WO2001017522A1 - Method of treating cancer using tetraethyl thiuram disulfide - Google Patents
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- WO2001017522A1 WO2001017522A1 PCT/US1999/027193 US9927193W WO0117522A1 WO 2001017522 A1 WO2001017522 A1 WO 2001017522A1 US 9927193 W US9927193 W US 9927193W WO 0117522 A1 WO0117522 A1 WO 0117522A1
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- A61K33/00—Medicinal preparations containing inorganic active ingredients
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- A61K33/26—Iron; Compounds thereof
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- A61K33/00—Medicinal preparations containing inorganic active ingredients
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- A61K33/30—Zinc; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/34—Copper; Compounds thereof
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- A61K33/36—Arsenic; Compounds thereof
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- A61K33/24—Heavy metals; Compounds thereof
- A61K33/38—Silver; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C12Y—ENZYMES
- C12Y116/00—Oxidoreductases oxidizing metal ions (1.16)
- C12Y116/03—Oxidoreductases oxidizing metal ions (1.16) with oxygen as acceptor (1.16.3)
- C12Y116/03001—Ferroxidase (1.16.3.1), i.e. ceruloplasmin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
Definitions
- This invention generally relates to methods of treating cancer, and particularly to methods of treating cancer using dithiocarbamate.
- antineoplastic agents have significant toxicities, such as bone marrow suppression, renal dysfunction, stomatitis, enteritis and hair loss, it would be of major advantage to have a relatively less toxic agent available for use alone or in combination with current drugs in order to better treat the patient without risking injury from the therapy itself.
- dithiocarbamates containing a reduced sulfhydryl group e.g., pyrrolidinedithioearbamate, (PDTC) have been shown to inhibit the proliferation of cultured vascular smooth muscle cells as well as cultured colorectal cancer cells. . ee, e.g., Tsai et al, J. Biol. Chem. 271 :3667-3670 (1996); Chinery et al., Nature Med. 11:1233-1241 (1997); Chinery et al, Cancer Res. 58:2323-2327 (1998). It has been suggested that antioxidants having a reduced sulfhydryl group may be used in treating colorectal cancer.
- PDTC pyrrolidinedithioearbamate
- Dithiocarbamates such as pyrrolidinedithioearbamate (PDTC) and diethyldithiocarbamate (DEDC) have also been reported to inhibit apoptosis in different cell types, e.g., in rat thymocytes and Jurkat T lymphocytes, in short-term incubations. See, e.g., Nobel et al. Chem. Res. Toxicol. 10(6):636-643 (1997). In this article, it is disclosed that copper is required in the inhibition of apoptosis by PDTC and DEDC.
- PDTC pyrrolidinedithioearbamate
- DEDC diethyldithiocarbamate
- thiuram disulfides such as disulfiram (oxidized disulfide of diethyldithiocarbamate which does not contain a reduced sulfhydryl group) are much more potent apoptosis inhibitors than PDTC or DEDC.
- disulfiram oxidized disulfide of diethyldithiocarbamate which does not contain a reduced sulfhydryl group
- disulfiram inhibition of thymocyte apoptosis is not dependent on copper.
- Disulfiram is used as a metal chelator to inactivate superoxide dismutase.
- Ascorbic acid is employed to inhibit catalase. It is disclosed that Meth A tumor cell proliferation is inhibited upon simultaneous addition of disulfiram and ascorbic acid. It is suggested that the combined use of disulfiram and ascorbic acid increases the intracellular oxygen free radicals within tumor cells.
- the present invention provides a method for treating established cancer using disulfiram, either alone, or in combination with a heavy metal ion or a stimulant of a heavy metal ion.
- thiuram disulfide alone exhibits potent inhibitory effect on established tumor cells in absence of catalase or ascorbic acid. Disulfiram is even effective in inhibiting the growth of established melanomas cells and non-small cell lung cancer cells, which are known to be poorly responsive to currently available antineoplastic agents.
- the antiproliferative and antineoplastic effect of disulfiram on established tumor cells is heavy metal ion-dependent.
- the tumor cell growth inhibition effect of disulfiram can be significantly enhanced by the addition of heavy metal ions such as copper, zinc, gold, and silver ions, or a heavy metal ion stimulant, e.g., ceruloplasmin or a cytokine which can induce an acute phase response in the tumor cells.
- this invention provides a method for treating established cancer in a patient by administering to the patient a therapeutically effective amount of a thiuram disulfide.
- a tetralkylthiuram disulfide such as tetraethylthiuram disulfide, better known as disulfiram is used.
- a method for treating established cancer in a patient comprising administering to the patient a therapeutically effective amount of a thiuram disulfide, preferably disulfiram, and a heavy metal ion.
- the heavy metal ion is administered as a complex or chelate with the thiuram disulfide.
- Suitable heavy metal ions include but are not limited to ions of arsenic, bismuth, cobalt, copper, chromium, gallium, gold, iron, manganese, nickel, silver, titanium, vanadium, selenium, and zinc.
- the thiuram disulfide and the heavy metal ion are administered in combination with another anticancer agent.
- a method for treating established cancer in a patient comprises administering to the patient a therapeutically effective amount of a thiuram disulfide and a cytokine such as interferon ⁇ , interferon ⁇ , interferon ⁇ , and IL-6.
- a thiuram disulfide administered is a tetraalkyl thiuram disulfide, preferably disulfiram.
- another anticancer agent can also be administered to the patient for a combination therapy.
- the method for treating established cancer in a patient comprises administering to the patient a therapeutically effective amount of a thiuram disulfide, preferably disulfiram, and ceruloplasmin.
- the present invention provides a pharmaceutical composition which comprises a pharmaceutically acceptable carrier, and a complex between a thiuram disulfide and a heavy metal ion.
- the composition can further contain another anticancer agent.
- the active compounds of this invention can be administered through a variety of administration routes. For example, they can be administered orally, intravenously, indermally, subcutaneously and topically.
- the present invention is effective for treating various types of cancer, including but not limited to melanoma, non-small cell lung cancer, small cell lung cancer, renal cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, ovarian cancer, uterine cancer, lymphoma, and prostate cancer.
- the present invention will be especially effective in treating melanoma, lung cancer, breast cancer, and prostate carcinoma.
- Thiuram disulfides such as disulfiram have been used clinically for many years in treating various other diseases such as alcohol abuse, and have been proved to be relatively non-toxic and safe. (Disulfiram is available as an oral formulation in the U.S. as Antabuse ® from Wyeth-Ayerst Laboratories, Philadelphia, PA).
- thiuram disulfides in this invention offers a readily available and easily used treatment for cancers in man and other mammals.
- Figure IB demonstrates that the cell-impermeate Cu 2+ chelator bathocuproine-disulfonic acid prevents growth inhibition by disulfiram.
- Figure 1C is a schematic diagram demonstrating that supplementation of growth medium with copper enhances the antiproliferative activity of disulfiram.
- Figure ID shows that ceruloplasmin can serve as a source of copper for enhancing the antiproliferative activity of disulfiram.
