WO2022065172A1 - Molécule pénétrant la membrane cellulaire ainsi qu'application de celle-ci, et procédé d'amélioration de pénétration de membrane cellulaire de molécule pénétrant la membrane cellulaire - Google Patents

Molécule pénétrant la membrane cellulaire ainsi qu'application de celle-ci, et procédé d'amélioration de pénétration de membrane cellulaire de molécule pénétrant la membrane cellulaire Download PDF

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Publication number
WO2022065172A1
WO2022065172A1 PCT/JP2021/034020 JP2021034020W WO2022065172A1 WO 2022065172 A1 WO2022065172 A1 WO 2022065172A1 JP 2021034020 W JP2021034020 W JP 2021034020W WO 2022065172 A1 WO2022065172 A1 WO 2022065172A1
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cell membrane
peptide
permeable molecule
acid
molecule according
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English (en)
Japanese (ja)
Inventor
千明 小宮
敏裕 鹿倉
恵太 井口
佑介 平山
俊秀 藤井
光昭 北野
達也 馬渡
寛士 北
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Kaneka Corp
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Kaneka Corp
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Priority claimed from JP2020159040A external-priority patent/JP2023162461A/ja
Priority claimed from JP2020159038A external-priority patent/JP2023162460A/ja
Application filed by Kaneka Corp filed Critical Kaneka Corp
Publication of WO2022065172A1 publication Critical patent/WO2022065172A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility

Definitions

  • the present invention relates to a cell membrane permeable molecule, a peptide complex containing the cell membrane permeable molecule and a method for producing the same, a peptide library containing the peptide complex, a method for screening a functional peptide using the peptide library, and a cell membrane permeation.
  • the present invention relates to a method for improving cell membrane permeability of a sex molecule.
  • Small molecule drugs with a molecular weight of up to about 500 have been developed as many drugs because of their low manufacturing cost and low immunogenicity.
  • this small molecule drug has low specificity, and it has become increasingly difficult to develop in recent years due to the problem of side effects due to off-targeting.
  • sales of high molecular weight drugs derived from antibodies and proteins having a molecular weight exceeding 10,000 are increasing because of their high specificity and few side effects.
  • the problems are that it is expensive, that it may cause immunogenicity due to its large molecular weight, and that it generally does not migrate into the cell, so that the molecules that can be the target are limited.
  • molecules with a molecular weight of about 500 to 5,000 such as peptides and nucleic acids, so-called medium-molecular-weight drugs, have the advantages of low-molecular-weight drugs that are inexpensive and have low immunogenicity, and their specificity to target molecules. It is expected to be a drug that has the advantages of high molecular weight drugs, such as high prices and few side effects.
  • peptides as medium-molecular-weight drugs, we have created a peptide (polypeptide) library consisting of various amino acid sequences and have high affinity for specific target molecules such as disease-related proteins. A method for selecting peptides is widely used.
  • a ribosome display method using a ribosome display complex containing a peptide chain, an mRNA molecule, and a ribosome is known (for example, patent). See Document 1).
  • the RD method is extremely excellent and useful because it is possible to prepare a peptide library of 1012 or more kinds in a few minutes simply by mixing an in vitro translation system and mRNA.
  • a medium-molecular-weight drug such as a peptide or nucleic acid targets an intracellular molecule
  • a means for permeating the cell membrane to reach the desired intracellular target molecule is required.
  • Many cells, such as cytoskeleton-related proteins and kinase-related factors, can be targeted in the treatment of various diseases. Therefore, the establishment of a useful intracellular introduction method leads to a significant expansion of the scope of application of medium-molecular-weight drugs.
  • Techniques for incorporating the active ingredient into cells include, for example, a method of fusing the amino acid sequence of a cell membrane penetrating peptide containing a large amount of basic amino acids, and a dendrimer which is a dendritic polymer having a regularly branched structure from the center.
  • the method to be used see, for example, Patent Document 2 and the like are known.
  • the present invention includes a cell membrane penetrating molecule having a function of incorporating an active ingredient into a cell and having low toxicity to a living body, a peptide complex containing the cell membrane penetrating molecule, a method for producing the same, and the peptide complex.
  • a peptide library a method for screening a functional peptide using the peptide library, and a cell membrane penetrating molecule that can efficiently take up an active ingredient into a cell even if the molecular weight is a small cell membrane penetrating molecule.
  • the purpose is to provide a method for improving sex.
  • a salt with an acid having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 It was found that it can be a cell membrane permeable molecule with low toxicity to the living body while having a function of taking up the active ingredient into the cell. Further, by using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid, the cell membrane becomes equal to or higher than the cell membrane-permeable molecule having a large number of structures contributing to cell membrane permeation. It was found that the permeability can be improved. (In the general formula (I), R represents a bond).
  • the present invention is based on the above-mentioned findings of the present inventors, and the means for solving the above-mentioned problems are as follows. That is, ⁇ 1> A cell membrane-permeable molecule having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 as a salt with an acid. (In the general formula (I), R represents a bond). ⁇ 2> A peptide complex comprising the peptide and the cell membrane-permeable molecule according to ⁇ 1>. ⁇ 3> A peptide library comprising the peptide complex described in ⁇ 2> above.
  • ⁇ 4> A method for producing a peptide complex, which comprises introducing the cell membrane-permeable molecule according to ⁇ 1> into a peptide.
  • ⁇ 5> A method for screening a functional peptide, which comprises screening a functional peptide using the peptide library according to the above ⁇ 3>.
  • ⁇ 6> A method for improving the cell membrane permeability of a cell membrane-permeable molecule, which comprises using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid. (In the general formula (I), R represents a bond).
  • the cell membrane-permeable molecule which is a cell membrane-permeable molecule having a function of taking up an active ingredient into a cell and having a function of taking up the active ingredient into the cell, which can solve the conventional problems and achieve the object, and the cell membrane permeation.
  • the cell membrane permeable molecule of the present invention has at least a structure represented by the following general formula (I) and is a salt with an acid having a pKa value of less than 4.7. (In the general formula (I), R represents a bond).
  • the portion other than R in the structure represented by the general formula (I) is a portion that contributes to cell membrane permeability.
  • the number of portions of the cell membrane-permeable molecule other than R in the structure represented by the general formula (I) is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably one. .. That is, the cell membrane permeable molecule of the present invention preferably has one dendritic structure represented by the general formula (I).
  • the R portion of the structure represented by the general formula (I) is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably bonded to a heteroatom-containing group.
  • the heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a peptide linking group linked to the peptide (A), the peptide linking group and the general formula (I). An embodiment consisting of a linking group for linking with the structure represented by), (B) an embodiment consisting of a peptide linking group for linking with a peptide, and the like.
  • the heteroatom-containing group may have a skeleton of a peptide or nucleic acid.
  • the length of the heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the lower limit of the number of atoms bonded linearly is preferably 1 atom or more.
  • the upper limit is preferably 50 atoms or less, more preferably 25 atoms or less. Examples of the number of atoms bonded to the linear chain of 25 or less include heteroatom-containing groups in the cell membrane-permeable molecule G3-DCX described later.
