WO2023068366A1 - Ozone-containing aqueous solution composition - Google Patents

Abstract

The present invention addresses mainly the problem of providing a novel composition or pharmaceutical that targets the mitochondrial network etc. and exhibits excellent anticancer activity while having a minimal effect on normal cells.　An example of the present invention is a composition comprising an aqueous solution that contains ozone and an activator (for example, a divalent iron salt, a flavin, or a nitrogen monoxide supplying agent), said composition being for inducing: fragmentation of mitochondria that have gathered uniformly in the vicinity of the cell nucleus of a cancer cell in a hypoxic state; accumulation of the fragmented mitochondria to one pole of the cell nucleus; and damage to the cell nucleus or death of the cancer cell after the accumulation.

Description

Ozone-containing aqueous solution composition

(Cross reference to related applications)
This application claims the benefit of Japanese Application No. 2021-172840 filed with the Japan Patent Office on October 22, 2021. The Japanese application is hereby incorporated by reference for all purposes as if the entire application documents (description, claims, drawings, abstract) were set forth herein. .
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.). . The macroscopic shape of mitochondria (mitochondrial network) is determined by the balance between fission and fusion, and these two opposing processes are both controlled by dynamin-related proteins (Drp) with guanosine triphosphate activity. Controlled by groups. Since homeostasis of mitochondrial dynamics is essential for energy supply, metabolic activity, and maintenance of mitochondrial DNA, its abnormalities lead to cellular dysfunction and death. Furthermore, mitochondria form a reticular network and are distributed throughout the cytoplasm (pan-cytoplasmic), or divided and distributed uniformly near the cell nucleus (nuclear peripheral clustering, PNMC: Perinuclear mitochondrial clustering). It exhibits a variety of intracellular distribution patterns, such as In recent years, it has become clear that the intracellular distribution of mitochondria is deeply involved in the plasticity, motility, invasiveness, survival and death of cancer cells (Non-Patent Documents 1 and 2). Therefore, 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.

However, since regulation of mitochondrial dynamics is essential not only for cancer cells but also for the survival of normal cells, indiscriminate deregulation may damage normal cells and cause side effects.

D. Pendin et al., Frontiers in Oncology, 2017 May 22; 7: 102. A. Altieri, British Journal of Cancer, 2017 Jul 25; 117(3): 301-305.

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.

As a result of intensive studies by the present inventors, the use of a composition containing ozone and an activator (also referred to as an adjuvant) 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.
[1] A composition comprising an aqueous solution containing ozone and an activator for killing cancer cells.
[2] 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.
[3] The above [1] or [2], wherein 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. ].
[4] The divalent iron salt is ferrous sulfate, ferrous chloride, ferrous bromide, or ammonium iron (II) sulfate, and 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.
[5] The 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.
[6] A composition obtained by bubbling an ozone-containing gas obtained by irradiating an oxygen-containing gas with UV-C in an aqueous solution containing an activator, or ozone obtained by silently discharging an oxygen-containing gas. 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].

[7] An anticancer agent comprising the composition according to any one of [1] to [6] above.
[8] The anticancer agent of [7] above, which is applied to epithelial cell-derived cancer, non-epithelial cell-derived cancer, leukemia, or lymphoma.
[9] 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.

[10] An ozone generator used for producing the composition according to any one of [1] to [6] above, comprising an air pump section and an ozone generation section, wherein the air pump section 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.
[12] The ozone generator according to [11] above, wherein the UV-C lamp has a spectral component with an emission wavelength of 185 nm.
[13] An ozone generator used for producing the composition described in [1] or [2] above, comprising a high-voltage power supply unit and a discharge unit, and generating an AC voltage obtained by the high-voltage power supply unit. An ozone generator, wherein an ozone-containing gas is generated from the oxygen-containing gas by applying an electric current to the oxygen-containing gas in the discharge section and causing discharge in the oxygen-containing gas.
[14] The ozone generator according to [13] above, wherein the discharge is a dielectric barrier discharge.

