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Title:
LIMONOID COMPOUNDS FOR TREATMENT OF CANCER
Document Type and Number:
WIPO Patent Application WO/2020/181173
Kind Code:
A1
Abstract:
Methods and compositions are provided for treatment of tumors, such as multi-drug resistant tumors. Purified limonoid compounds, such as limonin glucoside, in pharmaceutical or nutraceutical compositions, are administered for treatment or prevention of tumors in an individual. Tumors preventable or treatable with purified limonoid compounds may include elevated levels of gamma-glutamyl transferase and/or glutathione.

Inventors:
KELLEY DARSHAN (US)
Application Number:
US2020/021345
Publication Date:
September 10, 2020
Filing Date:
March 06, 2020
Export Citation:
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Assignee:
KELLEY DARSHAN SINGH (US)
International Classes:
A61K31/155; A61K31/366; A61P3/04
Domestic Patent References:
WO2018170405A12018-09-20
Foreign References:
US20150250811A12015-09-10
Other References:
NPL-READI ET AL.: "Inhibition of P-glycoprotein activity by limonin and other secondary metabolites from Citrus species in human colon and leukaemia cell lines", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 626, no. 2, January 2010 (2010-01-01), pages 139 - 145, XP026789397, Retrieved from the Internet DOI: 10.1016/j.ejphar.2009.09.040
Attorney, Agent or Firm:
MCGOWAN, Malcolm K. et al. (US)
Download PDF:
Claims:
CLAIMS

I claim:

1. A method for prevention of a malignant tumor comprising elevated gamma glutamyl transferase (GGT) and/or glutathione (GSH), said method comprising administering a

prophylactically effective amount of at least one purified limonoid compound or a pharmaceutically acceptable salt thereof to an individual in need thereof, wherein said administration prevents a tumor comprising elevated GGT and/or GSH from developing or recurring in said individual or prevents a tumor comprising elevated GGT and/or GSH from progressing to a drug resistant tumor in said individual.

2. A method according to claim 1, wherein said tumor comprising elevated GGT and/or GSH is a multi -drug resistant (MDR) tumor.

3. The method of claim 1, wherein said at least one purified limonoid compound comprises limonin, limonin glucoside (LG), deoxylimonin, limonin carboxymethoxime, nomilin, nomilin 17-b- D-glucopyranoside, or obacunone, or a prophylactically effective derivative thereof.

4. The method of claim 1, wherein said mammal is a human.

5. The method of claim 1, wherein said administration comprises a daily dose of about 70 mg to about 21 g, or about 1 mg/kg body weight (kgbw)/day to about 300 mg/kgbw/day, of said purified limonoid compound.

6. The method of claim 1, wherein said purified limonoid compound is administered in conjunction with one or more additional compound(s) for prevention of said tumor, wherein said limonoid compound and said one or more additional compound(s) comprise an additive or synergistic effect in prevention of said tumor in said individual.

7. The method of claim 6, wherein said one or more additional compound(s) comprises a chemotherapeutic compound, a flavonoid, vitamin E, retinoic acid, or curcumin.

8. The method of claim 6, wherein hepatotoxicity caused by said one or more additional compound(s) is lower in said individual in comparison to the hepatoxicity of said additional compound(s) in the absence of administration of said limonoid compound.

9. The method of claim 1, wherein said tumor comprises a lung, ovarian, breast, bone marrow, nasopharyngeal carcinoma, esophageal squamous cell, endometrial, hepatocellular, head or neck laryngeal, colon, prostate, hepatic cholangiocarcinoma tumor, or leukemia.

10. The method of claim 1, wherein said individual is in remission from cancer.

11. A method for treatment of a malignant tumor comprising elevated GGT and/or GSH, said method comprising administering a therapeutically effective amount of at least one purified limonoid compound or a pharmaceutically acceptable salt thereof to an individual in need thereof, wherein said administration ameliorates or eliminates at least one sign and/or symptom of said tumor in said individual.

12. A method according to claim 11, wherein said tumor comprising elevated GGT and/or GSH is a MDR tumor.

13. The method of claim 11, wherein said treatment comprises reduction of GGT activity and/or GSH level in the tumor, reduction in tumor size, prevention of metastasis, reduction of at least one circulating biomarker for said tumor, and/or improved response to one or more additional therapeutic compound(s).

14. The method of claim 11, wherein said at least one purified limonoid compound comprises limonin, limonin glucoside (LG), deoxylimonin, limonin carboxymethoxime, nomilin, nomilin 17-b- D-glucopyranoside, or obacunone, or a therapeutically effective derivative thereof.

15. The method of claim 11, wherein said mammal is a human.

16. The method of claim 11, wherein said administration comprises a daily dose of about 70 mg to about 21 g, or about 1 mg/kgbw/day to about 300 mg/kgbw/day, of said purified limonoid compound.

17. The method of claim 11, wherein said purified limonoid compound is administered in conjunction with one or more additional compound(s) for treatment of said tumor, wherein said limonoid compound and said one or more additional compound(s) comprise an additive or synergistic effect in treatment of said tumor in said individual.

18. The method of claim 17, wherein said one or more additional compound(s) comprises a chemotherapeutic compound, a flavonoid, vitamin E, retinoic acid, or curcumin.

19. The method of claim 17, wherein hepatotoxicity caused by said one or more additional compound(s) is lower in said individual in comparison to the hepatoxicity in the absence of administration of said limonoid compound.

20. The method of claim 11, wherein said tumor comprises a lung, ovarian, breast, bone marrow, nasopharyngeal carcinoma, esophageal squamous cell, endometrial, hepatocellular, head or neck laryngeal, colon, prostate, hepatic cholangiocarcinoma, or leukemia tumor.

Description:
LIMONOID COMPOUNDS FOR TREATMENT OF CANCER

FIELD OF THE INVENTION

[01] The invention relates to use of limonoid compounds for treatment of cancers, in particular cancers with elevated levels of gamma glutamyl transferase.

BACKGROUND

[02] Worldwide, a total of 18.1 million new cases of cancer and 9.6 million deaths from cancer were estimated for 2018 (Bray F, et. al. (2018), CA Cancer J Clin. 68:394-424). In both sexes combined, lung cancer is the most common cause of death, followed by colorectal, stomach, and liver cancers, respectively. In the United States, cancer is the second most common cause of death, after deaths from cardiovascular disease (CVD). Europe contains 9% of the world population but has 25% of the global cancer incidence (Ferlay J. et. al. (2018) European Journal of Cancer 103: 356-387). Cancer is expected to be the leading cause of death in every country of the world in the 21 st century (Bray F, et. al. (2018), supra).

[03] Effective treatment of metastatic cancers usually requires the use of toxic chemotherapy. In most cases, multiple dmgs are used to provide a multifaceted approach to target different aspects of cancer development and progression, and also because resistance to a single drug almost always develops. However, even with the use of multiple drug regimens, many tumors develop multiple drug resistance (MDR). Reduced uptake of the drug by cancer cells, metabolic changes in the cell that impair the cytotoxic effects of dmgs to kill the cancer cells, increased efflux of the dmgs from the cancer cells, and other mechanisms contribute to the development of MDR (Szakacs, G., et. al. (2006) Nature Reviews 5:219-234; Xia CQ et. al. (2012) Mol Pharmacol 82: 1008-1021; Stavrovskaya, A. A., et al. (2018) Biochemistry (Moscow) 83:779-786). Safe dmgs that can overcome MDR are needed to control dmg resistant and aggressive tumors.

[04] Glutathione (glutamylcysteinylglycine; GSH) is a tripeptide that is present in nearly all living cells and is the most abundant sulfhydryl compound present in animal tissues, mainly in the cytoplasm. It is the major cellular antioxidant and is cmcial in maintaining the balance between oxidation and reduction. GSH and the enzymes involved in its biosynthesis, catabolism and detoxification (e.g., disulfide oxidized glutathione (GSSG), gamma glutamyl transferase (GGT), glutathione reductase, glutathione-S-transferase (GST), glutathione peroxidase (GPX)) play an important role in several diseases including cancer. In cancer, these enzymes protect the tumor microenvironment against oxidative stress, cell death, and are important in tumor growth and development (Corso, C.R. and Acco, A. (2018) Clinical Reviews in Oncology and Hematology 128:43-57). Under redox stress, increased amounts of GSH are synthesized and cysteine becomes rate limiting for GSH synthesis. Resistance to a number of drugs (cisplatin, cyclophosphamide, melphalan, mechlorethamine, nitrosourea, and others) is associated with elevated intracellular levels of GSH (Hanigan, M.H. (2014) Advances in Cancer Research 122: 103-141). Pro-oxidant anticancer drugs increase oxidative stress, deplete GSH, and enhance the effectiveness of chemotherapeutic agents (Zymorzynski, S., et ah, (2015) Biomedical Research International 2015: 1-6).

[05] GSH is important in the detoxification of carcinogens; however, elevated levels of GSH induce resistance to chemotherapy in many types of tumors (Traverso, N., et al. (2013) Oxidative Medicine and Cellular Longevity 2013: 1-10). Ample evidence reveals that glutathione and glutathione-based systems play a critical role in cancer initiation, progression, and the development of MDR (Hatem E., et al. (2017 ) Antioxidant and Redox Signaling 27: 1217-1234; Hanigan M.H. (2014), supra, Nunes, S.C., et. al. (2018) Int. J. Mol. Sci. 19: 1882-1897; Traverso, N., et. al. (2013), supra). Increased levels of GSH and over expression of Glutathione 8-transferases (GSTs) are often associated with an increased resistance to cancer chemotherapeutic drugs via GSH conjugation and detoxification, involving the combined mechanisms of GSH, glutamine S- transferase (GST) and glutathione S -conjugate export pump (GS-X pump). An understanding of the regulation of the GSH system is important for development of effective treatments for cancer.