  • the hetero atom in the hetero atom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose. For example, at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. And so on.
  • the heteroatom may be only one kind or two or more kinds.
  • the content ratio of the heteroatom in the heteroatom-containing group is not particularly limited and may be appropriately selected depending on the intended purpose, but it may be 10% or more in terms of increasing the water solubility of the cell membrane-permeable molecule. preferable.
  • the upper limit of the content ratio of the heteroatom is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 60% or less, more preferably 50% or less.
  • the range of the content ratio of the heteroatom in the heteroatom-containing group is preferably 10% or more and 60% or less, and more preferably 10% or more and 50% or less.
  • the content ratio of the heteroatom in the heteroatom-containing group can be calculated as follows. It should be noted that all atoms include hydrogen atoms.
  • Content ratio of heteroatoms in heteroatom-containing groups (%) ⁇ (number of heteroatoms in heteroatom-containing groups) / (total number of heteroatom-containing groups) ⁇ ⁇ 100
  • the heteroatom-containing group preferably contains a repeating unit.
  • the repeating unit is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include alkylene oxide, alkyleneimine and alkylene sulfide.
  • the repeating unit may be only one kind or two or more kinds. Further, the repeating unit may have a substituent.
  • alkylene oxide is preferable from the viewpoint of availability and stability.
  • the alkylene oxide is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include at least one selected from the group consisting of methylene oxide, ethylene oxide and propylene oxide.
  • the number of the repeating units is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the lower limit of the repeating units is preferably 1 or more, and the upper limit is preferably 10 or less.
  • the substituent contained in the repeating unit is not particularly limited and may be appropriately selected depending on the intended purpose.
  • substituents include alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclyl. Hydroxy, mercapto, amino, carbonyl, amide, thioamide, urea, sulfonamide, carboxy and nitrile are preferred.
  • the peptide linking group contributes to the introduction of the cell membrane-permeable molecule into the peptide by reacting with the reactive amino acid residue in the peptide.
  • the reactive amino acid residue is an amino acid residue that reacts with the peptide linking group, may be an amino acid residue that directly reacts with the peptide linking group, or can react with the peptide linking group. It may be an amino acid residue modified to.
  • the group for linking the peptide is not particularly limited as long as it can be linked to the peptide, and can be appropriately selected depending on the intended purpose.
  • the thiol group of the cysteine residue and the side chain amino of the lysine residue can be selected.
  • examples thereof include a chain hydroxyl group (-OH), an ortho-position carbon of a tyrosine residue, and a group capable of forming a bond by reacting with a side chain sulfide group (-SMe) of a methionine residue.
  • Specific examples of the peptide linking group include, for example, an alkyl halide group, an activated carbonyl group, and an unsaturated hydrocarbon group described in paragraphs [0067] to [0076] of International Publication No.
  • Examples thereof include an epoxy group, a sulfonyl-containing group, an isocyanate group, a thioisocyanate group, a carben generating group, a disulfide bond-containing group, and a thiol group.
  • the peptide linking group contributes to the cyclization of the peptide by reacting with the reactive amino acid residue in the peptide (hereinafter, may be referred to as "cyclizing group").
  • the reactive amino acid residue that reacts with the cyclic group may be an amino acid residue that directly reacts with the cyclic group, or may be an amino acid residue modified so as to react with the cyclic group. May be good.
  • the cyclic group is not particularly limited and may be appropriately selected depending on the intended purpose, but an electron-withdrawing group is preferable.
  • the electron-withdrawing group is not particularly limited and may be appropriately selected depending on the intended purpose, but it preferably has a halogen.
  • the type of the halogen is not particularly limited and may be appropriately selected depending on the intended purpose, but for example, a chlorine atom, a bromine atom and an iodine atom are preferable.
  • the number of halogens is not particularly limited and may be appropriately selected depending on the intended purpose. However, the lower limit of the number of halogens is preferably 2 or more, and the upper limit is preferably 5 or less and 4 or less.
  • the electron-withdrawing group may have a substituent. If the electron-withdrawing group contains an unsaturated structure in its structure, the peptide is likely to be cyclized.
  • the unsaturated structure is not particularly limited, but is, for example, an aromatic ring structure, a heterocyclic structure, an alicyclic hydrocarbon structure, an alkenyl structure, an alkynyl structure, a carbonyl structure, a thiocarbonyl structure, an oxime structure, a cyano structure, or an isocyanate structure.
  • a benzyl halide is more preferable, and a 3,5-bis (halomethyl) benzyl group is particularly preferable.
  • the type of the halogen is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a chlorine atom, a bromine atom and an iodine atom are preferable, and a chlorine atom is more preferable.
  • Cyclization of a peptide with the cyclic group may be carried out by reacting the cyclic group with at least one group selected from the group consisting of a thiol group, an amino group and a hydroxy group contained in the peptide. preferable.
  • peptide linking group when the peptide and the cell membrane permeable molecule are bound by the oxime ligation method, those represented by the following structural formula can also be used as the peptide linking group.
  • the linking group is a group that links the peptide linking group and the structure represented by the general formula (I).
  • the structure of the linking group is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a structure containing the above-mentioned repeating unit.
  • the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
  • cell membrane permeable molecule examples include compounds represented by the following structural formulas.
  • a part of the structure of the cell membrane permeable molecule may be changed when it is in the form of a salt.
  • the cell membrane permeable molecule G3-DCX represented by the following structural formula is a cyclic group which is a 3,5-bis (chloromethyl) benzyl group as a heteroatom-containing group in the R portion in the general formula (I). This is an example having a peptide linking group) and a linking group. Since the cell membrane-permeable molecule G3-DCX has a cyclizing group, it is possible to cyclize the peptide and impart cell membrane permeability to the peptide in one step.
  • the cell membrane permeable molecule G3 represented by the following structural formula is an example in the general formula (I) having a peptide linking group capable of binding to a peptide by the oxime ligation method as a heteroatom-containing group in the R portion. Is.
  • the cell membrane permeable molecule of the present invention is a salt with an acid having a pK a (acid dissociation constant) value of less than 4.7.
  • the value of pK a1 which is the value of pK a in the first stage dissociation is taken as the value of pK a .
  • the acid having a pK a value of less than 4.7 is not particularly limited and may be appropriately selected depending on the intended purpose, but trifluoroacetic acid (-0) in that it is less toxic to the living body.
  • the lower limit of the pK a value is more than -8.0 (the pK a value is -8.) In that it is less toxic to the living body. (Greater than 0) is preferred, more than -3.0 (pK a value is greater than -3.0) is more preferred.
  • the upper limit of the pK a value for an acid having a pK a value of less than 4.7 is preferably 3.5 or less, and more preferably less than 3.3.
  • the cell membrane permeable molecule has a structure represented by the above-mentioned general formula (I), and is a salt with an acid having a pKa value of less than 4.7, as long as the effect of the present invention is not impaired. , Other configurations may be included.
  • the method for producing the cell membrane-permeable molecule is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out. For example, it can be produced by appropriately selecting a known chemical synthesis technique with reference to the method described in US Pat. No. 7,862,807.
