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WO2002067965A1 - Compositions a base de synergies de catechine-proanthocyanadine pour la prevention et le traitement du cancer - Google Patents

Compositions a base de synergies de catechine-proanthocyanadine pour la prevention et le traitement du cancer Download PDF

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Publication number
WO2002067965A1
WO2002067965A1 PCT/US2002/005294 US0205294W WO02067965A1 WO 2002067965 A1 WO2002067965 A1 WO 2002067965A1 US 0205294 W US0205294 W US 0205294W WO 02067965 A1 WO02067965 A1 WO 02067965A1
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proanthocyanadins
catechins
cancer
composition
supplement
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PCT/US2002/005294
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English (en)
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Dorothy M. Morre
James D. Morre
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Purdue Research Foundation
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Priority to US10/468,911 priority Critical patent/US20040142048A1/en
Publication of WO2002067965A1 publication Critical patent/WO2002067965A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/35—Caprifoliaceae (Honeysuckle family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/90—Smilacaceae (Catbrier family), e.g. greenbrier or sarsaparilla

Definitions

  • the present invention relates to novel methods and compositions which 10 utilize catechins, including but not limited to, epigallocatechin gallate (EGCg), epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin (EGC), which are found in varying levels in tea leaves, in combination with proanthocyanadins, including but not limited to, blue fruit extracts.
  • catechins including but not limited to, epigallocatechin gallate (EGCg), epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin (EGC), which are found in varying levels in tea leaves, in combination with proanthocyanadins, including but not limited to, blue fruit extracts.
  • the compositions of the invention contain various amounts of the catechins and proanthocyanadins, and optionally, other therapeutic agents.
  • the invention 15 also encompasses the varying modes of administration of the catechins and proanthocyanadins as a dietary or nutritional supplement or as a therapeutic compound.
  • Tea is generally in the form of black, oolong, and green tea, all originating from the tea plant, Camellia sinensis. Tea is cultivated in approximately thirty countries worldwide, and is consumed globally. Although the level of tea consumption varies around the world, it is believed that tea consumption is second only to water (Ahmad et al., 1998,
  • Green tea has been prized as a traditional tonic and has been widely consumed in East Asia. Recent studies have attempted to link green tea to antioxidant benefits including protection against the damage caused by cigarette smoke, pollution, stress, and other toxins (for an overview, see e.g., Mitscher, 1998, The Green Tea Book, Avery Publishing Group, Garden City Park, New York and Weisburger, 1997, Can. Lett.
  • the polyphenols also known as catechins.
  • the polyphenols describe a class of substituted phenolic compounds that are known as flavanols or catechins.
  • the polyphenols in green tea that have been identified are catechin (C), epicatechin (EC), gallocatechin (GC), gallocatechin gallate (GCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCg) (Fig. 1).
  • caffeine, theobromine, theophylline, and phenolic acids, such as gallic acid are also present as constituents of green tea in smaller quantities than the polyphenols (Ahmad et al., 1998, Nutrition and Chemical Toxicity, John Wiley and Sons, London, England, pp. 301-343).
  • EGCg Epigallocatechin gallate
  • the administration of a pharmacologically effective amount of EGCg has been alleged to reduce the incidence of lung cancer in a mammal (U.S. Patent No. 5,391,568).
  • a bioavailability study showed that frequent green tea consumption results in high levels of EGCg in various body organs, suggesting that green tea consumption may protect against cancers localized to different sites of the body (Sugunama et al., 1998, Carcinogenesis 19:1771-1776).
  • EGCg has been implicated in blocking DNA transcription of a number of genes in cancer cell lines.
  • EGCg inhibits the DNA and protein synthesis of the growth factor receptors epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGF-R), and fibroblast growth factor receptor (FGF-R) (Liang et al., 1997, J. Cell. Biochem. 67:55-65).
  • EGCg has also been implicated in blocking transcription of nitric oxide (NO) synthase by inhibiting the binding of transcription factor NFKB to the NO synthase promotor (Lin and Lin, 1997, Mol. Pharmacol. 52:465-472 and Chan et al., 1997, Biochem. Pharmacol.
  • NO nitric oxide
  • EGCg inhibits AP-1 transcriptional activity (Dong et al., 1997, Can. Res. 57:4414-4419).
  • Apoptosis differs from necrosis, and is regarded as an ideal mechanism for the elimination of cells.
  • EGCg induced apoptosis may result from either cell cycle arrest and/or H 2 O 2 production (Ahmad et al., 1997, J. Nat. Can. Inst. 89:1881- 1886; Fujiki et al., 1998, Mutat. Res. 402:307-310; and Yang et al., 1998, Carcinogenesis 19:611-616).
  • EGCg may be involved in the growth regulation of human epidermal
  • Green tea extract an important source of EGCg, has previously been reported to enhance the effect of the anti-cancer agents, e.g., adriamycin and doxorubicin (Sugiyama and Sadzuka, 1998, Can. Lett. 133:19-26 and Sadzuka et al, 1998, Clin. Can. Res. 4: 153-156).
  • Green tea in combination with adriamycin inhibits tumor growth in M5076 ovarian sarcoma cells, whereas adriamycin alone does not inhibit tumor growth in M5076 ovarian sarcoma cells (Sugiyama and Sadzuka, 1998, Can. Lett. 133:19-26).
  • Green tea extract in combination with doxorubicin, also enhances the inhibitory growth effect on Ehrlich ascites carcinoma tumors in tumor-bearing mice, presumably by increasing the concentration of doxorubicin concentration in the tumor, but not in normal tissue (Sadzuka et al., 1998, Clin. Can. Res. 4:153-156).
  • EGCg has also been shown to enhance the effect of cancer prevention drugs in vitro.
  • EGCg has been shown to enhance the apoptotic effect of sulindac and tamoxifin, presumably by EGCg enhancing the intracellular concentration of the cancer prevention drugs.
  • Both sulindac and tamoxifin induce apoptosis of human cancer cells and inhibit TNF ⁇ release from BALB/c- 3T3 cells (Piazza et al., 1995, Can. Res. 55:3110-3116; Chen et al., 1996, J. Cell. Biochem. 61:9-17; and Sugunama et al., 1996, Can. Res. 56:3711-3715).
  • Proanthocyanadins are dietary sources of polyphenols. As described by Fine in a review article, which is incorporated by reference in its entirety, considerable recent research has explored therapeutic applications of oligomeric proanthocyanidin complexes, which are naturally occurring plant metabolites widely available in fruits, vegetables, nuts, seeds, flowers, and bark and are primarily known for their antioxidant activity (Fine, 2000, Altern Med Rev 5(2): 144-51).
  • Oligomeric proanthocyanidins are naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, which have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress.
