WO2009087368A1

WO2009087368A1 - Compositions comprising ganoderma lucidum extracts and uses thereof

Info

Publication number: WO2009087368A1
Application number: PCT/GB2009/000021
Authority: WO
Inventors: Sue Watson; Kenneth Muir; Rajendra Kumari; Lee Yi Fang; De-an GUO
Original assignee: The University Of Nottingham
Priority date: 2008-01-04
Filing date: 2009-01-05
Publication date: 2009-07-16
Also published as: GB0800134D0

Abstract

A composition comprising one of, or a mixture of, an extract of a Ganoderma lucidum triterpene and an extract of a Ganoderma lucidum polysaccharide for one or more of the following: treating a pre-cancerous lesion; inhibiting cancer cell proliferation; for combining with one or more existing anti-cancer medicaments; and inhibiting angiogenesis.

Description

COMPOSITIONS COMPRISING GANODERMA LUCIDUM EXTRACTS AND USES THEREOF

The invention relates to a composition comprising Ganoderma lucidum extracts of polysaccharide and triterpene for inhibiting cancer cell proliferation, for inhibiting the growth of or causing diminution of a precancerous lesion, for inhibiting angiogenesis, and for enhancing the efficacy of other treatments. The invention also relates to the use of the composition for the manufacture of a medicament, and to a method of treating a cancer or a pre-cancerous lesion, of inhibiting angiogenesis, or a method of enhancing the efficacy of other treatments.

Ganoderma lucidum (G. lucidum) , also known as LingZhi in Chinese and Reishi in Japanese, is a popular medicinal fungus in use for 2000 years and described in Shen Nong's Herbal Classic as being applied in the treatment of various diseases. G. lucidum is widely distributed in both tropical and temperate geographic regions and grows as a parasite on a wide variety of trees. The wild form of G. lucidum is generally rare and today is artificially cultivated indoors or outdoors on a bed of logs or woodchips .

The anti-cancer properties of G. lucidum has attracted much attention. G. lucidum has been demonstrated to exert anti-cancer effects against leukemic cell lines (Muller, C.I. et al. , 2006, Leukaemia Research 30: 841-848), prostate cancer cell lines (Jiang, J. et al., International Journal of Oncology 24: 1093-1099; Sliva, D. et al. , 2002, Biochemical and Biophysical Research Communication 298: 603-612), breast cancer cell lines (Hu, H. et al. , 2002, International, Journal of Cancer 102:250-253; Jiang, J. , et al. International Journal of Oncology 29:695-703), colorectal cancer cell lines (Xie, J. T. et al. , 2006, Experimental Oncology 28: 25- 29) and lung cancer cell lines (Tang, W. et al. , 2006, Life Science 80: 205-211) .

Inhibition of cell proliferation by G. lucidum was observed in vitro by induction of apoptosis and cell cycle arrest in a dose-dependent manner

(Tang et al.) . The anti-cancer activity of a variant form of G. lucidum was demonstrated in allogeneic and syngeneic tumor bearing mice. The orally Ganoderma-fed tumor-bearing mice demonstrated a smaller tumor size and a longer life span (Zhu, X-L et al. , 2007, Journal of Ethnopharmacology 111: 219-226) .

Moreover, it was recently reported that G. lucidum inhibited the early event of angiogenesis in PC-3 prostate cancer cells by downregulating the secretion of VEGF and TGF-Bl (Stanley, G. et al. , 2005, Biochemical and Biophysical Research Communication 330: 46-52) . A similar anti- angiogenesis effect was also shown in lung cancer cells (Cao, Q-Z. , et al. , 2006, Life Science 78:1457-1463) and breast cancer cells (Sliva, D. , et al.) .

In MDA-MB-231 breast cancer cells, the suppression of cell migration has been shown to result from inhibition of transcription factors AP-I and NF-kappaB, factors playing an important role in many biological responses (Sliva, D., et al.) . NF-kappaB is known to be associated with tumor cell proliferation, invasion and angiogenesis. Activation of NF-kappaB promotes cell survival, and it is thought that inhibition of NF-kappaB suppresses cell proliferation and indirectly induces apoptosis providing a possible target for chemotherapeutic agents (Beg, A. A. et al. , 1996, Sciences 274:782-784).

Many chemical constituents of G. lucidum have been identified including its fruit body, mycelia and spore polysaccharides, proteins, nucleotides, B2009/00002!

3 fatty acids, sterols, cerebrosides and triterpenes. Polysaccharides and lanostane-based triterpenes isolated from G. lucidum have been attributed to Ganoderma's anticancer property (Russell, R. et al. , 2006, Phytochemistry 67: 1985-2001) . Triterpenes generally isolated from basidiocarps and spores are thought to have anti-cancer and anti-viral properties, while macromolecular polysaccharides isolated from basidiocarps, mycelium or culture medium have been shown to function as immuno-modulators (Russell, R. et al. , 2006, Phytochemistry 67: 1985-2001) . Water-soluble polysaccharide extracts of G. lucidum are credited with enhancing phagocytosis of macrophages (Jiang, Z. Y. , et al. , 2003, J. Microbiology 23:51-54) , playing a role in the induction of TNF-α expression and IL-I production (Lin, Z. B. 2005, J. Pharmacological Sciences 99: 144-153), promoting of maturation of dendritic cells (Lin, Z. B. , et al. , 2002, Immunology Letters 83: 163-169) and enhancing immunological effector cells in immuno-suppressed mice (Zhu, X-L, et al.).

According to a first aspect of the invention there is provided a composition comprising one of, or a mixture of, an extract of a Ganoderma lucidum polysaccharide and an extract of a Ganoderma lucidum triterpene for treating one or more of a pre-cancerous lesion, for inhibiting cancer cell proliferation, for combining with one or more existing anti-cancer medicaments, and for inhibiting angiogenesis .

