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WO1993016690A1 - Compositions cytoprotectrices contenant du pyruvate et des antioxydants - Google Patents

Compositions cytoprotectrices contenant du pyruvate et des antioxydants Download PDF

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Publication number
WO1993016690A1
WO1993016690A1 PCT/US1993/000260 US9300260W WO9316690A1 WO 1993016690 A1 WO1993016690 A1 WO 1993016690A1 US 9300260 W US9300260 W US 9300260W WO 9316690 A1 WO9316690 A1 WO 9316690A1
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WO
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Prior art keywords
cytoprotective
fatty acids
pyruvate
pyruvic acid
cells
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Application number
PCT/US1993/000260
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English (en)
Inventor
Alain Martin
Original Assignee
Warner-Lambert Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Warner-Lambert Company filed Critical Warner-Lambert Company
Priority to JP5514824A priority Critical patent/JPH07505145A/ja
Priority to EP93912064A priority patent/EP0627917A1/fr
Publication of WO1993016690A1 publication Critical patent/WO1993016690A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • 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
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/361—Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/365—Hydroxycarboxylic acids; Ketocarboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers

Definitions

  • This invention pertains to cytoprotective compositions for preventing and reducing injury to mammalian cells from a medicament having cytotoxic
  • the cytoprotective composition comprises (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and
  • the cytoprotective composition comprises pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof, (b) an antioxidant, and (c) a
  • the cytoprotective compositions may be incorporated into a wide variety of pharmaceutically acceptable carriers to prepare pharmaceutical products.
  • This invention also relates to methods for preparing and using the cytoprotective compositions and the pharmaceutical products in which the cytoprotective compositions may be used.
  • Cancer is a group of neoplastic diseases affecting different organs and systems in the body.
  • a common feature in all cancers is cellular mutation and abnormal and uncontrolled cell growth usually at a rate greater than that of normal body cells. Neither the etiology of cancer nor the manner in which cancer causes death is completely understood.
  • a problem with the chemotherapeutic treatment of cancer is that normal cells which proliferate rapidly, such as those in bone marrow, hair follicles, and the gastrointestinal tract, are often damaged or killed by 5 the anti-neoplastic agents.
  • This cytotoxicity problem occurs because the metabolism of cancer cells is similar to that of normal cells and anticancer agents lack specificity for cancer cells. Because most of the metabolic differences between normal and neoplastic cells 10 are quantitative, anticancer drugs are usually employed at or near the toxic range in order to obtain satisfactory therapeutic effects.
  • a cytoprotective step is desirable to protect the cells from the cytotoxic agent, resuscitate the injured cells, and help produce new cells to replace the dead cells.
  • Injured cells require low levels of oxygen in the initial stages of recovery to suppress 20 oxidative damage and higher levels of oxygen in the later stages of recovery to stimulate cellular viability and proliferation.
  • lipid peroxidation which involves the oxidative degradation of unsaturated lipids.
  • Lipid peroxidation is highly detrimental to membrane structure and function and can cause numerous cytopathological effects.
  • Cells defend against lipid peroxidation by producing radical scavengers such as superoxide dismutase, catalase, and peroxidase. Injured cells have a decreased ability to produce radical scavengers.
  • Excess hydrogen peroxide can react with DNA to cause backbone breakage, produce mutations, and alter and liberate bases.
  • Hydrogen peroxide can also react with pyrimidines to open the 5, 6-double bond, which reaction inhibits the ability of pyrimidines to hydrogen bond to complementary bases, Hallaender et al. (1971) .
  • Such oxidative biochemical injury can result in the loss of cellular membrane integrity, reduced enzyme activity, changes in transport kinetics, changes in membrane lipid content, and leakage of potassium ions, amino acids, and other cellular material.
  • Antioxidants have been shown to inhibit damage associated with active oxygen species. For example, pyruvate and other alp a-ketoacids have been reported to react rapidly and stoichiometrically with hydrogen peroxide to protect cells from cytolytic effects, O'Donnell-Tormev et al.. J. Exp. Med.. 165, pp. 500-514 (1987) .
  • United States patents nos. 3,920,835, 3,984,556, and 3,988,470, all issued to Van Scott et al.. disclose methods for treating acne, dandruff, and palmar keratosis, respectively, which consist of applying to the affected area a topical composition comprising from about 1% to about 20% of a lower aliphatic compound containing from two to six carbon atoms selected from the group consisting of alpha-hydroxyacids, alpha-ketoacids and esters thereof, and 3-hydroxybutryic acid in a pharmaceutically acceptable carrier.
  • the aliphatic compounds include pyruvic acid and lactic acid.
  • a topical 5 composition comprising from about 1% to about 20% of a - compound selected from the group consisting of amides and ammonium salts of alpha-hydroxyacids, Jeta-hydroxyacids, and alpha-ketoacids in a pharmaceutically acceptable carrier.
  • the compounds include the amides and ammonium 10 salts of pyruvic acid and lactic acid.
  • a topical composition comprising an effective amount of a compound selected from the group consisting of alpha-hydroxyacids, beta-hydroxyacids, and alpha-ketoacids in a pharmaceutically acceptable carrier.
  • the acidic compounds include pyruvic acid and lactic
  • United States patent no. 4,294,852 issued to Wildnauer et al.. discloses a composition for treating skin which comprises the alpha-hydroxyacids, beta- 25 hydroxyacids, and alpha-ketoacids disclosed above by Van Scott et al. in combination with C 3 -C 8 aliphatic alcohols.
  • Veech discloses an electrolyte solution which comprises a mixture of L-lactate and pyruvate in a ratio from 20:1 s to 1:1, respectively, or a mixture of D-Jeta- * hydroxybutyrate and acetoacetate, in a ratio from 6:1 to r 0.5:1, respectively.
  • Sodium pyruvate has been reported to reduce the number of erosions, ulcers, and hemorrhages on the gastric mucosa in guinea pigs and rats caused by acetylsalicylic acid.
  • the analgesic and antipyretic properties of acetylsalicylic acid were not impaired by sodium pyruvate, Puschmann, Arzneistoffforschun ⁇ . 33, pp. 410-415 and 415-416 (1983).
  • Pyruvate has been reported to produce a relative stabilization of left ventricular pressure and work parameter and to reduce the size of infarctions. Pyruvate improves resumption of spontaneous beating of the heart and restoration of normal rates and pressure development, Bun er et al.. J. Mol. Cell. Cardiol. , 18, pp. 423-438 (1986) , Mochizuki et al.. J. Phvsiol. (Paris) . 76, pp. 805-812 (1980) , Re itz et al. , Cardiovasc. Res.. 15, pp. 652-658 (1981) , Giannelli et al. , Ann. Thorac. Sur ⁇ . , 21. pp. 386-396 (1976) .
  • Sodium pyruvate has been reported to act as an antagonist to cyanide intoxification (presumably through the formation of a cyanohydrin) and to protect against the lethal effects of sodium sulfide and to retard the onset and development of functional, morphological, and biochemical measures of acrylamide neuropathy of axons, Schwartz et al.. Toxicol. APPI. Pharmacol.. 50. pp. 437- 442 (1979), Sabri et al.. Brain Res.. 483, pp. 1-11 (1989) .
  • a chemotherapeutic cure of advanced L1210 leukemia has been reported using sodium pyruvate to restore abnormally deformed red blood cells to normal.
  • the deformed red blood cells prevented adequate drug delivery to tumor cells, Cohen, Cancer Chemother. Pharmacol. , 5., pp. 175-179 (1981) .
  • Primary cultures of heterotopic tracheal transplant exposed in vivo to 7, 12-dimethyl- benz(a)anthracene were reported to be successfully maintained in enrichment medium supplemented with sodium pyruvate along with cultures of interleukin-2 stimulated peripheral blood lymphocytes, and plasmacytomas and hybridomas, pig embryos, and human blastocysts, Shacter. J. Immunol. Methods. 99. pp.
  • United States patents nos. 4,158,057, 4,351,835, 4,415,576, and 4,645,764, all issued to Stanko. disclose methods for preventing the accumulation of fat in the liver of a mammal due to the ingestion of alcohol, for controlling weight in a mammal, for inhibiting body fat while increasing protein concentration in a mammal, and for controlling the deposition of body fat in a living being, respectively.
  • the methods comprise administering to the mammal a therapeutic mixture of pyruvate and dihydroxyacetone, and optionally riboflavin.
  • 4,548,937 discloses a method for controlling the weight gain of a mammal which comprises administering to the mammal a therapeutically effective amount of pyruvate, and optionally riboflavin.
  • United States patent no. 4,812,479, issued to Stanko. discloses a method for controlling the weight gain of a mammal which comprises administering to the mammal a therapeutically effective amount of dihydroxyacetone, and optionally riboflavin and pyruvate.
