EP3630090A1 - Combination therapy for cancer using botanical compositions and enzalutamide - Google Patents

Abstract

Botanical compositions comprising non-alcoholic organic extracts of Ganoderma lucidum, Salvia miltiorrhiza, and Scutellaria barbata for use in conjunction with enzalutamidefor cancer therapy, are provided. Methods for treatment or therapy of prostate cancer in a human is provided, the method comprising: administering an effective amount of a botanical composition that is effective for reducing androgen receptor protein expression; and administering concurrently an effective amount of enzalutamide, wherein the concurrent administration of the enzalutamide and the botanical composition achieves a therapeutic effect that is more effective than either agent alone.

Description

COMBINATION THERAPY FOR CANCER USING BOTANICAL COMPOSITIONS

AND ENZALUTAMIDE

INVENTORS: James DAO and James DAO

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Patent Application Serial No.

62/511,358, filed May 26, 2017 and titled "COMBINATION THERAPY FOR CANCER USING BOTANICAL COMPOSITIONS AND ENZALUTAMIDE," the contents of which are incorporated herein in their entirety by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention relates generally to the field of compositions for treatment of cancer. More specifically, the invention provides multifunctional, multitargeted compositions of botanical extracts in combination with enzalutamide for the prevention and therapy of cancer, and specifically prostate cancer.

BACKGROUND OF THE INVENTION

[0003] Other than skin cancer, prostate cancer is the most common cancer in American men. The American Cancer Society's estimates for prostate cancer in the United States for 2017 are: about 161,360 new cases of prostate cancer; and about 26,730 deaths from prostate cancer. About 1 man in 7 will be diagnosed with prostate cancer during his lifetime. Prostate cancer develops mainly in older men. About 6 cases in 10 are diagnosed in men aged 65 or older, and it is rare before age 40. Prostate cancer is the third leading cause of cancer death in American men, behind lung cancer and colorectal cancer. About 1 man in 39 will die of prostate cancer.

[0004] Most prostate cancers are slow growing; however, there are cases of aggressive prostate cancers. The cancer cells may metastasize (spread) from the prostate tu ulher parts of the body, particularly the bones and lymph nodes. Prostate cancer may cause pain, difficulty in urinating, problems during sexual intercourse, or erectile dysfunction. Other symptoms can potentially develop during later stages of the disease.

[0005] Treatment options for prostate cancer with intent to cure are primarily surgery, radiation therapy, and proton therapy. Other treatments, such as hormonal therapy, chemotherapy, cryosurgery, and high intensity focused ultrasound (HIFU) also exist, depending on the clinical scenario and desired outcome.

[0006] Enzalutamide is an orally bioavailable, organic, non-steroidal small molecule targeting the androgen receptor (AR) with potential antineoplastic activity. Through a mechanism that is reported to be different from other approved AR antagonists, enzalutamide inhibits the activity of prostate cancer cell ARs, which may result in a reduction in prostate cancer cell proliferation and, correspondingly, a reduction in the serum prostate specific antigen (PSA) level. AR over- expression in prostate cancer represents a key mechanism associated with prostate cancer hormone resistance

[0007] Enzalutamide is marketed by Astellas Pharma Inc. with the brand name XTANDI^®. It is recommended in combination with a luteinizing hormone-releasing hormone analog or surgical castration. XTANDI (enzalutamide) capsules are indicated for the treatment of patients with metastatic castration-resistant prostate cancer (CRPC).

[0008] Researchers continue to look for foods (or substances in them) that can help lower prostate cancer risk. Scientists have found some substances in tomatoes (lycopenes) and soybeans (isoflavones) that might help prevent prostate cancer. Some studies have found that men with high levels of vitamin D seem to have a lower risk of developing the more lethal forms of prostate cancer. Overall though, studies have not found that vitamin D protects against prostate cancer.

[0009] There is a need for enhanced prostate cancer therapy regimens that increase effectiveness of the therapy while reducing side effects and toxicity resulting from the chemotherapeutic treatment.

[0010] Compositions of botanicals comprising therapeutically effective amounts of two or more of an extract of Ganoderma lucidum, an extract of Salvia miltlorrhiza and an extract of

Scutellaria barbata for prevention and therapy of cancer have been reported by Dao et al. (US Pat. App. Pub. No. 20050208070). SUMMARY OF THE INVENTION

[0011] The present invention relates to combinations of enzalutamide and compositions botanical extracts for treatment and theraopy of prostate cancer.

[0012] The compositions of botanical extracts can be used to reduce or alleviate the side affects when used with standard non-botanical chemotherapies. Side effects are reduced by inhibiting inflammatory responses, modulating immune responses, reducing oxidative stress, modulating immune responses, inhibiting viral and microbial infections, modulating cell proliferative responses or other biological responses. The compositions of the invention may also alleviate side affects of standard therapeutic agents by balancing general biological responses against perturbations in specific biological pathways due to treatment with the therapeutic agent.

[0013] In a preferred embodiment, the composition comprises combinations of two or more extracts of Ganoderma lucidum, Scutellaria barbata, Scutellaria baicalensis, Salvia miltiorrhiza, and optionally, Hippophae rhamnoides (sea buckthorn).

[0014] In one embodiment, this method comprises treatment or therapy of prostate cancer in a human is provided, the method comprising: administering an effective amount of a botanical composition that is effective for reducing androgen receptor protein expression; and

administering concurrently an effective amount of a compound having anti-androgen activity, wherein the concurrent administration of the compound and the botanical composition achieves more effective therapy than either agent alone. Botanical compositions comprising non-alcoholic organic extracts of Ganoderma lucidum, Salvia miltiorrhiza, and Scutellaria barbata in conjunction with enzalutamide therapy are used.

[0015] The present invention and other objects, features, and advantages of the present invention will become further apparent in the following Detailed Description of the Invention and the accompanying Figures and embodiments. BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1 shows the synergistic effect on inhibition of cell proliferation by a combination of Aneustat™(OMN54) and MDV3100^® (enzalutamide) on castration-resistant prostate cell (C4- 2 cell line) growth. [0017] Figure 2 shows the synergistic effect on inhibition of cell proliferation by a combination of Aneustat™(OMN54) and ENZA (MDV3100®; enzalutamide) on castration-resistant prostate cell (C4-2B MDV) growth. C4-2B MDVR cells were treated with enzalutamide (ENZA) or OMN54 or their combination as indicated in FBS condition, total cell number was determined after 5 days treatment.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0018] The term "plant" as used herein refers to seeds, leaves, stems, flowers, roots, berries, bark, or any other plant parts that are useful for the purposes described. For certain uses, it is preferred that the underground portion of the plant, such as the root and rhizoma, be utilized. The leaves, stems, seeds, flowers, berries, bark, or other plant parts, also have medicinal effects and can be used for preparing tea and other beverages, cream, and in food preparation.

[0019] The term "treatment" or "treating" or 'therapy" as used herein, for purposes of the specification and claims, includes preventing, inhibiting, curing, or alleviating.

[0020] By the term "administering," it is meant that the compositions are delivered to the host in such a manner that it can achieve the desired purpose. As mentioned the compositions can be administered by an effective route, such as orally, topically, rectally, etc.

[0021] "Synergism" may be measured by combination index (CI). The combination index method was described by Chou and Talalay. (Chou, T.-C. The median-effect principle and the combination index for quantitation of synergism and antagonism, p. 61-102. In T.-C. Chou and D. C. Rideout (ed.), Synergism and antagonism in chemotherapy. Academic Press, San Diego, Calif. (1991); Chou, T.-C, and P. Talalay. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs on enzyme inhibitors. Adv. Enzyme Regul. 22:27-55 (1984)). A Cl value of 0.90 or less is considered synergistic, with values of 0.85 being moderately synergistic and values below 0.75 being significantly synergistic. CI values of 0.90 to 1.10 are considered to be merely additive and higher values are antagonistic.

