WO2006060819A2 - Dhmeq as a sensitizing agent for chemotherapy and immunotherapy of resistant cancer cells - Google Patents

Abstract

The present invention for the first time identifies DHMEQ as a sensitizing agent for therapy (e.g., chemotherapy, hormonal therapy, radiotherapy and immunotherapy) of resistant and sensitive cells. The present invention therefore provides methods of treating drug and immunotherapy-sensitive cancers and treating drug and immunotherapy -resistant cancers with DHMEQ or structurally similar compounds either alone or in combination with chemotherapy, hormonal therapy, radiotherapy and immunotherapy agents.

Description

DHMEQ AS A SENSITIZING AGENT FOR CHEMOTHERAPY AND IMMUNOTHERAPY OF RESISTANT CANCER CELLS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No. 60/632,900 filed December 3, 2004 which is incorporated herein by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Grant No. DAMD 17-02-1- 0023, awarded by the US Department of Defense. The US Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention pertains to the treatment of cell proliferation disorders and, particularly cancer, using DHMEQ and similar compounds in combination with another therapeutic reagent. The provided compositions and methods provide for synergy and are also provide a means for overcoming resistance to cancer therapy reagents.

BACKGROUND OF THE INVENTION

[0004] Current cancer treatments consist of surgery, chemotherapy, hormonal therapy, radiotherapy and immunotherapy. While initially cancer patients respond to these treatments, many, however, experience relapse and recurrences. Relapse cancers usually develop resistance to further treatments unless accompanied by high-dose chemotherapy and bone marrow transplantation, and even in these cases the response rates are not high and significant morbidity is observed. There is a need of non-toxic alternative therapies to fight resistant and malignant tumors.

[0005] A new approach has been to develop drugs based on their molecular modes of action and based on a molecular understanding on the underlying mechanisms of resistance. A good example is the recently approved drug Gleevec in the treatment of acute myologeneous leukemia. Gleevec is a small chemical, and its mode of action is based on its ability to inhibit a specific intracellular target in the cancer cell. Also, a proteasome inhibitor, Velcade, has been designed to inhibit the proteasome in tumor cells and has been approved by the FDA this year for treatment of multiple myeloma. Currently, many other molecular targets are being investigated for the design of chemicals with anti-tumor specificity in different cancers.

[0006] Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been recently identified as important in promoting apoptosis in breast and colon adenocarcinoma. Apoptosis induced by TRAIL is mediated by two death receptors referred to as TRAIL- R1/DR4 and TRAIL-R2/DR5. TRAIL is a cytotoxic molecule that exerts selective antitumor cytotoxic activity with minimal toxicity to normal tissues. Further, TRAIL or agonist monoclonal antibody (mAb) to TRAIL receptors, DR4 and DR5, are currently being tested clinically.

[0007] The present invention reports the use of a chemical inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) (Chaicharoenpong et al., 2002), which has been reported to inhibit specifically the transcription factor NF-κB activity by blocking its translocation from the cytosol to the nucleus. The transcription factor NF-κB is constitutively active in many cancers and has been shown to regulate transcription of many genes involved in cell proliferation, cell survival and several gene products that inhibit cell death or apoptosis by various drugs and stimuli. Thus, NF-κB activity in cancer cells invariably regulates the response to various cytotoxic drugs. Therefore, inhibition of NF-κB can result in the inhibition of NF-κB -mediated effects and results in inhibition of cell proliferation and survival in some cancer cells but not all.

[0008] The ideal antineoplastic drug would kill cancer cells selectively, with a wide therapeutic index relative to its toxicity towards non-malignant cells. It would also retain its efficacy against malignant cells even after prolonged exposure to the drug. Unfortunately, none of the current chemotherapies possess an ideal profile. Most possess very narrow therapeutic indexes, and in practically every instance cancerous cells exposed to slightly sublethal concentrations, of a chemotherapeutic agent will develop resistance to such an agent, and quite often cross-resistance to several other antineoplastic agents.

[0009] This invention provides methods and compositions for treating cancer and other diseases based upon the discoveries that the combination of DHMEQ with various chemotherapeutic/immunotherapeutic drugs can act with synergy and also reverse resistance to therapies with other agents. Synergy was achieved for cytotoxicity and apoptosis in different models of cancer cells in vitro including cancer cells which were not responsive to DHMEQ alone or other therapeutics used alone.

BRIEF SUMMARY OF THE INVENTION

[0010] In a first aspect this invention provides combination therapies for treating cancer and cell proliferation disorders. The methods relate to the administration of DHMEQ or NF- KB inhibitory DHMEQ analogs in combination with another cancer therapy reagent. The cancer cells can be either therapy-sensitive or therapy resistant. Furthermore, the present application demonstrates that DHMEQ or NF- KB inhibitory DHMEQ analogs can act as a therapeutic agents to induce apoptosis in cancer cells after sensitization of the cells by an antibody or by various chemosensitizing reagents. These cancer cells can be either therapy- sensitive or therapy-resistant. Hence, this invention also provides methods of treating tumor cells of various origins which are refractory to chemotherapy. The methods can be generalized for multiple cancers and utilizes cancer therapy reagents (e.g., conventional approved drugs) in combination with DHMEQ, or with DHMEQ alone.

[0011] Accordingly, in this first aspect, the invention provides combination therapies for treating tumors or cancers by administering a first agent which is a cancer therapy reagent (e.g., chemotherapeutic agent, a hormone, an immunotherapeutic agent, or radiation) and a second agent which is a NF- KB inhibitor compound of the formula:

wherein R¹ represents a hydrogen atom or a lower alkanoyl group (e.g., C2-C6 or C2-C4 alkanoyl groups including acetyl, propionyl and butanoyl groups and isomer groups thereof with an acetyl group being a preferred alkanoyl group). The alkanoyl group may be substituted or unsubstituted. In some embodiments, the lower alkanoyl group is a C2-C4 alkanoyl group which may be substituted or unsubstituted and saturated or unsaturated. In exemplary embodiments, the lower alkanoyl group is a C2-C4 alkanoyl group which is unsubstituted and unsaturated. In the above formula, R² represents a group of the following formulae (A), (B), (C), (D), (E), (F), (G), or H.

wherein each R³ is independently selected from a lower alkyl group (e.g., methyl, ethyl, propyl, butyl groups and isomer groups thereof, with a methyl group and an ethyl group being preferred) or a homolog of the compound. In some embodiments, the R³ lower alkyl group can be optionally substituted. In other embodiments, the R³ lower alkyl groups can be unsubstituted and saturated or unsaturated. In further such embodiments, the R³ lower alkyl group is methyl or ethyl. In any of the above embodiments, wherein there is a plurality of R³ members, each R³ member can be the same or different from each other. The homologs of the compounds of Formula I are compounds of the above formula in which a ring hydrogen atom is replaced by a methyl or ethyl group. The compounds of Formula I for use according to the invention can inhibit NF-κB activity.

[0012] In an exemplary embodiment, the second agent is a compound is of the formula:

in which R¹ is as defined above. In a preferred embodiment where R¹ is H, the compound is

(-)DHMEQ.

[0013] As various optical isomers of the compounds of formula I are pharmacologically active as NF-κB inhibitors, the compounds may be administered as a racemic mixture and/or in varying enantiomeric ratios (e.g., enantiomeric ratios of 2%, 5%, 10%, 20% or 50% according to either isomer). In some embodiments, the isomers of the formulae

are preferred and used in enantiomeric ratios of at least 90%, at least 95%, at least 98%, or at least 99%.

[0014] For instance, either of the two optical isomers of DHMEQ:

(-)DHMEQ (+)DHMEQ

may be administered in the above ratios. However, in this embodiment, the (-) DHMEQ isomer is preferred as the second agent.

[0015] In some embodiments, the second agent is a salt of prodrug of the compound of formula I.

[0016] In some embodiments, the first agent is a cytotoxic antiproliferative agent. More particularly, in certain embodiments, the first agent is an alkylating drug, an antimetabolite, a microtubule inhibitor, a podophyllotoxin, a nitrosourea, a hormone therapy, a kinase inhibitor, or an antiangiogenic agent. In further embodiments, the first agent is carboplatin, doxorubicin, gemitabine HCl, temolozolamide, cyclophosphamide, methotrexate, paclitaxel, etoposide, carmustine, cisplatin, tamoxifen, or interferon. In a further embodiment, the second agent is a compound of Formula II or, more particularly, DHMEQ or (-)DHMEQ.

[0017] In some embodiments, the first agent is an agonist antibody against TRAIL receptors DR4 or DR5, a TRAIL receptor agonist, or TRAIL. In a further embodiment, the second agent is a compound of Formula II or, more particularly, DHMEQ or (-)DHMEQ.

[0018] In some embodiments, the first agent is riuximab, herceptin, or an anti-CD-22 antibody.

[0017] In certain embodiments, the subject to be treated has cancer. In some further embodiments, the tumor is a solid tumor (e.g., ovarian, breast, head and neck, prostate, glioma, colon, stomach, hepatic, renal, chondrocytoma, small cell lung carcinoma, non-small cell lung carcinoma, and melanoma), a mesothelioma, a lymphoma, or a leukemia. The cancer may be therapy resistant or therapy-sensitive. In some embodiments, the cancer is resistant to Rituximab or another antibody-mediated cancer immunotherapeutics or chemotherapeutic agent. [0019] In one embodiment, the amount of the administered first agent and amount of second agent are administered simultaneously or within a few minutes of each other. In some embodiments, the amount of first agent is administered before or after the amount of the second agent and the difference in time of administration is within an hour, a day, or a week. Such combination therapies may be repeated according to the response of the subject and be administered on daily, weekly or other periodic or variable schedules according to the status of the patient, the condition to be treated, the particular first and second agents used.

[0020] In a second aspect, the invention provides a therapy for preventing or treating therapy resistant tumor or cancer in a patient by administering a cancer therapy reagent and a compound of Formula I. In some embodiments, the subject is first diagnosed as having a therapy resistant tumor and then the combination therapy is administered. In some embodiments, the subject is a person at risk of developing a therapy resistant tumor for the first agent being administered.

[0021] In a third aspect, the invention provides a method of treating or preventing a disease state or disorder characterized by an abnormal or undesired level of cell proliferation by administration of a first agent useful in the treatment of disorder and a second agent which is a compound of formula I, or of any of the embodiments of such compounds disclosed above. In this aspect, the disorder can be, for example, an inflammatory disorder or a disorder mediated by proliferation of cells of the immune system. In further particular embodiments, the combination therapy compositions and methods of the present invention are useful for the treatment of rheumatoid arthritis, transplant rejection, multiple sclerosis, inflammatory bowel disease, lupus, graft v. host disease, T cell mediated hypersensitivity disease, psoriasis, Hashimoto's thyroiditis, and Guillain-Barre syndrome. Other conditions include restenosis, where vascular smooth muscle cells are involved, inflammatory disease states, where endothelial cells, inflammatory cells and glomerular cells are involved A wide variety of indications may be treated, either prophylactically or therapeutically, with the methods and compositions of the present invention. In this aspect, the invention also provides combination pharmaceutical compositions comprising an anti-inflammatory agent and/or an immunosuppressive agent in combination with a compound of Formula I.