  • Rats fed a calcium-oxalate lithogenic diet including sodium pyruvate were reported to develop fewer urinary calculi (stones) than control rats not given sodium pyruvate, Oqawa et al. , Hinyokika Kiyo. 32, pp. 1341-1347 (1986) .
  • United States patent no. 4,521,375 discloses a method for sterilizing surfaces which come into contact with living tissue. The method comprises sterilizing the surface with aqueous hydrogen peroxide and then neutralizing the surface with pyruvic acid.
  • United States patent no. 4,416,982 issued to Tauda et al.. discloses a method for decomposing hydrogen peroxide by reacting the hydrogen peroxide with a phenol or aniline derivative in the presence of peroxidase.
  • United States patent no. 4,696,917, issued to Lindstrom et al.. discloses an eye irrigation solution which comprises Eagle's Minimum Essential Medium with Earle's salts, chondroitin sulfate, a buffer solution, 2- mercaptoethanol, and a pyruvate.
  • the irrigation solution may optionally contain ascorbic acid and alpha- tocopherol.
  • United States patent no. 4,725,586, issued to Lindstrom et al.. discloses an irrigation solution which comprises a balanced salt solution, chondroitin sulfate, a buffer solution, 2-mercaptoethanol, sodium bicarbonate or dextrose, a pyruvate, a sodium phosphate buffer system, and cystine.
  • the irrigation solution may optionally contain ascorbic acid and gamma-tocopherol.
  • Bissett et al.. discloses a photoprotective composition comprising (a) a sorbohydroxamic acid, (b) an anti- inflammatory agent selected from steroidal anti- inflammatory agents and a natural anti-inflammatory agent, and (c) a topical carrier. Fatty acids may be present as an emollient.
  • United States patent no. 4,847,071, issued to Bissett et al.. discloses a photoprotective composition comprising (a) a tocopherol or tocopherol ester radical scavenger, (b) an anti- inflammatory agent selected from steroidal anti- inflammatory agents and a natural anti-inflammatory agent, and (c) a topical carrier.
  • United States patent no. 4,847,072, issued to Bissett et al.. discloses a topical composition comprising not more than 25% tocopherol sorbate in a topical carrier.
  • United States patent no. 4,533,637 discloses a culture medium which comprises a carbon source, a nucleic acid source precursor, amino acids, vitamins, minerals, a lipophilic nutrient, and serum albumin, and cyclodextrins.
  • the lipophilic substances include unsaturated fatty acids and lipophilic vitamins such as Vitamin A, D, and E. Ascorbic acid may also be present.
  • United Kingdom patent application no. 2,196,348A, to Kovar et al.. discloses a synthetic culture medium which comprises inorganic salts, monosaccharides, amino acids, vitamins, buffering agents, and optionally sodium pyruvate adding magnesium hydroxide or magnesium oxide to the emulsion.
  • the oil phase may include chicken fat.
  • Yu et al. discloses a method for stabilizing a water-in- oil emulsion which comprises adding magnesium hydroxide or magnesium oxide to the emulsion.
  • the oil phase may include chicken fat.
  • Preparat ⁇ on-H T J ' 1 M 1 has been reported to increase the rate of wound healing in artificially created rectal ulcers.
  • the active ingredients in Preparation-HTM are ski ⁇ respiratory factor and shark liver oil, Subramanyam et al.. Digestive Diseases and Sciences, 29, pp. 829-832 (1984) .
  • Robinson. discloses a controlled release treatment composition
  • a treating agent and a bioadhesive agent comprising a water-swellable but water-insoluble, fibrous cross-linked carboxy-functional polymer.
  • European patent application no. 0410696A1 discloses a mucoadhesive delivery system comprising a treating agent and a polyacrylic acid cross- linked with from about 1% to about 20% by weight of a polyhydroxy compound such as a sugar, cyclitol, or lower polyhydric alcohol.
  • compositions While the above therapeutic compositions are reported to inhibit the production of reactive oxygen intermediates, none of the above compositions are entirely satisfactory cytoprotective compositions. None of the compositions has the ability to simultaneously decrease cellular levels of hydrogen peroxide production, increase cellular resistance to cytotoxic agents, increase rates of cellular proliferation, and increase cellular viability to protect and resuscitate mammalian cells.
  • the present invention provides such improved therapeutic cytoprotective compositions without the disadvantages characteristic of previously known compositions.
  • the present invention pertains to cytoprotective compositions for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties.
  • the cytoprotective composition comprises (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof, and (b) an antioxidant.
  • the cytoprotective composition comprises (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cells.
  • the cytoprotective compositions of the present invention may be administered to cells concurrently with a cytotoxic agent.
  • the cytoprotective compositions may also be administered to cells prior to administration of a cytotoxic anticancer agent to selectively protect non- cancerous cells in the presence of cancerous cells.
  • the cytoprotective compositions may be incorporated into a wide variety of pharmaceutically acceptable carriers to prepare pharmaceutical products.
  • This invention also relates to methods for preparing and using the cytoprotective compositions and the pharmaceutical products in which the cytoprotective compositions may be used.
  • Figure 1 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells after 24 hours, as determined by tritiated thymidine incorporation assay, following treatment of the cells with different dosage levels of Doxorubicin.
  • Figure 1, bottom portion is a graph showing the viability of U937 monocytic leukemia tumor cells after 24 hours, as determined by exclusion of the vital dye trypan blue assay, following treatment of the cells with different dosage levels of Doxorubicin.
  • Figure 2 is a graph showing the viability of U937 monocytic leukemia tumor cells after 1 hour, as determined by exclusion of the vital dye trypan blue assay, following treatment of the cells with different dosage levels of Doxorubicin.
  • Figure 3 is a graph showing the viability of U937 monocytic leukemia tumor cells after 24 hours, as determined by tritiated thymidine incorporation assay, following treatment of the cells with the cytoprotective components of the present invention, alone and in combinations, at different dosage levels.
  • Figure 4 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate followed by administration of different dosage levels of Doxorubicin.
  • Figure 4, bottom portion is a graph showing the viability of peripheral blood onocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate followed by administration of different dosage levels of Doxorubicin.
  • Figure 5 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 5, bottom portion is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 6 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 10 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 6, bottom portion is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 10 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 7, top portion is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 7, bottom portion is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 8 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 8, bottom portion is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • FIG. 9 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 10 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 9, bottom portion is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 10 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 10 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 50 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 10, bottom portion is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 50 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 11 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 10 U Vitamin E and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 11, bottom portion is -a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 10 U Vitamin E and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 12 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 12, bottom portion is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E and 0.5% fatty acids followed by administration of different dosage levels of
  • Figure 13 is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate, 10 U Vitamin E, and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 13, bottom portion is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate, 10 U Vitamin E, and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 14 is a graph showing the viability of U937 monocytic leukemia tumor cells in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate, 50 U Vitamin E, and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 14, bottom portion is a graph showing the viability of peripheral blood monocytes in a wash-out study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate, 50 U Vitamin E, and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 15, top portion is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate followed by administration of different dosage levels of Doxorubicin.
  • Figure 15, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate followed by administration of different dosage levels of Doxorubicin.
  • Figure 16 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 16, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 17, top portion is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 10 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 17, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 10 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 18, top portion is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 18, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 19 is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 19, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 20 top portion, is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 10 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 20, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 10 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 21, top portion is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 50 U Vitamin E followed by administration of different dosage levels of Doxorubicin.
  • Figure 21, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate and 50 U Vitamin E followed by administration of different dosage levels of Doxorubici .
  • Figure 22, top portion is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 10 U Vitamin E and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 22, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 10 U Vitamin E and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 23, top portion is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 23, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 50 U Vitamin E and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 24, top portion is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate, 10 U Vitamin E, and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 24, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate, 10 U Vitamin E, and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 25, top portion is a graph showing the viability of U937 monocytic leukemia tumor cells in a co- culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate, 50 U Vitamin E, and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • Figure 25, bottom portion is a graph showing the viability of peripheral blood monocytes in a co-culture study, as determined by tritiated thymidine incorporation assay, after 24 hour pretreatment of the cells with 5 mM sodium pyruvate, 50 U Vitamin E, and 0.5% fatty acids followed by administration of different dosage levels of Doxorubicin.
  • cytoprotective compositions for protecting mammalian cells from a medicament having cytotoxic properties by preventing and reducing injury to the cells.
  • Cells treated with the cytoprotective compositions of the present invention show decreased levels of hydrogen peroxide production, increased resistance to cytotoxic agents, increased rates of proliferation, and increased viability.
  • the cytoprotective compositions may be administered to cells concurrently with a cytotoxic agent or the cytoprotective compositions may be administered to cells prior to administration of a cytotoxic anticancer agent to selectively protect non-cancerous cells in the presence of cancerous cells.
