HK1151731A - Treatment of breast cancer with a parp inhibitor alone or in combination with anti-tumor agents - Google Patents

Description

Treatment of breast cancer with PARP inhibitors alone or in combination with other anti-tumor agents

Cross referencing

The present application claims the following priority: U.S. provisional application No. 60/987,333 (attorney docket No. 28825-742.101) entitled "Treatment of Triple Negative reactive Cancer with a Combination of an antibiotic, a Platinum Complex, and a PARP Inhibitor", filed on 12.11.2007; U.S. provisional application No. 61/012,364 entitled "Treatment of Cancer with Combination of Topoisomerase Inhibitors and PARPINhibitors" filed on 7.12.2007 (attorney docket No. 28825-747.101); and U.S. provisional application No. 61/058,528 (attorney docket No. 28825-757.101) filed on 3.6.2008 and entitled "Treatment of Breast, Ovarian, and Ulterine center with a PARP Inhibitor", each of which is incorporated herein by reference.

Background

Cancer is a group of diseases characterized by uncontrolled cell growth. The annual incidence of cancer is estimated to exceed 130 million in the united states alone. Despite the use of surgery, radiation, chemotherapy, and hormones in the treatment of cancer, cancer remains the second leading cause of death in the united states. It is estimated that 560,000 americans die of cancer annually.

Cancer cells simultaneously activate several pathways that positively and negatively regulate cell growth and cell death. This feature suggests that modulation of cell death and survival signals may provide a new strategy for improving the efficacy of current chemotherapy.

Breast cancer is generally treated with a combination of surgery and adjuvant therapy. Surgery is to remove cancerous lesions, while adjuvant therapy (radiotherapy, chemotherapy, or a combination of both) is to combat any possible residual cancer cells after surgery. Breast cancer can be broadly classified according to the presence or absence of Hormone Receptors (HR). Hormone receptor positive (HR +) cancers are characterized by the expression of the Estrogen Receptor (ER), the estrogen receptor, or the Progesterone Receptor (PR), or both. Adjuvant treatment of ER + breast cancer typically involves chemotherapy with Selective Estrogen Receptor Modulators (SERMs) such as tamoxifen or raloxifene. Unfortunately, while approximately 70% of breast cancers are ER positive, the remaining 30% of HR negative breast cancers are not amenable to treatment with SERMs. Thus, other adjuvant chemotherapy, such as treatment with anthracyclines (alone or in combination with taxanes), has been attempted for ER negative breast cancer.

Anthracycline therapy is limited by life-long dose limits based on concerns about cardiotoxicity. For patients with metastatic breast cancer, treatment with gemcitabine and carboplatin, whether first-time with a taxane or once pre-treated with a taxane, is a conventional combination chemotherapy. Platinum drugs show promising antitumor activity for basal-like locally advanced breast cancer. For basal-like breast cancers, DNA damaging agents have considerable anti-tumor efficacy due to defects in the DNA repair pathways inherent to such breast cancers.

Despite the availability of antimetabolites such as gemcitabine and platinum complexes such as carboplatin, there is no accepted standard of care for ER-negative breast cancer. In particular, triple negative metastatic breast cancer (i.e., ER negative, and/or PR negative, and/or human epidermal growth factor receptor 2(HER2) negative breast cancer) is refractory to standard treatment, while SERM chemotherapy is completely ineffective. Thus, there is a need for effective treatments for cancer, particularly triple negative metastatic breast cancer.

While there are some limited treatment options for cancer, various variants of cancer, including triple negative breast cancer, are particularly difficult to treat because they are refractory to standard chemotherapy or hormonal therapy. Thus, there is a need for an effective treatment for cancer, particularly for various variants of cancer.

Summary of The Invention

In some embodiments, the present invention provides a method of treating breast cancer that is negative for at least one of ER, PR, or HER2 in a patient, comprising administering to the patient at least one PARP inhibitor. In some embodiments, the present invention provides a method of treating breast cancer that is negative for at least one of ER, PR, or HER2 in a patient in need thereof, comprising: (a) obtaining a sample from a patient; (b) the sample was tested to determine each of the following: whether the cancer is ER positive or ER negative; whether the cancer is PR positive or PR negative; whether the cancer is HER2 positive or HER2 negative; (c) treating the patient with at least one PARP inhibitor if the test indicates that the cancer is negative for at least one of ER, PR or HER 2. In some embodiments, the method further comprises treating the patient with at least one PARP inhibitor if two or more of the following conditions are met: (a) the cancer is ER negative, (b) the cancer is PR negative, (c) the cancer is HER2 negative. In some embodiments, the present invention provides methods of treating breast cancer that is negative for at least one of ER, PR, or HER2 in a patient, comprising: (a) testing a sample taken from the patient for PARP expression; and (b) administering at least one PARP inhibitor to the patient if PARP expression exceeds a predetermined level.

In the practice of any of the methods disclosed herein, at least one therapeutic effect is obtained, which is a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, stable disease, or complete response to pathology. In some embodiments, treatment with PARP inhibitors achieves comparable clinical benefit rates (CBR ═ CR + PR + SD ≧ 6 months) as compared to antineoplastic agent treatment. In some embodiments, the improvement in clinical benefit rate is at least about 30% compared to treatment with an antineoplastic agent alone. In some embodiments, the PARP inhibitor is a PARP-1 inhibitor. In some embodiments, the PARP-1 inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the PARP inhibitor is of formula (IIa) or a metabolite thereof:

formula (IIa)

Wherein, (1) R₁、R₂、R₃、R₄And R₅At least one of the substituents being always a sulfur-containing substituent, the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R ₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituent, and R₁、R₂、R₃、R₄And R₅At least one of these 5 substituents is always iodine, and where the iodine is always present with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The groups are adjacent; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group₁、R₂、R₃、R₄Or R₅The substituents are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The substituents are adjacent.

In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is stage I, stage II, or stage III. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2; and wherein the breast cancer is positive for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is deficient in homologous recombination DNA repair. In some embodiments, the breast cancer has impaired BRCA1 or BRCA2 function. In some embodiments, the treatment comprises a treatment cycle for at least 11 days, wherein on days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 1 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally, parenterally by injection or infusion, or by inhalation. In some embodiments, the treatment cycle is from about 11 days to about 30 days. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with at least one anti-neoplastic agent. The antineoplastic agent is an antineoplastic alkylating agent, an antineoplastic antimetabolite agent, an antineoplastic antibiotic, a plant-derived antineoplastic agent, an antineoplastic platinum complex, an antineoplastic camptothecin derivative, an antineoplastic tyrosine kinase inhibitor, a monoclonal antibody, an interferon, a biological response modifier, a hormonal antineoplastic agent, an antineoplastic virus agent, an angiogenesis inhibitor, a differentiation inducer, a PI3K/mTOR/AKT inhibitor, a cell cycle inhibitor, an apoptosis inhibitor, an hsp 90 inhibitor, a tubulin inhibitor, a DNA repair inhibitor, an antiangiogenic agent, a receptor tyrosine kinase inhibitor, a topoisomerase inhibitor, a taxane, a Her-2 targeting agent, a hormone antagonist, a growth factor receptor targeting agent, or a pharmaceutically acceptable salt thereof. In some embodiments, the antineoplastic agent is decitabine (citabine), capecitabine (capecitabine), valopicabine (valopicitabine), or gemcitabine (gemcitabine). In some embodiments, the antineoplastic agent is selected from the group consisting of Avastin (bevacizumab), Sutent (sunitinib), Nexavar (sorafenib), Recentin (cediranib), ABT-869, Axitinib (Axitinib), Irinotecan (Irinotecan), topotecan (topotecan), paclitaxel, docetaxel, lapatinib, trastuzumab (herceptin), lapatinib, tamoxifen, a steroidal aromatase inhibitor, a non-steroidal aromatase inhibitor, fulvestrant, an Epidermal Growth Factor Receptor (EGFR) inhibitor, cetuximab, panitumumab, an insulin-like growth factor 1 receptor (IGF1R) inhibitor, and CP-751871. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with one or more antineoplastic agents. In some embodiments, the anti-tumor agent is administered prior to, concurrently with, or after the PARP inhibitor is administered. In some embodiments, the method further comprises surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, DNA therapy, adjuvant therapy, neoadjuvant therapy, immunotherapy, nanotherapy or a combination thereof. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with gamma radiation. In some embodiments, the sample is a tissue or body fluid sample. In some embodiments, the sample is a tumor sample, a blood sample, a plasma sample, a peritoneal fluid sample, an exudate (exudate), or an effusion (effusion). In some embodiments, the method further comprises determining the expression of estrogen receptor, progesterone receptor, or human epidermal growth factor 2 receptor in a sample taken from the patient.

In some embodiments, the present invention provides methods of treating breast cancer in a patient comprising administering to the patient at least one PARP inhibitor in combination with at least one anti-neoplastic agent. In some embodiments, the present invention provides a method of treating breast cancer in a patient in need of treatment, comprising: (a) obtaining a sample from a patient; (b) the sample was tested to determine each of the following: whether the cancer is ER positive or ER negative; whether the cancer is PR positive or PR negative; whether the cancer is HER2 positive or HER2 negative; (c) if the test indicates that the cancer is negative for at least one of ER, PR, or HER2, treating the patient with a combination of therapeutic agents, wherein the therapeutic agents include at least one PARP inhibitor and at least one antineoplastic agent. In some embodiments, wherein the method further comprises treating the patient with a combination of therapeutic agents comprising at least one PARP inhibitor and at least one anti-neoplastic agent if two or more of the following conditions are met: (a) the cancer is ER negative, (b) the cancer is PR negative, (c) the cancer is HER2 negative. In some embodiments, the present invention provides a method of treating breast cancer in a patient, comprising: (a) testing a sample taken from the patient for PARP expression; and (b) administering to the patient at least one PARP inhibitor and at least one anti-neoplastic agent if the PARP expression exceeds a predetermined level.

In the practice of any of the methods disclosed herein, in some embodiments at least one therapeutic effect is obtained, which is a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, stable disease, or complete response to pathology. In some embodiments, the clinical benefit rate (CBR ≧ CR + PR + SD ≧ 6 months) is improved as compared to treatment with an anti-tumor agent but without a PARP inhibitor. In some embodiments, the improvement in clinical benefit rate is at least about 60%. In some embodiments, the PARP inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the PARP inhibitor is of formula (IIa) or a metabolite thereof:

formula (IIa)

Wherein: (1) r₁、R₂、R₃、R₄And R₅At least one of the substituents being always a sulfur-containing substituent, the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituent, and R ₁、R₂、R₃、R₄And R₅At least one of the 5 substituents always being presentIodine, and wherein said iodine is always with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The groups are adjacent; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent.

In some embodiments, the anti-neoplastic agent is an anti-tumor alkylating agent, an anti-tumor antimetabolite agent, an anti-tumor antibiotic, a plant-derived anti-neoplastic agent, an anti-tumor platinum complex, an anti-tumor camptothecin derivative, an anti-tumor tyrosine kinase inhibitor, a monoclonal antibody, an interferon, a biological response modifier, a hormonal anti-neoplastic agent, an anti-tumor viral agent, an angiogenesis inhibitor, a differentiation inducing agent, a PI3K/mTOR/AKT inhibitor, a cell cycle inhibitor, an apoptosis inhibitor, an hsp 90 inhibitor, a tubulin inhibitor, a DNA repair inhibitor, an anti-angiogenic agent, a receptor tyrosine kinase inhibitor, a topoisomerase inhibitor, a taxane, a Her-2 targeting agent, a hormone antagonist, a growth factor receptor targeting agent, or a pharmaceutically acceptable salt thereof. In some embodiments, the antineoplastic agent is decitabine, capecitabine, valopicitabine, or gemcitabine. In some embodiments, the antineoplastic agent is selected from the group consisting of Avastin (bevacizumab), Sutent (sunitinib), Nexavar (sorafenib), Recentin (cediranib), ABT-869, Axitinib (Axitinib), irinotecan, topotecan, paclitaxel, docetaxel, lapatinib, herceptin, lapatinib, tamoxifen, a steroid aromatase inhibitor, a non-steroid aromatase inhibitor, fulvestrant, an Epidermal Growth Factor Receptor (EGFR) inhibitor, cetuximab, panitumumab, an insulin-like growth factor 1 receptor (IGF1R) inhibitor, and CP-751871. In some embodiments, the method further comprises surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, viral therapy, DNA therapy, adjuvant therapy, neoadjuvant therapy, RNA therapy, immunotherapy, nanotherapy or a combination thereof. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with gamma radiation.

In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is stage I, stage II, or stage III. In some embodiments, the breast cancer is HR negative breast cancer. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2; and wherein the breast cancer is positive for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is deficient in homologous recombination DNA repair. In some embodiments, the breast cancer has impaired BRCA1 or BRCA2 function. In some embodiments, the treatment comprises a treatment cycle of at least 11 days, wherein: (a) on days 1 and 8 of the cycle, the patient receives about 100-5000mg/m²Gemcitabine of (1); (b) on days 1 and 8 of the cycle, the patient receives from about 10 to about 400mg/m²Carboplatin of (a); and (c) on days 1, 4, 8, and 11 of the cycle, the patient receives from about 1 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the treatment cycle is from about 11 days to about 30 days. In some embodiments, on days 1 and 8 of the cycle, the patient receives about 100-2,500mg/m ²And from about 10 to about 400mg/m of gemcitabine²Carboplatin of (a); and on days 1, 4, 8, and 11 of the cycle, the patient receives from about 1 to about 50mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof of comparable molar content. In some embodiments, on days 1 and 8 of the cycle, the patient receives about 500-2000mg/m²And from about 50 to about 400mg/m of gemcitabine²Carboplatin of (a); and on days 1, 4, 8 and 11 of the cycle, the patient receives from about 1 to about 50mg/kg of 4-iodo-3-nitrobenzamide, or a salt thereofThe molar content of the corresponding metabolites. In some embodiments, on days 1 and 8 of the cycle, the patient receives about 1000mg/m²Gemcitabine and carboplatin for about AUC 2 of (a); and on days 1, 4, 8 and 11 of the cycle, the patient receives about 1, 2, 3, 4, 6, 8 or 10, 12, 14, 16, 18 or 20mg/kg of 4-iodo-3-nitrobenzamide. In some embodiments, the antineoplastic agent is administered by parenteral injection or infusion. In some embodiments, the PARP inhibitor is 4-iodo-3-nitrobenzamide, which is administered orally, or parenterally by injection or infusion, or by inhalation. In some embodiments, the method further comprises administering the taxane to the patient by parenteral injection or infusion. In some embodiments, the sample is a tissue or body fluid sample. In some embodiments, the sample is a tumor sample, a blood sample, a plasma sample, a peritoneal fluid sample, an exudate or a effusion. In some embodiments, the method further comprises determining the expression of estrogen receptor, progesterone receptor, or human epidermal growth factor 2 receptor in a sample taken from the patient.

Reference to a reference

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be so incorporated.

Brief description of the drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

figure 1 shows the upregulation of PARP1 gene expression in human primary cancers. Horizontal line, median PARP1 expression; square, interquartile range; bar, standard deviation.

FIG. 2 shows the effect of 4-iodo-3-nitrobenzamide plus carboplatin or gemcitabine on TNBC (triple negative metastatic breast cancer) cell cycle progression in vitro. Viability of MDA-MB-463 TNBC cells was quantified by FACS (fluorescence activated cell sorter) analysis.

FIG. 3 shows the inhibition of PARP in Peripheral Mononuclear Blood Cells (PMBC) of patients receiving 4-iodo-3-nitrobenzamide treatment.

Figure 4 shows expression profiles of PARP1, ER, PR, and HER2 for human breast cancer tumor samples from metastatic TNBC secondary trials. Data were normalized to β -glucuronidase gene expression. The data represents the analysis of 50 clinical breast cancer samples and 19 normal breast samples. The vertical line represents median gene expression and the box represents the interquartile range.

FIG. 5 shows the PFS (survival without disease progression) Kaplan-Meier curve for metastatic TNBC patients, compared to gemcitabine/carboplatin alone treatment with 4-iodo-3-nitrobenzamide plus gemcitabine/carboplatin. The Kaplan-Meier method was used to summarize the PFS distribution for the two treatment groups. The data of the two groups were compared using a two-sided log-rank test (log-rank), a significance level of 5%. G/C, gemcitabine/carboplatin; G/C + BA, gemcitabine/carboplatin + 4-iodo-3-nitrobenzamide (BA).

FIG. 6 shows that 4-iodo-3-nitrobenzamide (BA) potentiates S-and G2/M cell cycle arrest and enhances the anti-proliferative effects of gamma radiation in human triple negative MDA-MB-468 breast cancer cells.

Detailed Description

In some embodiments, the present invention provides a method of treating breast cancer that is negative for at least one of ER, PR, or HER2 in a patient, comprising administering to the patient at least one PARP inhibitor. In some embodiments, at least one therapeutic effect is obtained that is a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, complete response to pathology, or stable disease. In some embodiments, treatment with PARP inhibitors achieves comparable clinical benefit rates (CBR ═ CR + PR + SD ≧ 6 months) as compared to antineoplastic agent treatment. In some embodiments, the improvement in clinical benefit rate is at least about 30%. In some embodiments, the PARP inhibitor is a PARP-1 inhibitor. In some embodiments, the PARP inhibitor is of formula (IIa) or a metabolite thereof:

Formula (IIa)

Wherein: (1) r₁、R₂、R₃、R₄And R₅At least one of the substituents being always a sulfur-containing substituent, the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituent, and R₁、R₂、R₃、R₄And R₅At least one of these 5 substituents is always iodine, and where the iodine is always present with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The substituents are adjacent; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, the PARP-1 inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof.

In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is stage I, stage II, or stage III. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2; and the breast cancer is positive for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is ER negative breast cancer. In some embodiments, the breast cancer is ER negative and HER2 positive. In some embodiments, the breast cancer is ER negative and PR positive. In some embodiments, the breast cancer is ER negative and both HER2 positive and PR positive. In some embodiments, the breast cancer is a PR negative breast cancer. In some embodiments, the breast cancer is PR negative and ER positive. In some embodiments, the breast cancer is PR negative and HER2 positive. In some embodiments, the breast cancer is PR negative and both ER positive and HER2 positive. In some embodiments, the breast cancer is HER2 negative breast cancer. In some embodiments, the breast cancer is HER2 negative and ER positive. In some embodiments, the breast cancer is HER2 negative and PR positive. In some embodiments, the breast cancer is HER2 negative and both ER positive and PR positive. In some embodiments, the breast cancer is ER negative and PR negative. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 positive. In some embodiments, the breast cancer is ER negative and HER2 negative. In some embodiments, the breast cancer is ER negative, HER2 negative, and PR positive. In some embodiments, the breast cancer is PR negative and HER2 negative. In some embodiments, the breast cancer is PR negative, HER2 negative, and ER positive. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 negative. In some embodiments, the breast cancer is deficient in homologous recombination DNA repair.

In some embodiments, the treatment comprises a treatment cycle of at least 11 days, and on days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 1 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally, parenterally by injection or infusion, or by inhalation. In some embodiments, the treatment cycle is from about 11 days to about 30 days.

In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with at least one anti-neoplastic agent. The antitumor agent is an antitumor alkylating agent, an antitumor antimetabolite agent, an antitumor antibiotic, a plant-derived antitumor agent, an antitumor organoplatinum compound, an antitumor camptothecin derivative, an antitumor tyrosine kinase inhibitor, a monoclonal antibody, an interferon, a biological response modifier, a hormonal antitumor agent, an antitumor virus agent, an angiogenesis inhibitor, a differentiation inducer, or other drugs exhibiting antitumor activity, or a pharmaceutically acceptable salt thereof. In some embodiments, the antineoplastic agent is decitabine, capecitabine, valopicitabine, or gemcitabine. In some embodiments, the antineoplastic agent is a platinum complex. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with one or more antineoplastic agents. The antineoplastic agent is administered prior to, concurrently with, or subsequent to the administration of the PARP inhibitor. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with an anti-angiogenic agent such as Avastin (bevacizumab). In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with a topoisomerase inhibitor, such as irinotecan or topotecan. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with a taxane such as paclitaxel or docetaxel. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with a Her-2 targeted drug such as trastuzumab (herceptin). In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with a hormonal therapy agent, such as the hormone antagonist tamoxifen. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with a drug targeting a growth factor receptor, including Epidermal Growth Factor Receptor (EGFR) and insulin-like growth factor 1(IGF-1) receptor (IGF1R) inhibitor. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with gamma radiation. In some embodiments, the method further comprises surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, adjuvant therapy, neoadjuvant therapy, RNA therapy, DNA therapy, viral therapy, immunotherapy, nanotherapy or a combination thereof.

Some embodiments described herein provide a method of treating breast cancer in a patient comprising administering to the patient at least one PARP inhibitor and at least one anti-neoplastic agent. In some embodiments, at least one therapeutic effect is obtained that is a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, complete response to pathology, or stable disease. In some embodiments, the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment with the antimetabolite and platinum complex but without the PARP inhibitor. In some embodiments, the improvement in clinical benefit rate is at least about 60%. In some embodiments, the PARP inhibitor is benzamide or a metabolite thereof. In some embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the platinum complex is selected from cisplatin, carboplatin, platinum oxalate (oxapeltin), and oxaliplatin. In some embodiments, the platinum complex is carboplatin. In some embodiments, the antimetabolite agent is decitabine. In some embodiments, the antimetabolite agent is selected from decitabine, capecitabine, gemcitabine, or valopicitabine. In some embodiments, the antimetabolite is gemcitabine. In some embodiments, the method further comprises administering a taxane to the patient. In some embodiments, the taxane is paclitaxel or docetaxel. In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is stage I, stage II, or stage III. In some embodiments, the breast cancer is HR negative breast cancer. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2; and the breast cancer is positive for at least one receptor of ER, PR or HER 2. In some embodiments, the breast cancer is HR negative breast cancer. In some embodiments, the breast cancer is ER negative breast cancer. In some embodiments, the breast cancer is ER negative and HER2 positive. In some embodiments, the breast cancer is ER negative and PR positive. In some embodiments, the breast cancer is ER negative and both HER2 positive and PR positive. In some embodiments, the breast cancer is a PR negative breast cancer. In some embodiments, the breast cancer is PR negative and ER positive. In some embodiments, the breast cancer is PR negative and HER2 positive. In some embodiments, the breast cancer is PR negative and both ER positive and HER2 positive. In some embodiments, the breast cancer is HER2 negative breast cancer. In some embodiments, the breast cancer is HER2 negative and ER positive. In some embodiments, the breast cancer is HER2 negative and PR positive. In some embodiments, the breast cancer is HER2 negative and both ER positive and PR positive. In some embodiments, the breast cancer is ER negative and PR negative. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 positive. In some embodiments, the breast cancer is ER negative and HER2 negative. In some embodiments, the breast cancer is ER negative, HER2 negative, and PR positive. In some embodiments, the breast cancer is PR negative and HER2 negative. In some embodiments, the breast cancer is PR negative, HER2 negative, and ER positive. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 negative. In some embodiments, the breast cancer is deficient in homologous recombination DNA repair.

In some embodiments, the method further comprises administering a PARP inhibitor in combination with an anti-neoplastic agent. In some embodiments, the antineoplastic agent is an antineoplastic alkylating agent, an antineoplastic antimetabolite agent, an antineoplastic antibiotic, a plant-derived antineoplastic agent, an antineoplastic platinum complex, an antineoplastic camptothecin derivative, an antineoplastic tyrosine kinase inhibitor, a monoclonal antibody, an interferon, a biological response modifier, a hormonal antineoplastic agent, an angiogenesis inhibitor, a differentiation inducer, or other drug exhibiting antineoplastic activity, or a pharmaceutically acceptable salt thereof. In some embodiments, the platinum complex is cisplatin, carboplatin, platinum oxalate, and oxaliplatin. In some embodiments, the anti-tumor antimetabolite agent is decitabine, capecitabine, gemcitabine, or valopicitabine. In some embodiments, the method further comprises administering to the patient a PARP inhibitor in combination with one or more antineoplastic agents. In some embodiments, the anti-tumor agent is administered prior to, concurrently with, or after the PARP inhibitor is administered. In some embodiments, the anti-neoplastic agent is an anti-angiogenic agent, such as Avastin (bevacizumab). In some embodiments, the antineoplastic agent is a topoisomerase inhibitor, including but not limited to irinotecan, topotecan, or camptothecin. In some embodiments, the antineoplastic agent is a taxane, including but not limited to paclitaxel or docetaxel. In some embodiments, the anti-tumor agent is a Her-2 targeted drug, such as trastuzumab (herceptin). In some embodiments, the antineoplastic agent is a hormone antagonist, such as tamoxifen. In some embodiments, the antineoplastic agent is a drug that targets a growth factor receptor. In some embodiments, the agent is an Epidermal Growth Factor Receptor (EGFR) inhibitor or an insulin-like growth factor 1(IGF-1) receptor (IGF1R) inhibitor. In other embodiments, the method further comprises surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, adjuvant therapy, neoadjuvant therapy, viral therapy, RNA therapy, immunotherapy, nanotherapy or a combination thereof.

In some embodiments, the treatment comprises a treatment cycle of at least 11 days, and on days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the treatment comprises a treatment cycle of at least 11 days, and on days 4, 8, and 11 of the treatment cycle, the patient receives from about 1 to about 50mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the treatment comprises a treatment cycle of at least 11 days, and on days 1, 4, 8, and 11 of the treatment cycle, the patient receives about 1, 2, 3, 4, 5, 6, 8, or 10, 12, 14, 16, 18, or 20mg/kg of 4-iodo-3-nitrobenzamide.

In some embodiments, the treatment comprises a treatment cycle of at least 11 days, wherein: (a) on days 1 and 8 of the cycle, patients receive about 100-2000mg/m²Gemcitabine of (1); (b) on days 1 and 8 of the cycle, the patient receives from about 10 to about 400mg/m²Carboplatin of (a); and (c) on days 1, 4, 8, and 11 of the cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, on days 1 and 8 of the cycle, the patient receives about 100-2,500mg/m ²Gemcitabine and carboplatin of about AUC 1-5 (about 10 to about 400 mg/m)²Carboplatin of (a); and on days 1, 4, 8, and 11 of the cycle, the patient receives from about 1 to about 50mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof of comparable molar content. In some embodiments, on days 1 and 8 of the cycle, the patient receives about 500-2000mg/m²And from about 50 to about 400mg/m of gemcitabine²Carboplatin of (a); and on days 1, 4, 8, and 11 of the cycle, the patient receives from about 1 to about 50mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof of comparable molar content. In some embodiments, on days 1 and 8 of the cycle, the patient receives about 1000mg/m²Gemcitabine and carboplatin for about AUC 2 of (a); and on days 1, 4, 8 and 11 of the cycle, the patient receives about 1, 2, 3, 4, 6, 8 or 10, 12, 14, 16, 18 or 20mg/kg of 4-iodo-3-nitrobenzamide.

Some embodiments described herein provide a method of treating breast cancer in a triple negative breast cancer patient, comprising administering to the patient about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content, on days 1, 4, 8, and 11 of a 21-day treatment cycle. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments provide a method of treating breast cancer in a triple negative breast cancer patient, comprising, within a 21 day treatment cycle: (a) on days 1 and 8 of the cycle, about 100-2000mg/m is administered to the patient²Gemcitabine of (1); (b) on days 1 and 8 of the cycle, the patient is administered AUC 0.1-10 of carboplatin (about 10 to 400 mg/m)²Carboplatin of (a); and (c) administering about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof in molar equivalent amounts, to the patient on days 1, 4, 8, and 11 of the cycle. In some embodiments, the gemcitabine is administered as an intravenous infusion. In some embodiments, the carboplatin is administered as an intravenous infusion. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion. In some embodiments, the gemcitabine is administered as an intravenous infusion. In some embodiments, the carboplatin is administered as an intravenous infusion. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments described herein provide a method of treating breast cancer in a triple negative breast cancer patient, comprising: (a) establishing a treatment cycle of about 10 days to about 30 days; (b) about 1mg/kg to about 50mg/kg of 4-iodo-3-nitrobenzamide, or its metabolite in molar equivalent, is administered to the patient daily on 1 to 10 separate days of the cycle. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments described herein provide a method of treating breast cancer in a triple negative breast cancer patient, comprising: (a) establishing a treatment cycle of about 10 days to about 30 days; (b) about 100 to about 5000mg/m is administered to the patient daily by intravenous infusion on 1 to 5 separate days of the cycle²Gemcitabine of (1); (c) AUC 1 to AUC 10 carboplatin (e.g., about 10 to about 400 mg/m) was administered to the patient daily by intravenous infusion on 1 to 5 separate days of the cycle²Carboplatin); and (d) administering to the patient about daily on 1 to 10 separate days of the cycle1mg/kg to about 50mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof of comparable molar content. In some embodiments, the gemcitabine is administered as an intravenous infusion. In some embodiments, the carboplatin is administered as an intravenous infusion. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments provide a method of treating breast cancer in a patient in need thereof, comprising: (a) obtaining a sample from a patient; (b) testing the sample to determine at least one of: (i) whether the cancer is ER positive or ER negative; (ii) whether the cancer is PR positive or PR negative; (iii) whether the cancer is HER2 positive or HER2 negative; (c) treating the patient with a combination of therapeutic agents if the test indicates that the cancer is ER negative, PR negative, or HER2 negative, wherein the therapeutic agents comprise at least one antimetabolite agent, at least one platinum complex, and at least one PARP inhibitor; and (d) selecting a different treatment modality if the test result does not indicate that the cancer is ER negative, PR negative, or HER2 negative.

Some embodiments include a method of treating breast cancer in a patient in need thereof, comprising: (a) obtaining a sample from a patient; (b) testing the sample to determine at least one of: (i) whether the cancer is ER positive or ER negative; (ii) whether the cancer is PR positive or PR negative; (iii) whether the cancer is HER2 positive or HER2 negative; (c) treating the patient with at least one PARP inhibitor if the test indicates that the cancer is ER negative, PR negative, or HER2 negative; and (d) selecting a different treatment modality if the test result does not indicate that the cancer is ER negative, PR negative, or HER2 negative.

In some embodiments, at least one therapeutic effect is obtained that is a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, complete response to pathology, or stable disease. In some embodiments, the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment without a PARP inhibitor. In some embodiments, the clinical benefit rate is at least about 30%. In some embodiments, the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment with the antimetabolite and platinum complex but without the PARP inhibitor. In some embodiments, the clinical benefit rate is at least about 60%. In some embodiments, the PARP inhibitor is a PARP-1 inhibitor. In other embodiments, the PARP inhibitor is benzamide or a metabolite thereof. In some embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the platinum complex is selected from cisplatin, carboplatin, platinum oxalate, and oxaliplatin. In some embodiments, the platinum complex is carboplatin. In some embodiments, the antimetabolite agent is decitabine. In some embodiments, the antimetabolite agent is selected from decitabine, capecitabine, gemcitabine, or valopicitabine. In some embodiments, the antimetabolite is gemcitabine. In some embodiments, the method further comprises administering a taxane to the patient. In some embodiments, the taxane is paclitaxel or docetaxel. In some embodiments, the sample is a tissue or body fluid sample. In some embodiments, the sample is a tumor sample, a blood sample, a plasma sample, a peritoneal fluid sample, an exudate or a effusion. In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is ER negative metastatic breast cancer. In some embodiments, the breast cancer is stage I, stage II, or stage III. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2; and the breast cancer is positive for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is ER negative and PR positive. In some embodiments, the breast cancer is ER negative and HER2 positive. In some embodiments, the breast cancer is ER negative and is both PR positive and HER2 positive. In some embodiments, the breast cancer is PR negative metastatic breast cancer. In some embodiments, the breast cancer is PR negative and ER positive. In some embodiments, the breast cancer is PR negative and HER2 positive. In some embodiments, the breast cancer is PR negative and both ER positive and HER2 positive. In some embodiments, the breast cancer is HER2 negative metastatic breast cancer. In some embodiments, the breast cancer is HER2 negative and ER positive. In some embodiments, the breast cancer is HER2 negative and PR positive. In some embodiments, the breast cancer is HER2 negative and both ER positive and PR positive. In some embodiments, the breast cancer is ER negative and PR negative. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 positive. In some embodiments, the breast cancer is ER negative and HER2 negative. In some embodiments, the breast cancer is ER negative, HER2 negative, and PR positive. In some embodiments, the breast cancer is PR negative and HER2 negative. In some embodiments, the breast cancer is PR negative, HER2 negative, and PR positive. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 negative.

