Composition for preventing and / or treating cardiotoxicity induced by anthracycline anticancer agent

Abstract

To provide a mitochondrial DNA nucleoid aggregation inhibitor.SOLUTION: The present invention relates to a mitochondrial DNA nucleoid aggregation inhibitor containing vitamin K1 and / or vitamin K2. The present invention also relates to a pharmaceutical composition for preventing and / or treating cardiotoxicity induced by use of an anthracycline anticancer agent, the pharmaceutical composition containing the nucleoid aggregation inhibitor, and also relates to a food composition for prevention and / or alleviation of side effects derived from an anthracycline anticancer agent, the food composition containing vitamin K1 and / or vitamin K2 as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an inhibitor of mitochondrial DNA nucleoid aggregation, which contains vitamin K1 and / or vitamin K2. The present invention also relates to a pharmaceutical composition containing the nucleoid aggregation inhibitor for preventing and / or treating cardiotoxicity induced by anthracycline anticancer drugs, and a food composition containing vitamin K1 and / or vitamin K2 as active ingredients for preventing and / or alleviating side effects caused by anthracycline anticancer drugs. [Background technology]

[0002] There has been much research and product development into cancer prevention or treatment drugs, and many drugs are actually used in clinical practice. Among them, anthracycline anticancer drugs are highly effective chemotherapeutic agents and are therefore used to treat many cancers, including leukemia, lymphoma, breast cancer, uterine cancer, ovarian cancer, bladder cancer, and lung cancer, and are often used in treatment regimens for childhood cancers.

[0003] On the other hand, side effects of anthracycline anticancer drugs are related to cardiac complications (e.g., cardiomyopathy, decreased left ventricular function, heart failure, etc.) caused by cardiotoxicity, but this cardiotoxicity is dose-dependent and may result from cumulative exposure. Although there are individual variations, progressive cardiac dysfunction occurs with increasing cumulative dose. For example, at 200 mg / m2 of doxorubicin, 2 Diastolic dysfunction occurs at 400-600 mg / m 2 Systolic dysfunction is thought to occur in this condition. Diastolic dysfunction alone is often asymptomatic, but systolic dysfunction is often accompanied by signs and symptoms of heart failure. Anthracycline anticancer drugs also affect cardiac progenitor cells, causing delayed cardiac dysfunction in children 10 to 20 years after treatment (Non-Patent Document 1). A recent trend in cancer treatment is to consider improving quality of life (QOL) as an important therapeutic effect in addition to extending the survival time of cancer patients, and how to reduce the side effects of anticancer drugs is an important issue.

[0004] To avoid the occurrence of such side effects or to alleviate the symptoms of side effects, it may be possible to address the issue by, for example, reducing the dose of the anticancer drug per administration. However, reducing the dose of the anticancer drug will reduce its anticancer effect and ultimately lead to a prolonged treatment period. Furthermore, administering an anticancer drug at a low dose over a long period of time may result in a total dose that is higher than normal, which may worsen the symptoms of side effects more than expected. Therefore, in order to avoid reducing the effectiveness of the anticancer drug, there is a strong demand for the development of drugs that can alleviate side effects without reducing the dose.

[0005] To date, strategies to prevent or reduce the incidence of cardiotoxicity caused by anthracyclines have included: (1) Use of anthracycline liposomal preparations (e.g., Doxil), (2) Concomitant use of antioxidants (e.g., vitamin E, piperidine nitroxide, probucol, etc.) or iron chelators (e.g., dexrazoxane), (3) Concomitant use of cardiovascular drugs (e.g., calcium channel blockers, beta-blockers (carvedilol), renin-angiotensin system inhibitors), (4) Targeted therapy, etc. However, problems remain, such as insufficient suppression of cardiotoxicity and the occurrence of other side effects (Non-Patent Documents 1 and 2). Specifically, dexrazoxane is the only drug approved for reducing cardiotoxicity and the onset of heart failure in cancer patients receiving anticancer drugs. However, despite its clinical efficacy, it is difficult to achieve the same results as anthracycline anticancer drugs, such as doxorubicin and epirubicin, at a dose of 300 mg / m 2 ~500mg / m 2 Dexrazoxane is only approved for the treatment of patients with metastatic breast cancer who have already received cumulative doses of dexrazoxane, and is not approved for use in children and adolescents. It has also been reported that the use of dexrazoxane has drawbacks, such as interfering with the antitumor activity of anthracyclines, inducing secondary malignancies, and inducing blood and bone marrow disorders.

[0006] Non-patent documents 3 and 4 each describe that DNA intercalators such as doxorubicin intercalate into mitochondrial DNA, thereby inducing enlargement of mitochondrial DNA nucleoids (aggregates or nucleoid clusters), and suggest that this is likely to be involved in significant clinical toxicity.

[0007] The present inventors have found that suppression of the mitochondrial fission factor Drp1 induces the aggregation of numerous nucleoids, forming large clusters, in association with mitochondrial fusion (Non-Patent Document 5). Furthermore, the present inventors have reported that, as predicted from the above findings, nucleoids aggregate intracellularly in the cardiac muscle of Drp1-deficient mice, leading to uneven distribution of the respiratory chain within mitochondria, resulting in decreased overall cardiac respiratory activity and impaired myofibril formation, leading to cardiac dysfunction and lethality in the neonatal period (Non-Patent Document 6). These results demonstrate, for the first time, that structural control of nucleoids is essential for neonatal cardiac growth in mice and that the coordinated dynamics of mitochondrial membranes and DNA play an important role in vivo.

[0008] Vitamin K, a fat-soluble vitamin, generally functions as a coenzyme for γ-glutamyl carboxylase (GGCX) and is known to be involved in blood coagulation and bone formation. Naturally occurring vitamin K is broadly classified into two types: vitamin K1 (phylloquinone, hereinafter also referred to as "PK"), synthesized by plants, and vitamin K2 (menaquinone-n (n = 1-14), hereinafter also referred to as "MK-n"), produced by microorganisms such as intestinal bacteria and found in large amounts in fermented foods and animal bodies. Vitamin K shares a common structure with 2-methyl-1,4-naphthoquinone (vitamin K3, menadione, hereinafter also referred to as "MD"), with side chains of varying lengths and degrees of unsaturation attached to the carbon-3 position (Non-Patent Document 7). However, to date, it has not been known that vitamin K has the effect of inhibiting the aggregation of mitochondrial DNA nucleoids, and there have been no reports showing that it has a preventive or therapeutic effect on cardiotoxicity induced by anthracycline anticancer drugs. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Kyorin Medical Association Journal, 2014, Vol.45, 31-34 [Non-patent document 2] Progress in Cardiovascular Diseases, 2007, Vol.49, 330-352 [Non-patent document 3] Oncogene, 2009, Vol.28, 3880-3891 [Non-patent document 4] Toxicol. Appl. Pharmacol., 2016, Vol.302, 31-40 [Non-patent document 5] Proc. Natl. Acad. Sci. USA, 2013, Vol.110, 11863-11868 [Non-patent document 6] Mol. Cell. Biol., 2015, Vol.35, 211-223 [Non-Patent Document 7] Chemistry and Biology, 2018, Vol.56, No.1, 26-32 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention addresses the issue of effectively preventing and / or treating (alleviating) cardiotoxicity caused by anthracycline anticancer drugs without reducing the dosage of the anticancer drugs and without significantly interfering with the anticancer effect. Specifically, the present invention aims to provide a pharmaceutical composition and a food composition for preventing and / or treating cardiotoxicity induced by anthracycline anticancer drugs. [Means for solving the problem]

[0011] As a result of intensive research into the above-mentioned problems, the present inventors have found that vitamin K1 and / or vitamin K2 inhibit mitochondrial DNA nucleoid aggregation, and have also discovered for the first time that the use of a pharmaceutical composition or food composition containing vitamin K1 and / or vitamin K2 as an active ingredient can effectively prevent and / or treat cardiotoxicity induced by anthracycline anticancer drugs, leading to the completion of the present invention. One embodiment of the present invention is, for example, as follows.

