WO2023068366A1 - Composition de solution aqueuse contenant de l'ozone - Google Patents

Composition de solution aqueuse contenant de l'ozone Download PDF

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WO2023068366A1
WO2023068366A1 PCT/JP2022/039317 JP2022039317W WO2023068366A1 WO 2023068366 A1 WO2023068366 A1 WO 2023068366A1 JP 2022039317 W JP2022039317 W JP 2022039317W WO 2023068366 A1 WO2023068366 A1 WO 2023068366A1
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ozone
cancer
containing gas
aqueous solution
composition
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PCT/JP2022/039317
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Japanese (ja)
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良弘 鈴木
真奈美 鈴木
静加 印南
洋 岡嶋
基 松永
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東京計器株式会社
良弘 鈴木
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Publication of WO2023068366A1 publication Critical patent/WO2023068366A1/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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • 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
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • 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
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • 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
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    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B13/00—Oxygen; Ozone; Oxides or hydroxides in general
    • C01B13/10—Preparation of ozone
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B13/00—Oxygen; Ozone; Oxides or hydroxides in general
    • C01B13/10—Preparation of ozone
    • C01B13/11—Preparation of ozone by electric discharge

Definitions

  • the present invention belongs to the technical field of anticancer agents. TECHNICAL FIELD The present invention relates to an anticancer agent capable of selectively killing substantially only cancer cells. The present invention also relates to ozone-containing aqueous compositions that can constitute such anticancer agents.
  • Eukaryotic cells including cancer cells, generally have intracellular organelles called mitochondria.
  • Mitochondria are organelles responsible for a variety of cellular functions, from energy production to macromolecular biosynthesis, redox (Redox) and calcium ion homeostasis. Mitochondria play a pivotal role in the regulation of cell proliferation, differentiation and death and are essential in cell death signaling pathways. Mitochondria are highly flexible and motile organelles that change their shape, size, and localization according to cellular conditions (energy requirements, intracellular calcium, reactive oxygen species (ROS) concentration, etc.). .
  • ROS reactive oxygen species
  • mitochondria mitochondria
  • Drp dynamin-related proteins
  • PNMC Perinuclear mitochondrial clustering
  • Non-Patent Documents 1 and 2 modulation of mitochondrial dynamics and intracellular distribution (hereinafter collectively referred to as dynamics) is an important target in the development of anticancer drugs as a powerful means of inducing cancer cell death.
  • the main object of the present invention is to provide a novel composition or drug that targets mitochondrial dynamics and exerts excellent anticancer activity while having minimal effect on normal cells.
  • composition containing ozone and an activator can selectively modulate mitochondrial dynamics in cancer cells, thereby The inventors have found that it is possible to selectively kill only cancer cells substantially without affecting normal cells, and have completed the present invention.
  • Examples of the present invention include the following.
  • a composition comprising an aqueous solution containing ozone and an activator for killing cancer cells.
  • a composition consisting of an aqueous solution containing ozone and an activator, comprising fragmentation of mitochondria uniformly aggregated near the cell nuclei of hypoxic cancer cells, and the cell nuclei of the fragmented mitochondria A composition for inducing accumulation in the upper pole and injury to the cell nucleus or cell death of the cancer cells after the accumulation.
  • the activator is one or more selected from the group consisting of ferrous salts, flavins, nitric oxide donors, and salinomycin and pharmaceutically acceptable salts thereof. ].
  • the divalent iron salt is ferrous sulfate, ferrous chloride, ferrous bromide, or ammonium iron (II) sulfate
  • the flavin is riboflavin, flavin mononucleotide (FMN), or flavin adenine dinucleotide (FAD), wherein the nitric oxide donor is nitrite ion, nitrate ion, organic nitrates, organic nitrites, metal nitrosyls, sydnonimines, S-nitrosothiol, or hydroxyimine
  • the composition according to any one of [1] to [3] above.
  • composition according to any one of [1] to [4] above, wherein the aqueous solution is an aqueous solution containing a mammalian cell culture medium or an infusion preparation.
  • the gas contained in the above [1] is a composition obtained by bubbling in an aqueous solution containing an activator, or a composition obtained by irradiating an aqueous solution containing an activator with low-temperature atmospheric air plasma. ] to [5].
  • An anticancer agent comprising the composition according to any one of [1] to [6] above.
  • the anticancer agent of [7] above which is applied to epithelial cell-derived cancer, non-epithelial cell-derived cancer, leukemia, or lymphoma.
  • the cancer derived from epithelial cells is lung cancer, breast cancer, pancreatic cancer, colon cancer, gastric cancer, prostate cancer, ovarian cancer, oral cancer, or cancer derived from other organs, and non-epithelial cells
  • the anticancer agent according to [8] above, wherein the originating cancer is osteosarcoma, angiosarcoma, fibrosarcoma, melanoma, neuroblastoma, glioma, or other cancers.
  • the oxygen-containing gas introduced from the outside of the apparatus is irradiated with ultraviolet rays in the ozone generating unit, and the oxygen-containing gas after the ultraviolet irradiation, which has become an ozone-containing gas, is sent outside the apparatus by the air supply function of the air pump unit.