[0022] In some embodiments of any of the above aspects, the subject is first diagnosed as having a therapy resistant cell proliferation disorder or condition and then the combination therapy is administered. In some embodiments, the subject is a person diagnosed at risk of developing the resistance to the first agent before the combination therapy comprising the first and second agents is administered.

[0023] In a fourth aspect, the invention provides compositions or kits for use according to the first aspect of the invention. Accordingly, the invention also provides pharmaceutical compositions or kits comprising a first agent and a second agent. The first and second agents are as identified for use in the combination therapies disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1. DHMEQ induces upregulation of the TRAIL (tumor necrosis factor- related apoptosis-inducing ligand) receptor DR5 leading to sensitization of the prostate carcinoma cell line PC-3 to TRAIL-induced apoptosis. The luciferase values represent the % of control and are the mean±SEM of 7 independent experiments. *p<0.03, ** p≤ 0.016, *** /7<0.001, control vs pDR5/-605 or pDR5/YYl mutant - transfected and treated cells.

[0025] Figure 2. DHMEQ sensitizes RAMOS-B-NHL cells to CDDP-induced apoptosis.

[0026] Figure 3. DHMEQ sensitizes RAMOS-B-NHL cells to NPI-0052-induced apoptosis.

[0027] Figure 4. DHMEQ sensitizes the highly drug-resistant and rituximab-resistant AIDS-B-NHL cell line 2F7 and 2F7RR-1 to taxol-induced apoptosis.

[0028] Figure 5. DHMEQ sensitizes the highly drug-resistant and rituximab-resistant AIDS-B-NHL cell line 2F7 and 2F7RR-1 to Etoposide (VP-lό)-induced apoptosis.

[0029] Figure 6. DHMEQ sensitizes the highly drug-resistant and rituximab-resistant AIDS-B-NHL cell line 2F7 and 2F7RR-1 to vincristine-induced apoptosis.

[0030] Figure 7. DHMEQ sensitizes the highly drug-resistant and rituximab-resistant AIDS-B-NHL cell line 2F7 and 2F7RR-1 to CDDP-induced apoptosis.

DETAILED DESCRIPTION OF THE INVENTION INTRODUCTION

[0031] This application provides methods and compositions for combination therapy of cancers and cell proliferative disorders based upon the use of DHMEQ or a DHMEQ-analog or homolog and another cancer therapy reagent. Conditions amenable to treatment by the methods and compositions of the present invention include any state of undesirable cell growth, including various neoplastic diseases, abnormal cellular proliferations and metastatic diseases, where any of a wide variety of cell types may be involved. [0032] The efficacy of the methods and compositions is supported by studies, as reported herein, on the use of combination treatment of DHMEQ and either chemotherapeutic or immunotherapeutic drugs on cancer cells and also on cancer cells that are resistant to either DHMEQ and drug treatment alone. Accordingly, DHMEQ can act synergistically when administered with a second cancer therapy reagent.

[0033] The NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) is structurally similar to the antibiotic epoxyquinomicin-C. DHMEQ inhibits TNF-α-induced activation of NF-κB and acts at the level of the nuclear transportation (Umezawa K, Chaicharoenpong C. MoI. Cells 14(2): 163-7 (2002); Chaicharoenpong C, Kato K, Umezawa K. Bioorg Med Chem. 10(12):3933-9 (2002); Ariga et al. J Biol Chem. 277(27):24625-30 (2002)). It has been reported that DHMEQ inhibited the growth of several prostate carcinoma cell lines in vitro and induced significant apoptosis after 48 h of culture. Further, intra-peritoneal administration of DHMEQ significantly inhibited pre-established prostate cancer cells' tumor cell growth in nude mice without side effects. (Kikushi et al., Can. Res. 63: 107 (2003)) Miyajima et al., Journal of Urology 169: 15559 (2003), reported that DHMEQ can prevent inflammatory renal responses in rats and indicateed that DHMEQ can prevent inflammation in kidney diseases. In unpublished data (personal communication with Dr. Umezawa, October, 2004 in Crete, Greece), DHMEQ used as single agent was tested for anti-tumor activity in mice for various human cancers including breast, bladder, kidney and multiple myeloma, and they observed a good anti-tumor response.

[0034] An NF-κB inhibitor is a compound that inhibits a biological activity of NF-κB. An inhibitor may bind to NF- KB, or a component thereof. An inhibitor may, for instance, inhibit translocation of NF- KB to the nucleus, inhibit NF- KB regulation of transcription of YYl, promote apoptosis, inhibit NF-κB dependent gene expression, inhibit NF-κB dependent stimulation by exogenous or endogenous agents, inhibit NF-κB complex formation with other factors which bind NF~κB). Accordingly, in some further embodiments, the compound of formula I is a compound which inhibits translocation of NF- KB to the nucleus, inhibits NF- KB regulation of YYl transcription, or sensitizes a cell to apoptosis-causing agents. The agent may bind to NF- KB. [0035] The chemical DHMEQ, through its inhibition of NF-κB activity can inhibit gene products that are transcriptionally regulated by NF-κB, including inflammatory cytokines, anti-apoptotic gene products, survival factors, etc. Through such mechanisms, resistant cancer cells undergo molecular and genetic modifications resulting in decreasing the threshold of resistance mechanisms and thus upon exposure to cytotoxic agents that previously were not effective, the combination results in significant cytotoxicity and apoptosis. Several cancer models have been examined in the laboratory in vitro and are provided herein.

[0036] The studies provided herein examine the effects of DHMEQ on resistance and synergy and the mechanisms by which DHMEQ acts to produce them. The studies look at the gene products whose expression is affected by DHMEQ. For example, the studies show that DHMEQ inhibits the expression of the anti-apoptotic gene product BC1-2/BC1-_XL, and through this selective downregulation, the tumor cells (lymphoma/carcinoma) are sensitized to drug-induced apoptosis.

[0037] Also, the studies demonstrate that DHMEQ inhibits the transcription repressor Yin- Yang 1 (YYl) which negatively regulates the transcription of death surface receptors such as Fas, TNF-Rl, DR4 and DR5 that are triggered by cytotoxic lymphocytes/macrophages expressing corresponding ligands. Thus, the upregulation of these receptors by DHMEQ sensitizes the tumor cells to immune-mediated apoptosis and immunotherapy.

[0038] The studies also demonstrate that DHMEQ upregulates the expression of Raf-kinase Inhibitor Protein (RKIP), which has been shown to inhibit survival pathways (Raf/ MEK/ ERK and NF-κB). Thus, treatment of tumor cells with DHMEQ and the induction of RKIP result in inhibition of the survival pathways and downstream gene products involved in cell survival and the cells are sensitized to drug/immune apoptosis. The studies also show that DHMEQ can reverse the resistance of non-Hodgkin's Lymphoma cells that are resistant to the FDA-approved rituximab (anti CD20 antibody) and sensitizes these cells to both drug and immune-apoptosis (see, Jazirehi et al., Cancer Research 65(1): 264 (2005) and 46^th ASH Annual Meeting and Exposition in San Diego, California (Dec. 4-7, 2004); see, Vega et al., J. Immunology 1755:2174-2183 (2005); see, Huerta-Yepez S, Vega M, Umezawa K, Baritaki S, Spandidos DA, Bonavida B. Chemo and immuno-sensitization of prostate cancer cell lines by the NF-κB inhibitor, DHMEQ. Presented at the 7^th International Symposium on Molecular Medicine in Crete, Greece. (Vega M. et al., J. Immunology 175:2174-2183 (2005)). Each of which is incorporated by reference in its entirety.

[0039] These results demonstrate that DHMEQ is a very active chemical that can sensitize drug and immune-resistant cancer cells to various apoptotic stimuli. DHMEQ also exhibits anti-inflammatory activity, thus inhibiting metastases. In summary, DHMEQ is a non-toxic chemical (in mice) that has the ability, when used at very low doses, to sensitize highly resistant tumor cells (primary, metastatic or selected for resistance to drugs and immunotherapy) to both chemotherapy and immunotherapy.

[0040] In addition, DHMEQ can also sensitize tumor cells to radiotherapy and hormonal therapy. DHMEQ acts at a central pathway of tumor cell survival by inhibiting NF-κB, which controls the transcription of genes encoding growth factors, cytokines, cell-adhesion molecules, and anti-apoptotic gene products, all of which favor tumor cell growth, metastases and resistance.

[0041] One method of practicing this invention is to utilize DHMEQ or other compounds of Formula I in combination with conventional and newly developed cytotoxic or immune drugs in the treatment of malignant resistant tumors. In addition, these compounds of Formula I can be used as single agent in vivo in sensitizing the immune-resistant tumor cells to host-immune anti-tumor cytotoxicity. Also, these compounds can be used as a chemo- preventive agent or during remission in cancer patients in that it can sensitize newly arising tumor cells to host-immune destruction.

[0042] There are many advantages on the utilization of these compounds compared to other therapeutic strategies that are aimed at inhibiting NF-κB or its gene products. The compounds are small chemicals that can reach both solid and non-solid tumors with minimal toxicity to normal tissues. They can be used at very small doses and the sensitizing effect is much more potent than other inhibitors tested. They have the advantage of sensitizing high refractory tumors to both chemotherapy and immunotherapy (with subtoxic doses and the combination results in significant synergy in cytotoxicity), findings that have not been tested by other inhibitors.

DEFINITIONS

[0043] The abbreviations used herein are conventional, unless otherwise defined. [0044] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Gennaro, A. R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J. G., Limbird, L. E., and Gilman, A. G., eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Weir, D. M., and Blackwell, C. C, eds. (1986) Handbook of Experimental Immunology, VoIs. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, VoIs. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, C. R., and Graham, A., eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag.

[0045] Unless otherwise stated, the following terms used in the specification and claims have the meanings given below.

[0046] It is noted here that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0047] The designation "first" and "second" with regard to the above agents is not meant to indicate the order of administration but only to indicate which agent is being referenced. Accordingly, the first agent and the second agent can be administered in any temporal sequence with respect to each other, including simultaneously. For instance, in exemplary embodiments, the second agent can be administered before the first agent. And, of course, they may be administered when co-formulated in single pharmaceutical preparation.