Table 1. Synergism/antagonism as a function of CI values

Combination Index (CI) Value Interpretation

>10 Very strong antagonism 3.3 - 10 Strong antagonism

1.45 - 3.3 Antagonism

1.2 - 1.45 Moderate antagonism

1.1 - 1.2 Slight antagonism

0.9 - 1.1 Additive

0.85 - 0.9 Slight synergism

0.7 - 0.85 Moderate synergism

0.3 - 0.7 Synergism

0.1 - 0.3 Strong synergism

< 0.1 Very strong synergism

[0022] It is noted that determination of synergy may be affected by biological variability, dosage, experimental conditions (temperature, pH, oxygen tension, etc.), treatment schedule and combination ratio.

[0023] A botanical composition drug include: bio-availability and minimal toxicity. Preferably the drug can be administered orally. The botanical composition provides a combination of multiple therapeutic functions to act simultaneously and synergistically on multiple biological targets. The botanical composition comprises low doses of individual therapeutic ingredients to minimize disruption of physiological homeostasis and development of drug resistance. Typically, a history of therapeutic efficacy and safety is considered in selecting nature-derived active ingredients.

Cancer:

[0024] Several cellular pathways have been implicated in cancer. Apoptotic pathway, mitogen- activated protein kinase (MAPK) Signaling Pathway, cell signaling via the phosphoinositide 3- kinase (PI3K) pathway, Signal transducers and activators of transcription (STAT) signaling pathway, p53 signaling pathway, Wnt signaling pathway, cyclooxygenase (COX) enzymes have all been known to contribute to several steps involved in tumor formation, such as neoplastic transformation, metastasis, and angiogenesis. Also, growth factors, oncogenes such as Ras mutations, tumor suppressor genes, androgen and estrogen receptors, co-activators & repressors, and numerous others pathways have been shown to affect tumor formation and carcinogenesis.

[0025] A botanical drug suitable for cancer would include functions to affect one or more of these pathways as well as functions generally related to alleviation of cancer conditions such as anti-inflammation, immune system modulation, anti-angiogenic, anti-metastatic, and the like.

[0026] A botanical drug suitable for a particular type of cancer will possess functionalities that also are directed to pathways unique to a type of cancer. For example, botanical drug suitable for the treatment of prostate cancer may include functionalities related to androgen receptors.

Preferably, the botanical drug is administered in combination with standard chemotherapy regimens to achi

[0027] Prostate cancer is a complex disease. A number of biological pathways have been implicated in prostate cancer development: growth factor activity, cell death (apoptosis), oncogenesis, tumor suppression, cell cycle modulation, cell surface modulation, androgen receptors, co-activators & repressors. Several conditions are associated with prostate disease: Benign prostate hyperplasia (BPH), prostatitis, prostatic intraepithelial neoplasia (PIN).

[0028] Benign prostatic hyperplasia (BPH) refers to the increase in size of the prostate in middle-aged and elderly men. BPH is characterized by hyperplasia of prostatic stromal and epithelial cells, resulting in the formation of large, fairly discrete nodules in the periurethral region of the prostate. Although prostate specific antigen levels may be elevated in these patients, because of increased organ volume and inflammation due to urinary tract infections, BPH is not considered to be a premalignant lesion.

[0029] Alpha blockers (a 1 -adrenergic receptor antagonists) provide symptomatic relief of BPH symptoms. When 5a-reductase inhibitors are used together with alpha blockers a reduction of BPH progression to acute urinary retention and surgery has been noted in patients with enlarged prostates. (Kaplan SA, McConnell JD, Roehrborn CG, et al (2006). Combination therapy with doxazosin and finasteride for benign prostatic hyperplasia in patients with lower urinary tract symptoms and a baseline total prostate volume of 25 ml or greater. J Urol 175(1): 217-20.)

[0030] Several botanicals show effectiveness in treating BPH and can be used as components of a botanical formulation. Serenoa repens (saw palmetto) fruit extracts are alleviating mild-to- moderate BPH symptoms with comparable efficacy to finasteride. (Wilt TJ, Ishani A, MacDonald R, (2002). Serenoa repens for benign prostatic hyperplasia. Cochrane Database Syst Rev 2002 (3), CD001423) Other botanicals effective in treating BPH include beta-sitosterol from Hypoxis rooperi (African star grass), pygeum (extracted from the bark of Prunus africana), Cucurbita pepo (pumpkin) seed and Urtica dioica (stinging nettle) root. (Wilt TJ, Ishani A, Rutks I, MacDonald R (2000) Phytotherapy for benign prostatic hyperplasia Public Health Nutr 3(4A):459-72). One double-blind trial has also supported the efficacy of rye flower pollen. (Buck AC, Cox R, Rees RWM, et al. (1990) Treatment of outflow tract obstruction due to benign prostatic hyperplasia with the pollen extract, Cernilton. A double-blind placebo-controlled study Br. J. Urol. 66:398-404).

[0031] Prostate cancer is classified as an adenocarcinoma, or glandular cancer, that begins when normal semen-secreting prostate gland cells mutate into cancer cells. Initially, small clumps of cancer cells remain confined to otherwise normal prostate glands, a condition known as carcinoma in situ or prostatic intraepithelial neoplasia (PIN). Although there is no clear evidence that ΡΓΝ is a cancer precursor, it is closely associated with cancer.

[0032] Prostate specific antigen (PSA) is a 34 kD glycoprotein manufactured almost exclusively by the prostate gland. Also known as kallikrein III, PSA is a serine protease. (Lilja H. (Nov 2003). "Biology of Prostate-Specific Antigen". Urology 62 ((5 Suppl 1)): 27-33).

[0033] PSA is often elevated in the presence of prostate cancer and in other non-malignant prostate disorders such as BPH. A blood test to measure PSA is the most effective test currently available for the early detection of prostate cancer. Higher than normal levels of PSA are associated with both localized and metastatic prostate cancer (CaP). However, PSA levels can change for many reasons other than cancer. Two common causes of high PSA levels in the absence of cancer are enlargement of the prostate (benign prostatic hypertrophy (BPH)) and infection in the prostate (prostatitis).

[0034] Thus, PSA is not a perfect test. Some men with prostate cancer do not have an elevated PSA, and most men with an elevated PSA do not have prostate cancer. Short of biopsy, no noninvasive tests provide a clear diagnosis of prostate cancer. Enzalutamide

[0035] Enzalutamide (brand name Xtandi) is a synthetic non-steroidal antiandrogen (NSAA) which was developed by the pharmaceutical company Medivation for the treatment of metastatic, castration-resistant prostate cancer. Enzalutamide is also known as MDV3100.

Medivation has reported up to an 89% decrease in serum prostate specific antigen (PSA) levels after a month of taking the drug. Research suggests that enzalutamide may also be effective in the treatment of certain types of breast cancer. In August 2012, the United States (U.S.) Food and Drug Administration (FDA) approved enzalutamide for the treatment of castration-resistant prostate cancer. Enzalutamide is represented by the chemical formula:

[0036] Enzalutamide is clinically active in metastatic castration-resistant prostate cancer. PSA level decreased more than 50% in 40 of 65 chemo-naive patients and 38 of 75 chemotherapy- treated patients. Median time to radiographic progression was 56 weeks for chemo-naive patients and 25 weeks for the post-chemotherapy population.

[0037] Enzalutamide is a diarylthiohydantoin derivative, and is structurally related to earlier NSAAs such as flutamide, nilutamide, and bicalutamide. However, unlike the first-generation NSAA bicalutamide, enzalutamide does not promote translocation of AR to the cell nucleus and in addition prevents binding of AR to deoxyribonucleic acid (DNA) and AR to coactivator proteins. (Tran C. et al., Science. 324 (5928): 787-90 (2009)). As such, it has been described as an AR signaling inhibitor in addition to antagonist. (Antonarakis ES (June 2013). Translational Andrology and Urology. 2 (2): 1 19-120).

