WO2016065353A1 - Combination therapy with fenofibrate and 2-deoxyglucose or 2-deoxymannose - Google Patents

Abstract

Combination therapy comprising (1) fenofibrate and (2) 2-deoxyglucose or 2-deoxymannose is disclosed. Methods of using the combination therapy in the treatment of hyperproliferative disease including cancer and pharmaceutical compositions comprising (1) fenofibrate and (2) 2-deoxyglucose or 2-deoxymannose are also disclosed.

Description

COMBINATION THERAPY WITH FENOFIBRATE AND 2-DEOXYGLUCOSE OR 2-

DEOXYMANNOSE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62/068,195, filed October 24, 2014, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present disclosure relates to combination therapy comprising fenofibrate and 2- deoxyglucose or 2-deoxymannose for use in treating cancer and other hyperproliferative diseases.

BACKGROUND

[0003] Fenofibrate (propan-2-yl 2-{4-[(4-chlorophenyl)carbonyl]phenoxy}-2- methylpropanoate; abbreviated FF) is a drug that has been prescribed for decades to treat high cholesterol. The majority of the FF administered is metabolized in vivo to fenofibric acid via hydrolysis of the carboxyl ester moiety. FF is thought to lower cholesterol and triglycerides by activating peroxisome proliferator- activated receptor alpha (PPARa), which in turn activates lipoprotein lipase, thereby increasing lipolysis and eliminating triglyceride-rich particles from the blood. FF is typically administered orally in a dosage of 40 mg to 120 mg per day for this indication.

[0004] Sugar analogs having a substitution of the hydroxyl group at the 2' position of the sugar molecule, e.g., 2-deoxyglucose compounds and 2-deoxymannose compounds, are widely used as medical imaging and diagnostic agents, e.g., to measure tissue uptake of glucose. Such sugar analogs cannot undergo further glycolysis. Examples of 2-deoxyglucose compounds include 2-deoxy-D-glucose (C₆H₁₂O₅), fluorodeoxyglucose (C₆H₁₁FO₅), and 2-deoxy-2- chloroglucose (C₆H₁₁CIO₅), and examples of 2-deoxymannose compounds include 2-deoxy-2- fluoromannose (C₆H₁₁FO₅) and 2-deoxy-2-chloromannose (C₆H₁₁CIO₅).

[0005] Both FF and 2-deoxyglucose or 2-deoxymannose have been extensively tested in human patients and are generally considered to be non-toxic, in contrast to conventional chemotherapeutic agents, whose cytotoxic effects allow for the killing of cancer cells. SUMMARY OF THE INVENTION

[0006] The present disclosure is related to combination therapy comprising (1) fenofibrate (FF) and (2) 2-deoxyglucose or 2-deoxymannose. In one aspect, a method of inhibiting cancer cell growth described herein comprises contacting a cancer cell with (1) FF and (2) 2- deoxyglucose or 2-deoxymannose in an amount effective to inhibit cancer cell growth. In a further aspect, the cancer cell is in vivo and the contacting step comprises administering FF and 2-deoxyglucose or 2-deoxymannose to a subject. In another aspect, the disclosure provides a method of treating or preventing a neoplastic, hyperplastic, or hyperproliferative disorder in a subject in need thereof comprising administering a therapeutically effective amount of FF and 2- deoxyglucose or 2-deoxymannose to the subject.

[0007] In various aspects of the methods of the present disclosure, FF and/or 2-deoxyglucose or 2-deoxymannose is administered orally, intravenously, intratumorally, topically, or intraperitoneally. In one aspect, the FF is administered in a daily dosage of about 1 mg to about 100 mg and/or the 2-deoxyglucose or 2-deoxymannose is administered in a daily dosage of about 1 mg/kg to about 60 mg/kg. In another aspect, the FF is administered in an amount effective to achieve a plasma FF concentration of about 10 μΜ to about 50 μΜ. Optionally, the FF is administered in an amount effective to achieve a plasma fenofibric acid concentration of less than about 10 μΜ. Optionally, the 2-deoxyglucose or 2-deoxymannose is selected from the group consisting of 2-deoxy-D-glucose, 2-deoxy-2-fluoroglucose, 2-deoxy-2-fluoromannose, 2- deoxy-2-chloromannose, and combinations thereof. In one aspect, the FF and/or the 2- deoxyglucose or 2-deoxymannose is administered in a sub-therapeutic amount. In one aspect, the FF and 2-deoxyglucose or 2-deoxymannose are administered concurrently. In another aspect, the FF is administered before the 2-deoxyglucose or 2-deoxymannose. In yet another aspect, the 2-deoxyglucose or 2-deoxymannose is administered before the FF.

[0008] In one aspect of the methods of the present disclosure, the amount of FF and 2- deoxyglucose or 2-deoxymannose is effective to induce apoptosis in cancer cells. In one aspect, the administration of FF and 2-deoxyglucose or 2-deoxymannose results in a synergistic increase in cancer cell death. In a related aspect, the 2-deoxyglucose is administered in an amount effective to increase the cytotoxicity of the FF compared to FF administered alone and/or the FF is administered in an amount effective to increase the cytotoxicity of the 2-deoxyglucose or 2- deoxymannose compared to 2-deoxyglucose or 2-deoxymannose administered alone. In one aspect, the administration of 2-deoxyglucose or 2-deoxymannose mitigates an insulin response. Optionally, the 2-deoxyglucose or 2-deoxymannose is administered gradually, e.g., via a slow- release pump or in a modified release formulation.

[0009] In another aspect, the FF is administered in a formulation that prevents conversion of the FF to fenofibric acid. Optionally, the formulation is a liposomal formulation. The present disclosure also provides a method of treating or preventing a neoplastic, hyperplastic, or hyperproliferative disorder in a subject in need thereof comprising administering a

therapeutically effective amount of FF in a formulation that prevents conversion of the FF to fenofibric acid to the subject. Optionally, the subject has multiple myeloma.

[0010] The present disclosure further provides a pharmaceutical composition comprising (1) FF and (2) 2-deoxyglucose or 2-deoxymannose. Optionally, the pharmaceutical composition comprises FF in a dosage amount of about 1 mg to about 100 mg and/or 2-deoxyglucose or 2- deoxymannose in a dosage amount of about 1 mg/kg to about 60 mg/kg. The present disclosure further provides kits comprising (1) FF and (2) 2-deoxyglucose or 2-deoxymannose and instructions for co-administering the FF and 2-deoxyglucose or 2-deoxymannose to a subject. In one aspect, the kit or pharmaceutical composition comprises FF and/or 2-deoxyglucose or 2- deoxymannose in a formulation to be administered orally, intravenously, intratumorally, topically, or intraperitoneally. In another aspect, the kit or pharmaceutical composition comprises FF in a formulation that prevents the conversion of FF to fenofibric acid (optionally, a liposomal formulation). In one aspect, a kit or composition provides 2-deoxyglucose or 2- deoxymannose in a modified-release formulation, such as a formulation suitable for use in a slow-release pump.

[0011] In various aspects of the present disclosure, combination therapy comprising FF and 2- deoxyglucose or 2-deoxymannose is administered to a subject having cancer. In one aspect, the cancer is selected from the group consisting of bone cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, multiple myeloma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, prostate cancer, sarcoma, skin cancer, testicular cancer, throat cancer, thyroid cancer. Optionally, the cancer is bone cancer, breast cancer, multiple myeloma, or skin cancer.

