[go: up one dir, main page]

WO2018203127A1 - Compositions for treatment of malignant tumors and precancerous conditions, methods of use thereof and methods for manufacturing medicaments - Google Patents

Compositions for treatment of malignant tumors and precancerous conditions, methods of use thereof and methods for manufacturing medicaments Download PDF

Info

Publication number
WO2018203127A1
WO2018203127A1 PCT/IB2018/000456 IB2018000456W WO2018203127A1 WO 2018203127 A1 WO2018203127 A1 WO 2018203127A1 IB 2018000456 W IB2018000456 W IB 2018000456W WO 2018203127 A1 WO2018203127 A1 WO 2018203127A1
Authority
WO
WIPO (PCT)
Prior art keywords
pyrithione
cancer
ascorbic acid
cells
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2018/000456
Other languages
French (fr)
Inventor
Masayuki Numata
Hung-Yi Fan
Aidi LI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Firmiana Health Sciences Inc
Original Assignee
Firmiana Health Sciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Firmiana Health Sciences Inc filed Critical Firmiana Health Sciences Inc
Priority to CN201880025684.7A priority Critical patent/CN111542325A/en
Priority to JP2018554579A priority patent/JP6676779B2/en
Publication of WO2018203127A1 publication Critical patent/WO2018203127A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/576—Six-membered rings
    • C07F9/58—Pyridine rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4415—Pyridoxine, i.e. Vitamin B6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/65—One oxygen atom attached in position 3 or 5
    • C07D213/66—One oxygen atom attached in position 3 or 5 having in position 3 an oxygen atom and in each of the positions 4 and 5 a carbon atom bound to an oxygen, sulphur, or nitrogen atom, e.g. pyridoxal
    • C07D213/67—2-Methyl-3-hydroxy-4,5-bis(hydroxy-methyl)pyridine, i.e. pyridoxine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/89—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members with hetero atoms directly attached to the ring nitrogen atom