  • infectious cell means a cell which has (a) injured membranes so that transport through the membranes is diminished resulting in an increase in toxins and normal cellular wastes inside the cell and a decrease in nutrients and other components necessary for cellular repair inside the cell, (b) an increase in concentration of oxygen radicals inside the cell because of the decreased ability of the cell to produce antioxidants and enzymes, and (c) damaged DNA, RNA, and ribosomes which must be repaired or replaced before normal cellular functions can be resumed.
  • resuscitation of injured mammalian cells means the reversal of cytotoxicity, the stabilization of the cellular membrane, an increase in the proliferation rate of the cell, and/or the normalization of cellular functions such as the secretion of growth factors, hormones, and the like.
  • cytotoxicity as used herein means a condition caused by a cytotoxic agent that injures the cell. Injured cells do not proliferate because injured cells expend all energy on cellular repair. Aiding cellular repair promotes cellular proliferation.
  • Epidermal keratinocytic cells and monocytic cells have multiple oxygen generating mechanisms and the degree to which each type of mechanism functions differs in each type of cell.
  • monocytes for example, the respiratory bursting process is more pronounced than in epidermal keratinocytes.
  • the components in the cytoprotective compositions of the present invention may vary depending upon the types of cells involved in the condition being treated.
  • the therapeutic cytoprotective composition for treating mammalian cells comprises (a) pyruvate, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids.
  • pyruvate or pyruvic acid
  • pyruvate can be transported inside a cell where it can act as an antioxidant to neutralize oxygen radicals in the cell.
  • Pyruvate can also be used inside the cell in the citric acid cycle to provide energy to increase cellular viability, and as a precursor in the synthesis of important biomolecules to promote cellular proliferation.
  • pyruvate can be used in the multifunction oxidase system to reverse cytotoxicity.
  • Antioxidants especially lipid-soluble antioxidants, can be absorbed into the cell membrane to neutralize oxygen radicals and thereby protect the membrane.
  • the combination of pyruvate inside the cell and an antioxidant in the cellular membrane functions in a synergistic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by use of either type of component alone.
  • the saturated and unsaturated fatty acids in the present invention are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cells.
  • the fatty acids in the cytoprotective composition which may be in the form of mono-, di-, and/or triglycerides or free fatty acids, are readily available for the repair of injured cells and the production of new cells to replace dead cells.
  • Cells injured by oxygen radicals need to produce unsaturated fatty acids to repair cellular membranes.
  • the production of unsaturated fatty acids by cells requires oxygen.
  • the injured cell needs high levels of oxygen to produce unsaturated fatty acids and at the same time needs to reduce the level of oxygen within the cell to reduce oxidative injury.
  • the need of the cell to produce unsaturated fatty acids is reduced and the need for high oxygen levels is also reduced.
  • the presence of mixtures of saturated and unsaturated fatty acids in the cytoprotective composition significantly enhances the ability of pyruvate and the antioxidant to inhibit reactive oxygen production.
  • unsaturated fatty acids By stabilizing the cellular membrane, unsaturated fatty acids also improve membrane function and enhance pyruvate transport into the cell.
  • unsaturated fatty acids also improve the repair of cellular membranes rate of the cells.
  • the three components in the cytoprotective composition function together in a synergistic manner to prevent and reduce injury to mammalian cells, increase the resuscitation rate of injured cells, and increase the production of new cells.
  • the therapeutic cytoprotective composition for treating mammalian cells comprises (a) pyruvate, (b) lactate, and (c) a mixture of saturated and unsaturated fatty acids.
  • lactate is employed instead of an antioxidant. Antioxidants react with, and neutralize, oxygen radicals after the radicals are already formed. Lactate, on the other hand, is a component in the cellular feedback mechanism and inhibits the respiratory bursting process to suppress the production of active oxygen species.
  • the combination of pyruvate to neutralize active oxygen species and lactate to suppress the respiratory bursting process functions in a synergistic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by use of either type of component alone.
  • the presence of mixtures of saturated and unsaturated fatty acids in the cytoprotective composition significantly enhances the ability of pyruvate and lactate to inhibit reactive oxygen production.
  • the three components in the cytoprotective composition in this embodiment function together in a synergistic manner to prevent and reduce injury to mammalian cells, and increase the proliferation and resuscitation rate of the cells.
  • the therapeutic cytoprotective composition for treating mammalian cells comprises (a) an antioxidant, and (b) a mixture of saturated and unsaturated fatty acids.
  • the presence of mixtures of saturated and unsaturated fatty acids in the cytoprotective composition in this embodiment significantly enhances the ability of the antioxidant to inhibit reactive oxygen production.
  • the combination of an antioxidant to neutralize active oxygen species and fatty acids to rebuild cellular membranes and reduce the need of the cell for oxygen functions in a synergistic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by either type of component alone.
  • the components in the cytoprotective composition in this embodiment function together in a synergistic manner to prevent and reduce injury to mammalian cells, and increase the proliferation and resuscitation rate of the cells.
  • the therapeutic cytoprotective composition for treating mammalian cells comprises (a) lactate, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids.
  • lactate is employed because the respiratory bursting process is more pronounced in monocytes than in epidermal keratinocytes.
  • the combination of lactate to suppress the respiratory bursting process and an antioxidant to neutralize active oxygen species functions in a synergistic manner to reduce hydrogen peroxide production in the cell to levels lower than can be achieved by either component alone.
  • the presence of mixtures of saturated and unsaturated fatty acids in the cytoprotective composition in this embodiment significantly enhances the ability of lactate and the antioxidant to inhibit reactive oxygen production.
  • the three components in the cytoprotective composition in this embodiment function together in a synergistic manner to prevent and reduce injury to mammalian cells, and increase the proliferation and resuscitation rate of the cells.
  • the therapeutic cytoprotective composition for treating mammalian cells comprises (a) pyruvate, and (b) an antioxidant.
  • the combination of pyruvate inside the cell and the antioxidant in the cellular membrane functions in a synergistic manner to reduce hydrogen peroxide production in the cell and thereby prevent injury to the cell.
  • the mixture of saturated and unsaturated fatty acids need not be employed in the cytoprotective composition.
  • the two components in the cytoprotective composition in this embodiment function together in a synergistic manner to prevent and reduce injury to mammalian cells.
  • the combination of ingredients set out in the above embodiments functions together in an enhanced manner to prevent and reduce injury to mammalian cells and to increase the proliferation and resuscitation rate of mammalian cells.
  • the therapeutic effect of the combination of the components in each of the above embodiments is markedly greater than that expected by the mere addition of the individual therapeutic components.
  • applicant's therapeutic cytoprotective compositions have the ability to decrease intracellular levels of hydrogen peroxide production, increase cellular resistance to cytotoxic agents, increase rates of cellular proliferation, and increase cellular viability.
  • the cells which may be treated with the cytoprotective compositions in the present invention are mammalian cells. Although applicant will describe the present cytoprotective compositions as useful for treating mammalian epidermal keratinocytes and mammalian monocytes, applicant contemplates that all mammalian cells which may be protected or resuscitated by applicant's cytoprotective compositions may be used in the present invention.
  • Keratinocytes are representative of normal mammalian cells and are the fastest proliferating cells in the body. The correlation between the reaction of keratinocytes to injury and therapy and that of mammalian cells in general is very high.
  • Monocytes are representative of specialized mammalian cells such as the white blood cells in the immune system and the organ cells in liver, kidney, heart, and brain. The mammalian cells may be treated in vivo and in vitro .
  • Epidermal keratinocytes are the specialized epithelial cells of the epidermis which synthesize keratin, a scleroprotein which is the principal constituent of epidermis, hair, nails, horny tissue, and the organic matrix of the enamel of teeth.
  • Mammalian epidermal keratinocytes constitute about 95% of the epidermal cells and together with melanocytes form the binary system of the epidermis. In its various successive stages, epidermal keratinocytes are also known as basal cells, prickle cells, and granular cells.
  • Monocytes are mononuclear phagocytic leukocytes which undergo respiratory bursting and are involved in reactive oxygen mediated damage within the epidermis.
  • Leukocytes are white blood cells or corpuscles which may be classified into two main groups: granular leukocytes (granulocytes) which are leukocytes with abundant granules in the cytoplasm and nongranular leukocytes (nongranulocytes) which are leukocytes without specific granules in the cytoplasm and which include the lymphocytes and monocytes.
  • Phagocyte cells are cells which ingest microorganisms or other cells and foreign particles.
  • Monocytes are also known as large mononuclear leukocytes, and hyaline or transitional leukocytes.
  • Pyruvic acid (2-oxopropanoic acid, alpha- ketopropionic acid, CH 3 COCOOH) or pyruvate (at physiological pH) is a fundamental intermediate in protein and carbohydrate metabolism and in the citric acid cycle.
  • the citric acid cycle (tricarboxylic acid cycle, Kreb's cycle) is the major reaction sequence which executes the reduction of oxygen to generate adenosine triphosphate (ATP) by oxidizing organic compounds in respiring tissues to provide electrons to the transport system.