[0012] The foregoing summary is not intended to define every aspect of the invention, and other features and advantages of the present disclosure will become apparent from the following detailed description, including the drawings. The present disclosure is intended to be related as a unified document, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, paragraph, or section of this disclosure. In addition, the disclosure includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the disclosure described or claimed with "a" or "an," it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning. With respect to elements described as one or more within a set, it should be understood that all combinations within the set are contemplated. If aspects of the disclosure are described as "comprising" a feature, embodiments also are contemplated "consisting of or "consisting essentially of the feature. Additional features and variations of the disclosure will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 depicts the cytotoxicity of fenofibrate (FF), 2-deoxy-D-glucose (2DG), and oligomycin (Olig), alone or in combination, in human osteosarcoma 143B cells. The x-axis depicts the drug treatment. The y-axis depicts the percentage of cells that are dead within the total cell population following treatment (Cell Death %).

[0014] Figure 2 depicts the cytotoxicity of FF, 2DG, and Olig, alone or in combination, in human breast cancer MCF7 cells. The x-axis depicts the drug treatment. The y-axis depicts the percentage of cells that are dead within the total cell population following treatment (Cell Death %).

[0015] Figure 3 depicts the cytotoxicity of FF, 2DG, and Olig, alone or in combination, in human breast cancer SKBR3 cells. The x-axis depicts the drug treatment. The y-axis depicts the percentage of cells that are dead within the total cell population following treatment (Cell Death %). [0016] Figure 4 depicts the cytotoxicity of FF, 2DG, fluorodeoxyglucose (FDG), and Olig, alone or in combination, in human melanoma NM2C5 cells. The x-axis depicts the drug treatment. The y-axis depicts the percentage of cells that are dead within the total cell population following treatment (Cell Death %).

[0017] Figure 5A depicts ATP levels in human melanoma NM2C5 cells treated with FF, 2DG, and Olig, alone or in combination. The x-axis depicts the drug treatment. The y-axis depicts the ATP level expressed as a percentage of the ATP level in control cells (ATP %).

Figure 5B depicts ATP levels measured after 5 and 24 h of drug exposure. P value was

***p<0.001 as compared to controls.

[0018] Figure 6 depicts the cytotoxicity of FF, 2DG, and FDG, alone or in combination, in human multiple myeloma cells. The x-axis depicts the drug treatment. The y-axis depicts the percentage of cells that are dead within the total cell population following treatment (Cell Death %).

[0019] Figure 7 shows apoptosis measured as cleaved caspase-3 (Casp3) in human melanoma NM2C5 cells treated with FF and 2DG, alone or in combination.

[0020] Figure 8 shows downregulation of pERK and the anti-apoptotic protein MCL-1 in human melanoma NM2C5 cells treated with FF, 2DG, and Olig, alone or in combination.

[0021] Figure 9 shows downregulation of the autophagy marker LC3BII in cells treated with FF and 2DG alone or in combination.

[0022] Figure 10 shows tumor volume in a mouse xenograft model of human melanoma cells treated with FF and 2DG, alone or in combination. The 2DG was administered intraperitoneally (IP) or using a continuous release pump (PUMP). The x-axis depicts the treatment period in days. The y-axis depicts tumor volume in cubic millimeters.

[0023] Figure 11 shows lactate levels in cells treated with FF or 2DG alone or in combination. The x-axis depicts the treatment. The y-axis depicts the Lactate %. Lactate levels in the medium were measured after 5 and 24 h of drug exposure. P value was *p<0.05 and **p<0.01 as compared to controls.

[0024] Figure 12A shows NM2C5 cells incubated with siRNA against either luciferase (SiLuciferase, as negative control) or Noxa (SiNoxa) for 24 h before addition of 40 μΜ of FF, 2mM of 2DG or a combination of both as indicated. Following 72 h of treatment, cell death analysis was performed. P value was **p<0.01 compared to FF combined with 2DG treatment in the control (SiLuciferase) group. Figure 12B shows Western blot analysis of Noxa protein levels in NM2C5 cells treated as indicated. β-Actin was used as a loading control.

[0025] Figure 13A shows NM2C5 cells were treated with 1 mM of mannose (Mann), 20 uM of Z-VAD-FMK (zVAD) or 1 mM of glucose (Glue) in the presence of 40 μΜ FF, 2 mM 2DG or combined, for 48 h followed by cell death analysis. P value was *p<0.05 as compared to controls. Figure 13B shows a Western blot of NM2C5 cells treated with 1 mM of Mann in the presence of 40 μΜ of FF and 2 mM of 2DG for 24 h to detect the level of p-eIF2a protein.

Figure 13C shows a Western blot of NM2C5 cells treated with 1 mM of Glue in the presence of 40 μΜ of FF and 2 mM of 2DG for 24 h to detect levels of p-AMPK and p-4EBPl proteins, β- Actin was used as a loading control.

DETAILED DESCRIPTION

[0026] The present disclosure relates to combination therapy comprising (1) fenofibrate (FF) and (2) 2-deoxyglucose or 2-deoxymannose. FF, 2-deoxyglucose, and 2-deoxymannose are considered non-toxic and have been administered to human patients for non-cancer indications for years. Considering the well-established safety profile of the compounds and because they have been shown to have limited efficacy in vivo when used alone in inhibiting tumor growth (see, e.g., Saidi et al., Molecular Cancer (2006) 5:13; Raez et al., Cancer Chemother Pharmacol (2013) 71:534-530), combination therapy comprising FF and 2-deoxyglucose or 2- deoxymannose has not previously been proposed for use in treating cancer and would not be expected to have any clinical advantages.

[0027] As described herein, combination therapy comprising FF and 2-deoxyglucose or 2- deoxymannose is surprisingly effective at inhibiting growth of and killing a wide variety of cancer cell types, even when FF and 2-deoxyglucose or 2-deoxymannose are administered at doses that are not therapeutically effective when administered alone. The synergistic cytotoxicity achieved using the combination of FF and 2-deoxyglucose or 2-deoxymannose according to the present disclosure allows for treatment of cancer and other hyperproliferative diseases using smaller doses of drug, thereby further minimizing potential side effects. [0028] The following definitions may be useful in aiding the skilled practitioner in understanding the disclosure. Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0029] The term "2-deoxyglucose" refers to a glucose molecule wherein the 2-hydroxyl group is substituted with hydrogen or another atom, e.g., a halogen atom. Examples of 2-deoxyglucose compounds include 2-deoxy-D-glucose ((4R,5S,6R)-6-(hydroxymethyl)oxane-2,4,5-triol;

abbreviated 2DG), fluorodeoxyglucose (2-deoxy-2-fluoroglucose; abbreviated FDG), and 2- deoxy-2-chloroglucose. The term "2-deoxymannose" refers to a mannose molecule wherein the 2-hydroxyl group is substituted with hydrogen or another atom, e.g., a halogen atom. Examples of 2-deoxymannose compounds include 2-deoxy-2-fluoro-D-mannose and 2-deoxy-2-chloro-D- mannose.