Definitions

  • compositions for treatment of malignant tumors and precancerous conditions comprising
  • the current invention discloses new compositions and therapeutic/preventative methods to suppress cancer cells growing/arising in the acidic environment.
  • chemotherapeutics aim to block rapid proliferation and cell division of cancer cells by introducing chemical damage to deoxyribonucleic acids (DNA).
  • DNA is a genetic blueprint for both cancer and normal cells, and these traditional cancer chemotherapeutics often also damage normal tissues and cause serious side effects.
  • Other chemotherapeutics target constituents of the cellular structure proteins that are involved with cell division and proliferation, but these agents also cause non-specific toxicity to non-cancer cells such as bone marrow and a subpopulation of avidly dividing gut cells.
  • Malignant tumors are not simple assemblies of cancer cells. Healthy tissues and cells surrounding tumor cells establish a unique environment (sometimes also referred to herein as the "tumor microenvironment"), which supports cancer cells, allowing them to grow, spread and metastasize.
  • One of the most important elements of the tumor microenvironment is the acid.
  • the tumor microenvironment is more acidic than tumor-free environment. Acidic metabolic products released to outside of the tumor cells provide the source of acid in the tumor microenvironment, which reduces the sensitivity to chemo- and radiation therapies, and stimulates tumor growth and spreading.
  • the acidic tumor microenvironment is a spectacular therapeutic target for malignant tumors. It is expected that a method that sensitively blocks proliferation of tumor cell s growing in the acid ic environment will become a promising new anticancer therapy.
  • precancerous conditions Disease conditions that themselves are not yet malignant, but have a high risk to develop into cancer are called precancerous conditions.
  • a highly acidic environment is established in precancerous lesions prior to the appearance of malignant tumors as a result of sustained and uncontrolled inflammation associated with precancerous conditions.
  • the acidic environment accelerates genetic alterations and cancer occurrence (Coussens and Werb, 2002).
  • malignant tumor cells grow more preferentially than non-malignant cells in the acidic environment, which further drives acidification.
  • excessive acid weakens the ability of immune cells, thereby increasing the chance of occurrence of malignant tumors (Lardner, 2001).
  • AKs Actinic Keratoses
  • HPV Human Papillomavirus
  • AKs are rough and scaly skin conditions caused by the excessive exposure to UV radiation, and are one of the most common diagnoses accounting for 10% of outpatients of dermatology clinics in the United States.
  • AKs are a frequent cause of skin cancers; approximately 65% of squamous cell carcinomas and 36% of basal cell carcinomas are caused by AKs according to one study (Criscione et al., 2009).
  • Cryosurgery and topical administration of anti-cancer agents are the major therapies for AKs.
  • AKs are not fatal before malignant tumors arise.
  • HPV infection occurs in the cervix, the outer surface area of the female genitalia, anus, penis and oral cavity.
  • WHO World Health Organization
  • HPV infection is known to cause cancers in these bodily parts, particularly HPV is involved with almost all cases of cervical cancer (http://www.who.int/mediacentre/factsheets/fs380/en/).
  • Cervical cancer is the fourth most common cancer in female and second most common cancer in developing countries, where more than 85% of cervical cancer occurs. While HPV vaccination and early diagnosis by cancer screening greatly reduced the risk of cervical cancer in developed countries, socio-economieal problems in less developed countries make adaptation of these methods difficult. Introduction of a facile and less expensive method is awaited.
  • 2006/0040980 discloses a method to use the formulation that combines zinc pyrithione and zinc salts as an anti-cancer and anti-angiogenic agent. More recent experimental studies further exploited this idea and proposed to deliver heavy metals such as copper (Liu et al., 2014), cadmium, platinum (Zhao et al., 2016b) and nickel (Zhao et al., 2016a) to leukemia, myeloma, lung cancer and liver cancer using a pyrithione salt as a vehicle. These previous arts have taught us the importance of heavy metals that pass through pyrithione pores in killing and blocking proliferation of malignant tumors.
  • the acidic tumor microenvironment is known to cause acquisition of resistance to many anti-cancer chemotherapeutic agents.
  • Currently available methods for preventing the development of cancer from precancerous conditions have limitations in terms of efficacy, safety and costs.
  • the present invention discloses compositions and therapeutic and preventative methods for malignant tumors, using acidity as a tool to potentiate the anti-cancer activity.
  • the disclosed methods and compositions are used to prevent the
  • any one or more of zinc pyrithione, sodium pyrithione, any other salt forms of pyrithione or salt- free forms of pyrithione in the manufacture of a medicament for the treatment of malignant tumors occurring in the skin, cervix, vagina, penis, anus and other bodily parts.
  • the pH of the formulation is maintained to be acidic in the presence of a pharmacologically acceptable buffer.
  • the approach efficiently inhibits malignant tumor proliferation under an acidic environment.
  • this approach may be combined with one or more conventional cancer therapies, such as chemotherapy, radiation therapy and surgery.
  • pyrithione incorporated with a chelator Ethylenediaminetetraacetic acid (EDTA), any one or more of Calcium EDTA, Disodium EDTA, Diammonium EDTA, Dipotassium EDTA, Disodiui 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 malignant tumors.
  • EDTA Ethylenediaminetetraacetic acid
  • pyrithione incorporated with pyridoxine, pyridoxal, pyridoxamine, and 5 '-phosphate esters thereof, collectively known as vitamin B6, in the manufacture of a medicament for the treatment and prevention of cancer or malignant tumors.
  • the approach improves and enhances the efficiency of inhibiting proliferation of malignant tumors under an acidic environment.
  • pyrithione incorporated 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, in the manufacture of a medicament for the treatment and prevention of cancer or malignant tumors.
  • ascorbic acid also known as L-ascorbic acid and vitamin C
  • any salt form of ascorbic acid or any chemical derivative of ascorbic acid in the manufacture of a medicament for the treatment and prevention of cancer or malignant tumors.
  • the approach improves and enhances the efficiency of inhibiting proliferation of malignant tumors under an acidic environment.
  • pyrithione in the manufacture of a medicament for the prevention of cancer arising from precancerous lesions such as Human Papillomavirus (HPV) infection in genital lesions and Actinic Keratoses (AKs) in skin.
  • HPV Human Papillomavirus
  • AKs Actinic Keratoses
  • acidic formulations are topically administered to cancer growing in the close proximity of the surface of body or bodily structure of organs.
  • Topical administration is a superior method to systemic administration in delivering pyrithione- containing formulations in many ways. Firstly, topical administration is safe because of the limited exposure of medicaments to blood and healthy bodily parts, thereby reducing the risk of adverse effects. Secondly, topical administration does not require a high level of technical expertise, and self administration is easier comparing to systemic administration. Thirdly, the acidic compositions disclosed by the current invention optimized for topical administration are efficiently delivered to cancer. Topical administration is also advantageous in delivering acidic formulations without influencing the acidity of blood and other bodily parts. Importantly, systemic acidification of the blood and entire body will cause heart failure. Certain
  • embodiments concern the use of administration methods that combine topical administration of pyrithione (and enhancers) and systemic administration of select enhancers (such as oral administration of vitamin B6 and intravenous administration of vitamin C), which further increase the efficacy of treatment.
  • Fig. 1 shows the chemical structure 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 the treatment with zinc pyrithione (Pyz), in two different pH media, on the viability of human brain tumor U87, breast cancer MDA-MB-231, cervical cancer HeLa, and colon cancer HT29 cells.
  • Cells were grown in the absence or presence of zinc pyrithione at the indicated doses for 3 days at 37°C. Cell viability was determined by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay.
  • MTT Methylthiazolyldiphenyl-tetrazolium bromide
  • Fig. 3 is a graph showing the effect of the treatment with sodium pyrithione (Pyn), in two different pH media, on the viability of human brain tumor U87, breast cancer MDA-MB- 231, cervical cancer HeLa, and colon cancer HT29 cells.
  • Cells were grown in the absence or presence of zinc pyrithione at the indicated doses for 3 days at 37°C. 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), in two different pH media, on HeLa cells.
  • Cells were grown in the absence or presence of zinc pyrithione at the indicated doses for 3 days at 37°C. Non-viable cell populations after treatment with Pyz and Pyn were determined by trypan-blue staining.
  • FIG. 5 are photographs showing the effect of the treatment with zinc pyrithione, in two different pH media, acidity-enhanced anti-proliferative effects of zinc pyrithione on brain tumor U87 cells grown in a spheroid 3D-culture.
  • Fig. 6 are photographs showing the effect of the zinc pyrithione (Pyz) and sodium pyrithione (Pyn), in two different pH media, on mitochondrial accumulation of superoxide in HeLa human cervical cancer cells.
  • Fig. 7 is a graph showing the effect of the treatment with erlotinib and gefitinib, in two different pH media, on the viability of human glioma U87, breast cancer MDA-MB-231, cervical cancer HeLa, and colon cancer HT29 cells. Cell viability was determined by MTT assay.
  • Fig. 8 is a graph showing the enhancement of anti-cancer effect of zinc pyrithione by ethylenediaminetetraacetic acid (EDTA) in human cervical cancer HeLa, brain tumor U87 and colon cancer HT29 cells. Cell viability was determined by MTT assay.
  • EDTA ethylenediaminetetraacetic acid
  • Fig. 9 is a graph showing the enhancement of anti-cancer effect of zinc pyrithione by pyridoxine in human cervical cancer HeLa cells. Cell viability was determined by MTT assay.
  • Fig. 10 presents a graph and photographs showing the enhancement of anti-cancer effect of zinc pyrithione and sodium pyrithione by sodium ascorbate and 3-o-ethyl ascorbic acid in human cervical cancer HeLa cells.
  • Cell viability was determined in 2D cultures (Fig 10A) and in a spheroid-3D culture (Fig. 10B).
  • Fig. 11 A is a graph showing the effect of topical administration of different formulations containing or not containing zinc pyrithione, 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate on the growth of human skin cancer cells implanted to immune-deficient mice.
  • Fig. 1 IB presents microscopic pictures of skin, liver and kidney showing that topical administration of formulations containing zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitate has no adverse effects on these tissues.
  • Table summarizes the hematological test results of the mice after topical administration of formulations containing zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitate.
  • pyrithione and “pyrithione compounds” are used interchangeably, referring to pyrithione and any salt thereof that are compatible with other ingredients of the formulation and compatible with administration to humans. Pyrithione is obtained from commercial sources most typically in a form of either zinc-salt 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 under different names such as 2-Mercaptopyridine-N-Oxide, 1- Hydroxy-2-pyridinethione, l-Hydroxypyridine-2-thione, Omedine, (2-Pyridilthio)-N-oxide, and 2-Pyridinethiol-l -oxide.
  • Chemical derivatives refer to compounds that have similar structure and function.
  • the sodium salt of pyrithione sodium pyrithione
  • Sodium pyrithione is commonly synthesized by reacting 2-chloropyridine-N-oxide with NaSH and NaOH (see, for example, the disclosures of U.S. Pat. No. 3,159,640).
  • the zinc salt of pyrithione may be synthesized by reacting pyrithione acid (i.e., 1- hydroxy-2-pyridinethione) or a soluble salt thereof with a zinc salt (e.g., ZnS0 4 , ZnCh) to form a zinc pyrithione precipitate (see, for example, U.S. Pat. No. 2,809,971).
  • pyrithione acid i.e., 1- hydroxy-2-pyridinethione
  • a zinc salt e.g., ZnS0 4 , ZnCh
  • Tumor(s) is a pathological terminology referring to changes recognized as swelling of a part of the body. Tumors may be either malignant - that show cancerous uncontrolled growth, or benign - that are generally harmless and curable (although certain types of cancer may arise from benign tumors if left untreated).
  • Malignant tumors are tumors that divide without control and can spread to nearby tissues directly or to remote organs through bloodstream or lymphatic systems, the process called metastasis.
  • Cancers are technically considered to be a group of malignant tumors that arise from epithelial cells of skin and organs. Malignant tumors arisen from non-epithelial cells such as blood, bone vessels, cartilages, muscles and supporting tissues that provide elasticity are technically excluded from cancer.
  • dermal malignant melanomas are derived from non-epithelial cells of the skin; therefore, malignant melanomas are not cancer.
  • malignant tumors, cancers and neoplasia are interchangeably used in a broad sense and, as such, melanomas are often referred to as a type of skin cancer.
  • cancer is understood in a broad sense and “cancer” is interchangeably used with "malignant tumor”.
  • Malignant tumors detected in the skin can be cutaneous squamous cell carcinoma, cutaneous melanoma, cutaneous adenocarcinoma, or any other forms of malignant tumors arising in or metastasized to skin regions.
  • Carcinomas herein refer to the pathological terminology of malignant tumors of epi thelial origin whereas melanomas are malignant tumors of non-epithelial origin.
  • malignant tumors detected in the skin are divided into basal cell carcinomas, squamous cell carcinomas and others that include malignant melanomas.
  • Malignant tumors detected in the uterus and vagina herein refer to squamous cell carcinoma, squamous adenocarcinoma, other malignant tumors, and metastatic tumors.
  • the term “treat” as used herein refers to provide a medical aid to patients for the purpose of ameliorating undesired medical conditions or preventing the worsening of such undesired conditions.
  • Anti-tumor effect “anti-cancer effect” and “anti-neoplastic effect” are interchangeably used, referring to slowing or regressing tumor growth, decreasing tumor size and prevention of spreading to other organs.
  • the compound is considered to be effective when the size of tumor is reduced.
  • the compound is considered to be effective when symptoms associated with cancer are relieved. In another aspect, the compound is considered to be effective when medical examinations show signs of reduction in tumor burden. Medical examinations include biochemical tests, tumor markers, diagnostic imaging, and histochemical examinations. In still another aspect, the compound is considered to be effective when the patients treated with the method show prolonged survival.
  • Transdermal administration herein refers to a broader definition that is used interchangeably with topical administration.
  • Topical administration includes all methods to deliver drugs through the surface of the body and the inner linings of the body passages, generally known as epithelial and mucosal tissues.
  • Excipients are organic or inorganic substances that do not interfere with active compounds and serve as carriers for active compounds.
  • Pharmacologically acceptable carriers include, but not limited to, water, polyethylene glycols (PEGs), salt solutions, lactose, amylose, alcohol, oils, fatty acids, gelatin, silicic acid, surfactants, viscous paraffin, hydroxymethyl- cellulose, polyvinylpyrrolidone, lubricants, and the like.
  • the current invention discloses a topical administration method and compositions of pyrithione for treating or preventing malignant tumors.
  • the formulation consists of pyrithione and chemical compounds that potentiate the anti-tumor activity of pyrithione under acidic conditions together with a carrier that does not interfere with the action of the compound.
  • the formulation is directly applied onto the bodily surface or the inner linings of the bodily structures.
  • Topical administration is effective for targeting therapeutic agents to malignant tumors growing close to the bodily surface or inner linings of bodily structures.
  • pyrithione efficiently kills tumor cells.
  • Some component(s) of the formulation may be administered by intravenous injection, subcutaneous injection, intramuscular injection, inhaling or oral intake.
  • the maximum pH of the formulation is 6.9, as anti-tumor activity of pyrithione is significantly more potent at pH 6.9 compared to pH 7.4 (see for example Fig. 10). It is more preferable that the maximum pH of the formulation is 6.4 as it has been found that anti-tumor activity by pyrithione is more potent at pH 6.4 than 6.9 (see for example Fig. 10). Even more preferably, the maximum pH of the formulation is 5.6 as it has been found that the pH 5.5 topical formulation containing pyrithione suppresses proliferation of implanted human skin cancer cells in mice (Fig. 11). A previous art U.S. Pat. No.
  • 6,455,076B 1 has recognized the importance of acids in enhancing the efficacy of active ingredients in cosmetic products.
  • the art further provided a method to prevent skin irritation. According to the art, irritation by acids equivalent to pH 2.0 is preventable. In another embodiment, accordingly, the minimum pH of the formation is 2.0.
  • EDTA substantially potentiates the anti-tumor activity of zinc pyrithione (Fig. 8).
  • EDTA is commonly used as a stabilizer of medical, cosmetic and food products that deprives and neutralizes zinc and other heavy metals with a given capacity.
  • pyrithione even in the absence of zinc and any other heavy metals, exerts potent acidity-enhanced anti-tumor activity, an unexpected finding in light of the previous arts that taught us the importance of heavy metals that pass through pyrithione (see “Background and description of related arts” [0009]).
  • Pyridoxine is one of the compounds showing vitamin B6 activity; other vitamin B6 compounds include pyridoxal, pyridoxamine, and 5'-phosphate esters thereof.
  • pyridoxine, pyridoxal, pyridoxamine, 5'-phosphate esters thereof, pyridoxine dicaprylate, pyridoxine dipalmitate or any other derivatives thereof is provided with zinc pyrithione (or any other metal pyrithione) or sodium pyrithione (or any other metal-free pyrithione).
  • sodium ascorbic acid particularly in combination with acidity substantially potentiates the anti-tumor activity of zinc pyrithione (Fig. 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- hydroxy group of the ascorbic acid, which makes this compound chemically stable and easily absorbed through the skin. It has been found that 3-o-ethyl ascorbic acid particularly in combination with acidity substantially potentiates the anti-tumor activity of zinc pyrithione (Fig. 10). It has been also found that 3-o-ethyl ascorbic acid particularly in combination with acidity substantially potentiates the anti-tumor activity of sodium pyrithione (Fig. 10).
  • Ascorbyl tetraisopalmitate is an oil-soluble derivative of ascorbic acid that is readily absorbed through the skin and effectively converted into ascorbic acid. As such, ascorbyl tetraisopalmitate and 3-o-ethyl ascorbic acid are commonly used for skin care consumer products. It has been found that topical application of a formulation containing a moderate concentration of zinc pyrithione (1 ⁇ ), 3-o-ethyl ascorbic acid (3 mM) and ascorbyl tetraisopalmitate (2 mM) inhibits proliferation of human skin cancer cells implanted into mouse skin (Fig. 11).
  • 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitate (or any other chemical derivatives of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid), ascorbic acid or a combination thereof is provided with zinc pyrithione (or any other metal pyrithione) or sodium pyrithione (or any other metal-free pyrithione) for topical administration.
  • compositions in the form of creams, ointments, lotions, gels, solid sticks, sprays, drops (eye drops, nose drops and alike) and occlusive devices.
  • These formulas may be oil-in-water emulsion, water-in-oil emulsion, viscous liquid, or water-soluble solution.
  • Occlusive devices such as a semi-permeable membrane covering a reservoir containing pyrithione and other active ingredients may be used to release pyrithione and other active ingredients such as chemotherapeutic agents, steroids and anti-inflammatory agents.
  • Pyrithione topical formula can be formulated according to the conventional methods using suitable excipients that include, for example, emulsifiers, surfactants, thickening agents, moisturizers, skin conditioning agents, skin protectants, and sun screen agents. Preservatives and
  • bacteriostatic agents such as methyl hydroxybenzoate, propyl hydroxybenzoate and chlorocresol may be optionally incorporated in the occlusive devices.
  • Direct application of the composition to cancer or pre-cancer lesions close to the bodily surface or close to the inner bodily structure may be carried out using pharmaceutically acceptable salts by means of injection by needles or similar devices, narrow jet propagated by high-pressure, externally applied electric field differences, ultrasound, laser beam, magnetic field and radiation.
  • Suppository forms may be used.
  • Suppository formulations may be prepared by mixing the compounds and compositions of the invention with a suitable excipient such as cocoa butter, polyethylene glycols with different molecular weights, glycerin and glycerinated gelatin. These excipients are solid at room temperature, but will melt at body temperature and release the compounds and compositions in the rectum, vagina or cervix.
  • chemical penetration enhancers and solubilization enhancers are incorporated into the formulation according to the known art (Williams and Barry, 2012).
  • Chemical penetration enhancers include unsaturated fatty acids such as oleic acid, docosahexaenoic acid, eicosapentanoic acid, terpenes, terpenoids, essential oils, azone, and pyrrolidones; some examples of solubilization enhancers include low doses of surfactants, cyclodextrins, and dimethyl sulfoxide (DMSO).
  • the formulation contains pyrithione at least 0.000317% by weight, and preferably at least 0.00634% by weight as the minimum concentration required to suppress growth of cancer cells in three-dimensional (3D) cultures is 0.0000634% (2 ⁇ ) (see Fig. 5).
  • the formulation contains at least 1% pyrithione.
  • the formulation contains pyrithione up to 5.5 % by weight (173 mM) as it has been found that emulsions containing 5.5 % zinc pyrithione is stable when applied onto the skin.
  • EDTA is added to the formulation in an amount of at least about 0.0003% by weight (10 ⁇ ) that is the minimum concentration required to enhance anticancer effects by zinc pyrithione (see Fig. 8).
  • the formulation contains EDTA at the maximum concentrations from 10 to 15% by weight as these are the amounts safely used in dentistry for a short-term dental treatment (see reference (Lanigan and Yamarik, 2002)). More preferably, the formulation contains EDTA at the maximum concentration of approximately 2%, as this is a typical maximum concentration of EDTA suitable for a long-term use in cosmetic formulations.
  • the topical formulation contains at least 0.00017% by weight
  • the formulation contains at least 0.0017% by weight (100 ⁇ ) of pyridoxine, as this is the concentration that shows even a higher potentcy.
  • the formation contains at least 0.017% by weight (1 mM) of pyridoxine, as this concentration shows acidity-dependent anti-tumor effects even in the absence of zinc pyrithione. Because of other factors such as skin permeability, diffusion, and chemical stability, it is likely that higher concentrations of pyridoxine are required to achieve the anticipated effect.
  • the previous art has unveiled cosmetic compositions containing from 0.001% to 15% of pyridoxine (U.S. Pat No. 20,060,018,860A1). It was noted however that formulations containing 10% by weight or a less amount of pyridoxine are more stable in the presence of other ingredients. Therefore, in one embodiment, the formulation contains 10% by weight or less amounts of pyridoxine. Oil-soluble pyridoxine derivatives such as pyridoxine dicaprylate and pyridoxine dipalmitate are easily absorbed through the bodily surface and show stable activity once absorbed.
  • pyridoxine dicaprylate, pyridoxine dipalmitate, or any other derivatives of pyridoxine are added to the formulation in an amount of at least 0.00017% by weight, preferably 0.0017% and more preferably 0.017%, and in an amount of less than 10% by weight.
  • the topical formulation contains at least 0.04% by weight (2 mM) of the composition of 3-o-ethyl ascorbic acid, as 2 mM 3-o-ethyl ascorbic acid in combination with pyrithione and acidity regresses growth of cancer cells in 2D cultures (see Fig. 10).
  • the formulation contains at least 0.2% (10 mM) by weight of 3-o-ethyl ascorbic acid, as 10 mM 3-o-ethyl ascorbic acid in combination with pyrithione and acidity regress growth of cancer cell s in 3D cultures.
  • the formulation contains at least 3% by weight of 3-o-ethyl ascorbic acid, as the cream formulation containing 3% 3-o-ethyl ascorbic acid in combination with zinc pyrithione and acidity significantly suppresses proliferation of human skin cancer cells implanted in mice (see Fig. 11).
  • the formulation also contains 2% by weight of ascorbyl tetraisopalmitate (see Example 11).
  • the formulation contains at least 3% of 3-o- ethyl ascorbic acid plus 2% or higher concentrations of any one, or two or more in combination, of ascorbyl tetraisopalmitate, ascorbyl palmitate and any other derivative of ascorbic acid. It was noted that formulations containing more than 20% by weight of 3-o-ethyl ascorbic acid is irritating at times to sensitive skins. Therefore, in one embodiment, the formulation contains less than 20%, preferably less than 10% by weight of pyridoxine.
