CN111542325A

CN111542325A - Compositions for treating malignant and precancerous tumors, methods of use thereof, and methods of making medicaments

Info

Publication number: CN111542325A
Application number: CN201880025684.7A
Authority: CN
Inventors: 沼田雅行; 范泓逸; 李爱迪
Original assignee: Wutong Tree Health Science Co
Current assignee: Wutong Tree Health Science Co
Priority date: 2017-05-03
Filing date: 2018-05-04
Publication date: 2020-08-14
Also published as: JP2019520310A; WO2018203127A1; JP6676779B2

Abstract

Compositions and methods for pyrithione compounds and pharmacologically acceptable salts thereof are described. The present invention provides topical methods of administration for treating skin cancer, treating cervical cancer, treating other cancers, preventing the development of skin cancer from premalignant lesions, preventing the development of cervical cancer from chronic Human Papillomavirus (HPV) infection, preventing the development of other cancers from chronic HPV infection. Also disclosed are compositions comprising at least one pyrithione compound, ethylenediaminetetraacetic acid (EDTA), ascorbic acid, any salt thereof and chemical derivatives thereof, pyridoxine and any derivatives thereof, and a pharmacologically acceptable buffer. The pH in these compositions was adjusted to 4.5 to 6.4.

Description

Compositions for treating malignant and precancerous tumors, methods of use thereof, and methods of making medicaments

Technical Field

Rapidly growing cancer cells produce acid due to their metabolism, and the released acid makes the environment around the cancer cells acidic. This makes the cancer environment more acidic than normal tissue. The present invention discloses a novel composition and therapeutic/prophylactic methods to inhibit the growth/appearance of cancer cells in an acidic environment.

Background

Malignant tumors have become a leading cause of death worldwide. According to the statistics of the international cancer research institute, there are over 1400 million new cancer cases worldwide in 2012, which is expected to increase to 2000 by 2030; likewise, 800 million people die globally from cancer in 2012, with an expected increase to 1300 or more thousands by 2030.

Chemotherapy, surgery and radiation are the most commonly used therapeutic strategies against malignant tumors. Despite major advances in the field of cancer research, there are still a number of problems to be solved by current methods of treatment. For example, many traditional chemotherapeutic agents aim to block the rapid proliferation and cell division of cancer cells by introducing chemical damage to deoxyribonucleic acid (DNA). DNA is a genetic model of cancer and normal cells, and these traditional cancer chemotherapeutics also often damage normal tissues and cause serious side effects. Other chemotherapeutic agents target components of cellular structural proteins involved in cell division and proliferation, but these agents also produce non-specific toxicity to non-cancer cells, such as bone marrow and strongly dividing intestinal cell subsets. Recently, there has been increasing interest in therapeutics that target specific molecules that are only present in cancer cells but not in normal cells, a good example being inhibitors against growth factor receptors such as erlotinib and gefitinib. However, not all cancers have sufficient levels of growth factor receptors. Furthermore, even cases with an initial good response to targeted therapy tend to develop resistance because activation of other mechanisms compensate for the function of blocked growth factor receptors. Although surgery can effectively remove isolated malignancies, recurrence and spread of cancer to other body sites resulting from surgery are major problems. In addition, surgery may result in bleeding, scarring, infection, and impaired organ function. Radiation is also associated with the risk of damaging normal tissues and the risk of secondary cancer and long-term cosmetic risks. Chemotherapeutic agents, particularly when administered by injection or orally, can cause a number of serious side effects as described above. There is an urgent need for a new anti-cancer therapy.

Malignant tumors are not a simple collection of cancer cells. The healthy tissue and cells surrounding the tumor cells establish a unique environment (sometimes also referred to as the "tumor microenvironment") that supports the cancer cells, allowing them to grow, spread and metastasize. One of the most important elements in the tumor microenvironment is acid. The tumor microenvironment is more acidic than the tumor-free environment. The acidic metabolites released outside the tumor cells provide a source of acid in the tumor microenvironment, which reduces sensitivity to chemotherapy and radiation therapy, and stimulates tumor growth and spread. The acidic tumor microenvironment is an attractive therapeutic target for malignancies. It is expected that a method of sensitively preventing proliferation of tumor cells growing in an acidic environment will become a promising new anticancer therapy.

A disease condition that has not worsened in itself but has a high risk of developing cancer is called a precancerous condition. Due to the persistent and uncontrolled inflammation associated with precancerous conditions, a highly acidic environment is established in precancerous lesions prior to the appearance of malignant tumors. The acidic environment accelerates genetic changes and carcinogenesis (Coussens and Werb, 2002). Once present, malignant tumor cells grow preferentially in an acidic environment over non-malignant cells, which further drives acidification. In addition to malignant tumors growing preferentially over non-malignant cells in an acidic tumor environment, excess acid impairs the ability of immune cells, thereby increasing the chances of malignancy development (Lardner, 2001).

Actinic Keratosis (AK) and Human Papilloma Virus (HPV) infection are good examples of precancerous conditions. AK is a rough and scaly skin condition caused by excessive exposure to ultraviolet radiation and is one of the most common diagnoses in outpatients of the american dermatology clinic. AKs are a common cause of skin cancer; according to one study, approximately 65% of squamous cell carcinomas and 36% of basal cell carcinomas are caused by AK (Criscione et al, 2009). Cryosurgery and topical administration of anticancer agents are the primary therapies for AK. However, AK was not lethal before the appearance of malignant tumors. The transformation process takes ten years or more, multiple AKs often appear in one patient, and it is difficult to predict which AKs will progress to aggressive cancer. These facts make the treatment of AKs a challenge. Less expensive and less invasive pyrithione therapy provides an excellent alternative to preventing skin cancer.

HPV infections occur in the external surface areas of the cervix, female genitalia, anus, penis, and oral cavity. According to the World Health Organization (WHO), HPV is the most common viral infection transmitted by sexual contact. HPV infection is known to cause cancer in these body parts, especially HPV involving almost all cervical cancer cases (http:// www.who.int/media/facesheets/fs 380/en /). Cervical cancer is the fourth most common cancer in women and the second most common cancer in developing countries, where over 85% of cervical cancer occurs. Although HPV vaccination and early diagnosis by cancer screening can greatly reduce the risk of developing cervical cancer in developed countries, the socioeconomic problems in less developed countries make these approaches poorly adaptable. A simple and inexpensive method is awaiting introduction.

Recent experimental studies have shown that zinc pyrithione effectively inhibits the growth of human oral cancer cells (Srivastava et al, 2015) and leukemias (malignancies derived from leukocytes) implanted in the skin of immunodeficient mice (Tailler et al, 2012). Both studies showed that the dose of pyrithione that stops cancer growth had no adverse effect on the host. Likewise, low concentrations of zinc pyrithione have been shown to be effective in killing prostate cancer cells, while 10 times higher doses of zinc pyrithione are needed to kill non-cancerous prostate cells (Carraway and Dobner, 2012). However, these prior art techniques do not provide methods or compositions for curing or preventing cancer.

Pyrithione is integrated into biological membranes and forms pores that allow zinc and other heavy metals to pass through. The use of zinc and other metals for anti-cancer therapy of cancer cell sensitivity to heavy metals is known. For example, U.S. patent publication No. 2011/0117210 describes the use of organic and inorganic salts of zinc as anticancer agents. U.S. patent No. 7,528,125 discloses a series of novel compounds chemically modified with pyrithione and suggests their use as a method for delivering zinc, copper, manganese, iron and other metal complexes to tumors. In addition, U.S. patent publication No. 2006/0040980 discloses a method of using a formulation that combines zinc pyrithione and a zinc salt as an anti-cancer and anti-angiogenic agent. Recent experimental studies further took advantage of this idea and proposed the use of heavy metals such as copper (Liu et al, 2014), cadmium, platinum (Zhao et al, 2016b) and nickel (Zhao et al, 2016a) for leukemia, myeloma, lung and liver cancers using pyrithione salts as carriers. These prior art have taught our importance of heavy metals to kill and block the proliferation of malignant tumors through the pores of pyrithione.

Disclosure of Invention

Technical problem

The acidic tumor microenvironment is known to cause acquisition of resistance to many anticancer chemotherapeutic agents. The currently available methods for preventing the development of cancer from the pre-cancerous state have limitations in terms of efficacy, safety and cost.

Solution to the problem

Compositions and methods for treating and preventing malignant tumors using acidity as a means for enhancing anticancer activity are disclosed. In some embodiments, the disclosed methods and compositions are used to prevent cancer progression from a precancerous condition by killing sporadic cancer cells produced in the precancerous condition.

According to one aspect of the present invention, disclosed herein is the use of any one or more of zinc pyrithione, sodium pyrithione, pyrithione in any other salt form, or pyrithione in a salt-free form, in the manufacture of a medicament for treating malignancies occurring on the skin, cervix, vagina, penis, anus, and other body sites. The pH of the formulation is kept acidic in the presence of a pharmacologically acceptable buffer. The method can effectively inhibit malignant tumor proliferation in acidic environment. In another aspect, the method may be combined with one or more conventional cancer therapies, such as chemotherapy, radiation therapy, and surgery.

According to another aspect of the invention, disclosed herein is the use of pyrithione, including the use of any one or more of the chelating agents ethylenediaminetetraacetic acid (EDTA), calcium EDTA, disodium EDTA, diammonium EDTA, dipotassium EDTA, disodium EDTA, TEA-EDTA, tetrasodium EDTA, tripotassium EDTA, trisodium EDTA, HEDTA, trisodium HEDTA, and any other salt form of EDTA, in the manufacture of a medicament for the treatment and prevention of cancer or malignancies. This constructs and improves known techniques relating to zinc-dependent and other heavy metal-dependent antitumor activity of unchelated pyrithione, enhancing the anticancer effect of pyrithione.

In accordance with another aspect of the present invention, there is disclosed the use of pyrithione in combination with pyridoxine, pyridoxal, pyridoxamine and 5' -phosphate esters thereof (collectively referred to as vitamin B6) for the manufacture of a medicament for the treatment and prevention of cancer or malignancies. The method improves and increases the efficiency of inhibiting malignant tumor proliferation in acidic environment.

According to another aspect of the present invention, disclosed herein is the use of pyrithione in combination with any one or more of ascorbic acid (also known as L-ascorbic acid and vitamin C), any salt form of ascorbic acid, or any chemical derivative of ascorbic acid, for the preparation of a medicament for the treatment and prevention of cancer or malignancies. The method improves and increases the efficiency of inhibiting malignant tumor proliferation in acidic environment.

In accordance with another aspect of the present invention, disclosed herein is the use of pyrithione in the manufacture of a medicament for preventing cancer caused by precancerous lesions, such as Human Papilloma Virus (HPV) infections and Actinic Keratoses (AKs) in genital lesions, on the skin.

