JP2019520310A - Therapeutic agent for cancer and precancerous lesion, method of treatment, and method of producing therapeutic agent - Google Patents

Abstract

The present invention discloses ingredients and methods using a pyrithione compound and a pharmaceutically acceptable pyrithione salt. The present invention relates to skin cancer treatment by topical therapy, cervical cancer treatment, treatment of other cancers, prevention of skin cancer onset from precancerous skin lesions, prevention of cervical cancer onset from papilloma virus infection, papilloma virus infection And disclose methods of preventing the onset of other cancers. Furthermore, in the present invention, a drug component containing at least one kind of pyrithione compound, EDTA, ascorbic acid, any of ascorbate, any ascorbic acid derivative, pyridoxine, pyridoxine derivative, a pharmacologically acceptable buffer, Disclose. The pH of the drug component is assumed to be between 4.5 and 6.4.

Description

  Rapidly proliferating cancer cells produce acids as a result of their metabolism, and the released acids acidify the environment surrounding the cancer. As a result, the environment surrounding cancer exhibits higher acidity than the environment surrounding normal tissue. The present invention discloses a drug formulation and treatment / prevention method for treating or preventing cancer that develops and grows in an acidic environment.

  Malignant tumors are one of the leading causes of death worldwide. According to statistics conducted by the International Organization for Cancer Research in 2012, the total number of cancer cases worldwide was more than 14 million in 2012, but it is expected to increase to more than 20 million in 2030. Be done. Similarly, the annual total number of cancer deaths worldwide was 8 million in 2012, which is expected to increase to over 13 million in 2030.

  Chemotherapy, surgery and radiation are the most common treatment strategies for malignant tumors. Although the cancer research area has made significant progress, current treatments have a number of problems that need to be improved. As one example, in many traditional chemotherapies, DNA (deoxyribonucleic acid) is chemically damaged to treat with the goal of inhibiting high proliferation activity and cell division of cancer cells. Such conventional chemotherapy often damages normal cells other than cancer cells and causes serious side effects because DNA is a blueprint of genetic information possessed by both cancer cells and normal cells. Other chemotherapeutic agents include those targeting cell growth proteins or cell division among cell constituent proteins, but these chemotherapeutic agents are also cancer cells such as bone marrow and enteric cells with extensive cell division. Exert non-specific toxicity on normal cells of More recently, therapeutic agents that target cancer specific molecules that are present only in cancer cells but not in normal cells have attracted attention. Cell growth factor receptor inhibitors erlotinib and gefitinib are good examples of therapeutic agents that target cancer specific molecules. However, not all cancer cells have sufficient levels of growth factor receptors. In addition, even in the case where treatment targeting cancer-specific molecular therapy initially produces a therapeutic effect, other mechanisms that complement the function of the suppressed growth factor receptor are activated and eventually the cancer cells acquire therapeutic resistance. There is a tendency to Surgical treatment may be effective in removing isolated malignancies, but recurrence and infiltration of other body parts of the cancer caused by surgical procedures is a major problem. In addition, surgery can cause bleeding, scarring, infections, and damage to normal organs. Radiation therapy is also at risk for normal tissue damage and secondary carcinogenesis, as well as long-term cosmetic risks. Chemotherapy can cause many of the serious side effects mentioned above, especially when given intravenously or orally. There is a strong need to develop new anti-cancer therapeutics.

  Malignant tumors are more than just a mass of cancer cells. The normal tissue and normal cells surrounding the tumor cells form unique environments (sometimes also called "tumor microenvironments") to promote the growth, invasion, and metastasis of cancer cells. Acid is one of the most important elements of the tumor microenvironment. Tumor microenvironments are more acidic than tumor free environments. Acidic metabolites released extracellularly from tumor cells are the source of acid in the tumor microenvironment, which results in decreased chemo- and radiation therapy treatment efficiency and further promotes tumor growth and invasion. Acidic environments are attractive targets for the treatment of malignancies. Therapeutics that effectively suppress tumor cell growth growing in an acidic environment are expected to be promising new treatments.

  Pathologies that are not cancer per se but are at high risk of developing cancer are called "precancerous lesions". As a result of the persistent and uncontrolled inflammatory response associated with precancerous lesions, precancerous lesions form a highly acidic environment prior to the onset of cancer. The acidic environment promotes the change of genetic information and the onset of cancer (Non Patent Literature 1: Coussens and Werb, 2002). Once malignant tumor cells are generated, acidification is further enhanced because malignant tumor cells proliferate more selectively than in normal cells in an acidic environment. In addition to the selective growth of malignant tumors over normal cells in an acidic environment, the possibility of canceration is also enhanced by the fact that the immune cell activity is attenuated by excess acid (Non-patent Document 2: Lardner, 2001) ).

  Nikko keratosis and papillomavirus infections are good examples of precancerous lesions. It is a rough, scaly lesion caused by excessive UV exposure and is one of the most frequently diagnosed skin diseases that accounts for 10 percent of dermatology outpatients in the United States. It has been reported that actinic keratosis often causes skin cancer, and according to some research results, 65 percent squamous cell carcinoma and 36 percent basal cell carcinoma may be caused by actinic keratosis (Non-patent Document 3) : Criscione et al., 2009). Cryosurgery and transdermal administration of anti-cancer chemotherapy are the main treatments for actinic keratosis. However, actinic keratosis is not a life-threatening disease before canceration. It takes 10 or more years for canceration, and often it is expected that multiple actinic keratosis will appear in the same patient and which actinic keratosis will progress to invasive cancer difficult. These facts make it difficult to treat actinic keratosis.

  Papilloma virus infection occurs in the cervix, female external genitalia, anus, penis and oral cavity. According to the World Health Organization (WHO), papilloma virus infection is the most frequent of viral infections transmitted by sexual contact (Non-patent Document 4). Although papilloma virus infection is known to cause cancer at these sites mentioned above, papilloma virus infection is involved in almost all cases, particularly in cervical cancer. Cervical cancer is the fourth most common cancer among women, but it is the second most common cancer among women in developing countries, accounting for over 85 percent of all patients. Although early diagnosis with papilloma virus vaccines and cancer screening has markedly reduced the frequency of cervical cancer in developed countries, adopting these methods in developing countries is difficult because of social and economic issues. A simpler and cheaper method is awaited.

  According to a recent experimental study, zinc pyrithione was implanted in the subcutaneous of an immunodeficient mouse in human oral cancer cells (Non-patent document 5: Srivastava et al., 2015) and human leukemia cells (leukocyte cell-derived malignancy) (Non-patent Document 6: Tailler et al., 2012) was suggested to effectively suppress the growth. Both studies revealed that at concentrations that inhibit cancer cell growth, pyrithione does not cause adverse effects in the host organism. Similarly, while low concentrations of zinc pyrithione effectively kill prostate cancer cells, it has been found that 10 times higher concentrations of zinc pyrithione are required to kill non-cancerous cells ( Non-Patent Document 7: Carraway and Dobner, 2012). However, these prior arts do not provide information on methods or drug components for treating or preventing cancer.

  Pyrithione is incorporated into biological membranes to form small holes that are permeable to zinc. It is known to use zinc and other metals for cancer treatment by utilizing the toxicity of cancer cells to heavy metals. For example, Patent Document 1 (US Patent Application Publication No. 2011/0017210) introduces a method of using zinc organic salts and zinc inorganic salts for cancer treatment. Patent Document 2 (US Patent No. 7,528,125) discloses a new compound in which a series of pyrithione is chemically modified, and it is used to treat zinc, copper, manganese, iron and other metal compounds as tumors. Disclosed methods of administration. Furthermore, Patent Document 3 (US Patent Application Publication No. 2006/0040980) disclosed an anti-cancer drug and a method of using zinc pyrithione and a zinc salt in combination as an anti-angiogenic drug. In more recent experimental research, this technology and idea are further developed, and copper (Platin et al., 2014: Liu et al., 2014), cadmium, platinum (Platy non-patent literature 9: Zhao et al., We have proposed a method of administering a wider range of heavy metals such as 2016b) and nickel (Zhao et al., 2016a) to leukemia, bone marrow tumor, myeloma, lung cancer and liver cancer. From these prior art, we have learned that heavy metals that pass through the holes formed in biological membranes by pyrithione have a cell killing effect and an antiproliferative effect on malignant tumors.

U.S. Patent Application Publication No. 2011/0017210 U.S. Patent No. 7,528,125 US Patent Application Publication No. 2006/0040980

Coussens, L. M., and Werb, Z. (2002). Inflammation and cancer. Nature 420, 860-867. Lardner, A. (2001). The effects of extracellular pH on immune function. Journal of leukemia biology 69, 522-530. Crincione, V. D., Weinstock, M. A., Naylor, M. F., Luque, C., Eide, M. J., and Bingham, S. F. (2009). Actinic keratoses. "Human papillomavirus (HPV) and cervical cancer", World Health Organization website, published February 15, 2018, http://www.who.int/mediacentre/factsheets/fs380/en/ Srivastava, G., Matta, A., Fu, G., Somasundaram, RT, Datti, A., Walfish, PG, and Ralhan, R. (2015). Anticancer activity of pyrithione zinc in oral cancer cells identified in small molescule screens and xenograft model: Implications for oral cancer therapy. Molecular oncology 9, 1720-1735. Tailer, M., Senovilla, L., Lainey, E., Thepot, S., Metivier, D., Sebert, M., Baud, V., Billot, K., Fenaux, P., and Galluzzi, L. (2012). Antineoplastic activity of ouabain and pyrithione zinc in acute myeloid leukemia. Oncogene 31, 3536-3546. Carraway, R. E., and Dobner, P. R. (2012). Zinc pyrithione causes ERK- and PKC-dependent necrosis distinct from TPEN-induced apoptosis in prostate cancer cells. Biochimica et Biophysica Acta (BBA) -Molecular Cell Research 1823, 544-557. Liu, N., Liu, C., Li, X., Liao, S., Song, W., Yang, C., Zhao, C., Huang, H., Guan, L., and Zhang, P. (2014). A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases. Scientific reports 4, 5240. Zhao, C., Chen, X., Zang, D., Lan, X., Liao, S., Yang, C., Zhang, P., Wu, J., Li, X., and Liu, N. (2016b). Platinum-containing compound platinum pyrithione is stronger and safer than cisplatin in cancer therapy. Biochemical Pharmacology in press. Zhao, C., Chen, X., Zang, D., Lan, X., Liao, S., Yang, C., Zhang, P., Wu, J., Li, X., and Liu, N. (2016a). A novel nickel complex works as a proteasomal deubiquitinase inhibitor for cancer therapy. Oncogene.

  Acidic tumor microenvironments are known to be responsible for resistance to many anti-cancer chemotherapeutic agents. In addition, methods that can currently be implemented to prevent canceration from precancerous lesions are limited in terms of efficiency, safety, and cost.

  The present invention discloses drug components and methods for enhancing anticancer activity using acidity to treat and prevent malignant tumors. In some embodiments, the disclosed therapeutic methods and drug components are used to prevent the progression of precancerous lesions to cancer by killing cancer cells that are sporadically developing in precancerous lesions. Be

  In one aspect of the present invention, zinc pyrithione, sodium pyrithione, other pyrithione salts, or any one or more of pyrithione free of salts, on the skin, cervix, vagina, penis, anus, and other body parts Disclosed is the manufacture and use as a medicament for the treatment of developed malignancies. The pH of the components is kept acidic using a pharmacologically acceptable pH adjuster. This method effectively suppresses the growth of malignant tumor in acidic environment. In another aspect, the method may be used in combination with existing anti-cancer treatments such as chemotherapy, radiation and surgery.

