WO2023112975A1 - Promoteur de la fonction lymphocytaire, agent destiné à une utilisation combinée avec un agent thérapeutique pour une thérapie anticancéreuse médiée par les lymphocytes, et promoteur de la fonction mitochondriale des lymphocytes - Google Patents

Promoteur de la fonction lymphocytaire, agent destiné à une utilisation combinée avec un agent thérapeutique pour une thérapie anticancéreuse médiée par les lymphocytes, et promoteur de la fonction mitochondriale des lymphocytes Download PDF

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WO2023112975A1
WO2023112975A1 PCT/JP2022/046124 JP2022046124W WO2023112975A1 WO 2023112975 A1 WO2023112975 A1 WO 2023112975A1 JP 2022046124 W JP2022046124 W JP 2022046124W WO 2023112975 A1 WO2023112975 A1 WO 2023112975A1
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cells
lymphocyte
therapy
group
lymphocytes
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PCT/JP2022/046124
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English (en)
Japanese (ja)
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章良 浅井
尚久 小郷
裕明 池田
大輔 村岡
洋介 道津
直純 原田
Original Assignee
一般社団法人ファルマバレープロジェクト支援機構
国立大学法人 長崎大学
ユナイテッド・イミュニティ株式会社
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Publication of WO2023112975A1 publication Critical patent/WO2023112975A1/fr

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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

Definitions

  • the present invention relates to a lymphocyte function enhancer, a combination drug with a therapeutic agent for lymphocyte-mediated cancer therapy, a lymphocyte mitochondrial function enhancer, and the like.
  • T cell-dependent cancers such as immune checkpoint inhibition therapy such as PD-1 inhibitory antibody and genetically modified T cell infusion therapy called CAR-T or TCR-T Immunotherapy
  • TCR T cell receptors
  • CAR chimeric antigen receptors
  • MHC major histocompatibility genes
  • tumor cells often have low levels of MHC and antigen expression, and in such cases T cells are weakly recognizing tumor cells and do not kill them, that is, they do not exert their effector functions. This phenomenon is thought to impair the effects of T cell-dependent cancer immunotherapy, and there is a need for a therapeutic technique that can overcome this phenomenon.
  • Non-Patent Document 1 reports that the Bcr-Abl tyrosine kinase inhibitor imatinib, which is used as a therapeutic agent for chronic myelogenous leukemia, enhances tumor immune response activity.
  • An object of the present invention is to provide a lymphocyte function enhancing agent.
  • the object of the present invention is to provide a lymphocyte function-enhancing agent based on a novel mechanism.
  • the present inventors have found that the compounds represented by the general formula (1), salts thereof, and solvates thereof contain at least one selected from the group consisting of The present inventors have found that the above problems can be solved by a lymphocyte function enhancing agent. Based on this finding, the inventors have further studied and completed the present invention. That is, the present invention includes the following aspects.
  • R 1 represents an alkyl group.
  • R 2 and R 3 are the same or different and represent a nitro group or a hydrogen atom (except when both R 2 and R 3 are hydrogen atoms).
  • Lymphocyte function enhancing agent containing at least one selected from the group consisting of the compound represented by, salts thereof, and solvates thereof.
  • Item 1A A method for enhancing lymphocyte function, comprising contacting lymphocytes with at least one selected from the group consisting of compounds represented by general formula (1), salts thereof, and solvates thereof.
  • Item 1A2 A method for enhancing lymphocyte function, comprising administering to a living body at least one selected from the group consisting of the compound represented by formula (1), salts thereof, and solvates thereof.
  • Section 1B A drug containing at least one selected from the group consisting of compounds represented by general formula (1), salts thereof, and solvates thereof, for use in lymphocyte hyperfunction.
  • Item 1C Use of a drug containing at least one selected from the group consisting of the compound represented by formula (1), salts thereof, and solvates thereof, for producing a lymphocyte function enhancing agent.
  • Item 1D Use of at least one selected from the group consisting of the compound represented by the general formula (1), salts thereof, and solvates thereof, as a lymphocyte function enhancing agent.
  • Item 2 The lymphocyte function enhancing agent according to Item 1 , wherein the alkyl group represented by R 1 has 8 to 20 carbon atoms.
  • Item 3 The lymphocyte function enhancing agent according to Item 1 or 2, wherein the lymphocytes are T cells.
  • Item 4. The lymphocyte function enhancing agent according to any one of Items 1 to 3, wherein the lymphocytes are CD8-positive T cells.
  • Item 5 The lymphocyte function enhancing agent according to any one of Items 1 to 4, wherein the lymphocytes are tumor-infiltrating lymphocytes.
  • Item 6. The agent for enhancing lymphocyte function according to any one of Items 1 to 5, for use in at least one selected from the group consisting of activation of lymphocytes, promotion of lymphocyte proliferation, and promotion of cytokine production.
  • Item 7. The agent for enhancing lymphocyte function according to any one of Items 1 to 6, for use in enhancing the mitochondrial function of lymphocytes.
  • Item 8 The lymphocyte function enhancing agent according to any one of Items 1 to 7, which is used in combination with lymphocyte-mediated cancer therapy.
  • the lymphocyte-mediated cancer therapy is immune checkpoint inhibition therapy, CAR-T therapy, TCR-T therapy, oncolytic virus therapy, cancer vaccine therapy, immunostimulatory antibody therapy, bispecific T cell induction Item 9.
  • Item 10 The lymphocyte function enhancing agent according to any one of Items 1 to 7, which is used for the prevention or treatment of infectious diseases.
  • R 1 represents an alkyl group.
  • R 2 and R 3 are the same or different and represent a nitro group or a hydrogen atom (except when both R 2 and R 3 are hydrogen atoms).
  • a combination drug with a therapeutic agent for lymphocyte-mediated cancer treatment comprising at least one selected from the group consisting of the compound represented by the following, salts thereof, and solvates thereof.
  • Item 11A Contacting lymphocytes with at least one selected from the group consisting of compounds represented by the general formula (1), salts thereof, and solvates thereof, and lymphocyte-mediated cancer treatment A method of treating cancer, comprising administering an agent.
  • Section 11B At least one selected from the group consisting of compounds represented by general formula (1), salts thereof, and solvates thereof for use in combination with therapeutic agents for lymphocyte-mediated cancer therapy Drugs containing.
  • Item 11C A drug containing at least one selected from the group consisting of the compound represented by the general formula (1), salts thereof, and solvates thereof, in combination with a therapeutic agent for lymphocyte-mediated cancer therapy use for the manufacture of
  • Item 11D Use of at least one selected from the group consisting of the compound represented by the general formula (1), salts thereof, and solvates thereof, in combination with a therapeutic agent for lymphocyte-mediated cancer therapy .