  • a method of forming a salt a crude product of a cell membrane permeable molecule that is not in the form of a salt is purified by HPLC using a mobile phase according to the type of the target salt, and the form of the target salt is obtained.
  • the method of making the cell membrane permeable molecule of the above the cell membrane permeable molecule in the form of a salt is passed through an anion exchange resin in which the counter ion is replaced with the ion of the target salt, and is eluted to form the target salt.
  • an anion exchange resin in which the counter ion is replaced with the ion of the target salt, and is eluted to form the target salt. Examples thereof include a method of making a cell membrane permeable molecule.
  • the method for confirming whether or not the obtained cell membrane permeable molecule has a desired structure is not particularly limited, and a known analytical method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance can be selected. Analytical methods such as spectroscopy, carbon 13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy, and liquid chromatography can be mentioned.
  • the cell membrane permeabilizing molecule of the present invention can impart excellent cell membrane permeability to active ingredients such as peptides, nucleic acids, proteins and complexes thereof while reducing toxicity to living organisms.
  • the peptide complex of the present invention comprises at least the peptide and the above-mentioned cell membrane permeable molecule of the present invention, and further contains other configurations as required.
  • the peptide is not particularly limited as long as the cell membrane permeable molecule can be introduced, and can be appropriately selected depending on the intended purpose. However, the peptide is cyclized by introducing the cell membrane permeable molecule. Is preferable.
  • the type of amino acid in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be a natural amino acid, an unnatural amino acid, or a D-form. It may be present or it may be L-form.
  • the peptide may be a modified peptide such as a lipopeptide.
  • the amino acid residue used for the introduction of the cell membrane permeable molecule (hereinafter, may be referred to as “reactive amino acid residue”) is not particularly limited and may be appropriately selected depending on the intended purpose, for example. , Cysteine residue, lysine residue, histidine residue, tryptophan residue, tyrosine residue, serine residue, threonine residue and the like.
  • the reactive amino acid residue may be, for example, a cysteine residue, a lysine residue, a serine residue, or a threonine residue. And so on. Further, a hydroxy group, a mercapto group, or an amino group in the amino acid residue may be used.
  • the reactive amino acid residue may be used alone or in combination of two or more.
  • the number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the number of the reactive amino acid residues in the peptide is preferably 2 or more.
  • the upper limit of the number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the position is not stable and it may be difficult to compare the characteristics of the peptide derived from the amino acid sequence, so 10 or less is preferable.
  • the reactive amino acid residue may be separately introduced into the peptide. preferable.
  • the position of the reactive amino acid residue in the peptide is not particularly limited and can be appropriately selected depending on the intended purpose.
  • RNA chain an mRNA molecule, a peptide chain which is a translation thereof (hereinafter, also referred to as “polypeptide chain”), and a ribosome display complex containing ribosome (hereinafter, referred to as “RD complex”).
  • RD complex a ribosome display complex containing ribosome
  • it is a portion protruding from the exit tunnel (exit tunnel) of the ribosome, specifically, the second position from the N-terminal to the 30th position (N) from the C-terminal. It is preferable to use it between the position 2nd from the terminal and the position 30th from the C end) in that the modification reaction by the linker molecule can be less likely to be sterically inhibited by the ribosome.
  • the position from the C-terminal the 50th position from the C-terminal is preferable, and the 100th position is more preferable.
  • the position can be appropriately set according to the chain length of the peptide, and is, for example, the 2nd to 1,000th position from the N-terminal.
  • the 2nd to 100th positions from the N-terminal are preferable, and the 2nd to 50th positions from the N-terminal are more preferable.
  • the method for producing the RD complex is not particularly limited, and a known method can be appropriately selected. Examples thereof include the method described in International Publication No. 2017/213158. It can also be manufactured using a commercially available kit.
  • the amino acid sequence of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, but one containing a random sequence at a specific position is preferable so as to be useful as a peptide library. From such a random sequence, a useful amino acid sequence can be identified according to a predetermined purpose.
  • the position of the random sequence in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the position is not particularly limited. It is preferably between the 2nd position from the N-terminal to the 30th position from the C-terminal (including the 2nd position from the N-terminal and the 30th position from the C-terminal). That is, the reactive amino acid residue is preferably contained in a random sequence. Therefore, the preferred position of the random sequence can be set from the same range as the preferred position of the reactive amino acid residue.
  • the number of the random sequences in the peptide may be one or two or more.
  • the upper limit of the number of the random sequences is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 or less.
  • the number of amino acid residues per random sequence is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 1 or more and 30 or less. The longer one random sequence and the larger the number of random sequences, the greater the diversity of the peptide library.
  • the peptide may further contain a sequence for purifying a polypeptide chain such as a FLAG® sequence or a polyHis sequence, a sequence selectively cleaved by a protease or the like, a spacer sequence, or the like.
  • a sequence for purifying a polypeptide chain such as a FLAG® sequence or a polyHis sequence, a sequence selectively cleaved by a protease or the like, a spacer sequence, or the like.
  • the number of amino acid residues of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 10 or more and 5,000 or less.
  • the lower limit of the number of amino acid residues of the peptide is preferably 150 or more, more preferably 200 or more.
  • the upper limit of the number of amino acid residues of the peptide is preferably 800 or less, more preferably 600 or less, and particularly preferably 500 or less.
  • the lower limit value and the upper limit value can be appropriately combined and selected.
  • the method for synthesizing the peptide is not particularly limited, and a known method can be appropriately selected.
  • the cell membrane permeable molecule is the cell membrane permeable molecule of the present invention described above.
  • the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
  • the other constitution of the peptide complex is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected depending on the intended purpose.
  • a luminescent substance such as a fluorescent substance, a dye, a radioactive substance, and the like. Examples include drugs, toxins, nucleic acids, amino acids, sugars, lipids, various polymers and the like. These may be used alone or in combination of two or more.
  • the fluorescent substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include fluorescent dyes such as fluorescein, rhodamine, coumarin, pyrene and cyanine.
  • the other constitution can be attached to the above-mentioned peptide, for example, directly or via a linking group or the like.
  • the peptide complex of the present invention has excellent cell permeability and low toxicity to the living body. Therefore, for example, by preparing a peptide complex library containing a random sequence and performing screening, it is useful to have excellent cell permeability, low toxicity to a living body, and high affinity for a target substance.
  • the amino acid sequence can be specified.
  • the method for producing a peptide complex of the present invention includes at least an introduction step of introducing the cell membrane permeable molecule of the present invention into a peptide, and further includes other steps as necessary.
  • the introduction step is a step of introducing the cell membrane-permeable molecule of the present invention into a peptide (hereinafter, may be referred to as "binding", “inserting", or “linking”).
  • a peptide hereinafter, may be referred to as "binding", "inserting", or “linking”
  • cell membrane permeability can be imparted to the peptide.
  • the cell membrane permeable molecule having the above-mentioned cyclization group is introduced, the cyclization of the peptide and the impartation of the cell membrane permeability to the peptide can be performed at the same time.
