JP2008545965A - Compositions and methods for the treatment of cancer - Google Patents

Abstract

  Disclosed herein are methods and compositions for the treatment of cancer. In particular, the present invention discloses inhibitors of the Fanconi anemia pathway and methods of using the same. Such inhibitors are useful for inhibiting DNA damage repair and can be useful, for example, in the treatment of cancer.

Description

RELATED APPLICATION This application claims priority based on US Provisional Application 60 / 684,136 filed May 24, 2005. All of the above applications are incorporated herein by reference.

Government Support This invention was made with government support under grant numbers DK43889 and HL52725 from the National Institutes of Health. The US government has certain rights in this invention.

The present invention relates generally to compositions and methods for the treatment of cancer.

BACKGROUND OF THE INVENTION Many types of cancer are resistant to effective chemotherapeutic treatment. In ovarian cancer, resistance is seen even with chemotherapeutic agents such as cisplatin. Cisplatin (cis-diamine dichloroplatinum or CDDP) was originally discovered in the late 1960s and is a cytotoxic drug used to treat many cancers, including ovarian cancer. Cisplatin acts by platinization of DNA, resulting in DNA cross-linking. Up to 50% of ovarian cancers are inherently resistant to conventional chemotherapeutic agents such as cisplatin or other similar platinum treatments. Many resistance mechanisms have been claimed. However, the exact mechanism underlying the intrinsic and extrinsic resistance to chemotherapy has not been elucidated. One way to overcome the resistance of chemotherapy involves the use of chemosensitizers. Chemical sensitizers generally inhibit the mechanism of resistance. Examples include verapamil, reserpine, tamoxifen and cremophor, inhibitors of efflux pumps (MDR1, P-glycoprotein) that confer multiple drug resistance. However, such chemical sensitizers are only effective in some tumors where drug excretion is the main resistance mechanism. In addition, many of these chemical sensitizers have undesirable side effects.

SUMMARY OF THE INVENTION In one aspect, the present invention provides a method for predicting whether a subject having a neoplastic disease or disorder will respond to a genotoxic anti-tumor agent. The method includes obtaining a biological sample from the subject and determining the degree of ubiquitination of the Fanconi anemia complementation group D2 (FANC D2) polypeptide in the biological sample. The degree of ubiquitination of the FANC D2 polypeptide in a subject's biological sample is less than about 70% when compared to a biological sample from a control subject, indicating that the subject is a genotoxic antitumor agent. Suggest that you will respond.

  In another aspect, the invention provides a method for predicting whether a subject having a neoplastic disease or condition will respond to a genotoxic anti-tumor agent. The method includes obtaining a biological sample from the subject and determining a FANC D2-containing focus in the biological sample. The difference in focus formation is that the subject-derived sample contains less than about 70% FANC D2-containing focus when compared to a biological sample from a control subject, which is a genotoxic anti-tumor agent Suggests that the subject will respond.

  In another embodiment, a method for identifying an inhibitor of a non-FA DNA damage repair pathway is provided. The method includes the following steps: (a) providing a control cell in which the Fanconi anemia (FA) pathway functions; (b) providing a test cell that is syngeneic with the test cell but lacks the FA pathway. (C) contacting the test and control cells with the test compound; and (d) comparing the sensitivity of the test and control cells to the test compound. The increased sensitivity of the test cell to the test compound indicates that the test compound is an inhibitor of the non-FA DNA damage repair pathway.

  In yet another aspect, a method of treating a neoplastic disease in a subject in need of treatment is provided. The method tests an effective amount of: (a) an inhibitor of the FA pathway or a pharmaceutically acceptable salt, ester, derivative, solvate or prodrug thereof, and (b) a genotoxic antitumor agent combination. Including administration to the body.

  In yet another aspect, a method of treating a neoplastic disease in a subject in need of treatment is provided. The method comprises an effective amount of: (a) an inhibitor of the FA pathway or a pharmaceutically acceptable salt, ester, derivative, solvate or prodrug thereof; and (b) an inhibitor of a non-FA-DNA damage repair pathway. Administering a combination of: to a subject.

  In another aspect, a method of treating a neoplastic disease in a subject in need of treatment is provided. The method comprises an effective amount of: (a) an inhibitor of the FA pathway or a pharmaceutically acceptable salt, ester, derivative, solvate or prodrug thereof; (b) an inhibitor of a non-FA-DNA damage repair pathway; And (c) administering a genotoxic anti-tumor agent or a pharmaceutically acceptable salt, ester, derivative, solvate or prodrug combination thereof to a subject.

  In another aspect, a method of increasing the susceptibility of a neoplastic disease to a genotoxic anti-tumor agent is provided. The method comprises administering an effective amount of an inhibitor of the FA pathway before, after or simultaneously with the administration of a therapeutically effective dose of the agent.

  In a final embodiment, a method is provided for increasing the susceptibility of a neoplastic disease to a genotoxic anti-tumor agent. The method comprises administering an effective amount of (a) an inhibitor of the FA pathway or a pharmaceutically acceptable salt, ester, derivative, solvate thereof before, after or simultaneously with the administration of a therapeutically effective dose of the agent. Or administering a combination of a prodrug and (b) an inhibitor of a non-FA DNA damage repair pathway.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based in part on a series of findings that reveal that the FA pathway plays a critical role in cancer susceptibility to antitumor agents. The role of the FA pathway in regulating the susceptibility of neoplastic diseases and / or cancer cells to antitumor agents has been elucidated using cell lines that are defective in components of the FA pathway and using inhibitors of the FA pathway. I came. Accordingly, in one embodiment, a method for treating a subject having a neoplastic disease is provided. One such method involves administering an effective amount of an inhibitor of the FA pathway in combination with a genotoxic anti-tumor agent. Another method involves administering an effective amount of an inhibitor of the FA pathway in combination with an inhibitor of the non-FA DNA damage repair pathway. Also provided are compositions useful for the treatment of neoplastic diseases, which comprise FA pathway inhibitors in combination with genotoxic anti-tumor agents and / or inhibitors of non-FA DNA damage repair pathways. Also provided are pharmaceutical compositions and kits thereof, which include FA pathway inhibitors and anti-tumor agents and / or inhibitors of non-FA DNA damage repair pathways.

  Also provided are methods for identifying agents that modulate the FA pathway. These methods are useful for identifying inhibitors of the FA pathway. Inhibitors identified in this way are potentially useful as chemical and / or radiosensitizers. Also provided in the present invention are methods for identifying inhibitors of non-FA DNA damage repair pathways used in combination with FA pathway inhibitors. Combining inhibitors can be useful for administration to patients receiving anti-tumor agents.

I. Definitions As used herein, the terms “neoplasm”, “neoplastic disease”, “neoplasia”, “cancer”, “tumor” and “proliferative disease” refer to the ability for self-proliferation. Different populations that are abnormal states or situations characterized by rapid proliferation of cell proliferation, usually partially or wholly lacking structural mechanisms and functional regulation of normal tissues Form. The aforementioned terms are meant to include hematopoietic tumors (eg, lymphoma or leukemia) and solid tumors (eg, sarcoma or carcinoma), pre-cancerous and cancerous growth or carcinogenic processes, metastatic tissue or malignant traits Includes all types of convertible cells, tissues or organs, regardless of histopathological type or invasive stage. A hematopoietic tumor is a malignant tumor that affects the hematopoietic mechanism (the mechanism involved in the formation of blood cells) and components of the immune system, and leukocytes (blood and bone marrow leukocytes) Including white blood cells and their precursors, and lymphoma (associated with lymphocytes) Solid tumors include sarcomas, which include muscle, cartilage, blood vessels, fibrous tissue, fat or bone In addition, solid tumors include carcinomas, which include epithelial structures (external epithelium (eg, digestive tract, lung and neck skin and epithelium) and various glands (eg, It is a malignant tumor arising from the inner epithelium lining the breast, pancreas, thyroid)). Examples of tumors that are particularly sensitive to treatment by the methods of the present invention include leukemia and hepatocellular carcinoma, sarcoma, vascular endothelial cancer, breast cancer, central nervous system cancer (eg astrocytoma, gliosarcoma, neuroblastoma) , Oligodendroglioma and glioblastoma), prostate cancer, lung and bronchial cancer, laryngeal cancer, esophageal cancer, colon cancer, colorectal cancer, gastrointestinal cancer, melanoma, ovarian cancer and endometrial cancer, kidney cancer and Includes bladder cancer, liver cancer, endocrine cancer (eg thyroid), and pancreatic cancer.

  “Genotoxic agent” or “genotoxic agent” refers to any compound or treatment that induces DNA damage when applied to a cell. Such agents can be chemical or radioactive substances. Genotoxic agents are those in which the main biological activity of a chemical (or metabolite) is a change in the information encoded in the DNA. Genotoxic agents may have different mechanisms of action, such as alkylating agents such as ethyl methanesulfonate (EMS), nitrosoguanine and vinyl chloride; bulky such as benzo (a) pyrene and aflatoxin B1 Addition products; active oxygens such as superoxide, hydroxy radicals; base analogs such as 5-bromouracil; intercalating agents such as acridine orange and ethidium bromide.

  As used herein, a “genotoxic anti-tumor agent” is a genotoxic agent used, for example, in chemotherapy to treat cancer. In particular, “genotoxic anti-tumor agents” include both chemical agents or other agents that cause damage to DNA. These agents include DNA alkylating agents, intercalating agents and the like. Non-limiting examples of “genotoxic antitumor agents” include 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU), busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, Contains daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide and topotecan. “Genotoxic antitumor agents” also include those types of radiation used to treat a subject with sufficient dose of radiation to cause DNA damage, particularly for the treatment of cancer.

  As used herein, “DNA damage” refers to chemical and / or physical modification of DNA in a cell, methylation, double-strand breaks by alkylation, cross-linking, thymidine dimer caused by ultraviolet light, And oxidative damage formed by the binding of oxygen radicals to DNA bases.

As used herein, “chemosensitizer” and “chemosensitizing agent” are used when a therapeutically effective amount is administered to a subject, such as a disease (neoplastic disease, In the treatment of benign and malignant tumors and cancerous cells, etc.) it refers to compounds that increase sensitivity to chemotherapeutic compounds and / or compounds that improve the therapeutic effects of the compounds. Increased sensitivity to chemotherapeutic compounds, including genotoxic anti-tumor agents, can be measured, for example, by measuring the decrease in cellular LD _{50 to} the compound in the presence of chemical sensitizers.

  Similarly, as used herein, “radiosensitizer” and “radiosensitizing agent” can be used when a therapeutically effective amount is administered to a subject, such as a disease (tumor). In the treatment of sexually transmitted diseases, benign and malignant tumors and cancerous cells), it refers to a compound that increases the sensitivity to radiation therapy (treatment with electromagnetic radiation) and / or a compound that improves the therapeutic effect of radiation therapy. Also contemplated are electromagnetic radiation treatments of other diseases not described herein.

  “Sample” or “biological sample” means any cell or tissue, or a composition comprising a cell or tissue, or an isolate from a subject. The sample can be from the heart, brain, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus, small intestine or colon. Another type of biological sample for use in detecting the presence or absence of DNA damage in a subject exposed to a genotoxic agent, such as radiation, a chemical, is a preparation containing leukocytes, such as peripheral blood, sputum, Saliva, urine and the like may be used.

  As used herein, “degree of ubiquitination” of a FANC D2 polypeptide usually refers to the level of FA pathway activation, which is the degree of monoubiquitination of the FANC D2 polypeptide in a subject or biological sample thereof. Measured by. As used herein, the “degree of ubiquitination” of a FANC D2 polypeptide includes the percentage of total FANC D2 polypeptide in a sample that is monoubiquitinated and can be expressed as a fraction or a percentage. As used herein, the “degree of ubiquitination” of a FANC D2 polypeptide can also be measured using any alternative method that detects activation of the FA pathway, including the degree of focus formation.

  As used herein, “degree of focus formation” refers to the total number or formation rate of FANC D2-containing focus in a sample. FANC D2-containing foci are nucleoprotein complexes that are formed in response to activation of the FA pathway, for example, upon exposure to genotoxic agents. A FANC D2-containing focus can be detected, for example, by immunofluorescence microscopy using a labeled antibody against the FANC D2 polypeptide, as further described herein. In certain cases, FANC D2-containing foci can also be detected in cells expressing functional fusion proteins comprising GFP and FANC D2 polypeptides. In these cells, FANC D2-containing foci can be detected using fluorescence microscopy without using anti-FANC D2 antibodies. The degree of focus formation can be normalized from one sample to another, eg, the total number of cells, the total number of intact nuclei, the total volume of the sample or the total mass of the sample.

  “Difference in focus formation” means the difference in the number, size or persistence of FANC D2-containing focus when the test sample is compared to either the control sample or the reference sample . Differences include an increase or decrease of 2 fold or more, or 2 fold or less, eg, 5, 10, 20, 100, 1000 fold or more compared to a control or reference sample. Differences also include increases or decreases that are greater than or equal to 5% or less, eg, 10%, 20%, 30%, 50%, 75%, 100%, relative to a control or reference sample.

  As used herein, “modulate” the formation of a FANC D2-containing focus refers to a change or alteration in the formation of a FANC D2-containing focus in a biological sample. Modulation may be an increase or decrease in the number, size or persistence of the focus within the biological sample, and more than 2 times or less than the control sample or reference sample, eg 5, 10, Including an increase or decrease of 20, 100, 1000 times or more. Also, the modulation may be an increase or decrease that is greater than or equal to 5% or less, eg 10%, 20%, 30%, 50%, 75%, 100% compared to the control sample or reference sample.

  As used herein, exposure to a “low level” genotoxic anti-tumor agent may result in doses of certain genotoxic anti-tumor agents at doses that result in no more than 20% of the FANC D2-containing focus in biological samples. Refers to exposure. Because of the large number of genotoxic anti-tumor agents to which samples can be exposed, and because of the different susceptibility of different samples to such genotoxic anti-tumor agents, indicate relative doses to the formation of a FANC D2-containing focus Is preferred over indicating the absolute dose of a particular genotoxic anti-tumor agent.