[0030] The terms "co-administering" and "combination therapy" mean that FF and 2- deoxyglucose or 2-deoxymannose are administered in a manner that permits the two compounds to exert physiological effects during an overlapping period of time. In combination therapy comprising FF and 2-deoxyglucose or 2-deoxymannose, the compounds may be administered in the same pharmaceutical composition or in separate compositions, via the same or different routes of administration. The FF and 2-deoxyglucose or 2-deoxymannose may be coadministered concurrently, i.e., simultaneously, or at different times, as long as the compounds exert physiological effects during an overlapping period of time. For example, the FF and 2- deoxyglucose or 2-deoxymannose may both be administered to a subject within a time period of about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 24, or about 48 hours. For example, 2-deoxyglucose or 2-deoxymannose can be administered as a continuous intravenous infusion or in a slow-release oral formulation once or twice a day, and FF can be given orally or intravenously once or twice a day. If the FF and 2-deoxyglucose or 2- deoxymannose are not co-administered concurrently, either the FF or 2-deoxyglucose or 2- deoxymannose may be administered first. As long as the subsequent compound is administered while a physiological effect of the first compound is present, the compounds are considered to be co-administered and used in combination therapy in accordance with the teachings of the disclosure. [0031] The terms "therapeutically effective amount" and "effective amount" refer to an amount of a single agent or combination therapy effective to achieve a desired biological, e.g., clinical, effect. A therapeutically effective amount varies with the nature of the disease being treated, the length of time that activity is desired, and the age and the condition of the subject. In one aspect, a therapeutically effective amount is an amount effective to inhibit growth of hyperproliferative cells, prevent cancer cell metastasis, and/or result in cancer cell death, e.g., via apoptosis or necrosis. A "sub-therapeutic amount" refers to an amount of a single agent that does not achieve a desired biological, e.g., clinical, effect. For example, a sub-therapeutic amount of FF or 2-deoxyglucose or 2-deoxymannose may be an amount that does not cause a significant increase in cancer cell death compared to control (untreated) cells.

[0032] The term "synergistic increase" refers to an improvement in a therapeutic effect from administration of combination therapy comprising FF and 2-deoxyglucose or 2-deoxymannose compared to the sum of the therapeutic effects of FF and 2-deoxyglucose or 2-deoxymannose alone. For example, if a dosage of FF administered alone achieves X% cell death and a dosage of 2-deoxyglucose or 2-deoxymannose administered alone achieves Y% cell death, then a combination of those doses of FF and 2-deoxyglucose or 2-deoxymannose that achieves greater than (X+Y)% cell death is considered to have a synergistic increase in cell death. In one aspect, a synergistic increase refers to a therapeutic effect resulting from combination therapy comprising FF and 2-deoxyglucose or 2-deoxymannose administered in sub-therapeutic amounts.

[0033] The term "administered gradually" refers to administration of a drug to a patient or release of a drug from a formulation in a manner that is slower compared to an intravenous or intraperitoneal bolus or immediate release formulation (i.e., a formulation wherein the drug is intended to be released immediately following administration to a subject). A "modified release formulation" refers to a formulation wherein the drug release rate differs from an immediate release formulation. Modified release formulations include extended-release, delayed-release, and targeted-release formulations, and methods for their preparation are known in the art.

[0034] The present disclosure provides methods of treatment comprising administering a therapeutically effective amount of (1) FF and (2) 2-deoxyglucose or 2-deoxymannose to a subject in need thereof. In one aspect, a method of inhibiting cancer cell growth comprises contacting a cancer cell with FF and 2-deoxyglucose or 2-deoxymannose in an amount effective to inhibit cancer cell growth. In a further aspect, the cancer cell is in vivo and the contacting step comprises administering FF and 2-deoxyglucose or 2-deoxymannose to a subject, e.g., a human patient. A method of treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder in a subject in need thereof is also provided and comprises administering a

therapeutically effective amount of FF and 2-deoxyglucose or 2-deoxymannose to the subject. The present disclosure also provides use of FF and 2-deoxyglucose or 2-deoxymannose for the preparation of a medicament, wherein the medicament comprises an amount of FF and 2- deoxyglucose or 2-deoxymannose that is effective for treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder.

[0035] In one aspect, the administration of FF and 2-deoxyglucose or 2-deoxymannose results in a synergistic increase in cell (e.g., cancer or other hyperproliferative cell) death, i.e., compared to the effects of FF and 2-deoxyglucose or 2-deoxymannose administered alone. In one aspect, the 2-deoxyglucose or 2-deoxymannose is administered in an amount effective to increase the cytotoxicity of the FF compared to FF administered alone. In another aspect, the FF is administered in an amount effective to increase the cytotoxicity of the 2-deoxyglucose or 2- deoxymannose compared to 2-deoxyglucose or 2-deoxymannose administered alone. In still another aspect, both the 2-deoxyglucose or 2-deoxymannose and the FF are administered in an amount effective to increase the cytotoxicity of the other compound compared to the other compound administered alone. Optionally, one or more additional compounds may be administered to increase the efficacy of combination therapy comprising FF and 2-deoxyglucose or 2-deoxymannose. Examples of such additional compounds include, but are not limited to, butylated hydroxyanisole (BHA), any inhibitor of the anti-apoptotic protein MCL-1 or its upstream regulator ERK, and any inhibitor of protein kinase B (also known as Akt). Examples of inhibitors of Akt include Akt Inhibitor X (ΑΓΧ) and curcumin.

[0036] In one aspect, the methods of the present disclosure comprise administering FF and/or

2-deoxyglucose or 2-deoxymannose in a sub-therapeutic amount, i.e., an amount that does not on its own achieve a desired biological effect. In one aspect, a sub-therapeutic dose of FF or 2- deoxyglucose or 2-deoxymannose has an anticancer effect comparable to a placebo. For example, a sub-therapeutic dose of FF or 2-deoxyglucose or 2-deoxymannose may achieve growth inhibition of cancer cells that is not significantly different than no treatment at all or may actually promote tumor growth. In one aspect, the FF is administered in a sub-therapeutic amount. In another aspect, the 2-deoxyglucose or 2-deoxymannose is administered in a subtherapeutic amount. In still another aspect, both the FF and 2-deoxyglucose or 2-deoxymannose are administered in a sub -therapeutic amount. According to the methods of the present disclosure, even when FF and/or 2-deoxyglucose or 2-deoxymannose is administered in a subtherapeutic amount, the combination of both compounds is still therapeutically effective.

[0037] In one aspect, the disease to be treated is cancer. Examples of treatable cancers include, but are not limited to, adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B- cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear-cell sarcoma of the kidney,

craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer,

hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangio sarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogeous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non- small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, meduUoblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular cancer, oligo astrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma periotonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, squamous carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.

[0038] For example, the methods of the present disclosure can be used to treat a cancer selected from the group consisting of bone cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, multiple myeloma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, prostate cancer, sarcoma, skin cancer, testicular cancer, throat cancer, and thyroid cancer. In some aspects, the disease to be treated is bone cancer, breast cancer, multiple myeloma, or skin cancer. One of ordinary skill will appreciate that treating a cancer does not require complete eradication of the cancer. Any beneficial physiologic response is contemplated, such as tumor shrinkage, tumor cell death, reduction or halting of metastasis, reduction in cancer cell markers, alleviation of symptoms and the like.

[0039] In another aspect, the disease to be treated is a benign hyperproliferative disorder including, but not limited to, benign soft tissue tumors, bone tumors, brain and spinal tumors, eyelid and orbital tumors, granuloma, lipoma, meningioma, multiple endocrine neoplasia, nasal polyps, pituitary tumors, prolactinoma, pseudotumor cerebri, seborrheic keratoses, stomach polyps, thyroid nodules, cystic neoplasms of the pancreas, hemangiomas, vocal cord nodules, polyps, and cysts, Castleman disease, chronic pilonidal disease, benign prostate hyperplasia, dermatofibroma, pilar cyst, pyogenic granuloma, and juvenile polyposis syndrome.