  • the cell membrane-permeable molecule may be introduced into at least one peptide in the reaction product, but it is preferable that the cell membrane-permeable molecule is introduced into all the peptides.
  • the peptide is the same as that described in the ⁇ Peptide> section of the above (peptide complex).
  • the peptide used in the introduction step may be an embodiment of a peptide library.
  • the peptide in this peptide library is in a state in which the cell membrane-permeable molecule of the present invention has not been introduced.
  • the cell membrane permeable molecule is the cell membrane permeable molecule of the present invention described above.
  • the method of introduction is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a method of reacting a peptide linking group in the cell membrane permeable molecule with a reactive amino acid residue in the peptide For example, a method of reacting the cell membrane permeable molecule with the peptide in the presence of a reducing agent can be mentioned.
  • the reducing agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tris (2-carboxyethyl) phosphine hydrochloride and the like.
  • the conditions such as the temperature and time of the reaction are not particularly limited and may be appropriately selected depending on the intended purpose.
  • the method for forming the peptide complex in the form of a salt is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out.
  • a known chemical synthesis technique can be appropriately selected and carried out.
  • the same method as the method described in 1) can be mentioned.
  • the method for confirming whether or not the obtained peptide complex has a desired structure is not particularly limited, and a known analytical method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance spectroscopy can be selected. Analytical methods such as method, carbon 13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy, and liquid chromatography can be mentioned.
  • the peptide library of the present invention contains at least the peptide complex of the present invention, and further contains other configurations as required. That is, the peptide library of the present invention contains a peptide complex into which the cell membrane permeable molecule of the present invention has been introduced.
  • the peptide library may consist only of the peptide complex of the present invention, or may contain a peptide into which the cell membrane permeable molecule has not been introduced.
  • the peptide library can be produced in the same manner as described above (method for producing a peptide complex).
  • the method for screening a functional peptide of the present invention includes at least a step of screening a functional peptide using the peptide library of the present invention, and further includes other steps as necessary.
  • the screening method is not particularly limited as long as the peptide library of the present invention is used, and a known method can be appropriately selected. For example, a desired target substance and the peptide library are mixed, a bound peptide complex (for example, an RD complex) is selected, RNA is dissociated from the RD complex, and DNA is prepared from the RNA.
  • a screening method based on the ribosome display method which repeats the steps of transcribing to mRNA and then producing an RD complex library again to screen for a functional peptide having an affinity for the target substance, can be mentioned. Further, a screening method using a phage display method, an mRNA display method, a DNA display method, a one-bead one-compound method, or the like can also be mentioned.
  • the screening method may include an introduction step of introducing the cell membrane-permeable molecule of the present invention into the selected peptide in a process of repeating the screening step or the like.
  • the introduction step can be performed in the same manner as the ⁇ introduction step> in the above-mentioned (method for producing a peptide complex).
  • the method for improving the cell membrane permeability of a cell membrane-permeable molecule of the present invention includes at least a salt forming step, and further includes other steps as necessary. ..
  • the salt forming step in the method for improving membrane permeability of the present invention is a step of converting a cell membrane-permeable molecule having a structure represented by the following general formula (I) into a salt with an acid. (In the general formula (I), R represents a bond).
  • the method for producing the cell membrane-permeable molecule having the structure represented by the general formula (I) is not particularly limited, and a known chemical synthesis technique can be appropriately selected and carried out. For example, it can be produced by appropriately selecting a known chemical synthesis technique with reference to the method described in US Pat. No. 7,862,807.
  • the method of using a cell membrane-permeable molecule having a structure represented by the general formula (I) as a salt with an acid is not particularly limited, and is a known chemical synthesis technique. Can be selected as appropriate. For example, a method of purifying a crude product of a cell membrane-permeable molecule that is not in the form of a salt by HPLC using a mobile phase according to the type of the target salt to obtain a cell membrane-permeable molecule in the form of the target salt.
  • a method of passing a cell membrane-permeable molecule in the form of a salt through an anion exchange resin in which a counterion is replaced with an ion of the target salt and eluting the molecule to obtain a cell membrane-permeable molecule in the form of the target salt, etc. Can be mentioned.
  • the cell membrane-permeable molecule produced in the salt-forming step of the method for improving membrane permeability of the present invention is a salt with an acid.
  • the type of the acid is not particularly limited and may be appropriately selected depending on the intended purpose. For example, trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, mesylic acid, tosylic acid, tartrate acid, citric acid, acetic acid. , Various amino acids and the like. Among these, acids other than acidic amino acids are preferable, and acids having a pKa (acid dissociation constant) value of ⁇ 8.0 or more and 4.7 or less are more preferable.
  • the value of pK a1 which is the value of pK a in the first stage dissociation is taken as the value of pK a .
  • the acid having a pK a value of ⁇ 8.0 or more and 4.7 or less is not particularly limited and may be appropriately selected depending on the intended purpose.
  • trifluoroacetic acid (-0.3), hydrochloric acid ( -8.0), sulfuric acid (-3.0), nitric acid (-1.3), phosphoric acid (2.1 (pK a1 )), mesylic acid (-2.6), tosylic acid (-2.8) ), Tartrate acid (3.2 (pK a1 )), citric acid (3.1 (pK a1 )), acetic acid (4.7) and the like.
  • the number in parentheses after the acid name represents the value of pK a . In the case of the value of pK a1 , it is described as pK a1 after the numerical value.
  • the cell membrane-permeable molecule produced by the method for improving membrane permeability of the present invention has a structure represented by the above-mentioned general formula (I) and is a salt with an acid as long as the effect of the present invention is not impaired. In, it may have other configurations.
  • the method for confirming whether or not the cell membrane permeable molecule has a desired structure is not particularly limited, and a known analytical method can be appropriately selected.
  • mass spectrometry and proton nuclear magnetic resonance spectroscopy can be selected.
  • the method for improving membrane permeability of the present invention it is possible to improve the cell membrane permeability of a cell membrane-permeable molecule having a small molecular weight. Therefore, by introducing the cell membrane-permeable molecule into an active ingredient such as a peptide, nucleic acid, or protein, the efficiency of incorporation into these cells can be improved.
  • the cell membrane permeable molecule is preferably one introduced into a peptide.
  • the present invention is characterized in that the peptide complex into which a cell membrane permeable molecule having a structure represented by the general formula (I) is introduced is used as a salt with an acid. It is also related to the method of improving cell membrane permeability.
  • the method for improving the cell membrane permeability of the peptide complex includes at least a salt forming step, and further includes other steps as necessary.
  • the salt forming step is a step of converting a peptide complex into which a cell membrane-permeable molecule having a structure represented by the general formula (I) into a salt with an acid, and the cell membrane-permeable molecule becomes a peptide. Except for the fact that it has been introduced, it can be carried out in the same manner as the salt forming step in the method for improving the cell membrane permeability of the cell membrane permeable molecule described above.
  • the peptide complex contains at least a peptide and a cell membrane-permeable molecule having a structure represented by the general formula (I), and further contains other configurations as necessary.