Some embodiments of the present invention provide methods for treating breast cancer in a patient in need thereof, comprising: (a) obtaining a sample from a patient; (b) the sample was tested to determine each of the following: (i) whether the cancer is ER positive or ER negative; (ii) whether the cancer is PR positive or PR negative; (iii) whether the cancer is HER2 positive or HER2 negative; (c) treating a patient with at least one PARP inhibitor if two or more of the following conditions are met: (i) the cancer is ER negative; (ii) the cancer is PR negative; or (iii) the cancer is HER2 negative; and (d) selecting a different treatment modality if at least two of the aforementioned conditions are not met. Some embodiments of the present invention provide methods for treating breast cancer in a patient in need thereof, comprising: (a) obtaining a sample from a patient; (b) the sample was tested to determine each of the following: (i) whether the cancer is ER positive or ER negative; (ii) whether the cancer is PR positive or PR negative; (iii) whether the cancer is HER2 positive or HER2 negative; (c) treating the patient with a combination of therapeutic agents if two or more of the following conditions are met, wherein the therapeutic agents comprise at least one antimetabolite agent, at least one platinum complex, and at least one PARP inhibitor: (i) the cancer is ER negative; (ii) the cancer is PR negative; or (iii) the cancer is HER2 negative; and (d) selecting a different treatment modality if at least two of the aforementioned conditions are not met. In some implementations In the protocol, at least one therapeutic effect is obtained, which is a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, complete response to pathology, or stable disease. In some embodiments, the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment without a PARP inhibitor. In some embodiments, the clinical benefit rate is at least about 30%. In some embodiments, the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment with the antimetabolite and platinum complex but without the PARP inhibitor. In some embodiments, the clinical benefit rate is at least about 60%. In some embodiments, the sample is a tissue or body fluid sample. In some embodiments, the sample is a tumor sample, a blood sample, a plasma sample, a peritoneal fluid sample, an exudate or a effusion. In some embodiments, the PARP inhibitor is a PARP-1 inhibitor. In other embodiments, the PARP inhibitor is benzamide or a metabolite thereof. In some embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the platinum complex is selected from cisplatin, carboplatin, platinum oxalate, and oxaliplatin. In some embodiments, the platinum complex is carboplatin. In some embodiments, the antimetabolite agent is decitabine. In some embodiments, the antimetabolite agent is decitabine, capecitabine, gemcitabine, or valopicitabine. In some embodiments, the antimetabolite is gemcitabine. In some embodiments, the method further comprises administering a taxane to the patient. In some embodiments, the taxane is paclitaxel or docetaxel. In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is stage I, stage II, or stage III. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2; and the breast cancer is positive for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is ER negative metastatic breast cancer. In some embodiments, the breast cancer is PR negative metastatic breast cancer. In some embodiments Breast cancer is HER2 negative metastatic breast cancer. In some embodiments, the breast cancer is ER negative and PR negative. In some embodiments, the breast cancer is ER negative and HER2 negative. In some embodiments, the breast cancer is PR negative and HER2 negative. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 negative. In some embodiments, treating comprises selecting a treatment cycle of at least 11 days, and: (a) on days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof of comparable molar content. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion. In some embodiments, treating comprises selecting a treatment cycle of at least 11 days, and: (a) on days 1 and 8 of the cycle, patients receive about 100-2000mg/m²Gemcitabine of (1); (b) on days 1 and 8 of the cycle, the patient receives from about 10 to about 400mg/m²Carboplatin of (a); and (c) on days 1, 4, 8, and 11 of the cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the gemcitabine is administered as an intravenous infusion. In some embodiments, the carboplatin is administered as an intravenous infusion. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments disclosed herein provide a method of treating breast cancer in a patient in need thereof, comprising: (a) obtaining a sample from a patient; (b) the sample was tested to determine each of the following: (i) whether the cancer is ER positive or ER negative; (ii) whether the cancer is PR positive or PR negative; and (iii) whether the cancer is HER2 positive or HER2 negative; (c) treating a patient with at least one PARP inhibitor if two or more of the following conditions are met: (i) the cancer is ER negative; (ii) the cancer is PR negative; or (iii) the cancer is HER2 negative; and (d) selecting a different treatment modality if two or more of conditions (i) to (iii) are not satisfied.

Some embodiments described herein provide a method of treating ER negative, PR negative, HER-2 negative metastatic breast cancer in a patient in need of treatment comprising administering to the patient at least one PARP inhibitor. In some embodiments, the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment without a PARP inhibitor. In some embodiments, the clinical benefit rate is at least about 30%. In some embodiments, two or more therapeutic compounds are administered to a patient in a unit dosage form. In some embodiments, the PARP inhibitor is a PARP-1 inhibitor. In other embodiments, the PARP inhibitor is benzamide or a metabolite thereof. In some embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is ER negative metastatic breast cancer. In some embodiments, the breast cancer is PR negative metastatic breast cancer. In some embodiments, the breast cancer is HER2 negative metastatic breast cancer. In some embodiments, the breast cancer is ER negative and PR negative. In some embodiments, the breast cancer is ER negative and HER2 negative. In some embodiments, the breast cancer is PR negative and HER2 negative. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 negative. In some embodiments, treating comprises selecting a treatment cycle of at least 11 days, at days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments provide a method of treating breast cancer in a patient, comprising: (a) testing a sample taken from the patient for PARP expression; and (b) administering at least one PARP inhibitor to the patient if PARP expression exceeds a predetermined level. In some embodiments, at least one therapeutic effect is obtained that is a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, complete response to pathology, or stable disease. In some embodiments, the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment without a PARP inhibitor. In some embodiments, the improvement in clinical benefit rate is at least about 30%. In some embodiments, the PARP inhibitor is a PARP-1 inhibitor. In other embodiments, the PARP inhibitor is benzamide or a metabolite thereof. In some embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the breast cancer is metastatic breast cancer.

In some embodiments, the method further comprises determining the expression of estrogen receptor, progesterone receptor, or human epidermal growth factor 2 receptor in a sample taken from the patient. In some embodiments, the breast cancer is HR negative breast cancer. In some embodiments, the breast cancer is ER negative breast cancer. In some embodiments, the breast cancer is a PR negative breast cancer. In some embodiments, the breast cancer is HER2 negative breast cancer. In some embodiments, the breast cancer is ER negative and PR negative. In some embodiments, the breast cancer is ER negative and HER2 negative. In some embodiments, the breast cancer is PR negative and HER2 negative. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 negative. In some embodiments, treating comprises selecting a treatment cycle of at least 11 days, at days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments disclosed herein provide a method of treating breast cancer in a patient in need thereof, comprising: (a) obtaining a sample from a patient; (b) the sample was tested to determine each of the following: (i) whether the cancer is ER positive or ER negative; (ii) whether the cancer is PR positive or PR negative; and (iii) whether the cancer is HER2 positive or HER2 negative; (c) treating the patient with a combination of therapeutic agents if two or more of the following conditions are met, wherein the therapeutic agents comprise at least one antimetabolite agent, at least one platinum complex, and at least one PARP inhibitor: (i) the cancer is ER negative; (ii) the cancer is PR negative; or (iii) the cancer is HER2 negative; and (d) selecting a different treatment modality if two or more of conditions (i) to (iii) are not satisfied. In some embodiments, the gemcitabine is administered as an intravenous infusion. In some embodiments, the carboplatin is administered as an intravenous infusion. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments described herein provide a method of treating ER negative, PR negative, HER-2 negative metastatic breast cancer in a patient in need of treatment comprising administering to the patient at least one antimetabolite agent, at least one platinum complex, and at least one PARP inhibitor. In some embodiments, the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment with the antimetabolite and platinum complex but without the PARP inhibitor. In some embodiments, the clinical benefit rate is at least about 60%. In some embodiments, two or more therapeutic compounds are administered to a patient in a unit dosage form. In some embodiments, the PARP inhibitor is benzamide or a metabolite thereof. In some embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the platinum complex is selected from cisplatin, carboplatin, platinum oxalate, and oxaliplatin. In some embodiments, the platinum complex is carboplatin. In some embodiments, the antimetabolite agent is decitabine. In some embodiments, the antimetabolite agent is selected from decitabine, capecitabine, gemcitabine, or valopicitabine. In some embodiments, the antimetabolite is gemcitabine. In some embodiments, the method further comprises administering a taxane to the patient. In some embodiments, the taxane is paclitaxel or docetaxel. In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the breast cancer is stage I, stage II, or stage III. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2; and the breast cancer is positive for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is ER negative metastatic Breast cancer. In some embodiments, the breast cancer is PR negative metastatic breast cancer. In some embodiments, the breast cancer is HER2 negative metastatic breast cancer. In some embodiments, the breast cancer is ER negative and PR negative. In some embodiments, the breast cancer is ER negative and HER2 negative. In some embodiments, the breast cancer is PR negative and HER2 negative. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 negative. In some embodiments, treating comprises selecting a treatment cycle of at least 11 days, and: (a) on days 1 and 8 of the cycle, patients receive about 100-2000mg/m²Gemcitabine of (1); (b) on days 1 and 8 of the cycle, the patient receives from about 10 to about 400mg/m²Carboplatin of (a); and (c) on days 1, 4, 8, and 11 of the cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the gemcitabine is administered as an intravenous infusion. In some embodiments, the carboplatin is administered as an intravenous infusion. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments provide a method of treating breast cancer in a patient, comprising: (a) testing a sample taken from the patient for PARP expression; and (b) administering to the patient at least one antimetabolite agent, at least one platinum complex, and at least one PARP inhibitor if PARP expression exceeds a predetermined level. In some embodiments, at least one therapeutic effect is obtained that is a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, complete response to pathology, or stable disease. In some embodiments, the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment with the antimetabolite and platinum complex but without the PARP inhibitor. In some embodiments, the improvement in clinical benefit rate is at least about 60%. In some embodiments, the PARP inhibitor is benzamide or a metabolite thereof. In some embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the platinum complex is selected from the group consisting of cisplatin, carboplatin, platinum oxalate, and olplatinum Thaliplatin. In some embodiments, the platinum complex is carboplatin. In some embodiments, the antimetabolite agent is decitabine. In some embodiments, the antimetabolite agent is selected from decitabine, capecitabine, gemcitabine, or valopicitabine. In some embodiments, the antimetabolite is gemcitabine. In some embodiments, the method further comprises administering a taxane to the patient. In some embodiments, the taxane is paclitaxel or docetaxel. In some embodiments, the breast cancer is metastatic breast cancer. In some embodiments, the method further comprises determining the expression of estrogen receptor, progesterone receptor, or human epidermal growth factor 2 receptor in a sample taken from the patient. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is negative for at least one of ER, PR, or HER 2; and the breast cancer is positive for at least one of ER, PR, or HER 2. In some embodiments, the breast cancer is HR negative breast cancer. In some embodiments, the breast cancer is ER negative breast cancer. In some embodiments, the breast cancer is a PR negative breast cancer. In some embodiments, the breast cancer is HER2 negative breast cancer. In some embodiments, the breast cancer is ER negative and PR negative. In some embodiments, the breast cancer is ER negative and HER2 negative. In some embodiments, the breast cancer is PR negative and HER2 negative. In some embodiments, the breast cancer is ER negative, PR negative, and HER2 negative. In some embodiments, treating comprises selecting a treatment cycle of at least 11 days, and: (a) on days 1 and 8 of the cycle, patients receive about 100-2000mg/m ²Gemcitabine of (1); (b) on days 1 and 8 of the cycle, the patient receives from about 10 to about 400mg/m²Carboplatin of (a); and (c) on days 1, 4, 8, and 11 of the cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof that is equivalent in molar content. In some embodiments, the gemcitabine is administered as an intravenous infusion. In some embodiments, the carboplatin is administered as an intravenous infusion. In some embodiments, the 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Accordingly, embodiments described herein include treating a patient with at least three chemically distinct agents, one of which is an antimetabolite agent, one of which is a platinum-containing complex, and one of which is a PARP inhibitor. In some embodiments, one or more such substances can be present in a wide variety of physical forms, such as free bases, salts (especially pharmaceutically acceptable salts), hydrates, polymorphs, solvates, metabolites, and the like. Unless otherwise defined herein, the use of chemical names is intended to include all physical forms of the named compounds. For example, if not further limited, the recitation of 4-iodo-3-nitrobenzamide is intended to include the free base in general, as well as all pharmaceutically acceptable salts, polymorphs, hydrates, metabolites, and the like. When it is intended that the disclosure or claims be limited to a particular physical form of a compound, this will be made clear in the paragraphs or claims referring to that compound.

In some embodiments, provided herein are methods of treating breast cancer comprising administering to a patient at least one taxane, at least one platinum complex, and at least one PARP inhibitor. In some embodiments, at least one therapeutic effect is obtained that is a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, complete response to pathology, or stable disease. In some embodiments, the PARP inhibitor is benzamide or a metabolite thereof. In some embodiments, the benzamide is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the platinum complex is selected from cisplatin, carboplatin, platinum oxalate, and oxaliplatin. In some embodiments, the platinum complex is carboplatin. In some embodiments, the taxane is paclitaxel or docetaxel. In some embodiments, the taxane is paclitaxel. In some embodiments, the method comprises the following for a treatment cycle of at least 11 days: (a) on day 1 of the cycle, about 10-200mg/m is administered to the patient²The paclitaxel of (a); (b) on day 1 of the cycle, about 10-400mg/m is administered to the patient ²Carboplatin of (a); and (c) on day 1 of the cycle and throughout the cycleAbout 1-100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof in molar equivalent amounts, is administered to the patient twice a week.

In some embodiments, the present disclosure provides a method of treating breast cancer in a patient, comprising: (a) obtaining a sample from a patient; (b) testing the sample to determine the level of PARP expression of the sample; (c) determining whether PARP expression exceeds a predetermined level, and if so, administering to the patient at least one taxane, at least one platinum complex, and at least one PARP inhibitor. In some embodiments, if the sample does not have PARP expression above a predetermined level, the method further comprises selecting a different treatment regimen. In some embodiments, the cancer is breast cancer that is negative for one or more hormone receptors. In some embodiments, the breast cancer is HER2 negative breast cancer. In some embodiments, the cancer is negative for Estrogen Receptor (ER), Progesterone Receptor (PR), or HER 2. In some embodiments, the cancer is positive for at least one hormone receptor or HER 2. In some embodiments, the taxane is cisplatin, carboplatin, platinum oxalate, or oxaliplatin. In some embodiments, the taxane is paclitaxel. In some embodiments, the platinum complex is cisplatin or carboplatin. In some embodiments, the platinum complex is carboplatin. In some embodiments, the PARP inhibitor is benzamide or a metabolite thereof. In some embodiments, the PARP inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof. In some embodiments, the sample is a tumor section or a body fluid.

Some embodiments described herein provide methods of treating breast cancer in a patient, comprising, within a 21 day treatment cycle: (a) on day 1 of the cycle, about 750mg/m is administered to the patient²The paclitaxel of (a); (b) on day 1 of the cycle, about 10-400mg/m is administered to the patient²Carboplatin of (a); and (c) administering about 1-100mg/kg of 4-iodo-3-nitrobenzamide to the patient on day 1 of the cycle and twice weekly thereafter. In some embodiments, paclitaxel is administered as an intravenous infusion. In some embodiments, the carboplatin is administered as an intravenous infusion. In some embodimentsThe 4-iodo-3-nitrobenzamide is administered orally or by intravenous infusion.

Some embodiments described herein provide a method of treating breast cancer in a patient, comprising: (a) establishing a treatment cycle of about 10 days to about 30 days; (b) about 100 to about 2000mg/m is administered to the patient daily by intravenous infusion on 1 to 5 separate days of the cycle²The paclitaxel of (a); (c) about 10-400mg/m is administered to the patient by intravenous infusion over about 10 to about 300 minutes per day on 1 to 5 separate days of the cycle²Carboplatin of (a); and (d) administering about 1mg/kg to about 8mg/kg of 4-iodo-3-nitrobenzamide to the patient over a period of about 10 to about 300 minutes per day on 1 to 10 separate days of the cycle.

Thus, embodiments described herein include treating a patient with at least three chemically distinct substances, one of which is a taxane (e.g., paclitaxel or docetaxel), one of which is a platinum-containing complex (e.g., cisplatin or carboplatin or cisplatin), and one of which is a PARP inhibitor (e.g., BA or its metabolites). In some embodiments, one or more of these substances can be present in a wide variety of physical forms, such as free bases, salts (especially pharmaceutically acceptable salts), hydrates, polymorphs, solvates, metabolites, and the like. Unless otherwise defined herein, the use of chemical names is intended to include all physical forms of the named compounds. For example, if not further limited, the recitation of 4-iodo-3-nitrobenzamide is intended to include the free base in general, as well as all pharmaceutically acceptable salts, polymorphs, hydrates, and metabolites thereof. When it is intended to limit the disclosure or claims to a particular physical form of a compound, this will be made clear in the paragraphs or claims referring to that compound.

The terms "effective amount" or "pharmaceutically effective amount" refer to an amount of a therapeutic agent sufficient to produce a desired biological, therapeutic and/or prophylactic effect. The result can be a reduction and/or alleviation of signs, symptoms, or causes, or any other desired change in a biological system. For example, an "effective amount" for treatment refers to the amount of a nitrobenzamide compound disclosed herein by itself, or a composition containing the nitrobenzamide compound, that is required to bring about a clinically meaningful reduction in the disease. Suitable effective amounts for any individual case can be determined by one of ordinary skill in the art using routine experimentation.

By "pharmaceutically acceptable" or "pharmacologically acceptable" is meant a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to an individual without causing significant undesirable biological effects or interacting in a deleterious manner with any of the components contained in the composition.

The term "treatment" and grammatical equivalents thereof as used herein includes achieving a therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit means the radical cure or improvement of the disease being treated. For example, for cancer patients, therapeutic benefit includes radical treatment or amelioration of the cancer being treated. Likewise, with the eradication or amelioration of one or more of the physiological symptoms associated with the disease, some therapeutic benefit is achieved such that an improvement in the condition is observed in the patient, although in practice the patient may still suffer from the disease. For prophylactic benefit, the methods of the invention or the compositions of the invention can be administered to a patient at risk of developing cancer or a patient reported to suffer from one or more physiological symptoms of such a condition, even though a diagnosis of the condition may not have been made.

Antitumor agent

Antineoplastic agents useful in the present invention include, but are not limited to, antineoplastic alkylating agents, antineoplastic antimetabolites, antineoplastic antibiotics, plant-derived antineoplastic agents, antineoplastic platinum complexes, antineoplastic camptothecin derivatives, antineoplastic tyrosine kinase inhibitors, monoclonal antibodies, interferons, biological response modifiers, and other drugs that exhibit antineoplastic activity, or pharmaceutically acceptable salts thereof.

In some embodiments, the antineoplastic agent is an alkylating agent. The term "alkylating agent" as used herein refers to a reagent that gives an alkyl group in an alkylation reaction in which a hydrogen atom of an organic compound is substituted with an alkyl group. Examples of antineoplastic alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, melphalan, busulfan, dibromomannitol, carboquone, thiotepa, ranimustine, nimustine, temozolomide, or carmustine.

In some embodiments, the antineoplastic agent is an antimetabolite. The term "antimetabolite" as used herein broadly includes substances that disturb normal metabolism and substances that inhibit an electron transfer system to thereby prevent the production of high-energy intermediates, because they have similarities in structure and function with metabolites important to living biological organisms, such as vitamins, coenzymes, amino acids, and sugars. Examples of antimetabolites having antitumor activity include, but are not limited to, methotrexate, 6-mercaptopurine nucleoside, mercaptopurine, 5-fluorouracil, tegafur, doxifluridine, carmofur, cytarabine arabinoside ester (cytarabine ocfosfae), enocitabine, S-1, gemcitabine, fludarabine or disodium pemetrexed, with 5-fluorouracil, S-1, gemcitabine and the like being preferred.

In some embodiments, the antineoplastic agent is an antitumor antibiotic. Examples of antitumor antibiotics include, but are not limited to, actinomycin D, doxorubicin, daunorubicin, neocarzinostain, bleomycin, pelomycin, mitomycin C, aclacinomycin, pirarubicin, epirubicin, netstastin, idarubicin, sirolimus, or valrubicin.

In some embodiments, the antineoplastic agent is a plant-derived antineoplastic agent. Examples of antineoplastic agents of plant origin include, but are not limited to, vincristine, vinblastine, vindesine, etoposide, sobuzolne, docetaxel, paclitaxel and vinorelbine, with docetaxel and paclitaxel being preferred.

In some embodiments, the antineoplastic agent is a camptothecin derivative that exhibits antineoplastic activity. Examples of antitumor camptothecin derivatives include, but are not limited to, camptothecin, 10-hydroxycamptothecin, topotecan, irinotecan, or 9-aminocamptothecin, with camptothecin, topotecan, and irinotecan being preferred. Moreover, irinotecan is metabolized in vivo and shows antitumor effects like SN-38. Camptothecin derivatives are believed to act by a mechanism and activity that is nearly identical to that of camptothecin (e.g., Nitta, et al, Gan to KagakuRyoho, 14, 850-857 (1987)).

In some embodiments, the antineoplastic agent is an organo-platinum compound or platinum coordination compound having antineoplastic activity. An organoplatinum compound herein refers to a platinum-containing compound that can provide platinum in ionic form. Preferred organo-platinum compounds include, but are not limited to, cisplatin; cis-diammine platinum (II) dihydrate-ion; chloro (diethyltriamine) -platinum (II) chloride; dichloro (ethylenediamine) -platinum (II); platinum (II) (carboplatin) bis-amine (1, 1-cyclobutanedicarboxylic acid); cis-spiroplatinum; iproplatin; platinum (II) diammine (2-ethylmalonate); ethylenediamine platinum malonate (II); hydration of (1, 2-diaminodicyclohexyl) platinum (II) sulfate; hydration of (1, 2-diaminodicyclohexyl) malonic acid platinum (II); (1, 2-diaminocyclohexane) malonic acid platinum (II); (4-carboxyphthalic acid) (1, 2-diaminocyclohexane) platinum (II); (1, 2-diaminocyclohexane) - (isocitric acid) platinum (II); (1, 2-diaminocyclohexane) oxalic acid platinum (II); ormaplatin; tetraplatin; carboplatin, nedaplatin, and oxaliplatin, preferably carboplatin or oxaliplatin. In addition, other antitumor organo-platinum compounds mentioned in the present specification are known and commercially available and/or can be prepared by one of ordinary skill in the art using routine techniques.

In some embodiments, the antineoplastic agent is an antineoplastic tyrosine kinase inhibitor. The term "tyrosine kinase inhibitor" as used herein refers to a chemical substance that inhibits "tyrosine kinases" by converting the lambda-phosphate of ATP to the hydroxyl group of a specific tyrosine in the protein. Examples of anti-tumor tyrosine kinase inhibitors include, but are not limited to, gefitinib, imatinib, erlotinib, Sutent, Nexavar, Recentin, ABT-869, and Axitinib.

In some embodiments, the antineoplastic agent is an antibody or binding portion of an antibody that exhibits anti-tumor activity. In some embodiments, the anti-neoplastic agent is a monoclonal antibody. Examples include, but are not limited to, abciximab, adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, daclizumab, eculizumab (eculizumab), efalizumab (efalizumab), ibritumomab tiumumab, infliximab, moluzumab-CD 3, natalizumab, omalizumab (omalizumab), palivizumab, panitumumab (panitumumab), ranibizumab (ranibizumab), gimumab ozogamicin, rituximab, tositumomab, trastuzumab, or any antibody fragment specific for an antigen.

In some embodiments, the antineoplastic agent is an interferon. Such interferons have antitumor activity, and are glycoproteins produced and secreted by most animal cells after viral infection. It has not only the effect of inhibiting the growth of viruses but also various immune effector mechanisms including the inhibition of cell (especially tumor cell) growth and the enhancement of natural killer cell activity, and thus is designated as a cytokine. Examples of anti-tumor active interferons include, but are not limited to, interferon alpha-2 a, interferon alpha-2 b, interferon beta, interferon gamma-1 a, and interferon gamma-n 1.

In some embodiments, the antineoplastic agent is a biological response modifier. It is generally a generic term for substances or drugs that regulate living organism defense mechanisms or biological responses, such as survival, growth and differentiation of tissue cells, to make them useful to individuals against tumors, infections or other diseases. Examples of biological response modifiers include, but are not limited to, coriolus versicolor polysaccharide, lentinan, cezopyran, streptococcus hemolyticus, and ubenimex.

In some embodiments, the antineoplastic agent includes, but is not limited to, mitoxantrone, L-asparaginase, procarbazine, dacarbazine, hydroxyurea, pentostatin, tretinoin, alfacapt (alefacept), alpha-dabbepotin (darbepoetin alfa), anastrozole, exemestane, bicalutamide, leuprorelin, flutamide, fulvestrant, pegaptanib sodium (pegaptanib octasodium), dinekin diftotx, aldesleukin, thyrotropin-alpha, arsenic trioxide, bortezomib (bortezomib), capecitabine, and goserelin.

The terms "antineoplastic alkylating agent", "antineoplastic antimetabolite", "antineoplastic antibiotic", "antineoplastic plant-derived antineoplastic agent", "antineoplastic platinum coordination compound", "antineoplastic camptothecin derivative", "antineoplastic tyrosine kinase inhibitor", "monoclonal antibody", "interferon", "biological response modifier" and "other antineoplastic agent" are known and commercially available or can be prepared by methods known per se, or well known, or conventional by those skilled in the art. The preparation of gefitinib is described, for example, in U.S. patent 5,770,599; the preparation of cetuximab is described, for example, in WO 96/40210; the preparation of bevacizumab is described, for example, in WO 94/10202; the preparation of oxaliplatin is described, for example, in U.S. Pat. Nos. 5,420,319 and 5,959,133; gemcitabine is prepared, for example, in U.S. patent nos. 5,434,254 and 5,223,608; and camptothecin preparation processes are described in us patents 5,162,532, 5,247,089, 5,191,082, 5,200,524, 5,243,050 and 5,321,140; the preparation of irinotecan is described, for example, in U.S. Pat. No.4,604,463; the preparation of topotecan is described, for example, in U.S. Pat. No. 5,734,056; the preparation of temozolomide is described, for example, in JP-B No. 4-5029; and a process for producing rituximab is described in, for example, JP-W No. 2-503143.

The above antineoplastic alkylating agents are commercially available as shown in the following examples: nitrogen mustard N-oxide available from mitsubishi pharma corp under the trade name Nitrorin; cyclophosphamide sold by Shionogi & co, ltd. under the trade name Endoxan; ifosfamide under the trade name Ifomide, available from Shionogi & co, ltd; melphalan available from GlaxoSmithKline corp under the trade name Alkeran; busulfan available from Takeda Pharmaceutical co., ltd. under the trade name of Mablin; dibromomannitol sold by Kyorin Pharmaceutical co., ltd. under the trade name Myebrol; carboquone available from Sankyo co, ltd. under the trade name Esquinon; thiotepa, available from Sumitomo Pharmaceutical co, ltd, under the trade name Tespamin; ramustine under the trade name Cymerin, by Mitsubishi Pharma corp; nimustine available from Sankyo co, ltd under the trade name of Nidran; temozolomide, marketed by schering corp under the trade name Temodar; and carmustine available from guilford pharmaceuticals inc.

The above antineoplastic antimetabolites are commercially available as shown in the following examples: methotrexate, available from takeda pharmaceutical co, ltd, under the trade name Methotrexate; 6-mercaptopurine glycoside available from Aventis corp under the trade name Thioinosine; mercaptopurine available from takeda pharmaceutical co, ltd under the trade name Leukerin; 5-fluorouracil available from Kyowa Hakko Kogyo Co., Ltd. under the trade name 5-FU; tegafur available from taiho pharmaceutical co, ltd. under the trade name Futraful; doxifluridine available from nippon roche co, ltd under the trade name Furutulon; carmofur available from yamanouchi pharmaceutical co., ltd. under the trade name Yamafur; cytarabine available from nippon shinyaku co, ltd. under the trade name Cylocide; cytarabine arabinoside esters available from Nippon kayaku co, ltd. under the trade name Strasid; enocitabine available from Asahi Kasei corp. under the trade name Sanrabin; s-1 available from Taiho Pharmaceutical Co., Ltd, under the trade name TS-1; gemcitabine available from Eli Lilly & Co. under the trade name Gemzar; fludarabine available from Nippon Schering co., ltd. under the trade name Fludara; and disodium pemetrexed with the trade name alimata available from Eli Lilly & co. The above antitumor antibiotics are commercially available as shown in the following examples: actinomycin D available from Banyu pharmaceutical co, ltd. under the trade name Cosmegen; doxorubicin, commercially available from Kyowa Hakko kogyoco, ltd. under the trade name adriacin; daunorubicin, available from Meiji Seika Kaisha ltd. under the trade name Daunomycin; neocarzinostatin, available from Yamanouchi pharmaceutical co, ltd, under the trade name Neocarzinostatin; bleomycin sold under the trade name Bleo from Nippon kayaku co, ltd; pelomomycin available from Nippon Kayaku Co, ltd. under the trade name Pepro; mitomycin C, available from Kyowa Hakko Kogyo co, ltd. under the trade name Mitomycin; aclacinomycin sold under the trade name Aclacinon, available from Yamanouchi Pharmaceutical co. Pirarubicin, available from Nippon Kayaku Co., Ltd, under the trade name Pinorubicin; epirubicin under the trade name Pharmorubicin, purchased from Pharmacia corp; neat stastatin ester available from Yamanouchi Pharmaceutical co., ltd. under the trade name Smancs; idarubicin, available from Pharmacia corp, under the trade name Idamycin; sirolimus available from Wyeth corp under the trade name Rapamune; and valrubicin under the trade name Valstar, available from Anthra Pharmaceuticals inc.

The above plant-derived antitumor agents are commercially available as shown in the following examples: vincristine available from Shionogi & co., ltd. under the trade name Oncovin; vinblastine available from kyorin pharmaceutical co, ltd. under the trade name Vinblastine; vindesine available from Shionogi & co, ltd. under the trade name feldsin; etoposide available from Nippon Kayaku co., ltd. under the trade name Lastet; sobuconazole under the trade name Perazolin, available from Zenyaku Kogyo Co., Ltd; docetaxel available from Aventis corp under the trade name taxotere; paclitaxel available from Bristol-Myers Squibb Co. under the trade name Taxol; and vinorelbine available from Kyowa Hakko Kogyo co, ltd. under the trade name Navelbine.

The above antitumor platinum coordination compounds are commercially available as shown in the following examples: cisplatin, available from Nippon Kayaku co., ltd., under the trade name Randa; carboplatin available from Bristol-Myers Squibb Co. under the trade name Parasplatin; nedaplatin available from Shionogi & co., ltd. under the trade name Aqupla; and oxaliplatin under the trade name Eloxatin, available from Sanofi-Synthelabo co.

The above antitumor camptothecin derivatives are commercially available as shown in the following examples: irinotecan with Campto as the trade name, purchased from Yakult Honsha co., ltd; topotecan purchased from GlaxoSmithKline corp. under the trade name Hycamtin; and camptothecin available from Aldrich Chemical co, inc.

