Patent Description:
The use of natural products is becoming increasingly popular with humans and companion animals. Some of these natural products are being incorporated into dietary supplements and medical foods. There is a need in the art for supplements which are useful as chemoprotective and/or antioxidant agents. In addition, there is a need in the art for pharmaceutical compositions and dietary supplements which are useful for conditions and disorders associated with the breast.

Chemoprotection through the use of natural products is evolving as a safe, effective, inexpensive, easily accessible, and practical means to prevent or reduce the occurrence of many conditions affecting humans and domesticated animals. It is known that carcinogens which can damage cells at the molecular level are often ingested and inhaled as non-toxic precursors. These non-toxic precursors may then convert into carcinogenic substances in the body. Chemoprotective agents, such as natural substances which can activate detoxifying enzymes or their co-factors, can counteract and allow for the elimination or potentiate the other naturally existing defenses such as the immune system.

Some natural products have antioxidant activity. Oxidative stress plays a major role in aging, the progression of neurodegenerative diseases as well as physiological trauma, such as ischemia. Antioxidant agents can reduce or inhibit the oxidation of vital biomolecules and may play a role in treating, preventing, or reducing the occurrence of cancer, coronary heart disease, stroke, and neurodegenerative diseases, Alzheimer's Disease, dementia, and stroke are examples of conditions affected by oxidative stress.

Cancers are largely thought to be a consequence of exposure to environmental challenges - whether from within (i.e. - estrogen, progesterone hormones) or externally (i.e. - bisphenol A (BPA) from plastic) - and chronic inflammation. Fortunately, the damage from environmental challenges can be negated via a complex network of Phase II chemoprotective enzymes found in many cell types of our body. It is well known that estrogens and their metabolites can lead to the proliferation of breast tissue and tumors. Worse, the quinone estrogen metabolites have the capacity to enter the breast tissue and migrate into the nucleus of ductal and glandular epithelial cells. There, they bind to DNA forming quinone estrogen DNA adducts which lead to downstream mutations. These mutations are thought to be responsible for the very foundation of a tumor: cancer initiation. Fortunately, a particular phase II enzyme,NAD(P)H:quinone oxidoreductase (NQO1) can take dangerous and highly reactive quinone estrogens and metabolize them to inert chemicals that can readily be removed from the body. Thus, a major mechanism to decrease cancer incidence is to induce protective Phase II enzymes including NQO1. Increased levels of NQO1 can be effective at treating, preventing, repairing, reducing the occurrence of, decreasing the symptoms associated with any conditions which are resulting from high levels of quinone estrogens. Examples of quinone estrogens include, but are not limited to catechol quinones of estrogen. Quinone estrogens are described in the following references: <NPL>; <NPL>; <NPL>; and <NPL>.

An example of a natural product thought to have chemoprotective and antioxidant properties is sulforaphane. Sulforaphane is an organosulfur compound which is also known as <NUM>-isothiocyanato-<NUM>-methylsulfinylbutane. The sulforaphane precursor, glucoraphanin, can be obtained from vegetables of the Brassicaceae family, such as broccoli, brussels sprouts, and cabbage. However, copious amounts of vegetables must be consumed in order to obtain levels adequate for chemoprevention. Glucoraphanin is converted into sulforaphane by a thioglucosidase enzyme called myrosinase, which occurs in a variety of exogenous sources such as Brassicaceae vegetables and endogenously in the gut microflora. However, upon ingestion of glucoraphanin, not all animals are capable of achieving its conversion to sulforaphane, most likely due to variations in microflora populations and overall health. In addition, in acidic environments such as the stomach, glucoraphanin can be converted to inert metabolites. The active metabolite, sulforaphane induces nuclear erythroid-<NUM>-related factor (Nrf2) which, in turn, upregulates the production of Phase II detoxification enzymes and cytoprotective enzymes such as glutathione S-transferases, NAD(P)H:quinone oxidoreductase (NQO1), and heme-oxygenase-<NUM> (HO-<NUM>). Sulforaphane has been thought to induce the production of these enzymes without significantly changing the synthesis of P-<NUM> cytochrome enzymes. The upregulation of Phase II enzymes is thought to play a role in a variety of biological activities, including the protection of the brain from cytotoxicity, the protection of the liver from the toxic effects of fat accumulation, and the detoxification of a variety of other tissues.

Sulforaphane and its precursor glucoraphanin have been studied extensively. <NPL>) discuss a clinical Phase I study determining the safety, tolerability, and metabolism of broccoli sprout glucosinolates and isothiocyanates. Shapiro et al. discuss a placebo-controlled, double-blind, randomized clinical study of sprout extracts containing either glucosinolates such as glucoraphanin or isothiocyanates such as sulforaphane in healthy human subjects. The study found that administration of these substances did not result in systematic, clinically significant, adverse effects. <NPL>) discuss the pharmacokinetics of broccoli sprout isiothiocyanates in humans.

A number of mushrooms have been used or studied for their medicinal effects. These "medicinal mushrooms" are thought to have beneficial properties, such as antiviral, antimicrobial, anticancer, antihyperglycemic, and/or anti-inflammatory activity. Examples of medicinal mushrooms include maitake, shiitake, reishi, cremini, almond, chestnut, wood ear, cloud ear, porcini, ink cap, yarta gunbu, enokitake, shemeji, tiger milk, morel, bamboo, golden oyster, pink oyster, king oyster, hiratake, cauliflower, white jelly, golden jelly, matsutake, Mexican truffle, and straw mushrooms.

Maitake mushrooms (Grifola frondosa) are edible mushroom consumed widely as food and used in traditional medicine to enhance immune function and to treat cancer. Maitake mushrooms, which contain glucans, are thought to have beneficial properties, such as antitumor and immunomodulatory effects. There exist standardized extracts from maitake mushroom that contain as active ingredients glucans such as protein-bound beta-glucans. Beta <NUM>,<NUM>-glucan, a protein bound polysaccharide, has been identified as an active constituent in maitake mushrooms. Maitake mushrooms have been demonstrated to have antitumor effects, inhibiting tumor metastasis in vitro. In one study, tumor regression or significant improvements in symptoms were observed in half of the subjects using maitake extract. In a study of postmenopausal breast cancer patients, oral administration of maitake extract was shown to have immunomodulatory effects.