  In another aspect of the present invention, pyrethion in addition to a chelating agent ethylenediaminetetraacetic acid (EDTA), EDTA calcium salt, EDTA disodium salt, EDTA ammonium salt, EDTA dipotassium salt, EDTA disodium salt, TEA-EDTA For the manufacture of a medicament for treating or preventing cancer or malignant tumor, containing EDTA tetrasodium salt, EDTA tetrapotassium salt, EDTA trisodium salt, HEDTA, HEDTA trisodium salt and other EDTA salts Disclose. This builds on the prior art of the anti-cancer effect of zinc and other metals depending on unchelated pyrithione, and by adding further improvements thereto, makes the anti-cancer action of pyrithione more effective.

  In yet another aspect of the present invention, pyrithione is used, and pyridoxine, pyridoxal, pyridoxamine, and their pentaphosphates together with pyrithione, which are generally collectively referred to as vitamin B6, for the treatment and prevention of malignant tumors. Disclosed for use in the manufacture of a medicament for use. This improves and promotes the growth inhibitory effect of the malignant tumor in an acidic environment.

  In still another aspect of the present invention, pyrithione is used, and L-ascorbic acid, ascorbic acid in a place known as vitamin C, any of ascorbic acid salts, any compound derived from ascorbic acid, or one of these compounds is used. It is disclosed that a kind or a combination thereof is added together with pyrithione for use in the manufacture of a medicament for the treatment and prevention of malignant tumor. This improves and promotes the growth inhibitory effect of the malignant tumor in an acidic environment.

  In yet another aspect of the present invention, the use of pyrithione in the manufacture of a medicament for preventing the onset of cancer from a precancerous lesion represented by genital infection of papilloma virus infection or actinic keratosis is disclosed. .

  According to the present invention, the fact that an anticancer drug component mainly composed of pyrithione is enhanced by an acid enables an efficient, simple, safe, and inexpensive cancer cell growth inhibitory effect.

  The concentration required for pyrithione to suppress cancer cell growth is because the existing anticancer agent known by the prior art suppresses cancer cell growth when the combination of the enhancer disclosed in the present application and the acidity of the drug component is used. It was found to be 100 to 1000 times lower than the required concentration. This clearly demonstrates the effectiveness of the disclosed method and drug component. Furthermore, the therapeutic method, drug component, and application to pharmaceutical production disclosed herein satisfy the technical need that can not be achieved by the existing technology, which selectively kills cancer cells growing in an acidic tumor microenvironment. It is a thing.

  In some embodiments, a formulation with acidity is administered locally to a cancer that develops near the body surface or near the organ surface. Topical administration is superior to systemic administration in many ways in administering a formulation containing pyrithione. First, the local administration method is safe because local administration has less chance of the prescribed drug coming into contact with blood or healthy organs, and it is less likely to cause adverse effects on healthy tissues. Second, local administration does not require a high level of technology, and administration by the affected individual is easier than systemic administration. Third, the acidic formulation optimized for topical administration disclosed in the present invention is that the active ingredient can be effectively delivered to the cancer. Furthermore, the local administration method is also advantageous in that it allows cancerous tissue to be reached without affecting the acid drug component in the whole body blood and the acidity of healthy organs. It is important to note that acidification of the blood and the whole body causes heart failure. In one embodiment, topical administration of pyrithione (and enhancer) is combined with systemic administration of certain enhancers (eg, oral administration of vitamin B6, intravenous injection of vitamin C), which further enhances the therapeutic effect. Do.

  Since the pyrithione and other pharmaceutical ingredients disclosed herein are widely used in industrial applications by the mass production techniques disclosed so far, medicines can be inexpensively used using the methods and pharmaceutical ingredients disclosed herein. It is possible to manufacture by skilled technique. This is particularly advantageous in the manufacture of medicines in countries where economic development is relatively imminent.

FIG. 1 shows the chemical structures of zinc pyrithione (sometimes abbreviated as "Pyz" in some cases) and sodium pyrithione (sometimes abbreviated as "Pyn" in some cases). FIG. 2 shows human brain tumor U87 cells, breast cancer MDA-MB-231 cells, cervical cancer HeLa cells, and large intestine when zinc pyrithione (Pyz) is treated in cell culture media (medium) having two different pHs. It is the graph which showed the effect on survival of cancer HT29 cells. The cells were cultured at 37 ° C. for 3 days in the presence or absence of the indicated concentration of zinc pyrithione. Cell viability was measured by methylthiozolyl diphenyl tetrazolium bromide (MTT) assay. Figure 3. Human brain tumor U87 cells, breast cancer MDA-MB-231 cells, cervical cancer HeLa cells, and colon cancer HT29 cells when sodium pyrithione (Pyn) is treated in cell cultures with two different pH. Is a graph showing the effect on survival of Cells were cultured at 37 ° C. for 3 days in the presence or absence of the indicated concentrations of sodium pyrithione. Cell viability was measured by MTT assay. FIG. 4 summarizes the cell-killing effects of sodium pyrithione (Pyn) and zinc pyrithione (Pyz) when treated on HeLa cells in cell culture media (medium) having two different pHs. Cells were cultured at 37 ° C. for 3 days in the presence or absence of the indicated concentrations of pyrithione. Non-viable cells were counted by trypan blue staining. FIG. 5 is a photograph showing the acid-induced antiproliferative effect observed when treating brain tumor U87 cells cultured with zinc pyrithione in a spheroid three-dimensional culture method in cell culture media (medium) having two different pHs. It is. Figure 6 shows that superoxide accumulated in mitochondria when treated with Hepatic cervical cancer in culture medium (medium) with zinc pyrithione (Pyz) or sodium pyrithione (Pyn) at two different pH levels. Is a photo showing Fig. 7 shows brain tumor U87 cells, breast cancer MDA-MB-231 cells, cervical cancer HeLa cells and colon cancer HT29 cells treated with erlotinib and gefitinib in cell culture media (medium) having two different pHs. The effects on survival rates are summarized. Cell viability was measured by MTT assay. FIG. 8 is a graph summarizing the effect of ethylenediaminetetraacetic acid (EDTA) on the anti-cancer effect of zinc pyrithione on human brain tumor U87 cells, cervical cancer HeLa cells, and colon cancer HT29 cells. Cell viability was measured by MTT assay. FIG. 9 is a graph summarizing the effect of pyridoxine on the anti-cancer effect of zinc pyrithione on human cervical cancer HeLa cells. Cell viability was measured by MTT assay. FIG. 10 is a graph and a photograph summarizing the effect of sodium ascorbic acid and 3-o-ethylascorbic acid on the anticancer effect of sodium pyrithione and zinc pyrithione on cervical cancer HeLa cells. Cell viability was examined by two-dimensional culture (FIG. 10A) and spheroid three-dimensional culture (FIG. 10B). FIG. 11A shows the effect of topical administration of a pharmaceutical preparation containing or not containing zinc pyrithione, 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate on the proliferation of human skin cancer cells subcutaneously implanted in immune deficient mice. Is a graph showing FIG. 11B shows that transdermal administration of a drug formulation containing zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitic acid does not exhibit adverse effects on normal skin, liver, and kidneys. It is.

Definitions Unless otherwise specified, "pyrithione" and "pyrithione compound" are used interchangeably and refer to any of pyrithione and pyrithione salts that can be administered to the human body without interfering with other formulation ingredients. Pyrithione is most commonly obtained from commercial sources as a zinc or sodium salt (see FIG. 1), but other salt forms such as nickel and platinum are also known. Pyrithione is 2-mercaptopyridine-N-oxide, 1-hydroxy-2-pyridinethione, 1-hydroxypyridine-2-thione, omedine, (2-pyridylthio) -N-oxide, 2-pyridinethiol-1-oxide and the like. Also known by the name. The derivatives mean compounds having similar structures and functions. The sodium salt of pyrithione (sodium pyrithione) is widely used in commercial products. Sodium pyrithione can be generally synthesized by reacting 2-chloropyridine-N-oxide with sodium hydrogen sulfide and sodium hydroxide (see, for example, U.S. Pat. No. 3,159,640). Zinc salts of pyrithione (zinc pyrithione) can be synthesized as zinc pyrithione precipitates formed by pyrithione acid (1-hydroxy-2-pyridinethione) or its water-soluble salts and zinc salts (eg zinc sulfate, zinc chloride) (See, eg, US Pat. No. 2,809,971).

  Tumor is a pathological term defined as swelling of body parts. There are two types of tumors, a type called "malignant tumor that grows indefinitely like cancer" and a type called benign and curable benign tumor that is generally harmless (however, some types of cancer originate from benign tumors). Some things do).

  Malignant tumors are tumors that can undergo unlimited cell division, direct invasion to neighboring tissues, and invasion to distant tissues via blood or lymph system called metastasis. Cancer is understood strictly as a group of malignancies derived from epithelial cells of skin and organs. On the other hand, malignant tumors derived from cells other than epithelial cells, such as blood, bone, blood vessels, cartilage, muscles and supporting tissues, are strictly excluded from cancer. For example, according to pathological definition, skin melanoma is not cancer because it is a malignant tumor derived from non-epithelial cells. However, in reality, the names malignancy, cancer, neopresiae are used interchangeably in the broadest sense, so melanoma is often referred to as part of skin cancer. Here, the term "cancer" is broadly understood to be compatible with "malignant tumor".

  Malignant tumors found in the skin include skin squamous cell carcinoma, skin melanoma (malignant melanoma), skin adenocarcinoma, and other malignancies that develop in the skin area or metastasize to the skin. Carcinoma (carcinoma) is a pathological term that refers to a malignancy of epithelial origin, and melanoma (malignant melanoma) is a malignancy of non-epithelial cell origin. In another classification, malignancies that develop in the skin are classified into others including basal cell carcinoma, squamous cell carcinoma, melanoma (malignant melanoma).

  Malignant tumors found in the uterus and vagina refer to squamous cell carcinoma, squamous adenocarcinoma, other malignant tumors, and metastatic tumors.

  As used herein, "treatment" refers to a medical procedure for a sick person for the purpose of improving from an undesirable medical condition or preventing the deterioration of such an undesirable condition. The terms "anti-tumor effect", "anti-cancer effect" and "anti-neoplastia effect" are used interchangeably to delay or eliminate tumor growth, reduce tumor size, and invade other organs Means to prevent. In one aspect, when a compound reduces the size of a tumor, it is determined that the compound was found to be effective. In another aspect, it is judged that the effect of the compound is recognized by the alleviation of symptoms associated with cancer. In yet another aspect, it is determined that the compound is effective as a result of a compound's medical examination indicating a decrease in tumor load. Medical examination includes biochemical examination, tumor marker examination, imaging diagnosis and histopathology examination. In yet another meaning, it is judged that the efficacy of the compound is recognized by observing the prolongation of the survival time of the affected person by administration of the compound.

  The term transdermal administration is more broadly defined herein and is used interchangeably with the term topical administration. Local administration generally refers to all methods involved in drug administration from the body surface of the drug, and drug administration from the inner peripheral part of the body vessel generally known as the epithelium and mucous membranes.

  An excipient is an organic / inorganic compound that acts as a drug carrier for these active ingredients without interfering with the active drug ingredients. Pharmaceutically acceptable drug carriers include water, polyethylene glycol (PEG), salt solution, lactose, amylose, alcohol, oils, fatty acid, gelatin, silicic acid, surfactant, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, Lubricants and the like are included.

Methods of treating malignancy and therapeutic agent components The malignancy has an acidic extracellular environment with a pH of 6.0 to 6.9 and sometimes lower pH, while the pH of non-cancer normal tissue is 7.3 To 7.4 (Parks et al., 2013). Previous experimental studies have attempted to reduce the acidity of the tumor environment and inhibit tumor growth by inhibiting the proton pump and the enzyme that produces acid. However, even if one of the mechanisms for acidifying the tumor environment is inhibited, other mechanisms that complement these are more activated, so the therapeutic effect that is expected with such a strategy Is not listed. The present invention, unlike such a strategy, is a method of efficiently and selectively killing cancer cells using the acidity of the tumor.