  • R 1 represents an alkyl group.
  • R 2 and R 3 are the same or different and represent a nitro group or a hydrogen atom (except when both R 2 and R 3 are hydrogen atoms).
  • a prophylactic or therapeutic agent for infectious diseases containing at least one selected from the group consisting of the compound represented by the following, salts thereof, and solvates thereof.
  • Section 12A A method for preventing or treating an infectious disease, comprising administering to a living body at least one selected from the group consisting of the compound represented by formula (1), salts thereof, and solvates thereof.
  • Section 12B A drug containing at least one selected from the group consisting of compounds represented by general formula (1), salts thereof, and solvates thereof, for use in the prevention or treatment of infectious diseases.
  • Item 12C Use of an agent containing at least one selected from the group consisting of the compound represented by formula (1), salts thereof, and solvates thereof, for the production of an agent for the prevention or treatment of infectious diseases.
  • Item 12D Use of at least one selected from the group consisting of compounds represented by general formula (1), salts thereof, and solvates thereof, as a prophylactic or therapeutic agent for infectious diseases.
  • R 1 represents an alkyl group.
  • R 2 and R 3 are the same or different and represent a nitro group or a hydrogen atom (except when both R 2 and R 3 are hydrogen atoms).
  • Lymphocyte mitochondrial function enhancing agent containing at least one selected from the group consisting of the compound represented by the following, salts thereof, and solvates thereof.
  • Item 13A A method for enhancing mitochondrial function of lymphocytes, comprising contacting lymphocytes with at least one selected from the group consisting of the compound represented by formula (1), salts thereof, and solvates thereof.
  • Item 13B A drug containing at least one selected from the group consisting of the compound represented by general formula (1), salts thereof, and solvates thereof, for use in enhancing the mitochondrial function of lymphocytes.
  • Item 13C Use of an agent containing at least one selected from the group consisting of the compound represented by the general formula (1), salts thereof, and solvates thereof, for producing an agent for enhancing the mitochondrial function of lymphocytes.
  • Item 13D Use of at least one selected from the group consisting of the compound represented by the general formula (1), salts thereof, and solvates thereof, as an agent for enhancing mitochondrial function of lymphocytes.
  • R 1 represents an alkyl group.
  • R 2 and R 3 are the same or different and represent a nitro group or a hydrogen atom (except when both R 2 and R 3 are hydrogen atoms).
  • a lymphocyte TCR repertoire diversity-increasing agent containing at least one selected from the group consisting of a compound represented by the following, salts thereof, and solvates thereof.
  • Item 14A A method for increasing TCR repertoire diversity of lymphocytes, comprising contacting lymphocytes with at least one selected from the group consisting of compounds represented by general formula (1), salts thereof, and solvates thereof. .
  • Section 14B A drug containing at least one selected from the group consisting of compounds represented by general formula (1), salts thereof, and solvates thereof, for use in increasing TCR repertoire diversity of lymphocytes.
  • Item 14C Use of an agent containing at least one selected from the group consisting of the compound represented by the general formula (1), salts thereof, and solvates thereof, for producing an agent for increasing TCR repertoire diversity of lymphocytes .
  • Item 14D Use of at least one selected from the group consisting of compounds represented by the general formula (1), salts thereof, and solvates thereof, as an agent for increasing TCR repertoire diversity of lymphocytes.
  • a lymphocyte function enhancing agent can be provided.
  • FIG. 2 shows flow cytometry results of Test Example 1.
  • FIG. Each plot represents a test compound, with circled and arrowed plots representing compound A and circled and unarrowed plots representing compound B.
  • 1 shows the results of flow cytometry in the concentration-dependent confirmation test of Compound A in Test Example 1.
  • FIG. 2 shows the results of WST-8 assay in Test Example 2.
  • FIG. The horizontal axis indicates the compound A concentration in the medium.
  • 3 shows flow cytometry results of Test Example 3.
  • FIG. Low indicates a medium concentration of 0.5 ⁇ M, and High indicates a medium concentration of 1.5 ⁇ M.
  • n.s. indicates P value by ANOVA test is greater than or equal to 0.05, * indicates P value by ANOVA test is less than 0.05, ** indicates P value by ANOVA test is less than 0.01.
  • FIG. 2 shows the results of flow cytometry when CT26 cells of Test Example 4 were used.
  • n.s. indicates a P value greater than or equal to 0.05 by ANOVA test, * indicates a P value less than 0.05 by ANOVA test.
  • 2 shows the results of flow cytometry when CMS5a cells of Test Example 4 were used. * indicates P-value less than 0.05 by ANOVA test, ** indicates P-value less than 0.01 by ANOVA test.
  • 2 shows the results of flow cytometry and tumor volume measurement in the colorectal cancer model of Test Example 5.
  • FIG. n.s. indicates a P value greater than or equal to 0.05 by ANOVA test, * indicates a P value less than 0.05 by ANOVA test. 2 shows the results of tumor volume measurement in the fibrosarcoma model of Test Example 5.
  • FIG. n.s. indicates a P value greater than or equal to 0.05 by ANOVA test
  • * indicates a P value less than 0.05 by ANOVA test.
  • the results of flow cytometry and tumor volume measurement in combination with the PD-1 inhibitor of Test Example 6 are shown.
  • n.s. indicates P-value by ANOVA test is greater than or equal to 0.05
  • * indicates P-value by ANOVA test is less than 0.05
  • ** indicates P-value by ANOVA test is less than 0.01
  • * ** indicates a P-value less than 0.001 by ANOVA test.
  • 2 shows the results of tumor volume measurement in combination with the TCR gene-modified T cell therapy of Test Example 6.
  • FIG. * indicates P value less than 0.05 by ANOVA test.
  • FIG. 2 shows the results of a mitochondrial membrane potential analysis test when spleen cells were used in Test Example 7.
  • FIG. A higher TMRE value on the vertical axis indicates a normal membrane potential.
  • FIG. 2 shows the results of a mitochondrial membrane potential analysis test when TIL was used in Test Example 7.
  • FIG. A higher TMRE value on the vertical axis indicates a normal membrane potential. * indicates P value less than 0.05 by ANOVA test.
  • 2 shows flow cytometry results of Test Example 8.
  • Lymphocyte function-enhancing agent in this specification, sometimes referred to as “agent of the present invention”
  • Lymphocyte function enhancing agent containing Lymphocyte function enhancing agent
  • R 1 represents an alkyl group.
  • the alkyl group represented by R 1 includes both linear and branched (preferably linear).
  • the number of carbon atoms in the alkyl group is not particularly limited, it is, for example, 1-30.