[0012] [1] A mitochondrial DNA nucleoid aggregation inhibitor containing vitamin K1 and / or vitamin K2 (hereinafter sometimes referred to as "the mitochondrial DNA nucleoid aggregation inhibitor of the present invention" or "the nucleoid aggregation inhibitor of the present invention"). [2] The nucleoid aggregation inhibitor according to [1] above, wherein vitamin K1 and / or vitamin K2 is phylloquinone or menaquinone-4. [3] A pharmaceutical composition for preventing and / or treating cardiotoxicity induced by an anthracycline anticancer drug, comprising the nucleoid aggregation inhibitor described in [1] or [2] above (hereinafter, also referred to as the "medicament of the present invention" or the "pharmaceutical composition of the present invention"). [4] The pharmaceutical composition according to [3] above, wherein the cardiotoxicity is cardiotoxicity caused by respiratory failure. [5] The pharmaceutical composition according to [3] above, wherein the nucleoid aggregation inhibitor is administered before the administration of an anthracycline anticancer drug. [6] The pharmaceutical composition according to any one of the above [3] to [5], wherein the anthracycline anticancer drug is doxorubicin. [7] A method for preventing and / or treating cardiotoxicity induced by an anthracycline anticancer drug, comprising administering a pharmaceutically effective amount of the agent according to [1] or [2] above to a subject suffering from cancer who is undergoing treatment with an anthracycline anticancer drug. [8] A method for preventing and / or treating side effects of an anthracycline anticancer drug, comprising administering a pharmaceutically effective amount of the agent according to [1] or [2] above to a subject suffering from cancer who is undergoing treatment with an anthracycline anticancer drug. [9] The method according to [7] or [8] above, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain tumor, breast cancer, esophageal cancer, colon cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, and thyroid cancer.

[10] A food composition for preventing and / or reducing side effects of anthracycline anticancer drugs, containing vitamin K1 and / or vitamin K2 as active ingredients (hereinafter sometimes referred to as the "food composition of the present invention").

[11] A food composition for preventing and / or reducing cardiotoxicity induced by anthracycline anticancer drugs, comprising vitamin K1 and / or vitamin K2 as active ingredients.

[12] The food composition according to

[10] or

[11] above, which is taken before administration of an anthracycline anticancer drug.

[13] A food composition according to any one of

[10] to

[12] above, which is labeled to "reduce the risks of anticancer drugs," "protect the body from anticancer drugs," and / or "relieve pain caused by anticancer drugs."

[14] The food composition according to any one of

[10] to

[13] above, characterized in that it is prepared as a supplement, a functional food, a health food, a special purpose food, a health food, a specified health food, or a nutrient-functional food.

[15] The food composition according to

[14] above, characterized in that it is prepared in the form of a tablet, pill, capsule, powder, granule, fine granule, troche, liquid, nasogastric tube feeding agent, or enteral nutrition agent.

[16] Use of vitamin K1 and / or vitamin K2 for producing a medicament for preventing and / or treating cardiotoxicity induced by anthracycline anticancer drugs.

[17] Vitamin K1 and / or vitamin K2 for use in the prevention and / or treatment of cardiotoxicity induced by anthracycline anticancer drugs.

[18] Vitamin K1 and / or vitamin K2 for use in the prevention and / or treatment of side effects of anthracycline anticancer drugs.

[19] Vitamin K1 and / or vitamin K2 for producing a nucleoid aggregation inhibitor.

[20] A method for screening a compound for preventing and / or treating cardiotoxicity induced by an anthracycline anticancer drug, comprising: (i) mixing a test compound with HeLa cells, adding an anthracycline anticancer drug thereto, staining the nucleoids of the treated HeLa cells with SYBR Green I, and evaluating the degree of nucleoid aggregation caused by the test compound based on comparison with a control group not containing the test compound; (ii) selecting a compound that inhibits nucleoid aggregation more than a control group based on the evaluation in (i) above; A screening method (hereinafter also referred to as "the screening method of the present invention") comprising the steps of: [Effects of the Invention]

[0013] According to the present invention, it is possible to provide an inhibitor of mitochondrial DNA nucleoid aggregation, which contains vitamin K1 and / or vitamin K2, which are safe vitamins. By applying the nucleoid aggregation inhibitor of the present invention to a subject suffering from cancer undergoing treatment with an anthracycline anticancer drug, it is possible to effectively prevent and / or treat cardiotoxicity induced by the anthracycline anticancer drug without interfering with the cell proliferation inhibitory effect of the anthracycline anticancer drug. In other words, according to the present invention, it is possible to provide a pharmaceutical composition or food composition for preventing and / or treating side effects (cardiotoxicity) of anthracycline anticancer drugs, which contains vitamin K1 and / or vitamin K2 as active ingredients. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1A shows a fluorescence micrograph of mitochondrial DNA and nucleoids stained with SYBR Green I and whole mitochondria stained with MitoTracker Red after the addition of doxorubicin (500 nM). Figure 1B shows a comparison of basal and maximal respiratory activity between doxorubicin (500 nM)-treated cells and untreated cells (DMSO only added) (control). [Figure 2] Figure 2A shows mice that were given corn oil (control), MK-4, or Mito-TEMPO daily for 7 days starting 1 week before DXR administration. On the 8th day, each group received DXR, followed by corn oil (control), MK-4, or Mito-TEMPO daily for another 4 days. On the 13th day, each group received DXR, and the mice were dissected on the 14th day. Fluorescence micrographs were taken of the mitochondrial DNA and nucleoids of cardiomyocytes stained with anti-DNA antibodies, and the entire mitochondrial inner membrane was stained with anti-cytochrome c antibodies. Figure 2B shows comparative data on the amount of mRNA encoding brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) for each group, after total RNA was extracted from the hearts of mice from each group. [Figure 3]Figure 3 shows the change in HeLa cell viability (%) over time for HeLa cells cultured for 24 hours, to which corn oil was added, followed by MK-4 24 hours later and DXR 24 hours later, followed by cells 48 hours later; HeLa cells cultured for 24 hours, to which corn oil was added, followed by DXR 24 hours later, followed by cells 48 hours later; and HeLa cells cultured for 24 hours, to which corn oil was added, followed by cells 72 hours later (control group). DETAILED DESCRIPTION OF THE INVENTION

[0015] The terms used herein are defined as follows: Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] As used herein, "vitamin K1" refers to a compound represented by the following formula:

[0017] [ka]

[0018] It is a single compound (phylloquinone: PK) found primarily in plant foods such as seaweed, green and yellow vegetables, and vegetable oils, and is the primary dietary source of vitamin K1 for humans, generally accounting for more than 90% of total vitamin K intake. PK is preferred for human use because it is safe even in cases of significant overdose.

[0019] As used herein, "vitamin K2" refers to a compound represented by the following formula:

[0020] [ka]

[0021] (wherein n is an integer of 1 to 14) (menaquinone-n (n = 1 to 14):MK-n). MK-4 is found in relatively high amounts in animal foods such as chicken meat and eggs, while menaquinones with long side chains, such as MK-7, MK-8, and MK-9, are found in large amounts in fermented foods such as cheese, yogurt, and natto. In addition to vitamin K derived from diet, menaquinones (mainly MK-10 and MK-11, with small amounts of MK-7, MK-8, MK-9, and MK-12) synthesized by intestinal bacteria are also known. It has also been reported that MD is produced as a metabolite during the small intestinal absorption of PK, transported to peripheral tissues via the blood, and converted to MK-4 (Non-Patent Document 7).

[0022] According to the Ministry of Health, Labor and Welfare's "Dietary Reference Intakes for Japanese (2020 edition)," the recommended intake of vitamin K (mainly consisting of vitamins K1 and K2) is 150 μg / day for both men and women aged 18 and over. Since there have been no reported cases of side effects even with the use of 45 mg / day of vitamin K2, no upper limit for vitamin K intake has been set in Japan's dietary reference intakes.

