WO2007041276A2 - Methodes permettant de moduler l'apoptose, la croissance cellulaire et l'expression des proteines au moyen de compositions a base de plantes - Google Patents

Methodes permettant de moduler l'apoptose, la croissance cellulaire et l'expression des proteines au moyen de compositions a base de plantes Download PDF

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WO2007041276A2
WO2007041276A2 PCT/US2006/038019 US2006038019W WO2007041276A2 WO 2007041276 A2 WO2007041276 A2 WO 2007041276A2 US 2006038019 W US2006038019 W US 2006038019W WO 2007041276 A2 WO2007041276 A2 WO 2007041276A2
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weight
extract
hydroalcoholic
supercritical
ginger
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PCT/US2006/038019
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WO2007041276A3 (fr
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Thomas Newmark
Debra L. Bemis
Aaron E. Katz
Paul Schulick
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New Chapter, Inc.
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    • 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)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
    • 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)
    • A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
    • 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)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • 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)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/539—Scutellaria (skullcap)
    • 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)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A61K36/704—Polygonum, e.g. knotweed
    • 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)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A61K36/718—Coptis (goldthread)
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9068—Zingiber, e.g. garden ginger

Definitions

  • the present inventive subject matter relates to novel methods for modulating -apoptosis, cell growth, and protein expression, comprising administration of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
  • a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
  • Prostate cancer is a serious and often life-threatening condition.
  • Prostate cancer which is characterized by rapidly-proliferating cell growth, continues to be the subject of worldwide research efforts directed toward the identification of therapeutic agents which are effective in the treatment thereof. Effective therapeutic agents prolong the survivability of the patient, inhibit the rapidly-proliferating cell growth associated with the disease, or effect a regression of the disease. Research in this area is primarily focused on identifying agents which are therapeutically effective in humans and other mammals.
  • prostate cancer With prostate cancer, as with all solid tumors, it is the metastatic encroachment of the tumor on other vital function that causes the demise of the patient. Approximately 10% of patients are diagnosed initially with metastatic disease. Ultimately, 30-40% of patients with this cancer will develop metastatic disease. Once metastasis occurs, the cancer follows a relentless progression unless interrupted by effective treatment. Prostate cancers are classified based on their aggressiveness and how different they are from the surrounding prostate tissue. There are several different ways to classify tumors; one of the more common is the Whitmore-Jewett system, in which tumors are rated as follows:
  • tumor is unable to be felt on physical examination, and is usually detected by accident after prostate surgery done for other reasons.
  • tumor is confined to the prostate and usually detected by physical examination or PSA testing.
  • cancer has metastasized to regional lymph nodes or other parts of the body, such as the bone and lungs for example.
  • prostate cancers are now found before they cause symptoms.
  • the symptoms listed below are possible indicators of prostate cancer: urinary hesitancy, urinary dribbling, urinary retention, pain with urination, pain with ejaculation, lower back pain, pain with bowel movement, excessive urination at night, incontinence, bone pain or tenderness, hematuria, abdominal pain, anemia, weight loss, and lethargy.
  • the appropriate treatment of prostate cancer is often controversial. Treatment options vary based on the stage of the tumor. In the early stages, surgical removal of the prostate and radiation therapy may be used to eradicate the tumor. Metastatic cancer of the prostate may be treated by hormonal manipulation, reducing the levels of testosterone by drugs or removal of the testes, or by chemotherapy.
  • Surgical removal of the prostate has several possible complications, including impotence and urinary incontinence. Removal of the testes alters hormone production and may be recommended for metastatic cancer, and has possible complications including loss of testosterone production, leading to problems with sexual function, osteoporosis, and loss of muscle mass. Radiation therapy has possible complications including loss of appetite, fatigue, skin reactions such as redness and irritation, rectal burning or injury, diarrhea, cystitis, and blood in the urine. Hormonal manipulation, which is mainly used to relieve symptoms without curing the prostate cancer, has possible complications including nausea and vomiting, hot flashes, anemia, lethargy, osteoporosis, reduced sexual desire, liver problems, diarrhea, enlarged breasts, and erectile dysfunction, along with the obvious lack of treatment of the disease itself. Chemotherapy, using medications such as mitoxantrone, prednisone, paclitaxel, docetaxel, estramustine, and adriamycin, has possible complications which are numerous and specific to a given chemotherapy drug.
  • Cyclooxy ⁇ enase Inhibitors Cyclooxygenase is an enzyme-protein complex with a variety of biochemical actions. There are at least three primary COX isoenzymes, COX-I, COX-2, and COX-3.
  • COX-I is a constitutive enzyme, produced at a reasonably consistent level at all times. It plays an important role in, for example, gastrointestinal protection, kidney function, and the aggregation of blood platelets.
  • COX-2 production is not constant; it varies depending on signals from various biochemical catalysts. For example, in the case of arthritis inflammation and pain, COX-2 responds to tissue damage by oxidizing arachidonic acid, creating prostaglandins which in turn produce local inflammation.
  • COX-3 has been identified relatively recently (Chandrasekharan, et al . , PNAS U.S.A., 99 (21) :1392 ⁇ -31 (2002)). In humans, COX-3 mRNA is expressed most abundantly in the cerebral cortex and heart tissues. COX-3 activity is selectively inhibited by analgesic/antipyretic drugs. It has been suggested that inhibition of COX-3 could represent a mechanism by which these drugs decrease pain and possibly fever.
  • Arachidonic acid and its precursor, linoleic acid are present in significant quantities in animal fats and a variety of vegetable oils, both of which are generally thought to be consumed in greater quantities in the typical Western diet in comparison to Eastern diets.
  • the elevated intake of these fatty acids provides increased substrate availability for cyclooxygenases (COX) , enzymes responsible for converting arachidonic acid into potent signaling molecules termed prostaglandins.
  • COX cyclooxygenases
  • prostaglandins In addition to their vital role as second messengers in many important biological pathways, prostaglandins have recently been implicated in tumor development, progression, and metastasis.
  • COX-1 cyclooxygenase-1
  • COX-2 cyclooxygenase-2
  • COX-2 inhibitory drugs have shown the ability to suppress prostate cancer cell growth in vitro, induce apoptosis, and suppress growth of human prostate tumor xenografts in immunodeficient mouse models or transgenic models of prostate cancer such as the TRAMP mouse (See, e.g. Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, and Levine AC: Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol.
  • COX-2 Inhibitors for Treating Cancer. It has been postulated that COX-2 inhibitors may be useful for treating cancer. Yet only a very few patents actually disclose the use of COX-2 inhibitors for treating any cancers.
  • U.S. Patent No. 5,466,823 to Talley, et al . (Pyrazol-1-yl) benzene sulfonamides are disclosed as inhibitors of cyclooxygenase-2, and for use in the treatment of inflammation, arthritis, and pain, and as being useful for preventing colon cancer. However, their use for actually treating colon cancer or for treating or preventing other neoplasias is not disclosed.
  • U.S. Patent No. 6,469,040 to Seibert, et al . discloses a method of using a specific, disclosed class of cyclooxygenase-2 inhibitor derivatives in preventing and treating epithelial cell neoplasia in a subject.
  • U.S. Patent No. 6,534,540 to Kindness, et al . discloses a combination of the proprietary HMG-CoA reductase inhibitor lovastatin and the proprietary COX-2 inhibitor rofecoxib for the treatment of cancer, especially prostate cancer, and a method of treatment of cancer, especially prostate cancer, by that combination.
  • Salicylic acid for example, is a traditional inflammatory inhibitor agent found in willow tree bark and the chemical derivative of this agent, aspirin, remains one of the most commonly used COX inhibitory substances in the world.
  • inventive methods and compositions which inhibit growth and promote apoptosis in prostate cancer cells and other cells.
  • inventive compositions inhibit cell growth and induce apoptosis in LNCaP cells, and surprisingly that these actions occur independently of COX-2 enzyme inhibition.
  • the present inventive subject matter relates to a method for modulating expression of one or more cellular proteins in a target cell, wherein said one or more proteins comprises Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
  • the present inventive subject matter further relates to a method for inhibiting cell growth, inducing apoptosis, or both, in a target cell, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry, wherein said administration modulates expression of one or more of cellular proteins comprising Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof.
  • the present inventive subject matter further relates to a method, in an individual in need thereof, for treating a disease, disorder, or condition associated with expression of one or more of cellular proteins comprising Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition for treating said disease, disorder, or condition, the composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
  • a composition for treating said disease, disorder, or condition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese
  • Figure 1 is a graph which depicts growth inhibition of LNCaP cells following exposure to the inventive compositions.
  • Figure 2 is a photograph which depicts a Western blot of apoptosis in LNCaP cells following treatment with the inventive compositions.
  • Figure 3A is a photograph which depicts COX-2 mRNA expression in LNCaP and human prostate epithelial cells (hPECs) following treatment with the inventive compositions.
  • Figure 3B is a graph which depicts flow cytometric analysis of LNCaP cells treated with the inventive compositions.
