WO2014172315A2 - Methods of treating proliferative disorders - Google Patents

Abstract

The invention is based on the discovery that elevated levels of oxidative stress shortly after administration of chemotherapeutic or radiation therapy in subjects having proliferative disorders is indicative of sensitivity to the administered chemotherapeutic or radiation therapy. Consequently, measurement of oxidative stress biomarker levels can inform whether a particular chemotherapeutic or radiation therapy is likely to be efficacious in treating a proliferative disorder before prolonged exposure to the therapy. This information allows a subject to avoid unnecessary and otherwise dangerous therapies and increases the likelihood that a selected therapeutic course will ultimately lead to treatment of a particular proliferative disorder.

Description

METHODS OF TREATING PROLIFERATIVE DISORDERS

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. Provisional Application Number 61/812,411, which was filed on April 16, 2013, and is hereby incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cancer represents the most important medical challenge of our times as it ranks first as a cause of death in the Western world. The control of cancer relies heavily on pharmacological agents (chemotherapy) and to a lesser extent on radiation therapy.

The response of a given individual to a particular treatment for cancer is often

unpredictable. This uncertainty originates to some extent from variations in drug disposition and is compounded by the significant heterogeneity of virtually all types of cancer. For example, breast cancer, the most common female solid tumor in the US (1), is a

heterogeneous group of diseases, being subdivided into those expressing estrogen (ER) and progesterone (PR) receptors; those with HER-2 amplification; and those without expression of ER, PR, or HER-2 amplification, known as "triple negative" breast cancer (1-3). These subtypes differ in their response to treatment and prognosis (3), with triple negative patients having the worst prognosis (4-9).

It is now clear that identifying responders and non-responders prior to initiating treatment with a given agent is critical to ensure therapeutic success and spare patients (and the health care system) the inconvenience and expense of a futile, and sometimes toxic, agent. The same applies to cancer treatment with radiation therapy. Thus, predictive biomarkers, defined as those associated with benefit from a particular therapy, are highly desirable. (Predictive biomarkers are distinct from prognostic biomarkers, which indicate the likelihood of recurrence or survival.) In the case of breast cancer, for example, testing for ER, PR, and human epidermal growth factor receptor (EGFR) type 2 to guide treatment decisions is standard of care. This rapidly evolving area underscores the enormous importance of identifying predictive biomarkers for a given drug used to treat breast cancer. As recently stated, "practitioners seek a predictive marker that indicates the likelihood that if "Ms Jones" expresses the marker will benefit more from a new treatment than from standard treatment, whereas if she does not express the marker she will derive little or no benefit from the new treatment" (10).

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B4245785.4 The currently used cancer drugs act through a variety of mechanisms that often form the basis of their classification. Although common mechanistic themes can be recognized even among structurally disparate compounds, no dominant (much less unifying) mechanism of action has been identified. The same applies to radiation therapy. Oxidative stress, however, may be a common mechanistic theme for several groups of cancer drugs and radiation therapy, and one that can be of great importance to cancer chemoprevention. At the heart of oxidative stress are the Reactive Oxygen and Nitrogen Species (RONS), which include oxygen radicals (e.g., 0₂ ^~) and non-radical derivatives of 0₂ (H₂0₂), and reactive nitrogen species (e.g. NO). Free radicals are highly reactive because they contain unpaired electrons.

Reactive oxygen and nitrogen species are produced continuously by the

mitochondria (0₂ ^~, H₂0₂, OH*) of most cells and also by cytochrome P450 (0₂ ^~, H₂0₂), macrophages (0₂ ^~, H₂0₂, NO*), and peroxisomes (H₂0₂) (13, 14). To contain RONS, the cell has invested heavily into a defense system, which includes: a) classic antioxidant enzymes: e.g., superoxide dismutase, catalase, glutathione (GSH) peroxidase, glutaredoxine and thioredoxin. b) non-classic antioxidant enzymes, e.g., heme oxygenase- 1. c) phase II detoxifying enzymes, such as GSH reductase and NQOl, and d) non-enzymatic

antioxidants: glutathione (GSH) and vitamins E and C; GSH, present in mammalian cells at mM concentrations, is the most important of them.

The biological behavior of RONS depends on their concentration: at low

concentrations they protect the cell, being part of its normal signaling network; at higher concentrations they can damage many biological molecules, such as DNA, proteins, and lipids; and at even higher concentrations, as when produced in response to anticancer agents, they initiate the death of the cancer cell and help control cancer (16, 17). The latter case has been thoroughly documented by us in studies of compounds with significant anticancer efficacy (17-19). For example, phospho-ibuprofen markedly suppressed the growth of colon cancer xenografts; induced apoptosis; inhibited cell proliferation; and increased the urinary levels of 15-F₂-isoprostane, a marker of oxidative stress (18).

Remarkably, oxidative stress was induced only in the tumors, and the apoptotic effect was restricted to xenografts.

Phospho-ibuprofen is a member of the novel phospho-modified compounds that include phospho-NSAIDs (non-steroidal anti-inflammatory drugs); phospho-valproic acid is an example of additional phospho-modified compounds with anticancer activity that are

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B4₂45785.4 not phospho-NSAIDs. An exemplary phospho-NSAID is phospho-aspirin, a derivative of aspirin synthesized using a general approach as taught, for example in U.S. Patent No. 8,236,820, U.S. Patent Application Publication Nos. US 2012-0316139, 2013-0225529, 2014-0088044, and 2014-0088045 and International Patent Application Publication Nos. WO2005065361 and WO2013130625 (11). Phospho-aspirin consists of aspirin and diethylphosphate-glycerol linked through a carboxylic ester (Fig. 1). In preclinical models, for example, phospho-aspirin strongly inhibits the growth of both cultured human breast cancer cell lines and of breast cancer xenografts in nude mice (Fig. 1); appears safe; and has oxidative stress as a key component of its mechanism of action. Like all phospho-NSAIDs, phospho-aspirin, is a new chemical entity, differing drastically from its parent compound in both structure and pharmacological properties. Phospho-valproic acid also acts by inducing oxidative stress.

RONS cannot be directly detected in humans because they have short lifetimes, thus creating the need to detect biomarkers of oxidative stress (21, 22). Due to their high chemical reactivity, RONS oxidize non-enzymatically nearly all cellular components; the products of such reactions can serve as biomarkers of oxidative stress. Although the levels of such oxidative products do not measure RONS levels per se, in general they are proportional to the RONS levels. Such biomarkers are preferable because they eliminate the variability introduced by the action of enzymes. Of note, in experimental animals and cells, RONS can be detected in cells, organs, and live animals by electron spin resonance spectroscopy (also known as electron paramagnetic resonance spectroscopy) especially when combined with spin-trapping techniques which are required to stabilize highly reactive free radicals. Several technical improvements have been described and may be applicable to this invention. In addition, RONS can be detected in cells, tissues and animals using appropriate molecular probes.

SUMMARY OF THE INVENTION

In general, the invention features measurement of oxidative stress biomarker levels to determine whether a particular chemotherapeutic or radiation therapy is likely to be efficacious in treating a proliferative disorder before prolonged exposure to the therapy. This information allows a subject to avoid unnecessary and otherwise dangerous therapies and increases the likelihood that a selected therapeutic course will ultimately lead to treatment of a particular proliferative disorder.

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B4245785.4 Thus, the invention features a method of predicting a response of a subject with a neoplastic disease to administration of a chemotherapeutic agent, comprising: (i) administering a probe agent to the subject; (ii) obtaining a test sample (e.g., a blood sample, stool, or urine sample) from the subject; (iii) measuring a level of a biomarker associated with oxidative stress in the test sample (e.g., one, two, three, four, five, or more biomarkers) in the test sample; and (iv) comparing the measured level to a reference value; wherein a measured level associated with elevated oxidative stress indicates that the neoplastic disease will respond to treatment with the chemotherapeutic agent. Steps (ii) and (iii) may be performed less than about three weeks (e.g., two weeks or less, less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s)) after step (i). Additionally, the invention features a method of treating cancer, comprising steps (i)-(iv) above, and administering one or more chemotherapeutic agents to said subject if the test sample has a measured level of the biomarker associated with elevated levels of oxidative stress, thereby treating the cancer. Similarly, the invention features a related method of treating cancer, comprising administering one or more chemotherapeutic agents to a subject identified as having a level of a biomarker associated with elevated levels of oxidative stress (e.g., by a method as described herein), thereby treating the cancer.