  • the number of carbon atoms is preferably 4-25, more preferably 8-20, still more preferably 10-16, still more preferably 10-14, from the viewpoint of lymphocyte hyperfunction.
  • R 2 and R 3 are the same or different and represent a nitro group or a hydrogen atom (except when both R 2 and R 3 are hydrogen atoms).
  • one of R2 and R3 is a nitro group and the other is a hydrogen atom.
  • R2 is a hydrogen atom and R3 is a nitro group.
  • the compound of the present invention is compound A or compound B below, more preferably compound A.
  • the salt of the compound of the present invention is not particularly limited as long as it is a pharmaceutically acceptable salt.
  • both acid salts and basic salts can be employed.
  • acid salts include inorganic salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; acetate, propionate, tartrate, fumarate, maleate, malic acid organic salts such as salts, citrates, methanesulfonates, and paratoluenesulfonates;
  • examples of basic salts include alkali metal salts such as sodium salts and potassium salts; Alkaline earth metal salts such as salts; Salts with ammonia; Morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, tri(hydroxyalkyl) Examples thereof include salts with organic amines such as amines.
  • solvate of the compound of the present invention and its salt is not particularly limited.
  • Solvents constituting the solvent field include, for example, water, pharmaceutically acceptable organic solvents (eg, ethanol, glycerol, acetic acid, etc.), and the like.
  • the compound of the present invention is a known compound, or is synthesized according to or according to a method known to those skilled in the art using a known compound as a starting material (for example, according to or according to a method for synthesizing a compound of the present invention which is a known compound). can be done.
  • the active ingredient of the present invention has lymphocyte hyperfunction. Therefore, the active ingredient of the present invention can be used as an active ingredient of medicines, reagents, etc. (herein sometimes referred to as "the drug of the present invention"), more specifically, as a lymphocyte function enhancing agent. It can be used as an active ingredient.
  • the lymphocytes targeted by the agents of the present invention are not particularly limited, but preferably include cells capable of exhibiting effector functions.
  • Lymphocytes preferably include T cells. More specifically, for example, CD8-positive T cells, CD4-positive T cells, CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, T cells prepared from iPS cells, ⁇ -T cells, ⁇ -T cells, NK cells, NKT cells and the like.
  • Target lymphocytes also include genetically modified lymphocytes such as CAR-T cells and TCR-T cells.
  • the target lymphocytes may be those existing in vivo or those existing in vitro (for example, cells collected from a living body or their proliferated cells, cell lines, or those prepared from ES cells or iPS cells). may be
  • the active ingredient of the present invention can exert lymphocyte hyperfunction on tumor-infiltrating lymphocytes, especially on tumor-reactive T cells. From this point of view, the lymphocytes can preferably be tumor-infiltrating lymphocytes.
  • Lymphocyte hyperfunction is not particularly limited as long as it leads to enhancement of lymphocyte function, especially function as effector cells. Lymphocyte hyperfunction includes, for example, lymphocyte activation, promotion of lymphocyte proliferation, promotion of cytokine production, and the like. From this point of view, the agent of the present invention or the active ingredient of the present invention can be used for at least one selected from the group consisting of activation of lymphocytes, promotion of lymphocyte proliferation, and promotion of cytokine production.
  • Lymphocyte activation is determined by various activation markers (for example, for T cells, CD25, GITR, CD223, PD-1, Tim-3, CD69, NKG2D, NKG2A, CD169, 4-1BB, KLRG, CD122, CTLA -4, CD95, GITR, CD40L, Trail, etc.) can be determined as an index.
  • activation markers for example, for T cells, CD25, GITR, CD223, PD-1, Tim-3, CD69, NKG2D, NKG2A, CD169, 4-1BB, KLRG, CD122, CTLA -4, CD95, GITR, CD40L, Trail, etc.
  • Cytokines include, for example, cytokines produced by various T cells (e.g., CTL, Th1, Th2, Th9, Th17, etc.), more specifically, for example, IFN- ⁇ , TNF- ⁇ , IL-2, Perforin, Granzyme , IL-17, IL-21, IL-22, IL-9, IL-10, IL-4, IL-5, IL-13 and the like.
  • the active ingredient of the present invention has a mitochondrial function-enhancing effect on lymphocytes. By this action, a lymphocyte hyperfunction action can be exhibited. From this point of view, the agent of the present invention or the active ingredient of the present invention can be used for enhancing mitochondrial function of lymphocytes.
  • mitochondrial hyperfunction is not particularly limited as long as it is a mitochondrial function that leads to lymphocyte hyperfunction.
  • mitochondrial membrane potential can be used as an index for determination.
  • Mitochondrial functions such as lymphocyte activation, proliferation, cytokine production and the like can be enhanced by improving the mitochondrial membrane potential or partially or completely releasing the suppressed state.
  • the active ingredient of the present invention has the action of increasing the diversity of the TCR repertoire of lymphocytes. This action increases the proportion of T cells with antigen-recognizing ability, increases the proportion of activatable lymphocytes, and can exert lymphocyte hyperfunction. From this point of view, the agent of the present invention or the active ingredient of the present invention can be used to increase the diversity of the TCR repertoire of lymphocytes.
  • the increased diversity of the TCR repertoire can be evaluated, for example, by TCR beta sequencing, more specifically, for example, by the method of Test Example 9.
  • the active ingredient of the present invention has a lymphocyte function-enhancing effect, it can be used in combination with a lymphocyte-mediated cancer therapy to improve the effect of the therapy.
  • the agent of the present invention or the active ingredient of the present invention is used in combination with lymphocyte-mediated cancer therapy, for example, in combination with a therapeutic agent for lymphocyte-mediated cancer therapy.
  • the timing of combined use is not particularly limited, and for example, it may be administered at the same time or on the same day as the therapeutic agent for lymphocyte-mediated cancer therapy, or on the day of administration of the therapeutic agent for lymphocyte-mediated cancer therapy. It may be one or more days (eg 2-14 days) before or after.
  • the lymphocyte-mediated cancer therapy is not particularly limited as long as it is a therapy involving attacking cancer cells by lymphocytes (preferably effector T cells).
  • T therapy preferably effector T cells
  • TCR-T therapy oncolytic virus therapy, cancer vaccine therapy, immunostimulatory antibody therapy, bispecific T cell induction antibody therapy, soluble T cell receptor drug therapy, radiation therapy, anticancer drug therapy etc.
  • Immune checkpoint inhibitors include, for example, anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies.
  • CAR-T cells/TCR-T cells include various antigens (e.g., ERK1, ERK2, MART-1/Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal-associated antigen (CRC) -C017-1A/GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate specific target membrane antigen (PSMA), T-cell receptor/CD3 ⁇ chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE -A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp
  • viruses with tumor specificity based on viruses with cytolytic activity such as herpes virus and adenovirus, can be widely used.