- Figure 2A shows the result of a control experiment, in which M1619 melanoma cells were treated with DMSO vehicle.
- Figure 2B demonstrates that apoptosis was induced in M1619 melanoma cells treated with 5 ⁇ M disulfiram.
- Figure 3 A is shows the flow cytometric analysis of the growth of unsynchronized Ml 619 melanoma cells in the presence of DMSO vehicle.
- Figure 3B is flow cytometric analysis of cells treated with 5 ⁇ M disulfiram, which shows that disulfiram reduces the number of cells in G 0 -G ⁇ and increases the portion in S phase of the cell cycle in M1619 melanoma cells.
- Figure 3C is flow cytometric analysis of cells treated with 5 ⁇ M disulfiram plus 250 ⁇ g/ml ceruloplasmin (Cerulo) which induce G 2 cell cycle arrest and apopotosis in M 1619 melanoma cells.
- Figure 4 demonstrates that antiproliferative activity of disulfiram is not mediated by nitric oxide.
- Figure 5 A is a photograph of Western blot analysis of cyclin A expression in Ml 619 cells treated with DMSO or disulfiram.
- Figure 5 A shows that disulfiram plus copper reduce expression of the cell-cycle protein cyclin A.
- Figure 5B is a photograph of a gel shift assay showing that disulfiram and copper inhibit transcription factor binding to the cyclic-AMP responsive element (CRE).
- CRE cyclic-AMP responsive element
- Figure 5C is a photograph of a gel shift assay in which disulfiram or disulfiram plus copper was directly added to the binding reaction.
- Figure 6A demonstrates that zinc potentiates the antiproliferative activity of disulfiram.
- Figure 6B demonstrates that the antiproliferative activity of disulfiram is enhanced by supplementation of medium with other heavy metals.
- Figure 6C demonstrates that complexes of disulfiram with gold exhibit enhanced antiproliferative activity.
- thiuram disulfides refers to compounds having the formula of:
- Ri, R 2 , R 3 , and R are same or different and represent hydrogen, and unsubstituted or substituted alkyl, alkenyl, alkynyl, aryl, alkoxy, and heteroaryl groups.
- the alkyl groups can include cycloalkyl and hetercycloalkyl groups.
- Ri, R 2 , and the N atom in the formula can together form an N-heterocyclic ring, which is, e.g., heterocycloalkyl or heterocycloaryl.
- R t and the N atom in the formula can together form an N-heterocyclic ring, which is, e.g., heterocycloalkyl or heterocycloaryl.
- thiuram disulfide is a disulfide form of dithiocarbamates which have a reduced sulfhydryl group. Many dithiocarbamates are known and synthesized in the art.
- Nonlimiting examples of dithiocarbamates include diethyldithiocarbamate, pyrrolidinedithioearbamate, N-methyl, N- ethyldithiocarbamates, hexamethylenedithiocarbamate, imadazolinedithiocarbamates, dibenzyldithiocarbamate, dimethylenedithiocarbamate, dipopyldithiocarbamate, dibutyldithiocarbamate, diamyldithiocarbamate, N-methyl, N-cyclopropylmethyldithiocarbamate, cyclohexylamyldithiocarbamate pentamethylenedithiocarbamate, dihydroxyethyldithiocarbamate, N-methylglucosamine dithiocarbamate, and salts and derivatives thereof.
- a sulfhydryl-containing dithiocarbamate can be oxidized to form a thiuram
- thiuram disulfides as defined above can be used.
- tetraalkylthiuram disulfide preferably tetraethylthiuram disulfide which is known as disulfiram
- Disulfiram has the following formula:
- Disulfiram has been used clinically in the treatment of alcohol abuse, in which disulfiram inhibits hepatic aldehyde dehydrogenase.
- Methods of making thiuram disulfides are generally known in the art. Exemplary methods are disclosed in, e.g., Thorn, et al., The Dithiocarbamates and Related Compounds, Elsevier, New York, 1962; and U.S. Patent Nos. 5,166,387, 4,144,272, 4,066,697, 1,782,111, and 1,796,977, all of which are incorporated herein by reference.
- treating cancer specifically refers to administering therapeutic agents to a patient diagnosed of cancer, i.e., having established cancer in the patient, to inhibit the further growth or spread of the malignant cells in the cancerous tissue, and/or to cause the death of the malignant cells.
- This invention provides a method for treating cancer in a patient.
- thiuram disulfides such as disulfiram
- heavy metal ions such as copper, zinc, gold, and silver ions significantly enhance the inhibitory effect of thiuram disulfides on tumor cells, while the depletion of such heavy metal ions prevents growth inhibition by disulfiram.
- a method for treating an established cancer in a patient is provided.
- a thiuram disulfide can be administered to a patient having established cancer to treat the cancer.
- the thiuram disulfide administered is a tetra alkyl thiuram disulfide such as tetra ethylthiuram disulfide, i.e., disulfiram.
- the method for treating cancer in a patient comprises administering to the patient a therapeutically effective amount of a thuram disulfide and a heavy metal ion.
- Non-limiting examples of heavy metal ions include ions of arsenic, bismuth, cobalt, copper, chromium, gallium, gold, iron, manganese, nickel, silver, titanium, vanadium, selenium and zinc. Preferably, gold, silver, zinc, selenium and copper ions are used. Sources of such heavy metal ions are known to the ordinary artisan. For example, such ions can be provided in a sulfate salt, or chloride salt form, or any other pharmaceutically suitable forms.
- One or more thiuram disulfide compounds and one or more heavy metal ions can be administered to a patient.
- the thiuram disulfide compound and the heavy metal ion can be administered in combination or separately. Preferably, they are administered as a chelating complex.
- thiuram disulfide compounds are excellent chelating agents and can chelate heavy metal ions to form chelates. Preparation of chelates of thiuram disulfide compounds and heavy metal ions are known to the ordinary artisan.
- chelates of disulfiram and copper, zinc, silver, or gold ions can be conveniently synthesized by mixing, in a suitable solvents, disulfiram with, e.g., CuS0 4 , ZnCl 2 , C 3 H 5 Ag0 3 , or HAuCl 4 3H 2 O to allow chelates to be formed.
- a suitable solvents e.g., CuS0 4 , ZnCl 2 , C 3 H 5 Ag0 3 , or HAuCl 4 3H 2 O.
- Other thiuram disulfide compound- heavy metal ion chelates are disclosed in, e.g., Burns et al., Adv. Inorg. Chem. Radiochem. 23:211-280 (1980), which is incorporated herein by reference.
- a method for treating cancer in a patient includes administering to the patient a therapeutically effective amount of a thiuram disulfide compound and an intracellular heavy metal ion stimulant, which can enhance the intracellular level of the above described heavy metal ions in the patient.
- Intracellular heavy metal ion carriers are known.
- ceruloplasmin can be administered to the patient to enhance the intracellular copper level.
- Other heavy metal ion carriers known in the art may also be administered in accordance with this aspect of the invention.
- the heavy metal ion carriers and the thiuram disulfide compound can be administered together or separately, and preferably in separate compositions.