  • ATP adenosine triphosphate
  • Acetyl coenzyme A (“active acetyl”) is oxidized in this process and is thereafter utilized in a variety of biological processes and is a precursor in the biosynthesis of many fatty acids and sterols.
  • the two major sources of acetyl coenzyme A are derived from the metabolism of glucose and fatty acids. Glycolysis consists of a series of transformations wherein each glucose molecule is transformed in the cellular cytoplasm into two molecules of pyruvic acid. Pyruvic acid may then enter the mitochondria where it is oxidized by coenzyme A in the presence of enzymes and cofactors to acetyl coenzyme A. Acetyl coenzyme A can then enter the citric acid cycle.
  • pyruvic acid derived from glycogen
  • Lactic acid is reoxidized and partially retransformed to glycogen during rest.
  • Pyruvate can also act as an antioxidant to neutralize oxygen radicals in the cell and can be used in the multifunction oxidase system to reverse cytotoxicity.
  • the pyruvate in the present invention may be selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof.
  • the pharmaceutically acceptable salts of pyruvic acid may be alkali salts and alkaline earth salts.
  • the pyruvate is selected from the group consisting of pyruvic acid, sodium pyruvate, potassium pyruvate, magnesium pyruvate. calcium pyruvate, zinc pyruvate, manganese pyruvate, and mixtures thereof.
  • the pyruvate is selected from the group of salts consisting of sodium pyruvate, potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate, and mixtures thereof. Most preferably, the pyruvate is sodium pyruvate.
  • the amount of pyruvate present in the cytoprotective compositions of the present invention is a therapeutically effective amount.
  • a therapeutically effective amount of pyruvate is that amount of pyruvate necessary to increase the proliferation and resuscitation rate of mammalian cells.
  • the exact amount of pyruvate is a matter of preference subject to such factors as the type of condition being treated as well as the other ingredients in the composition.
  • pyruvate is preferably present in the cytoprotective composition in an amount from about 10% to about 75%, preferably from about 20% to about 60%, and more preferably from about 25% to about 55%, by weight of the cytoprotective composition.
  • pyruvate is preferably present in the cytoprotective composition in an amount from about 10% to about 50%, preferably from about 20% to about 45%, and more preferably from about 25% to about 40%, by weight of the cytoprotective composition.
  • (+) alpha-hydroxypropionic acid, CH 3 CHOHCOOH) or lactate occurs in small quantities in the blood and muscle fluid of mammals. Lactic acid concentration increases in muscle and blood after vigorous activity. Lactate is a component in the cellular feedback mechanism and inhibits the natural respiratory bursting process of cells thereby suppressing the production of oxygen radicals.
  • the lactate in the present invention may be selected from the group consisting of lactic acid, pharmaceutically acceptable salts of lactic acid, and mixtures thereof.
  • the pharmaceutically acceptable salts of lactic acid may be alkali salts and alkaline earth salts.
  • the lactate is selected from the group consisting of lactic acid, sodium lactate, potassium lactate, magnesium lactate, calcium lactate, zinc lactate, manganese lactate, and mixtures thereof. More preferably, the lactate is selected from the group consisting of lactic acid, sodium lactate, potassium lactate, magnesium lactate, calcium lactate, zinc lactate, manganese lactate, and mixtures thereof. Most preferably, the lactate is lactic acid.
  • the amount of lactate present in the cytoprotective compositions of the present invention is a therapeutically effective amount.
  • a therapeutically effective amount of lactate is that amount of lactate necessary to increase the proliferation and resuscitation rate of mammalian cells.
  • a therapeutically effective amount of lactate is that amount necessary to suppress the respiratory bursting process of white blood cells to protect and resuscitate the mammalian cells.
  • a therapeutically effective amount of lactate in a composition is from about 5 to about 10 times the amount of lactate normally found in serum.
  • the exact amount of lactate is a matter of preference subject to such factors as the type of condition being treated as well as the other ingredients in the composition.
  • lactate is present in the cytoprotective composition in an amount from about 10% to about 50%, preferably from about 20% to about 45%, and more preferably from about 25% to about 40%, by weight of the cytoprotective composition.
  • Antioxidants are substances which inhibit oxidation or suppress reactions promoted by oxygen or peroxides. Antioxidants, especially lipid-soluble antioxidants, can be absorbed into the cellular membrane to neutralize oxygen radicals and thereby protect the membrane.
  • the antioxidants useful in the present invention may be selected from the group consisting of Vitamin A (retinol) , vitamin A 2 (3, 4-didehydroretinol) , all forms of carotene such as alpha-carotene, beta- carotene (beta, Jeta-carotene) , gramma-carotene, delta- carotene, Vitamin C (ascorbic acid, L-ascorbic acid) , all forms of tocopherol such as Vitamin E (alpha-tocopherol, 3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri- decyl)-2H-l-benzopyran-6-ol) , Jeta-tocopherol,
  • the amount of antioxidant present in the cytoprotective compositions of the present invention is a therapeutically effective amount.
  • a therapeutically effective amount of antioxidant is that amount of antioxidant necessary to increase the proliferation and resuscitation rate of mammalian cells.
  • the exact amount of antioxidant is a matter of preference subject to such factors as the type of condition being treated as well as the other ingredients in the composition.
  • the antioxidant is preferably present in the cytoprotective composition in an amount from about 10% to about 75%, preferably from about 20% to about 60%, and more preferably from about 25% to about 55%, by weight of the cytoprotective composition.
  • the antioxidant is preferably is present in the cytoprotective composition in an amount from about 10% to about 50%, preferably from about 20% to about 45%, and more preferably from about 25% to about 40%, by weight of the cytoprotective composition.
  • the mixture of saturated and unsaturated fatty acids in the present invention are those fatty acids required for the repair of mammalian cellular membranes and the production of new cells. Hence, the fatty acids are readily incorporated into the cell and are immediately available for the repair of injured cells and the proliferation of new cells.
  • the presence of the mixtures of saturated and unsaturated fatty acids in the cytoprotective compositions significantly enhances the ability of pyruvate, lactate, and the antioxidant to inhibit reactive oxygen production.
  • Fatty acids are carboxylic acid compounds found in animal and vegetable fat and oil. Fatty acids are classified as lipids and are composed of chains of alkyl groups containing from 4 to 22 carbon atoms and 0-3 double bonds and characterized by a terminal carboxyl group, -C00H. Fatty acids may be saturated or unsaturated and may be solid, semisolid, or liquid. The most common saturated fatty acids are butyric acid (C4) , lauric acid (C ⁇ 2 ) 1 palmitic acid (C_ ⁇ ) , and stearic acid (C ⁇ g) • Unsaturated fatty acids are usually derived from vegetables and consist of alkyl chains containing from 16 to 22 carbon atoms and 0-3 double bonds with the characteristic terminal carboxyl group. The most common unsaturated fatty acids are oleic acid, linoleic acid, and linolenic acid (all C_ Q acids) .
  • the mixture of saturated and unsaturated fatty acids required for the repair of mammalian cellular membranes in the present invention may be derived from animal fats and waxes.
  • Cells produce the chemical components and the energy required for cellular viability and store excess energy in the form of fat.
  • Fat is adipose tissue stored between organs of the body to furnish a reserve supply of energy.
  • the preferred animal fats and waxes have a fatty acid composition similar to that of human fat and the fat contained in human breast milk.
  • the preferred animal fats and waxes may be selected from the group consisting of human fat, chicken fat, cow fat (defined herein as a bovine domestic animal regardless of sex or age) , sheep fat, horse fat, pig fat, and whale fat.
  • the more preferred animal fats and waxes may be selected from the group consisting of human fat and chicken fat.
  • the most preferred animal fat is human fat.
  • Mixtures of other fats and waxes, such as vegetable waxes, marine oils (especially shark liver oil) , and synthetic waxes and oils, which have a fatty acid composition similar to that of animal fats and waxes, and preferably to that of human fats and waxes, may also be employed.
  • the mixture of saturated and unsaturated fatty acids may also be derived from animal and vegetable fats and waxes, and mixtures thereof.
  • the mixture of saturated and unsaturated fatty acids has a composition similar to that of human fat and comprises the following fatty acids: butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myri ⁇ tic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gaddoleic acid.
  • butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gaddoleic acid are present in the mixture in about the following percentages by weight, respectively (carbon chain number and number of unsaturations are shown parenthetically, respectively) : 0.2%-0.
  • the mixture of saturated and unsaturated fatty acids is typically chicken fat comprising the following fatty acids: lauric acid, myristic acid, myristoleic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid, margaroleic acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gaddoleic acid.