  • cancer vaccines for example, the various antigens described above can be widely used as they are or complexed with other substances.
  • immunostimulatory antibodies examples include anti-CD137 antibody, anti-CD40 antibody, anti-OX40 antibody, anti-CD127 antibody, and the like.
  • Anti-CD19/CD3 bispecific antibody anti-CD20/CD3 bispecific antibody, anti-CD33/CD3 bispecific antibody, anti-BCMA/CD3 bispecific antibody, anti-CD19/CD3 bispecific antibody, anti-CD33/CD3 bispecific antibody, anti-BCMA/CD3 bispecific antibody antibodies, anti-CD123/CD3 bispecific antibodies, and the like.
  • Soluble T-cell receptor drugs include gp100-specific soluble T-cell receptor, MAGE-A4-specific soluble T-cell receptor, PRAME-specific soluble T-cell receptor, NY-ESO-1-specific soluble T-cell receptor etc.
  • Anticancer agents include, for example, alkylating agents, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum agents, molecular target drugs, hormone agents, and biological agents.
  • alkylating agents examples include cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine.
  • Antimetabolites include, for example, enocitabine, carmofur, capecitabine, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, gemcitabine, cytarabine, cytarabine octophosphate, nerarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, Cladribine, doxifluridine, hydroxycarbamide, mercaptopurine and the like.
  • microtubule inhibitors examples include alkaloid anticancer agents such as vincristine, and taxane anticancer agents such as docetaxel and paclitaxel.
  • Antibiotic anticancer agents include, for example, mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, aclarubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, and dinostatin stimaramer.
  • topoisomerase inhibitors examples include CPT-11, irinotecan, and topotecan, which have topoisomerase I inhibitory activity, and etoposide and sobuzoxan, which have topoisomerase II inhibitory activity.
  • platinum agents examples include cisplatin, nedaplatin, oxaliplatin, and carboplatin.
  • Hormonal agents include, for example, dexamethasone, finasteride, tamoxifen, astrozole, exemestane, ethinylestradiol, chlormadinone, goserelin, bicalutamide, flutamide, brednisolone, leuprorelin, letrozole, estramustine, toremifene, fosfestrol, mitotane, Methyltestosterone, medroxyprogesterone, mepitiostane and the like.
  • biologics include interferon ⁇ , ⁇ and ⁇ , interleukin 2, ubenimex, and dried BCG.
  • molecular targeted drugs examples include rituximab, alemtuzumab, trastuzumab, cetuximab, panitumumab, imatinib, dasatinib, nilotinib, gefitinib, erlotinib, temsirolimus, bevacizumab, VEGF trap, sunitinib, sorafenib, tocituzumab, bortezomib, gemtuzumab o Zogamicin, ibritumomab ozogamicin , ibritumomab tiuxetan, tamibarotene, tretinoin and the like.
  • Human epidermal growth factor receptor 2 inhibitors such as vascular endothelial growth factor receptor 2 inhibitors ( ⁇ -VEGFR-2 antibodies); various tyrosine kinase inhibitors, such as MAP kinase inhibitors; cytokine-targeted inhibitors; Molecularly targeted agents such as proteasome inhibitors, antibody-anticancer drug combinations, and the like can also be included. These inhibitors also include antibodies.
  • the target cancer for lymphocyte-mediated cancer therapy is not particularly limited, and for example, various B-cell malignant lymphomas (B-cell acute lymphocytic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, , intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma, etc.), myeloproliferative disease, myelodysplastic/myeloproliferative neoplasms (CMML, JMML, CML, MDS/MPN-UC), myelodysplastic syndrome, acute myelogenous Leukemia, multiple myeloma, lung cancer, colon cancer, ovarian cancer, breast cancer, brain cancer, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, Examples include chondrosarcoma and rhabdomyosarcoma.
  • the active ingredient of the present invention Since the active ingredient of the present invention has a lymphocyte hyperfunction, it can exert a preventive or therapeutic effect on infectious diseases. From this point of view, the drug of the present invention or the active ingredient of the present invention can be used for prevention or treatment of infectious diseases. Examples of infectious diseases include bacterial infections, viral infections, and the like.
  • Gram-negative bacteria include, for example, Enterobacteriaceae (e.g., Escherichia, Klebsiella, Salmonella, Shigella, etc.), Acinetobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Moraxella Bacteria, Helicobacter, Campylobacter, Aeromonas, Vibrio (e.g. Vibrio cholerae, Vibrio parahaemolyticus), Haemophilus (e.g.
  • Gram-positive bacteria include, for example, staphylococci (e.g., Staphylococcus aureus, Staphylococcus epidermidis, etc.), enterococci (e.g., Enterococcus), streptococci (e.g., group A streptococci, group B streptococci, pneumonia Streptococcus violet), Bacillus (e.g. Bacillus cereus, Bacillus anthracis), Clostridia (e.g.
  • Tetanus Clostridium botulinum, Clostridium difficile
  • Corynebacterium e.g. Diphtheria
  • Listeria Listeria
  • Lactobacillus genus Bifidobacterium Propionibacterium (for example, P. acnes causing acne), actinomycetes, and the like.
  • viruses causing viral infections include influenza viruses (e.g., type A, type B, etc.), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster virus, mumps virus, arbovirus, respiratory syncytial virus, and SARS.
  • influenza viruses e.g., type A, type B, etc.
  • rubella virus e.g., Ebola virus, coronavirus, measles virus, varicella-zoster virus, mumps virus, arbovirus, respiratory syncytial virus, and SARS.
  • Viruses e.g., hepatitis B virus, hepatitis C virus, etc.
  • yellow fever virus AIDS virus, rabies virus, hantavirus, dengue virus, Nipah virus, enveloped viruses (viruses having an envelope) such as lyssa virus; Adenovirus , norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackievirus, human parvovirus, encephalomyocarditis virus, poliovirus, rhinovirus, and other non-enveloped viruses (viruses without envelopes).
  • the active ingredient of the present invention has a mitochondrial function-enhancing effect not only on lymphocytes, it is also useful for mitochondrial diseases, for example.
  • the subject or patient can be a mammal, including, for example, primates such as humans, and companion animals such as dogs and cats.
  • patient means a subject with a disease or pathological condition.
  • the subject to whom the active ingredient of the present invention is administered can be a subject with abnormal mitochondrial function, such as a mitochondrial disease patient.
  • a patient with a mitochondrial disease can be a patient with an abnormality in the mitochondrial genome or a patient with an abnormality in the nuclear genome.