  The term “modulator” refers to a compound (natural or non-natural) such as a biopolymer (eg, nucleic acid, protein, non-peptide or organic molecule), or a bacterial, plant, fungal or animal (particularly mammalian) cell or tissue, etc. This refers to extracts derived from biological materials, or even inorganic elements or molecules. Modulators can be included in the screening assays described herein to inhibit (directly or indirectly) a biological process or group of processes (eg, agonists, partial antagonists, partial agonists). , Antagonists, antitumor agents, cytotoxic agents, inhibitors of tumor metastasis or cell growth, cell growth promoters, etc.). The activity of the modulator may be known, unknown, or partially known. Such modulators can be screened using the methods described herein.

  The term “modulator candidate” refers to a compound that is tested as a putative modulator by one or more screening methods of the invention. Usually, various predefined concentrations such as 0.01 μM, 0.1 μM, 1.0 μM and 10.0 μM are used for screening, as described more fully below. A test compound control can include measuring a signal in the absence of the test compound, or comparing to a compound known to modulate the target.

  As used herein, “FA pathway inhibitors” and “FA pathway inhibitors” are (natural or non-natural) compounds such as biopolymers (eg, nucleic acids, proteins, non-peptides or organic molecules), or It refers to extracts derived from biological materials such as bacteria, plants, fungi or animal (especially mammalian) cells or tissues, or even inorganic elements or molecules. “FA pathway inhibitors” and “FA pathway inhibitors” generally refer to compounds that inhibit the ability of the FA pathway to repair DNA damage. Inhibition of the FA pathway by “FA pathway inhibitors” or “FA pathway inhibitors” can be assessed using the techniques described herein, including detection of FANC D2-containing focus and FANC D2 polypeptides. Detection of monoubiquitination of, but not limited to. As will be appreciated by those skilled in the art, the method contemplates any other method for the detection of currently known or future known FA pathway inhibitors. Inhibition may be a reduction in the number, size or persistence of FANC D2-containing foci and a reduction of more than 2-fold compared to a control or reference, e.g., 2, 5, 10, 20, 100, 1000-fold or more including. Inhibition may also be a decrease of 5% or more, eg, up to 5%, 10%, 20%, 30%, 50%, 75% or 100% compared to a control or reference. Furthermore, “FA pathway inhibitors” and “FA pathway inhibitors” as used herein include pharmaceutically acceptable salts, solvates, esters, derivatives or prodrugs.

  As used herein, “non-FA DNA damage repair pathway” includes direct deletion pathway, non-homologous end joining (NHEJ) pathway, base excision repair (BER) pathway, nucleotide excision repair (NER) pathway and mismatch repair (MR). Refers to any DNA damage repair pathway selected from the group consisting of pathways.

  The pharmaceutical compositions of the invention can be administered using any amount and any route of administration effective to improve the therapeutic effect of the drug. As used herein, a “therapeutically effective amount” when used in combination with a chemical or radiosensitizer, is a chemical sensitization sufficient to provide the desired effect on the target cell or tissue. Refers to the amount of sensitizer. The exact amount required will vary from subject to subject and will depend on species, age and general condition of the subject; individual chemical sensitizers;

  The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. This term explicitly excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers are pharmaceutically acceptable, such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents and preservatives. Including, but not limited to, excipients. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose, while corn starch and alginic acid are suitable disintegrants. Binders include starch and gelatin, while lubricants, when present, are usually magnesium stearate, stearic acid or talc. If desired, tablets may be coated with a substance such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

As used herein, “therapeutically effective dose” refers to the amount of a protein or antibody, antagonist or inhibitor thereof that prevents or ameliorates a symptom or condition (eg, a neoplastic disease). The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical means in cell cultures or laboratory animals, such as ED ₅₀ (therapeutically effective dose in 50% of the population) and LD ₅₀ (population of the population). 50% lethal dose). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio LD ₅₀ / ED _50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies is used to calculate the formulation range for human use. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dose varies within this range depending on the dose used, patient sensitivity and route of administration.

  The exact dose is chosen by the respective physician or veterinarian taking into account the patient to be treated. Dosage and administration are adjusted to provide the active moiety at a sufficient concentration or to maintain the desired effect. Additional factors that may be considered include the severity of the condition; age, weight and sex of the subject; dietary habits, time and frequency of administration, drug combination, response sensitivity and tolerance / response to treatment. Long-acting pharmaceutical compositions can be administered every 3-4 days, once a week or once every two weeks, based on the half-life and clearance rate of the individual formulation.

  The term “pharmaceutically acceptable salt” refers to both acid and base addition salts. The nature of the salt is not critical provided that it is pharmaceutically acceptable. Examples of acid addition salts are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, citric acid, tartaric acid, succinic acid, oxalic acid, malic acid, glutamic acid, propionic acid , Glycolic acid, gluconic acid, maleic acid, embonic acid (pamonic acid), methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, pantothenic acid, benzenesulfonic acid, toluenesulfonic acid, sulfanilic acid, mesylic acid, cyclohexyl Including, but not limited to, aminosulfonic acid, stearic acid, algenic acid, β-hydroxybutyric acid, malonic acid, galactaric acid, galacturonic acid and the like. Suitable pharmaceutically acceptable base addition salts are metal salts formed from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or N, N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, Examples include, but are not limited to, organic salts formed from ethylenediamine, N-methylglucamine, lysine, procaine and the like. Further examples of pharmaceutically acceptable salts are described in Journal of Pharmaceutical Sciences (1977) 66: 2. All these salts can be prepared from modulators of FANC D2-containing focus by treating the compound with the appropriate acid or base by conventional means.

II. FANC D2 focus
The cellular response to DNA damage is a complex network of cell cycle checkpoints, DNA repair and pathways that mediate apoptosis. A model obstacle to study these pathways has been the DNA double-strand breaks that rapidly induce cell cycle checkpoints and are repaired by many different pathways. In mammalian cells, both homologous and non-homologous recombination pathways are utilized. Detailed studies in mammalian cells have shown that DNA repair and cell cycle checkpoint protein complexes are rapidly localized to double-strand break sites induced by ionizing radiation. These proteins produce a focus that can be detected by immunofluorescence analysis.

  Fanconi anemia complementation group D2 (FANC D2) is a component of a protein complex involved in chromosome stability and repair. Fanconi anemia (FA) is a hereditary disease characterized in part by deficiencies in DNA repair mechanisms that increase an individual's risk for various cancers. In response to DNA damage, the FA complex activates FANC D2 and then binds to the breast cancer type I polypeptide (BRCA1). Activation of FANC D2 occurs by phosphorylation of serine 222 residue by telangiectasia ataxia mutation (ATM) kinase. Furthermore, activation via the FA pathway occurs via monoubiquitination of FANC D2 at lysine 561. In its unmodified form, FANC D2 exists diffusely throughout the nucleus. When ubiquitinated, FANC D2 forms a dot or focus in the nucleus. FANC D2 ubiquitination and subsequent formation of nuclear foci occur in response to DNA damage. By co-immunoprecipitation, Nakanishi et al. Discovered a constitutive interaction between FANC D2 and Nimygen-cutting syndrome 1 (NBS1), and these proteins interact in two different aggregates to check the S phase. Provided evidence of mediating resistance to Point and Mitomycin C-induced chromosomal damage (Nakanishi et al. (2002) Nat Cell Biol. 4: 913-20).

  At least two types of ionizing radiation-induced focus have been observed: one contains Rad51, BRCA1 and BRCA2 proteins and the other contains the Mre11-Rad50-NBS1 complex. The Rad51 focus contains the tumor suppressor proteins BRCA1 and BRCA2, and also appears in S phase without exogenous induction of DNA damage.

  The Mre11-Rad50-NBS1 focus can be detected as early as 10 minutes after irradiation and is clearly present at the DNA cleavage site during DNA repair. These foci also colocalize with the BRCA1 protein, which has been shown to be necessary for their formation, possibly through physical interaction with human Rad50 (hRad50). Furthermore, co-immunoprecipitation experiments performed with BRCA1 suggest that there are a number of additional proteins in this complex (referred to as the BRCA1-binding monitoring complex). These include the mismatch repair proteins Msh2, Msh6 and Mlh1, checkpoint kinase ATM, the product of the Bloom syndrome gene BLM, and the replication factor C. BRCA1, NBS1, and hMre11 have all been shown to be substrates for ATM kinases and become phosphorylated in response to the presence of DNA cleavage.

  The present invention relates to the discovery that cells exposed to genotoxic anti-tumor agents form FANC D2-containing foci. Currently, multiple DNA damage response proteins have been identified that form a nuclear focus, also called IRIF (ionizing radiation-induced focus), in response to DNA damage. Methods for detecting FANC D2-containing focus are described in U.S. Patent Application No. 10 / 165,099 and U.S. Patent Application No. 60 / 540,380, along with methods for detecting and quantifying the relative amount of ubiquitinated FANC D2 polypeptides, and The contents of which are hereby incorporated by reference in their entirety.

III. Detection method of FANC D2 activation
1. Detection using FANC D2 binding ligand
The whole cell concentration of FANC D2 protein does not change significantly in response to DNA damage. Rather, DNA damage results in recruitment of FANC D2-containing foci along with FANC D2 monoubiquitination. One skilled in the art understands that instead of measuring the presence of a FANC D2-containing focus, a ligand is used that specifically binds to monoubiquitinated FANC D2, but does not bind to non-ubiquitinated FANC D2. Will be done. In order to detect the presence of monoubiquitinated FANC D2, it is preferred to bind the ligand to the detectable label. The main advantage of using such ligands is that, as will be appreciated by those skilled in the art, the basal concentration of monoubiquitinated FANC D2 in cells with intact DNA is generally low, so that The concentration can be measured in a sample taken from a living subject using the concentration of monoubiquitinated FANC D2 as a surrogate marker. An antibody that specifically recognizes the monoubiquitinated form of FANC D2 (FANC D2-L) is quite useful as a rapid diagnostic agent. For example, this antibody is used for:

1) Immunohistochemistry (IH). This antibody can be used to examine tissue sections prepared from solid tumors (eg, breast cancer, ovarian cancer, lung cancer). A positive signal due to IH will predict that the tumor is resistant to cisplatin and related drugs.

2) FACS analysis. Peripheral blood lymphocytes (PBL) can be screened with this antibody. A positive signal suggests the presence of activated FANC D2, consistent with the recent exposure of the individual to IR or toxin. This antibody is therefore a useful extension of the radiation dosimeter assay described herein.

3) High-throughput assay for screening for inhibitors of purified FA complex. These inhibitors will interfere with the ability of the FA complex to monoubiquitinate FANC D2 in vitro. The novel monoclonal antibody will be a useful reagent for the detection of the final product. Additional methods for measuring FANc D2-containing focus using ligands that specifically recognize monoubiquitinated FANC D2 include immunoblot analysis using extracts of samples collected from live subjects or enzyme-linked immunosorbent assays (ELISA) or FACS analysis (Harlow et al., 1999, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, NY).

  A sensitive indicator of IR exposure is increased monoubiquitination of FANC D2. In non-injured cells, the ratio of FANC D2-L (monoubiquitinated isoform) to FANC D2-S (non-ubiquitinated isoform) is about 0.5 to 0.6. This ratio (L / S) is easily calculated by comparing the concentration of the L band with respect to the S band in Western blotting. A sensitive indicator of increased FANC D2 monoubiquitination and IR exposure is the conversion of the L / S ratio to 1.0 or higher.

2. Detection using GFP-FANC D2 fusion protein
Another method for detection of FANC D2 activation and focus formation is to use FANC D2 protein fused to a fluorescent protein (eg, GFP). A functional fusion protein of FANC D2 and GFP can form a focus when exposed to a genotoxic anti-tumor agent. These focus can then be viewed with a fluorescence microscope. Thus, the formation of FANC D2-containing foci can be measured as a surrogate marker for activation of the FA pathway in response to exposure to genotoxic anti-tumor agents. Methods for making such fusion protein constructs are described in US Patent Application No. 60 / 540,380, along with methods for detecting the formation of FANC D2-containing foci, which are hereby incorporated by reference.

IV. Identification of FA Pathway Inhibitors The present invention encompasses methods and compositions useful for the treatment of neoplastic diseases using FA pathway inhibitors. Inhibitors of the FA pathway are described in the methods described herein, as well as, for example, U.S. Patent Application No. 10 / 165,099 and U.S. Patent Application No. 60 / 540,380, the contents of which are incorporated herein by reference. ) Can also be identified by the method already described in (1). For example, inhibitors of the FA pathway can be systematically identified using a three-step method, as summarized in FIG.

  The first stage of the screening involves high-throughput methods for identifying agents that alter the formation of FANC D2-containing foci. For example, detection of FANC D2-containing focus by using a cell line expressing a FANC D2 ligand, such as an anti-FANC D2 antibody, or a functional eGFP-FANC D2 fusion protein is described in US patent application Ser. No. 10 / 165,099 and US patent application. No. 60 / 540,380, the contents of which are incorporated herein by reference. This method induces the formation of a FANC D2-containing focus at the same time before or after exposing cells or biological samples to genotoxic anti-tumor agents (eg, ionizing radiation (IR), mitomycin C or cisplatin) Contacting with a test compound at a dose to be administered. The number and size of FANC D2-containing foci in the cells are then measured and compared to control cells that have not been contacted with the test compound. A decrease in the number and / or size of FANC D2-containing foci compared to control cells suggests that the drug is an inhibitor of the FA pathway, whereas the number of FANC D2-containing foci compared to control cells and / or Or an increase in size suggests that the drug is an agonist of the FA pathway. Potential agonists and inhibitors identified in this way can be further tested to see if they exert a direct or indirect effect on the FA pathway, for example, directly damage DNA Can be determined (in the case of potential agonists of the FA pathway) or by reducing the effect of the genotoxic anti-tumor agent used in the screening.