In another aspect, the invention features a method of predicting a response of a subject with a neoplastic disease to administration of radiation therapy, comprising: (i) administering radiation therapy to the subject; (ii) obtaining a test sample (e.g., a blood sample, stool, or urine sample) from the subject; (iii) measuring a level of a biomarker associated with oxidative stress in the test sample (e.g., one, two, three, four, five, or more biomarkers) in the test sample; and (iv) comparing the measured level to a reference value; wherein a measured level associated with elevated oxidative stress indicates that the neoplastic disease will respond to radiation therapy. Steps (ii) and (iii) may be performed less than about three weeks (e.g., two weeks or less, less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s)) after step (i). Additionally, the invention features a method of treating cancer, comprising steps (i-iv), and administering radiation therapy to said subject if the test sample has a measured level of the biomarker associated with elevated levels of oxidative stress, thereby treating the cancer. Similarly, the invention features a related method of treating cancer, comprising administering radiation therapy to a subject identified as having a level of a biomarker associated with elevated levels of oxidative stress (e.g., by a method as described herein), thereby treating the cancer.

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B4245785.4 In another aspect, the invention features a method of determining whether a subject with a neoplastic disease is responding to treatment with radiation therapy or a

chemotherapeutic agent, comprising: (i) obtaining a test sample from the subject being treated with the radiation therapy or chemotherapeutic agent; (ii) measuring a level of a biomarker associated with oxidative stress in the test sample; and (iii) comparing the measured level to a reference value; wherein a measured level associated with elevated levels of oxidative stress indicates that the patient is responding to treatment with the chemotherapeutic agent. Steps (i) and (ii) may be performed less than about three weeks (e.g., two weeks or less, less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s)) after step (i).

In another aspect, the invention features a method of determining whether a subject with a neoplastic disease is responding to treatment with radiation therapy, comprising: (i) obtaining a test sample from the subject being treated with the radiation therapy; (ii) measuring a level of a biomarker associated with oxidative stress in the test sample; and (iii) comparing the measured level to a reference value; wherein a measured level associated with elevated levels of oxidative stress indicates that the patient is responding to treatment with radiation therapy. Steps (i) and (ii) may be performed less than about three weeks (e.g., two weeks or less, less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s)) after step (i). In any of the above methods, the reference value may be a standard value or range associated with a health condition, a measured level for the biomarker from a control, a measured level for the biomarker from a reference group having a known health state, or a measured level for the biomarker from the subject obtained prior to administering the anticancer agent or radiation therapy. The reference group may be a population of one or more individuals with a known disease state.

A chemotherapeutic agent of the invention can be any agent known or later found to induce oxidative stress, e.g., a phospho-NSAID (non-steroidal anti-inflammatory drug, e.g., phospho-aspirin, phospho-ibuprofen, phospho-fluribuprofen, and phospho-sulindac or any phospho-NSAID described herein), phospho-valproic acid, arsenic trioxide, emodin, anthracyclines, such as daunorubicin and doxorubicin, cisplatin, bortezomib, synthetic retinoids, imexon, 2-methoxyestradiol, tetrathiomolybdate, motexafin gadolinium, phenylethyl isothiocyanate, erlotinib or biological agents such as antibodies against signaling molecules, e.g., epidermal growth factor (EGFR) or vascular endothelial growth factor (VEGF).

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B4245785.4 In another aspect, the invention features a method of detecting the presence of cancer in a subject with a suspected neoplastic disease, comprising: (i) administering a probe agent to the subject; (ii) obtaining a test sample from the subject; (iii) measuring a level of a biomarker associated with oxidative stress in the test sample; and (iv) comparing the measured level to a reference value; wherein a measured level associated with elevated oxidative stress indicates that the patient has neoplastic disease.

Biomarkers of oxidative stress include F₂-isoprostanes (e.g., 5, 8, 12, or 15 F₂- isoprostane), malondialdehyde (MDA), dityrosine, 8-hydroxy-2'-deoxyguanosine (8-OH- dG), and glutathione (GSH). Other markers include, e.g., a change in expression of a gene associated with oxidative stress, e.g., NADPH oxidase, glutathione synthase, thioredoxin and all its iso forms (Trx-1, Trx-2 etc), thioredoxin reductase (TrxR), NADP, superoxide dismutase (SOD), catalase, glutathione peroxidase, glutaredoxine, heme oxygenase- 1, phase II detoxifying enzymes, such as GSH reductase and NQOl, as well as non-enzymatic antioxidants such as GSH and vitamins E and C. Gene expression can be measured by determining the amount of a corresponding nucleic acid (e.g., mRNA) or protein.

Other biomarkers of oxidative stress include, e.g., individual RONS species, e.g. NO, superoxide anion, H₂0₂ and others. These species, can, e.g., be assayed in test samples by such methods as, for example, electron spin resonance spectroscopy, especially with prior administration of appropriate compounds that facilitate their detection (spin-trapping). RONS, individually or in groups, can also be assayed by loading appropriate test samples, e.g., cells, tissues, or mammals with informative molecular probes, such as 2', 7' - dichlorofluorescein diacetate (DCFDA), a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species activity within the cell; diaminofluoresceins, e.g., DAF-2 for nitric oxide; mitoSox-Red for mitochondrial superoxide anion; dihydroethidium (DHE) for cytoplasmic superoxide anion; and others.

Also, biomarkers of oxidative stress include miRNAs.

Also, in any of the above methods, the control sample can be, e.g., obtained from the subject prior to step (i) and/or from the subject prior to any administration of the probe agent, chemotherapeutic agent, or radiation therapy to the subject.

By "test sample" or "sample" is meant a solid or fluid sample. Test samples may include cells, protein or membrane extracts of cells, blood or biological fluids. Solid test samples include samples taken from feces, the rectum, central nervous system, bone, breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid

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B4₂45785.4 tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, and the thymus. Biological fluid samples include samples taken from the blood, serum, cerebral spinal fluid (CSF), semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, stool, and tears. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the test sample is a breast, lung, colon, or prostate tissue sample obtained by needle biopsy.

Induction of oxidative stress can be indicated by an increase in presence of a particular biomarker (e.g., a protein, nucleic acid, or analyte) as compared to a control from a normal or reference sample (e.g., an increase of at least 0.1 fold, 0.2 fold, 0.5 fold, or 1.0 fold, e.g., from about 0.1-5.0 fold, from 0.2-fold to 2.0-fold, from 0.3-fold to 1.0-fold, from 0.3-fold to 0.7-fold) as compared to a control or a normal reference sample. An increase in gene expression or protein expression can be determined using any useful methods known in the art or described herein (e.g., as determined by PCR, gel electrophoresis, ELISA, or the like).

A decrease in presence of a particular biomarker (e.g., a protein, nucleic acid, or analyte) can also correspond to induction of oxidative stress where, e.g., the presence of a particular protein, nucleic acid, or analyte is negatively correlated with oxidative stress. In such cases, a decrease in presence of the particular protein, nucleic acid, or analyte as compared to a control from a normal or reference sample of at least 0.1 fold, 0.2 fold, 0.5 fold, or 1.0 fold, e.g., from about 0.1-5.0 fold, from 0.2-fold to 2.0-fold, from 0.3-fold to 1.0-fold, from 0.3-fold to 0.7-fold, would be indicative of an "increase" in oxidative stress.

The "reference value" may be obtained by measuring the level for the biomarker in a standard or reference sample, by obtaining a prerecorded value, or by calculating a value from an algorithm. A reference sample is any sample or standard that is used for comparison purposes. A standard may be obtained from a purified reference biomarker at a known concentration. A reference sample can be a sample taken from the same subject prior to the onset of a disorder (e.g., a proliferative disorder) or prior to the administration of a particular chemotherapeutic or radiation therapy. Alternatively, a reference sample can be obtained from samples from one or more subjects not having the disorder and/or not treated with a particular chemotherapeutic or radiation therapy or one or more subjects that

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B4245785.4 have been successfully treated for the disorder. A prerecorded value can be based on samples taken from a population of one or more individuals with a known disease state (e.g., individuals known not to have a proliferative disorder or to have been successfully treated for a proliferative disorder). Finally, one or more prerecorded values may be used to calculate the reference value in order to compensate for differences between subjects that correlate with biomarker levels.

By "subject" is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline.

By "treating" is meant administering a chemotherapeutic or radiation therapy to a subject already suffering from, or at risk of developing, a disorder (e.g., proliferative disease) to improve the subject's condition.

By "proliferative disorder" is meant a disorder associated with abnormal cell growth. Exemplary cell proliferative disorders include cancer (e.g., brain cancer, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, polycythemia vera, Hodgkin's disease, non-Hodgkin's disease, Waldenstrom's macroglobulinemia, heavy chain disease, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,

endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendriglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma, lung cancer, squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and colon cancer). It also includes conditions such as papillomas (both cutaneous and anogenital), actinic keratosis, eczema and also premalignant conditions such as adenomas of the colon, dysplastic lesions

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B4245785.4 in any human organ or tissue such as the skin, lung, breast, digestive system, urinary and genital systems.