[0040] In one aspect of the present methods, a therapeutically effective amount of

combination therapy described herein, typically formulated in accordance with pharmaceutical practice, is administered to a subject in need thereof. A particular administration regimen for a particular subject will depend, in part, upon the type and severity of the disease to be treated, the amount of combination therapy administered, the route(s) of administration, and the cause and extent of any side effects. The amount of combination therapy administered to a subject (e.g., a mammal, such as a human) in accordance with the invention should be sufficient to effect the desired response over a reasonable time frame. Dosage typically depends upon the route, timing, and frequency of administration. Accordingly, a clinician titers the dosage and modifies the route of administration to obtain the optimal therapeutic effect. The treatment period will depend on the particular condition and subject and may last one day to several days, weeks, months, or years.

[0041] The present disclosure also provides a pharmaceutical composition comprising (1) FF and (2) 2-deoxyglucose or 2-deoxymannose. In one aspect, the pharmaceutical composition comprises FF in a dosage amount of about 1 mg to about 100 mg and/or 2-deoxyglucose or 2- deoxymannose is a dosage amount of about 1 mg/kg to about 60 mg/kg based on the weight of the tumor or subject. In one aspect, the pharmaceutical composition is for use in the treatment of a neoplastic, hyperplastic, or hyperproliferative disease, such as cancer. The present disclosure also provides use of a composition described herein for the preparation of a medicament, wherein the medicament comprises an amount of the composition that is effective for treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder. In one aspect, the pharmaceutical composition comprises a pharmaceutically acceptable carrier including, but not limited to, water, saline, phosphate buffered saline, and commercial buffers. Preferably, the composition is sterile. Other excipients, including buffering agents, dispersing agents, and preservatives, are known in the art and may be included in the pharmaceutical composition. Further examples of components that may be employed in compositions are presented in Remington's Pharmaceutical Sciences, 16^th Ed. (1980) and 20^th Ed. (2000), Mack Publishing Company, Easton, Pa. A composition may be in any suitable dosage form including, but not limited to, tablets, capsules, implants, depots, liquids, patches, pumps, lozenges, creams, ointments, lotions, aerosols, and eye drops.

[0042] The present disclosure also provides a kit comprising (1) FF and (2) 2-deoxyglucose or 2-deoxymannose and instructions for co-administration of a therapeutically effective amount of the FF and 2-deoxyglucose to a subject having cancer. In one aspect, a kit or pharmaceutical composition of the present disclosure comprises FF and/or 2-deoxyglucose or 2-deoxymannose in a formulation to be administered orally, intravenously, intratumorally, topically or

intraperitoneally.

[0043] Suitable methods of administering FF and 2-deoxyglucose or 2-deoxymannose, for example, in a pharmaceutical composition of the present disclosure, are well-known in the art.

Although more than one route can be used to administer a compound, a particular route can provide a more immediate and more effective reaction than another route. Depending on the circumstances, FF and/or 2-deoxyglucose or 2-deoxymannose is introduced into tumor sites, applied or instilled into body cavities, absorbed through the skin or eye or mucous membranes, inhaled, ingested, and/or injected or otherwise introduced into circulation. FF and/or 2- deoxyglucose or 2-deoxymannose can be administered orally or injected intravenously or intraperitoneally, resulting in systemic uptake, or administered locally by directly contacting tumor cells. In certain circumstances, it will be desirable to deliver FF and/or 2-deoxyglucose or 2-deoxymannose through injection or infusion by intravenous, intratumoral, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intranasal, transdermal, enteral, topical, sublingual, urethral, vaginal, or rectal means; by controlled, delayed, gradual, sustained or otherwise modified release systems; or by implantation devices. If desired, FF and/or 2-deoxyglucose or 2-deoxymannose is administered regionally via intratumoral administration, intraocular administration, intrathecal administration, intracerebral (intra-parenchymal) administration, intracerebroventricular administration, topical administration, subcutaneous administration, or intraarterial, intravenous, or intraperitoneal administration targeting the region of interest. Alternatively, the combination therapy is administered locally via implantation of a matrix, membrane, pump, sponge, or another appropriate material loaded with FF and/or 2-deoxyglucose or 2-deoxymannose. Where an implantation device is used, the device is, in one aspect, implanted into any suitable site (e.g., tissue, organ, or cavity), and delivery of the desired compound is, for example, via diffusion, timed-release bolus, gradual administration, or continuous administration. In various aspects, the FF and/or 2-deoxyglucose or 2-deoxymannose may be administered orally, intravenously, intratumorally, topically or intraperitoneally.

[0044] In one aspect, the FF and 2-deoxyglucose or 2-deoxymannose are administered concurrently. In another aspect, the 2-deoxyglucose or 2-deoxymannose is administered before the FF. In still another aspect, the FF is administered before the 2-deoxyglucose or 2- deoxymannose. Purely by way of illustration, the methods of the present disclosure comprise administering, e.g., from about 1 mg/day to about 100 mg/day or more of FF and/or from about 1 mg/kg/day to about 60 mg/kg/day or more of 2-deoxyglucose or 2-deoxymannose, based on the weight of the tumor or subject, depending on the factors mentioned above. In some aspects, the daily dosage of FF ranges from about 50 mg to about 100 mg, about 10 mg to about 40 mg, about 1 mg to about 20 mg, or about 20 mg to about 50 mg. In related aspects, the daily dosage of 2-deoxyglucose or 2-deoxymannose ranges from about 1 mg/kg to about 25 mg/kg, about 10 mg/kg to about 40 mg/kg, about 5 mg/kg to about 20 mg/kg, or about 30 mg/kg to about 60 mg/kg. In one aspect, the methods comprise administering FF in amount effective to achieve a plasma FF concentration of about 10 μΜ to about 50 μΜ and/or 2-deoxyglucose or 2- deoxymannose in an amount effective to achieve a plasma 2-deoxyglucose or 2-deoxymannose concentration of about 50 μg/mL to about 400 μg/mL. In some aspects, the FF is administered in an amount effective to achieve a plasma FF concentration of about 10 μΜ to about 30 μΜ, for example, about 10 μΜ to about 20 μΜ, about 15 μΜ to about 40 μΜ, or about 25 μΜ to about 50 μΜ. In related aspects, the 2-deoxyglucose or 2-deoxymannose is administered in an amount effective to achieve a plasma 2-deoxyglucose or 2-deoxymannose concentration of about 100 μg/mL to about 200 μg/mL, or about 250 μg/mL to about 400 μg/mL, or about 40 μg/mL to about 150 μg/mL. The foregoing dosages for use in the methods and compositions of the present disclosure are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this invention.