  • lauric acid, myristic acid, myristoleic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid, margaroleic acid, stearic, oleic acid, linoleic acid, linolenic acid, arachidic acid, and gaddoleic acid are present in the mixture in about the following percentages by weight, respectively: 0.1% (C 12 ), 0.8% (C 14 ), 0.2% (C 14:1 ), 0.1% (C 15 ) , 25.3% (C 16 ) , 7.2% (C 16:1 ), 0.1% (C 17 ), 0.1% (C 17:1 ), 6.5% (C 18 ) , 37.7% ( c 18: l )- 20.6% (C 18:2 ), 0.8% (C 18:3 ), 0.2% (C 20 ) , and 0.3% (C 2 o:i) / all percentages +/- 10%.
  • fatty acids and percentages thereof present in the fatty acid mixture are given as an example.
  • the exact type of fatty acid present in the fatty acid mixture and the exact amount of fatty acid employed in the fatty acid mixture may be varied in order to obtain the result desired in the final product and such variations are now within the capabilities of those skilled in the art without the need for undue experimentation.
  • the amount of fatty acids present in the cytoprotective compositions of the present invention is a therapeutically effective amount.
  • a therapeutically effective amount of fatty acids is that amount of fatty acids necessary to increase the repair of cellular membranes and resuscitation rate of mammalian cells.
  • the exact amount of fatty acids employed is subject to such factors as the type and distribution of fatty acids employed in the mixture, the type of condition being treated, and the other ingredients in the composition.
  • the fatty acids are preferably present in the cytoprotective composition in an amount from about 10% to about 75%, preferably from about 20% to about 60%, and more preferably from about 25% to about 55%, by weight of the cytoprotective composition.
  • the fatty acids are preferably present in the cytoprotective composition in an amount from about 10% to about 50%, preferably from about 20% to about 45%, and more preferably from about 25% to about 40%, by weight of the cytoprotective composition.
  • the therapeutic cytoprotective compositions for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties may be selected from the group consisting of:
  • pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • lactate selected from the group consisting of lactic acid, pharmaceutically acceptable salts of lactic acid, and mixtures thereof;
  • the cytoprotective compositions for treating mammalian cells may be selected from the group consisting of: (1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • lactate selected from the group consisting of lactic acid, pharmaceutically acceptable salts of lactic acid, and mixtures thereof;
  • the cytoprotective compositions for treating mammalian cells may be selected from the group consisting of:
  • pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and (b) an antioxidant.
  • the cytoprotective compositions for treating mammalian cells comprise: (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • a cytoprotective composition is made by forming an admixture of the components of the composition.
  • a cytoprotective composition is made by forming an admixture of (a) a pyruvate, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids.
  • a cytoprotective composition is made by forming an admixture of (a) a pyruvate, (b) a lactate, and (c) a mixture of saturated and unsaturated fatty acids.
  • a cytoprotective composition is made by forming an admixture of (a) an antioxidant, and (b) a mixture of saturated and unsaturated fatty acids.
  • a cytoprotective composition is made by forming an admixture of (a) a lactate, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids.
  • a cytoprotective composition is made by forming an admixture of (a) a pyruvate, and (b) an antioxidant.
  • the admixture may be formed in a solvent such as water. If necessary, the pH of the solvent is adjusted to a range from about 3.5 to about 8.0, and preferably from about 4.5 to about 7.5, and more preferably about 6.0 to about 7.4. The admixture is then sterile filtered. Other ingredients may also be incorporated into the cytoprotective composition as dictated by the nature of the desired composition as well known by those having ordinary skill in the art. The ultimate cytoprotective compositions are readily prepared using methods generally known in the pharmaceutical arts.
  • the inventive therapeutic cytoprotective compositions may be stored for future use or may be formulated in effective amounts with a cytotoxic agent to form cytoprotective pharmaceutical compositions.
  • the combination of the cytoprotective compositions of the present invention and the medicament cytotoxic to cells provides a cytoprotective pharmaceutical composition having the ability to prevent and reduce injury to mammalian cells from the cytotoxic medicament and increase the resuscitation rate of injured mammalian cells.
  • the dose level of the cytotoxic medicament in the cytoprotective pharmaceutical composition may thereby be raised to higher than normal levels.
  • the cytotoxic agents which may be used in the cytoprotective pharmaceutical compositions of the present invention may be selected from a wide variety of medicaments.
  • medicaments taken on a long term regimen tend to cause liver, kidney, tissue, and other toxicity problems.
  • certain cytotoxic medicaments such as potent chemotherapeutic medicaments used to treat malignant tissues, are believed to stimulate release of significant amounts of reactive oxygen species by mammalian tissues which can cause oxidative injury.
  • Combination of the cytoprotective compositions of the present invention with such cytotoxic medicaments may inhibit induction of reactive oxygen production while simultaneously decreasing side effects of such medicaments. By decreasing the side effects of such medicaments, the dosage levels of the medicaments may be increased thereby increasing the therapeutic effect of the medicaments.
  • the cytoprotective compositions may be used in topical cytoprotective pharmaceutical compositions in combination with cytotoxic medicaments such as epithelial cell cohesiveness reducers such as tretinoin (Retin A) , dermatological abradants, and anti-inflammatories, to protect and enhance the resuscitation rate of the injured mammalian cells.
  • cytotoxic medicaments such as epithelial cell cohesiveness reducers such as tretinoin (Retin A) , dermatological abradants, and anti-inflammatories, to protect and enhance the resuscitation rate of the injured mammalian cells.
  • the cytoprotective compositions may also be used in ingestible cytoprotective pharmaceutical compositions in combination with medicaments that cause cytotoxic side effects such as anti-tumor, anti-viral, and antibacterial medicaments including the lipid regulating agents gemfibrozil and lovastatin, centrally acting anticholinesterases such as tacrine, chemotherapeutic medicaments such as the anthracycline antibiotic doxorubicin, gastric irritants such as acetylsalicylic acid and ibuprofen, to protect and enhance the resuscitation rate of the injured mammalian cells.
  • medicaments that cause cytotoxic side effects such as anti-tumor, anti-viral, and antibacterial medicaments including the lipid regulating agents gemfibrozil and lovastatin, centrally acting anticholinesterases such as tacrine, chemotherapeutic medicaments such as the anthracycline antibiotic doxorubicin, gastric irritants such as acetyls
  • the medicament having cytotoxic properties in the cytoprotective pharmaceutical compositions is selected from the group consisting of doxorubicin, gemfibrozil, lovastatin, and tacrine.
  • the medicament having cytotoxic properties in the cytoprotective pharmaceutical compositions is selected from the group consisting of doxorubicin, gemfibrozil, and tacrine.
  • the medicament having cytotoxic properties is doxorubicin.
  • Doxorubicin (Adriamycin) is a cytotoxic anthracycline antibiotic reported to produce regression in disseminated neoplastic conditions such as in various leukemias, tumors, neuroblastomas, sarcomas, and carcinomas.
  • Gemfibrozil is a lipid regulating agent which lowers elevated serum lipids primarily by decreasing serum triglyceride with a variable reduction in total serum cholesterol.
  • Lovastatin is a cholesterol lowering agent which inhibits the enzymatic biosynthesis of cholesterol.
  • Tacrine (Cognex, 1,2,3,4-tetrahydro-9-acridinamine) is a centrally active anticholinesterase useful as a cognition activator. Tacrine has undergone clinical trials for use in treating severe Alzheimer's disease (presenile dementia) .
  • the medicament having cytotoxic properties in the cytoprotective pharmaceutical compositions is an anticancer agent.
  • anticancer agents include chemically reactive drugs having nonspecific action, anti-metabolites, antibiotics, plant products, hormones, and other miscellaneous chemotherapeutic agents.
  • Chemically reactive drugs having nonspecific action include alkylating agents and N-alkyl-N-nitroso compounds. Examples of alkylating agents include nitrogen mustards, azridines (ethylenimines) , sulfonic acid esters, and epoxides.
  • Anti-metabolites are compounds that interfere with the formation or utilization of a normal cellular metabolite and include amino acid antagonists, vitamin and coenzyme antagonists, and antagonists of metabolites involved in nucleic acid synthesis such as glutamine antagonists, folic acid antagonists, pyrimidine antagonists, and purine antagonists.
  • Antibiotics are compounds produced by microorganisms that have the ability to inhibit the growth of other organisms and include actinomycins and related antibiotics, glutarimide antibiotics, sarkomycin, fumagillin, streptonigrin, tenuazonic acid, actinogan, peptinogan, and anthracyclic antibiotics such as doxorubicin.
  • Plant products include colchicine, podophyllotoxin, and vinca alkaloids.
  • Hormones include those steroids used in breast and prostate cancer and corticosteroids used in leukemias and lymphomas.
  • Other miscellaneous chemotherapeutic agents include urethan, hydroxyurea, and related compounds; thiosemicarbazones and related compounds; phthalanilide and related compounds; and triazenes and hydrazines.
  • the anticancer agent is an antibiotic.
  • the anticancer agent is doxorubicin.
