent des peptides à fusion de membrane dépendante du pH et des domaines de transduction de protéine (PTD). Le nombre d'acides aminés constituant les peptides à fusion de membrane n'est pas inférieur à 21.
PCT/JP2013/084963 2012-12-28 2013-12-26 Peptides pour le transfert de molécules cibles à l'intérieur de cellules Ceased WO2014104227A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011126974A1 (fr) * 2010-04-09 2011-10-13 Merck Sharp & Dohme Corp. Entités chimiques isolées inédites et procédés d'administration d'oligonucléotides
WO2013165816A2 (fr) * 2012-05-02 2013-11-07 Merck Sharp & Dohme Corp. Compositions de petit acide nucléique interférent (sina)
WO2013166155A1 (fr) * 2012-05-02 2013-11-07 Merck Sharp & Dohme Corp. Nouveaux conjugués contenant tétragalnac et peptide et procédés pour l'administration d'oligonucléotides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011126974A1 (fr) * 2010-04-09 2011-10-13 Merck Sharp & Dohme Corp. Entités chimiques isolées inédites et procédés d'administration d'oligonucléotides
WO2013165816A2 (fr) * 2012-05-02 2013-11-07 Merck Sharp & Dohme Corp. Compositions de petit acide nucléique interférent (sina)
WO2013166155A1 (fr) * 2012-05-02 2013-11-07 Merck Sharp & Dohme Corp. Nouveaux conjugués contenant tétragalnac et peptide et procédés pour l'administration d'oligonucléotides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEE YJ ET AL.: "Modeling of the endosomolytic activity of HA2-TAT peptides with red blood cells and ghosts", BIOCHEMISTRY, vol. 49, no. 36, 2010, pages 7854 - 7866 *
WADIA JS ET AL.: "Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis", NAT. MED., vol. 10, no. 3, 2004, pages 310 - 315 *

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