  • An ozone generator characterized by discharging to. [11] The ozone generator according to [10] above, wherein the ozone generator comprises a UV-C lamp.
  • a method for producing the composition comprising the steps of producing a gas and bubbling the produced ozone-containing gas into an aqueous solution containing an activator.
  • the ultraviolet rays are UV-C.
  • the UV-C has a spectral component with a wavelength of 185 nm.
  • the production method according to [15] above, wherein the oxygen-containing gas is a nitrogen-free gas.
  • the mitochondria are fragmented and the fragments are accumulated (aggregated) at one pole of the cell nucleus (unipolar mitochondrial nuclear marginal clustering, MPMC: Monopolar Perinuclear Mitochondrial Clustering). Cancer cells can then be selectively killed while leaving normal cells substantially unaffected.
  • MPMC Monopolar Perinuclear Mitochondrial Clustering
  • FIG. 1 is a conceptual diagram showing pan-cytoplasmic distribution of mitochondria, nuclear marginal clustering (PNMC), unipolar mitochondrial nuclear marginal clustering (MPMC), and the effect of the composition according to the present invention on cancer cells and normal cells.
  • PNMC nuclear marginal clustering
  • MPMC unipolar mitochondrial nuclear marginal clustering
  • FIG. Mitochondrial morphology, intracellular distribution, and nuclear morphology at each stage are shown.
  • BRIEF DESCRIPTION OF THE DRAWINGS It is a conceptual diagram which shows one aspect
  • FIG. 4 is a conceptual diagram showing another aspect of the configuration of the ozone generator according to the present invention.
  • FIG. 10 is a diagram showing the cell proliferation rate after administering APAM (7-50%) to human donors NOR-3, 143B, LM8, human oral squamous cell carcinoma cells SAS, and HOC-313 and culturing for 72 hours.
  • FIG. 2 shows membrane integrity and apoptosis after administration of APAM (25, 50%) or Gemicitabine (Gem 1 ⁇ M)) to human osteosarcoma cells 143B and mouse osteosarcoma cells LM8 and culturing for 24 hours.
  • Western blotting images of caspase-3 activation after administration of APAM (50%) to human osteosarcoma cells 143B and mouse osteosarcoma cells LM8 and culture for 0-24 hours are shown.
  • Fig. 10 shows the measurement results of tumor size (left figure) and mouse body weight (right figure) for each week when APAM (50%) was intravenously administered.
  • Human oral squamous cell carcinoma cells HOC-313 were administered APAM (25, 50%) and cultured for 2 hours. It is an image.
  • APAM superoxide
  • HOS hydrogen peroxide
  • OFOxiOrange hydroxyl radical
  • Human osteosarcoma cells HOS were administered with OBM (ozone-containing gas produced by UV irradiation bubbled into phenol red-free DMEM) and CPTIO (nitrogen monoxide specific scavenger Carboxy-PTIO) alone or in combination. , Cell proliferation rate after 18 hours of culture.
  • OBM ozone-containing gas produced by UV irradiation bubbled into phenol red-free DMEM
  • CPTIO nitrogen monoxide specific scavenger Carboxy-PTIO
  • Fig. 2 shows fluorescence microscope images of human osteosarcoma cells HOS administered with OBM (25, 50%) and OBW (25, 50%) and cultured for 18 hours to observe the morphology of cells, nuclei, and mitochondria.
  • FIG. 10 is a diagram showing the cell proliferation rate after administering OBM, OBW, or APAM to human osteosarcoma cells 143B and culturing for 72 hours.
  • FIG. 10 is a diagram showing the cell growth rate after administering OBW, Fe 2+ aqueous solution, or Fe 3+ aqueous solution alone or in combination to human osteosarcoma cells 143B and culturing them for 72 hours.
  • FIG. 10 is a diagram showing the cell growth rate after administering OBW, riboflavin (vitamin B 2 , VB2) aqueous solution, or NOR-3 aqueous solution alone or in combination to human osteosarcoma cells 143B and culturing for 72 hours.
  • FIG. 2 is a diagram showing cell growth rates after administering APAM (air plasma irradiation solution) to human osteosarcoma cells HOS and human lung fibroblasts (WI-38) and culturing for 72 hours. It is a phase contrast image (Phase Contrast, PC) by a fluorescence microscope of the cell morphology after administration of APAM (25, 50%) to human osteosarcoma cells HOS and culturing for 18 hours.
  • APAM air plasma irradiation solution
  • FIG. 10 is a diagram showing the cell proliferation rate after administering pOBM (a gas containing air-free ozone produced by silent discharge and bubbling air-free ozone-containing gas into phenol red-free DMEM) to human oral squamous cell carcinoma cells SAS and culturing them for 72 hours. .
  • pOBM a gas containing air-free ozone produced by silent discharge and bubbling air-free ozone-containing gas into phenol red-free DMEM
  • FIG. 2 is a diagram showing the cell growth rate after administering pOBM to human oral squamous cell carcinoma cells HOC-313 and culturing for 72 hours.
  • Cell proliferation rate after administration of pOBM, 2,2'-bipyridyl (BP), deferoxamine (DFO), Carboxy-PTIO (CPTIO), and catalase alone or in combination to HOC-313 and cultured for 72 hours It is a figure which shows.