  • the anticancer agent is doxorubicin.
  • the invention is directed at a cytoprotective pharmaceutical composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties which comprises:
  • a cytoprotective composition selected from the group consisting of: (1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • the cytoprotective compositions of the present invention may be combined in an immediate release form with an anticancer medicament having cytotoxic properties in a timed release form to provide a timed release cytoprotective pharmaceutical composition.
  • the timed release composition releases the cytoprotective composition substantially immediately and releases the cytotoxic chemotherapeutic medicament after a suitable period of time, for example from one to 24 hours after releasing the cytoprotective composition, to selectively protect non-cancerous cells in the presence of cancerous cells against the cytotoxic chemotherapeutic medicament.
  • Cancer cells unlike normal cells or benign tumor cells, exhibit the properties of invasion and metastasis and are highly anaplastic.
  • the invention is direct at a timed-release cytoprotective pharmaceutical composition for selectively protecting non-cancerous mammalian cells in the presence of cancerous mammalian cells from an anticancer medicament having cytotoxic properties which comprises:
  • a cytoprotective composition in immediate release form selected from the group consisting of: (1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • an anticancer medicament having cytotoxic properties in timed-release form; wherein the cytoprotective composition is released substantially immediately and the anticancer medicament is released after a period of time sufficient such that the cancerous cells have substantially metabolized the cytoprotective composition and the non- cancerous cells have not substantially metabolized the cytoprotective composition.
  • a suitable or sufficient period of time is that period of time wherein the cancerous cells have substantially metabolized the cytoprotective composition and the non-cancerous cells have not substantially metabolized the cytoprotective composition.
  • the period of time should not be so long that the non-cancerous cells substantially metabolize the cytoprotective composition and are unprotected.
  • the exact time is subject to such factors as the type and quantity of cytoprotective composition employed, the medicament having cytotoxic properties used, and the type of cancerous cells and non-cancerous cells being treated.
  • the period of time may be varied in order to obtain the result desired and such variations are within the capabilities of those skilled in the art without the need for undue experimentation.
  • the present invention extends to methods for making the cytoprotective pharmaceutical composition.
  • a cytoprotective pharmaceutical composition is made by forming an admixture of the components of the composition.
  • the cytoprotective compositions may be prepared using standard techniques and equipment known to those skilled in the art.
  • the apparatus useful in accordance with the present invention comprises apparatus well known in the chemical and biochemical arts, and therefore the selection of the specific apparatus will be apparent to the artisan.
  • a cytoprotective pharmaceutical composition is made by forming an admixture of the cytoprotective composition and the medicament having cytotoxic properties.
  • a timed-release cytoprotective pharmaceutical composition is made by forming an admixture of the cytoprotective composition in immediate release form and the anticancer medicament having cytotoxic properties . in timed-release form.
  • the present invention extends to methods for using the therapeutic cytoprotective compositions.
  • the cytoprotective compositions of the present invention may be administered to cells concurrently with a cytotoxic medicament.
  • the cytoprotective compositions of the present invention may be administered to cells prior to the administration of a cytotoxic anticancer medicament to selectively protect non-cancerous cells in the presence of cancerous cells against the anticancer agent.
  • the invention is directed at a method for protecting mammalian cells from a medicament having cytotoxic properties which comprises the steps of:
  • cytoprotective composition selected from the group consisting of: (1) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • step (C) administering the cytoprotective composition from step (A) and the medicament from step (B) concurrently to mammalian cells to protect the mammalian cells from the medicament having cytotoxic properties.
  • the invention is direct at a method for selectively protecting non- cancerous mammalian cells in the presence of cancerous mammalian cells from an anticancer medicament having cytotoxic properties which comprises the steps of:
  • pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • an antioxidant an antioxidant
  • step (C) administering the cytoprotective composition from step (A) and the medicament from step (B) concurrently to mammalian cells to selectively protect non-cancerous mammalian cells in the presence of cancerous mammalian cells from the anticancer medicament having cytotoxic properties; wherein the cytoprotective composition is released substantially immediately and the anticancer medicament is released after a period of time sufficient such that the cancerous cells have substantially metabolized the cytoprotective composition and the non- cancerous cells have not substantially metabolized the cytoprotective composition.
  • the invention is direct at a method for selectively protecting non-cancerous mammalian cells in the presence of cancerous mammalian cells from an anticancer medicament having cytotoxic properties which comprises the steps of:
  • a cytoprotective composition to prevent and reduce injury to the mammalian cells selected from the group consisting of:
  • pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • fatty acids are those fatty acids required for the resuscitation of injured mammalian cells; and (2) (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof; and
  • cytoprotective compositions of the present invention Methods for administering the cytoprotective compositions of the present invention to mammalian cells will vary depending upon the particular condition being treated and the cytotoxic agent employed. In general, the cytoprotective compositions will be administered in the same manner as the cytotoxic agent. Of course, the type of carrier will vary depending upon the mode of administration desired for the pharmaceutical composition as is conventional in the art.
  • the cytoprotective compositions of the present invention may be administered parenterally, in the form of sterile solutions or suspensions, such as intravenously, intramuscularly, or subcutaneously.
  • the cytoprotective compositions may also be administered topically.
  • Non-oral topical compositions employ non-oral topical vehicles, such as oils, petrolatum bases, emulsions, lotions, creams, gel formulations, foams, ointments, sprays, salves, and films, which are intended to be applied to the skin or body cavity and are not intended to be taken by mouth.
  • Oral topical compositions employ oral vehicles, such as outhwashes, rinses, oral sprays, suspensions, bioadhesives, and dental gels, which are intended to be taken by mouth but are not intended to be ingested.
  • the cytoprotective compositions may also be administered orally, in the form of pills, tablets, capsules, troches, and the like, as well as sublingually, rectally, or transcutaneously with a suitable pharmaceutically acceptable carrier for that particular mode of administration as is conventional in the art.
  • dosage unit forms refers to physically discrete units suitable for use as a unitary dosage, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier.
  • the cytoprotective compositions of the present invention may be incorporated into a sterile solution or suspension. These preparations should contain at least about 0.1% of the inventive composition, by weight, but this amount may be varied to between about 0.1% and about 50% of the inventive composition, by weight of the parental composition. The exact amount of the inventive composition present in such compositions is such that a suitable dosage level will be obtained. Preferred compositions and preparations according to the present invention are prepared so that a paranteral dosage unit contains from between about 0.5 milligrams to about 100 milligrams of the inventive composition.
  • Suitable carriers include propylene glycol- alcohol-water, isotonic water, sterile water for injection (USP) , emulphorTM-alcohol-water, cremophor-EL or other suitable carriers known to those skilled in the art.
  • the sterile solutions or suspensions may also include the following adjuvants: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium metabisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA) ; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.
  • the parental preparations may be enclosed in ampules, disposable syringes, or multiple
  • the therapeutic cytoprotective composition is incorporated into a non-oral topical vehicle which may be in the form of oils, petrolatum bases, emulsions, lotions, creams, gels formulations, foams, ointments, sprays, salves, and films, and the like.
  • Non-oral topical vehicles include water and pharmaceutically acceptable water-miscible organic solvents such as ethyl alcohol, isopropyl alcohol, propylene glycol, glycerin, and the like, and mixtures of these solvents.
  • Typical non-toxic non-oral topical vehicles known in the pharmaceutical arts may be used.
  • the non-oral topical cytoprotective compositions may also contain conventional additives employed in those products. Conventional additives include humectants, emollients, lubricants, stabilizers, dyes, and perfumes, providing the additives do not interfere with the therapeutic properties of the cytoprotective composition.
  • the cytoprotective composition is incorporated into an oral topical vehicle which may be in the form of a mouthwash, rinse, oral spray, suspension, dental gel, bioadhesive, and the like.
  • Typical non-toxic oral vehicles known in the pharmaceutical arts may be used in the present invention.
  • the preferred oral vehicles are water, ethanol, and water-ethanol mixtures.
  • the water-ethanol mixtures are generally employed in a weight ratio from about 1:1 to about 20:1, preferably from about 3:1 to about 20:1, and most preferably from about 3:1 to about 10:1, respectively.
  • the pH value of the oral vehicle is generally from about 4 to about 7, and preferably from about 5 to about 6.5.
  • the oral topical cytoprotective compositions may also contain conventional additives normally employed in those products.
  • Conventional additives include a fluorine providing compound, a sweetening agent, a flavoring agent, a coloring agent, a humectant, a buffer, and an emulsifier, providing the additives do not interfere with the therapeutic properties of the cytoprotective composition.
  • cytoprotective compositions of the present invention may be admixed with a topical vehicle to form a topical cytoprotective composition.
  • topical cytoprotective compositions will comprise the cytoprotective composition in an amount from about 0.1% to about 10% and a topical vehicle in a quantity sufficient to bring the total amount of composition to 100%, by weight of the topical cytoprotective composition.