[0038] When LNCaP cells (a prostate cancer cell line) engineered to express elevated levels of AR (as found in patients with advanced prostate cancer) were treated with enzalutamide, the expression of androgen-dependent genes PSA and TMPRSS2 was down regulated in contrast to bicalutamide where the expression was upregulated. In VCaP cells which over-express the AR, enzalutamide induced apoptosis whereas bicalutamide did not. Further, enzalutamide behaves as an antagonist of the W741C mutant AR in contrast to bicalutamide which behaves as a pure agonist when bound to the W741C mutant. Enzalutamide has only 2- to 3-fold lower affinity for the AR relative to the endogenous ligand dihydrotestosterone (DHT). Enzalutamide is reported to be a strong inducer of the enzyme CYP3A4 and a moderate inducer of CYP2C9 and

CYP2C19, and can affect the circulating concentrations of drugs that are metabolized by these enzymes. (McCutcheon SB (2013). J Adv Pract Oncol. 4 (3): 182-5.)

[0039] PREVAIL clinical trials funded by Medivation and Astellas Pharma Global

Development, showed Enzalutamide significantly decreased the risk of radiographic progression and death and delayed the initiation of chemotherapy in men with metastatic prostate cancer. (Beer TM et al, N Engl J Med 2014; 371:424-433).

[0040] AFFIRM Clinical Trials funded by Medivation and Astellas Pharma Global

Development, showed enzalutamide prolongs survival in men with castration-resistant prostate cancer after chemotherapy. (Scher HI et al., N Engl J Med 2012; 367: 1 187-1 197).

[0041] Enzalutamide has approximately 5- to 8-fold higher binding affinity for the androgen receptor (AR) compared to bicalutamide. As such, it has been described as an AR signaling inhibitor in addition to antagonist. Enzalutamide has only 2- to 3-fold lower affinity for the AR relative to the endogenous ligand dihydrotestosterone (DHT).

[0042] Also, unlike with the first-generation NSAAs (flutamide, nilutamide, and bicalutamide), there has been no evidence of hepatotoxicity or elevated liver enzymes in association with enzalutamide treatment in clinical trials.

Side effects of Enzalutamide Therapy

[0043] Notable side effects of enzalutamide seen in clinical trials have included gynecomastia, breast pain/tenderness, fatigue, diarrhea, hot flashes, headache, sexual dysfunction, and, less commonly, seizures. (Tombal B, et al. (November 2015). "Long-term Efficacy and Safety of Enzalutamide Monotherapy in Hormone-naive Prostate Cancer: 1- and 2- Year Open-label

Follow-up Results". European Urology. 68 (5): 787). Other "common" side effects reported in clinical trials have included neutropenia, visual hallucinations, anxiety, cognitive disorder, memory impairment, hypertension, dry skin, and pruritus (itching). Enzalutamide is regarded as having a moderate negative effect on sexual function and activity, significantly less than that of GnRH analogs but similar to that of other NSAAs such as bicalutamide. Enzalutamide is regarded as having a moderate negative effect on sexual function and activity, significantly less than that of GnRH analogues but similar to that of other NSAAs such as bicalutamide.

[0044] The most common adverse reactions (> 10%) that occurred more commonly (> 2% over placebo) in the XTANDI patients from the two placebo-controlled clinical trials were asthenia/fatigue, back pain, decreased appetite, constipation, arthralgia, diarrhea, hot flush, upper respiratory tract infection, peripheral edema, dyspnea, musculoskeletal pain, weight decreased, headache, hypertension, and dizziness/vertigo.

Dosing of Enzalutamide

[0045] According to the product insert of XTANDI® (Astellas Pharma Inc.) the enzalutamide is provided as 40 mg capsules and 160 mg (four 40 mg capsules) are administered daily, with or without food.

Botanical compositions for prostate cancer therapy

[0046] A botanical formulation presents an optimal first line therapy when high PSA levels are detected. Botanical compositions, such as those disclosed herein, have very low toxicity and yet are effective against prostate cancer.

[0047] The botanical composition was designed following demonstration of a number of desirable functions among the ingredients and in the assembled composition. A number of therapeutically active chemical entities are present in Ganoderma lucidum (#9), Scutellaria barbata (#15), and Salvia miltiorrhiza {#14): ganoderic acid H, crytotanshinone, tanshinone IIA, scutellarin tetramethyl ether, scutellarin, apigenin and wogonin.

[0048] A number of chemical entities are present in the botanical composition: adenosine, ganoderic acid A, oleic acid, tanshinone IIA, scutellarin, apigenin, luteolin, and wogonin. Each of these chemical entities is known to demonstrate one or moic of anti-viral, anti-inflammatory, immune modulatory, anti-angiogenic and anti-cancer/metastatic functions.

[0049] An exemplary combination of Ganoderma lucidum (#9), Scutellaria barbata (#15), and Salvia miltiorrhiza (#14) was designated OMN54, based on the synergism displayed by certain combinations of extracts of the three botanicals when each botanical is between 1% w/w and 90% w/w of the combined composition. Extracts of the botanicals were preferably made in organic medium, such as alcohol, and non-alcoholic media including ester, lipid and the like. In a preferred embodiment the extracts were made in ethyl acetate medium.

[0050] Significant synergism was expressed by botanical compositions comprised of the following three organic extracts at the specified amounts (w/w):

■ Ganoderma lucidum at 33-50% w/w. More specifically the Ganoderma lucidum extract is selected from 33%, 35%, 40%, 42%, 44%, 45%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5% and 50%.

^■ Scutellaria barbata at 33-50% w/w. More specifically the Scutellaria barbata extract is selected from 33%, 35%, 40%, 42%, 44%, 45%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5% and 50%.

^■ Salvia miltiorrhiza at 1-10% w/w. More specifically the Salvia miltiorrhiza extract is

selected from 1 %, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% and 10%.

[0051] In vitro studies have indicated that OMN54 did not modulate pro-inflammatory proteins in unstimulated PBMC (suggesting safety for long-term use). In the presence of inflammatory stimulus (PHA mitogen), OMN54 suppressed inflammatory signaling (significant antiinflammatory activity). The effect of OMN54 in stimulated and unstimulated cells was observed for a number of proteins comprising cytokines, chemokines and growth factors.

[0052] The human prostate cancer cell line, LNCaP, is androgen sensitive and PSA positive. OMN54 was tested for activity on LNCaP cell line, on the expression of specific genes associated with prostate cancer. High levels of Prostate Specific Antigen (PSA) are associated with prostate cancer. The significant suppression of the PSA transcript by OMN54 is considered beneficial in the treatment of prostate cancer.

[0053] OMN54 displays profound antiproliferative effect on prostate cancer cells, inducing the apoptosis of both androgen receptor (AR)-positive (LNCaP) and AR-negative (DU-145) prostate cancer cell lines. OMN54 also displays NF-kappa B inhibition. The anti-inflammatory and antiproliferative functions displayed by the botanical composition also are effective against BPH. Thus, following detection of high PSA levels and prior to invasive tests for diagnosis of cancer, a low toxicity botanical formulation which is effective against both BPH and neoplastic states like prostate cancer and PIN, provides a promising regimen for first intervention.

[0054] Other botanicals effective against prostate cancer include capsaicin, found in red peppers, which has a profound anti-proliferative effect on human prostate cancer cells in culture and in mouse xenografts. (Mori A et al. Cancer Res. 2006 Mar 15;66(6):3222-9). Capsaicin down- regulates PSA expression by direct inhibition of PSA transcription mediated by inhibition of NF- kappa B activation by preventing its nuclear migration. (Id.)

[0055] A 2006 study of green tea derivatives demonstrated prostate cancer prevention in patients at high risk for the disease. (Bettuzzi S, Brausi M, Rizzi F, Castagnetti G, Peracchia G, Corti A (2006). "Chemoprevention of human prostate cancer by oral administration of green tea catechins in volunteers with high-grade prostate intraepithelial neoplasia: a preliminary report from a one-year proof-of-principle study". Cancer Res 66 (2): 1234-40). A phytochemical called di-indolyl-methane found in cruciferous vegetables is suspected of having anti-androgenic and immune modulating properties.