  • the formulation contains 30% or less, preferably 20% or less, and more preferably 10% or less amounts of sodium ascorbate (or any other ascorbate salt). In another embodiment, the formulation contains 15% or less, preferably 10% or less, and more preferably 5% or less amounts of ascorbyl tetraisopalmitate (or any other derivative of ascorbic acid).
  • the formulation containing pyrithione and enhancers is topically administered one time every day.
  • the formulation containing pyrithione and enhancers is topically administered one time every day.
  • only 34% of zinc pyrithione remains in the treated skin area after 20 hours of topical application (see reference (Parekh et al., 1970)).
  • the previous art further teaches us that topical application of pyrithione with administration intervals shorter than 20 hours increases the effective concentration of pyrithione in the treated skin area. This means more than one time a day of administration increases the effective concentration of pyrithione in the skin.
  • the formulation containing pyrithione and enhancers is topically administered two times every day. In yet another embodiment, the formulation containing pyrithione and enhancers is administered topically three times every day. In one embodiment, the agents are administered until the desired therapeutic outcome has been achieved; where in a typical example, the pyrithione formulation may be delivered for a period that can range from two months to 36 months. In another embodiment, one day to four weeks of intervals in between each therapeutic cycle are introduced. In yet another embodiment, chemotherapy, radiation and/or surgery are carried out during intervals. Other interval schemes and therapeutic durations may be considered appropriate to one skilled in the art.
  • Another example of application of the topical administration of pyrithione is for treating cervical cancer, vaginal cancer and other malignant tumors arising in the cervix of the uterus and vagina.
  • Topical pyrithione administration may be employed for medical treatment of other malignant tumors that are readily accessible from the bodily surface.
  • Target diseases may include, but not limited to, malignant tumors arising in or metastasized to head and neck, oral cavity, anus, rectum, prostate, breast, bone, muscle and cartilage.
  • Topical administration infi ltrates the composition from the outer surface of a tumor.
  • Systemic administration delivers the composition to a tumor via tumor-nurturing blood vessels. Combining these two administration methods will maximize the effective concentration of the composition in a tumor.
  • Oral and intravenous administrations are widely used systemic administration routes to increase vitamin levels in the blood plasma.
  • pyridoxine, pyridoxal, pyridoxamine, or 5'-phosphate esters thereof is administered orally in a minimum amount of 600 mg/day in combination with topical administration of the formulation containing pyrithione and pyridoxine (or derivatives thereof).
  • Previous arts have taught us that a risk of neuropathy is associated with high levels of pyridoxine intake exceeding 1,000 mg daily (see reference (Bender, 1999)). Therefore, in another embodiment, pyridoxine, pyridoxal, pyridoxamine, or 5'-phosphate esters thereof is orally administered in a maximum amount of 1,000 mg/day in combination with topical administration of the formulation containing pyrithione and pyridoxine (or derivatives thereof).
  • pyridoxine, pyridoxal, pyridoxamine, or 5 '-phosphate esters thereof is orally administered twice a day, preferably three times a day, and even more preferably four times a day.
  • 3-o-ethyl ascorbic acid or any other chemical derivative of ascorbic acid
  • sodium ascorbate or any other salt of ascorbic acid
  • ascorbic acid is intravenously administered in combination with topical administration of the formulation containing pyrithione and any one, or two or more in combination, of 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.
  • intravenous administration of 5 g, but not 1 g or 3 g, of ascorbic acid can elevate the plasma concentration of ascorbic acid to 2 mM or higher. Therefore, in one embodiment, a minimum dose of 5 g 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 intravenously administered.
  • Intravenous administration of ascorbic acid up to 100 g is a safe and effective method to increase the plasma concentration of ascorbic acid according to the art.
  • a maximum dose of 100 g 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 intravenously infused.
  • 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 intravenously infused.
  • Previous arts have also taught us that dividing the daily dose of ascorbic acid into multiple administrations helps sustain the effective plasma concentration in the body. For example, intravenous infusion of ascorbic acid at a dose of 100 g, 50 g and 10 g establishes and sustains plasma ascorbic acid concentrations over 2 mM for approximately 5.5 hours, 3.5 hours and 1.3 hours, respectively.
  • intravenous administration of 50 g ascorbic acid twice a day and 10 g five times a day are more efficient methods than once a day 100 g administration in order to sustain plasma ascorbic acid concentrations over 2 mM.
  • 50 g 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 intravenously infused twice a day.
  • 10 g sodium ascorbate (or any other salt of ascorbic acid), 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid) is intravenously infused five times a day.
  • 33 g 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 intravenously infused three times a day. In one embodiment, one day to four weeks of intervals in between each therapeutic cycle are introduced. Other interval schemes and therapeutic durations may be considered appropriate to one skilled in the art.
  • standard dosages of pyrithione and other components of the formulation, and administration protocols aim to suppress tumor proliferation and induce regression of the malignant tumor.
  • standard dosages of pyrithione and other components of the formulation, and administration protocols used can vary depending on many variables such as clinical conditions, the type of malignant tumor, age, body weight of the recipient patient, the route of administration and other factors as determined by the experience of the clinical specialists conducting the treatment.
  • standard doses of pyrithione, other components of the formulation, and administration protocols can also vary depending on the type of the accompanied therapy, the degree of responsiveness and adverse side effects to the accompanied therapy. Regression of malignant tumor may be assessed with reference to the size of the tumor. Failure of malignant tumors to reoccur after the termination of treatment is another indication of regression.
  • Methods and compositions to prevent progression of malignant tumors from precancerous conditions [0072]
  • a unique nature of malignant tumors in the skin and cervix is that they frequently arise from non-cancerous inflammatory changes in these organs. It can be also said that the occurrence of malignant tumors in the skin and cervix is often predictable by the presence of abnormal conditions in these organs.
  • the acidic environment created by sustained and uncontrolled inflammation associated with precancerous lesions makes the use of pyrithione ideal to suppress the growth of newly arising malignant tumor cells.
  • methods of preventing cancer are disclosed where pyrithione-containing formulations are topically administered to the bodily surface or mucosa lesions that have a high probability of developing malignant tumors in the future. The disclosed method regresses newly occurring small numbers of malignant tumor cells before they form a visible tumor, taking advantage of the acidic pH of the disease lesion as well as acidic pH of pyrithione formula.
  • One area of application is preventing the progression of skin cancer from AKs and other precancerous conditions occurring in the skin.
  • Another area of application is preventing the progression of cervical cancer, vaginal cancer, anal cancer, penile cancer and oral cancer from chronic HPV infection.
  • Topical administration may be carried out by pharmaceutically acceptable salts in the form of creams, ointments, lotions, gels, solid sticks, sprays and occlusive devices.
  • These formulations may be oil-in-water emulsion, water-in-oil emulsion, viscous liquid, or water- soluble solution.
  • pH of the topical formulation is 6.9 or lower, preferably 6.4 or lower, and more preferably 5.6 or lower. In light of the extreme acidity in the precancerous lesion, it is even more preferable that pH of the formulation is 4.5 or lower. In another embodiment, the minimum pH of the formation is 2.0.
  • the formulation used for animal experiments contains 20 mM citrate buffer (see Examples 1 1). In one embodiment, the maximum concentration of citrate buffer in the formulation is 100 mM as this concentration will increase the pH stability; preferably 75 mM and even more preferably 50 mM as these concentrations increase the chemical stability of the composition.
  • the minimum concentration of citrate buffer is 5 mM, preferably 7.5 mM and more preferably 10 mM as these lower concentrations of buffer increases chemical stability of the composition.
  • phosphate buffer another physiological buffer that is safe to humans, is used.
  • Chemical penetration enhancers include unsaturated fatty acids such as oleic acid, docosahexaenoic acid,
  • solubilization enhancers include low doses of surfactants, cyclodextrins, and dimethyl sulfoxide (DMSO).
  • Suppository forms may be used.
  • Suppository formulations may be prepared by mixing the compounds and compositions of the invention with a suitable excipient such as cocoa butter, polyethylene glycols with different molecular weights, glycerin and glycerinated gelatin. These excipients are solid at room temperature, but will melt at body temperature and release the compounds and compositions in the rectum, vagina or cervix.
  • the topical formulation contains minimum 0.000317%, preferably 0.00634% and more preferably 1% by weight of zinc pyrithione, sodium pyrithione or any other form of pyrithione compounds. In another embodiment, the formulation contains maximum 5.5% by weight of zinc pyrithione, sodium pyrithione or any other form of pyrithione compounds.
  • EDTA is added to the formulation in an amount between
  • 0.0003% and 15% by weight preferably between 0.0003% and 10%, and more preferably between 0.0003% and 2%.
  • the topical formulation contains at least 0.00017% by weight, preferably at least 0.0017%, and even more preferably at least 0.017% of pyridoxine. In another embodiment, the formulation contains 10% by weight or less, preferably 5% or less, and even more preferably 2.5% or less amounts of pyridoxine. Oil-soluble pyridoxine derivatives such as pyridoxine dicaprylate and pyridoxine dipalmitate are easily absorbed through the bodily surface and show stable activity once absorbed. Pyridoxamine, pyridoxal 5'-phosphate and
  • the topical formulation contains at least 0.04% by weight, preferably at least 0.2%, more preferably at least 3% of the composition of 3-o-ethyl ascorbic acid (or any other derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid) or ascorbic acid.
  • the formulation contains at least 3% of 3-o-ethyl ascorbic (or any other water soluble derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid) or ascorbic acid plus 2% or higher concentrations of any one, or two or more in combination of ascorbyl tetraisopalmitate, ascorbyl palmitate, and any other lipophilic derivative of ascorbic acid.
  • the formulation contains less than 20%, preferably less than 10% by weight of pyridoxine.
  • pyridoxine, pyridoxal, pyridoxamine, or 5'-phosphate esters thereof in an amount from 0.1 mg/day to 1 g/day and more preferably from 10 mg/day to 300 mg/day is orally administered while the formulation containing pyrithione is topically administered.
  • the daily dose of pyridoxine, pyridoxal, pyridoxamine, or 5'- phosphate esters thereof is orally administered once a day.
  • pyridoxine, pyridoxal, pyridoxamine, or 5'-phosphate esters thereof is orally administered every eight hours three times a day.
  • 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 intravenously administered in combination with topical administration of the formulation containing pyrithione and one, or two or more in combination of 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.
  • a minimum daily dose of 5 g 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 intravenously administered.
  • a maximum daily dose of 100 g 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 intravenously infused.
  • the formulation contains at least 0.04% by weight, preferably at least 0.2%, and more preferably at least 3% of 3-o-ethyl ascorbic acid. Still more preferably, the formation contains at least 3% of 3-o-ethyl ascorbic acid and 2% or higher concentrations of any one, or two or more in combination of ascorbyl tetraisopalmitate, ascorbyl palmitate, and any other lipophil ic derivative of ascorbic acid. In another embodiment, the formulation contains 30% or less, preferably 20% or less, and more preferably 10% or a less amount of 3-o-ethyl ascrobic acid.
  • 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 intravenously administered in combination with topical administration of pyrithione-containing formulation.
  • the daily dose is administered once a day.
  • the daily dose is given in two injections a day.
  • the daily dose is given in three injections a day.
  • the agents are administered until the desired therapeutic outcome has been achieved.
  • one day to four weeks of intervals in between each therapeutic cycle are introduced. Other interval schemes and therapeutic durations may be considered appropriate to one skilled in the art.
  • the topical formulation containing pyrithione can be administered once a day, preferably twice a day, and more preferably three times a day through the bodily surface, genitalia or other organs.
  • the topical formulation containing pyrithione is administered daily.
  • the topical formulation containing pyrithione is administered three times a week.
  • administration consists of one to 96-week cycles, preferably two to 32- week cycles, and more preferably two to 16-week cycles.
  • one day to four weeks of intervals in between each therapeutic cycle are introduced. Other interval schemes and therapeutic durations may be considered appropriate to one skilled in the art.
  • dosage regiments are closely monitored by physicians. Durations of administration and periodic intervals will be also determined through preclinical and clinical investigations. One skilled in the art can also determine these variables by a number of variables including the age, body weight and clinical conditions. Clinical conditions are determined by signs, symptoms and routinely conducted medical test results. Additional methods for clinical assessment include pathological tests, colposcopy (vaginal and cervical examination by a special magnifying device), and serological examinations.
  • Example 1 Cell lines and reagents, colon cancer HT29 cell lines, breast cancer MDA-MB-
  • glioma U87 and skin cancer A2058 cells were obtained from American Type Culture Collection (ATCC). These cancer cells were trypsinized and resuspended in cryopreservation media containing 90% culture media consisting of Dalbecco's Modified Eagle's Media (DMEM) and 10% fetal bovine serum (FBS).
  • DMEM Dalbecco's Modified Eagle's Media
  • FBS fetal bovine serum
  • Human cervical cancer HeLa supplemented with 10% DMSO and stored in liquid nitrogen until use. Prior to experiments, cells were recovered from liquid nitrogen and grown in DMEM containing 10% FBS in the presence of 5% C0 2 in atmosphere. When cells reached approximately 80% confluency, cells were trypsinized and subdivided with a ratio of 1:4. For growing cells without exposure to test compounds, DMEM supplemented with 3.7 g/L sodium bicarbonate and pH was adjusted to 7.3, supplemented with 10% FBS, and cells were maintained at 37°C in 5% C0 2 incubator.
  • DMEM supplemented with 10 mM 1,4-Piperazinediethanesulfonic acid (PIPES, pK a 6.1-7.5) was adjusted to pH 6.4 and DMEM supplemented with 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) (HEPES, pKa 6.8-8.2) was adjusted to pH 7.4.
  • PIPES P6757, Lot#026K5416
  • HEPES H4034
  • PIPES (pK a 6.1-7.5) and HEPES, (pK a 6.8-8.2) were used as buffers to stabilize the pH to 6.4 and 7.4, respectively.
  • the two pH values 6.4 and 7.4 were adapted for the experiments to model a representative acidity of the tumor microenvironment and the normal environment, respectively.
  • MTT assay determines the cell viability by detecting cells' metabolic activity. Trypan-blue can enter only dead cells that lost their membrane integrity (but not live cells). Using this dye, only dead cells can be visualized. Cell-killing effect of zinc pyrithione (Pyz) and sodium pyrithione (Pyn) treatment at pH 7.4 and 6.4 on HeLa cell was determined by trypan blue staining assay. Ctr indicates the control treated with the same dose of vehicle that contains no drug.
  • Zinc pyrithione exhibits anti-proliferative effects on Spheroid 3D cultured brain tumor cells.
  • Spheroid culture allows us to assess the efficacy of anti-cancer agents in 3D culture that models the complexity of the real cancer (Friedrich et al., 2009). Because bioactivity of many compounds is influenced by the tumor microenvironment, an important question was whether pyrithione influences the growth of spheroids. To address this, collagen-embedded spheroids of brain tumor U87 cells were established under either acidic (pH 6.4) or neutral (pH 7.4) conditions, exposed to zinc pyrithione for three days at 37°C and tested the effect on spheroid growth.
  • % agarose solution heated to 60°C was mixed with an equal volume of culture media (DMEM pH 6.4 and 7.4 supplemented with 10% FBS), and 50 ⁇ of the mixture was transferred to each well of 96 plates. The plates were cooled at room temperature for 30 minutes until agarose solidifies, 1000 cells resuspended in 50 ⁇ of culture media were overlaid over the top of the agarose layer and centrifuged at l,500g for 5 minutes. Cells were cultured at 37°C for 3 days, spheroids formed on the agarose layer were grown in the presence or absence of different concentrations of zinc pyrithione for additional 3 days.
  • culture media DMEM pH 6.4 and 7.4 supplemented with 10% FBS
  • the size of the spheroids after a 3-day incubation with 1 ⁇ or 2 ⁇ of zinc pyrithione in the pH 6.4 culture medium was substantially smaller than that of untreated control spheroids.
  • the size of the spheroids exposed to 1 ⁇ or 2 ⁇ zinc pyrithione in the pH 7.4 culture medium for three days were almost identical to that of pyrithione-untreated control spheroids. The results are summarized in Fig. 5.
  • Acidity-potentiated anti-tumor activity of pyrithione causes accumulation of superoxide in mitochondria.
  • ROS reactive oxygen species
  • pyrithione photochemically decomposes hydroxyl radical and (pyridin-2-yl)sulfanyl radical (See reference (DeMatteo et al., 2005), we postulated pyrithione might influence mitochondrial superoxide production and clearance. To address this, cancer cells were treated with pyrithione for 2 hours and assessed by visualizing a fluorescent probe that specifically label mitochondria based on the level of superoxide.
  • HeLa human cervical cancer cells were seeded to 8-well glass-bottom chamber slide at 4000 cells/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS.
  • DMEM fetal bovine serum
  • pH 7.4 culture media containing 1.25 ⁇ , ⁇
  • MitoSOXTM (M31008, Invitrogen) and 2.5 ⁇ M DRAQ5TM (#62254, Thermo Scientific) for 5 min at 37°C. MitoSOXTM was used to quantitatively deterimne mitochondrial superoxide production whereas DRAQ5TM was used to fluorescently label the nucleus of the cell to visualize the cells under fluorescence microscope. After 5 min, cells were placed in NaCl-saline buffered (150 mM NaCl, 5 mM KC1, 2 mM CaCh, 1 mM MgCh, 20 mM HEPES, pH 7.4), and imaged by confocal microscopy.
  • NaCl-saline buffered 150 mM NaCl, 5 mM KC1, 2 mM CaCh, 1 mM MgCh, 20 mM HEPES, pH 7.4
  • Gefitinib and erlotinib are epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors that can specifically block proliferation of cancer cells, thereby providing a different mechanism of action from classical chemotherapy.
  • EGFR epidermal growth factor receptor
  • Gefitinib and erlotinib are used in clinic to treat malignant tumors such as lung cancer and malignant melanoma of skins. It is not known whether these clinically used molecular targeting therapeutic agents also exhibit acidity- enhanced anti-tumor activity.
  • Cervical cancer HeLa cells, colon cancer HT29 cells and brain tumor U87 cells were seeded onto 96-well dishes at 2,000 cells/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS in the presence of 5% C0 2 in atmosphere.
  • different concentrations of EDTA were added to the media together with zinc pyrithione.
  • Serum was excluded from the culture media in these experiments to avoid the undesirable inactivation of EDTA by metals and cations present in the serum.
  • pyrithione is an important component of zinc pyrithione for the anti-cancer activity, and removal of zinc by EDTA enhances such an activity. Enhancement by EDTA can be also said that the addition of EDTA lowers the dose of zinc pyrithione to achieve the same level of anti-cancer activity, providing the useful information for practical application.
  • Anti-cancer activity of zinc pyrithione (Pyz) and sodium pyrithione (Pyn) is enhanced by sodium ascorbate or 3-o-ethyl ascorbic acid under acidic conditions.
  • Sodium ascorbate (AscNa) in combination with acidity also showed significant enhancement of anti-cancer effects of zinc pyrithione (Pyz).
  • ZincNa zinc pyrithione
  • Spheroids in the suspension culture were grown in the absence or presence of zinc pyrithione, or 3-o-ethyl ascorbic acid, or in combination for 72 hours. After 3 days, Spheroids were transferred to another 96-wells. The plate was centrifugured at 1,500 g for 5 min and solution was carefully replaced with PBS without disturbing the spheroids at the bottom of the wells.
  • Direct application of a formulation containing zinc pyrithione, 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate regresses proliferation of human skin cancer cells implanted to mice.
  • 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate are derivatives of ascorbic acid that are readily absorbed through the skin and effectively converted into ascorbic acid.
  • Anti-cancer activity of topical application of formulations containing zinc pyrithione, 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate, or combination thereof was assessed in a mouse xenograft model of skin cancer.
  • mice C57BL/6 athymic nude mice, 6 weeks old male, were injected subcutaneously with 2.5x10 6 human skin cancer A2058 cells. Following 2-3 days of injection when the size of tumors reached approximately 3 mm 3 , mice were randomly assigned to 4 groups, 5 mice in each group, and the cream formulation was applied once a day directly onto the skin over the tumor area.
  • Citric acid 1M solution 0.7% Sodium citrate 1M solution: 1.3% Zinc pyrithione: 1%
  • the water phase components are combined and mixed while heating at 75°C until completely dissolved in a suitable container.
  • the oil phase components are combined and mixed while heating at 75°C until completely dissolved.
  • the oil phase mixture is then added to the water phase mixture and mixed well so as to form an emulsion.
  • the emulsion is then allowed to cool to about 50-55°C.
  • the emulsion is then milled until the product becomes uniform.
  • Citric acid 1M solution 0.7% Sodium citrate 1M solution: 1.3% Distilled water: to the total volume 100% b) Oil phase Behenyl alcohol: 3%
  • the water phase components are combined and mixed while heating at 75°C until completely dissolved in a suitable container.
  • the oil phase components are combined and mixed while heating at 75°C until completely dissolved.
  • the oil phase mixture is then added to the water phase mixture and mixed well so as to form an emulsion.
  • the emulsion is then allowed to cool to about 50-55°C.
  • the emulsion is then milled until the product becomes uniform.
  • the water phase components are combined and mixed while heating at 75°C until completely dissolved in a suitable container.
  • the oil phase components are combined and mixed while heating at 75°C until completely dissolved.
  • the oil phase mixture is then added to the water phase mixture and mixed well so as to form an emulsion.
  • the emulsion is then allowed to cool to about 50-55°C.
  • the emulsion is then milled until the product becomes uniform.
  • the tumor volume was determined according to the formula ab 2 /2 where a is the length and b is the width, the relative tumor volume of each treatment group was calculated and plotted (Fig. 11 A).
  • X-axis indicates days after transdermal administration starts;
  • Y-axis indicates relative tumor size.
  • the average tumor size of Group 3 mice (“Pyz+Asc" in Fig. 11A) was smaller than the tumor size of other groups. This result suggests that the topical application of the formulation containing both pyrithione and ascorbic acid derivatives is an effective method to reduce the tumor growth, but the formulations containing pyrithione alone or derivatives of ascorbic acid alone are not effective.
  • GR Granulocyte (numbers/L); GR% Granulocytes (%); HCT Hematocrit (%); HBG hemoglobin (g/L); LY Lymphocytes (numbers/L); LY% Lymphocytes (%); MCH Mean Corpuscular Hemoglobin (pg); MCHC Mean Corpuscular Hemoglobin Concentration (g/L); MO Monocyte (numbers/L); MO% Monocytes (%); MPV Mean Platelet Value (fL); PCT Procalcitonin (%); PDW Platelet Distribution Width (fL); PLT Platelet (g/L); RBC Red Blood Cell (numbers/L); RDW Red blood cell Distribution Width (%); WBC White Blood Cells (numbers/L)
  • Zinc pyrithione induces ERK-and PKC-dependent necrosis distinct from TPEN-induced apoptosis in prostate cancer cells.
  • a novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases. Scientific reports 4, 5240.
  • a novel nickel complex works as a proteasomal deubiquitinase inhibitor for cancer therapy. Oncogene.
  • Platinum-containing compound platinum pyrithione is stronger and safer than cisplatin in cancer therapy. Biochemical Pharmacology in press.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nutrition Science (AREA)
  • Physiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Dermatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

