Patent Description:
Various anticancer agents have been developed and introduced into market; however, they are to kill cancer cells or suppress proliferation thereof and inevitably produce side effects on normal cells. In addition, the pharmacological effects of a new anticancer agent are investigated in combination with an existing anticancer agent in clinical trials, but it is difficult to develop the new anticancer agents. In the circumstance, molecular target drugs (primarily, antibody drug) targeting a specific gene product has been developed.

For example, an anti-CD20 antibody (generic name: rituximab) is used for treating CD20-positive non-Hodgkin's lymphoma and few side effects are produced on cells which express no CD20 on the surface thereof.

An anti-VEGF antibody (generic name: bevacizumab) inhibits the function of VEGF, thereby suppressing angiogenesis and proliferation/metastasis of tumors.

An anti-HER2 antibody (generic name: trastuzumab) exhibits an anti-tumor effect against breast cancer cells or the like highly expressing HER2 protein on the surface thereof.

Molecular target drugs are also used in a cancer immunotherapy to kill cancer cells through in vivo immune response. An anti-CTLA4 antibody (generic name: ipilimumab) and an anti-PD-<NUM> antibody (generic name: nivolumab) cancel a function to suppress CTL.

(cytotoxic T cell)'s attack against cancer cells, thereby indirectly serving as an anticancer agent.

These molecular target drugs (antibodies) are mainly constituted of naturally occurring amino acids, and it is not necessary to care about the effect on metabolites thereof; however, the excessive administration of them may , in vivo, induce an anti-idiotype antibody (an antibody recognizing an antibody administered as a drug as a foreign substance and binding thereto). In addition to this problem, because of a macromolecule, molecular target drugs cannot enter into the cells.

Patent Document <NUM> discloses an aqueous solution containing ozone nanobubbles.

Patent Document <NUM> refers to nanobubble water in connection with preventing or treating cancer.

Patent Document <NUM> refers to a composition containing ozone nanobubbles, which are formed by crushing ozone nanobubbles and which are present in a stabilized manner. Particularly, the composition is for suppressing bladder cancer growth.

Non Patent Document <NUM> refers to the association of different ozone concentration with <NUM>-fluorouracil and cisplatin in human colon cancer cell in order to investigate possible anticancer synergistic effects.

Any subject-matter falling outside the scope of the claims is provided for information only.

The present invention provides an anticancer agent, which is easily absorbed in cells and metabolized in vivo without changing a chemical structure and induces no immune response, and wherein the agent can be used in combination with other anticancer agent(s). Solution to Problem.

As a result of intensive studies, the present inventors of the present application have found that ozone water storable for a long term has an anticancer effect; and furthermore, that the anticancer effect is significantly increased in combination with an existing anticancer agent.

A pharmaceutical composition according to the present invention can be easily absorbed in cells and metabolized in vivo without changing a chemical structure, exert an anticancer effect without inducing an immune response, and be used in combination with other anticancer agent(s).

The subject-matter of the present invention is set forth in the independent claim. Preferred embodiments are the subject-matter of the dependent claims. Hence, the present invention refers to the following subject-matter:
Ozone water comprising ozone nano-bubbles for use in treating or preventing a cancer,.

The ozone water for use according to the invention, wherein a concentration of ozone in the ozone water is <NUM> ppm or more, preferably <NUM> ppm or more and more preferably <NUM> ppm or more.

The ozone water for use according to the invention, wherein the ozone water comprises bittern-containing water.

The ozone water for use according to the invention, wherein the cancer is selected from the group consisting of lung cancer, bladder cancer, prostate cancer, ovarian cancer, esophageal cancer, stomach cancer, cervical cancer and malignant lymphoma.

The ozone water for use according to the invention, wherein the ozone water is to be orally, intravenously or intratumorally administered.

The ozone water for use according to the invention, wherein the ozone water is to be administered once every day or three times per week.

The "ozone water" refers to water in which ozone (O<NUM>) is dissolved. The ozone water for use according to the present invention is ozone nano-bubble water containing a high concentration of mineral, in which ozone nano-bubbles are maintained. The ozone water for use in the present invention comprises ozone nano-bubbles, further a Na ion, a Mg ion, a K ion and/or a Ca ion, and further at least one selected from the group consisting of a sulfur ion, a boron ion, a lithium ion, a silicon ion, a zinc ion, an iron ion and a strontium ion.

