WO2022054625A1 - Polyamide de pyrrole-imidazole présentant des propriétés de transport nucléaire améliorées - Google Patents

Polyamide de pyrrole-imidazole présentant des propriétés de transport nucléaire améliorées 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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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

L'invention concerne un complexe contenant un polyamide de pyrrole-imidazole qui reconnaît une séquence d'ADN particulière et se lie à celle-ci et contenant un peptide qui contient de 1 à 5 résidus d'arginine.
PCT/JP2021/031757 2020-09-11 2021-08-30 Polyamide de pyrrole-imidazole présentant des propriétés de transport nucléaire améliorées WO2022054625A1 (fr)

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