  The inventor screened agents that enhance the anti-cancer effect by cell culture medium having typical acidity in tumor microenvironment such as pH 6.9 and 6.4. As a result, it was surprisingly found that the anticancer activity of pyrithione is dramatically enhanced by the acidic cell culture medium (see FIGS. 1, 2, 3, 4, 5, 6). Anticancer activity of pyrithione in acidic environment was observed in cancer cells with different genetic backgrounds such as cervical cancer, brain tumor, colon cancer, breast cancer, etc. It was not observed in the factor receptor (Epidermal Growth Factor Receptor (EGFR)) tyrosine kinase specific inhibitor gefitinib and erlotinib (FIG. 7).

  The present invention discloses a topical administration of pyrithione and its components for the treatment or prevention of malignancy. The pharmaceutical formulation consists of pyrithione, a chemical component that promotes potentiation in an acidic environment, and a drug carrier that does not interfere with the medicinal component. The drug formulation is applied directly to the surface of the body or internal organ lumen. Local administration is effective in efficiently reaching a therapeutic agent to malignant tumor cells that grow near the body surface or near the lumen surface of a body organ. When the acidic pH of the drug formulation component is combined with the acidity of the tumor itself, pyrithione exerts an effective tumor cell killing effect. Some of the formulation ingredients may also be administered intravenously, subcutaneously, intramuscularly, by inhalation, orally.

  In certain embodiments, the pH of the drug formulation component is at a maximum of 6.9, which indicates that the antitumor effect of pyrithione is significantly more effective at pH 6.9 than at pH 7.4. Because it is high (see FIG. 10 as an example). More preferably, the pH of the drug formulation component is at a maximum of 6.4, because the antitumor effect of pyrithione is significantly higher in the case of pH 6.9 when it is pH 6.9. (See FIG. 10 as an example). More preferably, the pH of the drug formulation component is at a maximum of 5.6, which suppressed the growth of human skin cancer cells implanted in mice by topical administration of a pharmaceutical preparation comprising pyrithione adjusted to pH 5.5 (See FIG. 11). Previously disclosed U.S. Patent No. 6,455,076 recognizes the importance of the acid in promoting the efficiency of the active ingredient of a cosmetic product. This prior art further presented a method to prevent skin irritation. According to this prior art, it is possible to prevent skin irritation generated by acids equivalent to pH 2.0. For this reason, as a further embodiment, the pH of the drug formulation component is at least 2.0 or more.

  Compound screening found that EDTA significantly enhanced the antitumor effect of zinc pyrithione under acidic conditions (Figure 8). EDTA works to remove and neutralize zinc and other heavy metals within a certain tolerance range, and is widely used as a pharmaceutical, cosmetic and food stabilizer. This means that pyrithione exerts a strong antitumor effect even in the absence of zinc and other heavy metals, and it is important that heavy metals that are permeable to pyrithione be effective on the antitumor effect Can not be predicted from the prior art (see background and related art) that revealed In light of the discovery in the present application that sodium pyrithione exhibits a potent acid-induced anti-cancer effect similar to zinc pyrithione (see FIGS. 2 and 3), both zinc pyrithione and sodium pyrithione are used to treat malignancy. It means that it is effective. The antitumor effect of pyrithione which is not dependent on zinc (or any other heavy metal) disclosed in the present application may reduce the risk of heavy metal overdose by long-term administration. The use of zinc pyrithione (or any other metal loaded pyrithione) and the use of sodium pyrithione (or any other metal free pyrithione) in tumor therapy is disclosed herein.

  As a result of compound screening, it was found that combining the 10 to 100 μM concentration of pyridoxine with the acidity of the formulation component significantly enhanced the anti-cancer action of zinc pyrithione (FIG. 9). Furthermore, in the case of pyridoxine at a concentration of 1 mM, in combination with the acidity of the formulation component, it not only enhances the anticancer activity of zinc pyrithione, but pyridoxine alone exhibits acid-dependent anticancer activity even in the absence of pyrithione. It was discovered. Pyridoxine is one of the compounds having vitamin B6 activity, and other vitamin B6 compounds include pyridoxal, pyridoxamine, and their 5-position phosphate. In one embodiment, pyridoxine, pyridoxal, pyridoxamine, their 5-phosphate ester, pyridoxine dicaprylate, pyridoxine dipalmitate, any of these derivatives, zinc pyrithione (or any other metal-added pyrithione) Or shall be supplied with sodium pyrithione (or any metal free pyrithione).

  Sodium ascorbate was found to significantly enhance the anti-cancer action of zinc pyrithione, in particular in combination with acidifying the formulation components (FIG. 10). Sodium ascorbate is one of ascorbate and shows activity similar to ascorbic acid.

  3-o-ethylascorbic acid has an ethyl group, and the chemical stability is enhanced by the ether bond of this ethyl group with the 3-hydroxy group of ascorbic acid, so that percutaneous absorption is easily performed. become. 3-o-ethyl ascorbic acid was found to significantly enhance the anti-cancer action of zinc pyrithione, particularly by combining it with the acidity of the formulation components (FIG. 10). Furthermore, 3-o-ethyl ascorbic acid was found to significantly enhance the anticancer action of sodium pyrithione, particularly by combining it with acidifying the formulation components.

  Ascorbyl tetraisopalmitic acid is a lipid-soluble ascorbic acid derivative which is easily absorbed percutaneously and converted to ascorbic acid. Therefore, ascorbyl tetraisopalmitic acid and 3-o-ethyl ascorbic acid are widely used in skin care products. Topical administration of a drug formulation containing low concentrations of zinc pyrithione (1 μM), 3-o-ethyl ascorbic acid (3 mM) and ascorbyl tetraisopalmitic acid (2 mM) resulted in human skin cancer cells transplanted into the skin of mice It was found to suppress proliferation (Figure 11).

  Histological examination of the skin site, liver and kidney exposed to the topical drug formulation revealed no findings of tissue damage or tissue abnormality (micrograph shown in FIG. 11B). Furthermore, as the results summarized in the table show, no hematologic abnormalities were observed due to topical administration of pharmaceutical ingredients containing pyrithione and ascorbic acid derivative group. Summarizing these findings, it is suggested that the method disclosed herein does not cause serious side effects.

  In certain embodiments, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitic acid (or any other ascorbic acid derivative), sodium ascorbate (or any other ascorbate), ascorbic acid, or The combination is to be administered topically with zinc pyrithione (or pyrithione loaded with any other metal) or sodium pyrithione (or pyrithione without any metal).

  Direct administration of drug components to cancer cells in the vicinity of the surface of body surface or internal organs, and precancerous lesion site, using a pharmaceutically acceptable salt, cream, ointment, lotion, gel, solid stick, spray Drops (eye drops, nose drops, etc.), or in the form of a sealing device. These pharmaceutical formulations may be oil-in-water (O / W) emulsions, water-in-oil (W / O) emulsions, viscous solutions, or aqueous solutions. It is also possible to use a sealing device such as a semipermeable membrane covering a reservoir containing pyrithione and other active substances in order to release pharmacologically active substances such as pyrithione and anticancer drugs, steroids and anti-inflammatory agents. Pharmaceutical formulations for topical administration of pyrithione can be formulated by a general method using appropriate excipients, and the excipients include, for example, emulsifiers, surfactants, thickeners, humectants, Skin conditioners, skin protectants, sunscreens, etc. may be included. The closed topical administration device may optionally contain a preservative such as methylhydroxyparaben, propylhydroxyparaben, chlorocresol, or a bacterial growth inhibitor.

  The drug component can be a needle or a device based thereon, a high pressure stenosis jet administration method, an external electrolysis, or the like using a pharmaceutically acceptable salt for cancer near the body surface or near the surface of an internal organ, or a precancerous lesion site. Application of difference, ultrasound, laser beam, electromagnetic waves, radiation, etc. may be used for administration.

  Suppositories may be used for vaginal and rectal introduction. Suppository formulations may be prepared by blending the compounds disclosed herein with the formulation components and suitable excipients such as cocoa butter, polypropylene glycols of various molecular weights, glycerin, glycerinated gelatin and the like. These excipients are solid at room temperature but dissolve upon body temperature, releasing the compound and formulation components into the rectum, vagina, and cervix.

  In certain embodiments, skin permeability enhancers and solubility enhancers may be added to the formulation according to the prior art to promote absorption from the inner edges of body surfaces or structures (Williams and Barry, 2012 ). Skin permeability enhancers include unsaturated fatty acids such as oleic acid, docosahexaenoic acid, eicosapentaenoic acid, terpenes, terpenoids, essential oils, azones, pyrrolidones and the like. Examples of dissolution promoters include low concentration surfactants, cyclodextrin, dimethyl sulfoxide (DMSO) and the like.

  Zinc pyrithione at 0.00000317% weight concentration (100 nM) was found to suppress cancer cell growth by two-dimensional culture in combination with acid (see FIGS. 2, 3 and 6). According to the prior art, the transdermal absorption efficiency of pyrithione is said to be around 1% (see (Wedig et al., 1974)). From this, in one embodiment, at least 0.000317% by weight of pyrithione is included. Since 0.0000634% (2 μM) of pyrithione is required to suppress cancer cell growth by three-dimensional culture (see FIG. 5), it is preferable to contain at least 0.00634% by weight of pyrithione. The cream containing 1% by weight of zinc pyrithione showed a remarkable antitumor effect (FIG. 11). From this, it is further preferable to contain at least 1% pyrithione. As an emulsion containing 5.5% pyrithione was found to be stable, another embodiment would include up to 5.5% by weight pyrithione.

  In one embodiment, EDTA contains 0.0003% by weight (10 μM) (see FIG. 8), which is the lowest concentration necessary to enhance the anticancer effect of zinc pyrithione, as the lowest concentration. The drug formulation contains up to 10 to 15% by weight of EDTA, since this concentration of EDTA can be used safely for short-term dental treatment (Lanigan and Yamarik, 2002). More preferably, the drug formulation will contain up to about 2% by weight EDTA, as this concentration is the maximum concentration where it is suitable for long-term use as a cosmetic ingredient.

  In one embodiment, the topical drug formulation comprises a minimum of 0.00017% by weight (10 mM) pyridoxine, but this concentration is the minimum pyridoxine required to promote the acid-dependent antitumor effect of zinc pyrithione. It is based on concentration (FIG. 9). More preferably, the pharmaceutical formulation comprises a minimum of 0.0017% by weight (100 μM) of pyridoxine, but this concentration of pyridoxine is due to the higher potency. More preferably, the pharmaceutical formulation contains at least 0.017% by weight (1 mM) pyridoxine, but this concentration of pyridoxine exerts an acid-dependent antitumor effect even in the absence of zinc pyrithione. by. Due to other factors such as skin permeability, permeability, chemical stability, etc., it is considered that higher concentrations of pyridoxine are required to exert the effect expected in practice. For example, the prior art has disclosed cosmetic ingredients containing 0.001% to 15% pyridoxine (see US Patent Application Publication No. 2006/0018860). However, in the presence of other formulation components, formulations containing 10% by weight or less of pyridoxine have been found to be more stable. Thus, in one embodiment, the pharmaceutical formulation contains 10% by weight or less of pyridoxine. The fat-soluble pyridoxine derivatives pyridoxine dicaprylate and pyridoxine dipalmitate are easily absorbed from the body surface, and once absorbed, exhibit stable activity. In certain embodiments, the pyridoxine dicaprylate, pyridoxine dipalmitate, or other pyridoxine derivative contains at least 0.00017 wt% or more, preferably at least 0.0017 wt% or more, more preferably at least 0.017 wt% or more It shall be contained at a concentration of up to 10% by weight or less.