Advantageous effects of the invention

According to the present invention, we can effectively, simply, safely and inexpensively inhibit the proliferation of cancer cells by using an acid-enhanced antitumor composition comprising pyrithione as a main ingredient.

When combined with the presently disclosed enhancers and acidity, it was found that the concentration of pyrithione required to inhibit cancer cell proliferation is 100-fold to 1,000-fold lower than that required for anticancer agents known in the prior art, highlighting the substantial efficacy of the disclosed methods and compositions. Furthermore, the use of the presently disclosed methods, compositions, and prepared medicaments addresses an unmet need in the art by preferentially killing cancer cells growing in an acidic tumor microenvironment.

In some embodiments, the acidic formulation is administered topically to a cancer growing near a body surface or a body structure of an organ. Topical administration is an excellent method of systemic administration to deliver pyrithione-containing formulations in a variety of ways. First, topical administration is safe because the agent has limited exposure to blood and healthy body parts, thereby reducing the risk of adverse reactions. Secondly, local administration does not require a high level of technical expertise and is easier to self-administer than systemic administration. Third, the disclosed acidic compositions optimized for topical administration are effectively delivered to cancer. Topical administration is also advantageous in delivering acidic formulations without affecting the acidity of the blood and other body parts. Importantly, systemic acidification of the blood and the whole body will lead to heart failure. Certain embodiments relate to methods of administration using a combination of topical administration of pyrithione (and an enhancer) and systemic administration of selected enhancers (such as oral vitamin B6 and intravenous vitamin C), which further enhances therapeutic efficacy.

In view of the wide industrial application of pyrithione and other ingredients disclosed in the art, resulting from the prior art mass production of these ingredients, those skilled in the art are able to inexpensively produce medicaments and ingredients using the disclosed methods. This is particularly advantageous for the manufacture of medicaments in less developed countries.

Drawings

FIG. 1 shows the chemical structures of zinc pyrithione (sometimes abbreviated herein as "Pyz") and sodium pyrithione (sometimes abbreviated herein as "Pyn").

FIG. 2 is a graph showing the effect of treatment with zinc pyrithione (Pyz) in two different pH media on the survival of human brain tumor U87, breast cancer MDA-MB-231, cervical cancer HeLa and colon cancer HT29 cells. Cells were grown for 3 days at 37 ℃ in the absence or presence of the indicated dose of zinc pyrithione. Cell viability was determined by methylthiazolyldiphenyl-tetrazole bromide (MTT) assay.

FIG. 3 is a graph showing the effect of treatment with sodium pyrithione (Pyn) in two different pH media on the survival of human brain tumor U87, breast cancer MDA-MB-231, cervical cancer HeLa, and colon cancer HT29 cells. Cells were grown for 3 days at 37 ℃ in the absence or presence of the indicated dose of zinc pyrithione. Cell viability was determined by MTT assay.

Fig. 4 is a graph showing the cell killing effect of zinc pyrithione (Pyz) and sodium pyrithione (Pyn) on HeLa cells in two different pH media. Cells were grown for 3 days at 37 ℃ in the absence or presence of the indicated dose of zinc pyrithione. Non-viable cell populations treated with Pyz and Pyn were assayed by trypan blue staining.

Figure 5 is a photograph showing the effect of treatment with zinc pyrithione in two different pH media on the anti-proliferative effect of zinc pyrithione on acidity enhancement of brain tumor U87 cells grown in globular 3D culture.

Fig. 6 is a photograph showing the effect of zinc pyrithione (Pyz) and sodium pyrithione (Pyn) on mitochondrial accumulation of superoxide in HeLa human cervical cancer cells in two different pH media.

FIG. 7 is a graph showing the effect of erlotinib (Er) and gefitinib (Gef) on the survival of human glioma U87, breast cancer MDA-MB-231, cervical cancer HeLa and colon cancer HT29 cells in two different pH media. Cell viability was determined by MTT assay.

FIG. 8 is a graph showing that the anticancer effect of zinc pyrithione is enhanced by ethylenediaminetetraacetic acid (EDTA) in human cervical cancer HeLa, brain tumor U87, and colon cancer HT29 cells. Cell viability was determined by MTT assay.

Fig. 9 is a graph showing that pyridoxine enhances the anticancer effect of zinc pyrithione in human cervical cancer HeLa cells. Cell viability was determined by MTT assay.

FIG. 10 shows a graph and photograph of sodium ascorbate and 3-o-ethyl ascorbic acid enhancing the anticancer effects of zinc pyrithione and sodium pyrithione in human cervical cancer HeLa cells. Cell viability was determined in 2D cultures (fig. 10A) and spheroid-3D cultures (fig. 10B).

FIG. 11A is a graph showing the effect of topical administration of different formulations with and without zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitate on the growth of human skin cancer cells implanted in immunodeficient mice.

Figure 11B presents micrographs of skin, liver, and kidney showing that topical administration of formulations containing zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitate did not adversely affect these tissues.

The table summarizes the results of hematological testing of mice following topical administration of a formulation containing zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitate.

More detailed information is provided in the examples section.

Detailed Description

Definition of

Unless otherwise indicated, the terms "pyrithione" and "pyrithione compound" are used interchangeably to refer to pyrithione and any salts thereof, which are compatible with other ingredients of the formulation and with human administration. Pyrithione is available from commercial sources, most typically in the form of the zinc or sodium salt (see fig. 1), but other forms of pyrithione salts such as nickel and platinum pyrithione are also known. Pyrithione is also known by different names, such as 2-mercaptopyridine-N-oxide, 1-hydroxy-2-pyrithione, 1-hydroxypyridine-2-thione, Omedine, (2-pyridylthio)) -N-oxide and 2-pyridinethiol-oxide. Chemical derivatives refer to compounds having similar structures and functions. The sodium salt of pyrithione (sodium pyrithione) is widely used in commercial products. Sodium pyrithione is typically synthesized by reacting 2-chloropyridine-N-oxide with NaSH and NaOH (see, e.g., the disclosure of U.S. patent No. 3,159,640). The zinc salt of pyrithione (zinc pyrithione) may be prepared by reacting pyrithione acid (i.e., 1-hydroxy-2-pyrithione) or a soluble salt thereof with a zinc salt (e.g., ZnSO)₄ZnCl) to form a zinc pyrithione precipitate (see, e.g., U.S. patent No. 2,809,971).

Tumor is a pathological term that refers to changes in swelling that are considered to be part of the body. Tumors may be malignant-exhibiting uncontrolled growth of cancerous, or benign-generally harmless and curable (some types of cancer may arise from benign tumors if left untreated).

Malignant tumors are tumors that divide without control and can spread directly to nearby tissues or distant organs through the bloodstream or lymphatic system, a process known as metastasis. Technically, cancer is a group of malignant tumors that are produced by the epithelial cells of the skin and organs. Malignancies arising from non-epithelial cells such as blood, bone vessels, cartilage, muscle and supporting tissues are technically excluded from cancer. For example, according to the pathological definition, malignant melanoma of the dermis is derived from non-epithelial cells of the skin; thus, malignant melanoma is not cancer. In practice, however, malignant tumors, cancers and neoplasias are used interchangeably in a broad sense, and thus melanoma is often referred to as a skin cancer. Herein, the term "cancer" is to be understood in a broad sense, and "cancer" is used interchangeably with "malignancy".

The malignancy detected in the skin can be a cutaneous squamous cell carcinoma, a cutaneous melanoma, a cutaneous adenocarcinoma, or any other form of malignancy that occurs in or metastasizes to an area of skin. Cancer herein refers to the pathological term of an exogenous malignant tumor, whereas melanoma is a malignant tumor of non-epithelial origin. In another classification, malignant tumors detected in the skin are classified as basal cell carcinoma, squamous cell carcinoma, and other cancers including malignant melanoma.

Malignant tumors detected in the uterus and vagina are referred to herein as squamous cell carcinoma, squamous adenocarcinoma, other malignancies, and metastatic tumors.

The term "treating" as used herein refers to providing medical assistance to a patient to ameliorate an undesirable medical condition or to prevent such an undesirable condition from worsening. "anti-tumor effect," "anti-cancer effect," and "anti-tumor-formation effect" are used interchangeably and refer to slowing or regressing tumor growth, reducing tumor size, and preventing spread to other organs. In one aspect, the compound is considered effective when the size of the tumor decreases. On the other hand, the compound is considered effective when the symptoms associated with cancer are alleviated. On the other hand, the compound is considered effective when medical examination shows signs of reduced tumor burden. Physical examinations include biochemical examinations, tumor markers, diagnostic imaging, and histochemical examinations. In another aspect, the compound is considered effective when the patient treated with the method exhibits prolonged survival.

Transdermal administration herein refers to the broader definition used interchangeably with topical administration. Topical administration includes all methods of delivering agents through the body surface and lining of body passages, commonly referred to as epithelial and mucosal tissues.

Excipients are organic or inorganic substances which do not interfere with the active compound and act as carriers for the active compound. Pharmacologically acceptable carriers include, but are not limited to, water, polyethylene glycol (PEG), saline solutions, lactose, amylose, alcohols, oils, fatty acids, gelatin, silicic acid, surfactants, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, lubricants, and the like.

Therapeutic methods and compositions for malignant tumors

Malignant tumors establish an acidic extracellular pH of 6.0-6.9 and often even lower, while non-cancerous tissues have a pH of 7.3-7.4(Parks et al, 2013). Previous experimental studies attempted to inhibit tumor growth by blocking proton pumps or acid-producing enzymes to reduce the acidity of the tumor environment. However, this strategy has had limited success because inhibition of one of these mechanisms ultimately promotes other mechanisms that compensate for the inhibited proton pump or enzyme. In contrast, the present invention utilizes tumor acidity to effectively and selectively kill cancer cells.

The present inventors have investigated agents that enhance their anti-cancer effect by acidic media at pH6.9 and 6.4, which is the typical pH of the tumor microenvironment. Surprisingly, a significant enhancement in the antitumor activity of pyrithione at acidic pH was found (see figures 2, 3,4, 5 and 6). Antitumor activity of pyrithione under acidic conditions was observed in four genetically distinct cancer cells, from cervical cancer to brain tumor to colon cancer to breast cancer, while acidity-enhanced antitumor activity of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib was not observed (fig. 7).

The present invention discloses a topical administration method and a composition of pyrithione for the treatment or prevention of malignant tumors. The formulation consists of pyrithione and a chemical compound that enhances the antitumor activity of the pyrithione in combination with a carrier that does not interfere with the action of the compound under acidic conditions. The formulation is administered directly onto the body surface or the inner lining of the body structure. Topical administration is effective for targeting therapeutic agents to malignant tumors that grow near the surface of the body or the lining of body structures. Because of the acidic pH of the formulation and the inherent acidity of the tumor, pyrithione effectively kills tumor cells. Some components of the formulation may be administered by intravenous injection, subcutaneous injection, intramuscular injection, inhalation, or oral ingestion.