  The second stage of the screening involves the detection of ubiquitinated FANC D2 polypeptide. As described above, activation of the FA pathway results in monoubiquitination of the FANC D2 polypeptide. Thus, activation of the FA pathway can be measured by detecting the relative amount of ubiquitinated FANC D2 compared to non-ubiquitinated FANC D2 polypeptide. FANC D2 ubiquitination can be detected by performing immunoblot analysis of protein extracts. In immunoblot analysis, ubiquitinated FANC D2 migrates to higher molecular weight bands and can be detected using a labeled FANC D2 ligand (eg, an anti-FANC D2 antibody). Therefore, the second stage of the screening involves the inclusion of FANC D2 at the same time, before or after exposing cells or biological samples to genotoxic antitumor agents (eg, ionizing radiation (IR), mitomycin C or cisplatin). A method comprising contacting the test compound with a dose that induces focus formation. The amount of ubiquitinated FANC D2 polypeptide compared to non-ubiquitinated FANC D2 polypeptide is detected and compared to a control cell sample or biological sample that has not been contacted with the test compound. Differences in the relative amount of ubiquitinated FANC D2 compared to control cells suggests that the test compound is a modulator of the FA pathway. An increase in the relative amount of ubiquitinated FANC D2 polypeptide compared to control cells or biological samples suggests that it is an agonist of the FA pathway, whereas the relative amount of ubiquitinated FANC D2 polypeptides compared to control cells or biological samples A decrease in the amount suggests that it is an inhibitor of the FA pathway. As described above, potential agonists and inhibitors thus identified can be further tested to determine whether they exert a direct or indirect effect on the FA pathway, e.g., DNA It can be determined whether it is caused by direct damage (in the case of potential agonists of the FA pathway) or by reducing the effect of the genotoxic anti-tumor agent used in the screening.

  The third stage of the screening involves testing the sensitivity of compounds to genotoxic antitumor agents in vitro. It may be expected that contacting a cell or biological sample with an inhibitor of the FA pathway will increase the sensitivity of the sample / cell to genotoxic anti-tumor agents. Specific inhibition of the FA pathway by a test agent is expected to increase the degree of sensitivity compared to, for example, a cell line that has a particular defect in one or more components of the FA pathway. Cell lines useful for this type of assay include the ovarian cancer cell line, 2008, which lacks FANCF. 2008 cells deficient in FANCF are highly sensitive to genotoxic antitumor agents (see open squares in the graph of FIG. 8), and this sensitivity is restored to the wild-type level by overexpression of FANCF. (FIG. 8, white circle). Second, the role of FANCF in restoring genotoxic susceptibility to the wild-type level is abolished by contact with a test agent that inhibits the FA pathway (Figure 8, filled circles), while the FANCF transfection In the absence of susceptibility, genotoxic susceptibility to antitumor agents remains unaffected (FIG. 8, black squares).

  The three stages of the above screening provide a rational approach to quickly identify and characterize potential modulators of the FA pathway. It should be understood that the method for identifying a modulator is not limited to the specific embodiments of the invention described above, and that the embodiments can be varied and still fall within the scope of the invention. It is. Furthermore, the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

V. Inhibitors of the FA pathway The present invention contemplates the use of inhibitors of the FA pathway. Inhibitors of the FA pathway include any compound that, when administered before, after, or simultaneously with a genotoxic anti-tumor agent that normally causes the formation of a FANC D2-containing foci, results in inhibition of the formation of the FANC D2-containing foci. Examples of genotoxic anti-tumor agents that induce the formation of FANC D2-containing foci include, but are not limited to, ionizing radiation (IR) and DNA alkylating agents such as cisplatin or mitomycin C. Inhibition of the FA pathway can also be detected by measuring the relative amount of ubiquitinated FANC D2 polypeptide and non-ubiquitinated FANC D2 polypeptide in a sample with agents that normally induce ubiquitination. For example, detection of a FANC D2-containing focus using microscopic detection means is similar to determining the relative ubiquitination status of a FANC D2 polypeptide as in US patent application Ser. No. 10 / 165,099 (filed Jun. 6, 2002). ) And U.S. Patent Application No. 60 / 540,380 (filed January 30, 2004), the contents of which are incorporated herein by reference. That is, a FANC D2-containing focus can be detected using an anti-FANC D2 antibody using immunofluorescence microscopy. Alternatively, a fluorescent protein-labeled version of FANC D2 can be transfected into the cells of interest, and the formation of FANC D2-containing foci can be measured microscopically by detecting a fluorescent “focus”, which Is also described in US Patent Application No. 60 / 540,380. Compounds that inhibit the FA pathway, such as wortmannin and trichostatin A, have been previously disclosed, for example, in US Patent Application No. 60 / 540,380 (filed Jan. 30, 2004).

The present invention describes further illustrations of inhibitors of the FA pathway including curcumin, H-9 and Ulster Paulon, which were identified using the screening methods described herein.

H-9 kinase inhibitors, also known as N-2-aminoethyl-5-isoquinoline sulfonamides (Formula I), include PKA, PKG, PKC, calcium / calmodulin-dependent protein kinase and myosin light chain kinase Known as inhibitors of several kinases (Inagaki et al. (1985) J Biol. Chem. 260 (5): 2922-5; Ito et al. (1988) Int. J Immunopharmacol. 10: 211-216).

Alster Paulon (Formula II) is known to inhibit Cdk1 / cyclin B, Gsk-3B and Cdk5 (Sausville et al. (2000) Ann NY Acad. Sci. 910: 207-221; Schultz et al. (1999) J Med. Chem. 42: 2909-2919).

Curcumin (turmeric yellow, 1,7-bis (4'-hydroxy-3'-methoxyphenyl) -1,6-heptadiene-3,5-dione, also known as diferroylmethane) is a spice A low molecular weight polyphenol derived from a turmeric and involved in the regression of several solid tumors in humans (Cheng et al. (2001) Anticancer Res. 21: 2895-2900). Curcumin is safe in human clinical trials at doses up to 8000 mg / day (Cheng et al., Ibid). Recent studies suggest that curcumin may have activity in the treatment of other human diseases such as cystic fibrosis (Egan et al. (2004) Science 304: 600-602).

  Geldanamycin (formula IV) is a benzoquinone ansamycin antibiotic that binds to Hsp90 (heat shock protein 90) and alters its function. The present invention includes compositions and methods comprising geldanamycin and analogs thereof. Analogs of geldanamycin include 17- (allylamino) -17-demethoxy-geldanamycin (Schnur et al. (1995) J Med. Chem. 38: 3806-12; Dunn (2002) J. Natl. Cancer Inst 94, 1194-5). The present invention further contemplates compositions and methods comprising other inhibitors of HSP90, particularly benzoquinone ansamycin inhibitors of HSP90, coumarin derivatives (eg, as described in WO 00/53169).

  Other compounds that can inhibit the FA pathway include those listed in Table 2. Therefore, inhibitors of the FA pathway include Ulster Paulon, (+-) 13-HODE, nifedipine, penitrem A, geldanamycin, Go6976, leukotriene B3, trichostatin-A, AG-370, mitomycin C, amanitin (α Amanitin), HNMPA- (AM) 3, propidium iodide, DRB, ochratoxin, Ca-074-Me, K252c, wortmannin, actinomycin D, AG213, BAPTA-AM, curcumin, puromycin, bumetanide, methyladenine (3 -Methyladenine), H9, TPEN, spermine NONOate, PD00600, 5323069 and 1M556S.

VI. Inhibitor cells of other DNA damage repair pathways continuously undergo different types of DNA damage. These damages can be caused by exposure to various endogenous and exogenous drugs and radiation, including reactive oxygens such as superoxide (O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ). Furthermore, humans are constantly exposed to any and all carcinogens, many of which have the effect of causing damage to DNA. In humans, at least six different mechanisms have been shown to exist for DNA damage repair, depending on the type of damage received.

  Many cancers have a defect in at least one of the six major DNA damage repair pathways. In addition to causing increased genomic instability, disruption of any of these DNA repair mechanisms can result in increased sensitivity to genotoxic anti-tumor agents. Therefore, these cancers are highly dependent on one of the other five DNA damage pathways to survive. Thus, disruption of the second, non-FA DNA damage repair pathway in these neoplastic diseases (eg, by small molecule inhibitors) can lead to selective cancer cell death. That is, many cancers can have a preferential (major) DNA damage repair pathway. Since one DNA damage repair pathway is already disrupted or markedly reduced in cancer cells, it is redundant to the preferred pathway to maintain high growth rates and prevent DNA damage in these cells Burden. Thus, in cancer cells where the major DNA damage repair pathway is disrupted or reduced, disruption of the preferential pathway by exogenous inhibitors can have a profound cytotoxic effect on tumor cells, but , It can have a relatively low cytotoxic effect on surrounding normal cells.

  Loss of the FA / BRCA pathway results in chromosomal instability and increases cisplatin sensitivity, thus increasing activity in maintaining non-FA DNA damage repair pathways, including the base excision repair (BER) pathway. Thus, inhibitors of non-FA DNA damage repair pathways (eg BER) (such as PARP1 inhibitors or inhibitors of specific kinases in the BER pathway) are lethal to those cells, but normal (non-tumor) ) There will be little effect on the cells.

The present invention also contemplates the use of various other DNA damage repair pathway inhibitors. As mentioned above, there are several major pathways for DNA damage repair, including non-homologous end joining (NHEJ), base excision repair (BER) nucleotide excision repair (NER) and mismatch repair (MR). Including, but not limited to. These mechanisms are described, for example, by Hoeijmakers JHJ (2002) Nature 411: 366-374, Svejstrup JQ (2002) Nat Rev Mol Cell Biol. 3: 21-29 and Panasci, DNA Repair in Cancer Therapy Humana Press, 2004, Totowa, NJ, but is not limited to these (which are incorporated herein by reference).

A. Non-homologous end joining (NHEJ)
DNA double-strand breaks (DSBs) can be caused by any environment or other factors, including reactive oxygenates, ionizing radiation (IR) and certain anti-tumor agents such as bleomycin. Failure of DSB repair can have many consequences including mutations, chromosomal abnormalities and eventual cell death. Non-homologous end joining (NHEJ), also called illegitimate recombination, is one major pathway to repair DSB. Some members of the NHEJ pathway are shown in Table 1.

  DNA-dependent protein kinase (DNA-PK) is composed of a catalytic subunit (DNA-PKcs) and a regulatory subunit (Ku70 / Ku80 heterodimer). The DNA-PKcs subunit is a serine / threonine kinase and belongs to the phosphatidylinositol-3 kinase family. Ku80 / Ku70 heterodimer (Ku) exhibits sequence-dependent binding to double-stranded ends and recruits and activates the DNA-PKcs catalytic subunit upon binding to DNA. Some candidates for inhibitors of DNA-PK are, for example, viridine (Hanson, JR Nat. Prod. Rep., 12: 381-384, 1995), wortmannin, quercitins (Izzard et al. (1999) Cancer. Res., 59: 2581-2586), LY294002 (Vlahos et al. (1994) J. Biol. Chem., 269: 5241-5248), which are incorporated herein by reference. Other inhibitors of NHEJ include inhibitors of ATM, which are described in US Patent Application No. 2004/0002492, incorporated herein by reference.

B. Base excision repair (BER)
Single-stranded DNA break (SSB) is one of the most frequent disorders that occur in cellular DNA. SSB can occur spontaneously or as an intermediate in the enzymatic repair of base damage during base excision repair (BER) (Caldecott (2001) Bioessays 23 (5): 447-55). In this repair pathway, after removal of the damaged base by DNA glycosylase, the resulting aprin / apyrimidine (AP) site is first treated with ApeI AP endonuclease to release the 5 ′ deoxyribose-phosphate; The 3'β removal product is released by AP lyase activity. Subsequent removal of these AP sites by DNA polymerase β or PCNA-dependent polymerase fill-in either one nucleotide (for Pol β) or a longer repair patch (for Pol δ / ε). Allow recombination synthesis to occur, then they recombine (Wilson (1998) Mutat. Res. 407: 203-15). If the SSB site is not effectively processed and removed, it forms a population of damaged sites and a stopped replication fork, resulting in the formation of DSB with potentially lethal consequences for the cell (Chaudhry & Weinfeld ( 1997) J Biol Chem. 272: 15650-5; Harrison, Hatahet et al. (1998) Nucleic Acids Res. 26: 932-41).

  Poly (ADP ribose) polymerase (PARP) is a DNA-binding zinc finger protein that catalyzes the transport of ADP ribose residues from NAD + to itself and different chromatin components to form branched ADP ribose polymers. The enzymatic activity of PARP is induced during DNA damage, suggesting a role for PARP in DNA repair and DNA damage-induced cell death. A number of inhibitors of PARP have been disclosed, some of which are commercially available. For example, PJ-34 N- (6-oxo-5,6-dihydrophenanthridin-2-yl) -N, N-dimethylacetamide.HCl, INHBP 5-iodo-6-amino-1,2-benzopyrone, 3-aminobenzamide, benzamide, 4-amino-1,8-naphthalimide, 6 (5H) -phenanthridinone, 5-aminoisoquinolinone (5-AIQ); hydrochloride, 4-hydroxyquinazoline, 4- Quinazolinol, 1,5-isoquinolinediol, 5-hydroxy-1 (2H) -isoquinolinone, 3,4-dihydro-5- [4- (1-piperidinyl) butoxy] -1 (2H) -isoquinolinone (DPQ) All are available from Inotek Pharmaceuticals (Beverly, MA). GPI 15427 (Tentori et al. (2003) Proceedings of the AACR, 44, Abs No. 5466) and methoxyamine (Liuzzi et al. (1985) J Biol. Chem. 260, 5252-5258; Rosa et al. (1991) Nucleic Acids Res., 19, 5569-5574; and Horton et al. (2000) J. Biol. Chem., 275, 2211-2218) have been reported to promote anti-tumor effects of both chemotherapy and radiation therapy. I came.