  • the topical cytoprotective compositions will comprise the cytoprotective composition in an amount from about 0.1% to about 10%, and in a most preferred embodiment, the topical cytoprotective compositions will comprise the cytoprotective composition in an amount from about 0.1% to about 8%, and a topical vehicle in a quantity sufficient to bring the total amount of composition to 100%, by weight of the topical cytoprotective composition.
  • the present invention extends to methods for preparing the topical cytoprotective compositions.
  • the topical cytoprotective composition is prepared by admixing a therapeutically effective amount of the cytoprotective composition of the present invention, the cytotoxic agent, and a topical vehicle.
  • the final compositions are readily prepared using standard methods and apparatus generally known by those skilled in the pharmaceutical arts.
  • the apparatus useful in accordance with the present invention comprises mixing apparatus well known in the pharmaceutical arts, and therefore the selection of the specific apparatus will be apparent to the artisan.
  • the invention is directed at a cytoprotective pharmaceutical composition which comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of a cytoprotective composition for preventing and reducing injury to mammalian cells from a medicament having cytotoxic properties, wherein the cytoprotective composition is selected from the group consisting of:
  • pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • the invention is directed at a method for preparing a cytoprotective pharmaceutical composition for protecting mammalian cells from a medicament having cytotoxic properties which comprises the steps of:
  • pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof;
  • step (C) admixing the cytoprotective composition from step (A) and the pharmaceutically acceptable carrier from step (B) to form a pharmaceutical composition.
  • Peripheral blood was obtained from a normal healthy volunteer by venipuncture using an EDTA- containing Vacutainer (Becton Dickinson Mountain View, Ca.). A total of 10 ml of peripheral blood was mixed in a ratio of 1:1 with Dulbecco's Minimal Essential Medium
  • the mixture was divided into 2 ml portions and each portion was layered onto 6 ml of Ficoll-Hypaque gradient mixture (Pharmacy, Inc., Piscataway, N.J.) and centrifuged in a Beckman T-J6 refrigerated centrifuge for 30 minutes at 1500 rpm and 4° C. After the cells were washed twice with phosphate buffered saline, the cells were resuspended in Hank's Balanced Salt Solution without
  • Peripheral blood monocytes and U937 monocytic leukemia tumor cells were placed in sterile culture flasks and maintained in culture using Dulbecco's Minimal Essential Medium, with 10% fetal calf serum, supplemented with 2mM glutamine and Pen/Strep.
  • the cytotoxicity of the cytotoxic agent on the cells was analyzed by propidium iodide exclusion techniques and flow cytometric quantitation. Viability of the cells was quantified as the number of cells that excluded the vital dye trypan blue.
  • Sodium pyruvate was dissolved in distilled water and the solution was adjusted to pH 7.4 with IN sodium hydroxide solution. Solutions were sterile filtered. Stock solutions were prepared so that the vehicle would not be more than 1% of the total volume of the culture media.
  • a mixture of fatty acids derived from chicken fat was prepared by mixing 0.1% chicken fat with mineral oil to form an emulsified solution. Tween 80 was added to separate cultures of cells at similar concentrations and to examine possible vehicle effects.
  • Alpha-tocopherol phosphate (Sigma Chemical Company, St. Louis, MO) was added directly to the culture medium.
  • the results from the tritiated thymidine incorporation assay a measure of DNA synthesis and cellular proliferation, correlated directly with the results from the dye exclusion viability assay. Because the tritiated thymidine incorporation assay is a more quantitative assay, the tritiated thymidine incorporation assay was used for the remainder of the studies.
  • Doxorubicin is an anthracycline antibiotic used as a first line agent in a number of neoplastic conditions and is a well characterized cytotoxic agent.
  • Doses and times examined ranged from 0.1, 0.5, 1, 5, 10, 25, and 50 ug/ml of Doxorubicin for 20-60 minutes and 24 hours.
  • the range of optimal concentrations for cytotoxity of Doxorubicin was established for u937 monocytic tumor cells to be 0.5, 1 and 5 ug at 24 hours and 10 ug at 1-2 hours, see Figures 1 and 2.
  • the cytoprotective agents sodium pyruvate, Vitamin E, and fatty acids alone, and in combination, were examined for their ability to decrease the cytotoxity of Doxorubicin to U937 monocytic leukemia cells and normal peripheral blood monocytes.
  • Optimal concentrations of the single ingredients of sodium pyruvate, Vitamin E, and fatty acids were examined.
  • the optimal concentrations of the agents that were able to protect cells against Doxorubicin induced cytotoxity were as follows: 10-50 U Vitamin E, 0.5% fatty acids, and 5 mM of sodium pyruvate, see Figure 3.
  • cytotoxity of Doxorubicin on the cells was determined by viability assays which examined the differential ability of the cytoprotective compositions alone, and in combinations, to protect the normal cells from the cytotoxity of the chemotherapeutic agent.
  • the cells were isolated and examined for morphological evidence of cytotoxicity or prevention of cytotoxicity. These studies determined the cytoprotective effect of the single agents and the combination of agents on the normal and tumor cells. DNA synthesis studies using 3 H-thymidine (1 uCi/well) were carried out 4 hours prior to termination of the experiment to determine the effect of the formulations on the proliferation of the cells as a measure of the prevention of cytotoxicity and the extent of Doxorubicin- induced cytotoxicity. Propidium iodide exclusion analysis was carried out for direct quantitation of the cytotoxicity and the prevention of cytotoxicity. Each set of studies was performed in triplicate so that statistical analysis of the significant differences between the treatment groups could be conducted.
  • the effect of the cytoprotective agents on the co-culture of tumor and normal cells was very different from the effect of these agents on the individual cell types alone. An interaction between the normal cells and the tumor cells must cause the viability of the tumor cells to be significantly diminished.
  • the cytoprotective combination of 5 mM sodium pyruvate, 0.5% fatty acids. and 10 U Vitamin E provided significant protection to the normal peripheral monocytes and did not protect the tumor cells from the effects of the cytotoxic agent.
  • Vitamin E prevented cytotoxity to U937 tumor cells induced by 1 ug/ml Doxorubicin (42% to 62% viable) , see Figure 7.
  • the viability of cultured peripheral monocytes without Doxorubicin was 66% and increased to 75% with the cytoprotective combination of 5 mM sodium pyruvate, 10 U Vitamin E, and 0.5% fatty acids, see Figure 13.
  • the viability of cultured peripheral monocytes treated with 0.5 ug/ml Doxorubicin was 47% and increased to 63.5% when pretreated with the cytoprotective combination of 5 mM sodium pyruvate, 10 U Vitamin E, and 0.5% fatty acids. see Figure 13.
  • the viability of cultured U937 tumor cells without Doxorubicin was 67% and did not increase when treated with any of the agents, see Figure 13.
  • the viability of cultured U937 tumor cells with 0.5 ug/ml Doxorubicin treatment was 47% and the highest increase in viability occurred with pretreatment of 50 U Vitamin E and 0.5% fatty acids, see Figure 12.
  • the viability of cultured U937 tumor cells with 1 ug/ml Doxorubicin treatment was 45% and the highest increase in viability occurred with pretreatment of 10 U Vitamin E and 0.5% fatty acids, see Figure 12.
  • Optimal concentrations of the cytoprotective agents to prevent Doxorubicin-induced cytotoxity were found to be 5 mM sodium pyruvate, 10-50 U Vitamin E, and 0.5% fatty acids.
  • the cytoprotective combination of sodium pyruvate Vitamin E, and fatty acids and the combination of 5mM sodium pyruvate and 10 U Vitamin E protected the normal peripheral blood monocytes from Doxorubicin-induced cytotoxity, see Figure 13.
  • Vitamin E alone and fatty acids alone prevented the cytotoxity of Doxorubicin in U937 cells, see Figure 11.

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Abstract

La présente invention se rapporte à des compositions cytoprotectrices servant à prévenir et à réduire des lésions provoquées dans des cellules de mammmifères par un médicament présentant des caractéristiques cytotoxiques. Selon un mode de réalisation, la composition cytoprotectrice comprend a) un pyruvate choisi dans le groupe composé d'acide pyruvique, de sels pharmaceutiquement acceptables d'acide pyruvique et de mélanges de ceux-ci, et, b) un antioxydant. Selon un second mode de réalisation, la composition cytoprotectrice comprend un pyruvate choisi dans le groupe composé d'acide pyruvique, de sels pharmaceutiquement acceptables d'acide pyruvique et de mélanges de ceux-ci, b), un antioxydant, et c) un mélange d'acides gras saturés et insaturés, ces acides gras étant ceux requis pour réparer des membranes cellulaires et renouveler les cellules des mammifères. Ces compositions peuvent être incorporées dans une grande variété d'excipients pharmaceutiquement acceptables pour préparer des produits pharmaceutiques. Cette invention se rapporte également à des procédés de préparation et d'utilisation des compositions cytoprotectrices, et aux produits pharmaceutiques dans lesquels elles peuvent être utilisées.