- Ceruloplasmin is a protein naturally produced by human body and can be purified from human serum. This 132-kD glycoprotein, which carries 7 copper atoms complexed over three 42-45 kD domains, is an acute phase reactant and the major copper-carrying protein in human plasma. See Halliwell, et al. Methods Enzymol. 186:1-85(1990).
- Ceruloplasmin can be isolated from animal or human serum. Alternatively, genetical engineered ceruloplasmin can also be expressed in vitro in, e.g., bacteria, yeast, plant, animal or human cells and purified therefrom. Ceruloplasmin and thiuram disulfide are typically administered in different compositions. Thiuram disulfide and ceruloplasmin can be administered at about the same time, or at some time apart. For example, ceruloplasmin can be administered from about five minutes to about 12 hours before or after thiuram disulfide is administered to the patient.
- a cytokine is administered to the patient in addition to a thiuram disulfide compound.
- Suitable cytokines include, e.g., interferon ⁇ , interferon ⁇ , interferon ⁇ , and interleukin 6 (IL-6).
- IL-6 interleukin 6
- Such cytokines when administered to a patient, are capable of inducing an acute phase response in the body of the patient thus stimulating serum ceruloplasmin in the patient.
- the biochemical and physiological properties of such cytokines have been studied extensively in the art and are familiar to skilled artisans.
- the cytokines can be purified from human or animal serum. They can also be obtained by genetic engineering techniques.
- cytokines may also be used in this invention.
- Genetically or chemically modified cytokines can also be administered.
- certain peptidic cytokines have longer circulation time in animals when such cytokines are conjugated with a water soluble, non-immunogenic polymer such as polyethylene glycol.
- the cytokines are administered in a different composition from the thiuram disulfide compound.
- the cytokines and thiuram disulfide can be administered at about the same time, or at some time apart from each other.
- the cytokines can be administered from about 5 minutes to about 24 hours before or after the administration of thiuram disulfide.
- the method of this invention can be used in combination with a conventional anticancer therapy.
- the method of this invention can be complemented by a conventional radiation therapy or chemotherapy.
- the method of this invention comprises administering to a patient a thiuram disulfide compound and heavy metals, and another anticancer agent. Treatment by ceruloplasmin or a cytokine, and a thiuram disulfide compound can also be conducted along with the treatment with another anticancer agent.
- any anticancer agents known in the art can be used in this invention so long as it is pharmaceutically compatible with the thiuram disulfide compounds, heavy metal ions, ceruloplasmin, and/or cytokines used.
- pharmaceutically compatible it is intended that the other anticancer agent will not interact or react with the above composition, directly or indirectly, in such a way as to adversely affect the effect of the treatment of cancer, or to cause any significant adverse side reaction in the patient.
- anticancer agents known in the art include cisplatin, carmustine, herceptin, carboplatin, cyclophosphamide, nitrosoureas, fotemustine, vindesine, etoposide, daunorubicin, adriamycin, taxol, taxotere, fluorouracil, methotrexate, melphalan, bleomycin, salicylates, aspirin, piroxicam, ibuprofen, indomethacin, maprosyn, diclofenac, tolmetin, ketoprofen, nabumetone, oxaprozin, doxirubicin, nonselective cyclooxygenase inhibitors such as nonsteroidal anti-inflammatory agents (NSAIDS), and selective cyclooxygenase-2 (COX-2) inhibitors.
- NSAIDS nonsteroidal anti-inflammatory agents
- COX-2 selective cyclooxygenase-2
- the anticancer agent used can be administered simultaneously in the same pharmaceutical preparation with the thiuram disulfide compound, heavy metal ions, ceruloplasmin, and/or cytokines as described above.
- the anticancer agent can also be administered at about same time but by a separate administration.
- the anticancer agent can be administered at a different time from the administration of the thiuram disulfide compound, heavy metal ions, ceruloplasmin, and/or cytokines.
- the methods of this invention are suitable for treating cancers in animals, especially mammals such as canine, bovine, porcine, and other animals.
- the methods are used in treating human patients.
- the methods are useful for treating various types of cancer, including but not limited to melanoma, non-small cell lung cancer, small cell lung cancer, renal cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, ovarian cancer, uterine cancer, lymphoma, and prostate cancer.
- the present invention will be especially effective in treating melanoma, lung cancer, breast cancer, and prostate carcinoma.
- the active compounds of this invention are typically administered in a pharmaceutically acceptable carrier through any appropriate routes such as parenteral, intravenous, oral, intradermal, subcutaneous, or topical administration.
- the active compounds of this invention are administered at a therapeutically effective amount to achieve the desired therapeutic effect without causing any serious adverse effects in the patient treated.
- the thiuram disulfide compound disulfiram can be effective when administered at an amount within the conventional clinical ranges determined in the art. Typically, it can be effective at an amount of from about 125 to about 1000 mg per day, preferably from about 250 to about 500 mg per day. However, the amount can vary with the body weight of the patient treated.
- the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at predetermined intervals of time.
- the suitable dosage unit for each administration of disulfiram can be, e.g., from about 50 to about 1000 mg, preferably from about 250 to about 500 mg.
- the desirable peak plasma concentration of disulfiram generally is about 0.05 to about 10 ⁇ M, preferably about 0.5 to about 5 ⁇ M , in order to achieve a detectable therapeutic effect. However, a plasma concentration beyond such ranges may work as well.
- Disulfiram has been used clinically in treating alcohol abuse.
- a dosage form of disulfiram approved by the U.S. Food and Drug Administration is a dosage form of disulfiram approved by the U.S. Food and Drug Administration
- Disulfiram implanted subcutaneously for sustained release has also been shown to be effective at an amount of 800 to 1600 mg to achieve a suitable plasma concentration. This can be accomplished by using aseptic techniques to surgically implant disulfiram into the subcutaneous space of the anterior abdominal wall. See, e.g., Wilson et al., J. Clin. Psych. 45:242-247 (1984).
- a sustained release dosage formulation comprised to 80% poly(glycolic- co-L-lactic acid) and 20% disulfiram has also been described in Phillips et al., J.
- Disulfiram is relatively non-toxic, with an LDs 0 in rodents of 8.6 g/kg. See, e.g., The Merck Index, 10th Edition, Reference 3382, Merck & Co., Rahway, NJ, 1983, page 491. Disulfiram can be used in a similar dosage in the present invention.
- the therapeutically effective amount for other thiuram disulfide compounds may also be estimated or calculated based on the above dosage ranges of disulfiram and the molecular weights of disulfiram and the other thiuram disulfide compounds, or by other methods known in the art.
- Heavy metal ions can be administered separately as an aqueous solution in a pharmaceutically suitable salt form. However, they are preferably administered in a chelate form in which the ions are complexed with thiuram disulfide compounds.
- the amount of heavy metal ions to be used advantageously is proportional to the amount of thiuram disulfide compound to be administered based on the molar ratio between a heavy metal ion and thiuram disulfide compound in the chelate. Methods for preparing such chelates or complexes are known and the preferred methods are disclosed above and in the examples below.
- the therapeutically effective amount for IL-6 can be from about 1 to about 100 ⁇ g/kg per day, preferably from about 5 to about 50 ⁇ g/kg per day.