In one embodiment, the maximum pH of the formulation is 6.9, because the anti-tumor activity of pyrithione is significantly more effective at pH6.9 as compared to pH7.4 (see, e.g., fig. 10). More preferably, the maximum pH of the formulation is 6.4, as the anti-tumor activity of pyrithione has been found to be more effective at pH6.4 than pH6.9 (see, e.g., fig. 10). Even more preferably, the maximum pH of the formulation is 5.6, as topical formulations containing pyrithione at pH5.5 have been found to inhibit proliferation of implanted human skin cancer cells in mice (fig. 11). Previously, U.S. patent No. 6,455,076B has recognized the importance of acids in enhancing the efficacy of active ingredients in cosmetics. The art also provides a method of preventing skin irritation. According to the prior art, irritation of an acid corresponding to a pH of 2.0 can be prevented. Thus, in another embodiment, the minimum pH of the formation is 2.0.

By screening chemical compounds, it has been found that EDTA, particularly in combination with acidity, substantially enhances the antitumor activity of zinc pyrithione (fig. 8). EDTA is commonly used as a stabilizer in medical, cosmetic and food products, which loses and neutralizes zinc and other heavy metals at a given capacity. This indicates that pyrithione, even in the absence of zinc and any other heavy metal, exerts potent acidic enhanced antitumor activity, which is an unexpected finding in view of the prior art that teaches us the importance of heavy metals to pass pyrithione (see "background and description of related art" [0009 ]). This, together with the finding that sodium pyrithione acts as zinc pyrithione with effective acidic enhanced antitumor activity (see fig. 2 and 3), suggests the effectiveness of zinc pyrithione and sodium pyrithione in the treatment of malignancies. The zinc pyrithione (or any other metal) independent anti-tumor activity found may reduce the potential risk of metal overload associated with chronic administration. Disclosed herein is the use of zinc pyrithione (or any other metal pyrithione) and sodium pyrithione (or any other metal-free pyrithione) as an anti-tumor therapeutic approach.

By screening chemical compounds, it has been found that pyridoxine at 10 to 100 μ M significantly enhances the anti-tumor activity of zinc pyrithione when treated in combination with acidity (figure 9). It has also been found that 1mM pyridoxine not only enhances the antitumor activity of zinc pyrithione, but also exhibits acidity-dependent antitumor activity even in the absence of pyrithione, when treated in combination with acidity. Pyridoxine is one of the compounds that exhibits vitamin B6 activity; other vitamin B6 compounds include pyridoxal, pyridoxamine, and the 5' -phosphate esters thereof. In one embodiment, the pyridoxine, pyridoxal, pyridoxamine, its 5' -phosphate ester, pyridoxine dicaprylate, pyridoxine dipalmitate, or any other derivative thereof has zinc pyrithione (or any other metal pyrithione) or sodium pyrithione (or any other metal-free pyrithione).

It has been found that sodium ascorbate, particularly in combination with acidity, substantially enhances the antitumor activity of zinc pyrithione (figure 10). Sodium ascorbate is a salt of ascorbic acid and exhibits the same biological activity.

3-O-Ethyl ascorbic acid has an ethyl group that forms an ether group with the 3-hydroxyl group of ascorbic acid, which makes the compound chemically stable and readily absorbed through the skin. It has been found that 3-o-ethyl ascorbic acid, in particular in combination with acidity, substantially enhances the antitumor activity of zinc pyrithione (figure 10). It has been found that 3-o-ethyl ascorbic acid, in particular in combination with acidity, substantially enhances the antitumor activity of zinc pyrithione (figure 10).

Ascorbyl tetraisopalmitate is an oil-soluble derivative of ascorbic acid that is readily absorbed through the skin and efficiently converted to ascorbic acid. Thus, ascorbyl tetraisopalmitate and 3-o-ethyl ascorbic acid are commonly used in skin care consumer products. It has been found that topical administration of a formulation containing a moderate concentration of zinc pyrithione (1 μ M), 3-o-ethyl ascorbic acid (3 mM), and ascorbyl tetraisopalmitate (2 mM) inhibits proliferation of human skin cancer cells implanted in the skin of mice (FIG. 11).

Histological analysis of the skin areas exposed to the topical formulation, liver and kidney showed no signs of tissue damage or tissue abnormality (micrograph presented in fig. 11B). In addition, the results summarized in the table show no signs of blood abnormalities associated with topical administration of compositions containing pyrithione and an ascorbic acid derivative. Together, these findings indicate that no significant adverse effects are associated with the disclosed methods.

In one embodiment, zinc pyrithione (or any other metal pyrithione) or sodium pyrithione (or any other metal-free pyrithione) is provided to 3-o-ethyl ascorbic acid, tetraisopalmitate ascorbate (or any other chemical derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid), ascorbic acid, or a combination thereof for topical administration.

The compositions can be administered directly to cancers or precancerous lesions of body structures near the body surface or near organs using pharmaceutically acceptable salts in the form of creams, ointments, lotions, gels, solid sticks, sprays, drops (eye drops, nasal drops, etc.) and stoppers. These formulations may be oil-in-water emulsions, water-in-oil emulsions, viscous liquids, or water-soluble solutions. Stoppers such as semipermeable membranes covering reservoirs containing pyrithione and other active ingredients may be used to release the pyrithione and other active ingredients, such as chemotherapeutic agents, steroids, and anti-inflammatory agents. The pyridone topical formulations may be formulated according to conventional methods using suitable excipients, including, for example, emulsifiers, surfactants, thickeners, humectants, skin conditioners, skin protectants and sunscreens. Preservatives and bacteriostats such as methyl hydroxybenzoate, propyl hydroxybenzoate and chlorocresol may optionally be incorporated into the occluding device.

The compositions can be administered directly to the cancer or precancerous lesion near the body surface or near internal body structures using pharmaceutically acceptable salts injected through a needle or similar device, propagating narrow jets through high pressure, externally applied electric field differences, ultrasound, laser beams, magnetic fields, and radiation.

For vaginal and rectal applications, suppository forms may be used. Suppository formulations may be prepared by mixing the compounds and compositions of the present invention with suitable excipients such as cocoa butter, polyethylene glycols of different molecular weights, glycerin and glycerogelatin. These excipients are solid at room temperature, but melt at body temperature and release the compounds and compositions in the rectum, vagina or cervix.

In one embodiment, to increase the efficacy of absorption through the body surface or lining of the body structure, chemical penetration enhancers and solubilization enhancers are incorporated into the formulation according to known techniques (Williams and Barry, 2012). Chemical penetration enhancers include unsaturated fatty acids such as oleic acid, docosahexaenoic acid, eicosapentaenoic acid, terpenes, terpenoids, essential oils, azone, and pyrrolidone; some examples of solubilizing agents include low doses of surfactants, cyclodextrins, and dimethyl sulfoxide (DMSO).

0.00000317 wt% (100 nM) of zinc pyrithione in combination with acidity was found to inhibit the growth of cancer cells in two-dimensional (2D) cultures (see FIGS. 2, 3,4 and 6). According to the prior art, the transdermal absorption efficiency of pyrithione compounds is about 1% (see reference (Wedig et al, 1974.) thus, in one embodiment, the formulation contains at least 0.000317 wt%, preferably at least 0.00634 wt% of pyrithione, since the minimum concentration required to inhibit growth of cancer cells in three-dimensional (3D) cultures is 0.0000634% (2) μ M) (see fig. 5). It has been found that topical administration of a cream composition containing 1 wt% zinc pyrithione has significant anti-tumor activity (figure 11). Therefore, it is more preferred that the formulation contain at least 1% pyrithione. In another embodiment, the formulation contains up to 5.5% by weight (173 mM) of pyrithione, as it has been found that emulsions containing 5.5% zinc pyrithione are stable when administered to the skin.

In some embodiments, EDTA is added to the formulation in an amount of at least about 0.0003 weight percent (10 μ M), which is the minimum concentration required to enhance the anti-cancer effect of zinc pyrithione (see figure 8). The formulation contains EDTA at a maximum concentration of 10-15% by weight, since these are amounts that are safe for short-term dental treatment in dentistry (see reference (Lanigan and Yamarik, 2002)). More preferably, the formulation contains a maximum concentration of EDTA of about 2%, as this is a typical maximum concentration of EDTA suitable for long-term use in cosmetic formulations.

In one embodiment, the topical formulation contains at least 0.00017 wt% (10 μ M) pyridoxine as this is the minimum concentration that shows an acidity-dependent enhancement of the antitumor effect of zinc pyrithione (see figure 9). Preferably, the formulation contains at least 0.0017 wt% (100 μ M) pyridoxine as this is the concentration that shows even higher efficacy. Even more preferably, the formation contains at least 0.017 wt.% (1mM of pyridoxine) since this concentration shows an acidity-dependent anti-tumor effect even in the absence of zinc pyrithione. Due to other factors such as skin permeability, diffusion and chemical stability, higher concentrations of pyridoxine may be required to achieve the desired effect. For example, the prior art discloses cosmetic compositions containing 0.001% to 15% pyridoxine (U.S. patent No. 20,060,018,860a 1). However, it was noted that formulations containing 10 wt.% or less pyridoxine were more stable in the presence of other ingredients. Thus, in one embodiment, the formulation contains pyridoxine in an amount of 10% by weight or less. Oil-soluble pyridoxine derivatives such as pyridoxine dicaprylate and pyridoxine dipalmitate are readily absorbed through the body surface and exhibit stable activity once absorbed. In some embodiments, the pyridoxine dicaprylate, pyridoxine dipalmitate, or any other pyridoxine derivative is added to the formulation in an amount of at least 0.00017 weight%, preferably 0.0017 weight%, more preferably 0.017 weight%, and less than 10 weight%.

In one embodiment, the topical formulation contains at least 0.04 wt% (2 mM) of a 3-o-ethyl ascorbic acid composition, such as 2mM of 3-o-ethyl ascorbic acid in combination with pyrithione and acidity to allow regression of cancer cell growth in 2D cultures (see FIG. 10). Preferably, the formulation contains at least 0.2 wt% (10 mM) of 3-o-ethyl ascorbic acid, such as 10mM of 3-o-ethyl ascorbic acid in combination with pyrithione and acidity that allows regression of cancer cell growth in 3D cultures. More preferably, the formulation contains at least 3% by weight of 3-o-ethyl ascorbic acid, because the cream formulation containing 3% 3-o-ethyl ascorbic acid in combination with zinc pyrithione and acidity significantly inhibit the proliferation of human skin cancer cells implanted in mice (see fig. 11). In addition to 3-o-ethyl ascorbic acid and zinc pyrithione, the formulation also contained 2% by weight of ascorbyl tetraisopalmitate (see example 11). Thus, even more preferably, the formulation contains at least 3% 3-o-ethyl ascorbic acid plus any one or combination of two or more of ascorbyl tetraisopalmitate, ascorbyl palmitate and any other ascorbic acid derivative at a concentration of 2% or more. It is noteworthy that formulations containing more than 20% by weight of 3-o-ethyl ascorbic acid sometimes produce irritation to sensitive skin. Thus, in one embodiment, the formulation contains less than 20%, preferably less than 10% by weight pyridoxine.