The above anti-tumor tyrosine kinase inhibitors are commercially available as shown in the following examples: gefitinib, available from AstraZeneca corp, under the trade name Iressa; imatinib, available from Novartis AG under the trade name Gleevec; and erlotinib, available from OSI Pharmaceuticals inc, under the trade name Tarceva.

The monoclonal antibodies described above are commercially available as shown in the following examples: cetuximab available from Bristol-MyersSquibb co. under the trade name Erbitux; bevacizumab available from Genentech, inc under the trade name Avastin; rituximab available from Biogen Idec inc. under the trade name Rituxan; alemtuzumab available from Berlex inc. under the trade name Campath; and trastuzumab available from Chugai Pharmaceutical co, ltd under the trade name Herceptin.

The interferons mentioned above are commercially available as shown in the following examples: interferon alpha available from sumitomo pharmaceutical co, ltd. under the trade name Sumiferon; interferon alpha-2 a available from takeda pharmaceutical co, ltd under the trade name Canferon-a; interferon alpha-2 b available from Schering-Plough corp under the trade name of Intron a; interferon beta available from mochida pharmaceutical co, ltd. under the trade name ifn.beta.; interferon gamma-1 a, available from Shionogi & co, ltd. under the trade name imuomax-gamma; and interferon gamma-n 1 available from otsuka pharmaceutical co.

The biological response modifiers described above are commercially available as shown in the following examples: coriolus versicolor polysaccharide available from sankyoco, ltd under the trade name krestin; lentinan available from Aventis corp under the trade name Lentinan; a cilazapyran sold under the trade name soniiran, available from Kaken Seiyaku co., ltd; hemolytic streptococcus under the trade name Picibanil, available from Chugai Pharmaceutical co, ltd; and ubenimex under the trade name Bestatin, available from Nippon Kayaku co.

Such other antineoplastic agents are commercially available, as shown in the following examples: mitoxantrone available from wyeth lederle Japan, ltd. under the trade name Novantrone; l-asparaginase, available from KyowaHakko Kogyo co, ltd, under the trade name Leunase; procarbazine available from Nippon Roche co., ltd. under the trade name natula; dacarbazine available from KyowaHakko Kogyo co, ltd under the trade name Dacarbazine; hydroxyurea available from Bristol-Myers Squibb Co. under the trade name Hydrea; pentostatin under the trade name Coforin, purchased from KagakuOyobi Kessei ryohe Kenkyusho; retinoic acid available from Nippon Roche co, ltd. under the trade name Vesanoid; alfasiteds available from Biogen idecconc under the trade name Amevive; dabipoten α available from Amgen inc under the trade name Aranesp; anastrozole available from AstraZeneca corp under the trade name Arimidex; exemestane available from Pfizer inc under the trade name of amonasin; bicalutamide available from AstraZeneca corp. under the trade name Casodex; leuprolide available from takeda pharmaceutical co, ltd under the trade name leuprolin; flutamide available from Schering-Plough Corp. under the trade name Eulexin; fulvestrant available from astrazeneca corp. under the trade name Faslodex; macugen brand name pegaptanib sodium available from Gilead Sciences, inc; a dinierein-toxin linker under the trade name Ontak, available from Ligand Pharmaceuticals Inc.; aldesleukin available from Chiron corp under the trade name Proleukin; thyrotropin alpha available from Genzyme core under the trade name Thyrogen; arsenic trioxide available from Cell Therapeutics, inc, under the trade name Trisenox; bortezomib, available from Millennium Pharmaceuticals, inc. under the trade name velcro; capecitabine available from Hoffmann-La Roche, ltd. under the trade name Xeloda; and goserelin available from AstraZeneca Corp. under the trade name Zoladex. The term "antitumor agent" as used herein includes the above-mentioned antitumor alkylating agents, antitumor antimetabolites, antitumor antibiotics, plant-derived antitumor agents, antitumor platinum coordination compounds, antitumor camptothecin derivatives, antitumor tyrosine kinase inhibitors, monoclonal antibodies, interferons, biological response modifiers, and other antitumor drugs.

Other anti-tumor or anti-cancer agents may be used in combination with the benzopyrone compounds. Such suitable antineoplastic or anti-cancer agents include, but are not limited to, 13-cis-retinoic acid, 2-CdA, 2-chlorodeoxyadenosine, 5-azacitidine, 5-fluorouracil, 5-FU, 6-mercaptopurine, 6-MP, 6-TG, 6-thioguanine, Abraxane, isotretinoin, actinomycin-D, doxorubicin, fluorouracil, anagrelide, Ala-Cort, aldesleukin, alemtab, ALIMTA, alitame A acid, Alkaban-AQ, melphalan, all-trans retinoic acid, alpha interferon, altretamine, methotrexate, amifostine, aminoglutethimide, anagrelide, nilutamide, anastrozole, arabinosyl cytosine (Aranosylate), Ara-C, Alfatabestin (Aranesp), altrex, anastrozole, daclizine, dara, arazetane, aran (Arraon), arsenic trioxide, Asparaginase, ATRA, Avastin (bevacizumab), azacitidine, BCG, BCNU, bendamustine, bevacizumab, bexarotene, BEXXAR, bicalutamide, BiCNU, bleomycin sulfate, bleomycin, Bortezomib (Bortezomib), busulfan, Busulfex, C225, calcium folinate, Campath, Camptosar, camptothecin-11, capecitabine, Carac, carboplatin, Carmustine, Carmustine Wafer, bicalutamide, CC-5013, CCI-779, CCNU, CDDP, eNU, CeCeCeCeCeU, daunorubicin, cetuximab, chlorambucil, cisplatin, calcium folinate, droabine, cortisone, more Raw mycin, CPT-11, cyclophosphamide, aminoglutethimide, cytarabine liposome, Cytosar-U, Cytoxan, dacarbazine, Dacogen, actinomycin, alpha-bepotin (Darbepoetin alfa), Dasatinib (Dasatinib), daunorubicin, rubicin, daunorubicin hydrochloride, daunorubicin liposome, daunorubicin citrate liposome, dexamethasone, decitabine, Delta-cortex, prednisone, dinelaukin (Denileukin Diftitox), Depo Cyt^TMDexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone, dexrazoxane, DHAD, DIC, dexamethasone acetate (Diodex), docetaxel, Doxil, Droxina^TMDTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin, Alfateptin, Erbitux, Erbitux, Erlotinib, Erwinia L-asparaginase, estramustine, amifostine, Vanbrex, etoposide phosphate, flutamide, Evepitant, exemestane, Faleton, Faslodex, Freon, filgrastim, floxuridine, Fudalabine, Fluoroprole, Fluorouracil (Fluoromethyltestosterone, Flutaltamine, folinic acid, FUDR cream, fulvestrant, G-CSF, Gefitinib, gemcitabine, Gemcitabine, Selectine-chemotherapeutic drugs, Wei, Gliadel, GM-CSF, Waserpentin, Waxaglitazone, G-CSF, Mexateslin, Melamine, macrophage-stimulating factor, macrophage, and macrophage colony, Hydroxyurea, hydrocortisone acetate, hydrocortisone sodium phosphate, hydrocortisone succinate, hydrocortisone phosphate, hydroxyurea, temozolomide, zerumycin (Ibritumomab Tiuxetan), idarubicin, Ifex, IFN-alpha, ifosfamide, IL-11, IL-2, imatinib mesylate, dacarbazine, interferon alpha-2 b (polyethylene glycol conjugate), interleukin-2, interleukin-11, (interferon alpha-2 b), Iressa, irinotecan, Isotretinoin (isotretinin), azaepothilone (Ixabepilone), azaepothilone (Ixempra), kilasdrone (t), hydrogen Cortisone, lapatinib, L-asparaginase, LCR, Lenalidomide (Lenalidomide), letrozole, folinic acid (Leucovorin), lecorane, Leukenelin acetate, vincristine, cladribine, liposomal Ara-C, Liquid Pred, lomustine, L-PAM, L-sabcomeline, Lupron, leuprorelin acetate depot sustained release injection, Matulane, Maxidex, mechlorethamine hydrochloride, methylprednisolone, mernolone, megestrol acetate, melphalan, mercaptopurine, mesna, Mesnex, methotrexate, sodium methotrexate, methylprednisolone, Metaportone, mitomycin-C, mitoxantrone, M-prednisolone, MTC, Mergex, mustarne, mustara, Nestalactine, Nestalotilone, Nestalotide, Nestalactine, Myostatin, Nestarobine, Myostatin, Myostatic, Myostatin, youbujin, Nexavar, Nilandron, nilutamide, Nipent, Nitrogen Mustard (Nitrogen Mustard), Novaldex, norflurazon, octreotide acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevkin, Orapred, Orasone, oxaliplatin, paclitaxel, protein-bound paclitaxel, pamidronate, Panretin, platidine, prednisolone, PEG interferon, Peg filgrastim, polyethylene glycol-interferon, PEG-L-asparaginase, PEMETEXED, pentostatin, melphalan, cisplatin, AQ, prednisolone, prednisone, Prelone, procarbazine, PROCRIT, Proleleukin, Prolifepsipran 20 (with a calciphilin implant), mercaptopurine, loxide, rilamine hydrochloride (Rilomefloxacin, Rivelin), Revocane, Revtufen, Revoca, Rivelin, Revocat, Revocalisib, Revocat, Revocalisine, Relutropine, Reynaud, Refatin, Relutropine 20 (with an implant), Revocalin, Refatin, Relutroping, Refatin, Revalbut, Solu-Memrol, sorafenib, SPRYCEL, STI-571, streptozotocin, SU11248, Sunitinib, Sutent, tamoxifen, Tarceva, Targretin, Taxol, Taxotere, Temodar, temozolomide, Temsirolimus, teniposide, TESPA, thalidomide, Thalomid, TheraCys, thioguanine, thiophosphoramide (Thiophoamide), Thiopolex, sertepin, TICE, topotecan, toremifene, Torisel, tositumomab, trastuzumab, vitamine, and Thiophoramide Formic acid, Trexall^TMArsenic trioxide, TSPA, TYKERB, VCR, Vectibix, Velban, Velcade, VePesid, tretinoin, Viadur, Vidaza, vinblastine sulfate, Vincasar Pfs, vincristine, vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Veronica (Vumon), Hiluoda, streptozotocin, Zevalin (Zevalin), Debrezon, Norrehd, zoledronic acid, Zolinza, Zomet.

An antimetabolite agent:

antimetabolites are drugs that interfere with the normal metabolic processes of cells. Since cancer cells replicate rapidly, interfering with cellular metabolism will have a greater effect on cancer cells than on host cells. Gemcitabine (i.e., 4-amino-1- [3, 3-difluoro-4-hydroxy-5- (hydroxymethyl) tetrahydroxy furan-2-yl)]-1H-pyrimidin-2-one; EliLilly corporation as GEMZARTradename of (d) is a nucleoside analog that interferes with cell division by blocking DNA synthesis, apparently leading to cell death by an apoptotic mechanism. The dose of gemcitabine may be adjusted for a particular patient. In adults, gemcitabine will be dosed at about 100mg/m when used in combination with a platinum-containing drug and a PARP inhibitor ²To about 5000mg/m²In the range of about 100mg/m²To about 2000mg/m²In the range of about 750mg/m²To about 1500mg/m²In the range of about 900mg/m²To about 1400mg/m²In the range of (1), or 1250mg/m². Dimension mg/m²Means per unit surface area of the patient (m, m²) Dose of gemcitabine used (mg). Gemcitabine may be administered by Intravenous (IV) infusion, for example, over a period of about 10 to about 300 minutes, about 15 to about 180 minutes, about 20 to about 60 minutes, or about 10 minutes. In this context, the term "about" means approximately the normal amount used; in some embodiments, a tolerance of ± 10% or ± 5%.

Platinum complexes (platinum complexes):

platinum complexes are pharmaceutical compositions for the treatment of cancer, containing at least one platinum center complexed with at least one organic group. Carboplatin ((SP-4-2) -diamine [1, 1-cyclobutane dicarboxylate (2-) -O, O']Platinum) is a DNA alkylating agent, like cisplatin and oxaliplatin. The dose of carboplatin is determined by calculating the area under the plasma concentration curve (AUC) in a manner known to those skilled in the art of cancer chemotherapy, taking into account the creatinine clearance of the patient. In some embodiments, the dose of carboplatin used in combination therapy with an antimetabolite, such as gemcitabine, and a PARP inhibitor, such as 4-iodo-3-nitrobenzamide, is calculated to result in an AUC of about 0.1 to 6 mg/ml-min, about 1 to 3 mg/ml-min, about 1.5 to about 2.5 mg/ml-min, about 1.75 to about 2.25 mg/ml-min, or about 2 mg/ml-min. (e.g., AUC 2 is shorthand for 2 mg/ml-min.) in some embodiments, the dose of carboplatin used in combination therapy with a taxane (e.g., paclitaxel or docetaxel) and a PARP inhibitor (e.g., 4-iodo-3-nitrobenzamide) is calculated such that the AUC is about 0.1-6 mg/ml-min, about 1-3 mg/ml-min, about 1.5 to about 2.5 mg/ml-min, about 1.75 to about 2.25 mg/ml-min, or about 2 mg/ml-min. (e.g., AUC 2 is shorthand for 2 mg/ml-min.) in some embodiments, a suitable carboplatin dose is from about 10 to about 400mg/m ²E.g. about 360mg/m². Platinum complexes such as carboplatin are typically administered Intravenously (IV) over a period of about 10 to about 300 minutes, about 30 to about 180 minutes, about 45 to about 120 minutes, or about 60 minutes. In this context, the term "about" generally means "approximately. In some embodiments, "about" means ± 10% or ± 5%.

Topoisomerase inhibitors

In some embodiments, the methods of the present invention may comprise administering to a breast cancer patient an effective amount of a PARP inhibitor in combination with a topoisomerase inhibitor (e.g., irinotecan or topotecan).

Topoisomerase inhibitors designed to interfere with topoisomeraseDrugs for the action of enzymes (topoisomerases I and II), which are enzymes that control the alteration of DNA structure by catalyzing the cleavage and religation of the phosphodiester backbone of the DNA strand in the normal cell cycle (http:// en. wikipedia. org/wiki/topoisomerease _ inhibitor-cite _ note-url-lipids _ Medical _ solubility: topoisomerease _ inhibitor-1# note _ note-url-lipids _ dictionary: topoisomerease _ dictionary-1). Topoisomerase has become a popular target for cancer chemotherapy treatment. Topoisomerase inhibitors are thought to block the ligation step of the cell cycle, producing single and double strand breaks that disrupt the integrity of the genome. The introduction of these breaks subsequently leads to apoptosis and cell death. Topoisomerase inhibitors are generally classified according to the type of enzyme they inhibit. Topoisomerase I, the most common type of topoisomerase in eukaryotes, is the target of topotecan, irinotecan, lurtotecan and irinotecan, all of which are commercially available. Topotecan was purchased from GlaxoSmithKline under the trade name Hycamtim . Irinotecan was purchased from Pfizer under the tradename Ccamptosar. Lurtotecan is available as a liposomal formulation from Gilead Sciences inc. The topoisomerase inhibitor can be administered in an effective dose. In some embodiments, an effective dose for human treatment is from about 0.01 to about 10mg/m²The day is. The treatment may be repeated daily, biweekly, semi-weekly, or monthly. In some embodiments, the treatment cycle may be followed by a rest period of 1 day to several days, or 1 week to several weeks. In combination with a PARP-1 inhibitor, the PARP-1 inhibitor and the topoisomerase inhibitor may be administered on the same day or on different days.

Compounds targeting type II topoisomerases fall into 2 broad categories: topoisomerase poisons, which target the topoisomerase-DNA complex, and topoisomerase inhibitors, which disrupt catalytic conversion (catalyticturn over). Topology II (topo II) toxicants include, but are not limited to, eukaryotic type II topoisomerase inhibitors (topology II): amsacrine, etoposide phosphate, teniposide and doxorubicin. These drugs are anti-cancer treatments. Examples of topoisomerase inhibitors include ICRF-193. These inhibitors target the N-terminal atpase domain of topology II and prevent topology II conversion. The structure of the binding of this compound to the ATPase domain has been elucidated by Classen (Proceedings of the National Academy of science, 2004), indicating that the drug binds in a non-competitive manner and locks (locks down) the dimerization of the ATPase domain.

Anti-angiogenic agents

In some embodiments, the methods of the present invention may comprise administering to a breast cancer patient an effective amount of a PARP inhibitor in combination with an anti-angiogenic agent.

Angiogenesis inhibitors are substances that inhibit angiogenesis (the growth of new blood vessels). Each solid tumor (as opposed to leukemia) requires the generation of blood vessels to maintain its survival once it reaches a certain size. Tumors can only grow when they form new blood vessels. In adults, blood vessels are not usually formed everywhere unless active tissue repair is being performed. The angiogenesis inhibitor endostatin and related chemicals can inhibit the formation of blood vessels, thereby preventing the unlimited development of cancer. In patient trials, tumors lost viability and remained in that state even after the end of endostatin treatment. The side effects of this treatment are few, but the selectivity seems to be limited. Other angiogenesis inhibitors such as thalidomide and natural plant-based substances are also being actively studied.

Known inhibitors include the drug bevacizumab (Avastin), which binds to Vascular Endothelial Growth Factor (VEGF) and thereby inhibits its binding to receptors that promote angiogenesis. Other anti-angiogenic agents include, but are not limited to, carboxyamidotriazole, TNF-470, CM101, interferon- α, IL-12, platelet factor 4, suramin, SU5416, thrombospondin, vascular Production-inhibiting steroids (angiostatin) + heparin, cartilage-derived angiostatin, matrix metalloproteinase inhibitors, angiostatin, endostatin, 2-methoxyestradiol, ticagren, thrombospondin, prolactin, alpha-alpha_Vβ₃Inhibitors and linoamine.

Targeted Her-2 therapy

In some embodiments, the methods of the invention may comprise administering to a HER2 positive breast cancer patient an effective amount of a PARP inhibitor in combination with trastuzumab (herceptin).

Herceptin (Herceptin) (trastuzumab) is a targeted therapeutic for early HER-2 positive breast cancer. Herceptin is approved for the adjuvant treatment of HER-2 overexpression, node positive or node negative (ER/PR-negative or with a high risk profile) breast cancer. Herceptin can be used in several different ways: as part of a treatment regimen comprising doxorubicin, cyclophosphamide, and paclitaxel or docetaxel; (ii) use with docetaxel and carboplatin; or as a single drug following a multimodal anthracycline-based therapy. Herceptin has been approved for use in combination with paclitaxel for first-line treatment of HER-2 overexpressing metastatic breast cancer. Herceptin has been approved as a single drug for the treatment of HER-2 overexpressing breast cancer for treatment of patients receiving one or more chemotherapeutic regimens for metastatic disease.

Lapatinib or lapatinib ditosylate are orally active chemotherapeutic drugs for the treatment of solid tumors such as breast cancer. It was called small molecule GW572016 during development. Lapatinib can be prescribed to patients meeting criteria for a particular indication as part of a combination therapy for breast cancer. Pharmacologically, lapatinib is a dual tyrosine kinase inhibitor that interrupts the cellular signals that cause cancer. Lapatinib is used to treat breast cancer in women with HER-2 positive advanced breast cancer and whose disease has progressed following prior treatment with other chemotherapeutic drugs such as anthracyclines, taxane derivative drugs or trastuzumab (herceptin, Genentech).

Hormone therapy

In some embodiments, the methods of the present invention may comprise administering an effective amount of a PARP inhibitor in combination with hormone therapy to a breast cancer patient.

There are several hormones that attach to cancer cells and affect their proliferative capacity. The purpose of hormone therapy is to add, block or remove hormones. In the context of breast cancer, the estrogen and progesterone, which are female hormones, promote the growth of some breast cancer cells. Thus, hormone therapy is performed in these patients to block naturally occurring estrogens in the human body and to combat the growth of cancer. There are two types of hormone treatment for breast cancer: drugs that inhibit estrogen and progesterone from promoting breast cancer cell growth, and drugs or surgery that prevent hormone production by the ovary.

Common hormonal therapy drugs used to treat breast cancer include, but are not limited to, tamoxifen, toremifene, anastrozole, arnosine, frolon (Femara), and norrehd.

Tamoxifen-hormone antagonists

Tamoxifen (trade name Nolvadex) reduces the chance of recurrence of some early breast cancers and prevents the development of cancer in the unaffected breast. Tamoxifen also slows or stops the growth of cancer cells in vivo. In addition, tamoxifen may provide an alternative to looking to wait for or receive preventative mastectomy procedures in women at high risk of developing breast cancer. Tamoxifen is a drug known as a Selective Estrogen Receptor Modulator (SERM). In the breast, it acts as an antiestrogen. Estrogen promotes the growth of breast cancer cells, while tamoxifen prevents estrogen from attaching to estrogen receptors on these cells. As such, it is believed that the growth of breast cancer cells will cease. Tamoxifen is often used with chemotherapy and other breast cancer treatment methods. It is considered to be an option in the following cases: treatment of Ductal Carcinoma In Situ (DCIS) combined with mastectomy or mastectomy; adjuvant treatment of breast Lobular Carcinoma In Situ (LCIS) to reduce the risk of further breast cancer development; adjuvant treatment of metastatic breast cancer for men and women with estrogen receptor positive cancer; treatment of recurrent breast cancer; preventing the women with high risk of suffering from breast cancer from suffering from breast cancer.

Steroid and non-steroid aromatase inhibitors

Aromatase Inhibitors (AI) are a class of drugs that block aromatase and are used to treat breast and ovarian cancer in postmenopausal women. Aromatase inhibitors reduce the amount of estrogen in postmenopausal women with hormone receptor positive breast cancer. As estrogen is reduced in the body, the hormone receptors receive less growth signals and the progression of the cancer can slow or stop.

Aromatase inhibitor drugs include runing (chemical name: anastrozole), arnosine (chemical name: exemestane), and froron (chemical name: letrozole). Each drug is administered in pill form once daily for up to 5 years. However, in women with advanced (metastatic) disease, the drug should be continued as long as it works well.

Aromatase Inhibitors (AI) are classified into two types: irreversible steroid inhibitors, such as exemestane, permanently bound to an aromatase complex; and non-steroidal inhibitors (e.g., anastrozole, letrozole) that inhibit the enzyme in a reversible competitive manner.

Fulvestrant, also known as ICI 182,780 and "Faslodex", is a drug for the treatment of hormone receptor positive metastatic breast cancer in postmenopausal women with disease progression after anti-estrogen treatment. It is an estrogen receptor antagonist with no agonist effect, acting by simultaneously down-regulating and degrading the estrogen receptor. It is administered by once monthly injection.

Targeted therapy

In some embodiments, the methods of the invention may comprise administering to a breast cancer patient an effective amount of a PARP inhibitor in combination with a targeted growth factor receptor inhibitor, including but not limited to Epidermal Growth Factor Receptor (EGFR) and insulin-like growth factor I receptor (IGF 1R).

EGFR is overexpressed in some types of human cancer (including but not limited to lung and breast cancer) cells. Highly proliferating, invasive breast cancer cells often express abnormally high levels of EGFR, a phenomenon known to control cell differentiation and migration. The availability and FDA approval of specific EGFR tyrosine kinase inhibitors, such as gefitinib, further increases interest in EGFR. Inhibition of EGFR is an important anti-cancer treatment. Examples of EGFR inhibitors include, but are not limited to, cetuximab. This is an intravenous chimeric monoclonal antibody for the treatment of cancer, including but not limited to metastatic colorectal cancer and head and neck cancer. Panitumumab is another example of an EGFR inhibitor. This is a humanized monoclonal antibody against EGFR. Panitumumab has proven beneficial and better than supportive care when used alone in patients with advanced colon cancer, and the FDA in the united states has approved such use.

Activation of the type 1 insulin-like growth factor receptor (IGF1R) promotes cell proliferation and inhibits apoptosis of various cells. Transgenic mice expressing constitutively activated IGF1R or IGF-1 suffer from Breast tumors, and elevated levels of IGF1R were detected in primary Breast cancer (Yanochko et al. Breast cancer research 2006). Insulin-like growth factor 1 receptor (IGF1R) and HER2 have been shown to exhibit important signaling interactions in breast cancer. Specific inhibitors of one of these receptors may cross-inhibit the activity of the other receptor. Targeting these two receptors produces the greatest inhibition of the extracellular signal-regulated kinase 1/2 and the AKT signaling pathway downstream of them. Thus, such drug combinations may be clinically useful, even for tumors that are not refractory to single drug treatment, a specific example of which is the effect of the HER2/IGF1R inhibitor combination in MCF7 cells without overexpression of HER2 (Chakraborty AK, et al, Cancer res.2008 Mar 1; 68 (5): 1538-45). An example of an IGF1R inhibitor is CP-751871. CP-751871 is a human monoclonal antibody that selectively binds to IGF1R, thereby preventing IGF1 binding to the receptor and subsequent autophosphorylation of the receptor. Inhibition of IGF1R autophosphorylation can result in decreased receptor expression on IGF 1R-expressing tumor cells, decreased anti-apoptotic effects of IGF, and inhibition of tumor growth. IGF1R is a receptor tyrosine kinase expressed on most tumor cells and is involved in cell division, angiogenesis, and tumor cell survival.

PI3K/mTOR pathway

Deregulation of the phosphatidylinositol-3 kinase (PI3K) pathway is a common phenomenon in human cancers, and this deregulation can be due to inactivation of tumor suppressor factors (phosphatase and tensin homologues deleted on chromosome 10) or to activating mutations in p110- α. These hot spot mutations result in the oncogenic activity of the enzyme and contribute to the therapeutic resistance of the anti-HER 2 antibody trastuzumab. Therefore, this PI3K pathway is an attractive target for cancer therapy. The dual inhibitors of PI3K, NVP-BEZ235 and downstream mammalian target of rapamycin (mTOR), have been shown to inhibit activation of the downstream effectors Akt, S6 ribosomal protein and 4EBP1 in breast cancer cells. NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumor activity in Cancer cells containing wild-type and mutant P110-alpha (Violeta Serra, et al Cancer Research 68, 8022-.

Hsp90 inhibitors

These drugs target heat shock protein 90(hsp 90). Hsp90 is a chaperone protein, which normally functions to help other proteins form and maintain the desired shape in which they function. Chaperonins work by physical contact with other proteins. Hsp90 can also allow cancer cells to survive and even thrive despite genetic defects that often lead to cancer cell death. Thus, blocking the function of HSP90 and related chaperones may lead to cancer cell death, especially if the blocking of chaperone function is combined with other strategies that block cancer cell survival.

Tubulin inhibitors

Tubulin is a microtubule-forming protein that is a key component of the cytoskeleton (structural network). Microtubules are essential for cell division (mitosis), cell structure, transport, signaling, and activity. In view of their major role in mitosis, microtubules have become an important target for anticancer drugs-commonly referred to as antimitotic drugs, tubulin inhibitors and microtubule targeting drugs. These compounds bind to tubulin in microtubules and prevent the proliferation of cancer cells by interfering with microtubule formation required for cell division. This interference blocks the cell cycle sequence, leading to apoptosis.

Apoptosis inhibitors

Inhibitors of Apoptosis (IAPs) are a family of functionally and structurally related proteins, originally characterized in baculoviruses, that act as endogenous inhibitors of apoptosis. At least 6 members of the human IAP family, IAP homologues have been found in many organisms. 10058-F4 is a c-Myc inhibitor that induces cell cycle arrest and apoptosis. It is a cell permeable thiazolidinone that specifically inhibits the interaction of c-Myc-Max and prevents the transactivation of c-Myc target gene expression. 10058-F4 inhibits tumor cell growth in a c-Myc-dependent manner in vitro and in vivo. BI-6C9 is a tBod inhibitor and an anti-apoptotic agent. GNF-2 belongs to a new class of Bcr-abl inhibitors. GNF-2 appears to bind to the allosteric site myristoyl binding pocket, remote from the active site, thereby stabilizing the inactive form of the kinase. It has an IC at 267nM ₅₀Values inhibited the phosphorylation of Bcr-Abl, but did not inhibit 63 other kinases including native c-Abl and showed no toxicity at all to cells that did not express Bcr-Abl. GNF-2 shows great potential for a new class of inhibitors in the study of Bcr-Abl activity and in the treatment of treatment-resistant Chronic Myeloid Leukemia (CML) caused by Bcr-Abl oncoproteins. Pifithrin-alpha is p53 mediated apoptosis and p53 dependent gene transcription (e.g. cyclin G, p 21/waf)1 and mdm2 expression). Pifitrin-alpha increases the survival of cells following genotoxic stress such as UV radiation and treatment with cytotoxic compounds including doxorubicin, etoposide, paclitaxel and cytosine-beta-D-arabinofuranoside. Pifithrin-alpha can protect mice from lethal systemic gamma radiation, and the incidence of cancer is not increased.

PARP inhibition:

in some embodiments, the present invention provides methods of treating breast cancer that is negative for at least one of ER, PR, or HER2 by administering at least one PARP inhibitor to a patient in need of treatment. In other embodiments, the present invention provides methods of treating breast cancer by administering to a patient in need of treatment at least one PARP inhibitor as described herein in combination with at least one anti-neoplastic agent.

Without intending to be limited by any particular mechanism of action, it is believed that the compounds described herein have anti-cancer properties due to modulation of poly (ADP-ribose) polymerase (PARP) activity. This mechanism of action is related to the ability of PARP inhibitors to bind PARP and reduce its activity. PARP catalyzes the conversion of β -nicotinamide adenine dinucleotide (NAD +) to nicotinamide and poly-ADP-ribose (PAR). Both poly (ADP-ribose) and PARP are associated with transcription, cell proliferation, gene stability and carcinogenesis (Bouchard V.J. et al, Experimental health, Volume 31, Number 6, 6 months 2003, pp.446-454 (9); Herceg Z.; Wang Z. -Q.mutation Research/Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 6 months 2 days 2001, pp.97-110 (14)). Poly (ADP-ribose) polymerase 1(PARP1) is a key molecule in the repair of DNA Single Strand Breaks (SSBs) (de Murcia J, et al 1997, Proc Natl Acad SciUSA 94: 7303-. SSB knockout repair due to inhibition of PARP1 function results in DNA Double Strand Breaks (DSBs) that cause synthetic lethality in homologously directed DSB repair of defective cancer cells (Bryant HE, et al (2005) Nature 434: 913-.

BRCA1 and BRCA2 function as integral parts of the homologous recombination mechanism (HR) inseparable (Narod SA, Foulkes WD (2004) Nat Rev Cancer 4: 665-.

Cells deficient in BRCA1 or BRCA2 are deficient in double-strand break (DSB) repair by gene-switching based Homologous Recombination (HR) (Farmer H, et al (2005) Nature 434: 917-. Deficiency in the breast cancer susceptibility proteins BRCA1 or BRCA2 induces sensitivity of cells to inhibition of poly (ADP-ribose) polymerase (PARP) activity, leading to cell cycle arrest and apoptosis. It has been reported that the key role of BRCA1 and BRCA2 in double strand break repair via Homologous Recombination (HR) is the underlying cause of this sensitivity, and that the absence of RAD51, RAD54, DSS1, RPA1, NBS1, ATR, ATM, CHK1, CHK2, FANCD2, FANCA or FANCC induces this sensitivity (McCabe N. et al, Deficiency in the repair of DNA damage by homology binding and sensitivity binding (ADP-ribose) polymerase inhibition, cancer research 2006, vol.66, 8109, 8115). It has been suggested that for cancers with defects in BRCA1/2 or other HR pathway components, inhibition of PARP1 may be a specific treatment (Helleday T, et al, (2008) Nat Rev Cancer 8: 193-204). Triple negative tumors account for 15% of all breast cancers and often mask defects in the repair of DNA double strand breaks by Homologous Recombination (HR) mechanisms, such as BRCA1 dysfunction (Rottenberg S, et al Proc Natl Acad Sci usa.2008 Nov 4; 105 (44): 17079-84).