  • FIG. 10 is a diagram showing the cell proliferation rate after administering pOBM and NOR-3 aqueous solution alone or in combination to SAS and culturing for 72 hours.
  • the numbers in the figure are the ozone concentration (ppm) calculated from the dissolved ozone concentration of the undiluted solution.
  • FIG. 10 is a diagram showing the cell proliferation rate after administering pOBM and riboflavin (vitamin B 2 , VB2) aqueous solution alone or in combination to SAS and culturing for 72 hours.
  • the numbers in the figure are the ozone concentration (ppm) calculated from the dissolved ozone concentration of the undiluted solution.
  • FIG. 10 is a diagram showing the cell proliferation rate after administering pOBM and salinomycin sodium aqueous solution alone or in combination to HOC-313 and culturing for 72 hours.
  • composition according to the present invention is a composition comprising an aqueous solution containing ozone and an activator, and kills cancer cells. It is characterized by being used for
  • the composition of the present invention comprises fragmentation of mitochondria uniformly aggregated near the cell nucleus of hypoxic cancer cells, accumulation of the fragmented mitochondria in one pole on the cell nucleus, It can be used to induce damage to cell nuclei or cell death of cancer cells themselves after accumulation. Therefore, the composition of the present invention allows hypoxic cancer cells to escape from the state of nuclear marginal clustering (PNMC), the state of monopolar mitochondrial nuclear marginal clustering (MPMC), as well as cell nuclear injury or cell can lead to death.
  • PNMC nuclear marginal clustering
  • MPMC monopolar mitochondrial nuclear marginal clustering
  • the composition of the present invention can function as a PNMC deregulator or MPMC inducer for hypoxic cancer cells.
  • Normal cells on the other hand, cannot survive hypoxia in the first place and are not in the PNMC state under physiological conditions. Therefore, the composition of the present invention is selective, as it is believed to act only on cancer cells, with essentially no effect on normal cells.
  • PNMC hypoxia inducible transcription factors
  • the composition of the present invention targets such intracellular expansion and localization (positioning) of mitochondria.
  • the composition of the present invention fragmented the mitochondria of cancer cells to eliminate PNMC (Fig. 1B), and then aggregated the mitochondria to one pole on the cell nucleus to induce unipolar mitochondrial nuclear marginal clustering (MPMC). induce (Fig. 1C).
  • MPMC unipolar mitochondrial nuclear marginal clustering
  • Fig. 1C Loss of PNMC and MPMC induction lead to dehypoxia adaptation and nuclear injury and cell death.
  • PNMC is not essential for normal cells that cannot survive in hypoxic conditions, it is an intracellular phenomenon necessary for cancer cells to adapt to hypoxic conditions, and deregulation of PNMC is crucial for survival only in cancer cells. be an obstacle.
  • MPMC is initiated by mitochondrial morphological changes triggered by superoxide production in mitochondria, and this superoxide production is selectively observed in cancer cells. That is, the composition of the present invention can selectively damage or kill substantially only cancer cells.
  • the composition of the present invention contains ozone and an activator.
  • the activator is not particularly limited as long as it is a compound that can promote the cancer cell-killing effect of ozone, or a compound that can promote the disappearance of PNMC or the induction of MPMC by ozone, and is a compound having a function as a reducing agent. There may be.
  • the composition of the present invention contains, as an activator, one or more selected from the group consisting of ferric salts, flavins, nitric oxide donors, and salinomycin and pharmaceutically acceptable salts thereof. It is suitable to contain an appropriate amount.
  • the composition of the present invention may comprise an ozone-containing mammalian cell culture medium or infusion solution.
  • the concentration of dissolved ozone in the composition of the present invention is not particularly limited as long as the effect of the present invention is exhibited, but it is suitable, for example, within the range of 0.2 ppm to 5 ppm. Above all, it is preferably within the range of 0.3 ppm to 3 ppm, more preferably within the range of 1 ppm to 2 ppm. If the dissolved ozone concentration is less than 0.2 ppm, the effects of the present invention may not be exhibited, and if it exceeds 5 ppm, normal cells may be affected.
  • the dissolved ozone concentration can be measured by, for example, the 4-aminoantipyrine method, iodine-starch reaction, and ultraviolet method.
  • divalent iron salts examples include ferrous sulfate (FeSO 4 ), ferrous chloride (FeCl 2 ), ferrous bromide (FeBr 2 ), ammonium iron (II) sulfate ((NH 4 ) 2 Fe( SO 4 ) 2 ). Among them, ammonium iron(II) sulfate is preferred. These may be used singly or in any combination of two or more.
  • Flavins include, for example, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Among them, riboflavin is preferred. These may be used singly or in any combination of two or more.
  • Nitric oxide donors include, for example, nitrite ions, nitrate ions, organic nitrates, organic nitrites, metal nitrosyls, sydnonimines, S-nitrosothiols, and hydroxyimines. be done. These may be used singly or in any combination of two or more.
