WO2018203127A1 - Compositions pour le traitement de tumeurs malignes et d'états précancéreux, procédés d'utilisation de celles-ci et procédés de fabrication de médicaments - Google Patents

Compositions pour le traitement de tumeurs malignes et d'états précancéreux, procédés d'utilisation de celles-ci et procédés de fabrication de médicaments Download PDF

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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
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pyrithione
cancer
ascorbic acid
cells
tumor
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PCT/IB2018/000456
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English (en)
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Masayuki Numata
Hung-Yi Fan
Aidi LI
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Firmiana Health Sciences Inc.
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Priority to JP2018554579A priority Critical patent/JP6676779B2/ja
Priority to CN201880025684.7A priority patent/CN111542325A/zh
Publication of WO2018203127A1 publication Critical patent/WO2018203127A1/fr

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    • 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.

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Abstract

La présente invention concerne des compositions et des procédés d'utilisation de composés de pyrithione et des sels pharmacologiquement acceptables de ceux-ci. La présente invention concerne des procédés d'administration topique pour le traitement de cancers de la peau, le traitement du cancer du col de l'utérus, le traitement d'autres cancers, la prévention du développement du cancer de la peau à partir de conditions de peau précancéreuses, la prévention du développement du cancer du col de l'utérus à partir d'une infection par le papillomavirus humain (PVH) chronique, la prévention du développement d'autres cancers à partir d'une infection par le PVH chronique. La présente invention concerne également des compositions comprenant au moins un composé de pyrithione, de l'acide éthylènediaminetétracétique (EDTA), de l'acide ascorbique, tout sel et dérivé chimique de ceux-ci, de la pyridoxine et tout dérivé de celle-ci et des tampons pharmacologiquement acceptables. Dans ces compositions, le pH des compositions est ajusté entre 4,5 et 6,4.
PCT/IB2018/000456 2017-05-03 2018-05-04 Compositions pour le traitement de tumeurs malignes et d'états précancéreux, procédés d'utilisation de celles-ci et procédés de fabrication de médicaments WO2018203127A1 (fr)

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CN201880025684.7A CN111542325A (zh) 2017-05-03 2018-05-04 用于治疗恶性肿瘤和癌前病症的组合物及其使用方法和制备药剂的方法

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