  • the mitochondrial disease patient can be a hereditary mitochondrial disease patient or an acquired mitochondrial disease patient.
  • the subject to whom the active ingredient of the present invention is administered can be a subject with abnormal mitochondrial function and atrophied thymus. Atrophy of the thymus can be determined by comparison with normal thymuses possessed by individuals of the same age as the subject. According to the present invention, the subject to whom the active ingredient of the present invention is administered can be a subject with abnormal mitochondrial function and abnormal immune function. Abnormalities in mitochondrial function can be determined, for example, by comparing the mitochondrial membrane potential to the mitochondrial membrane potential of a healthy subject. Restoration of mitochondrial function in the thymus is thought to induce recovery of hypothymic function, improvement of thymic atrophy, and improvement of immune abnormalities.
  • the present invention provides a method for supplying activated mitochondria to the thymus, and the active ingredient of the present invention can be used in the treatment of individuals with abnormal mitochondrial function (e.g., patients with mitochondrial diseases or individuals aged 60 or older). It may also be useful to use in combination with
  • treatment includes mitigation (mitigation) of symptoms characteristic of the target disease or associated symptoms, prevention or delay of exacerbation of symptoms, and the like.
  • prevention means preventing or delaying the onset/development of a disease (disorder) or its symptoms, or reducing the risk of onset/development.
  • the drug of the present invention is not particularly limited as long as it contains the active ingredient of the present invention, and may further contain other ingredients as necessary.
  • Other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients.
  • Other components include additives as well as components having pharmacological action. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, perfumes, A chelating agent and the like are included.
  • the active ingredient of the present invention can exert the above effects and the like by itself. Therefore, the drug of the present invention can exert its desired effect without containing other ingredients having these effects and/or actions, but even if it contains other ingredients having pharmacological actions, good.
  • the mode of use of the drug of the present invention is not particularly limited, and an appropriate mode of use can be adopted according to its type.
  • the agent of the present invention can be used, for example, in vitro (e.g., added to the culture medium of cultured cells) or in vivo (e.g., administered to an animal), depending on its use. can also
  • Animals to which the agent of the present invention is applied are not particularly limited, but mammals include, for example, humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. Moreover, animal cells etc. are mentioned as a cell.
  • the types of cells are not particularly limited, such as blood cells, hematopoietic stem cells/progenitor cells, gametes (sperm, ovum), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells. , epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like.
  • the agent of the present invention can be in any dosage form, such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, Hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), oral dosage forms such as jelly, injection preparations (e.g., drip injections (e.g., intravenous drip preparations etc.), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external agents (e.g., ointments, poultices, lotions), suppository inhalants, eye agents, eye ointments, nasal drops
  • parenteral formulations such as drug, eardrops, and liposomes can be used.
  • the administration route of the agent of the present invention is not particularly limited as long as the desired effect is obtained, and is enteral administration such as oral administration, tube feeding, enema administration; intravenous administration, transarterial administration, intramuscular administration, Examples include parenteral administration such as intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intratumoral administration.
  • the content of the active ingredient in the drug of the present invention depends on the mode of use, the target of application, the condition of the target of application, etc., and is not limited, but is for example 0.0001 to 100% by weight, preferably 0.001 to 50% by weight. %.
  • the dosage is not particularly limited as long as it is an effective amount that exhibits efficacy. 1000 mg/kg body weight, preferably 0.5-500 mg/kg body weight per day, and 0.01-100 mg/kg body weight per day, preferably 0.05-50 mg/kg body weight for parenteral administration .
  • the above dosage can be adjusted appropriately depending on age, disease state, symptoms and the like.
  • Test example 1 Analysis of effects on T cells 1
  • the effects on T cell proliferation and activation were analyzed for each of a large number of test compounds.
  • a specific method is as follows. Spleens were isolated from DUC18 mice (transgenic mice for the murine T-cell receptor gene specific for the 9m peptide epitope derived from mutant ERK2 kinase), washed with RPMI1640 medium and exsanguinated. After grinding the spleen using a glass slide, released cells were collected in RPMI1640 medium. After centrifugation (300 ⁇ g, 5 min, 4° C.), the supernatant was removed and 2 mL of ACK solution was added to treat the cells for 1 min. 18 mL of RPMI1640 medium was added and centrifuged (300 xg, 5 minutes, 4°C).
  • the supernatant was removed and the cells were suspended in an appropriate amount of RPMI1640 medium. After counting the number of cells, the cells were suspended in RPMI1640 medium to a cell concentration of 1 ⁇ 10 7 cells/mL. The cell suspension was centrifuged to obtain 1 ⁇ 10 7 cells, the supernatant was removed, 1 ml of FarRed solution diluted 1000-fold with RPMI1640 medium was added, and the cells were allowed to stand at room temperature for 20 minutes. 9 mL of RPMI1640 medium containing 10% fetal calf serum (FCS) was added and centrifuged.
  • FCS fetal calf serum
  • the cells were adjusted to 1 ⁇ 10 6 cells/ml with RPMI1640 medium containing 10% FCS, and the cells were seeded in a 96-well plate at 100 ⁇ L/well. Each compound was adjusted to 6 ⁇ M in RPMI1640 medium containing 10% FCS and added at 50 ⁇ L/well, then 9m peptide was adjusted to 4 ⁇ g/ml in RPMI1640 medium containing 10% FCS and added at 50 ⁇ L/well. was added with Cells were transferred to a 96-well V-bottom microplate (Nunc), centrifuged (2000 rpm, 2 minutes, 4°C), and the supernatant was removed.
  • Each plot represents a test compound, with circled and arrowed plots representing compound A and circled and unarrowed plots representing compound B.
  • the structures of compound A and compound B are shown below.
  • Test example 2 Analysis of effects on T cells 2
  • spleen cells derived from BALB/c mice were treated with compound A, stimulated with anti-CD3 antibody and anti-CD28 antibody for 72 hours, and then subjected to WST-8 assay.
  • a specific method is as follows. Cell suspensions were seeded into 96-well clear flat-bottom microplates coated with anti-CD3 antibody. Anti-CD28 antibody (1 ⁇ g/mL) and compound A were added to 200 ⁇ L/well, and cultured at 37° C. in the presence of 5% CO 2 for 72 hours. Blank wells to which only the cell culture medium was added and control wells to which dimethylsulfoxide (DMSO) (0.1% final concentration), a solvent for the compound, was added were similarly prepared. After discarding the supernatant and washing once with 100 ⁇ L of PBS, 70 ⁇ L of assay medium (10% FBS, 1% penicillin-streptomycin-containing phenol red-free DMEM) was added.
