WO2022054625A1 - Pyrrole-imidazole polyamide having improved nuclear transportation properties - Google Patents

Pyrrole-imidazole polyamide having improved nuclear transportation properties Download PDF

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WO2022054625A1
WO2022054625A1 PCT/JP2021/031757 JP2021031757W WO2022054625A1 WO 2022054625 A1 WO2022054625 A1 WO 2022054625A1 JP 2021031757 W JP2021031757 W JP 2021031757W WO 2022054625 A1 WO2022054625 A1 WO 2022054625A1
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polyamide
inhibitor
sox2i
peptide
pharmaceutical composition
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French (fr)
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弘 杉山
ガネッシュ パンディアン ナマシバヤム
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国立大学法人京都大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A61K31/787—Polymers containing nitrogen containing heterocyclic rings having nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present disclosure relates to a pyrrole-imidazole polyamide having improved nuclear transferability and a pharmaceutical composition containing the pyrrole-imidazole polyamide.
  • Pyrrole-imidazole polyamide (hereinafter, also referred to as "PI polyamide” or “PIP”) is a synthetic ligand composed of N-methylpyrrole and N-methylimidazole linked by an amide bond, and is a base in the accessory groove of DNA. It is a compound that binds sequentially selectively.
  • PI polyamide ⁇ -aminobutyric acid ( ⁇ ) that connects two antiparallel PI polyamide chains and the C-terminus of one strand of the duplex and the N-terminus of the other strand.
  • ⁇ -aminobutyric acid ⁇
  • P / I pairs are C (cytosine) / G (guanine) base pairs and P / P pairs are A (adenine) / T (thymine) due to facing pyrrole (P) and imidazole (I) pairs.
  • the TA base pair can be specifically bound to any double-stranded DNA sequence by recognizing the G / C base pair by the I / P pair (Non-Patent Document 1). Therefore, by designing PI polyamides in which pyrrole and imidazole are linked in various orders, any region on the genome can be targeted.
  • PI polyamide To achieve biological activity in living cells, PI polyamide must be delivered into the cell nucleus and bound to nuclear DNA. However, some PI polyamide compounds have insufficient nuclear accumulation. Therefore, in recent years, the effects of the structural characteristics of PI polyamide on nuclear delivery have been extensively studied. In general, short-chain hairpin-type PI polyamides containing about 8 pyrrole / imidazole rings are suitable for efficient nuclear delivery, but cell uptake efficiency of long-chain PI polyamides containing more than 10 pyrrole / imidazole rings. Is low. On the other hand, long-chain PI polyamides are advantageous for binding affinity and sequence selectivity for DNA. Therefore, there is a demand for long-chain PI polyamides capable of stable and efficient nuclear delivery.
  • Non-Patent Document 2 It has not been applied to long-chain PI polyamides.
  • oligoarginine is widely used as a polymer cell delivery tool including proteins, nucleic acids, and liposomes. It has been reported that oligoarginine used as a delivery vector contains 6 to 12 arginine residues, and that a small number of arginine residues does not show efficient cell uptake (Non-Patent Documents 3 and 4). ).
  • the present disclosure is intended to provide PI polyamide with improved nuclear transferability.
  • the present inventors focused on the possibility of utilizing oligoarginine.
  • many coupling steps are required in solid phase synthesis, which complicates the synthesis work and makes the steps complicated.
  • the yield would decrease.
  • the arginine residue is positively charged under physiological pH, it was predicted that non-selective interaction with the negatively charged DNA phosphate skeleton would occur, impairing the sequence selectivity of PI polyamide. ..
  • the PI polyamide conjugated with a peptide containing 1 to 5 arginine residues of the present disclosure has significantly increased cell uptake efficiency and nuclear accumulation as compared with PI polyamide not conjugated with the arginine peptide. Furthermore, the concentration of the PI polyamide required for suppression of the target gene was significantly reduced. As described above, the PI polyamide conjugated with the arginine peptide of the present disclosure has increased cell membrane permeability, and the amount accumulated in the cell nucleus is increased, thereby expanding the use of PI polyamide in living cells. Furthermore, by using the PI polyamide of the present disclosure, it is possible to develop an efficient gene expression regulator, cell differentiation agent, and therapeutic agent for cancer, genetic disease, lifestyle-related disease and the like.
  • FIG. 1-1 shows the chemical structure of the PI polyamide used in the examples.
  • the legend of the schematic diagram shown in FIG. 1-1 is shown.
  • FIG. 2 shows the results of flow cytometric analysis for assessing cell uptake of SOX2i-TAMRA and SOX2i-R3-TAMRA.
  • FIG. 3 is a fluorescence image of HeLa cells treated with SOX2i-TAMRA or SOX2i-R3-TAMRA. The scale bar indicates 50 ⁇ m.
  • FIG. 4 shows the results of the electrophoretic mobility shift assay of SOX2i, SOX2i-R3, and Ctrl-R3.
  • FIG. 5 shows relative SOX2 RNA expression levels in 201B7 cells calculated using RT-qPCR. The average values calculated from the three culture wells are shown in a bar graph, and the individual values are represented by black dots. Each value is normalized to the mean value calculated from the control sample. HPRT1 and 18S were used as housekeeping genes in (A) and (B), respectively.
  • FIG. 6 shows the relative mRNA levels of PGC-1 ⁇ and PGC-1 ⁇ in PIP-introduced cells conjugated with Arg3 and Bi.
  • FIG. 7 shows the activation of mitochondria in low CD44 CD8 positive T cells by the addition of PIP conjugated with Arg3 and Bi.
  • Arg-BiR indicates a conjugate of PIP, Bi and Arg3 (Bi-R-Arg3)
  • BiR indicates a conjugate of PIP and Bi
  • Bi only indicates a bromodomain inhibitor. Indicated, "R only” indicates PIP.
  • FIG. 8 shows the activation of mitochondria in high CD44 CD8 positive T cells by the addition of PIP conjugated with Arg3 and Bi.
  • FIG. 9 schematically shows the schedule of the in vivo experiment of Example 5.
  • FIG. 10 shows the measurement result of the tumor size in Example 5.
  • the horizontal axis shows the number of days elapsed after tumor transplantation, and the vertical axis shows the tumor size.
  • FIG. 10 shows the measurement result of the tumor size in Example 5.
  • the horizontal axis shows the number of days elapsed after tumor transplantation, and the vertical axis shows the tumor size.
  • FIG. 11 shows the survival rate of mice in Example 5.
  • the horizontal axis shows the number of days elapsed after tumor transplantation, and the vertical axis shows the survival rate (%).
  • Ctrl IgG indicates an anti-PD-L1 monoclonal antibody isotype control-only administration group
  • PD-L1 indicates an anti-PD-L1 monoclonal antibody monotherapy group
  • PD-L1 + Bi-R-Arg3 Indicates a combination treatment group of an anti-PD-L1 monoclonal antibody and a conjugate of PIP, Bi and Arg3
  • Bi-R-Arg3 indicates a group of administration of only the conjugate of PIP, Bi and Arg3.
  • Bi indicates the administration group of the bromodomain inhibitor only
  • R indicates the administration group of PIP only.
  • PI Polyamide PI polyamide generally contains two antiparallel oriented chains consisting of N-methylpyrrole (P) residues and N-methylimidazole (I) residues, where P and I are amide-bonded to each other.
  • C ( O) -NH- "is linked [Trauger et al, Nature, 382, 559-61 (1996); White et al, Chem.Biol., 4,569-78 (1997); and Dervan, Bioorg. . Med. Chem., 9, 2215-35 (2001)].
  • anti-parallel means that two polyamide chains of PI polyamide are arranged in parallel, and the N- and C-termini of the two polyamide chains are oriented so as to be opposite to each other.
  • PI polyamide can take several forms, for example, hairpin type, cyclic, H-pin type, U-pin type and other PI polyamides are known.
  • the hairpin-type PI polyamide contains a ⁇ -aminobutyric acid moiety (hereinafter, also referred to as “ ⁇ -linker” or “ ⁇ -turn”) in addition to the above-mentioned two polyamide chains, and the C-terminal of one chain and the other.
  • the N-terminus of the chain is linked by a ⁇ -linker to form a U-shaped conformation (hairpin type).
  • P, I and the ⁇ -aminobutyric acid moiety are linked to each other by an amide bond.
  • the cyclic PI polyamide has a cyclic conformation in which the ends of the hairpin structure are ring-closed by a second ⁇ -turn.
  • H-pin and U-pin PI polyamides do not contain ⁇ -turns and are generally between the N-methyl groups of P in the center and between the N-methyl groups of P and I at the ends of the two polyamide chains, respectively. Are linked by an aliphatic linker.
  • hairpin type or cyclic PI polyamides are preferably used.
  • PI polyamide is a specific combination (P / I pair, I / P pair, or P) of a pair consisting of P and / or I facing each other between the above two polyamide chains (hereinafter, also referred to as "pyrrole-imidazole pair").
  • / P pair it binds to a specific base pair in DNA with high affinity.
  • a P / I pair can bind to a C / G base pair
  • an I / P pair can bind to a G / C base pair.
  • P / P pairs can bind to both A / T base pairs and T / A base pairs.
  • the PI polyamide may contain 3-hydroxypyrrole (Hp) and ⁇ -alanine residues in addition to P and I, and P can be replaced with Hp or ⁇ -alanine residues.
  • Hp / P pairs can bind to TA base pairs.
  • a P / Hp pair can bind to an A / T base pair.
  • the ⁇ -alanine / ⁇ -alanine pair can bind to T / A base pair or A / T base pair.
  • the ⁇ -alanine / I pair can bind to CG base pair.
  • the I / ⁇ -alanine pair can bind to a G ⁇ C base pair.
  • the ⁇ -alanine / P pair and the P / ⁇ -alanine pair can bind to TA base pair or AT base pair.
  • the ⁇ -turn moiety can bind to T / A base pair or A / T base pair.
  • Designing a PI polyamide that recognizes and binds to a desired DNA sequence by appropriately modifying the composition, order, combination, etc. of the pair formed by P, I, Hp, and / or ⁇ -alanine residues. Can be done.
  • the N-terminal side of the polyamide is designed to be on the 5'side of the target DNA sequence.
  • the methyl group on the nitrogen at the 1-position of P and I constituting the PI polyamide may be replaced with an alkyl group other than hydrogen or a methyl group.
  • alkyl groups other than methyl groups are linear, branched or cyclic saturated or unsaturated alkyl groups having 2 to 10 carbon atoms, preferably straight chain or branched chains having 2 to 5 carbon atoms.
  • Examples include linear, branched or cyclic saturated or unsaturated alkyl groups, such as ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl and the like.
  • the alkyl group including the methyl group may be substituted, and for example, methylene in the alkyl group may be substituted with oxygen or the like.
  • the 3-position of P constituting the PI polyamide may be substituted with a hydroxy group.
  • P or "pyrrole” and “I” or “imidazole” are N-substituted or N-unsubstituted pyrroles, 3-hydroxypyrroles, and N-substituted or as described above. Includes N-unsubstituted imidazole.
  • the DNA sequence targeted by PI polyamide is not particularly limited.
  • the PI polyamide may be designed to recognize a specific DNA sequence, that is, any desired DNA sequence, depending on its intended use.
  • a specific DNA sequence may be selected from, but not limited to, a sequence of a disease-related gene, a sequence of a gene to be modified or activated or suppressed, a repeat sequence such as a triple repeat sequence, or a related sequence thereof. good.
  • the length of a particular DNA sequence recognized and bound by PI polyamide is not limited, but is, for example, 2 to 30 base pairs, 3 to 25 base pairs, 4 to 20 base pairs, 5 to 15 base pairs, and the like. Alternatively, it may be a DNA sequence consisting of 6 to 10, 9 or 8 base pairs.
  • a longer DNA sequence containing the repeat sequence for example, a DNA sequence having 30 base pairs or more
  • it can also be targeted.
  • it is possible to target a DNA sequence longer than the conventional one by using a single PI polyamide.
  • the number and type of pyrrole-imidazole pairs formed by the P, I, Hp, and / or ⁇ -alanine residues constituting the PI polyamide used in the present disclosure is not particularly limited and is determined based on the target sequence. To. The number and type of pyrrole-imidazole pairs may be determined to recognize the specific DNA sequence described above. For example, in one embodiment, the number of pyrrole-imidazole pairs constituting the PI polyamide is 2 to 15, preferably 3 to 12, more preferably 4 to 10, for example 5, 6, 7, 8 or 9. It may be an individual.
  • a PI polyamide having a DNA sequence recognition moiety having a pyrrole-imidazole pair number of 5 or more it is preferably designed to contain a ⁇ -alanine residue.
  • the PI polyamide used in the present disclosure may be either a short-chain PI polyamide or a long-chain PI polyamide.
  • it may be a long-chain PI polyamide containing 5 or more pyrrole-imidazole pairs (that is, a long-chain PI polyamide containing 10 or more total P, I and ⁇ -alanine residues).
  • the term "pyrrole-imidazole pair” also includes a pair consisting of any combination of P, I, Hp, and ⁇ -alanine residues.
  • the ⁇ -turn moiety of the PI polyamide may be substituted, preferably having a substituent at the ⁇ - or ⁇ -position of the ⁇ -turn moiety, and more preferably the ⁇ -turn moiety. It may have a substituent at the ⁇ -position.
  • substituents include, but are not limited to, an amino group, an acetylamino group, a dimethylaminopropylamino group, a hydroxyl group, a methoxy group and the like.
  • the ⁇ -turn moiety may be an N- ⁇ -N- ⁇ -diaminobutyric acid residue or an N- ⁇ -N- ⁇ -diaminobutyric acid residue in which the ⁇ or ⁇ position is substituted with an amino group. good.
  • various functional groups or molecules may be added to the N-terminal and C-terminal of PI polyamide.
  • the functional groups or molecules to be added to the N-terminal and C-terminal of PI polyamide can be appropriately determined by those skilled in the art.
  • various functional groups can be added via an amide bond.
  • the functional group include, but are not limited to, a carboxyl group such as a ⁇ -alanine residue and a ⁇ -aminobutyric acid residue, an acetyl group, an amino group and the like.
  • an acetyl group may be added to the N-terminal.
  • a dimethylaminopropylamino group may be added to the C-terminal.
  • the N-terminal and C-terminal of PI polyamide may also be modified with a fluorescent group or a molecule such as biotin or isophthalic acid.
  • the fluorescent group includes, but is not limited to, fluorescein, rhodamine dye, TAMRA (5-carboxytetramethylrhodamine), cyanine dye, ATTO dye, Alexa Fluor dye, and BODIPY. Can be mentioned.
  • Fluorescein also includes fluorescein derivatives (eg, fluorescein isothiocyanate, etc.).
  • the PI polyamide may be a modified PI polyamide modified so as to maintain or improve the binding ability to DNA.
  • the PI polyamide modified product include a modified product in which an amino group is added to the ⁇ -position or ⁇ -position of the ⁇ -linker of PI polyamide, that is, an N- ⁇ -N- ⁇ -diaminobutyric acid residue or N- ⁇ -.
  • examples thereof include a modified product modified with a molecule of PI polyamide and a modified product obtained by modifying the C-terminal of PI polyamide with a molecule such as isophthalic acid.
  • the design method and manufacturing method of PI polyamide are known (see, for example, Japanese Patent No. 3045706, Japanese Patent Application Laid-Open No. 2001-136794, WO03 / 000683, Japanese Patent Application Laid-Open No. 2013-234135, JP-A-2014-173032).
  • it can be easily produced by automatic synthesis by a solid phase synthesis method (Fmoc solid phase synthesis method) using Fmoc (9-fluorenylmethoxycarbonyl). It can also be produced by a liquid phase synthesis method.
  • a "conjugate” (also referred to as a “complex”) is via a bond (eg, a covalent bond) sufficient to form a stable, larger construct. Refers to two or more molecules that are linked together to form the construct.
  • a complex containing the above PI polyamide and a peptide containing 1 to 5 arginine residues is provided. By introducing the arginine peptide into PI polyamide, the cell uptake efficiency and nuclear accumulation amount of PI polyamide are improved.
  • the "arginine peptide containing 1 to 5 arginine residues" used in the present disclosure may contain, for example, 2 to 4, preferably 3 arginine residues.
  • the arginine peptide is a peptide consisting of 1-5 arginine residues, for example a peptide consisting of 2-4, preferably 3 arginine residues.
  • the arginine contained in the above-mentioned arginine peptide may be either D-arginine or L-arginine, or may contain both.
  • the arginine peptide may be bound to any of the N-terminal, C-terminal, or ⁇ -turn moiety of PI polyamide. Further, it may be bonded to two or more positions selected from the N-terminal, C-terminal, and ⁇ -turn moiety.
  • the arginine peptide may be directly bound to PI polyamide via an amide bond, or may be bound via a linker.
  • the linker is not particularly limited as long as it does not interfere with the action of the arginine peptide and does not interfere with the recognition of the target sequence of PI polyamide.
  • Examples of the linker include, but are not limited to, ⁇ -alanine linker, polyether linker and the like, and the terminal of the linker is connected to PI polyamide and arginine peptide, for example, via an amide, ester, ether bond or the like. Be combined.
  • the above linker can be appropriately determined by those skilled in the art.
  • the arginine peptide When the arginine peptide is bound to the N-terminal or C-terminal of the PI polyamide in the complex of the PI polyamide and the arginine peptide, various functional groups or molecules are added to the N-terminal or C-terminal of the arginine peptide. You may.
  • the functional groups or molecules to be added to the N-terminal and C-terminal of the arginine peptide can be appropriately determined by those skilled in the art, and various functional groups can be added, for example, via an amide bond.
  • the functional group examples include carboxyl groups such as ⁇ -alanine residue and ⁇ -aminobutyric acid residue, and acetyl, as in the case of the functional group that can be added to the N-terminal or C-terminal of the above “1. PI polyamide”.
  • Groups, amino groups and the like can be mentioned.
  • an acetyl group may be added to the N-terminal.
  • a dimethylaminopropylamino group may be added to the C-terminal.
  • a functional molecule may be bound to the N-terminal or C-terminal of the arginine peptide conjugated to PI polyamide.
  • Conjugation (binding) of arginine peptide and PI polyamide can be performed by a known method.
  • an arginine peptide can be introduced during the synthesis of PI polyamide, and a conjugate of PI polyamide and arginine peptide can be easily produced by automatic synthesis by a solid phase synthesis method (Fmoc solid phase synthesis method).
  • the complex of PI polyamide and arginine peptide may be further conjugated (complexed) with a functional molecule.
  • the "functional molecule” is not particularly limited as long as it is a molecule having some function.
  • functional molecules include, but are not limited to, fluorescent molecules, biotin, polyethylene glycol, aminopolyethylene glycol and the like.
  • functional molecules include, but are not limited to, transcriptional modifiers that affect genes containing the sequence when PI polyamide binds to the target DNA sequence. Control substances, for example, transcription activators, transcription inhibitors and the like can be mentioned.
  • fluorescent molecules examples include fluorescein, fluorescein derivatives (for example, fluorescein isothiocyanate, etc.), rhodamine dyes, TAMRA (5-carboxytetramethylrhodamine), cyanine dyes, ATTO dyes, Alexa Fluor dyes, BODIPY, and the like. However, but not limited to these.
  • transcriptional activators include bromodomain inhibitors (eg, JQ1, 5-isoxazolyl-benzoimidazole compounds, I-BET762, OTX015, etc.), histone acetylase (HAT) activators [eg, N- (eg, N- (eg, N-). 4-Chloro-3- (trifluoromethyl) phenyl) -2-ethoxybenzamide (CTB), etc.], histone deacetylase (HDAC) inhibitor [for example, suberoylanilide hydroxamic acid (SAHA), etc.], etc.
  • HDAC histone deacetylase
  • SAHA suberoylanilide hydroxamic acid
  • the transcription inhibitor examples include, but are not limited to, HAT inhibitors, alkylating agents and the like.
  • the alkylating agent is not particularly limited, but is preferably one having low or no cytotoxicity in consideration of its use in a pharmaceutical composition described later.
  • alkylating agents include, but are not limited to, chlorambucil, duocarmycin, seco-CBI (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benzo].
  • alkylating agents include, but are not limited to, chlorambucil, duocarmycin, seco-CBI (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benzo].
  • e] Indole Indole
  • CBI 1,2,9,9a-tetrahydrocyclopropane
  • benzo benzo
  • pyrolobenzodiazepines nitrogen mustards and the like.
  • Chlorambucil, seco-CBI, and CBI given as examples of the above-mentioned alkylating agent are represented by the following chemical formulas.
  • the functional molecule may be attached to the N-terminus, C-terminus, or ⁇ -turn moiety of the PI polyamide, or to the N-terminus or C-terminus of the arginine peptide conjugated to the PI polyamide. May be good.
  • the functional molecule and the PI polyamide or arginine peptide may be directly bonded by, for example, an amide bond, a phosphodisulfide bond, an ester bond, a coordination bond, an ether bond, or the like, or may be bonded via a linker. ..
  • the linker is not particularly limited as long as it does not interfere with the action of the functional molecule and does not interfere with the target sequence recognition of PI polyamide and the action of the arginine peptide.
  • the linker may be a molecule containing a functional group that forms one or more bonds selected from the group consisting of amide bonds, phosphodisulfide bonds, ester bonds, coordination bonds, ether bonds and the like.
  • Examples of the linker include, but are not limited to, ⁇ -alanine linker, polyethylene glycol linker, peptide linker, alkyl linker, amino alkyl linker, polyether linker and the like.
  • Conjugation of the complex of PI polyamide and arginine peptide with a functional molecule can be performed according to a known coupling method or synthetic method.
  • the complex of PI polyamide and arginine peptide, or the complex of PI polyamide, arginine peptide and functional molecule is pharmacologically. It may be in the form of an acceptable salt.
  • the pharmacologically acceptable salt includes, for example, an inorganic acid salt such as a hydrochloride, a sulfate, a phosphate or a hydrobromide, or an acetate, a fumarate, a maleate, a oxalate, and the like. Examples thereof include organic acid salts such as citrate, methane sulfonate, benzene sulfonate or toluene sulfonate.
  • the PI polyamide conjugate is present in the form of an enantiomer or diastereomeric or a mixture thereof, wherein one or more selected from the PI polyamide, arginine peptide, functional molecule, and / or linker moiety is present. May be.
  • the substituent or arginine peptide or functional molecule may be in an R or S configuration. It may be bonded to the ⁇ -turn portion so as to take.
  • PI polyamide conjugates are obtained in the form of diastereomers or enantiomers, they can be separated by conventional methods well known in the art, such as chromatography or fractional crystallization.
  • an agent for activating mitochondria including a PI polyamide conjugate
  • Agents for activating mitochondria in the present disclosure preferably include a complex comprising the PI polyamide, arginine peptide, and functional molecule described above.
  • the functional molecule is, for example, a bromodomain inhibitor.
  • the agents for activating mitochondria in the present disclosure enhance the expression of PGC (peroxisome proliferator activated receptor gamma coactivator) -1 ⁇ and PGC-1 ⁇ , resulting in enhanced mitochondrial function.
  • compositions containing PI polyamide conjugates are provided.
  • the pharmaceutical composition in the present disclosure preferably comprises a complex containing the above PI polyamide, arginine peptide, and functional molecule.
  • the pharmaceutical composition in the present disclosure treats or prevents various diseases, for example, but is not limited to, tumors, cancers, mast cell diseases, allergies, immune diseases, triplet repeat diseases, etc., depending on the target sequence of PI polyamide contained therein. It can be used for the purpose of treating or preventing diseases such as repeat diseases, or modifying or activating or suppressing the expression of a desired gene.
  • the pharmaceutical composition in the present disclosure may be in either oral or parenteral dosage form.
  • dosage forms can be formulated according to a conventional method, and may contain a pharmaceutically acceptable carrier or additive.
  • Such carriers and additives include water, acetic acid, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, Carboxymethylstarch sodium, pectin, methylcellulose, ethylcellulose, xanthan gum, arabic rubber, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose , Surfactants and the like that are acceptable as pharmaceutical additives.
  • the additive is selected alone or in combination from the above depending on the dosage form of the pharmaceutical composition in the present disclosure.
  • the dosage form can be administered as tablets, capsules, fine granules, powders, granules, powders, liquids, syrups, sprays and the like, or in an appropriate dosage form.
  • injection type, coating agent, eye drop, external use, patch, suppository and the like can be mentioned.
  • injection type it can be administered systemically or locally by, for example, intravenous injection such as infusion, subcutaneous injection, intraperitoneal injection, intratumoral injection and the like.
  • the pharmaceutical composition in the present disclosure when used as an injectable preparation, is dissolved in a solvent (for example, physiological saline, buffer solution, dextrin solution, 0.1% acetic acid, etc.) and an appropriate additive (human) is used.
  • a solvent for example, physiological saline, buffer solution, dextrin solution, 0.1% acetic acid, etc.
  • it may be freeze-dried to form a dosage form that dissolves before use.
  • sugar alcohols such as mannitol and glucose and sugars can be used.
  • the subject to which the pharmaceutical composition in the present disclosure is administered includes, but is not limited to, any organism that utilizes double-stranded DNA for biological control, although it depends on the purpose of use thereof, for example, animals, particularly mammals ( For example, humans, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, baboons, etc.), preferably humans. In some embodiments, it may be administered to animals other than humans.
  • the method of administration of the pharmaceutical composition to a subject includes the species of the subject to be administered, the age, sex, and symptoms of the subject, and the health of the subject. It depends on the condition, the presence / absence and type of concomitant drug, and is appropriately determined by those skilled in the art.
  • the pharmaceutical composition in the present disclosure can be used in combination with other anticancer agents known in the art.
  • the mode of the combination is not limited, and can be appropriately carried out by those skilled in the art depending on the type of target cancer, the stage of treatment, and the like.
  • the pharmaceutical composition and other anticancer agents in the present disclosure may be administered to a subject at the same time or at different times.
  • the pharmaceutical composition and other anticancer agents in the present disclosure may be prepared as one combination drug containing each and administered to a subject, or may be prepared as separate agents and administered to a subject. You may.
  • the pharmaceutical composition and other anticancer agents in the present disclosure may be prepared as a kit separately comprising each.
  • an embodiment in which the pharmaceutical composition in the present disclosure and another anticancer agent are simultaneously administered for example, an embodiment in which one combination drug containing the pharmaceutical composition and the other anticancer agent in the present disclosure is administered to a subject. May be.
  • the pharmaceutical composition and other anticancer agents in the present disclosure are administered at different times
  • the pharmaceutical composition and other anticancer agents in the present disclosure are administered at different times. It may be possible, and for example, the pharmaceutical composition in the present disclosure and other anticancer agents may be administered from different administration routes.
  • anticancer agents used in combination are not limited, but are, for example, immune checkpoint inhibitors, alkylating agents, metabolic antagonists, anticancer antibiotics, plant alkaloids, antihormonal agents, platinum compounds, cytokines, etc.