  • Figure 4 is a photograph which depicts a Western blot of p21 and AR expression in LNCaP cells following treatment of LNCaP cells with the inventive compositions.
  • Figure 5 is a graph which depicts a Western blot of phospho- Stat3 and phospho-PKC ⁇ / ⁇ expression in LNCaP cells following treatment with the inventive compositions.
  • terapéuticaally effective amount refers to that amount of the extract which will contribute to the cancer-treating ability of the composition.
  • treating refers to partial or total inhibition of the growth, spreading, or metastasis of prostate neoplasia, as well as partial or total destruction of the cancer cells.
  • treating includes the reduction or elimination of prostate neoplasia, and also the reduction in the incidence of the disease.
  • preventing refers to either preventing the onset of prostate neoplasia, or preventing- the onset of a preclinically evident stage of prostate neoplasia in individuals at risk. Also intended to be- encompassed by this definition is the prevention of initiation for malignant cells, and the arrest or reversal of the progression of premalignant cells to malignant cells. "Preventing” also includes the prevention of growth or spreading of the prostate neoplasia. This includes prophylactic treatment of those at risk of developing a prostate neoplasia.
  • supercritical gas or “supercritical fluid” as used herein refers to a gas is that heated to a temperature critical point, over which the gas will maintain its gaseous state and not turn to a liquid regardless of pressure.
  • a gas heated to a temperature above its critical point will become very dense on compression, so that its characteristics resemble those of a fluid, but will not become liquid.
  • Carbon dioxide is commonly used in applications requiring a supercritical fluid.
  • the general properties of supercritical fluids and the general use of supercritical fluids in extraction processes are described in, e.g.
  • supercritical extraction refers to the technique in which hydrophobic compounds can be extracted from samples utilizing a supercritical fluid.
  • the solvation power of a supercritical fluid is increased as the pressure and temperature are increased above their critical points, producing an effective solvent for the isolation of hydrophobic molecules.
  • hydroalcoholic extraction refers to the technique in which hydrophillic compounds can be extracted from a sample utilizing a solution of alcohol and water, followed by evaporation of the solution to produce a extract consisting of dissolved solids.
  • neoplasia refers broadly to neoplastic, pre-malignant, and proliferative disease, including specifically benign, premalignant, or. malignant neoplasms in individuals with or without any prior history or diagnosis of neoplastic, pre-malignant, or proliferative disease.
  • neoplasia includes neoplasia that produce prostaglandins or express a cyclooxygenase, including both benign and cancerous tumors, growths, and polyps.
  • prostate neoplasia refers broadly to epithelial cancers, epitheliomas, carcinomas, sarcomas, or other malignant tumors or neoplasia of glandular origin in the prostate.
  • subject refers to any human or mammal subject who has a prostate neoplasia, preferably a human subject.
  • the subject is any human or animal subject, preferably a human subject, who is at risk for developing an epithelial cell-derived prostate neoplasia.
  • the subject may be at risk due to exposure to carcinogenic agents, being genetically predisposed to have a prostate neoplasia, and the like.
  • cyclooxygenase-2 inhibitor or "COX-2 inhibitor” as used herein refers to a compound or composition which is able to inhibit cyclooxygenase-2 without adverse inhibition of cyclooxygenase-1.
  • metastatic prostate cancer Although the occurrence rate of localized, latent forms of prostate cancer is consistent throughout the world, the occurrence of metastatic prostate cancer is much greater in western countries compared to eastern countries. This striking disparity suggests the involvement of environmental factors in the development of metastatic prostate cancer, and has prompted the initiation of several epidemiological studies which suggest a link between high fat diets and risk of metastatic prostate cancer.
  • arachidonic acid and its precursor, linoleic acid are present in significant quantities in animal fats and a variety of vegetable oils. Physiologically, these fatty acids are integral components of cellular membranes and also function as substrates for the production of an important group of potent, signaling lipids, termed eicosanoids.
  • Eicosanoids are known to be involved in the initiation of the inflammatory response, fever production, regulation of blood pressure, blood clotting, control of reproductive processes and tissue growth, and regulation of the sleep/wake cycle. Additionally, these powerful mediators and the enzymes that produce them, cyclooxygenases (COX) and lipoxygenases (LO) , have been implicated in development, progression, and metastasis of some tumors.
  • COX cyclooxygenases
  • LO lipoxygenases
  • inventive compositions represent a standardized herbal extract from ten different botanicals. Whereas each of the herbs that are used in the formulation of the inventive compositions are known to contain unique antiinflammatory and anti-cancer compounds, a dietary supplement with " a diverse chemical profile may better represent the benefits of an Eastern diet than any individual supplement alone.
  • One common property of each component of the inventive compositions is the ability to influence arachidonic acid metabolism. Cyclooxygenases, critical enzymatic mediators of the arachidonic acid metabolism/inflammatory cascade, have been increasingly under scrutiny as targets for the development of cancer preventative or therapeutic strategies.
  • COX-2 the inducible form of this family, as its expression and activity have been associated with the development and progression of various human cancers.
  • COX inhibitors have been shown to block cell growth and induce apoptosis in prostate cancer cells, as well as suppress tumor growth in prostate cancer xenograft models.
  • COX and COX-2 inhibitory agents have alternative mechanistic actions that are not dependent upon COX-2, as these agents have demonstrated growth inhibitory and pro-apoptotic activities in both COX-2 deficient cancer cell lines and tumor xenografts.
  • COX- independent activities include inhibition of cGMP-specific phosphodiesterases (PDE2, PDE5) , reduction of anti-apoptotic factor, BCL-xL, and inhibition of IKK ⁇ resulting in suppression of NF- ⁇ B signaling.
  • the encoded protein binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle progression at Gl.
  • the expression of the p21 gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle Gl phase arrest in response to a variety of stress stimuli.
  • the p21 protein can interact with proliferating cell nuclear antigen (PCNA) , a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair. This protein was reported to be specifically cleaved by CASP3-like caspases, which thus leads to a dramatic activation of CDK2, and may be instrumental in the execution of apoptosis following caspase activation.
  • PCNA proliferating cell nuclear antigen
  • Post-translational modifications to p21 include the following:
  • p21 protein is also known to interact with the following proteins: Casein kinase II, beta, Cyclin Dl, Tumor susceptibility gene 101, Zinc finger protein 356, DDX9 Proliferating cell nuclear antigen, DNA topoisomerase I, Cyclin dependent kinase 5, Replication factor A protein 1, Cyclin dependent kinase 4, PIMl, Proliferating cell nuclear antigen, Protein kinase C, eta, Cyclin El, Cyclin dependent kinase 2, SET protein, Cyclin Dl, Proliferating cell nuclear antigen, Cyclin dependent kinase 2, Proteasome subunit, alpha type 3, ADP ribosyl transferase, Proliferating cell nuclear antigen, GADD45 beta, BRCA2 and CDKNlA interacting protein, Cyclin dependent kinase 2, Proliferating cell nuclear antigen, Cyclin Bl, CDC2, DNA damage inducible transcript 1, GADD45 gamma, Cyclin dependent gamm
  • inventive compositions As well, Applicants' analysis of the effect of the inventive compositions on gene expression showed that it suppressed expression of AR in LNCaP cells.
  • Prostate cancer is a disease that is promoted by androgen action, and prostate cell growth and survival is supported by androgens.
  • inventive compositions like some other more specific COX inhibitory agents, down-regulates AR expression also suggests a potential mechanism through which the inventive compositions might specifically influence prostate cancer development and growth.
  • the exact mechanism of action involved in the reduction of AR expression in LNCaP cells following exposure to the inventive compositions is unknown at this time.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • APNs activated protein 1
  • the androgen receptor gene is more than 90 kb long and codes for a protein that has 3 major functional domains: the N-terminal domain, DNA-binding domain, and androgen-binding domain.
  • the protein functions as a steroid-hormone activated transcription factor. Upon binding the hormone ligand, the receptor dissociates from accessory proteins, translocates into the nucleus, dimerizes, and then stimulates transcription of androgen responsive genes.
  • This gene contains 2 polymorphic trinucleotide repeat segments that encode polyglutamine and polyglycine tracts in the N-terminal transactivation domain of its protein. Expansion of the polyglutamine tract causes spinal bulbar muscular atrophy (Kennedy disease) . Mutations in this gene are also associated with complete androgen insensitivity (CAIS) . Two alternatively spliced variants encoding distinct isoforms have been described.
  • CAIS complete androgen insensitivity
  • AR is also known to interact with the following proteins: RAN, member RAS oncogene family, Retinoic acid induced 17, Nuclear receptor corepressor 1, SRCl, Transcription factor HF, alpha subunit, AES, Cyclin Dl, Calreticulin, Insulin degrading enzyme, C20orfl4 protein, Ets transcription factor PDEF, TIF2, STAT3, Cyclin El, Squamous cell carcinoma antigen recognized by T cells 3, Protein inhibitor of activated STAT2, PDEF, PNRC, Transcriptional intermediary factor 1, alpha, ARA267, CAP binding protein complex interacting protein 1, Sex determining region Y protein, Death associated protein 3, PIASY, CDC 25B, SMAD4, Histone deacetylase 1, Supervillin, Nuclear hormone receptor TR4 , Ras related C3 botulinum substrate 3, Estrogen receptor alpha, Forkhead box Al, NCOA3, P160, Cyclin dependent kinase 9, HSP90A, Nuclear hormone receptor TR2, Ring finger protein 4,
  • Applicants also observed a markedly increased level of phospho-cJun, the active form of this protein, in cells treated with the inventive compositions compared to control cells which may play such a role in the reduction of AR expression levels in LNCaP cells treated with the inventive compositions.