[0023] Vitamin K is widely distributed in foods. Among animal foods, eggs and chicken are excellent sources, containing approximately 39 μg to 50 μg per 100 g of food. Among plant foods, spinach (boiled), komatsuna (boiled), chrysanthemum (boiled), daikon radish leaves (boiled), and broccoli (microwaved) contain approximately 220 μg to 460 μg per 100 g of food, while natto contains approximately 600 μg of vitamin K2 per 100 g. Furthermore, vegetable oils (olive oil, soybean oil, etc.) and products made from vegetable oils, such as margarine and salad dressing, are also excellent sources, containing approximately 10 μg to 300 μg of vitamin K per 100 g of oil.

[0024] As used herein, "containing vitamin K1 and / or vitamin K2" means containing either vitamin K1 or vitamin K2, or a mixture of vitamin K1 and vitamin K2. Vitamin K3 (MD), which is metabolized in the body of a subject (mammal), and converted to vitamin K2, may also be contained. Of these, those containing either vitamin K1 or vitamin K2 are preferred, and those containing vitamin K2 are more preferred.

[0025] As used herein, the term "mitochondrial DNA nucleoid" refers to a structure formed within the mitochondrial matrix together with DNA-binding proteins such as mitochondrial transcription factor A (TFAM), a transcription factor for mitochondrial DNA. For example, more than 1,000 nucleoid structures are observed in normal HeLa cells. Meanwhile, the present inventors have reported that when the mitochondrial fission factor Drp1 is inhibited, numerous nucleoids aggregate (assemble) to form large clusters in association with mitochondrial fusion due to impaired mitochondrial fission (Non-Patent Document 5).

[0026] As used herein, the term "mitochondrial DNA nucleoid aggregation inhibitor" refers to an agent that inhibits the aggregation of the nucleoids and maintains the nucleoids in a dispersed state.

[0027] As used herein, "respiratory failure" refers to a state in which nucleoids aggregate within cells due to mitochondrial division failure, resulting in uneven distribution of the respiratory chain within mitochondria and resulting in decreased respiratory activity throughout the tissue. As described in the Examples below, the present inventors have found for the first time that the addition of doxorubicin to cells results in the enlargement (aggregation) of mitochondrial DNA nucleoids, which in turn reduces respiratory activity.

[0028] As used herein, "cardiotoxicity caused by respiratory failure" means that when respiratory failure occurs in cardiomyocytes, the respiratory activity of the entire myocardium decreases, and at that time, myofibril formation becomes insufficient, thereby inducing cardiac dysfunction, i.e., cardiotoxicity.

[0029] The inhibitory effect (preventive and / or therapeutic effect) of "cardiotoxicity" can be evaluated by quantifying the blood levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), biomarkers for detecting cardiac dysfunction. ANP is primarily secreted by the atria, and BNP is primarily secreted by the ventricles. Blood levels of ANP and BNP increase with the onset of heart failure. In particular, BNP increases significantly as the severity of heart failure increases. Therefore, maintaining blood ANP and / or BNP levels at a level lower than that observed in the untreated setting (administration of an anthracycline anticancer drug alone) is considered to be effective in preventing and / or treating cardiotoxicity caused by an anthracycline anticancer drug. The preventive and / or therapeutic effect of cardiotoxicity can be evaluated not only by directly quantifying and comparing the blood levels of ANP and / or BNP, but also by quantifying and comparing the amounts of mRNA encoding ANP and / or BNP.

[0030] As used herein, "anthracycline anticancer agents" refer to compounds having an anthracene structural center. Examples of anthracycline anticancer agents include doxorubicin, aclarubicin, amrubicin, idarubicin, epirubicin, daunorubicin, pirarubicin, and valrubicin. In some embodiments, the anthracycline anticancer agent is doxorubicin.

[0031] As used herein, the term "side effects of anthracycline anticancer drugs" includes those generally recognized in the art, such as physical disorders such as loss of appetite, general fatigue, pain, dyspnea, skin symptoms, nausea, vomiting, diarrhea, fever, alopecia, anosmia, organ disorders (e.g., renal dysfunction (nephrotoxicity), cardiac dysfunction (cardiotoxicity), liver dysfunction (hepatotoxicity)), interstitial pneumonia, organ failure associated with peripheral circulatory failure, and bone marrow suppression. Mental distress associated with these physical disorders, such as anxiety, restlessness, loss of interest, emotional blunting, insomnia, feelings of alienation, fear, adjustment disorder, depression, delirium, and the like, are also encompassed as side effects. Cardiotoxicity, in particular, is a serious side effect of anthracycline anticancer drug therapy, is dose-dependent, and may result from cumulative exposure.

[0032] As used herein, "prevention" means inhibiting, suppressing, controlling, slowing down, or stopping the onset of clinical symptoms of a disease or the like in a subject who may develop the disease or the like but has not yet developed the disease, or who is concerned about the recurrence of the disease or the like after treatment of the disease or the like.

[0033] As used herein, "treatment" means curing, remission, alleviating, or delaying (suppressing) the worsening of clinical symptoms of a disease, illness, disorder, etc. (hereinafter referred to as "disease, etc.") in a subject who has developed the disease, etc.

[0034] As used herein, the term "prophylactic and / or therapeutic agent" includes pharmaceutical compositions or food compositions for prophylaxis and / or treatment.

[0035] As used herein, the term "pharmaceutically effective amount" refers to a dose of an active ingredient sufficient to achieve a preventive or therapeutic purpose in a subject.

[0036] As used herein, the term "subject" refers to a subject to which the pharmaceutical composition or food composition of the present invention is administered or consumed. Examples of the "subject" include mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, cows, horses, sheep, and monkeys, with humans being preferred.

[0037] As used herein, the "cancer" in "a subject suffering from cancer" is not particularly limited, but examples include bladder cancer, bone cancer, brain tumor, breast cancer, esophageal cancer, gastrointestinal cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, prostate cancer, sarcoma, stomach cancer, thyroid cancer, etc.

[0038] Mitochondrial DNA nucleoid aggregation inhibitor of the present invention As shown in Test Example 2 described below, vitamin K1 and / or vitamin K2 can inhibit mitochondrial DNA nucleoid aggregation induced by administration of doxorubicin and maintain the dispersed state, and therefore can be suitably used as an active ingredient of a mitochondrial DNA nucleoid aggregation inhibitor. Furthermore, a medicine containing the nucleoid aggregation inhibitor as an active ingredient can be suitably used as a medicine for preventing and / or treating cardiotoxicity caused by mitochondrial dysfunction induced by mitochondrial DNA nucleoid aggregation, particularly respiratory failure associated with the administration of anthracycline anticancer drugs.

[0039] The nucleoid aggregation inhibitor of the present invention may be either an agent composed only of vitamin K1 and / or vitamin K2, or a composition comprising vitamin K1 and / or vitamin K2 and a pharmaceutically acceptable carrier, etc. The nucleoid aggregation inhibitor of the present invention can be administered to a subject in a pharmaceutically effective amount. Examples of methods for administering the nucleoid aggregation inhibitor of the present invention include oral administration, intravenous administration, intraperitoneal administration, transdermal administration, intramuscular administration, intranasal administration, and mucosal administration, with oral administration being preferred.

[0040] The dosage (i.e., a pharmaceutically effective amount) of the nucleoid aggregation inhibitor of the present invention is not particularly limited and varies depending, for example, on the subject of administration, symptoms, the type and dosage of the anthracycline anticancer agent, dosage form, administration route, etc. However, when orally administered to a human adult patient, the daily dose is 0.5 mg or more and 1000 mg or less in terms of the active ingredients vitamin K1 and / or vitamin K2. The lower limit, converted into vitamin K1 and / or vitamin K2, is preferably 0.5 mg or more, more preferably 1 mg or more, and even more preferably 2 mg or more. Meanwhile, the upper limit, converted into vitamin K1 and / or vitamin K2, is preferably 500 mg or less, more preferably 100 mg or less.