  • One or more anti-cancer agents selected from the group consisting of formulations, molecular targeted agents, tumor immunotherapeutic agents, and cancer vaccines can be used.
  • the other anti-cancer agent may be an immune checkpoint inhibitor.
  • immune checkpoint inhibitor means a substance that inhibits the function of an immune checkpoint molecule.
  • An “immune checkpoint molecule” is a molecule that suppresses an immune response and / or an excessive immune response to self, eg, CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM3, etc.
  • BTLA, B7H3, B7H4, 2B4, CD160, A2aR, KIR, VISTA, TIGIT and the like can be mentioned, but the present invention is not limited thereto.
  • immune checkpoint inhibitors examples include anti-CTLA-4 antibody (eg, ipilimumab, tremellimumab, AGEN-1884), anti-PD-1 antibody (eg, nibolumab, REGN-2810, pembrolizumab, PDR-001, BGB-A317, etc.).
  • AMP-514 MEDI0680
  • BCD-100 IBI-308, JS-001, PF-06801591, TSR-042
  • anti-PD-L1 antibody eg, atezolizumab (RG7446, MPDL3280A
  • Abelumab PF-06834635, MSB0010718C.
  • the immune checkpoint inhibitor may be a PD-1 inhibitor or a PD-L1 inhibitor. In certain embodiments, the immune checkpoint inhibitor may be an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • the term "antibody” refers to a molecule that comprises a portion of an antibody as a component and retains its binding property to an antigen, such as Fab, Fab', F (ab') 2, Fv, or a single molecule. Chains Fv (scFv), Fab3, diabodies, triabodies, tetrabodies, minibodies, Bis-scFv, (scFv) 2-Fc, intact IgG and the like are included.
  • the antibody may be a human antibody or a humanized antibody.
  • the humanized antibody means an antibody in which the amino acid sequences of the CDRs of VH and VL of an antibody of a non-human animal are transplanted to appropriate positions of VH and VL of a human antibody.
  • the method of administration of the immune checkpoint inhibitor is not particularly limited, and the route of administration is generally oral or parenteral (for example, intravenous, subcutaneous, intracutaneous, intrathoracic, intraperitoneal, intramuscular, or intratissue). Can be done. In certain embodiments, the immune checkpoint inhibitor may be administered intravenously. The details of the dosage form suitable for these administration methods are as described above.
  • the dose and frequency of administration of the immune checkpoint inhibitor can be appropriately set by those skilled in the art according to the health condition, age, body weight, administration route, administration form, etc. of the subject so that the effective amount is administered to the subject.
  • the anti-PD-L1 antibody may be administered every 6 days, every 1 week, every 2 weeks, every 3 weeks or every 4 weeks.
  • the route of administration of the pharmaceutical composition in the present disclosure is not limited, and may be systemic administration or local administration.
  • the routes of administration include, for example, oral administration including sublingual administration, inhalation administration, direct administration to the target tissue by catheter or injection, intravenous administration including infusion, transdermal administration by patch, suppository, or nasogastric.
  • Parenteral administration such as administration by forced enteral nutrition using a tube, nasogastric tube, gastric fistula tube, or intestinal fistula tube can be mentioned.
  • the pharmaceutical composition in the present disclosure is preferably an agent for activating mitochondria. That is, the immune checkpoint inhibitor is used in combination with an agent for activating mitochondria.
  • it comprises a conjugate of PI polyamide, a triarginine peptide, and a bromodomain inhibitor that recognizes T), for example, the following formula: Includes conjugates indicated by.
  • the immune checkpoint inhibitor is preferably a PD-1 inhibitor or a PD-L1 inhibitor, and more preferably an anti-PD. -1 antibody or anti-PD-L1 antibody.
  • the combined use of an agent for activating mitochondria and an immune checkpoint inhibitor can enhance antitumor immunity.
  • the agent for activating mitochondria is an agent for enhancing the expression of at least one of PGC-1 ⁇ and PGC-1 ⁇ . Anything is fine, and it can be changed as appropriate.
  • Example 1 Synthesis of Complex (SOX2i-R3) and Control (Ctrl-R3) Containing PI Polyamide (SOX2i) and Triarginine Peptide Targeting SOX2 Binding Sequences SOX2i-R3, SOX2i, and Control (Ctrl-R3) ) was synthesized.
  • the structure of these PI polyamide conjugates is shown in FIG. 1-1.
  • the Fmoc-ImPy-OH dimer unit was used for the synthesis of SOX2i-R3.
  • the N-terminus was capped with an acetyl group by mixing with 20% acetaldehyde in N, N-dimethylformamide (DMF).
  • the synthesized compound was excised with N, N-dimethylaminopropylamine at 55 ° C. for 3 hours.
  • the reaction solution was poured into ether and the obtained solid was vacuum dried.
  • the protecting group (Pbf group) of the arginine residue of SOX2i-R3 and Trl-R3 was added to the deprotecting cocktail (trifluoroacetic acid: triisopropylsilane: water (95: 2.5: 2.5v / v) for 30 minutes at room temperature. %)) was removed.
  • Rough samples are purified by reverse phase HPLC using a JASCO HPLC system (JASCO Engineering UV2075 UV / vis detector and a PU-2089 plus gradient pump) with a preparative C18 (ODS) column (COSMOSIL 5C18-MS-II, 10IDx150 mm). did. H2O (+ 0.1% trifluoroacetic acid) and acetonitrile were used as mobile phases.
  • the coupling reaction was carried out by adding 1.3 eq 5-TAMRA-NHS ester and 3 eq N, N-diisopropylethylamine (DIEA) and shaking overnight at room temperature. Powdering, deprotection, purification and characterization were performed as described above.
  • Example 2 Confirmation of effect by triarginine SOX2i is a SOX2 downstream gene including SOX2 itself by inhibiting the binding of SOX2 to DNA in induced pluripotent stem cells (iPS cells) and human prostate and ovarian cancer cells. It was reported to suppress the expression of.
  • iPS cells induced pluripotent stem cells
  • SOX2i-R3 cell uptake and biological activity of SOX2i and SOX2i-R3 were investigated to clarify the effect of the triarginine peptide on PI polyamide.
  • JCRB9004 and 201B7 cells were obtained from JCRB Cell Bank (Japan) and RIKEN BRC (Japan), respectively.
  • HeLa cells were maintained in Dulbecco's modified Eagle's medium (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (Sigma) and 1% MEM non-essential amino acid solution (ThermoFisher Scientific).
  • 201B7 iPS cells were cultured on Matrigel Matrix (hESC-qualified, Corning) in mTeSR1 medium (Stemcell Technologies) supplemented with 0.5x penicillin / streptomycin (Nacalai Tesque). The cells were passaged using TrypLE Express Enzyme (without phenol red, Thermo Fisher Scientific) as a dissociation reagent, and seeded in a medium supplemented with 2.5 ⁇ M Y-27632 (Wako). Medium change to fresh medium (without Y-27632) was performed daily from the day following the passage.
  • Electrophoretic mobility shift assay Prepare DMSO solutions of SOX2i-R3, SOX2i and Trl-R3 and calculate the following using the maximum absorbance at 300-310 nm measured by Nanodrop ND-1000 (ThermoFisher Scientific).
  • a indicates the total number of pyrrole and imidazole rings and ⁇ -alanine residues
  • d indicates the optical path length (cm) (0.1 cm in the case of Nanodrop ND-1000)
  • Abs is at 300-310 nm. Shows maximum absorbance] The concentration was determined from.
  • Each template DNA (1 ⁇ M) was mixed with 1 ⁇ M SOX2i-R3, SOX2i or Ctrl-R3 at pH 7.0 in an aqueous buffer containing 10 mM sodium chloride, 10 mM sodium cacodylate and 2.5 v / v% DMSO. Prior to electrophoresis, the sample was annealed by heating to 95 ° C. for 3 minutes and cooled to 25 ° C. at a rate of ⁇ 0.5 ° C./5 seconds. Each 8 ⁇ L sample was mixed with 2 ⁇ L Novex Hi-Density TBE Simple Buffer (Thermo Fisher Scientific) and 1 ⁇ L of each loading mixture was loaded. Native-PAGE was performed in TBE buffer at 200 V for 60 minutes using a 20% polyacrylamide gel. The gel was stained with SYBR Gold (Thermo Fisher Scientific) and imaged with the FLA-3000 system (Fujifilm).
  • RT-qPCR Quantitative reverse transcription PCR
  • the reaction mixture was prepared using Thunderbird SYBR qPCR mix (Toyobo), a qPCR reaction was performed, and it was monitored on LightCyclor 480 System II (Roche). Relative expression levels of SOX2 were calculated from the mean Cp values of the three culture wells of each sample and normalized to the mean of the negative control. HPRT1 or 18S was used as the housekeeping gene. Table 1 shows the primer pairs used in the experiment.
  • SOX2i-R3-TAMRA was taken up by cells with higher efficiency than SOX2i-TAMRA. Therefore, it was shown that the introduction of three arginine residues into PI polyamide increased the cellular uptake of the PI polyamide.
  • dsDNA Two types of double-stranded DNA (dsDNA), namely 5'-CCGCAT AACAAAG TGCC-3'(SOX2-DNA; SEQ ID NO: 7) and 5'-CCTC AGCCGCC TTCC-3'(Ctrl-DNA; SEQ ID NO: 8).
  • Each of the dsDNAs contained one binding site for SOX2i-R3 and Ctrl-R3 (the binding site is underlined). Since the ⁇ -diaminobutyrate turn unit is known to have A / T and T / A selectivity, an A / T base pair was placed at the 5'end of the binding site.
  • dsDNA (1 ⁇ M) was incubated with each compound (1 ⁇ M) and unbound and bound DNA was separated by polyacrylamide gel. The results are shown in FIG.
  • SOX2i required 2 ⁇ M to down-regulate SOX2 expression by 60%, whereas for SOX2i-R3, only 100 nM was sufficient to achieve comparable suppression (FIG. 5A). .. Furthermore, little SOX2 expression was observed in cells treated with> 1 ⁇ M SOX2i-R3 (FIG. 5A). The significant improvement in the biological activity appears to be due to the efficient nuclear accumulation of SOX2i-R3. Furthermore, Ctrl-R3 had no effect on SOX2 expression. This supports the sequence-specific transcriptional repression by SOX2i-R3 (FIG. 5B).
  • a long-chain PI polyamide containing a total of 12 pyrrole / imidazole / ⁇ -alanine residues can be efficiently delivered into the cell nucleus. Furthermore, it was revealed that the concentration of PI polyamide compound required for transcription of the target gene was significantly reduced.
  • Example 3 Synthesis of PI Polyamide and Triarginine Peptide Containing Complex (Chb-M'-Arg3) Targeting RUNX Binding Sequence PI polyamide targeting RUNX binding sequence to RUNX binding sequence on DNA
  • PI polyamide that recognizes 5'-TGTGGT-3'as a consensus sequence of the RUNX binding site on the genome, chlorambucil as an alkylating agent, and a triarginine peptide.
  • a conjugate of PI polyamide and a triarginine peptide was synthesized by the Fmoc solid phase synthesis method, and then chlorambucil was introduced at the N-terminal of the conjugate.
  • a conjugate (Arg3-PIPM') of PI polyamide and triarginine peptide was synthesized using a PSSM-8 peptide automatic synthesizer. Specifically, 80 mg (59 ⁇ mol / 0) of Fmoc-D-Arg (Pbf) -Alko (0.59 mmol / g, 100-200 mesh, 1% DVB) resin was added to the reaction vessel (Small Libra tube) into which the synthetic sequence was input. .1 g) (Watanabe Kagaku Catalog, No. K01985) After setting the RV insert, 1 ml of NMP (N-methylpyrrolidone) was added while washing off the resin adhering to the wall surface, and left at room temperature for about 20 minutes. ..
  • NMP N-methylpyrrolidone
  • polyamide synthesis using PSSM-8 synthesizer 982 ⁇ l of the monomer solution was poured every minute into a 2 ml Eppen tube (2 ml sampling tube). It was set in the solid phase synthesizer in the order of sequence.
  • the polyamide sequence is as follows (synthesized from C-terminal to N-terminal). D-Arg (Pbf) (on resin) -D-Arg (Pbf) -D-Arg (Pbf) -bAl-Py-Py-PyPy- ⁇ Abu-Im-Dimer-Im-bAl (NH2)
  • freeze drying It was transferred to a 50 ml tube, put in about 20 ml, put in liquid nitrogen, frozen, and dried in a freeze dryer (EYELA FDU-1100) for 48 hours.
  • Dietil ether was added to a 50 ml tube, collected in a T sample stock tube of 1.5 ml (BMBio, catalogNo.T-202), and the operations of centrifugation and removal of the supernatant were repeated and dried with a desiccator.
  • a conjugate of PI polyamide (R) and a triarginine peptide was synthesized by the Fmoc solid phase synthesis method, and then a 5-isoxazolyl-benzoimidazole compound was used as a bromodomain inhibitor (Bi) with the above PI polyamide and the triarginine peptide. It was introduced at the N-terminal of the conjugate.
  • a conjugate (Arg3-PIPR) of PI polyamide and triarginine peptide was synthesized using a PSSM-8 peptide automatic synthesizer. Specifically, 80 mg (59 ⁇ mol / 0.1 g) of Fmoc-D-Arg (Pbf) -Alko (0.59 mmol / g, 100-200 mesh, 1% DVB) resin was added to the reaction vessel into which the synthetic sequence was input. After setting the RV insert, 1 ml of NMP was added while washing off the resin adhering to the wall surface for swelling, and the mixture was left at room temperature for about 20 minutes.
  • Pbf Fmoc-D-Arg
  • Fmoc-PyIm-OH (Dimer) synthesized in the liquid phase in advance was used and adjusted in the same manner as the monomer.
  • PGC (peroxisome proliferator activated receptor gamma coactivator) -1 ⁇ and PGC-1 ⁇ are proteins identified as transcription coactivators that bind to the transcription factor PPAR ⁇ , and their upregulation is known to result in enhanced mitochondrial function.
  • CD8 + T cells were obtained from B6 mice and siRNA against PGC-1 ⁇ or PGC-1 ⁇ was electroporated using a neon electroporation system (10 picomoles / 1 million cells). After electroporation, cells were incubated for 4 hours. Next, TCR stimulation was performed with anti-CD3 and anti-CD28 monoclonal antibodies, and at the same time, Bi-R-Arg3 was added in a dose-dependent manner and incubated for 48 hours. After 48 hours of stimulation, cells were collected, RNA was extracted and purified, and qPCR was performed as described in Example 2 (5). GAPDH was used as the loading control. The siRNA sequence and primer sequence used are shown in Table 4.
  • CD8 + T cells both low CD44 and high CD44
  • CD8 + T cells were harvested from B6 mice, T cells were stimulated with anti-CD3 / CD28 monoclonal antibodies for TCR stimulation, and the PIP, Bi and Arg3 conjugates synthesized above (BiR-Arg3), PIP and Bi conjugate (BiR), bromodomain inhibitor only (Bi), or PIP only (R) was added.
  • DMSO was added without adding any of the above compounds.
  • mitochondrial activation was assessed using mitochondrial dye staining. For staining, MitoTracker® Red, MitoTracker® Green, Mitochondrial SOX, and CellROX® (all manufactured by Thermo Fisher) were used as fluorescent probes.
  • Example 5 Enhancement of antitumor immunity by combined use of PD-1 blockade and Bi-R-Arg3 PGC-1 ⁇ / ⁇ forms a complex with various transcription factors (TF), and mitochondrial biosynthesis and metabolism.
  • TF transcription factors
  • OXPHOS Oxidative phosphorylation
  • FEO fatty acid oxidation
  • mitochondrial crystal rearrangement and other coactivators known to regulate the expression of genes involved.
  • blocking PD-1 induces the proliferation of short-term effector T cells having glycolytic metabolism. It has also been suggested that the lack of effector cells at the tumor site (because of their short life) may be one of the reasons for the non-responsiveness seen in some patients.
  • PI polyamide was injected every 3 days and anti-PD-L1 monoclonal antibody was injected every 6 days. Treatment was performed for 1 month, the size of the tumor was measured every other day using a caliper, and the tumor volume was calculated. In addition, the survival rate (%) of the mice after treatment was determined.
  • an anti-PD-L1 monoclonal antibody or an isotype control of an anti-PD-L1 monoclonal antibody (Rat IgG2b, k) was administered alone. Further, as a control, Bi-R-Arg3, R alone, or Bi alone was administered alone, and the same experiment was performed.
  • MC38 cells (mouse colorectal cancer cell line derived from C57BL / 6N background) were purchased from Kerafast Inc., U.S.
  • C57BL / 6N mice 6-8 week old females were purchased from CLEA Japan (Tokyo, Japan) and maintained under pathogen-free conditions.
  • Bi-R-Arg3 was synthesized according to the description in Example 4.
  • R is the PI polyamide shown in Example 4, and was synthesized according to a conventional method. Further, as Bi, 5-isoxazolyl-benzimidazole was used as in Example 4.
  • FIGS. 10-1 and 10-2 The results of measuring the tumor size are shown in FIGS. 10-1 and 10-2. Tumor sizes in groups treated with various PI polyamides were plotted. In the figure, the horizontal axis shows the elapsed date after tumor transplantation, and the vertical axis shows the tumor size. As is clear from FIGS. 10-1 and 10-2, Bi-R-Arg3, when used in combination with PD-1 blockade, caused more tumor degeneracy compared to PD-1 blockade alone.
  • mice The survival rate of mice is shown in FIG. As is clear from FIG. 11, Bi-R-Arg3 improved the survival rate when used in combination with PD-1 blockade as compared with PD-1 blockade alone.
  • Bi-R-Arg3 alone did not have a tumor degenerate effect and could not improve the survival rate of the tumor transplant host, as in the control Bi alone and R alone.
  • These in vivo data indicate that Bi-R-Arg3 has a synergistic effect with PD-1 blockade therapy and enhances antitumor immunity. That is, the antitumor immunity can be enhanced by using a drug for activating mitochondria and an immune checkpoint inhibitor in combination.

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Abstract

Provided is a complex containing a pyrrole-imidazole polyamide that recognizes a particular DNA sequence and binds thereto and containing peptide that contains 1-5 arginine residues.

Description

核移行性が改良されたピロール－イミダゾールポリアミドPyrrole-imidazole polyamide with improved nuclear transferability
　本開示は、核移行性が改良されたピロール－イミダゾールポリアミド、および該ピロール－イミダゾールポリアミドを含む医薬組成物に関する。 The present disclosure relates to a pyrrole-imidazole polyamide having improved nuclear transferability and a pharmaceutical composition containing the pyrrole-imidazole polyamide.
　ピロール－イミダゾールポリアミド（以下、「ＰＩポリアミド」または「ＰＩＰ」ともいう）は、アミド結合により連結されたＮ－メチルピロールとＮ－メチルイミダゾールから構成される合成リガンドであり、ＤＮＡの副溝に塩基配列選択的に結合する化合物である。いくつかの形態のＰＩポリアミドがあり、逆平行の２本のＰＩポリアミド鎖と、該２本鎖の一方の鎖のＣ末端と他方の鎖のＮ末端を連結しているγ－アミノ酪酸（γ－ターン）とを含むヘアピン型のＰＩポリアミドが最も一般的に用いられている。ＰＩポリアミドは、対面するピロール（Ｐ）およびイミダゾール（Ｉ）対により、Ｐ／Ｉ対がＣ（シトシン）・Ｇ（グアニン）塩基対を、Ｐ／Ｐ対がＡ（アデニン）・Ｔ（チミン）またはＴ・Ａ塩基対を、Ｉ／Ｐ対がＧ・Ｃ塩基対を認識し、これにより任意の二重鎖ＤＮＡ配列に特異的に結合することができる（非特許文献１）。したがって、ピロールおよびイミダゾールを様々な順序で連結させたＰＩポリアミドを設計することにより、ゲノム上のあらゆる領域を標的とすることができる。そのため、ＰＩポリアミドにより遺伝子発現を人為的にコントロールする目的で、またはＰＩポリアミドを利用してゲノム上の所望の標的部位へ薬剤等をインビボデリバリーする目的で、ＰＩポリアミドを基本骨格とした様々な化合物が開発されている。 Pyrrole-imidazole polyamide (hereinafter, also referred to as "PI polyamide" or "PIP") is a synthetic ligand composed of N-methylpyrrole and N-methylimidazole linked by an amide bond, and is a base in the accessory groove of DNA. It is a compound that binds sequentially selectively. There are several forms of PI polyamide, γ-aminobutyric acid (γ) that connects two antiparallel PI polyamide chains and the C-terminus of one strand of the duplex and the N-terminus of the other strand. -Hairpin-type PI polyamides containing (turn) and are most commonly used. In PI polyamide, P / I pairs are C (cytosine) / G (guanine) base pairs and P / P pairs are A (adenine) / T (thymine) due to facing pyrrole (P) and imidazole (I) pairs. Alternatively, the TA base pair can be specifically bound to any double-stranded DNA sequence by recognizing the G / C base pair by the I / P pair (Non-Patent Document 1). Therefore, by designing PI polyamides in which pyrrole and imidazole are linked in various orders, any region on the genome can be targeted. Therefore, for the purpose of artificially controlling gene expression with PI polyamide, or for the purpose of in vivo delivery of drugs and the like to a desired target site on the genome using PI polyamide, various compounds using PI polyamide as a basic skeleton. Has been developed.
　生きた細胞において生物学的活性を達成するには、ＰＩポリアミドは、細胞核中に送達されて、核ＤＮＡに結合されなければならない。しかしながら、ＰＩポリアミド化合物のなかには、核内蓄積が不十分なものもある。そこで、近年、ＰＩポリアミドの構造的特徴が核送達に与える影響について広範囲に研究されている。一般に、８個程度のピロール／イミダゾール環を含有する短鎖ヘアピン型ＰＩポリアミドは効率的な核送達に適しているが、１０個を超えるピロール／イミダゾール環を含有する長鎖ＰＩポリアミドの細胞取り込み効率は低い。その一方で、長鎖ＰＩポリアミドは、ＤＮＡに対する結合アフィニティーおよび配列選択性に有利である。したがって、安定かつ効率的な核内送達が可能な長鎖ＰＩポリアミドが求められている。８環のヘアピン型ＰＩポリアミドのγ－ジアミノ酪酸ターン部分にアリール基を導入することにより、ＰＩポリアミドの細胞取り込みが有意に増加したことが報告されているが（非特許文献２）、このアプローチは長鎖ＰＩポリアミドには応用されていない。 To achieve biological activity in living cells, PI polyamide must be delivered into the cell nucleus and bound to nuclear DNA. However, some PI polyamide compounds have insufficient nuclear accumulation. Therefore, in recent years, the effects of the structural characteristics of PI polyamide on nuclear delivery have been extensively studied. In general, short-chain hairpin-type PI polyamides containing about 8 pyrrole / imidazole rings are suitable for efficient nuclear delivery, but cell uptake efficiency of long-chain PI polyamides containing more than 10 pyrrole / imidazole rings. Is low. On the other hand, long-chain PI polyamides are advantageous for binding affinity and sequence selectivity for DNA. Therefore, there is a demand for long-chain PI polyamides capable of stable and efficient nuclear delivery. It has been reported that the introduction of an aryl group into the γ-diaminobutyric acid turn moiety of the 8-ring hairpin-type PI polyamide significantly increased the cellular uptake of the PI polyamide (Non-Patent Document 2). It has not been applied to long-chain PI polyamides.
　一方、オリゴアルギニンは、タンパク質、核酸、およびリポソームを包含する高分子の細胞デリバリーツールとして幅広く用いられている。デリバリーベクターとして使用されるオリゴアルギニンは６～１２個のアルギニン残基を含み、アルギニン残基の数が少ないと、効率的な細胞取り込みを示さないことが報告されている（非特許文献３および４）。 On the other hand, oligoarginine is widely used as a polymer cell delivery tool including proteins, nucleic acids, and liposomes. It has been reported that oligoarginine used as a delivery vector contains 6 to 12 arginine residues, and that a small number of arginine residues does not show efficient cell uptake (Non-Patent Documents 3 and 4). ).
　本開示は、核移行性が改良されたＰＩポリアミドを提供することを目的とする。本発明者らは、オリゴアルギニンの利用の可能性に着目した。しかしながら、ＰＩポリアミドと、６～１２個という長いアルギニンペプチドとのコンジュゲートを合成するには、固相合成において多くのカップリング工程が必要となるため、合成作業が煩雑になり、また、工程が多い結果、収率が減少することも予測された。さらに、アルギニン残基は、生理学的ｐＨ下で正に荷電しているため、負に荷電したＤＮＡホスフェート骨格との非選択的相互作用が生じ、ＰＩポリアミドの配列選択性を損なうことが予測された。 The present disclosure is intended to provide PI polyamide with improved nuclear transferability. The present inventors focused on the possibility of utilizing oligoarginine. However, in order to synthesize a conjugate of PI polyamide and a long arginine peptide of 6 to 12, many coupling steps are required in solid phase synthesis, which complicates the synthesis work and makes the steps complicated. As a result, it was predicted that the yield would decrease. Furthermore, since the arginine residue is positively charged under physiological pH, it was predicted that non-selective interaction with the negatively charged DNA phosphate skeleton would occur, impairing the sequence selectivity of PI polyamide. ..
　そこで、本発明者らは鋭意研究した結果、驚くべきことに、より少ない数のアルギニン残基を含むペプチドをＰＩポリアミドにコンジュゲートすることにより、該ＰＩポリアミドの核移行性が飛躍的に増大し、かつ、該ＰＩポリアミドの配列選択性にも影響を及ぼされないことを見出した。かくして、本発明を完成させた。 Therefore, as a result of diligent research by the present inventors, surprisingly, by conjugating a peptide containing a smaller number of arginine residues to PI polyamide, the nuclear transferability of the PI polyamide is dramatically increased. Moreover, it was found that the sequence selectivity of the PI polyamide was not affected. Thus, the present invention was completed.