  • JUN The oncogene JUN is the putative transforming gene of avian sarcoma virus 17; it appears to be derived from a gene of the chicken genome and has homologs in several other vertebrate species; the name JUN comes from the Japanese ' ju-nana, ' meaning the number 17.
  • JUN was originally thought to be identical to the transcription factor API.
  • API is not a single protein, but constitutes a group of related dimeric basic region-leucine zipper proteins that belong to the JUN, FOS
  • JUN 12-0-tetradecanoylphorbol-13-acetate (TPA) response elements or cAMP response elements.
  • JUN is the most potent transcriptional activator in its group, and its transcriptional activity is attenuated and sometimes antagonized by JUNB (165161) .
  • Post-translational modifications to c-Jun include the following : Residue Type Site U ⁇ stream Enzymes
  • c-Jun is also known to interact with the following proteins: Hematopoietically expressed homeobox, Retinoblastoma 1, Transcription factor 20, Glucocorticoid receptor, c-Fos Octamer binding transcription factor 1, High mobility group AT hook 1, Ribosomal protein L18a, c-Fos, TATA box binding protein, p52/54, Splicing factor HCCl, Runt related transcription factor 2, ERKl, DDX21, Heterogeneous nuclear ribonucleoprotein M, ETS2, MAPKAPK5, Ubiquitin conjugating enzyme E2I, COP9, subunit 5, CHOP, T antigen, DDX9, ATF4, TGIF, Glucocorticoid receptor, GATA binding protein 2, Direct, GILZ, BATF, Jun dimerization protein 2, Transcription initiation factor HB, PML, Nuclear transcription factor Y, alpha, Vitamin D receptor, Transcription factor SpI, C Ski, S
  • Applicants' phosphoprotein screening data reproducibly identified changes in the phosphorylation status of several prominent cell signaling molecules in LNCaP cells, including reduction in the phosphorylation of Stat3 and PKC ⁇ / ⁇ following treatment with the inventive compositions.
  • Stat3 is a latent transcription factor that mediates cytokine signals from the cell membrane to the nucleus and is activated by phosphorylation.
  • the protooncogene Stat3 transduces IL-6 signaling and is required for IL-6/gp 130-mediated transformation of normal cells.
  • Stat3 has been observed to be constitutively expressed in the majority of prostate tumors and prostate cancer cell lines, including LNCaP cells, at levels that appear to correlate to degree of malignancy and inhibition of Stat3 induces apoptosis of LNCaP cells.
  • the decrease in active Stat3 levels observed in the LNCaP cells following treatment with this herbal preparation may contribute to apoptosis induced by the inventive compositions.
  • STATs Signal Transducers and Activators of Transcription
  • JAK kinases transcription factors that are phosphorylated by JAK kinases in response to cytokine activation of a cell surface receptor tyrosine kinases.
  • the STATs dimerize and are localized to the nucleus where they activate transcription of cytokine-responsive genes.
  • Cytokines that activate STAT3 include growth hormone, IL-6 family cytokines, and G-CSF.
  • STAT3 induces progression through the cell cycle, prevents apoptosis and upregulates oncogenes, such as c-myc and bcl-X and may play a role in oncogenesis.
  • STAT3 has been shown to play a critical role in hematopoiesis . The importance of STAT3 is underscored by the failure of mice lacking STAT3 to survive embryogenesis. Crosstalk from pathways other than JAK kinases also leads to phosphorylation and activation of STAT3 as indicated by a role of mTOR (mammalian target of rapamycin, or p70 S6 kinase) and MAP kinase pathways in STAT3 activation and signaling.
  • mTOR mimalian target of rapamycin, or p70 S6 kinase
  • MAP kinase pathways in STAT3 activation and signaling.
  • the protein encoded by STAT3 gene is a member of the STAT protein family.
  • STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators.
  • This protein is activated through phosphorylation in response to various cytokines and growth factors including IFNs, EGF, IL5, IL6, HGF, LIF and BMP2.
  • This protein mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis.
  • the small GTPase Racl has been shown to bind and regulate the activity of this protein.
  • PIAS3 protein is a specific inhibitor of this protein.
  • Stat3 is also known to interact with the following proteins: FER, Janus kinase 1, Interferon, alpha receptor, SRCl, Thyroid stimulating hormone receptor, Janus kinase 2, ILl receptor accessory protein, STATl, Transcription factor 1, Glucocorticoid receptor, Interleukin 6 receptor, beta, ElA binding protein p300 SMADl, N-Myc interactor, TCPTP, Protein inhibitor of activated STAT3, SHINC2, Hepatocyte growth factor receptor, Protein disulfide isomerase A3, Interleukin 2 receptor, alpha, Ras related C3 botulinum toxin substrate 1, ElA binding protein p300, STAT3, Sam ⁇ 8, Androgen receptor, NFKB3, Stat3 Interacting Protein, BRCAl, SHP2, Interleukin 6 receptor, alpha, Zinc finger protein 467, Cell death regulatory protein GRIM 19, General transcription factor 2 I, Thymic stromal lymphopoietin, Growth hormone receptor, Janus kinas
  • PKC Active protein kinase C
  • phospho- protein kinase C is involved in a multitude of cellular responses including alterations in cell cycle progression, survival, and transformation.
  • the outcome of this signaling pathway appears to be dependent on many factors including the exact isozyme involved, as well as the cellular environment. In fact, depending on the time of PKC activation, the result may either promote or inhibit cell cycle progression.
  • Applicants observed a dramatic reduction in the level of phospho- PKC ⁇ / ⁇ in LNCaP cells following treatment with the inventive compositions which was concomitant with the induction of apoptosis by this agent. The significance of this outcome is yet to be understood, however elevated PKC expression has been correlated to the development of androgen independent prostate cancer. Additionally, patients with tumors demonstrating high levels of PKC expression have been found to have shorter survival time upon relapse. Although further experimentation is needed, inhibition of PKC ⁇ activation has been suggested to be a potentially effective drug target for the prevention of androgen insensitive disease.
  • PKC Protein kinase C
  • PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control.
  • PKC has the following substrates for its kinase activity:
  • Transient receptor potential cation channel subfamily V member 6 T Phosphorylation 702
  • N-methyl D-aspartate receptor subunit 2B Phosphorylation 1303 CD5 Phosphorylation 436 CD5 Phosphorylation 434
  • Phospholipase C beta 1 S Phosphorylation 887 Opioid receptor S Phosphorylation 344
  • Nitric oxide synthase 1 S Phosphorylation 852 HMG 17 S Phosphorylation 25 HMG 17 S Phosphorylation 29 HMG14 S Phosphorylation 7 Target Residue Activity Type Site HMG 14 S Phosphorylation 21 HMG14 ' S Phosphorylation 25
  • Multiprotein bridging factor 1 T Phosphorylation 91 Target Residue Activity Tvpe Site
  • PKC is also known to interact with the following proteins: Actin filament associated protein, A kinase anchor protein 12, Semenogelin I, Ser ⁇ enogelin II, Ezrin, Gamma aminobutyric acid receptor subunit rho 2, 8 Oxoguanine DNA glycosylase, Synapse associated protein 90 PDZ, Lamin Bl, Rho guanine nucleotide exchange factor 1, CD9, Myosin light chain kinase, smooth muscle and non-muscle isozymes, Arginine vasopressin receptor IA C terminal C terminal, Inositol-trisphosphate 3-kinase B, CDC42, Semenogelin I, Connexin 43, Guanine nucleotide binding protein, alpha 12, Solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter) member 1 0 0, Phospholipase Dl, 14-3-3 zeta 127-142, Integrin beta 1 313-3
  • phospho-Akt levels increased in the inventive compositions-treated LNCaP cells. This was unexpected as Akt activation is generally thought to result in a pro-cell survival response. However, it is possible that the observed elevation in phospho-Akt could be a- "last attempt" of the cancer cell to survive.
  • This sort of stress-mediated activation of the PI3K pathway has been observed in several other experimental systems, including renal tubular epithelial cells in which phospho-Akt levels were found to increase upon serum starvation and in mouse 3T3 fibroblasts stressed with cytotoxic agents such as H 2 O 2 . Activation of proliferative signaling mechanisms under stress conditions are quite possibly an attempt of the cells to sustain cell number.
  • the serine-threonine protein kinase encoded by the AKTl gene is v catalytically inactive in serum-starved primary and immortalized fibroblasts.