  • NOR-1 ( ⁇ )-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide
  • NOR -3 ( ⁇ )-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide
  • NOR-4 ( ⁇ )-N-[(E)- 4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl]-3-pyridinecarboxamide
  • NOR-5 ( ⁇ )-N-[(E)-4- Ethyl-3-[(Z)-hydroxyimino]-6-methyl-5-nitro-3-heptenyl]-3-pyridinecarboxamide
  • NOC-5 1-hydroxy-2-oxo-3-(3-aminopropyl )-3-isopropyl-1-triazene
  • NOC-7 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl )-3-
  • Salinomycin has the chemical name (3R,5S,6S,7S)-3-[(2S,5S,7R,9S,10S,12R,15R)-2-[(2R,5R,6S)-5-Ethyl-5 -hydroxy-6-methyltetrahydro-2H-pyran-2-yl]-15-hydroxy-2,10,12-trimethyl1,6,8-trioxadispiro[4.1.57.35]pentadec-13-en-9-yl]-6 -hydroxy-7-[(2R,3S,6R)-6-[(R)-1-(hydroxy-l2-methoxy)propyl]-3-methyltetrahydro-2Hpyran-2-yl]-5-methyloctan-4- It is a polyether ionophore antibiotic represented by one (CAS: No. 53003-10-4).
  • composition of the present invention can contain salinomycin or a pharmaceutically acceptable salt thereof as an activator.
  • Salinomycin or a pharmaceutically acceptable salt thereof can be in free form or a pharmaceutically acceptable salt thereof, a solvate such as a hydrate thereof, or an analogue thereof.
  • the pharmaceutically acceptable salt of salinomycin is not particularly limited as long as it is pharmaceutically acceptable and can form a salt of salinomycin, and examples thereof include base addition salts of salinomycin.
  • Examples of base addition salts include salts with inorganic bases and salts with organic bases.
  • Examples of salts with inorganic bases include salts with sodium, potassium, magnesium, calcium, aluminum and the like.
  • Examples of salts with organic bases include salts with methylamine, ethylamine, ethanolamine, lysine, ornithine and the like. Among these, the sodium salt of salinomycin is preferred.
  • composition of the present invention may contain one or more of the components used in mammalian cell culture media.
  • components include calcium chloride; potassium chloride; magnesium sulfate; is 10 to 600 mg/mL respectively.
  • vitamins such as calcium pantothenate, sodium pantothenate, choline chloride, inositol, niacin, pyridoxal, riboflavin, and thiamine (preferred contents of vitamins are 1 to 20 mg/mL.) and the like.
  • a mammalian cell culture medium can be used as the aqueous solution constituting the composition of the present invention.
  • examples of such media include MEM (Eagle's Minimum Essential Medium), GMEM (Glasgow's Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), and IMDM (Iscove's Modified Dulbecco's Medium).
  • MEM Eagle's Minimum Essential Medium
  • GMEM Gasgow's Minimum Essential Medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • IMDM Iscove's Modified Dulbecco's Medium
  • DMEM is preferable, and DMEM containing no phenol red is more preferable. Phenol red may, and preferably is not included in the compositions of the present invention.
  • an electrolyte solution can be used, and among them, an infusion preparation for mammals is preferable, and an infusion preparation for humans is more preferable. Infusion preparations are frequently used for replenishing water and electrolytes in humans and the like, and are preferable from the viewpoint of safety.
  • the infusion preparation is not particularly limited as long as it is used in medical practice. Examples include a hypotonic electrolyte solution such as recovery solution (No. 4 solution) and a solution to which glucose is added.
  • peripheral parenteral nutrition infusions containing glucose, electrolytes, amino acids and water-soluble vitamins include peripheral parenteral nutrition infusions containing glucose, electrolytes, amino acids and water-soluble vitamins, and high-calorie infusions containing glucose and electrolytes, as well as amino acids, vitamins, trace elements and the like. Any of these commercially available products can be used. Those containing the above-mentioned activator or reducing agent can be used as they are, and infusion solutions containing neither activating agents nor reducing agents can be used by adding activating agents or reducing agents.
  • Ozone bubbling liquid In one aspect of the composition of the present invention, an ozone-containing gas obtained by irradiating an oxygen-containing gas (such as air) with ultraviolet rays (such as UV-C) is bubbled in an aqueous solution containing the activator.
  • an oxygen-containing gas such as air
  • ultraviolet rays such as UV-C
  • an ozone-containing gas an ozone-containing gas obtained by silent discharge
  • an oxygen-containing gas for example, a mixed gas of argon and oxygen
  • pOBM aqueous solution containing the activator
  • Bubbling can be performed, for example, by inserting at least the tip of a nozzle into water or an aqueous solution and discharging ozone-containing gas from the tip of the nozzle.
  • the composition of the present invention can be a composition (APAM) obtained by irradiating an aqueous solution containing the activator with a cold atmospheric air plasma.
  • Low-temperature atmospheric air plasma refers to plasma generated at a low temperature of about room temperature under atmospheric pressure
  • irradiation liquid refers to an aqueous solution obtained by irradiating a liquid such as a cell culture medium, salt solution, or infusion solution with this plasma.
  • the cold atmospheric pressure air plasma irradiation liquid contains dissolved ozone.
  • anticancer agent according to the present invention contains the composition of the present invention.
  • concentration of ozone in the anticancer agent of the present invention is not particularly limited as long as it does not impair the effect of the present invention, but the appropriate and preferred amounts are the same as in the case of the composition of the present invention.