　すなわち、本開示は、例えば、以下の態様を提供する。
［１］特定のＤＮＡ配列を認識し、結合するＰＩポリアミドと、１～５個のアルギニン残基を含むペプチドとを含む複合体、
［２］ペプチドが３個のアルギニン残基を含む、上記［１］に記載の複合体、
［３］ピロール－イミダゾールポリアミドがヘアピン型または環状のＰＩポリアミドである、上記［１］または［２］に記載の複合体、
［４］機能性分子をさらに含む、上記［１］～［３］のいずれか１項に記載の複合体、
［５］機能性分子がエピジェネティックモディファイアーである、上記［４］に記載の複合体、
［６］機能性分子が、蛍光ラベル、アルキル化剤、ブロモドメイン阻害剤、ＨＤＡＣ阻害剤、ＨＡＴ活性化剤、およびＨＡＴ阻害剤からなる群から選択される、上記［４］に記載の複合体、
［７］上記［１］～［６］いずれか１項に記載の複合体を含む、医薬組成物、
［８］抗がん剤と併用される、上記［７］に記載の医薬組成物、
［９］抗がん剤が免疫チェックポイント阻害剤である、上記［８］に記載の医薬組成物、
［１０］免疫チェックポイント阻害剤がＰＤ－１阻害剤またはＰＤ－Ｌ１阻害剤である、上記［９］に記載の医薬組成物、
［１１］免疫チェックポイント阻害剤が抗ＰＤ－１抗体または抗ＰＤ－Ｌ１抗体である、上記［１０］に記載の医薬組成物、
［１２］下記式：
Figure JPOXMLDOC01-appb-C000002
 で示される、上記［６］に記載の複合体、
［１３］複合体がブロモドメイン阻害剤を含む、上記［９］～［１１］のいず１項に記載の医薬組成物、
［１４］上記［１２］に記載の複合体を含む、上記［１３］に記載の医薬組成物、
［１５］上記［１］～［６］いずれか１項に記載の複合体を対象に投与することを含む、疾患の治療または予防方法、
［１６］疾患ががんであり、抗がん剤と併用して、上記［１］～［６］いずれか１項に記載の複合体が対象に投与される、上記［１５］記載の方法、
［１７］抗がん剤が免疫チェックポイント阻害剤である、上記［１６］に記載の方法、
［１８］免疫チェックポイント阻害剤がＰＤ－１阻害剤またはＰＤ－Ｌ１阻害剤である、上記［１７］に記載の方法、
［１９］免疫チェックポイント阻害剤が抗ＰＤ－１抗体または抗ＰＤ－Ｌ１抗体である、上記［１８］に記載の方法、
［２０］複合体がブロモドメイン阻害剤を含む、上記［１６］～［１９］のいず１項に記載の方法、
［２１］上記［１２］に記載の複合体を投与することを含む、上記［２０］に記載の方法、
［２２］ＰＩポリアミドの核移行性を改良する方法であって、該ＰＩポリアミドを１～５個のアルギニン残基を含むペプチドと複合体化させることを特徴とする方法、
［２３］上記［１２］に記載の複合体を含み、ミトコンドリアを活性化させるための剤、
［２４］ミトコンドリアを活性化させるための剤と併用される、免疫チェックポイント阻害剤、
［２５］ミトコンドリアを活性化させるための剤が上記［１２］に記載の複合体を含む、上記［２４］に記載の免疫チェックポイント阻害剤、
［２６］免疫チェックポイント阻害剤がＰＤ－１阻害剤またはＰＤ－Ｌ１阻害剤である、上記［２４］または［２５］に記載の免疫チェックポイント阻害剤、
［２７］免疫チェックポイント阻害剤が抗ＰＤ－１抗体または抗ＰＤ－Ｌ１抗体である、上記［２６］に記載の免疫チェックポイント阻害剤。 That is, the present disclosure provides, for example, the following aspects.
[1] A complex containing a PI polyamide that recognizes and binds to a specific DNA sequence and a peptide containing 1 to 5 arginine residues.
[2] The complex according to the above [1], wherein the peptide contains three arginine residues.
[3] The complex according to the above [1] or [2], wherein the pyrrole-imidazole polyamide is a hairpin-type or cyclic PI polyamide.
[4] The complex according to any one of [1] to [3] above, further comprising a functional molecule.
[5] The complex according to the above [4], wherein the functional molecule is an epigenetic modifier.
[6] The complex according to the above [4], wherein the functional molecule is selected from the group consisting of a fluorescent label, an alkylating agent, a bromodomain inhibitor, an HDAC inhibitor, a HAT activator, and a HAT inhibitor. ,
[7] A pharmaceutical composition comprising the complex according to any one of the above [1] to [6].
[8] The pharmaceutical composition according to the above [7], which is used in combination with an anticancer agent.
[9] The pharmaceutical composition according to the above [8], wherein the anticancer agent is an immune checkpoint inhibitor.
[10] The pharmaceutical composition according to the above [9], wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
[11] The pharmaceutical composition according to the above [10], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
[12] The following formula:
Figure JPOXMLDOC01-appb-C000002
 The complex according to the above [6], which is indicated by.
[13] The pharmaceutical composition according to any one of [9] to [11] above, wherein the complex contains a bromodomain inhibitor.
[14] The pharmaceutical composition according to the above [13], which comprises the complex according to the above [12].
[15] A method for treating or preventing a disease, which comprises administering the complex according to any one of the above [1] to [6] to a subject.
[16] The method according to [15] above, wherein the disease is cancer and the complex according to any one of the above [1] to [6] is administered to a subject in combination with an anticancer drug.
[17] The method according to [16] above, wherein the anticancer agent is an immune checkpoint inhibitor.
[18] The method according to [17] above, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
[19] The method according to [18] above, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
[20] The method according to any one of [16] to [19] above, wherein the complex contains a bromodomain inhibitor.
[21] The method according to the above [20], which comprises administering the complex according to the above [12].
[22] A method for improving the nuclear transferability of PI polyamide, which comprises complexing the PI polyamide with a peptide containing 1 to 5 arginine residues.
[23] An agent containing the complex according to the above [12] and for activating mitochondria.
[24] Immune checkpoint inhibitors, used in combination with agents to activate mitochondria,
[25] The immune checkpoint inhibitor according to [24] above, wherein the agent for activating mitochondria comprises the complex described in [12] above.
[26] The immune checkpoint inhibitor according to the above [24] or [25], wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
[27] The immune checkpoint inhibitor according to the above [26], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
　本開示の１～５個のアルギニン残基を含むペプチドをコンジュゲートしたＰＩポリアミドは、該アルギニンペプチドをコンジュゲートしていないＰＩポリアミドと比べて、細胞取り込み効率および核内蓄積量が著しく増大しており、さらに、標的遺伝子の抑制に必要とされる該ＰＩポリアミド濃度は有意に減少された。このように、本開示のアルギニンペプチドをコンジュゲートしたＰＩポリアミドは、増大した細胞膜浸透性を有し、細胞核内蓄積量が増大することから、生存細胞におけるＰＩポリアミドの利用を拡大させる。さらに、本開示のＰＩポリアミドを用いれば、効率の良い遺伝子発現制御剤や細胞分化剤、および癌、遺伝病、生活習慣病等の治療薬を開発することができる。 The PI polyamide conjugated with a peptide containing 1 to 5 arginine residues of the present disclosure has significantly increased cell uptake efficiency and nuclear accumulation as compared with PI polyamide not conjugated with the arginine peptide. Furthermore, the concentration of the PI polyamide required for suppression of the target gene was significantly reduced. As described above, the PI polyamide conjugated with the arginine peptide of the present disclosure has increased cell membrane permeability, and the amount accumulated in the cell nucleus is increased, thereby expanding the use of PI polyamide in living cells. Furthermore, by using the PI polyamide of the present disclosure, it is possible to develop an efficient gene expression regulator, cell differentiation agent, and therapeutic agent for cancer, genetic disease, lifestyle-related disease and the like.
図１－１は、実施例で使用したＰＩポリアミドの化学構造を示す。トリアルギニンとコンジュゲートしたＰＩポリアミドによるＤＮＡ配列認識の概略図も示す（Ｗ＝ＡまたはＴ）。FIG. 1-1 shows the chemical structure of the PI polyamide used in the examples. A schematic diagram of DNA sequence recognition by PI polyamide conjugated with triarginine is also shown (W = A or T). 図１－１で示した概略図の凡例を示す。The legend of the schematic diagram shown in FIG. 1-1 is shown. 図２は、ＳＯＸ２ｉ－ＴＡＭＲＡおよびＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡの細胞取り込みを評価するためのフローサイトメトリー分析の結果を示す。FIG. 2 shows the results of flow cytometric analysis for assessing cell uptake of SOX2i-TAMRA and SOX2i-R3-TAMRA. 図３は、ＳＯＸ２ｉ－ＴＡＭＲＡまたはＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡで処理したＨｅＬａ細胞の蛍光画像である。スケールバーは５０μｍを示す。FIG. 3 is a fluorescence image of HeLa cells treated with SOX2i-TAMRA or SOX2i-R3-TAMRA. The scale bar indicates 50 μm. 図４は、ＳＯＸ２ｉ、ＳＯＸ２ｉ－Ｒ３、およびＣｔｒｌ－Ｒ３の電気泳動移動度シフトアッセイの結果を示す。図中、「Ｓ」は、ＳＯＸ２－ＤＮＡを示し、「Ｃ」は、Ｃｔｒｌ－ＤＮＡを示す。ＰＩＰ－ＤＮＡ複合体のシフトしたバンドを黒い矢印で示す。FIG. 4 shows the results of the electrophoretic mobility shift assay of SOX2i, SOX2i-R3, and Ctrl-R3. In the figure, "S" indicates SOX2-DNA and "C" indicates Ctrl-DNA. The shifted band of the PIP-DNA complex is indicated by a black arrow. 図５は、ＲＴ－ｑＰＣＲを用いて算出された、２０１Ｂ７細胞中の相対的ＳＯＸ２　ＲＮＡ発現レベルを示す。３つの培養ウェルから算出された平均値を棒グラフで示し、個々の値を黒色点で表す。各値は、コントロールサンプルから算出された平均値に対して正規化される。ＨＰＲＴ１および１８Ｓをそれぞれ、（Ａ）および（Ｂ）におけるハウスキーピング遺伝子として用いた。FIG. 5 shows relative SOX2 RNA expression levels in 201B7 cells calculated using RT-qPCR. The average values calculated from the three culture wells are shown in a bar graph, and the individual values are represented by black dots. Each value is normalized to the mean value calculated from the control sample. HPRT1 and 18S were used as housekeeping genes in (A) and (B), respectively. 図６は、Ａｒｇ３およびＢｉとコンジュゲートしたＰＩＰを導入した細胞におけるＰＧＣ－１αおよびＰＧＣ－１βの相対的ｍＲＮＡレベルを示す。図中、「Ａｒｇ３－ＢｉＲ」は、ＰＩＰ、ＢｉおよびＡｒｇ３のコンジュゲート（ＢｉＲ－Ａｒｇ３）を示す。FIG. 6 shows the relative mRNA levels of PGC-1α and PGC-1β in PIP-introduced cells conjugated with Arg3 and Bi. In the figure, "Arg3-BiR" indicates a conjugate of PIP, Bi and Arg3 (BiR-Arg3). 図７は、Ａｒｇ３およびＢｉとコンジュゲートしたＰＩＰの添加による、低ＣＤ４４ ＣＤ８陽性Ｔ細胞におけるミトコンドリアの活性化を示す。図中、「Ａｒｇ－ＢｉＲ」はＰＩＰ、ＢｉおよびＡｒｇ３のコンジュゲート（Ｂｉ－Ｒ－Ａｒｇ３）を示し、「ＢｉＲ」はＰＩＰとＢｉのコンジュゲートを示し、「Ｂｉ ｏｎｌｙ」はブロモドメイン阻害剤を示し、「Ｒ ｏｎｌｙ」はＰＩＰを示す。FIG. 7 shows the activation of mitochondria in low CD44 CD8 positive T cells by the addition of PIP conjugated with Arg3 and Bi. In the figure, "Arg-BiR" indicates a conjugate of PIP, Bi and Arg3 (Bi-R-Arg3), "BiR" indicates a conjugate of PIP and Bi, and "Bi only" indicates a bromodomain inhibitor. Indicated, "R only" indicates PIP. 図８は、Ａｒｇ３およびＢｉとコンジュゲートしたＰＩＰの添加による、高ＣＤ４４ ＣＤ８陽性Ｔ細胞におけるミトコンドリアの活性化を示す。図中、「Ａｒｇ－ＢｉＲ」はＰＩＰ、ＢｉおよびＡｒｇ３のコンジュゲート（Ｂｉ－Ｒ－Ａｒｇ３）を示し、「ＢｉＲ」はＰＩＰとＢｉのコンジュゲートを示し、「Ｂｉ ｏｎｌｙ」はブロモドメイン阻害剤を示し、「Ｒ ｏｎｌｙ」はＰＩＰを示す。FIG. 8 shows the activation of mitochondria in high CD44 CD8 positive T cells by the addition of PIP conjugated with Arg3 and Bi. In the figure, "Arg-BiR" indicates a conjugate of PIP, Bi and Arg3 (Bi-R-Arg3), "BiR" indicates a conjugate of PIP and Bi, and "Bi only" indicates a bromodomain inhibitor. Indicated, "R only" indicates PIP. 図９は、実施例５のｉｎ　ｖｉｖｏ実験のスケジュールを模式的に示す。FIG. 9 schematically shows the schedule of the in vivo experiment of Example 5. 図１０は、実施例５における腫瘍サイズの測定結果を示す。図中、横軸は腫瘍移植後の経過日数を示し、縦軸は腫瘍サイズを示す。図中、「Ｃｔｒｌ　ＩｇＧ」は抗ＰＤ－Ｌ１モノクローナル抗体のアイソタイプコントロールのみの投与群を示し、「ＰＤ－Ｌ１」は抗ＰＤ－Ｌ１モノクローナル抗体単独治療群を示し、「ＰＤ－Ｌ１＋Ｂｉ－Ｒ－Ａｒｇ３」は抗ＰＤ－Ｌ１モノクローナル抗体と、ＰＩＰ、ＢｉおよびＡｒｇ３のコンジュゲートとの併用治療群を示す。FIG. 10 shows the measurement result of the tumor size in Example 5. In the figure, the horizontal axis shows the number of days elapsed after tumor transplantation, and the vertical axis shows the tumor size. In the figure, "Ctrl IgG" indicates an anti-PD-L1 monoclonal antibody isotype control-only administration group, "PD-L1" indicates an anti-PD-L1 monoclonal antibody monotherapy group, and "PD-L1 + Bi-R-Arg3". "Shows a combination treatment group of an anti-PD-L1 monoclonal antibody and a conjugate of PIP, Bi and Arg3. 図１０は、実施例５における腫瘍サイズの測定結果を示す。図中、横軸は腫瘍移植後の経過日数を示し、縦軸は腫瘍サイズを示す。図中、「Ｃｔｒｌ　ＩｇＧ」は抗ＰＤ－Ｌ１モノクローナル抗体のアイソタイプコントロールのみの投与群を示し、「Ｂｉ－Ｒ－Ａｒｇ３」はＰＩＰ、ＢｉおよびＡｒｇ３のコンジュゲートのみの投与群を示し、「Ｂｉ」はブロモドメイン阻害剤のみの投与群を示し、「Ｒ」はＰＩＰのみの投与群を示す。FIG. 10 shows the measurement result of the tumor size in Example 5. In the figure, the horizontal axis shows the number of days elapsed after tumor transplantation, and the vertical axis shows the tumor size. In the figure, "Ctrl IgG" indicates a group in which only the isotype control of the anti-PD-L1 monoclonal antibody is administered, and "Bi-R-Arg3" indicates a group in which only the conjugate of PIP, Bi and Arg3 is administered, and "Bi". Indicates a group to which only the bromodomain inhibitor was administered, and "R" indicates a group to which only PIP was administered. 図１１は、実施例５におけるマウスの生存率を示す。図中、横軸は腫瘍移植後の経過日数を示し、縦軸は生存率（％）を示す。図中、「Ｃｔｒｌ　ＩｇＧ」は抗ＰＤ－Ｌ１モノクローナル抗体のアイソタイプコントロールのみの投与群を示し、「ＰＤ－Ｌ１」は抗ＰＤ－Ｌ１モノクローナル抗体単独治療群を示し、「ＰＤ－Ｌ１＋Ｂｉ－Ｒ－Ａｒｇ３」は抗ＰＤ－Ｌ１モノクローナル抗体と、ＰＩＰ、ＢｉおよびＡｒｇ３のコンジュゲートとの併用治療群を示し、「Ｂｉ－Ｒ－Ａｒｇ３」はＰＩＰ、ＢｉおよびＡｒｇ３のコンジュゲートのみの投与群を示し、「Ｂｉ」はブロモドメイン阻害剤のみの投与群を示し、「Ｒ」はＰＩＰのみの投与群を示す。FIG. 11 shows the survival rate of mice in Example 5. In the figure, the horizontal axis shows the number of days elapsed after tumor transplantation, and the vertical axis shows the survival rate (%). In the figure, "Ctrl IgG" indicates an anti-PD-L1 monoclonal antibody isotype control-only administration group, "PD-L1" indicates an anti-PD-L1 monoclonal antibody monotherapy group, and "PD-L1 + Bi-R-Arg3". "" Indicates a combination treatment group of an anti-PD-L1 monoclonal antibody and a conjugate of PIP, Bi and Arg3, and "Bi-R-Arg3" indicates a group of administration of only the conjugate of PIP, Bi and Arg3. "Bi" indicates the administration group of the bromodomain inhibitor only, and "R" indicates the administration group of PIP only.
１．ＰＩポリアミド
　ＰＩポリアミドは、一般に、Ｎ－メチルピロール（Ｐ）残基およびＮ－メチルイミダゾール（Ｉ）残基からなる逆平行に配向した２本の鎖を含み、ＰおよびＩは互いにアミド結合「－Ｃ（＝Ｏ）－ＮＨ－」で連結されている［Trauger et al, Nature, 382, 559-61(1996); White et al, Chem.Biol., 4,569-78(1997);およびDervan, Bioorg. Med. Chem., 9, 2215-35(2001)］。ここで「逆平行」とは、ＰＩポリアミドの２本のポリアミド鎖が平行に並んでおり、かつ、該２本のポリアミド鎖のＮおよびＣ末端が互いに逆向きになるよう配向されていることをいう。ＰＩポリアミドはいくつかの形態をとることができ、例えば、ヘアピン型、環状、Ｈ－ｐｉｎ型、Ｕ－ｐｉｎ型等のＰＩポリアミドが知られている。ヘアピン型ＰＩポリアミドは、上記した２本のポリアミド鎖の他にγ－アミノ酪酸部分（以下、「γ－リンカー」または「γ－ターン」ともいう）を含み、一方の鎖のＣ末端ともう一方の鎖のＮ末端がγ－リンカーによって連結されたＵ字型のコンフォメーション（ヘアピン型）をとる。ＰとＩとγ－アミノ酪酸部分とは互いにアミド結合で連結される。環状ＰＩポリアミドは、ヘアピン構造の末端を第２のγ－ターンによって閉環させて環状のコンフォメーションをとったものである。Ｈ－ｐｉｎ型およびＵ－ｐｉｎ型ＰＩポリアミドは、γ－ターンを含まず、一般に、それぞれ２本のポリアミド鎖の中央のＰのＮ－メチル基間および末端のＰおよびＩのＮ－メチル基間が脂肪族リンカーによって連結されている。本開示において、好ましくは、ヘアピン型または環状ＰＩポリアミドが使用される。 1. 1. PI Polyamide PI polyamide generally contains two antiparallel oriented chains consisting of N-methylpyrrole (P) residues and N-methylimidazole (I) residues, where P and I are amide-bonded to each other. C (= O) -NH- "is linked [Trauger et al, Nature, 382, 559-61 (1996); White et al, Chem.Biol., 4,569-78 (1997); and Dervan, Bioorg. . Med. Chem., 9, 2215-35 (2001)]. Here, "anti-parallel" means that two polyamide chains of PI polyamide are arranged in parallel, and the N- and C-termini of the two polyamide chains are oriented so as to be opposite to each other. say. PI polyamide can take several forms, for example, hairpin type, cyclic, H-pin type, U-pin type and other PI polyamides are known. The hairpin-type PI polyamide contains a γ-aminobutyric acid moiety (hereinafter, also referred to as “γ-linker” or “γ-turn”) in addition to the above-mentioned two polyamide chains, and the C-terminal of one chain and the other. The N-terminus of the chain is linked by a γ-linker to form a U-shaped conformation (hairpin type). P, I and the γ-aminobutyric acid moiety are linked to each other by an amide bond. The cyclic PI polyamide has a cyclic conformation in which the ends of the hairpin structure are ring-closed by a second γ-turn. H-pin and U-pin PI polyamides do not contain γ-turns and are generally between the N-methyl groups of P in the center and between the N-methyl groups of P and I at the ends of the two polyamide chains, respectively. Are linked by an aliphatic linker. In the present disclosure, hairpin type or cyclic PI polyamides are preferably used.
　ＰＩポリアミドは、上記２本のポリアミド鎖間で向かい合うＰおよび／またはＩからなる対（以下、「ピロール－イミダゾール対」ともいう）が特定の組み合わせ（Ｐ／Ｉ対、Ｉ／Ｐ対、またはＰ／Ｐ対）のときに、ＤＮＡ中の特定の塩基対に高い親和性で結合する。例えば、Ｐ／Ｉ対はＣ・Ｇ塩基対に結合することができ、Ｉ／Ｐ対はＧ・Ｃ塩基対に結合することができる。また、Ｐ／Ｐ対はＡ・Ｔ塩基対およびＴ・Ａ塩基対の両方に結合することができる。また、ＰＩポリアミドには、ＰおよびＩの他に、３－ヒドロキシピロール（Ｈｐ）およびβ－アラニン残基が含まれていてもよく、ＰをＨｐまたはβ－アラニン残基に置き換えることができる。Ｈｐ／Ｐ対は、Ｔ・Ａ塩基対に結合することができる。Ｐ／Ｈｐ対は、Ａ・Ｔ塩基対に結合することができる。β－アラニン／β－アラニン対は、Ｔ・Ａ塩基対またはＡ・Ｔ塩基対に結合することができる。β－アラニン／Ｉ対は、Ｃ・Ｇ塩基対に結合することができる。Ｉ／β－アラニン対は、Ｇ・Ｃ塩基対に結合することができる。β－アラニン／Ｐ対およびＰ／β－アラニン対は、Ｔ・Ａ塩基対またはＡ・Ｔ塩基対に結合することができる。さらに、γ－ターン部分は、Ｔ・Ａ塩基対またはＡ・Ｔ塩基対に結合することができる。Ｐ、Ｉ、Ｈｐ、および／またはβ－アラニン残基によって形成される対の構成、順序、および組み合わせ等を適宜変更することにより、所望のＤＮＡ配列を認識し、結合するＰＩポリアミドを設計することができる。好ましくは、ポリアミドのＮ末端側が標的ＤＮＡ配列の５’側にくるよう設計する。 PI polyamide is a specific combination (P / I pair, I / P pair, or P) of a pair consisting of P and / or I facing each other between the above two polyamide chains (hereinafter, also referred to as "pyrrole-imidazole pair"). / P pair), it binds to a specific base pair in DNA with high affinity. For example, a P / I pair can bind to a C / G base pair, and an I / P pair can bind to a G / C base pair. In addition, P / P pairs can bind to both A / T base pairs and T / A base pairs. Further, the PI polyamide may contain 3-hydroxypyrrole (Hp) and β-alanine residues in addition to P and I, and P can be replaced with Hp or β-alanine residues. Hp / P pairs can bind to TA base pairs. A P / Hp pair can bind to an A / T base pair. The β-alanine / β-alanine pair can bind to T / A base pair or A / T base pair. The β-alanine / I pair can bind to CG base pair. The I / β-alanine pair can bind to a G · C base pair. The β-alanine / P pair and the P / β-alanine pair can bind to TA base pair or AT base pair. In addition, the γ-turn moiety can bind to T / A base pair or A / T base pair. Designing a PI polyamide that recognizes and binds to a desired DNA sequence by appropriately modifying the composition, order, combination, etc. of the pair formed by P, I, Hp, and / or β-alanine residues. Can be done. Preferably, the N-terminal side of the polyamide is designed to be on the 5'side of the target DNA sequence.
　本開示において、ＰＩポリアミドを構成するＰおよびＩの１位の窒素上のメチル基は、水素またはメチル基以外のアルキル基で置き換わっていてもよい。メチル基以外のアルキル基の例としては、炭素数２～１０個の直鎖、分枝鎖または環状の飽和または不飽和アルキル基、好ましくは、炭素数２～５個の直鎖、分枝鎖直鎖、分枝鎖または環状の飽和または不飽和アルキル基が挙げられ、例えば、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、ｓｅｃ－ブチル、イソブチル、ｔｅｒｔ－ブチル等が挙げられる。また、メチル基を含め、アルキル基は置換されていてもよく、例えば、アルキル基中のメチレンは、酸素等で置換されていてもよい。さらに、上記したように、ＰＩポリアミドを構成するＰの３位はヒドロキシ基で置換されていてもよい。本明細書においてＰＩポリアミドに関して用いる場合、「Ｐ」または「ピロール」および「Ｉ」または「イミダゾール」なる語は、上記したようなＮ置換またはＮ非置換ピロール、３－ヒドロキシピロール、およびＮ置換またはＮ非置換イミダゾールを包含する。 In the present disclosure, the methyl group on the nitrogen at the 1-position of P and I constituting the PI polyamide may be replaced with an alkyl group other than hydrogen or a methyl group. Examples of alkyl groups other than methyl groups are linear, branched or cyclic saturated or unsaturated alkyl groups having 2 to 10 carbon atoms, preferably straight chain or branched chains having 2 to 5 carbon atoms. Examples include linear, branched or cyclic saturated or unsaturated alkyl groups, such as ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl and the like. Further, the alkyl group including the methyl group may be substituted, and for example, methylene in the alkyl group may be substituted with oxygen or the like. Further, as described above, the 3-position of P constituting the PI polyamide may be substituted with a hydroxy group. As used herein with respect to PI polyamide, the terms "P" or "pyrrole" and "I" or "imidazole" are N-substituted or N-unsubstituted pyrroles, 3-hydroxypyrroles, and N-substituted or as described above. Includes N-unsubstituted imidazole.
　本開示において、ＰＩポリアミドが標的とするＤＮＡ配列は特に限定されない。ＰＩポリアミドは、その使用目的に応じて、特定のＤＮＡ配列、すなわち、所望のいずれかのＤＮＡ配列を認識するように設計すればよい。例えば、特定のＤＮＡ配列は、限定するものではないが、疾患関連遺伝子の配列、修飾あるいは発現活性化または抑制したい遺伝子の配列、トリプレットリピート配列等のリピート配列、またはその関連配列から選択してもよい。 In the present disclosure, the DNA sequence targeted by PI polyamide is not particularly limited. The PI polyamide may be designed to recognize a specific DNA sequence, that is, any desired DNA sequence, depending on its intended use. For example, a specific DNA sequence may be selected from, but not limited to, a sequence of a disease-related gene, a sequence of a gene to be modified or activated or suppressed, a repeat sequence such as a triple repeat sequence, or a related sequence thereof. good.