PCT/US1993/000260 1992-02-25 1993-01-13 Compositions cytoprotectrices contenant du pyruvate et des antioxydants WO1993016690A1 (fr)

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WO1994015592A1 (fr) * 1993-01-05 1994-07-21 Thomas Najarian Compositions contenant des agents reduisant le taux de cholesterol et des antioxidants
WO1996006640A1 (fr) * 1994-08-30 1996-03-07 Warner-Lambert Company Composition bioadhesive cicatrisante
EP0804181A1 (fr) * 1995-09-19 1997-11-05 Cellular Sciences, Inc. Methode et composition de traitement de maladies mammaliennes provoquees par une reaction inflammatoire
WO1997041848A1 (fr) * 1996-05-08 1997-11-13 The Government Of The United States, Represented By The Secretary Of The Army Compositions pharmaceutiques contenant des carboxylates alpha-cetoniques
WO1998038993A1 (fr) * 1997-03-04 1998-09-11 Wisconsin Alumni Research Foundation Adhesif pour aluminium utilisant des inhibiteurs de corrosion a base d'aluminium-lithium
WO1998058627A1 (fr) * 1997-06-20 1998-12-30 Sabine Ebeling Composition dermoprotectrice
WO1999030706A1 (fr) * 1997-12-12 1999-06-24 Warner-Lambert Company Compositions antilipemiques combinant un inhibiteur de lipoproteine (a) et une statine
WO2000067782A2 (fr) * 1999-05-05 2000-11-16 Gestilab, Inc. Ceruloplasmine et composition antioxydante contenant cette substance, utilisations de ces dernieres en tant qu'agent neuroprotecteur
WO2001021742A1 (fr) * 1999-09-20 2001-03-29 Lipotech Partners Limited Partnership Compositions d'acides gras monoinsatures et procedes de fabrication de ces compositions
WO2003032941A2 (fr) * 2001-10-13 2003-04-24 Beiersdorf Ag Combinaison de principes actifs cosmetique et/ou dermatologique
WO2006073461A2 (fr) * 2004-05-05 2006-07-13 Clearant Inc. Procedes de sterilisation de melanges biologiques a l'aide d'acides alpha-cetoniques
WO2007099151A1 (fr) * 2006-03-01 2007-09-07 Etren Procédé et agents permettant de réduire le stress oxydatif
EP2018874A2 (fr) 1998-08-07 2009-01-28 Targacept, Inc. compositions pharmaceutiques pour la prévention et le traitement de troubles du système nerveux central comprenant un analogue de la nicotine et un inhibiteur de l'acétylcholinestérase
WO2011100373A1 (fr) 2010-02-09 2011-08-18 The Johns Hopkins University Procédés et compositions pour améliorer la fonction cognitive
WO2013188509A1 (fr) * 2012-06-15 2013-12-19 Creighton University Traitement d'une crise d'épilepsie
US10154988B2 (en) 2012-11-14 2018-12-18 The Johns Hopkins University Methods and compositions for treating schizophrenia
US10159648B2 (en) 2015-05-22 2018-12-25 Agenebio, Inc. Extended release pharmaceutical compositions of levetiracetam
US10806717B2 (en) 2013-03-15 2020-10-20 The Johns Hopkins University Methods and compositions for improving cognitive function
US11160785B2 (en) 2013-03-15 2021-11-02 Agenebio Inc. Methods and compositions for improving cognitive function

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WO1994015592A1 (fr) * 1993-01-05 1994-07-21 Thomas Najarian Compositions contenant des agents reduisant le taux de cholesterol et des antioxidants
WO1996006640A1 (fr) * 1994-08-30 1996-03-07 Warner-Lambert Company Composition bioadhesive cicatrisante
EP0804181A1 (fr) * 1995-09-19 1997-11-05 Cellular Sciences, Inc. Methode et composition de traitement de maladies mammaliennes provoquees par une reaction inflammatoire
EP0804181A4 (fr) * 1995-09-19 2005-02-02 Cellular Sciences Inc Methode et composition de traitement de maladies mammaliennes provoquees par une reaction inflammatoire
WO1997041848A1 (fr) * 1996-05-08 1997-11-13 The Government Of The United States, Represented By The Secretary Of The Army Compositions pharmaceutiques contenant des carboxylates alpha-cetoniques
WO1998038993A1 (fr) * 1997-03-04 1998-09-11 Wisconsin Alumni Research Foundation Adhesif pour aluminium utilisant des inhibiteurs de corrosion a base d'aluminium-lithium
US6133312A (en) * 1997-03-04 2000-10-17 Wisconsin Alumni Research Foundation Method of suppressing tumor growth with combinations of isoprenoids and statins
US6441029B1 (en) 1997-03-04 2002-08-27 Wisconsin Alumni Research Foundation Method of suppressing tumor growth with combinations of isoprenoids and statins
WO1998058627A1 (fr) * 1997-06-20 1998-12-30 Sabine Ebeling Composition dermoprotectrice
US6461624B2 (en) 1997-06-20 2002-10-08 Sabine Eggers Skin-protective composition
WO1999030706A1 (fr) * 1997-12-12 1999-06-24 Warner-Lambert Company Compositions antilipemiques combinant un inhibiteur de lipoproteine (a) et une statine
EP2018874A2 (fr) 1998-08-07 2009-01-28 Targacept, Inc. compositions pharmaceutiques pour la prévention et le traitement de troubles du système nerveux central comprenant un analogue de la nicotine et un inhibiteur de l'acétylcholinestérase
US6780842B2 (en) 1999-05-05 2004-08-24 Warner-Lambert Company Llc Ceruloplasmin and an antioxidant composition comprising the same and their uses as neuroprotective agent
WO2000067782A3 (fr) * 1999-05-05 2001-03-22 Gestilab Inc Ceruloplasmine et composition antioxydante contenant cette substance, utilisations de ces dernieres en tant qu'agent neuroprotecteur
WO2000067782A2 (fr) * 1999-05-05 2000-11-16 Gestilab, Inc. Ceruloplasmine et composition antioxydante contenant cette substance, utilisations de ces dernieres en tant qu'agent neuroprotecteur
WO2001021742A1 (fr) * 1999-09-20 2001-03-29 Lipotech Partners Limited Partnership Compositions d'acides gras monoinsatures et procedes de fabrication de ces compositions
WO2003032941A2 (fr) * 2001-10-13 2003-04-24 Beiersdorf Ag Combinaison de principes actifs cosmetique et/ou dermatologique
WO2003032941A3 (fr) * 2001-10-13 2004-07-08 Beiersdorf Ag Combinaison de principes actifs cosmetique et/ou dermatologique
WO2006073461A2 (fr) * 2004-05-05 2006-07-13 Clearant Inc. Procedes de sterilisation de melanges biologiques a l'aide d'acides alpha-cetoniques
WO2006073461A3 (fr) * 2004-05-05 2007-01-11 Clearant Inc Procedes de sterilisation de melanges biologiques a l'aide d'acides alpha-cetoniques
WO2007099151A1 (fr) * 2006-03-01 2007-09-07 Etren Procédé et agents permettant de réduire le stress oxydatif
WO2011100373A1 (fr) 2010-02-09 2011-08-18 The Johns Hopkins University Procédés et compositions pour améliorer la fonction cognitive
WO2013188509A1 (fr) * 2012-06-15 2013-12-19 Creighton University Traitement d'une crise d'épilepsie
US20150164857A1 (en) * 2012-06-15 2015-06-18 Creighton University Seizure therapy
US9700541B2 (en) 2012-06-15 2017-07-11 Creighton University Seizure therapy
US10154988B2 (en) 2012-11-14 2018-12-18 The Johns Hopkins University Methods and compositions for treating schizophrenia
US10624875B2 (en) 2012-11-14 2020-04-21 The Johns Hopkins University Methods and compositions for treating schizophrenia
US10806717B2 (en) 2013-03-15 2020-10-20 The Johns Hopkins University Methods and compositions for improving cognitive function
US11160785B2 (en) 2013-03-15 2021-11-02 Agenebio Inc. Methods and compositions for improving cognitive function
US10159648B2 (en) 2015-05-22 2018-12-25 Agenebio, Inc. Extended release pharmaceutical compositions of levetiracetam
US10925834B2 (en) 2015-05-22 2021-02-23 Agenebio, Inc. Extended release pharmaceutical compositions of levetiracetam

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AU3315397A (en) 1997-10-30
MX9301014A (es) 1993-09-01
CA2129732A1 (fr) 1993-09-02
ZA931297B (en) 1994-06-27
EP0627917A1 (fr) 1994-12-14
AU4275793A (en) 1993-09-13
JPH07505145A (ja) 1995-06-08

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