  • a novel grape seed proanthocyanidin extract has been shown to provide excellent protection against oxidative stress and free radical-mediated tissue injury (Bagchi et al., 2000, Toxicology 148(2-3): 187-97).
  • Bomser et al. has disclosed that a polyphenolic fraction from grape seeds results in the inhibition of TP A-induced tumor promotion in CD-I mouse epidermis (Bomser et al., 1999, Cancer Lett. 132(2): 151-7).
  • Proanthocyanadins are found in the extracts of blue fruits such as, but not limited to, wild blackberries (Rubus spp.) (Rosaceae), blue berries (Vaccinium sp.) (Ericaceae), elderberry (Sambucus canadensis) (Caprifoliaceae), choke cherry (Prunus virginian ⁇ ) (Rosaceae), pokeberry (Phytolacca americana) (Phytolaccaceae), wild grape (fox grape) (Vitis labrusca) (Vitaceae), and green briar (Smilax glauca) (Liliaceae). As described herein, proanthocyanadins, in combination with the green tea polyphenols, was shown to have potential utility for the prevention and treatment of cancer. 2.3. NADH oxidase
  • NADH NADH oxidase
  • NADH cell surface protein with hydroquinone
  • NOX protein is located at the external plasma membrane surface
  • CNOX was originally defined as a drug-indifferent constitutive NADH
  • cancer cells exhibit both drug-responsive and hormone and growth factor-indifferent (tNOX) as well as drug inhibited and hormone and growth factor dependent (CNOX) activities, non-transformed cells exhibit only the drug indifferent hormone- and drug-responsive CNOX.
  • tNOX drug-responsive and hormone and growth factor-indifferent
  • CNOX drug inhibited and hormone and growth factor dependent
  • the hormone-stimulated NADH oxidase activity of rat liver plasma membranes is not inhibited by cyanide (Morre, 1994, J. Bioenerg. Biomemb. 26: 421-433).
  • the enzyme also was distinguished from other oxidase activities by its response to several common oxidoreductase inhibitors, e.g., catalase, azide and chloroquine, as well as to various detergents e.g., sodium cholate, Triton X-100 and CHAPS (Morre and Brightman,
  • CNOX is a unique membrane-associated protein that is capable of oxidizing NADH but has an activity which is modulated by hormones and growth factors.
  • Cancer is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis). Clinical data and molecular biologic studies indicate that
  • cancer is a multistep process that begins with minor preneoplastic changes, which may under certain conditions progress to neoplasia.
  • Pre-malignant abnormal cell growth is exemplified by hyperplasia, metaplasia, or most particularly, dysplasia (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia,
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, but without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in
  • Atypical metaplasia involves a somewhat disorderly metaplastic epithelium.
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
  • Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism.
  • the neoplastic lesion may evolve clonally and develop an increasing capacity for invasion, growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host's immune surveillance (Roitt, Brostoff, and Kale, 1993, Immunology, 3rd ed., Mosby, St. Louis, pp. 17.1-17.12).
  • EGCg mediates its effects intracellularly, since EGCg incorporation into the cell seems to be a prerequisite for the inhibition of TNF ⁇ release.
  • the invention encompasses formulations comprising catechins and proanthocyanadins in ratios that result in synergistic properties.
  • the formulations are used as compositions for the prevention and treatment of cancer or as a dietary or nutritional supplement that protects white blood cells and maintains healthy blood levels.
  • Specific therapeutic regimens, pharmaceutical compositions, and kits are also provided by the invention.
  • the invention described herein comprises the administration of catechins in combination with proanthocyanadins, to a mammal as a dietary supplement.
  • the mammal is a human.
  • the invention described herein comprises the administration of a therapeutically effective amount of catechins in combination with proanthocyanadins, to a mammal in need of such therapy.
  • the mammal is a human.
  • the invention further encompasses the use of additional therapeutic agent(s) in combination therapy to treat cancer.
  • the catechins comprise epigallocatechin gallate (EGCg), epicatechin gallate (ECG), epigallocatechin (EGC), epicatechin (EC) or a combination thereof, in combination with one or more proanthocyanadins, such as, but not limited to, blue fruit extracts.
  • the disclosure is based, in part, on the discovery that catechins, proanthocyanadins, and other anti-cancer therapeutic agents, inhibit the activity of a cancer- specific protein, an isoform of NADH oxidase specific to cancer cells (tNOX).
  • tNOX an isoform of NADH oxidase specific to cancer cells
  • the inhibition of tNOX results in the inhibition of cell growth, and ultimately, apoptosis of the cancer cell, whereas normal cells (which lack tNOX but instead express the isoform CNOX) are less affected.
  • the invention provides a potent therapeutic effect with reduced or no adverse effects on normal, healthy cells.
  • one embodiment of the invention is directed to the administration of sustained release formulations so that a constant level of the catechins is maintained.
  • FIGS. 1A-1B Dose response of HeLa cell NADH oxidase activity (spectrophotometric assay) to juice of (A) greenbriar (Smilax glauca) and (B) pokeberry
  • FIGS. 2A-2B Dose response of the HeLa cell NADH oxidase activity (spectrophotometric assay) to juice of (A) wild and (B) cultivated blackberry (Rubus spp.).
  • FIGS. 3A-3D Dose response of the HeLa cell growth (96-well place assay) of juice of (A) pokeberry (Phytolacca americana), (B) green briar (Smilax glauca), (C) cultivated and (D) wild blackberry (Rubus spp.).
  • FIG. 4 Correlation between E dil 50 of tNOX inhibition with E dil 50 of growth inhibition (72 hours) of HeLa (human cervical carcinoma) cells in culture
  • FIG. 5 Synergy (o) between TegreenTM (A) and juice from wild blackberry ( ⁇ ) on growth inhibition of HeLa cells in culture determined by direct counting.
  • FIGS. 6A-6D Combinations between TegreenTM and juices from different fruits on growth inhibition of HeLa cells in culture determined by direct counts.
  • A choke cherry,
  • B greenbriar,
  • C pokeberry, and
  • D cultivated blackberry.
  • the present invention relates to compositions and methods for treatment and prevention of cancer.
  • the invention is based, in part, on the discovery that proanthocyanadins in blue fruits and catechins in tea, inhibit the activity of an isoform of NADH oxidase (tNOX) that is specific to cancer cells.
  • tNOX NADH oxidase
  • the inhibition of tNOX results in the inhibition of cell 30 growth, and ultimately, apoptosis of the cancer cell, whereas normal cells, which lack tNOX but express another isoform termed CNOX, are less affected.
  • the invention provides a selective and potent therapeutic effect with reduced or no adverse effects on normal, healthy cells.
  • the invention provides a composition comprising tea 35 catechins and proanthocyanadins.