　本開示において、ＰＩポリアミドが認識し、結合する特定のＤＮＡ配列の長さは、限定されないが、例えば２～３０塩基対、３～２５塩基対、４～２０塩基対、５～１５塩基対、または６～１０、９もしくは８塩基対からなるＤＮＡ配列であってもよい。トリプレットリピート配列等のリピート配列を標的とするＰＩポリアミドの場合、複数のＰＩポリアミドが標的配列に結合することより、当該リピート配列を含むさらに長いＤＮＡ配列（例えば、３０塩基対以上のＤＮＡ配列）を標的とすることもできる。後記するように、本開示の技術を用いれば、単独のＰＩポリアミドにより従来と比べて長いＤＮＡ配列を標的とすることができる。 In the present disclosure, the length of a particular DNA sequence recognized and bound by PI polyamide is not limited, but is, for example, 2 to 30 base pairs, 3 to 25 base pairs, 4 to 20 base pairs, 5 to 15 base pairs, and the like. Alternatively, it may be a DNA sequence consisting of 6 to 10, 9 or 8 base pairs. In the case of PI polyamide targeting a repeat sequence such as a triplet repeat sequence, a longer DNA sequence containing the repeat sequence (for example, a DNA sequence having 30 base pairs or more) can be obtained by binding a plurality of PI polyamides to the target sequence. It can also be targeted. As will be described later, by using the technique of the present disclosure, it is possible to target a DNA sequence longer than the conventional one by using a single PI polyamide.
　本開示において使用されるＰＩポリアミドを構成する、Ｐ、Ｉ、Ｈｐ、および／またはβアラニン残基によって形成されるピロール－イミダゾール対の数および種類は特に限定されず、標的配列に基づいて決定される。上記した特定のＤＮＡ配列を認識するように、ピロール－イミダゾール対の数および種類を決定すればよい。例えば、一実施形態において、ＰＩポリアミドを構成するピロール－イミダゾール対の数は、２～１５個、好ましくは３～１２個、より好ましくは４～１０個、例えば５、６、７、８または９個であってもよい。ピロール－イミダゾール対の数が５個以上のＤＮＡ配列認識部分を有するＰＩポリアミドを設計する場合は、好ましくはβアラニン残基を含むように設計される。本開示において使用されるＰＩポリアミドは、短鎖ＰＩポリアミドまたは長鎖ＰＩポリアミドのいずれであってもよい。例えば、５個以上のピロール－イミダゾール対を含有する長鎖ＰＩポリアミド（すなわち、含まれるＰ、Ｉおよびβアラニン残基の総数が１０個以上の長鎖ＰＩポリアミド）であってもよい。なお、本明細書において用いる場合、「ピロール－イミダゾール対」なる語は、Ｐ、Ｉ、Ｈｐ、およびβアラニン残基のいずれの組み合わせからなる対も包含する。 The number and type of pyrrole-imidazole pairs formed by the P, I, Hp, and / or β-alanine residues constituting the PI polyamide used in the present disclosure is not particularly limited and is determined based on the target sequence. To. The number and type of pyrrole-imidazole pairs may be determined to recognize the specific DNA sequence described above. For example, in one embodiment, the number of pyrrole-imidazole pairs constituting the PI polyamide is 2 to 15, preferably 3 to 12, more preferably 4 to 10, for example 5, 6, 7, 8 or 9. It may be an individual. When designing a PI polyamide having a DNA sequence recognition moiety having a pyrrole-imidazole pair number of 5 or more, it is preferably designed to contain a β-alanine residue. The PI polyamide used in the present disclosure may be either a short-chain PI polyamide or a long-chain PI polyamide. For example, it may be a long-chain PI polyamide containing 5 or more pyrrole-imidazole pairs (that is, a long-chain PI polyamide containing 10 or more total P, I and β-alanine residues). As used herein, the term "pyrrole-imidazole pair" also includes a pair consisting of any combination of P, I, Hp, and β-alanine residues.
　本開示において、ＰＩポリアミドのγ－ターン部分は置換されていてもよく、好ましくはγ－ターン部分のα位またはβ位に置換基を有していてもよく、さらに好ましくはγ－ターン部分のα位に置換基を有していてもよい。置換基としては、例えば、限定するものではないが、アミノ基、アセチルアミノ基、ジメチルアミノプロピルアミノ基、水酸基、メトキシ基等が挙げられる。例えば、γ－ターン部分は、α位またはβ位がアミノ基で置換された、Ｎ－α－Ｎ－γ－ジアミノ酪酸残基またはＮ－β－Ｎ－γ－ジアミノ酪酸残基であってもよい。 In the present disclosure, the γ-turn moiety of the PI polyamide may be substituted, preferably having a substituent at the α- or β-position of the γ-turn moiety, and more preferably the γ-turn moiety. It may have a substituent at the α-position. Examples of the substituent include, but are not limited to, an amino group, an acetylamino group, a dimethylaminopropylamino group, a hydroxyl group, a methoxy group and the like. For example, the γ-turn moiety may be an N-α-N-γ-diaminobutyric acid residue or an N-β-N-γ-diaminobutyric acid residue in which the α or β position is substituted with an amino group. good.
　本開示において、ＰＩポリアミドのＮ末端およびＣ末端には、種々の官能基または分子が付加されていてもよい。ＰＩポリアミドのＮ末端およびＣ末端に付加する官能基または分子は、当業者が適宜決定することができる。例えば、アミド結合を介して様々な官能基を付加することができる。該官能基の例としては、限定するものではないが、β－アラニン残基、γ－アミノ酪酸残基などのカルボキシル基、アセチル基、アミノ基等が挙げられる。例えば、限定するものではないが、Ｎ末端にはアセチル基が付加されていてもよい。例えば、限定するものではないが、Ｃ末端にはジメチルアミノプロピルアミノ基が付加されていてもよい。 In the present disclosure, various functional groups or molecules may be added to the N-terminal and C-terminal of PI polyamide. The functional groups or molecules to be added to the N-terminal and C-terminal of PI polyamide can be appropriately determined by those skilled in the art. For example, various functional groups can be added via an amide bond. Examples of the functional group include, but are not limited to, a carboxyl group such as a β-alanine residue and a γ-aminobutyric acid residue, an acetyl group, an amino group and the like. For example, but not limited to, an acetyl group may be added to the N-terminal. For example, but not limited to, a dimethylaminopropylamino group may be added to the C-terminal.
　ＰＩポリアミドのＮ末端およびＣ末端はまた、蛍光基やビオチン、イソフタル酸等の分子で修飾されていてもよい。本願明細書において、蛍光基としては、限定するものではないが、例えば、フルオレセイン、ローダミン系色素、ＴＡＭＲＡ（５－カルボキシテトラメチルローダミン）、シアニン系色素、ＡＴＴＯ系色素、Ａｌｅｘａ　Ｆｌｕｏｒ系色素、ＢＯＤＩＰＹが挙げられる。フルオレセインには、フルオレセイン誘導体（例えば、フルオレセインイソチオシアネート等）も含まれる。 The N-terminal and C-terminal of PI polyamide may also be modified with a fluorescent group or a molecule such as biotin or isophthalic acid. In the present specification, the fluorescent group includes, but is not limited to, fluorescein, rhodamine dye, TAMRA (5-carboxytetramethylrhodamine), cyanine dye, ATTO dye, Alexa Fluor dye, and BODIPY. Can be mentioned. Fluorescein also includes fluorescein derivatives (eg, fluorescein isothiocyanate, etc.).
　また、本開示において、ＰＩポリアミドは、ＤＮＡに対する結合能力を維持または向上するように修飾されたＰＩポリアミド修飾物であってもよい。該ＰＩポリアミド修飾物としては、例えば、ＰＩポリアミドのγ－リンカーのα位またはβ位にアミノ基を付加した修飾物、すなわち、Ｎ－α－Ｎ－γ－ジアミノ酪酸残基またはＮ－β－Ｎ－γ－ジアミノ酪酸残基からなるγ－リンカーを有する修飾物、該修飾物の上記アミノ基を蛍光基やビオチン等の分子で修飾した修飾物、ＰＩポリアミドのＮ末端を蛍光基やビオチン等の分子で修飾した修飾物、およびＰＩポリアミドのＣ末端をイソフタル酸等の分子で修飾した修飾物等が挙げられる。 Further, in the present disclosure, the PI polyamide may be a modified PI polyamide modified so as to maintain or improve the binding ability to DNA. Examples of the PI polyamide modified product include a modified product in which an amino group is added to the α-position or β-position of the γ-linker of PI polyamide, that is, an N-α-N-γ-diaminobutyric acid residue or N-β-. A modified product having a γ-linker composed of N-γ-diaminobutyric acid residues, a modified product obtained by modifying the amino group of the modified product with a molecule such as a fluorescent group or biotin, and an N-terminal of PI polyamide having a fluorescent group, biotin or the like. Examples thereof include a modified product modified with a molecule of PI polyamide and a modified product obtained by modifying the C-terminal of PI polyamide with a molecule such as isophthalic acid.
　ＰＩポリアミドの設計方法および製造方法は公知である（例えば、特許第３０４５７０６号、特開２００１－１３６９７４号、ＷＯ０３／０００６８３号、特開２０１３－２３４１３５号、特開２０１４－１７３０３２号参照）。例えば、Ｆｍｏｃ（９－フルオレニルメトキシカルボニル）を用いた固相合成法（Ｆｍｏｃ固相合成法）による自動合成によって簡便に製造することができる。また、液相合成法によって製造することもできる。 The design method and manufacturing method of PI polyamide are known (see, for example, Japanese Patent No. 3045706, Japanese Patent Application Laid-Open No. 2001-136794, WO03 / 000683, Japanese Patent Application Laid-Open No. 2013-234135, JP-A-2014-173032). For example, it can be easily produced by automatic synthesis by a solid phase synthesis method (Fmoc solid phase synthesis method) using Fmoc (9-fluorenylmethoxycarbonyl). It can also be produced by a liquid phase synthesis method.
２．ＰＩポリアミドコンジュゲート
　本明細書で使用される場合、「コンジュゲート」（または「複合体」ともいう）とは、安定したより大きな構築物を形成するのに十分な結合（例えば、共有結合）を介して連結されて、当該構築物を形成している２つまたはそれより多くの分子を指す。本開示において、上記ＰＩポリアミドと、１～５個のアルギニン残基を含むペプチドとを含む複合体が提供される。ＰＩポリアミドに該アルギニンペプチドを導入することによって、ＰＩポリアミドの細胞取り込み効率および核内蓄積量が向上する。したがって、本開示の技術を用いれば、従来、細胞取り込み効率が低いとされていた、長鎖ＰＩポリアミドやイミダゾール環を多く含むＰＩポリアミドであっても効率よく細胞内に送達させることができる。 2. 2. PI Polyamide Conjugates As used herein, a "conjugate" (also referred to as a "complex") is via a bond (eg, a covalent bond) sufficient to form a stable, larger construct. Refers to two or more molecules that are linked together to form the construct. In the present disclosure, a complex containing the above PI polyamide and a peptide containing 1 to 5 arginine residues is provided. By introducing the arginine peptide into PI polyamide, the cell uptake efficiency and nuclear accumulation amount of PI polyamide are improved. Therefore, by using the technique of the present disclosure, even a long-chain PI polyamide or a PI polyamide containing a large amount of an imidazole ring, which has been conventionally considered to have low cell uptake efficiency, can be efficiently delivered intracellularly.
　本開示において使用される「１～５個のアルギニン残基を含むアルギニンペプチド」は、例えば２～４個、好ましくは３個のアルギニン残基を含んでいてもよい。一の態様において、該アルギニンペプチドは、１～５個のアルギニン残基からなるペプチドであり、例えば２～４個、好ましくは３個のアルギニン残基からなるペプチドである。 The "arginine peptide containing 1 to 5 arginine residues" used in the present disclosure may contain, for example, 2 to 4, preferably 3 arginine residues. In one embodiment, the arginine peptide is a peptide consisting of 1-5 arginine residues, for example a peptide consisting of 2-4, preferably 3 arginine residues.
　上記アルギニンペプチドに含まれるアルギニンは、Ｄ－アルギニンまたはＬ－アルギニンのいずれであってもよく、またはその両方を含んでいてもよい。 The arginine contained in the above-mentioned arginine peptide may be either D-arginine or L-arginine, or may contain both.
　上記アルギニンペプチドは、ＰＩポリアミドのＮ末端、Ｃ末端、またはγ－ターン部分のいずれに結合していてもよい。また、Ｎ末端、Ｃ末端、およびγ－ターン部分から選ばれる２以上の位置に結合していてもよい。 The arginine peptide may be bound to any of the N-terminal, C-terminal, or γ-turn moiety of PI polyamide. Further, it may be bonded to two or more positions selected from the N-terminal, C-terminal, and γ-turn moiety.
　上記アルギニンペプチドは、アミド結合を介してＰＩポリアミドに直接結合していてもよく、またはリンカーを介して結合していてもよい。該リンカーとしては、アルギニンペプチドの作用を妨げず、かつＰＩポリアミドの標的配列認識を妨げないものであれば特に限定されない。リンカーの例としては、限定するものではないが、β－アラニンリンカー、ポリエーテルリンカー等が挙げられ、該リンカーの末端は、ＰＩポリアミドおよびアルギニンペプチドと例えばアミド、エステル、またはエーテル結合等を介して結合される。上記リンカーは当業者により適宜決定することができる。 The arginine peptide may be directly bound to PI polyamide via an amide bond, or may be bound via a linker. The linker is not particularly limited as long as it does not interfere with the action of the arginine peptide and does not interfere with the recognition of the target sequence of PI polyamide. Examples of the linker include, but are not limited to, β-alanine linker, polyether linker and the like, and the terminal of the linker is connected to PI polyamide and arginine peptide, for example, via an amide, ester, ether bond or the like. Be combined. The above linker can be appropriately determined by those skilled in the art.
　上記ＰＩポリアミドとアルギニンペプチドとの複合体においてアルギニンペプチドがＰＩポリアミドのＮ末端またはＣ末端に結合している場合、該アルギニンペプチドのＮ末端またはＣ末端には種々の官能基または分子が付加されていてもよい。該アルギニンペプチドのＮ末端およびＣ末端に付加する官能基または分子は、当業者が適宜決定することができ、例えば、アミド結合を介して様々な官能基を付加することができる。該官能基の例としては、上記「１．ＰＩポリアミド」のＮ末端またはＣ末端に付加できる官能基の例と同様に、β－アラニン残基、γ－アミノ酪酸残基などのカルボキシル基、アセチル基、アミノ基等が挙げられる。例えば、限定するものではないが、Ｎ末端にはアセチル基が付加されていてもよい。例えば、限定するものではないが、Ｃ末端にはジメチルアミノプロピルアミノ基が付加されていてもよい。あるいは、後記するように、ＰＩポリアミドにコンジュゲートしたアルギニンペプチドのＮ末端またはＣ末端に、機能性分子が結合していてもよい。 When the arginine peptide is bound to the N-terminal or C-terminal of the PI polyamide in the complex of the PI polyamide and the arginine peptide, various functional groups or molecules are added to the N-terminal or C-terminal of the arginine peptide. You may. The functional groups or molecules to be added to the N-terminal and C-terminal of the arginine peptide can be appropriately determined by those skilled in the art, and various functional groups can be added, for example, via an amide bond. Examples of the functional group include carboxyl groups such as β-alanine residue and γ-aminobutyric acid residue, and acetyl, as in the case of the functional group that can be added to the N-terminal or C-terminal of the above “1. PI polyamide”. Groups, amino groups and the like can be mentioned. For example, but not limited to, an acetyl group may be added to the N-terminal. For example, but not limited to, a dimethylaminopropylamino group may be added to the C-terminal. Alternatively, as described later, a functional molecule may be bound to the N-terminal or C-terminal of the arginine peptide conjugated to PI polyamide.
　アルギニンペプチドとＰＩポリアミドとのコンジュゲーション（結合）は、既知の方法により行うことができる。例えば、ＰＩポリアミドの合成時にアルギニンペプチドを導入することができ、固相合成法（Ｆｍｏｃ固相合成法）による自動合成によってＰＩポリアミドとアルギニンペプチドとのコンジュゲートを簡便に製造することができる。 Conjugation (binding) of arginine peptide and PI polyamide can be performed by a known method. For example, an arginine peptide can be introduced during the synthesis of PI polyamide, and a conjugate of PI polyamide and arginine peptide can be easily produced by automatic synthesis by a solid phase synthesis method (Fmoc solid phase synthesis method).
　本開示において、ＰＩポリアミドとアルギニンペプチドとの複合体は、さらに機能性分子とコンジュゲート（複合体化）していてもよい。本開示において、「機能性分子」は、何らかの機能を有する分子であれば特に限定されない。機能性分子の例としては、限定するものではないが、蛍光分子、ビオチン、ポリエチレングリコール、アミノポリエチレングリコール等が挙げられる。機能性分子のさらなる例としては、ＰＩポリアミドが標的ＤＮＡ配列に結合した際に該配列を含む遺伝子に影響を与える物質（エピジェネティックモディファイアー）が挙げられ、限定するものではないが、例えば、転写制御物質、例えば、転写活性化物質、転写抑制物質等が挙げられる。 In the present disclosure, the complex of PI polyamide and arginine peptide may be further conjugated (complexed) with a functional molecule. In the present disclosure, the "functional molecule" is not particularly limited as long as it is a molecule having some function. Examples of functional molecules include, but are not limited to, fluorescent molecules, biotin, polyethylene glycol, aminopolyethylene glycol and the like. Further examples of functional molecules include, but are not limited to, transcriptional modifiers that affect genes containing the sequence when PI polyamide binds to the target DNA sequence. Control substances, for example, transcription activators, transcription inhibitors and the like can be mentioned.
　蛍光分子の例としては、フルオレセイン、フルオレセイン誘導体（例えば、フルオレセインイソチオシアネート等）、ローダミン系色素、ＴＡＭＲＡ（５－カルボキシテトラメチルローダミン）、シアニン系色素、ＡＴＴＯ系色素、Ａｌｅｘａ　Ｆｌｕｏｒ系色素、ＢＯＤＩＰＹ等が挙げられるが、これらに限定されない。 Examples of fluorescent molecules include fluorescein, fluorescein derivatives (for example, fluorescein isothiocyanate, etc.), rhodamine dyes, TAMRA (5-carboxytetramethylrhodamine), cyanine dyes, ATTO dyes, Alexa Fluor dyes, BODIPY, and the like. However, but not limited to these.
　転写活性化物質の例としては、ブロモドメイン阻害剤（例えば、ＪＱ１、５－イソキサゾリル－ベンゾイミダゾール化合物、Ｉ－ＢＥＴ７６２、ＯＴＸ０１５等）、ヒストンアセチル化酵素（ＨＡＴ）活性化剤［例えば、Ｎ－（４－クロロ－３－（トリフルオロメチル）フェニル）－２－エトキシベンズアミド（ＣＴＢ）等］、ヒストン脱アセチル化酵素（ＨＤＡＣ）阻害剤［例えば、スベロイルアニリドヒドロキサム酸（ＳＡＨＡ）等］等が挙げられるが、これらに限定されない。 Examples of transcriptional activators include bromodomain inhibitors (eg, JQ1, 5-isoxazolyl-benzoimidazole compounds, I-BET762, OTX015, etc.), histone acetylase (HAT) activators [eg, N- (eg, N- (eg, N-). 4-Chloro-3- (trifluoromethyl) phenyl) -2-ethoxybenzamide (CTB), etc.], histone deacetylase (HDAC) inhibitor [for example, suberoylanilide hydroxamic acid (SAHA), etc.], etc. However, it is not limited to these.
　転写抑制物質の例としては、限定するものではないが、ＨＡＴ阻害剤、アルキル化剤等が挙げられる。アルキル化剤としては、特に限定されないが、後述する医薬組成物における使用を考慮して、細胞毒性が低いまたは無いものが好ましい。アルキル化剤の例としては、限定するものではないが、クロラムブシル（chlorambucil）、デュオカルマイシン（duocarmycin）、ｓｅｃｏ－ＣＢＩ（１－クロロメチル－５－ヒドロキシ－１，２－ジヒドロ－３Ｈ－ベンゾ［ｅ］インドール）、ＣＢＩ（１，２，９，９ａ－テトラヒドロシクロプロパン［ｃ］ベンゾ［ｅ］インドール－４－オン）、ピロロベンゾジアゼピン、ナイトロジェンマスタード等が挙げられる。ｓｅｃｏ－ＣＢＩまたはＣＢＩをＰＩポリアミドにコンジュゲートする場合は、ＰＩポリアミドとｓｅｃｏ－ＣＢＩまたはＣＢＩとの間にインドールを挿入してもよい。 Examples of the transcription inhibitor include, but are not limited to, HAT inhibitors, alkylating agents and the like. The alkylating agent is not particularly limited, but is preferably one having low or no cytotoxicity in consideration of its use in a pharmaceutical composition described later. Examples of alkylating agents include, but are not limited to, chlorambucil, duocarmycin, seco-CBI (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benzo]. e] Indole), CBI (1,2,9,9a-tetrahydrocyclopropane [c] benzo [e] indole-4-one), pyrolobenzodiazepines, nitrogen mustards and the like. When conjugating seco-CBI or CBI to PI polyamide, an indole may be inserted between PI polyamide and seco-CBI or CBI.
　上記アルキル化剤の例として挙げられたクロラムブシル、ｓｅｃｏ－ＣＢＩ、およびＣＢＩは、下記の化学式で表される。
Figure JPOXMLDOC01-appb-C000003
 Chlorambucil, seco-CBI, and CBI given as examples of the above-mentioned alkylating agent are represented by the following chemical formulas.
Figure JPOXMLDOC01-appb-C000003
　機能性分子は、ＰＩポリアミドのＮ末端、Ｃ末端、またはγ－ターン部分に結合していてもよく、あるいは、ＰＩポリアミドとコンジュゲートしているアルギニンペプチドのＮ末端またはＣ末端に結合していてもよい。機能性分子とＰＩポリアミドまたはアルギニンペプチドは、例えば、アミド結合、ホスホジスルフィド結合、エステル結合、配位結合、エーテル結合等によって直接結合していてもよく、またはリンカーを介して結合していてもよい。リンカーとしては、機能性分子の作用を妨げず、かつＰＩポリアミドの標的配列認識およびアルギニンペプチドの作用を妨げないものであれば特に限定されない。例えば、上記リンカーは、アミド結合、ホスホジスルフィド結合、エステル結合、配位結合、エーテル結合等からなる群から選択される１種類以上の結合を形成する官能基を含む分子であってもよい。リンカーの例としては、限定するものではないが、β－アラニンリンカー、ポリエチレングリコールリンカー、ペプチドリンカー、アルキルリンカー、アミノアルキルリンカー、ポリエーテルリンカー等が挙げられる。 The functional molecule may be attached to the N-terminus, C-terminus, or γ-turn moiety of the PI polyamide, or to the N-terminus or C-terminus of the arginine peptide conjugated to the PI polyamide. May be good. The functional molecule and the PI polyamide or arginine peptide may be directly bonded by, for example, an amide bond, a phosphodisulfide bond, an ester bond, a coordination bond, an ether bond, or the like, or may be bonded via a linker. .. The linker is not particularly limited as long as it does not interfere with the action of the functional molecule and does not interfere with the target sequence recognition of PI polyamide and the action of the arginine peptide. For example, the linker may be a molecule containing a functional group that forms one or more bonds selected from the group consisting of amide bonds, phosphodisulfide bonds, ester bonds, coordination bonds, ether bonds and the like. Examples of the linker include, but are not limited to, β-alanine linker, polyethylene glycol linker, peptide linker, alkyl linker, amino alkyl linker, polyether linker and the like.
　ＰＩポリアミドとアルギニンペプチドとの複合体と、機能性分子とのコンジュゲーションは、既知のカップリング方法または合成方法にしたがって行うことができる。 Conjugation of the complex of PI polyamide and arginine peptide with a functional molecule can be performed according to a known coupling method or synthetic method.
　本開示の一態様において、ＰＩポリアミドとアルギニンペプチドとの複合体、またはＰＩポリアミドとアルギニンペプチドと機能性分子との複合体（以下、まとめて「ＰＩポリアミドコンジュゲート」という）は、薬理学的に許容し得る塩の形態であってもよい。薬理学的に許容し得る塩としては、例えば、塩酸塩、硫酸塩、リン酸塩もしくは臭化水素酸塩等の無機酸塩、または酢酸塩、フマル酸塩、マレイン酸塩、シュウ酸塩、クエン酸塩、メタンスルホン酸塩、ベンゼンスルホン酸塩もしくはトルエンスルホン酸塩等の有機酸塩が挙げられる。 In one aspect of the present disclosure, the complex of PI polyamide and arginine peptide, or the complex of PI polyamide, arginine peptide and functional molecule (hereinafter collectively referred to as "PI polyamide conjugate") is pharmacologically. It may be in the form of an acceptable salt. The pharmacologically acceptable salt includes, for example, an inorganic acid salt such as a hydrochloride, a sulfate, a phosphate or a hydrobromide, or an acetate, a fumarate, a maleate, a oxalate, and the like. Examples thereof include organic acid salts such as citrate, methane sulfonate, benzene sulfonate or toluene sulfonate.
　本開示の一態様において、ＰＩポリアミドコンジュゲートは、ＰＩポリアミド、アルギニンペプチド、機能性分子、および／またはリンカー部分から選択される１以上が、エナンチオマーもしくはジアステレオマーの形態またはこれらの混合物として存在していてもよい。例えば、上記したように、ＰＩポリアミドがγ－ターン部分において置換基を有する場合またはアルギニンペプチドもしくは機能性分子とコンジュゲートする場合、該置換基またはアルギニンペプチドもしくは機能性分子は、Ｒ配置またはＳ配置を取るようにγ－ターン部分に結合していてもよい。ＰＩポリアミドコンジュゲートがジアステレオマーまたはエナンチオマーの形態で得られる場合、これらを当該技術で周知の慣用方法、例えば、クロマトグラフィーまたは分別結晶法等で分離することができる。 In one aspect of the present disclosure, the PI polyamide conjugate is present in the form of an enantiomer or diastereomeric or a mixture thereof, wherein one or more selected from the PI polyamide, arginine peptide, functional molecule, and / or linker moiety is present. May be. For example, as described above, if the PI polyamide has a substituent at the γ-turn moiety or is conjugated to an arginine peptide or functional molecule, the substituent or arginine peptide or functional molecule may be in an R or S configuration. It may be bonded to the γ-turn portion so as to take. When PI polyamide conjugates are obtained in the form of diastereomers or enantiomers, they can be separated by conventional methods well known in the art, such as chromatography or fractional crystallization.