In a second aspect, the invention provides a use of a composition comprising one of, or a mixture of, an extract of a Ganoderma lucidum polysaccharide and an extract of a Ganoderma lucidum triterpene in the preparation/manufacture of a medicament for treating one or more of a pre-cancerous lesion, for inhibiting cancer cell proliferation, for combining with one or more existing anti-cancer medicaments, and for inhibiting angiogenesis. According to a third aspect, the invention provides a method of treatment of one or more of a pre-cancerous lesion, a cancer and of angiogenesis, the method comprising administering to a subject a therapeutically effective amount of a composition comprising one of, or a mixture of, an extract of a Ganoderma lucidum polysaccharide and an extract of a Ganoderma lucidum triterpene.

The use of either of an extract of a G. lucidum polysaccharide or an extract of a G. lucidum triterpene to inhibit the growth of and cause diminution of pre-malignant cells such as pre-cancerous lesions or intraepithelial neoplasias, or to inhibit angiogenesis, or to act synergistically with other cancer treatments, particularly in a dose-dependent manner, is novel and surprising.

Similarly, the beneficial effects of a mixture of an extract of a G. lucidum polysaccharide and an extract of a G. lucidum triterpene on cancer cells, particularly in a dose-dependent manner, is both novel and surprising.

In various embodiments therefore, a composition comprising one of, or a mixture of, an extract of a Ganoderma lucidum polysaccharide and an extract of a Ganoderma lucidum triterpene, a use of said composition, and a method of treatment comprising administering said composition is provided, wherein the composition is combined with one or more anti- cancer medicaments, particularly wherein the one or more anti-cancer medicaments is a chemotherapeutic agent, and even more particularly wherein the chemotherapeutic agent is doxorubicin.

In yet other embodiments, said composition, use or method of treatment are provided, wherein the cancer cells are leukaemia, blood, prostate, lung, breast, cervical, vulva or colon cancer cells. Prostate cancer is one of the most common malignancies diagnosed in men and is the most common cancer found in men older than 60 years. A third or more of all men older than 50 years have a latent form of prostate cancer that may progress to life-threatening prostate cancer. It is therefore very important to develop therapies to treat or cure prostate cancer.

The terms "extract of a Ganoderma lucidum polysaccharide" , "a Ganoderma lucidum polysaccharide extract" and "polysaccharide extract" are used interchangeably herein and are intended to have the same meaning. Preferably an extract of a Ganoderma lucidum polysaccharide comprises glucan containing polysaccharides, more preferably branched

(l-3)-beta-D-glucan moiety containing polysaccharides (Chan, W. K. 2007 International Immunology Advance Access published July 2, 2007) .

Preferably an extract of a Ganoderma lucidum polysaccharide is obtained using the method described herein in Example 1.

The terms "extract of a Ganoderma lucidum triterpene", "a Ganoderma lucidum triterpene extract" and "triterpene extract" are used interchangeably herein and are intended to have the same meaning. Preferably an extract of a Ganoderma lucidum triterpene comprises at least the following six ganoderic acids: Ganoderic acid C2, Ganoderic acid B, Ganoderic acid AMI, Ganoderic acid K, Ganoderic acid H and Ganoderic acid D (Wang, X. et al. 2006 Journal of Pharmaceutical and Biomedical Analysis 41:838-844) .

Preferably an extract of a Ganoderma lucidum triterpene is obtained using the method described herein in Example 1. Preferably in a composition of the invention comprising either or both of an extract of a Ganoderma lucidum triterpene and an extract of a Ganoderma lucidum polysaccharide, no other Ganoderma lucidum extract is included.

In a further embodiment, the pre-cancerous lesion is an intra-epithelial neoplasia, particularly wherein the intra-epithelial neoplasia is of a tissue selected from blood, prostate, lung, breast, cervix, vulva or colon.

In yet other embodiments, said composition, use or method of treatment are provided, wherein the composition comprises polysaccharide and/or triterpene extracts in concentrations of between about 0 and about 600 μg/ml, and more particularly wherein the polysaccharide and/or triterpene extracts are present in concentrations of between about 0 and about 250 μg/ml, and even more particularly wherein the concentrations of polysaccharide and/or triterpene extracts are present in concentrations of between about 175 and about 250 μg/ml, and yet more particularly wherein the composition comprises polysaccharide extract in an amount of between about 50 and about 200 μg/ml and/or triterpene extract in an amount of between about 10 and about 200 μg/ml.

In another embodiment, said composition, use or method of treatment are provided, wherein the composition comprises a concentration of triterpene extract of between about 70 and about 175 μg/ml, or wherein the IC₅₀ of the concentration of either extract is between about 50 and about 600 μg/ml.

In a further embodiment, said composition, use or method of treatment are provided, wherein the composition further comprises between about 0.01 and about 2.0 μg/ml of doxorubicin, or further comprises between about 5 and about 50 μM EGCG. Compositions according to the invention may be for use in the treatment a pre-cancerous lesion such as an intra-epithelial neoplasia. The treatment may be achieved by inhibiting the growth of, and/or causing the diminution of, a pre-cancerous lesion such as an intra-epithelial neoplasia. The intra-epithelial neoplasia may be of a tissue selected from the blood, the prostate, the lung, the breast, the cervix, the vulva or the colon. Preferably the intra-epithelial neoplasia is of the prostate.

In one embodiment therefore, a composition, use or the method of treatment is provided, wherein the pre-cancerous lesion is an intra- epithelial neoplasia.

In a further embodiment, the intra-epithelial neoplasia is of a tissue selected from blood, prostate, lung, breast, cervix, vulva or colon. A composition according to the invention therefore may be used as a therapeutic composition to bring about the diminution of, or to inhibit an intra-epithelial neoplasia of the prostate, the blood, the lung, the breast, the cervix, the vulva or the colon. The composition may also be used for the preparation of a medicament for inhibiting the growth, or causing the diminution, of an intra-epithelial neoplasia.

In one embodiment, an effective composition is provided for inhibiting cancer cell proliferation, and/or for lessening the growth of pre-cancerous lesions and/or for inhibiting angiogenesis, comprising concentrations of G. lucidum polysaccharide and triterpene extracts of between about 0 and about 600 μg/ml, or of between about 0 and about 250 μg/ml.