Shiitake mushrooms (Lentinula edodes ) are edible mushrooms native to East Asia. Shiitake mushrooms contain mycochemicals, which are postulated to have antiviral, antibiotic, anti-inflammatory, antihypertensive and anticarcinogenic effects. This is thought to be largely a result of glucans, both alpha and beta glucans. Some shiitake mushroom extracts have alpha glucan content greater than <NUM>%. Additionally, lentinan (<NUM>,<NUM> beta-D-glucan), a polysaccharide isolated from shiitake, has been well studied and is thought to play a role in shiitake's beneficial effects. It has been shown to have anticancer effects in colon cancer cells, which may be due to its ability to suppress cytochrome P450 1A enzymes that are known to metabolize pro-carcinogens to active forms. Lentin, the protein component, has strong antifungal properties and has been found to inhibit proliferation of leukemic cells and suppress the activity of human immunodeficiency virus-<NUM> reverse transcriptase.

Reishi mushrooms (Ganoderma lucidum), also known as lingzki mushrooms, are edible mushrooms found in East Asia. Reishi mushrooms are thought to have anti-tumor, anti-cancer, immunomodulatory, and immunotherapeutic effects. Reishi mushrooms have a number of components which are thought to contribute to its activity, including glucan, such as beta-glucan, canthaxanthin, sterols, coumarin, ganoderic acid, and mannitol.

Baker's yeast (Saccaromyces cerevisiae) can be a source of glucans, in particular, beta-glucans. The active components of Baker's yeast can be extracted in a number of ways, such as the methods described in <NPL>, <CIT>; <CIT>; and <CIT>.

Glucans are described in the following references: <NPL>, and <NPL>.

<NPL>) discusses a study in Sprague-Dawley rats to determine the anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isiothiocyanates. The study determined that administration of sulforaphane was effective in blocking the formation of mammary tumors.

<NPL>) discusses a study in Sprague-Dawley rats to determine the effect of sulforaphane in chemoprevention in the breast. The study determined that oral administration of either sulforaphane resulted in a <NUM>-fold increase in NAD(P)H:quinone oxidoreductase (NQO1) enzymatic activity and a <NUM>-fold elevated immunostaining of the heme oxygenase-<NUM> (HO-<NUM>) enzyme in the mammary epithelium.

<NPL>) discusses a study regarding the effects of a freeze-dried aqueous extract of broccoli sprouts on bladder cancer development in rats. The study found that administration of the broccoli sprout extract resulted in a significant induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase <NUM> in the bladder, which are enzymes having protective activity against oxidants and carcinogens.

<NPL>) discloses a study determining the antiproliferative effect of an ethyl acetate fraction of shiitake mushrooms on human breast carcinoma cell lines (MDA-MB-<NUM> and MCF-<NUM>), a human nonmalignant breast epithelial cell line (MCF-10F), and two myeloma cell lines (RPMI08226 and IM-<NUM>). The study found that the inhibition of growth of tumor cells by the components in shiitake mushrooms may result from the induction of apoptosis.

<NPL>) discloses a study investigating the activation of natural killer (NK) cells and anticancer effects of an exo-biopolymer from rice bran cultured from Lentinus edodes. The study found that the exo-biopolymer may be effective for preventing and/or treating cancer through natural killer cell activation.

<NPL>) discusses the synergistic effect of the combination of interferon-α and maitake mushroom D-fraction (PDF), a bioactive mushroom extract on anticancer activity of interferon-α in bladder cancer T24 cells in vitro.

<NPL>) discusses a study assessing the anti-metastatic activity of a fraction of maitake mushrooms in a murine model of lung metastasis. The study found that the fraction inhibited tumor metastasis by activation of natural killer cells and antigen-presenting cells (APCs) and suppressing adhesion molecules such as ICAM-<NUM>, leading to the inhibition of tumor cell adhesion to vascular endothelial cells.

<CIT> discloses formulations comprising glucosinolates such as glucoraphanin, and myrosinase, wherein the formulation is encapsulated or coated. <CIT> discloses a dietary supplement composition having an ORAC value in excess of at least about <NUM>,<NUM> and which can be readily consumed in a small quantity. The composition comprises broccoli and a mushroom species selected from the group consisting of agaricus, cordyceps, maitake, reishi, shiitake, and combinations thereof.