[06] Gamma glutamyl transferase (GGT, also called gamma-glutyamyl transpeptidase) is an extracellular membrane enzyme that transfers the glutamyl group of GSH to an amino acid and forms cysteinylglycine and glutamyl amino acid (Pompella, A., et al. (2006) Biochemical Pharmacology 71 :231-238; Pompella, A., et al. (2007) Current Opinions in Pharmacology 7:360-366).

Cysteinylglycine can enter the cell and be reutilized for GSH synthesis or hydrolyzed to cysteine and glycine. GGT was originally viewed to decrease oxidative stress because it makes extracellular component amino acids available for intracellular GSH synthesis. However, the thiol group of cysteinylglycine acts as a pro-oxidant extracellularly, particularly in the presence of Fe 3+ and Cu 2+ , and is far more reactive than GSH (Mistry, D. et al. (2010) COPD 7:285-290; Pompella A., et al. (2007), supra). Cysteinylglycine binds to electrophilic and alkylating cancer drugs, making the tumors drug resistant.

[07] GGT expression was originally discovered in pre-neoplastic foci of non-expressing cells (rat liver, mouse skin, hamster tracheal epithelium) in response to tumor promotors that depleted GSH (Hanigan, M.H. (2014), supra). The same mechanism that was proposed to provide pre-neoplastic cells resistance to toxicity of promoting agents also confers resistance to pro-oxidant anticancer therapy in GGT positive tumors. The drug resistant cells with increased GSH synthesis are able to maintain intracellular GSH and avoid activation of cell death pathways. Thus, both GSH and GGT are normally protective agents. However, over production of both GSH and GGT imparts tumor drug resistance by altering the redox state. This decreases the potency of pro-oxidant drugs and detoxifies other drugs by making their sulfur adducts, providing cysteine and glutamine which are critical for the growth of rapidly growing tumors, activating drug efflux mechanisms and others.

[08] Serum levels of GGT are associated with pathological parameters and overall survival prognosis of human cancer patients following drug treatment (Xia, J., et al. (2016) Drug Discoveries and Therapeutics 10: 181-187; Kunutsor, S.K., et. al. (2015) Int. J. Cancer, 136: 1162-1170; Corti, A., et al. (2010) Anticancer Research 30: 1169-1182; Strasak, A.M., et. al. (2008) Int. J. Cancer

123: 1902-1906; Kazemi-Shirazi, L., et. al. (2007) Clinical Chemistry 53:940-946). Also, serum levels of GGT have been associated with severity of several human cancers including cervical (Polterauer,

S., et al. (2011) Gynecology Oncology 122:592-594), ovarian (Grimm, C., et al. (2013) British Jour. Cancer 109:610-614), nasopharyngeal carcinoma (Luo, M , et al. (2017) Oncotarget 8:67651-67662), esophageal squamous cell (Y ang, F., et al. (2015) Diseases of Esophagus 28:496-505), endometrial (Seebacher, Y., et al. (2012) British Jour. Cancer 106: 1551-1555), metastatic breast (Staudigl, C., et al. (2015) PLOS ONE, DOL 10. 1371/joumal pone.0125317), hepatocellular (Song, P,. et al. (2015) Medicine 94:e810), cholangiocarcinoma (Yin, X., et al. (2013) Eur. Jour. Gastroenterology and Hepatology 25: 1408-1414), leukemia (Sharma, P. and Karki, L. (2007) J Nepal Medical Assoc 46: 165-169) and others. Serum levels of GGT are also elevated in several other diseases, including cardiovascular, nonalcoholic fatty liver, alcoholic fatty liver, chronic kidney diseases, and others (U.S. Patent No. 9,066,965 B 1 and Kelley, D.S. (2015) Jour Functional Foods 12:271-281).

[09] Expression of GGT is increased in several human tumor tissues, including lungs, ovary, breast, brain, skin, liver, colon, pancreas, and leukemia cells (Pompella, A., et al. (2006), supra) and also in several cultured cell lines (Tew, K.D., et al. (1996) Molecular Pharmacology 50: 149-159), which suggests a role for this enzyme in conferring survival and growth advantage. Normal tissues expressing GGT include liver, kidney, prostate, pancreas, endometrial epithelium, follicular epithelium of thyroid. Carcinomas derived from GGT positive phenotype retained GGT positive phenotype. These included renal cell carcinoma, hepatocellular and cholangiocarcinoma, and carcinomas of the thyroid and prostate. Most tumors derived from kidney, liver, prostate, and thyroid were strongly positive for GGT, while pancreatic, ovarian, and uterine carcinomas showed a range of GGT expression. Normal tissues not expressing GGT include breast, lung, ovary, skin, and soft tissue sarcomas. Carcinomas of the lung and ovarian surface epithelium were typically positive for GGT, despite the absence of GGT in normal bronchial and ovarian surface epithelium. Both normal tissues and their tumors were negative for GGT in mesothelioma, melanoma, basal cell carcinoma, and most sarcomas (Hamgan, M.H. (2014), supra,· Hamgan, M.H., et al. (1999) Human Pathology 30:300-305; Hanigan, M.H. et.al., (1996) The Journal of Histochemistry and Cytochemistry 44: 1101-1108). Thus, GTT expression in different tumors ranged from high, variable, to undetectable.

[10] GGT expression has been implicated in drug resistance through elevation of intracellular GSH (Franzini, M., et al. (2006) European Journal of Cancer 42:2623-2630). However, the role of GGT in imparting tumor drug resistance and the mechanisms involved remain controversial (Franzini, M., et al. (2006), supra,· Fliedl, L., et al., (2014) Epub March 21, 2014,

http://dx.doi.org/lQ.14573/altex.1311 152: Daubeuf, S., et al., ( 2003) Biochemical Pharmacology 66: 595-604). GGT positive prostate tumors were significantly more resistant to the toxicity of cisplatin than GGT-negative tumors when injected into nude mice (Hanigan, M.H., et al., (1999), supra). Similarly, GGT transfection of melanoma cells increased their growth in vitro and in tumor xenografts, but it resulted in a decrease rather than increase of intracellular GSH (Franzini, M., et al. (2006), supra). In immortalized human kidney RPTEC-SV40 cells, low GGT activity improved cisplatin sensitivity, and the addition of GGT did not rescue but also increased cisplatin sensitivity (Fliedl, L , et al., (2014), supra. Adding GGT inhibitors as well as substrate (GSH) and product (cysteinyl-glycine) of GGT resulted in decreased sensitivity. It is a paradox that both reduced expression of GGT and addition of recombinant GGT decreased cisplatin sensitivity. Similarly, drug resistance in several human tumors is associated with GGT expression, but on the other hand, there are other human tumors that do not produce GGT and are drug resistant. The extracellular cysteinyl- glycine decreases tumor cell apoptosis in response to drag treatment by binding to the chemotherapy drags, and an increase in intracellular cysteine promotes tumor growth. It seems that a balance between the extracellular GGT activity and the intracellular concentration of cysteine determines tumor drag sensitivity. Tumor drag sensitivity is further complicated by the drag efflux proteins and the intra- and extracellular redox status.

[11] Currently there is an important critical need for drags of different mechanisms that are efficacious in surmounting MDR in human cancers. There is a need for new therapeutic methods to treat or prevent development of multi -drag resistance in tumors, in particular tumors with elevated levels of GGT or GSH.

BRIEF SUMMARY OF THE INVENTION

[12] Compositions and methods for prevention and treatment of cancer, such as multi-drug resistant tumors with elevated levels of GGT and/or GSH, are provided.

[13] In one aspect, methods are provided for prevention of a malignant tumor that includes elevated GGT and/or GSH. The methods include administering a prophylactically effective amount of at least one purified limonoid compound or a pharmaceutically acceptable salt thereof (e.g., one purified limonoid compound or a mixture of two or more purified limonoid compounds, or pharmaceutically acceptable salt(s) thereof), in a pharmaceutical or nutraceutical composition, to an individual in need thereof, e.g., a mammal, such as a human. Administration of the purified limonoid compound prevents a tumor with elevated GGT and/or GSH from developing or recurring in the individual or prevents a tumor with elevated GGT and/or GSH from progressing to a drug resistant tumor in the individual or prevents a drug resistant tumor from progressing to a MDR tumor in the individual. In one embodiment, the individual is in remission from cancer.

[14] In another aspect, methods are provided for treatment of a malignant tumor that includes elevated GGT and/or GSH. The methods include administering a therapeutically effective amount of at least one purified limonoid compound or a pharmaceutically acceptable salt thereof (e.g., one purified limonoid compound or a mixture of two or more purified limonoid compounds, or pharmaceutically acceptable salt(s) thereof), in a pharmaceutical or nutraceutical composition, to an individual in need thereof, e.g., a mammal, such as a human, Administration of the purified limonoid compound ameliorates or eliminates at least one sign and/or symptom of said tumor in said individual. In some embodiments, the tumor with elevated GGT and/or GSH is a drug resistant tumor. In one embodiment, the tumor is a MDR tumor. In some embodiments, treatment includes reduction of GGT activity and/or GSH level in the tumor, reduction in tumor size, prevention of metastasis, reduction of at least one circulating biomarker for the tumor, and/or improved response to one or more additional therapeutic compound(s).