The invention describes compositions and methods of use for pyrithione compounds and pharmacologically acceptable salts thereof. The invention provides topical administration methods for treating skin cancers, treating cervical cancer, treating other cancers, preventing skin cancer development from precancerous skin conditions, preventing cervical cancer development from chronic human papillomavirus (HPV) infection, preventing 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 and chemical derivatives thereof, pyridoxine and any derivative thereof and pharmacologically acceptable buffers. In these compositions, pH of the compositions is adjusted to between 4.5 and 6.4.

Description

Compositions for treatment of malignant tumors and precancerous conditions, methods of use thereof and methods for manufacturing medicaments
Technical Field
[001] Rapidly growing cancer cells produce acids as a result of their metabolism and the released acid makes the environment surrounding the cancer cells acidic. This makes the cancer environment more acidic than that of normal tissues. The current invention discloses new compositions and therapeutic/preventative methods to suppress cancer cells growing/arising in the acidic environment.
Background Art
[002] Malignant tumors have been a leading cause of death worldwide. According to the statistics from the International Agency for Research on Cancer, over 14 million new cancer cases were reported worldwide in 2012, which is expected to grow to over 20 million by 2030; likewise, eight million cancer deaths worldwide were reported in 2012, which is expected to grow to over 13 million by 2030.
[003] Chemotherapy, surgery, and radiation are the most commonly used therapeutic strategies against malignant tumors. In spite of the significant progress made in the field of cancer research, current therapeutic approaches have a number of issues that need to be resolved. For example, many traditional chemotherapeutics aim to block rapid proliferation and cell division of cancer cells by introducing chemical damage to deoxyribonucleic acids (DNA). DNA is a genetic blueprint for both cancer and normal cells, and these traditional cancer chemotherapeutics often also damage normal tissues and cause serious side effects. Other chemotherapeutics target constituents of the cellular structure proteins that are involved with cell division and proliferation, but these agents also cause non-specific toxicity to non-cancer cells such as bone marrow and a subpopulation of avidly dividing gut cells. More recently, therapeutic agents that target specific molecules present only in cancer cells but not in normal cells have attracted increasing attention, a good example being inhibitors against growth factor receptors such as erlotinib and gefitinib. However, not all cancers have adequate levels of growth factor receptors. Moreover, even cases that have initial good response to targeted therapy tend to develop resistance because of the activation of other mechanisms that compensate for the function of blocked growth factor receptors. Although surgery may effectively remove isolated malignant tumors, recurrence and the spreading of cancer to other bodily parts caused by the surgical procedure are major concerns. In addition, surgery may cause bleeding, scarring, infection and damage organ function. Radiation is also associated with a risk of damaging normal tissues and a risk of developing secondary cancer, as well as long-term cosmetic risks. Chemotherapeutics, particularly when given through injection or orally, can trigger a number of serious side effects as described above. There is a strong need for a new class of anti-cancer therapy.
[004] Malignant tumors are not simple assemblies of cancer cells. Healthy tissues and cells surrounding tumor cells establish a unique environment (sometimes also referred to herein as the "tumor microenvironment"), which supports cancer cells, allowing them to grow, spread and metastasize. One of the most important elements of the tumor microenvironment is the acid. The tumor microenvironment is more acidic than tumor-free environment. Acidic metabolic products released to outside of the tumor cells provide the source of acid in the tumor microenvironment, which reduces the sensitivity to chemo- and radiation therapies, and stimulates tumor growth and spreading. The acidic tumor microenvironment is a fascinating therapeutic target for malignant tumors. It is expected that a method that sensitively blocks proliferation of tumor cell s growing in the acid ic environment will become a promising new anticancer therapy.
[005] Disease conditions that themselves are not yet malignant, but have a high risk to develop into cancer are called precancerous conditions. A highly acidic environment is established in precancerous lesions prior to the appearance of malignant tumors as a result of sustained and uncontrolled inflammation associated with precancerous conditions. The acidic environment accelerates genetic alterations and cancer occurrence (Coussens and Werb, 2002). Once arisen, malignant tumor cells grow more preferentially than non-malignant cells in the acidic environment, which further drives acidification. In addition to the more preferential growth of malignant tumors than non-malignant cells in the acidic tumor environment, excessive acid weakens the ability of immune cells, thereby increasing the chance of occurrence of malignant tumors (Lardner, 2001).
[006] Actinic Keratoses (AKs) and Human Papillomavirus (HPV) infection are good examples of precancerous conditions. AKs are rough and scaly skin conditions caused by the excessive exposure to UV radiation, and are one of the most common diagnoses accounting for 10% of outpatients of dermatology clinics in the United States. AKs are a frequent cause of skin cancers; approximately 65% of squamous cell carcinomas and 36% of basal cell carcinomas are caused by AKs according to one study (Criscione et al., 2009). Cryosurgery and topical administration of anti-cancer agents are the major therapies for AKs. However, AKs are not fatal before malignant tumors arise. The transformation process takes ten years or longer, often multiple AKs appear in one patient, and it is hard to predict which AKs will progress to invasive cancers. These facts make the treatment of AKs a challenge. Less expensive and less invasive pyrithione therapy provides a superb alternative method to prevent skin cancer.
[007] HPV infection occurs in the cervix, the outer surface area of the female genitalia, anus, penis and oral cavity. According to World Health Organization (WHO), HPV is the most common viral infection transmitted through sexual contact. HPV infection is known to cause cancers in these bodily parts, particularly HPV is involved with almost all cases of cervical cancer (http://www.who.int/mediacentre/factsheets/fs380/en/). Cervical cancer is the fourth most common cancer in female and second most common cancer in developing countries, where more than 85% of cervical cancer occurs. While HPV vaccination and early diagnosis by cancer screening greatly reduced the risk of cervical cancer in developed countries, socio-economieal problems in less developed countries make adaptation of these methods difficult. Introduction of a facile and less expensive method is awaited.
[008] Recent experimental studies suggest that zinc pyrithione potently inhibits the growth of human oral cancer cells (Srivastava et al., 2015) and leukemia (malignant tumors originated from white blood cells) implanted into the skin of immune-deficient mice (Tailler et al., 2012). Both studies showed that pyrithione at the dose that stops the growth of cancer has no adverse effects to the host. Likewise, low concentrations of zinc pyrithione were shown to effectively kill prostate cancer cells whereas a 10-fold higher dose of zinc pyrithione was required to kill noncancerous prostate cells (Carraway and Dobner, 2012). However, these previous arts did not provide methods or compositions for curing or preventing cancer. [009] Pyrithione is integrated into biomembranes and forms small pores that allow zinc and other heavy metals to pass through. The use of zinc and other metals for anti-cancer therapy using the sensitivity of cancer cells to heavy metals is known. For example, Patent Pub. No. U.S. 2011/0117210 describes the use of organic and inorganic salts of zinc as anti-cancer agents. U.S. Pat. No. 7,528,125 disclosed a series of new compounds that are chemically modified from pyrithione and proposed to use them as a method to deliver zinc, copper, manganese, iron and other metal complexes to tumors. Further, Patent Pub. No. U.S. 2006/0040980 discloses a method to use the formulation that combines zinc pyrithione and zinc salts as an anti-cancer and anti-angiogenic agent. More recent experimental studies further exploited this idea and proposed to deliver heavy metals such as copper (Liu et al., 2014), cadmium, platinum (Zhao et al., 2016b) and nickel (Zhao et al., 2016a) to leukemia, myeloma, lung cancer and liver cancer using a pyrithione salt as a vehicle. These previous arts have taught us the importance of heavy metals that pass through pyrithione pores in killing and blocking proliferation of malignant tumors.
Summary of Invention
Technical Problem
[0010] The acidic tumor microenvironment is known to cause acquisition of resistance to many anti-cancer chemotherapeutic agents. Currently available methods for preventing the development of cancer from precancerous conditions have limitations in terms of efficacy, safety and costs.
Solution to Problem
[0011] The present invention discloses compositions and therapeutic and preventative methods for malignant tumors, using acidity as a tool to potentiate the anti-cancer activity. In some embodiments, the disclosed methods and compositions are used to prevent the
development of cancer from precancerous conditions by killing sporadic cancer cells arising in the precancerous lesion.
[0012] In accordance with an aspect of the present invention, disclosed herein is the use of any one or more of zinc pyrithione, sodium pyrithione, any other salt forms of pyrithione or salt- free forms of pyrithione, in the manufacture of a medicament for the treatment of malignant tumors occurring in the skin, cervix, vagina, penis, anus and other bodily parts. The pH of the formulation is maintained to be acidic in the presence of a pharmacologically acceptable buffer. The approach efficiently inhibits malignant tumor proliferation under an acidic environment. In another aspect, this approach may be combined with one or more conventional cancer therapies, such as chemotherapy, radiation therapy and surgery.
[0013] In accordance with another aspect of the present invention, disclosed herein is the use of pyrithione, incorporated with a chelator Ethylenediaminetetraacetic acid (EDTA), any one or more of Calcium EDTA, Disodium EDTA, Diammonium EDTA, Dipotassium EDTA, Disodiui 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 malignant tumors. This builds and improves upon the known art related to zinc-dependent and other heavy metal-dependent anti-tumor activity of unchelated pyrithione, enhancing pyrithione's anti-cancer effect.
[0014] In accordance with another aspect of the present invention, disclosed herein is the use of pyrithione, incorporated with pyridoxine, pyridoxal, pyridoxamine, and 5 '-phosphate esters thereof, collectively known as vitamin B6, in the manufacture of a medicament for the treatment and prevention of cancer or malignant tumors. The approach improves and enhances the efficiency of inhibiting proliferation of malignant tumors under an acidic environment.
[0015] In accordance with another aspect of the present invention, disclosed herein is the use of pyrithione, incorporated 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, in the manufacture of a medicament for the treatment and prevention of cancer or malignant tumors. The approach improves and enhances the efficiency of inhibiting proliferation of malignant tumors under an acidic environment.
[0016] In accordance with another aspect of the present invention, disclosed herein is the use of pyrithione in the manufacture of a medicament for the prevention of cancer arising from precancerous lesions such as Human Papillomavirus (HPV) infection in genital lesions and Actinic Keratoses (AKs) in skin.
Advantageous Effects of Invention [0017] According to the present inventions, we are able to effectively, simply, safely and inexpensively suppress proliferation of cancer cells by taking advantage of acidity-enhanced anti-tumor compositions including pyrithione as a main component.
[0018] The required concentrations of pyrithione to suppress proliferation of cancer cells, when combined with the currently disclosed enhancers and acidity, are found to be 100-fold to 1,000-fold lower than those required by anti-cancer agents known by the previous art, highlighting the substantial efficacy of the disclosed methods and compositions. Furthermore, the currently disclosed methods, compositions and the use for manufacturing medicaments resolve an unfulfilled need in the art by preferentially killing cancer cells growing in acidic tumor microenvironments.
[0019] In some embodiments, acidic formulations are topically administered to cancer growing in the close proximity of the surface of body or bodily structure of organs. Topical administration is a superior method to systemic administration in delivering pyrithione- containing formulations in many ways. Firstly, topical administration is safe because of the limited exposure of medicaments to blood and healthy bodily parts, thereby reducing the risk of adverse effects. Secondly, topical administration does not require a high level of technical expertise, and self administration is easier comparing to systemic administration. Thirdly, the acidic compositions disclosed by the current invention optimized for topical administration are efficiently delivered to cancer. Topical administration is also advantageous in delivering acidic formulations without influencing the acidity of blood and other bodily parts. Importantly, systemic acidification of the blood and entire body will cause heart failure. Certain
embodiments concern the use of administration methods that combine topical administration of pyrithione (and enhancers) and systemic administration of select enhancers (such as oral administration of vitamin B6 and intravenous administration of vitamin C), which further increase the efficacy of treatment.
[0020] In light of the widespread industrial use of pyrithione and other ingredients disclosed herein, which was enabled by the previous art for manufacturing these ingredients in bulk, it is within the skill in the art to inexpensively produce medicaments that use the disclosed methods and compositions. This is particularly advantageous for manufacturing medicaments in less developed countries. Brief Description of Drawings
[0021] Fig. 1 shows the chemical structure of zinc pyrithione (sometimes abbreviated herein as "Pyz") and sodium pyrithione (sometimes abbreviated herein as "Pyn").
[0022] Fig. 2 is a graph showing the effect of the treatment with zinc pyrithione (Pyz), in two different pH media, on the viability of human brain tumor U87, breast cancer MDA-MB-231, cervical cancer HeLa, and colon cancer HT29 cells. Cells were grown in the absence or presence of zinc pyrithione at the indicated doses for 3 days at 37°C. Cell viability was determined by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay.
[0023] Fig. 3 is a graph showing the effect of the treatment with sodium pyrithione (Pyn), in two different pH media, on the viability of human brain tumor U87, breast cancer MDA-MB- 231, cervical cancer HeLa, and colon cancer HT29 cells. Cells were grown in the absence or presence of zinc pyrithione at the indicated doses for 3 days at 37°C. Cell viability was determined by MTT assay.
[0024] Fig. 4 is a graph showing the cell-killing effect of zinc pyrithione (Pyz) and sodium pyrithione (Pyn), in two different pH media, on HeLa cells. Cells were grown in the absence or presence of zinc pyrithione at the indicated doses for 3 days at 37°C. Non-viable cell populations after treatment with Pyz and Pyn were determined by trypan-blue staining.
[0025] Fig. 5 are photographs showing the effect of the treatment with zinc pyrithione, in two different pH media, acidity-enhanced anti-proliferative effects of zinc pyrithione on brain tumor U87 cells grown in a spheroid 3D-culture.
[0026] Fig. 6 are photographs showing the effect of the zinc pyrithione (Pyz) and sodium pyrithione (Pyn), in two different pH media, on mitochondrial accumulation of superoxide in HeLa human cervical cancer cells.
[0027] Fig. 7 is a graph showing the effect of the treatment with erlotinib and gefitinib, in two different pH media, on the viability of human glioma U87, breast cancer MDA-MB-231, cervical cancer HeLa, and colon cancer HT29 cells. Cell viability was determined by MTT assay. [0028] Fig. 8 is a graph showing the enhancement of anti-cancer effect of zinc pyrithione by ethylenediaminetetraacetic acid (EDTA) in human cervical cancer HeLa, brain tumor U87 and colon cancer HT29 cells. Cell viability was determined by MTT assay.
[0029] Fig. 9 is a graph showing the enhancement of anti-cancer effect of zinc pyrithione by pyridoxine in human cervical cancer HeLa cells. Cell viability was determined by MTT assay.
[0030] Fig. 10 presents a graph and photographs showing the enhancement of anti-cancer effect of zinc pyrithione and sodium pyrithione by sodium ascorbate and 3-o-ethyl ascorbic acid in human cervical cancer HeLa cells. Cell viability was determined in 2D cultures (Fig 10A) and in a spheroid-3D culture (Fig. 10B).
[0031] Fig. 11 A is a graph showing the effect of topical administration of different formulations containing or not containing zinc pyrithione, 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate on the growth of human skin cancer cells implanted to immune-deficient mice.
[0032] Fig. 1 IB presents microscopic pictures of skin, liver and kidney showing that topical administration of formulations containing zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitate has no adverse effects on these tissues.
[0033] Table summarizes the hematological test results of the mice after topical administration of formulations containing zinc pyrithione, 3-o-ethyl ascorbic acid, and ascorbyl tetraisopalmitate.
[0034] More detailed information is provided in the Examples section.
Description of Embodiments
Definitions
[0035] Unless otherwise stated, the term "pyrithione" and "pyrithione compounds" are used interchangeably, referring to pyrithione and any salt thereof that are compatible with other ingredients of the formulation and compatible with administration to humans. Pyrithione is obtained from commercial sources most typically in a form of either zinc-salt 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 under different names such as 2-Mercaptopyridine-N-Oxide, 1- Hydroxy-2-pyridinethione, l-Hydroxypyridine-2-thione, Omedine, (2-Pyridilthio)-N-oxide, and 2-Pyridinethiol-l -oxide. Chemical derivatives refer to compounds that have similar structure and function. The sodium salt of pyrithione (sodium pyrithione) is a widely used in commercial products. Sodium pyrithione is commonly synthesized by reacting 2-chloropyridine-N-oxide with NaSH and NaOH (see, for example, the disclosures of U.S. Pat. No. 3,159,640). The zinc salt of pyrithione (zinc pyrithione) may be synthesized by reacting pyrithione acid (i.e., 1- hydroxy-2-pyridinethione) or a soluble salt thereof with a zinc salt (e.g., ZnS04, ZnCh) to form a zinc pyrithione precipitate (see, for example, U.S. Pat. No. 2,809,971).
[0036] Tumor(s) is a pathological terminology referring to changes recognized as swelling of a part of the body. Tumors may be either malignant - that show cancerous uncontrolled growth, or benign - that are generally harmless and curable (although certain types of cancer may arise from benign tumors if left untreated).
[0037] Malignant tumors are tumors that divide without control and can spread to nearby tissues directly or to remote organs through bloodstream or lymphatic systems, the process called metastasis. Cancers are technically considered to be a group of malignant tumors that arise from epithelial cells of skin and organs. Malignant tumors arisen from non-epithelial cells such as blood, bone vessels, cartilages, muscles and supporting tissues that provide elasticity are technically excluded from cancer. For example, according to the pathological definition, dermal malignant melanomas are derived from non-epithelial cells of the skin; therefore, malignant melanomas are not cancer. In reality, however, malignant tumors, cancers and neoplasia are interchangeably used in a broad sense and, as such, melanomas are often referred to as a type of skin cancer. Herein, the terminology "cancer" is understood in a broad sense and "cancer" is interchangeably used with "malignant tumor".
[0038] Malignant tumors detected in the skin can be cutaneous squamous cell carcinoma, cutaneous melanoma, cutaneous adenocarcinoma, or any other forms of malignant tumors arising in or metastasized to skin regions. Carcinomas herein refer to the pathological terminology of malignant tumors of epi thelial origin whereas melanomas are malignant tumors of non-epithelial origin. In another classification, malignant tumors detected in the skin are divided into basal cell carcinomas, squamous cell carcinomas and others that include malignant melanomas.
[0039] Malignant tumors detected in the uterus and vagina herein refer to squamous cell carcinoma, squamous adenocarcinoma, other malignant tumors, and metastatic tumors. [0040] The term "treat" as used herein refers to provide a medical aid to patients for the purpose of ameliorating undesired medical conditions or preventing the worsening of such undesired conditions. "Anti-tumor effect", "anti-cancer effect" and "anti-neoplastic effect" are interchangeably used, referring to slowing or regressing tumor growth, decreasing tumor size and prevention of spreading to other organs. In one aspect, the compound is considered to be effective when the size of tumor is reduced. In another aspect, the compound is considered to be effective when symptoms associated with cancer are relieved. In another aspect, the compound is considered to be effective when medical examinations show signs of reduction in tumor burden. Medical examinations include biochemical tests, tumor markers, diagnostic imaging, and histochemical examinations. In still another aspect, the compound is considered to be effective when the patients treated with the method show prolonged survival.
[0041] Transdermal administration herein refers to a broader definition that is used interchangeably with topical administration. Topical administration includes all methods to deliver drugs through the surface of the body and the inner linings of the body passages, generally known as epithelial and mucosal tissues.
[0042] Excipients are organic or inorganic substances that do not interfere with active compounds and serve as carriers for active compounds. Pharmacologically acceptable carriers include, but not limited to, water, polyethylene glycols (PEGs), salt solutions, lactose, amylose, alcohol, oils, fatty acids, gelatin, silicic acid, surfactants, viscous paraffin, hydroxymethyl- cellulose, polyvinylpyrrolidone, lubricants, and the like.
Therapeutic methods and compositions for malignant tumors