According to the invention there are provided compositions as described in the appended claims. The present invention provides a composition comprising a synergistic combination of: (i) sulforaphane or a derivative thereof selected from sulfoxythiocarbamate analogues of sulforaphane, <NUM>-methylsulfinylhexyl isothiocyanate (<NUM>-HITC), and compounds which comprise the structure of sulforaphane with different side chains and/or various lengths of spacers between the isothiocyanato and sulfoxide groups, and (ii) a mushroom (maitake or shiitake mushroom) extract or powder comprising one or more glucans; wherein the synergistic combination increases gene expression of NAD(P)H:quinone oxidoreductase <NUM> (NQO-<NUM>) in a subject. The present invention also provides a composition comprising a synergistic combination of: (i) sulforaphane or a derivative thereof selected from sulfoxythiocarbamate analogues of sulforaphane, <NUM>-methylsulfinylhexyl isothiocyanate (<NUM>-HITC), and compounds which comprise the structure of sulforaphane with different side chains and/or various lengths of spacers between the isothiocyanato and sulfoxide groups, and (ii) a mushroom (maitake or shiitake mushroom) extract or powder comprising one or more glucans, wherein the synergistic combination increases gene expression of NAD(P)H:quinone oxidoreductase <NUM> (NQO-<NUM>) in a subject for use in of treating, preventing, reducing the occurrence of, decreasing the symptoms associated with, and/or reducing secondary recurrences of, cancer, in particular breast cancer, prostate cancer, colon cancer, lung cancer, and bladder cancer in a subject. Increasing levels or increasing gene expression of NAD(P)H:quinone oxidoreductase <NUM> (NQO-<NUM>) in a subject, by administering to the subject: (i) sulforaphane or a derivative thereof, and (ii) a mushroom (maitake or shiitake mushroom extract) or powder, is disclosed. Also disclosed is a composition comprising: (i) sulforaphane or a derivative thereof, and (ii) a mushroom (maitake or shiitake mushroom) extract or powder for us in treating, preventing, reducing the occurrence of, decreasing the symptoms associated with, and/or reducing secondary recurrences of a disease or condition associated with elevated levels of quinone estrogen. The present invention also provides a composition comprising a synergistic combination of: (i) sulforaphane or a derivative thereof selected from sulfoxythiocarbamate analogues of sulforaphane, <NUM>-methylsulfinylhexyl isothiocyanate (<NUM>-HITC), and compounds which comprise the structure of sulforaphane with different side chains and/or various lengths of spacers between the isothiocyanato and sulfoxide groups, and (ii) one or more glucans; wherein the synergistic combination increases gene expression of NAD(P)H:quinone oxidoreductase <NUM> (NQO-<NUM>) in a subject. The present invention also provides a composition comprising a synergistic combination of: (i) sulforaphane or a derivative thereof selected from sulfoxythiocarbamate analogues of sulforaphane, <NUM>-methylsulfinylhexyl isothiocyanate (<NUM>-HITC), and compounds which comprise the structure of sulforaphane with different side chains and/or various lengths of spacers between the isothiocyanato and sulfoxide groups, and (ii) one or more glucans, wherein the synergistic combination increases gene expression of NAD(P)H:quinone oxidoreductase <NUM> (NQO-<NUM>) in a subject for use in of treating, preventing, reducing the occurrence of, decreasing the symptoms associated with, and/or reducing secondary recurrences of, cancer, in particular breast cancer, prostate cancer, colon cancer, lung cancer, and bladder cancer in a subject.

The present disclosure provides a composition comprising a synergistic combination of: (i) a broccoli extract or powder comprising a sulforaphane or a derivative thereof selected from sulfoxythiocarbamate analogues of sulforaphane, <NUM>-methylsulfinylhexyl isothiocyanate (<NUM>-HITC), and compounds which comprise the structure of sulforaphane with different side chains and/or various lengths of spacers between the isothiocyanato and sulfoxide groups, and (ii) a mushroom (maitake or shiitake mushroom) extract or powder comprising one or more glucans. The present invention also provides a composition comprising a synergistic combination of: (i) a broccoli extract or powder comprising a sulforaphane or a derivative thereof selected from sulfoxythiocarbamate analogues of sulforaphane, <NUM>-methylsulfinylhexyl isothiocyanate (<NUM>-HITC), and compounds which comprise the structure of sulforaphane with different side chains and/or various lengths of spacers between the isothiocyanato and sulfoxide groups, and (ii) a mushroom (maitake or shiitake mushroom) extract or powder comprising one or more glucans for use in treating, preventing, reducing the occurrence of, decreasing the symptoms associated with, and/or reducing secondary recurrences of, cancer, in particular breast cancer, prostate cancer, colon cancer, lung cancer, and bladder cancer in a subject. Increasing levels or increasing gene expression of NAD(P)H:quinone oxidoreductase <NUM> (NQO-<NUM>) in a subject, by administering to the subject: (i) a broccoli extract or powder, and (ii) a mushroom (maitake or shiitake mushroom) extract or powder, is disclosed. A composition comprising: (i) a broccoli extract or powder, and (ii) a mushroom (maitake or shiitake mushroom) extract or powder for use in treating, preventing, reducing the occurrence of, decreasing the symptoms associated with, and/or reducing secondary recurrences of a disease or condition associated with elevated levels of quinone estrogen is also disclosed.

The present invention relates to the combination of sulforaphane or a derivative thereof and a mushroom (maitake, shiitake mushroom extract or powder) comprising one or more glucans. The present invention also relates to the combination of a broccoli extract or powder comprising sulforaphane or a derivative thereof and a maitake or shiitake mushroom extract or powder comprising one or more glucans. The present invention also relates to the use of a mushroom extract or powder comprising one or more glucans, with sulforaphane or a derivative thereof, or a broccoli extract comprising sulforaphane or a derivative thereof. The present invention provides compositions relating to these combinations.

Methods comprising administering these combinations are disclosed. In some embodiments, the combination may be administered for treating, preventing, reducing the occurrence of, decreasing the symptoms associated with, and/or reducing secondary recurrences of, cancer, in particular breast cancer, prostate cancer, colon cancer, lung cancer, and bladder cancer in a subject, comprising administering to the subject. In some embodiments, the combination may be administered for increasing levels or increasing gene expression of NAD(P)H:quinone oxidoreductase <NUM> (NQO-<NUM>) in a subject. In some embodiments, the combination may be administered for treating, preventing, reducing the occurrence of, decreasing the symptoms associated with, and/or reducing secondary recurrences of a disease or condition associated with elevated levels of quinone estrogen.

Sulforaphane is also known as <NUM>-isothiocyanato-<NUM>-methylsulfinylbutane. Derivatives of sulforaphane include sulfoxythiocarbamate analogues of sulforaphane, <NUM>-methylsulfinylhexyl isothiocyanate (<NUM>-HITC), and compounds which comprise the structure of sulforaphane with different side chains and/or various lengths of spacers between the isothiocyanato and sulfoxide groups. Examples of derivatives of sulforaphane include those described in the following references: <NPL>; <NPL>; and <NPL>, and <NPL>.