Polysaccharide and triterpene extracts of G. lucidum may act alone, and together they may act additively and synergistically to inhibit cancer cell proliferation, and/to lessen the growth of pre-cancerous lesions and/or to inhibit angiogenesis.

Polysaccharide and triterpene extracts of G. lucidum may also act separately and together both additively and synergistically in combination with other compositions or treatment modalities, particularly conventional treatment modalities such as chemotherapeutics.

There are currently extremely limited therapeutic options available for the treatment of prostate intraepithelial neoplasia (PIN) , thus the therapeutic use of a composition according to the invention comprising one of, or a mixture of, G. lucidum extracts of polysaccharide and triterpene for the treatment of PIN provides valuable pharmaceutical and therapeutic approaches for treating precursor lesions of prostate cancer.

Compositions according to the invention are shown in the examples to enhance the efficacy of cancer treatments, and/or lessen the growth of a pre-cancerous lesion and/or inhibit angiogenesis, and/or act effectively in combination with another composition to enhance the efficacy of a cancer treatment, to lessen the growth of a pre-cancerous lesion or to inhibit angiogenesis .

Thus, a composition according to the invention may be used to treat a cancer in a patient already receiving a conventional anti-cancer treatment. Furthermore, compositions according to the invention are shown in the examples to have a synergistic effect when combined with conventional chemotherapeutics, such as doxorubicin, in the prevention of angiogenesis.

The polysaccharide extract and/or triterpene extract of G. lucidum used in compositions of the invention produce an inhibitory effect on cells from prostate cancer cell lines DU145 and LnCap, leukaemia cell lines HL60 and K562, lung cancer cell lines A549 and NCI-H460, breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-435, prostate intraepithelial neoplasia (pre-malignant) cell lines (PIN cell lines) WPE1-NA22, WPEl- NBIl and WPE1-NB14, and from oncogenes p53, c-myc, k-ras and Wnt, the extracts may also inhibit angiogenesis in human endothelial cells.

Many diseases/conditions are currently treated using more than one medicament in the treatment regime, either concomitantly administered or sequentially administered. It is therefore within the scope of the invention to use the compounds of the invention in therapeutic methods for the treatment of one of the above mentioned diseases/conditions in combination with one another, or as an adjunct to, or in conjunction with, other established therapies normally used in the treatment of said disease/condition. By analogy, it is also within the scope of the invention to use the compounds of the invention in combination with other therapeutically active compounds normally used in the treatment of one of the above-mentioned diseases/conditions in the manufacture of a medicament for the treatment said disease/condition.

Administration of other cancer therapeutic agents can occur prior to, concurrent with, or after administration with the G. lucidum extracts. Administration of the G. lucidum extracts can occur before, during or after surgical treatment, radiotherapy, hormone therapy, immunotherapy, hyperthermia, or other cancer treatment modalities. Administration of the G. lucidum extracts can occur daily, weekly, or monthly as needed.

According to another aspect the invention provides a pharmaceutical composition comprising the compounds of the invention, namely a G. lucidum polysaccharide extract and/or a G. lucidum triterpene extract . The compounds of the invention may be generally utilised as the free substance or as a pharmaceutically acceptable salt thereof.

In the present context, the term "pharmaceutically acceptable salt" is intended to indicate salts which are not harmful to a patient. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulphuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulphonic, ethanesulphonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulphonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p- aminobenzoic, glutamic, benzenesulphonic, p-toluenesulphonic acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated herein by reference. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethyl ammonium, ethylammonium, hydroxyethylammonium, diethyl ammonium, butylammonium, tetramethyl ammonium salts and the like.

The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19^th Edition, Gennaro, Ed. , Mack Publishing Co. , Easton, PA, 1995.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.

In addition, the compounds of the invention may form solvates with water or common organic solvents. Such solvates are also encompassed within the scope of the present invention.

Thus, in a further aspect, there is provided a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents.

The composition may further comprise a buffer system, preservative (s) , tonicity agent(s) , chelating agent(s) , stabilizer(s) and surfactant (s) , which is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000. The composition may also further comprise one or more therapeutic agents active against the same disease state.

Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenisation, en-capsulation, spray drying, microencapsulating, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Composition and Delivery (MacNally, EJ. , ed. Marcel Dekker, New York, 2000).

Administration of pharmaceutical compositions according to the invention may be through several routes of administration, for example, oral, rectal, nasal, pulmonary, topical (including buccal and sublingual) , transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition/disease to be treated and the active ingredient chosen.

For topical use, sprays, creams, ointments, jellies, gels, inhalants, dermal patches, implants, solutions of suspensions, etc. , containing the compounds of the present invention are contemplated. For the purpose of this application, topical applications shall include mouth washes and gargles .

Pharmaceutical compositions for oral administration include solid dosage forms such as hard or soft capsules, tablets, troches, dragees, pills, lozenges, powders and granules and liquid dosage forms for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups and elixirs, each containing a predetermined amount of the active ingredient, and which may include a suitable excipient.

Compositions intended for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically-acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.

The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in U.S. Patent Nos. 4,356,108; 4,166,452; and 4,265,874, incorporated herein by reference, to form osmotic therapeutic tablets for controlled release.

Formulations for oral use may also be presented as hard gelatine capsules where the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions may contain the active compounds in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose , methylcellulose , hydroxypropylmethylcellulose , sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavouring, and colouring agents may also be present. The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example a liquid paraffin, or a mixture thereof. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents .

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed using synthetic mono- or diglycerides . In addition, fatty acids such as oleic acid find use in the preparation of injectables. Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a solution or suspension for the administration of the prolactin receptor antagonist in the form of a nasal or pulmonal spray. As a still further option, the pharmaceutical compositions containing the compound of the invention can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration.

Pharmaceutical compositions for parenteral administration include sterile aqueous and non- aqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use.

The term "aqueous composition" is defined as a composition comprising at least 50 % w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 %w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50 %w/w water. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art. Depot injectable formulations are also contemplated as being within the scope of the present invention. When the compounds of the invention or composition thereof is used in combination with a second therapeutic agent active against the same disease state/condition, they may conveniently be administered alone or in combination, in either single or multiple doses, sequentially or simultaneously, by the same route of administration, or by a different route.