[15] In some embodiments of the methods of prevention or treatment described herein, elevated GGT and/or GSH refers to GGT activity and/or GSH level is at least about 10%, 15% or 20% or greater than the GGT activity or GSH level in a normal (i.e., non-malignant) tissue of the same tissue type, for example, the GGT activity and/or GSH level in an adjacent or adjoining non-malignant tissue of the same tissue type In some embodiments, the individual has a serum levels of GGT greater than about 60 U/l. In some embodiments, the tumor with elevated GGT and/or GSH is a multi-drug resistant (MDR) tumor.

[16] In some embodiments of the methods of prevention or treatment described herein, the tumor, if present, is a lung, ovarian, breast, bone marrow, nasopharyngeal carcinoma, esophageal squamous cell, endometrial, hepatocellular, head or neck laryngeal, colon, prostate, or cholangiocarcinoma tumor, or leukemia, although this list is not intended to be limiting.

[17] In some embodiments of the methods of prevention or treatment described herein, administration of the purified limonoid compound results in a reduction of GGT activity and/or GSH level in the tumor, if present, of about 1% to about 70%, in comparison to the tumor GGT activity and/or GSH level in the absence of administration of the purified limonoid compound. In some embodiments, administration of the purified limonoid compound results in a reduction of serum GGT level below about 60 U/l (e.g., reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more, in an individual who is predisposed to a development of a malignant tumor, an individual who is in remission from cancer, an individual who has a non-drug resistant tumor, or an individual who has a drag resistant, e.g., MDR, tumor).

[18] In some embodiments of the methods of prevention or treatment described herein, the purified limonoid compound(s) is/are selected from limonin, limonin glucoside (LG; limonin 17 b-D- glucopyranoside), deoxylimonin, limonin carboxymethoxime, nomilin, nomilin 17-b-ϋ- glucopyranoside, and obacunone, or combinations thereof, or therapeutically or prophylactically effective derivatives thereof, although this list of limonoid compounds is not intended to be limiting.

In some embodiments, the purified limonoid compound is in the form of a pharmaceutically acceptable salt, selected from acetate, citrate, carbonate, chloride, or other salts. [19] In some embodiments of the methods of prevention or treatment described herein, administration of the purified limonoid compound involves oral, intravenous, intraperitoneal, intramuscular, or topical administration, injection into tumor(s), inhalation, or a suppository. The daily dose of a purified limonoid compound in a method described herein may be about 70 mg to about 21 g (e.g., about 1 mg/kgbw/day to about 300 mg/kgbw/day).

[20] In some embodiments of the methods of prevention or treatment described herein, the punfied limonoid compound is administered in conjunction with one or more additional compound(s) for prevention of the tumor, wherein the purified limonoid compound and the one or more additional compound(s) have an additive or synergistic effect in prevention or treatment of the tumor in said individual. For example, the one or more additional compound(s) may be selected from a chemotherapeutic compound, a flavonoid, vitamin E, retinoic acid, and curcumin, or combinations thereof, although this list is not intended to be limiting. In one embodiment, the purified limonoid compound is administered sequentially, i.e., prior to or after administration of the one or more additional compound(s) for prevention or treatment of the tumor. In another embodiment, the purified limonoid compound is administered concurrently, i.e., at the same time or at substantially the same time as administration of the one more additional compound(s) for prevention or treatment of the tumor. In some embodiments, hepatotoxicity caused by the one or more additional compound(s), if any, is lower in the individual than the hepatoxicity of the additional compound(s) that would otherwise occur in the absence of administration of the limonoid compound. For example, hepatotoxicity of the additional compound(s) is reduced by about l% to about 70% in the individual in comparison to the hepatoxicity of the additional compound(s) in the absence of administration of the limonoid compound.

[21] In another aspect, pharmaceutical and nutraceutical compositions are provided, which include a prophylactically or therapeutically effective dose of a purified limonoid compound, including any of the limonoid compounds in any of the dosages described herein, formulated for administration to an individual for prevention or treatment of a malignant tumor. In some embodiments, the composition includes at least one anti-oxidant compound, such as, but not limited to, vitamin E, vitamin C, beta- carotene, a polyphenol compound, or a flavonoid compound, or combinations thereof.

[22] In some embodiments, the purified limonoid compound is selected from limonin, limonin glucoside (LG; limonin 17 b-D-glucopyranoside), deoxylimonin, limonin carboxymethoxime, nomilin, nomilin 17-p-D-glucopvranosidc. and obacunone, or combinations thereof, or therapeutically or prophylactically effective derivatives thereof, although this list is not intended to be limiting. In some embodiments, composition includes a daily dose of about 70 mg to about 21 g (e.g., about 1 mg/kgbw/day to about 300 mg/kgbw per day) of said limonoid compound, to be administered as a single unit dose or divided into multiple unit doses, such as 2, 3, or 4 doses per day. DETAILED DESCRIPTION

[23] Limonoids compounds are highly oxygenated triterpenoid compounds found in tissues of members of the Rutaceae and Meliaceae plant families. Limonin glucoside (LG) is an example of a limonoid compound.

[24] In a cross-over, placebo controlled, double-blind study, overweight/obese individuals either consumed a placebo or LG (0.5 g/d) drink for 8 weeks. At the end of the intervention, plasma GGT concentration was 33% lower when study participants consumed LG drinks than when they consumed placebo drinks. (Kelley, D.S., et al. (2015), supra, U.S. Patent No. 9,066,965 Bl). Plasma concentrations of alanine aminotransferase, alkaline phosphatase and complement C3 were also significantly lower in the LG period than placebo. Matrix metalloproteinase-9, free prostate-specific antigen, tumor necrosis factor-a were also significantly lower with LG than with placebo drink. In a rat model of hepatoxicity, limonin significantly decreased D-galactosamine- or ischemia reperfusion injury induced increase in GGT (Mahmoud, M.F., et al. (2014) Naunyn-Schmiedeberg's archiv. Pharmacol. 387:251-261; Mahmoud M.F., et al. (2014) European Journal of Pharmacology 740:676- 682). A number of other inhibitors of GGT have been tested (Hanigan, M.H. (2014), supra; Corti, A. (2010), supra,· none of them is safe, effective and approved for clinical use.

[25] Limonin/LG has been shown to decrease the incidence of chemically induced tumors of forestomach, lungs, skin, oral cavity, and colon in animal models (Lam, L.K.T., et al. (1994) Food Technology 48: 104-108; Miller, E.G., et al. (2004) J Agric Food Chem 52:4908-4912; Tanaka, T., et al. (2000) Carcinogenesis 22: 193-198; Shimizu, S., et al. (2015) J. Clin. Biochem. Nutr. 57:39-43). Results from studies conducted in animal models established that diets containing up to 3% limonin or LG were safe and had no adverse consequences (Manners, G.D. (2007) J. Agric. Food Chem 55:8285-8294). These limonoids also inhibited the growth of cultured leukemia, ovary, cervix, stomach, liver, colon adenocarcinoma, and neuroblastoma cell lines (Poulose, S.M., et al. (2006) Nutrition and Cancer 56: 103-112; Chidambara Murthy, K. N., et al. (2011) J. Agric. Food Chem. 59:2314-2323; Tian, Q., et al. (2001), Nutrition and Cancer 40: 180-184). However, GGT activity was not monitored in these cell lines and limonin has not been tested for treatment or prevention of human tumors with multi -drag resistance or having elevated GGT activity and/or GSH levels in vivo.

[26] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al.. Dictionary of Microbiology and Molecular Biology second ed., John Wiley and Sons, New York (1994), and Hale & Markham. The Harper Collins Dictionary of Biology. Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this invention. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[27] Numeric ranges provided herein are inclusive of the numbers defining the range. Definitions

[28] “A,”“an” and“the” include plural references unless the context clearly dictates otherwise.

[29] As used herein, the term“limonoid compound” refers to any limonoid based chemical compound. The term“limonoid compounds” refers inclusively to limonoid aglycones, limonoate A- ring lactones, limonoid glycosides including, but not limited to limonoid glucosides, limonoid carboxylic acids including, but not limited to limonoid glycoside mono- and di-carboxylic acids. Structures of several exemplary limonoid compounds can be found in U.S. Patent No. 9,066,965 B l, Manners, G.D., et. al. (2007) JAgric. Food Chemi 55:8285-8294, and Gualdani, R. et al. (2016) Molecules 21 : 1530-1569), which are incorporated by reference herein in their entireties.

[30] The term“limonin glucoside” or“limonin- 17-p-D-glucopyranoside” as used herein refers to, a limonoid glycoside having the chemical formula C 32 H 42 O 14 . The term“limonoid glycoside” refers to limonoid compounds that are carboxylic acid limonoids derived from hydrolyzed lactones, wherein the alcoholic oxygen of the hydrolyzed lactone ring is glycosolated. Thus, limonoid glycosides comprise derivatives of A-ring and D-ring d-hydroxy carboxylic acid forms of citrus limonoids. In general,“limonoid glycosides” are limonoid A-ring lactones that contain one or more sugar moieties attached via a, b-glycosidic linkage at C-17.

[31] “Limonoid glycosides” typically occur as limonoid glycoside carboxylic acids. Typically, limonoid glycoside carboxylic acids occur in two carboxylic acid forms; mono-carboxylic acids or mono-acids, and di-carboxylic acids, or di-acids.