In some embodiments, the composition comprises sulforaphane or a derivative thereof, preferably sulforaphane, in an amount of about <NUM> to about <NUM>. In some embodiments, compositions suitable for human use comprise about <NUM> to about <NUM>.

The disclosure provides methods comprising administration of sulforaphane or a derivative thereof to a subject, preferably sulforaphane, in an amount of about <NUM> to about <NUM> are disclosed. In some embodiments wherein the subject is human, the method comprises administration of about <NUM> to about <NUM>. Methods comprising administration of sulforaphane or a derivative thereof to a subject, preferably sulforaphane, in an amount of about <NUM>µg/kg to about <NUM>/kg, preferably about <NUM>µg/kg to about <NUM>/kg, more preferably about <NUM>µg/kg to about <NUM>/kg, more preferably about <NUM>µg/kg to about <NUM>/kg, and most preferably about <NUM>µg/kg to about <NUM>/kg, are disclosed. The method suitable for human use comprising administration of about <NUM>µg/kg to about <NUM>/kg, and more preferably about <NUM>/kg to about <NUM>/kg. The above amounts may refer to each dosage administration or a total daily dosage, is also disclosed. The total daily dosage refers to the total amount of a compound or ingredient which is administered to a subject in a twenty-four hour period.

A method comprising administration of more than one of a sulforaphane or a derivative thereof, is disclosed. In some embodiments, the compositions comprise more than one of a sulforaphane or a derivative thereof. For example, the methods or composition may comprise both sulforaphane and one or more derivatives thereof, or two or more derivatives. In some embodiments wherein the method or composition comprise more than one of a sulforaphane or a derivative thereof, the above amounts may refer to the amount of each sulforaphane or a derivative thereof, or the total amount of the more than one sulforaphane or derivative thereof.

The present disclosure also provides the use of a broccoli extract and/or powder, including for example broccoli seed and sprout extracts and powders. Methods of administration of broccoli extract and/or powder, are disclosed, and the present invention provides compositions comprising broccoli extract and/or powder. Examples of broccoli extracts and powders include for example those described in <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>, and <CIT>. Powders of broccoli may be obtained, for example, by air drying, freeze drying, drum drying, spray drying, heat drying and/or partial vacuum drying broccoli, preferably broccoli sprouts. In some embodiments, the compositions comprise use of about <NUM>µg to about <NUM>, more preferably about <NUM>µg to about <NUM>, even more preferably about <NUM>µg to about <NUM>, preferably about <NUM>µg to about <NUM>, more preferably about <NUM>µg to about <NUM>, and most preferably about <NUM> to about <NUM> of the broccoli extract. In some embodiments, the broccoli extract or powder is present in a composition or administered to a subject in amounts sufficient to provide sulforaphane in the amounts described above.

The sulforaphane or a derivative thereof may be obtained from any source, including for example one or more plants from the Brassicaceae (also known as Cruciferae) family. Examples of plants from the Brassicaceae family include the following: broccoli, Brussels sprouts, cauliflower, cabbage, horseradish, parsnip, radish, wasabi, watercress, and white mustard.

The present invention provides for the use of a mushroom extract or powder. In some embodiments, the mushrooms may comprise "medicinal mushrooms," including for example maitake, shiitake, reishi, cremini, almond, chestnut, wood ear, cloud ear, porcini, ink cap, yarta gunbu, enokitake, shemeji, tiger milk, morel, bamboo, golden oyster, pink oyster, king oyster, hiratake, cauliflower, white jelly, golden jelly, matsutake, Mexican truffle, and straw mushrooms. In preferred embodiments, the mushroom comprises maitake mushroom, shiitake mushroom, , and/or a mixture of one or more of these.

Maitake mushroom belongs to the species Grifola frondosa. Maitake mushroom may contain a number of fractions having biological activity. Examples of components found in maitake mushroom include: glucans (such as alpha-glucans and beta-glucans); lipids (such as octadecanoic and octadecadienoic acids); phospholipids (such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine and phosphatidic acid).

Shiitake mushroom belongs to the species Lentinula edodes. Shiitake mushroom may contain a number of fractions having biological activity. Examples of components found in shiitake mushrooms include glucans (such as alpha-glucans and beta-glucans), proteins (such as lentin); lipids (such as linoleic acid); and lignins.

Reishi mushrooms belong to the species Ganoderma lucidum. Reishi mushrooms may contain a number of fractions having biological activity. Examples of components found in reishi mushroom include: glucan (such as alpha-glucans and beta-glucans), canthaxanthin, sterols, coumarin, ganoderic acid, and mannitol.

In some preferred embodiments, the mushroom extract or powder comprises one or more glucans. A glucan is a polysaccharide of a D-glucose monomer linked by glycosidic bonds and may be in the alpha or beta form. In some embodiments, the glucan comprises one or more alpha-glucan and/or beta-glucans. Alpha-glucans include <NUM>,<NUM>-α-glucans and <NUM>,<NUM>-α -glucans and beta-glucans include <NUM>,<NUM>-β-glucans, <NUM>,<NUM>-β-glucans, and <NUM>,<NUM>-β-glucans. The glucans may be expressed in a variety of polymeric configurations. In preferred embodiments, the maitake mushroom extract or powder comprises <NUM>,<NUM>-β-glucans and/or <NUM>,<NUM>-β-glucans. In preferred embodiments, the shiitake mushroom extract or powder comprises <NUM>,<NUM>-α-glucans.