  • the invention provides a pharmaceutical composition comprising tea catechins, proanthocyanadins and a pharmaceutical carrier.
  • the invention provides a dietary supplement or nutritional composition comprising tea catechins and proanthocyanadins.
  • the invention further provides methods for preventing or treating cancer comprising administering compositions comprising tea catechins and proanthocyanadins.
  • the invention encompasses methods for preventing or treating cancer that comprises administering to a subject tea catechins adjunctively with proanthocyanadins such that both catechins and proanthocyanadins are present in vivo and in contact with cancer cells.
  • the cancer that is prevented or treated by the compositions and methods of the invention is a cancer that comprises cells that express tNOX.
  • the invention also provides methods for inhibiting the growth and/or proliferation of cancer cells and neoplastic cells comprising contacting the cancer cells and neoplastic cells with a composition comprising both tea catechins and proanthocyanadins.
  • the invention also encompasses methods for inhibiting the growth and/or proliferation of cancer and neoplastic cells comprising contacting the cells with tea catechins and with proanthocyanadins such that when the proantocyanidins are contacted with the cells, the tea catechins are still providing the anti-cancer activity to the cells, and vice versa.
  • the cancer cells and neoplastic cells that are inhibited by the catechin-proanthocyanadin mixtures comprise cells that express tNOX.
  • tea catechins are used either in the preparation of a composition of the invention that comprises both tea catechins and proantocyanidins, or in therapeutic or prophylactic methods in which tea catechins are administered adjunctively with proanthocyanidins.
  • Various formulations of tea catechins can be used as above-described.
  • the formulations used in the invention are based on green tea polyphenols typically found in green tea extracts which comprises 10-15% EGCg, 2-3% ECG, 2% EC, and 2-3% EGC (Suganuma et al., 1999, Can. Res. 59:4 . 4-47).
  • the present invention provides for a formulation in which EGCg constitutes at least 30%> of the total catechins. In a preferred embodiment, EGCg constitutes about 35%) to about 45%> of the total catechins. In a more preferred embodiment, EGCg constitutes about 40% of the total catechins.
  • EGCg be used in combination with other catechins, more specifically, those described infra.
  • the invention provides a formulation in which EGCg constitutes at least 30% of the total catechins and ECG constitutes at least 5%> of the total catechins.
  • EGCg constitutes about 35%o to about 45%) of the total catechins and ECG constitutes about 10% to about 20%> of the total catechins.
  • EGCg constitutes about 40%> of the total catechins and ECG constitutes about 15%> of the total catechins.
  • the invention provides a formulation in which
  • EGCg constitutes at least 30%> of the total catechins and EC constitutes at least 3%> of the total catechins. In a preferred embodiment, EGCg constitutes about 35% to about 45% of the total catechins and EC constitutes about 3%> to about 15%> of the total catechins. In a more preferred embodiment, EGCg constitutes about 40%> of the total catechins and EC constitutes about 7%> of the total catechins.
  • the invention provides a formulation in which
  • EGCg constitutes at least 0.01%> of the total catechins and EC constitutes an amount which is at least 10 fold greater than the EGCg content of the total catechins.
  • the total catechins may or may not include additional catechins such as ECG, EGC, and C, described above.
  • EC is present in an amount which is at least 100 fold greater than the EGCg content.
  • the EC content is at least 1000 fold greater than the EGCg content.
  • the amount of EGCg present in the catechin formulation is negligible.
  • the invention provides a formulation in which
  • EGCg constitutes at least 30% of the total catechins and EGC constitutes at least 1%> of the total catechins. In a preferred embodiment, EGCg constitutes about 35%> to about 45%> of the total catechins and EGC constitutes about 2%> to about 5% of the total catechins. In a more preferred embodiment, EGCg constitutes about 40%> of the total catechins and EGC constitutes about 3%> of the total catechins.
  • the invention provides a formulation in which
  • EGCg constitutes at least 30%> of the total catechins, EC constitutes at least 3%> of the total catechins, and ECG constitutes at least 5%> of the total catechins. In a preferred embodiment, EGCg constitutes about 35%> to about 45%) of the total catechins, EC constitutes about 3%> to about 15%> of the total catechins, and ECG constitutes about 10%> to about 20%) of the total catechins. In a more preferred embodiment, EGCg constitutes about 40%) of the total catechins, EC constitutes about 7%> of the total catechins. and ECG constitutes about 15%> of the total catechins.
  • the invention provides a formulation in which EGCg constitutes at least 30%> of the total catechins, EC constitutes at least 3%> of the total catechins, and EGC constitutes at least 1%> of the total catechins.
  • EGCg constitutes about 35%> to about 45%> of the total catechins
  • EC constitutes about 3%> to about 15%> of the total catechins
  • EGC constitutes about 2%> to about 5% of the total catechins.
  • EGCg constitutes about 40%) of the total catechins
  • EC constitutes about 1% of the total catechins
  • EGC constitutes about 3%> of the total catechins.
  • the invention provides a formulation in which EGCg constitutes at least 30%> of the total catechins, EC constitutes at least 3%> of the total catechins, ECG constitutes at least 5%> of the total catechins, and EGC constitutes at least 1% of the total catechins.
  • EGCg constitutes about 35 > to about 45%o of the total catechins
  • EC constitutes about 5%> to about 15%> of the total catechins
  • ECG constitutes about 10%> to about 20%> of the total catechins
  • EGC constitutes 2%> to about 5%> of the total catechins.
  • EGCg constitutes about 40%o of the total catechins, EC constitutes about 7%> of the total catechins. ECG constitutes about 15%) of the total catechins, and EGC constitutes about 3%o of the total catechins.
  • the invention provides a formulation in which EGCg constitutes at least 30%) of the total catechins, EC constitutes at least 3% of the total catechins, ECG constitutes at least 5%> of the total catechins, EGC constitutes at least 1%> of the total catechins, and C constitutes at least 5%> of the total catechins.
  • EGCg constitutes about 35%> to about 45% of the total catechins, EC constitutes about 5%> to about 15%) of the total catechins, ECG constitutes about 10%) to about 20%) of the total catechins, EGC constitutes 2%> to about 5%> of the total catechins, and C constitutes about 10%> to about 20%> of the total catechins.
  • EGCg constitutes about 40%> of the total catechins
  • EC constitutes about 7%> of the total catechins
  • ECG constitutes about 15%> of the total catechins
  • EGC constitutes about 3% of the total catechins
  • C constitutes about 15%> of the total catechins.
  • the invention provides a formulation which contains about 0.01%> of EGCg of the total catechins. In another embodiment, the formulations contain about 0.1%) of EGCg of the total catechins. In yet another embodiment, the formulation contains about 1.0% of EGCg of the total catechins. In yet another embodiment, the formulation contain about 5.0%) of EGCg of the total catechins. In another embodiment, the formulation contains less than 10 %> of EGCg of the total catechins. In another embodiment, the ratio of EC to EGCg concentration is about 10:1.