- Interferon ⁇ can be administered at from about 0.1 x 10 6 to about 10 x 10 6 international units per day, preferably from about 3 to about 8 x 10 6 international units per day, and the administration frequency can be from about three times per week to about once per day.
- Suitable dosage for interferon ⁇ can range from about 1 to about 200 ⁇ g per day, preferably from about 10 to about 100 ⁇ g per day administered once per week up to once per day.
- Interferon ⁇ can be administered at a dosage of from about 1 to about 1000 ⁇ g per day, preferably from about 50 to about 250 ⁇ g per day.
- Ceruloplasmin may be administered at an amount of from about 1 to about 100 mg per day, preferably from about 5 to about 30 mg per day.
- the therapeutically effective amount for each active compound can vary with factors including but not limited to the activity of the compound used, stability of the active compound in the patient's body, the severity of the conditions to be alleviated, the total weight of the patient treated, the route of administration, the ease of abso ⁇ tion, distribution, and excretion of the active compound by the body, the age and sensitivity of the patient to be treated, and the like, as will be apparent to a skilled artisan.
- the amount of administration can also be adjusted as the various factors change over time.
- the active compounds of this invention can be administered to a patient to be treated through any suitable routes of administration.
- the active compounds are delivered to the patient parenterally, i.e., intravenously or intramuscularly.
- the active compounds can be formulated into solutions or suspensions, or in lyophilized forms for conversion into solutions or suspensions before use.
- Sterile water, physiological saline, e.g., phosphate buffered saline (PBS) can be used conveniently as the pharmaceutically acceptable carriers or diluents.
- parenteral formulations including but not limited to acetates, citrates or phosphates buffers, sodium chloride, dextrose, fixed oils, glycerine, polyethylene glycol, propylene glycol, benzyl alcohol, methyl parabens, ascorbic acid, sodium bisulfite, and the like.
- the parenteral formulation can be stored in any conventional containers such as vials, ampoules, and syringes.
- the active compounds can also be delivered orally in enclosed gelatin capsules or compressed tablets.
- Capsules and tablets can be prepared in any conventional techniques.
- the active compounds can be inco ⁇ orated into a formulation which includes pharmaceutically acceptable carriers such as excipients (e.g., starch, lactose), binders (e.g., gelatin, cellulose, gum tragacanth), disintegrating agents (e.g., alginate, Primogel, and corn starch), lubricants (e.g., magnesium stearate, silicon dioxide), and sweetening or flavoring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint).
- Various coatings can also be prepared for the capsules and tablets to modify the flavors, tastes, colors, and shapes of the capsules and tablets.
- liquid carriers such as fatty oil can also be included in capsules.
- oral formulations such as chewing gum, suspension, syrup, wafer, elixir, and the like can also be prepared containing the active compounds used in this invention.
- Various modifying agents for flavors, tastes, colors, and shapes of the special forms can also be included.
- the active compounds can be dissolved in an acceptable lipophilic vegetable oil vehicle such as olive oil, corn oil and safflower oil.
- Topical formulations are generally known in the art including creams, gels, ointments, lotions, powders, pastes, suspensions, sprays, and aerosols.
- topical formulations include one or more thickening agents, humectants, and/or emollients including but not limited to xanthan gum, petrolatum, beeswax, or polyethylene glycol, sorbitol, mineral oil, lanolin, squalene, and the like.
- a special form of topical administration is delivery by a transdermal patch. Methods for preparing transdermal patches are disclosed, e.g., in Brown, et al, Annual Review of Medicine, 39:221-229 (1988), which is inco ⁇ orated herein by reference.
- the active compounds can also be delivered by subcutaneous implantation for sustained release. This may be accomplished by using aseptic techniques to surgically implant the active compounds in any suitable formulation into the subcutaneous space of the anterior abdominal wall. See, e.g., Wilson et al., J. Clin. Psych. 45:242-247 (1984). Sustained release can be achieved by inco ⁇ orating the active ingredients into a special carrier such as a hydrogel.
- a hydrogel is a network of high molecular weight biocompatible polymers, which can swell in water to form a gel like material.
- Hydrogels are generally known in the art. For example, hydrogels made of polyethylene glycols, or collagen, or poly(glycolic-co- L-lactic acid) are suitable for this invention. See, e.g., Phillips et al., J. Pharmaceut. Sci. 73:1718-1720 (1984).
- the active compounds can also be conjugated, i.e., covalently linked, to a water soluble non-immunogenic high molecular weight polymer to form a polymer conjugate.
- a water soluble non-immunogenic high molecular weight polymer e.g., polyethylene glycol
- polymers e.g., polyethylene glycol
- ADAGEN® PEGylated adenosine deaminase
- SCIDS severe combined immunodeficiency disease
- PEGylated L- asparaginase is being used to treat acute lymphoblastic leukemia (ALL).
- ALL acute lymphoblastic leukemia
- the covalent linkage between the polymer and the active compound is hydrolytically degradable and is susceptible to hydrolysis under physiological conditions.
- conjugates are known as "prodrugs" and the polymer in the conjugate can be readily cleaved off inside the body, releasing the free active compounds.
- microcapsules and nanocapsules generally known in the art, and hydrogels described above can all be utilized in oral, parenteral, topical, and subcutaneous administration of the active compounds.
- liposomes are micelles formed from various lipids such as cholesterol, phospholipids, fatty acids, and derivatives thereof. Active compounds can be enclosed within such micelles.
- Methods for preparing liposomal suspensions containing active ingredients therein are generally known in the art and are disclosed in, e.g., U.S. Pat. No. 4,522,811, which is inco ⁇ orated herein by reference.
- Several anticancer drugs delivered in the form of liposomes are known in the art and are commercially available from Liposome Inc. of Princeton, New Jersey, U.S.A. It has been shown that liposomal can reduce the toxicity of the active compounds, and increase their stability.
- the active compounds can also be administered in combination with other active agents that treats or prevents another disease or symptom in the patient treated.
- other active agents should not interfere with or adversely affect the effects of the active compounds of this invention on the cancer being treated.
- Such other active agents include but are not limited to antiviral agents, antibiotics, antifungal agents, anti-inflammation agents, antithrombotic agents, cardiovascular drugs, cholesterol lowering agents, hypertension drugs, and the like. It is to be understood that individuals placed on disulfiram therapy for their cancer must be warned against exposure to alcohol in any form, to avoid the precipitation of nausea and vomiting from buildup of acetaldehyde in the bloodstream. Subjects therefore must not only refrain from ingesting alcohol containing beverages, but should also not ingest over the counter formulations such as cough syrups containing alcohol or even use rubbing alcohol topically.
- the prostate adenocarcinoma cell line CRL 1435 (PC-3) was also cultured in RPMI 1640 with 10% FBS but passed with 0.05% trypsin and 0.53 mM EDTA.
- the squamous lung carcinoma NCI-H520 and the adenosquamous lung carcinoma NCI-H596 cell lines were grown in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES and 1.0 mM sodium pyruvate and passed with trypsin/EDTA.