[0045] In one aspect, the administration of 2-deoxyglucose or 2-deoxymannose mitigates an insulin response. Modified glucose molecules including 2-deoxyglucose or 2-deoxymannose can stimulate the release of insulin from the pancreas. The insulin response leads to adsorption of 2- deoxyglucose or 2-deoxymannose by fat and muscle cells, thereby reducing the amount delivered to target cells. In one aspect of the methods of the present disclosure, the 2- deoxyglucose or 2-deoxymannose is administered gradually, for example, via a slow-release pump or in a modified-release formulation. A slow-release pump, such as a continuous-release osmotic pump, or modified-release formulation gradually administers drug over an extended period of time. In one aspect, a kit or pharmaceutical composition comprises 2-deoxyglucose or 2-deoxymannose in a modified-release formulation. In one aspect, the 2-deoxyglucose or 2- deoxymannose is administered at a rate slower than 50 μg/mL/hour, for example, about 40 μg/mL/hour, about 30 μg/mL/hour, or about 20 μg/mL/hour. Such a gradual administration of 2- deoxyglucose or 2-deoxymannose mitigates, and may completely avoid, an insulin response that a single equivalent dose administered as a bolus can trigger (see U.S. Patent Application No. 12/994,265, incorporated herein by reference).

[0046] In one aspect of the present disclosure, FF is administered in a formulation that prevents conversion of the FF to fenofibric acid. FF is typically rapidly and extensively (up to

99%) metabolized to fenofibric acid in vivo. When FF is not converted to fenofibric acid, the anticancer effect of FF can be improved, particularly for cancer cells having an enlarged endoplasmic reticulum, such as multiple myeloma cells. A formulation that protects the FF and prevents its conversion to fenofibric acid, such as administering FF in a liposome, microparticle, or nanoparticle, could therefore improve therapeutic efficacy of the FF. In one aspect, a kit or pharmaceutical composition comprises FF in a formulation that prevents the conversion of FF to fenofibric acid. The present disclosure thus also provides a method of treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder in a subject in need thereof comprising administering a therapeutically effective amount of FF in a formulation that prevents conversion of the FF to fenofibric acid to the subject. In one aspect, the subject has multiple myeloma. In one aspect of the methods of the present disclosure, FF is administered in an amount effective to achieve a plasma fenofibric acid concentration of less than about 10 μΜ, for example, less than about 8 μΜ, less than about 5 μΜ, less than about 3 μΜ, or less than about 1 μΜ.

[0047] In one aspect, the methods and compositions of the present disclosure induce cytotoxic effects via the induction of apoptosis or necrosis. In one aspect, the administration of FF and 2- deoxyglucose or 2-deoxymannose is effective to induce apoptosis in cancer cells. Apoptotic cells may be identified by histological markers such as nuclear and cytoplasmic condensation and cellular fragmentation. Necrotic cells may be identified by histological markers such as cellular and organelle swelling, chromatin flocculation, loss of nuclear basophilia, degraded cytoplasmic structure, impaired organelle function, increased membrane permeability, and cytolysis. One mechanism for inducing apoptosis and/or necrosis involves the activation of initiator caspases (e.g., caspase-2, caspase-8, caspase-9, caspase-10), executioner caspases (e.g., caspase-3, caspase-6) and also pro-inflammatory caspases (e.g., caspase-1 and caspase-13). The role of the pro-inflammatory caspases in cell death is likely related to their ability to induce host inflammatory responses in vivo. Caspase activity can therefore be analyzed as a measure of apoptosis and/or necrosis. Other methods for detecting cell death via apoptosis and/or necrosis known in the art are also suitable for measuring the antitumor activity of the compositions of the present disclosure, including Tdt-mediated dUTP nick-end labeling (TUNEL) assays, in situ end labeling (ISEL) assays, DNA laddering assays, DNA fragmentation assays, fluorescence- activated cell sorting (FACS)/flow cytometry analysis, microscopy analysis, cell staining (e.g., using trypan blue, propidium iodide,7-actinomycin D, Annexin V, Hoescht, fluorescein diacetate-green, DAPI, and/or other dyes known in the art) assays, and enzyme-linked immunosorbent assays (ELISA). Apoptosis can also be promoted via a down-regulation of anti- apoptotic proteins such as MCL-1.

[0048] Tumor growth also can be analyzed to determine the antitumor activity of the combination therapy of the present disclosure. Tumor mass, volume, and/or length can be assessed using methods known in the art such as calipers, ultrasound imaging, computed tomography (CT) imaging, magnetic resonance imaging (MRI), optical imaging (e.g., bioluminescence and/or fluorescence imaging), digital subtraction angiography (DSA), positron emission tomography (PET) imaging and/or other imaging analysis. Tumor cell proliferation can also be analyzed using cellular assays that measure, e.g., DNA synthesis, metabolic activity, antigens associated with cell proliferation, and/or ATP. In various embodiments, the method of the present disclosure reduces the size of a tumor at least about 5% (e.g., at least about 10%, at least about 15%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%).

[0049] Without intending to be bound by theory, the unexpected therapeutic efficacy of combination therapy comprising FF and 2-deoxyglucose or 2-deoxymannose may be associated with a suppression of protective cellular responses to stress, including autophagy and anti- apoptosis pathways. Although FF is known to impair mitochondrial function, the combination of FF and 2-deoxyglucose is more toxic to cancer cells than 2-deoxyglucose and oligomycin (a more potent mitochondrial inhibitor) used together, indicating that the therapeutic efficacy of FF and 2-deoxyglucose or 2-deoxymannose is not merely due to the effect of FF on mitochondria. Additionally, studies involving other PPARa agonists (data not shown), indicate that the mechanism of action for the therapeutic efficacy of FF and 2-deoxyglucose or 2-deoxymannose is PPARa-independent.

[0050] The present disclosure will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

Example

[0051] The following examples describe in vitro and in vivo assays comparing the cytotoxic effects of FF and 2-deoxyglucose alone and in combination.

[0052] Materials and methods

[0053] Cell lines and reagents: NM2C5 human melanoma cells, 143B human osteosarcoma cells, MCF-7 and SKBR3 human breast cancer cells, and human multiple myeloma cells were purchased from ATCC (Manassas, VA). All cell lines were cultured in low glucose (1 mg/mL) DMEM growth medium supplemented with 10% FBS and penicillin/streptomycin (Invitrogen, Grand Island, NY). [0054] Drugs and antibodies: 2-deoxy-D-glucose, mannose, D-(+)-Glucose, rapamycin, and fenofibrate were purchased (Sigma- Aldrich, St. Louis, MO). General Caspase Inhibitor Z-VAD- FMK (FMK001) was obtained from R&D Systems (Minneapolis, MN). The following rabbit primary antibodies (Cell Signaling Technology, Danvers, MA) were used: LC3B (2775, which preferentially detects LC3B-II), Grp78 (3177), CHOP (2895), pAMPKa (Thrl72, 2535), p- p70S6K (Thr389, 9234), p-eIF2a (Ser51, 3597), p-4EBPl (Thr37/46 , 9459), Cleaved Caspase-3 (9664) and Mcl-1 (4572). Mouse anti-b-actin (A5441) primary antibody (Sigma- Aldrich), mouse anti-Noxa (OP180, Calbiochem), horseradish peroxidase-conjugated anti-rabbit (W4011) and anti-mouse (W4021) secondary antibody (Promega, Madison, WI) were also used.

[0055] Cytotoxicity assay: Cells were seeded onto 24- well plates and cultured overnight. Melanoma, osteosarcoma, and breast cancer cells were incubated for 48 hours with growth medium or 0.1% DMSO as controls, or with FF (40 μΜ), 2-deoxy-D-glucose (2DG; 2 mM), 2- deoxy-2-fluoroglucose (FDG; 2 mM), oligomycin (0.1 μg/mL), or a combination of the foregoing. Multiple myeloma cells were incubated for 24 hours with control medium or with 20 μΜ or 40 μΜ FF, 1 mM or 2 mM 2DG, 2 mM FDG, or a combination of the foregoing.