  • the ratio of EC to EGCg concentration is about 100:1. In yet another embodiment, the ratio of EC to EGCg concentration is about 1000:1.
  • the level of caffeine in the formulation is generally less than about 5%> and is preferably less than 0.5%) of the polyphenols.
  • the catechin formulations described above can be made by infusing natural tea (see, e.g., Wang et al., 1994, Cancer Research 54:3428-3435 or U.S. Patent No. 6,096,359, which is hereby incorporated by reference in its entirety) or by using tea concentrates that are commerically available (e.g., TegreenTM, Pharmanex, Brisbane, CA).
  • concentrations of the individual catechins in a formulation can be manipulated by adding purified catechins, which may be purchased (e.g., from Sigma, St.
  • proanthocyanadins are used either in the preparation of a composition of the invention that comprises both tea catechins and proantocyanidins, or in therapeutic or prophylactic methods in which proanthocyanidins are administered adjunctively with tea catechins.
  • Various formulations of proanthocyanidin can be used as above-described.
  • Proanthocyanadins are dietary sources of polyphenols. Oligomeric proanthocyanidins are naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, which have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. As described in U.S. Patent Nos . 5,646,178 and 5,650,432, which are incorporated by reference in their entireties, proanthocyanadins are known to have a variable number of flavonoid units. The proanthocyanidins can have 2 to 28 flavonoid units, preferably 2 to 15 flavonoid units, and most preferably 2 to 11 flavonoid units.
  • the flavonoid units include but are not limited to catechins, epicatechins, gallocatechins, galloepicatechins, flavanols, flavonols, flavandiols, leucocyanidins, leucodelphinidin anthocyanidins, or combinations thereof.
  • the flavonoid units can be singly or double linked to each other.
  • the proanthocyanadins used are present in blue fruits and include derivatives of proanthocyanadins, and related compounds resulting from the chemical breakdown of proanthocyanadins in blue fruits.
  • the term blue fruit extract refers to a preparation of blue fruits that comprises proanthocyanidins which can be obtained by methods such as, but not limited to, crushing, pressing, emulsifying, or chemically extracting a blue fruit.
  • the proanthocyanadin is derived from cultured blackberries (Rubus spp.) (Rosaceae).
  • the proanthocyanadin is derived from wild blackberries (Rubus spp.) (Rosaceae).
  • the proanthocyanadin is derived from blue berries (Vaccinium sp.) (Ericaceae).
  • the proanthocyanadin is derived from elderberry (Sambucus canadensis) (Caprifoliaceae). In another embodiment, the proanthocyanadin is derived from choke cherry (Prunus virginiana) (Rosaceae). In another embodiment, the proanthocyanadin is derived from pokeberry (Phytolacca americana) (Phytolaccaceae). In another embodiment, the proanthocyanadin is derived from wild grape (fox grape) (Vitis labrusca) (Vitaceae). In another embodiment, the proanthocyanadin is derived from green briar (Smilax glauca) (Liliaceae).
  • the proanthocyanadin can be obtained from a single or a mixture of blue fruits or blue fruit extracts.
  • the proanthocyanadin is derived from a concentrate of blue fruit, i.e., the concentrate contains more proanthocyanadins per unit mass than the fruit.
  • proanthocyanadins can be concentrated from blue fruits by first extracting active compounds from blue fruits with water or solvents such as, but not limited to, alcohols,acetone, acetonitrile, or ethyl acetate, or miscible mixtures of these solvents, water or methanol/water or acetone/water mixtures preferred, leaving a pulp or residue significantly depleted for the active compounds.
  • solvents such as, but not limited to, alcohols,acetone, acetonitrile, or ethyl acetate, or miscible mixtures of these solvents, water or methanol/water or acetone/water mixtures preferred, leaving a pulp or residue significantly depleted for the active compounds.
  • the blue fruits may be extracted with relatively non-polar organic solvents such as but not limited to, hexane, heptane, cyclohexane, methylene chloride, chloroform, or large molecular weight alcohols that contain more than 8 carbon atoms, and the like.
  • relatively non-polar organic solvents such as but not limited to, hexane, heptane, cyclohexane, methylene chloride, chloroform, or large molecular weight alcohols that contain more than 8 carbon atoms, and the like.
  • Such treatment extracts non-active materials, leaving a pulp or residue with increased concentrations of active substances.
  • This pulp or residue may be further processed by extraction with more polar solvents such as, but not limited to water, alcohols with fewer than 8 carbon atoms, acetone, acetonitrile, ethylacetate, or miscible mixtures of these solvents to further enrich the active fraction.
  • juice of blue fruits, diluted or concentrated can be used.
  • pokeberry, greenbriar, and choke cherry have a more potent effect on the inhibition of tNOX activity and the inhibition of HeLa cell growth than the other blue fruits.
  • the proanthocyanadins are isolated from pokeberry, greenbriar, and/or choke cherry.
  • all of the blue fruit extracts presented in Table 1 are effective at inhibiting tNOX activity and HeLa cell growth.
  • the invention is not to be limited to the blue fruits presented herein and the proanthocyanadins of the invention may include those obtained from other natural sources of proanthocyanadins.
  • the invention encompasses administration of the catechin formulations listed in Section 5.1.1 and proanthocyanadin formulations listed in Section 5.1.2 in combination.
  • the combination of catechins and proanthocyanadins possess a synergistic effect in the inhibition of tNOX activity and the inhibition of cancer cell growth, as demonstrated in the Example presented in Section 6.
  • synergistic refers to a combination which is more effective than the additive effects of any two or more single agents.
  • a determination of a synergistic interaction between catechins, proanthocyanadins, and optionally, one or more other anti-cancer or therapeutic agents may be based on the results obtained from the NOX assays described in Section 6 infra. The results of these assays are analyzed using Chou and Talalay's combination method and Dose-Effect Analysis with Microcomputers' software in order to obtain a Combination Index (Chou and Talalay, 1984, Adv. Enzyme Regul. 22:27-55 and Chou and Chou, 1987, software and manual, Elsevier Biosoft, Cambridge, UK, pp. 19-64). Combination Index values ⁇ 1 indicates synergy, values > 1 indicate antagonism and values equal to 1 indicate additive effects.
  • Adjunct administration of the tea catechins and proanthocyanadins of the invention means that the two are administered either as a mixture or sequentially.
  • the catechins may be administered before or after the proanthocyanadins, so long as the first administered agent is still providing anti-cancer activity in the animal when the second agent is administered.
  • Any of the modes of administration described infra may be used in combination to deliver the tea catechins and proanthocyanadins.