[0056] Botanicals are a valuable resource for the discovery and development of novel, naturally derived agents to treat human disease. Botanical extracts usually comprise multiple molecules and possess multiple functions useful in the treatment and prevention of disease. Botanical extracts also can function to maintain normal tissue homeostasis by affecting multiple biological pathways such as the inflammatory pathway, the immune response pathway and the oxidative stress response pathway. As a result, botanical extracts can alleviate the harmful side effects of many therapeutic agents used to treat multiple disease targets.

[0057] Botanicals have been demonstrated to be a successful source of anticancer compositions. Examples include Gynostemma pentaphyllum extract, Camellia sinensis (green tea) and

Crataegus pinnatifida (hawthorn berries) and a method of making the same are the subject of U.S. Pat. Nos. 5,910,308 and 6, 168,795. Some drugs, derived from plants that are currently used in cancer therapy were designed to perturb microtubule shortening (depolymerization) or lengthening (polymerization), such as paclitaxel, docetaxel, etoposide, vincristine, vinblastine, and vinorelbine (Compton, D. A., et al., (1999) Science 286:913-914). They share a common mechanism of action of binding to tubulin, the molecule of which microtubules are composed. (Compton, D. A., et al, (1999) Science 286:913-914). At least six plant-derived anticancer agents have received FDA approval (e.g., taxol, vinblastine, vincristine, topotecan, etoposide, teniposide). Other agents are being evaluated in clinical trials (e.g., camptothecin, 9AC, and irinotecan). Botanical extracts for the treatment cancer are described in U. S. Pat. Application Publications 20050214394 A 1 , 20050208070 A 1 and 20050196409 A 1.

[0058] The present invention provides novel compositions comprising botanical extracts to treat human diseases that are associated with multiple biological pathways in their pathologies. The compositions of the invention are comprised of two or more botanical extracts which work synergistically to modulate multiple biological pathways including but not limited to

inflammatory responses, immune responses, oxidative responses, viral and microbial infections, and cell proliferative responses.

[0059] (i) Ganoderma lucidum (Reishi): Ganoderma lucidum was praised for its effect of increasing memory and preventing forgetfulness in old age reported in Shen Nong Ben Cao Jing vol. 1 as early as 456-536 AD. Research on mice using orally or topically administered

Ganoderma lucidum suggests that Ganoderma lucidum has anti-inflammatory activity.

(Stavinoha, W., et al., (1995). Study of the anti-inflammatory efficacy of Ganoderma lucidum. In B.-K. Kim, & Y.S. Kim (Eds.), Recent Advances in Ganoderma lucidum research (pp. 3-7). Seoul Korea: The Pharmaceutical Society of Korea).

[0060] Applications of Ganoderma for (1) chemoprophylaxis of cancer in individuals at high risk for developing cancer (2) adjuvant use in the prevention of metastasis or recurrence of cancer (3) palliation of cancer related cachexia and pain and (4) adjunctive use with concurrent chemotherapy to reduce side-effects, maintain leukocyte counts and allow a more optimal dosing of chemo or radio therapeutics has been suggested (Chang, R. (1994) Effective Dose of

Ganoderma in Humans; Proceedings of Contributed Symposium 59A, B 5th International Mycological Congress, Vancouver: pp. 117-121). Since studies of human dosage were traditional and empirical, a proper dose range of Ganoderma for therapy was calculated using this data and pharmacokinetic principals. The calculations suggested that a (1) Ganoderma dried fruit body dose of 0.5 to 1 g per day for health maintenance (2) 2 to 5 g per day if there is chronic fatigue, stress, auto immune, or other chronic health problems (3) 5 to 10 g per day for serious illness. (Chang, R. (1993) Limitations and Potential applications of Ganoderma and related fungal polyglycans in clinical ontology; First International Conference on Mushroom Biology and Mushroom products: 96).

[0061] While Ganoderma lucidum is preferred, one skilled in the art would recognize that other species of Ganoderma may also be used in the present invention. For example, G. tsugae has been shown to modulate Thl/Th2 and macrophage responses in allergic murine model, and recombinantly expressed fungal immunomodulatory protein, FlP-gts, from G. tsugae inhibited telomerase activity in A549 human lung adenocarcinoma cell line (Lin, J.Y. et al., (2006) Food Chem. Toxicol.; Liao, C.H. et al., (2006) Mo. Carcinog. 45(4):220-9). Examples of other species of Ganoderma include, but are not limited to, G. applanatum, G. mongolicum, G. microsporum, G. subamboinense, G. pfeifferi, G. meredithae, G. oregonense (G. oregonse), G. resinaceum, G. oerstedii, G. ungulatum, G. mirabile, G. tsugae, G. sessile, G. valesiacum, G.fornicatum, G. carnosum, G. australe, and G. boninense.

[0062] (ii) Scutellaria barbata (Skullcap): Scutellaria barbata, a traditional Chinese medicine for liver, lung and rectal tumors, has been shown to inhibit mutagenesis, DNA binding and metabolism of aflatoxin B 1 (AFB 1) and cytochrome P450-linked aminopyrine N-demethylase (Wong B.Y. et al, (1993) Eur. J. Cancer Prev. 2(4):351-6; Wong B.Y. et al., (1992) Mutat. Res. 279(3):209- 16). Scutellaria barbata is also capable of enhancing macrophage function in vitro and inhibiting tumor growth in vivo (Wong B.Y. et al., (1996) Cancer Biother. Radiopharm. l l(l):51-6).

[0063] This herb contains vitamins C and E as well as calcium, potassium, magnesium, iron, zinc scutellarin, volatile oil, tannin and bitter principles. The scutellarin acts on the central nervous system. Scutellarin, an active ingredient from Scutellaria barbata has been purified by liquid chromatography (Wenzhu Zhang et al., (2003) J. of Liquid Chromatography & Related Technologies 26 (13):2133-40).

[0064] (iii) Scutellaria baicalensis: Scutellaria baicalensis has been shown to have antiproliferative and apoptotic activities against lymphocytic leukemia, lymphoma, and myeloma cell lines and possess anti-cancer activity on human malignant brain tumor cells (Kumagai, T. et al. (2006) Leuk. Res.; Scheck, A.C. et al., (2006) BMC Complement Altern. Med. 6:27). [0065] Scutellaria barbata should not be confused with Scutellaria baicalensis. Banzhilian, the whole plant of Scutellaria barbata, should not be confused with "scute," the common name referring to huangqin, the root of Scutellaria baicalensis. "Although both are of the same genus, Scutellaria barbata, for which the tops are used, has essential oils among the active components, while Scutellaria baicalensis relies primarily on flavonoids, particularly baicalin and baicalein. Scutellaria radix (root of Scutellaria baicalensis) and Scutellaria barbata comprise different sets of flavonoids and show different effects on proliferation of human leukemia cell line HL-60. Sonoda et al., J. Ethnopharm 91 :65-68 (2004)

[0066] While Scutellaria barbata and Scutellaria baicalensis are preferred, one skilled in the art would recognize that other species of Scutellaria may also be used in the present invention. For example, Scutellaria radix has been shown to suppress ethanol-induced caspase-11 expression and cell death in N(2)a cells, and Baicalein, a component of Scutellaria radix, leads to suppression of proliferation and induction of apoptosis in human myeloma cells (Kang, K. et al., (2005) Brain Res. Mol. Brain Res. 142(2): 139-45; Ma, Z. et al. (2005) Blood 105(8):3312-8). Examples of other species of Scutellaria include, but are not limited to, Scutellaria amabilis,

Scutellaria radix, Scutellaria rehderiana, and Scutellaria lateriflora. Preferred combinations are those where the extract from a particular species acts in synergy with extracts from other botanicals in the formulation or with other therapeutic agents in the composition.