Effective Dosages

The compounds of the invention, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease/condition being treated. The compound (s) may be administered therapeutically to achieve therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disease/condition being treated and/or eradication or amelioration of one or more of the systems associated with the underlying disorder. Therapeutic benefit also includes halting or slowing the progression of the disease, regardless of whether improvement is realised.

The exact dosage will depend upon the frequency and mode of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the disease/condition treated and any concomitant disease/condition to be treated and other factors evident to those skilled in the art. Determination of the effective dosage is well within the capabilities of those skilled in the art.

When a compound of the invention or a pharmaceutically acceptable salt, solvate or prodrug thereof is used in combination with a second therapeutic agent active against the same disease/condition the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. The invention will be further defined by reference to the following examples. It will be apparent to those skilled in the art that many modifications, both to compositions and to assay conditions may be practised without departing from the scope of the invention.

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which :-

Figure 1 (a) and (b) show the % cell viability of prostate cancer cell lines DU145, Lncap, and PC3M following treatment with various concentrations of G. lucidum polysaccharide extract.

Figure 2 (a) and (b) show the % cell viability of prostate cancer cell lines DU145, Lncap, and PC3M following treatment with various concentrations of G. lucidum triterpene extract.

Figure 3 (a) and (b) show the % cell viability of leukemic cancer cell lines HL60 and K562 following treatment with various concentrations of G. lucidum polysaccharide extract.

Figure 4 (a) and (b) show the % cell viability of leukemic cancer cell lines HL60 and K562 following treatment with various concentrations of G. lucidum triterpene extract.

Figure 5 (a) and (b) show the % cell viability of prostatic intra-epithelial neoplasia cell lines WPE1-NA22, WPEl-NBIl , and WPE1-NB14 following treatment with various concentrations of G. lucidum triterpene extract. Figure 6 shows the combined effect of G. lucidum polysaccharide extract and G. lucidum triterpene extract on cell proliferation of the prostate cancer cell line DU 145.

Figure 7 shows the combined effect of G. lucidum polysaccharide extract and G. lucidum triterpene extract on cell proliferation of the prostate cancer cell line LnC ap.

Figure 8 shows the combined effect of G. lucidum polysaccharide extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation of the prostate cancer cell line DU 145.

Figure 9 shows the combined effect of G. lucidum triterpene extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation of the prostate cancer cell line DU 145.

Figure 10 shows the combined effect of G. lucidum triterpene extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation of the prostate cancer cell line LnCap.

Figure 11 shows the combined effect of G. lucidum polysaccharide extract and G. lucidum triterpene extract on cell proliferation of the leukemic cell line HL60.

Figure 12 shows the combined effect of G. lucidum polysaccharide extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation of the leukemic cell line HL60.

Figure 13 shows the combined effect of G. lucidum triterpene extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation of the leukemic cell line HL60. Figure 14 shows the combined effect of G. lucidum polysaccharide extract and G. lucidum triterpene extract on cell proliferation of the leukemic cell line K562.

Figure 15 shows the combined effect of G. lucidum polysaccharide extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation of the leukemic cell line K562.

Figure 16 shows the combined effect of G. lucidum triterpene extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation of the leukemic cell line K562.

Figure 17 shows the combined effect of G. lucidum polysaccharide extract and G. lucidum triterpene extract on PIN cell line WPE1-NA22.

Figure 18 shows the combined effect of G. lucidum triterpene extract and doxorubicin on PIN cell line WPE1-NA22.

Figure 19 shows the combined effect of G. lucidum polysaccharide extract and G. lucidum triterpene extract on PIN cell line WPEl-NBIl.

Figure 20 shows the combined effect of G. lucidum triterpene extract and doxorubicin on PIN cell line WPEl-NBIl .

Figure 21 shows the combined effect of G. lucidum polysaccharide extract and G. lucidum triterpene extract on PIN cell line WPE1-NB14.

Figure 22 shows the combined effect of G. lucidum triterpene extract and doxorubicin on PIN cell line WPE1-NB14. Figure 23 shows the combined effect of G. lucidum triterpene extract and EGCG upon MCF-7 breast cancer cell line.

Figure 24 shows the combined effect of G. lucidum polysaccharide extract and EGCG upon MCF-7 breast cancer cell line.

Figure 25 shows a study of anti-angiogenic effect of polysaccharide extract and triterpene extract of G. lucidum on cultures of human endothelial cells at the earliest stages of tubule formation.

Figure 26 shows two 2D gel images of control and triterpene-treated WPEl -NB 14 cells. Gel 1 (left hand gel) shows the protein profile of a control group. Gel 2 (right hand gel) shows the protein profile of triterpene-treated WPE1-NB14 (150μg/ml of whole cell extract was loaded) . The cells were treated with 70μg/ml triterpene extract for 48 hours. Eight differentially expressed spots were circled for mass spectrometry .

Figure 27 shows Western blot analysis of vimentin and enolase a expression in prostate cancer cell line and PINS.

Figure 28 shows the results of an invasion assay, which demonstrates the invasive ability of RWPEl and WPE1-NB14. Invading cells were fixed, stained and counted.

Figure 29 shows the results of a wound healing assay. WPEl-NBIl cells were grown to confluency on a 6 well plate. A scratch was made through the cell layer using a pipette tip. (a) Cells in normal medium (K-FSM with EGF and BPE) . (b) Cells in growth factor starved medium, (c) Cells in medium containing 52.8μg/ml (IC₄₀) triterpene extract, (d) Cells in medium containing 33μg/ml (IC₂₅) triterpene extract. Pictures of the wound area were taken at 0, 6 and 24 hours.

EXAMPLE 1

Total extracts of polysaccharide and triterpene of G. lucidum were used. G. lucidum was obtained from the cultivation base of Green Valley Pharmaceutical Co. Ltd. (Shanghai, China).