  In certain embodiments, the topical pharmaceutical formulation contains a minimum of 0.04% by weight (2 mM) 3-o-ethyl ascorbic acid, which is 2 mM 3-o-ethyl ascorbic acid of pyrithione and acid This is because, in the presence, the growth of cancer cells under two-dimensional culture conditions is suppressed (see FIG. 10). More preferably, the topical drug formulation will contain at least 0.2% by weight (10 mM) 3-o-ethyl ascorbic acid, which is 10 mM 3-o-ethyl ascorbic acid in the presence of pyrithione and acid Under these conditions, the growth of cancer cells under three-dimensional culture conditions is suppressed (see FIG. 10). More preferably, the topical pharmaceutical formulation contains at least 3% by weight of 3-o-ethyl ascorbic acid, which is a human skin cancer in which 3% 3-o-ethyl ascorbic acid has been implanted in the mouse. This is because cell proliferation is efficiently suppressed in the presence of pyrithione and an acid (see FIG. 11). Besides 3% 3-o-ethyl ascorbic acid and zinc pyrithione, the formulation contains 2% by weight ascorbyl tetraisopalmitate (see Example 11). From this, it is more preferable that at least 3% by weight or more of 3-o-ethyl ascorbic acid, 2% by weight or more of ascorbyl tetraisopalmitate, ascorbyl palmitate, or any of the other ascorbic acid derivatives. Or one or a combination of two or more. The inventor has noticed that formulations containing more than 20% by weight of 3-o-ethyl ascorbic acid sometimes cause skin irritation to sensitive skin. From this, in one embodiment, 20% or less, more preferably 10% or less of pyridoxine is contained.

  Treatment of cancer cells in two-dimensional culture with 0.04 wt% (about 2 mM) sodium ascorbate with pyrithione and acid was found to suppress the growth of cancer cells (FIG. 10). In certain embodiments, the topically administered drug formulation comprises sodium ascorbate (or any other ascorbate salt), ascorbic acid, or ascorbyl tetraisopalmitate (or any other ascorbate) at a concentration of 0.04% or greater. Acid derivative). The formulation containing 30% sodium ascorbate or 15% ascorbyl tetraisopalmitate was stable and showed no skin hypersensitivity. Thus, in certain embodiments, the pharmaceutical formulation is sodium ascorbate (or any other concentration of 30% or less, more preferably 20% or less, even more preferably 10% or less). Ascorbate salt) shall be contained. In yet another embodiment, the pharmaceutical formulation comprises 15% or less, more preferably 10% or less, still more preferably 5% or less of ascorbyl tetraisopalmitate (or other ascorbic acid derivative) Shall be included.

  It was found that the topical administration of a formulation containing pyrithione to mouse skin once a day suppresses the growth of skin cancer (FIG. 11). Thus, in one embodiment, a pharmaceutical formulation containing pyrithione and an enhancer is to be administered topically once daily. According to known techniques, only 34% of zinc pyrithione remains in the area of skin administration 20 hours after topical administration (Parekh et al., 1970). Furthermore, this prior art has revealed that if topical re-administration of pyrithione occurs within 20 hours of dosing interval, the residual concentration of pyrithione in the area of skin administered is increased. This means that the effective concentration of pyrithione in the skin is increased by administration more than once a day. In another embodiment, a pharmaceutical formulation containing pyrithione and a potentiator should be topically administered twice daily. In yet another embodiment, a pharmaceutical formulation containing pyrithione and an enhancer should be topically administered three times a day. In certain embodiments, the drug is administered until the desired effect is obtained, but typically, the pyrithione drug formulation is administered for a period of 2 months to 36 months. In another embodiment, a one to four week washout period is provided between each treatment cycle. In yet another embodiment, chemotherapy, radiation and surgery are performed during this drug holiday. Other medication cycle regimes and duration of treatment may be considered as deemed appropriate by the skilled artisan.

  As an example of pyrithione local administration, it is applied to the treatment of malignancy on the skin.

  As another example of topical administration of pyrithione, it is applied to the treatment of cervical cancer, vaginal cancer, and other malignant tumors of the cervix and vaginal area.

  Local administration of pyrithione can be applied to other malignancy treatments accessible from the body surface. Target diseases include head and neck, oral cavity, anal, rectum, prostate, mammary gland, bone, muscle, cartilage, malignancies generated in cartilage, malignancy metastasized at these sites, but other malignancies are also targets. obtain.

  Local therapy penetrates the drug component from the outer surface of the tumor. Systemic therapy supplies the drug component to the tumor through the blood vessels that supply the tumor with blood flow. By combining these two administration methods, the intratumoral effective concentration of the drug component is maximized. Oral and intravenous administration methods are systemic administration methods widely used to increase serum levels of vitamins.

  Orally administered pyridoxine 600 mg rapidly enters the systemic circulation within 0.3 hours, and it has been revealed by the prior art that the serum concentration reaches 25 μM after 1.3 hours (see (Zempleni, 1995) ). The concentration of 25 μM is higher than 10 μM, which is the lowest concentration required to exert an anti-cancer effect when pyridoxine is administered with pyrithione and acid (FIG. 9). On the other hand, even if 100 mg of pyridoxine is administered by another common method of intravenous injection, the maximum serum concentration of pyridoxine achieved in 6 hours is only 0.37 μM. From these, in one embodiment, oral administration of a minimum daily dose of 600 mg of pyridoxine, pyridoxal, pyridoxamine, or the 5-position phosphorylated ester of these compounds is combined with topical administration of pyrithione and pyridoxine (or a derivative thereof) To be done. The prior art has shown an association between high doses of pyridoxine> 1,000 mg daily and the risk of neurological disorders (see (Bender, 1999)). Thus, in another embodiment, oral administration of pyridoxine, pyridoxal, pyridoxamine, or the 5-position phosphorylated ester of these compounds up to a maximum daily dose of 1000 mg concentration with topical administration of pyrithione and pyridoxine (or a derivative thereof) It is done in combination. Orally administered pyridoxine is rapidly degraded within one hour of the estimated half-life when entering the systemic circulation (see (Zempleni, 1995)), so it is desirable to divide the daily dose into multiple doses. In another embodiment, pyridoxine, pyridoxal, pyridoxamine, or the 5-position phosphorylated ester of these compounds are orally administered twice a day, more preferably three times a day, more preferably four times a day.

  The prior art shows that the maximum serum concentration of ascorbic acid that can be achieved by intravenous administration is 13,400 μM, whereas the maximum serum concentration of ascorbic acid that can be achieved by oral administration is only 220 μM. (See (Padayatty et al., 2004)). This is because intravenous administration of ascorbic acid can achieve the minimum serum concentration (1 to 10 mM) required to suppress cancer growth in the presence of pyrithione and acid, while oral administration can It shows that it does not reach (FIG. 10). In certain embodiments, intravenous administration of 3-o-ethyl ascorbic acid (or any of the other ascorbic acid derivatives), sodium ascorbate (or any of the other ascorbate salts) or ascorbic acid is pyrithione and 3-o -In combination with topical administration comprising one or more combinations of ethyl ascorbic acid (or any of the other ascorbic acid derivatives), sodium ascorbate (or any of the other ascorbate salts) or ascorbic acid Do. The prior art has shown that intravenous administration of 5 g of ascorbic acid can raise ascorbic acid serum concentrations to 2 mM or more, but similar effects can not be obtained with intravenous administration of 3 g or 1 g. Thus, in one embodiment, at least 5 g of ascorbic acid, sodium ascorbate (or any of the other ascorbate salts), or 3-o-ethyl ascorbic acid (or any of the other ascorbic acid derivatives) It shall be administered intravenously. Intravenous administration of ascorbic acid up to 100 g is known to be a safe and effective method for raising serum ascorbic acid concentrations. Thus, in certain embodiments, up to 100 g of ascorbic acid, sodium ascorbate (or any of the other ascorbate salts), or 3-o-ethyl ascorbic acid (or any of the other ascorbic acid derivatives) It shall be administered intravenously.

  The prior art also taught us that administering multiple daily doses of ascorbic acid in multiple doses is useful to maintain a constant level of serum effective concentration in the body. For example, intravenous infusion of 100 g, 50 g and 10 g of ascorbic acid keeps the serum ascorbic acid concentration at 2 mM or higher for about 5.5 hours, 3.5 hours and 1.3 hours, respectively. This is because injecting 50 g ascorbic acid twice a day intravenously, or injecting 10 g ascorbic acid five times a day is better than injecting 100 g ascorbic acid once a day. It means that it is more efficient in keeping the concentration of serum ascorbic acid at 2 mM or more. Thus, in one embodiment, 50 g of sodium ascorbate (or any of the other ascorbate salts), 3-o-ethyl ascorbic acid (or any of the other ascorbic acid derivatives) or ascorbic acid in one embodiment Infused intravenously twice a day. In another embodiment, one 10 g of sodium ascorbate (or other ascorbate) or 3-o-ethyl ascorbic acid (or other ascorbic acid derivative) is infused intravenously five times a day. In another embodiment, once 33 g of ascorbic acid, 3-o-ethyl ascorbic acid (or other ascorbic acid derivative), or sodium ascorbate (or other ascorbate) is intravenously administered three times a day inject. In one embodiment, a withdrawal period of 1 day to 4 weeks is provided between each treatment cycle. Other dosing cycle regimens and duration of treatment may be considered as deemed appropriate by the skilled artisan.

  In principle, standard concentrations and dosing protocols of pyrithione and other formulation ingredients are aimed at tumor growth suppression and tumor regression. For therapeutic applications, standard concentrations of pyrithione and other prescribed ingredients and the administration protocol used are: condition, type of malignancy, age, body weight of the affected person being treated, route of administration, etc., of the treating clinician It depends on the factor decided by experience. Standard concentrations and administration protocols for pyrithione and other prescribed components, when used in conjunction with other treatments, also depend on the type of treatment used, the therapeutic effect and the degree of side effects. Tumor regression of malignancy can be determined by tumor size. It may also be judged as tumor degeneracy if the malignant tumor does not relapse after the end of treatment.

Methods and drug components for preventing the progression of precancerous lesions to malignant tumors

  A unique feature of skin and cervical malignancies is that malignancies of these organs frequently develop from non-cancerous inflammatory conditions. This can also be said that the onset of malignancies in the skin and cervix can often be predicted by the aberrant state of these organs. The acidic environment caused by persistent and uncontrolled inflammation at the precancerous lesion site is ideal for using pyrithione to inhibit the growth of newly emerging malignancies here. In a further novel aspect, the present application discloses a method for preventing the onset of cancer from a body surface or a mucosal lesion which is likely to progress to a malignant tumor by using topical administration of a pharmaceutical preparation containing pyrithione. . The disclosed method takes advantage of the acid pH at the precancerous lesion site and the acid pH of the pyrithione formulation itself to denature a small number of malignant cells before they form a visible tumor mass.

  One application area is to prevent the development of skin cancer from actinic keratosis and other precancerous lesions of the skin.

  Another application field is to prevent the development of cervical cancer, vaginal cancer, anal cancer, penile cancer and oral cancer from chronic infection with papilloma virus.

  Topical administration may be carried out by incorporating pharmacologically acceptable salts into a cream, an ointment, a lotion, a gel, a solid stick, a spray, a sealing device. These pharmaceutical formulations may be oil-in-water (O / W) emulsions, water-in-oil (W / O) emulsions, viscous solutions, or aqueous solutions.

  In one embodiment, the pH of the topical drug formulation is 6.9 or less, more preferably 6.4 or less, and even more preferably 5.6 or less. Since the acidic environment is extremely strong at the precancerous lesion site, it is more desirable that the pH of the drug formulation be 4.5 or less. In yet another embodiment, the pH of the drug formulation is at least 2.0 or more. The drug formulation used in the animal studies of the present application contains 20 mM citrate buffer (see Example 11). In one embodiment, in order to enhance pH stability, it contains citrate buffer at a concentration of up to 100 mM in the drug formulation, but a more desirable maximum concentration is 75 mM, and a more desirable maximum concentration is 50 mM to maintain chemical stability. I assume. In another embodiment, the minimum concentration of citrate buffer is 5 mM, more preferably 7.5 mM, more preferably 10 mM. In some embodiments, phosphate buffer or other human-safe buffer is used.