Following the 24- or 48-hour exposure, attached cells and their respective culture media were collected and centrifuged at 400 g for 5 minutes. The pellets were then resuspended in Hanks Balanced Salt Solution (HBSS) and analyzed with a Vi-CELL® cell viability analyzer (Beckman Coulter, Brea, CA) based on trypan blue exclusion. Results were calculated as the percentage of dead cells out of the total cells counted.

[0056] Fluorescent microscopy: Cells were seeded onto 24- well plates and cultured for 18-22 h to approximately 60% confluence. After 24 h of drug exposure, cells were stained with DAPI (0.5 ug/ml) and visualized immediately with the Nikon Eclipse TE2000 microscope (Nikon, Melville, NY) to analyze changes in nuclear condensation and fragmentation. Microphotographs of the center of each well were taken at 60X magnification with the aid of imaging-capture software (NIS-Elements from Nikon, Melville, NY).

[0057] ATP quantification: Intracellular ATP levels were measured with the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI) according to the manufacturer's directions. Briefly, cells were seeded onto 96-well plates and cultured overnight to reach about 70% confluence. After 5 hours of drug exposure, cells were lysed in the same plate with the reagent included in the assay kit for 10 minutes. The mixtures were transferred onto opaque- walled 96-well plates, and luminescence produced from ATP-mediated chemical reaction was read by the luminescence module of the FLUOstar OPTIMA microplate reader (BMG

LABTECH, Ortenberg Germany). Readouts from control samples were set as 100% and those from all the other samples were presented as percentages of controls. Results were the averages of triplicate samples +/- SD from one representative experiment out of at least three independent ones. Short-time (5 h or 24 h) treatment was employed to minimize the toxicity and cell number loss caused by drug exposure.

[0058] Immunoblotting analysis: NCM25 cells were seeded onto six- well plates and cultured overnight to reach 40% to 70% confluence. Following drug exposure for 22 hours, cells were harvested and lysed with the lysis buffer (100 mM Tris-HCl at pH 7.4, 1% SDS, phosphatase inhibitor cocktail 2 and protease inhibitor cocktail) (Sigma- Aldrich, St. Louis, MO). Protein concentrations of each sample were determined using a Micro BCA™ Protein Assay Kit (Thermo Scientific, Rockford, IL) according to the manufacturer's directions, and equal amounts of proteins were loaded onto 4% to 15% Tris-HCl gradient gels (Bio-Rad, Hercules, CA). After SDS-PAGE, proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore), blocked with 5% milk, and probed with corresponding primary antibodies overnight (1 hour for β-actin). The membrane was washed and probed with secondary antibodies for 1 hour. The membrane was then incubated with SuperSignal West Pico or Femto

Chemiluminescent Substrate (Thermo Scientific), and signals were visualized on Blue Lite Autorad Films (ISCBio-Express, Kaysville, UT).

[0059] Oxygen consumption (mitochondrial respiration): Cells were grown in 75-cm² flasks until they were 70% to 80% confluent, and then were trypsinized. Cells (5 x 10⁶) were resuspended in 1 mL of RPMI, which did not contain glucose or fetal bovine serum, and 2DG or oligomycin. Oxygen consumption was measured with a Clark electrode (Hansatech) for 10 minutes, and the rate was expressed as (nmol oxygen)/min/10⁶ cells.

[0060] Lactate assay: At 24h after seeding, grown cells were rinsed twice with PBS, fresh medium was added to the control and test cultures, at the indicated drug concentrations, and the cells were re-incubated in 5% C0₂ for 5 or 24 h. Then, 0.5 ml of medium was removed from each culture and deproteinated by adding 1 ml of perchloric acid at 8% w/v, vortexing for 30 s, then placing this mixture in 4 °C for 5 min, and centrifuging at 1,500 g for 10 min. The supernatent was centrifuged three times more. To determine lactic acid, 0.025 ml of this supernatent was added to a reaction mixture containing 0.1 ml of lactic dehydrogenase (1,000 units/ml), 2 ml of glycine buffer (glycine, 0.6 mol/1, and hydrazine, pH 9.2), and 1.66 mg/ml NAD. Formation of NADH was measured with a Beckman DU 520 UV/VIS spectrophotometer at 340 nm, which directly corresponded to lactic acid levels as determined by a lactate standard curve. Readouts from control samples, normalized to cell number, were set at 100% and those from all the other samples were presented as percentages of controls. Samples were analyzed in triplicate.

[0061] siRNA transfection: Cells were seeded into 12 welled plates and cultured for 24 h to reach approximately 30% confluence using antibiotic-free media. Then, cells were transfected with either anti-Luc siRNA-1 (targeting luciferase) or Noxa siRNA (sc-37305, Santa Cruz Biotechnology, Inc.). Twenty-four hours after transfection, cells were treated for the indicated times and collected for immunoblotting or cytotoxicity analyses. The lowest concentrations of siRNAs were determined by dose response experiments which produced the most efficient knockdown with minimal toxicity.

[0062] Statistics: Statistical analyses were performed by two-tailed unpaired Student's t-test, and a P value less than 0.05 was considered significant.

[0063] Animal studies: NM2C5 cells (2 x 10⁶ cells in 20 iL of HBSS) were injected subcutaneously into single mammary fat pads of female nude CD-I mice. When the tumors reached a palpable size of 3 mm to 4 mm in diameter (approximately 14 days after implantation), mice were randomized into groups of 8 mice with comparable tumor size each and treated as follows: (1) 500 mg/kg of 2DG intraperitoneally every other day; (2) 100 mg/kg of FF by gavage every day; (3) ALZET® osmotic pumps (Durect Corporation, Cupertino, CA) filled with 2DG subcutaneously implanted in the mouse to deliver 41 μg/mL/hour of 2DG continuously; (4) combined FF and 2DG (intraperitonally or by pump); (5) saline injections or subcutaneously implanted pumps filled with saline (control). FF was formulated in 1% methylcellulose and 1% Tween-80, and 2DG was in PBS. To implant the pumps, mice were anesthetized by 62 mg of ketamine hydrochloride and 5 mg of Xylazine/kg i.p. Following the manufacturer's instructions, a skin incision was made in the back of the mouse. The pump filled with saline or 2DG was inserted and the incision was closed with 2 or 3 wound clips. Tumor growth was evaluated at least once per week by measuring tumor dimension with a digital caliper, and tumor volume was calculated based on ellipsoid dimensions (width x length x 0.5) until the experiment ended within two months.