[0048] DHMEQ refers to the chemical inhibitor dehydroxymethylepoxyquinomicin, an

NF-κB inhibitor based on the structure of antibiotic epoxyquionicin-C (Umezawa K,

Chaicharoenpong C. MoI. Cells 14(2): 163-7 (2002); Chaicharoenpong C, Kato K, Umezawa

K. Bioorg Med Chem. 10(12):3933-9 (2002), Ariga et al. J Biol Chem. 277(27):24625-30

(2002))).

[0049] "Cancer" refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia (including AML, ALL, and CML), and multiple myeloma.

[0050] "Therapy resistant" cancers, tumor cells, and tumors refers to cancers that have become resistant to both apoptosis-mediated (e.g., through death receptor cell signaling, for example, Fas ligand receptor, TRAIL receptors, TNF-Rl) and non-apoptosis mediated (e.g., antimetabolites, anti-angiogenic, etc.) cancer therapies. "Therapy sensitive" cancers are not resistant to therapy. Cancer therapies include chemotherapy, hormonal therapy, radiotherapy, and immunotherapy.

[0051] "Therapy-mediated or induced cytotoxicity" refers to all mechanisms by which cancer therapies kill or inhibit cancer cells, including but not limited to inhibition of proliferation, inhibition of angiogenesis, and cell death due to, for example, activation of apoptosis pathways (e.g., death receptor cell signaling, for example, Fas ligand receptor, TRAIL receptors, TNF-Rl). Cancer therapies include chemotherapy, immunotherapy, radiotherapy, and hormonal therapy.

[0052] "Therapeutic treatment" can refer to cancer or non-cancer therapy. "Cancer therapies" and "cancer therapy reagents" refer to apoptosis-mediated and non-apoptosis mediated cancer therapies that treat, prevent, or inhibit cancers, including chemotherapy, hormonal therapy (e.g., androgens, estrogens, antiestrogens (tamoxifen), progestins, thyroid hormones and adrenal cortical compounds), radiotherapy, and immunotherapy (e.g., ZEVALIN, BEXXAR, RITUXIN (rituximab), HERCEPTIN). Cancer therapies can be enhanced by administration with a sensitizing agent, as described herein, either before or with the cancer therapy.

[0053] A multitude of therapeutic agents have been developed over the past few decades for the treatment of various types of cancer. "Chemotherapeutic drugs" include conventional chemotherapeutic reagents such as alkylating agents, anti-metabolites, plant alkaloids, antibiotics, and miscellaneous compounds e.g., cis-platinum, CDDP, methotrexate, vincristine, adriamycin, bleomycin, and hydroxyurea. Chemotherapeutic drugs also include proteasome inhibitors such as salinosporamides (e.g., Salinosporamide A), bortezomib, PS- 519, and omuralide. The drugs can be administered alone or combination ("combination chemotherapy"). The most commonly used types of anticancer agents include: DNA- alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, a folate antagonist, and 5-fluorouracil, a pyrimidine antagonist), microtubule disruptors (e.g., vincristine, vinblastine, paclitaxel), DNA intercalators (e.g., doxorubicin, daunomycin, cisplatin), and hormone therapy (e.g., tamoxifen, flutamide). Other platinum coordination complexes that have been evaluated in clinical trials include carboplatin, tetraplatin, ormiplatin, iproplatin and oxaliplatin (see Kelland, Crit. Rev. Oncol. Hematol, 15: 191-219 (1993)).

[0054] By "sensitizingly effective amount or dose" or "sensitizingly sufficient amount or dose" herein is meant a dose that produces cancer cell or hyperproliferative cell sensitizing effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). For instance, sensitized cancer cells respond better to cancer therapy (are inhibited or killed faster or more often) than non-sensitized cells, as follows: Control samples (untreated with sensitizing agents) are assigned a relative cancer therapy response value of 100%. Sensitization is achieved when the cancer therapy response value relative to the control is about 110% or 120%, preferably 200%, more preferably 500- 1000% or more, i.e., at least about 10% more cells are killed or inhibited, or the cells are killed or inhibited at least about 10% faster. Cancer therapy response value refers to the amount of killing or inhibition of a cancer cell, or the speed of killing or inhibition of a cancer cell when it is treated with a cancer therapy. Some compounds are useful both as therapeutic reagents and as sensitizing reagents. Often, a lower dose (i.e., lower than the conventional therapeutic dose) or sub-toxic dose of such a reagent can be used to sensitize a cell. Often, when a cell is sensitized, a lower dose of the chemotherapeutic reagent can be used to achieve the same therapeutic effect as with a cell that has not been sensitized.

[0055] Sensitized proliferative cells respond better to therapy (are inhibited or killed faster or more often) than non-sensitized cells. Control samples (untreated with sensitizing agents) are assigned a relative therapy response value of 100%. Sensitization is achieved when the therapy response value relative to the control is about 110% or 120%, preferably 200%, more preferably 500-1000% or more, i.e., at least about 10% more cells are killed or inhibited, or the cells are killed or inhibited at least about 10% faster. Therapy response value refers to the amount of killing or inhibition of a proliferative cell, or the speed of killing or inhibition of a proliferative cell when it is treated. Some compounds are useful both as therapeutic reagents and as sensitizing reagents. Often, a lower dose (i.e., lower than the conventional therapeutic dose) or sub-toxic dose of such a reagent can be used to sensitize a cell. Often, when a cell is sensitized, a lower dose of the first immunosuppressive agent can be used to achieve the same therapeutic effect as with a cell that has not been sensitized.

[0056] By "therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" herein is meant a dose that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose can often be lower than the conventional therapeutically effective dose for non-sensitized cells.

[0057] The term "pharmaceutically acceptable salts" or "pharmaceutically acceptable carrier" is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds for use according to the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds for use according to the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.

[0058] Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like {see, e.g., Berge et ah, Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds for use according to the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for the present invention.

[0059] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0060] In addition to salt forms, the present invention can utilize compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds for use according to the present invention. Additionally, prodrugs can be converted to the compounds for use according to the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds for use according to the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Particular preferred drugs are prodrug esters.

[0061] The term "pro-drug ester," especially when referring to a pro-drug ester of the compound of Formula I refers to a chemical derivative of the compound that is rapidly transformed in vivo to yield the compound. The prodrug can, for example, be rapidly hydrolyzed in blood, the GI tract, or inside tissues. The term "pro-drug ester" refers to derivatives of the compounds disclosed herein formed by the addition of any of several ester- forming groups that are hydrolyzed under physiological conditions. Examples of pro-drug ester groups include, but are not limited to, acetoxymethyl, phthalidyl, indanyl pivoyloxymethyl, and methoxymethyl. Other examples of pro-drug ester groups can be found in, for example, T. Higuchi and V. Stella, in "Pro-drugs as Novel Delivery Systems", Vol. 14, A.C.S. Symposium Series, American Chemical Society (1975). [0062] Certain compounds for use according to the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds for use according to the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0063] Certain compounds for use according to the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention. The compounds for use according to this invention may contain one or more asymmetric carbon atoms and thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The term "stereoisomer" refers to chemical compounds which differ from each other only in the way that the different groups in the molecules are oriented in space. Stereoisomers have the same molecular weight, chemical composition, and constitution as another, but with the atoms grouped differently. That is, certain identical chemical moieties are at different orientations in space and, therefore, when pure, have the ability to rotate the plane of polarized light. However, some pure stereoisomers may have an optical rotation that is so slight that it is undetectable with present instrumentation. All such isomeric forms of these compounds are expressly included in the present invention.

[0064] Each stereogenic carbon may be of R or S configuration. Although the specific compounds exemplified in this application may be depicted in a particular configuration, compounds having either the opposite stereochemistry at any given chiral center or mixtures thereof are also envisioned. When chiral centers are found in the derivatives of this invention, it is to be understood that this invention encompasses all possible stereoisomers. The terms "optically pure compound" or "optically pure isomer" refers to a single stereoisomer of a chiral compound regardless of the configuration of the compound.

[0065] The compounds for use according to the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds for use according to the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0066] "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.

[0067] Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'_2> a dimer of Fab which itself is a light chain joined to V_H-C_HI by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'₂ dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature 348:552-554 (1990))

[0068] For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3^rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946,778, U.S. Patent No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al, Bio/Technology 10:779-783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al, Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al, EMBO J. 10:3655-3659 (1991); and Suresh et al, Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No. 4,676,980 , WO 91/00360; WO 92/200373; and EP 03089).

[0069] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239:1534-1536 (1988) and Presta, Cwrr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0070] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0071] As used herein, the term "alkyl" refers to a saturated or partially saturated monovalent straight or branched chain hydrocarbon group having from one to about 12 carbon atoms, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like. The alkyl is optionally substituted alkyl. As used herein, the term "lower alkyl" refers to alkyl groups having from 1 to about 6 carbon atoms. Exemplary lower alkyl groups are saturated and unsubstituted. Partially saturated alkyl groups can be alkenyl or alkynyl groups as set forth below.

[0072] As used herein, the term "substituted alkyl" refers to alkyl groups further bearing one or more substituents selected from hydroxy, keto, halo, alkoxy, mercapto, cyano, nitro, amino, carboxyl, sulfonyl, sulfonamide, sulfuryl, and the like.

[0073] As used herein, the term "alkenyl" refers to a partially saturated alkyl group. The alkenyl group may be a straight or branched chain hydrocarbyl groups having one or more carbon-carbon double bonds, and having in the range of about 2 up to 6 carbon atoms. The alkenyl group may be substituted and bear one or more substituents as set forth above. Alkenyl groups useful in the present invention include, but are not limited to, ethenyl, n- propenyl, isopropenyl, n-butenyl, isobutenyl, pentenyl, hexenyl, and the like.

[0074] As used herein, the term "alkynyl" refers to straight or branched chain hydrocarbyl alkyl groups having at least one carbon-carbon triple bond, and having in the range of about 2 up to 12 carbon atoms, and "substituted alkynyl" refers to alkynyl groups further bearing one or more substituents as set forth above. Alkynyl groups useful in the present invention include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. [0075] As used herein the term "alkanoyl" refers to a straight or branched chain alkyl group which is substituted with a carboxyl group. The alkanoyl group may be further substituted and bear one or more substituents as set forth above. A preferred alkanoyl group is unsubstituted acetyl.

[0076] \ <- is a symbol to indicate the position at which the R group is attached to the remainder of the compound of Formula I.

[0077] The subject of treatment is a mammal having the condition or disease to be treated or at a known elevated risk of occurrence or recurrence of the condition to be treated. The term "mammal" includes, without limitation, humans, domestic animals (e.g., dogs or cats), farm animals (cows, horses, or pigs), monkeys, rabbits, mice, and laboratory animals. Preferably, the subject is human. The subject may be refractory or resistant to therapy to the cancer therapy reagent or other agent to be co-administered with the compound of Formula I.