Extracts of polysaccharide and triterpene were prepared from G. lucidum according to the methods described in YANG et al. J Am Soc Mass Spectrom. 2007, 18, 927-939, as follows: Fruit bodies of G. lucidum (approximately 2 kg) were cut into small pieces and then pulverized. The powder was refluxed with 95% ethanol three times (12 L x 3) and the extract partitioned with petroleum ether and dichloromethane (1.5 L x 3) , respectively. The dichloromethane layer was concentrated under vacuum to yield approximately 15g of triterpene extract.

Fruit bodies of G. lucidum (approximately 100 g) were defatted with 95% alcohol and then refluxed with 20 vols. of water. The aqueous extract was fractionated into a polysaccharide fraction (alcohol insoluble) and non- polysaccharide fraction (alcohol soluble), further treated with trichloroacetic acid to remove proteins, then dialyzed against tap water for 2 days and distilled water for 1 day (molecular weight cut off 3000- 5000) . The retentate was washed sequentially with ethanol and acetone and concentrated under vacuum and freeze dried to obtain approximately

0.5g of polysaccharide extract.

The prostate cancer cell lines DU145 and LnCap were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS). PC3M cell lines were maintained in Minimum Essential Medium containing IX MEM non-essential amino acids, IX MEM vitamin, 1 mM sodium pyruvate and 10% FBS. HL60 was maintained in Iscove's Modified Dulbecco's Medium containing 20% FBS. K562 was maintained in Iscove's Modified Dulbecco's Medium containing 10% FBS. PIN cell lines were maintained in Keratinocyte-serum free medium with 2.5 μg Epidermal Growth Factor and 25mg Bovine Pituitary Extract.

The MCF-7, MDA-MB-231 and MDA-MB-435 beast cancer cell lines were maintained in RPMI growth medium containing 2mM L-glutamine and 10% heat-inactivated FBS. The lung cancer cell lines A549 and NCI- H460 lung cancer cell lines were maintained RPMI growth medium containing 2mM L-glutamine and 10% heat-inactivated FBS. Experiments on the breast cancer cell line, MCF-7, were carried out with both polysaccharide and triterpene extracts in combination with green tea catechin extract (EGCG), as well as with other cancer treatment modalities such as doxorubicin.

The anti-proliferative effect of polysaccharide and triterpene extracts were measured by culturing the treated cells with between 0 and 600 μg/ml of each extract for 72 hours and measuring their viability by the CellTiter-Blue^® assay. The IC₅₀ of each cell line was calculated as shown in the figures described below (the IC₅₀ represents half the maximal inhibitory concentration, or the concentration of an inhibitor that is required for 50% inhibition of its target) . Experiments with the breast cancer cell line, MCF-7, were carried out by culturing the cells with between 10 and 200 μg/ml of each extract, as well as experiments combining the extracts with between 5 and 50 μM EGCG.

The high-throughput screening Celltiter-Blue® fluorometric assay was used for estimating the number of viable cells. The indicator dye resazurin is able to measure the metabolic capacity of cells due to the fact that viable cells retain the ability to reduce resazurin into highly fluorescent resorufin.

Cells were harvested by following the standard protocol and seeded at 8xlO⁴ cells/ml into each well of a 384-well plate. On the following day, cells were treated with triterpene extract (0-600 μg/ml), polysaccharide extract (0-600 μg/ml) , the combination of triterpene extract and polysaccharide extract, or with the combination of one or both of the

G. lucidum extracts with the chemotherapeutic agent doxorubicin for 72 hours incubation at 37⁰C, or with EGCG.

A series of dilutions were made for each compound, each dilution having 6 replicates. Positive controls comprising doxorubicin treated cells, or EGCG treated cells, were included. Negative controls were water, saline, MeOH and medium only.

At the end of incubation, 10 μl CellTiter-Blue® reagent was added into each well. The plate was centrifuged at 500 rpm for 1 min followed by 1 minute shaking. The plate was then incubated at 37 ⁰C for 3-4 hours. The fluorescence signal was quantified at 570/600 nm using a FlexStation II reader.

EXAMPLE 2

Figure 1 shows the results of cell viability assays of G. lucidum polysaccharide extract treated prostate cancer cell lines DU145, PC3M and LnCap. (a) Cells were treated with G. lucidum polysaccharide extract (0-500 μg /ml) for 72h. The CellTiter-Blue® assay was performed and the number of viable cells was expressed as a percentage of untreated control cultures for each line. Results represent the mean ± S. D. of two experiments preformed in sextuplicates . (b) The IC₅₀ Of each cell line was calculated by performing a non-linear regression analysis.

In the following Examples 3-6, as with Example 2, cells were treated with an extract of either G. lucidum polysaccharide or G. lucidum triterpene in a concentration of 0-500 μg /ml for 72 hours, the CellTiter-Blue® assay was performed and the number of viable cells was expressed as a percentage of untreated control cultures for each line, results represent the mean ± S. D. of two experiments preformed in sextuplicates, and the IC₅₀ of each cell line was calculated by performing a non-linear regression analysis.

EXAMPLE 3

Figure 2 shows the results of cell viability assays of G. lucidum triterpene extract treated prostate cancer cell lines DU145, PC3M and LnCap.

EXAMPLE 4

Figure 3 shows the results of cell viability assays of G. lucidum polysaccharide extract treated leukemic cancer cell lines.

EXAMPLE 5

Figure 4 shows the results of cell viability studies of G. lucidum triterpene extract treated leukemic cancer cell lines.

EXAMPLE 6

Figure 5 shows the results of cell viability studies of G. lucidum triterpene extract treated prostatic intra-epithelial neoplasia cell lines. Such cell lines are pre-malignant human prostate cells which may be used as models of chemoprevention.

Table 1 shows the IC₅₀ for each cell line. From Table 1, it can be seen that both extracts of G. lucidum markedly suppress the proliferation of prostate and leukaemia cancer cells in a dose-dependent manner, and that the triterpene extract inhibits the growth of prostatic intra-epithelial neoplasia cells.