- the small cell lung carcinoma NCI-H82 was cultured as a suspension in RPMI 1640 with 10% FBS. All of the above were grown in a 37°C humidified environment containing 5% CO 2 /air.
- the breast carcinoma cell line MDA-MB-453 was grown in a 37° C humidified environment with free gas exchange with atmospheric air using Leibovitz's L-15 medium with 2 mM L-glutamine and 10% FBS and was passed with trypsin/EDTA.
- Proliferation of cultured cells was quantitated using a previously reported colorimetric method based upon metabolic reduction of the soluble yellow tetrazolium dye 3-[4,5-dimethylthiazol]- 2yl-2,5-diphenyl tetrazolium bromide (MTT) to its insoluble pu ⁇ le formazan by the action of mitochondrial succinyl dehydrogenase.
- MTT mitochondrial succinyl dehydrogenase
- cells were seeded into 24-well uncoated plastic plates (Costar) at 50,000 cells per well and cultured with respective media and mitogens. After 24-96 hr, medium was removed, cells were washed twice with 1 ml of sterile Dulbecco's modified phosphate buffered saline without Ca 2+ or Mg 2+ (DPBS), the medium was replaced with 1 ml/well fresh medium containing 100 ⁇ g/ml MTT, and plates were incubated an additional hour. MTT-containing medium was removed, 0.5 ml dimethylsulfoxide (DMSO) was added to each well, and the absorbance of the solubilized pu ⁇ le formazan dye was measured at 540 nm.
- DMSO dimethylsulfoxide
- Disulfiram was solubilized in dimethylsulfoxide (DMSO) so that the final concentration of DMSO was less than 0.3-0.5%. Equal volumes of DMSO were added to control experiments. Cell numbers were quantitated by the MTT assay 24-72 hours later. In some experiments disulfiram or PDTC were added immediately after cells were plated. In other experiments, cells were plated and allowed to grow for 24-72 hours before fresh media with disulfiram or PDTC was added, and cell numbers were studied by the MTT assay 24-72 hr later.
- DMSO dimethylsulfoxide
- Synergy was studied between disulfiram and N,N'-bis(2-chloroethyl-N-nitrosourea (carmustine or BCNU, 1.0 to 1,000 ⁇ M) or cisplatin (0.1 to 100 ⁇ g/ml) added to medium.
- the effect of metals on disulfiram was studied with 0.2 to 10 ⁇ M copper
- Disulfiram (5 ⁇ M), with or without 1.6 ⁇ M CuS0 , was added to cells grown to confluence on 100 x 15 mm plastic dishes, and cells were harvested 24 hours later for measurement of GSH as outlined below. Also, to assess whether a nonspecific antioxidant effect of disulfiram or PDTC might account for cellular growth inhibition, we studied the effect of the potent lipophilic antioxidant probucol (1.0 to 1,000 ⁇ M) on proliferation of malignant cell lines. Finally, the generation of intracellular oxidants in response to disulfiram (0.625 to 5 ⁇ M), copper (0.2 to 1.6 ⁇ M CuSO ) or 1.25 ⁇ M disulfiram plus various concentration of copper was measured directly, as outlined below.
- COX1 and COX2 inhibitors indomethacin (5 ⁇ g/ml) or sodium salicylate (1 mM).
- NO nitric oxide
- cells were cultured with or without addition of the impermeate Cu 2+ chelator bathocuprioinedisulfonic acid (BCPS, 100 ⁇ M) added to medium to sequester Cu in the extracellular compartment. Cells were also treated 12 hours with various concentration of disulfiram (0.625 to 5.0 ⁇ M) and intracellular copper levels were measured as outlined below.
- BCPS impermeate Cu 2+ chelator bathocuprioinedisulfonic acid
- cells were grown to confluence on 60 x 15 mm plastic Petri dishes and treated with 5 ⁇ M disulfiram or 5 ⁇ M disulfiram plus 1.6 ⁇ M CuSO 4 for 2 to 48 hours.
- Cells were lysed and levels of the pro-apoptotic protein p53, the anti-apoptotic protein Bcl-2, the cyclin inhibitor p21 WAF1/C ⁇ pl , and the cyclins A and Bl were measured by immunoblot assay as described below.
- Cytotoxicity and Apoptosis To assess for cytotoxicity, cells were plated at a density of 50,000 per well on 24 well plates and grown for 24 hours. Disulfiram was then added. After an additional 36 hr, medium was removed and replaced with DPBS containing 0.1% trypan blue. Cell death was assessed by counting the average number of trypan blue positive cells per 10X field in 5 random fields for 4 separate wells. To determine whether disulfiram induced apoptosis, cells grown to confluence on 35 mm Petri dishes or on glass slides were treated with disufiram or DMSO as vehicle.
- Apoptosis was studied by terminal deoxynucleotidyl transferase (TdT) dependent 3'-OH fluorescein end- labeling of DNA fragments, using a Fluorescein-FragELTM DNA fragmentation detection kit (Oncogene Research Products, Cambridge, MA). Apoptosis was also studied by visually assessing endonuclease dependent DNA fragmentation on ethidium bromide-stained agarose gels, as previously reported. See Dashtaki, et al. J. Pharmacol. Exper. Ther. 285:876-219 (1998).
- This method is based upon oxidation of dichlorofluorescin to 2',7'- dichlorofluorescein by H 2 0 2 in the presence of cellular peroxidases.
- Cells were plated in 24 well plastic plates at 50,000 cells per well and grown to confluence. Media was aspirated from wells and replaced with 100 ⁇ l medium containing 10 ⁇ M DCF-DA, and plates were incubated at 37° C for 30 min. The DCF-DA containing media was aspirated, cells were washed twice with media alone and 100 ⁇ l fresh media was added to wells.
- HTS 7000 fluorescence micro-plate reader
- Disulfiram (5 ⁇ M), with or without 1.6 ⁇ M CuS0 4 , was added to cells grown to confluence on 100 x 15 mm plastic dishes, and cells were harvested 24 hr later for measurement of GSH using the 5,5'-dithiobis(2-nitrobenzoic acid)-glutathione reductase recycling assay. See Anderson, M.E. Methods Enzymol. 113:548-555 (1985).
- Proteins in defrosted samples were separated by SDS-polyacrylamide gel electrophoresis on 12% polyacrylamide gels (15 ⁇ g protein/lane) and electrotransferred to 0.45 ⁇ m Hybond ECL nitrocellulose membranes (Amersham Life Sciences) using the wet transblot method in transfer buffer (0.025 M Tris, 0.192 M glycine, 2.6 mM SDS, and 20%[vol/vol] methanol; pH 8.8) at 100 volts for 1 hr. Blots were blocked overnight at 4° C with blocking buffer (PBS with 0.1% Tween 20) containing 5% fat-free milk powder (Carnation, Glendale, CA).
- transfer buffer 0.025 M Tris, 0.192 M glycine, 2.6 mM SDS, and 20%[vol/vol] methanol; pH 8.8
- Blots were blocked overnight at 4° C with blocking buffer (PBS with 0.1% Tween 20) containing 5% fat-free milk powder (Carnation, Glendale, CA).