[15] A production method for producing the composition according to any one of [1] to [6] above, wherein ozone is generated by irradiating an oxygen-containing gas with ultraviolet rays or by silent discharge to produce an ozone-containing composition. 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.
[16] The production method according to [15] above, wherein the ultraviolet rays are UV-C.
[17] The production method according to [16] above, wherein the UV-C has a spectral component with a wavelength of 185 nm.
[18] The production method according to [15] above, wherein the oxygen-containing gas is a nitrogen-free gas.
[19] The above [15] to [18], wherein the activator is one or more selected from the group consisting of ferric salts, flavins, nitric oxide donors, and salinomycin and pharmaceutically acceptable salts thereof. ] The manufacturing method as described in any one of ].
[20] The production method according to any one of [15] to [19] above, wherein the aqueous solution is an aqueous solution containing a mammalian cell culture medium or an infusion preparation.

According to the present invention, for cancer cells in which mitochondria are in a pancytoplasmic distribution or in a PNMC state, 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.

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. 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|mode of a structure of the ozone generator which concerns on this invention. 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 μM)) 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. BALB/cAJcl-nu/nu nude mice (8-week-old male) were transplanted with 143B human osteosarcoma cells (1×10 ⁶ cells/mouse) by intramedullary injection into the right tibia, 3 times a week starting 7 days after cell transplantation. 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. 1 is a fluorescence microscope image of superoxide (MitoSOX), hydrogen peroxide (Hydrop), and hydroxyl radical (OxiOrange) observed after administration of APAM (25, 50%) to human epidermal fibroblast HDF and culture for 2 hours. Human melanoma cells A2058, human osteosarcoma cells HOS, and human dermal fibroblasts (HDF) were administered APAM (25%) and cultured for 18 hours, after which the morphology of cells, nuclei, and mitochondria were observed with a fluorescence microscope. . The figure shows a superimposed image (MT/NU) of mitochondria (MT) and nuclei (NU) and an enlarged view thereof. 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. The numbers in the figure are the expected ozone concentration calculated from the dissolved ozone concentration (OBM 3.9 ppm) of the undiluted solution. 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. The 1st and 3rd figures from the top are Phase Contrast (PC) images, and the 2nd and 4th figures from the top are superimposed images (MT/NU) of mitochondria (MT) and nuclei (NU) and their enlarged views. indicates The numbers in the figure indicate the dissolved ozone concentration in the sample. 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. The numbers in the figure indicate the calculated dissolved ozone concentrations in the samples. It is a fluorescence microscopy image showing the morphology of nuclei and mitochondria after administration of APAM (50%) and tubulin synthesis inhibitor nocodazole (NC, 100 nM) alone or in combination to human osteosarcoma cells HOS and culturing for 18 hours. 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. . 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.

The present invention will be described in detail below.

1. Composition according to the present invention The composition according to the present invention (hereinafter referred to as "the composition of 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. That is, 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.

1.1 Induction of unipolar mitochondrial nuclear marginal clustering (MPMC) Cancer tissue often suffers from insufficient oxygen supply due to excessive cell proliferation and increased distance from blood vessels. and known to cause hypoxia. When hypoxia occurs, activation of hypoxia inducible transcription factors (HIFs) induces a hypoxic response. Under such hypoxic conditions, mitochondria diffusely present in the cytoplasm migrate to the periphery of the cell nucleus and become uniformly localized at the periphery of the cell nucleus. ) is known to be induced (Fig. 1A). PNMCs are oxidized domains of specific bases involved in binding to the dominant transcription factor (HIF-1α) for adaptation to hypoxic environments and the Hypoxia responsive element (HRE) essential for hypoxia-induced gene activation. is necessary for the formation of cancer cells and is involved in the survival of cancer cells.
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). Loss of PNMC and MPMC induction lead to dehypoxia adaptation and nuclear injury and cell death.
Although 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. In addition, 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.

1.2 Ingredients 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. Specifically, 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. In addition, 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.

Examples of divalent iron salts 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 (NO 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.

As such NO donors, for example, 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-methyl-1-triazene, NOC-12: 1-hydroxy -2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene, NOC-18: 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)- Mention may be made of 1-triazene, GSNO: S-nitrosoglutathione. Among them, NOR-3 is preferred. These may be used singly or in any combination of two or more.