[0078] The identification of subjects who are in need of treatment or prevention is well within the ability and knowledge of the clinician skilled in the art. For example, methods of identifying subjects who have, or are at risk of developing cancers, and their response to ongoing treatment are well known in the medical arts. In some embodiments, the subject is in remission. In other embodiments, the subject has had a recurrence of their cancer. Methods for identifying subjects at risk include family history of the development of a particular disease state and the presence of risk factors (e.g., occupational and environmental exposures, genetic histories) associated with the development of that disease state in the subject patient. More generally, a clinician skilled in the art can readily identify subjects in need of the instant methods, for example, by clinical tests, physical examination and other medical/family history.

[0079] In a particular embodiment, the compounds for use according to the present invention are useful for the treatment of cell proliferation disorders. These disorders include those mediated by a harmful proliferation of cells, particularly, harmful responses of the immune system. Conditions amenable to treatment by the compounds and compositions of the present invention include any state of undesirable cell growth, including various neoplastic diseases, abnormal cellular proliferations and metastatic diseases, where any of a wide variety of cell types may be involved, including cancers such as Kaposi's sarcoma, Wilms tumor, lymphoma, leukemia, myeloma, melanoma, breast, ovarian, lung, etc, psoriasis, etc. Other conditions include restenosis, where vascular smooth muscle cells are involved, and inflammatory disease states, where endothelial cells, inflammatory cells and glomerular cells are involved. The disorders may be acute or chronic. An inflammatory disorder includes atherosclerosis.

[0080] In a particular embodiments, the combination therapy compositions and methods of the present invention are useful for the treatment of arthritis, rheumatoid arthritis, psoriatic arthritis or osteoarthritis, organ transplant, acute transplant or heterograft and homograft rejection, ischemic and reperfusion injury, transplantation tolerance induction, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, lupus, graft vs. host diseases, T-cell mediated hypersensitivity diseases, contact hypersensitivity, delayed-type hypersensitivity, gluten-sensitive enteropathy, Type 1 diabetes, psoriasis, contact dermatitis, Hashimoto's thyroiditis, Sjogren's syndrome, autoimmune hyperthyroidism, Graves' Disease, Addison's disease, autoimmune polyglandular disease, autoimmune alopecia, vitiligo, autoimmune hypopituatarism, Guillain-Barre syndrome, autoimmune diseases, glomerulonephritis, serum sickness, uticaria, respiratory allergies, asthma, hayfever, allergic rhinitis, skin allergies, mycosis flugoides, acute inflammatory responses, dermatomyositis, and alopecia areata.

[0081] In such embodiments, the combination therapy involves administration and/or formulation of the compound of formula I with a first agent which is an immunosupressive agent, including but not limited to, cyclosporine, prednisone, azathioprine, tacrolimus and mycophenolate mofetil, dexamethasone, beclomethasone, methylprednisone, betamethasone, hydrocortisone, methotrexate, cyclosporin, rapamycin, antihistamine drugs, TNF antibodies, IL-I antibodies, soluble TNF receptors, soluble IL-I receptors, TNF or IL-I receptor antagonists, non-steroidal antiinflammatory agents, COX-2 inhibitors, and anti-cancer agents.

[0082] A wide variety of indications may be treated, either prophylactically or therapeutically, with the methods and combination pharmaceutical compositions of the present invention. For example, the subject methods and compositions have been found to be effective inducers of cell apoptosis. Apoptosis is effected by contacting a target cell, in or ex vivo, with an effective amount of the first agent and the second agent . The methods and compositions may be assayed for their ability to modulate apoptosis using cell and animal models to evaluate cell death and cytotoxicity. ADMINISTRATION AND PHARMACEUTICAL COMPOSITIONS

[0083] 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. The formulations, dosage regimens, and routes of administration for the first agent, or cancer therapy reagents, for use according to the invention can be the same as for their routine use in cancer therapy. With regard to the compounds of Formula I, there are many suitable formulations when they are administered as the sole active agent in a preparation or when they are administered in combination pharmaceutical composition with another active agent (the first agent or cancer therapy reagent) {see, e.g., Remington's Pharmaceutical Sciences, 17^th ed., 1989).

[0084] The compounds of Formula I, and the pharmaceutical compositions, generally, may be formulated into pharmaceutical compositions as natural or salt forms. Pharmaceutically acceptable non-toxic salts include the base addition salts (formed with free carboxyl or other anionic groups) which may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, procaine, and the like. Such salts may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as, for example, hydrochloric, sulfuric, or phosphoric acids, or organic acids such as acetic, p-toluenesulfonic, methanesulfonic acid, oxalic, tartaric, mandelic, and the like. Salts of the invention include amine salts formed by the protonation of an amino group with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like. Salts of the invention also include amine salts formed by the protonation of an amino group with suitable organic acids, such as p-toluenesulfonic acid, acetic acid, and the like. Additional excipients which are contemplated for use in the practice of the present invention are those available to those of ordinary skill in the art, for example, those found in the United States Pharmacopeia Vol. XXII and National Formulary Vol. XVII, U.S. Pharmacopeia Convention, Inc., Rockville, Md. (1989), the relevant contents of which is incorporated herein by reference.

[0085] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

[0086] The compound of choice, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0087] Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged nucleic acid with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the compound of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

[0088] In some embodiments, either the first or second agent, or both, can be formulated as a liposomal preparation or emulsion. Liposomes and emulsions are well known examples of delivery vehicles or carriers for drugs, particularly hydrophobic drugds. In a presently preferred embodiment, long-circulating, i.e., stealth liposomes can be employed. Such liposomes are generally described in Woodle, et ai, U.S. Patent No. 5,013,556. The compounds for use according to the present invention can also be administered by controlled release means and/or delivery devices such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719.

[0089] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0090] Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.

[0091] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The composition can, if desired, also contain other compatible therapeutic agents.

[0092] Preferred pharmaceutical preparations deliver one of the compounds for use according to the invention, optionally in combination with one or more therapeutic agents, in a sustained release formulation (e.g., the first and second agents may be co-formulated in one preparation). Typically, the compound of formula I is administered therapeutically as a sensitizing agent that increases the susceptibility of tumor cells to other cytotoxic cancer therapies, including chemotherapy, radiation therapy, immunotherapy and hormonal therapy. In some embodiments, the compound of formula I acts as a chemotherapeutic reagent after cellular sensitization using an antibody.

[0093] In therapeutic use for the treatment of cancer or other proliferative disorder, the compounds of formula I utilized in the pharmaceutical method of the invention can be administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily. A daily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound of additional active agents (e.g., cancer therapy reagents) being employed. The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also can be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.

Methods of Making the Compounds of Formula I.

[0094] Methods of making the compounds of formula I are disclosed in U.S. Patent No. 6,566,394, which is incorporated herein by reference, in its entirety. This reference teaches compounds of Formula I which are suitable for use according to the invention. This reference teaches DHMEQ can be made by the following synthetic routes:

Step a: Preparation ofN-(2-alkanoylbenzoyl)-2,5-dimethoxyaniline

[0095] In this step, 2,5-Dimethoxyaniline is first dissolved in a solvent (pyridine, etc.), and an ethyl acetate solution of the O-alkanoylsalicyloyl halide is added thereto at -78°C to 50⁰C, preferably under ice cooling, and the mixture is allowed to react with stirring. The reaction is quenched by addition of water and ethyl acetate is added. Then, the reaction mixture is sequentially washed with hydrochloric acid, water, a sodium hydrogen carbonate solution and, finally, water. Upon drying, the organic layer is concentrated under reduced pressure and dried under vacuum to obtain the N-(2-alkanoylbenzoyl)-2,5-dimethoxyaniline compound of formula (2). The compound can be used in the next step without purification.

Step b: Preparation of3-(O-alkanoylsalicyloyl)amino-4,4-dialkoxy-2,5- cyclohexadienone

[0096] The compound of formula (2) is next dissolved in a solvent such as methanol and diacetoxyiodobenzene is added thereto at -20⁰C to 50⁰C, with ice cooling. This mixture reacts at room temperature under stirring. Next, the mixture is concentrated under partial vacuum, and ethyl acetate is added. Then, the reaction mixture is washed with sodium hydrogen carbonate solution and saline. Next, the soluiton is concentrated under reduced pressure. The resulting residue is purified by column chromatography to obtain 3-(O- alkanoylsalicyloyl)amino-4,4-dialkoxy-2,5-cyclohexadienone (3).

Step c: Preparation ofSβ-epxoy^A-dialkoxyS-salicyloylamino-l-cyclohexenone

[0097] The purified compound of formula (3) is dissolved in a solvent (tetrahydrofuran, methanol, etc.), hydrogen peroxide water and sodium hydroxide are added thereto at -20⁰C to 50⁰C, with ice cooling, and the mixture reacts with stirring. Next, ethyl acetate is added and the mixture is sequentially washed with hydrochloric solution, aqueous sodium thiosulfate solution, and saline. Upon drying, the reaction mixture is further dried under vacuum. In order to remove the residual starting compound, the residue is dissolved in acetone, and p- toluenesulfonic acid is then added with stirring at room temperature. This procedure decomposes the starting compound. A residue is obtained by distilling off methanol under reduced pressure and ethyl acetate is added thereto. Next, the solution is washed with water. A residue is obtained by drying the ethyl acetate layer. This residue is purified by column chromatography to obtain 5,6-epxoy-4,4-dialkoxy-3-salicyloylamino-2-cyclohexenone compound represented by formula (4).

Step d: Preparation of 5,6-epoxy-2-salicyloylamino-2cyclohexen-l,4dione

[0098] The compound of formula (4) is dissolved in methylene chloride. Then, an inorganic acid or organic acid (trifluoroboron diethyl ether complex, etc.) is added with ice cooling, and the mixture reacts while stirring. A solvent (ethyl acetate, etc.) is next added to the reaction mixture, which is then washed with water. The ethyl acetate layer is concentrated and the resulting residue is washed with methanol to obtain 5,6-epoxy-2- salicyloylamino-2-cyclohexen-l,4-dione represented by formula (5).

Step e: Preparation ofSrf-epoxy^-hydroxyS-salicyloylamino^-cyclohexenone

[0099] The compound of formula (5) is suspended in a solvent (methanol, ethanol, THF, etc.). Next, a reducing agent (sodium borohydride, etc.) is added at -78⁰C to 5O⁰C, preferably under ice cooling. An organic solvent (ethyl acetate, methylene chloride, etc.) is added to the reaction mixture. This mixture is next sequentially washed with hydrochloric acid and water. After drying, the solvent layer is concentrated under reduced pressure, suspended, stirred and washed with methanol to obtain 5,6-epoxy-4-hydroxy-3-salicyloylamino-2-cyclohexenone (DHM2EQ) represented by formula (Ia).

[0100] Methods for separating the optical isomers of such compounds are well known to one of ordinary skill in the art and further disclosed in European Patent Application Publication No. EP 1600445 which is incorporated herein by reference.