- blots were incubated for 1 hr at room temperature with 2.0 ⁇ g/ml of primary antibody. After rinsing again as above, blots were incubated for 1 hr at room temperature with horseradish peroxidase(HRP)-conjugated secondary antibody diluted 1 :5,000 in blocking buffer. Immunoblots were rinsed again as above and detected via an enhanced chemiluminescence method (ECL Western blotting detection system, Amersham Life Science, Buckinghamshire, England). Autoradiographic film (X-OMAT AR, Eastman Kodak, Rochester, NY) was exposed to immunoblots for 10, 30, or 60 sec to obtain satisfactory images.
- HRP horseradish peroxidase
- Electrophoretic Mobility Shift Assays Nuclear protein was isolated and DNA binding reactions were performed as previously described in detail. See, Dashtaki, et al. J. Pharmacol. Exper. Ther. 285:876-219 (1998); Kennedy et al, Am. J. Respir. Cell Mol. Biol. 19:366-378 (1998).
- Nonidet P-40 NP-40
- NP-40 Nonidet P-40
- TCAACTCCCCTGAAAGGGTCCG-5' TCAACTCCCCTGAAAGGGTCCG-5'
- Samples were electrophoresed on a 5% nondenaturing polyacrylamide gel in Tris-glycine- EDTA (TGE, 120 mM glycine and 1 mM EDTA in 25 mM Tris, pH 8.5) buffer. Gels were dried and analyzed by autoradiography at -80° C using an image intensifier screen.
- Example 1 Disulfiram is antiproliferative against malignant human cell lines. M1619 human melanoma cell line was used to test the antiproliferative characteristics of disulfiram. Cells stimulated with 10% fetal bovine serum (FBS) were plated at a density of 50,000 cells per well. DMSO vehicle (5 ⁇ l per ml) or disulfiram (DS) at various concentrations was added to wells.
- FBS fetal bovine serum
- DMSO vehicle 5 ⁇ l per ml
- DS disulfiram
- a variety of other malignant human cell lines including M1585 melanoma, prostatic adenocarcinoma, nonsmall cell, small cell lung cancer, and adenocarcinoma of the breast were tested by the same method as described above.
- Cells stimulated with 10% fetal bovine serum (FBS) were plated at a density of 50,000 cells per well.
- FBS fetal bovine serum
- DMSO vehicle 5 ⁇ l per ml
- DS disulfiram
- DMSO vehicle (5 ⁇ l per ml) or disulfiram (DS) was added to wells at the indicated concentrations. After an additional 24 (lung) or 48 hours (breast), proliferation was quantitated as described above. Percent inhibition of growth was calculated as 100 x (1.0 - A 5 0 of MTT formazan in disulfiram treated cells/mean A 54 o of MTT formazan in DMSO vehicle treated cells). In some cell lines, a modest ( ⁇ 10%) but statistically significant inhibitory effect was observed with DMSO vehicle alone. The results are summarized in Table 1. Each value represents a mean of at least 4 experiments. A p ⁇ 0.01 compared to FBS + DMSO vehicle control.
- disulfiram was effective in inhibiting the growth of many different malignant cells. This was true whether disulfiram was added to culture media when cells were plated or later, after cell had grown for 24-48 hours.
- Adenosquamous lung carcinoma NCI- 47 ⁇ 4 A 57 ⁇ 4 A 50 ⁇ 3 A 50 ⁇ 4 A H596 Small cell lung carcinoma NCI-H82 68 ⁇ 3 A 76 ⁇ 6 A 76 ⁇ 5 A 72 ⁇ 3 A
- Example 2 Antiproliferative activity of disulfiram depends on complexation with copper.
- M1619 melanoma cells stimulated and plated as described in Example 1.
- 1.25 ⁇ M disulfiram (DS) or DMSO vehicle (5 ⁇ l per ml) was added to wells in the absence or presence of 50 or 100 ⁇ M bathocuproine-disulfonic acid (BPS). After 48 hours, proliferation was quantitated as described. *p ⁇ 0.001 compared to FBS + DMSO; +p ⁇ 0.001 compared to FBS + DS.
- the cell- impermeate Cu 2+ chelator bathocuproine-disulfonic acid prevents growth inhibition from disulfiram.
- Example 3 Copper enhances the antiproliferative activity of disulfiram.
- M1619 melanoma cells plated and stimulated as described in Example 1 were grown for 24 hr and supplemented with CuS0 or CuS0 4 plus 0.625 ⁇ M disulfiram. After an additional 24 hr proliferation was quantitated. As shown in Figure 1 C, supplementation of growth medium with copper enhances the antiproliferative activity of disulfiram. The addition of even 0.2 ⁇ M CuSO 4 to medium converts 0.625 ⁇ M disulfiram from a 50% inhibitory (IC 50 ) concentration into a 100%> inhibitory (IC100) concentration of drug. *p ⁇ 0.001 compared to no CuSO 4 .
- Ml 619 melanoma cells were plated, stimulated and grown for 24 hr in the presence or absence of 0.625 ⁇ M disulfiram or 5 ⁇ l/ml DMSO vehicle in the presence or absence of human ceruloplasmin (Cerulo) at a concentration representing the upper level in normal human serum (500 ⁇ g/ml). After 24 hours, proliferation was quantitated. *p ⁇ 0.001 compared to FBS + DMSO; +p ⁇ 0.001 compared to FBS + DS. As shown in Figure ID, ceruloplasmin can serve as a source of copper for enhancing the antiproliferative activity of disulfiram.
- Example 4 Disulfiram induces apoptosis.
- Ml 619 melanoma cells were grown to confluence on 35 mm Petri dishes or on glass slides and treated for 15 hours with disufiram or DMSO as vehicle.
- Apoptosis was studied by terminal deoxynucleotidyl transferase (TdT) dependent 3'-OH fluorescein end-labeling of DNA fragments, using a Fluorescein-FragELTM DNA fragmentation detection kit (Oncogene Research Products, Cambridge, MA).
- TdT terminal deoxynucleotidyl transferase
- a Fluorescein-FragELTM DNA fragmentation detection kit Oncogene Research Products, Cambridge, MA.
- apoptosis was induced in Ml 619 melanoma cells treated with 5 ⁇ M disulfiram.
- Disulfiram markedly increases 3'-OH fluorescein end-labeling of DNA fragments.
- Treatment of monolayers with even low doses of disulfiram markedly increased trypan blue dye uptake. 6 ⁇ 2, 8 ⁇ 3.6 and 94 ⁇ 18 trypan blue positive cells per well, respectively were observed for untreated, DMSO vehicle treated or H520 lung adenosquamous carcinoma cells treated with
- disulfiram also increased DNA laddering on ethidium bromide-stained agarose gels (data not shown).
- Example 5 Disulfiram and cooper induce G cell cycle arrest and apoptosis. Unsynchronized Ml 619 melanoma cells were incubated with DMSO
- Disulfiram increases the portion of cells in S phase.