  In one embodiment, skin permeability enhancers and solubility enhancers are added to the formulation according to known techniques (see (Williams and Barry, 2012). Skin permeability enhancers are used to increase the efficiency of absorption from the skin). And unsaturated fatty acids such as oleic acid, docosahexaenoic acid and eicosapentaenoic acid, terpenes, terpenoids, essential oils, azone, pyrrolidone etc. Examples of dissolution accelerators include low concentration surfactants, cyclodextrin, dimethyl sulfoxide and the like .

  Vaginal and rectal administration may use a suppository form. Suppository formulations can be made by incorporating the pharmaceutically active ingredients and pharmaceutical formulations of the present invention with cocoa butter, excipients such as polyethylene glycols of various molecular weights, glycerin, glycerinated gelatin and the like. Although these excipients are solid at room temperature, they dissolve upon body temperature, and the active ingredients and drug formulations are released into the rectum, vagina, and cervix.

  In certain embodiments, the topically administered drug formulation comprises zinc pyrithione, sodium pyrithione, or other pyrithione compounds at least 0.000317% by weight, more preferably at least 0.00634%, and even more preferably at least 1%. In another embodiment, the pharmaceutical formulation contains up to 5.5% by weight of zinc pyrithione, sodium pyrithione, or any other pyrithione compound.

  In certain embodiments, 0.0003% to 15%, more preferably 0.0003% to 10%, and even more preferably 0.0003% to 2% of EDTA is included in the drug formulation.

  In certain embodiments, the topically administered drug formulation contains at least 0.00017 wt%, more preferably at least 0.0017%, and even more preferably at least 0.017% pyridoxine. In another embodiment, the drug formulation contains at most 10% by weight or less, more preferably at most 5% or less, and even more preferably at most 2.5% or less of pyridoxine. The fat-soluble pyridoxine derivatives pyridoxine dicaprylate and pyridoxine dipalmitate are easily absorbed from the body surface, and once absorbed, exhibit stable activity. Pyridoxamine, pyridoxal pentaphosphate, and pyridoxamine pentaphosphate exhibit biological activities similar to pyridoxine. The fat-soluble pyridoxine derivatives pyridoxine dicaprylate and pyridoxine dipalmitate are easily absorbed from the skin and exhibit stable activity once absorbed. In one embodiment, these compounds are included in pharmaceutical formulations.

  In certain embodiments, the topically administered drug formulation comprises at least 0.04% by weight or more, more preferably at least 0.2% or more, still more preferably at least 3% or more of 3-o-ethyl ascorbic acid (or other ascorbic acid derivative Or sodium ascorbate (or any of the other ascorbate salts) or ascorbic acid. More desirably, the topically administered drug formulation comprises a minimum of 3% or more of 3-o-ethyl ascorbic acid (or any other ascorbic acid derivative), sodium ascorbate (or any other ascorbate) or ascorbic acid In addition to these, ascorbic acid tetraisopalmitate at a concentration of 2% or more, ascorbyl palmitate, and any one or more combinations of other fat-soluble ascorbic acid derivatives. In one embodiment, 20% by weight or less, more preferably 10% or less of pyridoxine is contained.

  In one embodiment, pyridoxine, pyridoxal, pyridoxamine, and oral administration of their 5-position phosphate ester in combination with topical administration of a pyrithione-containing formulation at 0.1 mg to 1 g daily, more preferably 10 mg to 300 mg daily Do. In one embodiment, pyridoxine, pyridoxal, pyridoxamine, and their 5-position phosphate ester are orally administered once a day. In another embodiment, pyridoxine, pyridoxamine, pyridoxal, pyridoxamine, and their 5-position phosphate esters are orally administered every 8 hours three times a day.

  In some embodiments, intravenous administration of 3-o-ethyl ascorbic acid (or any of the other ascorbic acid derivatives), sodium ascorbate (or any of the other ascorbate salts) or ascorbic acid, pyrithione and Drug containing 3-o-ethylascorbic acid (or any of other ascorbic acid derivatives), sodium ascorbate (or any of other ascorbate salts) or ascorbic acid either alone or in combination It is used in combination with topical administration of prescription. In one embodiment, ascorbic acid, sodium ascorbate (or any of the other ascorbates), or 3-o-ethyl ascorbic acid (or any of the other ascorbic acid derivatives), with a minimum dose of 5 g daily ) Intravenously. In another embodiment, ascorbic acid, sodium ascorbate (or any of the other ascorbates), or 3-o-ethyl ascorbic acid (or any of the other ascorbic acid derivatives, with a maximum dose of 100 g daily) Dose intravenously. In certain embodiments, the topical pharmaceutical formulation contains at least 0.04% by weight, more preferably at least 0.2%, and more preferably at least 3% of 3-o-ethyl ascorbic acid. Even more desirably, the topical drug formulation comprises at least a 3% concentration of 3-o-ethyl ascorbic acid and a minimum of 2% or more concentrations of ascorbyl tetraisopalmitate, ascorbyl palmitate, and other fat-soluble ascorbic acid derivatives. It contains any one or a combination of two or more. In another embodiment, the topical drug formulation contains 3-o-ethyl ascorbic acid up to a concentration of 30% or less, more desirably 20% or less, and even more desirably 10% or less.

  In some embodiments, 3-o-ethyl ascorbic acid (or any of the other ascorbic acid derivatives), sodium ascorbate (or any of the other ascorbate salts) or ascorbic acid intravenous administration comprises pyrithione Together with topical administration of the drug formulation. In one embodiment, the daily dose is administered in a single daily injection. In another embodiment, the daily dose is administered in twice daily injections. In yet another embodiment, the daily dose is administered in three injections daily. In one embodiment, the agent is administered until the desired effect is obtained. In another embodiment, a one to four week washout period is provided between each treatment cycle. Other dosing cycle regimes and duration of treatment may be considered as deemed appropriate by the skilled artisan.

  The topically administered pharmaceutical formulations containing pyrithione can be administered from the body surface, genitals or other organs once a day, more preferably twice a day, and more preferably three times a day. In certain embodiments, the pyrithione-containing topically administered drug formulation is administered daily. In another embodiment, the pyrithione-containing topically administered drug formulation is administered three times a week. In one embodiment, the drug administration period is from 1 week to 96 weeks, more preferably from 2 weeks to 32 weeks, still more preferably from 2 weeks to 16 weeks. In one embodiment, there is a one to four week washout period between each dosing cycle. Other medication and withdrawal cycle regimes and treatment periods may be considered as deemed appropriate by those skilled in the art.

  During the treatment period, the dosage is strictly supervised by the physician. Duration and interval of dosing can also be determined by clinical and preclinical studies. Those skilled in the art can judge these from many factors such as age, weight, clinical condition and the like. Clinical status is determined by signs, symptoms, and routine medical examinations. Other clinical diagnostic methods include pathological examination, colposcopy (examination with a special magnifier of the vagina and cervix) and serologic examination.

Example 1
Cell lines and cell reagents, colon cancer HT29 cells, breast cancer MDA-MB-231 cells, brain tumor U87 cells, skin cancer A2058 cells were obtained from the American Type Culture Collection (ATCC). After trypsinization, these cells were suspended in a cell culture medium composed of 90% Dulbecco's Modified Eagle's Media (DMEM) and 10% fetal bovine serum (FBS).

Cervical cancer HeLa cells were stored frozen in liquid nitrogen until used for experiments in the presence of 10% DMSO. Prior to the experiment, cells were removed from liquid nitrogen and cultured in an atmosphere containing 5% carbon dioxide using DMEM medium containing 10% fetal bovine serum (FBS). When the cell density reached about 80%, the cells were treated with trypsin and diluted and passaged at a ratio of 1: 4. Cells not exposed to the test compound were cultured in an atmosphere containing 5% carbon dioxide by adding 10% of fetal bovine serum to DMEM medium adjusted to pH 7.3 with 3.7 g / L sodium bicarbonate added . Pyrithione and other compounds, in order to examine the effects on cancer cell proliferation in pH and concentration dependence in DMEM medium 1,4 piperazinyl radiator down sulfone acid buffer _{(PIPES, pK a 6.1 ~ 7.5} ) the 10mM adjustment was added to a concentration pH 6.4, in DMEM medium 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid _{(HEPES, pK a 6.8 ~ 8.2} ) was added to a 10mM concentration pH7 Adjusted to .4. PIPES (P6757, Lot # 026K5416), HEPES (H4034, Lot # 087K54432), Sodium Pyrithione (2-Mercaptopyridine N-Oxide Sodium, H3261, Lot # 0655M4172V), Methylthiozolyldiphenyl-telolazolium bromide (MTT, M5655) , Sigma) were purchased from Sigma. Zinc pyrithione (PHR1401, Lot # LRAA8431) was purchased from Fluka. Erlotinib (# 10483, Lot # 0459700-31) and gefitinib (sc-202166, Lot # A0616) were purchased from Cayman Chemical and Santa Claus Biotechnology, respectively. Common chemical reagents were used as pure as possible, and other reagents were purchased from Sigma unless otherwise specified.

Example 2
The effect of treating cancer cells with zinc pyrithione (Pyz) at pH 7.4 and pH 6.4 conditions on cancer cell viability The malignant extracellular cell has a strong extracellular pH of 6.0 to 6.9 characteristic of malignant tumors. The extracellular pH of normal tissue is 7.3 to 7.4 while producing acidity (Parks et al., 2013). Contact of zinc pyrithione with 4 different human cancer cells such as cervical cancer HeLa cells, brain cancer U87 cells, colon cancer HT29 cells and breast cancer MDA-MB-231 cells shows that the survival rate of these cells is significantly reduced It was done. When treated with zinc pyrithione in an acidic medium at pH 6.4, cell viability decreased more significantly than when treated with a medium at pH 7.4. The chemical structure of zinc pyrithione is shown in FIG.

Cancer cells were seeded at 2,000 per well in 96-well culture dishes, and cultured overnight at 37 ° C. in an atmosphere containing 5% carbon dioxide in DMEM medium containing 10% fetal bovine serum. In order to investigate the pH-dependent anticancer activity of pyrithione and other compounds, pH = 6.4 DMEM medium was adjusted by adding PIPES to a concentration of 10 mM and adjusting the pH to 6.4. pH = 7.4 DMEM medium was adjusted by adding HEPES to a concentration of 10 mM and adjusting the pH to 7.4. PIPES (pK _a 6.1 ~ 7.5) and HEPES (pK _a 6.8 ~ 8.2) was used to adjust the pH value to 6.4 and 7.4 respectively. The pH of 6.4 was adopted as a representative pH value for modeling the acidity of the tumor environment, and the pH of 7.4 was adopted for each experiment as a representative pH value for modeling the acidity of a normal tissue environment. The cells were cultured for 72 hours at 37 ° C. in the presence of different concentrations of zinc pyrithione in pH = 6.4 DMEM medium or pH = 7.4 DMEM medium. The experiments were performed in bicarbonate-free media and in the absence of atmospheric carbon dioxide in order to minimize bicarbonate-mediated pH transfer due to proton transfer across the cell membrane. After 72 hours of exposure to zinc pyrithione, culture was continued by changing to a fresh medium containing 1 mg / mL MTT in DMEM containing 10% of 50 μL of fetal bovine serum per well. After culturing for 2 hours, the cells were thawed by adding 50 μL of medium volume and an equivalent volume of cell extract containing 20% SDS, 2% acetic acid, 2.5% hydrochloric acid, and 50% DMF as components. . After shaking the 96-well culture dish, it was allowed to react at 37 ° C. for 4 hours, and the absorbance at 570 nm was measured. After background subtraction, the relative survival rate was calculated using the measurement value of the non-drug-treated group as the control group. The statistical significance of different condition groups and each control group was examined by Student's t test respectively. A p-value of less than 0.05 was considered to be significantly different.