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).

The 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.

The composition of the present invention may contain one or more of the components used in mammalian cell culture media. Examples of such 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). Among them, 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.

As the aqueous solution that constitutes the composition of the present invention, for example, 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. Other examples 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.

1.3 Ozone bubbling liquid (OBM)
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. It can be a composition (OBM) consisting of: As another aspect, an ozone-containing gas (an ozone-containing gas obtained by silent discharge) is obtained by applying an alternating voltage to an oxygen-containing gas (for example, a mixed gas of argon and oxygen) to cause discharge in the oxygen-containing gas. ) is bubbled in an aqueous solution containing the activator (pOBM). 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.

1.4 Cold Atmospheric Air Plasma Irradiation Liquid (APAM)
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, and 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.

2. Anticancer agent according to the present invention The anticancer agent according to the present invention (hereinafter referred to as "anticancer agent of the present invention") contains the composition of the present invention. The 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. Among them, 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 (malignant tumors) 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, pirarubicin, idarubicin, aclarubicin, amrubicin, mitoxatron; Kinase inhibitors (e.g., gefitinib, erlotinib, osimertinib, afatinib, dacomitinib, imatinib, dasatinib, ponatinib, bosutinib, vandetanib, sunitinib, axitinib, pazopanib, lenvatinib, lapatinib, nintedanib, nilotinib, ibrutinib, giritinib, lectinib, crizotinib, lorlatinib), mTOR inhibitors (e.g., everolimus, sirolimus), proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib), olaparib, niraparib, sorafenib, vemurafenib, dabrafenib, trametinib, palbociclib, and abemaciclib. can be done. The combined use of the anticancer agent of the present invention and existing anticancer agents can exhibit synergistic antitumor effects by enhancing apoptosis and inducing non-apoptotic cell death. As a result, 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.

3 Ozone generator according to the present invention 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.

In addition, 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. By providing an ozone scrubber, 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.

For example, as shown in FIG. 2, 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. can be configured with In this case, 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. . For example, if an appropriate nozzle is employed at the tip of the gas discharge part in the bubbling part, 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.

After bubbling, 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, as another embodiment, 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. Suitably the discharge is a dielectric barrier discharge (DBD). The device of the present invention in this aspect may be provided with an air tank for fluid pressure control (pressure fluctuation reduction, etc.). Moreover, a pressure gauge, a flow meter, an air valve, a purge valve, etc. may be provided as necessary.

4 Method for producing the composition according to the present invention The method for producing the composition according to the present invention (hereinafter referred to as the "production method for 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.

4.1 Ozone Generating Step In one aspect of the production method of the present invention, ozone is generated by ultraviolet irradiation to produce an ozone-containing gas. For example, an ozone-containing gas can be produced by irradiating an oxygen-containing gas (such as air) with ultraviolet rays. It is important that 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. Among them, 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.

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.

In the production method of the present invention, as another aspect, ozone is generated by silent discharge (dielectric barrier discharge) to a gas containing oxygen to produce an ozone-containing gas. For example, 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. .

4.2 Bubbling Step In the production method of the present invention, 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.

There is no particular limitation on the pressure of the ozone-containing gas during bubbling, and it may be set as appropriate. 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.

The flow rate of the ozone-containing gas discharged from the nozzle during bubbling is not particularly limited and may be set as appropriate. 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 present invention will be specifically described below with reference to examples and test examples, but the present invention is not limited to the scope of the examples.

<Test procedure>
Test procedures common to each example are described below.

[1] Creation of air plasma A piezobrush (registered trademark) PZ2 (manufactured by Relyon, Germany) with a piezo element was used to create air plasma by taking in ambient air (creation conditions: output voltage: >20 kV, frequency: >50 kHz, electron density: 10 ^14-16 /cm)

[2] Preparation and dilution of air plasma irradiation solution (APAM) The prepared air plasma was irradiated to phenol red-free Dulbecco's modified Eagle medium (FR (-) DMEM) from 20 mm above for 1 minute per 1 mL of the medium. (APAM) was created. APAM was diluted with FR(-)DMEM.