In some embodiments, a maitake mushroom extract or powder may be used. In some embodiments, the maitake mushroom extract or powder is standardized to contain about <NUM>% to about <NUM>%, more preferably about <NUM>% to about <NUM>%, even more preferably about <NUM>% to about <NUM>%, and most preferably about <NUM>% to about <NUM>% of one or more glucans, preferably beta-glucans, and more preferably <NUM>,<NUM>-beta glucan and/or <NUM>,<NUM>-beta-glucan. Examples of maitake mushroom extracts and powders include those described in <CIT>; <CIT>, <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>. Powders of maitake mushroom may be obtained, for example, by air drying, freeze drying, drum drying, spray drying, heat drying and/or partial vacuum drying maitake mushrooms. In some embodiments, the composition comprises about <NUM>µg to about <NUM>, preferably about <NUM>µg to about <NUM>, and most preferably about <NUM>µg to about <NUM>. In some embodiments, compositions suitable for humans comprise about <NUM> to about <NUM> of maitake mushroom extract. In some embodiments, the method comprises administration of about <NUM>µg to about <NUM>, preferably about <NUM>µg to about <NUM>, and most preferably about <NUM>µg to about <NUM>. In some embodiments wherein the subject is human, the method comprises administration of about <NUM> to about <NUM> of maitake mushroom extract. The above amounts may refer to each dosage administration or a total daily dosage.

In some embodiments, a shiitake mushroom extract or powder may be used. In some embodiments, the shiitake mushroom extract or powder is standardized to contain about <NUM>% to about <NUM>%, preferably about <NUM>% to about <NUM>%, even more preferably about <NUM>% to about <NUM>%, and most preferably about <NUM>% to about <NUM>% of one or more glucans, preferably alpha-glucans, and more preferably <NUM>,<NUM>-alpha-glucan. Examples of shiitake mushroom extracts include those described in <CIT>; <CIT>; <CIT>, <CIT>, and <CIT>. Powders of maitake mushroom may be obtained, for example, by air drying, freeze drying, drum drying, spray drying, heat drying and/or partial vacuum drying maitake mushrooms. In some embodiments, the composition comprises about <NUM> to about <NUM>, preferably about <NUM> to about <NUM>, and most preferably about <NUM> to about <NUM>. In some embodiments, compositions suitable for humans comprise about <NUM> to about <NUM> of shiitake mushroom extract or powder. In some embodiments, the method comprises administration of about <NUM> to about <NUM>, preferably about <NUM> to about <NUM>, and most preferably about <NUM> to about <NUM>. In some preferred embodiments wherein the subject is human, the method comprises administration of about <NUM> to about <NUM> of shiitake mushroom extract or powder to a subject. The above amounts may refer to each dosage administration or a total daily dosage. The above amounts may refer to each dosage administration or a total daily dosage.

In some embodiments, the composition comprises use of one type or mushroom extract or powder, which is maitake mushroom extract or powder, or shiitake mushroom extract or powder. In some embodiments, the composition comprises use of a mixture of one or more types of mushroom extract or powder. In some embodiments, the comprises use of a mixture of one or more of the following: maitake mushroom extract or powder, shiitake mushroom extract or powder, and reishi mushroom extract or powder, with the proviso that at least one of maitake and shiitake mushroom extract or powder is present. The composition and method may comprise use of an extract or a powder, or a mixture of extracts and powders.

Methods further comprising administration of one or more additional components to the compositions of the present invention, are disclosed. The compositions of the present invention may further comprise one or more additional components. The additional components may include active pharmaceutical ingredients, nutritional supplements, and nutritional extracts. Examples of additional components include ursolic acid, quercetin or a derivative thereof, an aminosugar such as glucosamine, a glycosaminoglycan such as chondroitin, avocado/soybean unsaponifiables, vitamins such as vitamin K2, coffee fruit, magnesium, ursolic acid, proanthocyanidins, alpha- and beta-glucans, curcumin, phytosterols, phytostanols, and S-adenosylmethionine (SAMe). These additional components may be present in milk thistle (Silybum marianum) extract (silymarin), cranberry (Vaccinium macrocarpon) extract (proanthocyanidins, quercetin, and ursolic acid), turmeric (Curcuma longa). In some embodiments, the ratio of beta-glucan to sulforaphane or a derivative of (beta-glucan:sulforaphane or a derivative of) is about <NUM>:<NUM> to about <NUM>:<NUM>, preferably about <NUM>:<NUM> to about <NUM>:<NUM>, more preferably about <NUM>:<NUM> to about <NUM>:<NUM>, more preferably about <NUM>:<NUM> to about <NUM>:<NUM>, even more preferably about <NUM>:<NUM> to about <NUM>:<NUM>, and most preferably about <NUM>:<NUM> to about <NUM>:<NUM>. In some embodiments, the ratio of alpha-glucan to sulforaphane or a derivative of (alpha-glucan:sulforaphane or a derivative of) is about <NUM>:<NUM> to about <NUM>:<NUM>, preferably about <NUM>:<NUM> to about <NUM>:<NUM>, more preferably about <NUM>:<NUM> to about <NUM>:<NUM>, more preferably about <NUM>:<NUM> to about <NUM>:<NUM>, even more preferably about <NUM>:<NUM> to about <NUM>:<NUM>, and most preferably about <NUM>:<NUM> to about <NUM>:<NUM>.

In some embodiments, the composition comprises a unit dosage form, including pharmaceutical dosage forms suitable for oral, rectal, intravenous, subcutaneous, intramuscular, transdermal, transmucosal, and topical. In some preferred embodiments, the composition comprises an orally administrable dosage form or a rectally administrable dosage form. Examples of orally administrable dosage forms include a tablet, capsule, powder that can be dispersed in a beverage, a liquid such as a solution, suspension, or emulsion, a soft gel/chew capsule, a chewable bar, or other convenient dosage form known in the art. In preferred embodiments, the composition comprises a tablet, capsule, or soft chewable treat. The orally administrable dosage forms may be formulated for immediate release, extended release or delayed release.

In some embodiments, at least the sulforaphane or derivative thereof and/or the broccoli extract or powder are provided in a dosage form which allows for the release in an area of the gastrointestinal tract having a pH of at least <NUM> and preferably at least <NUM>, such as the small intestine, preferably the duodenum. In some embodiments, the mushroom extract or powder and/or any optional additional components are also released in an area of the gastrointestinal tract having a pH of at least <NUM> and preferably at least <NUM>, such as the small intestine, preferably the duodenum. The small intestine includes the duodenum, jejunum, and ileum.