[0041] The number of times the nucleoid aggregation inhibitor of the present invention is administered is not particularly limited, and may be, for example, 1 to 3 times a day, and may be administered before, after, or between meals. The administration period of the nucleoid aggregation inhibitor of the present invention is preferably started before (about 1 week before) administration of an anthracycline anticancer agent, and continued during the administration period of the anthracycline anticancer agent.

[0042] The pharmaceutical of the present invention (or the pharmaceutical composition of the present invention) As shown in Test Example 2 below, nucleoid aggregation was inhibited by administering vitamin K1 and / or vitamin K2 to mice before administering doxorubicin, and cardiac failure could be significantly prevented and suppressed. Furthermore, as shown in Test Example 3 below, doxorubicin treatment after addition of MK-4 did not inhibit the tumor cell proliferation inhibitory effect of doxorubicin. Therefore, a pharmaceutical (or pharmaceutical composition) containing vitamin K1 and / or vitamin K2 as an active ingredient can be suitably used as a pharmaceutical for preventing and / or treating side effects, particularly cardiotoxicity, induced by anthracycline anticancer drugs.

[0043] The medicament of the present invention may be either a medicament consisting only of vitamin K1 and / or vitamin K2, or a pharmaceutical composition comprising vitamin K1 and / or vitamin K2 and a pharmaceutically acceptable carrier, etc. The medicament or pharmaceutical composition of the present invention can be administered to a subject in a pharmaceutically effective amount. The medicament or pharmaceutical composition of the present invention can be administered, for example, by oral administration, intravenous administration, intraperitoneal administration, transdermal administration, intramuscular administration, intranasal administration, mucosal administration, etc., with oral administration being preferred.

[0044] Examples of pharmaceutically acceptable carriers include excipients (e.g., starch, corn starch, lactose, sugar, calcium carbonate, calcium phosphate, etc.), binders (e.g., starch, gum arabic, carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, etc.), lubricants (e.g., magnesium stearate, talc, hardened rapeseed oil, etc.), disintegrants (e.g., carboxymethylcellulose, talc, etc.), solvents (e.g., water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, thiamin mononitrate ... corn oil, olive oil, cottonseed oil, etc.), solubilizing agents (e.g., polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate, etc.), suspending agents (e.g., surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, etc.);Polysorbates, polyoxyethylene hydrogenated castor oil, etc.), isotonicity agents (e.g., sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.), buffers (e.g., buffer solutions such as phosphates, acetates, carbonates, and citrates), thickeners (e.g., sodium alginate, xanthan gum, sodium chondroitin sulfate, polyvinyl alcohol, povidone, etc.), preservatives (e.g., parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc.), antioxidants (e.g., sulfites, ascorbates (vitamin C), tocopherol (vitamin E), etc.), colorants ( Examples include water-soluble food tar dyes (e.g., food dyes such as Food Red No. 2 and No. 3, Food Yellow No. 4 and No. 5, and Food Blue No. 1 and No. 2), water-insoluble lake dyes (e.g., aluminum salts of the above-mentioned water-soluble food tar dyes), natural dyes (e.g., β-carotene, chlorophyll, and red iron oxide), sweeteners (e.g., saccharin sodium, dipotassium glycyrrhizinate, aspartame, and stevia), and coating substrates (e.g., polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose);

[0045] The pharmaceutical composition of the present invention can be prepared by mixing the above-mentioned components and then processing the mixture into preparations such as tablets, pills, capsules (including hard capsules, soft capsules, and microcapsules), powders, granules, fine granules, troches, liquids (including syrups, emulsions, and suspensions), nasal drops, and injections according to known methods.

[0046] Pharmaceutical compositions in the form of tablets, granules, or fine granules may be coated with the above-mentioned coating substrate by a method known per se for the purposes of taste masking, improving light stability, improving appearance, enteric coating, etc.

[0047] The content of vitamin K1 and / or vitamin K2 in the pharmaceutical (or pharmaceutical composition) of the present invention is not particularly limited and, for example, varies depending on the form of the formulation, but is 1% by weight or more and 100% by weight or less based on the total formulation. The lower limit is preferably 5% by weight or more, more preferably 30% by weight or more based on the total formulation. The upper limit is preferably 100% by weight or less, more preferably 95% by weight or less based on the total formulation.

[0048] The dosage of vitamin K1 and / or vitamin K2 (i.e., a pharmaceutically effective amount) is not particularly limited and varies depending on, for example, the subject of administration, symptoms, the type and dosage of the anthracycline anticancer agent, dosage form, administration route, etc., but when orally administered to a human adult patient, the daily dose is 0.5 mg or more and 1000 mg or less of the active ingredients vitamin K1 and / or vitamin K2. The lower limit, converted into vitamin K1 and / or vitamin K2, is preferably 0.5 mg or more, more preferably 1 mg or more, and even more preferably 2 mg or more. On the other hand, the upper limit, converted into vitamin K1 and / or vitamin K2, is preferably 500 mg or less, more preferably 100 mg or less.

[0049] The number of times vitamin K1 and / or vitamin K2 are administered is not particularly limited, and may be, for example, 1 to 3 times a day, and administration may be before, after, or between meals. The administration period of the nucleoid aggregation inhibitor of the present invention is preferably started before (about 1 week before) administration of an anthracycline anticancer agent, and continued during the administration period of the anthracycline anticancer agent.

[0050] Vitamin K1 and / or vitamin K2 can be used in combination with other therapies (e.g., chemotherapy, surgery (e.g., surgical resection, radiofrequency ablation, etc.), radiation therapy (e.g., X-rays, particle beam therapy, etc.)). This can enhance the therapeutic effects of cancer in subjects suffering from cancer (cancer patients) and reduce side effects. Of these, combination with chemotherapy is particularly preferred. Specific embodiments of combination with chemotherapy are shown below.

[0051] Vitamin K1 and / or vitamin K2 can be used in combination with other drugs (concomitant drugs) as long as their efficacy is not impaired. In this case, the timing of administration is not limited, and these drugs may be administered to a subject simultaneously or at different times. Furthermore, vitamin K1 and / or vitamin K2 and the concomitant drug may be administered in combination as a single preparation.

[0052] The dosage of the concomitant drug can be appropriately selected based on the clinically used dose, and the compounding ratio of vitamin K1 and / or vitamin K2 to the concomitant drug can be appropriately selected depending on the subject of administration, the administration route, the target disease, symptoms, the type of concomitant drug, etc.

[0053] When vitamin K1 and / or vitamin K2 are used to prevent cardiotoxicity induced by anthracycline anticancer drugs, concomitant medications include, for example, anticancer drugs other than anthracycline anticancer drugs (e.g., molecular targeted drugs, immune checkpoint inhibitors, chemotherapeutic agents), cardioprotective drugs (e.g., antioxidants, cardiovascular drugs), etc.

[0054] Examples of "molecular targeted drugs" include EGFR inhibitors such as afatinib, ertotinib, gefitinib, cetuximab, and panitumumab; ALK inhibitors such as crizotinib; EGFR / HER2 inhibitors such as lapatinib; HER2 inhibitors such as trastuzumab, trastuzumab emtansine, and betuzumab; bevacizumab, axitinib, sunitinib, sorafenib, pazopanib, regorafenib, These include angiogenesis inhibitors such as lenvatinib and ramucirumab; mTOR inhibitors such as eperolimus and temsirolimus; BCR-ABL inhibitors such as imatinib, dasatinib, and nilotinib; membranous differentiation antigen-targeting drugs such as ibritumovab tiuxetan, ofatumumab, rituximab, brentuximab vedotin, gemtuzumab ozogamicin, and mogamulizumab; denosumab; and bortezomib.

[0055] Examples of "immune checkpoint inhibitors" include anti-PD-1 antibodies such as nivolumab and pembrolizumab; anti-CTLA-4 antibodies such as ipilimumab; and anti-PD-L1 antibodies such as avelumab, atezolizumab, and durpalumab.