C. Nucleotide excision repair (NER)
Nucleotide excision repair (NER) acts on a variety of helix-distorted DNA lesions, most of which are caused by exogenous causes that interfere with normal base pairing. The primary function of NER in humans appears to be removal of ultraviolet (UV) -induced damage (eg, pyrimidine dimers). Members of the NER pathway have been identified and their deficiency can cause autosomal recessive disease called xeroderma pigmentosum (XP), which includes seven different genes: XPA, XPB, XPC, XPD, XPE, All of which function in the NER pathway, including XPF and XPG (Hoeijmakers (2001) Mutant Res. 485: 43-59).

  The eukaryotic NER contains two major branches: transcription-coupled repair (TCR) and genome-wide repair (GGR) (de Laat et al. (1999) Genes Dev. 13: 768-85, Tornaletti & Hanawalt (1999)). ) Biochimie. 81: 139-46). GGR is a slow and random process that examines the entire genome for damage, while TCR is highly specific and effective, concentrating on damage blocking RNA polymerase II. The two mechanisms differ in substrate specificity and substrate recognition. In GGR, the XPC-HR23B complex recognizes damage located in the non-transcribed region (Sugasawa et al. (2001) Genes Dev. 15: 507-21), whereas the termination of RNA polymerase II (RNAPII) is a recognition signal in TCR. It becomes. Although the molecular mechanism of RNAPII substitution is currently unclear, essential elements such as the cocaine syndrome proteins CSA, CSB, XPA-binding protein 2 (XAB2), TFIIH and XPG (Svejstrup 2002) may function in the TCR Have been identified. Next, in both GGR and TCR, a non-spiral structure is formed around the lesion when it is opened. This creates specific cleavage sites for XPG and ERCC1-XPF nucleases, and the resulting gap is filled by PCNA-dependent polymerase and closed by DNA ligase (de Laat et al., Ibid.).

D. Mismatch repair (MR)
Mismatch repair (MMR) removes both nucleotides mismatched by DNA polymerase and insertion / deletion loops caused by misalignment between repetitive sequences (Harfe & Jinks-Robertson (2000) Annu Rev Genet 34: 359-399). Initially, heterodimeric MSH complexes recognize nucleotide mismatches, followed by interactions with MLH1 / PMS2 and MLH1 / MLH3 complexes. Several proteins participate in the nucleotide removal and resynthesis process. Tumor cells deficient in mismatch repair have a much higher mutation frequency than normal cells (Parsons et al. (1993) Cell 75: 1227-1236, Bhattacharyya et al. (1994) Proc Natl Acad. Sci USA 91: 6319-6323) . At least six genes involved in mismatch repair, MSH2, MLH1, PMS2, MSH3, MSH6 and MLH3 have been identified in humans. Defects in these genes except MSH3 cause hereditary nonpolyposis colorectal cancer (HNPCC) (Hoeijmakers 2001).

  Other inhibitors for DNA damage repair have been disclosed, including aphidicolin (Gera (1993) J Immunol. 151: 3746-57), rapamycin (mTOR inhibitor, Sabers et al. (1995) J Biol. Chem. 270: 815- 22), AGT inhibitor 06-benzylguanine (Bronstein et al. (1992) Cancer Res. 52: 3851-6).

VII. Identification of Inhibitors of Non-FA DNA Damage Repair Pathways As noted above, in certain situations, cellular DNA damage repair pathways may be partially unnecessary. This is a problem in identifying agents that specifically block one pathway. Thus, inhibitors identified using cell-based methods with functional DNA damage repair pathways have numerous targets that are involved in multiple DNA damage repair pathways. Thus, using cell lines that lack one or more DNA damage repair pathways can greatly speed the identification of new specific inhibitors. Thus, according to one embodiment, a method of identifying an agent that inhibits a non-FA DNA damage repair pathway is provided. The method uses cells that are impaired in the FA pathway. The method includes contacting the cell with a drug and testing sensitivity to a genotoxic anti-tumor agent. An agent that increases sensitivity to a genotoxic anti-tumor agent in a test cell that contains a disorder of the FA pathway when compared to a control cell that contains a functional DNA damage repair pathway is Suggests that it inhibits the FA DNA damage repair pathway. In one embodiment, the test cell and the control cell are at least one component of the FA / BRCA pathway, e.g., in particular, FANCA, FANCB, FANCC, FANCD, FANCE, FANCF, FANCG, FANCL and ATR protein kinase. It is related except that it includes obstacles.

  According to one embodiment, the method includes comparing the susceptibility of two syngeneic cell lines differing in FA pathway function to genotoxic anti-tumor agents. In one embodiment, two syngeneic ovarian tumor lines, original 2008 cells and 2008 cells supplemented with FANCF cDNA are used. The original 2008 cells cannot express FANCF, these cells have disrupted the FA pathway, and they are hypersensitive to cisplatin. Complementing 2008 cells with FANCF cDNA restores the FA pathway in these cells. These control cells thus provide a basis for comparison. This syngeneic pair of cells is subjected to high-throughput chemical screening with a library of compounds (eg, kinase inhibitors). Agents that selectively kill 2008 cells (deficient in the FA / BRCA pathway) but do not kill 2008 control cells repaired with FANCF are candidates for inhibitors of the non-FA DNA damage repair pathway.

  The use of these syngeneic cell lines also allows the identification of gene products involved in DNA damage repair pathways other than the FA pathway. In one embodiment, genes that affect the viability of the original but not control cells are tested by systematic and extensive inhibition with siRNA libraries. For example, a barcoded siRNA library can be used for stable transfection of two cell lines. 2008 A gene that is required for cell viability but not for repaired cells. Genes important for DNA damage repair pathways other than the FA pathway (eg, the BER pathway), such as siRNA knockdown of such genes are lethal in the original 2008 cells, but in control 2008 transfected with FANCF cDNA It is expected to result in cells that are not.

  Agents that are identified in this way and can kill cells in which one or more DNA damage repair pathways are disrupted, but do not kill syngeneic cell lines in which disruption is restored can be used to treat cancer . Disruption of two or more of the six major DNA damage repair pathways can lead to cell death. Because many cancers already have one pathway that is knocked out or suppressed, a relatively non-toxic inhibitor to the second pathway (eg, the BER pathway) is a cancerous tumor even in the absence of chemotherapeutic agents. Can be sufficient to cause shrinkage. In addition, the use of a combination of two inhibitors in tumor cells whose primary DNA damage repair pathway is intact (eg, one inhibitor of the FA pathway and one inhibitor of the BER pathway) is the toxicity of such a combination. If it is not harmful to normal (non-cancerous) cells, it can be sufficient to cause significant tumor shrinkage. In such cases, prodrug methods that promote the uptake of these agents by cancer cells provide the necessary therapeutic index.

VIII. Disclosed herein antitumor agents by using combinations of anti-tumor agents in combination with an inhibitor of DNA damage repair pathways, a method of treating a patient having a neoplastic disease. Particularly useful anti-tumor agents include, but are not limited to, agents that cause DNA damage. These agents include DNA alkylating agents, intercalating agents and the like. Further contemplated is therefore the use of DNA damaging chemotherapeutic compounds, which include 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU), busulfan, carboplatin, carmustine, chlorambucil, cisplatin , Cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin, ifosfamide, irinotecan, lomustine, mechloretamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide and topotecan . In addition, the methods described herein also employ radiotherapy to treat neoplastic diseases. In one embodiment, the genotoxic anti-tumor agent does not inhibit DNA damage repair at the administered concentration.

IX. Identification of Antitumor Agent Responders The present invention is based on the surprising discovery that the effectiveness of the cellular FA pathway is strongly associated with its sensitivity to chemotherapeutic drugs. Accordingly, in one embodiment, the present invention provides a method for predicting whether a subject having a neoplastic disease or condition will respond to a genotoxic anti-tumor agent. The method includes obtaining a biological sample from the subject and determining the degree of ubiquitination of the Fanconi anemia complementation group D2 (FANC D2) polypeptide in the biological sample. A subject responds to a genotoxic antitumor agent if the degree of ubiquitination of the FANC D2 polypeptide in the subject's biological sample is less than about 70% when compared to a biological sample from a control subject. It suggests that you will do.

  In another aspect, the invention provides a method for predicting whether a subject having a neoplastic disease or condition will respond to a genotoxic anti-tumor agent. The method includes obtaining a biological sample from the subject and determining a FANC D2-containing focus in the biological sample. The difference in focus formation is that when compared to a biological sample from a control subject, the sample from the subject contains less than about 70% FANC D2-containing focus, which is a genotoxic anti-tumor agent. Suggest that the subject will respond.

  In one embodiment, the neoplastic disease is leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myelodysplasia, multiple myeloma, Hodgkin's disease or non-Hodgkin's lymphoma, small cell lung cancer or non-small Cell lung cancer, stomach cancer, intestinal cancer or colorectal cancer, prostate cancer, ovarian cancer or breast cancer, head cancer, brain or cervical cancer, cancer in the urethra, kidney or bladder cancer, malignant melanoma, liver cancer, uterine cancer or Selected from the group consisting of pancreatic cancer.

  According to these embodiments, the ability of a biological sample to activate the FA pathway is determined by measuring the level of FANC D2 monoubiquitination while identifying responders to chemotherapeutic agents, particularly genotoxic anti-tumor agents Decide to do. The anti-tumor agent can be anything used in the treatment of cancer, and in one embodiment, the mechanism of action of the anti-tumor agent is mediated by DNA damage. These compounds include, but are not limited to: 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU), busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, Daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide and topotecan and ionizing radiation.

  In certain embodiments, a patient or a biological sample obtained from a patient can be exposed to an anti-tumor agent prior to determining the extent of FANC D2 polypeptide ubiquitination. In one embodiment, the patient or biological sample obtained from the patient is exposed at a dose that is less than or equal to a therapeutically effective dose. In another embodiment, the exposure is 50% or less of the therapeutically effective dose of the anti-tumor agent.

  The degree of ubiquitination of the FANC D2 polypeptide can be compared to that of a control subject. As used herein, a control subject can be a single subject that has been previously determined to be normal for a response to an anti-tumor agent, or a number of normal subjects. Biological samples from either a single control subject or many control subjects can be used. In this embodiment, if the rate of FANC D2 ubiquitination is less than about 70% compared to the sample from the subject, the sample from the subject who received the same or equivalent dose of the antitumor agent as the test sample When compared, for example, less than 70%, less than 65%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less, the subject is anti-tumor Considered to be an agent responder. Further, in embodiments comprising exposing to an anti-tumor agent prior to determining the degree of ubiquitination of the FANC D2 polypeptide, the control sample is prepared prior to test sample preparation or test sample preparation. It can be prepared at the same time.

  In one embodiment, the subject or a biological sample taken from the subject can be treated with a genotoxic anti-tumor agent prior to measuring the effectiveness of the FA pathway. The dose of anti-tumor agent will be the amount necessary to induce FA pathway in normal subjects. Usually, the dose of antineoplastic agent is between about 5% and 100% of a standard therapeutically effective dose, more typically between 20% and 100%, and most typically about 35%. Will be between 100% and 100%.

  As described herein, there are a number of methods for measuring the extent of FANC D2 polypeptide ubiquitination in a biological sample. The degree of ubiquitination of the FANC D2 polypeptide can be measured using immunoblot analysis as described above. Alternatively, the formation of a FANC D2-containing focus can be detected as a surrogate marker for FANC D2 ubiquitination using, for example, immunofluorescence microscopy of biological samples.

  About 70% or less of ubiquitinated FANC D2 polypeptide formation compared to normal subjects, eg, 70% or less, 65% or less, 60% or less, 50% or less, 40% or less, 30% of normal subjects A subject is considered a responder if:

X. Treatment of Neoplastic Disease In certain embodiments, the subject or patient is administered a therapeutically effective dose of a genotoxic anti-tumor agent and an inhibitor of a non-FA DNA damage repair pathway (eg, the FA pathway). At the same time, administered before or after. Therapeutically effective doses of many antineoplastic agents are known and can be found, for example, in Cancer Chemotherapy and Biotherapy: Supervised by A Reference Guide Edition Number: 2 Tenenbaum, Saunders & CO (1994) Is incorporated herein by reference.

  Also provided herein are methods for treating a neoplastic disease in a subject in need of treatment for the neoplastic disease. In one embodiment, the method comprises administering to the subject an effective amount of an inhibitor of the FA pathway and a genotoxic anti-tumor agent. Antitumor agents include 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU), busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin , Ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide and topotecan, and ionizing radiation.

  In another aspect, a method of treating a neoplastic disease in a subject in need of treatment for the neoplastic disease is provided. The method includes administering to the subject an effective amount of an inhibitor of the FA pathway and an inhibitor of the non-FA DNA damage repair pathway. Non-FA DNA damage repair pathway inhibitors can be selected that inhibit any repair pathway, and PARP inhibitors, DNA-PK inhibitors, mTOR inhibitors, ERCC1 inhibitors, ERCC3 inhibitors, ERCC6 inhibitor, ATM inhibitor, XRCC4 inhibitor, Ku80 inhibitor, Ku70 inhibitor, XPA inhibitor, CHK1 inhibitor, CHK2 inhibitor, or a pharmaceutically acceptable salt, ester, derivative, solvate thereof or It can be selected from the group consisting of prodrugs. Inhibitors of the FA pathway can be administered before, simultaneously with, or after administering the inhibitor of the non-FA DNA damage repair pathway. Inhibitors can be administered parenterally, orally or directly to the tumor.

  Inhibitors of the FA pathway, like inhibitors of the non-FA DNA damage repair pathway, can act to increase the sensitivity of neoplastic diseases to genotoxic anti-tumor agents. Accordingly, in another aspect, a method for increasing the susceptibility of a neoplastic disease to a genotoxic anti-tumor agent is provided. The method includes administering a combination of an effective amount of an inhibitor of the FA pathway and an inhibitor of the non-FA DNA damage repair pathway before, after or simultaneously with the administration of a therapeutically effective dose of the agent. The method can be useful for the treatment of many types of neoplastic diseases and these are leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myelodysplasia, multiple myeloma , Hodgkin disease or non-Hodgkin lymphoma, small cell lung cancer or non-small cell lung cancer, stomach cancer, intestinal cancer or colorectal cancer, prostate cancer, ovarian cancer or breast cancer, head cancer, brain tumor or cervical cancer, cancer in the urethra, kidney Or it may be selected from the group consisting of bladder cancer, malignant melanoma, liver cancer, uterine cancer or pancreatic cancer.