The "fine bubbles" refer to floating minute air bubbles with a particle size of <NUM> or less. The "ultrafine bubbles" refer to floating minute air bubbles with a particle size of less than <NUM>. The "nano-bubbles" being comprised in the ozone water for use according to the present invention may be "ultrafine bubbles". The "nano-bubbles" may contain floating minute air bubbles with a particle size of <NUM> or more and less than <NUM>, preferably <NUM> or more and less than <NUM> and more preferably several tens to several hundreds nm.

The floating minute air bubbles can be produced by, e.g. an ejector system, cavitation system, swirling flow system or pressure dissolution method.

The "bittern" refers to a mixture of minerals (powder or liquid) produced from seawater and containing magnesium chloride as a main component.

The "cancer" refers to a "malignant tumor" in a broad sense, including "carcinoma", "sarcoma" and hematological malignancy such as leukemia. The malignant tumor refers to a cell population which infiltrates into the peripheral tissue or develops metastasis among cell populations (tumor, benign tumor and malignant tumor) uncontrollably self-proliferated by genetic mutation to. The malignant tumors are pathologically classified into.

The "cancer" as mentioned herein may include, but are not particularly limited to, carcinomas such as head and neck cancer (maxillary cancer, (upper, middle, lower) pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer), thoracic cancer (breast cancer, lung cancer (non-small cell lung cancer, small cell lung cancer)), digestive system cancer (esophageal cancer, stomach cancer, duodenal cancer, colorectal cancer (colon cancer, rectal cancer)), liver cancer (hepatocellular carcinoma, cholangiocellular carcinoma), gallbladder cancer, bile duct cancer, pancreatic cancer, anal cancer, urinary organ cancer (kidney cancer, ureteral cancer, bladder cancer, prostate cancer, penis cancer, testis (testicle) cancer), genital cancer (uterine cancer (cervical cancer, endometrial cancer), ovarian cancer, vulvar cancer, vaginal cancer) and skin cancer (basal cell carcinoma, squamous cell carcinoma).

Furthermore, the "cancer" as mentioned herein may include, but are not particularly limited to, fibrosarcoma, liposarcoma, myoma (e.g., leiomyosarcoma), hemangiosarcoma, Kaposi's sarcoma, lymphatic sarcoma, synovial sarcoma, osteosarcoma, extraosseous osteosarcoma and malignant peripheral nerve tumor.

Furthermore, the "cancer" as mentioned herein may include, but are not particularly limited to, hematological malignancies (for example, leukemia, malignant lymphoma, multiple myeloma).

In the present invention, the ozone water comprising ozone nano-bubbles, a Na ion, a Mg ion, a K ion and/or a Ca ion, and further at least one selected from the group consisting of a sulfur ion, a boron ion, a lithium ion, a silicon ion, a zinc ion, an iron ion and a strontium ion, is used in treating or preventing a cancer, and is to be administered sequentially with the other anticancer agent cisplatin.

The "anticancer agent" refers to a medicament for treating or preventing a cancer. The anticancer agents are classified, but are not particularly limited, into a molecular target drug, an alkylating agent, an antimetabolic drug, a plant alkaloid, an anticancer antibiotic, a platinum formulation, a hormonal agent, a biological response modifier and the like.

In the context of the present invention, the other anticancer agent is cisplatin.

The "molecular target drug" refers to a drug designed so as to target a specific gene product of cancer cells at a molecular level and efficiently acts on the targeted gene product. Examples of molecular target drugs (not claimed) include, but are not particularly limited to, ibritumomab tiuxetan, imatinib, everolimus, erlotinib, gefitinib, gemtuzumab ozogamicin, sunitinib, cetuximab, sorafenib, dasatinib, tamibarotene, trastuzumab, tretinoin, panitumumab, bevacizumab, bortezomib, lapatinib and rituximab. Further, ipilimumab and nivolumab fall within the molecular target drug, which cancel a function to suppress CTL (cytotoxic T cell)'s attack against cancer cells, thereby indirectly treating cancer with a cancer immunotherapy.

The "alkylating agent" functions to attach a mass of atomics named an alkyl group to the DNA of a cancer cell and allows the two helically twisted DNA strands to be abnormally joined to each other to prevent the DNAs from being coped. Examples of the alkylating agent include, but are not particularly limited to, ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan and ranimustine.