[0056] Examples of "chemotherapeutic agents" include alkylating agents such as busulfan, cyclophosphamide, dacarbazine, ifosfamide, melphalan, nimustine hydrochloride, procarbazine hydrochloride, ranimustine, temozolomide, and thiotepa; fluorouracil, mercaptopurine hydrate, cladripine, carmofur, cytarabine ocfosfate hydrate, cytarabine, doxifluridine, enocitabine, fludarabine phosphate; Antimetabolites such as gemcitabine hydrochloride, hydroxycarbamide, methotrexate, neolarabine, pemetrexed sodium hydrate, pentostatin, tegafur, tegafur / uracil combination, and tegafur / gimeracil / oteracil combination; vinca alkaloid drugs such as vinblastine hydrochloride, vincristine hydrochloride, vindesine hydrochloride, and vinorelbine tartrate; taxane drugs such as doxorubicin hydrate and paclitaxel; antitumor antibiotics such as leomycin hydrochloride and mitomycin C; topoisomerase inhibitors such as etoposide, irinotecan hydrochloride hydrate, nogitecan hydrochloride and sobuzoxane; platinum preparations such as carboplatin, cisplatin, nedaplatin and oxaliplatin; hormone preparations such as anastrozole, exemestane, letrozole, tamoxifen citrate, toremifene citrate, fadrozole hydrochloride hydrate, estramustine phosphate sodium, flutamide, goserelin acetate, leuprorelin acetate, medroxyprogesterone acetate and mepitiostane; anti-cancer streptococcal preparations such as picibanil; aceglatone, arsenic trioxide, Kawaratake polysaccharide powder, L-asparaginase, lentinan, levofolinate, mitotane, porfimer sodium, sizofiran, tamiparotene, tretinoin and ubenimex.

[0057] Examples of "cardioprotective drugs" include antioxidants such as ascorbic acid (vitamin C), α-tocopherol (vitamin E), glutathione, and probucol; and cardiovascular drugs such as calcium channel blockers (e.g., nifedipine, nicardipine, amlodipine, benidipine, barnidipine, manidipine, diltiazem, and verapamil), iloprost, phosphodiesterase-5 inhibitors (e.g., sildenafil), β-blockers (e.g., carvedilol), renin-angiotensin system inhibitors (e.g., losartan, valsartan, candesartan, captopril, enalapril, and aliskiren), and drugs for treating acute heart failure (e.g., ANP).

[0058] In some embodiments, the medicament (or pharmaceutical composition) of the present invention may be provided in the form of a kit, package (e.g., wrapping), or pharmaceutical set (and / or container) containing a written statement that the medicament (or pharmaceutical composition) can or should be used for preventing side effects, particularly cardiotoxicity, induced by anthracycline anticancer drugs. Such kits, packages, and pharmaceutical sets may include one or more containers filled with vitamin K1 and / or vitamin K2 and other drugs or substances (or ingredients). Examples of such kits, packages, and pharmaceutical sets include commercial kits, commercial packages, and commercial pharmaceutical sets appropriately targeted for the treatment and / or prevention of target diseases. The written statement included in such kits, packages, and pharmaceutical sets may include notices or inserts in the form mandated by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, indicating the government agency's approval of the manufacture, use, or sale of the product for human administration. The above kits, packages, and pharmaceutical sets include packaged products, and may also include structures configured for appropriate administration steps, or may include structures configured to achieve more desirable medical treatment and / or prevention, including the treatment and / or prevention of cancer.

[0059] Food composition of the present invention The food composition of the present invention may consist solely of vitamin K1 and / or vitamin K2, or may be a composition in which vitamin K1 and / or vitamin K2 is incorporated into a food (food additive, food, beverage, etc.). The food composition of the present invention is not particularly limited as long as it contains vitamin K1 and / or vitamin K2 and can be orally ingested by a subject, and there are no particular limitations on the type or form of the food composition. Furthermore, as shown in Test Example 2 below, cardiac failure was significantly suppressed by administering vitamin K1 and / or vitamin K2 to mice before administration of doxorubicin. Therefore, the food composition of the present invention can be used to prevent and / or alleviate side effects, particularly cardiotoxicity, induced by anthracycline anticancer drugs.

[0060] Examples of the food composition of the present invention include confectioneries such as lozenges, drops, candies, soda pops, gummies, and chewing gum; Western confectioneries such as cookies, crackers, biscuits, potato chips, bread, cakes, chocolates, donuts, puddings, and jellies; Japanese confectioneries such as rice crackers, yokan, daifuku, ohagi, manju, and castella; frozen desserts such as ice cream, popsicles, sherbet, and gelato; breads such as sliced ​​bread, French baguettes, and croissants; noodles such as udon, soba, Chinese noodles, and kishimen; fish paste products such as kamaboko, chikuwa, and fish sausage; meat products such as ham, sausage, hamburger steak, and corned beef; salt, pepper, miso, soy sauce, sauces, dressings, mayonnaise, ketchup, and sweeteners ( For example, condiments such as sugar, honey, powdered syrup, starch syrup, jam, marmalade, etc., and spices (e.g., mustard, pepper, etc.); teppanyaki foods such as Akashiyaki, takoyaki, monjayaki, okonomiyaki, yakisoba, and yakiudon; dairy products such as cheese, butter, margarine, and yogurt; various prepared foods such as natto, deep-fried tofu, tofu, konjac, dumplings, pickles, tsukudani (simmered foods in soy sauce), gyoza, shumai, croquettes, sandwiches, pizza, hamburgers, and salads; livestock products such as beef, pork, and chicken; seafood such as shrimp, scallops, clams, and kelp; various powders made from vegetables, fruits, plants, yeast, and algae; powdered oils and flavorings (vanilla, citrus fruits, bonito, etc.); vegetable oils (olive oil, soybean oil, etc.); and beverages.

[0061] Beverages include: food and drink such as soup and miso soup; powdered food and drink such as instant coffee, instant tea, instant milk, instant soup, and instant miso soup; alcoholic beverages such as whiskey, bourbon, spirits, liqueur, wine, fruit wine, sake, Chinese wine, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, happoshu, and chuhai; beverages containing fruit juice (e.g., apple, mandarin orange, grape, banana, pear, plum juice, etc.), and vegetable juice (e.g., Examples of non-alcoholic beverages include beverages containing vegetable juices (such as tomato, carrot, celery, cucumber, and watermelon), beverages containing fruit and vegetable juices, soft drinks, milk, soy milk, dairy drinks, drink-type yogurt, coffee, cocoa, tea drinks (black tea, green tea, barley tea, brown rice tea, sencha, gyokuro tea, roasted green tea, oolong tea, turmeric tea, pu-erh tea, rooibos tea, rose tea, chrysanthemum tea, and herbal teas (such as mint tea and jasmine tea)), energy drinks, sports drinks, and mineral water.

[0062] Suitable examples of such food compositions include foods or beverages to which vitamin K1 and / or vitamin K2 have been added, such as natto, yogurt, vegetable oil, jelly, tea drinks, alcoholic drinks, drops, candy, lemonade, cookies, crackers, biscuits, chocolate, cheese, butter, margarine, chewing gum, etc. When the above foods or beverages contain vitamin K1 and / or vitamin K2, the foods or beverages may be used as they are as food compositions for preventing and / or alleviating side effects, particularly cardiotoxicity, induced by anthracycline anticancer drugs, or may be used for the same purpose as vitamin K1 and / or vitamin K2-enriched food compositions by further adding vitamin K1 and / or vitamin K2 to the foods or beverages.

[0063] The food composition of the present invention may also be prepared as a functional food, health food, food for specified health uses, food for special dietary uses (e.g., food for sick people such as hospital food, sick food, and nursing care food), supplement, etc., and is preferably prepared as a food for specified health uses, food for special dietary uses, or supplement.