  • the anticancer agent of the present invention can be widely applied to all cases that can be called cancer (malignant tumor), regardless of whether they are solid cancers or blood cancers.
  • Examples of the application target of the anticancer agent of the present invention include epithelial cell-derived cancer (carcinoma), non-epithelial cell-derived cancer (sarcoma), leukemia, and lymphoma.
  • cancers derived from epithelial cells include, for example, lung cancer, breast cancer, pancreatic cancer, colon cancer, stomach cancer, prostate cancer, uterine cancer, ovarian cancer, oral cancer, and non-epithelial cancer.
  • Cell-derived cancers include, for example, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, melanoma, neuroblastoma, and glioblastoma.
  • Some cancers activate various defense pathways that suppress cell death by apoptosis and exhibit resistance to the cell-killing effects of anticancer drugs and radiation. Since the anticancer agent of the present invention can induce not only apoptosis but also multiple forms of non-apoptotic cell death, it can also be used to treat intractable cancers that are resistant to existing multimodal therapies. Examples of such intractable cancers include osteosarcoma, melanoma, pancreatic cancer, and glioblastoma.
  • the anticancer agent of the present invention can be used in combination with other anticancer agents.
  • Such other anticancer agents include, for example, nitrogen mustards such as cyclophosphamide, ifosfamide, melphalan, busulfan, and thiotepa; Alkylating drugs such as ureas; platinum compounds such as cisplatin, carboplatin, oxaliplatin, nedaplatin; antimetabolites such as 5-fluorouracil, cytarabine, gemcitabine, capecitabine, mercaptopurine, methotrexate, pemetrexed sodium; topoisomerase inhibitors such as zobzoxacin; microtubule inhibitors such as vinblastine, vincristine, vindesine, vinorelbine, paclitaxel, docetaxel; antibiotics such as mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin,
  • anticancer agent of the present invention can exhibit synergistic antitumor effects by enhancing apoptosis and inducing non-apoptotic cell death.
  • anticancer drugs with low tumor selectivity are expected to be used at a lower concentration and less side effects, and can be an excellent complementary therapy.
  • the dosage form of the anticancer agent of the present invention is not particularly limited, and for example, it can be formulated as an infusion, an injection, a spray, or an oral preparation. Among them, the forms of drops, injections, and sprays are preferable.
  • the ozone generator according to the present invention (hereinafter referred to as the "apparatus of the present invention") is, as one embodiment, for producing the composition of the present invention, as illustrated in FIG.
  • the ozone generator used has an air pump section and an ozone generation section, and the air introduced from the outside of the device by the intake function of the air pump section is irradiated with ultraviolet rays in the ozone generation section to become an ozone-containing gas. It is characterized in that the air that has been irradiated with the ultraviolet rays is discharged to the outside of the device by the air supply function of the air pump section.
  • the apparatus of the present invention may include an air tank for fluid pressure control (reduction of pressure fluctuations, etc.). Moreover, a pressure gauge, a flow meter, an air valve, a purge valve, etc. may be provided as necessary.
  • the air pump section is not particularly limited as long as it functions as a gas pump that has an intake function and an air supply function.
  • the ozone generator can be equipped with, for example, a UV-C lamp.
  • a UV-C lamp is required to have a spectrum with an emission wavelength of 185 nm, which reacts with oxygen molecules and contributes to the generation of ozone and active oxygen. Among them, those having spectral peaks at an emission wavelength of 185 nm, or at 185 nm and 254 nm are preferable from the viewpoint of sterilization of generated gas.
  • the UV-C lamp is preferable from the viewpoint of high stability and long life of the lamp, as long as it generates ultraviolet rays through electrodeless discharge using microwaves.
  • the pressure of the ozone-containing gas that can be discharged from the device of the present invention is not particularly limited, but it is suitable, for example, within the range of 25 kPa to 50 kPa. Above all, it is preferably in the range of 30 kPa to 45 kPa, more preferably in the range of 35 kPa to 40 kPa. From the viewpoint of controlling the pressure of the ozone-containing gas with high accuracy, the apparatus of the present invention preferably has a digital pressure gauge.
  • the device of the present invention can be equipped with an air bubbling section. If the ozone-containing gas supplied by the air pump section is discharged from the air bubbling section, the air bubbling section can be used for bubbling for producing the composition of the present invention. For example, an appropriate nozzle can be adopted for the air bubbling portion.
  • the flow rate of the ozone-containing gas released from the air bubbling part during bubbling is not particularly limited, but it is suitable, for example, within the range of 0.1 L/min to 10 L/min. Above all, it is preferably within the range of 1 L/min to 8 L/min, more preferably within the range of 2 L/min to 6 L/min.
  • the exhaust section according to the device of the present invention can be equipped with an ozone scrubber capable of removing ozone from the airflow before exhaust.
  • an ozone scrubber capable of removing ozone from the airflow before exhaust.
  • the apparatus of the present invention can exhaust air with high safety.
  • Manganese dioxide for example, is widely used as a catalyst for ozone scrubbers, and known scrubber materials such as manganese dioxide can also be used in the apparatus of the present invention.