- the combination of disulfiram and ceruloplasmin further increases the number of cells in S phase, induces G 2 cell cycle arrest and apoptosis.
- Figure 3 A shows the growth of unsynchronized Ml 619 melanoma cells in the presence of DMSO vehicle.
- Figure 3B shows that 5 ⁇ M disulfiram induces the number of cells in Go-Gi and increases the portion in S phase of the cell cycle in M1619 melanoma cells.
- Figure 3C shows that 5 ⁇ M disulfiram plus 250 ⁇ g/ml ceruloplasmin (Cerulo) induces G 2 cell cycle arrest and apopotosis in M1619 melanoma cells.
- Example 6 Disulfiram does not decrease proliferation through redox mechanisms.
- M1619 melanoma cells stimulated with 10% FBS were plated at a density of 50,000 cells per well, grown for 24 hours, and treated with 5 ⁇ M disulfiram or 5 ⁇ l/ml DMSO vehicle, in the presence or absence of the nitric oxide synthase inhibitor N ⁇ -nitro-Z--arginine (LNAME, 100 ⁇ M). After an additional 24 hours, proliferation was quantitated as described in Example 1. *p ⁇ 0.01 compared to DMSO; +p ⁇ 0.001 compared to DMSO.
- Figure 4 demonstrates that antiproliferative activity of disulfiram is not mediated by nitric oxide. While the nitric oxide synthase inhibitor N ⁇ -nitro-Z- arginine (LNAME) alone slightly enhanced cellular growth, LNAME did not reduce the antiproliferative effect of disulfiram. Thus, disulfiram does not appear to inhibit growth by adversely affecting cellular redox state.
- NAME nitric oxide synthase inhibitor N ⁇ -nitro-Z- arginine
- disulfiram failed to deplete GSH in M1619 cells (228 ⁇ 18 for FBS alone; 254 ⁇ 7 for DMSO vehicle control; 273 ⁇ 11 ⁇ M GSH ⁇ g cell protein for 5 ⁇ M disulfiram), and the combination of 5.0 ⁇ M disulfiram and 1.6 ⁇ M CuSO 4 even increased intracellular GSH (293 ⁇ 16 ⁇ M GSH/ ⁇ g cell protein; p ⁇ 0.05 compared to FBS alone).
- the potent antioxidant probucol did not significantly inhibit growth of any of our tumor cell lines.
- Example7 Disulfiram plus copper reduce expression of the cell-cycle protein cyclin A and inhibit DNA binding of transcription factors to DNA regulatory elements important for cyclin A expression.
- M1619 melanoma cells were plated at equal densities in 60 x 15 mm plastic dishes, grown to 80% confluence and treated with DMSO vehicle (5 ⁇ l/ml), disulfiram (5 ⁇ M), or the combination of disulfiram and CuS0 (1.6 ⁇ M). After the indicated times, cells were lysed and protein extracts were subjected to SDS- polyacrylamide gel electrophoresis (PAGE) followed by Western blotting using a rabbit polyclonal antibody. Typical experiments are shown for 2, 4, 8, 12, 24 and 48 hours of treatment.
- Figure 5 A shows that disulfiram plus copper reduce expression of the cell-cycle protein cyclin A, providing a potential proximate explanation for the antiproliferative effects of this drug-metal combination.
- levels of cyclin B 1 remained unchanged, and, in the cell lines we studied, disulfiram had no consistent effect on expression of the cell cycle inhibitor p21 WAF1 CIP1 (data not shown).
- Cyclin A expression is regulated in part by binding of c-Fos and CREB-1 protein heterodimers to a cyclic-AMP responsive element (CRE) in the cylin A promoter.
- CRE cyclic-AMP responsive element
- Figure 5B shows that the combination of disulfiram and copper essentially eliminates transcription factor binding to CRE after 6 hours of treatment, an expected result of which would be reduced transcription of the cyclin A gene.
- EMSAs for 2, 6, 12 or 24 hours of treatment fetal bovine serum (FBS) alone, lanes 1, 5, 9, and 13; FBS + DMSO vehicle, lanes 2, 6, 10,14; FBS + disulfiram, lanes 3, 7, 11,15; FBS + disulfiram + CuS0 4 , lanes 4, 8,12,16.
- FBS fetal bovine serum
- Example 8 Metals other than copper can enhance the antiproliferative activity of disulfiram.
- Wilson's disease the inherited disorder of copper overload. See Brewer, et al., J. Am. Coll. Nutr. 9:487-491 (1990); Reeves, et al., J. Nutr. 126:1701-1712 (1996).
- M1619 cells plated and stimulated as above were treated with FBS alone, DMSO vehicle (5 ⁇ l/ml), disulfiram (DS, 0.15 ⁇ M), 5 ⁇ M concentrations of metal salts (cupric sulfate, CuS0 4 ; silver lactate, C 3 H 5 Ag0 3 ; gold chloride,
- Example 9 Disulfiram potentiates the antineoplastic effect of ciplatin and carmustine.
- M1619 melanoma cells were cultured in 10% FBS and RPMI 1640 at a density of 50,000 cells/well in 24 well plates. After 48 hours cisplatin and 2.5 ⁇ M disulfiram or DMSO (5 ⁇ l per ml) were added to medium. After an additional 24 hours, proliferation was quantitated. Each bar represents mean MTT formazan absorbance in a minimum of 4 experiments. M1619 cells were cultured as above with addition of carmustine and 0.6 ⁇ M disulfiram or DMSO (5 ⁇ l per ml) to medium. After 24 hours, proliferation was quantitated.
- Table 2 shows that the combination of disulfiram and cisplatin or disulfiram and carmustine is significantly more antiproliferative against M 1619 cells than cisplatin or carmustine alone. Each number represents mean MTT formazan absorbance in a minimum of 4 experiments. ⁇ p ⁇ 0.05 compared to DMSO vehicle; B p ⁇ 0.01 compared to DMSO vehicle; c p ⁇ 0.001 compared to DMSO vehicle. Disulfiram was more potent as a growth inhibitor of neoplastic cell lines than its sulfhydryl-containing relative PDTC.
- the 50% inhibitor concentration (IC50) against Ml 585 melanoma cells was approximately 1.25 ⁇ M for PDTC but was only 0.3 ⁇ M for disulfiram. This suggests that the active antiproliferative construct of thiocarbamates might be the oxidized dimeric disulfide rather than the reduced thiol-containing monomeric form employed frequently as an antioxidant.