[3-1] Production of air-containing (AC) ozone gas 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.

[3-2] Production of air-free (AF) ozone gas Oxygen (0.4 L / min) is introduced into a DBD plasma probe (manufactured by Tokyo Keiki Co., Ltd.) to make it a gas (fluid), and an alternating voltage (100 V) is applied to the fluid. was applied to cause discharge to produce an ozone-containing gas. An outline of the apparatus and system is shown in FIG. 3 below.

[4] Preparation and dilution of ozone aqueous solution The prepared ozone-containing gas was blown into phenol red-free DMEM (FR(−)DMEM) or purified water at a pressure of 36 kPa and a flow rate of 4 L/min to prepare an ozone aqueous solution. Dissolved ozone concentration was measured by the 4-aminoantipyrine method. In the following, the aqueous ozone solutions prepared were OBM and pOBM, respectively, obtained by bubbling AC ozone and AF ozone into FR(−)DMEM, and OBW, by bubbling AC ozone into purified water. These were diluted with FR(-)DMEM or purified water, respectively.

[5] Measurement of Cell Proliferation Rate APAM and aqueous ozone solution were prepared according to the above test procedures [2] and [4], respectively. A water-insoluble test drug was dissolved in dimethylsulfoxide (DMSO) to a final concentration of 0.1% or less. Cells were suspended in DMEM containing 10% fetal calf serum (FCS/DMEM) and seeded (5 x ¹⁰⁴ cells/mL) in 96-well microplates and placed overnight in a _CO2 incubator (95% air/ After culturing in a 5% CO ₂ atmosphere), the test drug was administered and cultured for an additional 72 hours. Cell growth rate was measured using the water-soluble tetrazolium salt (WST-8) reduction method. This method utilizes the fact that a water-soluble tetrazolium salt produces a water-soluble formazan by mitochondrial oxidoreductase activity, and measures the cell growth rate by measuring the absorbance at a wavelength of 540 nm.

[6] Analysis of Cell Morphology and Nucleus ^, Tubulin, and Mitochondria Morphology and Subcellular Localization After culturing, the cells were treated with the test drug for 18 hours. After washing the cells, cell nuclei, tubulin, and mitochondria were stained with Hoechst 33342 (Hoe), Oregon Green Paclitaxel, and Mito Tracker Red (MTR), respectively, and the morphology was observed and photographed under a fluorescence microscope. At the same time, the cell morphology was observed and photographed. As controls, DMEM or dimethylsulfoxide (DMSO 0.1%) without plasma irradiation were administered (they had no effect on any observed subjects).

[7] Caspase activation, membrane integrity, apoptosis measurement A test drug was added to cells seeded (1×10 ⁵ cells/mL) in a 6-well microplate, and after 24 hours of culture, APC-labeled annexin V and 7- Amino-actinomycin D (7-AAD) (BD Biosciences) was added and measured on a FACS Celesta ^tm flowcytometer (BD Biosciences). Data were analyzed with Cell Quest Pro (Becton Dickinson Biosciences) and FlowJo software. Measurement was performed in triplicate, and annexin V-positive cells were defined as apoptotic cells.

[8] Analysis of intracellular ROS Cells were suspended in FCS/DMEM, seeded (5×10 ⁴ cells/mL) on a polylysine-coated 35 mm dish (Matsunami Glass), cultured overnight, and then treated with a test drug for 2 hours. . After washing the cells, superoxide was stained with Mito SOX ^tm Red (Thermo Fisher Scientific), hydrogen peroxide and hydroxyl radicals were stained with Hydrop ^tm and Oxi Orange ^tm (Goryo Kayaku), respectively, and stained with the EVOS FL Cell Imaging System (Thermo Fisher Scientific). observed and photographed. Data were analyzed with NIHImageJ software (NIH, Bethesda, MD, USA).