The "antimetabolic drug" refers to an anticancer agent, which is a substance analogous to a material for a nucleic acid in terms of chemical structure, suppresses synthesis of the DNA of cancer cells when they are divided/proliferated, thereby inhibiting metabolism of cancer cells and suppressing proliferation thereof. Examples of the antimetabolic drug include, but are not particularly limited to, enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine, cytarabine ocfosfate, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, doxyfluridine, nelarabine, hydroxycarbamide, fluorouracil (<NUM>-FU), fludarabine, pemetrexed, pentostatin, mercaptopurin and methotrexate.

The "plant alkaloid" refers to an anticancer agent using a highly toxic plant component. Examples of the plant alkaloid include, but are not limited to, a microtubule inhibitor and a topoisomerase inhibitor (for example, irinotecan, etoposide, eribulin, sobuzoxane, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine).

The "anticancer antibiotic" refers to an anti-tumor antibiotic produced from a mold or the like, contained in soil. Examples of the anticancer antibiotic include, but are not particularly limited to, actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, zinostatin stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, mitomycin C, mitoxantrone and liposomal doxorubicin.

The "platinum formulation" refers to an anticancer agent containing platinum. Examples of the platinum formulation include, but are not particularly limited to, oxaliplatin, carboplatin, cisplatin and nedaplatin.

The "hormonal agent" refers to an anticancer agent suppressing secretion or function of in vivo hormone. Examples of the hormonal agent include, but are not particularly limited to, anastrozole, exemestane, estramustine, ethinyl estradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, mepitiostane, leuprorelin and letrozole.

The "biological response modifier" refers to an anticancer agent inducing an in vivo biological reaction for treatment. Examples of the biological response modifier include interferon-α, interferon-β, interferon-y and interleukin.

Now, the present invention will be described in detail with reference to Examples; however, the present invention is not limited by these Examples.

Ozone water was prepared in accordance with a method described in International Publication No. <CIT>. More specifically, brine-containing water of a "deep-seawater bittern for professional use" (manufactured by Ako Kasei Co. ) was diluted three fold with water (tap water) and used in accordance with the description of Example <NUM>. The "deep-seawater bittern for professional use" contains <NUM> to <NUM> wt% of Mg ions in terms of MgCl<NUM>. Other than Mg ions, Ca ions (<NUM> to <NUM>/liter) and Na ions (<NUM> to <NUM>/liter) are contained.

In the diluted aqueous mineral solution, ozone micro-bubbles with a particle size of <NUM> to <NUM> are generated. The micro-bubbles generated in the solution first looks milky white and then turn transparent as the micro-bubbles are crushed over time. In this manner, ozone water having a constant ozone concentration of <NUM> ppm or more was obtained (Non Patent Document <NUM>).

The ozone water is available as NAnO3 from Opt Creation Inc.

The effect of stabilized ozone water NAnO3 on <NUM> types of cancer cell lines was investigated by 2D culture and 3D culture.

The following cell lines were obtained from ATCC (American Type Culture Collection) or JCRB cell bank and cultured in the mediums shown in Table <NUM>.

Each of the culture mediums (<NUM>µL) was added to each well of Imaging Plate (<NUM>-Well, Tissue Culture Treated (BD FALCON Cat#<NUM>). Thereafter, each of the cell suspensions (<NUM>µL) was seeded (<NUM> × <NUM><NUM> cells/well) and cultured in the presence of <NUM>% CO<NUM> at <NUM>.

<NUM> day after the initiation of culture, each of the following test-substance solutions (<NUM>µL) was added and the culturing was continued.

<NUM> days after the initiation of culture, ATP concentration was determined with CellTiter-Glo (R) Luminescent Cell Viability Assay (CTG) (Promega Cat#G7572) and based on the ATP standard curve. Provided that the survival rate at the ATP concentration of the negative control group (test-substance solution <NUM>) was regarded as <NUM>%, the survival rates of each test group was calculated (<FIG>).

Each of the culture mediums (<NUM>µL) was added to each well of NanoCulture Plate (MH pattern, low adhesive: ORGANOGENIX). Thereafter, each of the cell suspensions (<NUM>µL) was seeded at a density of <NUM> × <NUM><NUM> cells/well and cultured in the presence of <NUM>% CO<NUM> at <NUM>.

<NUM> days after the initiation of culture, each of the test-substance solutions (<NUM>µL) described in Table <NUM> was added and the culturing was continued.