In some embodiments, each of these components (i. e, sulforaphane or a derivative thereof, broccoli extract or powder, mushroom extract or powder, and/or additional components) are released simultaneously or concomitantly (i.e., within a short period of time of each other).

In some embodiments, the compositions may comprise orally administrable compositions which comprise enteric coated dosage forms or any dosage form which is resistant to degradation in an area of the gastrointestinal tract having pH below <NUM>, such as the stomach. For example, the orally administrable composition may comprise a tablet or capsule comprising an enteric coating. The enteric coating may comprise materials including, for example, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, methacrylic acid copolymer, methacrylic acid:acrylic ester copolymer, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose trimellitate, shellac, cellulose acetate trimellitate, carboxymethylethylcellulose, and mixtures thereof. The enteric coating may comprise any suitable enteric polymers known in the art. In some embodiments, one or more of the components in the composition may be embedded in a matrix of enteric polymers. In some embodiments, the orally administrable compositions comprise a capsule that dissolves slowly in gastric acid and travels to the small intestine, such as DRCAPS™ acid resistant capsules, which are marketed by CAPSUGEL® or any other acid resistant capsules.

In the most preferred form, the orally administrable composition is surrounded by a coating that does not dissolve unless the surrounding medium is at a pH of at least <NUM>, and more preferably at least <NUM>. Alternatively, a coating may be employed which controls the release by time, as opposed to pH, with the rate adjusted so that the components are not released until after the pH of the gastrointestinal tract has risen to at least <NUM>, and more preferably at least <NUM>. Thus, a time-release formulation may be used to prevent gastric presence of the sulforaphane. The coating layer(s) may be applied onto orally administrable composition using standard coating techniques. The enteric coating materials may be dissolved or dispersed in organic or aqueous solvents. The pH at which the enteric coat will dissolve can be controlled by a polymer, or combination of polymers, selected and/or ratio of pendant groups. For example, dissolution characteristics of the polymer film can be altered by the ratio of free carboxyl groups to ester groups. Enteric coating layers also contain pharmaceutically acceptable plasticizers such as triethyl citrate, dibutyl phthalate, triacetin, polyethylene glycols, polysorbates or other plasticizers. Additives such as dispersants, colorants, anti-adhering and antifoaming agents may also be included.

The compositions may contain one or more non-active pharmaceutical ingredients (also known generally as "excipients"). Non-active ingredients, for example, serve to solubilize, suspend, thicken, dilute, emulsify, stabilize, preserve, protect, color, flavor, and fashion the active ingredients into an applicable and efficacious preparation that is safe, convenient, and otherwise acceptable for use. The excipients are preferably pharmaceutically acceptable excipients. Examples of classes of pharmaceutically acceptable excipients include lubricants, buffering agents, stabilizers, blowing agents, pigments, coloring agents, flavoring agents, fillers, bulking agents, fragrances, release modifiers, adjuvants, plasticizers, flow accelerators, mold release agents, polyols, granulating agents, diluents, binders, buffers, absorbents, glidants, adhesives, anti-adherents, acidulants, softeners, resins, demulcents, solvents, surfactants, emulsifiers, elastomers and mixtures thereof.

In some embodiments, the combination of sulforaphane (or a derivative thereof) and a mushroom extract or powder (which contains glucans) demonstrates a synergistic effect. Synergy refers to the effect wherein a combination of two or more components provides a result which is greater than the sum of the effects produced by the agents when used alone. In preferred embodiments, the synergistic effect is greater than an additive effect.

In some embodiments, the combination of a sulforaphane (or a derivative thereof) and a mushroom extract or powder has a statistically significant and/or greater than additive effect than: (i) sulforaphane (or a derivative thereof) alone, and/or (ii) a mushroom extract or powder alone. In some embodiments, the combination of sulforaphane and glucan has a synergistic effect compared to sulforaphane alone, and glucan alone.

Methods of use, including methods of administration to a subject in need thereof, are disclosed. In some embodiments, the method comprises administration of the combination of a sulforaphane or a derivative thereof and a mushroom extract or powder. In some embodiments, the method comprises administration of the combination of a broccoli extract or powder and a mushroom extract or powder.

In some embodiments, the present invention relates to compositions of the present invention for use in treating, preventing, reducing the occurrence of, decreasing the symptoms associated with, and/or reducing secondary recurrences of, cancer, in particular breast cancer, prostate cancer, colon cancer, lung cancer, liver cancer, and bladder cancer in a subject. The use may be useful in reducing damage or slowing damage to tissues and organs, such as the breast, prostate, colon, lung, liver, and bladder. The present invention provides compositions for use in treating, preventing, decreasing the symptoms associated with, and/or reducing secondary recurrences of diseases and conditions associated with the reproductive system (including for example the breast and prostate), colon, liver, bladder, kidney, central nervous system, cardiovascular system, pulmonary system, genitourinary system, hematopoietic system, and joints. The present invention also provides compositions for use in treating, preventing, decreasing the symptoms associated with, and/or reducing secondary recurrences of cysts, such as benign cysts.

Increasing levels or increasing gene expression of NAD(P)H:quinone oxidoreductase <NUM> (NQO-<NUM>) in a subject is disclosed herein. This may also be useful in treating, preventing, decreasing the symptoms associated with, and/or reducing secondary recurrences of diseases and conditions which would be benefited from an increase in gene expression or levels of NQO-<NUM>. Examples of such diseases and conditions include cancer, myelodysplastic syndrome, cardiovascular disease, and tardive dyskinesia.