By "chemotherapeutic agent" is meant a compound effective to treat a proliferative disorder. Examples of chemotherapeutic agents are compounds that induce oxidative stress in the target cells or stromal cells of the proliferative disorder sensitive to the

chemotherapeutic agent. Chemotherapeutic agents include disclosed herein as well as a) alkylating agents, such as for example nitrosoureas and platinum; b) antimetabolites, such as folic acid analogs and purine and pyrimidine analogs; c) natural products, such as, for example, vinca alkaloids, taxanes, and camptothecins; d) hormones and antagonists, such as, for example, estrogens and anti-estrogens; and d) agents such as differentiating agents, protein tyrosine kinase inhibitors, immunomodulators , biological response modifiers, and monoclonal antibodies.

The term "probe agent" refers to any compound that elevates oxidative stress associated with neoplastic disease. All probe agents are predictive of whether one or more neoplastic diseases will respond to one or more chemotherapeutic agents. A probe agent is preferably the same agent as the chemotherapeutic agent, but may be different. Thus, the probe agent is predictive of whether a neoplastic disease will respond to treatment with the probe agent when administered as a chemotherapeutic agent. A probe agents may be predictive of the outcome of treatment other chemotherapeutic agents as well. Thus, a probe agent may be predictive of whether a neoplastic disease will respond to treatment with a different molecule when the different molecule is administered as a

chemotherapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Phospho-aspirin (PA) inhibits the growth of breast cancer xenografts. The chemotherapeutic effect of phospho-aspirin on subcutaneous MCF-7 xenografts (A; *, p<0.01, vs. control) and MDA-MB-231 xenografts (B; *, p<0.001, vs. control). All values are mean ±SEM; n=10-16 tumors/group. C: The structure of phospho-aspirin.

Fig. 2. Redox effects of phospho-aspirin in breast cancer. A: Phospho-aspirin induces RONS in MDA-MB-231 cells time-dependently (15 min-2 h). The antioxidant N-acetyl- cysteine (NAC) blocks it. Probes: MitoSox Red: mitochondrial superoxide anion (0₂ ^~); DCFDA: general RONS probe; DHE: cytoplasmic 0₂ ^~. B: Phospho-aspirin suppresses GSH levels in MDA-MB-231 cells; BHO, the GSH synthesis inhibitor, was the positive control. C: Phospho-aspirin inhibits TrxR activity in MDA-MB-231 xenografts. D: Phospho-aspirin

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B4₂45785.4 1.5xIC₅o for 2h suppresses the expression of Trx-1 and TrxR in MDA-MB-231 cells. E: Phospho-aspirin induces Racl/2/3 in MDA-MB-231 xenografts. All values: mean ± SEM. Fig. 3. Phospho-aspirin inhibits redox-sensitive signaling in MDA-MB-231 xenografts. A: Activated NF-kB (p-p65) levels determined by immunohistochemistry; representative images above; magnification 200X. B: Phospho-aspirin suppresses p-JNK. Immunoblot: each lane is from a single xenograft. Histogram: densitometry results from all animals in each group. All values: mean ± SEM.

Fig. 4. 15-F2-isoprostane is a rapidly responding predictive biomarker of phospho-aspirin in breast cancer. Left: Urine levels of 15-F₂-isoprostane vs. the volume of MDA-MB-231 xenografts in nude mice treated with phospho-aspirin for 25 days (Fig. 1). Their correlation is significant: the higher the levels, the smaller the tumor. Right: 15-F₂-isoprostane urine levels are markedly elevated following 2 daily doses of phospho-aspirin to nude mice with such breast cancer xenografts, compared to vehicle treated mice. Phospho-aspirin has no effect on 15-F₂-isoprostane in nude mice without xenografts (first column). Values: Mean ± SEM.

Fig. 5. Phospho-aspirin induces 8-OH-dG in MDA-MB-231 xenografts. A: Representative image of 8-OH-dG detected by immunohistochemistry. The photograph shows clearly delineated necrotic and unaffected areas: magnification 40X. B: Enlarged image from A; magnification 200X. C: Apoptosis from the same xenograft; TUNEL; 200X. D: Histogram of 8-OH-dG expression in vehicle and phospho-aspirin-treated groups. Values are mean ± SEM.

Fig. 6. MS spectra of product ions of 8-F₂-isoprostane. Top: 8-F₂-isoprostane generates abundant molecular ions at m/z 353.3 in electrospray negative-ion mode. Bottom: the above ion undergoes collision-induced fragmentation to generate an array of product ions, of which an ion at m/z 115.2 is the most abundant.

DETAILED DESCRIPTION OF THE INVENTION

In general, the invention is based on the discovery that elevated levels of oxidative stress biomarkers shortly after administration of chemotherapeutic or radiation therapy in subjects having proliferative disorders is indicative of sensitivity to the administered chemotherapeutic and/or radiation therapy. Consequently, measurement of oxidative stress biomarker levels can inform whether a particular chemotherapeutic or radiation therapy is likely to be efficacious in treating a proliferative disorder before prolonged exposure to the therapy. This information allows a subject to avoid unnecessary and otherwise dangerous

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B4₂45785.4 therapies and increases the likelihood that a selected therapeutic course will ultimately lead to treatment of a particular proliferative disorder.

Biomarkers of oxidative stress

Oxidative stress biomarkers have been validated in animal and clinical models (22, 23). Those detected in urine are preferable because a) urine collection is noninvasive; and b) urine is less liable than blood to artificially increase oxidative biomarkers during sample collection and storage. Examples of biomarkers that are suitable for the purposes of this invention are the following:

• F2-isoprostanes: They are produced by the non-enzymatic oxidation of arachidonic acid by RONS. F₂-isoprostanes are chemically stable and have been validated as sensitive biomarkers of oxidative stress in animal and clinical models and are the most extensively used (24-26). Since the oxygen molecule can be added to the 5, 8, 12 or 15 position of arachidonic acid, there are four regioisomers. Specific F₂-isoprostane isomers may be more or less sensitive to various outcomes, which is explained by the fact that different free radicals likely favor differential production of F₂-isoprostane isomers (27, 28).

• Malondialdehyde. The most representative aldehyde derivative of lipid peroxidation, malondialdehyde results from the peroxidation of polyunsaturated fatty acids and has been used extensively as an oxidation marker (29-31).

• Dityrosine: Proposed as a strong indicator of protein oxidation (32), dityrosine arises from the tyrosine radical, generated by the attack on proteins by many RONS, including hydroxyl radicals and peroxynitrite. Dityrosine, produced proportionally to the oxidative insult and the rate of radical formation, marks damaged proteins for degradation and is excreted in urine.

• 8-hydroxy-2'-deoxyguanosine (8-OH-dG). When RONS attack DNA, DNA is oxidized leading to purine and pyrimidine-derived lesions (33).

• 4-hydroxy-2-nonenal, or other nonenals, which are aldehyde derivative of lipid

peroxidation.

• Glutathione.

• Thioredoxin and all its isoforms.

• Thioredoxin reductase.

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B4₂45785.4 • NADPH oxidase and each of its component subunits: Rho guanosine triphosphatase (GTPase), usually Racl or Rac2 or Rac3; and the "phox" units gp91-PHOX , p22phox, p40phox, p47phox, and p67phox.

• GSH synthase.

• NADP, vitamins E and C.

• Superoxide dismutase, catalase, glutathione peroxidase, glutaredoxine, peroxiredoxine, heme oxygenase- 1, phase II detoxifying enzymes, such as GSH reductase and NQOl (NAD(P)H dehydrogenase, quinone 1).

• Individual RONS species, e.g. Of^", H₂0₂, OH*, H₂0₂, NO*, singlet oxygen.

• Additional oxidative stress biomarkers as detailed in publications cited herein.

In addition to the above, biomarkers of oxidative stress include expressed nucleic acids and proteins associated with oxidative stress (e.g., GSH, thioredoxin-1 (Trx-1), thioredoxin reductase (TrsR), and NADP). Such nucleic acid biomarkers can be measured by, e.g., polymerase chain reaction, next generation sequencing, oligonucleotide microarrays, and any other method known in the art. Protein biomarkers can be measured, e.g., by antibody binding (e.g., ELISA assay) or mass spectrometry.

Test samples used in the methods of the invention may include cells, protein or membrane extracts of cells, blood, or biological fluids. Examples of solid test samples include samples taken from feces, the rectum, central nervous system, bone, breast tissue, renal tissue, the uterus, the cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, ovarian tissue, adrenal tissue, testis tissue, the tonsils, and the thymus. Examples of biological fluid samples include samples taken from the blood, serum, CSF (cerebral spinal fluid), semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, stool, and tears. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the test sample is a breast, lung, colon, or prostate tissue sample obtained by needle biopsy.