TABLE 1

EXAMPLE 7

The combined anti-proliferative effect of polysaccharide extract and triterpene extract of G. lucidum was performed by culturing the prostate cancer cells with 0-300 μg/ml of polysaccharide extract and 0-150 μg/ml triterpene extract for 72h. The cell viability was again measured by the CellTiter-Blue® assay. A similar assay was also done to look at the combined effect of G. lucidum and the chemotherapeutic agent doxorubicin. The assay was also performed on leukaemia cancer cell lines.

Figure 6 shows the combined effect of G. lucidum polysaccharide extract and triterpene extract on cell proliferation of prostate cancer cell line DU145 treated with 0-150 μg/ml triterpene extract in combination with 0- 240 μg/ml of polysaccharide extract. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue^®. Different patterns of bar represent different concentrations of triterpene extract. % of cell viability was calculated by dividing the fluorescence reading (570/590 nm) of treated cell by the reading of medium control. The combination of the two extracts gave an additional 15% inhibition.

EXAMPLE 8

Figure 7 shows the combined effect of G. lucidum polysaccharide extract and triterpene extract on cell proliferation of prostate cancer cell line LnCap treated with 0-120 μg/ml triterpene extract in combination with 0-300 μg/ml of polysaccharide extract. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue^®. Different patterns of bar represent different concentrations of triterpene extract. % of cell viability was calculated by dividing the fluorescence reading (570/590nm) of treated cell by the reading of medium control. The combination of the two extracts resulted in an additive effect.

EXAMPLE 9

Figure 8 shows the combined effect of G. lucidum polysaccharide extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation. Prostate cancer cell line DU145 was treated with 0-0.039 μg/ml doxorubicin in combination with 0-240 μg/ml of polysaccharide extract. Cell viability was assessed 72 hours after the treatment by CellTiter- Blue^®. Different patterns of bar represent different concentrations of doxorubicin. % of cell viability was calculated by dividing the fluorescence reading (570/590 nm) of treated cell by the reading of medium control. The combination of polysaccharide extract and doxorubicin resulted in an additive effect.

EXAMPLE 10

Figure 9 shows the combined effect of G. lucidum triterpene extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation. Prostate cancer cell line DU145 was treated with 0-0.039 μg/ml doxorubicin in combination with 0-150 μg/ml of triterpene extract. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue^®. Different patterns of bar represent different concentrations of doxorubicin. % of cell viability was calculated by dividing the fluorescence reading (570/590 nm) of treated cell by the reading of medium control. The combination of triterpene extract and doxorubicin resulted in an additive effect.

EXAMPLE 11

Figure 10 shows the combined effect of G. lucidum triterpene extract and chemotherapeutic agent doxorubicin (Dox) on cell proliferation. Prostate cancer cell line LnCap was treated with 0-150 μg/ml triterpene extract in combination with 0-0.039 μg/ml doxorubicin. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue®. Different patterns of bar represent different concentrations of doxorubicin. % of cell viability was calculated by dividing fluorescence reading (570/590nm) of treated cell by the reading of medium control. The combination of triterpene extract and doxorubicin enhanced the anti-proliferative effect by 10%.

EXAMPLE 12

Figure 11 shows the combined effect of G. lucidum polysaccharide extract and triterpene extract on cell proliferation. Leukemic cell line HL60 was treated with 0-100 μg/ml polysaccharide extract in combination with 0- 120 μg/ml triterpene extract. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue^®. Different patterns of bar represent different concentrations of triterpene extract. % of cell viability was calculated by dividing fluorescence reading (570/590 nm) of treated cells by the reading of medium control. The combination of the two extracts of G. lucidum resulted in an additive effect.

EXAMPLE 13

Figure 12 shows the combined effect of G. lucidum polysaccharide extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation. Leukemic cell line HL60 was treated with 0-100 μg/ml polysaccharide in combination with 0-0.07 μg/ml doxorubicin. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue®. Different patterns of bar represent different concentrations of doxorubicin. % of cell viability was calculated by dividing fluorescence reading (570/590 nm) of treated cells by the reading of medium control. The combination of polysaccharide extract and doxorubicin enhanced the anti-proliferative effect by 16%.

EXAMPLE 14

Figure 13 shows the combined effect of G. lucidum triterpene extract and the chemotherapeutic agent doxorubicin (Dox) on cell proliferation. Leukemic cell line HL60 was treated with 0-120 μg/ml triterpene extract in combination with 0-0.07 μg/ml doxorubicin. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue^®. Different patterns of bar represent different concentrations of doxorubicin. % of cell viability was calculated by dividing fluorescence reading (570/590 nm) of treated cells by the reading of medium control. The combination of triterpene extract and doxorubicin enhanced anti-proliferative effect by 10%.

EXAMPLE 15

Figure 14 shows the combined effect of G. lucidum polysaccharide extract and triterpene extract on cell proliferation. Leukemic cell line K562 was treated with 0-360 μg/ml polysaccharide in combination with 0-250 μg/ml triterpene. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue®. Different patterns of bar represent different concentrations of doxorubicin. % of cell viability was calculated by dividing fluorescence reading (570/590 nm) of treated cells by the reading of medium control. The combination of the two extracts of G. lucidum enhanced the anti-proliferative effect by 10%.

EXAMPLE 16

Figure 15 shows the combined effect of G. lucidum polysaccharide extract and the chemotherapeutic agent doxorubicin on cell proliferation. Leukemic cell line K562 was treated with 0-360 μg/ml polysaccharide extract in combination with 0-0.2 μg/ml doxorubicin. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue®. Different patterns of bar represent different concentrations of doxorubicin. % of cell viability was calculated by dividing fluorescence reading (570/590 nm) of treated cells by the reading of medium control. EXAMPLE 17

Figure 16 shows the combined effect of G. lucidum triterpene extract and the chemotherapeutic agent doxorubicin on cell proliferation. Leukemic cell line K562 was treated with 0-240 μg/ml triterpene extract in combination with 0-0.2 μg/ml doxorubicin. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue^®. Different patterns of bar represent different concentrations of doxorubicin. % of cell viability was calculated by dividing the fluorescence reading (570/590nm) of treated cells by the reading of medium control. The combination of triterpene extract and doxorubicin enhanced the anti-proliferative effect by 6%.