  • assay medium 10% FBS, 1% penicillin-streptomycin-containing phenol red-free DMEM
  • Relative Growth (%) [OD (drug) - OD (blank)] / [OD (control) - OD (blank)] ⁇ 100.
  • Other conditions are as follows: anti-CD3 antibody: 0.33 ⁇ g/mL, mouse spleen cell seeding number: 1.5 ⁇ 10 5 cells/well.
  • Test example 3 Analysis of effects on T cells3
  • changes in activation markers other than CD25 and cytokines were investigated.
  • a specific method for analyzing changes in activation markers is as follows. Spleens were isolated from DUC18 mice, washed with RPMI1640 medium and debleed. After grinding the spleen using a glass slide, released cells were collected in RPMI1640 medium. After centrifugation (300 ⁇ g, 5 min, 4° C.), the supernatant was removed and 2 mL of ACK solution was added to treat the cells for 1 min. 18 mL of RPMI1640 medium was added and centrifuged (300 xg, 5 minutes, 4°C). The supernatant was removed and the cells were suspended in an appropriate amount of RPMI1640 medium.
  • the cells were adjusted to 1 ⁇ 10 6 cells/mL with RPMI1640 medium containing 10% FCS, and the cells were seeded in a 96-well plate at 100 ⁇ L/well.
  • Compound A was adjusted to 4 times the final concentration (Low 0.5 ⁇ M, High 1.5 ⁇ M) in RPMI medium containing 10% FCS, added at 50 ⁇ L/well, and then 9m peptide was added to RPMI containing 10% FCS. It was adjusted to 4 ⁇ g/mL with medium and added at 50 ⁇ L/well.
  • the proliferation analysis method is as follows. Spleens were isolated from DUC18 mice, washed with RPMI1640 medium and debleed. After grinding the spleen using a glass slide, released cells were collected in RPMI1640 medium. After centrifugation (300 ⁇ g, 5 min, 4° C.), the supernatant was removed and 2 mL of ACK solution was added to treat the cells for 1 min. 18 mL of RPMI1640 medium was added and centrifuged (300 xg, 5 minutes, 4°C). The supernatant was removed and the cells were suspended in an appropriate amount of RPMI1640 medium.
  • the cells were suspended in RPMI1640 medium to a cell concentration of 1 ⁇ 10 7 cells/mL.
  • the cell suspension was centrifuged to obtain 1 ⁇ 10 7 cells, the supernatant was removed, 1 mL of FarRed solution diluted 1000-fold with RPMI1640 was added, and the cells were allowed to stand at room temperature for 20 minutes.
  • 9 mL of RPMI1640 medium containing 10% FCS was added and centrifuged. The supernatant was removed, the cells were adjusted to 1 ⁇ 10 6 cells/mL with RPMI1640 medium containing 10% FCS, and the cells were seeded in a 96-well plate at 100 ⁇ L/well.
  • Compound A was adjusted to 4 times each final concentration (Low 0.5 ⁇ M, High 1.5 ⁇ M) in RPMI medium containing 10% FCS, added at 50 ⁇ L/well, and then 9m peptide was added to RPMI containing 10% FCS. It was adjusted to 4 ⁇ g/mL with medium and added at 50 ⁇ L/well. Transfer the cells to a 96-well V-bottom microplate (Nunc), centrifuge (2000 rpm, 2 minutes, 4°C), remove the supernatant, and add 50 ⁇ L of staining buffer (PBS containing 2% FCS) per well. ).
  • An APC-Cy7-labeled anti-CD8 antibody was added, mixed, and allowed to stand in the dark at 4°C for 15 minutes. After washing the cells twice with 2% FCS-containing PBS, they were resuspended in 200 ⁇ L of staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). Data was obtained using a flow cytometer (FACS Canto II, BD Biosciences) using the attached analysis software (FACSDiva), and then the data was analyzed separately using analysis software (FlowJo).
  • cytokines IFN- ⁇
  • Spleens were isolated from DUC18 mice, washed with RPMI1640 medium and debleed. After grinding the spleen using a glass slide, released cells were collected in RPMI1640 medium. After centrifugation (300 ⁇ g, 5 min, 4° C.), the supernatant was removed and 2 mL of ACK solution was added to treat the cells for 1 min. 18 mL of RPMI1640 medium was added and centrifuged (300 xg, 5 minutes, 4°C). The supernatant was removed and the cells were suspended in an appropriate amount of RPMI1640 medium.
  • the cells were suspended in RPMI1640 medium to a cell concentration of 1 ⁇ 10 7 cells/mL. The supernatant was removed, the cells were adjusted to 1 ⁇ 10 6 cells/mL with RPMI1640 medium containing 10% FCS, and the cells were seeded in a 96-well plate at 100 ⁇ L/well. Compound A was adjusted to 4 times the final concentration (Low 0.5 ⁇ M, High 1.5 ⁇ M) in RPMI medium containing 10% FCS, added at 50 ⁇ L/well, and then 9m peptide was added to RPMI containing 10% FCS. It was adjusted to 4 ⁇ g/mL with medium and added at 50 ⁇ L/well.
  • GoldiPlug (BD Biosciences) was added to a final concentration of 1/1000, and cultured at 37°C, 5% CO 2 for 6 hours. Collect the cells in a 96-well V-bottom microplate (Nunc), centrifuge (2000 rpm, 2 minutes, 4°C), remove the supernatant, and add 50 ⁇ L of staining buffer (2% fetal bovine PBS containing serum). A PE-labeled anti-CD8 antibody was added, mixed, and allowed to stand in the dark at 4°C for 15 minutes. After washing the cells twice with 200 ⁇ L of staining buffer, 100 ⁇ L of Cytofix/Cytoperm buffer (BD Biosciences) was added and gently mixed.
  • staining buffer 2% fetal bovine PBS containing serum
  • Test example 4 Analysis of effects on T cells4 If Compound A can activate T cells in the presence of antigenic stimulation, it should also promote the activation of T cells that infiltrate cancer tissues, receive natural antigenic stimulation, and are trying to attack cancer. be. To investigate this, the effect of compound A on CD8-positive killer T cells recovered from mouse-transplanted tumors was analyzed.
  • a specific method is as follows. Mouse colon cancer cell line CT26 or mouse fibrosarcoma cell line CMS5a cultured in a T75 flask (Corning) was detached using 0.5% trypsin-containing PBS and collected in 10% FCS-containing RPMI1640 medium. After centrifugation (300 ⁇ g, 5 minutes, 4°C), the supernatant was removed, washed twice with RPMI1640 medium, suspended in RPMI1640 medium at a concentration of 1 ⁇ 10 6 cells/100 ⁇ L, and 100 ⁇ L/individual was subcutaneously implanted into BALB/c mice at a dose of .