  • the apparatus of the present invention includes an ozone generating section, an air pump section, an air tank, a pressure gauge, a flow meter, and an air bubbling section in the gas introduction passage, and an ozone scrubber in the exhaust passage.
  • the ozone-containing gas is generated by irradiating the air introduced from the outside of the apparatus with ultraviolet rays in the ozone generating section, and the gas can be guided to the air bubbling section using an air pump and an air tank and discharged.
  • the nozzle can be inserted into the aqueous solution to effect bubbling.
  • the pressure control of the ozone-containing gas during bubbling can be performed using an air tank while monitoring the pressure and flow rate with a pressure gauge or a flow meter installed in the flow path. Also, if an air valve is appropriately installed in the flow path, the pressure and flow rate of the gas can be adjusted using this valve. If a purge valve is installed in the flow path of the purge gas from the air tank, it is possible to adjust the purge pressure and the like.
  • the gas can be guided to the ozone scrubber along the exhaust flow path and then exhausted to the outside of the device. Further, the purge gas from the air tank can also be structured to be led to the ozone scrubber. Leakage of ozone from the apparatus of the present invention can be minimized by forming each flow path and air bubbling portion into a sealed structure and guiding all of the released gas to the ozone scrubber.
  • the apparatus of the present invention is an ozone generator used for producing the composition of the present invention, as illustrated in FIG.
  • An alternating voltage obtained by a power source is applied to the oxygen-containing gas in the discharge unit to cause discharge in the oxygen-containing gas, thereby generating an ozone-containing gas from the oxygen-containing gas.
  • the discharge is a dielectric barrier discharge (DBD).
  • DBD dielectric barrier discharge
  • the device of the present invention in this aspect may be provided with an air tank for fluid pressure control (pressure fluctuation reduction, etc.).
  • a pressure gauge, a flow meter, an air valve, a purge valve, etc. may be provided as necessary.
  • the method for producing the composition according to the present invention is a production method for producing the composition of the present invention, comprising an oxygen-containing A process of producing ozone-containing gas by irradiating the gas with ultraviolet rays or silent discharge to produce ozone-containing gas (ozone-generating process), and a process of bubbling the ozone-containing gas produced in the ozone-generating process into water or an aqueous solution (bubbling process). ) and The production method of the present invention also includes a method of producing the composition of the present invention by bubbling the ozone-containing gas produced by the ozone generator of the present invention into water or an aqueous solution.
  • ozone is generated by ultraviolet irradiation to produce an ozone-containing gas.
  • an ozone-containing gas can be produced by irradiating an oxygen-containing gas (such as air) with ultraviolet rays.
  • an oxygen-containing gas such as air
  • the ultraviolet rays have a spectrum with an emission wavelength of 185 nm, which reacts with oxygen molecules and contributes to the generation of ozone and active oxygen. is preferred.
  • those having spectral peaks at a wavelength of 185 nm, or at 185 nm and 254 nm are preferable from the viewpoint of sterilization of the generated gas.
  • UV-C lamp As an ultraviolet irradiation method, for example, irradiation with a UV-C lamp can be mentioned.
  • the UV-C lamp is preferable from the viewpoint of high stability and long life of the lamp as long as it generates ultraviolet rays by, for example, electrodeless discharge using microwaves.
  • ozone is generated by silent discharge (dielectric barrier discharge) to a gas containing oxygen to produce an ozone-containing gas.
  • an ozone-containing gas can be produced by introducing an oxygen-containing gas (such as a mixed gas of argon and oxygen) into a dielectric barrier discharge (DBD) plasma probe and applying an alternating voltage to the gas.
  • an oxygen-containing gas such as a mixed gas of argon and oxygen
  • DBD dielectric barrier discharge
  • the composition of the present invention is produced by bubbling the ozone-containing gas produced in the ozone generating step into water or an aqueous solution. Bubbling can be performed, for example, by inserting at least the tip of a nozzle into water or an aqueous solution and discharging ozone-containing gas from the tip of the nozzle.
  • the pressure of the ozone-containing gas during bubbling is preferably in the range of 30 kPa to 45 kPa, more preferably in the range of 35 kPa to 40 kPa.
  • APAM air plasma irradiation solution
  • FR (-) DMEM phenol red-free Dulbecco's modified Eagle medium
  • APAM was created.
  • APAM was diluted with FR(-)DMEM.
  • An experimental ultraviolet generator (the device of the present invention: generating ultraviolet rays (wavelength 185 nm) by electrodeless discharge using microwaves, manufactured by Tokyo Keiki Co., Ltd.) It was operated at a wave power of 35 W and air was fed into it by a compressor to produce an ozone-containing gas.
  • An outline of the apparatus and system is shown in FIG. 2 below.
  • KDM30 manufactured by Krone was used as a pressure gauge in each system.
  • TBST Tris-buffered saline
  • the membrane was reacted with the primary antibody in TBST containing 2% non-fat dry milk at 4°C. reacted overnight. Subsequently, after washing twice with TBST, it was reacted with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. After washing the resulting membrane with TBST three times, the signal was detected with LAS-4000 (Fujifilm) using a chemiluminescent reagent (GE Healthcare).