In some embodiments, the present invention relates to compositions for use in treating, preventing, reducing the occurrence of, decreasing the symptoms associated with, and/or reducing secondary recurrences of a disease or condition associated with elevated levels of quinone estrogen. Examples of such diseases or conditions include cancer, myelodysplastic syndrome, cardiovascular disease, and tardive dyskinesia.

Methods relating to providing a beneficial effect on biomarkers, and treating, preventing, reducing the occurrence of, decreasing the symptoms associated with abnormal levels of these biomarkers, are disclosed. Examples of such biomarkers include NADPH-dependent enzymes, thioredoxin (TXN), thioredoxin reductase-<NUM> (Txnrd-<NUM>), glutamate-cysteine ligase subunit (GCLC), sulfotransferase 1A1 (SULT1A1), heme oxygenase-<NUM> (HMOX1), glutathione peroxidase-<NUM> (GPx-<NUM>), glutathione S-transferse theta <NUM> (GSTT2), microsomal glutathione S-transferase <NUM> (MGST1), aldehyde oxidase (AOX1), aldo-keto reductase 1B8 (Akr1b8), flavin-containing monooxygenase <NUM> (FMO2), Fc receptor region receptor III (Fcgr3), tryptase beta <NUM> (TPSB1), mast cell protease-<NUM> (Mcpt6), neurexin-<NUM>-alpha (NRXN-<NUM>), microphthalmia-associated transcription factor (MITF), type II iodothyronine deiodinase (DIO2), angiopoietin-<NUM> (Angpt14), cluster of differentiation (CD36), and Ntel. Diseases or conditions associated with elevated or abnormal levels of these biomarkers include, for example, cancer, pulmonary and central nervous system tuberculosis, multiple sclerosis, Crohn's disease, atherosclerosis, osteoarthritis, asthma, stroke, emphysema, diabetic nephropathy, chronic histiocytic intervillositis of the placenta, hypertension, abdominal aortic aneurysm, inflammatory bowel disease, chronic rhinosinusitis, coronary artery disease, and kidney disease.

Method comprising administering to a subject in need thereof a combination of sulforaphane and a mushroom extract or powder containing glucan, is disclosed. In preferred embodiments, the combinations demonstrate a synergistic effect.

In preferred embodiments, one or more components of the combinations (for example, the mushroom extract or powder; or the sulforaphane or derivative thereof and the mushroom extract or powder) are administered together in one composition or dosage form, or separately, preferably within a period in which their therapeutic properties overlap. In some embodiments, the components of the combinations may be administered in two or more orally administrable compositions or dosage forms. For example, in some embodiments, the sulforaphane or a derivative thereof is administered in one orally administrable dosage form, while the a mushroom extract or powder are administered in one or more separate or additional orally administrable dosage form(s). In preferred embodiments, the components of the combination are administered in one dosage form.

In some embodiments, the combination may be administered at a frequency of <NUM> to <NUM> times daily, preferably <NUM> to <NUM> times daily, more preferably <NUM> to <NUM> times daily, and most preferably <NUM> time daily.

The dosages disclosed in this application refer preferably to dosages suitable for humans. Dosage calculations can be determined by those of skilled in the art by evaluating body weight, surface area, metabolic rate, and species differences.

The term "subject" refers to any mammal. In preferred embodiments, the subjects are humans.

The following are exemplary formulation of the present invention:.

A Hydrophobic Interaction Chromatographic (HILIC) method was developed, comprising the following conditions:.

The above conditions allow separation of five typical Brassicaceae glucosinolates, including the sulforaphane precursor, glucoraphanin.

About <NUM> of broccoli seed extract containing about <NUM>% (w/w) glucoraphanin were subjected to hydrolysis by a fixed concentration of broccoli sprout-derived myrosinase in the presence of variable concentration of ascorbic acid, ranging from <NUM> to <NUM>µmoles/Liter. The reaction mixtures were thermostated at 38ºC; aliquots were withdrawn every <NUM> minutes for <NUM> minutes, and concentration of glucoraphanin determined chromatographically. The rate of glucoraphanin consumption was interpreted as the rate its conversion to sulforaphane. Graphical representation of glucoraphanin content reduction as a function of increasing ascorbic acid concentration results in a series of linear plots; the slopes of the linear regression lines reflect the rate of glucoraphanin consumption, in µmoles/minute. It is apparent that in the presence of <NUM>µmoles/Liter concentration of ascorbic acid, the reaction rate increased <NUM>-fold relative to that which proceeded in the absence of modulatory effects of ascorbic acid.

A two-part experiment was conducted to further elucidate the role of ascorbic acid in modulating myrosinase activity. All solutions were prepared in <NUM> Tris-buffered saline, at pH <NUM>, previously identified as an optimal for myrosinase activity; each sample tube had <NUM> of freeze-dried broccoli powder accurately weighed in as a source of myrosinase. Experiment was conducted at <NUM>ºC for <NUM> hours, with sample aliquots removed in <NUM>-minute increments, and both glucoraphanin and sulforaphane content assessed by HPLC. A strongly acidic "stop" solution was utilized to instantaneously inhibit further myrosinase activity in the removed aliquots. A control sample contained no ascorbic acid, and the enzymatic conversion proceeded unassisted by a co-factor.

PART <NUM>. In the presence of the fixed concentration of ascorbic acid, <NUM> mmol/Liter, an increasing amount of broccoli seed extract (about <NUM>% glucoraphanin, w/w) was added, ranging from <NUM> to <NUM>.

PART <NUM>. While keeping the amount of broccoli seed extract fixed at <NUM>, the concentration of ascorbic acid was varied from <NUM> mmol/Liter to <NUM> mmol/Liter.

The table below presents glucoraphanin and sulforaphane expressed in µmoles. It is apparent that within the first <NUM> minutes in almost all the reaction mixtures, conversion of glucoraphanin to sulforaphane was complete. However, careful examination of the enzymatic conversion occurring in the control sample, without the stimulating effects of ascorbic acid, reveals an equimolar conversion of glucoraphanin to sulforaphane, i.e., the amount of glucoraphanin consumed results in the equivalent amount of sulforaphane produced.