Therapy

The invention encompasses use of any chemotherapeutic agent now known, or later determined, to cause oxidative stress. The induction of oxidative stress by many classes of chemotherapeutic agents has also been well-recognized as a phenomenon that is critical to

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B4₂45785.4 their efficacy (reviewed, for example, in (49, 50)). Chemotherapeutic agents listed in these two publications include, among others, arsenic trioxide, emodin, anthracyclines, such as daunorubicin and doxorubicin, cisplatin, bortezomib, synthetic retinoids (50), as well as agents under clinical evaluation, such as, for example, imexon, 2-methoxyestradiol, tetrathiomolybdate, motexaphin gadolinium, phenylethyl isothiocyanate (49). Each of references 49 and 50 are specifically incorporated by reference in their entirety and, in particular, to include all of the chemotherapeutic agents disclosed therein.

In certain embodiments, the chemotherapeutic agent (and/or the probe agent) of the invention is a phospho-NSAID (non-steroidal anti-inflammatory drugs). Such drugs are disclosed, e.g., in U.S. Patent No. 8,236,820, U.S. Patent Application Publication Nos. 2012-0316139, 2013-225529, 2014-0088044, and 2014-0088045, and International Patent Application Publication Nos. WO 2005/065361 WO 2013/130625, WO 2009/023631, WO 2014/047569, and WO 2014/047592. Each of these patents and applications is incorporated by reference in its entirety such that, e.g., it is as if each compound recited in the above- publications is listed individually here. Particular examples of phospho-NSAIDs include phospho-aspirin, phospho-ibuprofen, phospho-fluribuprofen, and phospho-sulindac.

In certain embodiments, the agent has a structure of Formula (I):

Formula (I) or an enantiomer, a diastereomer, a racemate, a tautomer, salt, hydrate, cocrystal, or compositions thereof.

In Formula I, A is an optionally substituted aliphatic, heteroaliphatic, aromatic, heteroaromatic substituent or alkylaryl substituent having 1 to 100 carbon atoms or is selected from:

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B4245785.4

B4245785.4

Formul ormula A-XVI Formula A-XVII

D is absent or

X¹ and X² are independently selected from -0-, -NR⁵-, and -S-;

R¹ and R⁴ are independently selected from hydrogen and trifluoromethyl;

R² is selected from -SCH₃, -S(0)CH₃, and -S(0)₂CH₃;

R³ is selected from hydroxyl, Z, -X¹-(CH₂)4-Z,

R⁵ is selected from hydrogen and Ci_₆ alkyl;

Z is selected from:

Formula Z-VI Formula Z-VII

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B4₂45785.4

Formula Z-VIII

R⁶ and R⁷ are independently selected from hydrogen, Ci_₆-alkyl, and polyethylene glycol residue.

In some embodiments, X¹ is -NR⁵-, and R⁵ is selected from hydrogen, methyl, and ethyl.

In other embodiments, X¹ is -0-.

In certain embodiments, Z is OR⁷ , R⁶ is selected from ethyl and a polyethylene glycol residue, and R⁷ is selected from hydrogen and ethyl.

In still other embodiments, A is selected from:

Formula A-II Formula A-III Formula A-XII

16

B4245785.4 R¹ and R⁴ are independently selected from hydrogen and trifluoromethyl, and X is selected from -0-, -S-, and -NH-.

In some embodiments, X¹ is -0-, Z is -0-P(0)(CH₂CH₃)2, and A is:

In certain embodiments, X¹ is selected from -O- and -NH-, Z is -0-P(0)(CH₂CH₃)₂,

A is:

and R is selected from hydrogen and trifluoromethyl.

In other embodiments, X¹ and X² are independently selected from -O- and -NH-, Z

and R is selected from hydrogen and trifluoromethyl.

In some embodiments, X¹ and X² are independently selected from -0-, -S-, and - is:

17

B4₂45785.4 In some embodiments, X¹ is selected from -0-, -S-, and -NH-, Z is selected from - -P(0)(CH₂CH₃)₂ and -0N0₂, A is:

and R¹ is selected from hydrogen and trifluoromethyl, and X² is selected from -0-, -S- and - NH-. in embodiments, X¹ is selected from -O- and -NH-, Z is -0N0₂, and A is:

Accordingly, the compounds of Formula I include but are not limited to compounds of which the structures are shown below:

1 2

18

B4₂45785.4

B4245785.4

B4245785.4

B4245785.4

Formula (II)

or a pharmaceutically acceptable salt thereof.

In Formula II: Y¹ is a polyethylene glycol residue; R⁶ is selected from hydrogen, Ci_ 6-alkyl, and polyethylene glycol residue;

A is an optionally substituted aliphatic, heteroaliphatic, aromatic, heteroaromatic substituent or alkylaryl substituent having 1 to 100 carbon atoms or selected from:

Formula ula A-IV

Formul la A- VII

- VIII Formula A-IX Formula A-X

Formula A-XI Formula A-XII

22

B4245785.4

Formula A-XIV

Formula A-XVI Formula A-XVII

D is absent or

X¹ and X² are independently selected from -0-, -NR⁵-, and -S-;

R¹ and R⁴ are independently selected from hydrogen and trifluoromethyl;

R² is selected from -SCH₃, -S(0)CH₃, and -S(0)₂CH₃;

R³ is selected from hydroxyl, Z, and -X^B-Z;

R⁵ is selected from hydrogen and Ci_₆ alkyl;

B is selected from:

Formula B-I

a single bond, and an aliphatic group with 1 to 22 carbon atoms;

R⁸ is a C i_4 alkylene; and

R⁹ is hydrogen, Ci_₆-alkyl, halogenated Ci_₆-alkyl, Ci_₆-alkoxy, halogenated Ci_₆-alkoxy, -C(0)-Ci_₆-alkyl, -C(0)0-Ci_₆-alkyl, -OC(0)-Ci_₆-alkyl, -C(0)NH₂,

23

B4₂45785.4 -C(0)NH-Ci_6-alkyl, -S(0)-Ci_₆-alkyl, -S(0)₂-Ci_₆-alkyl, -S(0)₂NH-Ci_₆-alkyl, cyano, halo or hydroxyl.

In further embodiments, Y¹ is a polyethylene glycol residue described by

-0(CH₂CH₂0)_mR¹⁰, wherein m is 1 to 100 (e.g. 20 to 100, 20 to 50, 40 to 50), and R¹⁰ is selected from hydrogen, alkyl and alkoxy, and R⁶ is hydrogen.

In still other embodiments, Y¹ is -0(CH₂CH₂0)_mR¹⁰ wherein m is 45, R¹⁰ is - OCH₃, and R⁶ is hydrogen.

In some embodiments, X¹ is -0-.

In other embodiments, X¹ is -NR⁵- and R⁵ is selected from hydrogen, methyl, and ethyl.

In certain embodiments, B is -(CH₂)₄-.

In some embodiments, A is:

In a third aspect, the agent is a compound of general Formula III

Formula (III)

or a pharmaceutically acceptable salt thereof.

In Formula III: A is selected from:

24

B4₂45785.4

Formula A-III Formula A-V

Formula A- VI Formula A- VIII

Formula A-XI Formula A-XII

Formula A-XIII IV

-XV Formula A-XVI Formula A-XVII

Formula A-XVIII Formula A-XIX

25

785.4 D is absent or

X¹ and X² are independently selected from -0-, -NR⁵-, and -S-;

R¹ and R⁴ are independently selected from hydrogen and trifluoromethyl;

X³ is selected from -S- and -NH-;

R³ is selected from hydroxyl, Z, and -X^B-Z;

R⁵is selected from hydrogen and Ci_₆ alkyl;

B is selected from:

Formula B-I Formula B-II

a single bond, and an aliphatic group with 1 to 22 carbon atoms;

R⁸, R¹¹, and R¹² are the same or different Ci_₄ alkylene;

R⁹ is hydrogen, Ci_₆-alkyl, halogenated Ci_₆-alkyl, Ci_₆-alkoxy, halogenated Ci_₆-alkoxy, -C(0)-Ci_₆-alkyl, -C(0)0-Ci_₆-alkyl, -OC(0)-Ci_₆-alkyl, -C(0)NH₂,

-C(0)NH-Ci_6-alkyl, -S(0)-Ci_₆-alkyl, -S(0)₂-Ci_₆-alkyl, -S(0)₂NH-Ci_₆-alkyl, cyano, halo or hydroxy;

Z is selected from: - -OR« H-°^R6 O-N

7 , OR⁷ , OR⁷ , ⁰ , ^{¾ 0-} O ^{N 3}

Formula Z-I Formula Z-II Formula Z-III Formula Z-IV Formula Z-V

Formula Z-VI Formula Z-VII

26

B4₂45785.4

Formula Z-VIII

or B together with Z forms a structure:

Formula BZ-I

R⁶ and R⁷ are independently selected from hydrogen, Ci_6-alkyl, and polyethylene glycol residue; and

R¹³ is selected from hydrogen, an aliphatic group with 1 to 22 carbon atoms (e.g. Ci_ ₆-alkyl), and polyethylene glycol residue.