[32] The term“isolated” or“purified” refers to a material that is substantially or essentially free from components which are used to produce the material. For compositions disclosed herein, the term “isolated” refers to material that is substantially or essentially free from components which normally accompany the matenal in the mixture used to prepare the composition, for example, substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.

[33] “Isolated” and“pure” or“purified” are used interchangeably. Typically, isolated limonin glucoside or any limonoid compound used in a method as described herein, such as any of the limonoid compounds described herein, has a level of purity that, in exemplary embodiments, is expressed as a range. The lower end of the range of punty for the component is about 60%, about 70% or about 80% and the upper end of the range of purity is about 70%, about 80%, about 90% or more than about 90%. Thus, when limonin glucoside is more than about 90% pure, the purities are also typically expressed as a range. The lower end of the range of purity is about 90%, about 92%, about 94%, about 96% or about 98%. The upper end of the range of purity is about 92%, about 94%, about 96%, about 98% or about 100% purity. Purity is determined by any art-recognized method of analysis (e.g., HPLC, or a similar means). [34] The term“derived from” encompasses the terms“originated from,”“obtained from,” “obtainable from,”“isolated from,” and“created from,” and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material.

[35] The term“gamma glutamyl transferase” or“GGT” refers to EC number 2.3.2.2.

The term“glutathione” or“GSH” refers to a tripeptide with a gamma peptide linkage between the carboxyl group of the side chain of a glutamate residue and the amine group of a cysteine reside, wherein the carboxyl group of cysteine is attached via a normal peptide linkage to a glycine residue.

[36] The term“elevated GGT” as used herein refers to GGT activity in a malignant tumor that is at least about 10%, 15%, 20%, or greater above the GGT activity level in normal (non-malignant) tissue of the same tissue type, e.g., adjacent or adjoining non-malignant tissue. Also, serum levels of GGT above the upper normal level of 60 U/l are often associated with aggressive MDR tumors.

[37] The term“elevated GSH” as used herein refers to GSH activity in a malignant tumor that is at least 10%, 15%, 20%, or greater above the GSH level in normal (non-malignant) tissue of the same tissue type, e.g., adjacent or adjoining non-malignant tissue.

[38] The term“ameliorate” refers to any indicia of success in the treatment of a pathology or condition, e.g., a malignant tumor, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the pathology or condition more tolerable or bearable to the patient or subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being. The amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination or any other appropnate means known in the art.“Ameliorate” as used herein may also refer to the complete elimination of the symptoms.

[39] The term“to treat”,“treating”,“treatment” or“therapy” as used herein, refers to

reducing/decreasing or eliminating at least one symptom of a disease or disorder, e.g., a malignant tumor, including but not limited to remission, reduction in tumor size, reduction or prevention of metastasis, reduction in at least one biomarker associated with the tumor, or improvement of response to another therapeutic substance. Treatment of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, treatment refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, treatment refers to modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. Treatment in the methods described herein includes inhibiting growth of a tumor (slowing or arresting its development), providing relief from the symptoms or side-effects of the tumor (including palliative treatment), and causing regression or elimination of the tumor. [40] “Remission” refers to a decrease in or disappearance of signs and symptoms of cancer.

[41] The term“effective amount” or“an amount effective to” or a“therapeutically effective amount” or any grammatically equivalent term means the amount that, when administered to an animal or human for treating a disease or condition, is sufficient to effect treatment or therapy for that disease.“Therapeutically effective amount” means the amount of a compound that, i.e., a limonoid compound as described herein, that when administered to an individual for treating a disease or condition, is sufficient to effect such treatment for the disease or condition or to reduce severity of or eliminate at least one symptom of the disease or condition. “Therapeutically effective amount” means that amount of the compound that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. The“therapeutically effective amount” can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. Thus, a“therapeutically effective amount” or“effective amount” refers to that amount of the compound sufficient to result in an amelioration of one or more symptoms, for example, treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in effectiveness of treatment, healing, prevention or amelioration of such conditions, typically providing a statistically significant improvement in the treated patient population. When referencing an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When referring to a combination, a therapeutically effective dose or amount refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, including serially or simultaneously. In one embodiment, a therapeutically effective amount of purified limonoid compound ameliorates symptoms of a malignant tumor.

[42] “Prophylaxis” means a measure taken for the prevention of a disease or condition or at least one symptom thereof.“Prophylaxis” or“prevention,” in the context of the methods described herein, refers to preventing a malignant tumor from occurring in a subject that may be predisposed to cancer, but does not yet experience or exhibit symptoms of the disease, or prevention of progression of a tumor to a drug resistant or multi -drug resistant (MDR) tumor. Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of a disease or condition in question, e.g., an individual in cancer remission. Persons at risk of developing a particular disease or condition may include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the disease or condition.

[43] “Prophylactically effective amount” or any grammatically equivalent term means the amount that, when administered to an animal or human for treating a disease or condition, is sufficient to effect prevention or prophylaxis for that disease. “Prophylactically effective amount” means the amount of a compound that, i.e., a limonoid compound as described herein, that when administered to an individual for prevention of a disease or condition, is sufficient to effect such prevention of the disease or condition or to prevent development of at least one symptom of the disease or condition or effect development of the symptom at a lower level of severity than in the absence of administration of the compound. The“prophylactically effective amount” can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[44] “Preventing” or“prevention” refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, or causing the symptom to develop with less severity than in absence of the treatment). “Prevention” or“prophylaxis” may refer to delaying the onset of the disease or disorder, including the recurrence of cancer of a patient in remission.

[45] “Pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[46] “Pharmaceutically acceptable vehicle” or“pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient or carrier with which a limonoid compound as described herein is administered.

[47] As used herein, the expression“pharmaceutically acceptable carrier” refers to any material, which when combined with a limonoid compound, retains the limonoid compound activity and is non reactive with a subject's immune systems. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets including coated tablets and capsules. Typically such carriers contain excipients 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, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions including such carriers are formulated by well-known conventional methods.

[48] As used herein, the term“administering,” refers to suitable means of delivering a limonoid compound to an individual in need thereof. In an exemplary embodiment,“administering” a limonoid compound to an individual in need thereof refers to oral administration. In another exemplary embodiment,“administering” a limonoid compound to an individual in need thereof refers to administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, or the implantation of a slow-release device e.g., a mini -osmotic pump, to the individual. Administration is by any route including parenteral, and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Moreover, where injection is to treat a tumor, administration may be directly to the tumor and/or into tissues surrounding the tumor. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[49] “Pharmaceutical” refers to a composition or reagent, e.g. , an artificially synthesized composition or reagent, a method of use of the composition or reagent, and the like, that is capable of a pharmaceutical (e.g., medicinal) effect, and which is capable of being administered to a subject. “Pharmaceutical effect,” without limitation, can imply that the composition, reagent, or method is capable of stimulating a desired effect, (e.g., biochemical, genetic, cellular, physiological, or clinical effect), in at least one individual, such as a mammalian subject, for example, a human, in at least 5% of a population of subjects, in at least 10%, in at least 20%, in at least 30%, in at least 50% of subjects, or more. A pharmaceutical is typically a substance recognized by an official pharmacopoeia or formulary, and may be used, for example, in the diagnosis, cure, mitigation, treatment, or prevention of disease. A pharmaceutical is generally regarded to a substance (other than food) that is intended to affect a structure or function of the body. A pharmaceutical substance may be intended for use as a component of a medicine but is not itself a device or a component, part or accessory of a device. Biological products may be pharmaceuticals, but differences may exist regarding their manufacturing processes (e.g. , biological process versus chemical process).

[50] “Nutraceutical” refers to a composition that contains a naturally-occurring food substance or a component derived from a food substance that imparts not only a nutritional benefit but also a medicinal benefit for the prevention and/or treatment of disease.

[51] “MDR” or“multiple drug resistance” or“multi-drug resistance” refers to the resistance to multiple different drugs that are structurally and functionally distinct from a drug to which a tumor is originally resistant (Cui Q., et al. (2018) Drug Resistance Updates 41 : 1-25). Although not wishing to be bound by theory, MDR may be mediated by the overexpression of drug efflux transporters, mutations in some proteins, DNA damage or repair, and/or blockade of drug induced apoptosis and the presence of detoxifying systems.

[52] The term“unit dosage” or“unit dosage form” refers to a physically discrete units suitable as a unitary dosages for an individual to whom administered, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic or prophylactic effect, e.g., in association with a suitable pharmaceutically acceptable excipient.

[53] “Individual” or“subject” refers to humans and non-human mammals, including both human and veterinary subjects. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the individual is human.

[54] The term“enhanced” refers to any degree of betterment, augmentation embellishment, beautification, strengthening and/or improvement. For example the phrase“enhanced performance” indicates that performance is improved in one state, by comparison to another. [55] The term“improved” refers to a more desirable condition than previously existed, or alternatively, improved refers to state wherein a more desirable result is achieved under one set of conditions as compared with another. Improvement is demonstrated by any indicia of success, betterment, progression, or amelioration including any objective or subjective parameter such as abatement, remission, and/or diminishing of symptoms or an improvement in an individual's physical well-being. Improvement can be based on objective or subjective parameters; including the results of a physical examination.

Limonoid compounds

[56] Limonoid compounds are highly oxygenated triterpenoid compounds found in tissues from members of the Rutaceae and Meliaceae plant families. For example, members of the Family Rutaceae, genus Citrus comprise limonoids in their juice, fruit tissues, and seeds. Limonin glucoside (LG) is a limonoid compound.