[9] Protein Analysis by Western Blotting Cells were washed with calcium- and magnesium-free phosphate-buffered saline (PBS), and then washed with Cellytic M Lysis containing protease and protein phosphatase inhibitor cocktail (Sigma-Aldrich). It was dissolved with buffer (Merck) and the residue was removed by centrifugation. Supernatants were collected and protein was quantified by BCA protein assay (Thermo Fisher Scientific). SDS-conjugated proteins were separated by SDS-polyacrylamide gel electrophoresis using a 4-12% gradient gel (Invitrogen) and transferred to a PVDF membrane. After blocking for 30 minutes with Tris-buffered saline (TBST) containing 0.2% Tween-20 and 2% non-fat dry milk, 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).

[10] Measurement of Antitumor Effect on Mouse 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 μL) 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.

[Example 1]
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 μM)) 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.

[Example 4]
BALB/cAJcl-nu/nu nude 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.

[Example 7]
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 μM) 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. It induced a decrease in OBM cell proliferation rate. The action of OBM (25%) was markedly inhibited by CPTIO, whereas the action of OBM (50%) was not inhibited by CPTIO. These results demonstrate that OBM can specifically induce NO-mediated cell death at low concentrations.

[Example 8]
OBM (25, 50%) and OBW (25, 50%) were administered to human osteosarcoma cells HOS, and after 18 hours of culture, the morphology of cells, nuclei, and mitochondria was observed under a fluorescence microscope according to the test procedure described above [6]. (Fig. 12). Control cells (Ctrl) exhibited a spindle shape and adhered well to the glass substrate. Most of the nuclei were oval and the internal structure was visually recognized as a region with different staining properties. Mitochondria (MT) had a filamentous or reticular structure and were diffusely distributed around the nucleus into the cytoplasm or uniformly localized around the nucleus (PNMC morphology). Both OBM and OBW increased swollen cells and decreased adhesiveness in a concentration-dependent manner. The nucleus (NU) was shrunken or thinly fragmented, the internal structure obscured, and uniformly stained. OBM treatment resulted in mitochondria fission and fusion, resulting in a change to the MPMC morphology that was localized on one pole of the nucleus, whereas OBW treatment showed only fragmentation of mitochondria. These results indicate that OBM specifically induces MPMC morphology.

[Example 9]
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.

[Example 10]
Human osteosarcoma cells 143B were treated with OBW (12.5%, 25%, 50%), Fe ²⁺ aqueous solution (30 μM, 100 μM) with ammonium iron (II) sulfate hexahydrate as a solute, ammonium iron (III) sulfate 12 water An aqueous Fe ³⁺ solution (100 μM) containing a solute of solute was administered alone or in combination, and after 72 hours of culture, the cell proliferation rate was measured according to the test procedure [5] described above (FIG. 14). Fe ²⁺ enhances the effect of OBW in a concentration-dependent manner, whereas Fe ³⁺ does not. Since Fe ²⁺ is redox-active, whereas Fe ³⁺ is inactive, enhancement of the redox reaction with dissolved ozone is thought to lead to increased antitumor effects.

[Example 11]
OBW (12.5%, 25%, 50%), riboflavin (vitamin B ₂ , VB2) aqueous solution (15 μM), NOR-3 aqueous solution (30 μM) 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.

[Example 12]
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.

[Example 13]
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%).

[Example 14]
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.

[Example 15]
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). In addition, pOBM (12.5-50%), iron chelator 2,2'-bipyridyl (BP 100 μM), iron chelator (DFO 100 μM), NO-specific scavenging in cultured human squamous cell carcinoma cells HOC-313 Agents Carboxy-PTIO (CPTIO 30 μM) and catalase (Catalase 10 U/mL) were administered singly or in combination, and after 72 hours of culture, the cell proliferation rate was measured according to the above test procedure [5] (FIGS. 20 and 21). In both cases, control cells (Ctrl) were treated with FR (-) DMEM without ozone blowing.

These results demonstrate that pOBM has a strong anticancer effect. In addition, since the iron chelator suppressed the anticancer activity by about 50% and the catalase completely suppressed the anticancer activity, it was shown that pOBM induces cytotoxicity mediated by iron and H ₂ O ₂ .

[Example 16]
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 μM) 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.