[0067] (iv) Salvia miltiorrhiza (Dan Shen): There are over 900 species of salvia and many of them have histories of medicinal uses. Dan shen is used in traditional Chinese medicine to promote blood circulation and to remove blood stasis (Bensky D., Gamble A Chinese herbal Medicine Materia Medica 1987 Eastland Press: Seattle. 384). It increases the activity of SOD in platelets, thus providing protection against pulmonary embolism and inhibition of platelet aggregation. (Wang, X. et al., (1996) Zhongguo Zhong Yao Za Zhi 21:558-60). Salvia miltiorrhiza has been shown to lower cholesterol, reduce endothelial damage and to inhibit lipid peroxidation in hypercholesterolemic animals. This inhibition of oxidation of LDL may reduce atherosclerosis (Wu Y.J. et al., (1998) Arteriosclerosis Thromb Vase Biol 18:481-6). A Salvia miltiorrhiza constituent has been found to inhibit noradrenalin-induced contraction of the aortic strips through reduction in Ca²⁺ mobilization. This vasodilatory activity may explain the traditional use of Salvia miltiorrhiza in hypertension (Nagai M. et al., Biol Pharm Bull (1996) 19:228-32). Salvia miltiorrhiza has been shown to have a markedly superior effect to nitroglycerin, with a more persistent action and better improvement of cardiac function (Bai, Y.R. and Wang, S.Z., (1994) Zhongguo Zhong Xi Yi Jie He Za Zhi 14:24-5, 4).

[0068] Salvia miltiorrhiza is also the top ingredient in Dan Shen Compound. Dan Shen

Compound comprises four important herbs for the improvement of peripheral circulation and general wellbeing. The actions of Crataegus levigata are enhanced by the Chinese herb Salvia miltiorrhiza (Dan Shen), the Indian herb Coleus forskohlii and Valeriana officinalis. Chinese herbal medicine utilizes Salvia miltiorrhiza for women's irregularities, abdominal pain, insomnia, hives, hepatitis and mastitis.

[0069] (v) Hippophae rhamnoides (sea buckthorn): Sea buckthorn seed oil contains a high content of the two essential fatty acids, linoleic acid and a-linolenic acid, which are precursors of other polyunsaturated fatty acids such as arachidonic and eicosapentaenoic acids. The oil from the pulp/peel of seabuckthorn berries is rich in palmitoleic acid and oleic acid (Chen et al., "Chemical composition and characteristics of seabuckthorn fruit and its oil." Chem. Ind. Forest Prod. (Chinese) 10 (3), 163-175). The increase in the level of α-linolenic acid in plasma lipids showed a clear improving effect on AD symptoms (Yang et al., (2000) J. Nutr Biochem.

11(6) :338-340). These effects of α-linolenic acid may have been due to both changes in the eicosanoid composition and other mechanisms independent of eicosanoid synthesis (Kelley (1992) Nutrition, 8 (3), 215-2).

[0070] Antioxidant and immunomodulatory properties of sea buckthorn {Hippophae

rhamnoides) have been demonstrated using lymphocytes as a model system. (Geetha et al. J Ethnopharmacol 2002 Mar; 79(3):373-8). The antiulcerogenic effect of a hexane extract from Hippophae rhamnoides has also been demonstrated. (Suleyman H. et al., (2001) Phytother Res 15(7):625-7). Radioprotection by an herbal preparation of Hippophae rhamnoides against whole body lethal irradiation in mice suggests free radical scavenging, acceleration of stem cell proliferation and immunostimulation properties. (Goel H.C. et al., (2002) Phytomedicine

9(l): 15-25)

[0071] (vi) Camellia sinensis (Green tea): Dried leaves from the Camellia sinensis plant is processed into three types of tea: oolong tea, black tea, and green tea. Green tea extract is a bioflavonoid-rich, potent extract which is used primarily for fighting free radicals. It has a high content of polyphenols, which are a type of bioflavonoids. In making green tea, the tea leaves are stabilized by moist or dry heat which destroys the enzyme polyphenoloxidase and thus, prevents oxidation of polyphenols. These polyphenols are the main biologically active ingredients in green tea. In preferred embodiments, the green tea is Dragon Well tea or Lung Ching tea.

[0072] The polyphenols in green tea are catechins, with multiple linked ring-like structures. Polyphenols are a form of bioflavonoids with several phenol groups. They control both taste and biological action. Catechins, a chemical group of polyphenols possessing antioxidant properties (protecting cells from free radical-mediated damage), include epigallocatechin-3 gallate

(EGCG), epigallocatechin, and epicatechin-3-gallate. Recently, ECGC has been shown to be an inhibitor of urokinase (Jankun et al., (1997) Nature 387:561), and quinol-oxidase; enzymes that may be crucial for growth of tumor cells. Epigallocatechin-3 gallate (EGCG) also protects against digestive and respiratory infections.

[0073] Novel tumor inhibiting, immune boosting, inflammation reducing and anti-oxidative properties observed for compositions comprising a combination of two or more extracts of

Ganoderma lucidum, Scutellaria barbata, Scutellaria baicalensis, and Salvia miltiorrhiza and, optionally, Hippophae rhamnoides (seabuckthorn) and Camellia sinensis (green tea) and the synergistic effects demonstrated by novel combinations of two or more of these extracts used in the method according to the present invention are a likely result of combinations of one or more of saponins, flavonoids, and polyphenols present in the extracts.

Formulations of Botanical Compositions

[0074] The compositions of the present invention can be in any form which is effective, including, but not limited to dry powders, grounds, emulsions, extracts, and other conventional compositions. To extract or concentrate the effective ingredients of the compositions, typically the botanical part is contacted with a suitable solvent, such as water, alcohol, methanol, mixed solvents, or any other solvents. The choice of the solvent can be made routinely, e.g., based on the properties of the active ingredient that is to be extracted or concentrated by the solvent. Preferred active ingredients of the compositions crenulata include, but are not limited to, salidroside, tyrosol, β-sitosterol, gallic acid, pyrogallol, crenulatin, rhodionin, and/or rhodiosin. These ingredients can be extracted in the same step, e.g., using an alcoholic solvent, or they may be extracted individually, each time using a solvent which is especially effective for extracting the particular target ingredient from the plant. In certain embodiments, extraction can be performed by the following process: Milling the selected part, preferably root, to powder. The powder can be soaked in a desired solvent for an amount of time effective to extract the active agents from the compositions. The solution can be filtered and concentrated to produce a paste that contains a high concentration of the constituents extracted by the solvent. In some cases, the paste can be dried to produce a powder extract of the compositions crenulata. The content of active ingredient in the extract can be measured using HPLC, UV and other spectrometry methods.

[0075] The compositions of the present invention can be administered in any form by any effective route, including, e.g., oral, parenteral, enteral, intraperitoneal, topical, transdermal (e.g., using any standard patch), ophthalmic, nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intra-arterial, and intrathecal, etc. It can be administered alone, or in combination with any ingredient(s), active or inactive, including in a medicinal form, or as a food or beverage additive.

[0076] In preferred embodiments of the invention, the compositions are administered orally in any suitable form, including, e.g., whole plant, powdered or pulverized plant material, extract, pill, capsule, granule, tablet or a suspension.

[0077] The compositions can be combined with any pharmaceutically acceptable carrier. By the phrase, "pharmaceutically acceptable carriers," it is meant any pharmaceutical carrier, such as the standard carriers described, e.g., Remington's Pharmaceutical Science, 18th Edition, Mack Publishing company, 1990. Examples of suitable carriers are well known in the art and can include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion and various types of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets pharmaceutical and capsules. Typically such carriers contain excipients such as such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols. Such carriers can also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods. Generally excipients formulated with the compositions are suitable for oral administration and do not deleteriously react with it, or other active components.

[0078] Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy

methylcellulose and the like. Other additives include, e.g., antioxidants and preservatives, coloring, flavoring and diluting agents, emulsifying and suspending agents, such as acacia, agar, alginic acid, sodium alginate, bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose, cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, octoxynol 9, oleyl alcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate, sorbitan esters, stearyl alcohol, tragacanth, xanthan gum, and derivatives thereof, solvents, and miscellaneous ingredients such as microcrystalline cellulose, citric acid, dextrin, dextrose, liquid glucose, lactic acid, lactose, magnesium chloride, potassium metaphosphate, starch, and the like.