Methods for screening for NF-κB Inhibitory Activity

NF- KB Production Inhibitory Activity

[0101] The inhibitory effect of the compounds can be screened using a co-transfection bioassay as taught in U.S. Patent No. 6,566,394, which is incorporated herein by reference for all purposes. A reporter using luciferase DNA can be prepared and the NF-K.B inhibitory activity measured by use of promoter/reporter assay.

[0102] The plasmid for the luciferase assay can be obtained by coupling luciferase gene derived from lampyrid to 3xκB derived from the IgK gene and HSV-TK promoter. Further, for the β-galactosidase assay, a plasmid obtained by coupling β-galactosidase gene to the β- actin promoter can be used.

2) Transfection and Luciferase Assay [0103] Transfection can be performed by a DEAE-dextran method. 2 x 10⁶ cells were washed once with 1 x TBS((Tris-HCL (25 mM), NaCl (137 mM), KCl (5 mM), and Na₂ HPO₄ (0.5 mM)) and incubated in transfection buffer ((2 x TBS (200 μl), 100 x Ca²⁺Mg²⁺ ((CaCl₂.2H₂O) (78 mM, 4 μl), MgCl₂.6H₂O (76 mM)), and DEAE-dextran (1 mg/ml, 200 μl)) containing 1 μg of plasmid at room temperature for 30 minutes with tapping for every 10 minutes. Thereafter, the cells were washed with 1 x.TBS and inoculated at 37⁰C on a 12-well plate (Coster: N. Y., U.S.A.) at 1 x.lO⁶ cells/well. On the day next, the test compound scan be added in various concentrations. After 2 hours' incubation, TNFα (20 ng/ml) can be added and incubated for 6 hours. The cells can next be centrifuged at 3,500 rpm for 5 minutes. After removing the supernatant, 50 μl each of lysis buffer (Tris-HCl (25 mM, pH 7.8), DTT (2 mM), l,2-diaminocyclohexane-N,N',N',N-tetraacetic acid (2 mM), and 10% glycerol, 1% Triton X-100) can be added and the cells solubilized in ice for 30 minutes. This preparation can then be centrifuged at 15,000 rpm for 5 minutes and the supernatant used as a sample.

[0104] For 10 μl of the sample, 100 μl of a luminescent substrate solution (Tricine (20 mM), (MgCO₃)4Mg(OH)₂5H₂O (1.07 mM), MgSO₄ (2.67 mM), EDTA (0.1 mM), DTT (33.3 mM), Coenzyme A (270 μM), luciferin (470 μM), and ATP (530 μM)) was added and amount of luminescence measured. One of ordinary skill in the art would appreciate that the measured amounts can next be corrected by β-galactosidase assay results to obtain the value of the luciferase activity.

[0105] For example, 20 μl of a sample can be added to 230 μl of Z buffer (KCl (10 mM), MgSO₄ (1 mM), 2-mercaptoethanol (50 mM), and NaPO₄ (100 mM: pH 7.5)) and further 50 μl of o-nitrophenyl-.beta.-D-galactopyranoside (ONPG, Sigma) and a NaPO₄ (100 mM, pH 7.5) solution (2 mg/ml) added and the mixture incubated at 37.degree. C. When the solution turns yellow, 250 μl Of Na₂CO₃ (1 M) added and the optical density at absorption wavelength of 420 nm measured with a spectrophotometer.

EMSA

Nuclear proteins (5 μg) can be mixed for 30 min at room temperature with Biotin-labeled oligonucleotide probe NF-κB or YYl using EMSA Kit Panomicst (Panomics Inc., Redwood City, CA, USA) following the manufacturer's instructions and as described previously (Vega et al., Oncogene 23:3530-3540 (2004). 10 μl can be subjected to 5% polyacrylamide gel electrophoresis for 90 min in TBE buffer (Bio-Rad Laboratories) and transferred to Nylon membrane Hybond-Np (Amersham Pharmacia Biotech,Germany) using the Trans-Blots SD semi-dry Transfer cell System (Bio-Rad, Hercules, CA, USA). The membranes can be transferred to a UV Crosslinker FB-UVXL-1000 Fisher technology (Fisher Scientific, NY, USA) for 3 min. The detection can be carried out as per the manufacturer's instructions, after the membranes are exposed using Hyperfϊlm ECL (Amersham Pharmacia Biotech). The Relative concentrations can be assessed by densitometric analysis.

Nuclear Translocation

NF-κB inhibitory activity can be determined by assessing the ability of a compound to inhibit translocation of NF- KB to the cell nucleus. p65 is a component of NF- KB. Accordingly, GFP-labeled p65 can be used to assess the effect of an agent on the intracellular location of the NF-κB protein. Such methods can be performed as disclosed by Ariga et al. (Ariga et al., J. Biol. Chem. 277 (27):24625 (2002)).

[0106] Methods of screening the compounds of formula I, for NF- KB inhibitory activity are exemplified in the Examples.

CLINICAL TESTING.

[0107] The effectiveness of treatment may be determined by controlled clinical trials. For instance, patients having cancer with measurable or evaluable tumors can be studied. A measurable tumor is one that can be measured in at least two dimensions such as a lung tumor surrounded by aerated lung, a skin nodule, or a superficial lymph node. An evaluable tumor in one that can be measured in one dimension such as a lung tumor not completely surrounded by aerated lung or a palpable abdominal or soft tissue mass that can be measured in one dimension. Tumor markers which have been shown to be highly correlated with extent of disease will also be considered to provide an evaluable disease, such as PSA for prostate cancer, CA- 125 for ovarian cancer, CA- 15-3 for breast cancer, etc.

[0108] The tumor can be measured or evaluated before and after treatment by whatever means provides the most accurate measurement, such as CT scan, MRI scan, Ultrasonography, etc. New tumors or the lack thereof in previously irradiated fields can also be used to assess the anti-tumor response. The criteria for evaluating response will be similar to that of the WHO Handbook of Reporting Results of Cancer Treatment, WHO Offset Publication 1979, 49-World Health Organization, Geneva. The following results are defined for uni- and bi-dimensionally measurable tumors. Complete response: Complete disappearance of all clinically detectable malignant disease determined by two observations not less than four weeks apart.

Partial Response: (a) for bidimensionally measurable tumors, a decrease of at least 50% in the sum of the products of the largest perpendicular diameters of all measurable tumors as determined by two observations not less than four weeks apart, (b) for unidimensionally measurable tumors, a decrease by at least 50% in the sum of the largest diameters of all tumors as determined by two observations not less than four weeks apart. In cases where the patient has multiple tumors, It is not necessary for all tumors to have regressed to achieve a partial response as defined herein, but no tumor should have progressed and no new tumor should appear.

Stable disease: (a) for bidimensionally measurable tumors, less than a 50% decrease to less than a 25% increase in the sum of the products of the largest perpendicular diameters of all measurable tumors, (b) for unidimensionally measurable tumors, less than a 50% decrease to less than a 25 % increase in the sum of the diameters of all tumors. For (a) and (b) no new tumors should appear.

No clinical response, i.e. progressive disease in defined as an increase of more than 50% in the product of the largest perpendicular diameters for at least one bidimensionally measurable tumor, or an increase of more than 25% in measurable dimension of at least one unidimensionally measurable tumor.

[0109] Of course elimination or alleviation of other known signs or symptoms of cancer, especially those listed previously can also be used to evaluate the effectiveness of this invention.

[0110] The cancers should be evaluated, i.e. tumors measured, etc., no more than 14 days before the start of the treatment. Twenty eight days after this initial administration another administration period may be performed, and evaluations performed 28 days after the start of this second cycle. The treatment cycles may be continued until a clinical response is achieved or unacceptable toxicity is encountered.

[0111] Another aspect of this invention is the treatment of cancer with reduced side effects normally associated with the first agent alone. This objective can be achieved by administration of lower doses of the two active ingredients or by shorter duration of dosing brought about by the synergistic effect of the combination.

[0112] The following examples are offered to illustrate, but not to limit the claimed invention. EXAMPLES

EXAMPLE 1: Cells and Culture Conditions

[0113] The human androgen-independent PC-3 cell line can be obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells can be maintained as a monolayer in 80 mni2 plates in RPMI 1640 (Life Technologies, Bethesda, MD, USA), supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Life Technologies) (to ensure the absence of complement), 1% (v/v) penicillin (100 U/ml), 1% (v/v) streptomycin (100 U/ml), 1% (v/v) L-glutamine, 1% (v/v) pyruvate, and 1% nonessential amino acids (Invitrogen Life Technologies, Carlsbad, CA, USA). FBS can be charcoal-stripped to maintain CL-I cells in an androgen-free medium. The LNCaP cell medium can be supplemented with 0.1 nmol/1 R 1881 methyltrienolone (New Life Science Products, Boston, MA, USA). The cell cultures can be incubated at 37°C and 5% carbon dioxide.

EXAMPLE 2. Preparation of Plasmids

[0114] The pDR5 WT promoter luciferase (pDR5 promoter) reporter plasmid and the pDR5 promoter with the 5 '-deletion mutant -605 that includes the YYl binding site (pDR5/- 605) have been previously characterized (Yoshida, et al, 2001). The pDR5 plasmid missing the YYl binding sequence (pDR5-YYl mutant) can be generated by using the QuikChange site-directed mutagenesis method (Stratagene). The mutagenesis reaction contains the pDR5 plasmid as a template DNA and two complementary oligonucleotides, each containing the desired mutation surrounded by 15 bp of flanking sequence on both the 5' and the 3' sides. A PCR-based method can use the complementary primers pDR5-yyl F (5'-TGT CATG TACTGGGACTACAGGCC-3') and pDR5-yyl R (5'-

GGGAGGCTGAGGTGGGAGTATCTGC-S'). The PCRs contain 125 ng of each primer, IX PFU buffer [20 rnM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 2 mM MgSO4, 100 μg of bovine serum albumin/ml, 0.1% Triton X-100], a 2.5 μM concentration of each deoxynucleoside triphosphate, and Pf u polymerase. Cycling conditions are 95⁰C for 3 min, followed by 30 cycles of 95°C for 45 seg, 69°C for 1 min, and 72°C for 11 min. PCR products are purified by QIAquick PCR purification kit QIAGEN Inc (Valencia, CA. USA).

EXAMPLE 3. Luciferase DR5 promoter reporter assay [0115] PC-3 cells can be transfected by electroporation using pulses at 250 V/975 mF (Bio- Rad), with 20 μg of pDR5 promoter, pDR5-YYl mutant or pDR5/-605. After transfection, the cells can be allowed to recover overnight and were cultured in six-well plates. Cells were treated or left untreated. Cells can then be harvested in IX lysis buffer and luciferase activity can be measured according to the manufacturer's protocol (BD Biosciences, Palo Alto, CA, USA) using an analytical luminescence counter Monolith 2010. The assays can be performed in triplicate. Data can be normalized by protein concentration using Bio-Rad protein assay.