  • the present invention is to be understood as embracing all such regimens and the term "adjunct administration" is to be interpreted accordingly.
  • the tea catechins and proanthocyanadins are administered adjunctively as a mixture, they are preferably given in the form of a composition comprising both agents.
  • one embodiment of the invention provides for a pharmaceutical composition comprising tea catechins and proanthocyanadins, and optionally, a pharmaceutically acceptable carrier.
  • tea catechins and proanthocyanadins when administered adjunctively as a mixture, they are given in the form of a nutritional composition or dietary supplement comprising tea catechins and proanthocyanadins.
  • a sustained release catechin formulation as described infra in Section 5.2. and disclosed in U.S. Patent Application No. 09/637,840, which is hereby incorporated by reference in its entirety, is used in combination with a proanthocyanadin formulation, such as but not limited to an extract of one or more blue fruit(s).
  • the blue fruit extract is isolated from pokeberry, greenbriar, and/or choke cherry.
  • the sustained release formulation comprises both catechins and proanthocyanadins.
  • a catechin formulation as described in Section 5.1.1 and disclosed in U.S. Patent Application No. 09/537,211, which is hereby incorporated by reference in its entirety, is used in combination with a proanthocyanadin formulation, such as but not limited to an extract of one or more blue fruit(s).
  • a proanthocyanadin formulation such as but not limited to an extract of one or more blue fruit(s).
  • the blue fruit extract is isolated from pokeberry, greenbriar, or choke cherry.
  • the catechin formulation is naturally-occurring , i.e., endogenous to green tea.
  • the catechin formulation contains catechin ratios that are not native to green tea.
  • the methods of the invention also encompasses administrating the catechin formulations described in Section 5.1.1 and proanthocyanadin formulations, or pharmaceutically acceptable salts or derivatives thereof, described in Section 5.1.2 in combination with other therapeutic agents, such as anti-cancer drugs, and optionally, pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient, is chemically inert and is not toxic to the patient to whom it is administered.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases.
  • pharmaceutically acceptable derivative refers to any homolog, analog, or fragment corresponding to the formulations as described in Section 5.1 which exhibits anti-cancer activity and is non-toxic to the subject.
  • therapeutic agent or “anti-cancer agent” refers to any molecule, compound or treatment that assists in the treatment of a cancer or the diseases caused thereby.
  • the therapeutic agents include, but are not limited to adriamycin and adriamycin conjugates, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, methotrexate, fluorouacil, floxuridie, cytarabine, mercaptopurine, thioguanine, pentostatin, vinblastine, vincristine, etoposide, teniposide, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, interferon- alpha, cisplatin, carboplatin, mitoxantrone, hydroxyurea, procarbazine, mitotan
  • simalikalactone D and glaucarubolone and pharmaceutically acceptable derivatives thereof.
  • the therapeutic agents which have been shown to inhibit tNOX and cancer cell growth include adriamycin, bullatacin, simalikalactone D, and glaucarubolone, descriptions of which are provided in U.S. Patent No. 5,605,810, which is incorporated by reference in its entirety for all purposes.
  • Methods of the invention also encompasses administering the catechin formulations and proanthocyanadin formulations, to cancer patients undergoing chemotherapy and/or irradiation for a primary cancer.
  • use of the catechin formulations, anti-cancer agents, and combinations thereof provides a method for treating the metastasized, i.e. secondary cancer, in said patients.
  • the catechin and proanthocyanadin formulations of the invention can be administered with a monoclonal antibody directed against tNOX for the treatment or prevention of cancer.
  • a monoclonal antibody directed against tNOX for the treatment or prevention of cancer.
  • An example of such a monoclonal antibody to the human tNOX protein has been described and has been used in the expression cloning of tNOX from HeLa cells (Chueh et al., 1997, Arch. Biochem. Biophys. 342:38-44).
  • the invention further provides tea catechins and/or proanthocyanidins that are formulated as sustained release compositions.
  • sustained release formulation refers to any composition that provides slow, controlled, and/or timed release of one or more active ingredients.
  • the catechins are formulated as a sustained release formulation and are adjunctively administered with the proanthocyanadins.
  • the proanthocyanadins are formulated as a sustained release formulation and are adjunctively administered with the catechins.
  • both catechins and proanthocyanadins are formulated as sustained release formulations or as a single sustained release formulation.
  • the sustained release composition of the invention when administered to a human, results in circulating levels of the catechins, proanthocyanadins, or both at about 10 "9 and 10 "4 M for at least 48 hours.
  • the circulating levels of the catechins, proanthocyanadins. or both are preferably maintained at up to 10 "7 M for at least 48 hours in the sera.
  • the circulating levels of the catechins, proanthocyanadins, or both are preferably maintained at up to 10 "5 M for at least 48 hours in the sera.
  • the levels are either circulating in the patient systemically, or in a preferred embodiment, localized to the tumor, and in a most preferred embodiment, localized to the cell surface of the cancer cells.
  • the catechin and proanthocyanadin levels are maintained over a certain period of time as is desired and can be easily determined by one of skill in the art using this disclosure and available pharmaceutical compendia.
  • the invention includes a unique feature of administration comprising a sustained release formulation so a constant level of EGCg is maintained between 10 "8 and 10 "6 M between 48 to 96 hours in the sera.
  • sustained and/or timed release formulations may be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Patent Nos.: 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 4,710,384, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, the disclosures of which are each incorporated herein by reference.
  • compositions can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, may be readily selected for use with the compositions of the invention.
  • single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, caplets, powders, and the like, that are adapted for sustained release are encompassed by the present invention.
  • the sustained release formulation contains active ingredients such as, but not limited to, microcrystalline cellulose, maltodextrine, ethylcellulose, and magnesium stearate.
  • active ingredients such as, but not limited to, microcrystalline cellulose, maltodextrine, ethylcellulose, and magnesium stearate.
  • CapsuDar® SR Biodar, Yavne, Israel
  • microencapsulation which consists of the active ingredients microcrystalline cellulose, maltodextrine, ethylcellulose, and magnesium stearate.
  • the sustained release formulation is encapsulated by coating particles or granules of the composition of the invention with varying thicknesses of slowly soluble polymers or by microencapsulation.
  • the sustained release formulation is encapsulated with a coating material of varying thickness (e.g., about 1 micron to 200 microns) that allows the dissolution of the pharmaceutical composition about
  • the coating material is a food approved additive.
  • the coating material is sold under the trademark Eudragit RS or RL (Rohm Pharma, Germany).
  • the sustained release formulation is a matrix dissolution device, which is prepared by compressing the drug with a slowly soluble 0 polymer carrier into a tablet.