It was found that the combination of 0.04% (about 2 mM) sodium ascorbate with pyrithione and acidity inhibited the proliferation of cancer cells in 2D cultures (fig. 10). In some embodiments, sodium ascorbate (or any other ascorbate salt), ascorbic acid, or ascorbyl tetraisopalmitate (or any other derivative of ascorbic acid) is included in the topical formulation at a concentration of 0.04% or higher. It was noted that the formulations containing 30% sodium ascorbate or 15% ascorbyl tetraisopalmitate were stable and no signs of skin irritation were detected. Thus, in some embodiments, the formulation contains sodium ascorbate (or any other ascorbate salt) in an amount of 30% or less, preferably 20% or less, more preferably 10% or less. In another embodiment, the formulation contains ascorbyl tetraisopalmitate (or any other derivative of ascorbic acid) in an amount of 15% or less, preferably 10% or less, more preferably 5% or less.

Daily topical administration of the formulations containing pyrithione to mouse skin was found to regress the growth of skin cancer (fig. 11). Thus, in one embodiment, the formulation containing the pyrithione and the enhancer is topically administered once per day. According to known techniques, only 34% of the zinc pyrithione remains in the treated skin area after topical administration for 20 hours (see reference (Parekh et al, 1970)). The prior art further teaches that topical administration of pyrithione at intervals of less than 20 hours increases the effective concentration of pyrithione in the treated skin area. This means that more than one administration per day will increase the effective concentration of pyrithione in the skin. In another embodiment, the formulation containing pyrithione and an enhancer is topically administered twice daily. In another embodiment, the formulation containing the pyrithione and the enhancer is topically administered three times per day. In one embodiment, the agent is administered until the desired therapeutic result is achieved; in a typical example, the pyrithione formulation may be delivered for a period of time (which may be from two months to 36 months). In another embodiment, a one day to four week interval between each treatment cycle is introduced. In yet another embodiment, chemotherapy, radiation and/or surgery is performed during the interval. Other interval schemes and treatment durations may be considered appropriate by those skilled in the art.

One example of topical administration of pyrithione is for the treatment of malignant tumors in the skin.

Another example of an application for topical administration of pyrithione is for the treatment of cervical cancer, vaginal cancer, and other malignancies occurring in the uterus and vaginal cervix.

Topical pyrithione administration may be used in medical therapy for other malignancies that are accessible from the body surface. The target disease may include, but is not limited to, malignancies that occur or metastasize in the head and neck, mouth, anus, rectum, prostate, breast, bone, muscle and cartilage.

Topical administration permeates the composition from the outer surface of the tumor. Systemic administration delivers the composition to the tumor through tumor-cultured blood vessels. Combining these two methods of administration will maximize the effective concentration of the composition in the tumor. Oral and intravenous administration are widely used systemic routes of administration to increase the level of vitamins in the plasma.

Previous techniques have revealed that oral administration of 600mg of pyridoxine rapidly enters systemic circulation within 0.3 hours, and that the pyridoxine concentration in plasma reaches 25 μ M1.3 hours after administration (see reference (Zempleni, 1995)). This concentration (25 μ M) was higher than the minimum concentration (10 μ M) required for anticancer effect when combined with pyrithione and acidity (fig. 9). In contrast, intravenous administration of 100mg pyridoxine (another commonly used method of administration) only reaches a maximum plasma level of 0.37. mu.M 6 hours after administration. Thus, in one embodiment, pyridoxine, pyridoxal, pyridoxamine or a 5' -phosphate thereof is administered orally in a minimum amount of 600mg per day, while a formulation containing pyrithione and pyridoxine (or a derivative thereof) is administered topically. The prior art teaches that the risk of neuropathy is associated with high levels of pyridoxine intake of more than 1,000mg per day (see reference (Bender, 1999)). Thus, in another embodiment, pyridoxine, pyridoxal, pyridoxamine or a 5' -phosphate thereof is administered orally at a maximum of 1,000 mg/day, while a formulation containing pyrithione and pyridoxine (or a derivative thereof) is administered topically. Since pyridoxine given orally decays rapidly once it enters the systemic circulation, a half-life of less than 1 hour is estimated (see reference (zemplen, 1995)), it is desirable to divide the total daily dose into multiple intakes per day. In one embodiment, pyridoxine, pyridoxal, pyridoxamine or the 5' -phosphate thereof is administered orally twice a day, preferably three times a day, even more preferably four times a day.

The prior art has revealed that the maximum achievable plasma concentration of ascorbic acid by intravenous administration is 13,400 μ M, whereas the maximum achievable plasma concentration of ascorbic acid by oral administration is only 220 μ M (see reference (Padayatty et al, 2004)). This suggests that intravenous administration, but not oral administration, can determine the minimum plasma concentration (1 to 10 mM) required for ascorbic acid or its derivatives to inhibit cancer proliferation in combination with pyrithione and acidity (see fig. 10). In some embodiments, 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid), or ascorbic acid is administered intravenously, while a formulation containing any one or a combination of two or more of pyrithione and 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid), and ascorbic acid is administered topically. The prior art teaches that intravenous administration of 5g but not 1 or 3g of ascorbic acid can increase the plasma concentration of ascorbic acid to 2mM or higher. Thus, in one embodiment, a minimum dose of 5g of ascorbic acid, sodium ascorbate (or any other ascorbate salt) or 3-o-ethyl ascorbic acid (or any chemical derivative of ascorbic acid) is administered intravenously. According to the prior art, intravenous administration of up to 100g of ascorbic acid is a safe and effective way to increase the plasma concentration of ascorbic acid. Thus, in another embodiment, a maximum dose of 100g of ascorbic acid, sodium ascorbate (or any other ascorbate salt) or 3-o-ethyl ascorbic acid (or any chemical derivative of ascorbic acid) is infused intravenously.

The prior art also teaches that dividing the daily dose of ascorbic acid into multiple administrations helps to maintain an effective plasma concentration in vivo. For example, intravenous infusion of ascorbic acid at doses of 100g, 50g and 10g establishes and maintains plasma ascorbic acid concentrations above 2mM for about 5.5 hours, 3.5 hours and 1.3 hours, respectively. This means that intravenous administration of 50g ascorbic acid twice daily and 10g five times daily is a more effective method than administration of 100g daily to maintain plasma ascorbic acid concentrations above 2 mM. Thus, in one embodiment, 50g of sodium ascorbate (or any other salt of ascorbic acid), 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid), or ascorbic acid is infused intravenously twice daily. In another embodiment, 10g of sodium ascorbate (or any other salt of ascorbic acid), 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid) is infused intravenously five times a day. In another embodiment, 33g of sodium ascorbate (or any other salt of ascorbic acid), 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid), or ascorbic acid is infused intravenously three times a day. In one embodiment, a one day to four week interval between each treatment cycle is introduced. Other interval schemes and treatment durations may be considered appropriate by those skilled in the art.

Principally, standard doses of pyrithione and other components of the formulation and dosing regimens are intended to inhibit tumor proliferation and induce regression of malignant tumors. In therapeutic applications, the standard dose of pyrithione and other components of the formulation, as well as the dosage regimen employed, may vary depending upon a number of variables, such as clinical condition, type of malignancy, age, weight of the recipient patient, route of administration, and other factors, as determined by the experience of the clinical professional conducting the treatment. When combined with other therapies, the standard dose of pyrithione, other components of the formulation, and the dosing regimen may also vary depending on the type of concomitant therapy, the extent of response, and adverse side effects on the concomitant therapy. Regression of malignancy can be assessed with reference to the size of the tumor. The absence of recurrence of the malignancy after the end of treatment is another sign of regression.

Methods and compositions for preventing the progression of malignant tumors from precancerous lesions

A unique property of malignant tumors in the skin and cervix is that they are often caused by noncancerous inflammatory changes in these organs. It can also be said that the presence of abnormalities in these organs in general can be predictive of the occurrence of malignant tumors in the skin and cervix. The acidic environment created by the persistent and uncontrolled inflammation associated with precancerous lesions makes the use of pyrithione ideal for inhibiting the growth of new-born malignant tumor cells. In another aspect, a method of preventing cancer is disclosed wherein a pyrithione-containing formulation is topically administered to future body surface or mucosal lesions having a high probability of developing malignancy. The disclosed methods utilize the acidic pH of disease lesions and the acidic pH of pyrithione formulations to regress emerging small numbers of malignant tumor cells before they form visible tumors.

One field of application is the prevention of skin cancer from developing from AK and other pre-cancerous conditions occurring in the skin.

Another field of application is the prevention of the progression of cervical, vaginal, anal, penile and oral cancer from chronic HPV infection.

Topical administration may be carried out by means of pharmaceutically acceptable salts in the form of creams, ointments, lotions, gels, solid sticks, sprays and stoppers. These formulations may be oil-in-water emulsions, water-in-oil emulsions, viscous liquids, or water-soluble solutions.

In one embodiment, the pH of the topical formulation is 6.9 or less, preferably 6.4 or less, more preferably 5.6 or less. In view of the extreme acidity in precancerous lesions, it is even more preferred that the pH of the formulation is 4.5 or lower. Thus, in another embodiment, the minimum pH of the formation is 2.0. The formulation used for the animal experiments contained 20mM citrate buffer (see example 11). In one embodiment, the maximum concentration of citrate buffer in the formulation is 100mM, as this concentration will increase pH stability; preferably 75mM, even more preferably 50mM, as these concentrations increase the chemical stability of the composition. In another embodiment, the minimum concentration of citrate buffer is 5mM, preferably 7.5mM, more preferably 10mM, as these lower concentrations of buffer increase the chemical stability of the composition. In some embodiments, a phosphate buffer is used, which is another physiological buffer that is safe for humans.

In one embodiment, to increase efficacy of absorption through the skin, chemical penetration enhancers and solubilization enhancers are incorporated into the formulation according to known techniques (Williams and Barry, 2012). Chemical penetration enhancers include unsaturated fatty acids such as oleic acid, docosahexaenoic acid, eicosapentaenoic acid, terpenes, terpenoids, essential oils, azone, and pyrrolidone; some examples of solubilizing agents include low doses of surfactants, cyclodextrins, and dimethyl sulfoxide (DMSO).