EXAMPLE 4. Determination of apoptosis

[0116] After each treatment, the adherent cells and the floating cells can be recovered by centrifugation at 1800 rpm for 8 min. Afterwards, the cells can be washed once with ice-cold IX phosphate-buffered saline (PBS) and resuspended in 100 μl of the cytofix/cytoperm solution (PharMigen, San Diego, CA, USA) for 20 min. Thereafter, the samples can be washed twice with ice-cold IX perm/wash buffer solution (PharMingen) and stained with FITC-labeled anti-active caspase 3 mAb for 30 min (light protected). The samples can be subsequently washed once with IX perm/wash buffer solution and 250 ml of IX PBS added prior to flow cytometry analysis on a flow cytometer EPICSR XL-MCL (Coulter, Co. Miami, FL, USA), with the System II™ Software and the percent positive cells can berecorded. As a negative control, the cells can be stained with isotype control (pure IgG) under the same conditions described above.

EXAMPLE 5. DHMEQ induces upregulation of the TRAIL receptor DR5 leading to sensitization of the prostate carcinoma cell line PC-3 to TRAIL-induced apoptosis.

[0117] PC-3 cells were transfected with 1 μg of pDR5 luciferase reporter plasmids (pDR5 W/T, pDR5/YYl mutant or pDR5/-605) for 24 hr. After transfection the cells were treated or left untreated with 1 or 3 μg/ml of CDPP or DHMEQ for 18 hr. The promoter activity was determined by assessment of luciferase activation expressed as relative light units. The data (see Figure 1) show that PC-3 cells transfected with DR5 W/T plasmid resulted in basal luciferase activity (control) and treatment with CDDP significantly augmented luciferase activity in a dose-depended manner. PC-3 cells transfected with the deleted, or YYl mutated constructs also resulted in significant potentiation of the basal luciferase activity. However treatment of those transfectants with CDDP didn't reveal statistically significant enhancement of luciferase activity compared to non treated transfectants. DHMEQ also augmented the basal luciferase activity in the pDR5 W/T - transfected cells in a dose dependent manner, however as observed with CDDP, it didn't change significantly the DR5 promoter activity in any of the other transfectants.

[0118] This example demonstrates that DHMEQ upregulates the transcription and translation of the TRAIL receptor DR5 on the prostate carcinoma cell line, PC-3. The upregulation of DR5 correlates strongly with sensitization to TRAIL-induced apoptosis. The mechanism by which DHMEQ upregulates DR5, was shown to involve the activity of the transcription repressor, YYl, using a reporter system for the DR5 promoter. Accordingly, DHMEQ is a potent sensitizing agent to reverse TRAIL resistance in tumor cells.

EXAMPLE 6: DHMEQ reversal of CDDP resistance of lymphoma cells and results in significant potentiation of CDDP-induced apoptosis and synergy.

[0119] RAMOS-B-NHL tumor cells were treated with various concentrations of DHMEQ and also treated with CDDP (15 ug/mL) and incubated at 37 degrees for 24 hours. The cells were harvested and tested for apoptosis. The data (see Figure 2) indicates that DHMEQ sensitizes the drug-resistant RAMOS tumor cells to CDDP-induced apoptosis. The extent of the chemo-sensitizing effect of DHMEQ was a function of the concentration used.

[0120] These findings indicate the therapeutic potential of DHMEQ in the reversal of resistance to chemotherapeutic agents. Other studies have also shown that DHMEQ sensitizes tumor cells to other drugs (i.e., Adriamycin, Taxol, Vincristine, VP- 16, etc) (data not shown).

EXAMPLE 7. DHMEQ sensitizes RAMOS-B-NHL cells to NPI-0052-induced apoptosis.

[0121] RAMOS-B-NHL tumor cells were treated with DHMEQ (lOug/mL) alone or in combination with the proteasome inhibitor NPI-0052 (Nereus Pharmaceuticals, San Diego) at various concentrations. In addition, for control, the tumor cells were treated with DHMEQ and CDDP. The findings (see Figure 3) demonstrate that DHMEQ sensitizes the tumor cells to NPI-0052-induced apoptosis, and the extent of apoptosis was a function of the NPI-0052 concentration used.

[0122] These results show that the NFkB inhibitor, DHMEQ, when used in combination with the proteasome inhibitor, NPI-0052, results in significant apoptosis and synergy in a highly drug-resistant cell line. Each agent used alone had no effect. In addition, the concentration of NPI-0052 used was very small. The findings strongly indicate that the proteasome inhibitor, NPI-0052, is behaving much like a chemotherapeutic drug (see Figure 2) with its cytotoxicity manifested following sensitization of DHMEQ.

EXAMPLE 8. DHMEQ sensitizes the highly drug-resistant and rituximab-resistant AIDS-B-NHL cell line 2F7 and 2F7RR-1 to chemotherapeutic-induced apoptosis.

[0123] This example demonstrates that DHMEQ not only sensitizes the tumor cell line to various drug-induced apoptosis, but also sensitizes the highly drug and rituximab-resistant 2F7-RR1. Clearly in the clinic, NHL patients treated with rituximab develop resistance to rituximab and those patients are highly resistant to chemotherapeutic drugs. There are no current therapeutic modalities for the treatment of those patients. The findings here provide an alternative, in that, DHMEQ can be significantly used as a sensitizing agent, and in addition, the doses of drugs are sub-optimal compared to the conventional doses used as single agents.

[0124] The AIDS-B-NHL cell line 2F7 and the newly generated rituximab-resistant variant, 2F7-RR1 were treated with DHMEQ (lOug/mL) alone or in combination with various concentrations of taxol. The cells were incubated for 24 hr and harvested and examined for apoptosis by flow cytometry. The data (see Figure 4) demonstrates that DHMEQ sensitizes both the WT2F7 and the resistant 2F7-RR1 to taxol-induced apoptosis. The extent of apoptosis was a function of the concentration of taxol used. In all cases, the sensitization was synergistic and statistically significant.

[0125] Figure 5 shows that DHMEQ sensitizes the highly drug-resistant and rituximab- resistant AIDS-B-NHL cell line 2F7 and 2F7RR-1 to Etoposide (VP-16)-induced apoptosis. The AIDS-B-NHL cell line 2F7 and the newly generated rituximab-resistant variant, 2F7- RRl were treated with DHMEQ (lOug/mL) alone or in combination with various concentrations of VP- 16. The cells were incubated for 24 hr and harvested and examined for apoptosis by flow cytometry. The data demonstrates that DHMEQ sensitizes both the WT2F7 and the resistant 2F7-RR1 to VP-16-induced apoptosis. The extent of apoptosis was a function of the concentration of VP- 16 used. In all cases, the sensitization was synergistic and statistically significant.

[0126] Figure 6 shows that DHMEQ sensitizes the highly drug-resistant and rituximab- resistant AIDS-B-NHL cell line 2F7 and 2F7RR-1 to vincristine-induced apoptosis. The AIDS-B-NHL cell line 2F7 and the newly generated rituximab-resistant variant, 2F7-RR1 were treated with DHMEQ (10ug/mL) alone or in combination with various concentrations of vincristine. The cells were incubated for 24 hr and harvested and examined for apoptosis by flow cytometry. The data demonstrates that DHMEQ sensitizes both the WT2F7 and the resistant 2F7-RR1 to vincristine-induced apoptosis. The extent of apoptosis was a function of the concentration of vincristine used. In all cases, the sensitization was synergistic and statistically significant.

[0127] Figure 7 shows that DHMEQ sensitizes the highly drug-resistant and rituximab- resistant AIDS-B-NHL cell line 2F7 and 2F7RR-1 to CDDP-induced apoptosis. The AIDS- B-NHL cell line 2F7 and the newly generated rituximab-resistant variant, 2F7-RR1 were treated with DHMEQ (10ug/mL) alone or in combination with various concentrations of CDDP. The cells were incubated for 24 hr and harvested and examined for apoptosis by flow cytometry. The data demonstrates that DHMEQ sensitizes both the WT2F7 and the resistant 2F7-RR1 to CDDP-induced apoptosis. The extent of apoptosis was a function of the concentration of CDDP used. In all cases, the sensitization was synergistic and statistically significant.

EXAMPLE 9: Chemo- and immuno- sensitization of prostate cancer cell lines by the NF-κB inhibitor, DHMEQ

[0128] The present study was designed to investigate the role of DHMEQ as a sensitizing agent for both drug and immune-mediated apoptosis using prostate cancer cell lines as models. PC3 and CL-I prostate cancer cell lines were treated with various concentrations of DHMEQ (1-3 ug/mL) for 4h and followed by treatment with various concentrations of either CDDP or recombinant TRAIL for 18h. The cells were then assessed for apoptosis by caspase-3 activation by flow. The findings demonstrate that the combination treatment resulted in significant potentiation of apoptosis and synergy was achieved. The mechanism of DHMEQ mediated sensitization was examined. These findings demonstrate that DHMEQ inhibits NF-κB activity by EMSA and selectively downregulates BcI_xL expression, which is under the transcription regulation of NF-κB. The direct role of Bcl_xL in the sensitization was corroborated by the use of the chemical inhibitor 2MAM-A3 which inhibits BcI_xL, function. Accordingly, 2MAM-A3 sensitizes the tumor cells to both drug and TRAIL-induced apoptosis, like DHMEQ. These findings demonstrate that DHMEQ is a potent chemo and immunosensitizing agent and is a potential therapeutic agent in the reversal of tumor cell resistance when used in combination with drugs and/or immunotherapy. EXAMPLE 10. Sensitization of Rituximab-resistant Ramos RRl and Daudi RRl clones to various chemotherapeutic drugs by a novel Nuclear Factor-B Activation Inhibitor

[0129] This study examined the similarities and differences observed in wild type (wt) parental and rituximab-resistant Ramos and Daudi cells following treatment with either rituximab (20 mg/ml) or DHMEQ (10 mg/ml). In the wt cells, both agents inhibited NF-κB DNA-binding activity, down-regulated BCIX_L expression and sensitized the cells to drug- induced apoptosis. Rituximab inhibited both the NIK — IKB -α — IKK — NF-κB and the Raf- 1— MEK1/2— ERK1/2— AP-I signaling pathways, while DHMEQ specifically inhibited NF- KB activity. Rituximab-resistant Ramos and Daudi clones were generated by continuous exposure of the cells to step-wise increasing concentrations of rituximab for several weeks and clones isolated by limiting dilution. The CD20+ expressing Ramos RRl and Daudi RRl clones were analyzed for their response to treatment with rituximab or DHMEQ. Compared to the wt cells, the resistant clones exhibited hyper-activation of the NF-κB pathway, increased expression of BC1-_XL, and higher resistance to structurally and functionally distinct drugs; their treatment with DHMEQ (15 mg/ml), but not with rituximab, inhibited the activity of the hyper-activated NF-κB, reduced the over-expressed BcI -_xL and sensitized the highly drug-resistant Ramos RRl and Daudi RRl clones to various chemotherapeutic drug (CDDP, ADR, VP- 16, Taxol, etc.)-induced apoptosis. These findings demonstrate that DHMEQ can reverse the acquired drug-resistance phenotype in both parental and rituximab/drug-resistant NHL B-cell lines when used in combination with subtoxic concentrations of drugs. In vivo, DHMEQ inhibits subcutaneously transplanted prostate cancer xenografts in nude mice (Kikuchi et al., Cancer Res. 63: 107, 2003). Consequently, the present findings indicate the potential therapeutic application of DHMEQ in combination with drugs, in the treatment of rituximab/drug resistant B-NHL.