In still other embodiments, X¹ is -0-.

In certain embodiments, X¹ is -NR⁵- and R⁵ is selected from hydrogen, methyl, and ethyl.

In some embodiments, B is selected from:

In other embodiments, Z is sel CH₂CH₃)₂ and -ON0₂.

In further embodiments, BZ is

In certain embodiments, X¹ is selected from -O- and -NH-, B is selected from

is -OP(0)(OCH₂CH₃)₂, and A is:

27

B4₂45785.4

In some embodiments, X¹ is selected from -O- and -NH-, B is selected from and R³ is:

In some embodiments, wherein X¹ is selected from -O- and -NH-, B is selected from is -OP(0)(OCH₂CH₃)₂, A is:

and X is selected from -O- and -NH-.

In other embodiments, X¹ is selected from -O- and -NH-, B is selected from

(0)(OCH₂CH₃)₂, and A is:

In further embodiments, X¹ is selected from -O- and -NH-, B is selected from

Z is -OP(0)(OCH₂CH₃)₂, A is:

28

B4₂45785.4

and R³ is hydroxyl or selected from:

In certain embodiments, X¹ is selected from -O- and -NH-, B is selected from

Z is -OP(0)(OCH₂CH₃)₂, A is:

R is hydroxyl or selected from:

In some embodiments, X¹ is selected from -O- and -NH-, B is selected from

-OP(0)(OCH₂CH₃)₂, A is:

29

B4₂45785.4

and R is selected from hydrogen and trifluoromethyl.

In some embodiments, X¹ is selected from -O- and -NH-, B is selected from P(0)(OCH₂CH₃)₂, A is:

and R is selected from hydrogen and trifluoromethyl.

In other embodiments, X¹ is selected from -O- and -NH-, B is selected from is -OP(0)(OCH₂CH₃)₂, A is:

and X is selected from -0-, -S-, and -NH-.

In other embodiments, X¹ is selected from -O- and -NH-, B is selected from d Z , Z is selected from -OP(0)(OCH₂CH₃)₂ and -ON0₂, A is:

and X is selected from -0-, -S-, and -NH-.

In some embodiments, X¹ is selected from -O- and -NH-, B is -(CH₂)₄-, Z is - 0N0₂, A is:

30

B4₂45785.4

, R is selected from hydrogen and trifluoromethyl, and X is selected from -S-, and -NH-.

In other embodiments, X¹ is -NH-, A

R¹ is selected from hydrogen and trifluoromethyl, and X³ is selected from -S-, and -NH-.

Accordingly, the compounds of Formula III include but are not limited to compounds of which the structures are shown below:

22 23

28 29 30

31

B4245785.4

B4245785.4

B4245785.4

B4245785.4

B4245785.4

B4245785.4

B4245785.4

B4245785.4 84

89

39

B4245785.4

B4245785.4

In a fourth aspect the agent is a compound of general Formula IV

Formula (IV)

or a pharmaceutically acceptable salt thereof.

In Formula IV: A is an optionally substituted aliphatic, heteroaliphatic, aromatic, heteroaromatic substituent or alkylaryl substituent having 1 to 100 carbon atoms or selected from:

41

B4245785.4 V

Formula A-VIII Formula A-IX Formula A-X

Formula A-XI Formula A-XII

Formula A-XIII Formula A-XIV

42

45785.4 V Formula A-XVI Formula A-XVII

D is absent or

X² is selected from -0-, -NR⁵-, and -S-;

R¹ and R⁴ are independently selected from hydrogen and trifluoromethyl;

R² is selected from -SCH₃, -S(0)CH₃, and -S(0)₂CH₃;

R³ is selected from hydroxyl, Z, and -X^B-Z;

R⁵ is selected from methyl and ethyl;

B is selected from:

Formula B-I Formula B-II

a single bond, and an aliphatic group with 1 to 22 carbon atoms;

R⁸, R¹¹, and R¹² are the same or different Ci_₄ alkylene;

R⁹ is hydrogen, Ci_₆-alkyl, halogenated Ci_₆-alkyl, Ci_₆-alkoxy, halogenated Ci_₆-alkoxy, -C(0)-Ci_₆-alkyl, -C(0)0-Ci_₆-alkyl, -OC(0)-Ci_₆-alkyl, -C(0)NH₂,

-C(0)NH-Ci_₆-alkyl, -S(0)-Ci_₆-alkyl, -S(0)₂-Ci_₆-alkyl, -S(0)₂NH-Ci_₆-alkyl, cyano, halo or hydroxy;

Z is selected from:

Formula Z-I Formula Z-II Formula Z-III Formula Z-IV Formula Z-V

Formula Z-VI Formula Z-VII

43

B4₂45785.4

Formula Z-VIII

or B together with Z forms a structure:

Formula BZ-I

R⁶ and R⁷ are independently selected from hydrogen, Ci_6-alkyl, and polyethylene glycol residue; and

R¹³ is selected from hydrogen, an aliphatic group with 1 to 22 carbon atoms (e.g. Ci_ 6-alkyl), and polyethylene glycol residue.

In a fifth aspect, the agent is a compound having a structure selected from

44

B4245785.4

B4245785.4

Formula (V)

In Formula V: A is an optionally substituted aliphatic, heteroaliphatic, aromatic, heteroaromatic substituent or alkylaryl substituent having 1 to 100 carbon atoms;

X¹ is selected from -0-, -S-, and -NR⁵-;

R⁵ is selected from hydrogen and a Ci_₆ alkyl;

B is an aliphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, aralkyl, or

heteroaromatic group optionally substituted with one or more R¹⁵ moieties,

each R¹⁴ is independently, selected from hydrogen, halogen, hydroxyl, alkoxyl,-CN; an optionally substituted aliphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, aralkyl, heteroaromatic moiety; -OR^R, -S(=0)_nR^d, -NR^bR^c, -C(=0)R^a and -C(=0)OR^a; n is 0-2; R^a, for each occurrence, is independently selected from hydrogen and an optionally substituted aliphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, aralkyl, or a heteroaromatic moiety; each of R^b and R^c, for each occurrence, is independently selected from hydrogen; hydroxyl, S0₂R^d, and aliphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, aralkyl, heteroaromatic or an acyl moiety; R^d, for each occurrence, is independently selected from hydrogen, -N(R^e)₂, aliphatic, aryl and heteroaryl, R^e, for each occurrence, is independently hydrogen or aliphatic; and R^R is an optionally substituted aliphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, aralkyl, heteroaromatic or acyl moiety;

Z is selected from:

Formula Z-I Formula Z-II Formula Z-III Formula Z-IV Formula Z-V

Formu a Z-VI

46

B4₂45785.4

Formula Z-VIII

or B together with Z forms a structure:

Formula BZ-I

R⁶ and R⁷ are independently selected from hydrogen, Ci_6-alkyl, and polyethylene glycol residue; and

R¹³ is selected from hydrogen, an aliphatic group with 1 to 22 carbon atoms (e.g. Ci_ 6-alkyl), and polyethylene glycol residue;

or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound of Formula V is further described by Formula I, II, III, or IV or any specific compound described herein.

In another embodiment the compound of Formula V is a compound disclosed in US Patent No. 8,236,820, incorporated by reference. For example, the compound of Formula V can be selected from:

47

B4245785.4 120 121

132

In certain embodiments, the chemotherapeutic (and/or probe) agent comprises a phospho-nonsteroidal anti-inflammatory agent having one or more phosphate moieties (phospho-NSAIDs). Compounds that may be used in the present invention are disclosed in WO 2013/130625, WO 2009/023631, WO 2005/065361, and WO 2011/094589, which are incorporated herein by reference. Further incorporated herein by reference are US provisional application Serial No. 61/704,021, US application Serial No. 14/033,976, US application Serial No. 14/034,421, and US application Serial No. 14/033,932, which disclose other compounds which may be used herein.