  Inhibitors of the FA pathway are further useful as agents that increase the sensitivity of neoplastic diseases to genotoxic anti-tumor agents. Accordingly, in another aspect, the present invention provides a method of increasing the susceptibility of a neoplastic disease to a genotoxic anti-tumor agent. The method comprises administering an effective amount of an inhibitor of the FA pathway before, after or simultaneously with the administration of a therapeutically effective dose of a genotoxic anti-tumor agent. As noted above, FA pathway inhibitors can be administered before, simultaneously with, or after administration of non-FA DNA damage repair pathway inhibitors, and can be administered parenterally, orally or directly to the tumor. Can be administered. In one embodiment, the method includes administering an inhibitor of the non-FA DNA damage repair pathway in addition to the FA inhibitor and the genotoxic anti-tumor agent. Inhibitors of the non-FA DNA damage repair pathway can be administered before, after or simultaneously with the administration of a therapeutically effective dose of the FA pathway inhibitor and the genotoxic anti-tumor agent.

  The effectiveness of the compositions disclosed herein in preventing or treating neoplastic diseases can be tested, for example, in animal models of certain neoplastic diseases. Numerous examples of animal models are well known to those skilled in the art and are described, for example, in Holland, Mouse Models of Cancer (Wiley-Liss 2004); Teicher, Tumor Models in Cancer Research (Humana Press; 2001); Kallman, Rodent Tumor Models in Experimental Cancer Therapy (Mcgraw-Hill, TX, 1987); Hedrich, The Laboratory Mouse (Handbook of Experimental Animals) (Academic Press, 2004); and Arnold and Kopf-Maier, Immunodeficient Animals: Models for Cancer Research (Contributions And Oncology, Vol. 51) (Karger, 1996), the contents of which are hereby incorporated by reference in their entirety.

XI. Test compound of the present invention
Whether in vitro or in vivo, the present invention provides a method for screening for compositions that can inhibit the formation of FANC D2-containing foci and compositions that inhibit DNA damage repair pathways other than the FA pathway. Include. Modulator candidate compounds can be screened from large libraries of synthetic or natural compounds. Numerous means are currently used for the random and direct synthesis of compounds based on sugars, peptides and nucleic acids. Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH) and Microsource (New Milford, CT). . A rare chemical library is available from Aldrich (Milwaukee, Wis.). Combinatorial libraries can be obtained or prepared. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available, for example, from Pan Laboratories (Bothell, WA) or MycoSearch (NC) and are well known in the art. It can be easily produced by a known method. Furthermore, natural and synthetically generated libraries and compounds are readily modified by conventional chemical, physical and biochemical means.

  Useful compounds are organic compounds, usually including small molecule organic compounds, but can be found within a number of chemical species. The molecular weight of the low molecular weight organic compound is greater than 50 daltons and even less than about 2,500 daltons, preferably less than about 750 daltons, more preferably less than about 350 daltons. Examples of types include heterocycles, peptides, saccharides, steroids and the like. The compounds can be modified to improve efficacy, stability, drug compatibility, and the like. The structural identification of an agent can be used to identify, generate or screen additional agents. For example, when identifying a peptide drug, by functionalizing the amino terminus or carboxyl terminus, such as by using a non-natural amino acid such as a D-amino acid, particularly D-alanine (eg, the amino group is acylated or They can be modified in a variety of ways to improve stability, such as by alkylation, carboxyl group esterification or amidation).

  Modulator candidates that can be screened by the methods of the present invention include receptors, enzymes, ligands, regulators and structural proteins. Modulator candidates also include nucleoproteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, cell membrane related proteins, serum proteins, viral antigens, bacterial antigens, protozoan antigens and parasite antigens. Modulator candidates further include proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic acids (eg, RNA such as ribozymes or antisense nucleic acids). Proteins or polypeptides that can be screened using the methods of the present invention include hormones, growth factors, neurotransmitters, enzymes, coagulation factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, structures Includes proteins, viral antigens, parasite antigens, bacterial antigens and antibodies (see below).

  Candidate modulators that can be screened by the present invention are also designed to eliminate substances that are deficient in test cells or organisms, or substances that are clinically effective at concentrations higher than normal, as well as unwanted protein translation. Contained substances. The nucleic acids used in the present invention not only encode the modulator candidates described above, but can exclude or encode products that eliminate harmful proteins. Such nucleic acid sequences are antisense RNA and ribozymes and the DNA expression constructs that encode them. Note that as described below, antisense RNA molecules, ribozymes, or genes encoding them can be administered to a test cell or organism by nucleic acid delivery methods known in the art. By inactivating the nucleic acid sequence, a ribozyme or antisense RNA specific for the target mRNA can be encoded. Hammerhead ribozymes are the smallest known and can be produced in vitro or transported into cells (Sullivan (1994) J. Invest. Dermatol., 103: 85S-98S; Usman et al. (1996), Curr. Opin. Struct. Biol., 6: 527-533).

XII. In another embodiment of the pharmaceutical composition , the present invention provides an FA combined with an anti-tumor agent and / or an inhibitor of a non-FA DNA damage repair pathway as described above, and a pharmaceutically acceptable carrier as described below. The present invention relates to methods and pharmaceutical compositions comprising inhibitors of the pathway. Pharmaceutical compositions comprising inhibitors of the FA pathway are useful for the treatment of various diseases and conditions including cancer and may also be useful as protective agents against genotoxic anti-tumor agents.

In one embodiment, the present invention provides a method of treating a subject in need of treatment for a neoplastic disease comprising administering a combination of the following effective amounts:
a) inhibitors of the FA pathway, or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof, and
b) Genotoxic antitumor agents.

  Examples of inhibitors of the FA pathway include H-9, Ulster Paulon and curcumin. However, it will be understood by one of skill in the art that additional inhibitors of the FA pathway can be identified using, for example, the methods described herein. In this regard, inhibitors of the FA pathway can be small molecules and antibodies, ribozymes or siRNA molecules.

  This method is applicable to leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myelodysplasia, multiple myeloma, Hodgkin's disease or non-Hodgkin's lymphoma, small cell lung cancer or non-small cell lung cancer, gastric cancer, intestinal cancer Or various tumors including colorectal cancer, prostate cancer, ovarian cancer or breast cancer, head cancer, brain or cervical cancer, cancer in the urethra, kidney or bladder cancer, malignant melanoma, liver cancer, uterine cancer or pancreatic cancer Can be used to treat sexually transmitted diseases. In one embodiment, the method is used to treat ovarian cancer.

  The dose of an inhibitor of the FA pathway depends on several factors including solubility, bioavailability, plasma protein binding, renal clearance, and inhibition constant. An amount effective to achieve at least partial FA pathway inhibition in a particular therapeutic application is defined as an “effective dose”. The amount necessary to obtain this dose will depend on the severity of the disease and the general state of the patient's own immune system, but generally ranges from 0.005 to 5.0 mg per kilogram body weight, 0.05 A dose of ˜2.0 mg / kg / dose of inhibitor is usually used. Alternatively, the dose can be administered using a functional dose because activation of the FA pathway in the subject uses ubiquitination of the FANC D2 polypeptide using the methods described herein. This is because it can be determined experimentally. Thus, an “effective dose” of an inhibitor of the FA pathway reduces the level of FANC D2 ubiquitination to about 70% or less, more typically about 50% or less of a control sample when compared to a control sample. It may mean the dose required for In this regard, control samples are ideally taken from the same subject prior to administration of the inhibitor.

  The dose of the FA pathway inhibitor relative to the dose of the genotoxic anti-tumor agent can be expressed as a ratio. Inhibitors of the FA pathway are between about 100: 1 and about 1: 100, for example 1: 100, 1:50, 1:10, 1: 5, 1 for anti-tumor agents genotoxic on a molar basis. : 2, 1: 1, 2: 1, 5: 1, 10: 1, 20: 1, 50: 1 or 100: 1 ratio.

  Genotoxic antitumor agents are drugs used to treat neoplastic diseases, such as 1,3-bis (2-chloroethyl) -1-nitrosourea (BCNU), busulfan, carboplatin, carmustine, chlorambucil, Including cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide and topotecan.

  The doses of the above-described anti-tumor agents have been established for different types of neoplastic diseases. However, co-administration with inhibitors of the FA pathway can increase the sensitivity of neoplastic diseases to anti-tumor agents. Thus, the dose of antineoplastic agent can be less than the dose typically administered for a particular neoplastic disease. Lower doses can have the added benefit of reducing side effects. Usually, however, the dose of anti-tumor agent is expected to fall between about 20% to 100%, more typically between about 35% to 100% of the typical dose for a particular neoplastic disease.

In yet another embodiment, the invention provides a method of treating a subject in need of treatment for a neoplastic disease, comprising administering to the subject a combination of the following effective amounts:
(A) inhibitors of the FA pathway, or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof, and (b) inhibitors of the DNA damage repair pathway.

  Inhibitors of DNA damage repair pathway are PARP inhibitors, DNA-PK inhibitors, FA inhibitors, mTOR inhibitors, ERCC1 inhibitors, ERCC3 inhibitors, ERCC6 inhibitors, ATM inhibitors, XRCC4 inhibitors, Ku80 inhibitors , Ku70 inhibitor, XPA inhibitor, CHK1 inhibitor, CHK2 inhibitor, or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof.

  In one embodiment, the non-FA DNA damage repair pathway is a pathway other than the FA pathway. In one embodiment, the inhibitor targets a pathway selected from the group consisting of a non-homologous end-joining DNA damage repair pathway, a mismatch repair pathway, and a nucleotide excision pathway. In another embodiment, the inhibitor targets a non-homologous end-joining DNA damage repair pathway. In yet another embodiment, the inhibitor targets the direct elimination pathway. In another embodiment, the inhibitor targets the mismatch repair pathway. In yet another embodiment, the inhibitor targets the nucleotide excision repair pathway. In another embodiment, the inhibitor targets the base excision repair pathway.

  The ideal dose of an inhibitor of the DNA damage repair pathway will depend on the severity of the disease and the general condition of the patient's own immune system, as described above for inhibitors of the FA pathway, however, In general, doses of 0.05 to 2.0 mg / kg / dose of inhibitor are commonly used, ranging from 0.005 to 5.0 mg per kilogram of body weight. Alternatively, the appropriate dose can be determined empirically and inhibition of the DNA damage repair pathway can be measured using a biological sample taken from the subject. Thus, an “effective dose” of an inhibitor of a DNA damage repair pathway reduces the level of a particular pathway to about 70% or less, more typically about 50% or less of a control sample when compared to a control sample. It may mean the dose required for In this regard, control samples are ideally taken from the same subject prior to administration of the inhibitor.

In yet another embodiment, the invention provides a method of treating a subject in need of treatment for a neoplastic disease, comprising administering to the subject a combination of the following effective amounts:
(A) an inhibitor of the FA pathway, or a pharmaceutically acceptable salt, ester, derivative, solvate or prodrug thereof,
(B) inhibitors of non-FA DNA damage repair pathways, and (c) genotoxic agents, or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof.

  Inhibitors of the FA pathway, dosages and methods of administration thereof are as described above. Similarly, inhibitors of non-FA DNA damage repair pathways, as well as their doses and methods of administration, are as described above. However, as noted above, administration of inhibitors of the FA pathway, and further non-FA DNA damage repair pathways, can increase sensitivity to anti-tumor agents. Thus, the dose of antineoplastic agent can be less than the dose typically administered for a particular neoplastic disease. Lower doses can have the added benefit of reducing side effects. Usually, however, the dose of anti-tumor agent is expected to fall between about 20% to 100%, more typically between about 35% to 100% of the typical dose for a particular neoplastic disease.

  A compound of the present invention, or a pharmaceutically acceptable salt, ester, derivative, solvate or prodrug thereof, is administered orally, intravenously, intramuscularly for therapeutic or prophylactic treatment of a disease, It can be formulated for subcutaneous, topical and / or parenteral administration. For oral or parenteral administration, the compounds of the invention can be mixed with conventional pharmaceutical carriers and excipients such as tablets, capsules, elixirs, suspending agents, syrups, cachets, etc. Can be used. Compositions comprising compounds of the present invention may comprise from about 0.1% to about 99.9%, from about 1% to about 98%, from about 5% to about 95%, from about 10% to about 80% or from about 15% to about 60% by weight. % Active compound.

  The compounds of the invention can be administered at different times using the method of divided administration. For example, in certain situations, it may be advantageous to administer an inhibitor of the FA pathway prior to, simultaneously with, or after administration of a genotoxic anti-tumor agent or other agent. Similarly, the method of administering each compound will depend on the optimal means of administering them.

  The medicaments disclosed herein are prepared according to standard procedures and selected to reduce, prevent or eliminate cancer or to provide a protective effect against genotoxic anti-tumor agents such as ionizing radiation. (For example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA; and Goodman and Gilman, Pharmaceutical Basis for a general description of how to administer various antimicrobial agents to treat humans; of Therapeutics, Pergamon Press, New York, NY, the contents of which are incorporated herein by reference). The compositions of the invention can be delivered using controlled release delivery systems (eg, capsules) or sustained release delivery systems (eg, biodegradable matrices). Examples of delayed release delivery systems for drug delivery suitable for administration of the compositions of the present invention are described in US Pat. No. 4,452,775, US Pat. No. 5,239,660 and US Pat. No. 3,854,480.

  The pharmaceutically acceptable compositions of the present invention comprise one or more non-toxic pharmaceutically acceptable carriers and / or diluents that collectively refer to one or more compounds of the present invention as “carrier” materials herein. And / or adjuvants and / or excipients and optionally other active ingredients. The composition may include common carriers and excipients such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The composition may comprise croscarmellose sodium, microcrystalline cellulose, sodium starch glycolate, and alginic acid.