[57] Typically, limonoid compounds are classified as belonging to one of five basic structural categories: (1) neutral aglycones, e.g., limonin or nomilin; (2) monocarboxylic aglycones, e.g., limonoic acid A-ring lactone; (3) dicarboxylic aglycones, e.g., nomilinoic acid; (4) monocarboxylic glycosides, e.g., limonin glucoside, and (5) dicarboxylic glycosides, e.g., nomilinic acid glucoside (Herman Z., et al. (1992) In: Modem Methods of Plant Analysis New Series, Volume 14 Seed Analysis, Linskens, H. F. and Jackson, J. F. eds. Springer-Verlag Berlin, pgs 361-375).

[58] Some examples of exemplary limonoid compounds are provided in Manners (2007), supra and JAgric Food Chem 55:8285-8294 and Gualdani, R. et ah, (2016), supra, which are incorporated by reference herein.

[59] Nonlimiting examples of limonoid compounds that may be used in the methods disclosed herein include limonin, LG, limonin b-D-glucopyranoside, deoxylimonin, limonin

carboxymethoxime, nomilin, nomilin 17-p-D-gliicopyranosidc. and obacunone, and therapeutically or prophylactically effective derivatives thereof.

[60] In some embodiments, a limonin compound is administered as a pharmaceutically acceptable salt. Nonlimiting examples include acetate, citrate, carbonate, and chloride salts of limonin compounds, such as but not limited to acetate, citrate, carbonate, and chloride salts of any of the limonoid compounds described herein.

Sources of Limonin Glucoside

[61] Limonin glucoside may be obtained from any synthetic method (see, e.g., B. Heasley (2011) Eur. J. Org. Chem. 19-46; Yamashita et al. (2015 ) Angew. Chem. Int. Ed. 54:8538-8541) or any naturally-occurring material that includes limonoid compounds. Limonoid compounds may be purified from fruits, peels, and seeds of plants from the Families Rutaceae and Meliaceae. For example, Citrus fruit tissues and by-products of juice processing such as peels and molasses are ready sources of limonin glucoside.

[62] Limonin glucoside-containing material can be in any form, for example whole fresh fruit, whole dried fruit, particulate solid material, a liquefied suspension of solids, and/or particulate solid dried extract obtained from Citrus species or species of genera closely related to Citrus,· e.g., Citrus seeds, processed Citrus seed meal or extracts, filtrates or enzymatic digests of Citrus seeds or Citrus seed meal. In other embodiments the limonoid compound containing material is in the form of water soluble extracts, filtrates or enzymatic digests of limonoid compound-containing material, e.g., Citrus fruit components, Citrus juice processing by-products or processed Citrus including peel, pulp, core, seeds, juice, juice processing pulp wash, processed Citrus molasses and Citrus fruit sections or obtained from species of genera closely related to Citrus. Purification of limonin glucoside may be achieved using any method or combination of methods known in the art (see, e.g., Breksa III, A. P., Manners, G. D , & Ibarra Jr., P. (2008) Journal of the Science of Food and Agriculture 88: 2113- 2218; Breksa III, A. P., Dragull, K., & Wong RY (2008) Journal of Agricultural Food Chemistry 56: 5595-5598; Breksa III, A. P., Hidalgo M B., & Wong R Y (2008) Stability of limonin glucoside in beverage matrices, Journal of the Science of Food and Agriculture 88: 2194-2200; Breksa III, A. P. & Dragull, K. (2009) Food Chemistry 113: 1308-1313; Breksa III, A. P„ Hidalgo M B, Yuen M L (2009) Food Biochemistry 117: 739-744).

Methods

[63] Methods are provided herein for prevention or treatment of a malignant tumor (e.g., a tumor with elevated GGT and/or GSH), for example, a drug resistant or MDR tumor. A prophylactically or therapeutically effective amount of a purified limonoid compound is administered to an individual. The individual may be a mammal, e.g., a human.

[64] In one embodiment of a method for prevention, administration of the purified limonoid compound prevents the tumor from developing in the individual. The individual may be predisposed to development of a tumor, e.g., genetic predisposition (e.g., due to family history or genetic testing), obesity, due to screening for cancer, by virtue of environmental or toxic exposure (e.g., exposure to toxins or radiation), elevated plasma GGT, or an individual who previously had cancer, or the individual may be in cancer remission. In another embodiment of a method for prevention, administration of the limonoid compound prevents an existing drug sensitive tumor in the individual from progressing to a drug resistant tumor or prevents a drug sensitive tumor in the individual from progressing to a MDR tumor.

[65] In a method for treatment, administration of the purified limonoid compound ameliorates or eliminates at least one symptom of the tumor in the individual. Symptoms that are treated by the methods described herein may include, but are not limited to, reduction in GGT activity and/or GSH level in the tumor, tumor size, prevention or reduction of metastasis, reduction of at least one circulating or cellular (e.g., tumor cell surface) biomarker for the tumor, and/or improved response to one or more additional therapeutic compound or composition in comparison to the response to the compound or composition in the absence of administration of the limonoid compound.

[66] In some embodiments, the purified limonoid compound is limonin, LG (limonin 17 b-D- glucopyranoside), deoxylimonin, limonin carboxymethoxime, nomilin, nomilin 17-b-ϋ- glucopyranoside, or obacunone, or a therapeutically or prophylactically effective derivative thereof, or a pharmaceutically acceptable salt thereof. In one embodiment, the purified limonoid compound is LG. In some embodiments, the purified limonoid compound is formulated as a pharmaceutically acceptable salt such as, but not limited to, an acetate, citrate, carbonate, or chloride salt.

[67] In some embodiments, a mixture of two or more purified limonoid compounds is administered in a method of prevention or treatment as described herein. For example, a mixture of two or more purified limonoid compounds as described herein, wherein each of the purified limonoid compounds is in an amount that is about 5% to about 50% by weight of the total weight of limonoid compounds in a pharmaceutical or nutraceutical composition, may be administered. For example, a mixture of two or more purified limonoid compounds that includes limonin, LG (limonin 17 b-D- glucopyranoside), deoxylimonin, limonin carboxymethoxime, nomilin, nomilin 17-b-ϋ- glucopyranoside, or obacunone, or a therapeutically or prophylactically effective derivative thereof, wherein each of the purified limonoid compounds is in an amount that is about 5% to about 50% by weight of the total weight of limonoid compounds, may be administered.

[68] Tumors that are preventable or treatable by methods described herein include, but are not limited to, lung, ovarian, breast, bone marrow, nasopharyngeal carcinoma, esophageal, hepatocellular, head or neck laryngeal, colon, prostate, hepatic cholangiocarcinoma tumors, and leukemias. In some embodiments, the tumor is a lung, ovarian, breast, bone marrow, nasopharyngeal carcinoma, esophageal, hepatocellular, head or neck laryngeal, colon, prostate, hepatic cholangiocarcinoma, or leukemia tumor with elevated GGT and/or GSH.

[69] Conventionally, the tissue and tumor activity of GGT has been determined in tissue homogenate or by immunohistochemistry (see, e.g., Hanigan, M.H. and Frierson, J.R. (1996) The Journal of Histochemistry and Cytochemistry 44: 1101-1108; Wang, Q., et al. (2017) Oncotarget 8:36171-36184). However, recently a number of methods have been reported using bioluminogenic, florescence or optical probes, and near infrared to detect GGT activity in tumor and in normal tissue in situ (Li, S., et al. (2017) Biosensors and Bioelectronics 98:325-329; Li, L., et. al. (2018) Analytical and Bioanalytical Chemistry 410:6771-6777; Luo, Z., et al (2017) Chemistry A European Journal 23: 14778-14785; Luo, Z., et al (2019) CHEMBIOCHEM Minireviews 20:474-487; Bai, B., et. al. (2018) Chem Commun 54: 12393-12396).

[70] A number of analytical methods are available for the determination of GSH and GSSG in biological samples (Monostori, P., et al., (2009) Journal of Chromatography B 877:3331-3346; Bayram, B., et. al., (2014) JourAgric Food Chem 62:402-408; Guistarini, D., et. al., (2017) Free Radical Biology and Medicine 112:360-375). Any of the referenced methods may be used depending upon the availability and the samples to be analyzed.

[71] Although not wishing to be bound by theory, an increase in GSH is may be a contributing factor to tumor drug resistance by binding to and reacting with drugs, interacting with reactive oxygen species (ROS), preventing damage to proteins or DNA, and/or participating in DNA repair and the efflux of chemotherapeutic drugs (Traverso, N., et. al. (2013), supra ; Bansal, A. and Simon, M.C. (2018) Journal of Cell Biology 217:2291-2298). Ranges for GSH for most normal cells are in millimolar concentrations. For example, the GSH concentration in liver cells typically ranges between 5 and 10 mM (Wu, G., et. al. (2004) JNutr. 134:489-492). However, the levels of GSH in tumor cells including bone marrow, breast, colon, larynx, head and neck, lung, and other cancers are several fold higher when compared with the corresponding levels in normal tissues (Perry, R.R., et al (1993) Cancer 72:783-787; Wu, G. et. al. (2004), supra ; Baldiran, G.K., et al., (2004) Cell Biochemistry and Functions 22:343-352; Lu, S.C., et. al. (2009) Mol Aspects Med 30:42-49; Singh S., et al. (2012) Journal of Experimental Therapeutic and Oncology 4:303-316; Bansal, K. and Simon, M.C. (2018), supra. A relationship between increased GSH levels in tumors and resistance to cisplatin, , 4- hydroxyphosphmide, methotrexate, melphalan, nitrosourea, taxol, and other drugs has been reported (Bracht, K., et al., (2006 ) Anti cancer Drugs 17:41-51).