Particularly preferred for this purpose are phospho-ibuprofen I, phospho-ibuprofen glycerol II, phospho-ibuprofen glycerol amide III, phospho-ibuprofen amide IV, phospho- sulindac V, phospho-sulindac amide VI, phospho-aspirin VII, phospho-valproic acid VIII, the compounds IX and X, phospho-pentyl-ibuprofen XI, phospho-decyl-ibuprofen XII, phospho-hexyl-aspirin XIII, phosphor-butyl-aspirin XIV, and phospho-salicylic acid glycerol XV, the structures of which are shown below:

I

49

B4245785.4

B4245785.4 In one embodiment, the agent is a compound having a Formula VI:

O

A-U-X¹— B— Z

Formula VI

or an enantiomer, a diastereomer, a racemate, a tautomer, salt, hydrate, cocrystal, or compositions thereof, wherein

X¹ is selected from the group consisting of -0-, -S- and -NR¹-;

R¹ being hydrogen or C_1-100-alkyl, preferably Ci_₂2-alkyl, particularly preferred Ci_ _lo-alkyl;

A is an optionally substituted aliphatic, heteroaliphatic, aromatic, heteroaromatic substituent or alkylaryl substituent having in a preferred embodiment 1 to 100, and even more preferably 1 to 42 carbon atoms. Preferably, A is derived from among NSAIDs. In one of the referred embodiments, A is selected from the group consisting of:

!-ormu!a A-lil Formula A-IV Formula A-V

Formula A-vi!i Formula A-!X Formula A-X

51

B4245785.4

Formula A-X!

-orrrsuSa A-XII Formufa A-X!lf

wherein,

R being selected from hydrogen and trifluoromethyl;

R being selected from -X -C(0)-CH₃;

R¹¹ being selected from -SCH₃, -S(0)CH₃ and -S(0)₂CH₃;

R¹² being selected from hydroxy, -B-Z and Formula A-XII

whereby

X² is selected from the group consisting of -0-, -S- and -NR¹³-, wherein, R¹³ being hydrogen or Ci_₆-alkyl;

ed from the group consisting of

Formula B-! Formula B-IS

a single bond and an aliphatic substituent, preferably with 1 to 100, more preferred with 1 to 42 and particularly preferred with 1 to 22 carbon atoms,

wherein,

52

B4₂45785.4 R², R⁴ and R⁵ being the same or different Ci-3-alkylene;

R³ being hydrogen, Ci_₆-alkyl, halogenated Ci_6-alkyl, Ci_6-alkoxy, halogenated Ci_₆-alkoxy, -C(0)-Ci_₆-alkyl, -C(0)0-Ci_₆-alkyl, -OC(0)-Ci_₆-alkyl, - C(0)NH₂, -C(0)NH-Ci_₆-alkyl, -S(0)-Ci_₆-alkyl, -S(0)₂-Ci_₆-alkyl, -S(0)₂NH-Ci_₆- alkyl, cyano, halo or hydroxyl;

Z is selected independently from the group consisting of:

Formula Z-I Formula Z-II Formula Z-III Formula Z-IV Formula Z-V

Formula Z-VI Formula Z-VII

Formula Z-VIII wherein,

R⁶ being independently selected from hydrogen, C_1-100-alkyl, preferably Ci_₆- alkyl, and polyethylene glycol residue;

R⁷ being selected from hydrogen, C_1-100-alkyl, preferably Ci_₆-alkyl, and polyethylene glycol residue;

or B together with Z forms a structure:

53

B4₂45785.4 Formula BZ-I

wherein R⁶ being defined as above; and

R⁸ being independently selected from hydrogen, an aliphatic substituent with 1 to 22 carbon atoms, more preferred Ci_₆-alkyl and a polyethylene glycol residue

Preferably, the folic acid residue is selected from

Formula Z-VIII

In one embodiment, A is represented by Formula A-I or A-IV, XI is -O- and -B-Z is not -(CH₂)₄-0-P(0)(OC₂H₅)₂.

In another embodiment, A is represented by Formula A-II and XI is not -O- and/or - B- is an aliphatic substituent with 1 to 100, preferably with 1 to 42 carbon atoms.

In another embodiment, the agent is selected from compounds having Formula (VII)

Formula VII or an enantiomer, a diastereomer, a racemate, a tautomer, salt or hydrate thereof, wherein, m = 0 or 1 ;

X¹ and X² are independently selected from the group consisting of -0-, -S- and -

54

B4₂45785.4 NR¹-, R¹ being hydrogen or Ci_₆-alkyl;

B is an optionally substituted aliphatic, heteroaliphatic, aromatic, heteroaromatic or alkylaryl substituent having 1 to 40 carbon atoms;

Z¹ is selected from the group consisting of hydrogen, farnesyl and a folic acid residue;

Z² is selected from

Formula Z-I Formula Z-II

whereby

Z² is preferably represented by Formula Z-I;

R⁶ being independently selected from hydrogen, Ci_ioo-alkyl and a polyethylene glycol residue,

R⁷ being independently selected from hydrogen, Ci_ioo-alkyl and a polyethylene glycol residue; or

B together with Z² forms a structure

Formula BZ-I

R⁶ being defined as above, and

R⁸ being independently selected from hydrogen, an aliphatic substituent with 1 to 22 carbon atoms, more preferred Ci_₆-alkyl and a polyethylene glycol residue and

R⁹ being selected from hydrogen and trifluoromethyl.

A further aspect of the present invention, the agent is a compound of Formula VIII:

55

B4245785.4 Formula VIII or an enantiomer, a diastereomer, a racemate, a tautomer, salt or hydrate thereof, wherein X², B, Z² and R⁹ are as defined above.

In a specific embodiment, the composition further comprises

difluoromethylornithine or cimetidine.

In certain embodiments, the agent is a compound (e.g., an anti-cancer agent) having a structure of Formula IX:

G-X-L-Y-M

Formula IX

or an enantiomer, a diastereomer, a racemate, a tautomer, salt or hydrate thereof, wherein G is selected from one of the moieties shown below (G^G⁴¹);

56

B4245785.4

57

B4245785.4

58

B4245785.4

R¹ being hydrogen or C_1-100-alkyl, preferably Ci_22-alkyl, particularly preferred Ci_ lo-alkyl;

59

B4245785.4 L is selected from one of the moieties shown below (L^L³); wherein in L¹, p is from 1-100, preferably 1-22, most preferably 1-10;

Y is selected from -0-, -NR²-, or -S-;

R² being hydrogen or C_1-100-alkyl, preferably Ci_₂2-alkyl, particularly preferred Ci_i₀-alkyl; and

M is selected from one of the moieties shown below (M^M³); wherein in M² n is preferably 1-10:

In certain embodiments, the agent is a compound (e.g., an anti-cancer agent) having a structure of Formula X:

G-X-N

Formula X

or an enantiomer, a diastereomer, a racemate, a tautomer, salt or hydrate thereof, wherein G is selected from one of the moieties shown above (G^G⁴¹);

X is selected from -0-, -NR¹-, or -S-,

R¹ being hydrogen or C_1-100-alkyl, preferably Ci_₂₂-alkyl, particularly preferred C_1-10- alkyl; and

60

B4245785.4 The biomarkers of oxidative stress of the invention can also be used to determine sensitivity to radiation therapy. Radiation therapy is one of the clearest examples of antineoplastic treatment whose mechanism relies primarily on RONS (51). Mechanistically, radiation therapy alters cellular homeostasis by modifying the redox status of cancer cells (and normal cells, hence its side effects) and can trigger cell death by apoptosis through the mitochondrial pathway (52).

EXAMPLES

Phospho-aspirin strongly inhibits the growth of human breast cancer xenografts

We assessed the chemotherapeutic potential of phospho-aspirin using ER(+) and ER(-) subcutaneous breast cancer xenografts in nude mice (Fig. 1). In the ER(+) MCF-7 xenografts, phospho-aspirin 120 mg/kg, 5 d/wk orally potently inhibited MCF-7 xenograft growth, resulting in complete growth arrest. At the same dose, phospho-aspirin inhibited MDA-MB-231 (triple negative) xenograft growth significantly, starting on day 8 of treatment until the end of the study (p<0.001 for all time points). At sacrifice, phospho- aspirin inhibited tumor growth by 80% (vehicle = 308.5±36.2 mm³ vs. Phospho-aspirin =142.9±16.4 mm³; mean ± SEM for these and all subsequent values).

Induction of oxidative stress by phospho-aspirin

We determined the effect of phospho-aspirin on RONS levels by using various molecular probes: DCFDA (general RONS), DHE (cytoplasmic 0₂·^~), and MitoSox Red (mitochondrial 0₂·^") (Fig. 2). Phospho-aspirin 1.5xIC₅o increased significantly RONS levels in MDA-MB-231 cells in vitro. Compared to control, phospho-aspirin increased DCFDA by 76%, DHE by 51%, and MitoSox Red by 51%. Of note, pretreatment with the antioxidant N-acetyl-cysteine (NAC) 10 mM for 4 h partially abrogated mitochondrial 0₂·^~ induction. Phospho-aspirin had a significant effect on three major players in redox homeostasis, GSH, the thioredoxin system, and NADPH oxidase (Fig. 2B-D).