  • AKTl and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKTl. It was shown that the activation occurs through phosphatidylinositol 3-kinase.
  • AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKTl, which then phosphorylates and inactivates components of the apoptotic machinery.
  • T Phosphorylation 308 3 Phosphoinositide dependent protein kinase 1; Proteoglycan 2; Pyruvate dehydrogenase kinase, isoenzyme 1
  • T Dephosphorylation 308 Protein phosphatase 2 A, catalytic subunit, alpha isoform Y Phosphorylation 315 c-Src
  • Aktl also has the following substrates for its kir activity :
  • Nuclear receptor subfamily 4 group A, member 1 S Phosphorylation 351 ASKl S Phosphorylation 83
  • Aktl is also known to interact with the following proteins: Tumor necrosis factor ligand superfamily, member 11, Protein kinase C, zeta, HSP90A, HSP90B, Protein kinase C, theta, CTMP, ILK, GAB2, AFX 1, TCLlB oncogene, IRAKI, Keratin 10, IKK alpha, IMP dehydrogenase 2, TRIB3, Tuberin, TCLl protein, B-Raf, GrblO, MAPKAP kinase 2, IRS 1, MAP3K11, FTS, 3 Phosphoinositide dependent protein kinase 1, Mature T cell proliferation 1, Nuclear receptor subfamily 4, group A, member 1, T cell leukemia/lymphoma 6 protein, UDP-glucuronate decarboxylase
  • MAPK8 interacting protein 1, Plexin Al, Protein kinase C like
  • Tuberous sclerosis 1 gene MAP3K8, RAFl, PTPNl, 14-3-3 zeta, MAP2K4, Myosin II, and C terminal modulator protein.
  • inventive compositions demonstrate that the inventive compositions, unique herbal extract preparations, inhibit COX-I and COX-2 activity, strongly suppress cell growth and induce apoptosis in LNCaP cells. Cumulatively these results suggest that the inventive compositions would be expected to have value in chemoprevention or adjuvant therapy for prostate cancer patients.
  • Applicants have developed a mixture comprised of herbal extracts, and the mixture has COX-2 inhibitory activity.
  • compositions are unique, in that they are prepared via a supercritical CO 2 extraction process. Unlike traditional solvent based extraction methods, supercritical CO 2 extraction allows the natural products in the herbs to be obtained without leaving chemical residues behind in the preparation.
  • inventive compositions inhibit cell growth and induce apoptosis in LNCaP cells, and surprisingly, that these actions appear to occur independently of COX-2 enzyme inhibition.
  • compositions comprising a therapeutically effective amount of extracts of one or more of rosemary, turmeric, oregano, ginger, holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, or barberry, or combinations thereof, are effective in modulating expression of one or more cellular proteins in a target cell.
  • the present inventive subject matter relates to a method for modulating expression of one or more cellular proteins in a target cell, wherein said one or more proteins comprises Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and/or ginger; and/or therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and/or barberry.
  • said composition is administered orally.
  • the orally administered composition is in the form of one or more capsules, one or more tablets, or one or more pills
  • the composition comprises: (A) from about 4.5% to about 7.5%, and more preferably from about 5.5% to about 6.5%, by weight of the hydroalcoholic extract of ginger;
  • (C) from about 1.0% to about 1.5%, and more preferably from about 1.2% to about 1.4%, by weight of the supercritical extract of turmeric;
  • (D) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the supercritical extract of rosemary;
  • the hydroalcoholic extract of ginger used in the present invention is preferably prepared as follows.
  • the ginger rhizome which is preferably cryogenically ground to preserve heat sensitive components, is subjected to supercritical extraction, preferably with carbon dioxide, to obtain: (i) an oil extract, referred to herein as "the supercritical extract” of ginger, containing delicate lipophilic components, and (ii) an oil-free residue.
  • the oil-free residue is then extracted in a water/alcohol, preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water.
  • a water/alcohol preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water.
  • the alcohol/water liquid is then evaporated off, leaving a powdered extract residue, referred to herein as "the hydroalcoholic extract" of ginger .
  • the weight ratio of the supercritical extract of ginger to the hydroalcoholic extract of ginger is from about 0.9:1 to about 1.4:1.
  • the supercritical extracts of ginger, rosemary, turmeric and oregano used in the present invention can be prepared according to known supercritical extraction methods, such as disclosed, e.g., in E. Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer Verlag 4 1988, which is hereby incorporated by reference herein.
  • the hydroalcoholic extracts of rosemary, turmeric, holy basil, green tea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensis used in the present invention can be prepared according to conventional hydroalcoholic extraction techniques.
  • the hydroalcoholic extracts can be prepared by extracting the plant portion in a water/alcohol, preferably water/ethanol, mixture preferably composed of 60-80 parts alcohol and 40-20 parts water, and then evaporating off the water/alcohol liquid, leaving a powdered extract residue referred to herein as "the hydroalcoholic extract”.
  • the weight ratio of the hydroalcoholic extract of turmeric to the supercritical extract of turmeric is from about 8:1 to about 12:1.
  • the weight ratio of the supercritical extract of rosemary to the hydroalcoholic extract of rosemary is from about 1.6:1 to about 2.4:1.
  • the hydroalcoholic extract of ginger comprises from about 2.4% to about 3.6%, more preferably from about 2.7% to about 3.3%, and most preferably about 3.0%, by weight of pungent compounds.
  • the supercritical extract of ginger comprises from about 24% to about 36%, more preferably from about 27% to about 33%, and most preferably about 30%, by weight of pungent compounds; and from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of zingiberene.
  • the supercritical extract of turmeric comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of turmerones .
  • the supercritical extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants.
  • the supercritical extract of oregano comprises from about 0.64% to about 0.96%, more preferably from about 0.72% to about 0.88%, and most preferably about 0.8%, by weight of total phenolic antioxidants.
  • the hydroalcoholic extract of turmeric comprises from about 5.6% to about 8.4%, more preferably from about 6.3% to about 7.7%, and most preferably about 7%, by weight of curcumin.
  • the hydroalcoholic extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants.
  • the hydroalcoholic extract of holy basil comprises from about 1.6% to about 2.4%, more preferably from about 1.8% to about 2.2%, and most preferably about 2%, by- weight of ursolic acid.
  • the hydroalcoholic extract of green tea comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of polyphenols.
  • the hydroalcoholic extract of huzhang comprises from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of resveratrol.
  • the hydroalcoholic extract of Chinese goldthread comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
  • the hydroalcoholic extract of barberry comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
  • said composition comprises:
  • composition further comprises: (i) the supercritical extract of ginger and the post-supercritical hydroalcoholic extract of ginger at a weight ratio of from about 0.9 to about 1.4 parts of supercritical extract per 1 part of post-supercritical hydroalcoholic extract;
  • the composition is administered in a daily dosage of at least about 700 mg.
  • composition is administered on a daily basis for at least 4 weeks.
  • the inventive subject matter is further based on the discovery that a combination of certain herbs properly extracted and .blended in appropriate proportions can used for inhibiting cell growth, inducing apoptosis, or both, in a target cell.
  • compositions comprising a therapeutically effective amount of extracts of one or more of rosemary, turmeric, oregano, ginger, holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang,
  • Chinese goldthread, or barberry, or combinations thereof are effective in, are effective for inhibiting cell growth, inducing apoptosis, or both, in a target cell.
  • the present inventive subject matter relates to a method for inhibiting cell growth, inducing apoptosis, or both, in a target cell, comprising administration of an effective amount of a composition comprising therapeutically effective amount of extracts of one or more of rosemary, turmeric, oregano, ginger, holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang,
  • composition is administered orally.
  • orally administered composition is in the form of one or more capsules, one or more tablets, or one or more pills
  • composition comprises:
  • (C) from about 1.0% to about 1.5%, and more preferably from about 1.2% to about 1.4%, by weight of the supercritical extract of turmeric;
  • (D) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the supercritical extract of rosemary;
  • (H) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of holy basil; (I) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of green tea; (J) from about 8.0% to about 12.0%, and more preferably from about 9.0% to about 11.0%, by weight of the hydroalcoholic extract of huzhang; (K) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the hydroalcoholic extract of Chinese goldthread; (L) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the hydroalcoholic extract of barberry; and (M) from about 2.0% to about 3.0%, and more preferably from about 2.25% to about 2.75%, by weight of the hydroalcoholic extract of Scutellaria baicalensis.
  • the hydroalcoholic extract of ginger used in the present invention is preferably prepared as follows.
  • the ginger rhizome which is preferably cryogenically ground to preserve heat sensitive components, is subjected to supercritical extraction, preferably with carbon dioxide, to obtain: (i) an oil extract, referred to herein as "the supercritical extract” of ginger, containing delicate lipophilic components, and (ii) an oil-free residue.
  • the oil-free residue is then extracted in a water/alcohol, preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water.
  • the alcohol/water liquid is then evaporated off, leaving a powdered extract residue, referred to herein as "the hydroalcoholic extract” of ginger.
  • the weight ratio of the supercritical extract of ginger to the hydroalcoholic extract of ginger is from about 0.9:1 to about 1.4:1.