　本開示の一態様において、ＰＩポリアミドコンジュゲートを含むミトコンドリアを活性化させるための剤が提供される。本開示におけるミトコンドリアを活性化させるための剤は、好ましくは、上記ＰＩポリアミド、アルギニンペプチド、および機能性分子を含む複合体を含む。ここで、機能性分子は、例えば、ブロモドメイン阻害剤である。本開示におけるミトコンドリアを活性化させるための剤は、より好ましくは、５’－ＷＣＧＣＣＷ－３’配列（Ｗ＝ＡまたはＴ）を認識するＰＩポリアミドとトリアルギニンペプチドとブロモドメイン阻害剤とのコンジュゲートであり、例えば、下記式：
Figure JPOXMLDOC01-appb-C000004
 で示される。本開示におけるミトコンドリアを活性化させるための剤は、ＰＧＣ（peroxisome proliferator activated receptor gamma coactivator）－１αおよびＰＧＣ－１βの発現を亢進し、ミトコンドリアの機能増強をもたらす。 In one aspect of the present disclosure, an agent for activating mitochondria, including a PI polyamide conjugate, is provided. Agents for activating mitochondria in the present disclosure preferably include a complex comprising the PI polyamide, arginine peptide, and functional molecule described above. Here, the functional molecule is, for example, a bromodomain inhibitor. The agent for activating mitochondria in the present disclosure is more preferably a conjugate of PI polyamide, a triarginine peptide and a bromodomain inhibitor that recognizes the 5'-WCGCCW-3'sequence (W = A or T). For example, the following formula:
Figure JPOXMLDOC01-appb-C000004
 Indicated by. The agents for activating mitochondria in the present disclosure enhance the expression of PGC (peroxisome proliferator activated receptor gamma coactivator) -1α and PGC-1β, resulting in enhanced mitochondrial function.
３．ＰＩポリアミドコンジュゲートを含む医薬組成物
　本開示において、ＰＩポリアミドコンジュゲートを含む医薬組成物が提供される。本開示における医薬組成物は、好ましくは、上記ＰＩポリアミド、アルギニンペプチド、および機能性分子を含む複合体を含む。本開示における医薬組成物は、含まれるＰＩポリアミドの標的配列によって、様々な疾患の治療または予防、例えば限定するものではないが、腫瘍、癌、マスト細胞疾患、アレルギー、免疫疾患、トリプレットリピート病等のリピート病等の疾患の治療または予防、あるいは所望の遺伝子の修飾または発現活性化もしくは抑制等の目的で使用することができる。 3. 3. Pharmaceutical Compositions Containing PI Polyamide Conjugates In the present disclosure, pharmaceutical compositions containing PI polyamide conjugates are provided. The pharmaceutical composition in the present disclosure preferably comprises a complex containing the above PI polyamide, arginine peptide, and functional molecule. The pharmaceutical composition in the present disclosure treats or prevents various diseases, for example, but is not limited to, tumors, cancers, mast cell diseases, allergies, immune diseases, triplet repeat diseases, etc., depending on the target sequence of PI polyamide contained therein. It can be used for the purpose of treating or preventing diseases such as repeat diseases, or modifying or activating or suppressing the expression of a desired gene.
　本開示における医薬組成物は、経口投与および非経口投与のいずれの剤形でもよい。これらの剤形は常法にしたがって製剤化することができ、医薬的に許容される担体や添加物を含むものであってもよい。このような担体および添加物として、水、酢酸、医薬的に許容される有機溶剤、コラーゲン、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、カルボキシメチルセルロースナトリウム、ポリアクリル酸ナトリウム、アルギン酸ナトリウム、水溶性デキストラン、カルボキシメチルスターチナトリウム、ペクチン、メチルセルロース、エチルセルロース、キサンタンガム、アラビアゴム、カゼイン、寒天、ポリエチレングリコール、ジグリセリン、グリセリン、プロピレングリコール、ワセリン、パラフィン、ステアリルアルコール、ステアリン酸、ヒト血清アルブミン、マンニトール、ソルビトール、ラクトース、医薬添加物として許容される界面活性剤等が挙げられる。 The pharmaceutical composition in the present disclosure may be in either oral or parenteral dosage form. These dosage forms can be formulated according to a conventional method, and may contain a pharmaceutically acceptable carrier or additive. Such carriers and additives include water, acetic acid, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, Carboxymethylstarch sodium, pectin, methylcellulose, ethylcellulose, xanthan gum, arabic rubber, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose , Surfactants and the like that are acceptable as pharmaceutical additives.
　上記添加物は、本開示における医薬組成物の剤型に応じて上記の中から単独でまたは適宜組み合わせから選ばれる。剤形としては、経口投与の場合は、錠剤、カプセル剤、細粒剤、粉末剤、顆粒剤、散剤、液剤、シロップ剤、噴霧剤等として、または適当な剤型により投与が可能である。非経口投与の場合は、注射剤型、塗布剤、点眼剤、外用剤、貼付剤、座薬剤等が挙げられる。注射剤型の場合は、例えば点滴等の静脈内注射、皮下注射、腹腔内注射、腫瘍内注射等により全身または局部的に投与することができる。 The additive is selected alone or in combination from the above depending on the dosage form of the pharmaceutical composition in the present disclosure. In the case of oral administration, the dosage form can be administered as tablets, capsules, fine granules, powders, granules, powders, liquids, syrups, sprays and the like, or in an appropriate dosage form. In the case of parenteral administration, injection type, coating agent, eye drop, external use, patch, suppository and the like can be mentioned. In the case of the injection type, it can be administered systemically or locally by, for example, intravenous injection such as infusion, subcutaneous injection, intraperitoneal injection, intratumoral injection and the like.
　例えば、注射用製剤として使用する場合、本開示における医薬組成物を溶剤（例えば、生理食塩水、緩衝液、ブドウ糖溶液、０．１％酢酸等）に溶解し、これに適当な添加剤（ヒト血清アルブミン、ＰＥＧ、マンノース修飾デンドリマー、シクロデキストリン結合体等）を加えたものを使用することができる。あるいは、使用前に溶解する剤形とするために凍結乾燥したものであってもよい。凍結乾燥用賦形剤としては、例えば、マンニトール、ブドウ糖等の糖アルコールや糖類を使用することができる。 For example, when used as an injectable preparation, the pharmaceutical composition in the present disclosure is dissolved in a solvent (for example, physiological saline, buffer solution, dextrin solution, 0.1% acetic acid, etc.) and an appropriate additive (human) is used. Serum albumin, PEG, mannose-modified dendrimer, cyclodextrin conjugate, etc.) can be added. Alternatively, it may be freeze-dried to form a dosage form that dissolves before use. As the excipient for freeze-drying, for example, sugar alcohols such as mannitol and glucose and sugars can be used.
　本開示における医薬組成物を投与する対象は、その使用目的にもよるが、二本鎖ＤＮＡを生体制御に利用するあらゆる生物が挙げられ、限定するものではないが、例えば動物、特に哺乳動物（例、ヒト、ラット、マウス、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サル、ヒヒ等）、好ましくはヒトに投与することができる。また、ある実施態様においては、ヒトを除く動物に投与してもよい。 The subject to which the pharmaceutical composition in the present disclosure is administered includes, but is not limited to, any organism that utilizes double-stranded DNA for biological control, although it depends on the purpose of use thereof, for example, animals, particularly mammals ( For example, humans, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, baboons, etc.), preferably humans. In some embodiments, it may be administered to animals other than humans.
　本開示における医薬組成物の対象への投与方法（投与経路、投与量、１日の投与回数、投与のタイミング、等）は、投与対象の種、対象の年齢、性別、および症状、対象の健康状態、併用薬の有無および種類等によって異なり、当業者によって適宜決定される。 The method of administration of the pharmaceutical composition to a subject (administration route, dose, frequency of administration per day, timing of administration, etc.) in the present disclosure includes the species of the subject to be administered, the age, sex, and symptoms of the subject, and the health of the subject. It depends on the condition, the presence / absence and type of concomitant drug, and is appropriately determined by those skilled in the art.
　本開示における医薬組成物は、本分野において公知の他の抗がん剤と併用することができる。該併用の態様は限定されず、対象とするがんの種類や治療ステージ等に応じて、当業者が適宜実施することができる。本開示における医薬組成物と他の抗がん剤とは、同時又は異時に対象に投与されてよい。また、本開示における医薬組成物と他の抗がん剤とは、それぞれを含む一の配合剤として準備され、対象へ投与されてもよく、またはそれぞれ別々の剤として準備され、対象へ投与されてもよい。また、本開示における医薬組成物と他の抗がん剤とは、それぞれを別々に備えるキットとして準備されてもよい。 The pharmaceutical composition in the present disclosure can be used in combination with other anticancer agents known in the art. The mode of the combination is not limited, and can be appropriately carried out by those skilled in the art depending on the type of target cancer, the stage of treatment, and the like. The pharmaceutical composition and other anticancer agents in the present disclosure may be administered to a subject at the same time or at different times. In addition, the pharmaceutical composition and other anticancer agents in the present disclosure may be prepared as one combination drug containing each and administered to a subject, or may be prepared as separate agents and administered to a subject. You may. In addition, the pharmaceutical composition and other anticancer agents in the present disclosure may be prepared as a kit separately comprising each.
　本開示における医薬組成物と他の抗がん剤とを同時に投与する態様としては、例えば、本開示における医薬組成物と他の抗がん剤とを含む一の配合剤を対象へ投与する態様であってよい。 As an embodiment in which the pharmaceutical composition in the present disclosure and another anticancer agent are simultaneously administered, for example, an embodiment in which one combination drug containing the pharmaceutical composition and the other anticancer agent in the present disclosure is administered to a subject. May be.
　本開示における医薬組成物と他の抗がん剤とを異時に投与する態様としては、例えば、本開示における医薬組成物と他の抗がん剤とを、時間をずらしてそれぞれ投与する態様であってよく、また、例えば、本開示における医薬組成物と他の抗がん剤とを異なる投与経路から投与する態様であってよい。 As an embodiment in which the pharmaceutical composition and other anticancer agents in the present disclosure are administered at different times, for example, the pharmaceutical composition and other anticancer agents in the present disclosure are administered at different times. It may be possible, and for example, the pharmaceutical composition in the present disclosure and other anticancer agents may be administered from different administration routes.
　併用される他の抗がん剤は限定されないが、例えば、免疫チェックポイント阻害剤、アルキル化薬、代謝拮抗薬、抗がん性抗生物質、植物性アルカロイド薬、抗ホルモン薬、白金化合物、サイトカイン製剤、分子標的薬、腫瘍免疫治療薬、およびがんワクチンからなる群から選択される、１または複数の抗がん剤を用いることができる。ある実施態様では、他の抗がん剤は免疫チェックポイント阻害剤であってもよい。 Other anticancer agents used in combination are not limited, but are, for example, immune checkpoint inhibitors, alkylating agents, metabolic antagonists, anticancer antibiotics, plant alkaloids, antihormonal agents, platinum compounds, cytokines, etc. One or more anti-cancer agents selected from the group consisting of formulations, molecular targeted agents, tumor immunotherapeutic agents, and cancer vaccines can be used. In certain embodiments, the other anti-cancer agent may be an immune checkpoint inhibitor.
　本開示において、「免疫チェックポイント阻害剤」は、免疫チェックポイント分子の機能を阻害する物質を意味する。「免疫チェックポイント分子」は、自己に対する免疫応答および／または過剰な免疫反応を抑制する分子であり、例えば、ＣＴＬＡ－４、ＰＤ－１、ＰＤ－Ｌ１、ＰＤ－Ｌ２、ＬＡＧ－３、ＴＩＭ３、ＢＴＬＡ、Ｂ７Ｈ３、Ｂ７Ｈ４、２Ｂ４、ＣＤ１６０、Ａ２ａＲ、ＫＩＲ、ＶＩＳＴＡ、ＴＩＧＩＴ等が挙げられるが、これらに限定されない。免疫チェックポイント阻害剤として、例えば、抗ＣＴＬＡ－４抗体（例えば、イピリムマブ、トレメリムマブ、ＡＧＥＮ－１８８４）、抗ＰＤ－１抗体（例えば、ニボルマブ、ＲＥＧＮ－２８１０、ペムブロリズマブ、ＰＤＲ－００１、ＢＧＢ－Ａ３１７、ＡＭＰ－５１４（ＭＥＤＩ０６８０）、ＢＣＤ－１００、ＩＢＩ－３０８、ＪＳ－００１、ＰＦ－０６８０１５９１、ＴＳＲ－０４２）、抗ＰＤ－Ｌ１抗体（例えば、アテゾリズマブ（ＲＧ７４４６、ＭＰＤＬ３２８０Ａ）、アベルマブ（ＰＦ－０６８３４６３５、ＭＳＢ００１０７１８Ｃ）、デュルバルマブ（ＭＥＤＩ４７３６）、ＢＭＳ－９３６５５９、ＣＡ－１７０、ＬＹ－３３０００５４）、抗ＰＤ－Ｌ２抗体（例えば、ｒＨＩｇＭ１２Ｂ７）、ＰＤ－Ｌ１融合タンパク質、ＰＤ－Ｌ２融合タンパク質（例えば、ＡＭＰ－２２４）、抗Ｔｉｍ－３抗体（例えば、ＭＢＧ４５３）、抗ＬＡＧ－３抗体（例えば、ＢＭＳ－９８６０１６、ＬＡＧ５２５）、抗ＫＩＲ抗体（例えば、リリルマブ）等、並びに、これらの抗体の重鎖および軽鎖相補性決定領域（ＣＤＲ）を有する抗体、および、これらの抗体の重鎖可変領域（ＶＨ）および軽鎖可変領域（ＶＬ）を含む抗体が挙げられる。ある実施態様では、免疫チェックポイント阻害剤はＰＤ－１阻害剤またはＰＤ－Ｌ１阻害剤であってもよい。ある実施態様では、免疫チェックポイント阻害剤は抗ＰＤ－１抗体または抗ＰＤ－Ｌ１抗体であってもよい。 In the present disclosure, "immune checkpoint inhibitor" means a substance that inhibits the function of an immune checkpoint molecule. An "immune checkpoint molecule" is a molecule that suppresses an immune response and / or an excessive immune response to self, eg, CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM3, etc. BTLA, B7H3, B7H4, 2B4, CD160, A2aR, KIR, VISTA, TIGIT and the like can be mentioned, but the present invention is not limited thereto. Examples of immune checkpoint inhibitors include anti-CTLA-4 antibody (eg, ipilimumab, tremellimumab, AGEN-1884), anti-PD-1 antibody (eg, nibolumab, REGN-2810, pembrolizumab, PDR-001, BGB-A317, etc.). AMP-514 (MEDI0680), BCD-100, IBI-308, JS-001, PF-06801591, TSR-042), anti-PD-L1 antibody (eg, atezolizumab (RG7446, MPDL3280A), Abelumab (PF-06834635, MSB0010718C). ), Durvalumab (MEDI4736), BMS-936559, CA-170, LY-3300054), anti-PD-L2 antibody (eg, rHIgM12B7), PD-L1 fusion protein, PD-L2 fusion protein (eg, AMP-224), Anti-Tim-3 antibody (eg, MBG453), anti-LAG-3 antibody (eg, BMS-986016, LAG525), anti-KIR antibody (eg, lilylmab), etc., as well as heavy and light chain complementarity determination of these antibodies. Examples include antibodies having regions (CDRs) and antibodies comprising heavy chain variable regions (VH) and light chain variable regions (VL) of these antibodies. In certain embodiments, the immune checkpoint inhibitor may be a PD-1 inhibitor or a PD-L1 inhibitor. In certain embodiments, the immune checkpoint inhibitor may be an anti-PD-1 antibody or an anti-PD-L1 antibody.
　本開示において、「抗体」の用語には、抗体の一部分を構成要素として含み、抗原への結合性を保持する分子、例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ、または一本鎖Ｆｖ（ｓｃＦｖ）、Ｆａｂ３、ダイアボディ、トリアボディ、テトラボディ、ミニボディ、Ｂｉｓ－ｓｃＦｖ、（ｓｃＦｖ）２－Ｆｃ、インタクトＩｇＧ等が含まれる。抗体は、ヒト抗体またはヒト化抗体であってもよい。ヒト化抗体とは、ヒト以外の動物の抗体のＶＨおよびＶＬのＣＤＲのアミノ酸配列をヒト抗体のＶＨおよびＶＬの適切な位置に移植した抗体を意味する。 In the present disclosure, the term "antibody" refers to a molecule that comprises a portion of an antibody as a component and retains its binding property to an antigen, such as Fab, Fab', F (ab') 2, Fv, or a single molecule. Chains Fv (scFv), Fab3, diabodies, triabodies, tetrabodies, minibodies, Bis-scFv, (scFv) 2-Fc, intact IgG and the like are included. The antibody may be a human antibody or a humanized antibody. The humanized antibody means an antibody in which the amino acid sequences of the CDRs of VH and VL of an antibody of a non-human animal are transplanted to appropriate positions of VH and VL of a human antibody.
　免疫チェックポイント阻害剤の投与方法は特に限定されず、経口または非経口（例えば、静脈内、皮下、皮内、胸腔内、腹腔内、筋肉内、組織内）の一般的な投与経路を経ることができる。ある実施態様では、免疫チェックポイント阻害剤は静脈内投与されてもよい。これらの投与方法に適する剤形の詳細は、上記の通りである。 The method of administration of the immune checkpoint inhibitor is not particularly limited, and the route of administration is generally oral or parenteral (for example, intravenous, subcutaneous, intracutaneous, intrathoracic, intraperitoneal, intramuscular, or intratissue). Can be done. In certain embodiments, the immune checkpoint inhibitor may be administered intravenously. The details of the dosage form suitable for these administration methods are as described above.
　免疫チェックポイント阻害剤の投与量および投与回数は、有効量が対象に投与されるように、対象の健康状態、年齢、体重、投与経路、投与形態等に応じて当業者が適宜設定できる。例えば、抗ＰＤ－L１抗体を６日毎、１週毎、２週毎、３週毎または４週毎に投与し得る。 The dose and frequency of administration of the immune checkpoint inhibitor can be appropriately set by those skilled in the art according to the health condition, age, body weight, administration route, administration form, etc. of the subject so that the effective amount is administered to the subject. For example, the anti-PD-L1 antibody may be administered every 6 days, every 1 week, every 2 weeks, every 3 weeks or every 4 weeks.
　本開示における医薬組成物の投与経路は限定されず、全身投与であっても、局所投与であってもよい。投与経路としては、例えば、舌下投与を含む経口投与、吸入投与、カテーテルや注射による標的組織に対する直接投与、点滴を含む静脈内投与、貼付剤等による経皮投与、座薬、または、経鼻胃管、経鼻腸管、胃ろうチューブ、もしくは腸ろうチューブ等を用いる強制的経腸栄養法による投与等の非経口投与を挙げることができる。 The route of administration of the pharmaceutical composition in the present disclosure is not limited, and may be systemic administration or local administration. The routes of administration include, for example, oral administration including sublingual administration, inhalation administration, direct administration to the target tissue by catheter or injection, intravenous administration including infusion, transdermal administration by patch, suppository, or nasogastric. Parenteral administration such as administration by forced enteral nutrition using a tube, nasogastric tube, gastric fistula tube, or intestinal fistula tube can be mentioned.
　本開示における医薬組成物と免疫チェックポイント阻害剤とを併用する場合、医薬組成物は、好ましくは、ミトコンドリアを活性化させるための剤である。すなわち、免疫チェックポイント阻害剤は、ミトコンドリアを活性化させるための剤と併用される。この場合、本開示における医薬組成物は、例えば、ＰＧＣ－１αおよびＰＧＣ－１βの少なくとも一方の発現を増強させるための剤であり、より好ましくは、５’－ＷＣＧＣＣＷ－３’配列（Ｗ＝ＡまたはＴ）を認識するＰＩポリアミドとトリアルギニンペプチドとブロモドメイン阻害剤とのコンジュゲートを含み、例えば、下記式：
Figure JPOXMLDOC01-appb-C000005
 で示されるコンジュゲートを含む。また、本開示における医薬組成物と免疫チェックポイント阻害剤とを併用する場合、免疫チェックポイント阻害剤は、好ましくは、ＰＤ－１阻害剤またはＰＤ－Ｌ１阻害剤であり、より好ましくは、抗ＰＤ－１抗体または抗ＰＤ－Ｌ１抗体である。このように、ミトコンドリアを活性化させるための剤と免疫チェックポイント阻害剤とを併用することで、抗腫瘍免疫力を高めることができる。なお、ミトコンドリアを活性化させるための剤と免疫チェックポイント阻害剤とを併用する場合、ミトコンドリアを活性化させるための剤は、ＰＧＣ－１αおよびＰＧＣ－１βの少なくとも一方の発現を増強させるための剤であればよく、適宜変更可能である。 When the pharmaceutical composition in the present disclosure is used in combination with an immune checkpoint inhibitor, the pharmaceutical composition is preferably an agent for activating mitochondria. That is, the immune checkpoint inhibitor is used in combination with an agent for activating mitochondria. In this case, the pharmaceutical composition in the present disclosure is, for example, an agent for enhancing the expression of at least one of PGC-1α and PGC-1β, and more preferably, a 5'-WCGCCW-3'sequence (W = A). Alternatively, it comprises a conjugate of PI polyamide, a triarginine peptide, and a bromodomain inhibitor that recognizes T), for example, the following formula:
Figure JPOXMLDOC01-appb-C000005
 Includes conjugates indicated by. When the pharmaceutical composition in the present disclosure is used in combination with an immune checkpoint inhibitor, the immune checkpoint inhibitor is preferably a PD-1 inhibitor or a PD-L1 inhibitor, and more preferably an anti-PD. -1 antibody or anti-PD-L1 antibody. As described above, the combined use of an agent for activating mitochondria and an immune checkpoint inhibitor can enhance antitumor immunity. When an agent for activating mitochondria and an immune checkpoint inhibitor are used in combination, the agent for activating mitochondria is an agent for enhancing the expression of at least one of PGC-1α and PGC-1β. Anything is fine, and it can be changed as appropriate.
　以下、実施例を用いて本発明をさらに具体的に説明するが、本発明はかかる実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to such examples.
実施例１：ＳＯＸ２結合配列を標的とするＰＩポリアミド（ＳＯＸ２ｉ）とトリアルギニンペプチドを含む複合体（ＳＯＸ２ｉ－Ｒ３）およびコントロール（Ｃｔｒｌ－Ｒ３）の合成
　ＳＯＸ２ｉ－Ｒ３、ＳＯＸ２ｉ、およびコントロール（Ｃｔｒｌ－Ｒ３）を合成した。これらのＰＩポリアミドコンジュゲートの構造を図１－１に示す。Ｃｔｒｌ－Ｒ３は、ＳＯＸ２結合配列とは異なる配列を標的とする。ＳＯＸ２結合配列として、５’－ＷＷＣＷＷＷＧ－３’（Ｗ＝ＡまたはＴ）を選択した。Ｃｔｒｌ－Ｒ３の標的配列として、５’－ＷＧＣＣＧＣＣ－３’（Ｗ＝ＡまたはＴ）を選択した。Ｄ－アルギニンを用いた。 Example 1: Synthesis of Complex (SOX2i-R3) and Control (Ctrl-R3) Containing PI Polyamide (SOX2i) and Triarginine Peptide Targeting SOX2 Binding Sequences SOX2i-R3, SOX2i, and Control (Ctrl-R3) ) Was synthesized. The structure of these PI polyamide conjugates is shown in FIG. 1-1. Ctrl-R3 targets a sequence different from the SOX2 binding sequence. 5'-WWCWWWG-3'(W = A or T) was selected as the SOX2 binding sequence. 5'-WGCCGCC-3'(W = A or T) was selected as the target sequence for Ctrl-R3. D-arginine was used.
（１）一般的情報
　合成には、標準的な供給元から購入した試薬を用いた。固相合成は、コンピューターアシストオペレーションシステムを有する自動合成機ＰＳＳＭ－８（Shimadzu）を用いて行った。逆相高速液体クロマトグラフィー（ＨＰＬＣ）は、Ｃｈｅｍｃｏｂｏｎｄ　５－ＯＤＳ－Ｈ逆相カラム（４．６×１５０ｍｍ）を備えたＪＡＳＣＯ　ＨＰＬＣシステム（JASCO Engineering UV2075 UV/vis detector and a PU-2089 plus gradient pump）で行った。Ｈ２Ｏ（＋０．１％トリフルオロ酢酸）およびアセトニトリルを移動相として用い、プログラムは、流速１．０ｍＬ／分、４０分、０～１００％アセトニトリルの直線勾配であった。２５４ｎｍでの吸光度をモニターした。ＭＡＬＤＩ－ＴＯＦ質量分析をｍｉｃｒｏｆｌｅｘシステム（Bruker）を用いて行った。 (1) General information Reagents purchased from standard suppliers were used for synthesis. Solid-phase synthesis was performed using an automatic synthesizer PSSM-8 (Shimadzu) equipped with a computer-assisted operation system. Reverse Phase High Performance Liquid Chromatography (HPLC) is a JASCO HPLC system (JASCO Engineering UV2075 UV / vis detector and a PU-2089 plus gradient pump) equipped with a Chemcobond 5-ODS-H reverse phase column (4.6 x 150 mm). I went there. Using H2O (+ 0.1% trifluoroacetic acid) and acetonitrile as mobile phases, the program had a linear gradient of 0-100% acetonitrile at a flow rate of 1.0 mL / min, 40 min. Absorbance at 254 nm was monitored. MALDI-TOF mass spectrometry was performed using a microflex system (Bruker).