[Example 17]
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 μM) 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.

[Example 18]
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 μM) 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.

According to the composition of the present invention, etc., cancer cells can be selectively killed without substantially affecting normal cells. In addition, according to 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.

Claims (20)

A composition comprising an aqueous solution containing ozone and an activator for killing cancer cells. A composition comprising an aqueous solution containing ozone and an activator, comprising fragmentation of mitochondria uniformly aggregated near the cell nucleus of hypoxic cancer cells and a portion of the fragmented mitochondria on the cell nucleus. A composition for inducing accumulation to poles and injury to the cell nuclei or cell death of the cancer cells after the accumulation. 3. The composition according to claim 1 or 2, wherein the activator is one or more selected from the group consisting of ferric 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, and the flavin is riboflavin, flavin mononucleotide (FMN), or flavin adenine dinucleotide. (FAD), and the nitric oxide donating agent is organic nitrates, organic nitrites, metal nitrosyls, sydnonimines, S-nitrosothiols, or hydroxyimines. A composition according to any one of claims The composition according to any one of claims 1 to 4, wherein the aqueous solution is an aqueous solution containing a mammalian cell culture medium or an infusion formulation. The ozone-containing gas obtained by irradiating the oxygen-containing gas with UV-C is a composition obtained by bubbling in an aqueous solution containing an activator, or the ozone-containing gas obtained by silently discharging the oxygen-containing gas. , 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 pressure air plasma. A composition according to any one of the preceding claims. An anticancer agent comprising the composition according to any one of claims 1 to 6. 8. The anticancer agent according to claim 7, which is applied to cancer derived from epithelial cells, cancer derived from non-epithelial cells, leukemia, or lymphoma. The epithelial cell-derived cancer is lung cancer, breast cancer, pancreatic cancer, colon cancer, gastric cancer, prostate cancer, ovarian cancer, oral cancer, or other organ-derived cancer, and the non-epithelial cell-derived cancer is 9. The anticancer of claim 8, wherein the cancer is osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, melanoma, neuroblastoma, or glioma. cancer drug. An ozone generator used for producing the composition according to any one of claims 1 to 6, comprising an air pump section and an ozone generator section, wherein the air is introduced from the outside of the apparatus by the suction function of the air pump section. The ozone-containing gas is irradiated with ultraviolet rays in the ozone generating unit, and the oxygen-containing gas after the ultraviolet irradiation becomes the ozone-containing gas and is discharged outside the apparatus by the air supply function of the air pump unit. and an ozone generator. The ozone generator according to claim 10, wherein said ozone generator comprises a UV-C lamp. The ozone generator according to claim 11, wherein said UV-C lamp has a spectral component with an emission wavelength of 185 nm. 3. An ozone generator used for producing the composition according to claim 1 or 2, comprising a high-voltage power source and a discharger, wherein the AC high voltage obtained by the high-voltage power source is applied to oxygen in the discharger. 1. An ozone generator, characterized in that an ozone-containing gas is generated from an oxygen-containing gas by applying a voltage to the containing gas and causing a discharge in the oxygen-containing gas. 14. The ozone generator of claim 13, wherein said discharge is a dielectric barrier discharge. A production method for producing the composition according to any one of claims 1 to 6, comprising a step of producing ozone-containing gas by irradiating an oxygen-containing gas with ultraviolet rays or by silent discharge to generate ozone. and bubbling the produced ozone-containing gas into an aqueous solution containing an activator. The manufacturing method according to claim 15, wherein the ultraviolet rays are UV-C. 17. The manufacturing method according to claim 16, wherein the UV-C has a spectral component with a wavelength of 185 nm. 16. The manufacturing method according to claim 15, wherein the oxygen-containing gas is a nitrogen-free gas. Any one of claims 15 to 18, wherein the activator is one or more selected from the group consisting of ferric salts, flavins, nitric oxide donors, and salinomycin and pharmaceutically acceptable salts thereof. The manufacturing method described in . The production method according to any one of claims 15 to 19, wherein the aqueous solution is an aqueous solution containing a mammalian cell culture medium or an infusion preparation.

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