In one embodiment, the topical formulation contains a minimum of 0.000317 wt.%, preferably 0.00634 wt.%, more preferably 1 wt.% of zinc pyrithione, sodium pyrithione, or any other form of pyrithione compound. In another embodiment, the formulation contains up to 5.5% by weight of zinc pyrithione, sodium pyrithione, or any other form of pyrithione compound.

In some embodiments, EDTA is added to the formulation in an amount from 0.0003 to 15 wt%, preferably from 0.0003 to 10 wt%, more preferably from 0.0003 to 2 wt%.

In one embodiment, the topical formulation contains at least 0.00017%, preferably at least 0.0017%, even more preferably at least 0.017% by weight pyridoxine. In another embodiment, the formulation contains pyridoxine in an amount of 10% by weight or less, preferably 5% by weight or less, even more preferably 2.5% by weight or less. An oil-soluble pyridoxine derivative. Such as pyridoxine dicaprylate and pyridoxine dipalmitate, are readily absorbed through the body surface and exhibit stable activity once absorbed. Pyridoxamine, pyridoxal 5 '-phosphate and pyridoxamine 5' -phosphate show similar biological activities to pyridoxine. Lipophilic derivatives of pyridoxine, such as pyridoxine dicaprylate and pyridoxine dipalmitate, are readily absorbed through the skin and show longer stability after absorption. In some embodiments, these compounds are included in a formulation.

In one embodiment, the topical formulation contains at least 0.04%, preferably at least 0.2%, more preferably at least 3% by weight of the composition of 3-o-ethyl ascorbic acid (or any other derivative of ascorbic acid), sodium ascorbate (or any other ascorbate salt) or ascorbic acid. Even more preferably, the formulation contains at least 3% 3-o-ethyl ascorbic acid (or any other water soluble derivative of ascorbic acid), sodium ascorbate (or any other ascorbate salt) or ascorbic acid plus any one or combination of two or more of ascorbyl tetraisopalmitate, ascorbyl palmitate and any other lipophilic derivative of ascorbic acid at a concentration of 2% or higher. In one embodiment, the formulation contains less than 20%, preferably less than 10% by weight pyridoxine.

In some embodiments, pyridoxine, pyridoxal, pyridoxamine, or a 5' -phosphate ester thereof, is administered orally in an amount of from 0.1 mg/day to 1 g/day, more preferably from 10 mg/day to 300 mg/day, while the formulation containing pyrithione is administered topically. In one embodiment, the daily dose of pyridoxine, pyridoxal, pyridoxamine or the 5' -phosphate ester thereof is administered orally once daily. In another embodiment, pyridoxine, pyridoxal, pyridoxamine or the 5' -phosphate ester thereof is administered orally three times per day, every 8 hours.

In some embodiments, 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid), or ascorbic acid is administered intravenously, while a formulation containing any one or a combination of two or more of pyrithione and 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid), and ascorbic acid is administered topically. In one embodiment, a minimum daily dose of 5g of ascorbic acid, sodium ascorbate (or any other ascorbate salt) or 3-o-ethyl ascorbic acid (or any chemical derivative of ascorbic acid) is administered intravenously. In another embodiment, a maximum daily dose of 100g of ascorbic acid, sodium ascorbate (or any other salt of ascorbic acid) or 3-o-ethyl ascorbic acid (or any chemical derivative of ascorbic acid) is infused intravenously. In one embodiment, the formulation contains at least 0.04 wt.%, preferably at least 0.2 wt.%, more preferably at least 3 wt.% 3-o-ethyl ascorbic acid. More preferably, the formation contains at least 3% 3-o-ethyl ascorbic acid and 2% or more of any one or combination of two or more of ascorbyl tetraisopalmitate, ascorbyl palmitate and any other lipophilic derivative of ascorbic acid. In another embodiment, the formulation contains 3-o-ethyl ascorbic acid in an amount of 30% or less, preferably 20% or less, more preferably 10% or less.

In some embodiments, 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid), or ascorbic acid is administered intravenously with topical administration of a formulation containing pyrithione. In one embodiment, the daily dose is administered once daily. In another embodiment, the daily dose is administered as a twice daily injection. In another embodiment, the daily dose is administered as three injections per day. In one embodiment, the agent is administered until the desired therapeutic result is achieved. In another embodiment, a one day to four week interval between each treatment cycle is introduced. Other interval schemes and treatment durations may be considered appropriate by those skilled in the art.

The topical formulation containing pyrithione may be administered once a day, preferably twice a day, more preferably three times a day, through a body surface, genitalia, or other organ. In one embodiment, a topical formulation containing pyrithione is administered daily. In another embodiment, the topical formulation containing pyrithione is administered three times per week. In one embodiment, administration comprises a period of 1 to 96 weeks, preferably a period of 2 to 32 weeks, more preferably a period of 2 to 16 weeks. In some embodiments, a one-day to four-week interval between each treatment cycle is introduced. Other interval schemes and treatment durations may be considered appropriate by those skilled in the art.

The dosage regimen is closely monitored by the physician throughout the course of medical treatment. The duration of administration and the regular intervals will also be determined by preclinical and clinical studies. One skilled in the art can also determine these variables by a number of variables, including age, weight, and clinical condition. Clinical conditions are determined by signs, symptoms and results of routinely performed medical tests. Other methods of clinical evaluation include pathological examination, colposcopy (vaginal and cervical examination with special magnification equipment) and serological examination.

Examples

Example 1

Cell lines and reagents, colon cancer HT29 cell line, breast cancer MDA-MB-231, glioma U87 and skin cancer A2058 cells were obtained from the American Type Culture Collection (ATCC). These cancer cells were trypsinized and resuspended in cryopreservation media containing 90% media consisting of Dalbecco's Modified Eagle's Media (DMEM) and 10% Fetal Bovine Serum (FBS).

Human cervical cancer HeLa was supplemented with 10% DMSO and stored in liquid nitrogen until use. Prior to the experiment, cells were recovered from liquid nitrogen and in DMEM containing 10% FBS in atmospheric 5% CO₂And (4) growing. When the cells reached about 80% confluence, the cells were trypsinized and washed with 1: 4. To culture cells without exposure to test compounds, DMEM supplemented with 3.7g/L sodium bicarbonate and pH was adjusted to 7.3, supplemented with 10% FBS, and cells were maintained at 37 ℃ in 5% CO₂An incubator. To investigate the pH-dependent and dose-dependent effects of pyrithione and other agents on cancer cell proliferation, DMEM supplemented with 10mM of 1, 4-piperazine diethylsulfonic acid (PIPES, pKa6.1-7.5) was adjusted to pH6.4, and DMEM supplemented with 10mM of 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid) (HEPES, pKa6.8-8.2) was adjusted to pH 7.4. PIPES (P6757, batch 026K5416) and HEPES (H4034, batch 087K54432), sodium pyrithione (sodium 2-mercaptopyridine N-oxide, H3261, batch No.)0655M4172V) and methylthiazolyldiphenyl-tetrazole bromide (MTT, M5655, Sigma) were purchased from Sigma. Zinc pyrithione (PHR1401, batch LRAA8431) was purchased from Fluka. Erlotinib (#10483, batch 0459700-31) and gefitinib (sc-202166, batch A0616) were purchased from Cayman Chemical Company and Santa Cruz Biotechnology, respectively. The general chemical reagents used were the highest grade purchased from Sigma, unless otherwise indicated.

Example 2

Effect of zinc pyrithione (Pyz) treatment at ph7.4 and ph6.4 on cancer cell viability.

Malignant tumors establish a characteristic acidic extracellular pH of 6.0-6.9, while normal tissues have a neutral pH of 7.3-7.4(Parks et al, 2013). This indicates that exposure of zinc pyrithione to four different types of human cancer cells from cervical cancer (HeLa cells), brain tumors (U87 cells), colon cancer (HT29 cells), and breast cancer (MDA-MB-231 cells) results in a significant reduction in cell viability. When treated in acidic medium at ph7.4, treatment with zinc pyrithione significantly reduced cell viability compared to when treated at ph 7.4. The chemical structure of zinc pyrithione is shown in figure 1.

Cancer cells were seeded at 2,000/well onto 96-well culture dishes and 5% CO in the atmosphere₂Incubate overnight at 37 ℃ in DMEM supplemented with 10% FBS in the presence. To investigate the pH-dependent anticancer effect of pyrithione and other agents, DMEM medium was prepared by supplementing 10mM of 1, 4-piperazine-diethylsulfonic acid (PIPES) and adjusting pH to 6.4 at pH 6.4. DMEM medium at pH7.4 was prepared by supplementing 10mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) and adjusting pH to 7.4. PIPES (pKA6.1-7.5) and HEPES (pKA6.8-8.2) were used as buffers to stabilize the pH to 6.4 and 7.4, respectively. Two pH values of 6.4 and 7.4 were applied in the experiment to simulate the representative acidity of the tumor microenvironment and normal environment, respectively. Cells were treated with increasing concentrations of zinc pyrithione in DMEM at pH6.4 or DMEM at pH7.4 and cultured for 72 hours at 37 ℃. In culture medium and atmospheric CO₂In the absence of bicarbonate, to minimize pH fluctuations caused by bicarbonate-mediated proton movement across the biofilm. Upon exposure to zinc pyrithioneAfter 72 hours, the medium was replaced with 50 μ l/well DMEM containing 10% FBS, freshly supplemented with 1mg/mL MTT. After 2 hours of incubation, cells were lysed to an equal volume of medium by adding 50 μ l of extraction buffer dissolved in 50% DMF water solution with 20% SDS, 2% acetic acid and 2.5% HCl. After stirring, the 96-well plate was incubated at 37 ℃ for 4 hours, and the absorbance at 570nm was measured. After background subtraction, relative survival was determined and shown to correlate with readings from untreated samples of compound. Statistical significance between each condition and the corresponding control group was determined individually by Student's t-test. If the p-value is less than 0.05, the condition is considered significantly different from the control group.

When cancer cells were treated with 50nM zinc pyrithione in a medium adjusted to pH7.4 for 72 hours, the relative survival rate was about 80-90% compared to untreated cells. When these cancer cells were treated with 50nM zinc pyrithione in a moderately acidic medium adjusted to pH6.4, the survival rate was 5-20% of that of the untreated cells. For example, the relative survival rates of HeLa human cervical cancer cells treated with 50nM zinc pyrithione at pH7.4 and pH6.4 were 87 + -9% and 5.3 + -1.6%, respectively (p < 0.001). These new findings suggest that the acidic environment contributes to the increase in the anticancer efficacy of zinc pyrithione. The results are summarized in fig. 2. If the p-value is less than 0.05, the conditions are considered to be significantly different.