  • the supercritical extracts of ginger, rosemary, turmeric and oregano used in the present invention can be prepared according to known supercritical extraction methods, such as disclosed, e.g., in E. Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer Verlag 4 1988, which is hereby incorporated by reference herein.
  • the hydroalcoholic extracts of rosemary, turmeric, holy basil, green tea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensis used in the present invention can be prepared according to conventional hydroalcoholic extraction techniques.
  • the hydroalcoholic extracts can be prepared by extracting the plant portion in a water/alcohol, preferably water/ethanol, mixture preferably composed of 60-80 parts alcohol and 40-20 parts water, and then evaporating off the water/alcohol liquid, leaving a powdered extract residue referred to herein as "the hydroalcoholic extract”.
  • the weight ratio of the hydroalcoholic extract of turmeric to the supercritical extract of turmeric is from about 8 : 1 to about 12:1.
  • the weight ratio of the supercritical extract of rosemary to the hydroalcoholic extract of rosemary is from about 1.6:1 to about 2.4:1.
  • the hydroalcoholic extract of ginger comprises from about 2.4% to about 3.6%, more preferably from about 2.7% to about 3.3%, and most preferably about 3.0%, by weight of pungent compounds.
  • the supercritical extract of ginger comprises from about 24% to about 36%, more preferably from about 27% to about 33%, and most preferably about 30%, by weight of pungent compounds; and from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of zingiberene.
  • the supercritical extract of turmeric comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of turmerones .
  • the supercritical extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants.
  • the supercritical extract of oregano comprises from about 0.64% to about 0.96%, more preferably from about 0.72% to about 0.88%, and most preferably about 0.8%, by weight of total phenolic antioxidants.
  • the hydroalcoholic extract of turmeric comprises from about 5.6% to about 8.4%, more preferably from about 6.3% to about 7.7%, and most preferably about 7%, by weight of curcumin.
  • the hydroalcoholic extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants.
  • the hydroalcoholic extract of holy basil comprises from about 1.6% to about 2.4%, more preferably from about 1.8% to about 2.2%, and most preferably about 2%, by weight of ursolic acid.
  • the hydroalcoholic extract of green tea comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of polyphenols.
  • the hydroalcoholic extract of huzhang comprises from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of resveratrol.
  • the hydroalcoholic extract of Chinese goldthread comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
  • the hydroalcoholic extract of barberry comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
  • said composition comprises:
  • composition from about 2.0% to about 3.0% by weight of the hydroalcoholic extract of Scutellaria baicalensis; and wherein said composition further comprises:
  • the composition is administered in a daily dosage of at least about 700 mg.
  • composition is administered on a daily basis for at least 4 weeks.
  • the composition comprises an additional agent selected from the group consisting of antineoplastic agents, growth inhibiting agents, and ' nutrients .
  • antineoplastic agents available in commercial use, in clinical evaluation and in pre-clinical development, which optionally are selected for treatment of prostate neoplasia by combination drug chemotherapy.
  • Such antineoplastic agents fall into several major categories: antimetabolite agents, antibiotic-type agents, alkylating agents, hormonal agents, immunological agents, interferon-type agents, metallomatrix proteases, superoxide dismutase mimics or ⁇ v ⁇ 3 inhibitors.
  • said antineoplastic agent is selected from the group consisting of antimetabolite agents, antibiotic-type agents, alkylating agents, hormonal agents, immunological agents, interferon-type agents, metallomatrix proteases, superoxide dismutase mimics, and ⁇ v ⁇ 3 inhibitors .
  • antineoplastic agents which may be used in combination with an inventive composition consists of antimetabolite-type antineoplastic agents.
  • Suitable antimetabolite antineoplastic agents may be selected from the group consisting of 5-FU-fibrinogen, acanthifolic acid, aminothiadiazole, brequinar sodium, carmofur, Ciba-Geigy CGP-30694, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, doxifluridine, Wellcome EHNA, Merck & Co.
  • EX-015 benzrabine, floxuridine, fludarabine phosphate, 5-fluorouracil , N- (2 ' -furanidyl) -5-fluorouracil, Daiichi ' Seiyaku FO-152, isopropyl pyrrolizine, Lilly LY-188011, Lilly LY-264618, methobenzaprim, methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosine kinase inhibitors, tyrosine protein kinase inhibitors, Taiho U
  • a second class of antineoplastic agents which may be used in combination with an inventive composition consists of alkylating-type antineoplastic agents.
  • Suitable alkylating-type antineoplastic agents may be selected from the group consisting of Shionogi 254-S, aldo-phosphamide analogues, altretamine, anaxirone, Boehringer Mannheim BBR-2207, bestrabucil, budotitane, Wakunaga CA-102, carboplatin, carmustine, Chinoin-139, Chinoin-153, chlorambucil, cisplatin, cyclophosphamide, American Cyanamid CL-286558, Sanofi CY-233, cyplatate, Degussa D-19-384, Sumimoto DACHP (Myr) 2, diphenylspiromustine, diplatinum cytostatic, Erba distamycin derivatives, Chugai DWA-2114R, ITI E09, elmus
  • a third class of antineoplastic agents which may be used in combination with an inventive composition consists of antibiotic-type antineoplastic agents.
  • Suitable antibiotic-type antineoplastic agents may be selected from the group consisting of Taiho 4181-A, aclarubicin, actinomycin D, actinoplanone, Erbamont ADR-456, aeroplysinin derivative, Ajinomoto AN-201-II, Ajinomoto AN-3, Nippon Soda anisomycins, anthracycline, azino-mycin-A, bisucaberin, Bristol-Myers BL-6859, Bristol-Myers BMY-25067, Bristol-Myers BMY-25551, Bristol-Myers BMY-26605, Bristol-Myers BMY-27557, Bristol-Myers BMY-28438, bleomycin sulfate, bryostatin-1, Taiho C-1027, calichemycin, chromoximycin, dactin
  • a fourth class of antineoplastic agents which may be used in combination with an inventive composition consists of a miscellaneous family of antineoplastic agents selected from the group consisting of alpha-carotene, alpha-difluoromethyl-arginine, acitretin, Biotec AD-5, Kyorin AHC-52, alstonine, amonafide, amphethinile, amsacrine, Angiostat, ankinomycin, anti-neoplaston AlO, antineoplaston A2, antineoplaston A3, antineoplaston A5, antineoplaston AS2-1, Henkel APD, aphidicolin glycinate, asparaginase, Avarol, baccharin, batracylin, benfluron, benzotript, Ipsen-Beaufour BIM-23015, bisantrene, Bristo-Myers BMY-40481, Vestar boron-10, bromofosfamide, Wellcome BW-50
  • radioprotective agents which may be used in the combination chemotherapy of this invention are AD-5, adchnon, amifostine analogues, detox, dimesna, 1-102, MM-159, N-acylated-dehydroalanines, TGF-Genentech, tiprotimod, amifostine, WR-151327, FUT-187, ketoprofen transdermal, nabumetone, superoxide dismutase (Chiron) , and superoxide dismutase Enzon.
  • said antineoplastic agent is selected from the group consisting of 5-FU-fibrinogen, acanthifolic acid, aminothiadiazole, brequinar sodium, carmofur, Ciba-Geigy CGP-30694, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, doxifluridine, Wellcome EHNA, Merck & Co.
  • EX-015 benzrabine, floxuridine, fludarabine phosphate, 5-fluorouracil, N- (2 ' -furanidyl) -5-fluorouracil, Daiichi Seiyaku FO-152, isopropyl pyrrolizine, Lilly LY-188011, Lilly LY-264618, methobenzaprim, methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosine kinase inhibitors, tyrosine protein kinase inhibitors, Taiho UFT,
  • a benefit provided by the inventive compositions is the utilization of supercritical extraction, an innovative technology for extracting herbs at low temperature without the use of industrial chemical solvents. Such extraction process allows for the highest potency of active compounds in the extracts, as much as 250 times the potency of the original fresh plant material.
  • Table I is a preferred embodiment of the orally administered composition, excluding inactive ingredients, as used in the inventive methods.
  • the amounts recited in Table I represent the preferred dosage of the ingredients listed.
  • Turmeric hydroalcoholic (7% curcumin - 7 mg) rhizome 100
  • Green tea hydroalcoholic 45% polyphenols - 45 mg
  • Huzhang hydroalcoholic 8% resveratrol - 6.4 mg
  • the composition set forth in Table I also includes extra virgin olive oil and yellow beeswax.
  • inventive methods use a therapeutically effective amount of the active compositions indicated above.
  • This effective amount will generally comprise from about 0.1 mg to about 100 mg of the active agent per kilogram of patient body weight per day. This effective amount can vary depending upon the physical status of the patient and other factors well known in the art.
  • this dosage of active agent can be administered in a single or multiple dosage units to provide the desired therapeutic effect. If desired, other therapeutic agents can be employed in conjunction with those provided by the present inventive subject matter.