<NUM> days after the initiation of culture, ATP concentration was determined with CellTiter-Glo (R) Luminescent Cell Viability Assay (CTG) (Promega Cat#G7572) and based on the ATP standard curve. Provided that the ATP concentration of the negative control group (test-substance solution <NUM>) was regarded as a survival rate of <NUM>%, the survival rates of each test group was calculated (<FIG>).

As a result, both in 2D culture and 3D culture, the survival rates of a group (test-substance solution <NUM>: NAnO3 concentration with a final of <NUM>%) and a vehicle group (test-substance solution <NUM>: sea-water mineral solution with a final concentration of <NUM>%) decreased by <NUM>% or less as compared with the negative control group (test-substance solution <NUM>) in the cancer cell lines (including human lung adenocarcinoma cell line A549, human breast cancer cell line BT474, human pancreatic cancer cell line MIAPaCa-<NUM>, human stomach cancer cell line MKN-<NUM>, human hepatoma cell line HepG2, human prostate cancer cell line DU-<NUM> and human colorectal cancer cell line HT29),. The survival rates of these cancer cells decreased in a dose-dependent manner. The effect of the vehicle group (test-substance solution <NUM>, seawater mineral solution with a final concentration of <NUM>%) to reduce survival rates of various cancer cells was considered to be equivalent to that of a group (test-substance solution <NUM>: NAnO3 with a final concentration of <NUM>%).

From the above results, it was observed that the growth suppression effect of NAnO3 on a cancer cell line varies in a dose-dependent manner under the test conditions; however, no significant difference was observed between test-substance solution <NUM> (<NUM>% NAnO3) and test-substance solution <NUM> (<NUM>% sea-water mineral solution).

The anti-tumor effect of a combination of NAnO3 and an existing anticancer agent (doxorubicin, gemcitabine, or cisplatin) against MIAPaCa-<NUM> cells (see, Table <NUM>) was examined. Only the combination with cisplatin is part of the invention. The examples concerning combinations with other anticancer agents are reference examples.

The following anticancer agents were used.

A cell suspension was prepared such that a MIAPaCa-<NUM> viable cell count became <NUM> × <NUM><NUM> cells/mL. The cell suspension (<NUM>) was seeded in each of wells of a <NUM> well plate, allowed to stand in a CO<NUM> incubator set at <NUM> and <NUM>% CO<NUM> and cultured for <NUM> hours or more to allow the cells to adhere to the bottom of the wells. After the completion of culture, the medium was removed from the wells and the following test-substance solutions (<NUM>) was added. The same operation was also repeated after <NUM> hours and <NUM> hours.

DMEM medium contains a <NUM> v/v% FBS (Hana-nesco Bio) and <NUM> × Penicillin-Streptomycin-Glutamine (GIBCO Cat#<NUM>). The <NUM> v/v% physiological saline /DMEM medium was prepared by adding physiological saline (<NUM>) to DMEM medium (<NUM>). The <NUM> v/v% NAnO3/DMEM medium was prepared by adding NAnO3 (<NUM>) to DMEM medium (<NUM>). The anticancer agent/DMEM medium was prepared by diluting an anticancer agent <NUM> fold with physiological saline, and then adding DMEM medium (<NUM>) to <NUM> of the solution. The anticancer agent + <NUM> v/v% NAnO3/DMEM medium was prepared by diluting an anticancer agent <NUM> fold with NAnO3, and thereafter, adding DMEM medium (<NUM>) to the solution (<NUM>).

After the completion of culture, the medium was removed and washing with PBS (-) was made. To each of the wells of the plate, a <NUM>% neutral red (NR) (Kanto Chemical Co. , Lot No.806W2294) D-MEM solution (<NUM>) was added and the plate was allowed to stand in a CO<NUM> incubator for <NUM> hours. After <NUM> hours, the NR solution was removed and washing was made with PBS (-). To each well, <NUM> of a fixation/extraction liquid (ethanol <NUM>% (v/v) glacial acetic acid <NUM>% (v/v) aqueous solution) was added and shaking was made for <NUM> minutes. Thereafter, the value of the absorbance was measured by using a microplate reader (EL × <NUM>, BIO-TEC INSTRUMENTS, INC. , wavelength: <NUM>).

Provided that the absorbance of DMEM medium (test-substance solution <NUM>) was regarded as <NUM>%, the cell survival rate (%) of each test group was obtained (<FIG>).