Example 3

Effect of sodium pyrithione (Pyn) treatment at ph7.4 and ph6.4 on cancer cell viability.

It has been proposed that zinc pyrithione or other heavy metals chemically conjugated to pyrithione cause anticancer activity. On the other hand, it has also been proposed that metal-free pyrithione impart anti-inflammatory and anti-infective activity. Therefore, an important next question for developing a new anticancer agent treatment is whether pyrithione causes an increased anti-proliferative effect due to acidity, or whether zinc causes an increased anti-proliferative effect due to acidity. To answer this question, the effect of sodium pyrithione on cancer cell proliferation was investigated.

This indicates that exposure of zinc pyrithione to four different types of human cancer cells from cervical cancer (HeLa cells), brain tumors (U87 cells), colon cancer (HT29 cells), and breast cancer (MDA-MB-231 cells) results in a significant reduction in cell viability. Treatment with sodium pyrithione significantly reduced cell viability when treated in an acidic medium at ph6.4 as compared to when treated at ph 7.4.

Cancer cells were seeded at 2,000/well onto 96-well culture dishes and 5% CO in the atmosphere₂Incubate overnight at 37 ℃ in DMEM supplemented with 10% FBS in the presence. To investigate the pH-dependent anticancer effect of sodium pyrithione, DMEM medium was prepared by supplementing 10mM PIPES and adjusting pH to 6.4 at pH 6.4. DMEM medium at pH7.4 was prepared by supplementing 10mM HEPES and adjusting pH to 7.4. Cells were treated with increasing concentrations of sodium pyrithione in pH6.4 DMEM or pH7.4 DMEM medium and cultured at 37 ℃ for 72 hours. In culture medium and atmospheric CO₂In the absence of bicarbonate, to minimize pH fluctuations caused by bicarbonate-mediated proton movement across the biofilm. After 72 hours of exposure to sodium pyrithione, the MTT assay was performed as in example 2. Statistical significance between each condition and the corresponding control group was determined individually by Student's t-test. If the/v value is less than 0.05, the condition is considered to be significantly different from the control group.

When cancer cells were treated with 100nM sodium pyrithione in a medium adjusted to pH7.4 for 72 hours, the relative survival rate was about 75% compared to untreated cells. When these cancer cells were treated with 100nM sodium pyrithione in a medium acidic medium adjusted to pH6.4, the viability ranged from 5-20% of the untreated cells. For example, the relative survival of HeLa cells of cervical cancer treated with 100nM zinc pyrithione at pH7.4 and pH6.4 was 90 + -9% and 4.9 + -1.7%, respectively (p < 0.001). The conditions are considered to be significantly different if the p-value is less than 0.05.

These new findings suggest that the acidic environment contributes to the increase in the anticancer efficacy of zinc pyrithione. The results are summarized in fig. 3.

Example 4

The MTT assay determines cell viability by measuring the metabolic activity of the cells. Trypan blue can only enter dead cells (but not live cells) that lose their membrane integrity. With this dye only dead cells were visible. Cell killing of zinc pyrithione (Pyz) and sodium pyrithione (Pyn) treated HeLa cells at ph7.4 and ph6.4 was determined by trypan blue staining assay. Ctr represents a control group treated with the same dose of vehicle without agent.

Cancer cells were seeded at 20,000/well onto 12-well culture dishes and 5% CO in the atmosphere₂Incubate overnight at 37 ℃ in DMEM supplemented with 10% FBS in the presence. To investigate the pH-dependent anticancer effects of zinc pyrithione and sodium pyrithione, DMEM medium was prepared by supplementing 10mM HEPES and adjusting pH to 7.4 at pH 7.4. DMEM medium at pH6.4 was prepared by supplementing 10mM PIPES and adjusting pH to 6.4. Cells were treated with increasing concentrations of zinc pyrithione and sodium pyrithione in DMEM at pH7.4 or 6.4 and cultured at 37 ℃ for 72 hours. Bicarbonate removal from DMEM at pH7.4 and DMEM medium at pH6.4 and in the absence of atmospheric CO₂In order to minimize pH fluctuations caused by bicarbonate-mediated proton movement across the biofilm. After 72 hours of exposure to zinc pyrithione or zinc pyrithione, cells were harvested and an equal volume of 0.4% trypan blue-PBS solution was added. Total cells and number of cells stained with trypan blue were scored in each image and the percentage of trypan blue positive cell population was determined. Four images were taken for each condition and the mean trypan blue positive population and standard deviation expressed as error bars were calculated. The results are summarized in fig. 4.

The results tell us that the cell viability determined from the trypan blue unstained population and the cell viability determined by MTT assay are comparable, supporting the idea that cell killing by pyrithione is more sensitive when treated in ph6.4 medium than in ph7.4 medium.

Example 5

Zinc pyrithione exhibits an antiproliferative effect on spherical 3D cultured brain tumor cells.

When tumors develop from semi-suspension cultured cells in the presence of collagen gels, which are components of the extracellular matrix (ECM), the tumors that appear in the ECM matrix are called "spheroids," reproducing the tumor growth (3D) microenvironment in three dimensions. Spherical culture enabled us to evaluate the efficacy of anticancer agents in 3D culture, mimicking the complexity of real cancers (Friedrich et al, 2009). Since the biological activity of many compounds is influenced by the tumor microenvironment, an important issue is whether pyrithione affects spheroid growth. To address this problem, collagen-embedded spheroids of brain tumor U87 cells were established under either acidic (ph6.4) or neutral (ph7.4) conditions, exposed to zinc pyrithione at 37 ℃ for three days and tested for effect on spheroid growth.

The 1% agarose solution heated to 60 ℃ was mixed with an equal volume of medium (DMEM ph6.4 and 7.4 supplemented with 10% FBS) and 50 μ l of the mixture was transferred to each well of 96 plates. The plate was cooled at room temperature for 30 minutes until the agarose solidified, 1000 cells resuspended in 50. mu.l of medium were overlaid on top of the agarose layer and centrifuged at 1,500g for 5 minutes. Cells were cultured at 37 ℃ for 3 days, and spheroids formed on the agarose layer were grown for 3 more days in the presence or absence of varying concentrations of zinc pyrithione. The size of spheroids after 3 days of incubation with 1 μ M or 2 μ M zinc pyrithione in pH6.4 medium was significantly smaller than that of untreated control spheroids. In sharp contrast, the size of spheroids exposed to 1 μ M or 2 μ M zinc pyrithione in pH7.4 medium for 3 days was almost the same as the size of control spheroids that were not treated with pyrithione. The results are summarized in fig. 5.

Example 6

The acidic enhanced antitumor activity of pyrithione results in the accumulation of superoxide in mitochondria.

Free radical forming compounds, such as hydrogen peroxide and superoxide, are produced during cellular metabolism. These Reactive Oxygen Species (ROS) pose a direct threat to cells when untransformed. It has been shown that loss of mitochondrial function by external stress or toxins promotes the accumulation of ROS in organelles. While most normal tissues are able to neutralize ROS to prevent further cellular damage, cancer cells have been shown to be more sensitive to intracellular ROS fluctuations and less resistant to ROS accumulation (see reference (Liou and Storz, 2010)). Since mercaptopyridone photochemically decomposes hydroxyl groups and (pyridin-2-yl) sulfanyl groups (see reference (DeMatteo et al, 2005)), we hypothesized that pyrithione may affect mitochondrial superoxide generation and clearance. To address this problem, cancer cells were treated with pyrithione for 2 hours and evaluated by visualizing fluorescent probes that specifically label mitochondria based on superoxide levels.

HeLa human cervical cancer cells were seeded at 4000 cells/well onto 8-well glass bottom chamber slides and incubated overnight at 37 ℃ in DMEM supplemented with 10% FBS. To investigate whether exposure of pyrithione to acidic or neutral external pH affects mitochondrial accumulation of superoxide, cells were first incubated with DMEM at pH6.4 or pH7.4 alone or in the presence of zinc pyrithione or zinc pyrithione for 2 hours at 37 ℃. The cells were then washed and incubated with a solution containing 1.25. mu.M mitoSOX^TM(M31008, Invitrogen) and 2.5. mu. MDRAQ5^TM(#62254, Thermo Scientific) was incubated at 37 ℃ for 5 minutes in pH7.4 medium. Mitosox^TMFor the quantitative determination of mitochondrial superoxide production, DRAQ5^TMFor fluorescent labeling of cell nuclei for observation of cells under a fluorescent microscope. After 5 minutes, the cells were placed in buffered NaCl-saline (150mM NaCl, 5mM KCl, 2mM CaCl, 1mM MgCl, 20mM HEPES, pH7.4) and imaged by confocal microscopy.

Accumulation of superoxide in mitochondria is one of the mechanisms by which cancer cells are killed. MitoSOX by using a fluorescent probe that sensitively and specifically detects superoxide in mitochondria^TMIt was found that treatment of cancer cells with 200nM zinc pyrithione or 400nM sodium pyrithione for 2 hours in acidic medium at pH6.4 resulted in significant accumulation of superoxide in the mitochondria, while treatment of cancer cells with 200nM zinc pyrithione or 400nM sodium pyrithione for 2 hours in conventional medium at pH7.4 did not result in accumulation of superoxide in the mitochondria (FIG. 6). Exposure of cancer cells to acidic medium at pH6.4 in the absence of pyrithione did not cause the accumulation of superoxide. These results indicate that contact of pyrithione with cancer cells under acidic conditions triggers the accumulation of superoxide in the mitochondria.

Example 7

The anticancer activity of gefitinib and erlotinib is not enhanced by the acidity of the medium.

Gefitinib and erlotinib are Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors that can specifically block the proliferation of cancer cells, thereby providing a different mechanism of action than classical chemotherapy. Gefitinib and erlotinib are used clinically for the treatment of malignant tumors, such as lung cancer and cutaneous malignant melanoma. It is not clear whether these clinically used molecularly targeted therapeutics also exhibit acidity-enhanced antitumor activity.

Cervical cancer HeLa cells were seeded at 2,000/well onto 96-well culture dishes and 5% CO in the atmosphere₂Incubate overnight at 37 ℃ in DMEM supplemented with 10% FBS in the presence. Cells were treated with increasing concentrations of gefitinib or erlotinib in pH6.4 DMEM or pH7.4 DMEM medium and incubated for 72 hours at 37 ℃. Bicarbonate removal from pH6.4 DMEM and pH7.4 DMEM media and in the absence of atmospheric CO₂Experiments were performed to minimize pH fluctuations caused by bicarbonate-mediated proton movement across the biofilm. The MTT assay was performed as in example 2 72 hours after exposure to gefitinib or erlotinib.