EXAMPLE 11. Inhibition of constitutive NF-κB or YYl activity sensitizes prostate cancer cells to TRAIL-induced apoptosis via upregulation of DR5 expression

[0130] Treatment of the androgen-independent, p53- PC-3 prostate cancer cells with CDDP or the NO donor, DETANONOate, resulted in inhibition of NF-κB activity, upregulation of DR5 expression and sensitization to TRAIL-induced apoptosis. This study explored mechanisms of negative regulation of DR5 expression by NF-κB. [0131] Apoptosis was assessed by PI (propidium Iodide) staining and by activated caspase- 3; DNA-binding activity by EMSA; transcription by luciferase reporter systems; and protein expression by flow cytometry and Western analysis.

[0132] Inhibition of NF-κB activity by CDDP and DETANANOate, as measured by NF-κB reporters, was corroborated in PC-3 cells. Treatment of PC-3 with the NF-κB inhibitor DHMEQ mimicked CDDP and DETANONOate effects and resulted in upregulation of DR5 expression and sensitization to TRAIL-induced apoptosis. DHMEQ similarly augmented Raf-1 Kinase Inhibitor Protein (RKIP) promoter activity. RKIP has been reported to inhibit multiple signaling pathways including NF-κB (Yeung et al., MoI Cell Biol., 2001, 21: 7207). Regulation of the human DR5 transcription was then examined using serial deletions of the WT human promoter (Yoshida et al., FEBS Lett., 2001, 507: 381-5). The basal activity of the DR5 promoter was significantly augmented by deletion of the -1224 to -605 region, which contains the putative DNA-binding site for the transcription repressor YYl. Similar results were obtained with cells transfected with a mutant of YYl DNA-binding site at -800 to -796. The direct role of YYl in downregulation of DR5 expression was further shown by transfection of YYl siRNA.

[0133] The findings reveal for the first time that CDDP or DETANONOate-mediated sensitization of tumor cells to TRAIL-induced apoptosis results from the induction of RKIP and inhibition of NF-κB-dependent transcription of YYl. Inhibition YYl upregulates DR5 expression and sensitizes the tumor cells to TRAIL-induced apoptosis. These findings identify new targets for therapeutic intervention for reversal of tumor cells resistance to TRAIL.

EXAMPLE 12. Sensitization of rituximab-sensitive and rituximab-resistant B-NHL cell lines/clones to TRAIL-induced apoptosis by Bortezomib and NFKB inhibitors

[0134] Patients with B-NHL respond initially to conventional chemotherapy and/or to immunotherapy with rituximab (alone or in combination with chemotherapy). However, patients develop resistance to these modalities and novel approaches to therapy are needed. [0135] Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been recently identified as important in promoting apoptosis in breast and colon adenocarcinoma. Apoptosis induced by TRAIL is mediated by two death receptors referred to as TRAIL- R1/DR4 and TRAIL-R2/DR5. TRAIL is a cytotoxic molecule that exerts selective antitumor cytotoxic activity with minimal toxicity to normal tissues. Further, TRAIL or agonist monoclonal antibody (mAb) to TRAE. receptors, DR4 and DR5, are currently being tested clinically.

[0136] The present study investigated the sensitivity of B-NHL cell lines to TRAIL- mediated apoptosis using the AIDS-related NHL (ARL) B-cell line, 2F7, and the B-NHL cell lines, Ramos and Daudi. Also, to recapitulate various aspects of acquired rituximab- resistance, rituximab-resistant (RR) clones were generated from the parental wild type (wt) cells. Rituximab failed to chemo-sensitize the RR clones and the clones exhibited higher resistance to various drugs (e.g., CDDP, VP-16, ADR, Vincristine, Taxol)(as noted above in Example 2)) and to TRAIL (1-250 ng/ml-18 h) compared to the wt cells as analyzed by DNA fragmention assay. The findings demonstrate that the wild type and RRl cells were resistant to TRAIL-mediated apoptosis at a wide range of TRAIL concentrations.

[0137] Turning next to means of reversing BKTRAIL resistance, we and others have reported that inhibition of NF- activity can sensitize TRAIL-resistant tumor cells to TRAIL- induced apoptosis. The effect of the proteasome and NF-κB inhibitor, Bortezomib (Velcade), Bay 11-7085 and the specific NF- KB inhibitor DHMEQ (Kikuchi et. al, Cancer Research 2003; 63: 107) was studied. Pretreatment of the NHL tumor cells with Bortezomib, Bay 11- 7085 or DHMEQ for 6 h followed by treatment with TRAIL for 18h resulted in significant augmentation of apoptosis and synergy was achieved. Both the rituximab-sensitive and rituximab-resistant tumor cells were sensitized by these inhibitors, though higher concentrations were required for sensitization of the RR clones. Interestingly, detailed analysis of the signaling pathways in the RR clones revealed constitutive hyper-activation of the NF-KB survival pathway leading to over-expression of anti-apoptotic gene products BcI- 2, Bcl-xL and McI-I.

[0138] Accordingly, patients with resistant B-NHL can be treated with combination of TRAIL/anti-DR4 or DR5 mAb and NF- KB inhibitors. Alternatively, these patients can be treated with agents that up-regulate TRAIL expression on host effectors (e.g., T cells, NK cells) in combination with NF-κB inhibitors.

EXAMPLE 13. New Targets identified for therapeutic intervention in the reversal of rituximab and drug resistant AIDS-B-NHL

[0139] In order to examine the mechanism of NHL resistance to rituximab, rituximab- resistant clones of 2F7 (2F7RR) were developed in the laboratory. Their response was compared with the wild type to rituximab treatment alone and with the combination of chemotherapeutic drugs. Unlike the wild type 2F7, rituximab treatment failed to sensitize 2F7 RRl to drug-induced apoptosis, failed to modulate the p38MAPK/NF-κB/YYl/STAT3 signaling pathways, did not inhibit Bcl-2 expression, and failed to chemosensitize the tumor cells. The studies examining the effect of various chemical inhibitors of this signaling pathway on chemosensitization demonstrate that treatment with the proteasome inhibitor Bortezomib or the NF-κB inhibitor DHMEQ significantly sensitized 2F7-RR1 cells to drug - induced apoptosis. These inhibitors also resulted in the inhibition of NF-κB, YYl and downregulated Bcl-2 expression. These findings demonstrate that rituximab and drug- resistant clone 2F7-RR1 can be sensitized to reverse chemoresistance. The findings also identify intracellular targets whose modification can reverse resistance. Such targets include the p38 MAPK pathway, the transcription factors NF-KB, YYl, or STAT3 and also inhibitors of Bcl-2 expression and/or activity. These findings also indicate that combination treatment of currently used drugs such as Bortezomib and chemotherapeutic drugs have a potential for the treatment of rituximab and drug-resistant AIDS-B-NHL.

EXAMPLE 14. Reversal of rituximab-resistant AIDS-B-NHL clone to chemotherapeutic drug-induced apoptosis by Bortezomib and DHMEQ

[0140] The mechanisms underlying the failure of B-NHL cancer patients to respond to treatment with rituximab, alone or in combination with chemotherapy, are not known. In efforts to address this issue, rituximab-resistant clones of the AIDS NHL cell line, (2F7RR) were generated. Recent findings have demonstrated that treatment of the wild type (wt) 2F7 with rituximab sensitized the tumor cells to various chemotherapeutic drug-induced apoptosis. Chemosensitization was the result of rituximab-mediated inhibition of the p38 MAPK signaling pathway and the selective inhibition of the anti-apoptotic Bcl-2 gene product (Vega et. al., Oncogene 23:4993, 2004). Analysis of one clone, 2F7RR1, revealed that the cells have diminished surface CD20 expression and failed to respond to CDC and to apoptosis following cross-linking. In addition, the cells were resistant to rituximab-mediated chemosensitization. In contrast to wt2F7, molecular analysis of the 2F7RR1 clone revealed that rituximab failed to inhibit p-Lyn, p38-MAPK, BcIXL, and Bcl-2. In addition, rituximab failed to inhibit the transcription factors NF-κB, YYl, SP-I, and STAT3. Noteworthy, 2F7RR1 exhibited higher resistance to drug-induced apoptosis compared to wt2F7 and showed overexpression of Bcl-2. Previous findings with the wt2F7 demonstrated that Bcl-2 was responsible for chemoresistance.

[0141] Accordingly, whether inhibition of Bcl-2 in 2F7RR1 can reverse chemoresistance was studied. Since Bcl-2 is under the transcriptional regulation of NF-«B, the effect of the NF-κB inhibitors Bortezomib and DHMEQ (DHMEQ was a kind gift from Dr.K. Umezawa Keio University, Japan) (Kikuchi, et al., Cancer Review, 2003, 63: 107) was studied. The findings revealed that treatment of 2F7RR1 with these inhibitors resulted in the reversal of resistance to a number of chemotherapeutic drugs (examples: taxol, vincristine, ADR, CDDP, VP16, etc.). The chemo-sensitization by Bortezomib and DHMEQ was equivalent and the combination treatment of each of these inhibitors with the drug These studies present evidence of synergythat rituximab and that drug-resistant tumor cells may be sensitized to chemotherapeutic drug-induced apoptosis via inhibition of NF- KB or Bcl-2. These findings also indicate that Bortezomib and DHMEQ may be clinically relevant in the treatment of rituximab and drug-resistant AIDS-B-NHL.