Inhibiting the activity of PARP molecules includes decreasing the activity of these molecules. The term "inhibit" and grammatical variations thereof such as "inhibited" is not intended to require a complete reduction in PARP activity. In some embodiments, the reduction in activity of the molecule is at least about 50%, at least about 75%, at least about 90%, or at least about 95% compared to the absence of inhibition, e.g., compared to the absence of a nitrobenzamide inhibitor of the present invention. In some embodiments, inhibition refers to an observable or measurable decrease in activity. In some cases of treatment, the inhibition is sufficient to produce a therapeutic and/or prophylactic benefit in the condition being treated. The phrase "does not inhibit" and grammatical variations thereof does not require a complete absence of effect on activity. For example, it refers to a situation wherein PARP activity is reduced by less than about 20%, less than about 10%, preferably less than about 5%, in the presence of an inhibitor such as a nitrobenzamide of the present invention.

Poly (ADP-ribose) polymerase (PARP) is an important enzyme in DNA repair and therefore has potential utility in chemotherapy resistance. Targeting PARP is believed to interrupt the DNA repair process, thereby enhancing taxane-mediated, antimetabolite-mediated, topoisomerase inhibitor-mediated, and growth factor receptor inhibitor, such as IGF1R inhibitor-mediated, and/or platinum complex-mediated DNA replication and/or repair in tumor cells. PARP inhibitors may also be highly active in those patients with breast cancer or those deficient in other DNA repair pathways with impaired BRCA1 and BRCA2 function. Primary breast cancers that are ER, PR and HER2 negative show up-regulation of PARP. This breast cancer subtype has a 9-fold risk of developing BRCA-1 mutations and may have further defects in the fanconi anemia DNA repair pathway.

4-iodo-3-nitrobenzamide (BA) is a small molecule that acts on tumor cells but has no toxic effect on normal cells. BA is believed to exert its anti-tumor effect through inhibition of PARP. BA is very lipophilic and it is distributed rapidly and widely into various tissues, including the brain and cerebrospinal fluid (CSF). It is actively resistant in vitro against a wide range of cancer cells, including drug resistant cell lines. One skilled in the art will recognize that BA may be administered in any form that is pharmaceutically acceptable, such as in the form of a pharmaceutically acceptable salt, solvate, or complex. Furthermore, since BA is capable of tautomerization in solution, the tautomeric forms of BA, together with salts, solvates or complexes, are included within the term BA (or the corresponding 4-iodo-3-nitrobenzamide). In some embodiments, BA may be administered in combination with a cyclodextrin, such as hydroxypropyl- β -cyclodextrin. However, one skilled in the art will recognize that other active and inactive agents may also be used in combination with BA; unless otherwise indicated, the recitation of BA shall include all its pharmaceutically acceptable forms.

Basal-like breast cancer has a higher tendency to metastasize to the brain; BA is known to cross the blood-brain barrier. While not wishing to be bound by any particular theory, it is believed that BA achieves its anti-tumor effect by inhibiting the function of PARP. In some embodiments, BA may be used to treat triple negative metastatic breast tumors. In some embodiments, BA can be used to treat breast tumors that are negative for at least one of ER, PR, and Her 2. In some embodiments, BA can be used to treat breast tumors in which at least two of ER, PR, and HER2 are negative, e.g., ER negative, PR negative, and HER-2 positive; or ER-positive, PR-negative, and Her-2-negative; or ER negative, PR positive and Her-2 negative.

In some embodiments, BA may be used in combination with an anti-tumor agent to treat breast tumors. In some embodiments, the antineoplastic agent is an antineoplastic alkylating agent, an antineoplastic antimetabolite agent, an antineoplastic antibiotic, a plant-derived antineoplastic agent, an antineoplastic platinum complex, an antineoplastic camptothecin derivative, an antineoplastic tyrosine kinase inhibitor, a monoclonal antibody, an interferon, a biological response modifier, a hormonal antineoplastic agent, an antineoplastic virus agent, an angiogenesis inhibitor, a differentiation inducer, or other drug exhibiting antineoplastic activity, or a pharmaceutically acceptable salt thereof. In other embodiments, BA may be used in combination with an antimetabolite such as gemcitabine and a platinum complex such as carboplatin to treat breast tumors. In other embodiments, BA may be used in combination with a taxane, such as paclitaxel, and a platinum complex, such as carboplatin, to treat metastatic breast tumors. In other embodiments, BA may be used in combination with an antimetabolite such as gemcitabine and a platinum complex such as carboplatin to treat breast tumors. In other embodiments, BA may be used in combination with a taxane, such as paclitaxel, and a platinum complex, such as carboplatin, to treat metastatic breast tumors. In other embodiments, BA may be used in combination with an anti-angiogenic agent to treat breast tumors. In other embodiments, BA may be used in combination with a topoisomerase inhibitor, such as irinotecan or topotecan, to treat breast tumors. In other embodiments, BA may be used in combination with hormone therapy to treat breast tumors. In other embodiments, BA may be used in combination with growth factor receptor inhibitors (including but not limited to EGFR and IGF1R inhibitors) to treat breast tumors. In some embodiments, the breast cancer is triple negative metastatic breast cancer. In some embodiments, the breast tumor is negative for at least one of ER, PR, or HER 2. In some embodiments, the breast tumor is negative for at least two of ER, PR, or HER 2. In some embodiments, the breast tumor is negative for at least one of ER, PR, or HER2 and positive for at least one of ER, PR, or HER 2. In some embodiments, the breast tumor is negative for at least two of ER, PR, and HER2, e.g., ER negative, PR negative, and HER-2 positive; or ER-positive, PR-negative, and Her-2-negative; or ER negative, PR positive and Her-2 negative.

The dose of PARP inhibitor may vary depending on the age, height, weight, general health of the patient, etc. In some embodiments, the dosage of BA ranges from about 1mg/kg to about 100mg/kg, about 2mg/kg to about 50mg/kg, about 2mg/kg, about 4mg/kg, about 6mg/kg, about 8mg/kg, about 10mg/kg, about 12mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 50mg/kg, about 60mg/kg, about 75mg/kg, about 90mg/kg, about 1 to about 25mg/kg, about 2 to about 70mg/kg, about 4 to about 100mg, about 4 to about 25mg/kg, about 4 to about 20mg/kg, about 50 to about 100mg/kg, or about 25 to about 75 mg/kg. BA may be administered intravenously, e.g., by Intravenous (IV) infusion over a period of about 10 to about 300 minutes, about 30 to about 180 minutes, about 45 to about 120 minutes, or about 60 minutes (i.e., about 1 hour). In some embodiments, BA may also be administered orally. In this context, the term "about" generally means "approximately. In some embodiments, "about" means ± 10% or ± 5%.

The synthesis of BA (4-iodo-3-nitrobenzamide) is described in U.S. Pat. No. 5,464,871, which is incorporated herein by reference in its entirety. BA may be prepared at a concentration of 10mg/mL and may be packaged in a convenient form such as a 10mL vial.

BA Metabolites (BA Metabolites):

"BA" as used herein means 4-iodo-3-nitrobenzamide; "BNO" means 4-iodo-3-nitrosobenzamide; "BNHOH" means 4-iodo-3-hydroxyanilinamide.

The precursor compounds useful in the present invention have the formula (Ia)

Wherein R is₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen, at least one of the 5 substituents is always nitro, and at least one substituent adjacent to nitro is always iodo, and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs or prodrugs thereof. R₁、R₂、R₃、R₄And R₅Halogen, such as chlorine, fluorine or bromine substituents are also possible.

Preferred precursor compounds of formula Ia are:

4-iodo-3-nitrobenzamides

(BA)

Some metabolites that may be used in the present invention are compounds of formula (IIa):

wherein: (1) r₁、R₂、R₃、R₄And R₅At least one of the substituents always being a sulfur-containing substituent, and the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C) ₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituent, and R₁、R₂、R₃、R₄And R₅At least one of these 5 substituents is always iodine, and where the iodine is always present with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The groups are adjacent; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent.

The following components are preferred metabolic compounds, each represented by the following chemical structural formula:

R₆selected from hydrogen, (C)₁-C₈) Alkyl, (C)₁-C₈) Alkoxy, isoquinolinone, indole, thiazole, oxazole, oxadiazole, thiophene or phenyl

Although not limited to any particular mechanism, examples of the metabolism of MS292 by nitroreductase or glutathione conjugation mechanisms are provided below:

Mechanism of nitroreductase

BA glutathione conjugation and metabolism:

the present invention provides the use of the aforementioned nitrobenzoyl metabolizing compounds for the treatment of breast cancer, including breast cancer that is negative for one or more of ER, PR, and/or HER 2.

Nitrobenzoyl metabolizing compounds have been reported to be selectively cytotoxic to malignant cancer cells, but non-toxic to non-malignant cancer cells. See Rice et al, proc.natl.acad.sci.usa89: 7703-7707(1992), which is incorporated herein in its entirety. In one embodiment, the nitrobenzoyl metabolizing compound used in the method of the invention may exhibit more selective toxicity to tumor cells relative to non-tumor cells.

In some embodiments, the present invention provides methods of treating breast cancer that is negative for at least one of ER, PR, or HER2 by administering at least one PARP inhibitor to a subject in need of treatment. In other embodiments, the present invention provides methods of treating breast cancer by administering to a subject in need of treatment at least one PARP inhibitor and at least one anti-neoplastic agent. In some embodiments, the metabolites of the invention are administered to a patient in need of such treatment in combination with chemotherapy using at least one metabolite (e.g., a citabine, such as gemcitabine) and at least one platinum complex (e.g., carboplatin, cisplatin, etc.). In other embodiments, the metabolites of the invention are administered to a patient in need of such treatment in combination with chemotherapy with at least one taxane (e.g., paclitaxel or docetaxel) in addition to at least one platinum complex (e.g., carboplatin, cisplatin, etc.). The dosage of such metabolites may range from about 0.0004 to about 0.5mmol/kg (millimoles of metabolite per kilogram of patient body weight), which corresponds to a range of about 0.1 to about 100mg/kg BA on a molar basis. Other effective dosages of metabolites range from 0.0024 to 0.5mmol/kg and from 0.0048 to 0.25 mmol/kg. Such doses may be administered once daily, once every other day, twice weekly, biweekly, monthly or other suitable regimen. For the metabolite, administration may be substantially the same as for BA, such as oral, intravenous, intraperitoneal and the like.

Taxane (b):

taxanes are drugs obtained from the branches, needles and bark of the pacific yew tree (Taxus brevifolia). In particular, paclitaxel is derived from 10-deacetylbaccatin by known synthetic methods. Taxanes such as paclitaxel and its derivatives docetaxel show antitumor activity against various types of tumors. Taxanes interfere with normal microtubule growth functions by destabilizing the microtubule structure, thereby disrupting the ability of cells to utilize their cytoskeleton in a normal manner. In particular, taxanes bind to the beta subunit of tubulin as a microtubule building block. The resulting taxane/microtubule complex is unable to disintegrate, resulting in abnormal cell function and eventual cell death. Paclitaxel induces programmed cell death (apoptosis) in cancer cells by binding to an apoptosis-inhibiting protein named Bcl-2 (B-cell leukemia-2), thereby preventing Bcl-2 from inhibiting apoptosis. Thus, paclitaxel has been shown to be effective in treating a wide variety of cancers by down-regulating cell division by disrupting normal cytoskeletal alignment during cell division and inducing apoptosis through an anti-Bcl-2 mechanism.

The dose of paclitaxel may vary depending on the patient's height, weight, health, tumor size and development status, etc. In some embodiments, the dose of paclitaxel ranges from about 100 to about 1500mg/m ²About 200 to about 1250mg/m²About 500 to about 1000mg/m²About 700 to 800mg/m²Or about 750mg/m²Paclitaxel administered over a period of up to about 10 hours, up to about 8 hours, or up to about 6 hours. In this context, the term "about" means approximately the normal amount used; in some implementationsWithin the protocol, tolerance limits of. + -. 10% or. + -. 5% are indicated.

Examples of taxanes include, but are not limited to, docetaxel, paclitaxel, and albumin-bound paclitaxel (Abraxane).

Combination therapy (combination therapy)

In some embodiments of the invention, the methods of the invention further comprise treating breast cancer by administering to the subject a PARP inhibitor (with or without at least one anti-neoplastic agent) in combination with other anti-cancer treatments including, but not limited to: surgery, radiation therapy (such as X-rays), gene therapy, immunotherapy, DNA therapy, adjuvant therapy, neoadjuvant therapy, viral therapy, RNA therapy, or nanotherapy.

When the combination therapy further includes a non-drug therapy, the non-drug therapy can be performed at any time so long as the beneficial effect is achieved by the combined action of the therapeutic agent and the non-drug therapy. For example, in appropriate cases, beneficial effects are still obtained when the non-drug treatment is temporarily removed from the administered therapeutic agent for a significant period of time. The conjugate (conjugate) and the other pharmacologically active agent may be administered to the patient simultaneously, sequentially or in combination. It will be appreciated that when a combination of the invention is used, the compound of the invention and the other pharmacologically active agent may be in the same pharmaceutically acceptable carrier and therefore administered simultaneously. They may be in separate pharmaceutical carriers for simultaneous administration, such as conventional oral dosage forms. The term "combination" also relates to the situation wherein the compounds are provided in separate dosage forms and administered sequentially.

Radiation therapy

Radiotherapy (or radiotherapy) is the medical use of ionizing radiation as part of cancer treatment to control malignant cells. Radiation therapy can be used for curative or adjuvant cancer treatment. It is used as a palliative therapy (where no cure is possible and the goal is local disease control or symptom relief) or as a therapeutic treatment (where the treatment has a survival benefit and it can be cured). Radiation therapy is used to treat malignant tumors and can be used as a primary therapy (primary therapy). Radiation therapy is also commonly combined with surgery, chemotherapy, hormonal therapy, or some combination thereof. Most common types of cancer can be treated in some way with radiation therapy. The precise therapeutic objective (curative, adjuvant, neoadjuvant, curative or palliative) will depend on the type, location and stage of the tumor, as well as the overall health of the patient.

Radiation therapy is commonly used for cancerous tumors. The radiation area may also include draining lymph nodes if they are clinically or radiologically associated with a tumor, or if there is a risk of sub-clinical malignant spread. It is desirable to include a margin of normal tissue around the tumor to account for the daily variability and the movement of the internal tumor.

Radiation therapy works by destroying the DNA of cells. The damage is produced by direct or indirect ionization of the atoms making up the DNA strand by a photon, electron, proton, neutron, or ion beam. Direct ionization occurs due to ionization of water, forming free radicals, particularly hydroxyl radicals, which then damage DNA. In the most common form of radiation therapy, the majority of the radiation effect is through free radicals. Since cells have mechanisms for repairing DNA damage, DNA damage on both strands has proven to be the most effective technique for altering cellular properties. Since cancer cells are generally undifferentiated and stem cell-like, they replicate more and have a reduced ability to repair sublethal damage compared to most healthy differentiated cells. DNA damage is transmitted through cell division, accumulating damage into cancer cells, causing them to die or replicate more slowly. Proton radiation therapy stops precisely at the tumor by emitting protons of varying energies.

Gamma radiation is also used to treat some types of cancer, including breast cancer. In a process known as gamma knife surgery, a plurality of concentrated gamma ray beams are directed at growth to kill cancer cells. The beams from different angles concentrate the radiation on growth while minimizing damage to surrounding tissue.

In some embodiments, gamma-rays are used to treat triple negative breast cancer, as exemplified in example 9.

Gene therapy medicine

Gene therapy drugs insert gene copies into specific sites of patient cells and are capable of targeting cancer and non-cancer cells. The goal of gene therapy is to replace the altered gene with a functional gene to stimulate the patient's immune response to cancer, to make cancer cells more sensitive to chemotherapy, to place a "suicide" gene in cancer cells, or to inhibit angiogenesis. The gene may be delivered to the target cell using a virus, liposome, or other carrier or carrier. This can be done by injecting the gene-vector composition directly into the patient, or by reinfusing in vitro infected cells back into the patient. Such compositions are suitable for use in the present invention.

Adjuvant therapy (adjuvant therapy)

Adjuvant therapy is treatment given after the primary treatment regimen to improve the chances of a cure. Adjuvant therapy may include chemotherapy, radiation therapy, hormonal therapy, or biological therapy.

Since the primary purpose of adjuvant therapy is to kill any cancer cells that may have spread, the therapy is generally systemic (using substances that are transported through the blood, reaching and affecting the cancer cells throughout the body). Adjuvant treatment of breast cancer involves chemotherapy or hormone therapy, either alone or in combination:

Adjuvant chemotherapy utilizes drugs to kill cancer cells. Studies have shown that the use of chemotherapy as an adjunct treatment to early stage breast cancer helps to prevent the primary cancer recurrence. Adjuvant chemotherapy is often a combination of anticancer drugs that has proven to be more effective than a single anticancer drug.

Adjuvant hormone therapy removes the female hormone estrogen in cancer cells, which is required for the proliferation of some breast cancer cells. In the most frequent case, adjuvant hormone therapy is treatment with tamoxifen drugs. Studies have shown that tamoxifen, when used as an adjuvant treatment for early breast cancer, helps prevent the recurrence of the original cancer and also helps prevent the development of new cancer in the other breast.

Ovaries are the main source of estrogen before menopause. For pre-menopausal women with breast cancer, adjuvant hormone therapy may involve the use of tamoxifen to remove the estrogen from the tumor cells. Drugs that inhibit estrogen production by the ovaries are under investigation. Alternatively, surgery may be performed to remove the ovaries.

Radiation therapy is sometimes used as a topical adjuvant therapy. When radiation therapy is performed before or after a mastectomy, it is considered an adjuvant therapy. The goal of this treatment is to destroy breast cancer cells that have spread to adjacent parts of the body, such as the chest wall or lymph nodes. When radiation therapy is performed after breast conservation surgery, it is an integral part of the primary treatment, rather than an adjuvant treatment.

New adjuvant therapy (neoadjuvant therapy)

Neoadjuvant therapy refers to therapy provided prior to the primary therapy. Examples of new adjunctive therapies include chemotherapy, radiation therapy and hormone therapy. In the process of treating breast cancer, the new adjuvant therapy enables the breast conservation operation to be adopted for patients with large-area breast cancer.

Oncolytic viral therapy (oncocytic viral therapy)

Viral therapy for cancer uses a class of viruses known as oncolytic viruses. Oncolytic viruses are viruses that are capable of infecting and lysing cancer cells while being harmless to normal cells, making them potentially useful in cancer therapy. Replication of oncolytic viruses promotes destruction of tumor cells, and also produces dose amplification at the tumor site. They can also be used as vectors for anticancer genes, delivering them specifically to the tumor site.

The 2 main approaches to generating tumor selectivity are: transduction targeted and non-transduction targeted. Transduction targeting involves altering the specificity of viral capsid proteins, thus increasing target cell entry while decreasing non-target cell entry. Non-transduction targeting involves altering the viral genome such that it is only able to replicate in cancer cells. This can be achieved by transcriptional targeting, where genes essential for viral replication are placed under the control of tumor specific promoters, or by attenuation (attenuation), which involves the introduction of deletions into the viral genome to eliminate functions not essential in cancer cells but essential in normal cells. There are other less well-known methods.

Chen et al (2001) used CV706 (prostate-specific adenovirus) in combination with radiation therapy on prostate cancer in mice. The combination treatment resulted in a synergistic increase in cell death and a significant increase in the scale of viral release (the number of viral particles released from each cell lysis).

ONYX-015 has been tested in combination with chemotherapy. Combination therapy provided better response than each individual treatment, but the results have not been fully documented. ONYX-015 has been shown to hold promise in combination with radiation therapy.

Intravenous administration of viral agents can be particularly effective against metastatic cancers that are particularly difficult to treat with routine therapy. However, blood-borne viruses may be inactivated by antibodies and rapidly cleared from the bloodstream by, for example, kupffer cells (the very active phagocytic cells in the liver, which are responsible for adenovirus clearance). Evading the immune system until the tumor is destroyed may be the biggest obstacle to the success of oncolytic virus therapy. To date, none of the techniques used to evade the immune system have been entirely satisfactory. It is in conjunction with conventional cancer treatments that oncolytic viral formulations show the most promise, as combination therapy works synergistically without significant adverse effects.

The specificity and adaptability of oncolytic viruses means that they have the potential to treat a wide range of cancers, including breast cancer, with minimal side effects. Oncolytic viruses have the potential to address the problem of selective killing of cancer cells.

Nano-therapy

Nano-sized particles have novel optical, electronic, and structural properties that are not available in single molecules or bulk solids. When linked to a tumor targeting moiety such as a tumor specific ligand or monoclonal antibody, these nanoparticles can be used to target cancer specific receptors, tumor antigens (biomarkers) and tumor vasculature with high affinity and accuracy. Formulations and methods of preparation for cancer nanotherapeutics are described in U.S. patent 7179484, and articles m.n.khalid, p.simard, d.hoarau, a.dragomir, j.leroux, Long circulating Poly (Ethylene Glycol) purified lipin and delivery drug to Solid turbines, Pharmaceutical Research, 23(4), 2006, all of which are incorporated herein in their entirety by reference.

RNA therapy

RNA including but not limited to siRNA, shRNA or microrna can be used to modulate gene expression and treat cancer. A double-stranded oligonucleotide is formed by assembling 2 different oligonucleotide sequences, wherein the oligonucleotide sequence of one strand is complementary to the oligonucleotide sequence of the second strand; the double-stranded oligonucleotide is typically assembled from 2 separate oligonucleotides (e.g., siRNA), or from a single molecule that folds upon itself to form a double-stranded structure (e.g., shRNA or small hairpin RNA). These double-stranded oligonucleotides known in the art all have the common feature that the double strands each have a different nucleotide sequence, wherein only one nucleotide sequence region (guide sequence or antisense sequence) has complementarity to the target nucleic acid sequence and the other strand (sense sequence) comprises a nucleotide sequence that is homologous to the target nucleic acid sequence.

Micrornas (mirnas) are single-stranded RNA molecules of about 21-23 nucleotides in length that regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into proteins (non-coding RNA); whereas they are processed from a primary transcript called pri-miRNA into a short stem-loop structure called pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to down-regulate gene expression.

Some RNA inhibitors can be used to inhibit the expression or translation of messenger RNA ("mRNA") associated with a cancer phenotype. Examples of such agents suitable for use herein include, but are not limited to, short interfering RNA ("siRNA"), ribozymes, and antisense oligonucleotides. Specific examples of RNA inhibitors suitable for use herein include, but are not limited to, Cand5, Sima-027, Fumivir, and angiozyme.

Small molecule enzyme inhibitors

Some small molecule therapeutic drugs are capable of targeting tyrosine kinase activity or downstream signaling signals of some cellular receptors, such as epidermal growth factor receptor ("EGFR") or vascular endothelial growth factor receptor ("VEGFR"). This targeting by small molecule therapy can produce an anti-cancer effect. Examples of such agents suitable for use herein include, but are not limited to, imatinib, gefitinib, erlotinib, lapatinib, canertinib, ZD6474, sorafenib (BAY 43-9006), ERB-569, and analogs and derivatives thereof.

Anti-metastatic agent

The process by which cancer cells spread from the site of the original tumor to other sites in the body is called metastasis. Some drugs have anti-metastatic properties and are designed to inhibit cancer cell spread. Examples of such agents suitable for use herein include, but are not limited to, marimastat, bevacizumab, trastuzumab, rituximab, erlotinib, MMI-166, GRN163L, huntingpeptides (hunter-killers), tissue inhibitors of the metalloprotease class (TIMPs), their analogs, derivatives, and variants.

Chemical preventive agent

Some drugs may be used to prevent the initial occurrence of cancer, or to prevent relapse or metastasis. Administration of these chemopreventive agents in combination with the disclosed edenside-NSAID conjugates can be used as a treatment and prevention of recurrence of cancer. Examples of chemopreventive drugs suitable for use herein include, but are not limited to, tamoxifen, raloxifene, tibolone, bisphosphonates, ibandronate, estrogen receptor modulators, aromatase inhibitors (letrozole, anastrozole), luteinizing hormone-releasing hormone agonists, goserelin, vitamin A, retinal, retinoic acid, fenretinide, 9-cis-retinoid acid, 13-cis-retinoid, all-trans-retinoic acid, isotretinoin, tretinoin, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, cyclooxygenase inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs), aspirin, ibuprofen, celecoxib, polyphenols, dithioE, green tea extracts, polyphenols, glucaric acid, interferon-alpha, anethole pentalene (anethole dithione), Zinc, pyridoxine, finasteride, doxazosin, selenium, indole-3-carbaldehyde, α -difluoromethylornithine, a carotenoid, β -carotene, lycopene, an antioxidant, coenzyme Q10, a flavonoid, quercetin, turmeric, catechin, epigallocatechin gallate, N-acetyl cysteine, indole-3-methanol, inositol hexaphosphate, isoflavone, gluconic acid, rosemary, soy, sabal and calcium. Other examples of chemopreventive drugs suitable for use in the present invention are cancer vaccines. These can be prepared by immunizing a patient with all or part of the cancer cell types targeted by the vaccine plating process.

The clinical curative effect is as follows:

staging of breast cancer:

stage 0 is used to describe non-invasive breast cancers such as DCIS and LCIS. At stage 0, there is no evidence that cancerous or non-cancerous abnormal cells have broken through the portion of the breast from which they originated, or have spread or invaded adjacent normal tissue.

Stage I describes invasive breast cancer (cancer cells breaking through or invading adjacent normal tissue) with tumor sizes up to 2 cm and no involvement of lymph nodes.

Stage II is divided into two subclasses, IIA and IIB. Stage IIA describes an invasive breast cancer in which no tumor can be found in the breast, but cancer cells are found in the axillary lymph nodes (axillary lymph nodes), or the tumor is 2 cm or less in size and has spread to the axillary lymph nodes, or the tumor is greater than 2 cm but not greater than 5 cm and has not spread to the axillary lymph nodes. Stage IIB describes an invasive breast cancer in which the tumor is greater than 2 cm but not greater than 5 cm and has spread to the axillary lymph nodes, or greater than 5 cm but has not spread to the axillary lymph nodes.

Stage III is divided into three subclasses IIIA, IIIB and IIIC. Stage IIIA describes an invasive breast cancer in which no tumor is found in the breast. Cancer is found in the axillary lymph nodes, which adhere to clumps or to other structures, or the cancer may have spread to the lymph nodes near the sternum, or the tumor is 5 cm or less and has spread to the axillary lymph nodes, which adhere to clumps or to other structures, or the tumor is greater than 5 cm and has spread to the axillary lymph nodes, which adhere to clumps or to other structures. Stage IIIB describes invasive breast cancer in which the tumor may be of any size and has spread to the chest wall and/or breast epidermis and may have spread to axillary lymph nodes, lymph nodes adhering to clumps or other structures, or the cancer may have spread to lymph nodes near the sternum. Stage IIIC describes an invasive breast cancer in which the breast may not have evidence of cancer, or if there is a tumor, it may be of any size and may have spread to the chest wall and/or breast epidermis, or cancer cells have spread to lymph nodes above or below the clavicle, and cancer may have spread to axillary lymph nodes or to lymph nodes near the sternum.

Clinical efficacy can be measured by any method known in the art. In some embodiments, the clinical treatment efficacy described herein can be determined by measuring the Clinical Benefit Rate (CBR). Clinical benefit rate is measured by determining the sum of the percentage of patients in Complete Remission (CR), the percentage of patients in Partial Remission (PR), and the percentage of patients with Stable Disease (SD) at a time at least 6 months after the end of treatment. The abbreviation of this formula is CBR ≧ CR + PR + SD ≧ 6 months. The CBR for treatment with gemcitabine and carboplatin combination was 45%.Thus, triple combination therapy with antimetabolites, platinum complexes, and PARP inhibitors (e.g., gemcitabine, carboplatin, and BA; CBR_GCB) Can be combined with gemcitabine and Carboplatin (CBR)_GC) In comparison to the dual combination therapy. In some embodiments, CBR_GCBAt least about 60%. In some embodiments, the CBR is at least about 30%, at least about 40%, or at least about 50%. CBR with paclitaxel and carboplatin combination treatment was 45%. Thus, CBR (e.g., paclitaxel, carboplatin, and BA; CBR) treated with a triple combination of taxane, platinum complex, and PARP inhibitor_GCB) CBR (CBR) for treatment with paclitaxel and carboplatin double combinations_GC) And (6) comparing. In some embodiments, CBR _GCBAt least about 60%.

In some embodiments, the CBR is at least about 30%, at least about 40%, or at least about 50%. In some embodiments, the therapeutic effect comprises a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, stable disease, or a pathologically complete response.

Neoadjuvant chemotherapy is currently widely used to treat large areas of breast cancer that are localized or likely to undergo surgery. Randomized trials have shown that neoadjuvant chemotherapy reduces the need for mastectomy (Powles et al, 1995; Fisher et al, 1998), with overall survival rates similar to adjuvant chemotherapy (Fisher et al, 1998). Survival in women who achieved no evidence of residual tumor histology (i.e., a pathological complete response (pCR)) after chemotherapy at surgery was significantly improved (Bonadonna et al, 1998; Fisher et al, 1998; Kuerer et al, 1999), and pCR was often used as an early surrogate indicator of therapeutic efficacy. However, there is no standard grading of the pathological response of breast tumors to neoadjuvant chemotherapy and many different grading systems have been proposed (Chevallier et al, 1993; Sataloff et al, 1995; Fisher et al, 1997; Honkoop et al, 1998; Kuerer et al, 1998; Ogston et al, 2003). Most, but not all, of these staging regimens include both no residual disease in any form and residual in situ Ductal Carcinoma (DCIS) but no invasive disease in the definition of pCR.

In some embodiments disclosed herein, the method comprises predetermining a cancer treatable with a PARP modulator. Some such methods include: the method comprises the steps of identifying a level of PARP in a breast cancer sample of the patient, determining whether the level of PARP expression in the sample is above a predetermined value, and if the level of PARP expression is above the predetermined value, treating the patient with a combination of a taxane (e.g., paclitaxel), a platinum complex (e.g., carboplatin) and a PARP inhibitor such as BA. In other embodiments, the methods comprise identifying a level of PARP in a breast cancer sample of the patient, determining whether the expression level of PARP in the sample is above a predetermined value, and if the expression of PARP is above the predetermined value, treating the patient with a PARP inhibitor, such as BA. In other embodiments, the method comprises predetermining cancer treatment with a PARP modulator. Some such methods include identifying a level of PARP in a breast cancer sample of the patient, determining whether the expression level of PARP in the sample is above a predetermined value, and if the expression of PARP is above the predetermined value, treating the patient with an anti-metabolite (e.g., gemcitabine), platinum complex (e.g., carboplatin), and PARP inhibitor, e.g., BA combination.

Breast tumors develop in women who inherit a deficiency in either the BRCA1 or BRCA2 gene because the tumor cells lose specific mechanisms for repairing damaged DNA. BRCA1 and BRCA2 are important for repair of DNA double strand breaks by homologous recombination, and mutations in these genes predispose to breast and other cancers. PARP is involved in the repair of base excision, a repair pathway for single strand breaks in DNA. BRCA1 or BRCA2 incompetence sensitizes cells to inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis (Jones C, Plummer ER. PARPinhibitors and cancer therapy-results and potential applications. Br JRadiol.2008 Oct; 81 Spec No 1: S2-5; drive Y, Calvert H. the potential of PARPinhibitors in genetic breakdown and over care cans. Ann Y Acad Sci.2008 Sep; 1138: 136-45; Farmer H et al. Targeting the DNA repair in BRCA mutated as a thermal adaptation. Nature.Apegr 14; 7035-917).