  Tablet binders that may be included are gum arabic, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Providone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose.

  Lubricants that can be used include magnesium stearate or other metal stearates, stearic acid, silicone solutions, talc, waxes, oils and colloidal silica.

  Flavoring agents such as peppermint, winter green oil, cherry flavoring agents may also be used. It may also be desirable to add a colorant to make the dosage form look more aesthetic or to help identify products containing the compounds of the invention.

  Solid preparations such as tablets and capsules are particularly useful for oral use. Sustained release formulations or enteric coating formulations can also be devised. Suspensions, syrups and chewable tablets are particularly suitable for pediatric and elderly use. Pharmaceutical compositions for oral administration are, for example, in the form of tablets, capsules, suspensions or solutions. The pharmaceutical composition is preferably made in the form of a dosage unit containing a therapeutically effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, tablets and capsules contain, in addition to the active ingredient, binders such as gum arabic, gelatin, polyvinylpyrrolidone, sorbitol or tragacanth; fillers such as calcium phosphate, glycine, lactose, corn starch, sorbitol or Sucrose; Lubricants such as magnesium stearate, polyethylene glycol, silica or talc: may include conventional carriers such as disintegrants such as potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid formulations are generally in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs, such as suspending agents, emulsifiers, non-aqueous solvents, preservatives, colorants and flavoring agents. Conventional additives may be included. Examples of additives for liquid formulations are gum arabic, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hardened edible fats, lecithin, methylcellulose, methyl or propyl parahydroxybenzoic acid , Propylene glycol, sorbitol or sorbic acid.

  For intravenous (iv) use, the compounds of the invention can be dissolved or suspended in any commonly used intravenous infusion and can be administered by injection. Intravenous infusion includes, but is not limited to, saline or Ringer's solution.

  Formulations for parental administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions or suspensions can be prepared from sterile powders or granules having one or more carriers described for use in oral dosage formulations. The compound can be dissolved in polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, sodium chloride and / or various buffers.

  For intramuscular formulations, sterile formulations of the compounds of the invention or suitable soluble salts forming the compounds of the invention can be prepared in pharmaceutical diluents such as water for injection (WFI), saline or 5% glucose. Can be administered dissolved. Suitable insoluble forms of the compounds can be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base (eg, esters of long chain fatty acids such as ethyl oleate).

  For topical use, the compounds of the invention can also be prepared in a form suitable for use on the skin or nasal and throat mucosa, creams, ointments, liquid sprays or inhalants, lozenges or throat applications. It can take the form of an agent. Such topical formulations can further contain a compound such as dimethyl sulfoxide (DMSO), which can facilitate surface penetration of the active ingredient.

  For use in the eye or ear, the compounds of the invention are in the form of a liquid or semi-liquid that is formulated in a hydrophobic or hydrophilic base, such as an ointment, cream, lotion, coating or powder. Can be provided.

  For rectal administration, the compounds of the invention may be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or other glycerides.

  Alternatively, the compounds of the invention may be in powder form for reconstitution with a suitable pharmaceutically acceptable carrier at the time of delivery. In another embodiment, the dosage unit form of the compound may be a solution of the compound or a salt thereof in a suitable diluent in a sterile sealed ampoule.

  The amount of a compound of the invention in a dosage unit includes a therapeutically effective amount of at least one active compound of the invention, and this amount can vary depending on the recipient subject, the route of administration and the frequency of administration. A subject refers to animals such as sheep or mammals including humans.

  In accordance with this aspect of the invention, the novel compositions disclosed herein are placed in a pharmaceutically acceptable carrier and are received by known drug delivery methods (including human subjects). ). In general, the method of the present invention for delivering the composition of the present invention in vivo substantially modifies only that the agent is replaced by a compound of the present invention in a method recognized in the art. Thus, drug delivery methods recognized in the art are used.

  The compounds of the present invention provide a method for treating pre-cancerous or cancerous conditions, or for use as a protective agent against genotoxic anti-tumor agents. The term “unit dose” as used herein refers to the amount of a therapeutically effective amount of a compound of the invention that induces the desired therapeutic response. The term “treatment” prevents the occurrence of a precancerous condition or cancerous condition, or controls or eliminates a precancerous or cancerous condition, in a subject in a therapeutically effective amount of at least one compound of the invention. Defined as administering for both. The term “desired therapeutic response” refers to treating a recipient subject with a compound of the present invention to restore, stop or prevent the precancerous or cancerous state of the recipient subject.

  The compounds of the present invention may be administered as a single daily dose or as multiple doses per day. Treatment regimes may require administration for extended periods of time, eg, several days or 2-4 weeks. The amount per dose or the total dose depends on the nature and severity of the condition, the age and general condition of the recipient subject, the tolerance of the recipient subject to the compound and the type of cancer, the sensitivity of the cancer to the therapeutic agent, and other It will depend on factors such as the combination of therapeutic agents (if used), the dose and type of therapeutic agent used.

  The compounds of the invention can also be administered in the diet or feed of a patient or animal. The animal feed can be a regular food product that can be compounded or added to the premix.

  The compounds of the invention may be taken together or separately in combination with any known clinically approved agent to treat recipient subjects in need of such treatment.

  Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. Further, disclosures relating to the methods and embodiments described herein include Chinomas et al., Mol. Cancer Ther. 5 (4): 952-961 (2006) and Taniguchi and D'Andrea, published electronically in Blood (2006). Both can be found in DOI 10.1182 / blood-2005-10-4240, both of which are hereby incorporated by reference in their entirety. All publications, patent applications, patents and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Example 1: Method
Cell lines and cell cultures
Hela cells, PD20 (FA-D2) fibroblasts and GM6914 (FA-A) fibroblasts were cultured as previously described (Taniguchi et al. (2002) Cell. 109: 459-472). The cells were cultured briefly in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal calf serum (FCS). FANCF-deficient ovarian tumor lines (2008) and FANCF cDNA repair 2008 cells have already been described. The breast cancer cell line MCF7 was obtained from the American Type Culture Collection (Manassas, Virginia). OVCAR5 and OVCAR8 were grown as previously described.

Plasmids and retrovirus-infected retroviral expression vectors, pMMP-puro (Ory et al. (1996) Proc Natl Acad. Sci USA. 93: 11400-11406) and pMMP-puro-FANCD2 have already been described (Timmers et al. (2001) ) Mol Cell. 7: 241-248; Garcia-Higuera et al. (2001) Mol Cell. 7: 249-262). pMMPpuro EGFP-FANCD2 was constructed by adding an EGFP cDNA sequence (from pEGFP-N1 (Clontech)) to the N-terminus of the FANCD2 cDNA sequence. The cDNA insert was confirmed by direct DNA sequencing. Generation of pMMP retroviral supernatant and infection of fibroblasts (PD20 fibroblast) was performed as previously described (Naf et al. (1998) Mol Cell Biol, 18: 5952-5960). After 48 hours, cells were trypsinized and selected in medium containing puromycin (1 μg / mL). Dead cells were removed and viable cells were grown under continuous selection in puromycin. Subcloning of PD20 fibroblasts infected with pMMPpuroEGFP-FANCD2 was performed by limiting dilution and clones that showed clear EGFP-FANCD2 focus formation upon IR treatment (15 Gy, 10 hours) were screened for drugs Selected for the experiment (PD20F-EGFP-FANCD2 clone 7).

Cytotoxicity analysis Human cells (Hela cells, PD20 fibroblasts, or 2008 cells; shown in Taniguchi et al. (2003) Nat Med. 9: 568-574) in DMEM-15% FCS (5 ml) in a 12-well plate Were seeded at 9 × 10 ⁴ cells / well. After cells are allowed to attach for 16-24 hours, the medium contains cisplatin (CDDP) or MMC (Sigma) at various concentrations, with or without different concentrations or the same concentration of kinase inhibitor or curcumin (Sigma). Replaced with DMEM-15% FCS. Cells were incubated for 1 day at 37 ° C. The medium was removed and the wells were washed once with phosphate buffered saline (PBS) before fresh addition of DMEM-15% FCS. After incubation for 5-6 days, the monolayer was washed twice with (PBS) and then fixed in 10% (vol / vol) methanol and 10% (vol / vol) acetic acid at 23 ° C. for 5-10 hours . Attached colonies were stained with 1% (wt / vol) crystal violet (Sigma) in methanol (0.5 ml per well) at 23 ° C. for 2-10 minutes. Plates were washed with distilled water and adsorbed by gentle agitation with methanol containing 0.1% (wt / vol) sodium dodecyl sulfate SDS (0.5 ml per well) at 23 ° C. for 1-4 hours The dye was resolubilized. Staining solution (150 μl) was transferred to a 96-well plate and diluted with methanol (1: 3). The crystal violet concentration was measured photometrically (595 nm) with a 3550 type microplate measuring device (Bio-Rad). For quantification, absorbance measurements at 595 nm were standardized with measurements obtained from untreated cells (CDDP concentration = 0 nM), where the cell survival yield was considered to be 100%.

Immunofluorescence microscopy cells were seeded on 4-well chamber slides (Falcon) and cultured for 16-24 hours. Slides were washed with PBS, adherent cells were fixed in paraformaldehyde (4% (wt / vol) in PBS) for 20 minutes at 23 ° C., and Triton X-100 (0.3% (vol / vol in PBS) )) For 10 minutes at 23 ° C. Stain with primary (purified anti-FANCD2 affinity) antibody and secondary (fluorescein-conjugated goat anti-rabbit) antibody for 2 hours at 23 ° C, then 5 minutes at 23 ° C with DAPI (4 ', 6-diamidino-2- Counterstaining was performed with phenylindole dihydrochloride; 10 μg / ml in PBS (Sigma). Slides were mounted on Vectashield (Vector Laboratories) and analyzed with a fluorescence microscope.

High-throughput screening for small molecule inhibitors of the FA / BRCA pathway First, we transduced PD20 (FA-D2) fibroblasts with pMMP-GFP-FANCD2-puro retroviral supernatant and 20 Of independent puromycin resistant colonies were selected. Judging by fluorescence microscopy, one clone (clone 7) had a low expression level of GFP-FANCD2 in the nucleus, but formed a vivid GFP focus in response to IR (2Gy). Clone 7 was used for the following experiments.

  Clone 7 cells were seeded in 384-well plates for high throughput screening. One hour after seeding, compounds from a commercial library (Kau et al. (2003) Cancer Cell. 4: 463-476) were added to each well at a single concentration of about 20-40 μM. . Library compounds were added to duplicate plates. After 12 hours of incubation, the plates were irradiated with radiation (15 Gy) and then incubated for an additional 12 hours to fix the cells for GFP microscopy. Micrographs were taken for each well and wells that had a significant (50%) reduction in GFP focus were identified by visual inspection.

Immunoblot whole cell lysates were electrophoresed by SDS-PAGE (7% polyacrylamide bisacrylamide gel for ATR and FANCD2 analysis and 10% for Chk1 and RPA1 analysis). The protein was transferred to nitrocellulose, blocked and incubated with primary antibody as described (Andreassen et al. (2004) Genes. Dev. 18: 1958-1963). Antibodies are anti-FANCD2 (E35, 1: 1000; Garcia-Higuera et al., Ibid), anti-ATR (1: 1000 Santa Cruz), anti-phosphate-S345-Chk1 (1: 1000, Cell Signaling) and anti-Chk1 (1 : 500 Santa Cruz). Membranes were washed, incubated with HRP-conjugated secondary antibody (Amersham) and detected by chemiluminescence (Amersham) as described (Taniguchi et al. (2002) Cell. 109: 459-472).

In vitro kinase assay for ATR
In vitro kinase assays with Flag-ATR and kinase-dead Flag-ATR have already been described (Andreassen et al., Ibid.).

Example 2: Identification and Characterization of Potential Inhibitors of FANC D2 Ubiquitination and Focus Formation Using the above microscopy, 489 known organisms from the Institute of Chemistry and Cell Biology (ICCB), Harvard Medical collection Active compounds were screened for inhibition of IR-mediated FANC D2 focus formation. A number of positive reactions were identified using primary screening using high-throughput fluorescence microscopy to identify agents that block the formation of FANC D2-containing foci when exposed to ionizing radiation. A number of compounds were identified as described in Table 2.

  In addition to the above compounds, 5,056 compounds from various commercial sets of ICCB were screened, leading to the identification of PD00600, 5323069 and 1M556S as potential positives. Many of the positive reactions identified above include Ulster Paulon, H-9, curcumin, geldanamycin, AG370, Go6976, spermine NONOate, PD00600, nifedipine, α-amanitin, K252c, 5323069, 1M566S, In addition, the ability to inhibit monoubiquitination of FANC D2 polypeptides and / or the ability to confer cisplatin hypersensitivity to 2008 cells expressing functional FANC F was tested. The following examples describe the effects of three compounds, H-9, curcumin and Ulster Paulon, in inhibiting the FA pathway.

Example 3: Identification and characterization of H-9 as an inhibitor of FANC D2 ubiquitination and focus formation
H-9 was identified as an inhibitor of the FA pathway using high throughput screening as described above. As shown in the fluorescence microscope of FIG. 7, H-9 inhibited the formation of FANC D2 focus in the range of 50-100 μM. H-9 treatment was found to reduce the overall level of FANC D2 monoubiquitination, as shown in a secondary screen using immunoblot analysis to determine the relative pool of monoubiquitinated FANC D2 (FIG. 8). H-9 had no effect on ATM-dependent phosphorylation of FANC D2, but inhibited ATR-dependent phosphorylation of CHK1. Finally, the supersensitivity of FANCF F deficient 2008 cells is restored to the wild-type level when transduced with FANCF cDNA, which can be similar in 2008 + FANCF cells by contact with curcumin (Fig. 9). Taken together, these results suggest that H-9 directly or indirectly blocks the FA / BRCA pathway by inhibiting ATP kinase.