  • the peptide in the method for improving the cell membrane permeability of the peptide complex is the same as that described in the item of ⁇ Peptide> in the above-mentioned (peptide complex).
  • the cell membrane-permeable molecule in the method for improving the cell membrane permeability of the peptide complex is a cell membrane-permeable molecule having a structure represented by the general formula (I) in the above-mentioned method for improving the membrane permeability.
  • the structure of the site that reacts with the reactive amino acid residue in the peptide may change.
  • binding also referred to as “binding”, “inserting”, or “linking”
  • linking The method for introducing the cell membrane-permeable molecule into the peptide (hereinafter, also referred to as “binding”, “inserting”, or “linking”) is described in the above-mentioned (method for producing a peptide complex). It is the same as that described in the item of ⁇ Introduction process>.
  • the peptide used in the peptide complex may be in the form of a peptide library. That is, as the peptide before the introduction of the above-mentioned cell membrane permeable molecule, a peptide in the form of a peptide library can also be used.
  • the other steps in the method for improving the cell membrane permeability of the peptide complex are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose.
  • Example A1 Synthesis of cell membrane permeable molecule G3-DHX trifluoroacetic acid salt
  • G3-DHX trifluoroacetic acid salt which is an example of a cell membrane permeable molecule, was synthesized as follows.
  • the compound C3 represented by the above structural formula was synthesized by the above reaction formula. Specifically, a methylene chloride solution (15 mL) of compound C1 (500 mg, 0.42 mmol) was cooled to 0 ° C., and compound C2 (manufactured by Tokyo Kasei Kogyo Co., Ltd., product number A2293) (117.5 mg, 0.504 mmol) was cooled. ), 1-Hydroxybenzotriazole (HOBT) (85.1 mg, 0.630 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC / HCl) (120 mg, 0.630 mmol).
  • HOBT 1-Hydroxybenzotriazole
  • EDC / HCl 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
  • the mixture was stirred at 25 ° C. for 17 hours.
  • the identification data of the compound C3 by 1H NMR were as follows.
  • the compound C4 represented by the above structural formula was synthesized by the above reaction formula. Specifically, nitrogen gas bubbling was performed on a solution consisting of compound C3 (533 mg, 0.379 mmol), compound DBXA (188 mg, 0.568 mmol) and THF (40 mL) to create a nitrogen atmosphere. A copper sulfate aqueous solution (200 mM; 1.89 mL, 0.379 mmol) and an ascorbic acid sodium aqueous solution (100 mM; 7.58 mL, 0.758 mmol) were added thereto, and the mixture was stirred at 25 ° C. for 3 hours.
  • the compound G3-DCX represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound C4 (497 mg, 0.286 mmol) and a 4N dioxane hydrochloride solution (30 mL) were mixed, and the mixture was stirred at 25 ° C. for 46 hours. After the reaction, a white solid was precipitated. The supernatant was removed to obtain a crude product of compound G3-DCX. Purification by preparative HPLC (high performance liquid chromatography) gave compound G3-DCX as a white solid (116 mg, 0.110 mmol, yield 54%).
  • the identification data of the compound G3-DCX by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry were as follows.
  • MALDI-TOF MS C 24 H 51 N 14 O 8 Calculated value ([M + H] + ) 1047.502, measured value 1047.953
  • the cell membrane permeable molecule G3-DHX trifluoroacetate represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound G3-DCX (506 mg, 0.480 mmol) and H2O (6 mL) were mixed and stirred at 80 ° C. for 2 hours. The reaction solution was concentrated to obtain a crude product of compound G3-DHX. Purification by HPLC (high performance liquid chromatography) using a mobile phase containing trifluoroacetic acid (TFA) gave the cell membrane permeable molecule G3-DHX trifluoroacetate as a white solid (224.6 mg, 0.169 mmol, Yield 35%).
  • HPLC high performance liquid chromatography
  • TSA trifluoroacetic acid
  • the identification data of the G3-DHX trifluoroacetic acid salt by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry were as follows. MALDI-TOF MS C 42 H 74 N 16 O 13 Calculated value ([M + H] + ) 1011.153, measured value 1011.550.
  • G3-DHX hydrochloride which is an example of a cell membrane permeable molecule, was prepared as follows.
  • An anion exchange resin in which G3-DHX trifluoroacetate (19.7 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (5 mg / mL) and counterions were replaced with chloride ions (19.7 mg). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The resulting solution was lyophilized to give G3-DHX hydrochloride (17.8 mg) as a white to off-white powder.
  • G3-DHX nitrate which is an example of a cell membrane permeable molecule
  • An anion exchange resin (diamond) obtained by dissolving G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 in ultrapure water (2 mg / mL) and substituting counterions with nitrate ions. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX nitrate (0.9 mg) as a white to off-white powder.
  • G3-DHX sulfate which is an example of a cell membrane permeable molecule
  • An anion exchange resin (diamond) obtained by dissolving G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 in ultrapure water (2 mg / mL) and substituting counterions with sulfate ions. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX sulfate (1.0 mg) as a white to off-white powder.
  • G3-DHX phosphate which is an example of a cell membrane permeable molecule
  • An anion exchange resin in which G3-DHX trifluoroacetate (1.5 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and counterions were replaced with phosphate ions (2 mg / mL). I was familiar with Diaion PA306S). Ultrapure water was used for elution. The resulting solution was lyophilized to give G3-DHX phosphate (1.4 mg) as a white to off-white powder.
  • G3-DHX mesylate which is an example of a cell membrane permeable molecule
  • G3-DHX tosylate which is an example of a cell membrane permeable molecule
  • G3-DHX tartrate which is an example of a cell membrane permeable molecule
  • An anion exchange resin (diamond) in which G3-DHX trifluoroacetate (2.2 mg) synthesized in the same manner as in Example A1 was dissolved in ultrapure water (2 mg / mL) and the counterion was replaced with tartrate ion. Ion PA306S). Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain G3-DHX tartrate (1.8 mg) as a pale yellow powder.
  • G3-DHX citrate which is an example of a cell membrane permeable molecule
  • Example A10 Synthesis of peptide complex G3-DCX-P trifluoroacetic acid salt
  • a peptide complex G3-DCX-P trifluoroacetate which is an example of a complex of a cell membrane permeable molecule and a peptide, was synthesized as follows.
  • the resin on which the peptide was formed was converted to TFA / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (volume ratio)). After soaking for 3 hours, the peptide was excised from the resin. The obtained peptide was purified by HPLC and freeze-dried to obtain a peptide having the above sequence (hereinafter, may be referred to as "P"; see the following structural formula).
  • the identification data of the peptide P by electrospray ionization mass spectrometry (ESI-MS) were as follows. ESI-MS C 72 H 92 N 16 O 22 O 3 Calculated value ([M + 2H] 2+ ) 815.295, measured value 814.67
  • the peptide complex G3-DCX-P trifluoroacetic acid salt represented by the above structural formula was synthesized by the above reaction formula. Specifically, the peptide P (11.0 mg, 6.75 ⁇ mol) synthesized above was dissolved in a mixed solution of 20 mM ammonium bicarbonate buffer (11.8 mL) and acetonitrile (MeCN) (1.7 mL). To this solution was added tris (2-carboxyethyl) phosphine (TCEP) (500 mM in H2O ; 14.8 ⁇ L, 7.4 ⁇ mol) and stirred at 25 ° C. for 15 minutes.