  • the coated particles have a size range between about 0.1 to about 300 microns, as disclosed in U.S. Patent Nos. 4,710,384 and 5,354,556, which are incorporated herein by reference in their entireties. Each of the particles is in the form of a micromatrix, with the active ingredient uniformly distributed throughout the polymer. 5 Sustained release formulations such as those described in U.S. Patent No.
  • plasticizer in the coating in order to permit sufficient flexibility to prevent substantial breakage during compression.
  • the specific amount of plasticizer varies depending on the nature of the coating and the particular plasticizer used. The amount may
  • the thickness of the coating may be increased slightly to make up for an increase in the amount
  • plasticizer in such an embodiment will be present in an amount of about 15 to 30 percent of the sustained release material in the coating, preferably 20 to 25 percent and the amount of coating will be from 10 to 25 percent of the weight of active material, preferably 15 to 20 percent. Any conventional pharmaceutically acceptable plasticizer may be incorporated into the coating.
  • Target Cancers that can be prevented or treated by the methods of the present invention include, but not limited to human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal
  • the cancer is one where circulating levels of tNOX are present in the sera of patients suffering from said cancer, e.g., rectal carcinoma, colon carcinoma, breast carcinoma, ovarian carcinoma, small cell lung carcinoma, colon carcinoma, chronic lymphocytic carcinoma, hairy cell leukemia, osophogeal carcinoma, prostate carcinoma, breast cancer, myeloma, and lymphoma, see e.g., U.S. Patent No. 5,605,810, which is incorporated by reference in its entirety.
  • the patient already has cancer and is undergoing treatment for said cancer.
  • the patient already has cancer but no metastasis, i.e., secondary cancer.
  • the patient already has cancer plus a metastatic cancer.
  • the patient having a cancer is immunosuppressed by reason of having undergone anti-cancer therapy (e.g., chemotherapy or radiation) prior to administration of the catechin complexes of the invention.
  • the patient is a post-treatment or post-operative cancer patient.
  • the cancer is a tumor.
  • the tumor is a tumor of epithelial tissue, lymphoid tissue, connective tissue, bone, or central nervous system.
  • the catechins and proanthocyanadins of the invention can be formulated as a sustained and/or timed release formulation as described in Section 5.2.
  • the levels of circulating catechin and proanthocyanadin compositions must be maintained above some minimum therapeutic dose to reduce the number of cancer cells or to prevent cancer.
  • the reduction in the number of cancer cells can be the result of cell death or apoptosis.
  • the reduction in the number of cancer cells can be a result of inhibition of cell growth, or cell growth arrest.
  • sustained release preparations may include: (1) extended activity of the composition; (2) reduced dosage frequency; and (3) increased patient compliance.
  • sustained release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the composition, and thus can affect the occurrence of side effects.
  • the sustained release formulations of the invention are designed to initially release an amount of the therapeutic composition that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of compositions to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level in the body, the therapeutic composition must be released from the dosage form at a rate that will replace the composition being metabolized and excreted from the body.
  • sustained release of an active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
  • sustained release component in the context of the present invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, microspheres, or the like, or a combination thereof, that facilitates the sustained release of the active ingredient.
  • compositions are water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, if the resulting composition has poor solubility in aqueous solvents, then it may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol.
  • a non-ionic surfactant such as Tween, or polyethylene glycol.
  • the compounds and their physiologically acceptable solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration or, in the case of tumors, directly injected into a solid tumor.
  • the composition may be in liquid form, (e.g. , solutions, syrups or suspensions), or may be presented as a drug product (e.g., capsule or powder) for reconstitution with water or other suitable vehicle before use.
  • liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, or fractionated
  • compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • the composition may take the form of a capsule or powder to be dissolved in a liquid for oral consumption.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the compounds of the present invention are formulated as controlled release powders of discrete microparticles which can be readily formulated in liquid form.
  • the sustained release powder is formulated as controlled release powders of discrete microparticles which can be readily formulated in liquid form.
  • 5 comprises particles containing an active ingredient and optionally, an excipient with at least one non-toxic polymer.
  • the powder can be dispersed or suspended in a liquid vehicle and will maintain its sustained release characteristics for a useful period of time. These dispersions or suspensions have both chemical stability and stability in terms of dissolution rate.
  • 10 powder may contain an excipient comprising a polymer, which may be soluble, insoluble, permeable, impermeable, or biodegradable.
  • the polymers may be polymers or copolymers.
  • the polymer may be a natural or synthetic polymer. Natural polymers include polypeptides (e.g., zein), polysaccharides (e.g., cellulose), and alginic acid. Representative synthetic polymers include those described, but not limited to, those described in column 3, lines 33-
  • Particularly suitable polymers include those described, but not limited to, those described in column 3, line 46-column 4, line 8 of U.S. Patent No. 5,354,556 which is incorporated by reference in its entirety.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • compositions of the invention may be formulated for parenteral administration, e.g., by intramuscular injections or implants for subcutaneous tissues and various body cavities and transdermal devices.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • the compositions may 30 take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • intramuscular injections are formulated as 35 aqueous or oil suspensions.
  • the sustained release effect is due to, in part, a reduction in solubility of the active compound upon complexation or a decrease in dissolution rate.
  • oil solutions and suspensions wherein the release rate of an active compound is determined by partitioning of the active compound out of the oil into the surrounding aqueous medium. Only active compounds which are oil soluble and have the desired partition characteristics are suitable.
  • Oils that may be used for intramuscular injection include, but are not limited to, sesame, olive, arachnis, maize, almond, cottonseed, and castor oil.
  • a highly developed form of drug delivery that imparts sustained release over periods of time ranging from days to years is to implant a drug-bearing polymeric device subcutaneously or in various body cavities.
  • the polymer material used in an implant which must be biocompatible and nontoxic, include but are not limited to hydrogels, silicones, polyethylenes, ethylene-vinyl acetate copolymers, or biodegradable polymers.
  • compositions may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • kits for carrying out the therapeutic regimens of the invention comprise in one or more containers having therapeutically or prophylactically effective amounts of the catechin and proanthocyanadin compositions in pharmaceutically acceptable form.
  • the catechin and proanthocyanadin composition in a vial of a kit of the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid.
  • the composition may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g. , saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for injection purposes.
  • kits of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad. Instructions are optionally included for administration of catechin/proanthocyanadin composition by a clinician or by the patient.
  • a formulation comprising catechins and proanthocyanadins may be used as a dietary or nutritional supplement.
  • the total daily dose ranges of the active catechins and proanthocyanadins for the conditions described herein are generally from about 10 mg to about 800 mg of catechins and from about 5 to 400 mg of proanthocyanadins administered in divided doses administered parenterally or orally.
  • a preferred total daily dose is from about 50 mg to about 400 mg of the active catechins and from about 25 to 200 mg of proanthocyanadins.