[0064] Results and Discussion

[0065] When combination therapy comprising FF and 2DG or FDG was administered, significant and synergistic toxicity was observed in a variety of cancer cell types at doses for which either compound, when administered alone, resulted in little to no toxicity (Figures 1-4 and 6). In human osteosarcoma cells, FF or 2DG administered alone killed less than 5% of the cells. In contrast, the combination of FF and 2DG resulted in about 35% cell death, a seven-fold increase compared to FF or 2DG alone (Figure 1). Similarly, in breast cancer cells, the combination of FF and 2DG achieved a more than two-fold (Figure 2) or more than three-fold (Figure 3) increase in cell death compared to 2DG or FF alone. In human melanoma cells, the combination of FF and either 2DG or FDG achieved the greatest cell killing of all the therapies tested (Figure 4). FF, 2DG, or FDG administered alone killed 10% or less of the cells. In contrast, the combination of FF and 2DG resulted in greater than 30% cell death, a more than three-fold increase compared to FF or 2DG alone. The combination of FF and FDG achieved greater than 40% cell death, a more than five-fold increase compared to FF or FDG alone. In multiple myeloma cells, the combination of 40 μΜ FF with 1 mM or 2 mM 2DG or FDG achieved a synergistic increase in cell death compared to the therapeutic effects of FF, 2DG, and FDG alone (Figure 6). Using morphological analyses, it was found that FF and 2DG induced cell death in the form of both necrosis (i.e., faintly stained nuclear "ghosts," indistinct vacuolated cytoplasm which appear light due to ruptured plasma membranes) and apoptosis (i.e., cells in early stage of apoptosis show aggregated chromatin abutting the nuclear membrane and condensed, basophilic cytoplasm). The morphological studies confirmed the absence of toxicity with either 2DG or FF alone, and synergistic increases in overall cell death at 24h when the two were combined.

[0066] The mitochondrial inhibitor oligomcyin, when used in combination with 2DG, was not as effective as combination therapy comprising 2DG and FF (Figures 1-4). Oligomycin is a more rapid and potent inhibitor of mitochondrial respiration than FF. Table 1 shows the rate of 0₂ consumption as a measure of mitochondrial respiration.

Table 1

[0067] The results using oligomycin indicated that FF was not sensitizing cells to 2DG by blocking mitochondrial function. In the intact melanoma cell line NM2C5 tested, the effects of FF on respiration as measured by oxygen consumption could not be detected at early time points (5 min) but at later times (5 h) and (24 h) modest reductive effects were observed. The result led to investigation of the ability of FF to convert NM2C5 metabolism from aerobic to anaerobic. It is well established that when mitochondrial function is inhibited, cells increase glycolysis and are forced to rely on this energy producing pathway for survival. Under these conditions pyruvate can no longer be efficiently oxidized by mitochondria, which results in a significant increase in lactate. At an early time point (5 h), FF induced a 50% increase in lactate which increases further at 24 h (100%) (Figure 11). Moreover, as expected, 2DG alone lowered lactate about 50% at both time points, and when combined with FF, lactate levels stimulated by FF alone were similarly decreased. The results indicated that the low dose of 2DG (2mM) used in the experiments was sufficient to inhibit glycolysis and that the FF-induced increase in lactate at 5 h could be due to FF's modest effects on mitochondrial oxygen consumption.

[0068] At 5 h, when FF was combined with 2DG, ATP was lowered by 64% of control and even more reduced at 24 h (71% ) (Figure 5B). The severe drop in ATP leading to significant energy stress correlated with the necrotic form of cell death observed in the morphological analyses. Moreover, ATP was found to be lowered similarly by oligomycin and FF when either are combined with 2DG, even though FF and 2DG in combination was significantly more toxic than oligomycin and 2DG together (Figure 5A). The results indicated that the synergistic cytoxicity of FF and 2DG was not simply due to the mechanism of FF blocking mitochondrial function and making the cell dependent on glycolysis and therefore sensitive to 2DG.

[0069] The combination of FF and 2DG induced apoptosis in human melanoma cells, as evidenced by caspase 3 cleavage, whereas FF or 2DG alone did not (Figure 7). 2DG has been shown to increase ER stress and the subsequent unfolded protein response (UPR) markers GRP 78 and CHOP. When melanoma cells were co-treated with 2DG and FF, GRP78 was attenuated (Figure 7), further confirming that FF converted cells from aerobic to anaerobic metabolism. CHOP was also reduced by the combination of 2DG and FF. However, phosphorylation of eIF2a, indicative of ER stress, as well as the energy stress sensor pAMPK , were both found to be increased, leading to the question of whether down-regulation of GRP 78 by the combination of FF and 2DG could result from the markedly lowered levels of ATP detected, leading to increased levels of p-AMPK and resulting in attenuation of mTOR (as measured by reduced phosphorylation of its downstream targets (P70S6K and 4EBP1).

[0070] To directly test the role of mTOR in lowering GRP78, melanoma cells were co-treated with 2DG and rapamycin. GRP78 induced by 2DG alone was completely inhibited when cells were co-treated with rapamycin, although there is no apparent cell toxicity. This result indicated that under conditions of energy stress (lowered ATP), components of the UPR required to respond to ER stress, i.e. GRP 78 and CHOP are compromised. The increased levels of p-eIF2a detected under these conditions could be indicative of greater ER stress resulting from the blockage of mTOR-mediated protein translation of GRP78, required for protein folding.

[0071] Additionally, the combination of FF and 2DG down-regulated the anti-apoptosis protein MCL-1 more than FF or 2DG alone, and also lowered levels of pERK, an upstream kinase known to regulate MCL-1 (Figure 8). MCL-1 has been reported to correlate with survival in numerous cell types. MCL-1 is the protein target of the pro-apoptotic BH3-only protein Noxa, and the combination of FF and 2DG resulted in increased Noxa. In order to determine whether changes in levels of these apoptotic proteins were involved with the toxicity induced, cells treated with 2DG and FF were co-treated with siRNA specific to Noxa. Significant but not complete protection from cell death was achieved with this treatment (Figure 12A), although levels of MCL-1 did not appear to recover (Figure 12B). Thus, the 2DG and FF-induced reduction in MCL-1 levels was more likely mediated by translational inhibition (mTOR and or eIF2a) due to metabolic and/or ER stress, and not by upregulation of Noxa. In support of this interpretation, when the toxicity induced by FF and 2DG was rescued by either mannose or glucose (Figure 13A), the functionality of eIF2cc and mTOR (4EBP1) were found to be recovered (Figures 13B and 13C), corresponding with increases in MCL-1. These results indicated that the levels of MCL-lare regulated by both ER and energy stress. Overall, the data demonstrated that combining FF and 2DG resulted in marked reduction of this critical anti- apoptotic protein. The effect of the combination of 2DG and FF on inhibiting the pERK/MCL-1 pathway contributes to the impaired growth of tumor cells.

[0072] The combination of FF and 2DG also inhibited the autophagy marker LC3BII (Figure 9). Autophagy is the controlled degradation of cellular components and has a protective effect in response to cellular stress. The inhibition of such an important biological process may contribute to the therapeutic efficacy of the combination therapy. However, because the combination therapy was well-tolerated and significantly lowered tumor volume, the inhibition of autophagy did not appear to be detrimental enough to normal cells to cause undue or treatment-restrictive toxicity.

[0073] In a xenograft mouse model of human melanoma, when 2DG was combined with FF, there was a significant and synergistic increase in tumor volume control compared to untreated animals or those treated with FF or 2DG alone (Figure 10). Mice treated with FF exhibited increases in tumor volume comparable to untreated animals. Better tumor volume control was seen with combination therapy comprising FF and 2DG when 2DG was administered via slow- release pump compared to intraperitoneal injection. Moreover, the total dose per week, 462 mg/kg, delivered by this method was more than three times lower than the total dose previously shown to have activity when animals were treated by IP injection three times per week (Maschek et al., Cancer Res (2004) 64(l):31-4). Combination therapy comprising FF and 2DG decreased tumor volume by more than 50%, with the combination of 2DG administered gradually and FF achieving a three-fold greater reduction in tumor volume compared to the control animals.