Patients deficient in the BRCA gene may have upregulated PARP levels. Upregulation of PARP can be an indicator of defective DNA repair pathways and unidentified BRCA-like genetic defects. Evaluation of PARP gene expression and impaired DNA repair, particularly defective homologous recombination DNA repair, can be used as an indicator of tumor sensitivity to PARP inhibitors. Thus, in some embodiments, breast cancer treatment can be enhanced by identifying early onset of cancer in patients deficient in BRCA and homologous recombinant DNA repair by measuring PARP levels, as well as by determining HR and/or HER2 status of the cancer. If PARP levels are upregulated, patients with defects in BRCA and homologous recombination DNA repair that can be treated with PARP inhibitors can be identified. In addition, patients deficient in these homologous recombination DNA repair are treatable with PARP inhibitors.

In some embodiments, a sample is taken from a patient with a breast lesion or hyperplasia suspected of being cancerous. While such a sample may be any biological tissue, in most cases the sample will be a portion of a suspected breast lesion, whether collected by way of a minimally invasive biopsy or therapeutic procedure (e.g., lumpectomy, mastectomy, partial or modified mastectomy or radical mastectomy, hysterectomy, or oophorectomy). Such samples may also include all or a portion of one or more lymph nodes extracted during a treatment procedure. PARP expression can then be analyzed. In some embodiments, if PARP expression is above a predetermined level (e.g., upregulated relative to normal tissue), the patient may be treated with a PARP inhibitor in combination with an antimetabolite and a platinum agent. In other embodiments, if PARP expression is above a predetermined level (e.g., upregulated relative to normal tissue), the patient may be treated with a PARP inhibitor, including a PARP inhibitor such as BA. Thus, it should be understood that while the embodiments described herein are directed to treatment of triple negative metastatic breast cancer, in some embodiments, the breast cancer does not necessarily have these characteristics, so long as the PARP upregulation threshold is reached.

In some embodiments, the tumor deficient in homologous recombination is identified by assessing the expression level of PARP. If an upregulation of PARP is observed, such tumors can be treated with PARP inhibitors. Another embodiment is a method of treating a cancer deficient in homologous recombination comprising assessing the expression level of PARP and, if overexpression is observed, treating the cancer with a PARP inhibitor.

Sample collection, preparation and separation

Biological samples can be collected from a variety of sources from a patient, including bodily fluid samples or tissue samples. The samples collected can be human normal and tumor samples, nipple aspirates (nipple aspirants). Samples can be collected repeatedly from an individual over a period of time (e.g., about 1 time a day, 1 time a week, 1 time a month, 1 time a half year, or 1 time a year). A variety of samples obtained from an individual over a period of time can be used to verify the results of early detection and/or to identify changes in biological models due to, for example, disease progression, drug treatment, and the like.

Sample preparation and isolation may involve any method, depending on the type of sample collected and/or the analysis of PARP. These methods include, by way of example only, concentration, dilution, pH adjustment, removal of highly abundant polypeptides (e.g., albumin, gamma globulin, transferrin, and the like), addition of preservatives and calibrators, addition of protease inhibitors, addition of denaturants, desalting of the sample, concentration of sample proteins, extraction and purification of lipids.

Sample preparation can also isolate molecules that complex non-covalently with other proteins (e.g., carrier proteins). The method may isolate those molecules that bind to a particular carrier protein (e.g., albumin), or use a more general method, such as releasing the bound molecules from all carrier proteins by protein denaturation (e.g., using an acid), and then removing the carrier protein.

Removal of unwanted proteins (e.g., highly abundant, non-beneficial, or undetectable proteins) from a sample can be performed using high affinity reagents, high molecular weight filtration membranes, ultracentrifugation, and/or electrodialysis. High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to highly abundant proteins. Sample preparation may also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques. Molecular weight filtration membranes include membranes that separate molecules by size and molecular weight. The membrane may also be used for reverse osmosis, nanofiltration, ultrafiltration and microfiltration.

Ultracentrifugation is a method of removing unwanted polypeptides from a sample. Ultracentrifugation centrifuges samples at about 15,000-60,000rpm while monitoring particle deposition (or lack thereof) with an optical system. Electrodialysis is a process using an electrically or semi-permeable membrane, in which ions are transferred from one solution to another through the semi-permeable membrane under the influence of a potential gradient. Electrodialysis is useful for concentrating, removing, or separating electrolytes because the membranes used in electrodialysis can have the ability to selectively transfer ions of positive or negative charge, repelling ions of opposite charge, and allowing species to pass through the semi-permeable membrane depending on size and charge.

Separation and purification in the present invention may include any method known in the art, such as capillary electrophoresis (e.g., in a capillary or on a chip) or chromatography (e.g., in a capillary, in a column, or on a chip). Electrophoresis is a method that can be used to separate ionic molecules under the influence of an electric field. Electrophoresis may be performed in a gel, in a capillary, or in a microchannel of a chip. Examples of gels for electrophoresis include starch, acrylamide, polyethylene oxide, agarose, or combinations thereof. The gel may be modified by the following method: self-crosslinking, addition of detergents or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or of substrates (zymography) and introduction of a pH gradient. Examples of capillaries for electrophoresis include capillaries interfaced with electrospray.

Capillary Electrophoresis (CE) is preferred for separating complex hydrophilic molecules and highly charged solutes. CE technology is also applicable to microfluidic chips. Depending on the type of capillary and buffer used, CE can be further divided into various separation techniques such as Capillary Zone Electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (ctipp), and Capillary Electrochromatography (CEC). Embodiments of coupling the CE technique with electrospray ionization include the use of a volatile solution, e.g., an aqueous mixture comprising a volatile acid and/or base and an organic substance (such as an alcohol or acetonitrile).

Capillary isotachophoresis (cITP) is a technique in which analytes move through a capillary at a constant velocity but are separated due to differences in their respective mobilities. Capillary Zone Electrophoresis (CZE), also known as Free Solution CE (FSCE), is based on the differences in electrophoretic mobility of substances (determined by the charge on the molecule) and the friction encountered by the molecule during movement (which is generally proportional to the size of the molecule). Capillary isoelectric focusing (CIEF) allows weakly ionizable amphipathic molecules to be separated by electrophoresis in a pH gradient. CEC is a hybrid technology between traditional High Performance Liquid Chromatography (HPLC) and CE.

Separation and purification techniques used in the present invention include any chromatography known in the art. Chromatography may be based on differences in adsorption and elution of some analytes, or the partitioning of analytes between a mobile phase and a stationary phase. Different examples of chromatography include, but are not limited to, Liquid Chromatography (LC), Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), and the like.

Identification of PARP levels

Poly (ADP-ribose) polymerase (PARP) is also known as poly (ADP-ribose) synthetase and poly ADP-ribose transferase. PARP catalyzes the formation of mono-and poly (ADP-ribose) polymers, which can attach to cellular proteins (as well as themselves) thereby altering the activity of these proteins. This enzyme plays a role in transcriptional regulation, cell proliferation, and chromatin remodeling (for a review, see: D.D' animals et al, "Poly (ADP-differentiation interactions in thermal regulation of nuclear functions", biochem. J.342249-268 (1999)).

PARP comprises an N-terminal DNA binding domain, a self-modifying domain and a C-terminal catalytic domain and a variety of cellular proteins interact with PARP. The N-terminal DNA binding domain comprises two zinc finger motifs. Transcription enhancer factor-1 (TEF-1), retinoid X receptor alpha, DNA polymerase alpha, X-ray repair cross-complementing factor-1 (XRCC1), and PARP itself interact with PARP within this binding domain. The self-modifying domain comprises the BRCT motif, a protein-protein interaction module. This motif was originally found at the C-terminus of BRCA1 (breast cancer susceptibility protein 1) and is present in a variety of proteins involved in DNA repair, recombination and cell cycle checkpoint control. Octameric transcription factor-1 (Oct-1), Yin Yang (YY)1 and ubiquitin 9(ubc9) containing the POU homeodomain are capable of interacting with the BRCT motif in PARP.

More than 15 members of the PARP gene family are present in the mammalian genome. PARP family proteins and poly (ADP-ribose) glycohydrolases (PARGs), which degrade poly (ADP-ribose) to ADP-ribose, can be involved in a variety of cellular regulatory functions, including DNA damage response and transcriptional regulation, and are associated with carcinogenesis and cancer biology in many respects.

Several PARP family proteins have been identified. Tankyrase has been found to be an interacting protein of telomere regulatory factor 1(TRF-1) and is involved in telomere regulation. Vault PARP (Vault PARP) (VPARP) is a component of the Vault complex (Vault complex) which acts as a nuclear-cytoplasmic carrier. PARP-2, PARP-3 and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin inducible PARP (TiPARP) have been identified. Thus, poly (ADP-ribose) metabolism can be associated with a variety of cellular regulatory functions.

One member of this gene family is PARP-1. The PARP-1 gene product is expressed at high levels in the nucleus and is activated depending on DNA damage. Without being bound by any theory, it is believed that PARP-1 binds to a single or double stranded break of DNA via the amino terminal DNA binding domain. This binding activates the carboxy-terminal catalytic domain and results in the formation of a polymer of ADP-ribose on the target molecule. Due to the centrally located self-modifying domain, PARP-1 itself is the target for poly ADP-ribosylation. Nuclear glycosylation of PARP-1 results in the separation of the PARP-1 molecule from the DNA. The entire process of binding, ribosylation and isolation occurs very rapidly. It has been proposed that transient binding of PARP-1 to the site of DNA damage results in the recruitment of the DNA repair system (machinery), or may act to inhibit recombination for a sufficient period of time to recruit the repair system.

The ADP-ribose source of the PARP reaction is Nicotinamide Adenine Dinucleotide (NAD). NAD is synthesized in cells from the cellular ATP pool, and thus high levels of activation of PARP activity can rapidly lead to depletion of the cellular energy pool. Induction of PARP activity has been shown to lead to cell death, which is associated with depletion of cellular NAD and ATP pools. PARP activity is induced in many instances of oxidative stress or during inflammation. For example, during reperfusion of ischemic tissue, reactive nitric oxide is produced, and nitric oxide causes the production of other reactive oxygen species, including hydrogen peroxide, peroxynitrate, and hydroxyl radicals. These latter substances can directly damage DNA, and the resulting damage induces activation of PARP activity. In general, it appears that sufficient activation of PARP activity occurs such that the cellular energy pool is depleted and the cells die. A similar mechanism is thought to occur during inflammation when endothelial cells and pro-inflammatory cells synthesize nitric oxide, which produces oxidative DNA damage in surrounding cells and subsequently activates PARP activity. Cell death resulting from PARP activation is believed to be a major contributor to the extent of tissue injury (due to ischemia-reperfusion or inflammation).

In some embodiments, the level of PARP in the sample from the patient is compared to a predetermined standard sample. The sample from the patient is typically from a diseased tissue, such as a cancer cell or tissue. The standard sample may be from the same patient or from a different subject. The standard sample is typically a normal, non-diseased sample. However, in some embodiments, such as for staging a disease or for assessing treatment efficacy, the standard sample is from a diseased tissue. The standard sample may be a combination of samples from several different subjects. In some embodiments, the level of PARP from the patient is compared to a predetermined level. The predetermined level is typically from a normal sample. As described herein, a "predetermined level of PARP" can be a level of PARP for use, for example, in assessing a patient that can be selected for treatment, assessing a response to treatment with a PARP inhibitor, assessing a response to treatment with a combination of a PARP inhibitor and a second therapeutic agent, and/or diagnosing cancer, inflammation, pain, and/or a related condition in a patient. The predetermined level of PARP may be determined in a population of patients with or without cancer. The predetermined level of PARP may be a single value, equally available for each patient, or the predetermined level of PARP may vary depending on the particular subpopulation of patients. For example, a male may have a different predetermined PARP level than a female; non-smokers may have a different predetermined level of PARP than smokers. The age, weight, and height of the patient may affect the predetermined PARP level of the individual. Also, the predetermined PARP level may be a level determined individually for each patient. The predetermined PARP level may be any suitable criterion. For example, the predetermined level of PARP may be from the same or different person as the person undergoing the patient selection assessment. In one embodiment, the predetermined level of PARP may be from a previous evaluation of the same patient. In this way, the progress of the patient selection may be monitored over time. Moreover, the criteria may be derived from an evaluation of one or more other persons (e.g., a selected group of persons). In this scenario, the degree of selection of the person whose selectivity is being evaluated may be compared to suitable other persons having similar conditions as the person of interest, such as those suffering from similar or the same conditions.

In some embodiments of the invention, the change in PARP from the predetermined level is about 0.5 fold, about 1.0 fold, about 1.5 fold, about 2.0 fold, 2.5 fold, about 3.0 fold, 3.5 fold, about 4.0 fold, about 4.5 fold, about 5.0 fold. In some embodiments, the fold change is about less than 1, about less than 5, about less than 10, about less than 20, about less than 30, about less than 40, or about less than 50. In other embodiments, the change in PARP level relative to the predetermined level is greater than about 1, greater than about 5, greater than about 10, greater than about 20, greater than about 30, greater than about 40, or greater than about 50. Preferred fold changes relative to the predetermined level are about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, and about 3.0.

Analysis of PARP levels in patients is particularly valuable and informative because it allows physicians to more effectively select the best treatment based on up-or down-regulated levels of PARP, as well as to use more robust treatments and treatment regimens. More aggressive treatments, or a combination of treatments and treatment regimens, can offset the poor prognosis and overall survival of the patient. With this information, medical practitioners can choose to provide some type of treatment, such as treatment with PARP inhibitors and/or more potent therapies.

In detecting PARP levels in a patient, over time (which may be days, weeks, months, and in some cases years, or intervals thereof), a sample of the patient's bodily fluid (e.g., serum or plasma) may be collected at intervals (the intervals being determined by a practitioner (e.g., a physician or clinician)) to determine the level of PARP and compared to the level of normal individuals over the course of treatment. For example, patient samples may be collected and monitored monthly, every 2 months, or a combination of 1, 2, or 3 month intervals in accordance with the present invention. Furthermore, during monitoring, the PARP levels of a patient obtained over time can be conveniently compared to each other and to the PARP values of normal controls, thereby providing the patient's own PARP values as an internal or personal control for long-term PARP monitoring.

PARP analysis technique

Analysis of PARP may include analysis of PARP gene expression, including analysis of DNA, RNA, analysis of PARP levels and/or analysis of PARP activity, including analysis of mono and poly ADP ribosylation levels. Without limiting the scope of the present invention, any technique known in the art can be used for the analysis of PARP and they are within the scope of the present invention. Some examples of such detection techniques are given below, but these examples in no way limit the wide variety of detection techniques that can be used in the present invention.

Gene expression profiling: gene expression analysis methods include polynucleotide hybridization analysis based methods, polynucleotide sequencing based polynucleotide nucleotide methods, and polynucleotide nucleotide and proteomics based methods. The most commonly used Methods for quantifying mRNA expression in samples known in the art include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106: 247-283 (1999)); RNase protection assays (Hod, Biotechniques 13: 852-854(1992)) and PCR-based methods such as reverse transcription-polymerase chain reaction (RT-PCR) technology (Weis et al, Trends in Genetics 8: 263-264 (1992)). Alternatively, various antibodies that recognize specific duplexes including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes may be used. Representative sequencing-based gene expression analysis methods include gene expression Sequencing (SAGE), Massively Parallel Signal Sequencing (MPSS) -based gene expression analysis, comparative genomic hybridization techniques (CGH), chromatin immunoprecipitation (ChIP), Single Nucleotide Polymorphisms (SNPs) and SNP arrays, Fluorescence In Situ Hybridization (FISH), protein binding arrays and DNA microarrays (also commonly referred to as gene or genomic chips, DNA chips, or gene microarrays), and RNA microarrays.

Reverse transcriptase PCR (RT-PCR): one of the most sensitive and flexible methods of gene expression analysis based on quantitative PCR technology is RT-PCR, which can be used to compare mRNA levels in different sample populations, normal tissues and tumor tissues (treated or not) to characterize gene expression patterns, to distinguish closely related mrnas, and to analyze RNA structure.

The first step is to isolate mRNA from the target sample. For example, the starting material can typically be total RNA isolated from a human tumor or tumor cell line, and the corresponding normal tissue or cell line, respectively. Thus, RNA can be isolated from a wide variety of normal and diseased cells and tissues, such as tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, and the like, or tumor cell lines. If the mRNA source is a primary tumor, mRNA can be extracted from frozen or sequestered fixed tissue, such as paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples. General methods for extracting mRNA are well known in the art and are disclosed in standard textbooks of Molecular Biology, including Ausubel et al, Current Protocols of Molecular Biology, John Wiley and sons (1997).

In particular, RNA isolation can be performed using the purification kit, the buffer kit and the protease provided by the manufacturer according to the manufacturer's instructions. For example, RNA prepared from tumors can be isolated by cesium chloride density gradient centrifugation. Since RNA cannot be used as a template for PCR, the first step in gene expression analysis using RT-PCR is to reverse transcribe the RNA template into cDNA, which is then exponentially amplified in a PCR reaction. The two most commonly used reverse transcriptases are the avilo myeloblastic leukemia virus reverse transcriptase (AMV-RT) and the Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is usually primed with specific primers, random hexamers, or oligo dT primers, depending on the context and goal of the expression assay. The derived cDNA can then be used as a template in subsequent PCR reactions.

In order to minimize the effect of errors and variations between different samples, RT-PCR is usually performed using internal standards. The ideal internal standard is expressed at a constant level between different tissues and is not affected by experimental treatments. The most commonly used RNAs for gene expression pattern normalization are the mRNA for the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β -actin.

A recent variation of RT-PCR technology is real-time quantitative PCR, which measures PCR product accumulation by means of a double-labeled fluorescent probe. The real-time quantitative PCR technique is compatible with both quantitative competitive PCR and quantitative comparative PCR. In quantitative competitive PCR, the internal competitor for each target sequence was used for normalization. Quantitative comparative PCR uses either a normalization gene contained in the sample, or a housekeeping gene of RT-PCR.

Fluorescence microscopy: some embodiments of the invention include fluorescence microscopy for the analysis of PARP. Fluorescence microscopy enables the observed structural molecular composition to be identified with highly chemically specific fluorescently labeled probes such as antibodies. This can be achieved by conjugating the fluorophore directly to the protein and introducing it back into the cell. Fluorescent analogs can function like the native protein and thus can be used to reveal the intracellular distribution and expression of the protein. Along with NMR, infrared spectroscopy, circular dichroism and other techniques, the intrinsic fluorescence decay of proteins and their associated fluorescence anisotropy observations, collision quenching and resonance energy transfer are all techniques for protein detection. The natural fluorescent protein can be used as a fluorescent probe. Aequorin produces a naturally occurring fluorescent protein known as Green Fluorescent Protein (GFP). The fusion of these fluorescent probes to the target protein enables visualization by fluorescence microscopy and quantification by flow cytometry.

By way of example only, some probes are labels such as fluorescein and its derivatives, carboxyfluorescein, rhodamine and its derivatives, atropic labels, fluorescent red, fluorescent orange: cy3/cy5 alternatives, long-lived lanthanide complexes, long wavelength (up to 800nm) labels, DY anthocyanidin labels, and phycobiliproteins. By way of example only, some probes are conjugates, such as isothiocyanate conjugates, biotin protein conjugates, and biotin conjugates. By way of example only, some probes are enzyme substrates, such as fluorescent and chromogenic substrates. By way of example only, some probes are fluorescent dyes, such as FITC (green fluorescence, excitation/emission wavelength of 506/529nm), rhodamine B (orange fluorescence, excitation/emission wavelength of 560/584nm), and nile blue a (red fluorescence, excitation/emission of 636/686 nm). Fluorescent nanoparticles can be used in various types of immunoassays. Fluorescent nanoparticles are based on different materials such as polyacrylonitrile, polystyrene, etc. Fluorescent molecular rotors are microenvironment-limited sensors that fluoresce when their rotation is limited. Several examples of molecular limitations include increasing dye (aggregation), binding to antibodies, or limitation by actin polymerization. IEF (isoelectric focusing) is an analytical tool for the separation of ampholytes, mainly proteins. An advantage of using fluorescent IEF-labeled IEF-gel electrophoresis is that it is possible to directly observe the formation of the gradient. Fluorescent IEF-labels can also be detected by UV absorption at 280nm (20 ℃).

Libraries of peptides can be synthesized on solid supports and the solid supports individually selected for subsequent staining by use of a colored acceptor. If the receptors do not show any color, their bound antibodies can be stained. The method can be used not only for protein receptors, but also for screening binding ligands for synthetic artificial receptors and for screening novel metal binding ligands. Automated methods of HTS and FACS (fluorescence activated cell sorter) can also be used. The FACS machine first passes the cells through a capillary tube and separates the cells by detecting their fluorescence intensity.

And (3) immunoassay: some embodiments of the invention include immunoassays for the analysis of PARP. In western blot assays, such as electrophoretic separation of proteins, individual proteins can be identified by their antibodies. The immunoassay may be a competitive binding immunoassay in which the analyte competes with the labeled antigen for a limited number of antibody molecules (e.g., radioimmunoassay, EMIT). The immunoassay may also be noncompetitive, in which antibodies are present in excess and labeled. As the analyte-antigen complex increases, the amount of labeled antibody-antigen complex may also increase (e.g., ELISA). If they are produced by injecting an antigen into an experimental animal, or monoclonal antibodies, if they are produced by cell fusion and cell culture techniques, the antibodies may be polyclonal antibodies. In immunoassays, antibodies can be used as reagents specific for analyte antigens.

Without limiting the scope and content of the invention, some types of immunoassays are, by way of example only, RIA (radioimmunoassay), enzyme immunoassays such as ELISA (enzyme-linked immunosorbent assay), EMIT (enzyme-enhanced immunoassay), Microparticle Enzyme Immunoassay (MEIA), LIA (luminescence immunoassay) and FIA (fluorescence immunoassay). These techniques can be used to detect biological substances in nasal samples. The antibody can be used as a primary antibody or a secondary antibody. They may be labelled with a radioisotope (e.g. 125I), a fluorescent dye (e.g. FITC) or an enzyme that catalyses a fluorescent or luminescent reaction (e.g. HRP or AP).

Biotin or vitamin H is a coenzyme that inherits specific affinity for avidin and streptavidin. This interaction makes biotinylated peptides a useful tool for qualitative and quantitative testing in various biotechnological assays. In order to improve biotin/streptavidin recognition by minimizing steric hindrance, it may be necessary to enlarge the distance between biotin and the peptide itself. This can be achieved by coupling a spacer molecule (such as 6-nitrohexanoic acid) between the biotin and the peptide.

Biotin quantification of biotinylated proteins provides a sensitive fluorescence assay to accurately determine the number of biotin labels on the protein. Biotinylated peptides are widely used in a variety of biomedical screening systems that require immobilization of at least one of the interacting substances on streptavidin-coated beads, membranes, slides, or microtiter plates. This assay is based on the displacement of a ligand labeled with a quencher dye from the biotin binding site of an agent. To expose any biotin groups in the multi-labeled protein that are sterically limited and inaccessible to the reagent, the protein may be treated with a protease to digest the protein.

EMIT is a competitive binding immunoassay, avoiding the usual separation steps. This is an immunoassay in which the protein is enzyme-labeled, and the enzyme-protein-antibody complex is enzyme-inactive, enabling quantification of label-free protein. Some embodiments of the invention include an ELISA to analyze PARP. ELISA is based on selective antibodies attached to a solid support and combined with enzyme reactions to produce a system capable of detecting low levels of protein. It is also known as enzyme immunoassay or EIA. The protein is detected by an antibody against the protein, in other words, it is an antigen of the antibody. Monoclonal antibodies are frequently used.

Such tests may require the antibody to be immobilized on a solid surface, such as the inner surface of a test tube, and the same antibody to be conjugated to an enzyme to be prepared. The enzyme may be an enzyme (e.g.beta-galactosidase) which produces a coloured product from a colourless substrate. Such testing can be accomplished, for example, by filling a tube with a solution of the antigen to be detected (e.g., protein). Any antigen molecule present may bind to the immobilized antibody molecule. Antibody-enzyme conjugates may be added to the reaction mixture. The antibody portion of the conjugate binds to any antigen molecule previously bound, creating an antibody-antigen-antibody "sandwich". After washing off any unbound conjugate, a substrate solution may be added. After a certain time, the reaction is terminated (for example by adding 1N NaOH) and the concentration of the coloured product formed is measured spectrophotometrically. The intensity of the color is directly proportional to the concentration of the bound antigen.

ELISA methods can also be used to measure antibody concentrations, in which case the culture wells are coated with the corresponding antigen. A solution containing the antibody (e.g., serum) may be added. After it has had sufficient time to bind to the immobilized antigen, an enzyme-conjugated anti-immunoglobulin consisting of the antibody being tested may be added. After washing away unreacted reagents, a substrate may be added. The intensity of the color produced is directly proportional to the amount of enzyme-labeled antibody bound (and thus the concentration of antibody detected).

Some embodiments of the invention include radioimmunoassays to analyze PARP. Radioisotopes can be used to study the in vivo metabolism, distribution and binding of small amounts of compounds. Using in vivo radioisotopes¹H、¹²C、³¹P、³²S and¹²⁷i, e.g.³H、¹⁴C、³²P、³⁵S and¹²⁵I. in the receptor immobilization method on a 96-well plate, a receptor is immobilized in each well by an antibody or a chemical method, and a radiolabeled ligand is added to each well to induce binding. Unbound ligand may be washed away and the standard may then be determined by quantitative analysis of radioactivity bound to or washed out of the ligand. Then, the addition of the screening target compound induces a competitive binding reaction with the receptor. If the compound exhibits a higher affinity for the receptor than a standard radioligand, then most of the radioligand will not bind to the receptor and can remain in solution. Thus, by analyzing the amount of bound radioligand (or washed-off ligand), the affinity of the test compound for the receptor can be demonstrated.

When receptors cannot be immobilized on 96-well plates or when ligand binding needs to be performed in solution phase, a membrane filtration method may need to be employed. In other words, after the ligand-receptor binding reaction in solution, if the reaction solution is filtered through nitrocellulose filter paper, small molecules including the ligand may pass through it, and only the protein receptor may remain on the paper. Only the ligand that binds strongly to the receptor may remain on the filter paper and the relative affinity of the added compound can be determined by quantitative analysis of standard radioligands.

Some embodiments of the invention include a fluorescence immunoassay for analyzing PARP. Fluorescence-based immunological methods are based on competitive binding between labeled and unlabeled ligands at highly specific receptor binding sites. Fluorescence techniques can be used in immunoassays based on the change in fluorescence lifetime as a function of analyte concentration. This technique can be used in conjunction with a short-lived dye such as Fluorescein Isothiocyanate (FITC) (the donor), whose fluorescence can be quenched by energy transferred to eosin (the acceptor). Many photoluminescent compounds can be used, such as cyanine, oxazine, thiazine, porphyrin, phthalocyanine, fluorescent ir-luminescent polynuclear aromatics, phycobiliprotein, squarylium and organometallic complexes, hydrocarbons and azo dyes.

Fluorescence-based immunological methods are, for example, heterogeneous or homogeneous. Heterogeneous immunoassays involve physical separation between bound and free labeled analytes. The analyte or antibody may be attached to a solid surface. This technique may be competitive (for higher selectivity) or non-competitive (for higher sensitivity). Detection may be direct (using only one antibody) or indirect (using a second antibody). Homogeneous immunoassays do not involve physical separation. The dual antibody fluorophore-labeled antigen participates in an equilibrium reaction with an antibody directed to both the antigen and the fluorophore. Labeled and unlabeled antigens may compete with limited anti-antigen antibodies.

Some fluoroimmunoassay methods include simple fluorescent labeling, Fluorescence Resonance Energy Transfer (FRET), Time Resolved Fluorescence (TRF), and Scanning Probe Microscopy (SPM). Simple fluorescent labeling methods can be used to determine receptor-ligand binding and enzyme activity by using the associated fluorescence and as a fluorescent indicator of various physiological changes in vivo, such as pH, ionic concentration and voltage. TRF is a method of selectively measuring lanthanide fluorescence after the emission of other fluorescent molecules has ended. TRF can be used with FRET and the lanthanide series can be either a donor or an acceptor. In scanning probe microscopy, at least one monoclonal antibody is attached to the solid surface, for example during the capture phase, and scanning probe microscopy is used to detect antigen/antibody complexes that may be present on the solid surface. The use of scanning tunneling microscopy eliminates the need for labels that are commonly used in many immunoassay systems to detect antigen/antibody complexes.

The protein identification method comprises the following steps: by way of example only, protein identification methods include low-throughput sequencing by Edman degradation, mass spectrometry techniques, peptide mass fingerprinting, resequencing, and antibody-based analysis. The protein quantitative analysis method comprises fluorescent dye gel staining, labeling or chemical modification methods (i.e. isotope-coded affinity tags (ICATS), binding fraction diagonal chromatography (cofardic)). The purified protein can also be used for the determination of three-dimensional crystal structures, and the method can be used for simulating intermolecular interactions. Common methods for determining three-dimensional crystal structure include X-ray crystallography and nuclear magnetic resonance spectroscopy. Characteristic indications of the three-dimensional structure of proteins can be detected by mass spectrometry. By coupling those portions of the protein that are spatially close but are far apart in sequence by chemical cross-linking, information about the overall structure can be inferred. By tracking the exchange of amide protons with deuterium in the solvent, it is possible to probe the likelihood of the solvent approaching various parts of the protein.

In one embodiment, Fluorescence Activated Cell Sorting (FACS) is used to identify PARP expressing cells. FACS is a special type of flow cytometer. It provides a method of sorting heterogeneous mixtures of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell. It provides a means of quantitatively recording fluorescent signals from individual cells and physically separating cells of particular interest. In another embodiment, expression of PARP is assessed using a microfluidic-based device.

Mass spectrometry can also be used to characterize PARP of patient samples. Two methods of whole protein ionization are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). First, intact proteins are ionized in either of the two methods described above and then introduced into a mass analyzer. Second, the protease is digested into smaller peptides with reagents such as trypsin or pepsin. Other proteolytic digestants may also be used. The collected peptide product is then introduced into a mass analyzer. This method is commonly referred to as a "bottom-up" protein analysis mode.

Whole protein mass spectrometry is performed using time of flight (TOF) mass spectrometry or fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. The instrument used for peptide mass spectrometry is quadrupole ion trap mass spectrometry. Multi-stage quadrupole-time-of-flight and MALDI time-of-flight instruments can also be used for this application.

There are two methods for isolating proteins or their enzymatically digested peptide products. The first method separates whole proteins and is called two-dimensional gel electrophoresis. The second method, high performance liquid chromatography, was used to separate the peptide product after enzymatic digestion. In some cases, it may be necessary to combine these two techniques.

Mass spectrometers can be used to identify proteins in two ways. Peptide mass spectrometry uses the mass of a proteolytic peptide, which is generated as a result of digestion of a series of known proteins, as input to search a database of predicted masses. If a protein sequence in the reference series matches a significant number of predicted masses to the experimental values, there is some evidence that this protein is present in the original sample.

Tandem mass spectrometry is also a method for identifying proteins. Collision induced dissociation is used in most applications to generate a set of fragments from a particular peptide ion. This cleavage process mainly results in cleavage products that break along peptide bonds.

Many different algorithms for identifying peptides and proteins have been described for tandem mass spectrometry (MS/MS), peptide sequencing from new sequences and sequence tag-based searches. One option that integrates the features of comprehensive data analysis is PEAKS. Other existing mass spectrometry software includes: peptide fragment fingerprints SEQUEST, Mascot, OMSSA and X! Tandem).