  • the dosages of proanthocyanadins described herein are also understand by one of skill in the art to be an equivalent amount of blue fruit extract.
  • a total daily dose of a formulation may be used as a dietary supplement is about 10 mg to about 800 mg of active catechins and from about 5 to 400 mg of proanthocyanadins administered twice daily (e.g., in the morning and the evening) at a dose of about 5 mg to about 400 mg and from about 2 to 200 mg of proanthocyanadins.
  • the dosage forms and compositions may comprise any of the forms and compositions described supra.
  • the formulation comprising catechins and proanthocyanadins is a tablet, capsule, gel, or a liquid-soluble powder.
  • the magnitude of a therapeutic dose of catechins and proanthocyanadins in the acute or chronic management of cancer will vary with the severity of the condition to be treated and the route of administration.
  • the dose, and dose frequency will also vary according to the age, body weight, condition and response of the individual patient, and the particular catechin and proanthocyanadin combination used. All combinations described in the specification are encompassed as therapeutic, active catechin and proanthocyanadin mixtures and it is understood that one of skill in the art would be able to determine a proper dosage of particular catechin and proanthocyanadin mixtures using the parameters provided in the invention.
  • one of ordinary skill in the art would be able to vary the dose of the proanthocyanadins relative to the amounts of catechins present, based on the guidance provided throughout the invention, particularly as described in Example 6.
  • the total daily dose ranges of the active catechins for the conditions described herein are generally from about 10 mg to about 1000 mg and from about 5 to 500 mg of proanthocyanadins administered in divided doses administered parenterally or orally or topically.
  • a preferred total daily dose is from about 200 mg to about 600 mg of the active catechins and from about 100 to 300 mg of proanthocyanadins.
  • the daily dose ranges of catechins for the conditions described herein are generally from about 10 to about 100 mg per kg weight and from about 5 to 50 mg of proanthocyanadins.
  • the catechin and proanthocamidin formulation of the invention is given daily until remission, followed by two to ten additional cycles, each lasting about 60 days in duration.
  • a sustained release formulation is preferred so that a fairly constant level of catechins is provided over the course of treatment, which is generally at least 48 hours and preferably at least 96 hours per cycle.
  • the formulation may be administered for as long as necessary to achieve the desired therapeutic effect.
  • a suitable dosage range for use is, e.g. , from 1 to about 10 mg per kg body weight of catechins and from about 0.5 to 5 mg per kg body weight of proanthocyanadins total daily.
  • a preferred dosing regimen involves intravenous infusion of about 1 to about 10 mg per kg body weight of catechins and from about 0.5 to 5 mg per kg body weight of proanthocyanadins extract. This daily treatment protocol is repeated once per month until the tumor growth tumor is inhibited or when the tumor shows signs of regression.
  • the effect of the therapy with catechins and proanthocyanadins on cancer treatment can be monitored by any methods known in the art, including but not limited to monitoring circulating tNOX activity in patient sera, as well as more traditional approaches such as determining levels of tumor specific antigens and putative biomarkers, e.g., carcinoembryonic antigens (CEA), alpha-fetoprotein; and changes in morphology and/or size using computed tomographic scan and/or sonogram. Desirable blood levels may be maintained by a continuous infusion of catechins and proanthocyanadins as ascertained by plasma levels. It should be noted that the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects, if any).
  • CEA carcinoembryonic antigens
  • alpha-fetoprotein alpha-fetoprotein
  • Desirable blood levels may be maintained by a continuous infusion of catechins and proanthocyanadins as as
  • Dosage forms include tablets, troches, cachet, dispersions, suspensions, solutions, capsules, gel caps, caplets, compressed tablets, sustained release devices, patches, and the like.
  • compositions of the present invention comprise catechins and proanthocyanadins as the active ingredients, as well as pharmaceutically acceptable salts thereof, and may also contain a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases.
  • the pharmaceutical compositions include compositions suitable for oral and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous, and other injectables) routes, although the most suitable route in any given case will depend on the nature and severity of the condition being treated.
  • catechin and proanthocyanadin carrier could be delivered via charged and uncharged matrices used as drug delivery devices such as cellulose acetate membranes, also through targeted delivery systems such as fusogenic liposomes attached to antibodies or specific antigens.
  • catechins and proanthocyanadins can be combined as the active ingredient(s) in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including tablets, capsules, powders, intravenous injections or infusions).
  • any of the usual pharmaceutical media may be employed, e.g., water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like; in the case of oral liquid preparations, e.g., suspensions, solutions, elixirs, liposomes and aerosols; starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like in the case of oral solid preparations e.g., powders, capsules, and tablets.
  • oral liquid preparations e.g., suspensions, solutions, elixirs, liposomes and aerosols
  • starches sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like in the case of oral solid preparations e.g., powders, capsules, and tablets.
  • compositions for parenteral dosage form such as intravenous injection or infusion
  • similar pharmaceutical media e.g., water, glycols, oils, buffers, sugar, preservatives and the like know to those skilled in the art.
  • parenteral compositions include, but are not limited to Dextrose 5% (w/v), normal saline or other solutions.
  • the total dose of the catechins and proanthocyanadins may be administered in a vial of intravenous fluid, e.g., ranging from about 0.01 to about 100 mg per kg body weight of catechins and from about
  • the volume of dilution fluid will vary according to the total dose administered and over the length of the period of time of administration.
  • An exemplary course of treatment of a patient with cancer or solid cancer can involve daily administration by intravenous infusion of catechins and proanthocyanadins in
  • an aqueous solution at a daily dose of about 1 to about 10 mg of the catechins and from about 0.5 to 5 mg of proanthocyanadins per kg of body weight of the patient.
  • the course of treatment may be repeated for up to ten times over approximately 10 months with a break of about three to six weeks in between courses.
  • the post-remission course of treatment involves infusion of catechins at a daily dose of about 0.01 to about 1 mg and from about
  • the invention encompasses the daily dose ranges of catechins for the conditions described herein are generally from about 0.1 to about 15 mg and from about 0.05 to 8 mg of proanthocyanadins per kg body weight administered in
  • the catechin and proanthocyanadin formulation of the invention is given daily, or until remission, followed by two to ten additional cycles, each lasting about 60 days in duration.
  • a sustained release formulation is preferred so that a fairly constant level of catechins and proanthocyanadins is provided over the course of treatment, which is generally at least
  • the formulation may be administered for as long as necessary to achieve the desired therapeutic effect.