[0079] The botanical compositions can also be formulated with other active ingredients, such as anti-oxidants, vitamins (A, C, ascorbic acid, B's, such as B l, thiamine, B6, pyridoxine, B complex, biotin, choline, nicotinic acid, pantothenic acid, B 12, cyanocobalamin, and/or B2, D, D2, D3, calciferol, E, such as tocopherol, riboflavin, K, Kl, K2). Preferred compounds, include, e.g. creatine monohydrate, pyruvate, L-Carnitine, a-lipoic acid, Phytin or Phytic acid, Co

Enzyme Q10, NADH, NAD, D-ribose, amino acids such as L-glutamine, Lysine, chrysin; pre- hormones such as 4-androstenedione, 5-androstenedione, 4(or 5-)androstenediol, 19-nor-4 (or 5- )-androstenedione, 19-nor-4 (or 5-)-androstenediol, Beta-ecdysterone, and 5-Methyl-7-Methoxy Isoflavone. Preferred active ingredients include, e.g., pine pollen, fructus lycii, Hippophae rhamnoides, Ligusticum, Acanthopanax, Astragalus, Ephedra, codonopsis, polygola tenuifolia Willd, Lilium, Sparganium, ginseng, panax notogiseng, Garcinia, Guggle, Grape Seed Extract or powder, and/or Ginkgo Biloba.

[0080] Other plants and herbs which can be formulated with the compositions of the present invention includes those mentioned in various text and publications, e.g., E.S. Ayensu, Medicinal Plants of West Africa, Reference Publications, Algonac, Mich. (1978); L. Boulos, Medicinal Plants of North Africa, Reference Publications Inc., Algonac, Mich. (1983); and N. C. Shah, (1982) J. Ethnopharm, 6:294-5.

[0081] A botanical formulation may comprise biologies and chemical entities, in addition to or in the place of, botanical extracts. Examples of biologies that may comprise a botanical composition include but are not limited to blood and blood products, cells, tissues and organs, gene therapy vectors, viral and bacterial vaccines, therapeutic products produced through biotechnology such as antibodies, monoclonal antibodies, and the like.

[0082] Pharmaceutically active agents that can comprise a botanical composition include, but are not limited to antioxidants, anticarcinogens, anti-inflammatory agents, hormones and hormone antagonists, anti-hypertensive agents, anti-inflammatory agents, tranquilizers, cardiotonic agents, antidepressants, corticosteroids, anti-ulcer agents, anti-allergy agents and anti-obesity agents, antibiotics, antibacterial agents, bacterial agents, and other medically useful drugs such as those identified in, e.g., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing

Company, 1990. A preferred composition of the present invention comprises, about 1%-100%, preferably about 20-70% of the botanical extract and, optionally, a pharmaceutically-acceptable excipient. Another preferred composition of the present invention comprises, about l%-99%, preferably about 20-70% of botanical extracts, 0.1-99%, preferably 1-10% of one or more pharmaceutically active agents and, optionally, a pharmaceutically-acceptable excipient.

[0083] In some embodiments, the botanical composition comprises a chemotherapeutic agent either in a single formulation or separately administered as part of a therapeutic regimen.

[0084] According to the instant invention, it has been surprisingly observed that concurrent treatment with Enzalutamide and OMN54 results in more effective remediation of prostate cancer symptoms with significantly reduced side effects and synergistic anticancer activity.

[0085] The instant invention relates to the administration of enzalutamide and the botanical compositions (such as OMN54) concurrently. By concurrently, it is understood that the botanical composition is administered simultaneously, or the same day, or within 24 hours of

enzalutamide.

[0086] In some embodiments, the botanical composition and enzalutamide is formulated as a tablet dosage form comprising: a) a first part comprising a capsule of enzalutamide, wherein the capsule is formulated with one or more other pharmaceutically acceptable excipients; and b) a second part comprising a composition comprising two or more extracts of Ganoderma lucidum, Scutellaria barbata, Scutellaria baicalensis, Salvia miltiorrhiza, and optionally other pharmaceutically acceptable excipients. In some embodiments, the dosage form comprises a bilayered dosage form. In some aspects the dosage form is coated with one or more enteric polymers, pharmaceutically acceptable seal coat polymers or rate controlling polymers. In some aspects, the two active ingredients are provided as a dispersion provided in a single capsule, granule, mini-tablet or tablet form.

[0087] In some aspects, enzalutamide and/or the botanical extracts composition are present in an immediate release, delayed release, sustained release, extended release, controlled release or modified release form.

[0088] In some aspects, the botanical extracts are formulated with excipients comprising one or more of an emulsifying agent, an antioxidant and a diluent. In some embodiments, the emulsifying agent is one or more of Cremophor EL, oleic acid and labrasol; the antioxidant is selected from ascorbic acid and alpha tocopherol; and the diluent is soya oil.

[0089] In other embodiments, the invention relates to a kit containing separate dosage forms for each active ingredient, for example, comprising enzalutamide provided in a capsule form and the botanical extracts provided in a second capsule form. In some embodiments, the capsule and the tablet are provided in a single package (such as a blister pack).

[0090] The present invention relates to methods of administering the compositions, e.g., to provide anti-inflammatory effects, to reduce inflammation, to provide antioxidant effects, to protect against oxidation, to provide antiproliferative effects, to provide anti-cancer effects, to promote DNA repair, to provide anti-radiation effects, to protect against radiation, and other conditions and diseases as mentioned herein.

[0091] An effective amount of the compositions are administered to such a host. Effective amounts are such amounts which are useful to achieve the desired effect, preferably a beneficial or therapeutic effect as described above. Such amount can be determined routinely, e.g., by performing a dose-response experiment in which varying doses are administered to cells, tissues, animal models (such as rats or mice in maze-testing, swimming tests, toxicity tests, memory tests as performed by standard psychological testing, etc.) to determine an effective amount in achieving an effect. Amounts are selected based on various factors, including the milieu to which the virus is administered (e.g., a patient with cancer, animal model, tissue culture cells, etc.), the site of the cells to be treated, the age, health, gender, and weight of a patient or animal to be treated, etc. Useful amounts include, 10 milligrams- 100 grams, preferably, e.g., 100 milligrams- 10 grams, 250 milligrams-2.5 grams, 1 gm, 2 gm, 3 gm, 500 milligrams- 1.25 grams, etc., per dosage of different forms of the compositions such as the botanical powder, botanical extract paste or powder, tea and beverages prepared to contain the effective ingredients of the compositions, and injections, depending upon the need of the recipients and the method of preparation.

[0092] The liquid, pharmaceutically active formulation comprises a pharmaceutically active botanical composition in a liquid diluent or carrier. The active ingredient may be dissolved or dispersed in the liquid diluent or carrier, which may be a water miscible or water immiscible medium. Examples of liquid diluents or carriers include the following three classes: (a) Water miscible carriers: Propylene Glycol, Polyethylene Glycol, Water, Solketal, Glycofurol,

Dimethylisosorbide, Nonionic surface active agents; (b) Oils and Organic carriers: Fractionated Coconut Oil, Sesame Oil, Soya Bean Oil, Vegetable Oil, Liquid Paraffin, Isopropylmyristate, Triacetin; and (c) Semi-solid carriers: High molecular weight polyethylene glycols, and White soft paraffin.