  • tumors were isolated from mice, disrupted using Gentle MACS (Miltenyi), and suspended in RPMI1640 medium.
  • Collagenase D final concentration 1 mg/mL, Roche
  • RPMI1640 medium After passing through a filtration filter (22 ⁇ m, BD Bioscience), the supernatant was removed after centrifugation (1200 rpm, 5 minutes, 4°C), and 2 mL of erythrocyte lysing solution was added to treat the cells for 1 minute. . 18 mL of RPMI1640 medium was added and centrifuged (1200 rpm, 5 minutes, 4°C).
  • TIL tumor infiltrating lymphocytes
  • a 24-well culture plate (Nunc) coated with anti-CD3 antibody at 0.1 ⁇ g/well was seeded with TIL, and compound A was added to a prescribed concentration (Low 0.5 ⁇ M, High 1.5 ⁇ M), and incubated at 37°C. Cultured at 5% CO2 . After 24 or 72 hours, harvest the cells in a 96-well V-bottom microplate (Nunc), centrifuge (2000 rpm, 2 min, 4°C), remove the supernatant, and add 50 ⁇ L of staining solution per well. It was suspended in a buffer (PBS containing 2% FCS).
  • Test example 5 Analysis of effect on tumor 1 As in Test Example 4, if Compound A infiltrates cancer tissue and receives natural antigen stimulation, promoting the activation of T cells that are trying to attack cancer, T cells are effective against cancer. should be able to target and inhibit tumor growth. In order to investigate this, a direct administration experiment of Compound A to mouse transplanted tumors was performed.
  • a specific method is as follows. Mouse colon cancer cell line CT26 or mouse fibrosarcoma cell line CMS5a cultured in a T75 flask (Corning) was detached using PBS containing 0.5% trypsin and collected in RPMI1640 medium containing 10% FBS. After centrifugation (300 ⁇ g, 5 minutes, 4°C), the supernatant was removed, washed twice with RPMI1640 medium, suspended in RPMI1640 medium at a concentration of 1 ⁇ 10 6 cells/100 ⁇ L, and 100 ⁇ L/individual was subcutaneously implanted into BALB/c mice at a dose of . Starting 6 days after transplantation, compound A was intratumorally administered for 5 consecutive days.
  • the major axis and minor axis of the tumor were measured over time, and the therapeutic effect was evaluated by calculating the tumor size (mm 3 ) as major axis x minor axis 2/2 .
  • the effect of intratumoral administration of Compound A on TIL was also examined.
  • Tumors were isolated from mice 72 hours after the last administration, disrupted using Gentle MACS (Miltenyi) and suspended in RPMI1 640 medium. Collagenase D (final concentration 1 mg/mL, Roche) was added, reacted at 37°C for 30 minutes, and then disrupted again using Gentle MACS.
  • TIL tumor infiltrating lymphocytes
  • Test example 6 Analysis of effect on tumor 2 The effect of combined use of compound A in lymphocyte-mediated cancer therapy (PD-1 inhibitor or TCR gene-modified T cell therapy) was analyzed.
  • Anti-PD-1 antibody (RMP1-14) was intraperitoneally administered at 200 ⁇ g diluted in PBS on days 10, 12 and 14 after tumor transplantation.
  • the major axis and minor axis of the tumor were measured over time, and the therapeutic effect was evaluated by calculating the tumor size (mm 3 ) as major axis x minor axis 2/2 .
  • a CMS5a cell line (presenting a 9m peptide epitope derived from an endogenous mutant ERK2 kinase) cultured in a T75 flask (Corning) was detached using PBS containing 0.5% trypsin and collected in RPMI1640 medium containing 10% FBS. bottom. After centrifugation (300 ⁇ g, 5 minutes, 4°C), the supernatant was removed, washed twice with RPMI1640 medium, suspended in RPMI1640 medium at a concentration of 1 ⁇ 10 6 cells/100 ⁇ L, and 100 ⁇ L/individual was subcutaneously implanted into BALB/c mice at a dose of .
  • CD8-positive T cells derived from the spleen of DUC18 mice were injected. It was suspended in RPMI1640 medium at a concentration and administered to BALB/c mice via the tail vein at a dose of 200 ⁇ L/individual. Compound A was also administered intratumorally for 5 consecutive days, starting 6 days after tumor implantation. The major axis and minor axis of the tumor were measured over time, and the therapeutic effect was evaluated by calculating the tumor size (mm 3 ) as major axis x minor axis 2/2 .
  • the CD8-positive T cells used for administration were separated by the following procedure. Spleens were isolated from DUC18 mice, washed with RPMI1640 medium and debleed. After grinding the spleen using a glass slide, released cells were collected in RPMI1640 medium. After centrifugation (300 ⁇ g, 5 min, 4° C.), the supernatant was removed and 2 mL of ACK solution was added to treat the cells for 1 min. 18 mL of RPMI1640 medium was added and centrifuged (300 xg, 5 minutes, 4°C). The supernatant was removed and the cells were suspended in an appropriate amount of RPMI1640 medium.
  • Test example 7 Analysis of mechanism of action 1 TMRE (tetramethylrhodamine ethyl ester) was used to examine the effect of compound A on mitochondrial function.
  • TMRE tetramethylrhodamine ethyl ester
  • mitochondria with membrane potential TMRE accumulates to saturation levels and emits reddish-orange fluorescence.
  • mitochondria lose their membrane potential due to apoptosis or metabolic stress the fluorescent dye diffuses throughout the cell, resulting in a marked decrease in fluorescence intensity.
  • the effect of compound A on the mitochondrial membrane potential was analyzed. As cells, both spleen cells and TILs were used.
  • spleen cells were isolated from DUC18 mice, washed with RPMI1640 medium and debleed. After grinding the spleen using a glass slide, released cells were collected in RPMI1640 medium. After centrifugation (300 ⁇ g, 5 min, 4° C.), the supernatant was removed and 2 mL of ACK solution was added to treat the cells for 1 min. 18 mL of RPMI1640 medium was added and centrifuged (300 xg, 5 minutes, 4°C). The supernatant was removed and the cells were suspended in an appropriate amount of RPMI1640 medium.
  • the cells were adjusted to 1 ⁇ 10 6 cells/mL with RPMI1640 medium containing 10% FCS, and the cells were seeded in a 96-well plate at 100 ⁇ L/well.
  • Compound A was adjusted to 4 times the final concentration (Low 0.5 ⁇ M, High 1.5 ⁇ M) in RPMI medium containing 10% FCS, added at 50 ⁇ L/well, and then 9m peptide was added to RPMI containing 10% FCS. It was adjusted to 4 ⁇ g/ml with medium and added at 50 ⁇ L/well.