  • a suitable dosage range for use is, e.g., from about 0.01 to about 1.5 mg per kg body weight of catechins and from about 0.005 to 1 mg of proanthocyanadins
  • a preferred dosing regimen involves intravenous infusion of the active catechins of the invention, as described above, in the amount of about 0.01 to about 10 mg and from about 0.005 to 5 mg of proanthocyanadins per kg body weight per day. This daily treatment protocol is repeated once per month until
  • the invention also encompasses methods for monitoring patient response to tea catechins and proanthocyanadins.
  • the response of neoplastic cells to the subject compositions may be monitored by assaying the blood or urine of the patient for the NOX activity that is responsive to the catechin and proanthocyanadin compositions, i.e., tNOX.
  • Various assays may be used to monitor activity, such as a NOX assay for neoplasia determination see e.g., U.S. Patent No. 5,605,810.
  • an effective dosage of the subject compositions may be administered in accordance with the requirement of an individual patient.
  • HeLa (ATCC CCL2) cells were grown in 150 cm 2 flasks in Minimal Essential Medium (Gibco), pH 7.4, at 37 °C with 10%> bovine calf serum (heat inactivated, plus 50 mg/1 gentamicin sulfate (Sigma).
  • NADH oxidase activity was monitored and measured by virtue of the decrease in absorbance at 340 nm wavelength.
  • a millimolar extinction coefficient of 6.22 for NADH was used to calculate rates of NADH oxidation. Additional tests that may be used include those such as those described by Morre, 1994, Bioenerg. Biomemb. 26:421, and Chueh et al., 1997, J. Biol. Chem. 272:11221, which are incorporated by reference in their entireties.
  • the blue fruits are crushed (e.g., with a garlic press) to obtain the blue fruit extracts, and the subsequent dilutions of the extracts are indicated as ratios (e.g., 1 :500 indicating a 500 part dilution of the extract in water or a buffer) in the figures and table.
  • proanthocyanadins i.e., the blue fruit extracts
  • tNOX NADH oxidase activity
  • HeLa cells human cervical carcinoma cells
  • Figures 5 and 6 a synergistic effect exists between tea catechins and the proanthocyanadins of the blue fruit extracts.
  • Figures 1, 2, and 3 the juice of greenbriar (Smilax glauca), pokeberry (Phytolacca americana), and wild and cultivated blackberry (Rubus spp.) inhibit both tNOX activity and the growth of cultured HeLa cells.
  • FIG. 4 and Table 1 indicate that pokeberry, greenbriar, and choke cherry are more effective than the other blue fruits tested for inhibiting tNOX activity and HeLa cell growth among the blue fruits tested. Despite the varying specific activities of the blue fruit extracts that are tested, all of the blue fruit extracts that are tested are effective for inhibiting tNOX activity and HeLa cell growth.
  • Figures 5 and 6 illustrate the effect on HeLa cell growth by a combination of a commercially supplied tea concentrate (TegreenTM, Pharmanex, Brisbane, CA) and the extract from wild and cultured blackberry, choke cherry, greenbriar, and pokeberry.
  • TegreenTM commercially supplied tea concentrate
  • Table 1 Edil 50 (aqueous dilution to give 50% inhibition) for inhibition of tNOX activity and growth of cultured HeLa (human cervical carcinoma cells) in culture (96 well plate assay) for juices from eight blue fruits.

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Abstract

La présente invention concerne des méthodes et des compositions permettant d'empêcher ou de traiter le cancer consistant à administrer d'une combinaison de catéchines et de proanthocyanadines. Les compositions de catéchines comprennent, mais pas seulement, du gallate d'épigallocatéchine (EGCg), de l'épicatéchine (EC), du gallate d'épicatéchine (ECG), de l'épigallocatéchine (EGC). Les compositions de proanthocyanadines comprennent, mais pas seulement, des extraits de fruits bleus. Les compositions uniques selon l'invention contiennent diverses combinaisons desdites catéchines et proanthocyanadines, combinées les unes avec les autres ou avec d'autres agents thérapeutiques et sont utilisées pour traiter les cancers primitifs ou métastatiques chez l'homme. L'invention concerne également divers modes d'administration desdits composés thérapeutiques, notamment des formulations qui peuvent être utilisées comme un complément diététique ou nutritionnel ou comme un composé thérapeutique.
PCT/US2002/005294 2001-02-22 2002-02-22 Compositions a base de synergies de catechine-proanthocyanadine pour la prevention et le traitement du cancer WO2002067965A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008054639A3 (fr) * 2006-10-30 2008-08-07 Insite Vision Inc Réactifs et procédés de traitement de maladies et d'infections oculaires
US8044095B2 (en) 2004-07-27 2011-10-25 Genprofiler S.R.L. Mixture of catechins or rather polyphenols extracted from chinese green tea or other vegetables for the prevention of prostate cancer and for the treatment of prostate hypertrophy (BPH)
WO2012038694A1 (fr) 2010-09-20 2012-03-29 Bionature E.A. Ltd. Composés anticancéreux
CN113358592A (zh) * 2020-03-04 2021-09-07 福建医科大学 基于海藻酸钠-铂纳米粒子氧化酶活性的原花青素检测方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780060A (en) * 1994-02-02 1998-07-14 Centre National De La Recherche Scientifique Microcapsules with a wall of crosslinked plant polyphenols and compositions containing them
US5972985A (en) * 1997-11-03 1999-10-26 Cytos Pharmaceuticals, Llc Histidine containing nutriceutical compositions
US5989557A (en) * 1995-03-14 1999-11-23 Indena S.P.A. Process for extracting polyphenol fractions of tea and compositions produced therewith

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780060A (en) * 1994-02-02 1998-07-14 Centre National De La Recherche Scientifique Microcapsules with a wall of crosslinked plant polyphenols and compositions containing them
US5989557A (en) * 1995-03-14 1999-11-23 Indena S.P.A. Process for extracting polyphenol fractions of tea and compositions produced therewith
US5972985A (en) * 1997-11-03 1999-10-26 Cytos Pharmaceuticals, Llc Histidine containing nutriceutical compositions

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8044095B2 (en) 2004-07-27 2011-10-25 Genprofiler S.R.L. Mixture of catechins or rather polyphenols extracted from chinese green tea or other vegetables for the prevention of prostate cancer and for the treatment of prostate hypertrophy (BPH)
WO2008054639A3 (fr) * 2006-10-30 2008-08-07 Insite Vision Inc Réactifs et procédés de traitement de maladies et d'infections oculaires
WO2012038694A1 (fr) 2010-09-20 2012-03-29 Bionature E.A. Ltd. Composés anticancéreux
CN113358592A (zh) * 2020-03-04 2021-09-07 福建医科大学 基于海藻酸钠-铂纳米粒子氧化酶活性的原花青素检测方法
CN113358592B (zh) * 2020-03-04 2023-06-06 福建医科大学 基于海藻酸钠-铂纳米粒子氧化酶活性的原花青素检测方法

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