  • mice Female Transplanted Tumor BALB/cAJcl-nu/nu nude mice (Clea Japan) were bred at 22-24° C. under a 12-hour light-dark cycle with a normal diet and water ad libitum. Mice (8-week-old male) were anesthetized with isoflurane and oxygen, and cancer cells (1 ⁇ 10 6 cells/mouse) suspended in 0.1 mL of DMEM were intramedullary injected into the right tibia and implanted. Seven days after cell transplantation, the test drug (200 ⁇ L) was intravenously administered three times a week. Tumor size and mouse body weight were measured every week. After 5 weeks, the mice were sacrificed and the tumors were excised and measured.
  • APAM was prepared according to the above test procedures [1] and [2], was used as a stock solution (100%), dissolved ozone concentration was measured and diluted with FR( ⁇ ) DMEM.
  • APAM (7-50%) was administered to cultured human osteosarcoma cells HOS, 143B, LM8, human oral squamous cell carcinoma cells SAS, and HOC-313, and after culturing for 72 hours, the cell growth rate was measured according to the above test procedure [5 ] (Fig. 4).
  • APAM significantly inhibited the proliferation of all cells in a concentration-dependent manner. ***P ⁇ 0.001; NS, not significant vs. Ctrl.
  • Example 2 Human osteosarcoma cells 143B and mouse osteosarcoma cells LM8 were treated with APAM (25, 50%) or Gemicitabine (Gem 1 ⁇ M)) and cultured for 24 hours, membrane integrity and apoptosis were measured according to the test procedure described above [7]. (Fig. 5). APAM dose-dependently increased apoptosis in LM8, but not in 143B.
  • Example 3 Human osteosarcoma cells 143B and mouse osteosarcoma cells LM8 were administered APAM (50%) and cultured for 0 to 24 hours (0, 1, 6, 12, 24 hours). It was measured by Western blotting according to the test procedure described above [9] (Fig. 6). APAM caused caspase-3 activation in LM8 but not in 143B. The results of [Examples 1-3] show that APAM can induce apoptotic as well as non-apoptotic cell death.
  • mice 8-week-old male were anesthetized with isoflurane and oxygen and suspended in 0.1 mL of DMEM. It was injected and implanted. Seven days after cell transplantation, APAM (50%) was intravenously administered three times a week, and tumor size and mouse body weight were measured every week according to the test procedure [10] above. After 5 weeks, the mice were sacrificed and the tumor was excised and measured (Fig. 7). APAM markedly suppressed tumor growth, but no adverse events such as weight loss in mice were observed. These results demonstrate that APAM exerts anti-tumor effects without side effects in animal models.
  • Example 5 Human oral squamous cell carcinoma cells HOC-313 and human epidermal fibroblasts (HDF) were administered APAM (25, 50%), cultured for 2 hours, superoxide (MitoSOX), hydrogen peroxide ( Hydrop), hydroxyl radicals (OxiOrange) were measured according to the above test procedure [8] ( Figures 8 and 9). APAM increased ROS in HOC-313 (Fig. 8) but not in HDF (Fig. 9). These results indicate that oxidative stress by APAM occurs selectively in tumors.
  • Example 6 Human melanoma cells A2058, osteosarcoma cells HOS and human dermal fibroblasts (HDF) were treated with APAM (25%) and cultured for 18 hours. It was observed with a fluorescence microscope (Fig. 10). In A2058 and HOS, APAM administration resulted in MPMC morphology in which mitochondria were fissioned and fused and localized to one pole of the nucleus, but these changes were not observed in HDF. These results indicate that APAM induces MPMC morphology in a tumor-selective manner.
  • OBM was prepared according to the test procedure [4] above, was used as a stock solution (100%), dissolved ozone concentration was measured and diluted with FR(-) DMEM. The stock solution is made into an ozone solution by blowing ozone (AC ozone) into the solvent for 1 minute per 1 mL.
  • OBM (6.25-50%) and nitric oxide (NO)-specific scavenging agent
  • Carboxy-PTIO (CPTIO 30 ⁇ M) were administered to cultured human osteosarcoma cells HOS alone or in combination. Proliferation rates were measured according to the test procedure [5] above ( Figure 11). Control cells (Ctrl) were treated with FR(-) DMEM without ozone blowing.
  • OBM 25, 50%
  • OBW 25, 50%
  • MT Mitochondria
  • OBM (12.5%, 25%, 50%), OBW (12.5%, 25%, 50%), APAM (25%, 50%) were administered to human osteosarcoma cells 143B and cultured for 72 hours.
  • Cell proliferation rate was measured according to the test procedure [5] described above (Fig. 13).
  • OBM exhibits much stronger anti-tumor effects than OBW. Since OBM has a much higher dissolved ozone concentration than OBW, there is a high possibility that there are differences in the solubility of ozone gas and the stabilization of dissolved ozone. For 143B, OBM has a higher anti-tumor effect than APAM.
  • OBW (12.5%, 25%, 50%), riboflavin (vitamin B 2 , VB2) aqueous solution (15 ⁇ M), NOR-3 aqueous solution (30 ⁇ M) alone or in combination was administered to human osteosarcoma cells 143B and cultured for 72 hours. Cell proliferation rate was later measured according to the test procedure [5] described above (Fig. 15).