Example 4: Identification and characterization of Alster Paulon as an inhibitor of FANC D2 ubiquitination and focus formation Similarly, Alster Paulon was identified as a potential inhibitor of the FA pathway using high-throughput screening. did. Alster Paulon is known to inhibit Cdk1 / cyclin B, Gsk-3B and Cdk-5 (Sausville et al. (2000) Ann NY Acad. Sci. 910: 207-221; Schultz et al. (1999) J Med. Chem. 42: 2909-2919). Alster Paulon inhibited the formation of FANC D2 focus at a concentration of 10 μM (FIG. 10). Similar to H-9, Ulster Paulon inhibited FANC D2 monoubiquitination and inhibited ATR-dependent phosphorylation of Chk1 (FIG. 11).

Example 5: Identification and characterization of curcumin as an inhibitor of FANC D2 ubiquitination and focus formation The natural compound curcumin also caused a dose-dependent decrease in FANC D2 focus formation in screening assays (data is Not shown). Furthermore, in the 3-20 μM range, curcumin caused a dose-dependent decrease in FANC D2 monoubiquitination on Hela cells and cisplatin-exposed Hela cells (FIG. 10). Curcumin also causes a dose-dependent decrease in ATR-mediated Chk1 phosphorylation (FIG. 11), further suggesting that curcumin blocks events upstream of the FA / BRCA pathway.

  We next tested the ability of curcumin to enhance the activity of cisplatin in a cytotoxicity assay. Curcumin sensitized FANCF-repaired 2008 cells to cisplatin, but was less effective for chemical sensitization of the original 2008 cells (FIG. 12). Curcumin sensitized cells at a dose range of 3-20 μM. This concentration range correlated with the curcumin dose range required for inhibition of the FA / BRCA pathway. Similar results were observed when cells were pretreated with curcumin for 24 hours before adding cisplatin. Taken together, these results suggest that curcumin synergizes with cisplatin in promoting ovarian tumor cytoxicity, and that this curcumin effect correlates with inhibition of the FA / BRCA pathway.

  Next, the synergistic effect of curcumin in breast cancer cell lines was evaluated to see if the cytotoxic effect of cisplatin was enhanced in cisplatin resistant cell lines. That is, curcumin was tested for the effect of sensitizing the breast cancer cell line, MCF7 (ATCC), to cisplatin. These results were similar to those seen with ovarian tumor cell lines. Both cisplatin and carboplatin showed increased cytotoxicity with the addition of curcumin (5-20 μM). These doses also lead to a decrease in FANC D2 monoubiquitination in this cell line, suggesting inhibition of the FA / BRCA pathway (FIG. 12). Chk1 phosphorylation was inhibited in a curcumin dose-dependent manner in all cell lines 2008; 2008 + F and MCF7 (FIG. 12).

  Curcumin's chemosensitizing activity was further tested for its specificity for cisplatin. The ovarian tumor cell line SKOV3 is sensitive to cisplatin and taxol (Yang and Page (1995) Oncol Res. 7: 619-24). Curcumin synergized with cisplatin in killing these cells. On the other hand, curcumin had no effect on the dose-dependent taxol cytotoxic properties of these cells.

Example 6: Repair of cisplatin hypersensitivity in ovarian cancer cell line 2008 by FANCF complementation To determine whether these inhibitors are cisplatin sensitizers, two ovarian tumor lines-cisplatin sensitive and FANCF deficient An original 2008 strain and a 2008 strain repaired with FANCF cDNA were used. The original 2008 strain is deficient in FANCF due to epigenetic silencing of the FANCF gene (Taniguchi et al. (2003) Nat Rev Cancer. 3: 23-34). Ulster Paulon increased the cytotoxicity of cisplatin in FANCF cDNA repaired cells but not in the original 2008 cells, indicating that this kinase inhibitor is a potential cisplatin sensitizer, It is suggested to act via inhibition of the FA pathway (FIG. 11). Curcumin sensitized FANCF repaired 2008 cells to cisplatin, but was less effective for chemical sensitization of the original 2008 cells (Figure 9 and Table 3). Curcumin sensitized cells at a dose range of 3-20 μM, which corresponded to the curcumin dose range required for inhibition of the FA / BRCA pathway. Similar results were observed when cells were pretreated with curcumin for 24 hours before adding cisplatin (data not shown). Taken together, these results suggest that curcumin synergizes with cisplatin in increasing ovarian cancer cytotoxicity and that this curcumin effect correlates with inhibition of the FA / BRCA pathway.

Example 7: Determination of maximum tolerated dose for intracerebroventricular administration of FA pathway inhibitors
The FA pathway inhibitors curcumin and Ulster Paulon are dissolved in 25% DMSO to 2.5 mM, which is the maximum concentration compatible with intracerebroventricular administration. Considering that the total ventricular system of mice consists of only 20 μl, the maximum concentration reached (50 μM) exceeded that required for FA pathway inhibition. Similarly, both AMD3100 and O6 benzylguanine are sufficiently soluble in solvents to allow estimated intraventricular concentrations to exceed those required for in vitro efficacy. To test each compound, a group of 6 mice is implanted with a ventricular catheter (4 mm forward of the lambda suture, 0.7 mm side of the midline and 2.5 mm below the dura mater). The catheter is connected to a subcutaneous Alzette osmotic pump type 1007D (90 μl volume delivered at 0.5 μl / hour) containing 2.5 mM, 1.25 mM, 0.625 mM, 0.313 mM, 0.151 mM of each compound or vehicle medium. Mice are observed daily for neuropathy during injection and dissected 21 days after surgery. Intracellular toxicity is assessed by staining brain sections with 1) whether the lateral ventricle and catheter are continuous, and 2) staining with hematoxylin and eosin in addition to TUNEL staining. The maximum dose delivered without clinical or histological signs of injury is selected for the next efficacy experiment described below.

Example 8: In vivo tumoricidal effect of systemic BCNU administration by intraventricular administration of an FA pathway inhibitor The mouse xenograft bioluminescence model used herein uses the U87 GMB cell line (ATCC). Has been transduced by retrovirus with the coding sequence of luciferase in the pMMP vector. An established U87-luciferase cell line is harvested at mid-logarithmic growth phase, resuspended at 50,000 cells in 10 μl PBS and introduced into the mouse brain using stereotaxic guidance (2 mm side of the cross stitch) And back, 3 mm below the dura mater). Mice are injected intraperitoneally with D-luciferin (Xenogen, Alameda, CA) and imaged with the IVIS imaging system (Xenogen) on the 5th and 10th postoperative days.

For each compound tested, a group of 30 mice were transplanted with U87-luciferase and investigated on day 5 and day 10 after surgery, of which 20-26 mice were transplanted with tumors transplanted. Engraftment is expected (Rubin et al. (2003) Proc. Natl. Acad. Sci. USA, 100: 13513-13518). Mice engrafted with a U87-luciferase tumor are then surgically implanted with a ventricular catheter and Alzette pump 1007D. Then stratify these mice into four groups:
Group 1: treated with control vehicle (25% DMSO);
Group 2: treated with intraperitoneal BCNU (15 mg / kg);
Group 3: treated by intraperitoneal administration of the maximum tolerated dose of FA inhibitor; and Group 4: treated by intraperitoneal administration of BCNU (15 mg / kg) in addition to the maximum tolerated dose of FA inhibitor.

  By placing the ventricular catheter on the opposite side of the tumor implantation site, the effects of tumor growth in the stereotaxic position are minimized. Intraperitoneal BCNU injection is performed 4 days after intracerebroventricular administration to allow sensitization. Mice are imaged 15 and 20 days after the initial tumor implantation. Comparison of tumor growth is determined using the LIVING IMAGE software package (Xenogen).

Example 9: Effect of FA inhibitors on sensitization of ovarian tumors to animal model anti-tumor agents Many animal models of ovarian cancer are known in the art. For example, Connolly et al. ((2003) Cancer Research, 63, 1389-1397), which is incorporated herein by reference, shows the epithelial ovarian cancer in mice by chimeric expression of the SV40 tag under the control of the MISIIR promoter. A method of generating is disclosed. In another illustration (Liu et al. (2004) Cancer Research 64, 1655-1663), which is also incorporated herein by reference, the human HRAS or KRAS oncogene can be transferred to immortalized human ovarian surface epithelial cells. An introduction is disclosed, which forms a subcutaneous tumor after injection into immunodeficient mice. These mouse models provide a useful tool to test the effectiveness of FA inhibitors in sensitizing ovarian tumors to antitumor agents. Use 6 mice per group. To test the efficacy of cisplatin, the following groups were used alone or in combination with the FA inhibitor Ulster Paulon:
Group 1: treated with control vehicle;
Group 2: treated with 4 mg / kg cisplatin;
Group 3: treated with Ulster Paulon 5 mg / kg;
Group 4: Treated with 4 mg / kg cisplatin and Ulster Paulon 5 mg / kg. Repeat this cycle after 2 days.

  All treatments begin 1 week after tumor inoculation. Mice are treated for a total of 10 cycles and dissected for tumor nodules 2 weeks (day 50) after withdrawal of drug treatment. Dissect and count the number of tumor nodules in the abdominal cavity, measure tumor diameter, measure ascites volume, and qualitatively observe the color of the peritoneal wall as an indicator of the degree of tumor-induced vascularization To evaluate the anti-tumor activity of each group. Toxicity is assessed by qualitative observation of the overall appearance and behavior of mice prior to dissection and by measuring body weight at various intervals during the course of treatment.

It will be apparent to those skilled in the art that the effectiveness of other FA inhibitors such as curcumin, Ulster Paulon and H-9 can be tested using this means. Curcumin is known to be safe at high doses with an LD ₅₀ greater than 10,000 mg / kg. Curcumin can be administered orally, intraperitoneally or intravesically. In one illustration, intraperitoneal administration of 100 mg / kg to 300 mg / kg of curcumin is performed and tested in mice alone or in combination with an anti-tumor agent (eg, cisplatin) as described above. In another example, curcumin is administered intravesically at the maximum possible dose determined using the methods described above.

Example 10: Efficacy of a combination of a FA inhibitor and a DNA damage repair pathway inhibitor in the treatment of ovarian tumors
The effectiveness of the combination of FA inhibitor and DNA damage repair pathway inhibitor is tested essentially as described in Example 6 above. That is, the effectiveness of FA inhibitors (eg, curcumin, H-9 or Ulster Paulon) in the treatment of ovarian tumors is tested alone or in combination with DNA damage repair pathway inhibitors. In one example, the following groups are tested:
Group 1: treated with control vehicle;
Group 2: treated with Ulster Paulon at 5 mg / kg;
Group 3: treated with methoxyamine at 2 mg / kg;
Group 4: Treated with Ulster Paulon at 5 mg / kg and with methoxyamine at 2 mg / kg. Repeat this cycle after 2 days.
Progress is observed as described above.

Example 11: Combination of FA inhibitor and DNA damage repair pathway inhibitor in sensitization of ovarian tumors to antitumor agents In this example, an FA pathway inhibitor such as curcumin, H-9 or Ulster Paulon and methoxyamine The ability in combination with a DNA damage repair pathway inhibitor such as in tumor sensitization to anti-tumor agents is tested essentially as described above using animal models. However, the dose of antitumor agent administered can be varied to determine whether sensitization will lower the total dose of antitumor agent needed to treat the tumor. The following groups of mice are tested:
Group 1: treated with control vehicle;
Group 2: treated with cisplatin 0 mg / kg; Ulster Paulon 5 mg / kg and methoxyamine 2 mg / kg;
Group 3: treated with cisplatin 1 mg / kg; Ulster Paulon 5 mg / kg and methoxyamine 2 mg / kg;
Group 4: treated with cisplatin 2 mg / kg; Ulster Paulon 5 mg / kg and methoxyamine 2 mg / kg;
Group 5: treated with cisplatin 4 mg / kg; Ulster Paulon 5 mg / kg and methoxyamine 2 mg / kg;
Progress is observed as described above.

Example 12: Clinical evaluation of treatment of recurrent Muller tube malignancy with curcumin and carboplatin A phase I open-label, dose escalation safety study was conducted in patients with relapsed cancer originating from the Muller tube. It is performed less than 12 months before chemotherapy with platinum. Curcumin is administered orally the night before, just before, the night and the next morning before intravenous administration of carboplatin AUC 5. The treatment cycle is 28 days, followed by a 28-day observation period following carboplatin administration starting on day 1. Decisions regarding dose escalation and dose-limiting toxicity are made at the end of the 4-week cycle. Patients with no signs of disease progression and tolerating treatment are eligible for further cycles of curcumin / carboplatin treatment.

  First, 3 patients are placed in the first dose level. The initial dose level will be carboplatin AUC 5 and curcumin 900 mg. If no one has dose limiting toxicity (DLT), then the three patients are given dose level 2. If DLT occurs at any dose level for one person, add 3 additional patients to that dose level. If DLT occurs at that dose level for two people, an excess of the maximum tolerated dose (MTD) is declared and MTD is defined as the pre-dose level. There is no escalation of dose between patients.

As the first dose in a drug administration experiment , carboplatin AUC 5 is infused over 60 minutes and curcumin 900 mg is taken orally. During the experiment, only the curcumin dose is gradually increased while the carboplatin dose is kept constant based on the patient's renal function. The cycle was determined as 28-day intervals, and one treatment of carboplatin on day 1 of the cycle and the day before carboplatin on day 1 (day 0), just before, day 1 night and day 2 In the morning, include one course of curcumin administered a total of 4 times during each cycle. The dose of curcumin will be incrementally increased for additional patient populations until DLT and MTD are determined.

Carboplatin administration Carboplatin is injected intravenously over 1 hour. The dose of carboplatin is calculated using the culvert equation based on creatinine clearance as follows:
Total dose (mg) = target AUC (mg / ml per minute) x (estimated GFR + 25)
Carboplatin dose is in mg / m ² without, calculated in mg. The initial target AUC for carboplatin treatment in this study is AUC = 5. Creatinine clearance (CrCL) can be measured or estimated using the Jelliffe equation.

Jelliffe formula for women:

^* Ages are rounded to the nearest tenth Personal selection of premedication for carboplatin antiemetics is acceptable. Standard premedications include zufuran, atiban and decadron.