[0064] The food composition of the present invention may be in the form of, for example, tablets, pills, capsules (including hard capsules, soft capsules, and microcapsules), powders, granules, fine granules, troches, liquids (including syrups, emulsions, and suspensions), nasogastric tube feeding preparations, enteral nutritional preparations, etc., with tablets or capsules being preferred. Another preferred embodiment of the food composition of the present invention is a food or beverage to which vitamin K1 and / or vitamin K2 has been added.

[0065] The food composition of the present invention is preferably a food for specified health uses, a food for special dietary uses, or a supplement in the form of a tablet or capsule.

[0066] In this specification, supplements do not only mean nutritional supplements, nutritional functional foods, etc. for supplementing nutrients, etc., but also health supplements, health functional foods, etc. that have functions that are useful for maintaining, restoring, improving, etc. health.

[0067] The food compositions of the present invention can be produced, for example, by adding vitamin K1 and / or vitamin K2 to food by known methods. Specifically, tablet food compositions can be produced by adding and mixing ingredients such as vitamin K1 and / or vitamin K2, excipients (e.g., lactose, sucrose, mannitol, corn starch, etc.), sweeteners, and flavorings, and then molding the mixture into a tablet shape using a tablet press or other device. If necessary, other ingredients (e.g., vitamins such as vitamin C, minerals such as iron, dietary fiber, crystalline cellulose, rapeseed (hardened rapeseed oil), and other additives) can also be added. Capsule food compositions can be produced, for example, by filling liquid, suspension, paste, powder, or granular food compositions containing vitamin K1 and / or vitamin K2 into capsules or by encapsulating them with a capsule base.

[0068] The food composition of the present invention can contain physiologically acceptable carriers in addition to commonly used food ingredients, food additives, various nutrients, vitamins, flavoring substances (e.g., cheese, chocolate, etc.), as long as the effects of the present invention are not impaired. Physiologically acceptable carriers include various conventional organic or inorganic carrier substances, such as excipients, binders, disintegrants, lubricants, colorants, sweeteners, preservatives, antioxidants, thickeners, and emulsifiers. Food additives include colorants, sweeteners, preservatives, antioxidants, and flavoring agents. The food composition may also contain other ingredients, such as minerals such as iron and calcium, and dietary fibers such as pectin, carrageenan, and mannan.

[0069] Examples of the excipient, binder, disintegrant, lubricant, solvent, solubilizer, suspending agent, buffer, thickener, colorant, sweetener, preservative, and antioxidant include those similar to those used in the pharmaceutical composition of the present invention described above.

[0070] Vitamins may be water-soluble or fat-soluble, and examples include retinol palmitate, α-tocopherol (vitamin E), bisbentiamine, riboflavin, pyridoxine hydrochloride, cyanocobalamin, sodium ascorbate (vitamin C), cholecalciferol, nicotinamide, calcium pantothenate, folic acid, biotin, choline bitartrate, niacin, and nicotinamide mononucleotide (NMN).

[0071] Food compositions in the form of tablets, granules, or fine granules may be coated with a coating substrate by a method known per se for the purposes of taste masking, improving light stability, improving appearance, enteric coating, etc. Examples of the coating substrate include the same ones as those used for the pharmaceutical composition of the present invention described above, and coating can be carried out in the same manner.

[0072] The content of vitamin K1 and / or vitamin K2 in the food composition of the present invention is not particularly limited and is, for example, 0.1% by weight or more and 50% by weight or less of the total food composition. The lower limit is preferably 0.5% by weight or more, more preferably 1.0% by weight or more, of the total food composition. Meanwhile, the upper limit is preferably 20% by weight or less, more preferably 10% by weight or less, of the total food composition.

[0073] The food composition thus obtained is safe and can be continuously given to subjects, particularly preferably humans.

[0074] The intake amount of the food composition of the present invention is not particularly limited, as long as the vitamin K1 and / or vitamin K2 is within the range of an effective amount for preventing and / or alleviating side effects, particularly cardiotoxicity, caused by anthracycline anticancer drugs. For example, when the food composition of the present invention is ingested by an adult for the purpose of preventing or alleviating side effects caused by anthracycline anticancer drugs, the intake amount of vitamin K1 and / or vitamin K2 will vary depending on the subject, the type and dosage of the anthracycline anticancer drug, the intake form, the amount of intake, etc., but will be 0.5 mg / day or more and 1000 mg / day or less in terms of the active ingredients, vitamin K1 and / or vitamin K2. The lower limit, calculated as vitamin K1 and / or vitamin K2, is preferably 0.5 mg / day or more, more preferably 1 mg / day or more, and even more preferably 2 mg / day or more. Meanwhile, the upper limit, calculated as vitamin K1 and / or vitamin K2, is preferably 500 mg / day or less, more preferably 100 mg / day or less. The above intake amount is also preferable from the viewpoint of achieving the desired effect without affecting palatability or food intake. Similar amounts can be administered to other mammals.

[0075] The number of times vitamin K1 and / or vitamin K2 should be taken is not particularly limited, and may be, for example, 1 to 3 times a day, and may be administered before, after, or between meals. However, it is preferable to start taking vitamin K1 and / or vitamin K2 before (about 1 week before) administration of an anthracycline anticancer drug and continue taking vitamin K1 and / or vitamin K2 during the administration of the anthracycline anticancer drug.

[0076] Vitamin K1 and / or vitamin K2 can be used in combination with other therapies (e.g., chemotherapy, surgery (e.g., surgical resection, radiofrequency ablation, etc.), radiation therapy (e.g., X-rays, particle beam therapy, etc.)). This can enhance the therapeutic effects of cancer in subjects suffering from cancer (cancer patients) and reduce side effects. Of these, combination with chemotherapy is particularly preferred. Specific embodiments of combination with chemotherapy are shown below.

[0077] The food composition of the present invention may be used alone or in combination with other therapies (i.e., the above-mentioned chemotherapy, surgery, radiation therapy, etc.). Specifically, for example, it can be used in combination with other pharmaceutical compositions, food compositions, or feeds that have cardioprotective effects. Combining it with other therapies can further enhance the cancer therapeutic effects and side effect reduction effects of anthracycline anticancer drugs.

[0078] The food compositions of the present invention also include those classified as health foods, functional foods, foods for specified health uses, health-related foods, foods with disease risk reduction claims, or special dietary foods (e.g., foods for sick people such as hospital food, sick food, and nursing care food). Examples of disease risk reduction claims include claims such as "for reducing the risks of anticancer drugs," "protecting the body from anticancer drugs," and / or "relieving pain caused by anticancer drugs." Thus, the food compositions of the present invention are, for example, foods and beverages containing vitamin K1 and / or vitamin K2 and labeled to reduce the risks of anticancer drugs.

[0079] Here, the functional claims attached to these food compositions may be made on the product itself, container, packaging, instructions, attached documents, or promotional materials.

[0080] Screening method of the present invention The results of Test Examples 1 and 2 described below show that administration of an anthracycline anticancer drug (doxorubicin) induces aggregation of mitochondrial DNA nucleoids, and that inhibition of mitochondrial DNA nucleoid aggregation reduces the cardiotoxicity induced by anthracycline anticancer drugs. Therefore, the following method can be used as a screening method for candidate compounds for the active ingredients of the pharmaceutical or food composition of the present invention.