Proteins can also be quantitatively analyzed by mass spectrometry. Typically, a stable (e.g., non-radioactive) heavier carbon (C13) or nitrogen (N15) isotope is added to one sample, while the other sample is labeled with a lighter isotope (e.g., C12 and N14). The two samples were mixed prior to analysis. Due to their differences in mass, peptides derived from different species can be distinguished. The peak intensity ratio corresponds to the relative abundance ratio of the peptide (and protein). Isotopic labeling methods are SILAC (stable isotopic labeling with amino acids during cell culture), trypsin catalyzed O18 labeling, ICAT (isotopically coded affinity labeling), ITRAQ (isotopic labeling for relative and absolute quantification). "semi-quantitative" mass spectrometry can be performed with the sample unlabeled. Typically, this is done using MALDI analysis (using linear mode). The peak intensity, or peak area, of each molecule (typically a protein) is related to the amount of protein in the sample. However, the individual signals depend on the major structure of the protein, the complexity of the sample, and the set-up of the instrument.

N-terminal sequencing facilitates identification of unknown proteins, confirmation of recombinant protein identity and fidelity (reading frame, translation starting point, etc.), interpretation of NMR and crystal structure data, display of degree of identity between proteins, or provide data for design of synthetic peptides for antibody production, etc. N-terminal sequencing utilizes Edman degradation chemistry to remove amino acid residues from the N-terminus of a protein in order and identify them by reverse phase HPLC. Sensitivity can reach the level of hundreds of femtomoles, and long sequence reads (20-40 residues) can often be obtained from tens of picomoles of starting material. Pure proteins (> 90%) can yield data that is easily interpretable, but less pure protein mixtures can also provide useful data, depending on the exact interpretation of the data. N-terminally modified (in particular acetylated) proteins cannot be sequenced directly, since the absence of free primary amino groups hampers edman chemistry. However, limited hydrolysis of the blocking protein (e.g., with cyanogen bromide) may allow the production of amino acid mixtures in each instrumental cycle, and database analysis can be performed to interpret meaningful sequence information. C-terminal sequencing is a post-translational modification that affects the structure and activity of proteins. A wide variety of conditions can be associated with impaired protein processing, and C-terminal sequencing provides another means to study protein structure and processing mechanisms.

Examples

Example 1: PARP1 expression in IDC breast cancer

Previous studies have shown an increase in PARP activity in ovarian, hepatocellular and rectal tumors and in human peripheral blood lymphocytes from patients with leukemia, as compared to normal healthy control tissue (Yalcintepe L, et al Braz J Med Biol Res 2005; 38: 361-5.Singh N. et al Cancer Lett 1991; 58: 131-5; Nomura F, et al J Gastroenterol Heapatol 2000; 15: 529-35). The present invention uses a gene expression database to examine the regulation of the PARP1 gene in 2000 various primary malignant tissues and normal human tissues. Although PARP1 expression and activity was very low and uniform in most normal human tissues and organs, it was upregulated in selected tumor cells and primary human malignancies, with the most significant differences in breast, ovarian, lung and uterine cancers (figure 1).

Tissue sample

Samples were harvested as part of a normal surgical procedure and snap frozen within 30 minutes after excision. Internal pathology review and validation was performed on the samples analyzed. Hematoxylin and eosin (H & E) stained slides prepared with adjacent tissues were used to confirm and classify diagnostic categories and to assess tumor cell composition. Expression of ER, PR and HER2 was determined using immunohistochemistry and fluorescence in situ hybridization. These results, as well as ancillary pathological and clinical data, were annotated with sample lists and regulatory databases (Ascenta, bioexpressistantas; GeneLogic, inc., Gaithersburg, MD).

RNA extraction and expression profiles

RNA extraction and hybridization was performed as described in Hansel et al. Array data quality was evaluated using an array high throughput application (Ascenta, bioixpress Gene Logic, Gaithersburg MD and Affymetrix, Santa Clara, CA) that evaluated the data against a variety of objective criteria including 5 '/3' GAPDH ratio, signal/noise ratio, and background, among other additional measures. GeneChip analysis was performed using Affymetrix microarray analysis software package version 5.0, Data Mining Tool 2.0, and microarray database software (Affymetrix, Santa Clara, Calif.). All genes represented on GeneChip were normalized and scaled to signal intensity 100.

Microarray data analysis

Pathological normal tissue samples were used to determine baseline expression of PARP1 mRNA. The mean and 90%, 95%, 99% and 99.9% Upper Confidence Limits (UCL) were calculated for each predictor. Because we evaluated the likelihood that each sample, outside of a normal set of samples, was within the baseline distribution, the prediction interval of the mean was chosen instead of the confidence interval to estimate the expected range of future individual magnitudes. Prediction interval ofDefinition of formula (I), wherein Is the average of normal breast samples; s is the standard deviation, n is the number of samples, and A is the 100 th (1- (p/2)) percentile of the Student' S t-distribution with n-1 degrees of freedom.

Baseline expression of PARP1 was determined using pathological normal tissue samples. The samples are divided into subclasses according to characteristics including tumor stage, smoking status, CA125 status or age. Each tumor sample was evaluated according to 90%, 95%, 99% or 99.9% UCL analysis. Analysis was performed using SAS v8.2 for Windows (www.sas.com).

Pearson correlations were calculated for the 11 probe sets (probe sets) compared to PARP 1. The correlation was based on a complete set of 194 samples. Pearson product-difference correlation is defined by

WhereinIs the average of a set of PARP1 probes andis the average of the set of probes to which PARP1 is related. Statistical significance is represented by the formulaDetermining, wherein r is correlation and n is the number of samples. The resulting values are considered to have a t-distribution of n-2 degrees of freedom.

Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR)

Multiplex RT-PCR was performed as described previously (Khan et al, 2007) using 25ng total RNA per sample. The multiplex assay used in this study was designed to detect RNA from Formalin Fixed Paraffin Embedded (FFPE) samples or from frozen tissues. The concentration of RNA was determined using a RiboGreen RNA quantification kit (Invitrogen) with a Wallac Victor 21420 multiple marker counter (multilable counter). RNA samples from each sample were analyzed on an Agilent Bioanalyzer according to the instructions of the Agilent 2100 Bioanalyzer. Reverse Transcription (RT) reactions were performed with Applied Biosystems 9700 as described previously. PCR reactions were performed for each cDNA using Applied Biosystems 9700. The RT reactions were blended with kanamycin RNA to monitor the efficiency of the RT and PCR reactions. Controls used included positive control RNA, no template control, and no reverse transcriptase control. The PCR reaction was analyzed by capillary electrophoresis. The fluorescently labeled PCR reaction products were diluted, combined with GenomeLab standard-400 (Beckman-Coulter), denatured, and analyzed using the CEQ 8800 gene analysis system. The expression of each target gene relative to the expression of β -Glucuronidase (GUSB) within the same reaction was reported as the mean and standard deviation of 3 independent assessments for each sample.

Breast cancer patients with Invasive Ductal Carcinoma (IDC) had a 1.8-fold increase in mean PARP1 expression compared to normal breast tissue (P <. 00001). Importantly, PARP1 overexpression occurs most frequently in breast cancer tissues that are ER, PR, or HER2 negative (table 1).

TABLE 1 PARP1 overexpression in IDC mammary tissue

IDC subtype	n	PARP1 overexpressed samples%
			Is normal	68	2.9％
IDC	169	30.2％
			ER+	35	22.9％
ER-	18	55.6％
			PR+	26	23.1％
PR-	20	45.0％
			HER2+	24	29.2％

HER2-	10	70.0％
			ER+/PR+	26	23.1％
ER-/PR-	8	62.5％
			ER+/PR-	8	25.0％

PARP1 overexpression was defined as samples that exceeded the 95% upper confidence limit in normal breast tissue distribution

Example 2: combination of 4-iodo-3-nitrobenzamide (BA) with chemotherapy

Cell culture

Breast cancer cells were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cell culture dish 10 for each P100 cell⁵Individual cell or individual P60 cell culture dish 10⁴Individual cells were plated in the presence of different concentrations of compound or DMSO control. After treatment, adherent cell numbers were measured using a Coulter counter by staining with 1% methylene blue. Methylene blue is dissolved in a 50% -50% mixture of methanol and water. Cells were plated in 24-well or 96-well plates and treated as planned, the medium aspirated, the cells washed with PBS, fixed in methanol for 5-10 minutes, the methanol aspirated and the plates allowed to dry completely. Methylene blue solution was added to the wells and the plates were incubated for 5 minutes. Remove staining solution and use dH ₂O washing the plate to washNo longer blue. After the plate was completely dried, a small amount of 1N HCl was added to each well to extract methylene blue. The OD reading at 600nm and a calibration curve were used to determine cell number.

Compound (I)

Compounds were dissolved directly from dry powder into 10mM stock in DMSO for each independent experiment. Control experiments were performed with matching volumes/concentrations of vehicle (DMSO); in these controls, the growth or cell cycle distribution of the cells showed no change.

PI rejection, cell cycle and TUNEL experiments

After drug addition and incubation, cells were trypsinized and aliquots were taken for counting and PI (propidium iodide) rejection experiments. A portion of the cells were centrifuged and resuspended in 0.5ml ice-cold PBS containing 5. mu.g/ml PI. The remaining portion of cells was fixed in ice-cold 70% ethanol and stored in a refrigerator overnight. For cell cycle analysis, cells were stained with Propidium Iodide (PI) by standard methods. Cellular DNA content was determined by flow cytometry using BD LSRII FACS and the percentage of cells at G1, S or G2/M was determined using ModFit software.

Cells were labeled for apoptosis using a "fluorescein IN situ cell death detection kit" (Roche Diagnostics Corporation, Roche Applied Science, Indianapolis, IN). Briefly, the fixed cells were centrifuged and washed once in Phosphate Buffered Saline (PBS) containing 1% Bovine Serum Albumin (BSA), then resuspended in 2ml of permeabilization buffer (0.1% Triton X-100 and 0.1% sodium citrate in PBS) at room temperature for 25 minutes and washed twice in 0.2ml PBS/1% BSA. Cells were resuspended in 50. mu.l TUNEL reaction mix (TdT enzyme and labeling solution) and incubated in an incubator at 37 ℃ in a humidified dark environment for 60 minutes. The labeled cells were washed once in PBS/1% BSA and then resuspended in 0.5ml ice-cold PBS containing 1. mu.g/ml 4', 6-diamidino-2-phenylindole (DAPI) for at least 30 minutes. All cell samples were analyzed with BD LSR II (BDBiosciences, San Jose, CA).

Bromomdeoxyuridine (BrdU) labeling assay

Add 50 μ l BrdU (Sigma Chemical co., st. louis, MO) stock solution (1mM) to yield a final concentration of 10 μ M BrdU. Cells were incubated at 37 ℃ for 30 minutes and fixed in ice-cold 70% ethanol and then stored overnight in a cold room (4 ℃). The fixed cells were centrifuged and washed once in 2ml PBS, then resuspended in 0.7ml denaturant (0.2mg/ml pepsin 2NHCl) at 37 ℃ for 15 minutes in the dark, suspended with 1.04ml 1M Tris buffer (Trizma base, sigma chemical Co.) and washed in 2ml PBS. Subsequently, the cells were resuspended in 100. mu.l of anti-BrdU antibody (DakoCytomation, Carpinteria, Calif.) diluted 1: 100 in TBFP osmotic buffer (0.5% Tween-20, 1% bovine serum albumin and 1% fetal bovine serum in PBS) and incubated for 25 minutes at room temperature in the dark and washed in 2ml PBS. Primary anti-labeled cells were resuspended in 100. mu.l Alexa Fluor F (ab ') 2 fragment of goat anti-mouse IgG (H + L) (2mg/mL) diluted 1: 200 in TBFP permeation buffer (Molecular Probes, Eugene, OR) and incubated for 25 min at room temperature in the dark and washed in 2mL PBS, followed by resuspension in 0.5mL ice-cold PBS containing 1. mu.g/mL 4', 6-diamidino-2-phenylindole (DAPI) for at least 30 min. All cell samples were analyzed with BD LSR II (BD Biosciences, San Jose, CA).

Combinations of 4-iodo-3-nitrobenzamide (BA) with various chemotherapeutic drugs have been tested in vitro and in vivo cancer models. Evaluation of BA in combination with gemcitabine or carboplatin in MDA-MB-468 breast cancer cell line derived from patients with triple negative metastatic adenocarcinoma showed that BA potentiated S-and G2/M cell cycle arrest and enhanced cytotoxic effects induced by carboplatin or gemcitabine (figure 2).

BA activity in combination with gemcitabine and carboplatin was evaluated on a human triple negative metastatic breast cancer MDA-MB-231 xenograft model in nu/nu nude mice. BA increased the activity of the gemcitabine and carboplatin combination and produced 4 Partial Remissions (PR) and 2 Complete Remissions (CR) and 1 tumor-free survivors (TFS) 35 days after drug administration (table 2). BA was well tolerated in combination with gemcitabine and carboplatin.

Table 2: in vivo Activity of BA in combination with Gemcitabine/Carboplatin in an MDA-MB-231 xenograft model of triple negative breast cancer

Treatment of	Partial relief	Complete relief	Survivors without tumor
				Control	0	0	0
Gemcitabine (15 mg/kg; intraperitoneal; 1X 4 intraperitoneal every 3 days) + Carboplatin (10 mg/kg; intraperitoneal; 1X 3 times a week)	4	0	0
				BS1-201(50 mg/kg; intraperitoneal; 1 time per two weeks) + Gemcitabine (15 mg/kg; intraperitoneal; 1 time per 3 days x 4 intraperitoneal) + Carboplatin (10 mg/kg; intraperitoneal; 1 time per week x 3)	4	2	1

Thus, 4-iodo-3-nitrobenzamide (BA) may potentiate the activity of a variety of cytotoxic chemotherapeutic agents, including carboplatin and gemcitabine.

Example 3: combination of 4-iodo-3-nitrobenzamide (BA) with irinotecan

Breast cancer cells were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cell culture dish 10 for each P100 cell⁵Individual cell or in P60 cell culture dishes 10⁴Individual cells were plated in the presence of different concentrations of compound or DMSO control. After treatment, adherent cell numbers were measured using a Coulter counter by staining with 1% methylene blue. Methylene blue is a 50% -50% mixture dissolved in methanol and water. Cells were plated in 24-well or 96-well plates and treated as planned, the medium aspirated, the cells washed with PBS, fixed in methanol for 5-10 minutes, the methanol aspirated and the plates allowed to dry completely. Methylene blue solution was added to the wells and the plates were incubated for 5 minutes. Remove staining solution and use dH₂The plates were washed until the wash was no longer blue. After the plate was completely dried, a small amount of 1N HCl was added to each well to extract methylene blue. The OD reading at 600nm and a calibration curve were used to determine cell number.

Compounds were dissolved directly from dry powder into 10mM stock in DMSO for each independent experiment. Control experiments were performed with matching volumes/concentrations of vehicle (DMSO); in these controls, the growth or cell cycle distribution of the cells showed no change.

The PI exclusion assay, cell cycle assay, TUNEL assay and BrdU labeling assay were performed as described above in example 2.

Combinations of 4-iodo-3-nitrobenzamide (BA) with irinotecan were tested at various concentrations in an in vitro cancer model. The combination of BA and irinotecan was evaluated in patient-derived MDA-MB-468 triple negative breast cancer cell lines with triple negative metastatic adenocarcinoma, showing that BA potentiates S-and G2/M cell cycle arrest and enhances the cytotoxic effects induced by irinotecan (table 3).

Table 3: cell cycle regulation of triple negative MDA-MB-468 breast cancer treated with 4-iodo-3-nitrobenzamide (BA) in combination with irinotecan

	G1	S	G2/M	Live cells,% control
					Irinotecan
Irinotecan 0uM + BA uM
					0	64.40	24.18	11.42	100.0
50	65.50	23.48	11.02	91.5
					100	57.72	26.93	15.34	67.5
Irinotecan 5uM + BA uM
					0	38.17	33.77	28.05	48.6
50	24.94	41.85	33.21	31.6

100

9.28

51.43

39.29

23.4

Thus, 4-iodo-3-nitrobenzamide (BA) may potentiate the activity of a variety of cytotoxic chemotherapeutic agents including carboplatin, gemcitabine and irinotecan.

Example 4: 4-iodo-3-nitrobenzamide (BA) and IGF1R inhibitor picropodophyllin (PPP) In combination with (1)

Breast cancer cells were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cell culture dish 10 for each P100 cell ⁵Individual cell or in P60 cell culture dishes 10⁴Individual cells were plated in the presence of different concentrations of compound or DMSO control. After treatment, adherent cell numbers were measured using a Coulter counter by staining with 1% methylene blue. Methylene blue is dissolved in a 50% -50% mixture of methanol and water. Cells were plated in 24-well or 96-well plates and treated as planned, the medium aspirated, the cells washed with PBS, fixed in methanol for 5-10 minutes, the methanol aspirated and the plates allowed to dry completely. Methylene blue solution was added to the wells and the plates were incubated for 5 minutes. Remove staining solution and use dH₂The plates were washed until the wash was no longer blue. After the plate was completely dried, a small amount of 1N HCl was added to each well to extract methylene blue. Use ofOD readings at 600nm and a calibration curve to determine cell number.

Compounds were dissolved directly from dry powder into 10mM stock in DMSO for each independent experiment. Control experiments were performed with matching volumes/concentrations of vehicle (DMSO); in these controls, the growth or cell cycle distribution of the cells showed no change.

The PI exclusion assay, cell cycle assay, TUNEL assay and BrdU labeling assay were performed as described above in example 2.

Combinations of 4-iodo-3-nitrobenzamide (BA) with the insulin-like growth factor 1 receptor (IGF1R) inhibitor picropodophyllin (PPP) were tested at various concentrations in an in vitro cancer model. Evaluation of the combination of BA and PPP in MDA-MB-468 triple negative breast cancer cell lines obtained from patients with triple negative metastatic adenocarcinoma revealed that BA potentiates S-and G2/M cell cycle arrest and enhances the cytotoxic effects induced by PPP (table 4).

Table 4: cell cycle regulation of triple negative MDA-MB-468 breast cancer treated with 4-iodo-3-nitrobenzamide (BA) in combination with IGF1R inhibitor picropodophyllin (PPP)

	G1	S	G2/M	% live cell control
					PPP 0nM+201uM
0	50.96	30.37	16.04	100
					50	50.20	31.34	15.21	82
100	40.63	34.52	20.16	61
					PPP 200nM+201uM
0	51.42	30.22	15.01	89
					50	49.75	31.41	15.10	77

100	37.51	35.58	21.30	59
					PPP 400nM+201uM
0	37.29	25.32	20.17	60
					50	32.88	28.47	22.37	42
100	23.62	31.78	29.98	32

Thus, 4-iodo-3-nitrobenzamide (BA) may potentiate the activity of growth factor receptor targeted inhibitors including picropodophyllin (PPP).

Example 5: treatment of triple negative breast cancer with BA

A multicenter, open label, randomized study was performed to demonstrate the therapeutic efficacy of treatment of triple negative metastatic breast cancer with 4-iodo-3-nitrobenzamide (BA).

The research aims are as follows: the main objectives of this study are as follows:

clinical benefit rate (CBR ═ CR + PR + SD > 6 months): it was determined that BA would produce 30% or more CBR compared to 45% CBR associated with gemcitabine and carboplatin treatment.

Further investigation of the safety and tolerability of BA

Secondary objectives of the study were as follows:

overall Response Rate (ORR)

Progression-free survival (PFS)

Evaluation of toxicity associated with each group

Exploratory objects (exploratory objects) of the present study are as follows:

characterization of the inhibitory Effect of BA on PARP Activity

Characterisation of PARP Activity in historical (historic) tumor tissue samples

Study of BRCA status in triple negative breast cancer

Study of response in subjects with cancer and known BRCA mutations compared to subjects without such mutations

Classification of breast cancer tissue as basal or luminal (luminal)

Research and design: open label, 2-cohort randomized safety and efficacy study, in which up to 90 patients (45 per cohort) were randomized to:

study group 1: gemcitabine (1000 mg/m) on days 1 and 8 of a 21-day cycle²(ii) a 30 min intravenous infusion) and carboplatin (AUC 2; 60 minute intravenous infusion); or

Study group 2: 4-iodo-3-nitrobenzamide (4mg/kg, 1 hr intravenous infusion) on days 1, 4, 8 and 11 of each 21-day cycle

Patients randomized to study cohort 2 will withdraw from the study as disease progresses

And (3) cross grouping: patients randomized to study group 1 could be swapped to receive continued treatment with gemcitabine/carboplatin in combination with 4-iodo-3-nitrobenzamide as disease progressed

Sample amount: up to 90 subjects, up to 45 subjects per cohort were participating in the study. Subjects will be randomly assigned, with up to 45 subjects per group in group 1 or group 2.

Population of subjects:

inclusion criteria were:

At least 18 years old

Metastatic breast cancer with measurable disease according to RECIST criteria (stage IV)

0-2 courses of antecedent chemotherapy in metastatic cases. Allowing advanced adjuvant/neoadjuvant therapy.

Histologically, the primary or metastatic site, breast cancer confirmed by immunohistochemistry (0, 1) as ER-negative, PR-negative and no HER-2 overexpression, or no gene amplification by FISH on primary tumors or metastatic lesions.

Prior chemotherapy was terminated at least 3 weeks before study participation.

Patients may receive therapy with adjuvant or metastatic events, however, if bisphosphonates are taken, bone lesions cannot be used to assess progression or remission.

Radiation therapy must end at least 2 weeks before study participation and radiation lesions may not be the measurable disease agent.

Patients may have CNS metastases if stable (no evidence of disease progression) for at least 3 months after topical treatment.

ECOG Performance status 0-1

Sufficient organ function is defined as follows: ANC greater than or equal to 1,5000/mm³Platelets greater than or equal to 100,000/mm³Creatinine clearance greater than 50 mL/min, ALT and AST below 2.5 times the Upper Limit of Normal (ULN) (or below 5 times ULN in the case of liver metastasis); the total bilirubin is less than 1.5 mg/dL.

Tissue blocks available for PARP studies are recommended, although the absence of tissue blocks does not preclude patient involvement.

Pregnant or lactating women will be excluded. Women of child-bearing age must obtain a documented negative pregnancy test within 2 weeks of study participation and agree to take acceptable contraceptive measures for the duration of study therapy.

Signed written informed consent approved by IRB.

Exclusion criteria:

lesions identifiable by PET alone

More than 2 prior chemotherapy courses (including adjuvant therapy). A sequential treatment regimen, such as AC-paclitaxel treatment, would be considered one regimen.

Prior treatments with gemcitabine, carboplatin, cisplatin or 4-iodo-3-nitrobenzamide have been received.

May affect significant medical conditions involved in the study (uncontrolled pulmonary, renal or hepatic dysfunction, uncontrolled infection).

A history of apparently uncontrolled heart disease; i.e. uncontrolled hypertension, unstable angina, recent myocardial infarction (within the previous 6 months), uncontrolled congestive heart failure and cardiomyopathies that are symptomatic or asymptomatic but with a drop in ejection fraction below 45%.

Researchers feel that other obvious co-existence conditions that may detract from effective and safe participation in the study.

Subjects who have enrolled in another study drug device trial or are receiving other study drugs.

Concomitant or anticipatory (within 7 days of study day 1) anticoagulant therapy (allowing low dose for catheter maintenance)

Prescribed concomitant medication

Not allowing simultaneous radiotherapy throughout the study

Can not comply with the research requirements

Screening trials and evaluations were only performed after written informed consent approved by the ethics committee (IRB) was obtained from each subject. Procedures will be performed within 14 days of administration (day 1), unless otherwise noted.

And (3) clinical evaluation: complete history, physical examination, ECOG status, height, weight, vital signs and concomitant medication records.

Laboratory studies: hematology (with variability, reticulocyte count, and platelets); prothrombin Time (PT) and Partial Thromboplastin Time (PTT); comprehensive chemical items (sodium, potassium, chloride, CO)₂Creatinine, calcium, phosphorus, magnesium, BUN, uric acid, albumin, AST, ALT, alkaline phosphatase, total bilirubin and cholesterol, HDL and LDL), urinalysis with microscopic examination, PARP inhibition in PBMC, pregnancy serum or urine examination in women of child bearing age. If a separate informed consent is signed, a BRCA profile will be obtained. This information can also be extracted from the subject's medical history. The clinical period is as follows: measurable disease is imaged by Computed Tomography (CT) or Magnetic Resonance Imaging (MRI).

Treatment: patients eligible for admission were enrolled in the study and randomly assigned to cohort 1 or cohort 2.

Study group 1: gemcitabine (1000 mg/m) on days 1 and 8 of a 21-day cycle²(ii) a 30 min intravenous infusion) and carboplatin (AUC 2; 60 minute intravenous infusion); or

Study group 2: 4-iodo-3-nitrobenzamide (4mg/kg, 1 hr intravenous infusion) on days 1, 4, 8 and 11 of each 21-day cycle

And (3) cross grouping: patients randomized to study group 1 could be swapped to receive continued treatment with gemcitabine/carboplatin in combination with 4-iodo-3-nitrobenzamide as disease progressed

Pre-and post-dose trials will be performed as outlined in the study protocol.

Dosing of both treatment groups will be repeated at a 21 day cycle.

Subjects may participate in the study until they experience drug intolerance or disease progression or withdraw consent. Subjects who reach CR will receive an additional 4 cycles. Subjects who discontinue treatment prior to PD should undergo routine phase assessments of each regimen up to the PD time. Once the subject discontinues treatment, the assessment of survival and overall response rate for disease-free progression will continue in 3 month intervals until disease progression or death.

In addition to the initial classification made at baseline, a first planned tumor response measurement for measurable disease would be taken after cycle 2 and then every other treatment cycle (approximately every 6-8 weeks). Tumor response according to the criteria for improved response assessment of solid tumors (RECIST) will be used to determine disease progression by CT or MRI (the same techniques used during screening must be used).

And (4) ending treatment: all subjects should end the course of treatment no more than 30 days after the last dose of 4-iodo-3-nitrobenzamide as indicated in the protocol. In addition, if not assessed within 30 days prior to the last dose of 4-iodo-3-nitrobenzamide, subjects will obtain an overall assessment of tumor response by clinical imaging.

And (3) safety evaluation: safety will be assessed by standard clinical and laboratory tests (hematology, blood chemistry and urinalysis). Toxicity ratings are defined by the National Cancer Institute, version 3.0 CTCAE.

Pharmacokinetics/pharmacodynamics

Blood samples for PK and pharmacodynamic analysis will only be obtained from subjects enrolled in study group 2, which includes cross-group subjects.

PK samples will be collected during cycle 1, prior to dosing and immediately after the end of infusion on day 1 and day 11.

Pharmacodynamic or PARP samples will be collected on cycle 1, day 1, 4, 8 and 11 prior to dosing. Samples were taken only on day 1 after dosing.

Sites that could not have PK or pharmacodynamic sample collection performed as specified would be allowed to participate in the study and the protocol for those sites would be revised accordingly.

The curative effect is as follows: tumors will be evaluated by standard methods (e.g., CT) at baseline and then approximately every 6-8 weeks in the absence of clinically significant disease progression.

Statistical method

The main objective of this study was to estimate the Clinical Benefit Rate (CBR) in the BA cohort. In each of the two groups, the primary efficacy endpoint (CBR) will be evaluated and a 90% confidence interval for the exact two terms calculated. CBR in both groups will be compared at a 5% significance level using the one-sided Fisher exact test. Minor and exploratory efficacy endpoints of survival and overall survival with no disease progression will be estimated and 95% confidence intervals will be calculated using the Kaplan-Meier method. The distribution of disease progression free survival and overall survival in the two groups will be compared using a time series test (log-rank test). Analysis of PARP inhibition data will be exploratory and descriptive in nature. For the primary safety endpoint, AEs and Severe Adverse Events (SAE) will be tabulated by study group, system organ category, and preferred terms. Laboratory test results after the first cycle will be summarized in terms of deviations from baseline values.

Follow-up: follow-up information was obtained on day 90 and every 90 days (+ -20 days) after the last dose of study drug.

Laboratory evaluation-blood and urine samples will be prepared for hematology, serum chemistry and urine analysis using standard procedures. The experimental items are defined as follows:

hematology: differential WBC count, RBC count, hemoglobin, hematocrit, and platelet count

Serum chemistry: albumin, ALP, ALT, AST, BUN, calcium, carbon dioxide, chloride, creatinine, gamma-glutamyl transferase, glucose, lactate dehydrogenase, phosphorus, potassium, sodium, total bilirubin, and total protein.

And (3) urinalysis: appearance, color, pH, specific gravity, ketones, protein, glucose, bilirubin, nitrite, urobilinogen and occult blood (the sediment microscopy test will be performed only when the results of the urine analysis test strip evaluation are positive)

Pharmacokinetic blood samples will only be obtained from subjects enrolled in study group 2 or cross-distributed to study group 2. Samples will be collected immediately prior to dosing and immediately after the end of each infusion on day 1 and day 11 of cycle 1.

Biomarkers are indicators of objective measurement and evaluation of normal biological processes, pathological processes, or pharmacological responses to therapeutic interventions. In oncology, molecular alterations that underlie the tumorigenic processes are of particular interest, where the molecular alterations can identify cancer subtypes, stage disease, assess the amount of tumor growth or predict disease progression, metastasis and response to BA.

The functional activity of PARP before and after BA treatment will be determined in Peripheral Blood Mononuclear Cells (PBMCs) using the PARP activity assay. PBMCs will be prepared from 5mL blood samples according to the procedures detailed in the research manual and will be measured for PARP activity/inhibition.

For detailed collection, handling and transportation procedures of all PARP samples, reference is made to the research manual that will be provided to each site.

Breast cancer (BRCA) gene testing is a blood test that examines specific alterations (mutations) in genes that help control normal cell growth (BRCA1 and BRCA 2). Women with BRCA mutations show a 36% to 85% incidence of breast cancer and a 16% to 60% incidence of ovarian cancer. Administration of PARP inhibitors to women with BRCA mutations may prove beneficial. This study was a preliminary attempt to determine any association between BRCA status and BA treatment response.

To achieve this, the BRCA status of all subjects should be determined (if not yet known). The subject needs to sign an independent informed consent. As this is not an inclusion criterion for the study, potential subjects who did not agree to receive this test would not be excluded from participating in the study for this reason.

In each of the two groups, the primary efficacy endpoint (CBR) will be estimated and the 90% confidence interval for the exact two terms will be calculated. CBR in both groups will be compared at a 5% significance level using the one-sided Fisher exact test. Temporal tests will be used to compare the secondary and exploratory efficacy endpoints of disease-free progression survival and overall survival in both groups.

Tumor response data will be reported in the form of a descriptive list for all subjects in the safety population with the aim of determining whether BA treatment has had a measurable clinical effect (e.g., time versus disease progression) and should continue after the first 8 weeks. The response data will use the modified RECIST classification.

The PARP inhibition assay will be exploratory and descriptive in nature, as the case may be. Statistical groups considering differences in PARP inhibition will be compared to any pharmacogenomic results (e.g., BRCA) from samples taken before, during, and after BA treatment.

Safety analysis will be completed for all subjects receiving at least 1 dose of BA.

The BA used in this study will be formulated at a concentration of 10mg/mL in 10mM phosphate buffer (pH 7.4) containing 25% hydroxypropyl β -cyclodextrin.

Evaluation criteria for solid tumor response (response evaluation criteria in solid tumors) (RECIST):

qualification of

Only patients with measurable disease at baseline should be included in the protocol where objective tumor response is the primary endpoint.

Measurable disease-the presence of at least one measurable lesion. If the measurable disease is limited to isolated lesions, its tumor characteristics should be confirmed cytologically/histologically.

Measurable lesions-lesions that can be measured accurately in at least one dimension, the maximum diameter of the lesion being ≧ 20mm using conventional techniques, or ≧ 10mm using helical CT scanning.

Unmeasurable lesions-all other lesions including microscopic lesions (maximum diameter < 20mm using conventional techniques or maximum diameter < 10mm using spiral CT scan), i.e. bone lesions, pia-mater disease, ascites, pleural/pericardial effusion, inflammatory breast disease, skin/pulmonary lymphangitis, cystic lesions and also abdominal masses not confirmed and subsequently confirmed by imaging techniques; and is

All measurements should be taken using a ruler or caliper and recorded in metric notation. All baseline assessments should be as close as possible to the start of treatment and must not be performed more than 4 weeks prior to the start of treatment.