EXAMPLE 15. Chemosensitization of drug-resistant Ramos B-NHL to drug-induced apoptosis: YYl expression is decreased in response to cytoskeletal-interacting drugs

[0142] The transcription factor Yin Yang 1 (YYl) regulates cellular differentiation, hematopoiesis, response to apoptotic stimuli, pathogenesis of cancer and its increased expression is associated with inhibition of differentiation of progenitor cells. We and others have previously shown that expression levels of YYl also correlate with drug sensitivity in cancer cells. A comparison between the wild type (wt) Ramos, with the recently generated rituximab-resistant Ramos (Ramos RR) clones, revealed that, unlike wt Ramos, Ramos RRl cannot be chemosensitized by rituximab and exhibited higher drug resistance. Further, there was enhanced YYl expression in Ramos RRl. Turning to the hypothesis that overexpression of YYl may be, in part, responsible for drug-resistance in Ramos RRl and its inhibition can reverse resistance, this study investigated whether the heightened expression of YYl in Ramos RRl cells can be reversed by a panel of drugs used in combination. Ramos and Ramos RRl cells were treated with vincristine, VP-16, CDDP, and ADR, and the NF-κB inhibitors Bortezomib and DHMEQ. Treatment of Ramos RRl with the NF-κB inhibitor, in combination with any of the above chemotherapeutic drugs, reversed the acquired drug- resistance and synergy was achieved. Noteworthy, in Ramos RRl cells, only vincristine (or in combination with NF-κB inhibitors) significantly abrogated or diminished YYl expression. Similarly, in the prostatic cell line PC-3, 2-methoxyestradiol, another cytoskeletal interacting drug, resulted in marked reduction of YYl expression level and activity (assessed by a luciferase reporter assay). These results indicate that YYl overexpression may regulate the resistance of B-NHL to a selected group of drugs but not all drugs. The studies also indicate that agents that can modulate YYl expression/activity may be potential therapeutics when used in combination with chemotherapeutic drugs in the treatment of drug and rituximab- resistant B-NHL.

EXAMPLE 16. Hyperactivation of the ERK1/2 MAPK and NF-κB signaling pathways in rituximab-resistant non-Hodgkin's lymphoma (NHL) ramos RRl and daudi RRl clones: pivotal roles in chemoresistance

[0143] The efficacy of rituximab (anti-CD20 mAb) (alone and/or combined with drugs) in eradicating tumor cells in vitro and in vivo is well documented. Nonetheless, a subset of patients does not respond or relapse after rituximab therapy. To recapitulate the non- responders and/or relapsed situations, rituximab-refractory (RR) Ramos and Daudi clones were generated by growing the cells in the presence of step- wise increasing concentrations of rituximab followed by multiple rounds of limiting dilution assay. Single cells were then propagated as clones. This study characterized representative clones, Ramos RRl and Daudi RRl, and examined the differences observed in RR clones compared to the wild type (wt) cells following treatment with either rituximab or pharmacological inhibitors. The clones exhibited the following properties: A partial decrease in surface CD20 expression and resistance to complement-dependent cytotoxicity. Rituximab (in both monomeric and cross- linked forms) was incapable of inhibition of cellular proliferation, induction of apoptosis or modification of signaling pathways {Jazirehi et ai, Cancer Res, 64: 7117, 2004). Both the ERK 1/2 and NF-κB pathways were hyperactivated in the clones with selective over- expression of Bcl-_xL, and consequently, the clones exhibited higher resistance to a wide array of drugs. Chemo-sensitizing agents namely rituximab, PD098059 (ERK1/2 inhibitor), DHMEQ (novel NF-κB inhibitor) or bortezomib (Velcade; proteasome inhibitor) were analyzed. The chemical inhibitors, but not rituximab, inhibited the activity of the hyperactivated ERK1/2 and NF-κB pathways, reduced the over-expressed Bcl-_xL and sensitized the highly drug-resistant clones to low concentrations of structurally and functionally distinct drugs (including topoisomerase II inhibitor, DNA damaging agents and microtubule poisons), albeit to varying degrees. These novel findings reveal that pharmacological inhibitors, used in combination with subtoxic concentrations of drugs, can reverse the acquired drug-resistance phenotype in the rituximab-refractory NHL clones and indicate their potential therapeutic application in the treatment of rituximab/drug refractory B-NHL.

[0144] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be indicateed to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of treating, preventing or inhibiting cancer, the method comprising the step of administering to a subject a therapeutically effective amount of a cancer therapy reagent and a sensitizingly effective amount of a compound of Formula I:

wherein R¹ represents a hydrogen atom or a lower alkanoyl group and R² represents a radical of the following formulae (A), (B), (C), (D), (E), (F), (G), or (H):

wherein each R³ is independently a lower alkyl group.

2. The method of claim 1, wherein R is methyl or ethyl.

3. The method of claim 1, wherein R¹ is Cl to C4 alkanoyl.

4. The method of claim 2, wherein R¹ represents a hydrogen atom.

5. The method of claim 1 , wherein the compound is of the formula:

6. The method of claim 1, wherein the compound is dehydroxymethylepoxyquinomicin (DHMEQ) .

7. The method of claim 1, wherein the cancer therapy reagent is a chemotherapeutic reagent, an immunotherapeutic reagent, a radiotherapeutic reagent, or a hormonal therapeutic reagent.

8. The method of claim 1, wherein the cancer is selected from the group consisting of non-Hodgkin's lymphoma, B-acute lymphoblastic lymphoma, prostate cancer, ovarian cancer, renal cancer, lung cancer, breast cancer, colon cancer, leukemia, multiple myeloma and hepatocarcinoma.

10. The method of claim 9, wherein the cancer is lymphoma.

11. The method of claim 9, wherein the cancer is non-Hodgkin's lymphoma.

12. The method of claim 1, wherein the cancer therapy reagent induces apoptosis.

13. The method of claim 1, wherein the cancer therapy reagent is a chemotherapeutic reagent, an immunotherapeutic reagent, a radiotherapeutic reagent, or a hormonal therapeutic reagent.

14. The method of claim 13, wherein the cancer therapy reagent is rituximab immunotherapy.

15. The method of claim 1, wherein the cancer is therapy-resistant.

16. The method of claim 15, wherein the therapy is selected from the group consisting of immunotherapy, chemotherapy, radiotherapy, and hormonal therapy.

17. The method of claim 1, wherein the cancer is therapy-sensitive.

18. The method of claim 1, wherein the therapeutically effective amount of a cancer therapy reagent and the sensitizingly effective amount of a compound of Formula I are administered concurrently.

19. The method of claim 18, wherein the cancer therapy reagent comprises bortezomib.

20. The method of claim 18, wherein the cancer therapy reagent is a chemotherapeutic reagent, an immunotherapeutic reagent, a radiotherapeutic reagent, or a hormonal therapeutic reagent.

21. The method of claim 1, wherein the therapeutically effective amount of a cancer therapy reagent and the sensitizingly effective amount of a compound of Formula I are administered sequentially. 22. The method of claim 1, wherein the subject is a human.

23. The method of claim 10, wherein the lymphoma is therapy resistant.

24. The method of claim 23, wherein the lymphoma is rituximab therapy resistant.

25. The method of claim 1, wherein the cancer therapy reagent comprises an alkylating agent, an anti-metabolite, or a proteasome inhibitor, TRAIL or anti-DR4 or DR5 mAb, or a salinosporamide.

26. The method of claim 1, wherein the cancer therapy reagent comprises cis- platinum, CDDP, methotrexate, vincristine, adriamycin, bleomycin, Salinosporamide A, bortezomib, etoposide, taxol, tamoxifen, PS-519, or omuralide.

27. The method of claim 1, wherein the cancer therapy reagent comprises a microtubule inhibitor, a podophyllotoxin, a nitrosourea, a hormone therapy, a kinase inhibitor, or an antiangiogenic agent.

28. The method of claim 1, wherein the the cancer therapy reagent comprises carboplatin, doxorubicin, gemcitabine, temolozolamide, cyclophosphamide, methotrexate, paclitaxel, or carmustine.

29. The method of claim 1, wherein the method treats, prevents, or inhibits a cancer with proteasome inhibitor therapy.

30. A composition comprising a therapeutically effective amount of a cancer therapy reagent and a sensitizingly effective amount of a compound of Formula I:

wherein R¹ represents a hydrogen atom or a lower alkanoyl group and R² represents a radical of the following formulae (A), (B), (C), (D), (E), (F), (G), or (H):

wherein each R³ is independently lower alkyl, in a physiologically acceptable excipient.

31. The composition of claim 1, wherein the compound of formula I is

DHMEQ. 32. The composition of claim 1, wherein the cancer therapy reagent is rituximab.

33. A kit comprising a therapeutically effective amount of a cancer therapy reagent and a sensitizingly effective amount of a compound of Formula I:

wherein R¹ represents a hydrogen atom or a lower alkanoyl group and R² represents a radical of the following formulae (A), (B), (C), (D), (E), (F), (G), or (H):

wherein each R³ is independently lower alkyl.

34. The kit of claim 33, wherein the cancer therapy reagent is rituximab.

35. The kit of claim 34, wherein the compound of formula I is DHMEQ.

36. A method of treating therapy resistant cancer, the method comprising the step of administering to a subject in need thereof a therapeutically acceptable dose of a compound of Formula I:

wherein R¹ represents a hydrogen atom or a lower alkanoyl group and R² represents a radical of the following formulae (A), (B), (C), (D), (E), (F), (G), or (H):

wherein each R is independently lower alkyl.

37. The method of claim 36, wherein the compound of formula I is DHMEQ.

38. The method of claim 37, wherein the therapy resistant cancer is selected from the group consisting of human cancers and carcinomas, including kidney, breast, lung, kidney, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, esophagus, and liver cancer, lymphoma, including non-Hodgkins and Hodgkins lymphoma, leukemia, and multiple myeloma.

39. The method of claim 37, further comprising administering DHMEQ in combination with chemotherapy, immunotherapy, hormonal therapy, or radiotherapy regimes, either at the same time or in serial doses.

40. The method of claim 37, comprising administering DHMEQ alone without additional cancer therapies.

41. A method of preventing therapy resistant cancer, the method comprising the step of administering a therapeutically acceptable dose of a compound of Formula I:

wherein R¹ represents a hydrogen atom or a lower alkanoyl group and R² represents a radical of the following formulae (A), (B), (C), (D), (E), (F), (G), or (H):

wherein each R is independently lower alkyl.

42. The method of claim 41, wherein the compound of formula I is DHMEQ.

43. The method of claim 41, wherein the subject is in remission. 44. A method of treating, preventing or inhibiting an inflammatory disorder or cell proliferation disorder mediated by proliferation of cells of the immune system, the method comprising the step of administering to a subject a therapeutically effective amount of an immunosuppressive agent and a sensitizingly effective amount of a compound of Formula I:

wherein R¹ represents a hydrogen atom or a lower alkanoyl group and R² represents a radical of the following formulae (A), (B), (C), (D), (E), (F), (G), or (H):

wherein each R is independently a lower alkyl group.

45. The method of claim 44, wherein the compound of formula I is DHMEQ.

46. The method of claim 45, wherein the disorder is inflammation, host versus graft rejection, or an autoimmune response.