[0081] That is, a method for screening a compound for preventing cardiotoxicity induced by an anthracycline anticancer drug, comprising: (i) mixing a test compound with HeLa cells, adding an anthracycline anticancer drug thereto, staining the nucleoids of the treated HeLa cells with SYBR Green I, and evaluating the degree of nucleoid aggregation caused by the test compound based on comparison with a control group not containing the test compound; (ii) selecting a compound that inhibits nucleoid aggregation more than a control group based on the evaluation in (i) above; A screening method comprising:

[0082] For candidate compounds selected by such a screening method, the preventive and / or therapeutic effects on cardiotoxicity can be easily evaluated by measuring the atrial natriuretic peptide (ANP) level, which is a biomarker for heart failure as described in Test Example 2 below. [Example]

[0083] The present invention will be described in more detail below based on test examples and formulation examples, but the present invention is not limited to these test examples and formulation examples and may be modified within the scope of the present invention. Furthermore, reagents, devices, materials, etc. used in the present invention are commercially available unless otherwise specified. % means % by weight unless otherwise specified. Other abbreviations used in the text have the following meanings. DXR: Doxorubicin MK-4: Menaquinone-4 DMSO: dimethyl sulfoxide Mito-TEMPO: (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride ANP: atrial natriuretic peptide BNP: Brain natriuretic peptide DMEM: Dulbecco's modified Eagle's medium FCCP: Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone FBS: fetal bovine serum HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

[0084] [Test Example 1: Doxorubicin (DXR)-induced enlargement of mitochondrial DNA nucleoids and reduction of respiratory activity] In Test Example 1, HeLa cells were used to examine the structural changes in the mitochondrial DNA nucleoid and the changes in respiratory activity caused by the addition of DXR.

[0085] (Experimental Method) HeLa cells were cultured in Dulbecco's modified Eagle's medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum (Invitrogen) at 37°C in a 5% CO2-injected incubator on glass-bottom dishes or plastic-bottom 96-well plates. DXR (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in DMSO to prepare a 10 mM stock solution. This solution was added to the culture medium of cultured cells to a final DXR concentration of 500 nM, and the cells were treated for 24 hours (DXR-treated cells). DMSO alone was added to the culture medium of cultured cells (untreated cells) as a control.

[0086] (Fluorescence micrograph) Mitochondria and nucleoids of untreated and DXR-treated cells were stained with SYBR Green I, and whole mitochondria were stained with MitoTracker Red. Cells were incubated with 10,000-fold diluted MitoTracker® Red CMXRos (INVITROGEN by ThermoFisher Scientific) for 10 minutes, followed by 100,000-fold diluted SYBR® Green I Nucleic Acid Gel Stain (10,000x concentrated) (INVITROGEN by ThermoFisher Scientific) dissolved in DMSO for 5 minutes at 37°C. Cells were then washed twice with 10% FBS in DMEM (DMEM High Glucose, Fujifilm Wako Pure Chemical Industries, Ltd.) and cultured in FluoroBrite® DMEM (GIBCO) containing 50 mM HEPES buffer (pH 7.4) for 30 minutes. Each cell was observed under a fluorescence microscope (Keyence All-in-one Fluorescence Microscope (BZ-X710)).

[0087] (Measurement of respiratory activity) Untreated and DXR-treated cells were seeded into three separate wells of an XFe24 cell culture plate. The following day, the cells were washed twice with fresh medium and then incubated in pre-warmed Agilent Seahorse XF Assay Media for Cell Mito Stress Test (25 mM glucose, 1 mM pyruvate, 2 mM glutamine, 1% FBS) at 37°C in a CO2-free incubator for 1 hour. Oxygen consumption was measured at 37°C in untreated and DXR-treated cells using an Agilent Seahorse XFe24 Analyzer (Agilent Technologies). Oxygen consumption was measured at steady state (basal respiratory activity) and maximal respiratory activity in the presence of 1 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone / antimycin A. Results were normalized by protein amount per well using the Pierce® BCA Protein Assay Kit (ThermoFisher Scientific). Respiratory activity was quantified after each run according to the manufacturer's instructions. *** P values ​​were determined by one-way analysis of variance (ANOVA) test and Tukey-Kramer test.

[0088] (result) As shown in Figure 1A, the addition of DXR caused mitochondrial DNA nucleoids to aggregate and become larger than in the control. Furthermore, as shown in Figure 1B, the addition of DXR caused the enlargement of mitochondrial DNA nucleoids, which significantly reduced basal and maximum respiratory activity (oxygen consumption (pmol / min / μg)).

[0089] [Test Example 2: Inhibitory effect of administration of vitamin K2 (MK-4) before administration of doxorubicin (DXR) on mitochondrial DNA nucleoid aggregation and associated cardiotoxicity inhibitory effect] In Test Example 2, 8-week-old C57BL / 6J male mice (manufactured by CLEA Japan, Inc.) were used to examine the preventive and / or therapeutic effects of cardiotoxicity by measuring the structural changes in mitochondrial DNA nucleoids caused by administration of MK-4, Mito-TEMPO, or corn oil (control) before doxorubicin administration, as well as the quantification of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which are biomarkers for detecting heart failure, and the amount of mRNA encoding natriuretic peptides (ANP / β-actin and BNP / β-actin).

[0090] (Experimental Method) DXR (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in DMSO to make a 10 mM stock solution. MK-4 (Tokyo Chemical Industry Co., Ltd.) was dissolved in 99.5% ethanol to make a 10 mM stock solution. Mito-TEMPO hydrate (Cayman Chemical) was dissolved in DMSO to a final concentration of 100 μM. Eight-week-old C57BL / 6J male mice (CLEA Japan, Inc.) (n = 3 per group) were intraperitoneally injected daily for 7 days with 50 mg / kg MK-4 or 0.1 mg / kg Mito-TEMPO, or control mice with corn oil. On day 8, mice were intraperitoneally injected with 12.5 mg / kg DXR diluted with equal volumes of DMSO and corn oil. For the next 4 days, mice were intraperitoneally injected with 50 mg / kg MK-4 or 0.1 mg / kg Mito-TEMPO, or control mice with corn oil. On day 13, mice were intraperitoneally injected with 12.5 mg / kg DXR. The following day (day 14), all mice in each group were sampled. Whole heart samples were collected for tissue sectioning and RNA extraction. To measure ANP and BNP levels, total RNA was extracted from mouse hearts using TRIzol® Reagent (ThermoFisher Scientific). Tissue sections were homogenized twice at 3800 rpm for 30 seconds using a MicroSmash® MS-100R (Takara Tomy). Subsequent conversion to cDNA was performed using the ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.) according to the manufacturer's protocol. Natriuretic peptide quantification was then performed using the StepOnePlus Real-Time PCR System (ThermoFisher Scientific) and PowerUp® SYBR® Green Master Mix (ThermoFisher Scientific) according to the manufacturer's protocol. Normalization was performed using β-actin.

[0091] (Staining of cardiomyocytes and fluorescence microscopy observation) To stain mitochondria and nucleoids in mouse myocardial samples, whole hearts were removed from mice and wiped clean. The samples were then embedded in Tissue-Tek OCT Compound (Sakura Finetech) and frozen in liquid nitrogen. These frozen samples were sectioned at 5 μm thickness using a CM3050S cryostat (Leica Biosystems). The myocardial sections were then fixed with 4% paraformaldehyde solution for 20 minutes at room temperature. They were then blocked with 2% bovine serum albumin in PBS for 30 minutes at room temperature. Mitochondria were stained with anti-cytochrome c antibody (BD Pharmigen), and mitochondrial DNA and nucleoids were stained with anti-DNA antibody (PROGEN), each incubated for 1 hour at room temperature. The sections were reacted with goat anti-mouse IgM (heavy chain) cross-adsorbed secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-21042) and goat anti-mouse IgG1 cross-adsorbed secondary antibody, Alexa Fluor 568 (Thermo Fisher Scientific, A-21124) as secondary antibodies. Finally, the sections were stained with the Vector TrueVIEW® Autofluorescence Quenching Kit with DAPI (Funakoshi Co., Ltd.) and mounted on slides with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Funakoshi Co., Ltd.). Prior to observation, the samples were left at room temperature for 2 hours in the dark. Immunofluorescent staining and nuclei were observed under a fluorescence microscope (KEYENCE All-in-one Fluorescence Microscope (BZ-X710)). To detect reactive oxygen species in mouse hearts, cardiac tissue sections were prepared as described above, and excess OCT compound was thoroughly washed off with PBS. Dihydroethidium (Fujifilm Wako Pure Chemical Industries, Ltd.) diluted to a final concentration of 2 μM with FluoroBrite® DMEM (GIBCO) was then applied to the washed tissue sections, which were then incubated at 37°C for 30 minutes in the dark. Finally, the sections were mounted on glass slides using VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Funakoshi Corporation). Sections were observed under a fluorescence microscope (KEYENCE All-in-one Fluorescence Microscope BZ-X710). ** P value and **** P values ​​were determined by one-way ANOVA test.