Lowered levels of MCL-1 as well as increased caspase 3 were found in the samples of tumor tissue from 2DG and FF treated animals. This latter result contrasted to the finding that in animals treated with 2DG alone, a decrease in MCL-1 or markers of cell death was not detected in the tumors, even though tumor volume and size were reduced (albeit less than in the animals receiving 2DG combined with FF). Thus, the in vivo results replicated the in vitro findings, demonstrating that 2DG alone is non-toxic yet growth inhibitory, but combining 2DG with FF induces tumor cell death. Moreover, the results showing that 2DG, or other sugars such as 2- FDG, combined with FF induce cytotoxicity indicate that this treatment successfully eradicates tumor cells without requiring prolonged, continued treatment.

[0074] The foregoing Example demonstrates that combination therapy comprising FF and 2- deoxyglucose, two drugs considered non-toxic that are currently used for non-cancer indications, is effective at inhibiting tumor growth in vivo and exhibits synergistic cytotoxicity against a variety of cancer types. By increasing energy as well as ER stress in tumor cells that are already under more stress than normal cells, the combination of FF and 2DG provides a universal means by which different cancer types may be targeted. Moreover, 2DG as a dual energy and ER stress-inducing agent, is particularly well- suited to take advantage of this natural window of selectivity that heightened tumor stress provides. In addition, increased glucose metabolism, which has been demonstrated by numerous PET scans to be a feature of most tumors and more recently shown to be driven by major oncogenes, presents a universal target that can be exploited by 2DG. The methods and compositions of the present disclosure achieve therapeutic efficacy, even at sub-therapeutic doses of FF and 2-deoxyglucose or 2-deoxymannose, and provide an improved therapeutic option useful for treating cancer.

[0075] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. 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 disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED:

1. A method of inhibiting cancer cell growth comprising contacting a cancer cell with (1) fenofibrate and (2) 2-deoxyglucose or 2-deoxymannose in an amount effective to inhibit cancer cell growth.

2. The method of claim 1, wherein the cancer cell is in vivo and the contacting step comprises administering fenofibrate and 2-deoxyglucose or 2-deoxymannose to a subject.

3. A method of treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder in a subject in need thereof comprising administering a therapeutically effective amount of (1) fenofibrate and (2) 2-deoxyglucose or 2-deoxymannose to the subject.

4. The method of any of claims 1-3, wherein the fenofibrate is administered in a subtherapeutic amount.

5. The method of any of claims 1-4, wherein the 2-deoxyglucose or 2-deoxymannose is administered in a sub-therapeutic amount.

6. The method of any of claims 1-5, wherein the fenofibrate is administered in a daily dosage of about 1 mg to about lOOmg and/or the 2-deoxyglucose or 2-deoxymannose is administered in a daily dosage of about 1 mg/kg to about 60 mg/kg.

7. The method of any of claims 1-6, wherein the 2-deoxyglucose or 2-deoxymannose is selected from 2-deoxy-D-glucose, 2-deoxy-2-fluoroglucose, 2-deoxy-2-chloroglucose, 2-deoxy- 2-fluoromannose, 2-deoxy-2-chloromannose, and combinations thereof.

8. The method of any of claims 2-7, wherein the subject has cancer.

9. The method of any of claims 1-8, wherein the cancer is selected from the group consisting of bone cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, multiple myeloma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, prostate cancer, sarcoma, skin cancer, testicular cancer, throat cancer, and thyroid cancer.

10. The method of any of claims 1-9, wherein the cancer is selected from the group consisting of bone cancer, breast cancer, multiple myeloma, and skin cancer.

11. The method of any of claims 1-10, wherein the amount of fenofibrate and 2- deoxyglucose or 2-deoxymannose is effective to induce apoptosis in cancer cells.

12. The method of any of claims 1-11, wherein the administration of fenofibrate and 2- deoxyglucose or 2-deoxymannose results in a synergistic increase in cancer cell death.

13. The method of any of claims 2-12, wherein the administration of 2-deoxyglucose or 2- deoxymannose mitigates an insulin response.

14. The method of any of claims 1-13, wherein the 2-deoxyglucose or 2-deoxymannose is administered in an amount effective to increase the cytotoxicity of the fenofibrate compared to fenofibrate administered alone.

15. The method of any of claims 1-14, wherein the fenofibrate is administered in an amount effective to increase the cytotoxicity of the 2-deoxyglucose or 2-deoxymannose compared to the 2-deoxyglucose or 2-deoxymannose administered alone.

16. The method of any of claims 2-15, wherein the fenofibrate and/or the 2-deoxyglucose or 2-deoxymannose is administered orally, intravenously, intratumorally, topically or

intraperitoneally.

17. The method of any of claims 2-16, wherein the 2-deoxyglucose or 2- deoxymannose is administered gradually.

18. The method of any of claims 2-17, wherein the 2-deoxyglucose or 2-deoxymannose is administered via a slow-release pump or in a modified-release formulation.

19. The method of any of claims 2-18, wherein the fenofibrate and 2-deoxyglucose or 2- deoxymannose are administered concurrently.

20. The method of any of claims 2-18, wherein the 2-deoxyglucose or 2-deoxymannose is administered before the fenofibrate.

21. The method of any of claims 2-18, wherein the fenofibrate is administered before the 2- deoxyglucose or 2-deoxymannose.

22. The method of any of claims 2-21, wherein the fenofibrate is administered in a formulation that prevents conversion of the fenofibrate to fenofibric acid in the plasma.

23. A method of treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder in a subject in need thereof comprising administering a therapeutically effective amount of fenofibrate in a formulation that prevents conversion of the fenofibrate to fenofibric acid to a subject.

24. The method of claim 23, wherein the subject has multiple myeloma.

25. The method of any of claims 22-24, wherein the fenofibrate is administered in a liposome, microparticle, or nanoparticle.

26. The method of any of claims 2-25, wherein the fenofibrate is administered in an amount effective to achieve a plasma fenofibrate concentration of about 10 μΜ to about 50 μΜ.

27. The method of any of claims 2-26, wherein the fenofibrate is administered in an amount effective to achieve a plasma fenofibric acid concentration of less than about 10 μΜ.

28. A kit comprising (1) fenofibrate and (2) 2-deoxyglucose or 2-deoxymannose and instructions for co-administration of a therapeutically effective amount of the fenofibrate and 2- deoxyglucose or 2-deoxymannose to a subject having cancer.

29. A pharmaceutical composition comprising (1) fenofibrate and (2) 2-deoxyglucose or 2- deoxymannose.

30. The pharmaceutical composition of claim 29, comprising fenofibrate in a dosage amount of about lmg to about 100 mg and/or 2-deoxyglucose or 2-deoxymannose in a dosage amount of about 1 mg/kg to about 60 mg/kg.

31. The kit or pharmaceutical composition of any of claims 29-30, wherein the fenofibrate and/or 2-deoxyglucose or 2-deoxymannose is in a formulation to be administered orally, intravenously, intratumorally, topically or intraperitoneally.

32. The kit or pharmaceutical composition of any of claims 29-31, wherein the fenofibrate is in a formulation that prevents the conversion of fenofibrate to fenofibric acid.

33. The kit or pharmaceutical composition of any of claims 29-32, comprising 2- deoxyglucose or 2-deoxymannose in a modified-release formulation.

34. Use of (1) fenofibrate and (2) 2-deoxyglucose or 2-deoxymannose for the manufacture of a medicament for use in treating cancer.

35. Fenofibrate in combination with 2-deoxyglucose or 2-deoxymannose for use in treatment of cancer.