  When cancer cells are treated with 50 nM zinc pyrithione in a culture medium (medium) adjusted to pH 7.4 for 72 hours, the relative cell survival rate to cells not treated with zinc pyrithione is about 80 − It was 90%. In contrast, when these cancer cells are treated with 50 nM zinc pyrithione for 72 hours in a weakly acidic culture medium (medium) adjusted to pH 6.4, relative to the group of cells not treated with zinc pyrithione Cell viability was about 5-20%. For example, when human cervical cancer HeLa cells were treated with 50 nM zinc pyrithione at pH 7.4, the relative survival rate to the group of cells not treated with zinc pyrithione was 87 ± 9%, whereas When 50 nM zinc pyrithione was treated at pH 6.4, the relative viability to cells not treated with zinc pyrithione was 5.3 ± 1.6% (p <0.001). These new findings indicate that the acidic environment enhances the efficacy of zinc pyrithione's anti-cancer action. The results are summarized in FIG. A P value of 0.05 or less is considered a significant difference.

Example 3
The effect of treating cancer cells with sodium pyrithione at pH 7.4 and pH 6.4 conditions on cancer cell survival rate It has been suggested that zinc pyrithione or other heavy metal addition type pyrithione exhibits an anticancer effect. On the other hand, it has been suggested that pyrithione to which no metal is added also has anti-inflammatory and anti-infective effects. From these, the next important issue that is needed to develop new methods of anti-cancer treatment is whether acid-enhanced anti-cancer action is due to pyrithione or zinc? It is. To answer this question, we examined the effect of sodium pyrithione on cancer cell growth.

  In the present application, sodium pyrithione is exposed to four different cancer cells: human cervical cancer cells (HeLa cells), brain tumor cells (U87 cells), colon cancer cells (HT29 cells), and breast cancer cells (MDA-MB-231 cells). Then, it was shown that the cell survival rate is significantly reduced. When sodium pyrithione was treated in acidic medium at pH 6.4, a more pronounced decrease in cell viability was observed compared to treatment in non-acidic medium at pH 7.4.

  Cancer cells were seeded at 2,000 per well of a 96-well culture dish, and cultured in air at 37 ° C. containing 5% carbon dioxide overnight in DMEM medium containing 10% fetal bovine serum. In order to investigate the pH-dependent anticancer activity of sodium pyrithione, pH = 6.4 DMEM medium was adjusted by adding PIPES to a concentration of 10 mM and adjusting the pH to 6.4. pH = 7.4 DMEM medium was adjusted by adding HEPES to a concentration of 10 mM and adjusting the pH to 7.4. Cells were cultured in pH = 6.4 DMEM medium or pH = 7.4 DMEM medium for 72 hours at 37 ° C. in the presence of sodium pyrithione at different concentrations from low concentration to high concentration. The experiments were conducted under bicarbonate-free conditions, using bicarbonate-free media, to minimize bicarbonate-mediated pH shift due to proton transfer through the cell membrane. After exposing the cells to sodium pyrithione for 72 hours, MTT assay was performed according to Example 2. The statistical significance of the different experimental condition groups and the control group was examined by Student's t-test. Significant differences are considered with p-values of 0.05 or less.

  When cancer cells were treated with 100 nM sodium pyrithione for 72 hours in a medium adjusted to pH 7.4, the relative cell viability was about 75% compared to cells not treated with sodium pyrithione . When these cancer cells were treated with 100 nM sodium pyrithione in pH 6.4 acidic medium for 72 hours, the relative cell viability was about 5% to 20% as compared to the cells not treated with sodium pyrithione. . For example, when human cervical cancer HeLa cells are treated with 100 nM zinc pyrithione at pH 7.4 or pH 6.4, the relative viability is 90 ± 9%, respectively, as compared to the group of cells not treated with sodium pyrithione. It was 4.9 ± 1.7% (p <0.001). A P value of 0.05 or less is considered a significant difference.

  These new findings indicate that the acidic environment has enhanced the anti-cancer efficacy of sodium pyrithione. The results are summarized in FIG.

Example 4
MTT assay measures cell viability by detecting cellular metabolic activity. Trypan blue can penetrate into the interior of the cell from the deintegrated biomembrane of dead cells (but not into the interior of living cells). Thus, only dead cells can be visualized using trypan blue. The cell killing effect was determined by trypan blue staining assay when HeLa cells were treated with zinc pyrithione (Pyz) or sodium pyrithione (Pyn) at pH 7.4 or pH 6.4. Ctr represents a control group cell treated with a drug-free medium containing only a solvent at the same concentration as the drug-treated group.

  The cancer cells were seeded at 20,000 per well in 12-well culture dishes, and cultured overnight at 37 ° C. in an atmosphere containing 5% carbon dioxide in DMEM medium containing 10% fetal bovine serum. In order to examine the pH-dependent anticancer activity of zinc pyrithione and sodium pyrithione, pH = 7.4 DMEM medium was adjusted by adding HEPES to a concentration of 10 mM and adjusting the pH to 7.4. pH = 6.4 DMEM medium was adjusted by adding PIPES to a concentration of 10 mM and adjusting the pH to 6.4. The cells were cultured for 72 hours at 37 ° C. in pH = 6.4 DMEM medium or pH = 7.4 DMEM medium in the presence of zinc pyrithione or sodium pyrithione at different concentrations from low concentration to high concentration. In order to minimize bicarbonate-mediated pH shift due to proton transfer across the cell membrane, experiments were performed in the absence of bicarbonate in the medium and carbon dioxide in the atmosphere. The cells were exposed to zinc pyrithione or sodium pyrithione for 72 hours, then suspended and collected, and a PBS solution containing 0.4% trypan blue was added to the cell suspension in an equal amount. On the recording photograph, the total number of cells and the number of cells stained with trypan blue were counted, and the ratio of cells stained with trypan blue was calculated. Four photographs were taken for one condition, and the average ratio of cells stained with trypan blue and the standard deviation indicated by the error bar are shown on the graph. The results are as summarized in FIG.

  The cell viability determined by not staining with trypan blue substantially matches the cell viability determined by MTT assay. This supports that when pyrithione is treated in pH 6.4 medium pyrithione is more sensitive to the cell killing effect than when treated in pH 7.4 medium.

Example 5
Zinc pyrithione exerts an antiproliferative effect on spheroid three-dimensional cultured brain tumor cells.

  A tumor mass is formed when the extracellular matrix collagen gel is added to the medium and the tumor cells are cultured in a semi-floating state. Tumors that develop in such extracellular matrix are called "spheroids" and are used as a model to reproduce the tumor microenvironment in three dimensions. The spheroid culture method makes it possible to analyze the effect of anticancer drugs in a three-dimensional culture that reproduces the complexity of the actual cancer (Friedrich et al., 2009). Since the bioactivity of many compounds is influenced by the tumor microenvironment, the question of whether pyrithione affects the growth of spheroids is of importance. To answer this question, brain tumor U87 cell spheroids made in the presence of collagen are made under acidic conditions (pH 6.4) or neutral conditions (pH 7.4) and treated with zinc pyrithione at 37 ° C for 3 days To measure the growth of spheroids.

  1% agar solution was dissolved by heating at 60 ° C, and equal volume of culture solution (10% fetal bovine serum added to pH 6.4 DMEM medium, or 10% fetal bovine serum added to pH 7.4 DMEM medium) The mixture to which was added was transferred 50 μl per well of the 96 well culture plate. The plate was cooled at room temperature for 30 minutes until the agar solidified, and a suspension of 1,000 cells in 50 μl of culture was overlaid thereon. The cells were cultured at 37 ° C. for 3 days, and the spheroids formed on the agar layer were cultured for 3 days in the presence or absence of different concentrations of zinc pyrithione. The size of spheroids treated with 1 μM or 2 μM of zinc pyrithione for 3 days with pH 6.4 culture medium is comparable to that of control group treated with pyrithione-free pH 6.4 culture medium for 3 days Was significantly smaller. In contrast to this, the spheroid size treated with 1 μM or 2 μM zinc pyrithione for 3 days with pH 7.4 culture medium is the spheroid size of the control group treated for 3 days with pyrithione-free culture medium. Almost no difference was found compared to the size of. The results are summarized in FIG.

Example 6
Acid-enhanced anti-cancer action of pyrithione causes superoxide accumulation in mitochondria.

  Free radical producing substances such as hydrogen peroxide and superoxide are produced in the process of cell metabolism. These reactive oxygen species (ROS) pose an immediate risk to cells if left unconverted. It has been previously shown that mitochondrial dysfunction due to external stress or toxin promotes the accumulation of reactive oxygen species in intracellular organelles. While most normal tissues can neutralize reactive oxygen species and block further cell damage development, cancer cells are more sensitive to increasing or decreasing concentrations of intracellular reactive oxygen species and are more reactive to reactive oxygen species accumulation. Resistance has also been shown to be low (see (Liou and Storz, 2010)). Since pyrithione photochemically degrades hydroxyradial and (pyridin-2-yl) sulfanyl group (see (DeMatteo et al., 2005)), we are involved in the production and removal of superoxide in mitochondria. I hypothesized that it might be. In order to verify this, after treating cancer cells with pyrithione for 2 hours, analysis was performed using a fluorescent probe that is visualized according to the concentration of mitochondrial superoxide.

HeLa human cervical cancer cells were seeded at 4,000 / well on an 8-well glass-bottomed chamber slide and cultured overnight at 37 ° C. in DMEM medium containing 10% fetal bovine serum (FBS) The When pyrithione is treated in an acidic or neutral extracellular environment, cells are either with or without zinc pyrithione or sodium pyrithione to determine what effect on mitochondrial superoxide accumulation, pH = 6.4 or The cells were cultured at 37 ° C. for 2 hours in DMEM medium of pH = 7.4. After cell washing, it treated ^{1.25 μM MitoSOX TM (M31008, Invitrogen} ) and ^{2.5 μM DRAQ5 TM (# 62254,} Thermo Scientific) 37 ℃ 5 min at pH7.4 medium containing. MitoSOX ^TM in order to quantitatively measure the superoxide production of mitochondria, DRAQ5 ^TM in order to visualize the cells under a fluorescence microscope by fluorescent staining nuclei were used, respectively. After 5 minutes, cells were observed with a confocal microscope while being infiltrated with NaCl-saline buffer (150 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 1 mM magnesium chloride, 20 mM HEPES, pH 7.4) .

Mitochondrial accumulation of superoxide is one of the mechanisms for killing cancer cells. By using sensitive and specific detectable MitoSOX ^TM superoxide in mitochondria, when the Soddy um pyrithione of 200nM zinc pyrithione or 400nM cancer cells treated for 2 hours in an acidic medium pH 6.4, drama It was confirmed that superoxide accumulated in mitochondria (Fig. 6). On the other hand, when cancer cells were treated with 200 nM of zinc pyrithione or 400 nM of sodium pyrithione in a normal medium at pH 7.4 for 2 hours, accumulation of superoxide in mitochondria could not be confirmed (FIG. 6). Even if the cancer cells were exposed to pyrithione-free acidic medium at pH 6.4 for 2 hours, no mitochondrial accumulation of superoxide was observed. From these results, it is suggested that contacting pyrithione with cancer cells in an acidic environment causes mitochondrial accumulation of superoxide.

Example 7
The anti-cancer effects of gefitinib and erlotinib are not enhanced by the acidic medium.

  Gefitinib and erlotinib are epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors that can specifically suppress the growth of cancer cells, thereby making them different from classical anticancer chemotherapeutic agents Provide a mechanism of action. Gefitinib and erlotinib are used clinically in the treatment of malignancies such as lung cancer and malignant melanoma of the skin (melanoma). It is not known whether the anti-cancer effect of the molecular target therapeutic agent used in these clinics is enhanced by acidity.