A cell suspension was prepared such that MIAPaCa-<NUM> viable cell count became <NUM> × <NUM><NUM> cells/mL. The cell suspension (<NUM>) was seeded in each of wells of a <NUM> well plate, allowed to stand in a CO<NUM> incubator set at <NUM> and <NUM>% CO<NUM> and cultured for <NUM> hours or more to allow the cells to adhere to the bottom of the plate. After the completion of culture, the medium was removed from the plate and the following test-substance solutions were added.

Test-substance solutions <NUM> to <NUM> were prepared in the same manner as in Section <NUM> and used. Test-substance solutions <NUM> to <NUM> were prepared by adding each anticancer agent/DMEM medium (<NUM>) and, in several minutes later, adding <NUM> of NAnO3. The same operation was also repeated after <NUM> hours and <NUM> hours.

After the completion of culture, the medium was removed and washing with PBS (-) was made. To each of the wells of the plate, a <NUM>% neutral red (NR) (Kanto Chemical Co. , Lot No.806W2294) D-MEM solution (<NUM>) was added and the plate was allowed to stand in a CO2 incubator for <NUM> hours. After <NUM> hours, the NR solution was removed and washing was made with PBS (-). To each plate, <NUM> of a fixation/extraction liquid (ethanol <NUM>% (v/v), glacial acetic acid <NUM>% (v/v) aqueous solution) was added and shaking was made for <NUM> minutes. Thereafter, the value of the absorbance was measured by using a microplate reader (EL × <NUM>, BIO-TEC INSTRUMENTS, INC. , wavelength: <NUM>).

As a result, in the experiment of concomitant administration, no difference was observed in the cell survival rate between the anticancer agents prepared with physiological saline and the anticancer agents prepared with NAnO3. Because of this, in the experiment of sequential administration, the exposure to the anticancer agents prepared with physiological saline was carried out, and then the exposure to the <NUM>% anticancer agents prepared with NAnO3 were carried out to examine the cell survival rate. As a result, the cell survival rate of groups exposed only to doxorubicin, gemcitabine and cisplatin were <NUM>%, <NUM>% and <NUM>%, respectively. In contrast, the cell survival rate of groups exposed also to NAnO3 were <NUM>%, <NUM>% and <NUM>%, respectively. In this case, the apparent suppression was observed. Furthermore, cisplatin exhibited higher suppression than NAnO3 alone exhibiting a cell survival rate of <NUM>%.

From the above results, in simultaneous exposure cases where cancer cells were exposed to each anticancer agent prepared with NAnO3, the proliferation of the cancer cells was not suppressed. In contrast, in the cases where the cancer cells were exposed to anticancer agents, and thereafter, exposed to NAnO3, the proliferation of the cells was apparently suppressed compared to the exposure to anticancer agents alone. In particular, in a combination use with cisplatin, it was presumed that a synergistic effect was produced.

To examine the anti-tumor effect of NAnO3 in vivo, NAnO3 was administered to MIAPaCa-<NUM> (pancreatic cancer cells) transplanted mice and its pharmacological effect was examined.

Animals (<NUM> nude mice (BALB/c-nu (BALB/cAnN. Cg-foxn1nu/CrlCrlj), female, <NUM> weeks old)) were purchased from Japan Charles River. At the day (Day <NUM> of the transplation) after the completion of the acclimatization period for a week, MIAPaCa-<NUM> cells (<NUM> × <NUM><NUM> cells per mouse) were subcutaneously transplanted to the back of all of the animals in accordance with a routine method using Matrigel (Corning Matrigel basal membrane matrix). Day <NUM> after the completion of transplantation, individuals having a tumor volume of <NUM><NUM> or more were selected and grouped into six (<NUM> mice/group) so as to have the same average tumor volume. NAnO3 (<NUM> ≈ <NUM>) was forcibly administered via the oral route once a day for <NUM> days or intermittently (three times per week) administered intravenously and intratumorally. During the breeding period, the mice were raised in accordance with a routine method and the body weight and the tumor diameter were periodically measured. The experimental regimen is shown in Table <NUM>.

Body weights of individuals of each group were measured on Day <NUM>, <NUM>, <NUM> and <NUM> after the initiation of experiment (Table <NUM> and <FIG>). As a result, the body weight changes shown in all test groups compared to Group <NUM> (control) in all the periods were almost the same. In any of the test groups, no abnormality was observed in general conditions. Photographs of mice on Day <NUM> belonging to each of test groups are shown in <FIG>.