Unlike pyrithione, no acidity-enhanced anticancer effect was observed when cancer cells were exposed to gefitinib or erlotinib. For example, when HeLa cells were exposed to 20 μ M gefitinib in a medium adjusted to pH7.4 and 6.4 for 72 hours, the relative cell viability determined by MTT assay was 54 ± 16% and 61 ± 9%, respectively. In another example, relative cell viability was 39 ± 8% and 46 ± 14% when HeLa cells were exposed to 20 μ M erlotinib for 72 hours in media adjusted to pH7.4 and 6.4, respectively. The discovery that the anticancer effects of gefitinib and erlotinib are not affected by acidity underscores the uniqueness of the acidity-enhanced anticancer effect of pyrithione. The results are summarized in fig. 7.

Example 8

Since many previous studies have shown that the anticancer activity of zinc pyrithione is mediated by zinc toxicity, it is now surprising to find that sodium pyrithione such as zinc pyrithione is effective in reducing cancer cell viability. Therefore, we further explored the possibility that zinc-free pyrithione may also be toxic to cancer cells, in addition to the zinc-mediated toxicity disclosed in the prior art. To address this problem, the potential effect of the metal chelator EDTA on the killing of cancer cells by zinc pyrithione was investigated.

Cervical cancer HeLa cells, colon cancer HT29 cells and brain tumor U87 cells were seeded at 2,000 cells/well on 96-well culture dishes and 5% CO in the atmosphere₂Incubate overnight at 37 ℃ in DMEM supplemented with 10% FBS in the presence. Colon cancer HT29 cells and brain tumor U87 cells were treated with increasing concentrations of zinc pyrithione in serum-Free (FBS) DMEM medium at 37 ℃ for 72 hours, while cervical cancer HeLa cells were cultured for 20 hours, which showed the same effect on other cells. In some experiments, varying concentrations of EDTA were added to the medium along with zinc pyrithione. Serum was excluded from the medium in these experiments to avoid unwanted inactivation of EDTA by metals and cations present in the serum. Bicarbonate was also removed from DMEM medium without serum pH7.4 and in the absence of atmospheric CO₂The experiment was performed under the condition of (1). Following incubation with zinc pyrithione with or without EDTA or EDTA alone, the MTT assay was performed as in example 2.

Referring to the results in fig. 6, it was found that zinc pyrithione inhibited cancer cell viability more significantly when the cancer cells were simultaneously exposed to EDTA. For example, exposure of 50nM zinc pyrithione to brain tumor U87 cells in serum-free medium for 20 hours inhibited cell proliferation and viability of 60 ± 7% pyrithione-untreated U87 cells. In the presence of 10 μ M and 20 μ M EDTA, 50nM of zinc pyrithione further inhibited cell proliferation to 30 ± 4% and 19 ± 11%, respectively, whereas EDTA exposure alone only resulted in a slight 10% inhibition of cell proliferation. These surprising results indicate that pyrithione is an important component of zinc pyrithione for anti-cancer activity, and that removal of zinc by EDTA enhances this activity. Enhancement of EDTA it can also be said that the addition of EDTA reduces the dosage of zinc pyrithione to achieve the same level of anti-cancer activity, providing useful information for practical applications.

Example 9

Pyridoxine enhances the anticancer activity of zinc pyrithione (Pyz) under acidic conditions.

Cervical cancer HeLa cells were seeded at 2,000/well onto 96-well culture dishes and 5% CO in the atmosphere₂Incubate overnight at 37 ℃ in DMEM supplemented with 10% FBS in the presence. Cells were treated with increasing concentrations of zinc pyrithione in DMEM at pH6.9 or DMEM medium without serum (FBS) at pH7.4 for 24 hours. In some experiments, pyridoxine was added to the medium at different concentrations along with zinc pyrithione. Bicarbonate removal from DMEM media at pH6.9 and pH7.4 and in the absence of atmospheric CO₂In order to minimize pH fluctuations caused by bicarbonate-mediated proton movement across the biofilm. After exposure to zinc pyrithione with or without pyridoxine, the cells were incubated with a medium at pH7.4 at 37 ℃ for an additional 48 hours. MTT assay was performed as in example 2.

Although 10 μ M and 100 μ M pyridoxine (as labeled B6 in fig. 9) was added to non-acidic pH7.4 medium along with 50nM zinc pyrithione, without affecting cell viability, 10 μ M and 100 μ M pyridoxine were added to acidic pH6.9 medium along with 50nM zinc pyrithione, reducing cell viability to 72 ± 4% and 40 ± 12%, respectively (fig. 9). Addition of 10 μ M and 100 μ M pyridoxine and 50nM zinc pyrithione to acidic medium at pH6.4 further reduced cell viability to 1.4 ± 0.6% and 2.5 ± 0.9%, respectively. Addition of pyridoxine alone at 10 μ M and 100 μ M to acidic or non-acidic medium did not reduce cell viability (figure 9). These results indicate that both 10. mu.M and 100. mu.M nvridoxin enhance antitumor activity by combining zinc pyrithione with acidity. Pyridoxine at 1mM also showed an enhanced antitumor activity of zinc pyrithione in combination with acidity. Addition of 1mM pyridoxine and 50nM zinc pyrithione to non-acidic pH7.4 medium resulted in cell viability of 71.5 ± 13%. Addition of 1mM pyridoxine and 50nM zinc pyrithione to acidic pH6.9 medium resulted in cell viability of 5.1 ± 3%. Addition of 1mM pyridoxine and 50nM zinc pyrithione to acidic pH6.4 medium resulted in cell viability of 1.2 ± 0.8%. These results indicate that the combination of pyridoxine, pyrithione and acid exhibits potent anticancer activity. Interestingly, the addition of 1mM pyridoxine to pH7.4, 6.9 and 6.4 medium in the absence of zinc pyrithione resulted in cell viability of 72.9 ± 8%, 21.6 ± 6% and 28.4 ± 3%, respectively. This result indicates that, in addition to potent pyrithione and acidity-dependent antitumor activity, 1mM pyridoxine itself has moderate acidity-dependent antitumor activity.

Example 10

Sodium ascorbate or 3-o-ethyl ascorbic acid in acidic conditions enhances the anticancer activity of zinc pyrithione (Pyz) and sodium pyrithione (Pyn).

Cervical cancer HeLa cells were seeded at 2,000/well onto 96-well culture dishes and 5% CO in the atmosphere₂Incubate overnight at 37 ℃ in DMEM supplemented with 10% FBS in the presence. Cells were treated with increasing concentrations of zinc pyrithione in DMEM with pH6.4 serum Free (FBS), DMEM with pH6.9 serum (FBS), or DMEM medium with pH7.4 serum Free (FBS) for 24 hours. In some experiments, sodium ascorbate or 3-o-ethyl ascorbic acid was added to the medium at different concentrations along with zinc pyrithione. Removing bicarbonate from the culture medium and in the absence of atmospheric CO₂Experiments were performed to minimize pH fluctuations caused by bicarbonate-mediated proton movement across the biofilm. After exposure to zinc pyrithione with or without sodium ascorbate or 3-o-ethyl ascorbate, cells were incubated with pH7.4 medium at 37 ℃ for an additional 48 hours. MTT assay was performed as in example 2.

It has been found that the addition of 2mM 3-o-ethyl ascorbic acid (AscE) and 5nM and 10nM zinc pyrithione (Pyz) to acidic medium pH6.9 reduced cell viability to 91 + -11% and 43 + -11%, respectively, compared to untreated controls. Addition of 2mM of 3-o-ethyl ascorbic acid together with 5nM and 10nM of zinc pyrithione to a medium at pH6.4 more effectively reduced cell viability to 22 ± 10 or 11 ± 10%, respectively. In contrast, when up to 10nM of zinc pyrithione alone was added, no decrease in cell viability was observed (fig. 10A). Furthermore, the addition of 2mM 3-o-ethyl ascorbic acid and up to 10nM zinc pyrithione to non-acidic medium pH7.4 did not reduce cell viability (FIG. 10). These results indicate that 3-o-ethyl ascorbic acid enhances the antitumor activity of zinc pyrithione in an acidity-dependent manner.

Similarly, 2mM 3-o-ethyl ascorbic acid (AscE) was added to 10nM and 20nM sodium pyrithione (Pyn) with acidic medium pH6.9, reducing cell viability to 96 + -5% and 48 + -7%, respectively, compared to untreated controls. The addition of 2mM 3-o-ethyl ascorbic acid to acidic medium pH6.4, together with 10nM and 20nM sodium pyrithione (Pyn), more effectively reduced cell viability to 17 + -5% and 7 + -3%, respectively. In contrast, when up to 20nM sodium pyrithione was added to acidic medium alone or in combination with 2mM 3-o-ethyl ascorbic acid to non-acidic medium, no reduction in cell viability was observed.

The combination of sodium ascorbate (AscNa) and acidity also showed a significant enhancement of the anticancer effect of zinc pyrithione (Pyz). For example, it has been found that adding 1mM sodium ascorbate and 10nM zinc pyrithione to acidic medium at pH6.9 moderately reduced cell viability to 85 ± 5%, while adding 1mM sodium ascorbate and 10nM zinc pyrithione to more acidic medium at pH6.4 was more effective in reducing cell viability to 46 ± 5% compared to untreated controls (fig. 10A). A similar but more pronounced effect was observed when 1mM sodium ascorbate and 20nM zinc pyrithione were added to acidic media at ph6.9 and ph 6.4. In contrast, when up to 10nM of zinc pyrithione (Pyz) alone was added to acidic pH6.4 medium or 10nM of zinc pyrithione (Pyz), and in combination with 1mM sodium ascorbate, was added to non-acidic pH7.4 medium, there was no significant difference in cell viability compared to the untreated control group (fig. 10A). These results indicate that ascorbic acid enhances the antitumor activity of zinc pyrithione in an acidity-dependent manner.

The antiproliferative effect of pyrithione on brain tumor cells in 3D cultures as measured by a spheroid culture system was enhanced by 3-o-ethyl ascorbic acid.

A 1% agarose solution heated to 60 ℃ was mixed with an equal volume of medium (dmemphph 7.4 supplemented with 10% FBS) and 50 μ Ι of the mixture was transferred to each well of a 96-well plate. The plate was cooled at room temperature for 30 minutes until the agarose solidified, 1000 cells resuspended in 50. mu.l of medium were overlaid on top of the agarose layer and centrifuged at 1,500g for 5 minutes. The cells were cultured at 37 ℃ for 24 hours. Spheroids in suspension cultures were grown in the absence or presence of zinc pyrithione or 3-o-ethyl ascorbic acid, or a combination of 72 hours. After 3 days, spheroids were transferred to another 96 wells. Plates were centrifuged at 1,500g for 5 minutes and the solution carefully replaced with PBS without disturbing the spheroids at the bottom of the wells. The plate was centrifuged again at 1,500g for 5 minutes and PBS was replaced with 0.1M sodium acetate pH 5.0, 0.1% w/v Triton X-100 and 5mM p-nitrophenyl phosphate (#34045, Thermo) buffer as fresh supplement and incubated at 37 ℃ for 2 hours. After 2 hours, the reaction was stopped by adding 10M NaOH, and the absorbance at 450nM was measured.