[0043] Malignant tumors establish the acidic extracellular pH of 6.0-6.9 and often times even lower, whereas pH of non-cancerous tissues is 7.3-7.4 (Parks et al., 2013). Previous experimental studies attempted to suppress tumor growth by reducing the acidity of tumor environment by blocking either proton-pumps or acid-producing enzymes. However, this strategy brought about limited success because inhibiting one of these mechanisms eventually elevates other mechanisms that compensate the inhibited proton pumps or enzyme. The current invention instead takes advantage of the tumor acidity to efficiently and selectively kill cancer cells. [0044] The current inventors have searched agents whose anti-cancer effect is enhanced by acidic culture media of pH 6.9 and 6.4, the typical pH of tumor microenvironment. The dramatic enhancement of anti-tumor activity of pyrithione at acidic pH has surprisingly been found (see Figs. 2, 3, 4, 5 and 6). The anti-tumor activity of pyrithione under acidic conditions was observed in four genetically different cancer cells, ranging from cervical cancer to brain tumor to colon cancer to breast cancer whereas the acidity-enhanced anti-tumor activity was not observed by epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib (Fig. 7).
[0045] The current invention discloses a topical administration method and compositions of pyrithione for treating or preventing malignant tumors. The formulation consists of pyrithione and chemical compounds that potentiate the anti-tumor activity of pyrithione under acidic conditions together with a carrier that does not interfere with the action of the compound. The formulation is directly applied onto the bodily surface or the inner linings of the bodily structures. Topical administration is effective for targeting therapeutic agents to malignant tumors growing close to the bodily surface or inner linings of bodily structures. As a result of the acidic pH of the formula together with the intrinsic acidity of the tumor, pyrithione efficiently kills tumor cells. Some component(s) of the formulation may be administered by intravenous injection, subcutaneous injection, intramuscular injection, inhaling or oral intake.
[0046] In one embodiment, the maximum pH of the formulation is 6.9, as anti-tumor activity of pyrithione is significantly more potent at pH 6.9 compared to pH 7.4 (see for example Fig. 10). It is more preferable that the maximum pH of the formulation is 6.4 as it has been found that anti-tumor activity by pyrithione is more potent at pH 6.4 than 6.9 (see for example Fig. 10). Even more preferably, the maximum pH of the formulation is 5.6 as it has been found that the pH 5.5 topical formulation containing pyrithione suppresses proliferation of implanted human skin cancer cells in mice (Fig. 11). A previous art U.S. Pat. No. 6,455,076B 1 has recognized the importance of acids in enhancing the efficacy of active ingredients in cosmetic products. The art further provided a method to prevent skin irritation. According to the art, irritation by acids equivalent to pH 2.0 is preventable. In another embodiment, accordingly, the minimum pH of the formation is 2.0.
[0047] Through screening chemical compounds, it has been found that EDTA, particularly in combination with acidity, substantially potentiates the anti-tumor activity of zinc pyrithione (Fig. 8). EDTA is commonly used as a stabilizer of medical, cosmetic and food products that deprives and neutralizes zinc and other heavy metals with a given capacity. This indicates that pyrithione, even in the absence of zinc and any other heavy metals, exerts potent acidity-enhanced anti-tumor activity, an unexpected finding in light of the previous arts that taught us the importance of heavy metals that pass through pyrithione (see "Background and description of related arts" [0009]). This, together with the finding that sodium pyrithione exerts potent acidity-enhanced anti-tumor activity as zinc pyrithione (refer to Figs. 2 and 3), indicates the usefulness of both zinc pyrithione and sodium pyrithione for treating malignant tumors. The discovered zinc (or any other metal)-independent anti-tumor activity of pyrithione may reduce the potential risk of metal-overloads associated with long-term administration. The use of both zinc pyrithione (or any other metal pyrithione) and sodium pyrithione (or any other metal-free pyrithione) as anti-tumor therapeutic methods is disclosed herein.
[0048] Through screening chemical compounds, it has been found that from 10 to 100 μΜ of pyridoxine when treated in combination with acidity substantially potentiates the antitumor activity of zinc pyrithione (Fig. 9). It has been also found that 1 mM pyridoxine when treated in combination with acidity not only potentiates the anti-tumor activity of zinc pyrithione, but also shows acidity-dependent anti-tumor activity even in the absence of pyrithione.
Pyridoxine is one of the compounds showing vitamin B6 activity; other vitamin B6 compounds include pyridoxal, pyridoxamine, and 5'-phosphate esters thereof. In one embodiment, pyridoxine, pyridoxal, pyridoxamine, 5'-phosphate esters thereof, pyridoxine dicaprylate, pyridoxine dipalmitate or any other derivatives thereof is provided with zinc pyrithione (or any other metal pyrithione) or sodium pyrithione (or any other metal-free pyrithione).
[0049] It has been found that sodium ascorbic acid particularly in combination with acidity substantially potentiates the anti-tumor activity of zinc pyrithione (Fig. 10). Sodium ascorbate is a salt of ascorbic acid and exhibits the same biological activity.
[0050] 3-o-ethyl ascorbic acid has an ethyl group that forms an ether group with the 3- hydroxy group of the ascorbic acid, which makes this compound chemically stable and easily absorbed through the skin. It has been found that 3-o-ethyl ascorbic acid particularly in combination with acidity substantially potentiates the anti-tumor activity of zinc pyrithione (Fig. 10). It has been also found that 3-o-ethyl ascorbic acid particularly in combination with acidity substantially potentiates the anti-tumor activity of sodium pyrithione (Fig. 10). [0051] Ascorbyl tetraisopalmitate is an oil-soluble derivative of ascorbic acid that is readily absorbed through the skin and effectively converted into ascorbic acid. As such, ascorbyl tetraisopalmitate and 3-o-ethyl ascorbic acid are commonly used for skin care consumer products. It has been found that topical application of a formulation containing a moderate concentration of zinc pyrithione (1 μΜ), 3-o-ethyl ascorbic acid (3 mM) and ascorbyl tetraisopalmitate (2 mM) inhibits proliferation of human skin cancer cells implanted into mouse skin (Fig. 11).
[0052] Histological analyses of the skin area exposed to the topical formulation, liver and kidney showed no sign on tissue damage or tissue abnormalities (microscopic pictures presented in Fig. 1 IB). Furthermore, results summarized in Table show no sign of blood abnormality associated with the topical administration of the compositions containing pyrithione and ascorbic acid derivatives. These findings collectively suggest that no apparent adverse effect is associated with the disclosed method.
[0053] In one embodiment, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitate (or any other chemical derivatives of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid), ascorbic acid or a combination thereof is provided with zinc pyrithione (or any other metal pyrithione) or sodium pyrithione (or any other metal-free pyrithione) for topical administration.
[0054] Direct application of the composition to cancer or pre-cancer lesions close to the bodily surface or close to the bodily structure of organs may be carried out using
pharmaceutically acceptable salts in the form of creams, ointments, lotions, gels, solid sticks, sprays, drops (eye drops, nose drops and alike) and occlusive devices. These formulas may be oil-in-water emulsion, water-in-oil emulsion, viscous liquid, or water-soluble solution.
Occlusive devices such as a semi-permeable membrane covering a reservoir containing pyrithione and other active ingredients may be used to release pyrithione and other active ingredients such as chemotherapeutic agents, steroids and anti-inflammatory agents. Pyrithione topical formula can be formulated according to the conventional methods using suitable excipients that include, for example, emulsifiers, surfactants, thickening agents, moisturizers, skin conditioning agents, skin protectants, and sun screen agents. Preservatives and
bacteriostatic agents such as methyl hydroxybenzoate, propyl hydroxybenzoate and chlorocresol may be optionally incorporated in the occlusive devices. [0055] Direct application of the composition to cancer or pre-cancer lesions close to the bodily surface or close to the inner bodily structure may be carried out using pharmaceutically acceptable salts by means of injection by needles or similar devices, narrow jet propagated by high-pressure, externally applied electric field differences, ultrasound, laser beam, magnetic field and radiation.
[0056] For vaginal and rectal applications, suppository forms may be used. Suppository formulations may be prepared by mixing the compounds and compositions of the invention with a suitable excipient such as cocoa butter, polyethylene glycols with different molecular weights, glycerin and glycerinated gelatin. These excipients are solid at room temperature, but will melt at body temperature and release the compounds and compositions in the rectum, vagina or cervix.
[0057] In one embodiment, in order to increase the efficacy of absorption through the bodily surface or inner linings of bodily structures, chemical penetration enhancers and solubilization enhancers are incorporated into the formulation according to the known art (Williams and Barry, 2012). Chemical penetration enhancers include unsaturated fatty acids such as oleic acid, docosahexaenoic acid, eicosapentanoic acid, terpenes, terpenoids, essential oils, azone, and pyrrolidones; some examples of solubilization enhancers include low doses of surfactants, cyclodextrins, and dimethyl sulfoxide (DMSO).
[0058] It was found that 0.00000317% by weight ( 100 nM) of zinc pyrithione in combination with acidity suppresses growth of cancer cells in two-dimensional (2D) cultures (see Figs. 2, 3, 4, and 6). According to the previous art, the efficiency of transdermal absorption of pyrithione compounds is around 1% (see references (Wedig et al., 1974)..). As such, in one embodiment, the formulation contains pyrithione at least 0.000317% by weight, and preferably at least 0.00634% by weight as the minimum concentration required to suppress growth of cancer cells in three-dimensional (3D) cultures is 0.0000634% (2 μΜ) (see Fig. 5). It has been found that topical application of a cream composition containing 1% zinc pyrithione by weight has a significant anti-tumor activity (Fig. 11). Accordingly, it is more preferable that the formulation contains at least 1% pyrithione. In another embodiment, the formulation contains pyrithione up to 5.5 % by weight (173 mM) as it has been found that emulsions containing 5.5 % zinc pyrithione is stable when applied onto the skin. [0059] In some embodiments, EDTA is added to the formulation in an amount of at least about 0.0003% by weight (10 μΜ) that is the minimum concentration required to enhance anticancer effects by zinc pyrithione (see Fig. 8). The formulation contains EDTA at the maximum concentrations from 10 to 15% by weight as these are the amounts safely used in dentistry for a short-term dental treatment (see reference (Lanigan and Yamarik, 2002)). More preferably, the formulation contains EDTA at the maximum concentration of approximately 2%, as this is a typical maximum concentration of EDTA suitable for a long-term use in cosmetic formulations.
[0060] In one embodiment, the topical formulation contains at least 0.00017% by weight
(10 μΜ), of pyridoxine, as this is the minimum concentration that shows the acidity-dependent enhancement of anti-tumor effects by zinc pyrithione (see Fig. 9). Preferably, the formulation contains at least 0.0017% by weight (100 μΜ) of pyridoxine, as this is the concentration that shows even a higher potentcy. Even more preferably, the formation contains at least 0.017% by weight (1 mM) of pyridoxine, as this concentration shows acidity-dependent anti-tumor effects even in the absence of zinc pyrithione. Because of other factors such as skin permeability, diffusion, and chemical stability, it is likely that higher concentrations of pyridoxine are required to achieve the anticipated effect. For example, the previous art has unveiled cosmetic compositions containing from 0.001% to 15% of pyridoxine (U.S. Pat No. 20,060,018,860A1). It was noted however that formulations containing 10% by weight or a less amount of pyridoxine are more stable in the presence of other ingredients. Therefore, in one embodiment, the formulation contains 10% by weight or less amounts of pyridoxine. Oil-soluble pyridoxine derivatives such as pyridoxine dicaprylate and pyridoxine dipalmitate are easily absorbed through the bodily surface and show stable activity once absorbed. In some embodiments, pyridoxine dicaprylate, pyridoxine dipalmitate, or any other derivatives of pyridoxine are added to the formulation in an amount of at least 0.00017% by weight, preferably 0.0017% and more preferably 0.017%, and in an amount of less than 10% by weight.
[0061] In one embodiment, the topical formulation contains at least 0.04% by weight (2 mM) of the composition of 3-o-ethyl ascorbic acid, as 2 mM 3-o-ethyl ascorbic acid in combination with pyrithione and acidity regresses growth of cancer cells in 2D cultures (see Fig. 10). Preferably, the formulation contains at least 0.2% (10 mM) by weight of 3-o-ethyl ascorbic acid, as 10 mM 3-o-ethyl ascorbic acid in combination with pyrithione and acidity regress growth of cancer cell s in 3D cultures. More preferably, the formulation contains at least 3% by weight of 3-o-ethyl ascorbic acid, as the cream formulation containing 3% 3-o-ethyl ascorbic acid in combination with zinc pyrithione and acidity significantly suppresses proliferation of human skin cancer cells implanted in mice (see Fig. 11). Aside from 3-o-ethyl ascorbic acid and zinc pyrithione, the formulation also contains 2% by weight of ascorbyl tetraisopalmitate (see Example 11). Accordingly, even more preferably, the formulation contains at least 3% of 3-o- ethyl ascorbic acid plus 2% or higher concentrations of any one, or two or more in combination, of ascorbyl tetraisopalmitate, ascorbyl palmitate and any other derivative of ascorbic acid. It was noted that formulations containing more than 20% by weight of 3-o-ethyl ascorbic acid is irritating at times to sensitive skins. Therefore, in one embodiment, the formulation contains less than 20%, preferably less than 10% by weight of pyridoxine.
[0062] It was discovered that 0.04% (approximately 2 mM) sodium ascorbate in combination with pyrithione and acidity regresses proliferation of cancer cells in 2D cultures (Fig. 10). In some embodiments, 0.04% or higher concentrations of 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. It was noted that formulations containing 30% sodium ascorbate or 15% ascorbyl tetraisopalmitate are stable and no sign of skin irritations is detected. As such in some embodiments, the formulation contains 30% or less, preferably 20% or less, and more preferably 10% or less amounts of sodium ascorbate (or any other ascorbate salt). In another embodiment, the formulation contains 15% or less, preferably 10% or less, and more preferably 5% or less amounts of ascorbyl tetraisopalmitate (or any other derivative of ascorbic acid).
[0063] It was found that once a day of topical administration of the pyrithione-containing formulation to the mouse skin regress growth of skin cancer (Fig. 11). Therefore, in one embodiment, the formulation containing pyrithione and enhancers is topically administered one time every day. According to the known art, only 34% of zinc pyrithione remains in the treated skin area after 20 hours of topical application (see reference (Parekh et al., 1970)). The previous art further teaches us that topical application of pyrithione with administration intervals shorter than 20 hours increases the effective concentration of pyrithione in the treated skin area. This means more than one time a day of administration increases the effective concentration of pyrithione in the skin. In another embodiment, the formulation containing pyrithione and enhancers is topically administered two times every day. In yet another embodiment, the formulation containing pyrithione and enhancers is administered topically three times every day. In one embodiment, the agents are administered until the desired therapeutic outcome has been achieved; where in a typical example, the pyrithione formulation may be delivered for a period that can range from two months to 36 months. In another embodiment, one day to four weeks of intervals in between each therapeutic cycle are introduced. In yet another embodiment, chemotherapy, radiation and/or surgery are carried out during intervals. Other interval schemes and therapeutic durations may be considered appropriate to one skilled in the art.
[0064] One example of application of the topical administration of pyrithione i s for treating malignant tumors in the skin.
[0065] Another example of application of the topical administration of pyrithione is for treating cervical cancer, vaginal cancer and other malignant tumors arising in the cervix of the uterus and vagina.
[0066] Topical pyrithione administration may be employed for medical treatment of other malignant tumors that are readily accessible from the bodily surface. Target diseases may include, but not limited to, malignant tumors arising in or metastasized to head and neck, oral cavity, anus, rectum, prostate, breast, bone, muscle and cartilage.
[0067] Topical administration infi ltrates the composition from the outer surface of a tumor. Systemic administration delivers the composition to a tumor via tumor-nurturing blood vessels. Combining these two administration methods will maximize the effective concentration of the composition in a tumor. Oral and intravenous administrations are widely used systemic administration routes to increase vitamin levels in the blood plasma.
[0068] Previous arts have revealed that orally administered 600 mg pyridoxine rapidly enters the systemic circulation within 0.3 hours and the pyridoxine concentration in blood plasma reaches 25 μΜ after 1.3 hours of administration (see reference (Zempleni, 1995)). This concentration (25 μΜ) is higher than the minimum concentration (10 μΜ) required for anticancer effects when combined with pyrithione and acidity (Fig. 9). In contrast, intravenous administration of 100 mg pyridoxine, another commonly used administration method, achieves only a maximum plasma level of 0.37 μΜ after 6 hours of administration. Therefore, in one embodiment, pyridoxine, pyridoxal, pyridoxamine, or 5'-phosphate esters thereof is administered orally in a minimum amount of 600 mg/day in combination with topical administration of the formulation containing pyrithione and pyridoxine (or derivatives thereof). Previous arts have taught us that a risk of neuropathy is associated with high levels of pyridoxine intake exceeding 1,000 mg daily (see reference (Bender, 1999)). Therefore, in another embodiment, pyridoxine, pyridoxal, pyridoxamine, or 5'-phosphate esters thereof is orally administered in a maximum amount of 1,000 mg/day in combination with topical administration of the formulation containing pyrithione and pyridoxine (or derivatives thereof). As orally administered pyridoxine decays rapidly once entering the systemic circulation with an estimated half life of less than one hour (see reference (Zempleni, 1995)), it is desirable to divide the total daily dose into multiple intakes a day. In one embodiment, pyridoxine, pyridoxal, pyridoxamine, or 5 '-phosphate esters thereof is orally administered twice a day, preferably three times a day, and even more preferably four times a day.
[0069] The previous art has revealed that the maximum achievable plasma concentration of ascorbic acid by intravenous administration is 13,400 μΜ whereas the maximum achievable plasma concentration of ascorbic acid by oral administration is only 220 μΜ (see reference (Padayatty et al., 2004)) This indicates that intravenous administration, but not oral
administration, can establish the minimum plasma concentrations (from 1 to 10 mM) needed for ascorbic acid or its derivatives to suppress proliferation of cancer 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 intravenously administered in combination with topical administration of the formulation containing pyrithione and any one, or two or more in combination, of 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. The previous art teaches us that intravenous administration of 5 g, but not 1 g or 3 g, of ascorbic acid can elevate the plasma concentration of ascorbic acid to 2 mM or higher. Therefore, in one embodiment, a minimum dose of 5 g 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 intravenously administered. Intravenous administration of ascorbic acid up to 100 g is a safe and effective method to increase the plasma concentration of ascorbic acid according to the art. Therefore, in another embodiment, a maximum dose of 100 g 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 intravenously infused. [0070] Previous arts have also taught us that dividing the daily dose of ascorbic acid into multiple administrations helps sustain the effective plasma concentration in the body. For example, intravenous infusion of ascorbic acid at a dose of 100 g, 50 g and 10 g establishes and sustains plasma ascorbic acid concentrations over 2 mM for approximately 5.5 hours, 3.5 hours and 1.3 hours, respectively. This means that intravenous administration of 50 g ascorbic acid twice a day and 10 g five times a day are more efficient methods than once a day 100 g administration in order to sustain plasma ascorbic acid concentrations over 2 mM. Accordingly, in one embodiment, 50 g 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 intravenously infused twice a day. In another embodiment, 10 g sodium ascorbate (or any other salt of ascorbic acid), 3-o-ethyl ascorbic acid (or any other chemical derivative of ascorbic acid) is intravenously infused five times a day. In yet another embodiment, 33 g 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 intravenously infused three times a day. In one embodiment, one day to four weeks of intervals in between each therapeutic cycle are introduced. Other interval schemes and therapeutic durations may be considered appropriate to one skilled in the art.
[0071] Principally, standard dosages of pyrithione and other components of the formulation, and administration protocols aim to suppress tumor proliferation and induce regression of the malignant tumor. In therapeutic applications, standard dosages of pyrithione and other components of the formulation, and administration protocols used can vary depending on many variables such as clinical conditions, the type of malignant tumor, age, body weight of the recipient patient, the route of administration and other factors as determined by the experience of the clinical specialists conducting the treatment. When combined with other therapies, standard doses of pyrithione, other components of the formulation, and administration protocols can also vary depending on the type of the accompanied therapy, the degree of responsiveness and adverse side effects to the accompanied therapy. Regression of malignant tumor may be assessed with reference to the size of the tumor. Failure of malignant tumors to reoccur after the termination of treatment is another indication of regression.
Methods and compositions to prevent progression of malignant tumors from precancerous conditions [0072] A unique nature of malignant tumors in the skin and cervix is that they frequently arise from non-cancerous inflammatory changes in these organs. It can be also said that the occurrence of malignant tumors in the skin and cervix is often predictable by the presence of abnormal conditions in these organs. The acidic environment created by sustained and uncontrolled inflammation associated with precancerous lesions makes the use of pyrithione ideal to suppress the growth of newly arising malignant tumor cells. In a further aspect, methods of preventing cancer are disclosed where pyrithione-containing formulations are topically administered to the bodily surface or mucosa lesions that have a high probability of developing malignant tumors in the future. The disclosed method regresses newly occurring small numbers of malignant tumor cells before they form a visible tumor, taking advantage of the acidic pH of the disease lesion as well as acidic pH of pyrithione formula.