[0093] In some embodiments, one or more emulsifiers or surfactants are included in the formulation. Suitable emulsifiers which can be used include one or more of fatty acids such as oleic acid, polyoxyethylene glycerol esters of fatty acids, such as Tagats; polooxylated castor oil, ethylene glycol esters, such as glycol stearate and distearate; propylene glycol esters, such as propylene glycol myristate; glyceryl esters of fatty acids, such as glyceryl stearates and monostcarates; sorbitan esters, such as spans and tweens; polyglyceryl esters, such as polyglyceryl 4-oleate; fatty alcohol ethoxylates, such as Brij type emulsifiers; ethoxylated propoxylated block copolymers, such as poloxamers; polyethylene glycol esters of fatty acids, such as Labrafils, Labrafacs, and Labrasols; cremophores; glycerol monocaprylate/caprate, such as Campmul CM 10; Gelucire, Capryol, Captex, Acconon, transcutol, triacetin, and the like. In some embodiments, antioxidants and/or diluents are used in the formulation. [0094] Compositions of the present invention comprise effective amounts of a combination of two or more extracts of Ganoderma lucidum, Scutellaria barbata, Scutellaria baicalensis, Salvia miltiorrhiza, and optionally, Hippophae rhamnoides (sea buckthorn) that exhibit synergy.

[0095] In one aspect of the invention, the botanical composition comprises effective amounts of extracts of Ganoderma lucidum, Scutellaria barbata, and Salvia miltiorrhiza. The dosage of the composition can be readily determined by one of skill in the art based on the effective concentrations of compositions shown to display the various properties described herein.

[0096] Compositions comprising different ratios of the individual extracts can similarly be determined. For example, a composition may exhibit anti-inflammatory effects at one concentration or ratios of combinations of extracts and varying degrees of cytotoxic effects at other concentrations or ratios of combinations of extracts. Any ratio of extracts of two or more of Ganoderma lucidum, Scutellaria barbata, Scutellaria baicalensis, and Salvia miltiorrhiza can be used in the compositions of the invention. It is preferred that each extract is present in the composition in equal amounts or at about 1 % to about 90% of the total composition. In some embodiments of the invention, a particular extract comprises at least 1 %, 1.5%, 2%, 5%, 10%, 15%, 25%, 33%, 40%, 45%, 47.5%, 48.5%, 49.5%, 50%, 60%, 66%, 75%, 90% or 98% by weight of the composition. In one embodiment the OMN54 comprises about 1-3% Salvia miltiorrhiza, and approximately equal amounts (45-50%) of Scutellaria barbata and Ganoderma lucidum.

[0097] In a further embodiment, the compositions of the present invention comprise botanical compounds that are useful in compositions to be administered in conjunction with therapeutic agents for the treatment of disease. These compositions exhibit synergistic action with the therapeutic agent based on their anti-inflammatory, antioxidant, immune modulating, antiviral, antibacterial, antiproliferative activity or any combination of activities thereof.

[0098] The compositions demonstrate antioxidant activity which prevents damage to chromosomes/genes, reduces effect of mutagens, alleviates side-effects of chemotherapeutic agents, alleviates side-effects of hormone therapeutic agents, and enhances cell repair mechanisms. [0099] The compositions further demonstrate immune system boosting activity which facilitates elimination of (i) damaged cells or (ii) cells with damaged genes. Further, the compositions provide general benefits of improving immune condition (passive immunotherapy).

[0100] The botanical sources of the extracts are botanicals that are essentially nontoxic with a long history of usage of the individual compounds/extracts. Anti-mutagenic properties as evidenced by Ames test results (together with increased sensitivity by synergism) reduce levels of chemotherapeutic agents necessary for treatment resulting in reduced toxicity for patients.

[0101] The botanical compositions demonstrate the ability to enhanced cell cycling which could make the botanical composition of the invention a powerful adjuvant to chemotherapy (e.g., with enzalutamide), hormonal therapy, or radiation therapy by increasing effectiveness and reducing necessary dosages of chemotherapeutic agents and hormone therapeutic agents.

[0102] Quality control. IC₅₀ based compositions can be standardized based on specific activities of defined properties.

[0103] The compositions are also suited for convenient (oral) drug delivery. Compositions are extracts made with hot water, alcoholic solvents (ethanol) and non-alocoholic organic solvents (ester, lipid, ethyl acetate, etc.).

[0104] Overall the botanical compositions show mostly cytostatic effect with very weak cytotoxic effects in the compositions of the invention. Histopathology of cells treated with the compositions of the invention indicates minimal retention of dead cancer cells which enhance recovery following cancer therapy.

EXAMPLES

[0105] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever.

[0106] The following combinations of extracts were used throughout the examples: Ganoderma lucidum, Scutellaria barbata, optionally Scutellaria baicalensis and Salvia miltiorrhiza extracts when combined in ratios that exhibit synergism are referred to as OMN54 in the specification. [0107] In addition, the compositions of the invention may include, optionally, Panax Quinquefolium (Western ginseng), Camellia sinensis (green tea), and Hippophae rhamnoides (sea buckthorn).

[0108] One skilled in the art would appreciate that while the following examples are illustrative of the invention, any cell line may be used. For example, although not limiting, cells may be obtained from ATCC, Rockville, Md.

[0109] One skilled in the art would also appreciate that while the foregoing examples are illustrative of the invention, multiple prostate cancer in vivo models may be used. For example, CaP xenografts in mice may be utilized. Additionally, a Pten knockout mouse strain, in which heterozygous mice develop tumors of the uterus, prostate, thyroid, colon, and adrenal medulla, may be obtained from the Mouse Models of Human Cancers Consortium (Podsypanina K. et al., (1999) Proc. Nat'l Acad. Sci. USA 96: 1563-1568).

Example 1: Methods for Preparation of Botanical Extracts

[0110] The compositions of the present invention may be administered as dried herbs. Botanical preparations contain phytochemicals some of which are soluble in aqueous media while others are relatively more soluble in organic (alcohol, lipid) media. Different extraction methods were used and tested for the ability to extract effective ingredients from the herbs. Extraction methods include: Aqueous (hot water) extraction; Organic (lipid fraction) extraction; non-alcoholic organic (ethyl acetate) extraction; and alcohol (ethanol) Extraction.

[0111] Products are prepared from herbs or herb blends by extraction with solvent (hot water, 80% ethanol, or ethyl acetate) under reflux for 30-60 minutes, separated by filtration to obtain a filtrate, and air dried for further analysis. The filtrates were combined, diluted or concentrated prior to determination of activities.

Example 2: Effect of treatment with a combination of OMNS4 with MDV3100^®

(enzalutamide) on Prostate Cancer Cells

[0112] Figure 1 shows the synergistic effect on inhibition of cell proliferation by a combination of Aneustat™(OMN54) and MDV3100^® (enzalutamide) on castration-resistant prostate cell growth. C4-2 cells were cultured in FBS conditions and treated with either OMN54, MDV3100 alone or in combination for 48 h. The cell number was counted. It shows synergistic effect using OMN54 and 40 μΜ MDV3100.

[0113] From the data, OMN54 is more effective than MDV in term of cell killing in vitro. This may be due to the following: MDV3100 specifically targets AR, while OMN54 targets multiple pathways including AR, IL-6, Stat3, Akt, etc. The unit for OMN54 is microgram. OMN50 is 50 μg and OMN100 is 100 μg. The data showed that there is a synergistic effect when OMN54 is combined with MDV at 40 microM. No additive effect was observed when MDV is at 20 μΜ or below. Therefore, the data of MDV10. 20 microM shown here is necessary to document that there is no additive effect at these doses when combined with OMN54. The ones with red circle are the data showing synergistic effect.

Example 3: Establishment of a human prostate cancer tissue xenograft/mouse model

[0114] A more predictive human tumor tissue xenograft model was used to assess efficacy of the botanical compositions. (Clin Cancer Research 2006: 12(13); 4043-4054) Human tumor grafts are better than cell line injections and human tumors grafted on kidney capsule have more immediate blood supply than subcutaneous grafts, thus more biological diversity is retained. The xenograft model more closely resembles clinical cancer than traditional in vivo models

[0115] Preclinical testing of prostate cancer therapeutics has been largely carried out using xenograft models in which human prostate cancer cell lines have been subcutaneously injected into immunodeficient mice. However, cancer cell xenografts may not accurately mimic the behavior of prostate tumors in vivo. In fact, cancer cell line xenograft models have a poor record of accurately predicting the clinical efficacy of anticancer agents. A novel xenograft model was established for a variety of pre-cancerous and cancerous human tissues, including prostate cancer tissue. Most importantly, the xenografts in the model retain the histological characteristics of the parental tissue. For selected types of cancer that the xenografts respond to therapy in a manner similar to that observed in patients. For example, prostate cancer tissue grown in SCED mice showed a dramatic response to androgen ablation therapy as regularly found in the clinic.