The same assessment method and the same technique should be used to characterize each lesion found and reported at baseline and during follow-up.

Clinical lesions are considered measurable only when they are superficial (e.g., skin nodules and palpable lymph nodes). For the case of skin lesions, color photographic recording is recommended, including a ruler to assess lesion size.

Measuring method

CT and MRI are currently the best and reproducible methods available for measuring selected target lesions for response assessment. Conventional CT and MRI should result in a continuous slice thickness of 10mm or less. The spiral CT should be performed using a 5mm continuous reconstruction algorithm. The above is applicable to tumors of the chest, abdomen and pelvis. Head and neck tumors and tumors of the extremities usually require special approaches.

A lesion on an X-ray chest sheet may also be considered measurable if it is clearly shown and surrounded by inflated lung tissue. However, CT is preferred.

When the main objective of the study is objective response assessment, no Ultrasound (US) should be used to measure tumor lesions. However, it can be used as a backup for clinical measurements of accessible superficial lymph nodes, subcutaneous lesions and thyroid nodules. Ultrasound can also be used to confirm whether superficial lesions, usually assessed by clinical examination, have completely disappeared.

There is still no adequate and widely-confirmed endoscopy and laparoscopy available for objective tumor assessment. Their use in this particular environment requires complex equipment and a high level of expertise, but only some centres have such conditions. Thus, the application of such techniques to evaluate objective tumor responses should be limited to central validation purposes. However, these techniques can be used to confirm the complete pathological response when obtaining a biopsy sample.

Tumor markers alone cannot be used to assess response. If the initial marker levels are above the upper normal limit, they must be reduced to normal levels in order to consider the patient as a complete clinical response when all lesions have disappeared.

In some rare cases, cytology and histology may be used to distinguish between PR and CR (e.g., to distinguish between residual benign lesions and residual malignant lesions after treatment of some tumors, such as germ cell tumors).

Baseline recording of "target" and "non-target" lesions

All measurable lesions representing all organs involved, up to 5 lesions per organ, and 10 lesions in total, should be identified as target lesions and recorded and measured at baseline.

The target lesions should be selected according to their size (the lesion with the largest diameter) and whether they are suitable for accurate measurement with repeatable measurement means (imaging techniques or clinical measurements).

The sum of the maximum diameters (LD) of all target lesions will be calculated and reported as the baseline LD sum. This baseline sum of LDs will be used as a reference to characterize objective tumors.

All other lesions (or disease sites) should be identified as non-target lesions and should also be recorded at baseline. Measurements of these lesions are not required, but it should be noted whether each of these lesions is present throughout the follow-up procedure.

Response Standard (response criterion)

Evaluation of target lesions:

complete Response (CR): all target lesions disappeared

Partial Response (PR): the total LD of the target lesion is reduced by at least 30 percent by taking the total LD of the baseline as a reference

Progressive Disease (PD): the sum of LD of the target lesions is increased by at least 20% with reference to the minimum sum of LD recorded since the start of treatment or the appearance of one or more new lesions

Stable Disease (SD): reference to the minimum sum of LD since the start of treatment, i.e., not sufficiently diminished to qualify as PR, or sufficiently increased to qualify as PD

Evaluation of non-target lesions

Complete Remission (CR): disappearance of all non-target lesions and normalization of tumor marker levels

Incomplete remission/Stable Disease (SD): one or more non-target lesions persist or/and the level of tumor markers persist above the normal limit.

Progression of Disease (PD): appearance of one or more new lesions and/or significant progression of existing non-target lesions (1)

Although the apparent development of "non-target" lesions is only an exception, in this environment the opinion of the treating physician should be taken as the main and the development should be confirmed later by a panel of review experts (or research moderator).

Evaluation of optimal Total response (overall response)

The best overall response is the best response record from the start of treatment until disease progression/recurrence (the smallest magnitude recorded since the start of treatment is taken as a reference for PD). Generally, the conclusion of the patient's best response will depend on whether the measurement is completed and the validation criteria are met.

Non-target lesions of target lesions overall response of new lesions (overall)

response)

CR CR No CR

Incomplete remission of CR (incomplete no PR)

response)/SD

PR non-PD No PR

SD non-PD No SD

PD any or none of PD

Any PD with or without PD

Any one has PD

Patients requiring an overall worsening of health condition who require discontinuation of therapy without objective evidence of disease progression at that time should be classified as having "marked symptomatic exacerbation". All effort should be made to document objective disease progression even after discontinuation of treatment.

In some cases, it may be difficult to distinguish disease from normal tissue. When the assessment of complete remission is dependent on this determinant, it is recommended that the residual lesion (fine needle aspiration/biopsy) should be studied to confirm the complete remission condition.

Confirmation of

The main objective of validating objective responses is to avoid overestimating the observed response rate (response). Where validation of a response is not feasible, it should be clarified that the response is not validated when the results of such studies are reported.

In order to characterize a condition as PR or CR, changes in tumor magnitude must be confirmed by repeated assessments that should be performed no less than 4 weeks after the response criteria are first met. Longer intervals as determined by the study protocol may also be considered appropriate.

In the case of SD, subsequent measurements must meet SD standards at least once after participation in the study at a minimum interval defined by the study protocol (typically not less than 6-8 weeks).

Duration of total mitigation (duration of over response)

The overall duration of response is the first day from the time the measurement criteria met CR or PR (whichever condition was recorded first) until objective recording of relapse or PD, with the minimum magnitude recorded since treatment initiation as a reference for PD.

Duration of stable disease condition

SD was measured from the start of treatment until the criteria for disease progression were reached, with the minimum magnitude recorded since the start of treatment as a reference.

The clinical relevance of SD duration varies with different tumor types and grades. It is therefore highly recommended to specify in the scheme the minimum time interval required between two measurements in order to determine the SD. This time interval should take into account the expected clinical benefit that such a condition may have on the study population.

Response review (response review)

For trials where response rate is the primary objective, it is strongly recommended that all responses should be reviewed by experts independent of the study at the completion of the study. Simultaneous review of the patient's archives and radiological images is the best approach.

Reporting of results

All patients included in the study must be evaluated for response to treatment even if there is a significant deviation in the treatment regimen or they are not eligible. Each patient will be classified into one of the following categories: 1) complete remission, 2) partial remission, 3) stable disease, 4) disease progression, 5) early death due to malignant disease, 6) early death due to toxicity, 7) early death due to other causes, or 9) unknown (unapproved, inadequate data).

All patients meeting the eligibility criteria should be included in the primary analysis results of the response rate. Patients belonging to response classes 4-9 should be considered non-responsive to treatment (disease progression). Thus, improper treatment planning or drug administration does not result in exclusion from the results of the response rate analysis. The exact definition of categories 4-9 will vary from protocol to protocol.

All conclusions should be based on all eligible patients.

Subsequently, excluding those patients who have identified significant deviations in the regimen (e.g., early death due to other causes, early treatment discontinuation, significant regimen violation, etc.), a secondary analysis can be performed on a patient subgroup basis. However, these secondary analyses may not be the basis for drawing conclusions about the efficacy of treatment, and reasons for excluding patients from the analysis should be clearly reported.

A 95% confidence interval should be specified.

Example 6: treatment of breast cancer with BA

Phase 1b, open label, dose escalation studies evaluated the safety of BA (2.0, 2.8, 4.0, 5.6, 8.0, and 11.2mg/kg) in combination with chemotherapy regimens (topotecan, gemcitabine, temozolomide, and carboplatin + paclitaxel) in subjects with advanced breast tumors. Evaluation of PBMCs from patients showed significant and prolonged PARP inhibition following multiple administrations of 2.8mg/kg or higher BA doses (figure 3).

Well tolerated combinations of BA with each cytotoxic treatment regimen were identified. Any toxicity observed is consistent with known and expected side effects of each chemotherapeutic regimen. There is no evidence that the addition of BA treatment in any of the tested cytotoxic protocols would potentiate known toxicity or increase its expected toxicity frequency. Biologically relevant doses (2.8mg/kg) were identified that elicited significant and persistent PARP inhibition at effective preclinical blood concentrations. Evidence indicates that approximately 80% of subjects have stable disease for 2 treatment cycles or longer, indicating potential clinical benefit.

Example 7: phase 2 study of treatment of triple negative metastatic breast cancer (TNBC) with BA alone or in combination with gemcitabine/carboplatin

Phase 2, open label, 2-cohort randomized safety and efficacy trials, investigated whether inhibition of PARP activity by combination treatment of BA with gemcitabine/carboplatin improved clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) in metastatic TNBC breast cancer patients compared to standard chemotherapy alone. The conclusion examined was that the CBR of TNBC subjects reached 60% after the addition of BA to gemcitabine/carboplatin compared to 45% when gemcitabine/carboplatin alone was used.

Terminal point

Primary endpoint

Clinical benefit rate (CBR ═ CR + PR + SD ≥ 6 months)

Safety and tolerability of BA

Secondary endpoint

Overall Response Rate (ORR)

Survival without disease Progression (PFS)

Exploratory endpoint

Identification of PARP Gene expression and pharmacogenomics of the obtained tumor samples

BRCA status

Response of subjects with cancer and these mutations compared to subjects without known BRCA mutations

Classification of Breast cancer tissue as basal or luminal

Dosage/time schedule

Subjects were randomly assigned to:

test group 1: gemcitabine (1000 mg/m) on days 1 and 8 of a 21-day cycle²(ii) a 30 min intravenous infusion) + carboplatin (AUC 2; 60 minute intravenous infusion)

Test group 2: gemcitabine (1000 mg/m) on days 1 and 8 of a 21-day cycle²(ii) a 30 min intravenous infusion) + carboplatin (AUC 2; 60 minute intravenous infusion); on days 1, 4, 8 and 11 of the cycle, additional BA (5.6 mg/kg; 1 hour intravenous infusion)

All dosing cycles were repeated every 21 days.

Subjects randomized to cohort 2 terminated the trial once the disease progressed. Once the disease progressed, subjects randomized to cohort 1 were cross-treated with BA in combination with gemcitabine/carboplatin.

Principal qualification criteria

Metastatic breast cancer with measurable disease according to RECIST criteria (stage IV)

0-2 prior courses of chemotherapy in the case of metastases; allowing advanced adjuvant/neoadjuvant therapy

Histologically documented (primary or metastatic site) breast cancer confirmed by immunohistochemistry (0, 1) as ER-negative, PR-negative and no HER-2 overexpression, or confirmed by FISH as no gene amplification.

·ECOG 0-1

Study population

To date, 85 patients were enrolled at 23 study sites (table 5).

Table 5: patient statistical characterization of phase II study

Grouping A: gemcitabine/carboplatin

Grouping B: BSI-201+ Gemcitabine/Carboplatin

^*Based on available data

ER, PR and HER2 expression profiles

The study content was based on traditional histological tests at the study site. Paraffin-embedded sections of the original biopsy were obtained from patients enrolled in clinical trials and identified the gene status as TNBC markers (including ER, PR and HER2) as well as PARP1, Top2A and Ki-67. The method is based on optimized multiplex quantitative RT-PCR for quantitative assessment of gene expression in formalin-fixed and paraffin-embedded (FFPE) tissues. Samples from clinical trials were compared to obtain control samples representing FFPE normal and tumor tissues independently. Furthermore, a series of samples were obtained from patients who were documented to be overexpressing the HER2 gene.

Tissue sample

Samples were harvested as part of a normal surgical procedure and snap frozen within 30 minutes after excision. Internal pathology review and validation was performed on the samples analyzed. Hematoxylin and eosin (H & E) stained slides from adjacent tissues were used to confirm and classify diagnostic categories and to assess tumor cytology. Expression of ER, PR and HER2 was determined using immunohistochemistry and fluorescence in situ hybridization. These results, as well as ancillary pathological and clinical data, were annotated with sample lists and regulatory databases (Ascenta, BioExpress databases; GeneLogic, inc., Gaithersburg, MD).

RNA extraction and expression profiles

RNA extraction and hybridization was performed as described in Hansel et al. Array data quality was evaluated using an array high throughput application (Ascenta, bioixpress Gene Logic, Gaithersburg MD and Affymetrix, Santa Clara, CA) that evaluated the data against a variety of objective criteria including 5 '/3' GAPDH ratio, signal/noise ratio, and background, among other additional measures. GeneChip analysis was performed using Affymetrix microarray analysis software package version 5.0, Data Mining Tool 2.0, and microarray database software (Affymetrix, Santa Clara, Calif.). All genes represented on GeneChip were normalized and scaled to signal intensity 100.

Microarray data analysis

Pathological normal tissue samples were used to determine baseline expression of PARP1 mRNA. The mean and 90%, 95%, 99% and 99.9% Upper Confidence Limits (UCL) were calculated for each predictor. Since we evaluated the likelihood that a single sample outside of a normal set of samples was within the baseline distribution, the mean was chosenPrediction intervals of values rather than confidence intervals estimate expected ranges of future individual magnitudes. Prediction interval ofDefinition of formula (I), whereinIs the average of normal breast samples; s is the standard deviation, n is the number of samples, and A is the 100 th (1- (p/2)) percentile of the Student' S t-distribution with n-1 degrees of freedom.

Baseline expression of PARP1 was determined using pathological normal tissue samples. The samples are divided into subclasses according to characteristics including tumor stage, smoking status, CA125 status or age. Each tumor sample was evaluated according to 90%, 95%, 99% or 99.9% UCL analysis. Analysis was performed using SAS v8.2 for Windows (www.sas.com).

Pearson correlations were calculated for the 11 probe sets compared to PARP 1. The correlation was based on a complete set of 194 samples. Pearson product-difference correlation is defined as

WhereinIs the mean of the PARP probe set andis the average of the set of probes to which PARP1 is related. Statistical significance is represented by the formulaDetermining, wherein r is correlation and n is the number of samples. The resulting values assume a t-distribution with n-2 degrees of freedom.

Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR)

Multiplex RT-PCR was performed as described previously (Khan et al, 2007) using 25ng total RNA per sample. The multiplex assay used in this study was designed to detect RNA from Formalin Fixed Paraffin Embedded (FFPE) samples or from frozen tissues. The concentration of RNA was determined using a RiboGreen RNA quantification kit (Invitrogen) in a Wallac victor 21420 multi-marker counter. RNA samples from each sample were analyzed on an Agilent Bioanalyzer according to the instructions of the Agilent 2100 Bioanalyzer. Reverse Transcription (RT) reactions were performed with Applied Biosystems9700 as described previously. PCR reactions were performed for each cDNA using Applied Biosystems 9700. The RT reactions were blended with kanamycin RNA to monitor the efficiency of the RT and PCR reactions. Controls used included positive control RNA, no template control, and no reverse transcriptase control. The PCR reactions were analyzed by capillary electrophoresis. Fluorescently labeled PCR reaction products were diluted, combined with Genome Lab standard-400 (Beckman-Coulter), denatured, and analyzed using the CEQ 8800 gene analysis system. The expression of each target gene relative to the expression of β -Glucuronidase (GUSB) within the same reaction was reported as the mean and standard deviation of 3 independent assessments for each sample.

Figure 4 shows the results of the first 50 patients enrolled in the trial. Although the "triple negative" classification of patients is based on results of ER, PR and HER2 obtained using conventional clinical methods, these results show that both ER and PR gene expression is lower than in normal tissues. HER2 expression was comparable to normal, but significantly different from patients overexpressing PARP1, gene expression was significantly elevated, confirming our previous observations.

Preliminary results

Safety feature

The reduction in dose was similar in both groups, whether it was the percentage of patients with dose reduction or the total number of patients with dose reduction (table 6).

Table 6: dose reduction

	Grouping A: gemcitabine/carboplatin	Grouping B: BSI-201+ Gemcitabine/Carboplatin
			Patients with reduced dose, N/N (%)	15/39(38.5％)	11/39(28.2％)
Reduce the total number	20	19

Gemcitabine/carboplatin dose reduction as defined by the algorithm in the protocol

In the chemotherapy-only cohort, 15 (38.9%) of 39 subjects had dose reductions, and in the BA + chemotherapy cohort, 11 (28.2%) of 39 subjects had dose reductions. Overall, there were 20 dose reductions for the gemcitabine/carboplatin group and 19 dose reductions for the BA + gemcitabine/carboplatin group. The safety of adding BA in gemcitabine/carboplatin was further confirmed considering that the dose given in cohort B was approximately three times that of cohort a.

The evaluation of adverse reactions (AE) showed that the two study groups were comparable (risk ratio 1.0, not corrected for the total time of the study; table 7).

TABLE 7 adverse reactions

Disorders of the kidney and urinary system	4	2			6	4				4
											Disorders of the reproductive system and of the breast	2				2	1				1
Respiratory, thoracic and diaphragm disorders	13	5	2		20	4	2			6

Disorders of the skin and subcutaneous tissue	9	6			15	7	1			8
											Surgical and medical procedures			1		1	2				2
Disorders of the vascular system	6	2			8	2				2
											Total of	107	70	29	9	215	93	39	15	4	151
Ratio of risks										1.0

Therapeutic effect

Patients of trial cohort a (gemcitabine/carboplatin only) showed evidence of disease progression much earlier than patients randomized to trial cohort B (gemcitabine/carboplatin + BA). Approximately 50% of subjects in group a showed disease progression at the end of cycle 2, while only 15% of subjects in group B showed disease progression at the same time.

Formal statistical analysis was performed using available preliminary data. This analysis shows that patients receiving BA plus gemcitabine/carboplatin had a significantly longer median PFS compared to patients receiving gemcitabine/carboplatin alone (day 211 vs. day 67; P < 0.0001; FIG. 5).

Preliminary assessments of Clinical Benefit Rate (CBR) (CBR-120 and CBR-180, respectively) were performed for patients enrolled in the study for 120 days and 180 days, respectively, and are shown in Table 8.

Table 8: preliminary assessment of clinical efficacy

Terminal point	Grouping A: gemcitabine/carboplatin	Grouping B: Gemcitabine/Carboplatin + BSI-201	P
				Median PFS	67 days	211 days	＜0.0001
CBR-180a	5/20(25％)	10/20(50％)	0.1908
				CBR-180b	6/20(30％)	14/20(70％)	0.0256

^aThe first 40 patients enrolled, SD (180 days) + PR + CR;^bthe first 40 patients enrolled, SD (120 days) + PR + CR

The determination of PR includes confirmed and unconfirmed responses.

The results show that the gemcitabine/carboplatin + BA grouping has a higher CBR trend.

Conclusion of preliminary study

Based on the above results, the following conclusions can be drawn for the phase 2 metastatic TNBC study:

patients who received the appropriate protocol were enrolled. Using traditional Immunohistochemistry (IHC) and gene expression profiling, the results of the genomic expression profiling of 50 patients initially enrolled in the trial showed that these patients were indeed both ER negative and PR negative and did not overexpress HER 2.

The patient cases of both study groups were comparable.

Omicron statistics showed that the median age and performance of each group were similar.

The degree of antecedent chemotherapy in the transferred cases was similar for both groups. The data for the first 69 patients showed no significant difference in pretreatment status for any of that cohort. The BA cohort had more patients receiving 2 courses of prior chemotherapy, suggesting that these subjects may be more resistant to chemotherapy than subjects receiving gemcitabine/carboplatin alone.

The cases of dose reduction were similar for both groups, whether in percentage of patients with reduced dose or total population with reduced dose. In the chemotherapy-only cohort, 15 (38.9%) of 39 subjects had dose reductions, and in the BA + chemotherapy cohort, 11 (28.2%) of 39 subjects had dose reductions. Overall, there were 20 dose reductions for the gemcitabine/carboplatin group and 19 dose reductions for the BA + gemcitabine/carboplatin group.

The AE ratios of the two groups are similar, supporting the conclusion that: the addition of BA to gemcitabine/carboplatin did not potentiate known toxicity or cause any new toxicity.

Patients receiving BA plus gemcitabine/carboplatin showed significant clinical benefit compared to those receiving gemcitabine/carboplatin alone based on median analysis of the progression-free survival status (day 211 vs. day 67; P < 0.0001). These results represent a significant improvement in PFS status compared to other metastatic TNBC studies.

Analysis of the clinical benefit rates of the first 40 patients enrolled in the trial showed a trend towards improvement upon the addition of BA to gemcitabine/carboplatin. This effect is believed to become more pronounced as the study matures.

Example 8: treatment of breast cancer with paclitaxel, carboplatin and BA combinations

Patients had triple negative metastatic breast cancer and were documented as having progressed. Histological confirmation of the primary tumor is required.

All patients had measurable disease. Measurable disease refers to a condition having at least one dimension that can be accurately measured in at least one direction (the longest dimension to be recorded). Each lesion must be greater than or equal to 20mm when measured by conventional techniques including palpation, X-ray plain film, CT and MRI, or greater than or equal to 10mm when measured by spiral CT.

Patients will have at least one "target lesion" for assessing a response to the present protocol, as defined by RECIST (section 8.1). Tumors within the previously irradiated region will be referred to as "non-target" lesions unless the lesion lasts for at least 90 days after the end of radiation treatment as documented by progression of the disease or as confirmed by biopsy. In addition, the patient must have recovered from recent surgery, radiation therapy, or other treatment and should be free of active infections that require the use of antibiotics.

Any hormone therapy for malignancy must be discontinued at least one week prior to enrollment. Allowing hormone replacement therapy to continue.

The patient must have enough:

bone marrow function: platelet count greater than or equal to 100,000/microliter and ANC count greater than or equal to 1,500/microliter, corresponding to CTCAE version 3.0, grade 1.

Renal function: creatinine less than or equal to 1.5 times Upper Limit of Normal (ULN), CTCAE version 3.0, grade 1.

Liver function: bilirubin is less than or equal to 1.5XULN (CTCAE version 3.0, grade 1). SGOT and alkaline phosphatase are less than or equal to 2.5XULN (CTCAE version 3.0, grade 1).

Nerve function: neuropathy (sensory and motor) is less than or equal to CTCAE version 3.0, grade 1.

Patients who may be pregnant must have negative serum pregnancy test results and take effective contraceptive measures before taking part in the study.

Non-eligible patients:

patients receiving advanced cytotoxic chemotherapy for breast cancer control.

Patients with a history of other invasive malignancies will be excluded if there is any evidence of other malignancies within the last five years, except for non-melanoma skin cancers and other specific malignancies noted in sections 3.23 and 3.24. Patients are also excluded if their previous cancer treatment conflicts with the treatment of the trial regimen.

Patients who have received advanced radiation therapy in any part of the abdominal cavity or pelvis other than for breast cancer therapy for the past 5 years will be excluded. Advanced radiation of localized breast, head and neck or skin cancers is permissible as long as treatment has ended more than three years prior to registration and the patient's disease has not recurred or metastasized.

The patient may have received prior adjuvant chemotherapy for localized breast cancer, as long as treatment has ended three years or more prior to registration and the patient's disease has not relapsed or metastasized.

Symptomatic or untreated brain metastatic tumors require concurrent therapy including, but not limited to, surgery, radiation therapy, and corticosteroid therapy.

Myocardial Infarction (MI), unstable angina, Congestive Heart Failure (CHF) above class II, New York Heart Association (NYHA) standard, or uncontrolled hypertension within 6 months on study day 1.

There is a history of epilepsy or antiepileptic drugs are currently being taken.

Research mode

Carboplatin (Parasplatin)，NSC#241240)

The formula is as follows: carboplatin was provided as a sterile lyophilized powder, contained in single dose vials containing 50mg, 150mg, and 450mg of carboplatin for intravenous infusion. Each vial contained equal parts by weight of carboplatin and mannitol.

Solution preparation: immediately prior to use, the contents of each vial must be prepared as a solution using usp injection grade sterile water, 5% dextrose in water, or usp 0.9% sodium chloride injection according to the following table:

vial content dilution volume

50mg 5ml

150mg 15ml

450mg 45ml

These dilutions will result in a carboplatin concentration of 10 mg/ml.

Note: aluminum can react with carboplatin leading to the formation and failure of precipitates. Thus, needles or intravenous drug delivery devices containing aluminum parts that may come into contact with the drug must not be used for the preparation or administration of carboplatin.

And (3) storage: the unopened carboplatin vial was stable when stored under controlled ambient temperature and light conditions, with the lifetime indicated in the package.

Stability: the carboplatin solution remained stable for 8 hours at room temperature when prepared as indicated. Since the formulation does not contain an antimicrobial preservative, it is recommended that the carboplatin solution be discarded 8 hours after dilution.

The supplier: commercially available from Bristol-Myers Squibb Company.

Taxol (Taxol)，NSC#673089)

The formula is as follows: paclitaxel is a poorly soluble plant product extracted from Taxus baccata. Improving solubility requires a mixed solvent system that is further diluted with 0.9% sodium chloride or 5% aqueous dextrose.

Paclitaxel was provided as a sterile concentrated solution at 6mg/ml in 5ml vials (30 mg/vial) in 50% polyoxyethylated castor oil (Cremophor EL) and United states Pharmacopeia grade 50% anhydrous ethanol. The vial must be diluted immediately prior to clinical use. It may also be provided in the form of 100mg and 300mg vials.

Solution preparation: paclitaxel will be diluted in an appropriate dose in 500-. Paclitaxel must be prepared in glass or polyolefin containers, since the Cremophor solvent that dissolves paclitaxel leaches the diethylhexyl phthalate (DEHP) plasticizer from polyvinyl chloride (PVC) bags and intravenous tubing.

Note: after paclitaxel was prepared, a small amount of fiber formation was observed in the solution (within acceptable limits specified in the usp small volume injection microparticle test). Thus, in-line filtration is necessary for paclitaxel solution administration. A hydrophilic microporous filter (such as IVEX-II, IVEX-HP or the like) having a pore size of no greater than 0.22 μm should be placed in the intravenous route, remote from the infusion pump, to achieve in-line filtration. Although the formation of microparticles does not indicate drug failure, a solution with too much microparticle formation should not be used.

And (3) storage: intact vials can be stored in the original package at a temperature range of 20-25 ℃ (36-77 ° f). Freezing or cold storage does not cause adverse reaction to the stability of the product.

Stability: although paclitaxel solutions (0.3-1.2mg/ml) were physically and chemically stable for 27 hours at ambient temperature (about 25 ℃) and room lighting when prepared according to the above procedure, all paclitaxel solutions were slightly hazy to the extent that it was proportional to the drug concentration and the length of time after preparation.

The supplier: commercially available from Bristol-Myers Squibb Company.

Administration: paclitaxel will be administered as a 3 hour continuous intravenous infusion at the appropriate dosage and dilution conditions. Paclitaxel will be administered by infusion control devices (pumps) using non-polyvinyl chloride tubing and connectors, such as intravenous injection devices (polyethylene or polyolefin) for intravenous infusion of nitroglycerin. No other drugs should be infused through the line where the paclitaxel administration is taking place. See section 5.2.

BA (4-iodo-3-nitrobenzamide)

BA will be produced and packaged on behalf of BiPar scientific and distributed using a BiPar approved clinical trial drug distribution program. BA will be provided in a 10ml bottle with a single input of liquid sterile product. BA was prepared in 25% hydroxypropyl-beta-cyclodextrin/10 mM phosphate buffer solution at pH 7.4 and an active ingredient concentration of 10 mg/ml. Each bottle contains an extractable amount of not less than 9.0 ml. The information on the study drug label will comply with ICH requirements of the U.S. Food and Drug Administration (FDA). The bulk bottles of BA will be shipped in cartons of 10 bottles each and identified with a single label. The tag will contain the following information: warning words in the united states for research drugs, study number, product name, concentration, storage precautions, date of retest, and name of the sponsor of the study.

Solution preparation: BA will be prepared and administered intravenously as follows for one hour:

the baseline body weight of the subject was multiplied by the dose level to calculate the amount of BA required for administration (4 mg/kg). For example

Subject baseline body weight 70kg

The dosage is 4mg/kg

The required dose is 280mg BA (4mg/kgx70kg)

The desired BA dose was divided by the concentration of BA in the vial (10mg/ml) to determine the ml of BA drug required for administration. For example:

280mg÷10mg/mL＝28mL

the number of BA vials was counted at 10ml per vial to obtain the required volume. (in this example, 3 vials would be required.) to obtain the desired volume of BA, more vials may be used if necessary.

The appropriate volume of BA medication is drawn from the vial with a syringe and set aside in preparation for an iv bag as described below.

It is recommended that the iv bag be filled with a total of 250ml of solution for a one hour administration time. 0.9% physiological saline or D5W was used as an intravenous infusion solution. If infusion is initiated with an intravenous bag containing more than 250ml of the solution, the excess solution is removed and discarded, and the entire drug is added to the solution. The calculated amount of BA drug was injected into the iv bag and sufficient mixing was ensured. An iv line was attached and replaced with the solution. Note: an empty iv bag can also be used, the calculated BA amount injected, then 0.9% saline or 5DW added until a total volume of 250ml is reached. This may be useful for BA volumes greater than 50 ml.

And (3) storage: the BA vial must be stored at a temperature of 2-8 deg.C in the dark. The medicine bottle is stored in the original paper box and is put into a temperature control device at 2-8 ℃. If desired, BA can be stored at 25 ℃ for 24 hours. If it was determined that BA had not been previously processed under these storage conditions, please contact BiPar corporation immediately. Without authorization from the BiPar company, drugs that are not stored under recommended storage conditions are not used.

Stability: BA should be administered within 8 hours after preparation. The infusion solution should be kept at ambient temperature (room temperature) prior to administration to the subject.

The supplier: BiPar Sciences Inc.

Treatment planning

Infusion of 175mg/m was followed by carboplatin infusion for 30 minutes (AUC ═ 6.0) every 21 days on day 1²Paclitaxel, at 3 hours, was dosed with BA at 4mg/ml/kg twice weekly from day 1, at 1 hour (BA dosing must be at least 2 days apart) until disease progression or adverse effect limits further treatment. This three week period is considered a treatment cycle. The number of cycles after which a complete clinical response is achieved will be determined by the treating physician. Patients who do not meet the criteria for disease progression (local response or stable disease) should continue to receive study treatment until toxicity limits.

Dose of carboplatin administered: the dose was calculated according to the Calvert (Calvert) formula using the Glomerular Filtration Rate (GFR) assessed according to the Jelliffe (Jelliffe) formula, as the area of interest under the concentration curve (AUC) times time. The initial dose will be AUC 6 over 30 min infusion.

The initial dose of carboplatin must be calculated using GFR. In the absence of new renal dysfunction or other grade 2 (serum creatinine > 1.5 × ULN) nephrotoxicity above or equal to CTCAE version 3.0, the dose of carboplatin will not be recalculated for subsequent cycles, but will be adjusted as specified.

For patients with abnormally low serum creatinine (less than or equal to 0.6mg/dl), a minimum of 0.6mg/dl should be used to estimate creatinine clearance due to reduced protein intake and/or reduced muscle mass. It can also be used to make a preliminary assessment of GFR if there is a more appropriate baseline creatinine value over a 4 week treatment period.

The karfft formula: carboplatin dose (mg) target AUC x (GFR + 25).

For the purposes of this protocol, GFR is considered equivalent to creatinine clearance. Creatinine clearance (Ccr) was estimated according to the jarovist method using the following formula: {98- [0.8 (age-20) ] } Ccr ═ 0.9 × Scr, where: ccr is the estimated creatinine clearance (ml/min); age (20-80) of the patient; serum creatinine (mg/dl). In the absence of new renal dysfunction or serum creatinine above 1.5 × ULN (CTCAE version 3.0, grade 2), the dose of carboplatin will not be recalculated for subsequent cycles, but will be adjusted according to the indicated hematologic criteria or otherwise.