  • compositions which are preferably delivered to the patient by means of a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier Such carriers are well known in the art and generally will be in either solid or liquid form.
  • Solid form pharmaceutical preparations which may be prepared according to the present inventive subject matter include powders, tablets, dispersible granules, capsules, and cachets. In general, solid form preparations will comprise from about 5% to about 90% by weight of the active agent.
  • a solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents; it can also be encapsulating material.
  • the carrier is a finely divided solid which is in admixture with the viscous active compound.
  • the active compound is mixed with a carrier having the necessary binding properties in suitable proportions and compacted to the shape and size desired.
  • suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • the term "preparation" is intended to include the formulation of the active compound with encapsulating materials as a carrier which may provide a capsule in which the active component (with or without other carriers) is surrounded by carrier, which is thus in association with it.
  • cachets are included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. If desired for reasons of convenience or patient acceptance, pharmaceutical tablets prepared according to the inventive subject matter may be provided in chewable form, using techniques well known in the art.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration.
  • liquid forms include solutions, suspensions, and emulsions.
  • solid form preparations are most conveniently provided in unit dose form and as such are used to provide a single liquid dosage unit.
  • sufficient solid may be provided so that after conversion to liquid form, multiple individual liquid doses may be obtained by measuring predetermined volumes of the liquid form preparation as with a syringe, teaspoon, or other volumetric container.
  • the solid form preparations intended to be converted to liquid form may contain, in addition to the active material, flavorants, colorants, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • the liquid utilized for preparing useful liquid form preparations may be water, isotonic water, ethanol, glycerine, propylene glycol, and the like as well as mixtures thereof. Naturally, the liquid utilized will be chosen with regard to the route of administration. For example, liquid preparations containing large amounts of ethanol are not suitable for parenteral use.
  • the pharmaceutical preparation may also be in a unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, for example, packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can also be a capsule, cachet, or tablet itself or it can be the appropriate number of any of these in packaged form.
  • the pharmaceutical preparations of the inventive subject matter may include one or more preservatives well known in the art, such as benzoic acid, sorbic acid, methylparaben, propylparaben and ethylenediaminetetraacetic acid (EDTA) .
  • Preservatives are generally present in amounts up to about 1% and preferably from about 0.05 to about 0.5% by weight of the pharmaceutical composition.
  • Useful buffers for purposes of the inventive subject matter include citric acid-sodium citrate, phosphoric acid-sodium phosphate, and acetic acid-sodium acetate in amounts up to about 1% and preferably from about 0.05 to about 0.5% by weight of the pharmaceutical composition.
  • Useful suspending agents or thickeners include cellulosics like methylcellulose, carageenans like alginic acid and its derivatives, xanthan gums, gelatin, acacia, and microcrystalline cellulose in amounts up to about 20% and preferably from about 1% to about 15% by weight of the pharmaceutical composition.
  • Sweeteners which may be employed include those sweeteners, both natural and artificial, well known in the art.
  • Sweetening agents such as monosaccharides, disaccharides and polysaccharides such as xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, partially hydrolyzed starch or corn syrup solids and sugar alcohols such as sorbitol, xylitol, mannitol and mixtures thereof may be utilized in amounts from about 10% to about 60% and preferably from about 20% to about 50% by weight of the pharmaceutical composition.
  • Water soluble artificial sweeteners such as saccharin and saccharin salts such as sodium or calcium, cyclamate salts, acesulfame-K, aspartame and the like and mixtures thereof may be utilized in amounts from about 0.001% to about 5% by weight of the composition.
  • Flavorants which may be employed in the pharmaceutical products of the inventive subject matter include both natural and artificial flavors, and mints such as peppermint, menthol, vanilla, artificial vanilla, chocolate, artificial chocolate, cinnamon, various fruit flavors, both individually and mixed, in amounts from about 0.5% to about 5% by weight of the pharmaceutical composition.
  • Colorants useful in the present inventive subject matter include pigments which may be incorporated in amounts of up to about 6% by weight of the composition.
  • a preferred pigment, titanium dioxide, may be incorporated in amounts up to about 1%.
  • the colorants may include other dyes suitable for food, drug and cosmetic applications, known as F.D.&C. dyes and the like. Such dyes are generally present in amounts up to about 0.25% and preferably from about 0.05% to about 0.2% by weight of the pharmaceutical composition.
  • F.D.&C. dyes and the like Such dyes are generally present in amounts up to about 0.25% and preferably from about 0.05% to about 0.2% by weight of the pharmaceutical composition.
  • a full recitation of all F.D.&C. and D.&C. dyes and their corresponding chemical structures may be found in the Kirk-Othmer Encyclopedia of Chemical Technology, in Volume 5, at pages 857-884, which text is accordingly incorporated herein by reference.
  • solubilizers include alcohol, propylene glycol, polyethylene glycol and the like and may be used to solubilize the flavors.
  • Solubilizing agents are generally present in amounts up to about 10%; preferably from about 2% to about 5% by weight of the pharmaceutical composition.
  • Lubricating agents which may be used when desired in the instant compositions include silicone oils or fluids such as substituted and unsubstituted polysiloxanes, e.g., dimethyl polysiloxane, also known as dimethicone. Other well known lubricating agents may be employed.
  • inventive methods use compositions which will display significant adverse interactions with other synthetic or naturally occurring substances.
  • a compound of the present inventive subject matter may be administered in combination with other compounds and compositions useful for treating prostate neoplasia.
  • inventive methods use compositions which may be administered in combination with other inventive compositions, other antineoplastic substances, and the like.
  • the optimal pharmaceutical formulations will be determined by one skilled in the art depending upon considerations such as the route of administration and desired dosage. See, for example, "Remington's Pharmaceutical Sciences", 18th ed. (1990, Mack Publishing Co., Easton, PA 18042), pp. 1435-1712, which is hereby incorporated by reference in its entirety. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present therapeutic agents of the inventive subject matter.
  • the compounds and compositions are preferably administered orally in the form of capsules, tablets, aqueous suspensions, or solutions.
  • Tablets may contain carriers such as lactose and corn starch, and/or lubricating agents such as magnesium stearate.
  • Capsules may contain diluents including lactose and dried corn starch.
  • Aqueous suspensions may contain emulsifying and suspending agents combined with the active ingredient.
  • the oral dosage forms may further contain sweetening, flavoring, coloring agents, or combinations thereof. Delivery in an enterically coated tablet, caplet, or capsule, to further enhance stability and provide release in the intestinal tract to improve absorption, is the best mode of administration currently contemplated. Dosacre
  • Dosage levels on the order of about 0.001 mg to about 100 mg per kilogram body weight of the active ingredient compounds or compositions are useful in the treatment of the above conditions, with preferred levels ranging from 200mg per day to i ⁇ OOmg per day.
  • the compounds and compositions of the present inventive subject matter may usually be given in two or three doses daily. Starting with a low dose (200-300mg) twice daily and slowly working up to higher doses if needed is a preferred strategy.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration .
  • a specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; drug combination; the severity of the particular disorder being treated; and the form of administration.
  • One of ordinary skill in the art would appreciate the variability of such factors and would be able to establish specific dose levels using no more than routine experimentation.
  • inventive compositions were provided by the manufacturer (New Chapter, Inc., Brattleboro, VT) . As described in greater detail in Example 1 and elsewhere in this application, the inventive compositions are an encapsulated olive oil based suspension and experiments were conducted using the liquid material that was directly removed from the capsules. For all of Applicants' experiments, the liquid was dissolved in DMSO at a 1/10 dilution and filtered through a 0.2 ⁇ m filter. For descriptive purposes the inventive compositions solution is expressed as total ⁇ l/ml of the inventive compositions. Compositional analysis of the extract reveals that the inventive compositions contain approximately 13 ⁇ M curcumin. All controls contained DMSO at similar concentrations. NS-398, indomethacin, and PGE 2 were obtained from Cayman Chemical Company (Ann Arbor, MI) . Curcumin was obtained from J. T. Baker (Phillipsburg, NJ) .
  • COX (ovine) Inhibitor Screening Assay The final dilutions of the inventive compositions tested in the assays were 0.45 ⁇ l/ml and 0.90 ⁇ l/ml which contain approximately 6 nM and 12 nM curcumin, respectively. Absorbance of the samples correlating to the activity of COX-I or COX-2 was detected at 600 nm using a 96 well spectrophotometric plate reader (Tecan, SLT Spectra) . This assay was completed in duplicate.
  • Cell Cultures The androgen sensitive human prostate cancer cell line LNCaP was purchased from the American Type Culture Collection (Manassas, VA) .
  • LNCaP cells were maintained in RPMI- 1640 media supplemented with 10% fetal bovine serum (FBS), L- glutamine, antibiotics (Gibco® Invitrogen Corporation) , and the synthetic androgen R1881 (0.05 nM; Perkin Elmer Life Sciences). The cells were maintained at 37 0 C in a humidified atmosphere of 95% air and 5% CO 2 .