  • TCEP (2-carboxyethyl) phosphine
  • Example A11 Preparation of peptide complex G3-DCX-P hydrochloride
  • the peptide complex G3-DCX-P trifluoroacetate (1.1 mg) synthesized in the same manner as in Example A10 was dissolved in ultrapure water (0.5 mg / mL), and the counterion was converted to chloride ion.
  • a substituted anion exchange resin (Diaion PA306S) was added. After shaking for 1 hour, the anion exchange resin was filtered off and eluted with ultrapure water. The obtained solution was freeze-dried to obtain G3-DCX-P hydrochloride (0.6 mg) as a white to off-white powder.
  • mice Toxicity evaluation of cell membrane permeable molecule
  • a 7-week-old mouse female, 15 to 25 g, Charles River Laboratories, Japan
  • a single dose was administered, and general symptoms and life / death were observed over time for 30 minutes after administration, and then general symptoms and life / death were continuously observed at a pace of once / day for 7 days.
  • Various acid salts of the cell membrane permeable molecule G3-DHX and placebo buffer control were administered to 3 mice in each group and observed.
  • body weight was measured on the day of administration and 1, 2, and 7 days after administration to investigate a significant increase or decrease in body weight. Whenever a fatal case was found, an autopsy was performed.
  • G3-DHX tosylate, G3-DHX mesylate, G3-DHX phosphate, G3-DHX citrate, or G3-DHX tartrate was administered at a dose of 10 mg / kg.
  • no symptoms such as walking disorders or respiratory irregularities or significant increase or decrease in body weight were observed in any of the individuals.
  • survival was confirmed for 7 days after administration.
  • G3-DHX trifluoroacetic acid salt no significant increase or decrease in body weight was observed, such as gait disturbance and respiratory irregularity, regardless of the dose of 10 mg / kg or 20 mg / kg.
  • the individual to which G3-DHX hydrochloride was administered had a slight gait disorder immediately after the administration, but recovered after 3 minutes. No symptoms were observed for the following 7 days, and there was no significant increase or decrease in body weight.
  • Individuals treated with G3-DHX sulfate showed respiratory irregularities immediately after administration, but recovered 3 minutes later. Abnormal symptoms were observed for the following 7 days, and there was no significant increase or decrease in body weight.
  • the individual to which G3-DHX acetate was administered at a dose of 10 mg / kg showed tremor immediately after the administration and died 2 minutes after the administration.
  • G3-DHX acetate has the highest toxicity after single intravenous administration to mice and is comparable to octaarginine (see below for the structural formula) (Marcel Grogg et al., Cell Penetration, Herbicidal Activity, and in- vivo-Toxicity of Oligo-Arginine Radivatives and of Novel Guandinium-Rich Compounds Dived from the Biopolymer Cyanophycin, Heil.
  • G3-DHX trifluoroacetate, G3-DHX hydrochloride, G3-DHX sulfate, G3-DHX phosphate, G3-DHX mesylate, G3-DHX tosylate, G3-DHX tartrate. , And G3-DHX citrate were found to be less toxic than G3-DHX acetate. From the pK a value of each acid and the toxicity result of each acid salt, it can be seen that acetic acid having a pK a value of 4.7 is the most toxic, and an acid having a pK a value smaller than acetic acid is less toxic. ..
  • the cell membrane permeable molecule becomes less toxic by using a salt with an acid having a pKa value of less than 4.7.
  • a salt with an acid having a pKa value of less than 4.7 In addition, with regard to G3-DHX hydrochloride and G3-DHX sulfate, slight symptoms were observed immediately after administration, and from the viewpoint of low toxicity, salts with acids exceeding pK a -8.0 were observed. It has been found to be preferable, and salts with acids above pK a -3.0 are more preferred.
  • Test Example A2 Evaluation of cell membrane permeability of peptide complex
  • HeLa cells Human cervix adenocarcinoma cell were cultured for 2 hours under the above conditions.
  • FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum), 2% (v / v) GlutaMax (manufactured by Thermo Fisher) added) was used. Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected.
  • the peptide P synthesized in Example A10 hardly permeates the membrane 2 hours after the start of culture, whereas the peptide complex G3-DCX-P trifluoroacetate synthesized in Example A10 has no membrane permeation. It was permeated through the membrane. From this, it was demonstrated that the cell membrane permeability was obtained by introducing the cell membrane permeability molecule of the present invention into the peptide.
  • Test Example A3 Evaluation of cell membrane permeability of peptide complex hydrochloride 5% in a cell culture medium containing 2 ⁇ M of either the peptide complex G3-DCX-P trifluoroacetate synthesized in Example A10 or the peptide complex G3-DCX-P hydrochloride synthesized in Example A11.
  • HeLa cells Human cervix adenocarcinoma cell
  • FluoroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum), 2% (v / v) GlutaMax (manufactured by Thermo Fisher) added) was used. Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected.
  • the P hydrochloride was permeated to the same extent. From this, it was demonstrated that even if the salt morphology of the peptide complex into which the cell membrane-permeable molecule of the present invention was introduced was changed, the same degree of cell membrane permeability was exhibited.
  • Example B1 Synthesis of cell membrane permeable molecule G3-DCX trifluoroacetic acid salt
  • ⁇ Synthesis of compound G3-DCX> -Synthesis of compound C1- In the same manner as in-Synthesis of compound C1 in Example A1 described above, compound C1 represented by the above-mentioned structural formula was synthesized.
  • Example B2 Synthesis of peptide complex G3-DCX-P trifluoroacetic acid salt
  • the peptide complex G3-DCX-P trifluoroacetic acid salt was synthesized in the same manner as in Example A10 described above.
  • Example B3 Preparation of peptide complex G3-DCX-P hydrochloride
  • the peptide complex G3-DCX-P hydrochloride was synthesized in the same manner as in Example A11 described above.
  • Example B4 Preparation of peptide complex G3-DCX-P acetate
  • the peptide complex G3-DCX-P acetate was synthesized in the same manner as in Reference Example A2 described above.
  • Example B5 Preparation of peptide complex G3-DCX-P nitrate
  • the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which counterions were replaced with nitrate ions. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P nitrate as a white to off-white powder.
  • anion exchange resin diaion PA306S
  • Example B6 Preparation of peptide complex G3-DCX-P sulfate
  • the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which counterions were replaced with sulfate ions. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P sulfate as a white to off-white powder.
  • anion exchange resin diaion PA306S
  • Example B7 Preparation of peptide complex G3-DCX-P phosphate
  • the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which counterions were replaced with phosphate ions. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P phosphate as a white to off-white color powder.
  • anion exchange resin diaion PA306S
  • Example B8 Preparation of peptide complex G3-DCX-P mesylate
  • the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with a mesylate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P mesylate as a colorless liquid.