In the examples above, the combination of G. lucidum polysaccharide extract and triterpene extract in combination with the chemotherapeutic agent doxorubicin can be seen to enhance the anti-proliferative effect on cancer cells.

EXAMPLE 18

In Figure 17, the combined effect of G. lucidum polysaccharide extract and triterpene extract on cell proliferation is shown. PIN cell line WPEl- NA22 was treated with 0-250 μg/ml polysaccharide in combination with 0-90 μg /ml triterpene. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue®. Different patterns of bar represent different concentrations of polysaccharide extract. % of cell viability was calculated by dividing fluorescence reading (570/590nm) of treated cells by the reading of medium control. The combination of the two extracts of G. lucidum resulted in an additive effect.

EXAMPLE 19 In Figure 18, the combined effect of G. lucidum triterpene extract and doxorubicin (Dox) on cell proliferation is shown. PIN cell line WPEl- NA22 was treated with 0-90 μg/ml triterpene extract in combination with 0.078-0.156μg/ml doxorubicin. Cell viability was assessed 72 hours after the treatment by CeIlT iter-Blue®. Different patterns of bar represent different concentration of Dox. % of cell viability was calculated by dividing fluorescence reading (570/590nm) of treated cells by the reading of medium control. The combination of two compounds enhanced the antiproliferative effect by 11%.

EXAMPLE 20

In Figure 19, the combined effect of G. lucidum polysaccharide extract and triterpene extract on cell proliferation is shown. PIN cell line WPEl- NBIl was treated with 0-90μg/ml triterpene extract in combination with 0-250μg/ml polysaccharide extract. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue®. Different patterns of bar represent different concentrations of polysaccharide. % of cell viability was calculated by dividing the fluorescence reading (570/590nm) of treated cells by the reading of medium control.

EXAMPLE 21

In Figure 20, the combined effect of G. lucidum triterpene extract and doxorubicin (Dox) on cell proliferation is shown. PIN cell line WPEl-

NBIl was treated with 0-90 μg/ml triterpene extract in combination with

0-0.156μg/ml dox. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue®. Different patterns of bar represent different concentrations of Dox. % of cell viability was calculated by dividing fluorescence reading (570/590nm) of treated cells by the reading of medium control. The combination of the two compounds resulted in an additive effect.

EXAMPLE 22

In Figure 21, the combined effect of G. lucidum polysaccharide extract and triterpene extract on cell proliferation is shown. PIN cell line WPEl- NB14 was treated with 0-90μg/ml triterpene extract in combination with 0-250μg/ml polysaccharide extract. Cell viability was assessed 72 hours after the treatment by CeIlT iter-Blue®. Different patterns of bar represent different concentrations of polysaccharide extract. % of cell viability was calculated by dividing fluorescence reading (570/590nm) of treated cells by the reading of medium control.

EXAMPLE 23

In Figure 22, the combined effect of G. lucidum triterpene extract and Doxorubicin (Dox) on cell proliferation is shown. PIN cell line WPEl- NB14 was treated with 0-90μg/ml triterpene extract in combination with 0-0.156μg/ml Dox. Cell viability was assessed 72 hours after the treatment by CellTiter-Blue^®. Different patterns of bar represent different concentrations of Dox. % of cell viability was calculated by dividing fluorescence reading (570/590nm) of treated cells by the reading of medium control. The combination of the two compounds enhanced the anti-proliferative effect by 12%.

A summary of the additive and enhanced effects of the combinational assays are shown in Table 2. TABLE 2

EXAMPLE 24

MCF-7 breast cancer cell line cells were treated with extracts of between 10 and 200 μg/ml triterpene, and between 10 and 200 μg/ml polysaccharide extracts, alone or in combination with green tea catechins (EGCG). Cell viability was assessed 72 hours after the treatment by CeIlT iter-Blue^®. Figures 23 and 24 show the effects, respectively, of triterpene and polysaccharide extracts both alone and in combination with EGCG upon MCF-7 breast cancer cell lines. EXAMPLE 25

Angiogenesis assays were carried out to measure the anti-angiogenic effect of G. lucidum extracts on human endothelial cells. The TCS angiokit^® used for the assays provides growing cultures of human endothelial cells at the earliest stages of tubule formation in a 24 well plate. In the assays, the number of tubules and branches formed in each well of each treatment were counted manually. The numbers shown in Table 3 below are the average of two counts from two wells of the same treatment. The average branches formed per tubule indicates the degree of inhibition. The protocol used was as follows: 125 μg/ml-500 μg/ml of polysaccharide extract and 50 μg/ml-100 μg/ml of triterpene extract were added to the cultures in the indicated wells. The tubules were quantified at Day 9 following the fixation and staining with CD31 staining kit (TCS). A positive control VEGF and negative control Suramin were also included.

Figure 25 shows the anti-angiogenic effect for G. lucidum polysaccharide extract and triterpene extract on cultures of human endothelial cells at the earliest stages of tubule formation, (a) Medium only control, (b) VEGF 2ng/ml positive control, (c) Suramin 2OuM negative control, (d) polysaccharide extract 150 μg/ml, (e) polysaccharide extract 250 μg/ml, (g) triterpene extract 100 μg/ml, (h) triterpene extract 50 μg/ml, (i) methanol 1% control, (j) doxorubicin 0.0039 μg/ml, (k) doxorubicin 0.039 μg/ml and polysaccharide extract 250 μg/ml and (1) doxorubicin 0.039 μg/ml and triterpene extract 100 μg/ml.

Both polysaccharide and triterpene extracts show a significant inhibition of tubule formation compared to the controls. This inhibitive effect is even stronger than the negative control Suramin. The triterpene extract showed a killing effect toward human endothelial cells at 100 μg/ml. The morphology of endothelial cells changed after 24 hours treatment with 100 μg/ml of triterpene extract.

TABLE 3

EXAMPLE 26

In this example G. lucidum triterpene extract is shown to inhibit PIN growth. In vitro results show that PIN cells lines are relatively sensitive to G. lucidum triterpene extract, and have a lower IC₅₀ compared to prostate cancer cells lines. In vivo the triterpene extract inhibits growth of a PIN cell line by 30% (p < 0.001) . This demonstrates that G. lucidum triterpene extract may be used in the prevention of progression of PIN cells to prostate cancer.