  • FBS fetal bovine serum
  • L- glutamine L- glutamine
  • antibiotics Gibco® Invitrogen Corporation
  • R1881 0.05 nM; Perkin Elmer Life Sciences
  • LNCaP cells were seeded in 12 well plates at a density of 38,000 cells per well in a final volume of 1 ml. Twenty four hours after seeding, the medium was removed and replaced with fresh medium containing the same concentration of DMSO (0.1%) as a vehicle control or with medium containing the inventive compositions at concentrations of 0.1 ⁇ l/ml, 0.05 ⁇ l/ml or 0.001 ⁇ l/ml in a final volume of 1 ml. The cultures were maintained in the incubator for a period of 3 days. At the end of the 24, 48, and 72 hour time periods, cells were harvested, trypsinized, and counted using a hemocytometer .
  • LNCaP cells were treated with 0.1 ⁇ l/ml the inventive compositions as described previously for 72 hours.
  • As a positive control LNCaP cells were treated for 24 hours with 10 nM TPA, a known inducer of apoptosis in this cell line. Cells were then trypsinized, counted, and processed according to the manufacturer.
  • RT-PCR of COX-2 in LNCaP Cells To determine if the COX-2 message is expressed in LNCaP cells, RT-PCR was conducted using two different sets of COX-2 specific primers (See HIa T and Neilson K: Human cyclooxygenase-2 cDNA. PNAS. 89, 7384-7388, 1992, and Liu XH, Yao S, Kirschenbaum A, and Levine AC: NS398, a selective cyclooxy-genase-2 inhibitor, induces apoptosis and down-regulation bcl-2 expression in LNCaP cells. Cancer Res. 58, 4245-4249, 1998) .
  • cDNA from normal human prostate epithelial cells (Cambrex, Walkersville, MD) was also analyzed for comparative purposes.
  • the PCR reaction was conducted using DNA Taq polymerase (New England Biolabs) as follows: (1) 95°C for 3 minutes, (2) 95°C for 20 seconds, 50°C for 30 seconds, 72°C for 40 seconds for 42 cycles, and (3) 72°C for 10 minutes. Following the reaction, samples were electrophoresed on a 1% agarose gel containing ethidium bromide, and amplicons were visualized under UV light.
  • PGE 2 Add-back Assay To determine the involvement of cyclooxygenase inhibition in the induction of apoptosis by the inventive compositions, LNCaP cells were treated with the herbal COX inhibitor in conjunction with PGE 2 , the main prostaglandin produced following COX activity. LNCaP cells were plated to 60% confluency in 25cm 2 flasks and exposed to 0.1 ⁇ l/ml of the inventive compositions alone or in combination with freshly diluted PGE 2 (1 nM or 10 nM) for 24 hours. The cells were then collected and processed for flow cytometric analysis, as follows. Adherent cells were trypsinized and pooled with the cells in suspension, centrifuged and washed thrice with ice cold PBS.
  • the cell count of each sample was adjusted to 500,000 cells per ml and fixed in a 2:1 ratio (vol/vol) in chilled ethanol overnight before staining with propidium iodide (PI) in the presence of RNAse.
  • Cell cycle distribution was analyzed on a Becton Dickinson Flow Cytometer (Becton Dickinson, San Jose, CA) , and at least 10,000 cells were analyzed for each experimental condition. Data analysis was performed using the "CellQuest" cell cycle analysis software. The percentage of the cell population that partitioned out into the SubGO fraction, indicating cells undergoing apoptosis, was determined for each treatment.
  • Protein lysates were prepared in a Ix lysis buffer plus protease and phosphatase inhibitors (20 mM MOPS, 5 mM EDTA, 2 mM EGTA, 30 mM NaF, 20 mM Na 4 P 2 O 7/ 1 mM Na 3 VO 4 , 1 mM phenlymethylsulfonylfluoride, 40 mM ⁇ - glycerophosphate, 5 ⁇ M pepstatin A, 10 ⁇ M leupetin, and 0.5% Nonidet P-40) .
  • the harvested lysates were then quantitated as described previously, diluted in SDS-PAGE sample buffer and submitted to the Company for analysis. The screening assay was repeated twice using two different batches of treated cells to verify the results.
  • the p21 (clone 187) and androgen receptor (clone 441) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at dilutions of 1/100 and 1/250, respectively. All antibodies were diluted in blocking buffer (6% nonfat dry milk in TBST) .
  • inventive compositions are prepared by methqds known in the art, and disclosed in Applicant Newmark' s U.S. Patent No. 6,387,416.
  • the preparation of the component elements of the inventive compositions is summarized as follows:
  • the hydroalcoholic extract of ginger used in the inventive compositions is preferably prepared as follows.
  • the ginger rhizome which is preferably cryogenically ground to preserve heat sensitive components, is subjected to supercritical extraction to obtain: (i) an oil extract, referred to herein as "the supercritical extract” of ginger, containing delicate lipophilic components, and (ii) an oil-free residue.
  • the oil-free residue is then extracted in a water/alcohol, preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water.
  • the alcohol/water liquid is then evaporated off, leaving a powdered extract residue, referred to herein as "the hydroalcoholic extract” of ginger.
  • composition of this invention will preferably contain the supercritical extract and the hydroalcoholic extract of ginger at a weight ratio of preferably from about 0.9 to about 1.4 parts, more preferably from about 1.1 to about 1.3 parts, most preferably about 1.17 parts, of supercritical extract per 1 part post-supercritical hydroalcoholic extract.
  • the supercritical extracts of ginger, rosemary, turmeric and oregano used in the inventive compositions can be prepared according to known supercritical extraction methods, such as disclosed, e.g., in E. Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer Verlag 4 1988, which is hereby incorporated by reference herein.
  • the hydroalcoholic extracts of rosemary, turmeric, holy basil, green tea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensis used in the inventive compositions can be prepared according to conventional hydroalcoholic extraction techniques.
  • the hydroalcoholic extracts can be prepared by extracting the plant portion in a water/alcohol
  • the hydroalcoholic extract of turmeric and the supercritical extract of turmeric will preferably be present at a weight ratio of preferably from about 8 to about 12 parts, more preferably from about 9 parts to about 11 parts, most preferably about 10 parts, of hydroalcoholic extract per 1 part of supercritical extract.
  • composition of this invention will preferably contain the supercritical extract of rosemary and the hydroalcoholic extract of rosemary at a weight ratio of preferably from about 1.6 to about 2.4 parts, more preferably from about 1.8 to about 2.2 parts, most preferably about 2.0 parts, of supercritical extract per 1 part of hydroalcoholic extract.
  • the hydroalcoholic extract of ginger used in the inventive compositions will preferably contain from about 2.4% to about 3.6%, more preferably from about 2.7% to about 3.3%, most preferably about 3.0%, by weight of pungent compounds (e.g., shogaol) .
  • the supercritical extract of ginger used in the inventive compositions will contain preferably from about 24% to about 36%, more preferably from about 27% to about 33%, most preferably about 30%, by weight of pungent compounds (e.g., shogaol) and preferably from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, most preferably about 8%, by weight of zingiberene .
  • the supercritical extract of turmeric used in the inventive compositions will contain preferably from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, most preferably about 45%, by weight of turmerones.
  • the supercritical extract of rosemary used in the inventive compositions will contain preferably from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, most preferably about 23%, by weight of total phenolic antioxidants ("TPA”) .
  • the supercritical extract of oregano used in the inventive compositions will contain preferably from about 0.64% to about 0.96%, more preferably from about 0.72% to about 0.88%, most preferably about 0.8%, by weight of TPA.
  • the hydroalcoholic extract of turmeric used in the inventive compositions will contain preferably from about 5.6% to about 8.4%, more preferably from about 6.3% to about 7.7%, most preferably about 7%, by weight of curcumin.
  • the hydroalcoholic extract of rosemary used in the inventive compositions will contain preferably from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, most preferably about 23%, by weight of TPA.
  • the hydroalcoholic extract of holy basil used in the inventive compositions will contain preferably from about 1.6% to about 2.4%, more preferably from about 1.8% to about 2.2%, most preferably about 2%, by weight of ursolic acid.
  • the hydroalcoholic extract of green tea used in the inventive compositions will contain preferably from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, most preferably about 45%, by weight of polyphonies.
  • the hydroalcoholic extract of huzhang used in the inventive compositions will contain preferably from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, most preferably about 8%, by weight of resveratrol.
  • the hydroalcoholic extract of Chinese goldthread used in the inventive compositions will contain preferably from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, most preferably about 6%, by weight of berberine.
  • the hydroalcoholic extract of barberry used in the inventive compositions will contain preferably from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, most preferably about 6%, by weight of berberine.
  • the Inventive Compositions inhibit COX-I and COX-2 enzyme activities
  • inventive compositions were analyzed using a colorimetric screening assay with purified ovine COX-I or COX-2 enzymes. Results shown in Table 1 demonstrate that the two different concentrations of the inventive compositions tested significantly inhibited COX-2 activity to an extent that was greater than the IC 50 of NS-398, a specific COX-2 inhibitor. The inventive compositions also inhibited COX-I activity in a similar manner to the IC 50 of indomethacin. These results suggest that the inventive compositions together are a general COX-inhibitory agent. The results are of this example are shown in Table II. Table II
  • the inventive compositions decrease the in vitro growth of the prostate cancer cell line , LNCaP , by affecting proliferation and apoptosis
  • the inventive compositions (0.1 ⁇ l/ml) were found to significantly inhibit the growth of LNCaP cells over a 72 hour time period compared to untreated, control cells (p ⁇ O.Ol) .