  • anion exchange resin diaion PA306S
  • Example B9 Preparation of peptide complex G3-DCX-P tosylate
  • the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with tosylate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P tosylate as a white powder.
  • anion exchange resin diaion PA306S
  • Example B10 Preparation of peptide complex G3-DCX-P tartrate
  • the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water and passed through an anion exchange resin (diaion PA306S) in which the counterion was replaced with tartrate ion. rice field. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P tartrate as a pale yellow powder.
  • anion exchange resin diaion PA306S
  • Example B11 Preparation of peptide complex G3-DCX-P citrate
  • the peptide complex G3-DCX-P trifluoroacetate synthesized in the same manner as in Example B2 was dissolved in ultrapure water to form an anion exchange resin (diaion PA306S) in which the counterion was replaced with citrate ion. got through. Ultrapure water was used for elution. The obtained solution was freeze-dried to obtain a peptide complex G3-DCX-P citrate as a pale yellow powder.
  • anion exchange resin diaion PA306S
  • Example B12 Synthesis of peptide complex G3-P trifluoroacetate
  • the compound K1 represented by the following structural formula was synthesized according to the method described in The Journal of Organometallic Chemistry 2013, 17-24.
  • “Fmoc” in the structural formula of a compound represents "9-fluorenylmethyloxycarbonyl group”.
  • the compound K3 represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound K1 (50 mg, 0.159 mmol) was dissolved in methylene chloride (5 mL) and cooled to 0 ° C. Here, compounds K2 (284 mg, 0.239 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride synthesized by the same method as described in US Pat. No. 7,862,807 are used. Salt (EDC / HCl) (45.8 mg, 0.239 mmol) and 1-hydroxybenzotriazole (HOBT) (32.2 mg, 0.239 mmol) were added and stirred at 0 ° C. for 12 hours.
  • EDC / HCl 45.8 mg, 0.239 mmol
  • HOBT 1-hydroxybenzotriazole
  • the identification data of the compound K4 by 1H NMR were as follows.
  • the cell membrane permeable molecule G3 represented by the above structural formula was synthesized by the above reaction formula. Specifically, a dioxane hydrochloride solution (4N; 1.0 mL) was added to compound K4 to prepare a solution, and the mixture was stirred at 25 ° C. for 2 hours. The precipitated white solid was obtained by centrifugation and further washed with diethyl ether (3 times at 3 mL) to obtain a cell membrane permeable molecule G3 (90 mg, quantitative yield).
  • the identification data of the cell membrane-permeable molecule G3 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows. MALDI-TOF MS C 24 H 50 N 14 O 8 Calculated value ([M + H] + ) 663.401, measured value 663.402
  • the resin on which the peptide was formed was used as trifluoroacetic acid (TFA) / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (92.5 / 2.5 / 2.5 / 2.5).
  • TFA trifluoroacetic acid
  • the peptide was excised from the resin by immersing it in (volume ratio)) for 3 hours.
  • the obtained peptide (55.0 mg, 30.0 ⁇ mol) was dissolved in DMF (2.5 mL), and 1,3-dibromo-2-propanol (20 mM DMF solution; 1.5 mL, 30 ⁇ mol) and N-methylmorpholine ( 10 mM DMF solution; 6.0 mL, 60 ⁇ mol) was added and stirred at 25 ° C. for 1 hour. Diethyl ether (100 mL) was added thereto to remove the supernatant, and the residue was washed with diethyl ether (100 mL) to obtain compound K5 as a white solid (54.6 mg, 28.9 ⁇ mol, yield 97%). ..
  • CMLYIVPYFSVGC in the structural formula of compound K5 represents the amino acid sequence of the peptide.
  • the identification data of the compound K5 by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows. MALDI-TOF MS C 94 H 128 N 15 O 20 S 3 + Calculated value ([M + H] + ) 1882.862, Measured value 1883.140
  • the compound Fmoc-G3-P represented by the above structural formula was synthesized by the above reaction formula. Specifically, compound K5 (30 mg, 0.0159 mmol), cell membrane permeable molecule G3 (52.7 mg, 0.0796 mmol), DMF (0.5 mL) and water (0.05 mL) were mixed and 25 ° C. Was stirred for 24 hours. Diethyl ether (5 mL) was added and centrifugation was performed to remove the supernatant. The residue was washed with diethyl ether (3 times with 3 mL) to obtain a crude product of compound Fmoc-G3-P. The compound Fmoc-G3-P (3.8 mg, 0.00150 mmol, yield 9%) was obtained by purification by reverse phase HPLC (high performance liquid chromatography) and freeze-drying.
  • HPLC high performance liquid chromatography
  • the peptide complex G3-P trifluoroacetic acid salt represented by the above structural formula was synthesized by the above reaction formula. Specifically, the compound Fmoc-G3-P synthesized above and a 20% piperidine / DMF solution (1.0 mL) were mixed and stirred at 25 ° C. for 2 hours using a vortex mixer. The solvent was volatilized by spraying nitrogen gas. The residue was washed with diethyl ether (1 mL 3 times).
  • fluorescein isothiocyanate (FITC) (0.8 mg, 0.002 mmol), diisopropylethylamine (DIPEA) (0.022 mL, 0.0124 mmol) and DMF (0.5 mL) are mixed and stirred at 25 ° C. for 20 hours. did. Diethyl ether (15 mL) was added and centrifugation was performed to remove the supernatant. The residue was washed with diethyl ether (twice at 5 mL) to give the peptide complex G3-P crude product.
  • DIPEA diisopropylethylamine
  • DMF 0.5 mL
  • the peptide complex G3-P trifluoroacetic acid salt (1.6 mg, 0.00059 mmol, yield 40%) was obtained as a white solid by purification by HPLC using a mobile phase containing TFA and freeze-drying.
  • the identification data of the peptide complex G3-P trifluoroacetic acid salt by MALDI-TOF MS were as follows. MALDI-TOF MS C 124 H 177 N 30 O 30 S 4 Calculated value ([M + H] + ) 2694.212, measured value 2694.208
  • CMLYIVPYFSVGC in the following structural formula represents the amino acid sequence of the peptide.
  • CMLYIVPYFSVGC in the following structural formula represents the amino acid sequence of the peptide.
  • Test Example B1 Evaluation of cell membrane permeability of peptide complex
  • a cell culture medium under the conditions of 5% (v / v) CO 2 and 37 ° C.
  • the cell membrane permeability of the peptide complex increases in the order of G3-P, G6-P and G9-P, and the larger the number of dendritic skeletons having guanidyl groups, the more the membrane.
  • the permeability was high.
  • the cell membrane permeability of G3-P trifluoroacetic acid salt 4 hours after the start of culture was significantly higher than that of G6-P and G9-P. From this, it was clarified that G3-P exhibits cell membrane permeability equal to or higher than that of G6-P and G9-P by taking the form of a salt with an acid.