[0073] One area of application is preventing the progression of skin cancer from AKs and other precancerous conditions occurring in the skin.
[0074] Another area of application is preventing the progression of cervical cancer, vaginal cancer, anal cancer, penile cancer and oral cancer from chronic HPV infection.
[0075] Topical administration may be carried out by pharmaceutically acceptable salts in the form of creams, ointments, lotions, gels, solid sticks, sprays and occlusive devices. These formulations may be oil-in-water emulsion, water-in-oil emulsion, viscous liquid, or water- soluble solution.
[0076] In one embodiment, pH of the topical formulation is 6.9 or lower, preferably 6.4 or lower, and more preferably 5.6 or lower. In light of the extreme acidity in the precancerous lesion, it is even more preferable that pH of the formulation is 4.5 or lower. In another embodiment, the minimum pH of the formation is 2.0. The formulation used for animal experiments contains 20 mM citrate buffer (see Examples 1 1). In one embodiment, the maximum concentration of citrate buffer in the formulation is 100 mM as this concentration will increase the pH stability; preferably 75 mM and even more preferably 50 mM as these concentrations increase the chemical stability of the composition. In another embodiment, the minimum concentration of citrate buffer is 5 mM, preferably 7.5 mM and more preferably 10 mM as these lower concentrations of buffer increases chemical stability of the composition. In some embodiments, phosphate buffer, another physiological buffer that is safe to humans, is used. [0077] In one embodiment, in order to increase the efficacy of absorption through skin, chemical penetration enhancers and solubilization enhancers are incorporated into the
formulation according to the known art (Williams and Barry, 2012). Chemical penetration enhancers include unsaturated fatty acids such as oleic acid, docosahexaenoic acid,
eicosapentanoic acid, terpenes, terpenoids, essential oils, azone, and pyrrolidones; some examples of solubilization enhancers include low doses of surfactants, cyclodextrins, and dimethyl sulfoxide (DMSO).
[0078] For vaginal and rectal applications, suppository forms may be used. Suppository formulations may be prepared by mixing the compounds and compositions of the invention with a suitable excipient such as cocoa butter, polyethylene glycols with different molecular weights, glycerin and glycerinated gelatin. These excipients are solid at room temperature, but will melt at body temperature and release the compounds and compositions in the rectum, vagina or cervix.
[0079] In one embodiment, the topical formulation contains minimum 0.000317%, preferably 0.00634% and more preferably 1% by weight of zinc pyrithione, sodium pyrithione or any other form of pyrithione compounds. In another embodiment, the formulation contains maximum 5.5% by weight of zinc pyrithione, sodium pyrithione or any other form of pyrithione compounds.
[0080] In some embodiments, EDTA is added to the formulation in an amount between
0.0003% and 15% by weight, preferably between 0.0003% and 10%, and more preferably between 0.0003% and 2%.
[0081] In one embodiment, the topical formulation contains at least 0.00017% by weight, preferably at least 0.0017%, and even more preferably at least 0.017% of pyridoxine. In another embodiment, the formulation contains 10% by weight or less, preferably 5% or less, and even more preferably 2.5% or less amounts of pyridoxine. Oil-soluble pyridoxine derivatives such as pyridoxine dicaprylate and pyridoxine dipalmitate are easily absorbed through the bodily surface and show stable activity once absorbed. Pyridoxamine, pyridoxal 5'-phosphate and
pyridoxamine 5 '-phosphate show similar biological activity as pyridoxine. Lipophilic derivatives of pyridoxine such as pyridoxine dicaprylate and pyridoxine dipalmitate are easily absorbed through the skin and show a longer stability after absorption. In some embodiments, these compounds are included in the formulation. [0082] In one embodiment, the topical formulation contains at least 0.04% by weight, preferably at least 0.2%, more preferably at least 3% of the composition of 3-o-ethyl ascorbic acid (or any other derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid) or ascorbic acid. Even more preferably, the formulation contains at least 3% of 3-o-ethyl ascorbic (or any other water soluble derivative of ascorbic acid), sodium ascorbate (or any other salt of ascorbic acid) or ascorbic acid plus 2% or higher concentrations of any one, or two or more in combination of ascorbyl tetraisopalmitate, ascorbyl palmitate, and any other lipophilic derivative of ascorbic acid. In one embodiment, the formulation contains less than 20%, preferably less than 10% by weight of pyridoxine.
[0083] In some embodiments, pyridoxine, pyridoxal, pyridoxamine, or 5'-phosphate esters thereof in an amount from 0.1 mg/day to 1 g/day and more preferably from 10 mg/day to 300 mg/day is orally administered while the formulation containing pyrithione is topically administered. In one embodiment, the daily dose of pyridoxine, pyridoxal, pyridoxamine, or 5'- phosphate esters thereof is orally administered once a day. In another embodiment, pyridoxine, pyridoxal, pyridoxamine, or 5'-phosphate esters thereof is orally administered every eight hours three times a day.
[0084] 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 intravenously administered in combination with topical administration of the formulation containing pyrithione and one, or two or more in combination of 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. In one embodiment, a minimum daily dose of 5 g 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 intravenously administered. In another embodiment, a maximum daily dose of 100 g 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 intravenously infused. In one embodiment, the formulation contains at least 0.04% by weight, preferably at least 0.2%, and more preferably at least 3% of 3-o-ethyl ascorbic acid. Still more preferably, the formation contains at least 3% of 3-o-ethyl ascorbic acid and 2% or higher concentrations of any one, or two or more in combination of ascorbyl tetraisopalmitate, ascorbyl palmitate, and any other lipophil ic derivative of ascorbic acid. In another embodiment, the formulation contains 30% or less, preferably 20% or less, and more preferably 10% or a less amount of 3-o-ethyl ascrobic acid.
[0085] 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 intravenously administered in combination with topical administration of pyrithione-containing formulation. In one embodiment, the daily dose is administered once a day. In another embodiment, the daily dose is given in two injections a day. In yet another embodiment, the daily dose is given in three injections a day. In one embodiment, the agents are administered until the desired therapeutic outcome has been achieved. In another embodiment, one day to four weeks of intervals in between each therapeutic cycle are introduced. Other interval schemes and therapeutic durations may be considered appropriate to one skilled in the art.
[0086] The topical formulation containing pyrithione can be administered once a day, preferably twice a day, and more preferably three times a day through the bodily surface, genitalia or other organs. In one embodiment, the topical formulation containing pyrithione is administered daily. In another embodiment, the topical formulation containing pyrithione is administered three times a week. In one embodiment, administration consists of one to 96-week cycles, preferably two to 32- week cycles, and more preferably two to 16-week cycles. In some embodiments, one day to four weeks of intervals in between each therapeutic cycle are introduced. Other interval schemes and therapeutic durations may be considered appropriate to one skilled in the art.
[0087] Throughout the course of medical treatment, dosage regiments are closely monitored by physicians. Durations of administration and periodic intervals will be also determined through preclinical and clinical investigations. One skilled in the art can also determine these variables by a number of variables including the age, body weight and clinical conditions. Clinical conditions are determined by signs, symptoms and routinely conducted medical test results. Additional methods for clinical assessment include pathological tests, colposcopy (vaginal and cervical examination by a special magnifying device), and serological examinations.
Examples
Example 1 [0088] Cell lines and reagents, colon cancer HT29 cell lines, breast cancer MDA-MB-
231, glioma U87 and skin cancer A2058 cells were obtained from American Type Culture Collection (ATCC). These cancer cells were trypsinized and resuspended in cryopreservation media containing 90% culture media consisting of Dalbecco's Modified Eagle's Media (DMEM) and 10% fetal bovine serum (FBS).
[0089] Human cervical cancer HeLa supplemented with 10% DMSO and stored in liquid nitrogen until use. Prior to experiments, cells were recovered from liquid nitrogen and grown in DMEM containing 10% FBS in the presence of 5% C02 in atmosphere. When cells reached approximately 80% confluency, cells were trypsinized and subdivided with a ratio of 1:4. For growing cells without exposure to test compounds, DMEM supplemented with 3.7 g/L sodium bicarbonate and pH was adjusted to 7.3, supplemented with 10% FBS, and cells were maintained at 37°C in 5% C02 incubator. For investigating pH-dependent and dose-dependent effects of pyrithione and other reagents on cancer cell proliferation, DMEM supplemented with 10 mM 1,4-Piperazinediethanesulfonic acid (PIPES, pKa 6.1-7.5) was adjusted to pH 6.4 and DMEM supplemented with 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) (HEPES, pKa 6.8-8.2) was adjusted to pH 7.4. PIPES (P6757, Lot#026K5416) and HEPES (H4034,
Lot#087K54432), sodium pyrithione (2-Mercaptopyridine N-oxide sodium, H3261,
Lot#0655M4172V) and Methylthiazolyldiphenyl-tetrazolium bromide (MTT, M5655, Sigma) were purchased from Sigma. Zinc pyrithione (PHR1401, Lot#LRAA8431) was purchased from Fluka. Erlotinib (#10483, Lot#0459700-31) and gefitinib (sc-202166, Lot#A0616) were purchased from Cayman Chemical Company and Santa Cruz Biotechnology, respectively. General chemical reagents used were of the highest grade possible purchased from Sigma unless otherwise specified.
Example 2
[0090] Effects of zinc pyrithione (Pyz) treatment at pH 7.4 and 6.4 on cancer cell viabilities.
[0091] Malignant tumors establish characteristically acidic extracellular pH of 6.0-6.9, whereas normal tissues have a neutral pH of 7.3-7.4 (Parks et ah, 2013). Here it is shown that the exposure of zinc pyrithione to four different types of human cancer cells originated from cervical cancer (HeLa cells), brain tumor (U87 cells), colon cancer (HT29 cells) and breast cancer (MDA-MB-231 cells) causes significant reduction in cell viability. Treatment with zinc pyrithione substantially diminished cell viabi lity when treated in acidic media of pH 6.4 comparing to when treated in pH 7.4. The chemical structure of zinc pyrithione is provided in Fig. 1.
[0092] Cancer cells were seeded onto 96-well dishes at 2,000/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS in the presence of 5% C02 in atmosphere. For investigating pH-dependent anti-cancer effects of pyrithione and other reagents, pH=6.4 DMEM medium was prepared by supplementing 10 mM of 1,4-Piperazinediethanesulfonic acid (PIPES) and adjusting pH to 6.4. The pH=7.4 DMEM medium was prepared by supplementing 10 mM of 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) and adjusting pH to 7.4. PIPES, (pKa 6.1-7.5) and HEPES, (pKa 6.8-8.2) were used as buffers to stabilize the pH to 6.4 and 7.4, respectively. The two pH values 6.4 and 7.4 were adapted for the experiments to model a representative acidity of the tumor microenvironment and the normal environment, respectively. Cells were treated with increasing concentrations of zinc pyrithione in either pH=6.4 DMEM or pH=7.4 DMEM medium and cultured for 72 hours at 37°C. Experiments were carried out in the absence of bicarbonate in the media and atmospheric CO2 to minimize the fluctuation of pH caused by bicarbonate-mediated proton movements across biomembranes. After exposure to zinc pyrithione for 72 hours, culture media were replaced with 50 μΙ,ΛνβΙΙ DMEM containing 10% FBS that was freshly supplemented with 1 mg/mL MTT. Following the 2-hour incubation, cells were lysed by adding 50 μΙ,ΛνβΙΙ extraction buffer composed of 20% SDS, 2% acetic acid and 2.5% HC1 dissolved in 50% DMF in water to an equal volume as culture media. After agitation, 96-well plates were incubated at 37°C for 4 hours and absorbance at 570 nm was measured. Following background subtraction, relative viabilities were determined and expressed in relation to the reading of compound-untreated samples. Statistical significance between each condition and respective control was individually determined by Student's t-test. Conditions were considered significantly different from control if p-value was less than 0.05.
[0093] When cancer cells were treated with 50 nM zinc pyrithione in culture media whose pH was adjusted to 7.4 for 72 hours, relative viabilities were approximately 80-90% comparing to untreated cells. When 50 nM zinc pyrithione was treated to these cancer cells in moderately acidic media whose pH was adjusted to 6.4, viability rates were 5-20% of untreated cells. For example, relative viability rates of HeLa human cervical cancer cells treated with 50 nM zinc pyrithione at pH 7.4 and pH 6.4 were 87±9% and 5.3±1.6% (p < 0.001), respectively. These new findings teach us that the acidic environment helps to increase anti-cancer potency of zinc pyrithione. The results are summarized in Fig 2. Conditions are considered significantly different if p- value was less than 0.05.
Example 3
[0094] Effects of sodium pyrithione (Pyn) treatment at pH 7.4 and 6.4 on cancer cell viabilities.
[0095] It has been suggested that zinc pyrithione or other heavy metal chemically conjugated with pyrithione causes anti-cancer activity. On the other hand, metal-free pyrithione was also suggested to confer anti-inflammatory and anti-infectious activities. Therefore, an important next question in order to develop a new method for anti-cancer medical treatment is whether pyrithione causes the acidity-enhanced anti-proliferative effect or zinc causes the acidity-enhanced anti-proliferative effect. To answer this question, the effect of sodium pyrithione on cancer cell proliferation was examined.
[0096] Here it is shown that the exposure of sodium pyrithione to four different types of human cancer cells originated from human cervical cancer (HeLa cells), brain tumor (U87 cells), colon cancer (HT29 cells) and breast cancer (MDA-MB-231 cells) causes significant reduction in cell viability. Treatment with sodium pyrithione substantially diminished cell viability when treated in acidic media of pH 6.4, comparing to when treated in pH 7.4.
[0097] Cancer cells were seeded onto 96-weIl dishes at 2,000/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS in the presence of 5% C02 in atmosphere. For investigating pH-dependent anti-cancer effects of sodium pyrithione, pH=6.4 DMEM medium was prepared by supplementing 10 raM of PIPES and adjusting pH to 6.4. The pH=7.4 DMEM medium was prepared by supplementing 10 mM HEPES and adjusting pH to 7.4. Cells were treated with increasing concentrations of sodium pyrithione in either pH=6.4 DMEM or pH=7.4 DMEM medium and cultured for 72 hours at 37°C. Experiments were carried out in the absence of bicarbonate in the media and atmospheric CO2 to minimize the fluctuation of pH caused by bicarbonate-mediated proton movements across biomembranes. After exposure to sodium pyrithione for 72 hours, MTT assay was carried out as in Example 2. Statistical significance between each condition and respective control was individually determined by Student's t-test. Conditions were considered significantly different from control if /vvalues were less than 0.05.
[0098] When cancer cells were treated with 100 nM sodium pyrithione in culture media whose pH was adjusted to 7.4 for 72 hours, relative viabilities were approximately 75% comparing to untreated cells. When 100 nM sodium pyrithione was treated to these cancer cells in moderately acidic media whose pH was adjusted to 6.4, viability rates were 5-20% of untreated cells. For example, relative viability rates of cervical cancer HeLa cells treated with 100 nM zinc pyrithione at pH 7.4 and pH 6.4 were 90±9% and 4.9±1.7% (p < 0.001), respectively. Conditions were considered significantly different if /^-values were less than 0.05.
[0099] These new findings teach us that the acidic environment helps to increase anticancer potency of sodium pyrithione. The results are summarized in Fig. 3.
Example 4
[00100] MTT assay determines the cell viability by detecting cells' metabolic activity. Trypan-blue can enter only dead cells that lost their membrane integrity (but not live cells). Using this dye, only dead cells can be visualized. Cell-killing effect of zinc pyrithione (Pyz) and sodium pyrithione (Pyn) treatment at pH 7.4 and 6.4 on HeLa cell was determined by trypan blue staining assay. Ctr indicates the control treated with the same dose of vehicle that contains no drug.
[00101] Cancer cells were seeded onto 12-well dishes at 20,000/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS in the presence of 5% C02 in atmosphere. For investigating pH-dependent anti-cancer effects of zinc pyrithione and sodium pyrithione, pH=7.4 DMEM medium was prepared by supplementing 10 raM of HEPES and adjusting pH to 7.4. The pH=6.4 DMEM medium was prepared by supplementing 10 mM PIPES and adjusting pH to 6.4. Cells were treated with increasing concentrations of zinc pyrithione and sodium pyrithione in either pH=7.4 DMEM or pH=6.4 DMEM medium and cultured for 72 hours at 37°C. Bicarbonate was eliminated from pH=7.4 DMEM and pH=6.4 DMEM media and experiments were carried out in the absence of atmospheric C02 to minimize the fluctuation of pH caused by bicarbonate-mediated proton movements across biomembranes. After exposure to zinc pyrithione or sodium pyrithione for 72 hours, cells were collected and equivalent volume of 0.4% trypan blue-PBS solution was added. Numbers of total cells and cells stained with trypan-blue in each image were scored, and percentages of trypan-blue positive cells populations were determined. Four images were taken per condition and average trypan- blue positive populations and standard deviations expressed as error bars were calculated. The results are summarized in Fig 4.
[00102] The result teaches us that cell viabilities determined by trypan-blue unstained populations and cell viabilities determined by MTT assays are comparable, supporting the notion that cell-killing by pyrithione is more sensitive when treated in pH 6.4 media than in pH 7.4 media.
Example 5
[00103] Zinc pyrithione exhibits anti-proliferative effects on Spheroid 3D cultured brain tumor cells.
[00104] When tumors are developed from culture cells in semi-suspension in the presence of collagen gel as extracellular matrix (ECM) components, tumors arisen in the ECM matrix are called "spheroids", recapitulate the tumor growth in the three dimensional (3D)
microenvironment. Spheroid culture allows us to assess the efficacy of anti-cancer agents in 3D culture that models the complexity of the real cancer (Friedrich et al., 2009). Because bioactivity of many compounds is influenced by the tumor microenvironment, an important question was whether pyrithione influences the growth of spheroids. To address this, collagen-embedded spheroids of brain tumor U87 cells were established under either acidic (pH 6.4) or neutral (pH 7.4) conditions, exposed to zinc pyrithione for three days at 37°C and tested the effect on spheroid growth.
[00105] One % agarose solution heated to 60°C was mixed with an equal volume of culture media (DMEM pH 6.4 and 7.4 supplemented with 10% FBS), and 50 μΐ of the mixture was transferred to each well of 96 plates. The plates were cooled at room temperature for 30 minutes until agarose solidifies, 1000 cells resuspended in 50 μΐ of culture media were overlaid over the top of the agarose layer and centrifuged at l,500g for 5 minutes. Cells were cultured at 37°C for 3 days, spheroids formed on the agarose layer were grown in the presence or absence of different concentrations of zinc pyrithione for additional 3 days. The size of the spheroids after a 3-day incubation with 1 μΜ or 2 μΜ of zinc pyrithione in the pH 6.4 culture medium was substantially smaller than that of untreated control spheroids. In sharp contrast, the size of the spheroids exposed to 1 μΜ or 2 μΜ zinc pyrithione in the pH 7.4 culture medium for three days were almost identical to that of pyrithione-untreated control spheroids. The results are summarized in Fig. 5.
Example 6
[00106] Acidity-potentiated anti-tumor activity of pyrithione causes accumulation of superoxide in mitochondria.
Free radical forming compounds such as hydrogen peroxide and superoxide are generated during cellular metabolic processes. These reactive oxygen species (ROS) present immediate threats to cells when left unconverted. Loss of mitochondria functions by external stress or toxins have been shown to facilitate accumulation of ROS in the organelle. While majority of normal tissues are capable of neutralizing ROS to prevent further cellular damage, it has been shown that cancer cells are more sensitive to intracellular ROS fluctuation and have lower tolerance for ROS accumulation (See reference (Liou and Storz, 2010)). As pyrithione photochemically decomposes hydroxyl radical and (pyridin-2-yl)sulfanyl radical (See reference (DeMatteo et al., 2005), we postulated pyrithione might influence mitochondrial superoxide production and clearance. To address this, cancer cells were treated with pyrithione for 2 hours and assessed by visualizing a fluorescent probe that specifically label mitochondria based on the level of superoxide.
[00107] HeLa human cervical cancer cells were seeded to 8-well glass-bottom chamber slide at 4000 cells/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS. To investigate whether exposure of pyrithione under acidic or neutral external pH influences mitochondrial accumulation of superoxide, cells were first incubated with pH=6.4 or pH=7.4 DMEM alone or in the presence of zinc pyrithione or sodium pyrithione for 2 hours at 37°C. Cells were then washed and incubated with pH 7.4 culture media containing 1.25 μ,Μ
MitoSOX™ (M31008, Invitrogen) and 2.5 \M DRAQ5™ (#62254, Thermo Scientific) for 5 min at 37°C. MitoSOX™ was used to quantitatively deterimne mitochondrial superoxide production whereas DRAQ5™ was used to fluorescently label the nucleus of the cell to visualize the cells under fluorescence microscope. After 5 min, cells were placed in NaCl-saline buffered (150 mM NaCl, 5 mM KC1, 2 mM CaCh, 1 mM MgCh, 20 mM HEPES, pH 7.4), and imaged by confocal microscopy.
[00108] Accumulation of superoxide in mitochondria is one of the mechanisms that kill cancer cells. By using MitoSOX™, a fluorescence probe that sensitively and specifically detects superoxide in mitochondria, it has been discovered that treatment of cancer cells with 200 nM zinc pyrithione or 400 nM sodium pyrithione for 2 hours in an acidic culture medium of pH 6.4 causes dramatic accumulation of superoxide in mitochondria, whereas treatment of cancer cells with 200 nM zinc pyrithione or 400 nM sodium pyrithione for 2 hours in a regular culture medium of pH 7.4 does not cause accumulation of superoxide in mitochondria (Fig. 6).