  • the redox-active riboflavin (vitamin B 2 ) and the nitric oxide NO donor NOR-3 also showed synergistic anti-tumor effects when administered in combination with OBW. It can be said that the effect is almost equivalent to that of OBM.
  • APAM (6.25-50%) was administered to human osteosarcoma cells HOS and human lung fibroblasts (WI-38), and after culturing for 72 hours, the cell proliferation rate was measured according to the above test procedure [5] (Fig. 16). ). APAM ( ⁇ 25%) significantly decreased HOS viability in a concentration-dependent manner, but had no effect on HDF viability at all concentrations tested. These results demonstrate that APAM exerts tumor-selective cytotoxicity.
  • APAM (25%, 50%) was administered to human osteosarcoma cells HOS, and after culturing for 18 hours, the cell morphology was observed with a fluorescence microscope using a phase contrast image (Phase Contrast, PC) according to the above test procedure [6] (Fig. 17). ). It induced an increase in swollen cells and destruction of cytoplasm and cell membrane in an APAM concentration-dependent manner. Ozone (8 ppm) was detected in the APAM stock solution (100%).
  • APAM (50%) and tubulin synthesis inhibitor nocodazole (NC, 100 nM) were administered alone or in combination to human osteosarcoma cells HOS, and after 18 hours of culture, the morphology of nuclei and mitochondria was examined by fluorescence microscopy according to the above test procedure [6]. (Fig. 18). APAM administration induced mitochondrial fission, fusion, and MPMC morphology, and NC inhibited these effects. These results indicate that MPMC induction is due to migration of mitochondria through microtubules.
  • pOBM was prepared according to the test procedure [4] above, was used as a stock solution (100%), dissolved ozone concentration was measured and diluted with FR( ⁇ ) DMEM. The stock solution is made into an ozone solution by blowing ozone (AF ozone) into the solvent for 3 minutes per 10 mL. pOBM (12.5 to 50%) was administered to cultured human squamous cell carcinoma cells SAS, and the cell proliferation rate after 72 hours of culture was measured according to the above test procedure [5] (Fig. 19).
  • pOBM was prepared according to the test procedure [4] above, was used as a stock solution (100%), dissolved ozone concentration was measured and diluted with FR( ⁇ ) DMEM. The stock solution is made into an ozone solution by blowing ozone (AF ozone) into the solvent for 1 minute per 10 mL.
  • Human squamous cell carcinoma cells SAS were administered pOBM (12.5%, 25%, 50%) and NOR-3 aqueous solution (NOR3 100 ⁇ M) alone or in combination, and after 72 hours of culture, the cell proliferation rate was measured by the above test. It was measured according to procedure [5] (Fig. 22). In both cases, control cells (Ctrl) were treated with FR (-) DMEM without ozone blowing.
  • the NO donor NOR-3 showed synergistic anti-tumor effects when co-administered with pOBM.
  • pOBM was prepared according to the test procedure [4] above, was used as a stock solution (100%), dissolved ozone concentration was measured and diluted with FR( ⁇ ) DMEM. The stock solution is made into an ozone solution by blowing ozone (AF ozone) into the solvent for 1 minute per 10 mL.
  • pOBM (12.5%, 25%, 50%) and riboflavin (vitamin B 2 , VB2) aqueous solution (15 ⁇ M) were administered alone or in combination to human squamous cell carcinoma cells SAS. It was measured according to the test procedure [5] (Fig. 23). Riboflavin (vitamin B2 ), which is redox-active, showed synergistic anti-tumor effects when co-administered with pOBM.
  • pOBM was prepared according to the test procedure [4] above, was used as a stock solution (100%), dissolved ozone concentration was measured and diluted with FR( ⁇ ) DMEM. The stock solution is made into an ozone solution by blowing ozone (AF ozone) into the solvent for 3 minutes per 10 mL.
  • Human squamous cell carcinoma cells HOC-313 were administered pOBM (25%) and salinomycin sodium aqueous solution (salinomycin 2.5 ⁇ M) alone or in combination, and after 72 hours of culture, the cell growth rate was measured according to the above test procedure [5]. (Fig. 24). Salinomycin showed an adjuvant effect on pOBM.
  • cancer cells can be selectively killed without substantially affecting normal cells.
  • the apparatus and the like of the present invention such a composition can be easily produced. Therefore, the present invention is useful in the medical industry centering on anticancer agents and production equipment thereof.

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Abstract

La présente invention aborde principalement le problème consistant à fournir une nouvelle composition ou un produit pharmaceutique qui cible le réseau mitochondrial, etc. et qui présente une excellente activité anticancéreuse tout en ayant un effet minimal sur les cellules normales.　Un exemple de la présente invention est une composition comprenant une solution aqueuse qui contient de l'ozone et un activateur (par exemple, un sel de fer divalent, une flavine, ou un agent d'alimentation en monoxyde d'azote), ladite composition étant destinée à induire : la fragmentation de mitochondries qui ont été rassemblées uniformément à proximité du noyau cellulaire d'une cellule cancéreuse dans un état hypoxique ; l'accumulation des mitochondries fragmentées sur un pôle du noyau cellulaire ; et l'endommagement du noyau cellulaire ou de la mort de la cellule cancéreuse après l'accumulation.
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