[72] In some embodiments, a tumor that is preventable or treatable by methods described herein is resistant to one or more dmg(s), such as chemotherapeutic drug(s). In one embodiment, the tumor is MDR.

[73] In methods of treatment described herein, administration of the purified limonoid compound results in a reduction of GGT and/or GSH of about 1% to about 70%. In some embodiments, administration of the purified limonoid compound results in a reduction of GGT and/or GSH, e.g, a reduction of about 1% to about 70%, thereby restoring or imparting sensitivity of a tumor to one or more dmg(s), such as chemotherapeutic dmg(s).

[74] In one embodiment, administration of the purified limonoid compound renders a MDR tumor with elevated GGT and/or GSH sensitive to one or more dmg(s), such as chemotherapeutic dmg(s), thereby ameliorating or eliminating at least one symptom of the tumor. In another embodiment, administration of the purified limonoid compound prevents growth and/or metastasis of a MDR tumor with elevated GGT and/or GSH in an individual. In another embodiment, administration of the purified limonoid compound eliminates a MDR tumor with elevated GGT and/or GSH in an individual, thereby rendering the individual in remission from the tumor. In an embodiment of a method of prevention as described herein, administration of the purified limonoid compound prevents development of a MDR tumor with elevated GGT and/or GSH in an individual. [75] Nonlimiting examples of drug(s), e.g., chemotherapeutic drug(s), to which tumor sensitivity may be established or reestablished via a method described herein include doxorubicin, taxol, vinca alkaloids, 5-flourouricil; methotrexate, cisplatin, cyclophosphamide, melphalan, mechlorethamine, nitrosourea, l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU), bortezomib, quinone containing drugs, sulhydryl -reactive chemotherapeutic drugs, and other drugs used to treat MDR tumors with elevated GSH (Hanigan, M.H. (2014), supra ; Cui, Q. et al. (2018) Drug Resistance Updates 41 : 1-25; Traverso, N., et. al. (2013), supra, Singh, S., et. al. (2012), supra, Balendiran, G.K., et. al. (2004) Cell

Biochemistry and Function 22:343-352).

[76] In some embodiments, administration of purified limonoid compound in a method as described herein reduces the level of one or more biomarker(s) indicative of cancer or a predisposition to cancer in an individual, including but not limited to, alphafetoprotein (AFP) (e.g., indicative of liver cancer), carcinoembryonic antigen (CEA) (e.g., indicative of colon cancer), prostate specific antigen (PSA) (e.g., indicative of prostate cancer), or p53 or HER2 antibodies (e.g., indicative of breast cancer). In some embodiments, administration of purified limonoid compound in a method as described herein reduces circulating levels of DNA, mRNA, and/or micro RNA, and/or changes total and/or differential cell count in an individual, e.g., indicative of tumor burden.

[77] In some embodiments, administration of purified limonoid compound in a method as described herein reduces the level of one or more biomolecule that mediates tumor drug resistance, including but not limited to a drug efflux protein, such as P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), or breast cancer resistance protein (BCRP/ABCG2), or suppresses the level of a cellular signaling pathway, such as the Wnt signaling pathway.

[78] The limonoid compound may be administered by any suitable method depending on the type of tumor, health status of the individual, and other factors which may be determined by a medical professional. In some embodiments, the purified limonoid compound is administered via oral, intravenous, intraperitoneal, intramuscular, or topical administration, via injection into a tumor, via inhalation, or via a suppository.

[79] In some embodiments of methods of treatment as described herein, purified limonoid compound is administered in conjunction with one or more additional compound(s) for treatment of a tumor, such as chemotherapeutic compound(s), flavonoid(s), vitamin E, retinoic acid, or curcumin, wherein the purified limonoid compound and the additional compound(s) have an additive or synergistic effect. The purified limonoid compound and the additional compound(s) may be administered together or separately, at the same time or at different times, i.e., the limonoid compound may be administered prior to or after administration of additional compound(s) or concurrently with administration of additional compound(s). In some embodiments, administration of the purified limonoid compound may reduce hepatotoxicity of the additional compound(s), e.g., by any of at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, or any of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% to about 70%.

[80] Typical dosages of purified limonoid compound include, but are not limited to, about 70 mg to about 21 g per day (e.g., about 1 to about 300 mg/kilogram of body weight (kgbw)/day). For example, about 70 mg to about 21 g of purified limonoid compound may be administered in a single daily dose, or divided into multiple doses per day, e.g., 2, 3, 4 doses per day.

[81] In some embodiments of therapeutic or prophylactic methods described herein, any of about 70 mg to about 250 mg, about 250 mg to about 500 mg, about 500 mg to about 750 mg, about 750 mg to about 1000 mg, about 1 g to about 1.5 g, about 1.5 g to about 2 g, about 2 g to about 2.5 g, about

2.5 g to about 3 g, about 3 g to about 3.5 g, about 3.5 g to about 4 g, about 4 g to about 4.5 g, about

4.5 g to about 5 g, about 5 g to about 5.5 g, about 5.5 g to about 6 g, about 6 g to about 6.5 g, about

6.5 g to about 7 g, about 7.5 g, about 7.5 g to about 8 g, about 7.5 g to about 8 g, about 8 g to about

8.5 g, about 8.5 g to about 9 g, about 9 g to about 9.5 g, about 9.5 g to about 10 g, about 10 g to about

10.5 g, about 10.5 g to about 11 g, about 11 g to about 11.5 g, about 11.5 g to about 12 g, about 12 g to about 12.5 g, about 12.5 g to about 13 g, about 13 g to about 13.5 g, about 13.5 g to about 14 g, about 14 g to about 14.5 g, about 14.5 g to about 15 g, about 15 g to about 15.5 g, about 15.5 g to about 16 g, about 16 g to about 16.5 g, about 16.5 g to about 17 g, about 17 g to about 17.5 g, about

17.5 g to about 18 g, about 18 g to about 18.5 g, about 18.5 g to about 19 g, about 19 g to about 19.5 g, about 19.5 g to about 20 g, about 20 g to about 20.5 g, about 20.5 g to about 21 g, about 70 mg to about 500 mg, about 100 mg to 1000 mg, about 500 mg to about 2.5 g, about 1 g to about 5 g, about 1 g to about 10 g, about 5 g to about 21 g, or about 10 g to about 21 g of purified limonoid compound may be administered per day. For example, in some embodiments of the therapeutic or prophylactic methods described herein, any of about 1 mg/kgbw to about 5 mg/kgbw, about 5 mg/kgbw to about 10 mg/kgbw, about 10 mg/kgbw to about 20 mg/kgbw, about 20 mg/kgbw to about 30 mg/kgbw, about 30 mg kgbw to about 40 mg/kgbw, about 40 mg/kgbw to about 50 mg/kgbw, about 50 mg/kgbw to about 60 mg/kgbw, about 60 mg/kgbw to about 70 mg/kgbw, about 70 mg/kgbw to about 80 mg/kgbw, about 80 mg/kgbw to about 90 mg/kgbw, about 90 mg/kgbw to about 100 mg/kgbw, about 100 mg/kgbw to about 110 mg/kgbw, about 110 mg/kgbw to about 120 mg/kgbw, about 120 mg/kgbw to about 130 mg/kgbw, about 130 mg/kgbw to about 140 mg/kgbw, about 140 mg/kgbw to about 150 mg/kgbw, about 150 mg/kgbw to about 160 mg/kgbw, about 160 mg/kgbw to about 170 mg/kgbw, about 170 mg/kgbw to about 180 mg/kgbw, about 180 mg/kgbw to about 190 mg/kgbw, about 190 mg/kgbw to about 200 mg/kgbw, about 200 mg/kgbw to about 210 mg/kgbw, about 210 mg/kgbw to about 220 mg/kgbw, about 220 mg/kgbw to about 230 mg/kgbw, about 230 mg/kgbw to about 240 mg/kgbw, about 240 mg/kgbw to about 250 mg/kgbw, about 250 mg/kgbw to about 260 mg/kgbw, about 260 mg/kgbw to about 270 mg/kgbw, about 270 mg/kgbw to about 280 mg/kgbw, about 280 mg/kgbw to about 290 mg/kgbw, about 290 mg/kgbw to about 300 mg/kgbw, about 1 mg/kgbw to about 50 mg/kgbw, about 1 mg/kgbw to about 100 mg/kgbw, about 10 mg kgbw to about 100 mg/kgbw, about 50 mg/kgbw to about 250 mg/kgbw, about 75 mg/kgbw to about 150 mg/kgbw, about 100 mg/kgbw to about 150 mg/kgbw, about 150 mg/kgbw to about 300 mg/kgbw, or about 100 mg/kgbw to about 300 mg/kgbw of purified limonoid compound may be administered per day.