  • Lower doses tested did not have a significant growth inhibitory effect on the LNCaP cells.
  • LNCaP cells were seeded at a density of 38,000 cells per well in 12-well plates and treated in duplicate with 0.1 ⁇ l/ml, 0.05 ⁇ l/ml, or 0.001 ⁇ l/ml of the inventive compositions, or vehicle control (0.1% DMSO). Five separate cell counts of each well were obtained for all treatments at 24, 48, and 72 hrs .
  • Caspase-3 activity in LNCaP cells was increased by 17-fold [SEM ⁇ 0.110) following a 72 hour incubation with the inventive compositions (0.1 ⁇ l/ml).
  • a known inducer of apoptosis in the LNCaP cell line, TPA was used as a positive control in this experiment and induced caspase-3 activity by 12-fold [SEM ⁇ 0.005) over control levels.
  • the presence of PARP cleavage products and elevated caspase-3 activity indicate that the inventive compositions are inducing apoptosis in LNCaP cells.
  • LNCaP cells express COX-2 enzyme
  • Applicants also determined if the LNCaP cells that were employed in our experiments showed evidence for the expression of this enzyme.
  • Applicants used two different oligonucleotide primer sets specific for human COX-2 in an RT-PCR procedure and applied these primers to cDNA prepared either from LNCaP cell RNA or from cultured normal human prostate epithelial cell (hPEC) RNA.
  • COX-2 expression is not detectable in LNCaP cells, and inhibition of COX activity does not appear to be responsible for apoptosis induced by the inventive compositions.
  • compositions increase expression of the cell cycle inhibitory protein r p21, and decrease expression of androgen receptor , phosphorylated Stat3 and phosphorylated PKC ⁇ p in LNCaP cells
  • Cells were treated with the inventive compositions (0.1 ⁇ l/ml) for 24 hours.
  • Cell lysates were prepared as described above, followed by SDS PAGE and Western analysis.
  • Membranes were probed with either p21 or AR antibody, then stripped and re-probed with ⁇ -actin antibody as a lane loading control. Detection of ⁇ -actin was utilized for normalization.
  • Tsujii M and DuBois RN Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 83, 493-501, 1995. Tsujii M, Kawano S, and DuBois RN: Cyclooxygenase-2 expression in colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA. 94, 3336-3340, 1997.
  • Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, and DuBois RN Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93, 705-716, 1998.
  • Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, and Levine AC Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol. 164, 820-825, 2000.
  • Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, and DuBois RN Up-regulation of cyclooxygenase-2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 101, 1183-1188, 1994.
  • Kargman SL, O'Neill GP, Vickers PJ, Evans JF, Mancini JA, and Jothy S Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res. 55, 2556-2559, 1995.
  • Cyclooxygenase-2 is up-regulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma. Cancer Res. 61, 8617-
  • Hsu AL, Ching TT, Wang DS, Song X, Rangnekar VM, and Chen CS The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem. 275, 11397-11403, 2000.
  • Gupta S Adhami VM, Subbarayan M, MacLennan GT, Lewin JS, Hafeli UO, Fu P, and Mukhtar H: Suppression of prostate carcinogenesis by dietary supplementation of celecoxib in transgenic adenocarcinoma of the mouse prostate model. Cancer Res. 64, 3334-3343, 2004.
  • KeIm MA, Nair MG, Strasburg GM, and DeWitt DL Antioxidant and cyclooxygenase inhibitory phenolic compounds from Ocimum sanctum Linn. Phytomedicine 7, 7-13, 2000.
  • Exisulind (sulindac sulfone) suppresses growth of human prostate cancer in a nude mouse xenograft model by increasing apoptosis.
  • Tegeder I, Pfeilschifter J, and Geisslinger G Cyclooxygenase-independent actions of cyclooxygenase inhibitors . FASEB J. 15, 2057-2072, 2001.
  • Kirschenbaum A Klausner AP, Lee R, Unger P, Yao S, Liu XH, and Levine AC: Expression of cyclooxygenase-1 and cyclooxygenase- 2 in the human prostate. Urology 56, 671-676, 2000. Subbarayan V, Sabichi AL, Llansa N, Lippman SM, and Menter DG: Differential expression of cyclooxygenase-2 and its regulation by tumor necrosis factor-alpha in normal and malignant prostate cells. Cancer Res. 61, 2720-2726, 2001.
  • Nishino T, Pusey CD, and Domin J Elevated Akt phosphorylation as an indicator of renal tubular epithelial cell stress. J Biol Chem. 211 , 33943-33949, 2002.
  • Shaw M, Cohen P, and Alessi DR The activation of protein kinase B by H 2 O 2 or heat shock is mediated by phosphoinositide 3- kinase and not by mitogen-activated protein kinase-activated protein kinase-2. Blochem J. 336, 241-246, 1998.
  • Kimmey MB Cardioprotective effects and gastrointestinal risks of aspirin: maintaining the delicate balance. Am J Med. 117(Suppl 5A), 72S-78S, 2004.

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Abstract

La présente invention concerne des méthodes permettant de moduler l'apoptose, la croissance cellulaire et l'expression des protéines, laquelle méthode consiste à administrer des quantités thérapeutiquement efficaces d'extraits supercritiques de romarin, de curcuma, d'origan et de gingembre; et des quantités thérapeutiquement efficaces d'extraits hydro-alcooliques d'ocimum sanctus, de gingembre, de curcuma, de Scutellaria baicalensis, de romarin, de thé vert, de huzhang, de coptide chinois, et d'épine vinette.
PCT/US2006/038019 2005-09-29 2006-09-29 Methodes permettant de moduler l'apoptose, la croissance cellulaire et l'expression des proteines au moyen de compositions a base de plantes WO2007041276A2 (fr)

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US10226503B2 (en) 2014-08-28 2019-03-12 Caliway Biopharmaceuticals Co., Ltd. Plant extract composition for reducing topical fat and promoting weight loss as well as applications thereof
CN110585443A (zh) * 2019-09-04 2019-12-20 中国人民解放军陆军军医大学第一附属医院 一种抑制胶质瘤侵袭性生长的复合物及其应用
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US11318110B2 (en) 2015-08-28 2022-05-03 Caliway Biopharmaceuticals Co., Ltd. Pharmaceutical composition for reducing local fat and uses thereof
US11433034B2 (en) 2015-08-28 2022-09-06 Caliway Biopharmaceuticals Co., Ltd. Pharmaceutical composition for reducing local fat and uses thereof

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9987325B2 (en) 2014-08-28 2018-06-05 Caliway Biopharmaceuticals Co., Ltd. Plant extract composition for reducing localized fat and promoting weight loss as well as application thereof
AU2015309351B2 (en) * 2014-08-28 2019-01-03 Caliway Biopharmaceuticals Co., Ltd. Composition and medicinal product for reducing body weight and body fat, and use of said product
US10226503B2 (en) 2014-08-28 2019-03-12 Caliway Biopharmaceuticals Co., Ltd. Plant extract composition for reducing topical fat and promoting weight loss as well as applications thereof
US10537605B2 (en) 2014-08-28 2020-01-21 Caliway Biopharmaceuticals Co., Ltd. Composition of plant extract and its pharmaceutical composition and application thereof
US10537548B2 (en) 2014-08-28 2020-01-21 Caliway Biopharmaceuticals Co., Ltd. Composition and medical product for reducing body weight and body fat, and use of said product
US11318110B2 (en) 2015-08-28 2022-05-03 Caliway Biopharmaceuticals Co., Ltd. Pharmaceutical composition for reducing local fat and uses thereof
US11433034B2 (en) 2015-08-28 2022-09-06 Caliway Biopharmaceuticals Co., Ltd. Pharmaceutical composition for reducing local fat and uses thereof
WO2017205389A1 (fr) * 2016-05-23 2017-11-30 Yale University Amélioration de l'indice thérapeutique d'inhibiteurs des points de contrôle anti-immunitaire par utilisation d'un traitement d'association comprenant un extrait de phy906, un extrait de scutellaria baicalensis georgi (s) ou un composé provenant de ces extraits
JP2019519525A (ja) * 2016-05-23 2019-07-11 イエール ユニバーシティ Ｐｈｙ９０６抽出物、スクテラリア・バイカレンシス・ジョージ（ｓ）抽出物、またはそのような抽出物に由来する化合物を含む併用療法を用いることによる、抗免疫チェックポイント阻害剤の治療指数の改善方法
CN110585443A (zh) * 2019-09-04 2019-12-20 中国人民解放军陆军军医大学第一附属医院 一种抑制胶质瘤侵袭性生长的复合物及其应用

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