Proposed chemotherapy management methods: the protocol may be implemented in an outpatient setting. Paclitaxel will be infused for 3 hours followed by 30 minutes of carboplatin followed by 1 hour of BA. BA will be administered as an intravenous infusion (1 hour infusion) twice weekly during the study. The administration of BA must be at least 2 days apart (e.g., on monday/thursday, monday/friday, or tuesday/friday). For paclitaxel and carboplatin treatment on day 1, an antiemetic regimen was proposed. The antiemetic regimen used should be based on consensus criteria of peer review. For treatment with BA alone, the use of prophylactic antiemetics is not required.

Preparation course of paclitaxel: in this study, paclitaxel will be administered as an infusion for 3 hours. For all cycles of paclitaxel administration, a preliminary course of paclitaxel therapy is recommended to reduce the risks associated with allergic reactions. The treatment course should include dexamethasone (intravenous infusion or oral administration), antihistamine H1 (such as diphenhydramine), and antihistamine H2 (such as cimetidine, ranitidine, or famotidine).

The maximum body surface area for dose calculation will be 2.0m²。

If the side effects are not severe, the patient may use the study medication indefinitely, as determined by the study moderator. Patients who achieve a complete clinical response may continue for further cycles at the discretion of the treating physician.

Evaluation criteria

Release Parameters (Parameters of Response) -RECIST criteria

Measurable disease refers to a condition having at least one dimension that can be accurately measured in at least one direction (the longest dimension to be recorded). Each lesion must be greater than or equal to 20mm when measured by conventional techniques including palpation, X-ray plain film, CT and MRI, or greater than or equal to 10mm when measured by spiral CT.

Baseline recording of "target" and "non-target" lesions

All measurable lesions representing all organs involved, up to 5 lesions per organ, and 10 lesions in total, should be identified as target lesions and recorded and measured at baseline. The target lesions should be selected according to their size (with the largest size lesion) and whether they are suitable for accurate measurement with repeatable measurement means (imaging techniques or clinical measurements). The sum of the maximum size (LD) of all target lesions will be calculated and reported as the baseline LD sum. This baseline LD sum will serve as a reference to further characterize the objective tumor response of the measurable disease.

All other lesions (or disease sites) should be identified as non-target lesions and should also be recorded at baseline. No measurements need to be taken, but these lesions should be tracked and recorded as "present" or "absent".

The baseline assessment of all disease conditions should be as close as possible to the assessment at the start of treatment and must not be performed 4 weeks prior to the start of treatment.

Best response (best response)

The longest dimension of each lesion needs to be measured in order to track the condition. From these changes in the sum of sizes, changes in tumor size and therapeutic efficacy can be estimated. All diseases must be assessed using the same technique as a benchmark. The above-mentioned changes in a case should be reported as the best response achieved after the patient has participated in the study.

Complete Remission (CR) refers to the disappearance of all target and non-target lesions, with no evidence of new lesions in two disease assessments separated by at least 4 weeks.

Partial Remission (PR) means a decrease of at least 30% in the sum of the Longest Dimensions (LD) of all measurable target lesions, referenced to the sum of baseline LD. There was no clear progression of non-target lesions nor new lesions. Two disease assessments are required to be at least 4 weeks apart. In the case where the only target lesion is an isolated pelvic mass (not measurable with radiographic imaging) measured by physical examination, a 50% reduction in LD is required.

By "exacerbated disease" is meant an increase in the sum of LD of the target lesion of at least 20% with reference to the minimum sum of LD, or the appearance of new lesions within 8 weeks of study participation. According to the opinion of the treating physician, the clear progression of non-target lesions (rather than pleural effusion without evidence of cytological evidence of tumor origin) within 8 weeks of study participation was also considered an exacerbated disease (in which case an explanation must be provided). In the case where the only target lesion is an isolated pelvic mass (not measurable with radiographic imaging) measured by physical examination, a 50% rise in LD is required.

The worsening of symptoms is defined as a general worsening of the health condition due to the disease, requiring changes in therapy without objective evidence of disease progression.

Stable disease refers to any condition that does not meet the above criteria.

Failure to assess remission is defined as the failure to repeat tumor assessment after study treatment has begun for reasons unrelated to symptoms or signs of disease.

Progression (study of measurable disease) is defined as any of the following:

the sum of LD of the target lesions was increased by at least 20% with reference to the smallest sum of LD recorded since study entry.

With reference to the minimum sum of LDs recorded since study participation, a 50% rise in LD is required in cases where the only target lesion is an isolated pelvic mass (not measurable by radiographic imaging) measured by physical examination.

One or more new lesions appear.

Death due to disease but no objective record of previous disease progression.

Since the disease causes a general deterioration in health status, there is a need for changing therapies without objective evidence of disease progression.

According to the opinion of the treating physician, there is a clear progression of non-target lesions, rather than pleural effusion without cytological evidence of tumor origin (in which case an explanation must be provided).

Recurrence (study of non-measurable disease) refers to evidence of increased clinical, radiological, or histological disease since participation in clinical studies.

Survival refers to the length of life observed from study participation until death or the last exposure.

"Progression-Free Survival" (study in which disease can be measured) refers to the period from study participation until disease Progression, death or last exposure.

"Recurrence-Free Survival" (study of unmeasurable disease) refers to the period from study participation until disease Recurrence, death or last exposure.

Subjective parameters including performance status, specific symptoms and side effects were graded according to CTCAE version 3.0.

Duration of study

Patients will receive treatment until disease progression or intolerable toxic intervention. Patients may reject study treatment at any time. Patients who have a complete clinical response after treatment will continue to receive more cycles of treatment at the discretion of the treating physician. Patients with a stable local response or condition should continue to receive treatment unless intolerable toxicity prevents further treatment.

All patients will receive treatment (and fill out all required case reports) until disease progression or study withdrawal. Patients will then receive follow-up visits (including physical examination and medical history) every 3 months for the first two years, and every 6 months for the following 3 years. During this 5 year, delayed toxicity will be monitored and patients will be informed of survival. Patients must submit questionnaires to the GOG statistics and data center unless consent is withdrawn.

Example 9: combination of 4-iodo-3-nitrobenzamide (BA) with gamma radiation

Triple negative breast cancer cells MDA-MB-468 were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cell culture dish 10 for each P100 cell⁵Individual cell or in P60 cell culture dishes 10⁴Individual cells were plated in the presence of different concentrations of compound or DMSO control. After treatment, adherent cell numbers were measured using a Coulter counter by staining with 1% methylene blue. Methylene blue is dissolved in a 50% -50% mixture of methanol and water. Cells were plated in 24-well or 96-well plates and treated as planned, the medium aspirated, the cells washed with PBS, fixed in methanol for 5-10 minutes, the methanol aspirated and the plates allowed to dry completely. Methylene blue solution was added to the wells and the plates were incubated for 5 minutes. Remove staining solution and use dH₂The plates were washed until the wash was no longer blue. After the plate was completely dried, a small amount of 1N HCl was added to each well to extract methylene blue. The OD reading at 600nm and a calibration curve were used to determine cell number.

The BA compound was dissolved directly from the dry powder to a 10mM stock in DMSO for each independent experiment. Control experiments were performed with matching volumes/concentrations of vehicle (DMSO); in these controls, the growth or cell cycle distribution of the cells showed no change.

The PI exclusion assay, cell cycle assay, TUNEL assay and BrdU labeling assay were performed as described above in example 2.

MDA-MB-468 cancer cells were treated with 3 Gray gamma irradiation with or without 100. mu.M BA. As shown in FIG. 6, BA potentiates S-and G2/M cell cycle arrest and enhances the anti-proliferative effects of gamma radiation in human triple negative MDA-MB-468 breast cancer cells.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and alternatives will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention. Methods and structures within the scope of these claims and equivalents thereof are also intended to be encompassed by this scope.

Claims (121)

1. A method of treating breast cancer which is negative for at least one receptor of ER, PR or HER2 in a patient comprising administering to the patient at least one PARP inhibitor.

2. The method of claim 1, wherein at least one therapeutic effect is obtained, said at least one therapeutic effect being breast tumor size reduction, metastasis reduction, complete remission, partial remission, stable disease, or complete response to pathology.

3. The method of claim 1 wherein comparable clinical benefit rates (CBR ═ CR + PR + SD ≧ 6 months) are achieved with PARP inhibitors as compared to antineoplastic agent treatment.

4. The method of claim 3, wherein the improvement in clinical benefit rate is at least about 30% compared to treatment with the anti-neoplastic agent alone.

5. The method of claim 1 wherein the PARP inhibitor is a PARP-1 inhibitor.

6. The method of claim 5, wherein the PARP-1 inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof.

7. The method of claim 1, wherein the PARP inhibitor is of formula (IIa) or a metabolite thereof; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof:

Formula (IIa)

Wherein, (1) R₁、R₂、R₃、R₄And R₅At least one of the substituents being always a sulfur-containing substituent, the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituent, and R₁、R₂、R₃、R₄And R₅At least one of the 5 substituents is always iodine, and said iodine is always associated with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The substituents are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent.

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

9. The method of claim 1, wherein the breast cancer is stage I, stage II, or stage III.

10. The method of claim 1, wherein the breast cancer is negative for at least one of ER, PR, or HER 2; and wherein the breast cancer is positive for at least one of ER, PR, or HER 2.

11. The method of claim 1, wherein the breast cancer is deficient in homologous recombination DNA repair.

12. The method of claim 1, wherein the breast cancer has impaired BRCA1 or BRCA2 function.

13. The method of claim 1, wherein treating comprises a treatment cycle of at least 11 days, wherein on days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 1 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a molar equivalent thereof, of a metabolite.

14. The method of claim 13, wherein the 4-iodo-3-nitrobenzamide is administered orally, or parenterally by injection or infusion, or by inhalation.

15. The method of claim 13, wherein the treatment cycle is from about 11 days to about 30 days.

16. The method of claim 1 further comprising administering to the patient a PARP inhibitor in combination with at least one anti-neoplastic agent.

17. The method of claim 16, wherein the anti-neoplastic agent is an anti-tumor alkylating agent, an anti-tumor antimetabolite agent, an anti-tumor antibiotic, a plant-derived anti-neoplastic agent, an anti-tumor platinum complex, an anti-tumor camptothecin derivative, an anti-tumor tyrosine kinase inhibitor, a monoclonal antibody, an interferon, a biological response modifier, a hormonal anti-neoplastic agent, an anti-tumor viral agent, an angiogenesis inhibitor, a differentiation inducing agent, a PI3K/mTOR/AKT inhibitor, a cell cycle inhibitor, an apoptosis inhibitor, an hsp 90 inhibitor, a tubulin inhibitor, a DNA repair inhibitor, an anti-angiogenic agent, a receptor tyrosine kinase inhibitor, a topoisomerase inhibitor, a taxane, a Her-2 targeting agent, a hormone antagonist, a growth factor receptor targeting agent, or a pharmaceutically acceptable salt thereof.

18. The method of claim 16 wherein the anti-neoplastic agent is decitabine, capecitabine, valopicitabine, or gemcitabine.

19. The method of claim 16, wherein the antineoplastic agent is selected from the group consisting of bevacizumab, sunitinib, sorafenib, cediranib, ABT-869, axitinib, irinotecan, topotecan, paclitaxel, docetaxel, lapatinib, trastuzumab, lapatinib, tamoxifen, a steroid aromatase inhibitor, a non-steroid aromatase inhibitor, fulvestrant, an Epidermal Growth Factor Receptor (EGFR) inhibitor, cetuximab, panitumumab, an insulin-like growth factor 1 receptor (IGF1R) inhibitor, and CP-751871.

20. The method of claim 16 further comprising administering to the patient a PARP inhibitor in combination with at least one anti-neoplastic agent.

21. The method of claim 16 wherein the anti-neoplastic agent is administered prior to, concurrently with, or subsequent to the administration of the PARP inhibitor.

22. The method of claim 1, further comprising surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, DNA therapy, adjuvant therapy, neoadjuvant therapy, immunotherapy, nanotherapy or a combination thereof.

23. The method of claim 1 further comprising administering to the patient a PARP inhibitor in combination with gamma radiation.

24. A method of treating breast cancer that is negative for at least one of ER, PR, or HER2 in a patient in need thereof, comprising:

(a) obtaining a sample from a patient;

(b) the sample was tested to determine each

(i) Whether the cancer is ER positive or ER negative;

(iii) whether the cancer is PR positive or PR negative;

(iii) whether the cancer is HER2 positive or HER2 negative;

(c) treating the patient with at least one PARP inhibitor if the test indicates that the cancer is negative for at least one of ER, PR, or HER 2.

25. The method of claim 24, further comprising treating the patient with at least one PARP inhibitor if the following two or more conditions are met:

(a) the cancer is ER-negative and the cancer is,

(b) the cancer is negative for PR and has a positive effect,

(c) the cancer was HER2 negative.

26. The method of claim 24, wherein at least one therapeutic effect is obtained, said at least one therapeutic effect being a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, stable disease, or complete response to pathology.

27. The method of claim 24, wherein the PARP inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof.

28. The method of claim 24, wherein the PARP inhibitor is of formula (IIa) or a metabolite thereof; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof:

formula (IIa)

Wherein: (1) r₁、R₂、R₃、R₄And R₅At least one of the substituents being always a sulfur-containing substituent, the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₃、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituent, and R₁、R₂、R₃、R₄And R₅At least one of the 5 substituents is always iodine, and wherein said iodine is always present with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent; in some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent.

29. The method of claim 24, wherein the sample is a tissue or body fluid sample.

30. The method of claim 29, wherein the sample is a tumor sample, a blood sample, a plasma sample, a peritoneal fluid sample, an exudate or a effusion.

31. The method of claim 24, wherein the breast cancer is metastatic breast cancer.

32. The method of claim 24, wherein the breast cancer is stage I, II or III.

33. The method of claim 24, wherein the breast cancer is negative for at least one of ER, PR, or HER 2; and wherein the breast cancer is positive for at least one of ER, PR, or HER 2.

34. The method of claim 24, wherein the breast cancer is deficient in homologous recombination DNA repair.

35. The method of claim 24, wherein the breast cancer has impaired BRCA1 or BRCA2 function.

36. The method of claim 24, wherein treating comprises selecting a treatment cycle of at least 11 days, and on days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a molar equivalent thereof, of a metabolite.

37. The method of claim 24, wherein the 4-iodo-3-nitrobenzamide is administered orally, or parenterally by injection or infusion, or by inhalation.

38. A method of treating breast cancer that is negative for at least one of ER, PR, or HER2 in a patient, comprising:

(a) testing a sample taken from the patient for PARP expression; and

(b) administering at least one PARP inhibitor to the patient if the PARP expression exceeds a predetermined level.

39. The method of claim 38, wherein at least one therapeutic effect is obtained, said at least one therapeutic effect being tumor size reduction, metastasis reduction, complete remission, partial remission, stable disease, or complete response to pathology.

40. The method of claim 38, wherein the PARP inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof.

41. The method of claim 38, wherein the PARP inhibitor is of formula (IIa) or a metabolite thereof; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof:

formula (IIa)

Wherein: (1) r₁、R₂、R₃、R₄And R₅At least one of the substituents being always a sulfur-containing substituent, the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R ₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituent, and R₁、R₂、R₃、R₄And R₅At least one of these 5 substituents is always iodine, and where the iodine is always present with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent.

42. The method of claim 38, wherein the breast cancer is metastatic breast cancer.

43. The method of claim 38, wherein the breast cancer is stage I, II, or III.

44. The method of claim 38, further comprising determining the expression of estrogen receptor, progesterone receptor, or human epidermal growth factor 2 receptor in a sample taken from the patient.

45. The method of claim 38, wherein the breast cancer is negative for at least one of ER, PR, or HER 2; and wherein the breast cancer is positive for at least one of ER, PR, or HER 2.

46. The method of claim 38, wherein the breast cancer is deficient in homologous recombination DNA repair.

47. The method of claim 38, wherein the breast cancer has impaired BRCA1 or BRCA2 function.

48. The method of claim 38, wherein the sample is a tissue or body fluid sample.

49. The method of claim 38, wherein treating comprises selecting a treatment cycle of at least 11 days, and on days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a molar equivalent thereof, of a metabolite.

50. The method of claim 49, wherein the 4-iodo-3-nitrobenzamide is administered orally, or parenterally by injection or infusion, or by inhalation.

51. A method of treating breast cancer in a patient comprising administering to the patient at least one PARP inhibitor in combination with at least one anti-neoplastic agent.

52. The method of claim 51, wherein at least one therapeutic effect is obtained, said at least one therapeutic effect being a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, stable disease, or a complete response to pathology.

53. The method of claim 51 wherein the clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment with an anti-neoplastic agent but without a PARP inhibitor.

54. The method of claim 53, wherein the improvement in clinical benefit rate is at least about 60%.

55. The method of claim 51, wherein the PARP inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof.

56. The method of claim 51, wherein the PARP inhibitor is of formula (IIa) or a metabolite thereof; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof:

formula (IIa)

Wherein: (1) r₁、R₂、R₃、R₄And R₅At least one of the substituents is always a sulfur-containing substituent, and the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituent, and R₁、R₂、R₃、R₄And R₅At least one of these 5 substituents is always iodine, and where the iodine is always present with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group ₁、R₂、R₃、R₄Or R₅The substituents are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent.

57. The method of claim 51, wherein the anti-neoplastic agent is an anti-tumor alkylating agent, an anti-tumor antimetabolite agent, an anti-tumor antibiotic, a plant-derived anti-neoplastic agent, an anti-tumor platinum complex, an anti-tumor camptothecin derivative, an anti-tumor tyrosine kinase inhibitor, a monoclonal antibody, an interferon, a biological response modifier, a hormonal anti-neoplastic agent, an anti-tumor viral agent, an angiogenesis inhibitor, a differentiation inducing agent, a PI3K/mTOR/AKT inhibitor, a cell cycle inhibitor, an apoptosis inhibitor, an hsp 90 inhibitor, a tubulin inhibitor, a DNA repair inhibitor, an anti-angiogenic agent, a receptor tyrosine kinase inhibitor, a topoisomerase inhibitor, a taxane, a Her-2 targeting agent, a hormone antagonist, a growth factor receptor targeting agent, or a pharmaceutically acceptable salt thereof.

58. The method of claim 51 wherein the anti-neoplastic agent is decitabine, capecitabine, valopicitabine, or gemcitabine.

59. The method of claim 51, wherein the anti-neoplastic agent is selected from the group consisting of bevacizumab, sunitinib, sorafenib, cediranib, ABT-869, axitinib, irinotecan, topotecan, paclitaxel, docetaxel, lapatinib, trastuzumab, lapatinib, tamoxifen, steroid aromatase inhibitors, non-steroid aromatase inhibitors, fulvestrant, an Epidermal Growth Factor Receptor (EGFR) inhibitor, cetuximab, panitumumab, an insulin-like growth factor 1 receptor (IGF1R) inhibitor, and CP-751871.

60. The method of claim 51, further comprising surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, viral therapy, DNA therapy, adjuvant therapy, neoadjuvant therapy, RNA therapy, immunotherapy, nanotherapy or a combination thereof.

61. The method of claim 51 further comprising administering to the patient a PARP inhibitor in combination with gamma radiation.

62. The method of claim 51, wherein said breast cancer is metastatic breast cancer.

63. The method of claim 51, wherein the breast cancer is stage I, stage II, or stage III.

64. The method of claim 51, wherein the breast cancer is HR negative breast cancer.

65. The method of claim 51, wherein the breast cancer is negative for at least one of ER, PR, or HER 2.

66. The method of claim 51, wherein the breast cancer is negative for at least one of ER, PR, or HER 2; and wherein the breast cancer is positive for at least one of ER, PR, or HER 2.

67. The method of claim 51, wherein the breast cancer is deficient in homologous recombination DNA repair.

68. The method of claim 51, wherein the breast cancer has impaired BRCA1 or BRCA2 function.

69. The method of claim 51, wherein treating comprises a treatment cycle of at least 11 days, wherein:

(a) on days 1 and 8 of the cycle, patients received approximately 100-5000mg/m²Gemcitabine of (1);

(b) on days 1 and 8 of the cycle, the patient receives from about 10 to about 400mg/m²Carboplatin of (a); and

(c) on days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 1 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof of comparable molar content.

70. The method of claim 69, wherein the treatment cycle is from about 11 days to about 30 days.

71. The method of claim 69 wherein the patient receives about 100 and 2500mg/m of the antibody on days 1 and 8 of the cycle²And from about 10 to about 400mg/m of gemcitabine²Carboplatin of (a); and on days 1, 4, 8, and 11 of the cycle, the patient receives from about 1 to about 50mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof of comparable molar content.

72. The method of claim 69 wherein the patient receives about 500-2000mg/m on days 1 and 8 of the cycle²And from about 50 to about 400mg/m of gemcitabine²Carboplatin of (a); and on days 1, 4, 8 and 11 of the cycle, the patient receives from about 1 to about 50mg/kg of 4-iodine -3-nitrobenzamide or metabolites thereof in molar equivalent amounts.

73. The method of claim 69, wherein the patient receives about 1000mg/m on days 1 and 8 of the cycle²Gemcitabine and carboplatin for about AUC 2 of (a); and on days 1, 4, 8 and 11 of the cycle, the patient receives about 1, 2, 3, 4, 6, 8 or 10, 12, 14, 16, 18 or 20mg/kg of 4-iodo-3-nitrobenzamide.

74. The method of claim 51 wherein the antineoplastic agent is administered by parenteral injection or infusion.

75. The method of claim 51, wherein the PARP inhibitor is 4-iodo-3-nitrobenzamide administered orally, or parenterally by injection or infusion, or by inhalation.

76. The method of claim 51, further comprising administering to the patient a taxane by parenteral injection or infusion.

77. A method of treating breast cancer in a patient in need of treatment comprising:

(a) obtaining a sample from a patient;

(b) the sample was tested to determine each of the following:

(i) whether the cancer is ER positive or ER negative;

(ii) whether the cancer is PR positive or PR negative;

(iii) whether the cancer is HER2 positive or HER2 negative;

(c) treating the patient with a combination of therapeutic agents if the test indicates that at least one of the ER, PR, or HER2 is negative for the cancer, wherein the therapeutic agents include at least one PARP inhibitor and at least one antineoplastic agent.

78. The method of claim 77, further comprising treating the patient with a combination of therapeutic agents if two or more of the following conditions are met, wherein the therapeutic agents comprise at least one PARP inhibitor and at least one antineoplastic agent:

(a) the cancer is ER-negative and the cancer is,

(b) the cancer is negative for PR and has a positive effect,

(c) the cancer was HER2 negative.

79. The method of claim 77, wherein at least one therapeutic effect is obtained, said at least one therapeutic effect being a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, stable disease, or a complete response to pathology.

80. The method of claim 77, wherein clinical benefit rate (CBR ═ CR + PR + SD ≧ 6 months) is improved as compared to treatment with an anti-neoplastic agent but without a PARP inhibitor.

81. The method of claim 80, wherein the clinical benefit rate is at least about 60%.

82. The method of claim 77, wherein the PARP inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof.

83. The method of claim 77, wherein the PARP inhibitor is of formula (IIa) or a metabolite thereof; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof:

Formula (IIa)

Wherein: (1) r₁、R₂、R₃、R₄And R₅At least one of the substituents is always a sulfur-containing substituent, and the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituent, and R₁、R₂、R₃、R₄And R₅At least one of these 5 substituents is always iodine, and where the iodine is always present with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The substituents are adjacent.

84. The method of claim 77, wherein the anti-neoplastic agent is an anti-tumor alkylating agent, an anti-tumor antimetabolite agent, an anti-tumor antibiotic, a plant-derived anti-neoplastic agent, an anti-tumor platinum complex, an anti-tumor camptothecin derivative, an anti-tumor tyrosine kinase inhibitor, a monoclonal antibody, an interferon, a biological response modifier, a hormonal anti-neoplastic agent, an anti-tumor viral agent, an angiogenesis inhibitor, a differentiation inducing agent, a PI3K/mTOR/AKT inhibitor, a cell cycle inhibitor, an apoptosis inhibitor, an hsp 90 inhibitor, a tubulin inhibitor, a DNA repair inhibitor, an anti-angiogenic agent, a receptor tyrosine kinase inhibitor, a topoisomerase inhibitor, a taxane, a Her-2 targeting agent, a hormone antagonist, a growth factor receptor targeting agent, or a pharmaceutically acceptable salt thereof.

85. The method of claim 77, wherein the anti-neoplastic agent is decitabine, capecitabine, valopicitabine, or gemcitabine.

86. The method of claim 77, wherein the anti-neoplastic agent is selected from the group consisting of bevacizumab, sunitinib, sorafenib, cediranib, ABT-869, axitinib, irinotecan, topotecan, paclitaxel, docetaxel, lapatinib, trastuzumab, lapatinib, tamoxifen, steroid aromatase inhibitors, non-steroid aromatase inhibitors, fulvestrant, an Epidermal Growth Factor Receptor (EGFR) inhibitor, cetuximab, panitumumab, an insulin-like growth factor 1 receptor (IGF1R) inhibitor, and CP-751871.

87. The method of claim 77, further comprising surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, adjuvant therapy, neoadjuvant therapy, viral therapy, RNA therapy, immunotherapy, nanotherapy or a combination thereof.

88. The method of claim 77, further comprising administering to the patient a PARP inhibitor in combination with gamma radiation.

89. The method of claim 77, wherein the sample is a tissue or body fluid sample.

90. The method of claim 77, wherein the sample is a tumor sample, a blood sample, a plasma sample, a peritoneal fluid sample, an exudate or a effusion.

91. The method of claim 77, wherein said breast cancer is metastatic breast cancer.

92. The method of claim 77, wherein said breast cancer is stage I, stage II or stage III.

93. The method of claim 77, wherein the breast cancer is negative for at least one of ER, PR, or HER 2.

94. The method of claim 77, wherein the breast cancer is negative for at least one of ER, PR, or HER 2; and wherein the breast cancer is positive for at least one of ER, PR, or HER 2.

95. The method of claim 77, wherein said breast cancer is deficient in homologous recombination DNA repair.

96. The method of claim 77, wherein the breast cancer has impaired BRCA1 or BRCA2 function.

97. The method of claim 77 wherein the antineoplastic agent is administered by parenteral injection or infusion.

98. The method of claim 77, wherein the 4-iodo-3-nitrobenzamide is administered orally, or parenterally by injection or infusion, or by inhalation.

99. A method of treating breast cancer in a patient, comprising:

(a) testing a sample taken from the patient for PARP expression; and

(b) administering to the patient at least one PARP inhibitor and at least one anti-neoplastic agent if the PARP expression exceeds a predetermined level.

100. The method of claim 99, wherein at least one therapeutic effect is obtained, said at least one therapeutic effect being a reduction in breast tumor size, a reduction in metastasis, complete remission, partial remission, stable disease, or complete response to pathology.

101. The method of claim 99 wherein the clinical benefit rate (CBR ═ CR + PR + SD > 6 months) is improved as compared to treatment with an anti-tumor agent but without a PARP inhibitor.

102. The method of claim 101, wherein the improvement in clinical benefit rate is at least about 60%.

103. The method of claim 99, wherein the PARP inhibitor is 4-iodo-3-nitrobenzamide or a metabolite thereof.

104. The method of claim 99, wherein the PARP inhibitor is of formula (IIa) or a metabolite thereof; and pharmaceutically acceptable salts, solvates, isomers, tautomers, metabolites, analogs, or prodrugs thereof:

formula (IIa)

Wherein: (1) r₁、R₂、R₃、R₄And R₅At least one of the substituents is always a sulfur-containing substituent, and the remaining substituents R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, hydroxyl, amino, nitro, iodine, bromine, fluorine, chlorine, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₃-C₇) Cycloalkyl and phenyl, wherein R ₁、R₂、R₃、R₄And R₅At least two of the 5 substituents are always hydrogen; or (2) R₁、R₂、R₃、R₄And R₅At least one of the substituents being other than a sulphur-containing substituentAnd R is₁、R₂、R₃、R₄And R₅At least one of these 5 substituents is always iodine, and where the iodine is always present with R as nitro, nitroso, hydroxyamino, hydroxy or amino₁、R₂、R₃、R₄Or R₅The substituents are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, hydroxy, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent. In some embodiments, in the compounds of (2), the iodo group is always with R as a nitroso, hydroxyamino, or amino group₁、R₂、R₃、R₄Or R₅The groups are adjacent.

105. The method of claim 99, wherein the anti-neoplastic agent is an antineoplastic alkylating agent, an antineoplastic antimetabolite agent, an antineoplastic antibiotic, a plant-derived anti-neoplastic agent, an antineoplastic platinum complex, an antineoplastic camptothecin derivative, an antineoplastic tyrosine kinase inhibitor, a monoclonal antibody, an interferon, a biological response modifier, a hormonal antineoplastic agent, an antineoplastic virus agent, an angiogenesis inhibitor, a differentiation inducing agent, a PI3K/mTOR/AKT inhibitor, a cell cycle inhibitor, an apoptosis inhibitor, an hsp 90 inhibitor, a tubulin inhibitor, a DNA repair inhibitor, an antiangiogenic agent, a receptor tyrosine kinase inhibitor, a topoisomerase inhibitor, a taxane, a Her-2 targeting agent, a hormone antagonist, a growth factor receptor targeting agent, or a pharmaceutically acceptable salt thereof.

106. The method of claim 99 wherein the anti-neoplastic agent is decitabine, capecitabine, valopicitabine, or gemcitabine.

107. The method of claim 99, wherein the anti-neoplastic agent is selected from the group consisting of bevacizumab, sunitinib, sorafenib, cediranib, ABT-869, axitinib, irinotecan, topotecan, paclitaxel, docetaxel, lapatinib, trastuzumab, lapatinib, tamoxifen, steroid aromatase inhibitors, non-steroid aromatase inhibitors, fulvestrant, an Epidermal Growth Factor Receptor (EGFR) inhibitor, cetuximab, panitumumab, an insulin-like growth factor 1 receptor (IGF1R) inhibitor, and CP-751871.

108. The method of claim 99, further comprising surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, adjuvant therapy, neoadjuvant therapy, viral therapy, RNA therapy, immunotherapy, nanotherapy or a combination thereof.

109. The method of claim 99 further comprising administering to the patient a PARP inhibitor in combination with gamma radiation.

110. The method of claim 99, wherein the sample is a tissue or body fluid sample.

111. The method of claim 99, wherein the sample is a tumor sample, a blood sample, a plasma sample, a peritoneal fluid sample, an exudate or a effusion.

112. The method of claim 99, wherein said breast cancer is metastatic breast cancer.

113. The method of claim 99, wherein the breast cancer is stage I, II, or III.

114. The method of claim 99, further comprising determining the expression of estrogen receptor, progesterone receptor, or human epidermal growth factor 2 receptor in a sample obtained from the patient.

115. The method of claim 99, wherein the breast cancer is negative for at least one of ER, PR, or HER 2.

116. The method of claim 99, wherein the breast cancer is negative for at least one of ER, PR, or HER 2; and wherein the breast cancer is positive for at least one of ER, PR, or HER 2.

117. The method of claim 99, wherein the breast cancer is deficient in homologous recombination DNA repair.

118. The method of claim 99, wherein the breast cancer has impaired BRCA1 or BRCA2 function.

119. The method of claim 99, wherein treatment comprises a treatment cycle of at least 11 days, and:

(a) on days 1 and 8 of the cycle, patients receive about 100-2000mg/m²Gemcitabine of (1);

(b) on days 1 and 8 of the cycle, the patient receives from about 10 to about 400mg/m ²Carboplatin of (a); and

(c) on days 1, 4, 8, and 11 of the treatment cycle, the patient receives from about 10 to about 100mg/kg of 4-iodo-3-nitrobenzamide, or a metabolite thereof of comparable molar content.

120. The method of claim 99 wherein the antineoplastic agent is administered by parenteral injection or infusion.

121. The method of claim 99, wherein the 4-iodo-3-nitrobenzamide is administered orally, or parenterally by injection or infusion, or by inhalation.