Transplantation sites were observed visually and by touch, and the major axis and minor axis of tumors were measured by an electronic caliper. Tumor volumes were calculated (table <NUM>, <FIG>) in accordance with the following expression: <MAT>
<IMG>.

Average tumor volume of Group <NUM> (control) was <NUM> ± <NUM><NUM> at the time of grouping and increased up to <NUM> ± <NUM><NUM> at the end of the administration period. From this, it was determined that MIAPaCa-<NUM> pancreatic cancer cells transplanted were successfully proliferated. The average tumor volume of oral administration group <NUM> (a dose of <NUM>/kg) were <NUM> ± <NUM><NUM> (grouping time) and <NUM> ± <NUM><NUM> (the end of the administration period); that of oral administration group <NUM> (a dose <NUM>/kg), were <NUM> ± <NUM><NUM> (grouping time) and <NUM> ± <NUM><NUM> (the end of the administration period); and furthermore, that of oral administration group <NUM> (a dose of <NUM>/kg) were <NUM> ± <NUM><NUM> (grouping time) and <NUM> ± <NUM><NUM> (the end of the administration period). No significant difference was observed; however, an increase of tumor volume tends to be suppressed in a dose-dependent manner. In intravenous administration group <NUM>, the average tumor volume was <NUM> ± <NUM><NUM> (grouping time) and <NUM> ± <NUM><NUM> (the end of the administration period). A tendency of significant growth suppression was shown. In intratumoral group <NUM>, the average tumor volume was <NUM> ± <NUM><NUM> (grouping time) and <NUM> ± <NUM><NUM> (the end of the administration period). Tendency of growth suppression was shown compared to Group <NUM> (control). Particularly, a significant reduction of tumor volume was confirmed in a single case at the time of measurement on Day <NUM>.

From the above results, NAnO3 exhibits a cancer cell growth suppression effect by any of oral administration, intravenous administration, and intratumoral administration, and significant side effects such as weight loss were not observed.

Based on the results of Example <NUM>, <NUM> nude mice (female, <NUM> weeks old (BALB/c-nu)), to which MIAPaCa-<NUM> human pancreatic cancer cells were transplanted, were intratumorally administered with NAnO3 (<NUM>µL/body) three times per week. <NUM> days after the completion of administration, the tumor tissues were excised out and subjected to histopathological examination. The rate of cells positive to gamma-H2AX was calculated.

The tumor tissues were fixed with <NUM>% neutral formalin and embedded in paraffin in accordance with a routine method, and subjected to immunohistochemical staining using an anti-gamma-H2AX antibody (Histone H2AX phosphorylated antibody (Ser <NUM>), OxiSelect (R) DNA double strand cleavage staining kit, company: Cell Biolab). The obtained specimens were observed through objective lens (<NUM> times magnification) and <NUM> sites per specimen were randomly selected and photographed. Thereafter, the number of cells where double stranded DNA was cleaved was counted (Table <NUM>).

Gamma-H2AX (phosphorylated histone protein) is a marker for double stranded DNA cleavage. From the above results, intratumoral double stranded DNA cleavage was confirmed even if it is extremely slight. It is so far known that reactive oxygen species such as ozone strongly damages DNA. Various anticancer agents such as cisplatin and doxorubicin are deemed to work based on the mechanism of damaging DNA. However, these drugs have a problem of giving damage to normal cells. In contrast, NAnO3 was found to exert an anticancer effect without virtually damaging DNA.

The ozone water for use according to the invention of the present application is easily absorbed in cells and metabolized in vivo without changing a chemical structure; and exerts an anticancer effect without inducing an immune response and can be used in combination with other anticancer agent(s). If it is desired to enhance the efficacy including DNA damage of the composition, the concentrations of minerals and ozone can be changed or light, ultrasonic wave and the like can be used in combination (Non Patent Document <NUM>). It has been currently found that Inducer, reactive oxygen (ROS: Reactive Oxygen Species), is important for activating drugs including nivolumab (Non Patent Document <NUM>).

Claim 1:
Ozone water comprising ozone nano-bubbles for use in treating or preventing a cancer,
wherein the ozone water comprises a Na ion, a Mg ion, a K ion and/or a Ca ion,
wherein the ozone water further comprises at least one selected from the group consisting of a sulfur ion, a boron ion, a lithium ion, a silicon ion, a zinc ion, an iron ion and a strontium ion,
wherein the ozone water is to be administered sequentially with other anticancer agent(s), and
wherein said other anticancer agent(s) is cisplatin.