By comparing the proteomic profile (Figure 26) of non-treated (left hand gel) and triterpene extract-treated (right hand gel) PIN cells (WPEl- NB 14) , two protein targets were identified. The two protein targets were selected from 8 spots indentified in the proteomic profile (Figure 26) and were analysed by mass spectrometry. Peptides were extracted from the eight the selected spots on the 2D gels by trypsin digestion and the proteins were identified using mass spectrometry. The data were submitted for protein homology identification using the MASCOT 2.1 search engine (Matrix Science) against the Homo sapiens (human, 202375 sequences) of NCBInr database (6388671 sequences; 2180845802 residues) . From the results of mass spectrometry, two interesting protein targets were found; Vimentin (spot 2) , an intermediate filament protein, which is a known epithelial-mesenchymal transition (EMT) marker, and enolase a (spot 6) .

EMT is normally defined as a phenotype state during which epithelial cells start to become invasive by acquisition of a mesenchymal phenotype.

Overexpression of vimentin has been shown to contribute to prostate cancer invasion and metastasis. The data in Figure 26 shows that vimentin is down regulated in triterpene extract-treated PIN cells. In addition, enolase a, a glycolytic enzyme, plays multiple functions in several physiological processes. Enolase a has an alternative translated form, named MBP-I which binds to the c-myc promoter and down regulates c- myc transcriptions, c-myc is related to the regulation of EMT. Figure 26 shows enolase a is unregulated in triterpene extract treated PIN cells. All together, the results indicate that the mechanism of action of the triterpene extract in PIN cells may relate to the regulation of EMT. To determine the effect of triterpene extract on the vimentin and enolase a protein expression level in PIN cells and prostate cancer cells, western blot analyses were carried out. As shown in Figure 27, treatment of PIN cells with triterpene extract resulted in down regulation of vimentin. On the other hand, enolase a was unregulated post treatment and peaked at 48hours. These results confirmed the proteomic observations of Figure 26.

EXAMPLE 27

Figure 28 shows the results of invasion assays using RWPEl and WPEl- NB14 cells lines. The invasive ability of RWPEl and WPE1-NB14 cells was assessed by use of a BD Biocoat Invasion Chamber. Cells were incubated with or without triterpene extract for 48 hours. The invading cells were then fixed, stained and counted. As shown in Figure 28 triterpene extract inhibited the invasion of RWPEl and WPE1-NB14 by 46% and 51 respectively at 70μg/ml.

EXAMPLE 28

Figure 29 shows the results of a wound healing assay using WPEl-NBIl PIN cells. Wound healing assays are used to study cell migration in vitro . The method mimics cell migration during wound healing in vivo . A wound was created on the monolayer of WPEl-NBIl cells. Cell movement into the wound area of triterpene-treated cells and control- treated cells as 0, 6 and 24 hours was compared. As shown in Figure 29 the wound was completely healed in cells in medium with growth factors, however, cells in the growth factor starved medium and triterpene extract containing medium showed a slower rate of wound healing. Examples 26, 27 and 28 demonstrate that G. lucidum triterpene extract has an anti-proliferative effect in both malignant and premalignant prostate cell lines. The mechanism of action of triterpene extract may relate to regulation of EMT by downregulation of vimentin and upregulation of enolase a. The results illustrate that triterpene extract may be used in the prevention of progression of PIN cells to prostate cancer. Accordingly, triterpene extract may be used as an anti-metastatic agent.

Claims

1. A composition comprising one of, or a mixture of, an extract of a Ganoderma lucidum triterpene and an extract of a Ganoderma lucidum polysaccharide for one or more of the following: treating a pre-cancerous lesion; inhibiting cancer cell proliferation; for combining with one or more existing anti-cancer medicaments; and inhibiting angiogenesis.

2. The use of the composition of claim 1 for the manufacture of a medicament for one or more of the following: treating a pre-cancerous lesion; inhibiting cancer cell proliferation; combining with one or more existing anti-cancer medicaments; and inhibiting angiogenesis.

3. A method of treatment of one or more of a pre-cancerous lesion, a cancer, or angiogenesis, the method comprising administering to a subject a therapeutically effective amount of the composition of claim 1.

4. The composition of claim 1, the use of claim 2, or the method of treatment of claim 3, wherein the composition is combined with one or more anti-cancer medicaments.

5. The composition, use or method of treatment of claim 4, wherein the one or more anti-cancer medicaments is a chemotherapeutic.

6. The composition, use or method of treatment of claim 5, wherein the chemotherapeutic is doxorubicin.

7. The composition, use or method of treatment of any preceding claims, wherein the cancer cells are leukaemia, blood, prostate, lung, breast, cervical, vulva or colon cancer cells.

8. The composition of claim 1, the use of claim 2, or the method of treatment of claim 3, wherein the pre-cancerous lesion is an intraepithelial neoplasia.

9. The composition, use or method of treatment of claim 8, wherein the intra-epithelial neoplasia is of a tissue selected from blood, prostate, lung, breast, cervix, vulva or colon.

10. The composition, use or method of treatment of any preceding claims, wherein the composition comprises polysaccharide and triterpene extracts in concentrations of between 0 and 600 μg/ml.

11. The composition, use or method of treatment of claim 10, wherein the concentrations of polysaccharide and triterpene extract are present in concentrations of between 175 and 250 μg/ml.

12. The composition, use or method of treatment of claim 10, wherein the composition comprises polysaccharide in an amount of between 50 and 200 μg/ml and triterpene in an amount of between 10 and 200 μg/ml.

13. The composition, use or method of treatment of claim 10, wherein the concentration of triterpene in the composition is between 70 and 175 μg/ml.

14. The composition, use or method of treatment of any preceding claims, wherein the composition further comprises between 0.01 and 2.0 μg/ml of doxorubicin.

15. The composition, use or method of treatment of any preceding claims, wherein the composition further comprises between 5 and 50 μM EGCG.