  Cervical carcinoma HeLa cells are seeded at 2,000 cells per well of a 96-well dish, in DMEM medium containing 10% concentration of fetal bovine serum (FBS) at 37 ° C. in air containing 5% carbon dioxide Culture overnight. The cells were treated with different concentrations of gefitinib or erlotinib from low to high concentration in pH 6.4 DMEM medium or pH 7.4 DMEM medium. In order to minimize pH fluctuations due to proton transfer through the cell membrane by bicarbonate, carbon dioxide was added to the atmosphere in pH = 6.4 or pH = 7.4 DMEM medium without bicarbonate. The experiment was conducted under the conditions not included. After 72 hours of exposure to gefitinib or erlotinib, MTT assay was performed according to Example 2.

  Unlike pyrithione, no potentiating effect of acid on anticancer activity was observed with gefitinib or erlotinib. As an example, when HeLa cells are treated with 20 μM gefitinib for 72 hours in a medium adjusted to pH 7.4 or pH 6.4, the cell viability detected by MTT assay is 54 ± 16%, 61 ± 9, respectively. %Met. As another example, when HeLa cells are treated for 72 hours in a medium adjusted to pH 7.4 or pH 6.4 with 20 μM erlotinib, the cell viability detected by MTT assay is 39 ± 8%, respectively. It was 46 ± 14%. The finding that the anti-cancer effects of gefitinib and erlotinib are not affected by the acidity represents the specificity of the acid-enhancing effect of pyrithione's anti-cancer action. The results are summarized in FIG.

Example 8
Since many previous studies have shown that zinc pyrithione's anti-cancer action is caused by zinc, the discovery in the present application that sodium pyrithione also reduces the survival rate of cancer cells like zinc pyrithione is a surprise. Thus, the inventor further explored the possibility that pyrithione without zinc has toxicity to cancer cells, other than zinc-mediated toxicity already disclosed by the prior art. In order to investigate this, the effect of metal chelating agent EDTA on the cancer cell effect by zinc pyrithione was examined.

  Cervical cancer HeLa cells, colon cancer HT29 cells, brain tumor U87 cells are seeded at 2,000 per well in a 96-well dish, 5% carbon dioxide in DMEM medium containing 10% fetal bovine serum (FBS) Culture was carried out overnight at 37 ° C. in the atmosphere contained. Colorectal cancer HT29 cells and brain tumor U87 cells were treated with different concentrations of zinc pyrithione in pH = 7.4 DMEM medium without fetal bovine serum for 20 hours at 37 ° C., whereas cervical cancer HeLa cells were treated with this. The treatment was performed for 72 hours with almost the same effect as in. In some experiments, different concentrations of EDTA were added to the medium along with zinc pyrithione. These experiments were performed in the absence of fetal bovine serum to avoid the undesirable situation that metals and cations present in the serum inactivate EDTA. Bicarbonate was also excluded from pH = 7.4 DMEM medium and experiments were performed in the absence of atmospheric carbon dioxide. MTT assay was performed according to Example 2 after the combination treatment with zinc pyrithione and EDTA, the treatment with zinc pyrithione alone, or the treatment with EDTA alone was performed.

  As shown in FIG. 6, it was found that cancer pyritethione treatment in the presence of EDTA more strongly suppressed the survival rate of cancer cells. As an example, when the brain tumor U87 cells were treated with 50 nM zinc pyrithione in a medium without fetal calf serum, the cell viability was 60 ± 7% of the viability of U87 cells in the control group without drug treatment. . The cell viability decreased to 30 ± 4% when treated with 10 μM EDTA with 50 nM zinc pyrithione, and further decreased to 19 ± 11% when 20 μM EDTA was added. On the other hand, when treated with only the concentration of EDTA, only a slight effect of 10% or less was observed. This surprising result suggests that pyrithione is an important component of the anti-cancer action of zinc pyrithione, and removal of zinc by EDTA enhances the anti-cancer action. The enhancing action by EDTA addition means that the addition of EDTA further lowers the concentration of zinc pyrithione required for anti-cancer action, and provides a useful situation for practical application.

Example 9
The anticancer activity of zinc pyrithione (Pyz) is potentiated by pyridoxine under acidic conditions.

  Cervical cancer HeLa cells are seeded at 2,000 per well of a 96-well culture dish, and in DMEM medium containing 10% concentration of fetal bovine serum (FBS) in air containing 5% carbon dioxide 37 Incubate overnight at ° C. The cells were cultured with different concentrations of zinc pyrithione from low concentration to high concentration for 24 hours in DMEM medium without fetal bovine serum at pH = 6.9 or pH = 7.4. In one experiment, different concentrations of pyridoxine were treated with zinc pyrithione. In order to minimize pH fluctuations due to proton transfer across the cell membrane, which is mediated by bicarbonate, experiments were carried out in DMEM medium with pH = 6.9 or pH = 7.4 without bicarbonate in the atmosphere. It was performed on the condition which does not contain carbon dioxide. The cells were treated with zinc pyrithione with pyridoxine or with zinc pyrithione alone, and then cultured for an additional 48 hours at 37 ° C. in pH = 7.4 medium. MTT assay was performed according to Example 2.

  Treatment of 10 μM and 100 μM pyridoxine (labeled as B6 in FIG. 9) with 50 nM zinc pyrithione in non-acidic medium at pH = 7.4 had no effect on cell viability, whereas Treatment of 50 nM zinc pyrithione with 10 μM pyridoxine in 6.9 acidic medium reduced the viability to 72 ± 4%, and treatment with 100 μM pyridoxine reduced the viability to 40 ± 12% (FIG. 9). Treatment with 10 μM or 100 μM pyridoxine together with 50 nM zinc pyrithione in acidic medium at pH = 6.4 further reduces the survival rate to 1.4 ± 0.6% and 2.5 ± 0.9%, respectively. (FIG. 9). The addition of only 10 μM or 100 μM pyridoxine to the acid medium or non-acid medium had no effect on cell viability (FIG. 9). These results suggest that 10 μM and 100 μM concentrations of pyridoxine enhance the anti-cancer action caused by the combination of zinc pyrithione and an acid. 1 mM pyridoxine also enhanced the anti-cancer effect of the combination of zinc pyrithione and acid. When 1 mM pyridoxine was added together with 50 nM zinc pyrithione and treated in non-acid medium at pH = 7.4, the cell viability was 71.5 ± 13%. When 1 mM pyridoxine was added together with 50 nM zinc pyrithione and treated in an acidic medium at pH = 6.9, the cell viability was 5.1 ± 3%. When 1 mM pyridoxine was added together with 50 nM zinc pyrithione and treated in an acidic medium at pH = 6.4, cell viability was 1.2 ± 0.8%. These results indicate that pyridoxine and pyrithione in combination with acidity exhibit an effective anti-cancer effect. Interestingly, when 1 mM pyridoxine is added to the medium at pH = 7.4, 6.9 and 6.4 in the absence of pyrithione, the cell viability is 72.9 ± 8% and 21.6 ± 6%, respectively. , And 28.4 ± 3%. This result suggests that 1 mM pyridoxine has a strong acid-resistant anticancer activity which is moderately dependent on 1 mM pyrithione in addition to the potent anticancer activity caused by the combination of pyrithione and an acid.

Example 10
The anticancer activity of zinc pyrithione (Pyz) and sodium pyrithione (Pyn) is enhanced by sodium ascorbate or 3-o-ethyl ascorbic acid under acidic conditions.

  Cervical cancer HeLa cells were seeded at 2,000 per well in 96-well dishes and cultured overnight at 37 ° C. in an atmosphere containing 5% carbon dioxide in DMEM medium containing 10% serum (FBS) . Different concentrations of zinc pyrithione from low to high concentration in cells, DMEM without serum (FBS) at pH = 6.4, DMEM without serum (FBS) at pH = 6.9, or pH = 7.4 The cells were treated for 24 hours in DMEM medium containing no serum (FBS). In some experiments, different concentrations of sodium ascorbate or 3-o-ethyl ascorbic acid were added to the medium along with zinc pyrithione. In order to minimize bicarbonate-mediated pH shift due to proton transfer across the cell membrane, experiments were performed in bicarbonate-free medium and in the absence of atmospheric carbon dioxide. After treating the cells with zinc pyrithione with sodium ascorbate or 3-o-ethyl ascorbic acid, or with zinc pyrithione alone, the cells were cultured for an additional 48 hours at 37 ° C. in pH = 7.4 medium. MTT assay was performed according to Example 2.

  When 2mM 3-o-ethylascorbic acid (AscE) is added to the medium at pH 6.9 together with 5nM and 10nM zinc pyrithione (Pyz), the cell viability is lower than that of the non-drug-treated control group. It was found to decrease to 91 ± 11% and 43 ± 11%. When 2mM 3-o-ethylascorbic acid (AscE) is added to the medium at pH = 6.4 with 5nM and 10nM zinc pyrithione (Pyz), cell viability is higher than that in the non-drug treated control group. It was found to decrease to 22 ± 10% and 11 ± 10% respectively. In contrast, addition of zinc pyrithione only at a concentration of 10 nM or less did not affect cell viability (FIG. 10A). Moreover, the cell viability did not decrease even if 2 mM 3-o-ethyl ascorbic acid and zinc pyrithione at a concentration of 10 nM or less were both added to the non-acid medium at pH 7.4. These results suggest that 3-o-ethylascorbic acid enhances the anti-cancer action of zinc pyrithione in an acid-dependent manner.

  Similarly, when 2 mM 3-o-ethyl ascorbic acid (AscE) is added together with 10 nM and 20 nM sodium pyrithione (Pyn) in acidic medium at pH 6.9, the cell viability is compared to the non-drug treated control group. It is reduced to 96 ± 5 and 48 ± 7%, respectively. When 2mM 3-o-ethylascorbic acid (AscE) is added together with 10nM and 20nM sodium pyrithione (Pyn) in acidic medium at pH 6.4, cell viability is lower than that of the non-drug treated control group, respectively. A further significant decrease was seen at 17 ± 5% and 7 ± 3%. On the other hand, even if only sodium pyrithione at a concentration of 20 nM or less is added to the acidic medium, or together with 2 mM 3-o-ethyl ascorbic acid and sodium pyrithione at a concentration of 20 nM or less in non-acidic medium However, no decrease in cell viability was observed.

  The combination of sodium ascorbate (AscNa) and acid also enhanced the anti-cancer action of zinc pyrithione (Pyz). As an example, when 1 mM sodium ascorbate was added together with 10 nM zinc pyrithione in an acidic medium at pH = 6.9, cell viability was moderately decreased to 85 ± 5% as compared to untreated control group. On the other hand, adding 10 nM zinc pyrithione with 1 mM sodium ascorbate in pH = 6.4 acidic medium can further reduce the cell viability by 46 ± 5% compared to untreated control group. It was found (FIG. 10A). A similar but more pronounced effect was seen when both 1 mM sodium ascorbate and 20 nM zinc pyrithione were added to the acidic medium at pH 6.9 and 6.4. On the other hand, even when zinc pyrithione (Pyz) alone at a concentration of 10 nM or less was added to the acidic medium, no significant change in cell viability was observed as compared to the non-drug-treated control group, and zinc pyrithione (Pyz) No significant change in cell viability was observed compared to the non-drug-treated control group even when both lM and 1 mM sodium ascorbate (AscNa) were added to the non-acid medium at pH = 7.4 (Fig. 10A). These results suggest that sodium ascorbate enhances the anti-cancer activity of zinc pyrithione in an acid-dependent manner.

  It was determined by the spheroid culture method that the anticancer effect of pyrithione on three-dimensional cultured brain tumor cells is enhanced by 3-o-ethyl ascorbic acid.