Spheroid cultures incubated with 500nM zinc pyrithione alone (Pyz) were found to inhibit cell viability to 88 ± 8% compared to untreated spheroids (fig. 10B). In contrast, the addition of zinc pyridone containing 5mM and 10mM of 3-o-ethyl ascorbic acid (AscE) further inhibited cell viability by 76. + -. 4% and 63. + -. 7%, respectively. These results indicate that 3-o-ethyl ascorbic acid enhances the antiproliferative activity of zinc pyrithione in 3D cultures. Furthermore, when 10mM of 3-o-ethyl ascorbic acid was added in combination with zinc pyrithione, the 3D spheroid structure became fragile, resulting in the dissociation of some cell populations from the periphery of the spheroid (see figure 10B, right panel).

Example 11

Direct application of a formulation containing zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitate proliferates human skin cancer cells implanted in mice.

3-O-Ethyl ascorbic acid and ascorbyl tetraisopalmitate are derivatives of ascorbic acid that are readily absorbed through the skin and efficiently converted to ascorbic acid. The anticancer activity of formulations containing zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitate, or combinations thereof, administered topically was evaluated in a mouse xenograft model of skin cancer.

6 weeks old male C57BL/6 athymic nude mice were injected subcutaneously with 2.5 × 10⁶Personal skin cancer a2058 cells. After 2-3 days of injection when the tumor size reached about 3mm3, the mice were randomly divided into 4 groups of 5 mice each, and the cream formulation was administered directly to the skin over the tumor area once a day. Group 1 (Pyz), cream containing 1% zinc pyrithione; group 2 (Asc), a cream containing 3% 3-o-ethyl ascorbic acid and 2% ascorbyl tetraisopalmitate; group 3 (Pyz + Asc), cream containing 1% zinc pyrithione, 3% 3-ethyl ascorbic acid and 2% ascorbyl tetraisopalmitate; group 4 (vehicle), carrier only. The composition of the formulation is shown below.

Composition of the formulation used in group 1.

a) Aqueous phase

Glycerol: 5 percent of

Polysorbate 20: 1 percent of

Citric acid 1M solution: 0.7 percent

Sodium citrate 1M solution: 1.3 percent of

Zinc pyrithione: 1 percent of

Distilled water: the total amount is 100%

b) Oil phase

Behenyl alcohol: 3 percent of

Cetyl alcohol: 3 percent of

Stearic acid glyceride: 1 percent of

Stearic acid: 1 percent of

Sorbitol stearate: 1 percent of

Cetyl palmitate: 1 percent of

Dimethyl silicone oil: 1 percent of

c) Cooling phase

Cyclomethicone: 5 percent of

d) The pH was adjusted to 5.5 with HCl or NaOH.

The aqueous phase components were combined and mixed while heating at 75 ℃ until completely dissolved in a suitable container. In a separate container, the oil phase components were mixed and mixed while heating at 75 ℃ until completely dissolved. The oil phase mixture was then added to the water phase mixture and mixed thoroughly to form an emulsion. The emulsion was then cooled to about 50-55 ℃. The emulsion was then ground until the product became homogeneous.

Composition of the formulation used in group 2.

a) Aqueous phase

Glycerol: 5 percent of

Polysorbate 20: 1 percent of

Citric acid 1M solution: 0.7 percent

Sodium citrate 1M solution: 1.3 percent of

Distilled water: to the total amount of 100%

b) Oil phase

Behenyl alcohol: 3 percent of

Cetyl alcohol: 3 percent of

Stearic acid glyceride: 1 percent of

Stearic acid: 1 percent of

Sorbitol stearate: 1 percent of

Cetyl palmitate: 1 percent of

Dimethyl silicone oil: 1 percent of

c) Cooling phase

Cyclomethicone: 5 percent of

3-o-ethyl ascorbic acid: 3 percent of

Ascorbyl tetraisopalmitate: 2 percent of

d) The pH was adjusted to 5.5 with HCl or NaOH.

The aqueous phase components were combined and mixed while heating at 75 ℃ until completely dissolved in a suitable container. In a separate container, the oil phase components were combined and mixed while heating at 75 ℃ until completely dissolved. The oil phase mixture was then added to the water phase mixture and mixed thoroughly to form an emulsion. The emulsion was then cooled to about 50-55 ℃. The emulsion was then ground until the product became homogeneous.

Composition of the formulation used in group 3.

a) Aqueous phase

Glycerol: 5 percent of

Polysorbate 20: 1 percent of

Citric acid 1M solution: 0.7 percent

Sodium citrate 1M solution: 1.3 percent of

Zinc pyrithione: 1 percent of

Distilled water: adding to make the total volume 100%

b) Oil phase

Behenyl alcohol: 3 percent of

Cetyl alcohol: 3 percent of

Stearic acid glyceride: 1 percent of

Stearic acid: 1 percent of

Sorbitol stearate: 1 percent of

Cetyl palmitate: 1 percent of

Dimethyl silicone oil: 1 percent of

c) Cooling phase

Cyclomethicone: 5 percent of

3-o-ethyl ascorbic acid: 3 percent of

Ascorbyl tetraisopalmitate: 2 percent of

d) The pH was adjusted to 5.5 with HCl or NaOH.

Composition of the formulation used in group 4.

a) Aqueous phase

Glycerol: 5 percent of

Polysorbate 20: 1 percent of

Citric acid 1M solution: 0.7 percent

Sodium citrate 1M solution: 1.3 percent of

Distilled water: adding to make the total volume 100%

b) Oil phase

Behenyl alcohol: 3 percent of

Cetyl alcohol: 3 percent of

Stearic acid glyceride: 1 percent of

Stearic acid: 1 percent of

Sorbitol stearate: 1 percent of

Cetyl palmitate: 1 percent of

Dimethyl silicone oil: 1 percent of

c) Cooling phase

Cyclomethicone: 5 percent of

d) The pH was adjusted to 5.5 with HCl or NaOH.

According to formula ab²The tumor volume was determined, where a is the length and b is the width, and the relative tumor volume for each treatment group was calculated and plotted (fig. 11A). The X axis represents the number of days after transdermal administration begins; the Y-axis represents relative tumor size. At the end of the experiment, the mean tumor size of group 3 mice ("Pyz + Asc" in fig. 11A) was smaller than the tumor size of the other groups. This result indicates that topical administration of formulations containing pyrithione and an ascorbic acid derivative is an effective method for reducing tumor growth, but formulations containing pyrithione alone or an ascorbic acid derivative alone are ineffective.

At the end of the experiment, histological samples of the skin areas in contact with the formulation, liver and kidney were prepared according to standard procedures. Sections were stained with hematoxylin and eosin. A micrograph of the histological sample is shown in fig. 11B. No tissue damage, abnormal cell death or any other adverse reaction was observed in group 3 (PyZ + Asc; mice treated with cream containing pyrithione, 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate) and group 4 (vehicle; mice treated with control cream containing no pyridone, 3-o-ethyl ascorbic acid or ascorbyl tetraisopalmitate).

Blood was extracted from representative animals of each group on the day of sacrifice and hematological testing was performed. The results are summarized in the table. All values were within the normal range except for low MCH, low MCV and high PLT observed in all groups, which may be caused by cancer growth in these animals. In addition to the slight increase in% granulocytes in the "Asc" animals, there were no outliers associated with the particular treatment group, which was considered irrelevant, since the granulocytes number of this animal was within the normal range. Ref reference; GR granulocytes (number/L); GR% granulocytes (%); HCT hematocrit (%); HBG hemoglobin (g/L); LY lymphocytes (number/L); LY% lymphocytes (%); MCH mean corpuscular hemoglobin (pg); MCHC mean corpuscular hemoglobin concentration (g/L); MO monocytes (number/L); MO% monocytes (%); MPV mean platelet value (fL); procalcitonin PCT (%); PDW platelet distribution width (fL); PLT platelets (g/L); RBC red blood cells (number/L); RDW red blood cell distribution width (%); WBC white blood cell (number/L)

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Claims

1. A composition for preventing and/or treating malignant tumors comprising at least one or more active ingredients selected from the group consisting of pyrithione, a pyrithione salt, a vitamin B6 compound, and a phosphorylated form of a vitamin B6 compound.

2. A composition for treating a precancerous condition comprising at least one or more active ingredients selected from the group consisting of pyrithione, a pyrithione salt, a vitamin B6 compound, and a phosphorylated form of a vitamin B6 compound.

3. The composition according to claim 1 or 2, wherein the active ingredient is one or more selected from the group consisting of pyrithione and a pyrithione salt, the composition further comprising at least one or more selected from the group consisting of EDTA, EDTA salts, ascorbic acid salts, and ascorbic acid compounds.

4. The composition of any one of claims 1-3, wherein the pH is 6.9 or less in the presence of a buffer.

5. A method for preventing and/or treating malignant tumors comprising the step of administering at least one or more active ingredients selected from the group consisting of pyrithione, a pyrithione salt, a vitamin B6 compound, and a phosphorylated form of a vitamin B6 compound.

6. Methods of treating pre-cancerous conditions using at least one or more active ingredients selected from the group consisting of pyrithione, a pyrithione salt, a vitamin B6 compound, and a phosphorylated form of a vitamin B6 compound.

7. The method according to claim 5 or 6, wherein the active ingredient is one or more selected from the group consisting of pyrithione and a pyrithione salt, and wherein the step uses an active ingredient having at least one or more selected from the group consisting of EDTA, EDTA salts, ascorbic acid, ascorbate salts, and ascorbate compounds.

8. 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.

9. Use of one or more compounds selected from the group consisting of pyrithione, a pyrithione salt, a vitamin B6 compound, and a phosphorylated form of a vitamin B6 compound, for the preparation of a formulation for the prevention and/or treatment of a malignant tumor.

10. Use of one or more compounds selected from the group consisting of pyrithione, a pyrithione salt, a vitamin B6 compound, and a phosphorylated form of a vitamin B6 compound in the preparation of a formulation for treating a precancerous condition.

11. Use according to claim 9 or 10 wherein one or more is selected from the group consisting of pyrithione and a pyrithione salt, and at least one or more is selected from the group consisting of EDTA, EDTA salts, ascorbic acid salts and ascorbic acid compounds.

12. Use according to any one of claims 7 to 11, wherein the pH is 6.9 or lower.