GSH: Phospho-aspirin decreased the levels of cellular GSH (50%> decrease at lxIC50). Co-incubation of phospho-aspirin with BSO, an inhibitor of GSH synthesis (35), synergistically induced RONS levels and inhibited cell growth.

• The thioredoxin system, composed of thioredoxin- 1 (Trx-1), thioredoxin reductase (TrxR), and NADP, plays a crucial part in the maintenance of redox homeostasis in cells by reducing oxidized proteins (36-40). Trx-1 is overexpressed in breast cancer (34). Phospho-aspirin targeted the major components of the thioredoxin system.

Phospho-aspirin a) inhibited TrxR activity in MDA-MB-231 cells in culture and in

61

B4₂45785.4 xenografts by 54% and 41%, respectively (p<0.02-0.04), without affecting its levels; and b) reduced Trx-1 levels in MDA-MB-231 cells after 1 hr of treatment with 1.5xIC₅o phospho-aspirin (Fig. 2).

• NADPH oxidase is a major source of RONS in cells. Its activation is regulated by Rac isoforms (numbered 1-3), Rho-like small GTPases (35)(41). Binding of GTP with Racl/2/3 and their subsequent activation are implicated in RONS production during various cellular responses (36). Phospho-aspirin induced RONS, at least in part, by activating the NADPH oxidase. For example, in MDA-MB-231 cells in culture or in xenografts, total Rac 1/2/3 levels were significantly enhanced by phospho-aspirin.

The induction of oxidative stress by phospho-aspirin was biologically relevant (Fig. 3). For example, phospho-aspirin inhibited the NF-κΒ and AP-1 pathways, whose transcriptional activities are sensitive to redox changes (37). In cultured MDA-MB-231 cells, phospho-aspirin inhibited NF-KB-DNA binding, and in MDA-MB-231 xenografts, it reduced the levels of activated NF-κΒ (its p-p65 subunit) by 44% (p<0.009), compared to the control group. Phospho-aspirin also concentration-dependently inhibited AP-l-DNA binding.

Oxidation biomarkers are predictive biomarkers of phospho-aspirin

In nude mice with MCF-7 xenografts treated with phospho-aspirin (or vehicle), we determined baseline levels of 15-F2-isoprostane in the urine of these mice and again on day 4 of treatment, and determined their change from baseline to be 10.6±1.5 ng/mg creatinine. This change was significantly correlated with tumor volume on day 10 (r=-0.817; p<0.03). Similarly, the corresponding change in the urine levels of dityrosine (97.1±17.65 fluorescence units/mg creatinine) correlated significantly with tumor volume on day 10 (r=- 0.798; p<0.04). These changes in the levels of 15-F₂-isoprostane in the urine and dityrosine on day 4, each independently correlated significantly with the inhibitory effect of phospho- aspirin on xenograft growth on day 21 as well (r= -0.711 and r=-0.683, respectively; p<0.05 for both).

Urinary markers respond rapidly to phospho-aspirin

For predictive biomarkers to be clinically useful, they must respond rapidly to the test drug. Thus, we treated nude mice bearing MDA-MB-231 xenografts with phospho- aspirin 120 mg/kg/day for 2 days. We collected 24 hr urine prior to starting treatment and during day 2 of treatment. We used two control groups: a) mice with MDA-MB-231

62

B4₂45785.4 xenografts treated with vehicle; and b) mice with no tumors treated with phospho-aspirin as above. For each animal, we determined the change in the levels of 15-F2-isoprostane from baseline (paired samples). As shown in Fig. 4 (right panel), phospho-aspirin dramatically increased the levels of 15-F₂-isoprostane in urine within 48 hours. This effect was limited to tumor bearing mice (no change in those without xenografts), indicating that xenografts are the origin of the elevated 15-F₂-isoprostane. Similar results were obtained from the same animals for dityrosine: Phospho-aspirin nearly tripled the amount of dityrosine in urine compared to baseline, whereas the vehicle had no effect (% of paired baseline urine sample: vehicle-treated=101±5; Phospho-aspirin treated=283±3; p<0.001).

Phospho-aspirin induces 8-OH-dG

We evaluated the effect of phospho-aspirin on 8-OH-dG on the breast cancer xenografts treated with phospho-aspirin by immunohistochemistry using a specific mAb. As shown in Fig. 5, phospho-aspirin nearly tripled the amount of 8-OH-dG in the xenografts (% 8-OH-dG(+) cells: controls=11.6±2.7 vs. phospho-aspirin=32.1±6.4;

p=0.009). Of great interest is the fact that 8-OH-dG is produced in areas of the xenograft that are necrotic, in contrast to unaffected areas. This was confirmed by staining a consecutive tissue section with TUNEL (Fig. 5C). This finding is in agreement with the concept that induction of strong oxidative stress by phospho-aspirin leads to cell death by apoptosis and/or necrosis (16).

Phospho-sulindac and phospho-ibuprofen administered to mice with lung cancer xenografts increase urinary 15-F₂-isoprostane levels

Two groups of nude mice (n=8 for each) bearing subcutaneous xenografts of A549 human lung cancer cells (average tumor size 152 mm³) were treated with phospho-sulindac (carboxylic ester, butane spacer) 200 mg/kg body weight once daily administered intraperitoneally (dissolved in corn oil) or vehicle. On day 3 of treatment, each mouse was placed in a metabolic cage and urine was collected for 24 hours. The group of mice treated with phospho-sulindac had over 4-fold increased levels of 15-F₂-isoprostane in their urine (control= 5.0 ± 1.4 vs. phospho-sulindac=22.3 ± 2.9 ng/mg creatinine; p<0.01). These findings indicate that brief treatment of lung cancer tumors with phospho-sulindac generates a strong response in an oxidative stress marker in urine.

We also studied five groups of nude mice (n=6 for each) bearing orthotopic (i.e., in the lungs) xenografts of A549 cells (injected intravenously, they homed to the lungs). These mice were treated with phospho-sulindac (carboxylic ester), or phospho-sulindac amide

63

B4₂45785.4 (butane linker), or phospho-sulindac amide (glycerol linker), each at 200 mg/kg body weight, or ibuprofen amide (butane linker) at 150 mg/kg body weight, or vehicle. All were administered once daily intraperitoneally (dissolved in corn oil) for 21 days. On day 21, 24- hour urine samples were collected as above and 15-F₂-isoprostane levels were determined by ELISA (Oxford Biomedical Research).

Compared to control, each of these compounds induced a marked and statistically significant increase in the urine levels of 15-F₂-isoprostane (p<0.01-0.03 for all), as follows: vehicle control = 3.9 ± 0.3; phospho-sulindac (carboxylic ester) = 14.3 ± 3.6; phospho-sulindac amide (butane linker) = 30.6 ± 5.2; phospho-sulindac amide (glycerol linker) = 24.7 ± 4.2; ibuprofen amide (butane linker) = 25.5 ± 5.1 ng/mg creatinine. These increases range between 3.7 and 7.8-fold over control, indicating that these compounds induce strong oxidative stress; and also suggesting that this effect represents a generalized response of cancer to this class of compounds. Of note, each of these compounds was also effective in inhibiting tumor growth, compared to controls.

Analytical methods for the determination of OS biomarkers in urine

• F₂-isoprostanes: The urine samples are purified and concentrated by solid phase

extraction, and are then subjected to LC-MS/MS analysis (42). The LC-MS system consists of Thermo TSQ Quantum Access triple quadruple mass spectrometer interfaced by an electrospray ionization probe with an Ultimate 3000 HPLC system. The four regioisomers of F₂-isoprostanes are identified and quantified based on their distinct retention times and ion fragmentation patterns. Fig. 6 provides an example of our methodology. 15-F₂-isoprostane can also be assayed by an ELISA assay (Cayman Chemical).

• MDA: MDA reacts with thiobarbituric acid to form a highly fluorescent adduct, which is separated by HPLC and quantified by a fluorescence detection (43). Briefly, urine is diluted 12-fold in 0.2% thiobarbituric acid solution pH 3.5. After heating at 95°C for 60 min, the reaction solution is centrifuged and loaded on HPLC system. The reaction adduct is separated on a reverse-phase column and monitored by fluorescence detector at 515/553 nm.