（２）ＳＯＸ２ｉ－Ｒ３、ＳＯＸ２ｉ、およびＣｔｒｌ－Ｒ３の合成
　ＳＯＸ２ｉ－Ｒ３、ＳＯＸ２ｉ、およびＣｔｒｌ－Ｒ３の合成には、既知のＦｍｏｃ固相合成を用いた。各Ｆｍｏｃモノマー単位（Ｆｍｏｃ－Ｄ－Ａｒｇ（Ｐｂｆ）－ＯＨ、Ｆｍｏｃ－β－アラニン－ＯＨ、Ｆｍｏｃ－γアミノ酪酸（γＡｂｕ）－ＯＨ、Ｆｍｏｃ－Ｎ－メチルピロール（Ｐｙ）－ＯＨ、およびＦｍｏｃ－Ｎ－メチルイミダゾール（Ｉｍ）－ＯＨ）を連続的に、ＳＯＸ２ｉ－Ｒ３およびＣｔｒｌ－Ｒ３の合成用Ｆｍｏｃ－ＤＡｒｇ（Ｐｂｆ）－Ａｌｋｏ樹脂、およびＳＯＸ２ｉの合成用ＰｙＩｍ－結合型（coupled）クリア樹脂に導入した。また、合成効率を改善するために、Ｆｍｏｃ－ＩｍＰｙ－ＯＨダイマー単位をＳＯＸ２ｉ－Ｒ３の合成に用いた。Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）中の２０％アセトアルデヒドと混合することによって、Ｎ末端をアセチル基でキャップした。合成された化合物は、５５℃で３時間、Ｎ，Ｎ－ジメチルアミノプロピルアミンを用いて切り出した。反応溶液をエーテル中に流入し、得られた固体を真空乾燥させた。ＳＯＸ２ｉ－Ｒ３およびＣｔｒｌ－Ｒ３のアルギニン残基の保護基（Ｐｂｆ基）を、室温で３０分間、脱保護カクテル（トリフルオロ酢酸：トリイソプロピルシラン：水（９５：２．５：２．５ｖ／ｖ％））を用いて除去した。粗サンプルを、分取Ｃ１８（ＯＤＳ）カラム（ＣＯＳＭＯＳＩＬ　５Ｃ１８－ＭＳ－ＩＩ，１０ＩＤｘ１５０ｍｍ）を有するＪＡＳＣＯ　ＨＰＬＣシステム（JASCO Engineering UV2075 UV/vis detector and a PU-2089 plus gradient pump）を用いる逆相ＨＰＬＣによって精製した。Ｈ２Ｏ（＋０．１％トリフルオロ酢酸）およびアセトニトリルを移動相として用いた。ＨＰＬＣおよびＭＡＬＤＩ－ＴＯＦ質量分析は、精製化合物の特徴づけのために行った。
ＳＯＸ２ｉ－Ｒ３：分析HPLCtR=15.6分、C96H133N40O18 +のMALDI-TOF MS m/z [M+H]+計算値2134.07、実測値2134.06
ＳＯＸ２ｉ：分析HPLCtR=16.9分、C75H92N27O14 +のMALDI-TOF MS m/z [M+H]+計算値1594.73、実測値1594.64
Ｃｔｒｌ－Ｒ３：分析HPLCtR=15.8分、C92H129N44O18 +のMALDI-TOF MS m/z [M+H]+計算値2138.05、実測値2138.04 (2) Synthesis of SOX2i-R3, SOX2i, and Ctrl-R3 Known Fmoc solid-phase synthesis was used for the synthesis of SOX2i-R3, SOX2i, and Ctrl-R3. Each Fmoc monomer unit (Fmoc-D-Arg (Pbf) -OH, Fmoc-β-alanine-OH, Fmoc-γ-aminobutyric acid (γAbu) -OH, Fmoc-N-methylpyrrole (Py) -OH, and Fmoc- N-Methylimidazole (Im) -OH) was continuously added to the Fmoc-DArg (Pbf) -Alko resin for synthesis of SOX2i-R3 and Ctrl-R3, and the PyIm-coupled clear resin for synthesis of SOX2i. Introduced. Further, in order to improve the synthesis efficiency, the Fmoc-ImPy-OH dimer unit was used for the synthesis of SOX2i-R3. The N-terminus was capped with an acetyl group by mixing with 20% acetaldehyde in N, N-dimethylformamide (DMF). The synthesized compound was excised with N, N-dimethylaminopropylamine at 55 ° C. for 3 hours. The reaction solution was poured into ether and the obtained solid was vacuum dried. The protecting group (Pbf group) of the arginine residue of SOX2i-R3 and Trl-R3 was added to the deprotecting cocktail (trifluoroacetic acid: triisopropylsilane: water (95: 2.5: 2.5v / v) for 30 minutes at room temperature. %)) Was removed. Rough samples are purified by reverse phase HPLC using a JASCO HPLC system (JASCO Engineering UV2075 UV / vis detector and a PU-2089 plus gradient pump) with a preparative C18 (ODS) column (COSMOSIL 5C18-MS-II, 10IDx150 mm). did. H2O (+ 0.1% trifluoroacetic acid) and acetonitrile were used as mobile phases. HPLC and MALDI-TOF mass spectrometry were performed to characterize the purified compound.
SOX2i-R3: Analytical HPLCt R = 15.6 minutes, C 96 H 133 N 40 O 18 + MALDI-TOF MS m / z [M + H] + calculated value 2134.07, measured value 2134.06
SOX2i: Analysis HPLCt R = 16.9 minutes, C 75 H 92 N 27 O 14 + MALDI-TOF MS m / z [M + H] + calculated value 1594.73, measured value 1594.64
Ctrl-R3: Analysis HPLCt R = 15.8 minutes, C 92 H 129 N 44 O 18 + MALDI-TOF MS m / z [M + H] + calculated value 2138.05, measured value 2138.04
（３）ＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡおよびＳＯＸ２ｉ－ＴＡＭＲＡの合成
　上記（２）の固相合成後、５５℃で３時間、３，３’－ジアミノ－Ｎ－メチルジイソプロピルアミンを用いて、化合物を樹脂から切断した。粗サンプルを、４．３ｇ　Ｃ１８　ＲｅｄｉＳｅｐ　Ｒｆ逆相フラッシュカラムを備えたＣｏｍｂｉＦｌａｓｈ　Ｒｆシステム（Teledyne Isco, Inc）を用いる逆相カラムクロマトグラフィーによって精製した。Ｈ２Ｏ（＋０．１％トリフルオロ酢酸）およびアセトニトリルを移動相として用いた。カップリング反応は、１．３当量の５－ＴＡＭＲＡ－ＮＨＳエステルおよび３当量のＮ，Ｎ－ジイソプロピルエチルアミン（ＤＩＥＡ）を加え、室温で一晩振盪させることによって行った。粉末化、脱保護、精製および特徴づけは上記の通りに行った。
ＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡ:分析HPLCtR=16.3分、C123H158N43O22 +のMALDI-TOF MS m/z [M+H]+計算値2589.26、実測値2588.89
ＳＯＸ２ｉ－ＴＡＭＲＡ: 分析HPLCtR=18.2分、C102H117N30O18 +のMALDI-TOF MS m/z [M+H]+計算値2049.92、実測値2049.91 (3) Synthesis of SOX2i-R3-TAMRA and SOX2i-TAMRA After the solid-phase synthesis of (2) above, the compound was prepared from the resin using 3,3'-diamino-N-methyldiisopropylamine at 55 ° C. for 3 hours. I disconnected. Crude samples were purified by reverse phase column chromatography using a CombiFlash Rf system (Teledyne Isco, Inc) equipped with a 4.3 g C18 RediSep Rf reverse phase flash column. H2O (+ 0.1% trifluoroacetic acid) and acetonitrile were used as mobile phases. The coupling reaction was carried out by adding 1.3 eq 5-TAMRA-NHS ester and 3 eq N, N-diisopropylethylamine (DIEA) and shaking overnight at room temperature. Powdering, deprotection, purification and characterization were performed as described above.
SOX2i-R3-TAMRA: Analysis HPLCt R = 16.3 minutes, C 123 H 158 N 43 O 22 + MALDI-TOF MS m / z [M + H] + calculated value 2589.26, measured value 2588.89
SOX2i-TAMRA: Analytical HPLCt R = 18.2 minutes, C 102 H 117 N 30 O 18 + MALDI-TOF MS m / z [M + H] + calculated value 2049.92, measured value 2049.91
実施例２：トリアルギニンによる効果の確認
　ＳＯＸ２ｉは、人工多能性幹細胞（ｉＰＳ細胞）ならびにヒト前立腺および卵巣癌細胞においてＳＯＸ２のＤＮＡへの結合を阻害することにより、ＳＯＸ２自体を含め、ＳＯＸ２下流遺伝子の発現を抑制することが報告された。本実施例では、ＰＩポリアミドにおけるトリアルギニンペプチドの効果を明らかにするために、ＳＯＸ２ｉおよびＳＯＸ２ｉ－Ｒ３の細胞取り込みおよび生物活性を調べた。 Example 2: Confirmation of effect by triarginine SOX2i is a SOX2 downstream gene including SOX2 itself by inhibiting the binding of SOX2 to DNA in induced pluripotent stem cells (iPS cells) and human prostate and ovarian cancer cells. It was reported to suppress the expression of. In this example, the cell uptake and biological activity of SOX2i and SOX2i-R3 were investigated to clarify the effect of the triarginine peptide on PI polyamide.
（１）細胞培養
　ＨｅＬａ（ＪＣＲＢ９００４）および２０１Ｂ７細胞はそれぞれ、ＪＣＲＢ　Ｃｅｌｌ　Ｂａｎｋ（日本）およびＲＩＫＥＮ　ＢＲＣ（日本）から入手した。ＨｅＬａ細胞は、１０％胎仔ウシ血清（Sigma）および１％ＭＥＭ非必須アミノ酸溶液（ThermoFisher Scientific）を補足したダルベッコ改変イーグル培地（ThermoFisher Scientific）中で維持した。 (1) Cell culture HeLa (JCRB9004) and 201B7 cells were obtained from JCRB Cell Bank (Japan) and RIKEN BRC (Japan), respectively. HeLa cells were maintained in Dulbecco's modified Eagle's medium (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (Sigma) and 1% MEM non-essential amino acid solution (ThermoFisher Scientific).
　２０１Ｂ７　ｉＰＳ細胞は、０．５ｘペニシリン／ストレプトマイシン（Nacalai Tesque）を補足したｍＴｅＳＲ１培地（Stemcell Technologies）中、Ｍａｔｒｉｇｅｌ　Ｍａｔｒｉｘ（hESC-qualified, Corning）上で培養した。細胞は、解離試薬としてＴｒｙｐＬＥ　Ｅｘｐｒｅｓｓ　Ｅｎｚｙｍｅ（フェノールレッド無し，ThermoFisher Scientific）を用いて継代し、２．５μＭのＹ－２７６３２（Wako）を補足した培地中に播種した。新鮮な培地（Ｙ－２７６３２無し）への培地交換は、継代の翌日から毎日行った。 201B7 iPS cells were cultured on Matrigel Matrix (hESC-qualified, Corning) in mTeSR1 medium (Stemcell Technologies) supplemented with 0.5x penicillin / streptomycin (Nacalai Tesque). The cells were passaged using TrypLE Express Enzyme (without phenol red, Thermo Fisher Scientific) as a dissociation reagent, and seeded in a medium supplemented with 2.5 μM Y-27632 (Wako). Medium change to fresh medium (without Y-27632) was performed daily from the day following the passage.
（２）生存細胞画像化
　実験の１日前に、指数関数的に増殖しているＨｅＬａ細胞をトリプシン処理し、１．５ｘ１０４細胞／ウェルの密度で８ウェルのｉｂｉ－ｔｒｅａｔ　μ－ｓｌｉｄｅｓ（ibidi）に播種した。ＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡおよびＳＯＸ２ｉ－ＴＡＭＲＡのジメチルスルホキシド（ＤＭＳＯ）溶液を調製し、９１０００Ｍ－１ｃｍ－１の吸光係数を用いて５６５ｎｍ付近のλｍａｘにて濃度を決定した。培地を２％ＦＢＳ（Sigma）および５μＭの各化合物（ＤＭＳＯ：０．５％）を含有するＯＰＴＩ－ＭＥＭ（ThermoFisher Scientific）に交換し、細胞を２４時間処理した。処理後、培養培地を除去し、１０％ＦＢＳ（Sigma）およびＨｏｅｃｈｓｔ３３３４２（１μｇ／ｍＬ）を補足したＦｌｕｏｒｏＢｒｉｔｅ　ＤＭＥＭ（ThermoFisher Scientific）を加え、１０分間インキュベートした。ＦｌｕｏｒｏＢｒｉｔｅ　ＤＭＥＭで細胞を２回洗浄し、ＦＶ１２００レーザー走査型顕微鏡（Olympus）によって画像化した。同じ画像化条件を全てのサンプルに適用した。画像分析は、ＦＶ１０－ＡＳＷ（Olympus）を用いて行った。 (2) Survival cell imaging One day before the experiment, exponentially proliferating HeLa cells were trypsinized and 8 wells of ibi-treat μ-slides (ibidi) at a density of 1.5x10 4 cells / well. Was sown in. Dimethyl sulfoxide (DMSO) solutions of SOX2i-R3-TAMRA and SOX2i-TAMRA were prepared and their concentrations were determined at λmax near 565 nm using an absorption coefficient of 91000M -1 cm -1 . The medium was replaced with OPTI-MEM (Thermo Fisher Scientific) containing 2% FBS (Sigma) and 5 μM each compound (DMSO: 0.5%) and the cells were treated for 24 hours. After treatment, the culture medium was removed, FluoroBrite DMEM (ThermoFisher Scientific) supplemented with 10% FBS (Sigma) and Hoechst 33342 (1 μg / mL) was added, and the mixture was incubated for 10 minutes. Cells were washed twice with FluoroBrite DMEM and imaged with an FV1200 laser scanning microscope (Olympus). The same imaging conditions were applied to all samples. Image analysis was performed using FV10-ASW (Olympus).
（３）フローサイトメトリー分析
　実験の１日前に、指数関数的に増殖しているＨｅＬａ細胞をトリプシン処理し、５ｘ１０４細胞／ウェルの密度で１２ウェルプレート（NIPPON Genetics)上に播種した。培地を２％ＦＢＳ（Sigma）および５μＭの各化合物（ＤＭＳＯ：０．５％）を含有するＯＰＴＩ－ＭＥＭ（ThermoFisher Scientific）に交換し、細胞を２４時間処理した。処理後、トリプシン処理によって細胞をプレートから分離させ、２回Ｄ－ＰＢＳで洗浄した。細胞ストレーナキャップを有するポリスチレンチューブ（Corning）を用いて細胞集塊を除去後、ＢＤ　ＦＡＣＳ　ＡｒｉａＩＩ（BD Biosciences）を用いてサンプルを分析した。各サンプルについて２００００事象を記録し、ＦＳＣ（前方散乱）／ＳＳＣ（側方散乱）プロットを集団ゲーティングに用いて、細胞破片および電気的ノイズから得られるデータ点を除去した。選抜後、ネガティブコントロールサンプルの１３，０８２事象、ＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡ処理サンプルの１２，３３０事象、およびＳＯＸ２ｉ－ＴＡＭＲＡ処理サンプルの１３，８３４事象のＴＡＭＲＡのシグナル強度をヒストグラムにプロットした。 (3) Flow Cytometry Analysis One day prior to the experiment, exponentially growing HeLa cells were trypsinized and seeded on 12-well plates (NIPPON Genetics) at a density of 5x10 4 cells / well. The medium was replaced with OPTI-MEM (Thermo Fisher Scientific) containing 2% FBS (Sigma) and 5 μM each compound (DMSO: 0.5%) and the cells were treated for 24 hours. After treatment, cells were separated from the plate by trypsin treatment and washed twice with D-PBS. After removing cell clumps using a polystyrene tube (Corning) with a cell strainer cap, samples were analyzed using BD FACS MariaII (BD Biosciences). 20000 events were recorded for each sample and FSC (forward scatter) / SSC (side scatter) plots were used for population gating to remove data points from cell debris and electrical noise. After selection, the TAMRA signal intensities of 13,082 events in the negative control sample, 12,330 events in the SOX2i-R3-TAMRA treated sample, and 13,834 events in the SOX2i-TAMRA treated sample were plotted in a histogram.
（４）電気泳動移動度シフトアッセイ
　ＳＯＸ２ｉ－Ｒ３、ＳＯＸ２ｉおよびＣｔｒｌ－Ｒ３のＤＭＳＯ溶液を調製し、Ｎａｎｏｄｒｏｐ　ＮＤ－１０００（ThermoFisher Scientific）によって測定された３００－３１０ｎｍにおける最大吸光度を用いて、下記の計算式：
Figure JPOXMLDOC01-appb-M000006
 ［式中、ａはピロールおよびイミダゾール環およびβ－アラニン残基の総数を示し、ｄは光路長（ｃｍ）を示し（Ｎａｎｏｄｒｏｐ　ＮＤ－１０００の場合、０．１ｃｍ）、Ａｂｓは、３００－３１０ｎｍにおける最大吸光度を示す］
から濃度を決定した。 (4) Electrophoretic mobility shift assay Prepare DMSO solutions of SOX2i-R3, SOX2i and Trl-R3 and calculate the following using the maximum absorbance at 300-310 nm measured by Nanodrop ND-1000 (ThermoFisher Scientific). formula:
Figure JPOXMLDOC01-appb-M000006
 [In the formula, a indicates the total number of pyrrole and imidazole rings and β-alanine residues, d indicates the optical path length (cm) (0.1 cm in the case of Nanodrop ND-1000), and Abs is at 300-310 nm. Shows maximum absorbance]
The concentration was determined from.
　１０ｍＭ塩化ナトリウム、１０ｍＭカコジル酸ナトリウムおよび２．５ｖ／ｖ％　ＤＭＳＯを含有する水性バッファー中、ｐＨ７．０で、各鋳型ＤＮＡ（１μＭ）を１μＭのＳＯＸ２ｉ－Ｒ３、ＳＯＸ２ｉまたはＣｔｒｌ－Ｒ３と混合した。電気泳動の前に、サンプルを９５℃に３分間加熱することによってアニールし、－０．５℃／５秒の速度で２５℃に冷却した。８μＬの各サンプルを２μＬのＮｏｖｅｘ　Ｈｉ－Ｄｅｎｓｉｔｙ　ＴＢＥ　Ｓａｍｐｌｅ　Ｂｕｆｆｅｒ（ThermoFisher Scientific）と混合し、１μＬの各ローディング混合物をロードした。２０％ポリアクリルアミドゲルを用いて、２００Ｖで６０分間、ＴＢＥバッファー中でＮａｔｉｖｅ－ＰＡＧＥを行った。ゲルをＳＹＢＲ　Ｇｏｌｄ（ThermoFisher Scientific）で染色し、ＦＬＡ－３０００システム（Fujifilm）で画像化した。 Each template DNA (1 μM) was mixed with 1 μM SOX2i-R3, SOX2i or Ctrl-R3 at pH 7.0 in an aqueous buffer containing 10 mM sodium chloride, 10 mM sodium cacodylate and 2.5 v / v% DMSO. Prior to electrophoresis, the sample was annealed by heating to 95 ° C. for 3 minutes and cooled to 25 ° C. at a rate of −0.5 ° C./5 seconds. Each 8 μL sample was mixed with 2 μL Novex Hi-Density TBE Simple Buffer (Thermo Fisher Scientific) and 1 μL of each loading mixture was loaded. Native-PAGE was performed in TBE buffer at 200 V for 60 minutes using a 20% polyacrylamide gel. The gel was stained with SYBR Gold (Thermo Fisher Scientific) and imaged with the FLA-3000 system (Fujifilm).
（５）定量逆転写ＰＣＲ（ＲＴ－ｑＰＣＲ）分析
　実験の１日前に、２０１Ｂ７　ｉＰＳ細胞を、２．５μＭのＹ－２７６３２を補足したｍＴｅＳＲ１培地中、マトリゲルでコートした２４ウェルプレート（Greiner）上に６×１０５細胞／ウェル濃度で播種した。培地を、各化合物および０．１％ＤＭＳＯを含有する分化培地（０．２％ＦＢＳおよび１ｘＬ－グルタミンを補足したＡｄｖａｎｃｅｄ　ＲＰＭＩ１６４０（ThermoFisher Scientific））に置き換えた。４８時間後、ＦａｓｔＧｅｎｅ　ＲＮＡ　Ｂａｓｉｃ　Ｋｉｔ（NIPPON Genetics）を用いて全ＲＮＡを各ウェルから抽出し、ＲｅｖｅｒＴｒａ　Ａｃｅ　ｑＰＣＲ　ＲＴ　Ｍａｓｔｅｒ　Ｍｉｘ　ｗｉｔｈ　ｇＤＮＡ　Ｒｅｍｏｖｅｒ（Toyobo）を用いて、製造者の指示書にしたがって５００ｎｇの全ＲＮＡから逆転写を行った。反応混合物は、Ｔｈｕｎｄｅｒｂｉｒｄ　ＳＹＢＲ　ｑＰＣＲ　ｍｉｘ（Toyobo）を用いて調製し、ｑＰＣＲ反応を行い、ＬｉｇｈｔＣｙｃｌｅｒ　４８０　Ｓｙｓｔｅｍ　ＩＩ（Roche）上でモニターした。ＳＯＸ２の相対的発現レベルを、各サンプルの３つの培養ウェルの平均Ｃｐ値から算出し、ネガティブコントロールの平均値に対して正規化した。ＨＰＲＴ１または１８Ｓをハウスキーピング遺伝子として用いた。実験に用いたプライマー対を表１に示す。 (5) Quantitative reverse transcription PCR (RT-qPCR) analysis One day prior to the experiment, 201B7 iPS cells were placed on a Matrigel-coated 24-well plate (Greiner) in mTeSR1 medium supplemented with 2.5 μM Y-27632. The seeds were seeded at a concentration of 6 × 10 5 cells / well. The medium was replaced with a differentiation medium containing each compound and 0.1% DMSO (Advanced RPMI1640 (ThermoFisher Scientific) supplemented with 0.2% FBS and 1xL-glutamine). After 48 hours, total RNA was extracted from each well using FastGene RNA Basic Kit (NIPPON Genetics) and 500 ng of total RNA was extracted from each well using ReverseTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). Reverse transcription was performed from RNA. The reaction mixture was prepared using Thunderbird SYBR qPCR mix (Toyobo), a qPCR reaction was performed, and it was monitored on LightCyclor 480 System II (Roche). Relative expression levels of SOX2 were calculated from the mean Cp values of the three culture wells of each sample and normalized to the mean of the negative control. HPRT1 or 18S was used as the housekeeping gene. Table 1 shows the primer pairs used in the experiment.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
（６）細胞取り込み試験
　実施例１で調製した、ＴＡＭＲＡ（carboxy tetramethyl-rhodamine）で蛍光標識したＰＩポリアミド化合物（図１－１）を用いてフローサイトメトリーを行い、それらの細胞取り込み効率を調べた。上記（３）に記載のとおり、ＨｅＬａ細胞を５μＭの各化合物（ＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡまたはＳＯＸ２ｉ－ＴＡＭＲＡ）で２４時間処理し、トリプシン処理およびリン酸バッファーセーライン洗浄後、フローサイトメトリーアッセイに付し、各細胞からＴＡＭＲＡのシグナル強度を測定した。結果を図２に示す。 (6) Cell uptake test Flow cytometry was performed using the PI polyamide compound (Fig. 1-1) fluorescently labeled with TAMRA (carboxy tetramethyl-rhodamine) prepared in Example 1 to examine their cell uptake efficiency. .. As described in (3) above, HeLa cells are treated with 5 μM compounds (SOX2i-R3-TAMRA or SOX2i-TAMRA) for 24 hours, treated with trypsin and washed with phosphate buffer sailline, and then subjected to a flow cytometry assay. Then, the signal intensity of TAMRA was measured from each cell. The results are shown in FIG.
　図２から明らかなように、ＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡは、ＳＯＸ２ｉ－ＴＡＭＲＡよりも高い効率で細胞に取り込まれた。したがって、ＰＩポリアミドへの３つのアルギニン残基の導入が該ＰＩポリアミドの細胞取り込みを増大させたことが示された。 As is clear from FIG. 2, SOX2i-R3-TAMRA was taken up by cells with higher efficiency than SOX2i-TAMRA. Therefore, it was shown that the introduction of three arginine residues into PI polyamide increased the cellular uptake of the PI polyamide.
　さらに、細胞中に取り込まれたＰＩポリアミド化合物の細胞内分布を、上記（２）に記載のとおり生存細胞の蛍光画像化により調べた。結果を図３に示す。 Furthermore, the intracellular distribution of the PI polyamide compound incorporated into the cells was examined by fluorescence imaging of living cells as described in (2) above. The results are shown in FIG.
　ＳＯＸ２ｉ－ＴＡＭＲＡではシグナルがほとんど観察されなかったが、ＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡは細胞内に強力なシグナルをもたらした。したがって、ＳＯＸ２ｉ－Ｒ３－ＴＡＭＲＡは主に核内に蓄積することがわかった。これらの結果は、フローサイトメトリーアッセイ結果と合わせると、トリアルギニン部分がＰＩポリアミドの細胞取り込みおよび核内蓄積を増大させることを示す。 Almost no signal was observed in SOX2i-TAMRA, but SOX2i-R3-TAMRA brought a strong signal into the cell. Therefore, it was found that SOX2i-R3-TAMRA mainly accumulates in the nucleus. These results, combined with the results of the flow cytometry assay, show that the triarginine moiety increases the cellular uptake and nuclear accumulation of PI polyamide.
（７）ＰＩポリアミドの配列選択性試験
　ＰＩポリアミドの配列選択性に対するアルギニンペプチドの影響の有無を明らかにするために、ＳＯＸ２ｉとは異なる配列を標的とするＰＩＰ－トリアルギニンコンジュゲート（Ｃｔｒｌ－Ｒ３、図１－１）を用いて、そのＤＮＡ結合を電気泳動移動度シフトアッセイによって調べた。 (7) Sequence selectivity test of PI polyamide In order to clarify the influence of arginine peptide on the sequence selectivity of PI polyamide, PIP-trialginine conjugate (Ctrl-R3,) targeting a sequence different from SOX2i, Using FIG. 1-1), the DNA binding was examined by an electrophoretic mobility shift assay.
　２種類の二本鎖ＤＮＡ（ｄｓＤＮＡ）、すなわち、５’－ＣＣＧＣＡＴＡＡＣＡＡＡＧＴＧＣＣ－３’（ＳＯＸ２－ＤＮＡ；配列番号７）および５’－ＣＣＴＣＡＧＣＣＧＣＣＴＴＣＣ－３’（Ｃｔｒｌ－ＤＮＡ；配列番号８）を調製した。該ｄｓＤＮＡはそれぞれ、ＳＯＸ２ｉ－Ｒ３およびＣｔｒｌ－Ｒ３の１つの結合部位を含有した（結合部位に下線を付す）。γ－ジアミノ酪酸ターン単位はＡ／ＴおよびＴ／Ａ選択性を有することが知られているので、結合部位の５’末端にＡ／Ｔ塩基対を配置した。上記（４）に記載のとおり、ｄｓＤＮＡ（１μＭ）を各化合物（１μＭ）と共にインキュベートし、非結合および結合ＤＮＡをポリアクリルアミドゲルによって分離した。結果を図４に示す。 Two types of double-stranded DNA (dsDNA), namely 5'-CCGCAT AACAAAG TGCC-3'(SOX2-DNA; SEQ ID NO: 7) and 5'-CCTC AGCCGCC TTCC-3'(Ctrl-DNA; SEQ ID NO: 8). Was prepared. Each of the dsDNAs contained one binding site for SOX2i-R3 and Ctrl-R3 (the binding site is underlined). Since the γ-diaminobutyrate turn unit is known to have A / T and T / A selectivity, an A / T base pair was placed at the 5'end of the binding site. As described in (4) above, dsDNA (1 μM) was incubated with each compound (1 μM) and unbound and bound DNA was separated by polyacrylamide gel. The results are shown in FIG.