Exposure of the cancer cells to the acidic culture medium of pH 6.4 in the absence of pyrithione does not cause the accumulation of superoxide. These results suggest that the contact of pyrithione to cancer cells under acidic conditions triggers accumulation of superoxide in mitochondria.
Example 7
[00109] Anti-cancer activity of gefitinib and erlotinib is not enhanced by acidity of culture media.
[00110] Gefitinib and erlotinib are epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors that can specifically block proliferation of cancer cells, thereby providing a different mechanism of action from classical chemotherapy. Gefitinib and erlotinib are used in clinic to treat malignant tumors such as lung cancer and malignant melanoma of skins. It is not known whether these clinically used molecular targeting therapeutic agents also exhibit acidity- enhanced anti-tumor activity.
[00111] Cervical cancer HeLa cells were seeded onto 96-well dishes at 2,000/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS in the presence of 5% C02 in atmosphere. Cells were treated with increasing concentrations of gefitinib or erlotinib in either pH=6.4 DMEM or pH=7.4 DMEM medium and cultured for 72 hours at 37°C .
Bicarbonate was eliminated from pH=6.4 DMEM and pH=7.4 DMEM media and experiments were carried out in the absence of atmospheric C02 to minimize the fluctuation of pH caused by bicarbonate-mediated proton movements across biomembranes. After exposure to gefitinib or erlotinib for 72 hours, MTT assay was carried out as in Example 2. [00112] Unlike pyrithione, the acidity-enhanced anti-cancer effects were not observed when cancer cells were exposed to gefitinib or erlotinib. For example, when HeLa cells were exposed to 20 μΜ gefitinib for 72 hours in culture media whose pH was adjusted to 7.4 and 6.4, relative cell viability rates determined by MTT assays were 54±16% and 61±9%, respectively. In another example, when HeLa cells were exposed to 20 μΜ erlotinib for 72 hours in culture media whose pH was adjusted to 7.4 and 6.4, relative cell viability rates were 39±8% and 46±14%, respectively. The finding that anti-cancer effects of gefitinib and erlotonib are not influenced by acidity underscores the uniqueness of acidity-enhanced anti-cancer effect of pyrithione. The results are summarized in Fig. 7.
Example 8
[00113] As a number of previous studies have indicated that anti-cancer activity of zinc pyrithione is mediated by zinc toxicity, the current finding that sodium pyrithione like zinc pyrithione effectively reduces cancer cell viability was surprising. We therefore further explored the possibility that zinc-free pyrithione, aside from the zinc-mediated toxicity that was unveiled by previous arts, can also exert toxicity to cancer cells. To address this, potential influence of a metal chelator EDTA on cancer cell killing by zinc pyrithione was examined.
[00114] Cervical cancer HeLa cells, colon cancer HT29 cells and brain tumor U87 cells were seeded onto 96-well dishes at 2,000 cells/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS in the presence of 5% C02 in atmosphere. Colon cancer HT29 cells and brain tumor U87 cells were treated with increasing concentrations of zinc pyrithione in the serum (FBS)-free pH=7.4 DMEM medium for 72 hours at 37°C whereas cervical cancer HeLa cells were incubated for 20 hours, which showed equivalent effects to other cells. In some experiments, different concentrations of EDTA were added to the media together with zinc pyrithione. Serum was excluded from the culture media in these experiments to avoid the undesirable inactivation of EDTA by metals and cations present in the serum. Bicarbonate was also eliminated from serum-free pH=7.4 DMEM media and experiments were carried out in the absence of atmospheric CO2. After the incubation with zinc pyrithione plus or minus EDTA or EDTA alone, MTT assay was carried out as in Example 2.
[00115] Referring to the results in Fig. 6, it was found that the suppression of cancer cell viability by zinc pyrithione was more prominent when cancer cells were concomitantly exposed to EDTA. For example, exposure of 50 nM zinc pyrithione to brain tumor U87 cells for 20 hours in serum-free culture media suppressed cell proliferation and viability to 60±7% of pyrithione- untreated U87 cells. In the presence of 10 μΜ and 20 μΜ EDTA, 50 nM zinc pyrithione further suppressed cell proliferation up to 30±4% and 19±1 1%, respectively, while exposure of EDTA alone caused only a slight suppression in cell proliferation by up to 10%. These surprising results suggest that pyrithione is an important component of zinc pyrithione for the anti-cancer activity, and removal of zinc by EDTA enhances such an activity. Enhancement by EDTA can be also said that the addition of EDTA lowers the dose of zinc pyrithione to achieve the same level of anti-cancer activity, providing the useful information for practical application.
Example 9
[00116] Anti-cancer activity of zinc pyrithione (Pyz) is enhanced by pyridoxine under acidic conditions.
[00117] Cervical cancer HeLa cells were seeded onto 96-well dishes at 2,000/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS in the presence of 5% CO2 in atmosphere. Cells were treated with increasing concentrations of zinc pyrithione in either pH=6.9 DMEM or pH=7.4 serum (FBS)-free DMEM medium for 24 hours. In some experiments, different concentrations of pyridoxine were added to the media together with zinc pyrithione. Bicarbonate was eliminated from pH=6.9 DMEM and pH=7.4 DMEM media and experiments were carried out in the absence of atmospheric C02 to minimize the fluctuation of pH caused by bicarbonate-mediated proton movements across biomembranes. After exposure to zinc pyrithione with or without pyridoxine, cells were incubated with pH=7.4 culture media at 37°C for additional 48 hours. MTT assay was carried out as in Example 2.
[00118] While adding 10 μΜ and 100 μΜ of pyridoxine (as labeled B6 in Fig. 9) to non- acidic pH=7.4 culture media together with 50 nM zinc pyrithione does not influence cell viability, adding 10 μΜ and 100 μΜ of pyridoxine to an acidic culture medium of pH=6.9 together with 50 nM zinc pyrithione reduced cell viability to 72±4% and 40±12%, respectively (Fig. 9). Adding 10 μΜ and 100 μΜ of pyridoxine to an acidic culture medium of pH=6.4 together with 50 nM zinc pyrithione further reduced cell viability to 1.4±0.6% and 2.5±0.9%, respectively. Adding 10 μΜ and 100 μΜ of pyridoxine alone to acidic or non-acidic culture media did not reduce cell viability (Fig. 9). These results suggest that 10 μΜ and 100 μΜ nvridoxine potentiates anti-tumor activity by zinc pyrithione in combination with acidity. One mM pyridoxine also showed enhancement of anti-tumor activity by zinc pyrithione in combination with acidity. Adding 1 mM pyridoxine and 50 nM zinc pyrithione to non-acidic pH=7.4 culture media resulted in cell viability of 71.5±13%. Adding 1 mM pyridoxine and 50 nM zinc pyrithione to acidic pH=6.9 culture media resulted in cell viability of 5.1±3%. Adding 1 mM pyridoxine and 50 nM zinc pyrithione to acidic pH=6.4 culture media resulted in cell viability of 1.2±0.8%. These results show that pyridoxine, pyrithione and acidity in combination show efficient anti-cancer activity. Interestingly, adding 1 mM pyridoxine to pH=7.4, 6.9 and 6.4 cultured media in the absence of zinc pyrithione resulted in 72.9±8%, 21.6±6% and 28.4±3% celt viability, respectively. This result suggests that 1 mM pyridoxine itself has a moderate acidity- dependent anti-tumor activity in addition to the potent pyrithione and acidity dependent antitumor activity.
Example 10
[00119] Anti-cancer activity of zinc pyrithione (Pyz) and sodium pyrithione (Pyn) is enhanced by sodium ascorbate or 3-o-ethyl ascorbic acid under acidic conditions.
[00120] Cervical cancer HeLa cells were seeded onto 96-well dishes at 2,000/well and incubated at 37°C overnight in DMEM supplemented with 10% FBS in the presence of 5% C02 in atmosphere. Cells were treated with increasing concentrations of zinc pyrithione in either pH=6.4 serum (FBS)-free DMEM, pH=6.9 serum (FBS)-free DMEM, or pH=7.4 serum (FBS)- free DMEM medium for 24 hours. In some experiments, different concentrations of sodium ascorbic acid or 3-o-ethyl ascorbic acid were added to the media together with zinc pyrithione. Bicarbonate was eliminated from culture media and experiments were carried out in the absence of atmospheric CO2 to minimize the fluctuation of pH caused by bicarbonate-mediated proton movements across biomembranes. After exposure to zinc pyrithione with or without sodium ascorbic acid or 3-o-ethyl ascorbic acid, cells were incubated with pH=7.4 culture media at 37°C for additional 48 hours. MTT assay was carried out as in Example 2.
[00121] It has been found that adding 2 mM 3-o-ethyl ascorbic acid (AscE) to an acidic culture medium of pH 6.9 together with 5 nM and 10 nM zinc pyrithione (Pyz) reduced cell viability to 91±1 1% and 43 ±1 1%, respectively, compared to untreated control. Adding 2 mM 3- o-ethyl ascorbic acid to a pH=6.4 culture medium together with 5 nM and 10 nM zinc pyrithione more efficiently reduced cell viability to 22±10 or 11±10%, respectively. In comparison, no reduction in cell viability was observed when up to 10 nM zinc pyrithione alone was added (Fig. 10A). In addition, adding 2 mM 3-o-ethyl ascorbic acid to the non-acidic culture media of pH 7.4 together with up to 10 nM zinc pyrithione did not reduce cell viability (Fig. 10). These results suggest that 3-o-ethyl ascorbic acid potentiates anti-tumor activity by zinc pyrithione in an acidity dependent manner.
[00122] Similarly, adding 2 mM 3-o-ethyl ascorbic acid (AscE) to an acidic culture medium of pH 6.9 together with 10 nM and 20 nM sodium pyrithione (Pyn) reduced cell viability to 96±5% and 48±7%, respectively, compared to untreated control. Adding 2 mM 2 mM 3-o-ethyl ascorbic acid to an acidic culture medium of pH 6.4 together with 10 nM and 20 nM sodium pyrithione (Pyn) more efficiently reduced cell viability to 17±5% and 7±3%, respectively. In contrast, when up to 20 nM sodium pyrithione was either added to the acidic culture medium alone or added in combination with 2 mM 3-o-ethyl ascorbic acid to non-acidic culture medium, no reduction in cell viability was observed.
[00123] Sodium ascorbate (AscNa) in combination with acidity also showed significant enhancement of anti-cancer effects of zinc pyrithione (Pyz). For example, it has been found that adding 1 mM sodium ascorbate to an acidic culture medium of pH=6.9 together with 10 nM zinc pyrithione moderately reduced cell viability to 85±5% compared to untreated control, whereas adding 1 mM sodium ascorbate to a more acidic culture medium of pH=6.4 together with 10 nM zinc pyrithione more efficiently reduced cell viability to 46±5% compared to untreated control (Fig. 10A). Similar but more prominent effects have been observed when 1 mM sodium ascorbate and 20 nM zinc pyrithione were added to the acidic media of pH 6.9 and 6.4. In contrast, when up to 10 nM zinc pyrithione (Pyz) alone was added to acidic pH 6.4 media or 10 nM zinc pyrithione (Pyz) in combination with 1 mM sodium ascorbate was added to non-acidic pH=7.4 culture media, cell viability was not significantly different compared to untreated control (Fig. 10A). These results suggest that ascorbic acid potentiates anti-tumor activity by zinc pyrithione in an acidity dependent manner.
[00124] Anti-proliferative effect of pyrithione on brain tumor cells in 3D cultures as determined by Spheroid culture systems is enhanced by 3-o-ethyl ascorbic acid. [00125] One % agarose solution heated to 60°C was mixed with an equal volume of culture media (DMEM pH 7.4 supplemented with 10% FBS), and 50 μΐ of the mixture was transferred to each well of 96-well plates. The plates were cooled at room temperature for 30 minutes until agarose solidifies, 1000 cells resuspended in 50 μΐ of culture media were overlaid over the top of the agarose layer and centrifuged at l,500g for 5 minutes. Cells were cultured at 37°C for 24 hours. Spheroids in the suspension culture were grown in the absence or presence of zinc pyrithione, or 3-o-ethyl ascorbic acid, or in combination for 72 hours. After 3 days, Spheroids were transferred to another 96-wells. The plate was centrifugured at 1,500 g for 5 min and solution was carefully replaced with PBS without disturbing the spheroids at the bottom of the wells. The plate was centrifuged at 1,500 g for 5 min again and PBS was replaced with buffer composed of 0.1 M pH=5.0 sodium acetate, 0.1% w/v Triton X-100, and freshly supplemented 5mM p-Nitrophenyl phosphate (#34045, Thermo Scentific), and incubated at 37°C for 2 hours. After 2 hours, 10M NaOH was added to terminate the reaction and the absorbance at 450nm was measured.
[00126] It has been found that spheroid culture incubated with 500 nM zinc pyrithione (Pyz) alone suppressed cell viability to 88±8% compared to untreated spheroids (Fig. 10B). In contrast, adding zinc pyirthione with 5 mM and 10 mM of 3-o-ethyl ascorbic acid (AscE) further suppressed cell viability to 76±4% and 63±7%, respectively. These results suggest that 3-o-ethyl ascorbic acid enhances anti-poliferative activity of zinc pyrithione in 3D cultures. Moreover, when 10 mM 3-o-ethyl ascorbic acid in combination with zinc pyrithione is added, the 3D Spheroid structure became fragile, resulting in some cell populations to dissociate from the periphery of spheroid (see Fig. 10B, right panels).
Example 11
Direct application of a formulation containing zinc pyrithione, 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate regresses proliferation of human skin cancer cells implanted to mice.
[00127] 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate are derivatives of ascorbic acid that are readily absorbed through the skin and effectively converted into ascorbic acid. Anti-cancer activity of topical application of formulations containing zinc pyrithione, 3-o-ethyl ascorbic acid and ascorbyl tetraisopalmitate, or combination thereof was assessed in a mouse xenograft model of skin cancer.
[00128] C57BL/6 athymic nude mice, 6 weeks old male, were injected subcutaneously with 2.5x106 human skin cancer A2058 cells. Following 2-3 days of injection when the size of tumors reached approximately 3 mm3, mice were randomly assigned to 4 groups, 5 mice in each group, and the cream formulation was applied once a day directly onto the skin over the tumor area. Group 1 (Pyz), cream containing 1% zinc pyrithione; Group 2 (Asc), cream containing 3% 3-o-ethyl ascorbic acid and 2% ascorbyl tetraisopalmitate; Group 3 (Pyz+Asc), cream containing 1% zinc pyrithione, 3% 3-o-ethyl ascorbic acid and 2% ascorbyl tetraisopalmitate; Group 4 (Vehicle), carrier only. The compositions of the formulations are shown below.
[00129] Composition of the formulation used for Group 1. a) Water phase Glycerol: 5% Polysorbate 20: 1%
Citric acid 1M solution: 0.7% Sodium citrate 1M solution: 1.3% Zinc pyrithione: 1%
Distilled water: to the total volume 100% b) Oil phase Behenyl alcohol: 3% Cetyl alcohol: 3% Glyceryl stearate: 1% Stearic acid: 1% Sorbitate stearate: 1% Cetyl palmitate: 1% Dimethicone: 1% c) Cooling phase Cyclomethicone: 5% d) pH is adjusted to 5.5 by HC1 or NaOH.
The water phase components are combined and mixed while heating at 75°C until completely dissolved in a suitable container. In a separate container, the oil phase components are combined and mixed while heating at 75°C until completely dissolved. The oil phase mixture is then added to the water phase mixture and mixed well so as to form an emulsion. The emulsion is then allowed to cool to about 50-55°C. The emulsion is then milled until the product becomes uniform.
[00130] Composition of the formulation used for Group 2. a) Water phase Glycerol: 5% Polysorbate 20: 1%
Citric acid 1M solution: 0.7% Sodium citrate 1M solution: 1.3% Distilled water: to the total volume 100% b) Oil phase Behenyl alcohol: 3%
Figure imgf000040_0001
The water phase components are combined and mixed while heating at 75°C until completely dissolved in a suitable container. In a separate container, the oil phase components are combined and mixed while heating at 75°C until completely dissolved. The oil phase mixture is then added to the water phase mixture and mixed well so as to form an emulsion. The emulsion is then allowed to cool to about 50-55°C. The emulsion is then milled until the product becomes uniform.
[00131] Composition of the formulation used for Group 3. a) Water phase Glycerol: 5% Polysorbate 20: 1%
Figure imgf000041_0001
The ware: ptep Pomp^a¾ ruv ef.«¾N.s½a mtoii *hik teispg st ?5:X' mtil compfeiely dissoivfd'in s suitable cwi&iftsr, hi u separate c«nisfnervihe oil pta® mmpomnts m& mmmmd and mked wftlfe hmhg at 7S.T mttti awpteteh $«fv«¾t Thz <M |>.ha«« m&tu*e¾f ibgivsddcd to the water phase mixture and mixed well so as to form an emulsion. The emulsion Is then allowed to cool so about 50-55ºC The emulsion is then milled until the product becomes uniform.
Figure imgf000042_0001
yc onKi iConi?: d) pH is adjusted to 5.5 by HC1 or NaOH.
The water phase components are combined and mixed while heating at 75°C until completely dissolved in a suitable container. In a separate container, the oil phase components are combined and mixed while heating at 75°C until completely dissolved. The oil phase mixture is then added to the water phase mixture and mixed well so as to form an emulsion. The emulsion is then allowed to cool to about 50-55°C. The emulsion is then milled until the product becomes uniform.
[00133] The tumor volume was determined according to the formula ab2/2 where a is the length and b is the width, the relative tumor volume of each treatment group was calculated and plotted (Fig. 11 A). X-axis indicates days after transdermal administration starts; Y-axis indicates relative tumor size. At the end point of the experiment, the average tumor size of Group 3 mice ("Pyz+Asc" in Fig. 11A) was smaller than the tumor size of other groups. This result suggests that the topical application of the formulation containing both pyrithione and ascorbic acid derivatives is an effective method to reduce the tumor growth, but the formulations containing pyrithione alone or derivatives of ascorbic acid alone are not effective.
[00134] At the end point of the experiment, histological samples of the skin areas that were in contact with the formulations, liver and kidney were prepared according to the standard procedure. Sections were stained with hematoxylin and eosin. Microscopic pictures of histological samples are presented in Fig. 1 IB. No sign of tissue damages, abnormal cell deaths or any other adverse effects were observed in both Group 3 (PyZ+Asc; mice treated with cream containing pyrithione, 3-o-ethyl ascrobic acid, and ascorbyl tetraisopalmitate) and Group 4 (Vehicle; mice treated with control cream that does not contain pyrihione, 3-o-ethyl ascrobic acid, or ascorbyl tetraisopalmitate).
[00135] Blood was extracted from a representative animal from each group on the day of sacrifice and subjected to hematological testing. The results are summarized in the table. All values are within a normal range except for the low MCH, low MCV and high PLT observed in all groups, which is likely caused by growing cancer in these animals. There are no abnormal values associated with specific treatment group(s) except for a slight elevation of granulocyte % in the "Asc" animal, which is regarded as irrelevant as the number of granulocytes of this animal is within a normal range. Ref Reference; GR Granulocyte (numbers/L); GR% Granulocytes (%); HCT Hematocrit (%); HBG hemoglobin (g/L); LY Lymphocytes (numbers/L); LY% Lymphocytes (%); MCH Mean Corpuscular Hemoglobin (pg); MCHC Mean Corpuscular Hemoglobin Concentration (g/L); MO Monocyte (numbers/L); MO% Monocytes (%); MPV Mean Platelet Value (fL); PCT Procalcitonin (%); PDW Platelet Distribution Width (fL); PLT Platelet (g/L); RBC Red Blood Cell (numbers/L); RDW Red blood cell Distribution Width (%); WBC White Blood Cells (numbers/L)
[00136] Table
Figure imgf000044_0001
[00137]
References
Bender, D.A. (1999). Non-nutritional uses of vitamin B 6. British Journal of Nutrition 81, 7-20.
Carraway, R.E., and Dobner, P.R. (2012). Zinc pyrithione induces ERK-and PKC-dependent necrosis distinct from TPEN-induced apoptosis in prostate cancer cells. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1823, 544-557.
Coussens, L.M., and Werb, Z. (2002). Inflammation and cancer. Nature 420, 860-867.
Criscione, V.D., Weinstock, M.A., Naylor, M.F., Luque, C, Eide, M.J., and Bingham, S.F. (2009). Actinic keratoses. Cancer 115, 2523-2530. DeMatteo, M.P., Poole, J.S., Shi, X., Sachdeva, R., Hatcher, P.G., Hadad, CM., and Platz, M.S. (2005). On the electrophilicity of hydroxyl radical: a laser flash photolysis and computational study. Journal of the American Chemical Society 127, 7094-7109.
Friedrich, J., Seidel, C, Ebner, R., and Kunz-Schughart, L.A. (2009). Spheroid-based drug screen: considerations and practical approach. Nature protocols 4, 309-324.
Lanigan, R., and Yamarik, T. (2002). Final report on the safety assessment of EDTA, calcium disodium EDTA, diammonium EDTA, dipotassium EDTA, disodium EDTA, TEA-EDTA, tetrasodium EDTA, tripotassium EDTA, trisodium EDTA, HEDTA, and trisodium HEDTA. International journal of toxicology 21, 95-142.
Lardner, A. (2001). The effects of extracellular pH on immune function. Journal of leukocyte biology 69, 522-530.
Liou, G.-Y., and Storz, P. (2010). Reactive oxygen species in cancer. Free radical research 44, 479-496.
Liu, N., Liu, C, Li, X., Liao, S., Song, W., Yang, C, Zhao, C, Huang, H., Guan, L., and Zhang, P. (2014). A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases. Scientific reports 4, 5240.
Padayatty, S.J., Sun, H., Wang, Y., Riordan, H.D., Hewitt, S.M., Katz, A., Wesley, R.A., and Levine, M. (2004). Vitamin C pharmacokinetics: implications for oral and intravenous use. Annals of internal medicine 140, 533-537.
Parekh, C, Min, B., and Golberg, L. (1970). Experimental studies of sodium pyridinethione. I. Percutaneous absorption in laboratory animals. Food and cosmetics toxicology 8, 147-160.
Parks, S.K., Chiche, J., and Pouyss6gur, J. (2013). Disrupting proton dynamics and energy metabolism for cancer therapy. Nature Reviews Cancer 13, 61 1-623.
Srivastava, G., Matta, A., Fu, G., Somasundaram, R.T., Datti, A., Walfish, P.G., and Ralhan, R. (2015). Anticancer activity of pyrithione zinc in oral cancer cells identified in small molecule screens and xenograft model: Implications for oral cancer therapy. Molecular oncology 9, 1720-
Tailler, M., Senovilla, L., Lainey, E., Thepot, S., Metivier, D., Sebert, M., Baud, V., Billot, K., Fenaux, P., and Galluzzi, L. (2012). Antineoplastic activity of ouabain and pyrithione zinc in acute myeloid leukemia. Oncogene 31, 3536-3546.
Wedig, J., Goldhamer, R., and Henderson, R. (1974). PERCUTANEOUS ABSORPTION, DISTRIBUTION, AND EXCRETION OF 3 2-6-C-14 PYRIDINETHIONES IN FEMALE YORKSHIRE PIGS. Paper presented at: Pharmacologist (AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3995 USA).
Williams, A.C., and Barry, B.W. (2012). Penetration enhancers. Advanced drug delivery reviews 64, 128-137. Zempleni, J. (1995). Pharmacokinetics of vitamin B6 supplements in humans. Journal of the American College of Nutrition 14, 579-586.
Zhao, C, Chen, X., Zang, D., Lan, X., Liao, S., Yang, C, Zhang, P., Wu, J., Li, X., and Liu, N. (2016a). A novel nickel complex works as a proteasomal deubiquitinase inhibitor for cancer therapy. Oncogene.
Zhao, C, Chen, X., Zang, D., Lan, X., Liao, S., Yang, C, Zhang, P., Wu, J., Li, X., and Liu, N. (2016b). Platinum-containing compound platinum pyrithione is stronger and safer than cisplatin in cancer therapy. Biochemical Pharmacology in press.