• Dityrosine: Dityrosine can be directly measured, as itself generates fluorescence at 315/410 nm (44). Briefly, urine is diluted 20-fold in 50 mM phosphate buffer pH 7.4, containing 6 M urea. Fluorescence intensity is measured at 315/410 nm, after 30 min at room temperature.

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B4₂45785.4 • 8-OH-dG is analyzed using an ELISA kit from Cayman Chemicals (Ann Arbor, MI). Table 1 summarizes the performance characteristics of analytical methods as described above.

Table 1. Performance characteristics of analytical methods

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B4245785.4 References

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B4245785.4 OTHER EMBODIMENTS

All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.

Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of medicine, pharmacology, or related fields are intended to be within the scope of the invention.

What is claimed is:

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Claims

1. A method of predicting a response of a subject with a neoplastic disease to administration of a chemotherapeutic agent, comprising:

(i) administering a probe agent to the subject;

(ii) obtaining a test sample from the subject;

(iii) measuring a level of a biomarker associated with oxidative stress in the test sample; and

(iv) comparing the measured level to a reference value,

wherein a measured level associated with elevated oxidative stress indicates that the neoplastic disease will respond to treatment with the chemotherapeutic agent.

2. A method of treating cancer, comprising:

performing the method of claim 1 ; and

administering one or more chemotherapeutic agents to said subject if the test sample has a measured level of the biomarker associated with elevated levels of oxidative stress.

3. A method of predicting a response of a subject with a neoplastic disease to administration of radiation therapy, comprising:

(i) administering radiation therapy to the subject;

(ii) obtaining a test sample from the subject;

(iii) measuring a level of a biomarker associated with oxidative stress in the test sample; and

(iv) comparing the measured level to a reference value,

wherein a measured level associated with elevated oxidative stress indicates that the neoplastic disease will respond to radiation therapy.

4. A method of treating cancer, comprising:

performing the method of claim 3; and

administering radiation therapy to said subject if the test sample has a measured level of the biomarker associated with elevated levels of oxidative stress.

5. The method according to any preceding claim, wherein the test sample is obtained from the subject less than about three weeks after the radiation therapy or probe agent is administered to the subject.

6. The method according to any preceding claim, wherein the test sample is obtained from the subject less than 5, 4, 3, 2 or 1 days after the radiation therapy or probe agent is administered to the subject.

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7. A method of determining whether a subject with a neoplastic disease is responding to treatment with radiation therapy or a chemotherapeutic agent, comprising:

(i) obtaining a test sample from the subject being treated with the radiation therapy or chemotherapeutic agent;

(ii) measuring a level of a biomarker associated with oxidative stress in the test sample; and

(iii) comparing the measured level to a reference value,

wherein a measured level associated with elevated levels of oxidative stress indicates that the patient is responding to treatment with the chemotherapeutic agent.

8. The method according to claim 7, wherein the test sample is obtained from the subject less than about three weeks after the radiation therapy or chemotherapeutic agent is administered to the subject.

9. The method according to claim 7, wherein the test sample is obtained from the subject less than 5, 4, 3, 2 or 1 days after the radiation therapy or chemotherapeutic agent is administered to the subject.

10. The method according to any preceding claim, wherein said reference value is: a standard value or range associated with a health condition;

a measured level for the biomarker from a control;

a measured level for the biomarker from a reference group having a known health state; or

a measured level for the biomarker from the subject obtained prior to administering the anticancer agent or radiation therapy.

11. The method according to claim 10, wherein:

said reference value is the measured level for the biomarker from a reference group; and

said reference group is a population of one or more individuals with a known disease state.

12. A method of predicting the response of a subject with a neoplastic disease to administration of radiation therapy or a chemotherapeutic agent, comprising:

obtaining a reference sample from the subject prior to administering radiation therapy or a probe agent;

administering radiation therapy or a probe agent to the subject;

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B4245785.4 obtaining a test sample from the subject after administering radiation therapy or the probe agent;

measuring a first level of a biomarker associated with oxidative stress in the reference sample;

measuring a second level of the same biomarker in the test sample; and

comparing the first measured level to the second measured level,

wherein a change between the first measured level and the second measured level associated with elevated oxidative stress indicates that the neoplastic disease will respond to treatment with radiation therapy or the chemotherapeutic agent.

13. The method according to claim 12, wherein the test sample is obtained from the subject less than about three weeks after the radiation therapy or probe agent is

administered to the subject.

14. The method according to claim 12 or 13, wherein the test sample is obtained from the subject less than 5, 4, 3, 2 or 1 days after the radiation therapy or probe agent is administered to the subject.

15. The method according to any preceding claim, wherein more than one

chemotherapeutic agent is administered to the subject.

16. The method according to any preceding claim, wherein the probe agent and/or chemotherapeutic agent is selected from a) alkylating agents, such as for example nitrosoureas and platinum; b) antimetabolites, such as folic acid analogs and purine and pyrimidine analogs; c) natural products, such as, for example, vinca alkaloids, taxanes, and camptothecins; d) hormones and antagonists, such as, for example, estrogens and anti- estrogens; and d) agents such as differentiating agents, protein tyrosine kinase inhibitors, immunomodulators, biological response modifiers, and monoclonal antibodies.

17. The method according to any one of claims 1-15, wherein said probe agent and/or chemotherapeutic agent is selected from phospho-valproic acid or other similarly modified anticancer agents, arsenic trioxide, emodin, anthracyclines, such as daunorubicin and doxorubicin, cisplatin, bortezomib, synthetic retinoids, imexon, 2-methoxyestradiol, tetrathiomolybdate, motexaphin gadolinium, phenylethyl isothiocyanate, erlotinib, and antibodies against epidermal growth factor (EGFR) or vascular endothelial growth factor (VEGF).

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18. A method of detecting the presence of cancer in a subject with a suspected neoplastic disease, comprising:

(i) administering a probe agent to the subject;

(ii) obtaining a test sample from the subject;

(iii) measuring a level of a biomarker associated with oxidative stress in the test sample; and

(iv) comparing the measured level to a reference value,

wherein a measured level associated with elevated oxidative stress indicates that the patient has neoplastic disease.

19. The method according to any preceding claim, wherein said chemotherapeutic agent and/or said probe agent is a phospho- non-steroidal anti-inflammatory drug (phospho- NSAID).

20. The method of claim 19, wherein said phospho-NSAID is selected from phospho- aspirin, phospho-ibuprofen, phospho-flurbiprofen, phospho-valproic acid, and phospho- sulindac.

21. A method of determining whether a subject with a neoplastic disease is responding to treatment with radiation therapy, comprising:

(i) obtaining a test sample from the subject receiving radiation therapy;

(ii) measuring a level of a biomarker associated with oxidative stress in the test sample; and

(iii) comparing the measured level to a reference value,

wherein a measured level associated with elevated levels of oxidative stress indicates that the patient is responding to treatment with radiation therapy.

22. The method according to any preceding claim, wherein more than one test sample is obtained from the subject.

23. The method according to any preceding claim, wherein more than one measured level is obtained for the biomarkers in the test sample.

24. The method according to any preceding claim, further comprising comparing more than one measured level and more than one reference value.

25. The method of any preceding claim, further comprising measuring levels of at least two biomarkers associated with oxidative stress in one or more test samples.

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26. The method of any preceding claim, wherein said biomarker is selected from an F₂- isoprostane, malondialdehyde, glutathione (GSH), dityrosine, and 8-hydroxy-2'- deoxyguanosine (8-OH-dG).

27. The method of claim 22 wherein said F₂-isoprostane is selected from 5, 8, 12, and 15 F₂-isoprostane.

28. The method according to any one of claims 1-25, wherein said biomarker is a change in the expression of a gene.

29. The method according to any one of claims 1-25 and 28, wherein said biomarker is a change in the expression of a gene associated with oxidative stress.

30. The method of claim 28 or 29, wherein said gene is selected from NADPH oxidase, GSH synthase, thioredoxin (Trx-1 or Trx-2), thioredoxin reductase, NADP, superoxide dismutase, catalase, glutathione peroxidase, glutaredoxine, heme oxygenase- 1, and phase II detoxifying enzymes, such as glutathione reductase and NQOl .

31. The method according to any one of claims 28-30, wherein said change is the difference in nucleic acid encoding said gene between the test sample and reference value.

32. The method according to any one of claims 28-31 , wherein said change is the difference in protein encoded by said gene between the test sample and reference value.

33. The method according to any one of claims 1-25, wherein said biomarker is a reactive oxygen species or a reactive nitrogen species.

34. The method according to any one of claims 1-25 and 33, wherein said biomarker is NO, superoxide anion, or H₂0₂.

35. The method according to any preceding claim, wherein said reference sample and/or test sample is a blood sample, a saliva sample, or a urine sample.

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