In the Part <NUM> of the experiment, the modulatory effect of the increasing concentration of ascorbic acid on the activity of myrosinase was assessed. An initial, apparently linear, increase in myrosinase-promoted conversion of glucoraphanin to sulforaphane is observed to about <NUM> mmol/L of ascorbic acid concentration, followed subsequently by a considerable leveling off.

Finally, examination of sulforaphane yield of after <NUM> minutes within the PART <NUM> of the experiment, reveals that in the presence of <NUM> mmol/Liter of ascorbic acid, the fixed amount of myrosinase contained in <NUM> of freeze-dried broccoli sprout powder is capable of generating at least <NUM>µmoles of sulforaphane, in a predictably linear fashion. <FIG>, <FIG>, <FIG>, and <FIG> demonstrate the results of this study.

Simulated Intestinal Fluid (SIF) powder, a commercially supplied concentrate closely approximating the human intestinal content in terms of composition, pH and ionic strength, was used. The experiment utilized a USP Dissolution Apparatus <NUM> (paddles), where into six dissolution vessels <NUM> of Simulated Intestinal Fluid was dispensed, along with <NUM> of freeze-dried broccoli sprout powder as a source of myrosinase. In vessels <NUM>-<NUM>, the concentration of ascorbic acid was varied from <NUM> to <NUM> mmol/Liter; in vessel <NUM>, in addition to <NUM> mmol/Liter ascorbic acid, <NUM> of pancreatin (8x USP) was suspended; in vessel <NUM>, in addition to <NUM> mmol/Liter ascorbic acid, and <NUM> of pancreatin (8x USP), a doubled amount of freeze-dried broccoli sprout powder (<NUM>) was added. After vessels were brought to <NUM>ºC, <NUM> of glucoraphanin-rich (<NUM>%, w/w) broccoli seed extract was added to each, and the resulting suspensions were stirred at <NUM> RPM for <NUM> hours. Aliquots were withdrawn every <NUM> minutes, and assayed for sulforaphane. <FIG> shows direct correlation between larger yield of sulforaphane and higher concentrations of ascorbic acid, especially at the earlier stages of the experiment.

The following study was conducted to determine the effect of the combination of sulforaphane and a maitake mushroom extract containing <NUM>% b-glucans on gene expression of Nad(P)H:quinone oxidoreductase <NUM> (NQO-<NUM>). NQO-<NUM> encodes a protein that is able to metabolize estrogen quinones, preventing them from forming DNA adducts that cause mutations and ultimately carcinogenesis. An increase in NQO-<NUM> expression is favorable for breast, colon, liver, lung, skin and prostate health.

In the study, the macrophage cell line RAW <NUM> was treated with DMSO (vehicle control), sulforaphane (SFN), maitake mushroom extract having about <NUM>% beta-glucan content (Maitake), or the combination of sulforaphane and maitake mushroom extract, for <NUM> hours. In particular, the cells were treated with one of the following: (i) DMSO (vehicle control), (ii) <NUM> SFN, (iii) <NUM>µg/mL Maitake, (iv) <NUM>µg/mL Maitake, (v) <NUM>µg/mL Maitake, (vi) <NUM> SFN and <NUM>µg/mL Maitake, (vii) <NUM> SFN and <NUM>µg/mL Maitake, and (viii) <NUM> SFN and <NUM>µg/mL Maitake. Gene expression of NQO-<NUM> gene expression was analyzed via quantitative RT-PCR. The results, which are depicted in <FIG>, show the following:.

The results demonstrate that the combination of sulforaphane and maitake mushroom extract had a synergistic effect compared to each component alone. This effect was found to be more than merely additive.

The following study was conducted to determine the effect of the combination of sulforaphane and a shiitake mushroom extract containing <NUM>% alpha glucans on gene expression of Nad(P)H:quinonequinone oxidoreductase <NUM> (NQO-<NUM>).

In the study, the macrophage cell line RAW <NUM> was treated with DMSO (vehicle control), sulforaphane (SFN), shiitake mushroom extract having at least <NUM>% alpha-glucan content (Shiitake), or the combination of sulforaphane and shiitake mushroom extract, for <NUM> hours. In particular, the cells were treated with one of the following: (i) DMSO (vehicle control), (ii) <NUM> SFN, (iii) <NUM>µg/mL Shiitake, (iv) <NUM>µg/mL Shiitake, (v) <NUM>µg/mL Shiitake, (vi) <NUM> SFN and <NUM>µg/mL Shiitake, (vii) <NUM> SFN and <NUM>µg/mL Shiitake, and (viii) <NUM> SFN and <NUM>µg/mL Shiitake. Gene expression of NQO-<NUM> gene expression was analyzed via quantitative RT-PCR. The results, which are depicted in <FIG>, show the following:.

The results demonstrate that the combination of sulforaphane and shiitake mushroom extract had a synergistic effect compared to each component alone. This effect was found to be more than merely additive.

A subject presents with breast cancer and is suffering from symptoms including damaged breast tissue and breast pain. She is administered a tablet containing glucoraphanin, myrosinase, ascorbic acid, and a maitake mushroom extract. The tablet is an enteric coated formulation which releases the contents in the small intestine. After one month of daily administration of the tablet, the subject experiences modulation of surrogate biomarkers including NQO-<NUM> which correlate with improved in symptoms.

Claim 1:
An orally administrable composition comprising a synergistic combination of:
a sulforaphane or a derivative thereof selected from sulfoxythiocarbamate analogues of sulforaphane, <NUM>-methylsulfinylhexyl isothiocyanate (<NUM>-HITC), and compounds which comprise the structure of sulforaphane with different side chains and/or various lengths of spacers between the isothiocyanato and sulfoxide groups in an amount of about <NUM> to about <NUM>; and
a maitake or shiitake mushroom extract or powder comprising one or more glucans.