Claims

Claims
Claim 1
Preventative and/or therapeutic compositions for malignant tumors comprising at least one or more active ingredient(s) selected from pyrithione, pyrithione salts, vitamin B6 compounds, and phosphorylated forms of vitamin B6 compounds.
Claim 2
Therapeutic compositions for precancerous conditions comprising at least one or more active ingredient(s) selected from pyrithione, pyrithione salts, vitamin B6 compounds, and phosphorylated forms of vitamin B6 compounds.
Claim 3
The composition(s) according to claim 1 or 2, wherein the active ingredient(s) is one or more selected from pyrithione and pyrithione salts, the composition(s) further comprising at least one or more selected from EDTA, EDTA salts, ascorbic acid, ascorbic salts, and ascorbic acid compounds.
Claim 4
The composition(s) according to any one of claims 1 to 3, wherein pH is 6.9 or lower in the presence of a buffer.
Claim 5
Methods for preventing and/or treating malignant tumors comprising the step of using at least one or more active ingredient(s) selected from pyrithione, pyrithione salts, vitamin B6 compounds, and phosphorylated forms of vitamin B6 compounds.
Claim 6
Methods for treating precancerous conditions using at least one or more active ingredient(s) selected from pyrithione, pyrithione salts, vitamin B6 compounds, and phosphorylated forms of vitamin B6 compounds. Claim 7
The method according to claim 5 or 6, wherein the active ingredient(s) is one or more selected from pyrithione and pyrithione salts, and wherein the step is using the active ingredient(s) with at least one or more selected from EDTA, EDTA salts, ascorbic acid, ascorbic salts, and ascorbic acid compounds.
Claim 8
The method according to any one of claims 5 to 7, wherein the active ingredient(s) is used under a condition of pH of 6.9 or lower.
Claim 9
The use of one or more selected from pyrithione, pyrithione salts, vitamin B6 compounds, and phosphorylated forms of vitamin B6 compounds in producing preventative and/or therapeutic formulations for malignant tumors.
Claim 10
The use of one or more selected from pyrithione, pyrithione salts, vitamin B6 compounds, and phosphorylated forms of vitamin B6 compounds in producing therapeutic formulations for precancerous conditions.
Claim 1 1
The use according to claim 9 or 10, wherein one or more selected from pyrithione and pyrithione salts, together with at least one or more selected from EDTA, EDTA salts, ascorbic acid, ascorbic salts, and ascorbic acid compounds.
Claim 12
The use according to any one of claims 7 to 1 1 , wherein pH is 6.9 or lower.
PCT/IB2018/000456 2017-05-03 2018-05-04 Compositions for treatment of malignant tumors and precancerous conditions, methods of use thereof and methods for manufacturing medicaments Ceased WO2018203127A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201880025684.7A CN111542325A (en) 2017-05-03 2018-05-04 Compositions for the treatment of malignancies and precancerous conditions, methods of their use, and methods of making medicaments
JP2018554579A JP6676779B2 (en) 2017-05-03 2018-05-04 Therapeutic agent for cancer and precancerous lesion, treatment method, and method for producing therapeutic agent

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762501025P 2017-05-03 2017-05-03
US62/501,025 2017-05-03

Publications (1)

Publication Number Publication Date
WO2018203127A1 true WO2018203127A1 (en) 2018-11-08

Family

ID=64016903

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2018/000456 Ceased WO2018203127A1 (en) 2017-05-03 2018-05-04 Compositions for treatment of malignant tumors and precancerous conditions, methods of use thereof and methods for manufacturing medicaments

Country Status (3)

Country Link
JP (1) JP6676779B2 (en)
CN (1) CN111542325A (en)
WO (1) WO2018203127A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112336865A (en) * 2019-08-08 2021-02-09 厦门大学 Application of zinc pyrithione in treatment of lung cancer

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113116811A (en) * 2020-01-14 2021-07-16 中国科学院上海药物研究所 Injectable long-acting analgesic pharmaceutical composition and preparation method and application thereof
CN113116813B (en) * 2020-01-14 2022-11-29 中国科学院上海药物研究所 Depot ropivacaine pharmaceutical composition, preparation method and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006021008A2 (en) * 2004-08-20 2006-02-23 Lind Stuart E Ionophores as cancer chemotherapeutic agents
US7528125B2 (en) * 2006-03-20 2009-05-05 Jonathan L. Sessler Water-soluble zinc ionophores, zinc chelators, and/or zinc complexes and use for treating cancer
US20110117210A1 (en) * 2009-11-17 2011-05-19 Andrey Ugolkov Therapeutic treatment of human cancers using simple salts of zinc
US20130167255A1 (en) * 2010-07-29 2013-06-27 Kyoto University Screening method for anticancer drugs

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100056825A1 (en) * 2008-08-28 2010-03-04 Telik, Inc. Formulations of canfosfamide and their preparation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006021008A2 (en) * 2004-08-20 2006-02-23 Lind Stuart E Ionophores as cancer chemotherapeutic agents
US7528125B2 (en) * 2006-03-20 2009-05-05 Jonathan L. Sessler Water-soluble zinc ionophores, zinc chelators, and/or zinc complexes and use for treating cancer
US20110117210A1 (en) * 2009-11-17 2011-05-19 Andrey Ugolkov Therapeutic treatment of human cancers using simple salts of zinc
US20130167255A1 (en) * 2010-07-29 2013-06-27 Kyoto University Screening method for anticancer drugs

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
SRIVASTAVA ET AL.: "Anticancer activity of pyrithione zinc in oral cancer cells identified in small molecule screens and xenograft model: Implications for oral cancer therapy", MOL. ONCOL., vol. 9, 2015, pages 1720 - 1735, XP029279058 *
TAILLER ET AL.: "Antineoplastic activity of ouabain and pyrithione zinc in acute myeloid leukemia", ONCOGENE, vol. 31, no. 30, 21 November 2011 (2011-11-21), pages 3536 - 3546, XP055549120, Retrieved from the Internet <URL:https://www.nature.com/articles/onc2011521.pdf> *
ZHAO ET AL.: "A novel nickel complex works as a proteasomal deubiquitinase inhibitor for cancer therapy", ONCOGENE, vol. 35, 18 April 2016 (2016-04-18), pages 5916 - 5927, XP055549123, Retrieved from the Internet <URL:https://www.nature.com/articles/onc2016114.pdf> *
ZHAO ET AL.: "Platinum-containing compound platinum pyrithione is stronger and safer than cisplatin in cancer therapy", BIOCHEM. PHARMACOL., vol. 116, 2 July 2016 (2016-07-02), pages 22 - 38, XP029696548 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112336865A (en) * 2019-08-08 2021-02-09 厦门大学 Application of zinc pyrithione in treatment of lung cancer
WO2021023290A1 (en) * 2019-08-08 2021-02-11 厦门大学 Application of zinc pyrithione in treatment of lung cancer

Also Published As

Publication number Publication date
CN111542325A (en) 2020-08-14
JP2019520310A (en) 2019-07-18
JP6676779B2 (en) 2020-04-08

Similar Documents

Publication Publication Date Title
Wu et al. Aloe-emodin (AE) nanoparticles suppresses proliferation and induces apoptosis in human lung squamous carcinoma via ROS generation in vitro and in vivo
US9220680B2 (en) Compositions and methods for localized drug delivery through mammary papillae
US20020177583A1 (en) Methods and compositions for the treatment of human and animal cancers
US20110117210A1 (en) Therapeutic treatment of human cancers using simple salts of zinc
HK1221638A1 (en) Treatment of cancer using coenzyme q10 combination therapies
Kretzer et al. Simvastatin increases the antineoplastic actions of paclitaxel carried in lipid nanoemulsions in melanoma-bearing mice
Jivan et al. Disulfiram with or without metformin inhibits oesophageal squamous cell carcinoma in vivo
Wang et al. ROS generation and autophagosome accumulation contribute to the DMAMCL-induced inhibition of glioma cell proliferation by regulating the ROS/MAPK signaling pathway and suppressing the Akt/mTOR signaling pathway
Huda et al. Ionic liquid-mediated delivery of a BCL-2 inhibitor for topical treatment of skin melanoma
CN103462896A (en) Methods and use of inducing apoptosis in cancer cells
WO2018203127A1 (en) Compositions for treatment of malignant tumors and precancerous conditions, methods of use thereof and methods for manufacturing medicaments
Ma et al. Dissolving microneedle-based cascade-activation nanoplatform for enhanced photodynamic therapy of skin cancer
WO2015172712A1 (en) Pharmaceutical composition for injection with synergistic effect of vitamin c and antitumour drugs
Zhang et al. Research progress on ferroptosis and nanotechnology-based treatment in triple-negative breast cancer
Wei et al. Nucleus-targeting Oxaplatin (IV) prodrug Amphiphile for enhanced chemotherapy and immunotherapy
JP2012520319A (en) Treatment of pancreatic cancer
Wang et al. Suppression of cancer proliferation and metastasis by a versatile nanomedicine integrating photodynamic therapy, photothermal therapy, and enzyme inhibition
Hamam Curcumin: New weapon against cancer
Zhou et al. Aptamer-conjugated dual-loaded liposomes exhibit synergistic effects in hepatic tumor treatment
KR102694803B1 (en) Combination of A-nor-5α-androstane compound drugs and anticancer agents
Hao et al. Novel fluorinated docetaxel analog for anti-hepatoma: Molecular docking and biological evaluation
WO2019032411A1 (en) Compositions and methods for inducing apoptosis in anaerobic cells and related clinical methods for treating cancer and pathogenic infections
Wang et al. Prediction and verification of targets for α-hederin/oxaliplatin dual-loaded rHDL modified liposomes: Reversing effector T-cells dysfunction and improving anti-COAD efficiency in vitro and in vivo
US11318161B2 (en) Methods of treating basal cell carcinoma and glioblastoma
CN102920657B (en) 16-pregnadiene alcohol ketone submicron emulsion injection and preparation method thereof

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2018554579

Country of ref document: JP

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18794758

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18794758

Country of ref document: EP

Kind code of ref document: A1