　ＳＯＸ２ｉの場合、ＳＯＸ２－ＤＮＡの完全なバンドシフトが観察されたが、Ｃｔｒｌ－ＤＮＡに対する結合は起こらなかった。ＳＯＸ２ｉ－Ｒ３の場合、いくつかの遊離ＳＯＸ２－ＤＮＡが残存したものの、ＳＯＸ２ｉの場合と同様の配列選択性結合が観察された。一方、Ｃｔｒｌ－Ｒ３はＣｔｒｌ－ＤＮＡのみに結合した。したがって、アルギニンペプチド部分は、ｄｓＤＮＡとの限定的な相互作用を有し、ＰＩポリアミドの配列選択性を損なわないことが分かった。 In the case of SOX2i, a complete band shift of SOX2-DNA was observed, but no binding to Ctrl-DNA occurred. In the case of SOX2i-R3, although some free SOX2-DNA remained, sequence-selective binding similar to that in SOX2i was observed. On the other hand, Ctrl-R3 bound only to Ctrl-DNA. Therefore, it was found that the arginine peptide moiety has a limited interaction with dsDNA and does not impair the sequence selectivity of PI polyamide.
（８）標的遺伝子抑制試験
　ＳＯＸ２ｉは、ＳＯＸ２自体とその下流遺伝子のＲＮＡ発現を抑制することが報告されているので、ＲＴ－ｑＰＣＲを行って、内在性遺伝子発現に対するＳＯＸ２ｉ－Ｒ３の影響を調べた。上記（５）に記載のとおり、２０１Ｂ７細胞を各化合物（ＳＯＸ２ｉ、ＳＯＸ２ｉ－Ｒ３、またはＣｔｒｌ－Ｒ３）で４８時間処理し、ＳＯＸ２　ＲＮＡ発現レベルを定量した。結果を図５に示す。 (8) Target gene suppression test Since it has been reported that SOX2i suppresses RNA expression of SOX2 itself and its downstream genes, RT-qPCR was performed to investigate the effect of SOX2i-R3 on endogenous gene expression. .. As described in (5) above, 201B7 cells were treated with each compound (SOX2i, SOX2i-R3, or Ctrl-R3) for 48 hours to quantify SOX2 RNA expression levels. The results are shown in FIG.
　興味深いことに、ＳＯＸ２発現を６０％ダウンレギュレートするのにＳＯＸ２ｉは２μＭ必要であったが、ＳＯＸ２ｉ－Ｒ３の場合は、同等な抑制を達成するのにたった１００ｎＭで十分であった（図５Ａ）。さらに、＞１μＭのＳＯＸ２ｉ－Ｒ３で処理した細胞では、ＳＯＸ２発現はほとんど観察されなかった（図５Ａ）。該生物学的活性における有意な改善は、ＳＯＸ２ｉ－Ｒ３の効率的な核内蓄積に起因するものと思われる。さらに、Ｃｔｒｌ－Ｒ３は、ＳＯＸ２発現に何の影響も及ぼさなかった。このことにより、ＳＯＸ２ｉ－Ｒ３による配列特異的転写抑制が裏付けられる（図５Ｂ）。 Interestingly, SOX2i required 2 μM to down-regulate SOX2 expression by 60%, whereas for SOX2i-R3, only 100 nM was sufficient to achieve comparable suppression (FIG. 5A). .. Furthermore, little SOX2 expression was observed in cells treated with> 1 μM SOX2i-R3 (FIG. 5A). The significant improvement in the biological activity appears to be due to the efficient nuclear accumulation of SOX2i-R3. Furthermore, Ctrl-R3 had no effect on SOX2 expression. This supports the sequence-specific transcriptional repression by SOX2i-R3 (FIG. 5B).
　まとめると、本実施例により、短いアルギニンペプチドをペプチドベクターとして用いることにより、総数１２個のピロール／イミダゾール／βアラニン残基を含む長鎖ＰＩポリアミドを細胞核内に効率的に送達することができ、さらに、標的遺伝子の転写に必要とされるＰＩポリアミド化合物の濃度を有意に減少させることが明らかとなった。 In summary, according to this example, by using a short arginine peptide as a peptide vector, a long-chain PI polyamide containing a total of 12 pyrrole / imidazole / β-alanine residues can be efficiently delivered into the cell nucleus. Furthermore, it was revealed that the concentration of PI polyamide compound required for transcription of the target gene was significantly reduced.
実施例３：ＲＵＮＸ結合配列を標的とするＰＩポリアミドとトリアルギニンペプチドを含む複合体（Ｃｈｂ－Ｍ’－Ａｒｇ３）の合成
　ＲＵＮＸ結合配列を標的とするＰＩポリアミドが、ＤＮＡ上のＲＵＮＸ結合配列へのＲＵＮＸファミリーメンバーの結合を阻害し、それによりＲＵＮＸファミリーの活性を阻害し、ひいては抗腫瘍効果や抗アレルギー効果等をもたらすことは、以前の研究によって明らかになっている（例えば、ＷＯ２０１８／０２１２００）。そこで、ゲノム上のＲＵＮＸ結合部位のコンセンサス配列として５’－ＴＧＴＧＧＴ－３’を認識するＰＩポリアミドと、アルキル化剤としてクロラムブシルと、トリアルギニンペプチドとのコンジュゲートを合成した。実施例１と同様に、ＰＩポリアミドとトリアルギニンペプチドのコンジュゲートをＦｍｏｃ固相合成法によって合成し、次いで、該コンジュゲートのＮ末端にクロラムブシルを導入した。 Example 3: Synthesis of PI Polyamide and Triarginine Peptide Containing Complex (Chb-M'-Arg3) Targeting RUNX Binding Sequence PI polyamide targeting RUNX binding sequence to RUNX binding sequence on DNA Previous studies have shown that it inhibits the binding of RUNX family members, thereby inhibiting the activity of the RUNX family, which in turn results in antitumor and antiallergic effects (eg, WO2018 / 021200). Therefore, we synthesized a PI polyamide that recognizes 5'-TGTGGT-3'as a consensus sequence of the RUNX binding site on the genome, chlorambucil as an alkylating agent, and a triarginine peptide. Similar to Example 1, a conjugate of PI polyamide and a triarginine peptide was synthesized by the Fmoc solid phase synthesis method, and then chlorambucil was introduced at the N-terminal of the conjugate.
　実施例１と同様に、ＰＳＳＭ－８ペプチド自動合成機を用いて、ＰＩポリアミドとトリアルギニンペプチドのコンジュゲート（Ａｒｇ３－ＰＩＰＭ’）の合成を行った。具体的には、合成シーケンスを入力したリアクションベッセル（Small Libra tube）にＦｍｏｃ－Ｄ－Ａｒｇ（Ｐｂｆ）－Ａｌｋｏ（０．５９ｍｍｏｌ／ｇ，１００－２００ｍｅｓｈ，１％ＤＶＢ）レジンを８０ｍｇ（５９μｍｏｌ／０．１ｇ）（渡辺化学カタログ，Ｎｏ．Ｋ０１９８５）入れ、ＲＶインサートをセット後、膨潤させるため１ｍｌのＮＭＰ（Ｎ－メチルピロリドン）を壁面に付着したレジンを洗い落としながら加え、２０分程度室温で放置した。 Similar to Example 1, a conjugate (Arg3-PIPM') of PI polyamide and triarginine peptide was synthesized using a PSSM-8 peptide automatic synthesizer. Specifically, 80 mg (59 μmol / 0) of Fmoc-D-Arg (Pbf) -Alko (0.59 mmol / g, 100-200 mesh, 1% DVB) resin was added to the reaction vessel (Small Libra tube) into which the synthetic sequence was input. .1 g) (Watanabe Kagaku Catalog, No. K01985) After setting the RV insert, 1 ml of NMP (N-methylpyrrolidone) was added while washing off the resin adhering to the wall surface, and left at room temperature for about 20 minutes. ..
（ＨＣＴＵ溶液の調製）
　１５ｍｌ遠心管を使用して、（５－クロロ－１－［ビス（ジメチルアミノ）メチリウミル］－１Ｈ－ベンゾトリアゾール－３－オキシド ヘキサフルオロホスフェート）ＨＣＴＵ　７９ｍｇ×９（カップリング数）＝７１１ｍｇを秤量後、１ｍｌ×９（カップリング数）＝９ｍｌのＮＭＰに溶かした。水道水入り超音波洗浄機に１５ｍｌ遠心管を入れ、１０分程度超音波をあてながら、溶解させた。 (Preparation of HCTU solution)
After weighing (5-chloro-1- [bis (dimethylamino) methyliumyl] -1H-benzotriazole-3-oxide hexafluorophosphate) HCTU 79 mg x 9 (coupling number) = 711 mg using a 15 ml centrifuge tube. It was dissolved in 1 ml × 9 (number of couplings) = 9 ml of NMP. A 15 ml centrifuge tube was placed in an ultrasonic cleaner containing tap water, and the mixture was dissolved while applying ultrasonic waves for about 10 minutes.
（ポリアミドモノマー溶液の調製）
　１５ｍｌ遠心管を使用して、モノマー毎に以下の指定量を秤量後、先程調製したＨＣＴＵ溶液に溶かした。超音波洗浄機に１５ｍｌ遠心管をいれ、１０分程度放置した。Ｆｍｏｃ－Ｐｙ－ＯＨ、Ｆｍｏｃ－Ｉｍ－ＯＨは不溶物をろ過するため、５ｍｌチップに脱脂綿をつめ、溶液をピペットマンで押し、新しい１５ｍｌ遠心管に入れた。１カップリングあたりの必要量２１１μｍｏｌとなるように秤量した。（合成機に使用するレジン量、そのレジンの担持量を入力、８０ｍｇ，０．６６０ｍｍｏｌ／ｇとしたとき、４倍当量のＦｍｏｃ体が２１１μｍｏｌとなる。）
Ｆｍｏｃ－Ｐｙ－ＯＨ　７６．５４ｍｇ　Ｍｗ：３６２．３８
Ｆｍｏｃ－γＡｂｕ－ＯＨ　６８．７２ｍｇ　Ｍｗ：３２５．３６
Ｆｍｏｃ－Ｉｍ－ＯＨ　７６．７５ｍｇ　Ｍｗ：３６３．３７
Ｆｍｏｃ－βアラニン－ＯＨ　６５．７６ｍｇ　Ｍｗ：３１１．３３ (Preparation of polyamide monomer solution)
The following specified amounts were weighed for each monomer using a 15 ml centrifuge tube, and then dissolved in the HCTU solution prepared earlier. A 15 ml centrifuge tube was placed in an ultrasonic cleaner and left for about 10 minutes. Fmoc-Py-OH and Fmoc-Im-OH were filled with absorbent cotton in a 5 ml tip to filter insoluble matter, and the solution was pressed with a pipette man and placed in a new 15 ml centrifuge tube. Weighed so that the required amount per coupling was 211 μmol. (When the amount of resin used in the synthesizer and the amount of the resin supported are input to 80 mg and 0.660 mmol / g, the Fmoc body having a quadruple equivalent is 211 μmol.)
Fmoc-Py-OH 76.54mg Mw: 362.38
Fmoc-γAbu-OH 68.72mg Mw: 325.36
Fmoc-Im-OH 76.75mg Mw: 363.37
Fmoc-β-alanine-OH 65.76 mg Mw: 311.33
（ポリアミド合成用反応溶液調製）
　褐色スクリュー管ｎｏ．６（３０ｍｍ×６５ｍｍ）にそれぞれ、Ｆｍｏｃ脱保護用反応液としてＤＭＦ中ピペリジン２０％溶液を２０ｍｌ（１回の脱保護に、ＤＭＦ中ピペリジン２０％溶液５００μｌ×２を使用し、ピペリジンは９８％から２０％となるようにＤＭＦで希釈した。１００ｍｌメスシリンダーを使用）、活性化剤として、ＤＭＦ中１０％ＤＩＥＡ溶液を５．２８ｍｌ入れ、合成機にセットした。 (Preparation of reaction solution for polyamide synthesis)
Brown screw tube no. 20 ml of 20% piperidine solution in DMF as a reaction solution for Fmoc deprotection is used for each of 6 (30 mm × 65 mm) (500 μl × 2 of 20% piperidine solution in DMF is used for one deprotection, and piperidine is from 98%. Diluted with DMF to 20% (using a 100 ml female cylinder), 5.28 ml of 10% DIEA solution in DMF was added as an activator and set in a synthesizer.
（ＰＳＳＭ－８合成機を用いたポリアミド合成）
　２ｍｌのエッペンチューブ（2ml sampling tube）にモノマー溶液を９８２μｌ毎分注した。シーケンス順に固相合成機にセットした。ポリアミドのシーケンスは以下（Ｃ末端からＮ末端へ合成）である。
Ｄ－Ａｒｇ（Ｐｂｆ）（ｏｎ　ｒｅｓｉｎ）－Ｄ－Ａｒｇ（Ｐｂｆ）－Ｄ－Ａｒｇ（Ｐｂｆ）－ｂＡｌ－Ｐｙ－Ｐｙ－ＰｙＰｙ－γＡｂｕ－Ｉｍ－Ｄｉｍｅｒ－Ｉｍ－ｂＡｌ（ＮＨ２） (Polyamide synthesis using PSSM-8 synthesizer)
982 μl of the monomer solution was poured every minute into a 2 ml Eppen tube (2 ml sampling tube). It was set in the solid phase synthesizer in the order of sequence. The polyamide sequence is as follows (synthesized from C-terminal to N-terminal).
D-Arg (Pbf) (on resin) -D-Arg (Pbf) -D-Arg (Pbf) -bAl-Py-Py-PyPy-γAbu-Im-Dimer-Im-bAl (NH2)
（切り出し工程）
　リアクションベッセルを合成機から取り出し、キャップ後１ｍｌ（Ｎ，Ｎ－ジメチル－１，３－プロパンジアミン）Ｄｐを加え、５５℃、３時間、８８０ｒｐｍ、振とう器で攪拌させた。ジエチルエーテル４０ｍｌを５０ｍｌ遠心管に入れ、先の反応液を一滴ずつ加え、粉体化させた。レジンについた残りの溶液は塩化メチレンを数滴（２ｍｌガラスピペット）レジンに加えて、洗い、濃縮し再びジエチルエーテルを用いて粉体化させた。１．５ｍｌチューブに粉体を移し上澄みを除いた後、１０分程度、デジケーターで乾燥させた。 (Cutout process)
The reaction vessel was removed from the synthesizer, 1 ml (N, N-dimethyl-1,3-propanediamine) Dp was added after the cap, and the mixture was stirred at 55 ° C. for 3 hours at 880 rpm with a shaker. 40 ml of diethyl ether was placed in a 50 ml centrifuge tube, and the above reaction solution was added drop by drop to make a powder. The remaining solution on the resin was made by adding a few drops of methylene chloride (2 ml glass pipette) to the resin, washing, concentrating and powdering again with diethyl ether. The powder was transferred to a 1.5 ml tube to remove the supernatant, and then dried with a desiccator for about 10 minutes.
（Ａｒｇ３－ＰＩＰＭ’のＮ末端にクロラムブシルを導入する）
　１．５ｍｌチューブに以下のものを秤量し、室温で１．５時間攪拌した。 (Introduce chlorambucil at the N-terminus of Arg3-PIPM')
The following items were weighed in a 1.5 ml tube and stirred at room temperature for 1.5 hours.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　ジエチルエーテル４０ｍｌを５０ｍｌ遠心管に入れ、先の反応液を一滴ずつ加え、粉体化させた。１００００ｒｐｍで５分遠心した。上澄みを除去し、ＤＭＦ１ｍｌを加え、再びジエチルエーテル４０ｍｌに滴下し、遠心この操作を２回繰り返した。１．５ｍｌチューブに粉体を移した。 40 ml of diethyl ether was placed in a 50 ml centrifuge tube, and the above reaction solution was added drop by drop to make a powder. Centrifuged at 10000 rpm for 5 minutes. The supernatant was removed, 1 ml of DMF was added, the mixture was added dropwise to 40 ml of diethyl ether again, and centrifugation was repeated twice. The powder was transferred to a 1.5 ml tube.
（Ｄ－Ａｒｇ（Ｐｂｆ）を脱保護する）
　ＴＦＡ：ｍＱ水：ＴＩＳ（９５：２．５：２．５）、９５０μｌ、２５μｌ、２５μＬを粉体に加え、室温で１時間攪拌させた。ジエチルエーテル４０ｍｌに滴下し粉体化し、デジケーターで乾燥させた。 (Deprotecting D-Arg (Pbf))
TFA: mQ water: TIS (95: 2.5: 2.5), 950 μl, 25 μl, 25 μL was added to the powder and stirred at room temperature for 1 hour. It was added dropwise to 40 ml of diethyl ether, powdered, and dried with a desiccator.
（ＨＰＬＣ精製）
　ＨＰＬＣ高速液体クロマトグラフィーはＪＡＣＳＯ（ＰＵ－２０８９Ｐｌｕｓ）、カラムは５Ｃ１８－ＭＳ－ＩＩ（COSMOSIL catalog No. 3455-91）を使用した。１．５ｍｌチューブに入った粉体ＣｈｂＭ’Ａｒｇ３にＤＭＦ（８００μＬ）を加え、ボルテックスにて溶解させた。３２－４２％アセトニトリル／０．１％ＴＦＡ（トリフルオロ酢酸）水、３０分、流速１８ｍｌ／分に設定し、目的物に由来する２５４ｎｍの吸収ピークを回収した。その後速やかに、スクリュー管（３０ｍｍ×６５ｍｍ）に移し、コンビニエバポｃ１８（（株）バイオクロマト）を使用し、３０℃、１時間でアセトニトリルを蒸発させた。 (HPLC purification)
JACSO (PU-2089Plus) was used for HPLC high performance liquid chromatography, and 5C18-MS-II (COSMOSIL catalog No. 3455-91) was used for the column. DMF (800 μL) was added to the powder ChbM'Arg3 in a 1.5 ml tube, and the mixture was dissolved by vortexing. 32-42% acetonitrile / 0.1% TFA (trifluoroacetic acid) water was set at 30 minutes and a flow rate of 18 ml / min, and the absorption peak at 254 nm derived from the target product was recovered. Immediately thereafter, the mixture was transferred to a screw tube (30 mm × 65 mm), and acetonitrile was evaporated at 30 ° C. for 1 hour using a convenience store evaporation c18 (Biochromatography Co., Ltd.).
（凍結乾燥）
　５０ｍｌチューブに移し、２０ｍｌ程度入れ、液体窒素に入れ、凍結させ、凍結乾燥機（ＥＹＥＬＡ　ＦＤＵ－１１００）で４８時間、乾燥させた。 (freeze drying)
It was transferred to a 50 ml tube, put in about 20 ml, put in liquid nitrogen, frozen, and dried in a freeze dryer (EYELA FDU-1100) for 48 hours.
（粉体の回収）
　ジエチールエーテルを５０ｍｌチューブに加え、Ｔサンプルストックチューブ１．５ｍｌ（BMBio、catlogNo.T-202）に回収し、遠心、上澄みを除く操作を繰り返し、デシケーターで乾燥させた。 (Powder recovery)
Dietil ether was added to a 50 ml tube, collected in a T sample stock tube of 1.5 ml (BMBio, catalogNo.T-202), and the operations of centrifugation and removal of the supernatant were repeated and dried with a desiccator.
Ｃｈｂ－Ｍ’－Ａｒｇ３
C92H129Cl2N37O15のMALDI-TOFMS m/z [M＋H]+計算値2062.99、実測値2064.11
Figure JPOXMLDOC01-appb-C000009
 Chb-M'-Arg3
C 92 H 129 Cl 2 N 37 O 15 MALDI-TOFMS m / z [M + H] + calculated value 2062.99, measured value 2064.11
Figure JPOXMLDOC01-appb-C000009
実施例４：ＰＩポリアミドとＢｉとトリアルギニンペプチドを含む複合体（Ｂｉ－Ｒ－Ａｒｇ３）の合成および生物学的活性の確認
　５’－ＷＣＧＣＣＷ－３’配列（Ｗ＝ＡまたはＴ）を認識するＰＩポリアミド（Ｒ）とトリアルギニンペプチドとのコンジュゲートをＦｍｏｃ固相合成法によって合成し、次いで、ブロモドメイン阻害剤（Ｂｉ）として５－イソキサゾリル－ベンゾイミダゾール化合物を上記ＰＩポリアミドとトリアルギニンペプチドとのコンジュゲートのＮ末端に導入した。 Example 4: Synthesis of Complex (Bi-R-Arg3) Containing PI Polyamide, Bi and Triarginine Peptide and Confirmation of Biological Activity Recognizing 5'-WCGCCW-3'Sequence (W = A or T) A conjugate of PI polyamide (R) and a triarginine peptide was synthesized by the Fmoc solid phase synthesis method, and then a 5-isoxazolyl-benzoimidazole compound was used as a bromodomain inhibitor (Bi) with the above PI polyamide and the triarginine peptide. It was introduced at the N-terminal of the conjugate.
　実施例１と同様に、ＰＳＳＭ－８ペプチド自動合成機を用いて、ＰＩポリアミドとトリアルギニンペプチドのコンジュゲート（Ａｒｇ３－ＰＩＰＲ）の合成を行った。具体的には、合成シーケンスを入力したリアクションベッセルにＦｍｏｃ－Ｄ－Ａｒｇ（Ｐｂｆ）－Ａｌｋｏ（０．５９ｍｍｏｌ／ｇ，１００－２００ｍｅｓｈ，１％ＤＶＢ）レジン８０ｍｇ（５９μｍｏｌ／０．１ｇ）を入れ、ＲＶインサートをセット後、膨潤させるため１ｍｌのＮＭＰを壁面に付着したレジンを洗い落としながら加え、２０分程度室温で放置した。 Similar to Example 1, a conjugate (Arg3-PIPR) of PI polyamide and triarginine peptide was synthesized using a PSSM-8 peptide automatic synthesizer. Specifically, 80 mg (59 μmol / 0.1 g) of Fmoc-D-Arg (Pbf) -Alko (0.59 mmol / g, 100-200 mesh, 1% DVB) resin was added to the reaction vessel into which the synthetic sequence was input. After setting the RV insert, 1 ml of NMP was added while washing off the resin adhering to the wall surface for swelling, and the mixture was left at room temperature for about 20 minutes.
（ＨＣＴＵ溶液の調製）
　１５ｍｌ遠心管を使用して、ＨＣＴＵ　７９ｍｇ×１２（カップリング数）＝９４８ｍｇを秤量後、１ｍｌ×１２（カップリング数）＝１２ｍｌのＮＭＰに溶かした。水道水入り超音波洗浄機に１５ｍｌ遠心管をいれ、１０分程度超音波をあてながら、溶解させた。 (Preparation of HCTU solution)
Using a 15 ml centrifuge tube, HCTU 79 mg × 12 (coupling number) = 948 mg was weighed and then dissolved in 1 ml × 12 (coupling number) = 12 ml NMP. A 15 ml centrifuge tube was placed in an ultrasonic cleaner containing tap water, and the mixture was dissolved while applying ultrasonic waves for about 10 minutes.
（ポリアミドモノマー溶液の調製）
　１５ｍｌ遠心管を使用して、モノマー毎に以下の指定量を秤量後、先程調製したＨＣＴＵ溶液に溶かした。超音波洗浄機に１５ｍｌ遠心管をいれ、１０分程度放置した。Ｆｍｏｃ－Ｐｙ－ＯＨ、Ｆｍｏｃ－Ｉｍ－ＯＨは不溶物をろ過するため、５ｍｌチップに脱脂綿をつめ、溶液をピペットマンで押し、新しい１５ｍｌ遠心管に入れた。１カップリングあたりの必要量２１１μｍｏｌとなるように秤量した。（合成機に使用するレジン量、そのｒｅｓｉｎの担持量を入力、８０ｍｇ，０．６６０ｍｍｏｌ／ｇとしたとき、４倍当量のＦｍｏｃ体が２１１μｍｏｌとなる。）
Ｆｍｏｃ－Ｐｙ－ＯＨ　７６．５４ｍｇ　Ｍｗ：３６２．３８
Ｆｍｏｃ－γＡｂｕ－ＯＨ　６８．７２ｍｇ　Ｍｗ：３２５．３６
Ｆｍｏｃ－Ｉｍ－ＯＨ　７６．７５ｍｇ　Ｍｗ：３６３．３７
Ｆｍｏｃ－βＡｌａ－ＯＨ　６５．７６ｍｇ　Ｍｗ：３１１．３３
Ｆｍｏｃ－Ｄ－Ａｒｇ（Ｐｂｆ）－ＯＨ　１３６．８９ｍｇ　Ｍｗ：６４８．７８
Ｆｍｏｃ－ｍｉｎｉＰＥＧ－ＯＨ　８１．４ｍｇ　Ｍｗ：３８５．４１ (Preparation of polyamide monomer solution)
The following specified amounts were weighed for each monomer using a 15 ml centrifuge tube, and then dissolved in the HCTU solution prepared earlier. A 15 ml centrifuge tube was placed in an ultrasonic cleaner and left for about 10 minutes. Fmoc-Py-OH and Fmoc-Im-OH were filled with absorbent cotton in a 5 ml tip to filter insoluble matter, and the solution was pressed with a pipette man and placed in a new 15 ml centrifuge tube. Weighed so that the required amount per coupling was 211 μmol. (When the amount of resin used in the synthesizer and the amount of resin supported thereof are input and set to 80 mg and 0.660 mmol / g, a 4-fold equivalent amount of Fmoc body is 211 μmol.)
Fmoc-Py-OH 76.54mg Mw: 362.38
Fmoc-γAbu-OH 68.72mg Mw: 325.36
Fmoc-Im-OH 76.75mg Mw: 363.37
Fmoc-βAla-OH 65.76 mg Mw: 311.33
Fmoc-D-Arg (Pbf) -OH 136.89 mg Mw: 648.78
Fmoc-miniPEG-OH 81.4mg Mw: 385.41
（ポリアミドダイマー溶液の調製）
　Ｆｍｏｃ－Ｉｍ－ＯＨの後のＦｍｏｃ－Ｐｙ－ＯＨは固相合成機ではカップリングしないため、予め液相で合成したＦｍｏｃ－ＰｙＩｍ－ＯＨ（Ｄｉｍｅｒ）を使用し、モノマー同様に調整した。
Ｆｍｏｃ－ＰｙＩｍ－ＯＨ　１０２．７５ｍｇ　Ｍｗ：４８６．４８ (Preparation of polyamide dimer solution)
Since Fmoc-Py-OH after Fmoc-Im-OH is not coupled by the solid phase synthesizer, Fmoc-PyIm-OH (Dimer) synthesized in the liquid phase in advance was used and adjusted in the same manner as the monomer.