  • TMRE was added to each well at a final concentration of 0.1 ⁇ M, and the cells were further cultured for 30 minutes.
  • the specific method when using TIL is as follows.
  • the CMS5a cell line cultured in a T75 flask (Corning) was detached using 0.5% trypsin-containing PBS, and collected in 10% FBS-containing RPMI1640 medium. After centrifugation (300 ⁇ g, 5 minutes, 4°C), the supernatant was removed, washed twice with RPMI1640 medium, suspended in RPMI1640 medium at a concentration of 1 ⁇ 10 6 cells/100 ⁇ L, and 100 ⁇ L/individual was subcutaneously implanted into BALB/c mice at a dose of .
  • Compound A was administered intratumorally 7 days after transplantation.
  • Tumors were isolated from mice one day after administration, disrupted using Gentle MACS (Miltenyi), and suspended in RPMI1640 medium. Collagenase D (final concentration 1 mg/mL, Roche) was added, reacted at 37°C for 30 minutes, and then disrupted again using Gentle MACS. After passing through a filtration filter (22 ⁇ m, BD Bioscience), the supernatant was removed after centrifugation (1200 rpm, 5 minutes, 4°C), and 2 mL of erythrocyte lysing solution was added to treat the cells for 1 minute. . 18 mL of RPMI1640 medium was added and centrifuged (1200 rpm, 5 minutes, 4°C).
  • the cells were suspended in RPMI1640 medium and used as tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the cells were transferred to a 96-well V-bottom microplate (Nunc), TMRE was added to each well at a final concentration of 0.1 ⁇ M, and cultured for 30 minutes. Thereafter, the cells were centrifuged (2000 rpm, 2 minutes, 4°C), the supernatant was removed, and the cells were suspended in 50 ⁇ L of staining buffer (2% FCS-containing PBS) per well.
  • An APC-Cy7-labeled anti-CD8 antibody was added, mixed, and allowed to stand in the dark at 4°C for 15 minutes.
  • Test example 8 Analysis of mechanism of action 2 It was investigated whether the activation of T cells by compound A could be canceled by various mitochondrial function inhibitors (Rotenone and Antimycin).
  • a specific method is as follows. Spleens were isolated from DUC18 mice, washed with RPMI1640 medium and debleed. After grinding the spleen using a glass slide, released cells were collected in RPMI1640 medium. After centrifugation (300 ⁇ g, 5 min, 4° C.), the supernatant was removed and 2 mL of ACK solution was added to treat the cells for 1 min. 18 mL of RPMI1640 medium was added and centrifuged (300 xg, 5 minutes, 4°C). The supernatant was removed and the cells were suspended in an appropriate amount of RPMI1640 medium.
  • the number of cells was adjusted to 1 ⁇ 10 6 cells/mL with RPMI1640 medium containing 10% FCS, and the cells were seeded in a 96-well plate at 100 ⁇ l/well.
  • Compound A was adjusted to 4 times the final concentration (Low 0.5 ⁇ M, High 1.5 ⁇ M) in RPMI medium containing 10% FCS, added at 50 ⁇ L/well, and then 9m peptide was added to RPMI containing 10% FCS. It was adjusted to 4 ⁇ g/mL with medium and added at 50 ⁇ L/well.
  • Rotenone and Antimycin were added to final concentrations of 1 ⁇ M (high) and 0.5 ⁇ M (Low) and cultured for 72 hours.
  • Test example 9 Analysis of mechanism of action 3 From the results of the test examples described above, it was considered that compound A may activate low-activity tumor antigen-specific CD8 T cells and expand the diversity of the TIL (tumor-infiltrating T cell) TCR repertoire. Therefore, the effect of compound A on the diversity of the TCR repertoire of T cells was investigated.
  • Mouse tumor cell line CT26 was inoculated subcutaneously into BALB/c mice.
  • PD-1+ CD8 TILs were isolated and their TCR repertoires were analyzed by TCR beta sequencing. Experiments were repeated at least 2-3 times.

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Abstract

L'invention concerne un promoteur de la fonction lymphocytaire. Le promoteur de fonction lymphocytaire contient au moins un élément choisi dans le groupe constitué par un composé représenté par la formule générale (1), un sel de ce dernier, et un solvate correspondant.
PCT/JP2022/046124 2021-12-16 2022-12-15 Promoteur de la fonction lymphocytaire, agent destiné à une utilisation combinée avec un agent thérapeutique pour une thérapie anticancéreuse médiée par les lymphocytes, et promoteur de la fonction mitochondriale des lymphocytes WO2023112975A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0381276A (ja) * 1989-08-24 1991-04-05 Fumio Yoneda ５−デアザフラビン系化合物を有効成分とする制癌剤
JP2000212087A (ja) * 1999-01-20 2000-08-02 Fujimoto Brothers:Kk ニトロ―５―デアザフラビン誘導体を有効成分とする放射線増感剤。

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0381276A (ja) * 1989-08-24 1991-04-05 Fumio Yoneda ５−デアザフラビン系化合物を有効成分とする制癌剤
JP2000212087A (ja) * 1999-01-20 2000-08-02 Fujimoto Brothers:Kk ニトロ―５―デアザフラビン誘導体を有効成分とする放射線増感剤。

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DOTSU, Yosuke et al. Chemical augmentation of mitochondrial electron transport chains tunes T cell activation threshold in tumors. J. Immunother. Cancer. February 2022, vol. 10(2):e003958, pages 1-14 *
KAWAMOTO, T. IKEUCHI, Y. HIRAKI, J. EIKYU, Y. SHIMIZU, K. TOMISHIMA, M. BESSHO, K. YONEDA, F. MIKATA, Y. NISHIDA, M. IKEHARA, K. S: "Synthesis and Evaluation of Nitro 5-Deazaflavins as Novel Bioreductive Antitumor Agents", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM NL, vol. 5, no. 18, 21 September 1995 (1995-09-21), Amsterdam NL , pages 2109 - 2114, XP004135314, ISSN: 0960-894X, DOI: 10.1016/0960-894X(95)00353-U *
KAWAMOTO, T. IKEUCHI, Y. HIRAKI, J. EIKYU, Y. SHIMIZU, K. TOMISHIMA, M. BESSHO, K. YONEDA, F. MIKATA, Y. NISHIDA, M. IKEHARA, K.: "Evaluation of Differential Hypoxic Cytotoxicity and Electrochemical Studies of Nitro 5-Deazaflavins", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM NL, vol. 5, no. 18, 21 September 1995 (1995-09-21), Amsterdam NL , pages 2115 - 2118, XP004135315, ISSN: 0960-894X, DOI: 10.1016/0960-894X(95)00352-T *

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