[82] In one embodiment, an individual at risk of developing a tumor or in remission may be administered about 70 mg to about 3.5 g of purified limonoid compound per day (e.g., about 1 to about 50 mg/kgbw/day). For example, any of about 70 mg to about 250 mg, about 250 mg to about 500 mg, about 500 mg to about 750 mg, about 750 mg to about 1000 mg, about 1 g to about 1.5 g, about 1.5 g to about 2 g, about 2 g to about 2.5 g, about 2.5 g to about 3 g, about 3 g to about 3.5 g, about 70 mg to about 500 mg, about 100 mg to 1000 mg, about 500 mg to about 2.5 g, about 1 g to about 3.5 g of purified limonoid compound may be administered per day. For example, any of about 1 mg/kgbw to about 5 mg/kgbw, about 5 mg/kgbw to about 10 mg/kgbw, about 10 mg/kgbw to about 20 mg/kgbw, about 20 mg/kgbw to about 30 mg/kgbw, about 30 mg/kgbw to about 40 mg/kgbw, about 40 mg/kgbw to about 50 mg/kgbw, about 1 mg/kgbw to about 10 mg/kgbw, about 1 mg/kgbw to about 25 mg/kgbw, about 10 mg/kgbw to about 30 mg/kgbw, about 10 mg/kgbw to about 50 mg/kgbw, or about 25 mg/kgbw to about 50 mg/kgbw of purified limonoid compound may be administered per day.

[83] In another embodiment, an individual with a drug-susceptible tumor who is not receiving chemotherapy is administered about 1.4 g to about 21g of purified limonoid compound per day (e.g, about 20 to about 300 mg/kgbw/day), e.g., in a method to prevent the tumor from becoming drug resistant, e.g., MDR. For example, any of about 1.4 g to about 1.5 g to about 2 g, about 2 g to about

2.5 g, about 2.5 g to about 3 g, about 3 g to about 3.5 g, about 3.5 g to about 4 g, about 4 g to about

4.5 g, about 4.5 g to about 5 g, about 5 g to about 5.5 g, about 5.5 g to about 6 g, about 6 g to about

6.5 g, about 6.5 g to about 7 g, about 7.5 g, about 7.5 g to about 8 g, about 7.5 g to about 8 g, about 8 g to about 8.5 g, about 8.5 g to about 9 g, about 9 g to about 9.5 g, about 9.5 g to about 10 g, about 10 g to about 10.5 g, about 10.5 g to about 11 g, about 11 g to about 11.5 g, about 11.5 g to about 12 g, about 12 g to about 12.5 g, about 12.5 g to about 13 g, about 13 g to about 13.5 g, about 13.5 g to about 14 g, about 14 g to about 14.5 g, about 14.5 g to about 15 g, about 15 g to about 15.5 g, about

15.5 g to about 16 g, about 16 g to about 16.5 g, about 16.5 g to about 17 g, about 17 g to about 17.5 g, about 17.5 g to about 18 g, about 18 g to about 18.5 g, about 18.5 g to about 19 g, about 19 g to about 19.5 g, about 19.5 g to about 20 g, about 20 g to about 20.5 g, about 20.5 g to about 21 g, about 70 mg to about 500 mg, about 100 mg to 1000 mg, about 500 mg to about 2.5 g, about 1 g to about 5 g, about 1 g to about 10 g, about 5 g to about 21 g, or about 10 g to about 21 g of purified limonoid compound may be administered per day. For example, any of about 20 mg/kgbw to about 30 mg/kgbw, about 30 mg/kgbw to about 40 mg/kgbw, about 40 mg/kgbw to about 50 mg/kgbw, about 50 mg/kgbw to about 60 mg/kgbw, about 60 mg/kgbw to about 70 mg/kgbw, about 70 mg/kgbw to about 80 mg/kgbw, about 80 mg/kgbw to about 90 mg kgbw, about 90 mg/kgbw to about 100 mg/kgbw, about 100 mg/kgbw to about 110 mg/kgbw, about 110 mg/kgbw to about 120 mg/kgbw, about 120 mg/kgbw to about 130 mg/kgbw, about 130 mg/kgbw to about 140 mg/kgbw, about 140 mg/kgbw to about 150 mg/kgbw, about 150 mg/kgbw to about 160 mg/kgbw, about 160 mg/kgbw to about 170 mg/kgbw, about 170 mg/kgbw to about 180 mg/kgbw, about 180 mg/kgbw to about 190 mg/kgbw, about 190 mg/kgbw to about 200 mg/kgbw, about 200 mg/kgbw to about 210 mg/kgbw, about 210 mg/kgbw to about 220 mg/kgbw, about 220 mg/kgbw to about 230 mg/kgbw, about 230 mg/kgbw to about 240 mg/kgbw, about 240 mg/kgbw to about 250 mg/kgbw, about 250 mg/kgbw to about 260 mg/kgbw, about 260 mg/kgbw to about 270 mg/kgbw, about 270 mg/kgbw to about 280 mg/kgbw, about 280 mg/kgbw to about 290 mg/kgbw, about 290 mg/kgbw to about 300 mg/kgbw, about 1 mg/kgbw to about 50 mg/kgbw, about 1 mg/kgbw to about 100 mg/kgbw, about 10 mg/kgbw to about 100 mg/kgbw, about 50 mg/kgbw to about 250 mg/kgbw, about 75 mg/kgbw to about 150 mg/kgbw, about 100 mg/kgbw to about 150 mg/kgbw, about 150 mg/kgbw to about 300 mg/kgbw, or about 100 mg/kgbw to about 300 mg/kgbw of purified limonoid compound may be administered per day.

[84] In another embodiment, an individual who is being administered chemotherapy is administered about 2.5 g to about 21 g of purified limonoid compound per day (e.g., about 35 to about 300 mg/kgbw/day), e.g., the day or evening before chemotherapy. For example, about 2.5 g to about 3 g, about 3 g to about 3.5 g, about 3.5 g to about 4 g, about 4 g to about 4.5 g, about 4.5 g to about 5 g, about 5 g to about 5.5 g, about 5.5 g to about 6 g, about 6 g to about 6.5 g, about 6.5 g to about 7 g, about 7.5 g, about 7.5 g to about 8 g, about 7.5 g to about 8 g, about 8 g to about 8.5 g, about 8.5 g to about 9 g, about 9 g to about 9.5 g, about 9.5 g to about 10 g, about 10 g to about 10.5 g, about 10.5 g to about 11 g, about 11 g to about 11.5 g, about 11.5 g to about 12 g, about 12 g to about 12.5 g, about 12.5 g to about 13 g, about 13 g to about 13.5 g, about 13.5 g to about 14 g, about 14 g to about 14.5 g, about 14.5 g to about 15 g, about 15 g to about 15.5 g, about 15.5 g to about 16 g, about 16 g to about 16.5 g, about 16.5 g to about 17 g, about 17 g to about 17.5 g, about 17.5 g to about 18 g, about 18 g to about 18.5 g, about 18.5 g to about 19 g, about 19 g to about 19.5 g, about 19.5 g to about 20 g, about 20 g to about 20.5 g, about 20.5 g to about 21 g, about 70 mg to about 500 mg, about 100 mg to 1000 mg, about 500 mg to about 2.5 g, about 1 g to about 5 g, about 1 g to about 10 g, about 5 g to about 21 g, or about 10 g to about 21 g of punfied limonoid compound may be administered per day. For example, about 35 mg/kgbw to about 40 mg/kgbw, about 40 mg/kgbw to about 50 mg/kgbw, about 50 mg/kgbw to about 60 mg/kgbw, about 60 mg kgbw to about 70 mg/kgbw, about 70 mg/kgbw to about 80 mg/kgbw, about 80 mg/kgbw to about 90 mg/kgbw, about 90 mg/kgbw to about 100 mg/kgbw, about 100 mg/kgbw to about 110 mg/kgbw, about 110 mg/kgbw to about 120 mg/kgbw, about 120 mg/kgbw to about 130 mg/kgbw, about 130 mg/kgbw to about 140 mg/kgbw, about 140 mg/kgbw to about 150 mg/kgbw, about 150 mg/kgbw to about 160 mg/kgbw, about 160 mg/kgbw to about 170 mg/kgbw, about 170 mg/kgbw to about 180 mg/kgbw, about 180 mg/kgbw to about 190 mg/kgbw, about 190 mg/kgbw to about 200 mg/kgbw, about 200 mg/kgbw to about 210 mg/kgbw, about 210 mg/kgbw to about 220 mg/kgbw, about 220 mg/kgbw to about 230 mg/kgbw, about 230 mg/kgbw to about 240 mg/kgbw, about 240 mg/kgbw to about 250 mg/kgbw, about 250 mg/kgbw to about 260 mg/kgbw, about 260 mg/kgbw to about 270 mg/kgbw, about 270 mg/kgbw to about 280 mg/kgbw, about 280 mg/kgbw to about 290 mg/kgbw, about 290 mg/kgbw to about 300 mg/kgbw, about 1 mg/kgbw to about 50 mg/kgbw, about 1 mg/kgbw to about 100 mg/kgbw, about 10 mg/kgbw to about 100 mg/kgbw, about 50 mg/kgbw to about 250 mg/kgbw, about 75 mg/kgbw to about 150 mg/kgbw, about 100 mg/kgbw to about 150 mg/kgbw, about 150 mg/kgbw to about 300 mg/kgbw, or about 100 mg/kgbw to about 300 mg/kgbw of purified limonoid compound may be administered per day.