Fmoc-PyIm-OH 102.75mg Mw: 486.48
（ポリアミド合成用反応溶液調製）
　褐色スクリュー管ｎｏ．６（３０ｍｍ×６５ｍｍ）にそれぞれ、Ｆｍｏｃ脱保護用反応液としてＤＭＦ中のピペリジン２０％溶液を２５．４ｍｌ（１回の脱保護にＤＭＦ中のピペリジン２０％溶液を５００μｌ×２使用，ピペリジンは９８％から２０％となるようにＤＭＦで希釈した。１００ｍｌメスシリンダーを使用）、活性化剤として、ＤＭＦ中１０％ＤＩＥＡ溶液６．３７ｍｌを入れ、合成機にセットした。 (Preparation of reaction solution for polyamide synthesis)
Brown screw tube no. 25.4 ml of 20% piperidine solution in DMF was used as the reaction solution for Fmoc deprotection in 6 (30 mm x 65 mm) (500 μl x 2 of 20% piperidine solution in DMF was used for one deprotection, and the piperidine was 98. Diluted with DMF from% to 20% (using a 100 ml female cylinder), 6.37 ml of 10% DIEA solution in DMF was added as an activator and set in a synthesizer.
（ＰＳＳＭ－８合成機を用いたポリアミド合成）
　２ｍｌのエッペンチューブ（2ml sampling tube）にモノマー溶液を９８２μｌ毎分注した。シーケンス順に固相合成機にセットした。ポリアミドのシーケンスは以下（Ｃ末端からＮ末端へ合成）である。
Ｄ－Ａｒｇ（Ｐｂｆ）（ｏｎ　ｒｅｓｉｎ）－Ｄ－Ａｒｇ（Ｐｂｆ）－Ｄ－Ａｒｇ（Ｐｂｆ）－ｂＡｌ－Ｐｙ－Ｐｙ－Ｄｉｍｅｒ－γＡｂｕ－Ｄｉｍｅｒ－Ｉｍ－Ｉｍ－ｍｉｎｉＰＥＧ－ｍｉｎｉＰＥＧ（ＮＨ２） (Polyamide synthesis using PSSM-8 synthesizer)
982 μl of the monomer solution was poured every minute into a 2 ml Eppen tube (2 ml sampling tube). It was set in the solid phase synthesizer in the order of sequence. The polyamide sequence is as follows (synthesized from C-terminal to N-terminal).
D-Arg (Pbf) (on resin) -D-Arg (Pbf) -D-Arg (Pbf) -bAl-Py-Py-Dimer-γAbu-Dimer-Im-Im-miniPEG-miniPEG (NH 2 )
（切り出し工程）
　リアクションベッセルを合成機から取り出し、キャップ後１ｍｌ Ｄｐを加え、５５℃、３時間、８８０ｒｐｍ、振とう器で攪拌させた。ジエチルエーテル４０ｍｌを５０ｍｌ遠心管に入れ、先の反応液を一滴ずつ加え、粉体化させた。レジンについた残りの溶液は塩化メチレンを数滴（２ｍｌガラスピペット）レジンに加えて、洗い、（濃縮し再び）ジエチルエーテルを用いて粉体化させた。１．５ｍｌチューブに粉体を移し上澄みを除いた後、１０分程度、デジケーターで乾燥させた。 (Cutout process)
The reaction vessel was removed from the synthesizer, 1 ml Dp was added after the cap, and the mixture was stirred at 55 ° C. for 3 hours at 880 rpm with a shaker. 40 ml of diethyl ether was placed in a 50 ml centrifuge tube, and the above reaction solution was added drop by drop to make a powder. The remaining solution on the resin was washed by adding a few drops (2 ml glass pipette) of methylene chloride to the resin and powdered (concentrated again) with diethyl ether. The powder was transferred to a 1.5 ml tube to remove the supernatant, and then dried with a desiccator for about 10 minutes.
（Ａｒｇ３－ＰＩＰＲのＮ末端にＢｉを導入する）
　１．５ｍｌチューブに以下のものを秤量し、室温で１．５時間攪拌した。 (Introduce Bi to the N-terminus of Arg3-PIPR)
The following items were weighed in a 1.5 ml tube and stirred at room temperature for 1.5 hours.
Ｂｉ
Figure JPOXMLDOC01-appb-C000010
 Bi
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
　ジエチルエーテル４０ｍｌを５０ｍｌ遠心管に入れ、先の反応液を一滴ずつ加え、粉体化させた。１００００ｒｐｍで５分遠心した。上澄を除き、ＤＭＦ１ｍｌを加え、再びジエチルエーテル４０ｍｌに滴下し、遠心この操作を２回繰り返した。１．５ｍｌチューブに粉体を移した。 40 ml of diethyl ether was placed in a 50 ml centrifuge tube, and the above reaction solution was added drop by drop to make a powder. Centrifuged at 10000 rpm for 5 minutes. After removing the supernatant, 1 ml of DMF was added, the solution was added dropwise to 40 ml of diethyl ether again, and centrifugation was repeated twice. The powder was transferred to a 1.5 ml tube.
（Ｄ－Ａｒｇ（Ｐｂｆ）を脱保護する）
　ＴＦＡ：ｍＱ水：ＴＩＳ（９５：２．５：２．５）、９５０μｌ、２５μｌ、２５μＬを粉体に加え、室温で１時間攪拌させた。ジエチルエーテル４０ｍｌに滴下し粉体化し、デジケーターで乾燥させた。 (Deprotecting D-Arg (Pbf))
TFA: mQ water: TIS (95: 2.5: 2.5), 950 μl, 25 μl, 25 μL was added to the powder and stirred at room temperature for 1 hour. It was added dropwise to 40 ml of diethyl ether, powdered, and dried with a desiccator.
（フラッシュカラム精製）
　ＣｏｍｂｉＦｌａｓｈ　Ｒｆ、カラムはＣ１８ＲｅｄｉＳｅｐＲｆ　４．３Ｇｒａｍ　Ｆｌａｓｈ　Ｃｏｌｕｍｎを使用した。１．５ｍｌチューブに入った粉体Ｂｉ－ＲＡｒｇ３にＤＭＦ（８００μＬ）を加え、ボルテックスにて溶解させた。Ｃ１８シリカに吸着させ、０－３５％アセトニトリル／０．１％ＴＦＡ水、２５分、流速１８ｍｌ／分に設定し、目的物に由来する２５４ｎｍの吸収ピークを回収した。その後速やかに、スクリュー管（３０ｍｍ×６５ｍｍ）に移し、コンビニエバポｃ１８（株バイオクロマト）を使用し、５５℃、１時間でアセトニトリルを蒸発、濃縮させた。 (Flash column purification)
CombiFlash Rf, C18RediSepRf 4.3Gram Flash Volume was used as the column. DMF (800 μL) was added to the powder Bi-RArg3 contained in a 1.5 ml tube, and the mixture was dissolved by vortexing. It was adsorbed on C18 silica, set to 0-35% acetonitrile / 0.1% TFA water, 25 minutes, and a flow rate of 18 ml / min, and the absorption peak at 254 nm derived from the target product was recovered. Immediately thereafter, the mixture was transferred to a screw tube (30 mm × 65 mm), and acetonitrile was evaporated and concentrated at 55 ° C. for 1 hour using a convenience evaporative c18 (strain biochromatography).
（凍結乾燥）
　風袋を計量したＴサンプルストックチューブ１．５ｍｌ（BMBBio,catlogNo.T-202）に濃縮させたサンプルを入れ、液体窒素に入れ、凍結させ、凍結乾燥機（EYELA FDU-1100）で１５時間、乾燥させた。 (freeze drying)
Put the concentrated sample in 1.5 ml (BMBBio, catalogNo.T-202) of T sample stock tube weighed with a tare, put it in liquid nitrogen, freeze it, and dry it in a lyophilizer (EYELA FDU-1100) for 15 hours. I let you.
Ｂｉ－Ｒ－Ａｒｇ３
C119H679N43O24のMALDI-TOFMS m/z [M＋H]+計算値25783.32、実測値2586.145
Figure JPOXMLDOC01-appb-C000012
 Bi-R-Arg3
C 119 H 679 N 43 O 24 MALDI-TOFMS m / z [M + H] + calculated value 25783.32, measured value 2586.145
Figure JPOXMLDOC01-appb-C000012
　ＰＧＣ（peroxisome proliferator activated receptor gamma coactivator）－１αおよびＰＧＣ－１βは、転写因子ＰＰＡＲγに結合する転写コアクチベーターとして同定されたタンパク質であり、これらの発現亢進はミトコンドリアの機能増強をもたらすことが知られている。ＣＤ８＋Ｔ細胞をＢ６マウスから取得し、ネオンエレクトロポレーションシステム（１０ピコモル／１００万細胞）を用いて、ＰＧＣ－１αまたはＰＧＣ－１βに対するｓｉＲＮＡをエレクトロポレートした。エレクトロポレーション後、細胞を４時間インキュベートした。次に、抗－ＣＤ３および抗－ＣＤ２８モノクローナル抗体を用いてＴＣＲ刺激を行い、同時に、Ｂｉ－Ｒ－Ａｒｇ３を用量依存的に加え、４８時間インキュベートした。４８時間刺激後、細胞を収集し、ＲＮＡを抽出および精製し、実施例２（５）の記載と同様にｑＰＣＲを行った。ＧＡＰＤＨをローディングコントロールとして用いた。使用したｓｉＲＮＡ配列およびプライマー配列を表４に示す。 PGC (peroxisome proliferator activated receptor gamma coactivator) -1α and PGC-1β are proteins identified as transcription coactivators that bind to the transcription factor PPARγ, and their upregulation is known to result in enhanced mitochondrial function. ing. CD8 + T cells were obtained from B6 mice and siRNA against PGC-1α or PGC-1β was electroporated using a neon electroporation system (10 picomoles / 1 million cells). After electroporation, cells were incubated for 4 hours. Next, TCR stimulation was performed with anti-CD3 and anti-CD28 monoclonal antibodies, and at the same time, Bi-R-Arg3 was added in a dose-dependent manner and incubated for 48 hours. After 48 hours of stimulation, cells were collected, RNA was extracted and purified, and qPCR was performed as described in Example 2 (5). GAPDH was used as the loading control. The siRNA sequence and primer sequence used are shown in Table 4.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　結果を図６に示す。図中、「Ａｒｇ３－ＢｉＲ」は、ＰＩＰ、ＢｉおよびＡｒｇ３のコンジュゲート（Ｂｉ－Ｒ　Ａｒｇ３）を示す。図６から明らかなように、Ｂｉ－ＲがｓｉＲＮＡによるノックダウンをレスキューし（ＰＧＣ－１α／βの発現を増強し）、トリアルギニンペプチドがＢｉ－Ｒの効果を高めた。 The results are shown in Fig. 6. In the figure, "Arg3-BiR" indicates a conjugate of PIP, Bi and Arg3 (Bi-R Arg3). As is clear from FIG. 6, Bi-R rescued knockdown by siRNA (enhanced expression of PGC-1α / β), and triarginine peptide enhanced the effect of Bi-R.
　次に、上記ＣＤ８+Ｔ細胞においてミトコンドリア染色を行った。ＣＤ８＋Ｔ細胞（低ＣＤ４４と高ＣＤ４４の両方）をＢ６マウスから採取し、Ｔ細胞をＴＣＲ刺激のために抗ＣＤ３／ＣＤ２８モノクローナル抗体で刺激し、上記で合成したＰＩＰ、ＢｉおよびＡｒｇ３のコンジュゲート（ＢｉＲ－Ａｒｇ３）、ＰＩＰとＢｉのコンジュゲート（ＢｉＲ）、ブロモドメイン阻害剤のみ（Ｂｉ）、またはＰＩＰのみ（Ｒ）を加えた。また、コントロールとして、上記のいずれの化合物も加えず、ＤＭＳＯを加えた。４８時間のインキュベーション後、ミトコンドリアの活性化を、ミトコンドリア色素染色を使用して評価した。染色には、蛍光プローブとして、ＭｉｔｏＴｒａｃｋｅｒ（登録商標）Ｒｅｄ、ＭｉｔｏＴｒａｃｋｅｒ（登録商標）Ｇｒｅｅｎ、Ｍｉｔｏｃｈｏｎｄｒｉａｌ　ＳＯＸ、およびＣｅｌｌＲＯＸ（登録商標）（いずれもThermoFisher製）を使用した。 Next, mitochondrial staining was performed on the above CD8 + T cells. CD8 + T cells (both low CD44 and high CD44) were harvested from B6 mice, T cells were stimulated with anti-CD3 / CD28 monoclonal antibodies for TCR stimulation, and the PIP, Bi and Arg3 conjugates synthesized above (BiR-Arg3), PIP and Bi conjugate (BiR), bromodomain inhibitor only (Bi), or PIP only (R) was added. Further, as a control, DMSO was added without adding any of the above compounds. After 48 hours of incubation, mitochondrial activation was assessed using mitochondrial dye staining. For staining, MitoTracker® Red, MitoTracker® Green, Mitochondrial SOX, and CellROX® (all manufactured by Thermo Fisher) were used as fluorescent probes.
　結果を図７および図８に示す。図中、「Ａｒｇ－ＢｉＲ」はＢｉＲ－Ａｒｇ３コンジュゲートの添加を示し、「ＢｉＲ」はＰＩＰとＢｉのコンジュゲートの添加を示し、「Ｂｉ ｏｎｌｙ」はブロモドメイン阻害剤の単独添加を示し、「Ｒ ｏｎｌｙ」はＰＩＰの単独添加を示す。図７および図８から明らかなように、ＰＩＰ－Ａｒｇ３－Ｂｉコンジュゲートを細胞に添加することにより、ミトコンドリアを活性化したことが分かった。 The results are shown in FIGS. 7 and 8. In the figure, "Arg-BiR" indicates the addition of the BiR-Arg3 conjugate, "BiR" indicates the addition of the PIP and Bi conjugate, "Bi only" indicates the addition of the bromodomain inhibitor alone, and " "R only" indicates the single addition of PIP. As is clear from FIGS. 7 and 8, it was found that mitochondria were activated by adding the PIP-Arg3-Bi conjugate to the cells.
実施例５：ＰＤ－１遮断とＢｉ－Ｒ－Ａｒｇ３との併用による抗腫瘍免疫の増強
　ＰＧＣ－１α／βは、さまざまな転写因子（ＴＦ）と複合体を形成し、ミトコンドリアの生合成、代謝、酸化的リン酸化（ＯＸＰＨＯＳ）、脂肪酸酸化（ＦＡＯ）、ミトコンドリアのクリスタ再編成などに関連する遺伝子の発現を制御することで知られるコアクチベーターである。一方、ＰＤ－１を遮断すると、解糖系代謝を持つ短期的なエフェクターＴ細胞の増殖が誘導されることが知られている。また、腫瘍部位にエフェクター細胞が少ない（短命であるため）ことが、一部の患者に見られる無反応性の理由の１つである可能性があることが示唆されている。さらに、長寿命のＴ細胞を生成するＯＸＰＨＯＳとＦＡＯを誘導することが、抗腫瘍免疫を高めるために重要であるとの報告もある。このようなことから、本実施例では、Ｂｉ－Ｒ－Ａｒｇ３によるＣＤ８＋Ｔ細胞におけるミトコンドリアの活性化が、ＰＤ－１遮断療法と併用することで、抗腫瘍免疫力を高めることができるかを調べた。 Example 5: Enhancement of antitumor immunity by combined use of PD-1 blockade and Bi-R-Arg3 PGC-1α / β forms a complex with various transcription factors (TF), and mitochondrial biosynthesis and metabolism. , Oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), mitochondrial crystal rearrangement, and other coactivators known to regulate the expression of genes involved. On the other hand, it is known that blocking PD-1 induces the proliferation of short-term effector T cells having glycolytic metabolism. It has also been suggested that the lack of effector cells at the tumor site (because of their short life) may be one of the reasons for the non-responsiveness seen in some patients. Furthermore, it has been reported that inducing OXPHOS and FAO, which produce long-lived T cells, is important for enhancing antitumor immunity. Therefore, in this example, whether the activation of mitochondria in CD8 + T cells by Bi-R-Arg3 can enhance antitumor immunity when used in combination with PD-1 blockade therapy. Examined.
（実験スケジュール）
　図９に示すスケジュールに従って、抗ＰＤ－Ｌ１モノクローナル抗体を用いたＰＤ－１遮断と、Ｂｉ－Ｒ－Ａｒｇ３との併用療法を行った。マウス大腸がん細胞株のＭＣ３８細胞（０．５×１０６）をＣ５７ＢＬ／６Ｎマウスの右脇腹に皮内注射（ｉ．ｄ．）した。６日目に治療を開始した。６日目に、抗ＰＤ－Ｌ１モノクローナル抗体（４０μｇ／マウス、クローン１０Ｆ．９Ｇ２，BioXcell, Catalog# BE0101）とＰＩポリアミド（１ｍｇ／ｋｇ（事前に最適化した用量））を腹腔内（ｉ．ｐ．）に注射した。ＰＩポリアミドは３日毎に、抗ＰＤ－Ｌ１モノクローナル抗体は６日毎に注射した。治療を１ヶ月間行い、腫瘍の大きさをカリパスを用いて隔日で測定し、腫瘍体積を算出した。加えて、治療後のマウスの生存率（％）を求めた。また、コントロール群において、抗ＰＤ－Ｌ１モノクローナル抗体、または抗ＰＤ－Ｌ１モノクローナル抗体のアイソタイプコントロール（Rat IgG2b, k）を単独投与した。さらにコントロールとして、Ｂｉ－Ｒ－Ａｒｇ３、Ｒのみ、またはＢｉのみを単独投与して同様の実験を行った。 (Experimental schedule)
According to the schedule shown in FIG. 9, PD-1 blockade using an anti-PD-L1 monoclonal antibody and combination therapy with Bi-R-Arg3 were performed. MC38 cells (0.5 × 106 ) of a mouse colorectal cancer cell line were injected intradermally (id) into the right flank of C57BL / 6N mice. Treatment was started on the 6th day. On day 6, anti-PD-L1 monoclonal antibody (40 μg / mouse, clone 10F.9G2, BioXcell, Catalog # BE0101) and PI polyamide (1 mg / kg (pre-optimized dose)) were placed intraperitoneally (ip). .) Was injected. PI polyamide was injected every 3 days and anti-PD-L1 monoclonal antibody was injected every 6 days. Treatment was performed for 1 month, the size of the tumor was measured every other day using a caliper, and the tumor volume was calculated. In addition, the survival rate (%) of the mice after treatment was determined. In addition, in the control group, an anti-PD-L1 monoclonal antibody or an isotype control of an anti-PD-L1 monoclonal antibody (Rat IgG2b, k) was administered alone. Further, as a control, Bi-R-Arg3, R alone, or Bi alone was administered alone, and the same experiment was performed.
　ＭＣ３８細胞（Ｃ５７ＢＬ／６Ｎバックグラウンド由来のマウス大腸がん細胞株）は、Kerafast Inc., U.S.から購入した。Ｃ５７ＢＬ／６Ｎマウスは、６-８週齢の雌をCLEA Japan（Tokyo, Japan）から購入し、病原体フリー条件下で維持した。Ｂｉ－Ｒ－Ａｒｇ３は、実施例４に記載にしたがって合成した。Ｒは実施例４に示されるＰＩポリアミドであり、常法にしたがって合成した。また、Ｂｉとして、実施例４と同様に、５－イソキサゾリル－ベンゾイミダゾールを用いた。 MC38 cells (mouse colorectal cancer cell line derived from C57BL / 6N background) were purchased from Kerafast Inc., U.S. For C57BL / 6N mice, 6-8 week old females were purchased from CLEA Japan (Tokyo, Japan) and maintained under pathogen-free conditions. Bi-R-Arg3 was synthesized according to the description in Example 4. R is the PI polyamide shown in Example 4, and was synthesized according to a conventional method. Further, as Bi, 5-isoxazolyl-benzimidazole was used as in Example 4.
（腫瘍サイズの測定結果）
　腫瘍サイズを測定した結果を図１０－１および図１０－２に示す。各種ＰＩポリアミドで処理したグループにおける腫瘍サイズをプロットした。図中、横軸は腫瘍移植後の経過日を示し、縦軸は腫瘍サイズを示す。図１０－１および図１０－２から明らかなように、Ｂｉ－Ｒ－Ａｒｇ３は、ＰＤ－１遮断との併用によって、ＰＤ－１遮断単独と比較してより腫瘍を縮退させた。 (Measurement result of tumor size)
The results of measuring the tumor size are shown in FIGS. 10-1 and 10-2. Tumor sizes in groups treated with various PI polyamides were plotted. In the figure, the horizontal axis shows the elapsed date after tumor transplantation, and the vertical axis shows the tumor size. As is clear from FIGS. 10-1 and 10-2, Bi-R-Arg3, when used in combination with PD-1 blockade, caused more tumor degeneracy compared to PD-1 blockade alone.
（生存率の測定結果）
　マウスの生存率を図１１に示す。図１１から明らかなように、Ｂｉ－Ｒ－Ａｒｇ３は、ＰＤ－１遮断との併用によって、ＰＤ－１遮断単独と比較して生存率を改善させた。 (Measurement result of survival rate)
The survival rate of mice is shown in FIG. As is clear from FIG. 11, Bi-R-Arg3 improved the survival rate when used in combination with PD-1 blockade as compared with PD-1 blockade alone.
　Ｂｉ－Ｒ－Ａｒｇ３単独では、コントロールのＢｉ単独、Ｒ単独と同様に、腫瘍縮退効果を有さず、腫瘍移植宿主の生存率を向上することができなかった。これらのｉｎ　ｖｉｖｏデータは、Ｂｉ－Ｒ－Ａｒｇ３がＰＤ－１遮断療法と相乗効果を有し、抗腫瘍免疫を増強することを示す。すなわち、ミトコンドリアを活性化させるための剤と免疫チェックポイント阻害剤とを併用することで、抗腫瘍免疫力を高めることができる。 Bi-R-Arg3 alone did not have a tumor degenerate effect and could not improve the survival rate of the tumor transplant host, as in the control Bi alone and R alone. These in vivo data indicate that Bi-R-Arg3 has a synergistic effect with PD-1 blockade therapy and enhances antitumor immunity. That is, the antitumor immunity can be enhanced by using a drug for activating mitochondria and an immune checkpoint inhibitor in combination.
SEQ ID NO:1; HPRT1 forward primer
SEQ ID NO:2; HPRT1 reverse primer
SEQ ID NO:3; SOX2 forward primer
SEQ ID NO:4; SOX2 reverse primer
SEQ ID NO:5; 18S forward primer
SEQ ID NO:6; 18S reverse primer
SEQ ID NO:7; SOX2-DNA
SEQ ID NO:8; Ctrl-DNA
SEQ ID NO:9; siRNA sequence for PGC-1a
SEQ ID NO:10; siRNA sequence for PGC-1b
SEQ ID NO:11; mPGC-1a forward primer
SEQ ID NO:12; mPGC-1a reverse primer
SEQ ID NO:13; mPGC-1b forward primer
SEQ ID NO:14; mPGC-1b reverse primer SEQ ID NO: 1; HPRT1 forward primer
SEQ ID NO: 2; HPRT1 reverse primer
SEQ ID NO: 3; SOX2 forward primer
SEQ ID NO: 4; SOX2 reverse primer
SEQ ID NO: 5; 18S forward primer
SEQ ID NO: 6; 18S reverse primer
SEQ ID NO: 7; SOX2-DNA
SEQ ID NO: 8; Ctrl-DNA
SEQ ID NO: 9; siRNA sequence for PGC-1a
SEQ ID NO: 10; siRNA sequence for PGC-1b
SEQ ID NO: 11; mPGC-1a forward primer
SEQ ID NO: 12; mPGC-1a reverse primer
SEQ ID NO: 13; mPGC-1b forward primer
SEQ ID NO: 14; mPGC-1b reverse primer

Claims (14)

  1. 　特定のＤＮＡ配列を認識し、結合するピロール－イミダゾールポリアミドと、１～５個のアルギニン残基を含むペプチドとを含む複合体。 A complex containing a pyrrole-imidazole polyamide that recognizes and binds to a specific DNA sequence and a peptide containing 1 to 5 arginine residues.
  2. 　ペプチドが３個のアルギニン残基を含む、請求項１に記載の複合体。 The complex according to claim 1, wherein the peptide contains 3 arginine residues.
  3. 　ピロール－イミダゾールポリアミドがヘアピン型または環状のピロール－イミダゾールポリアミドである、請求項１または２に記載の複合体。 The complex according to claim 1 or 2, wherein the pyrrole-imidazole polyamide is a hairpin-type or cyclic pyrrole-imidazole polyamide.
  4. 　機能性分子をさらに含む、請求項１～３のいずれか１項に記載の複合体。 The complex according to any one of claims 1 to 3, further comprising a functional molecule.
  5. 　機能性分子がエピジェネティックモディファイアーである、請求項４に記載の複合体。 The complex according to claim 4, wherein the functional molecule is an epigenetic modifier.
  6. 　機能性分子が、蛍光ラベル、アルキル化剤、ブロモドメイン阻害剤、ＨＤＡＣ阻害剤、ＨＡＴ活性化剤、およびＨＡＴ阻害剤からなる群から選択される、請求項４に記載の複合体。 The complex according to claim 4, wherein the functional molecule is selected from the group consisting of a fluorescent label, an alkylating agent, a bromodomain inhibitor, an HDAC inhibitor, a HAT activator, and a HAT inhibitor.
  7. 　請求項１～６いずれか１項に記載の複合体を含む、医薬組成物。 A pharmaceutical composition comprising the complex according to any one of claims 1 to 6.
  8. 　抗がん剤と併用される、請求項７に記載の医薬組成物。 The pharmaceutical composition according to claim 7, which is used in combination with an anticancer agent.
  9. 　抗がん剤が免疫チェックポイント阻害剤である、請求項８に記載の医薬組成物。 The pharmaceutical composition according to claim 8, wherein the anticancer agent is an immune checkpoint inhibitor.
  10. 　免疫チェックポイント阻害剤がＰＤ－１阻害剤またはＰＤ－Ｌ１阻害剤である、請求項９に記載の医薬組成物。 The pharmaceutical composition according to claim 9, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  11. 　免疫チェックポイント阻害剤が抗ＰＤ－１抗体または抗ＰＤ－Ｌ１抗体である、請求項１０に記載の医薬組成物。 The pharmaceutical composition according to claim 10, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
  12. 　下記式：
    Figure JPOXMLDOC01-appb-C000001
     で示される、請求項６に記載の複合体。     The following formula:
    Figure JPOXMLDOC01-appb-C000001
     The complex according to claim 6.
  13. 　請求項１２に記載の複合体を含み、ミトコンドリアを活性化させるための剤。 An agent for activating mitochondria, which comprises the complex according to claim 12.
  14. 　ミトコンドリアを活性化させるための剤と併用される、免疫チェックポイント阻害剤。 An immune checkpoint inhibitor used in combination with an agent for activating mitochondria.
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