[0092] (result) As shown in Figure 2A, pre-administration of MK-4 inhibited DXR-induced mitochondrial DNA nucleoid aggregation, whereas pre-administration of the antioxidant Mito-TEMPO did not. On the other hand, as shown in Figure 2A, it was confirmed that reactive oxygen species generated by DXR treatment were eliminated by pre-administration of MK-4 or Mito-TEMPO. Furthermore, as shown in Figure 2B, pre-administration of MK-4 significantly reduced the levels of mRNA encoding ANP and BNP, which are indicators of DXR-induced cardiotoxicity, whereas pre-administration of Mito-TEMPO slightly reduced the level of mRNA encoding ANP but hardly reduced the level of mRNA encoding BNP. These results confirm that DXR-induced cardiotoxicity is not sufficiently prevented or alleviated by antioxidants, but is effectively prevented and / or alleviated by pre-administration of MK-4. Furthermore, the results in Figure 2 also confirm that the degree of inhibition of mitochondrial DNA nucleoid aggregation reflects the strength of the preventive and / or therapeutic effect of cardiotoxicity.

[0093] [Test Example 3: Effect of pre-administration of vitamin K2 (MK-4) on the anti-cancer effect of doxorubicin (DXR)] In Test Example 3, using HeLa cells, it was investigated whether prior administration of MK-4 affects the cell proliferation inhibitory action of DXR.

[0094] (Experimental Method) HeLa cells (1×10 5 ) were seeded onto a 35 mm dish and cultured for 24 hours. The cells were then incubated with 100 μM MK-4 or corn oil for 24 hours. 500 nM DXR was added, and the cells were removed by trypsinization every day and counted using a CellDrop BF counter (Scrum Co., Ltd.). The survival rate relative to control cells (treated with corn oil) is shown in the graph. *** P values ​​were determined by one-way ANOVA test.

[0095] (result) As shown in Figure 3, the MK-4-DXR administration group was confirmed to have the same cell proliferation inhibitory effect as the DXR administration group, and it was confirmed that prior administration of MK-4 does not affect the cell proliferation inhibitory effect of DXR.

[0096] These results demonstrate that administration of vitamin K2 and / or vitamin K1, which is metabolized in vivo and converted to vitamin K2, prior to administration of anthracycline anticancer drugs significantly suppresses mitochondrial DNA nucleoid aggregation, resulting in excellent preventive and / or therapeutic effects against cardiotoxicity. Furthermore, because administration of vitamin K1 and / or vitamin K2 does not affect the cell proliferation inhibitory effect of anthracycline anticancer drugs, it is possible to provide pharmaceuticals and food compositions useful as preventive and / or therapeutic agents against cardiotoxicity induced by anthracycline anticancer drugs.

[0097] Examples of the formulation of the present invention include the following formulations, but the present invention is not limited to these formulation examples.

[0098] Formulation Example 1 (Tablet Production) 1) MK-4 40g 2) 50g corn starch 3) 40g of crystalline cellulose 4) Hydroxypropyl cellulose 17g 5) Magnesium stearate 3g 1000 tablets total 150g The total amount of 1), 2), and 3) and 12 g of 4) are mixed with water, vacuum dried, and then sized. 5 g of 4) and 3 g of 5) are mixed with this sized powder and compressed into tablets using a tablet press. In this way, 1,000 tablets containing 40 mg of MK-4 per tablet are obtained.

[0099] Formulation Example 2 (Tablet Production) 1) MK-4 18g 2) 100g starch 3) Microcrystalline cellulose 62g 4) Mannitol 15g 5) Vitamin C 50g 6) Magnesium stearate 5g 1000 tablets total 250g The total amount of 1), 2), and 40 g of 3) were mixed with water, vacuum dried, and then sized. The sized powder was mixed with the total amount of 4), 5), and 6), and 22 g of 3), and the mixture was compressed into tablets using a tablet press. In this way, 1,000 tablets containing 18 mg of MK-4 per tablet were obtained.

[0100] Formulation Example 3 (Tablet Production) 1) MK-4 18g 2) 100g starch 3) Microcrystalline cellulose 62g 4) Soybean oil 15g 5) Vitamin C 50g 6) Magnesium stearate 5g 1000 tablets total 250g The total amount of 1), 2), and 40 g of 3) were mixed with water, vacuum dried, and then sized. The sized powder was mixed with the total amount of 4), 5), and 6), and 22 g of 3), and the mixture was compressed into tablets using a tablet press. In this way, 1,000 tablets containing 18 mg of MK-4 per tablet were obtained.

[0101] Formulation Example 4 (Production of Capsules) 1) MK-4 40mg 2) Microcrystalline cellulose 10mg 3) Lactose 20mg Total 70mg 1), 2) and 3) are mixed and filled into a gelatin capsule.

[0102] Formulation Example 5 (Production of Capsules) 1) MK-4 40mg 2) Corn starch 30mg 3) Lactose 30mg Total 100mg 1), 2) and 3) are mixed and filled into a gelatin capsule. [Industrial Applicability]

[0103] The present invention provides an inhibitor of mitochondrial DNA nucleoid aggregation, containing vitamin K1 and / or vitamin K2, a safe vitamin. By administering the nucleoid aggregation inhibitor of the present invention to a subject suffering from cancer undergoing treatment with an anthracycline anticancer drug, the inhibitor can effectively prevent and / or treat cardiotoxicity induced by the anthracycline anticancer drug without interfering with the anthracycline anticancer drug's inhibitory effect on cancer cell proliferation. Specifically, the present invention provides a pharmaceutical composition or food composition containing vitamin K1 and / or vitamin K2 as an active ingredient for preventing and / or treating (alleviating) the side effects (cardiotoxicity) of anthracycline anticancer drugs. The present invention also provides a simple and effective method for screening compounds for preventing and / or treating cardiotoxicity induced by anthracycline anticancer drugs.

Claims

1. Vitamin K １ and / or vitamin K ２ A mitochondrial DNA nucleoid aggregation inhibitor comprising:

2. Vitamin K １ and / or vitamin K ２ The nucleoid aggregation inhibitor according to claim 1, wherein is phylloquinone or menaquinone-4.

3. A pharmaceutical composition for preventing and / or treating cardiotoxicity induced by anthracycline anticancer drugs, comprising the nucleoid aggregation inhibitor according to claim 1 or 2.

4. The pharmaceutical composition according to claim 3, wherein the cardiotoxicity is cardiotoxicity caused by respiratory failure.

5. The pharmaceutical composition according to claim 3, wherein the nucleoid aggregation inhibitor is administered before the administration of an anthracycline anticancer drug.

6. The pharmaceutical composition according to any one of claims 3 to 5, wherein the anthracycline anticancer drug is doxorubicin.

7. Vitamin K １ and / or vitamin K ２ A food composition for preventing and / or reducing cardiotoxicity induced by anthracycline anticancer drugs, comprising as an active ingredient:

8. The food composition according to claim 7, which is taken before administration of an anthracycline anticancer drug.

9. A method for screening a compound for preventing and / or treating cardiotoxicity induced by an anthracycline anticancer drug, comprising: (i) mixing a test compound with HeLa cells, adding an anthracycline anticancer drug thereto, staining the nucleoids of the treated HeLa cells with SYBR Green I, and evaluating the degree of nucleoid aggregation caused by the test compound based on comparison with a control group not containing the test compound; (ii) selecting a compound that inhibits nucleoid aggregation more than a control group based on the evaluation in (i) above; A screening method comprising:

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