[0116] Xenografts of human prostate cancer cell line DU145 were grown in vivo. One of two tissue xenografts grew to the size of a walnut. Tumors are grafted to the renal site survive and retain their original histopathology and differentiation marker profile, even after serial passages. The prostate cancer tissue very rapidly grows in SCID mice with a doubling time about 5 days. Cytogenetic analyses show some abnormal chromosomes. Not only are there translocations, there are also deletions and duplication of chromosomal segments (Note: since each chromosome has its own display color, more than one color along the length of a chromosome indicates a translocation). The Spectral Karyotyping (SKY) analysis shows that the tissue of a DU145 cancer xenograft contained only a low number of karyotypic alterations, although the cancer is highly advanced.

Example 4: Efficacy study on prostate cancer cell line (DU145)

[0117] Tumor xenografts from a prostate cell line DU145 were cut into 2 mm³ pieces and grafted into SCID/nod mice. Treatment was started at day 13 (mean volume = 15.6 mm³). The mice were divided into 3 equal groups for treatment with saline; Ganoderma lucidum, Salvia miltiorrhiza, and Scutellaria barbata at 3.3 IC50; and estramustine sodium phosphate (EMCYT^®) and anti-cancer drug (E+D). The combination of Ganoderma lucidum, Salvia miltiorrhiza, and Scutellaria barbata showed a significant inhibitory effect comparable to the E+D regimen.

OMN54 significantly inhibited growth of DU145 (andro gen-independent) human prostate cancer tumors in vivo. Importantly, this effect on tumor volume was comparable to that of standard chemotherapy which is associated with significant toxicities to patients.

Example 5: In vivo anti-prostate tumor activity assay

[0118] 6-week old male nude mice (BALB/c-nu/nu) are used for the experimental animal model. Animals are cut open from the abdomen and inoculated with the human prostate cancer cell lines-LNCaP cells (2x 10⁶ cells/50 l/Hanks Buffered Saline Solution/mice) from back side of the prostate with a 30g needle. The cut abdomens are then sutured with 5-0 thread. After 2 weeks with regular feeding, blood is drawn from each animal for measuring the serum PSA value. The compositions of the invention are administered orally to the mice as follows; 3 groups of 6 mice received 43.65, 14.4, or 4.3 mg/animal/day for 21 days. Age-matched control mice arc treated with saline for the same period.

[0119] Grafts are then harvested to determine the effect on tumor volume, histology, apoptosis index (Tunel assay) and proliferation index (proliferation marker Ki67 staining). The TUNEL assay detects apoptosis-induced DNA fragmentation through a quantitative fluorescence assay. Terminal deoxynucleotidyl transferase (TdT) catalyzes the incorporation of bromo-deoxyuridine (BrdU) residues into the fragmenting nuclear DNA at the 3'-hydroxyl ends by nicked end labeling. A TRITC-conjugated anti-BrdU antibody can then label the 3'-hydroxyl ends for detection.

[0120] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0121] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. A method for treatment or reducing the severity of cancer in a subject, the method comprising:

administering an effective amount of a botanical composition comprising two or more extracts in organic medium of Ganoderma lucidum, Salvia miltiorrhiza, and Scutellaria barbata, wherein each extract comprises from about 1 to about 90 percent w/w and wherein the botanical composition is effective for reducing androgen receptor protein expression; and

co-administering an effective amount of enzalutamide,

wherein the co-administration of the compound and the botanical composition achieves more effective therapy than administration of either agent alone.

2. The method of claim 1, wherein the enzalutamide is administered at 160 mg per day.

3. The method of claim 2, wherein the enzalutamide is administered at 40, or 80 or 120 mg per day.

4. The method of claim 1, wherein the co-adminstration is administration of the botanical composition and the administration of the enzalutamide is between 0 and 24 hours apart.

5. The method of claim 1, wherein each botanical extract is a non-alcoholic organic extract.

6. The method of claim 5, wherein the extract is made with ethyl acetate ester.

7. The method of claim 1, wherein co-administration of the enzalutamide and the botanical composition results in reduced toxicity and side-effects compared to administration of chemical compound alone.

8. The method of claim 1, wherein the enzalutamide and the botanical composition are administered in a single dosage form.

9. The method of claim 1, wherein the enzalutamide and the botanical composition are administered in a plurality of dosage forms.

10. The method of claim 1, wherein the enzalutamide and the botanical composition are administered orally.

11. The method of claim 1 , wherein the cancer is a castration-resistant prostate cancer.

12. The method of claim 1, wherein the cancer is an androgen-sensitive prostate cancer.

13. The method of claim 1, wherein the cancer is breast cancer.

14. The method of claim 1, wherein the enzalutamide is administered in a sub-therapeutic dose of less than 160 mg per day.

15. A capsule dosage form comprising:

a) a first amount of enzalutamide formulated with one or more other pharmaceutically acceptable excipients; and

b) a second amount of a composition comprising two or more organic extracts of

Ganoderma luciduni, Scutellaria barbata, Scutellaria baicalensis, Salvia miltiorrhiza, and optionally other pharmaceutically acceptable excipients.

16. The dosage form of claim 15, wherein the excipients comprise one or more of an emulsifying agent, an antioxidant and a diluent.

17. The dosage form of claim 15, wherein:

the emulsifying agent is one or more of Cremophor EL, oleic acid and labrasol;

the antioxidant is selected from ascorbic acid and alpha tocopherol; and

the diluent is soya oil.

18. A kit comprising:

a dosage form comprising an effective amount of a botanical composition comprising two or more extracts prepared in a non-alcoholic organic medium of Ganoderma lucidum, Salvia miltiorrhiza, and Scutellaria barbata, wherein each extract comprises from about 1 to about 90 percent w/w and wherein the botanical composition is effective for reducing androgen receptor protein expression;

a dosage form comprising an effective amount of enzalutamide; and

optionally one or more pharmaceutically acceptable excipients.

19. The kit of claim 18, wherein both dosage forms are for oral administration.

20. The kit of claim 18, wherein the pharmaceutically acceptable excipients comprise one or more of an emulsifying agent, an antioxidant and a diluent.

21. The kit of claim 20, wherein the emulsifier is selected from one or more of fatty acids, polyoxyethylene glycerol esters of fatty acids, polooxylated castor oil, ethylene glycol esters, propylene glycol esters glyceryl esters of fatty acids, sorbitan esters, polyglyceryl esters, fatty alcohol ethoxylates, ethoxylated propoxylated block copolymers, polyethylene glycol esters of fatty acids, cremophores, glycerol monocaprylate/caprate, Gelucire, Capryol, Captex, Acconon, transcutol, and triacetin.

22. The kit of claim 20, wherein

the emulsifying agent is one or more of Cremophor EL, oleic acid and labrasol;

the antioxidant is selected from ascorbic acid and alpha tocopherol; and

the diluent is soya oil.

23. The dosage form of claims 15 or 18 that comprises the botanical composition, comprising an effective amount of Ganoderma lucidum extract selected from 33%, 35%, 40%, 42%, 44%, 45%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5% and 50%.

24. The dosage form of claims 15 or 18 that comprises the botanical composition, comprising an effective amount of Scutellaria barbata extract selected from 33%, 35%, 40%, 42%, 44%, 45%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5% and 50%.

25. The dosage form of claims 15 or 18 that comprises the botanical composition, comprising an effective amount of Salvia miltiorrhiza extract selected from 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% and 10%.