  • Molecules with a molecular weight of more than 4,000 Da may cause immunogenicity, and it is desirable that the cell membrane-permeable molecule used in combination with a peptide having a molecular weight of more than 1,000 has a smaller molecular weight. Therefore, in the manufacture of pharmaceutical products, it is preferable to use G3-P having a smaller molecular weight than G6-P or G9-P. Further, as described above, by forming G3-P in the form of a salt with an acid, it is possible to remarkably improve the membrane permeability, and overcome the problem of lack of cell membrane permeability that G3-P has been a problem. It can be said that it was possible.
  • the method for improving the cell membrane permeability of the cell membrane-permeable molecule of the present invention is a useful method capable of improving the cell membrane permeability of the peptide complex containing the peptide as a pharmaceutical product.
  • Examples of aspects of the present invention include the following.
  • ⁇ 1> A cell membrane-permeable molecule having a structure represented by the following general formula (I) and having a pKa value of less than 4.7 as a salt with an acid. (In the general formula (I), R represents a bond).
  • ⁇ 2> The cell membrane-permeable molecule according to ⁇ 1>, wherein the R portion of the structure represented by the general formula (I) is bonded to a heteroatom-containing group.
  • ⁇ 3> The cell membrane-permeable molecule according to ⁇ 2>, wherein the heteroatom content in the heteroatom-containing group is 10% or more.
  • ⁇ 4> The above-mentioned ⁇ 2> to ⁇ 3>, wherein the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. It is a cell membrane permeable molecule.
  • ⁇ 5> The cell membrane-permeable molecule according to any one of ⁇ 2> to ⁇ 4>, wherein the heteroatom-containing group contains a repeating unit.
  • ⁇ 6> The cell membrane permeable molecule according to ⁇ 5>, wherein the repeating unit is an alkylene oxide.
  • the acid having a pKa value of less than 4.7 is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, mesylic acid, tosylic acid, tartaric acid and citric acid.
  • ⁇ 9> A peptide complex comprising the peptide and the cell membrane-permeable molecule according to any one of ⁇ 1> to ⁇ 8>.
  • a peptide library comprising the peptide complex according to ⁇ 9>.
  • a method for producing a peptide complex which comprises introducing the cell membrane-permeable molecule according to any one of ⁇ 1> to ⁇ 8> into a peptide.
  • a method for screening a functional peptide which comprises screening a functional peptide using the peptide library according to ⁇ 10>.
  • ⁇ 14> The method for screening a functional peptide according to ⁇ 13>, which comprises introducing the cell membrane permeable molecule according to any one of ⁇ 1> to ⁇ 8> into the peptide.
  • a method for improving the cell membrane permeability of a cell membrane-permeable molecule which comprises using a cell membrane-permeable molecule having a structure represented by the following general formula (I) as a salt with an acid. (In the general formula (I), R represents a bond).
  • R represents a bond.
  • ⁇ 17> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to ⁇ 16>, wherein the heteroatom content in the heteroatom-containing group is 10% or more.
  • the hetero atom in the hetero atom-containing group is at least one selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom and a phosphorus atom. It is a method for improving the cell membrane permeability of a cell membrane permeability molecule.
  • ⁇ 19> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to any one of ⁇ 16> to ⁇ 18>, wherein the heteroatom-containing group contains a repeating unit.
  • ⁇ 20> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to ⁇ 19>, wherein the repeating unit is an alkylene oxide.
  • ⁇ 21> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to ⁇ 20>, wherein the alkylene oxide is at least one selected from the group consisting of methylene oxide, ethylene oxide, and propylene oxide.
  • ⁇ 22> The method for improving the cell membrane permeability of the cell membrane-permeable molecule according to any one of ⁇ 15> to ⁇ 21>, wherein the cell membrane-permeable molecule is introduced into a peptide.

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Abstract

L'invention concerne une molécule pénétrant la membrane cellulaire qui possède une structure représentée par la formule générale (I), et qui consiste en un sel d'un acide de valeur ｐＫａ inférieure à ４,７. L'invention concerne également un procédé d'amélioration de pénétration de membrane cellulaire de molécule pénétrant la membrane cellulaire qui inclut une étape au cours de laquelle la molécule pénétrant la membrane cellulaire qui possède une structure représentée par la formule générale (I), est transformée en sel d'acide.
PCT/JP2021/034020 2020-09-23 2021-09-16 Molécule pénétrant la membrane cellulaire ainsi qu'application de celle-ci, et procédé d'amélioration de pénétration de membrane cellulaire de molécule pénétrant la membrane cellulaire Ceased WO2022065172A1 (fr)

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JP2020159040A JP2023162461A (ja) 2020-09-23 2020-09-23 細胞膜透過性分子の細胞膜透過性の向上方法
JP2020159038A JP2023162460A (ja) 2020-09-23 2020-09-23 細胞膜透過性分子及びその利用
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115960162A (zh) * 2023-02-02 2023-04-14 山西医科大学 一种膜扰动抗肿瘤纳米脂肽及其制备方法和应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080221043A1 (en) * 2006-08-23 2008-09-11 Vanderbilt University Dendritic molecular intracellular transporters and methods of making and using same
US7862807B2 (en) * 2002-07-19 2011-01-04 University Of California, San Diego Dendrimers as molecular translocators
WO2019213662A1 (fr) * 2018-05-04 2019-11-07 Ohio State Innovation Foundation Motifs non peptidiques de pénétration cellulaire
JP2020502051A (ja) * 2016-11-09 2020-01-23 オハイオ・ステイト・イノベーション・ファウンデーション ジスルフィド含有細胞膜透過ペプチド並びにその製造方法及び使用方法
WO2020195303A1 (fr) * 2019-03-28 2020-10-01 株式会社カネカ Complexe peptidique et son procédé de production, et utilisation dudit complexe peptidique
WO2020195302A1 (fr) * 2019-03-28 2020-10-01 株式会社カネカ Molécule de liaison pour produire un peptide en forme d'anneau perméable à la membrane cellulaire et son utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7862807B2 (en) * 2002-07-19 2011-01-04 University Of California, San Diego Dendrimers as molecular translocators
US20080221043A1 (en) * 2006-08-23 2008-09-11 Vanderbilt University Dendritic molecular intracellular transporters and methods of making and using same
JP2020502051A (ja) * 2016-11-09 2020-01-23 オハイオ・ステイト・イノベーション・ファウンデーション ジスルフィド含有細胞膜透過ペプチド並びにその製造方法及び使用方法
WO2019213662A1 (fr) * 2018-05-04 2019-11-07 Ohio State Innovation Foundation Motifs non peptidiques de pénétration cellulaire
WO2020195303A1 (fr) * 2019-03-28 2020-10-01 株式会社カネカ Complexe peptidique et son procédé de production, et utilisation dudit complexe peptidique
WO2020195302A1 (fr) * 2019-03-28 2020-10-01 株式会社カネカ Molécule de liaison pour produire un peptide en forme d'anneau perméable à la membrane cellulaire et son utilisation

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115960162A (zh) * 2023-02-02 2023-04-14 山西医科大学 一种膜扰动抗肿瘤纳米脂肽及其制备方法和应用

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