[85] Optionally, about 1 g to about 5 g (e.g., any of about 1 g to about 1.5 g, about 1.5 g to about 2 g, about 2 g to about 2.5 g, about 2.5 g to about 3 g, about 3 g to about 3.5 g, about 3.5 g to about 4 g, about 4 g to about 4.5 g, about 4.5 g to about 5 g, about 1 g to about 2.5 g, about 1.5 g to about 3 g, about 2 g to about 4 g, or about 2.5 g to about 5 g) of purified limonoid compound per day may be administered in between chemotherapy treatments. For example, about 14 mg/kgbw to about 70 mg/kgbw (e.g., any of about 14 g to about 14.5 g, about 14.5 g to about 15 g, about 15 g to about 15 5 g, about 15.5 g to about 16 g, about 16 g to about 16.5 g, about 16.5 g to about 17 g, about 17 g to about 17.5 g, about 17.5 g to about 18 g, about 18 g to about 18.5 g, about 18.5 g to about 19 g, about 19 g to about 19.5 g, about 19.5 g to about 20 g, about 20 g to about 20.5 g, about 20.5 g to about 21 g, about 70 mg to about 500 mg, about 100 mg to 1000 mg, about 500 mg to about 2.5 g, about 1 g to about 5 g, about 1 g to about 10 g, about 5 g to about 21 g, or about 10 g to about 21 g of purified limonoid compound may be administered per day. For example, about 35 mg/kgbw to about 40 mg/kgbw, about 40 mg/kgbw to about 50 mg/kgbw, about 50 mg/kgbw to about 60 mg/kgbw, about 60 mg/kgbw to about 70 mg/kgbw, about 14 mg/kgbw to about 20 mg/kgbw, about 14 mg/kgbw to about 35 mg/kgbw, about 30 to about 70 mg/kgbw, about 20 mg/kgbw to about 50 mg/kgbw, about 30 mg/kgbw to about 60 mg/kgbw, or about 40 mg/kgbw to about 70 mg/kgbw) of purified limonoid compound per day may be administered in between chemotherapy treatments.

Compositions

[86] Compositions are provided that include a purified limonoid compound, formulated for use in a method as disclosed described herein. In some embodiments, the composition includes purified limonin, LG (limonin b-D-glucopyranoside), deoxylimonin, limonin carboxymethoxime, nomilin, nomilin 17-p-D-glucopyranoside. or obacunone, or a therapeutically or prophylactically effective derivative thereof. In other embodiments, the composition includes the limonin compound in combination with one or more other substance(s) that are associated with the source of the limonoid, for example, a Citrus extract, e.g., from juice, fruit tissues, and seeds of a Citrus plant species, or substance(s) associated with chemical synthesis of the limonin compound. In some embodiments, the limonin compound is in the form of a pharmaceutically acceptable salt, such as, but not limited to, an acetate, citrate, carbonate, or chloride salt.

[87] A limonin compound, e.g., LG, used in the methods disclosed herein can be administered by any means known in the art, e.g., orally, parenterally, topically, or by local administration, such as, e.g., transdermally. The methods disclosed herein provide for therapeutic treatments including prophylactic treatments. A limonin compound in a pharmaceutical or nutraceutical formulation can be administered in a variety of unit dosage forms depending upon whether the disease condition is being treated after a subject has already acquired the disease, or if the subject is at risk of the disease but does not yet manifest symptoms of the disease (prophylactic administration), the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, 17th ed. Edited by Alfonso R. Gennaro. Mack Publishing Co., 20th and Northampton Streets, Easton, Pa. (“Remington's”). A person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine a therapeutically or prophylactically effective amount of a limonin compound for practice of the methods disclosed herein. For example, a purified limonin compound, e.g., LG, may be more effective at higher or lower doses. By evaluating a patient using the methods disclosed herein, a skilled practitioner will be able to determine whether a patient is responding to treatment and will know how to adjust the dosage levels accordingly.

[88] In some embodiments, the composition is a pharmaceutical or nutraceutical composition, and includes at least one pharmaceutically acceptable excipient. Such compositions can be prepared in a manner well known in the pharmaceutical art and include at least one active compound, i.e., a limonoid compound as described herein. In one embodiment, the composition includes a parenteral carrier. In another embodiment, the composition includes an oral carrier. In yet another embodiment, the composition includes an intravenous (iv) carrier.

[89] Purified limonin compound pharmaceutical or nutraceutical formulations can be prepared according to any method known to the art for the manufacture of pharmaceuticals or nutraceutical s. Such compositions can contain sweetening agents, flavoring agents, coloring agents, and/or preserving agents. Any purified limonin compound formulation can be admixed with nontoxic pharmaceutically acceptable excipient(s) which are suitable for manufacture.

[90] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition

Accordingly, there are a wide variety of suitable formulations of pharmaceutical or nutraceutical compositions of the present invention (see, e.g., Remington's, supra).

[91] In some embodiments, the composition is formulated for delivery to a desired site of action within an individual to whom it is administered. For example, the composition may be formulated for administration to a tumor (e.g., a tumor with elevated GGT and/or GSFI), for example, a drug resistant or MDR tumor.

[92] When employed as pharmaceuticals or nutraceuticals, i.e., for treatment or prophylaxis of a disease or condition, the compositions described herein are typically administered in the form of a pharmaceutical or nutraceutical composition.

[93] Generally, the compositions are administered in an effective amount, i.e., a therapeutically or prophylactically effective amount. The amount of the active agent, i.e., a limonoid compound as described herein, actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the activity of the limonoid compound administered, the age, weight, and response of the individual patient, the severity of the patient’s symptoms, and the like.

[94] In general, a limonin compound may be administered as pharmaceutical and/or nutraceutical compositions by any method known in the art for administering therapeutic drugs. The compositions can be administered by a variety of routes including oral, rectal, topical, transdermal, subcutaneous, intravenous, intraperitoneal, intramuscular, intranasal, or direct injection into a tumor. Depending on the intended route of delivery, the compositions are preferably formulated as either oral or injectable compositions or as salves, as lotions or as patches topical or transdermal administration.

[95] Compositions may take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropnate compositions; and comprise a limonin compound in combination with at least one pharmaceutically acceptable excipient. Limonin compositions are typically provided in a container or other packaging that is protected from exposure to light, such as an opaque or dark colored container. Suitable excipients are well known to persons of ordinary skill in the art, and they, and the methods of formulating the compositions, may be found in such standard references as Remington’s, supra. Suitable liquid carriers, especially for injectable solutions, include water, aqueous saline solution, aqueous dextrose solution, and glycols.

[96] Aqueous suspensions typically comprise a limonin compound in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may include a suspending agent, such as, e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.

[97] Oil suspensions can be formulated by suspending purified limonin compound in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant, such as, but not limited to, vitamin E, vitamin C, beta-carotene, a polyphenol, or a flavonoid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281 :93-102. The pharmaceutical formulations of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.

[98] The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In some embodiments of such compositions, active agent, i.e., a limonoid compound as described herein, may be a minor component (about 0.1% to about 50% by weight, or about 1% to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.

[99] Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. In some embodiments, the composition includes one or more anti-oxidant compound. Nonlimiting examples of such anti-oxidants include vitamin E, vitamin C, beta-carotene, polyphenols, and flavonoids.

[100] Injectable compositions are typically based upon injectable sterile saline or phosphate- buffered saline or other injectable carriers known in the art. As described above, the active agent, in such compositions, i.e., a limonoid compound as described herein, is typically a minor component, often being about 0.05% to 10% by weight, with the remainder being the injectable carrier and the like.

[101] In another embodiment, the purified limonoid compound formulations are useful for intravenous (IV) administration. The formulations for administration will commonly include a solution of the limonin compound dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of purified limonin glucoside in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol.

[102] The above-described components for orally administrable, or injectable administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Remington’s, supra, which is incorporated herein by reference.

[103] The limonoid compound described herein can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can be found in Remington’s Pharmaceutical Sciences.

[104] Compositions herein may be formulated for treatment or prophylaxis of one or more cancer conditions, such as a malignant tumor, for example, a lung, ovarian, breast, bone marrow, nasopharyngeal carcinoma, esophageal squamous cell, endometrial, hepatocellular, head or neck laryngeal, or intrahepatic cholangiocarcinoma tumor. In some embodiments, the tumor is a drug resistant tumor. In certain embodiments, the tumor is a MDR tumor.

[105] In some embodiments, the composition includes at least one additional compound, such as, for example, a chemotherapeutic compound, a flavonoid, or a tocotrienol, in addition to the limonoid compound, for treatment or prophylaxis of a malignant tumor (e.g., a tumor with elevated GGT and/or GSH), for example, a drug resistant or MDR tumor.

[106] After a pharmaceutical or nutraceutical containing a purified limonoid compound has been formulated in an acceptable carrier, it can be placed in an appropriate container and labeled for treatment of an indicated condition, e.g., a malignant tumor (e.g., a tumor with elevated GGT and/or GSH), for example, a drug resistant or MDR tumor. For administration of purified limonin compound, such labeling would include, e.g., instructions concerning the amount, frequency and method of administration.

[107] Compositions herein may include a therapeutically or prophylactically effective dose of about 70 mg to 21 grams/day (about 1 to about 300 mg/kgbw/day) of a purified limonoid compound as described herein, or any of the specific dosage ranges disclosed above, which may be formulated for administration in a single daily dose, multiple daily doses, or as bi-weekly, or weekly doses. A person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine a therapeutically or prophylactically effective amount of a purified limonin glucoside for practice as disclosed herein.

[108] Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope of the invention. Therefore, the description should not be construed as limiting the scope of the invention, which is delineated in the appended claims.

[109] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.