  After mixing an equal volume of medium (pH 7.4 DMEM medium supplemented with 10% fetal calf serum) mixed with 1% agarose solution heated and dissolved at 60 ° C., this mixture is added to one well of 96-well culture plate 50 μl of each was dispensed. The plate was cooled at room temperature for 30 minutes to solidify the agarose, and 50 μl of culture medium containing 1,000 cells was overlaid on the agarose layer and centrifuged at 1,500 g for 5 minutes. After incubation for 24 hours at 37 ° C., the spheroids contain different concentrations of zinc pyrithione, 3-o-ethyl ascorbic acid, or conditions containing zinc pyrithione and 3-o-ethyl ascorbic acid, or conditions without any drug, Culture for another 72 hours. The spheroids were then transferred to another 96 well. Plates were centrifuged at 1,500 g for 5 minutes and drug solution was replaced with PBS without stirring cells at the bottom of the wells. The plate is again centrifuged at 1,500 g for 5 minutes and PBS is mixed with 0.1 M pH = 5.0 sodium acetate and 0.1 wt% Triton X-100 without agitation of the cells at the bottom of the wells. The buffer solution was replaced with 5 mM p-nitrophenyl phosphate (# 34045, Thermo Fisher Science Co., Ltd.) added immediately before the experiment, and cultured at 37 ° C. for 2 hours. After 2 hours, 10 molar NaOH solution was added dropwise to stop the reaction, and the absorbance at 450 nm was measured.

  The viability of spheroids treated with only 500 nM zinc pyrithione (Pyz) was reduced to 88 ± 8% compared to untreated spheroids (FIG. 10B). In contrast, when 5 mM or 10 mM 3-o-ethylascorbic acid (AscE) was added together with zinc pyrithione, the cell viability further decreased to 76 ± 4% and 63 ± 7%, respectively. These results suggest that 3-o-ethyl ascorbic acid promotes the effect of suppressing cell growth in three-dimensional culture. Furthermore, when 10 mM 3-o-ethyl ascorbic acid was added together with zinc pyrithione, embrittlement of the three-dimensional spheroid structure occurred, and scattered cells were scattered from the periphery of the spheroid (FIG. 10B, right panel) See).

Example 11
Topical administration of a formulation containing zinc pyrithione, 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate inhibited the growth of human skin cancer cells implanted subcutaneously in mice.

  3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitic acid are derivatives of ascorbic acid which are rapidly absorbed from the skin and converted to ascorbic acid. The anti-cancer effects of topical administration of drug formulations containing zinc pyrithione, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitic acid, or combinations of these were examined in a mouse transplant model of skin cancer.

Six-week-old male C57BL / 6 athymic mice were subcutaneously implanted with 2.5 × 10 ⁶ human skin cancer A2058 cells. Two to three days after transplantation, when the size of the tumor is about 3 mm ³ , the mice are randomly divided into 4 groups of 5 mice each, and the drug formulation in the form of a cream is once a day, the skin just above the tumor Applied to the site of The first group (Pyz) contains only 1% of zinc pyrithione, the second group (Asc) contains 3% 3-o-ethyl ascorbic acid and 2% ascorbyl tetraisopalmitate, Three groups (Pyz + Asc) contain 1% zinc pyrithione and 3% 3-o-ethyl ascorbic acid and 2% ascorbyl tetraisopalmitate, and a fourth group (Vehicle) contains only carrier , Was administered topically. The prescription components are as follows.

Prescription ingredients used in Group 1 a) Water layer Glycerol: 5%
Polysorbate 20: 1%
Citric acid 1 molar solution: 0.7%
Sodium citrate 1 molar solution: 1.3%
Zinc pyrithione: 1%
Distilled water: The amount necessary to make the total volume 100% b) Oil phase Beninyl alcohol: 3%
Setanol: 3%
Glyceryl stearate: 1%
Stearic acid: 1%
Sorbitan stearate: 1%
1% cetyl palmitate
Dimethicone: 1%
c) Cooling layer cyclomethicone: 5%
d) adjust pH to 5.5 with HCl or NaOH

The aqueous layer components were stirred with heating at 75 ° C. until completely dissolved in a suitable container. The oil layer components were stirred with heating at 75 ° C until completely dissolved in a separate container. The oil layer components were added to the aqueous layer components and stirred until they were emulsified. The emulsion was cooled to 50-55 ° C. and stirring was continued until it was completely homogenized.

Prescription ingredients used in group 2 a) Water layer Glycerol: 5%
Polysorbate 20: 1%
Citric acid 1 molar solution: 0.7%
Sodium citrate 1 molar solution: 1.3%
Distilled water: The amount necessary to make the total volume 100% b) Oil phase Beninyl alcohol: 3%
Setanol: 3%
Glyceryl stearate: 1%
Stearic acid: 1%
Sorbitan stearate: 1%
1% cetyl palmitate
Dimethicone: 1%
c) Cooling layer cyclomethicone: 5%
3-o-ethyl ascorbic acid: 3%
Ascorbyl tetraisopalmitate: 2%
d) adjust pH to 5.5 with HCl or NaOH

The aqueous layer components were stirred with heating at 75 ° C. until completely dissolved in a suitable container. The oil layer components were stirred with heating at 75 ° C until completely dissolved in a separate container. The oil layer components were added to the aqueous layer components and stirred until they were emulsified. The emulsion was cooled to 50-55 ° C. and stirring was continued until it was completely homogenized.

Prescription ingredients used in group 3 a) Water layer Glycerol: 5%
Polysorbate 20: 1%
Zinc pyrithione: 1%
Citric acid 1 molar solution: 0.7%
Sodium citrate 1 molar solution: 1.3%
Distilled water: The amount necessary to make the total volume 100% b) Oil phase Beninyl alcohol: 3%
Setanol: 3%
Glyceryl stearate: 1%
Stearic acid: 1%
Sorbitan stearate: 1%
1% cetyl palmitate
Dimethicone: 1%
c) Cooling layer cyclomethicone: 5%
3-o-ethyl ascorbic acid: 3%
Ascorbyl tetraisopalmitate: 2%
d) adjust pH to 5.5 with HCl or NaOH

The aqueous layer components were stirred with heating at 75 ° C. until completely dissolved in a suitable container. The oil layer components were stirred with heating at 75 ° C until completely dissolved in a separate container. The oil layer components were added to the aqueous layer components and stirred until they were emulsified. The emulsion was cooled to 50-55 ° C. and stirring was continued until it was completely homogenized.

Formulated ingredients used in Group 4 a) Water layer Glycerol: 5%
Polysorbate 20: 1%
Citric acid 1 molar solution: 0.7%
Sodium citrate 1 molar solution: 1.3%
Distilled water: The amount necessary to make the total volume 100% b) Oil phase Beninyl alcohol: 3%
Setanol: 3%
Glyceryl stearate: 1%
Stearic acid: 1%
Sorbitan stearate: 1%
1% cetyl palmitate
Dimethicone: 1%
c) Cooling layer cyclomethicone: 5%
d) adjust pH to 5.5 with HCl or NaOH

The aqueous layer components were stirred with heating at 75 ° C. until completely dissolved in a suitable container. The oil layer components were stirred with heating at 75 ° C until completely dissolved in a separate container. The oil layer components were added to the aqueous layer components and stirred until they were emulsified. The emulsion was cooled to 50-55 ° C. and stirring was continued until it was completely homogenized.

Tumor volume a: tumor diameter, b: on the basis of the tumor short diameter is calculated by the calculation formula ab ^2/2, relative tumor volume of each treatment group is shown on the computation graph (Figure 11A). The horizontal axis of the graph shows the number of days after transdermal administration, and the vertical axis shows the relative tumor volume. The mean tumor size of group 3 mice at the end of the experiment ("Pyz + Asc" in FIG. 11A) was smaller than the mean tumor size of the other group mice. The results show that a formulation containing both pyrithione and an ascorbic acid derivative is an effective method for suppressing tumor growth, but not pyrithione alone or an ascorbic acid derivative alone.

  At the end of the experiment, mouse skin areas in contact with the drug formulation, liver and kidney tissue samples were prepared by the commonly used method. The sections were stained with hematoxylin and eosin. A photomicrograph of the tissue sample is shown in FIG. 11B. Findings suggestive of tissue damage, abnormal cell death, or other side effects include group 3 (PyZ + Asc; mice treated with cream formulations containing pyrithione, 3-o-ethylascorbic acid, ascorbyl tetraisopalmitate), and None was found in Group 4 (Vehicle; mice treated with a cream formulation containing neither pyrithione, 3-o-ethyl ascorbic acid nor ascorbyl tetraisopalmitate).

  On the day the animals were processed, blood sampling was performed and the results were tabulated. Mean erythrocyte hemoglobin (MCH) reduction, mean erythrocyte volume (MCV) reduction, and platelet (PLT) increase were observed in all prescription groups, probably for transplanted cancer cells, but in all other test items There were no abnormalities found. Although there was a slight% increase in granulocytes in the group formulated with only ascorbic acid ("Asc"), the% increase in granulocytes is not considered significant because the granulocyte count is normal. There was no blood abnormality specific to any of the prescribed groups other than granulocyte% increase. Ref: normal value; GR: granulocyte count (number per liter); GR%: granulocyte (%); HCT: hematocrit (%); HGB: hemoglobin (g / L); LY: lymphocyte count; : Lymphocytes (%); MCH: Mean erythrocyte volume (pg); MCHC: Mean erythrocyte volume concentration (g / L); MO: Monocyte count (number per liter); MO%: Monocytes (%); MPV : Average platelet volume (fL); PCT: Procalcitonin (%); PDW: Platelet distribution width (fL); PLT: Platelet (g / L); RBC: Red blood cell count (number per liter); RDW: Red blood cell distribution width (%); WBC: White blood cell count (number per liter)

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Claims (12)

  A composition for preventing and / or treating a malignant tumor, which comprises any one or more active ingredients selected from pyrithione, pyrithione salts, vitamin B6s, and phosphated vitamin B6s.   A preventive composition for a precancerous lesion, which comprises any one or more active ingredients selected from pyrithione, pyrithione salts, vitamin B6s, and phosphated vitamin B6s. The active ingredient is any one or more selected from pyrithione and pyrithione salts,
The composition according to claim 1 or 2, further comprising EDTA, and any one or more selected from EDTA salt, ascorbic acid, ascorbate, and ascorbic acid compounds.   The composition according to any one of claims 1 to 3, wherein the pH in the presence of a buffer is 6.9 or less.   A method for preventing and / or treating malignancy using any one or more active ingredients selected from pyrithione, pyrithione salts, vitamin B6s, and phosphated vitamin B6s.   A method for preventing a precancerous lesion, which comprises using any one or more active ingredients selected from pyrithione, pyrithione salts, vitamin B6s, and phosphated vitamin B6s. The active ingredient is any one or more selected from pyrithione and pyrithione salts,
The method according to claim 5 or 6, wherein the active ingredient is used together with EDTA and any one or more selected from EDTA salt, ascorbic acid, ascorbate and ascorbic acid compounds.   The method according to any one of claims 5 to 7, wherein the active ingredient is used at a pH of 6.9 or less.   Use of any one or more selected from pyrithione, pyrithione salts, vitamin B6s, and phosphated vitamin B6s in the manufacture of a medicament for preventing and / or treating a malignant tumor.   Use of any one or more selected from pyrithione, pyrithione salts, vitamin B 6s, and phosphated vitamin B 6s in the manufacture of a medicament for the prevention of precancerous lesions.   The combination of any one or more selected from the pyrithione and the pyrithione salt, and any one or more selected from EDTA, EDTA salt, ascorbic acid, ascorbate, and ascorbic acid compound. Use according to 9 or 10. 12. The use according to any of claims 9 to 11, wherein the pH is 6.9 or less.