Abstract
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AT99963914T ATE300291T1 (en) | 1999-09-08 | 1999-11-15 | TREATING CANCER WITH TETRAETHYLTHIURAME DISULFIDE |
AU20255/00A AU782029B2 (en) | 1999-09-08 | 1999-11-15 | Method of treating cancer using tetraethyl thiuram disulfide |
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2081094A (en) * | 1980-08-08 | 1982-02-17 | Univ Minnesota | The use of dithiocarbamic compounds in cancer therapy |
US4762705A (en) * | 1981-11-10 | 1988-08-09 | Adolf W. Schwimmer | Cancer therapy with interferon |
JPH04202139A (en) * | 1990-11-30 | 1992-07-22 | Tosoh Corp | Vascularization suppressing agent |
WO1999034763A2 (en) * | 1998-01-11 | 1999-07-15 | Yeda Research And Development Co. Ltd. | Pharmaceutical compositions comprising thiram for inhibiting angiogenesis |
WO1999034784A2 (en) * | 1998-01-11 | 1999-07-15 | Yeda Research And Development Co. Ltd. | Pharmaceutical compositions comprising disulfiram for inhibiting angiogenesis |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4148885A (en) | 1973-09-25 | 1979-04-10 | Institut Merieux | Immunostimulant medicine |
US4594238A (en) | 1980-08-08 | 1986-06-10 | Regents Of University Of Minnesota | Inhibition of undesired effect of platinum compounds |
US4581224A (en) | 1980-08-08 | 1986-04-08 | Regents Of The University Of Minnesota | Inhibition of undesired effect of platinum compounds |
US4645661A (en) | 1984-06-29 | 1987-02-24 | St. Jude Children's Research Hospital | Method for alleviating cisplatin-induced nephrotoxicity and dithiocarbamate compounds for effecting same |
US5035878A (en) | 1988-09-12 | 1991-07-30 | University Of Rochester | Use of dithiocarbamates to counteract myelosuppression |
US5187193A (en) | 1988-09-12 | 1993-02-16 | University Of Rochester | Method for stimulating transplanted bone marrow cells |
WO1993008842A1 (en) | 1991-11-08 | 1993-05-13 | Somatogen, Inc. | Hemoglobins as drug delivery agents |
US5821260A (en) | 1992-10-30 | 1998-10-13 | Emory University | Treatment for atherosclerosis and other cardiovascular and inflammatory diseases |
US5380747A (en) | 1992-10-30 | 1995-01-10 | Emory University | Treatment for atherosclerosis and other cardiovascular and inflammatory diseases |
US5807884A (en) | 1992-10-30 | 1998-09-15 | Emory University | Treatment for atherosclerosis and other cardiovascular and inflammatory diseases |
US5786344A (en) | 1994-07-05 | 1998-07-28 | Arch Development Corporation | Camptothecin drug combinations and methods with reduced side effects |
US5792787A (en) | 1995-06-07 | 1998-08-11 | Emory University | Treatment for atherosclerosis and other cardiovascular and inflammatory diseases |
EP0996440A1 (en) | 1996-11-06 | 2000-05-03 | Morris D. Faiman | Method for treatment of glutamate related disorders |
-
1999
- 1999-09-08 US US09/392,122 patent/US6589987B2/en not_active Expired - Lifetime
- 1999-11-15 AU AU20255/00A patent/AU782029B2/en not_active Expired
- 1999-11-15 EP EP99963914A patent/EP1214063B1/en not_active Expired - Lifetime
- 1999-11-15 WO PCT/US1999/027193 patent/WO2001017522A1/en active IP Right Grant
- 1999-11-15 ES ES99963914T patent/ES2244237T3/en not_active Expired - Lifetime
- 1999-11-15 AT AT99963914T patent/ATE300291T1/en not_active IP Right Cessation
- 1999-11-15 CA CA2384059A patent/CA2384059C/en not_active Expired - Lifetime
- 1999-11-15 DE DE69926407T patent/DE69926407T2/en not_active Expired - Lifetime
- 1999-11-15 JP JP2001521313A patent/JP2003514769A/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2081094A (en) * | 1980-08-08 | 1982-02-17 | Univ Minnesota | The use of dithiocarbamic compounds in cancer therapy |
US4762705A (en) * | 1981-11-10 | 1988-08-09 | Adolf W. Schwimmer | Cancer therapy with interferon |
JPH04202139A (en) * | 1990-11-30 | 1992-07-22 | Tosoh Corp | Vascularization suppressing agent |
WO1999034763A2 (en) * | 1998-01-11 | 1999-07-15 | Yeda Research And Development Co. Ltd. | Pharmaceutical compositions comprising thiram for inhibiting angiogenesis |
WO1999034784A2 (en) * | 1998-01-11 | 1999-07-15 | Yeda Research And Development Co. Ltd. | Pharmaceutical compositions comprising disulfiram for inhibiting angiogenesis |
Non-Patent Citations (8)
Title |
---|
BOROVANSKY J ET AL: "Cytotoxic interactions of Zn2+ in vitro: Melanoma cells are more susceptible than melanocytes", MELANOMA RESEARCH, vol. 7, December 1997 (1997-12-01), pages 449-453, XP000885402 * |
DATABASE WPI Derwent World Patents Index; AN 1992-295332, XP002135748, "Angiogenesis inhibitory agents comprising interleukin- 6 used for treating cancer and inflammation" * |
DORR, ROBERT T.: "A review of the modulation of cisplatin toxicities by chemoprotectants", PLATINUM OTHER MET. COORD. COMPD. CANCER CHEMOTHER. 2, [PROC. INT. SYMP.], 7TH (1996), MEETING DATE 1995, 131-154. EDITOR(S): PINEDO, HERBERT M.;SCHORNAGEL, JAN H. PUBLISHER: PLENUM, NEW YORK, N. Y., XP000885450 * |
HABS, M. ET AL: "Antitumor actitivy of new nitrosoureas on Yoshida sarcoma ascites cells in vivo", FORTSCHR. ONKOL. (1983), 10(CONTROL TUMOUR GROWTH ITS BIOL. BASES), 438-44, XP000885514 * |
MASHIBA H: "Inhibition of Meth-A tumor cell proliferation in combined use of disulfiram with catalase", TOXICOLOGY LETTERS, vol. 61, 1992, pages 75 - 80, XP000882693 * |
SCHIRMER H K A ET AL: "Disulfiram and tumor inhibition", TRANSACTIONS OF AMERICAN ASSOCIATION OF GENITO-URINARY SURGEONS, vol. 58, 1966, pages 63 - 65, XP002108887 * |
STEWART D J: "Phase I study of the combination of disulfiram with cisplatin", J CLIN. ONCOL., vol. 10, no. 6, 1987, pages 517 - 519, XP002108886 * |
TROMBETTA L D: "Protective effects of glutathione on diethyldithiocarbamate (DDC) cytotoxicity: A possible mechanism", TOXICOLOGY AND APPLIED PHARMACOLOGY, vol. 93, 1988, pages 154 - 164, XP000886148 * |
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Also Published As
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AU2025500A (en) | 2001-04-10 |
CA2384059C (en) | 2010-06-22 |
ES2244237T3 (en) | 2005-12-01 |
DE69926407D1 (en) | 2005-09-01 |
EP1214063B1 (en) | 2005-07-27 |
CA2384059A1 (en) | 2001-03-15 |
ATE300291T1 (en) | 2005-08-15 |
AU782029B2 (en) | 2005-06-30 |
EP1214063A1 (en) | 2002-06-19 |
US20030065026A1 (en) | 2003-04-03 |
US6589987B2 (en) | 2003-07-08 |
JP2003514769A (en) | 2003-04-22 |
DE69926407T2 (en) | 2006-05-24 |
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