Definition of Dose Limiting Toxicity (DLT) The determination of DLT for the purpose of assessing dose escalation is defined as follows using the NCI CTC version 3.0 criteria taking into account the known and approved carboplatin toxicity. Toxicity achieved without premedication is not considered to be DLT.
Any nausea or vomiting with premedication of maximum antiemetics> Stage 3
All other drug-dependent non-hematological toxicity> Stage 3
Hemotoxicity Neutrophil count <500 cells / μl> 7 days After administration of curcumin / carboplatin, any febrile neutropenia with neutrophil count <500 cells / μl (T defined as> 101 ° F)
Stage 3 with a platelet count <10,000 cells / μl, or signs of bleeding requiring blood products or platelet transfusions.
Hemoglobin with erythropoietin co-administration> Stage 4 toxicity

Evaluation of response Patients with measurable disease will be evaluated by standard criteria. Patients are reassessed after every 2 cycles of carboplatin / curcumin. In addition to the baseline / screening scan, a confirmation scan is obtained 4 weeks after the initial documentation of the objective response.

Definition responses and progression are evaluated in this experiment using the new international standards proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) Committee (JNCI 92 (3): 205-216, 2000). The change in the longest diameter of the tumor lesion is used for the RECIST criteria. Lesions are measurable or non-measurable using the criteria listed below.

Guidelines for assessing measurable disease:
At baseline, tumor lesions are classified as follows:
(1) Measurable-Lesions that can be accurately measured in at least one dimension with conventional technology 20mm or spiral CT 10mm. Or (2) Not measurable—all other lesions Record all measurements in metric notation. All baseline assessments should be as detailed as possible at the beginning of treatment and not more than 4 weeks before the start of treatment. Diseases that cannot be measured include: bone lesions, leptomeningeal disease, ascites, pleural / pericardial effusion, abdominal masses not confirmed and observed by imaging techniques, cystic lesions.

The same assessment methods and techniques are used at baseline and during the observation period to characterize the specific identification of the assay and each reported lesion. Evaluation based on imaging is preferred as an evaluation by clinical experiments when both methods are used to evaluate the anti-tumor effects of treatment.

Clinical Experiments Clinically detected lesions are considered measurable when they are on the surface (eg, skin nodules and palpable lymphadenopathy). All skin lesions are recorded with a color photograph, which includes a ruler that measures the size of the lesion.

If a chest X-ray can be clearly measured, a chest X-ray lesion is acceptable as a measurable lesion, but CT is preferable.

Computed tomography (CT) and magnetic resonance imaging (MRI)
CT and MRI are the most useful (and most reproducible) methods for measuring target lesions selected for response assessment. Conventional CT and MRI are performed on serial sections with a slice thickness of 10 mm or less.

Ultrasound ultrasound is not used to measure tumor lesions. Ultrasound can be considered as an alternative method for clinical measurements for superficial palpable lymphadenopathy and subcutaneous tissue lesions.

Tumor markers Tumor markers are not used alone to assess response. However, if the marker exceeds the upper limit from the beginning, when all tumor lesions have disappeared, the patient needs to recover to normal levels, which is considered a clinically complete response.

Cytology, Histology These techniques are used to distinguish between partial response (PR) and complete remission (CR) in rare cases where the remaining lesions of the tumor type may contain benign components.

  Cytologic confirmation of any exudate of tumor origin that appears or worsens during treatment when a measurable tumor meets the criteria for responding or stable disease is a response to stable or progressive disease. It is essential to distinguish.

Tumor response assessment
Documenting “target” and “non-target” lesions Up to 5 lesions per organ and all measurable lesions totaling 10 lesions are typical of all included organs and identified as target lesions And recorded and measured as a baseline. Target lesions are selected on the basis of their size (longest diameter) and suitability for accurate repeated measurements (either imaging techniques or clinical). The sum of major diameters of all target lesions is calculated and recorded as the baseline sum of major diameters. The baseline major axis sum is used as a reference to characterize the tumor response of interest.

  All other lesions (or sites of disease) are identified as non-target lesions and are also recorded as a baseline. Measurement of these lesions is not necessary, but whether each is present should be noted during the observation period.

Response criteria
Target lesion assessment criteria originated from the original WHO handbook, taking into account the measurement of the longest diameter of all target lesions only: see complete response—disappearance of all target lesions; partial response—baseline length sum Assuming that the sum of the major axis of the target lesion is reduced by at least 30%; progressive disease—at least 20 % Increase; stable disease—referring to the smallest sum of longest diameters from the start of treatment, there is no decrease sufficient to fit partial remission, nor increase enough to fit progressive disease.

Evaluation of non-target lesions The criteria used to determine a subject's tumor response to non-target lesions are: complete remission-disappearance of all non-target lesions and normalization of tumor marker levels; incomplete remission / stable disease-1 or more Non-target lesions persist and / or maintain tumor marker levels above the upper limit of normal values; and progressive disease—the appearance of one or more new lesions and / or a clear progression of existing non-target lesions.

FIG. 1 outlines an illustration of the flow of a screen for identifying FA pathway inhibitors and agonists. FIG. 2 outlines a high-throughput scheme for identifying FA pathway agonists and antagonists using fluorescence microscopy. FIG. 3 is a schematic diagram showing protein components identified in the FA pathway of DNA damage repair. FIG. 4 shows a schematic of the eGFP-FANC D2 fusion construct and ubiquitination of transfected PD20F, GM6914 and HeLa cells when exposed to ionizing radiation (IR), HU or mitomycin C (MMC). FIG. 5 is a fluorescence micrograph showing fluorescence signals emitted from PD20 and GM6914 cells transfected with eGFP-FANC D2. In addition, GM6914 transfected with FANCA exhibits a punctate FANC D2-containing focus when exposed to ionizing radiation. FIG. 6 outlines a method for screening for FA inhibitors by fluorescence microscopy. FIG. 7 is a fluorescence micrograph showing IR-mediated inhibition of FANC D2-containing foci formation when eGFP-FANC D2-containing cells are exposed to the kinase inhibitor H-9. Figure 8 shows IR-induced inhibition of FANC D2 monoubiquitination (top two panels), ATR kinase phosphorylation (middle panel), Chk1 phosphorylation (fourth panel) and Chk1 polypeptide levels (bottom panel). The immunoblot analysis which shows the effect of H-9 in a panel) is shown. The graph shows the sensitivity of 2008 cells to cisplatin enhanced by H-9. The 2008 cells are inherently sensitive to cisplatin because they lack FANCF (open squares). This sensitivity to cisplatin is reduced by FANCF transfection (2008 + F, open circles). Treatment of 2008 + F cells with H-9 restores sensitivity to cisplatin (filled circles), whereas untransfected cells do not affect sensitivity to cisplatin (filled squares). FIG. 9 shows inhibition of cisplatin-dependent FANC D2 monoubiquitination by curcumin treatment. Graph (9B) shows sensitization of FANCF repaired 2008 cells to cisplatin, and shows that the sensitizing effect on chemical sensitization of the original 2008 cells is reduced by curcumin treatment. FIG. 10 shows the effect of curcumin on FANCD2 focus and sensitivity to cisplatin. (A) Curcumin sensitizes human tumor cells to cisplatin. In this experiment, we compared 2008 cells (an ovarian tumor line that is defective in the FA pathway because it lacks the FANCF protein) and 2008 cells repaired with FANCF cDNA. Cells were pretreated with or without curcumin (20 μM) for 24 hours as indicated and then cells were exposed to cisplatin in increasing doses. Importantly, repaired 2008 cells are sensitized to cisplatin by pretreatment with curcumin. (B) Pretreatment of clone 7 cells with curcumin suppresses FANCD2 focus assembly. FIG. 11 shows an immunoblot analysis showing the effect of Ulster Paulon on the inhibition of IR-induced monoubiquitination of FANC D2. Alster Paulon is a Cdk1 inhibitor and does not inhibit, but rather promotes IR-induced D2 phosphorylation in T691. Alster Paulon inhibits IR-induced monoubiquitination of FANC D2 and phosphorylation (at serine 345) of Cdk1 in ovarian cancer cell lines. FIG. 12 is a photomicrograph showing the inhibition of IR-mediated FANC D2 and BRCA1-containing focus in cells treated with Ulster Paulon. FIG. 13 shows that treating cells with spermine NONOate induces phosphorylation and monoubiquitination of FANC D2 and phosphorylation of Chk1 in the absence of IR. The graph shows that when transfected with FANC F, the sensitivity of 2008 cells (2008 + F) to spermine NONOate is slightly reduced when compared to vector-transfected 2008 cells (2008 + vec). Show. FIG. 14 shows that treating cells with spermine NONOate induces phosphorylation and monoubiquitination of FANC D2 and phosphorylation of Chk1 in the absence of IR. FIG. 15 is a photomicrograph showing that spermine NONOate induces phosphorylation of histone H2AX in the absence and presence of IR. FIG. 16 shows an immunoblot analysis showing that geldanamycin (Hsp90 inhibitor) inhibits IR-induced D2 monoubiquitination in HeLa cells. Geldanamycin also causes a decrease in Chk1 expression. The graph shows that transfection of 2008 cells with FANC F does not change the sensitivity of the cells to geldanamycin. FIG. 17 is a photomicrograph showing inhibition of IR-mediated FANC D2 and BRCA1-containing focus in cells treated with geldanamycin. FIG. 18 shows the effect of Go6976, a PKC, Chk1 inhibitor, on IR-induced monoubiquitination of FANC D2 in HeLa cells. Go6976 promotes phosphorylation of Chk1 in HeLa cells. However, transfection of 2008 cells with FANC F does not affect sensitivity to Go6976. FIG. 19 is a photomicrograph showing inhibition of formation of IR-mediated FANC D2-containing focus in cells treated with Go6976. FIG. 20 is an immunoblot panel showing the effect of AG370, a PDGFR kinase inhibitor, on the inhibition of IR-induced monoubiquitination of FANC D2 in HeLa cells. The effect of AG370 on Chk1 is not clear. FIG. 21 is a photomicrograph showing inhibition of IR-mediated FANC D2 and BRCA1-containing focus in cells treated with AG370. FIG. 22 shows that inactivation of the FA pathway results in increased sensitivity to DNA crosslinkers including BCNU. (A) Syngeneic PD20 (circle) and retrovirus repaired PD20 (square) showed different susceptibility to BCNU. Survival was plotted on the Y axis. Similar data were obtained using 2008 (FANCF deficient cell line) and 2008 supplemented with FANF. (B) FA route model. (C) BCNU induced FANCD2 monoubiquitination only in the normal FA pathway (lane 4). 2008 and 2008 supplemented with FANCF by retrovirus were either not treated or treated with BCNU. Whole cell lysates were fractionated by SDS-PAGE and immunoblotted with FANCD2 antiserum. Similar data was obtained using PD20 and PD20 repaired with FANCD2. (D) The HT15 GBM cell line did not undergo FANCD2 monoubiquitination when treated with BCNU. Other GBM strains tested (LN308, LN428, A172, T98G are shown above; CRL1620, CRL2020, U87, U343 (not shown)) received appropriate FANCD2 monoubiquitination when treated with BCNU. (E) Not receiving FANCD2 monoubiquitination in the HT15 cell line correlated with increased sensitivity to BCNU. Survival was plotted on the Y axis.

Claims (66)

  A method for predicting whether a subject having a neoplastic disease responds to a genotoxic anti-tumor agent, comprising determining the size or number of FANC D2-containing foci in a sample from the subject, Wherein the subject is a genotoxic anti-tumor agent if the number or size of the focus in the sample is less than about 70% of the number or size of the focus in a sample from a control subject. The method that is expected to respond.   2. The method of claim 1, wherein the number or size of the focus is determined using an antibody specific for monoubiquitinated FANC D2.   2. The method of claim 1, wherein the subject is exposed to a genotoxic anti-tumor agent prior to taking a sample from the subject.   4. The method of claim 3, wherein the exposure is below a therapeutically effective dose.   4. The method of claim 3, wherein the exposure is about 50% or less of a therapeutically effective dose.   The method of claim 1, wherein the sample is exposed to a genotoxic antitumor agent prior to determining the number or size of the focus.   A method for predicting whether a subject having a neoplastic disease responds to a genotoxic anti-tumor agent, comprising determining the degree of ubiquitination of a FANC D2 polypeptide in a sample from the subject, Here, a subject is predicted to respond to a genotoxic anti-tumor agent if the degree of ubiquitination of the FANC D2 polypeptide in the sample is less than about 70% when compared to a sample from a control subject. How to be.   8. The method of claim 7, wherein the degree of ubiquitination of the FANC D2 polypeptide is determined by a method comprising immunoblot analysis, ELISA or FACS using an antibody specific for monoubiquitinated FANC D2.   9. The method of claim 8, wherein the ratio of FANC D2-L to FANC D2-S is determined.   8. The method of claim 7, wherein the subject is exposed to a genotoxic anti-tumor agent prior to taking a sample from the subject.   12. The method of claim 10, wherein the exposure is below a therapeutically effective dose.   11. The method of claim 10, wherein the exposure is about 50% or less of a therapeutically effective dose.   8. The method of claim 7, wherein the sample is exposed to a genotoxic anti-tumor agent prior to determining the degree of ubiquitination.   Genotoxic antitumor agents include 1,3-bis (2-chloroethyl) -1-nitrosourea, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin A method according to any one of the preceding claims, selected from the group consisting of:, ifosfamide, irinotecan, lomustine, mechloretamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide, topotecan and ionizing radiation. A method for identifying an inhibitor of a Fanconi anemia DNA repair pathway comprising:
(A) contacting the cell with a test compound;
(B) contacting the cell with a genotoxic anti-tumor compound before, after or simultaneously with step (a);
(C) quantifying intracellular FANC D2-containing focus; where the test compound is identified as an inhibitor of the Fanconi anemia DNA repair pathway when the amount of focus is less than in the control cell, wherein the control Cells have been contacted with the genotoxic antitumor agent but not with the test compound;
(D) examining the degree of monoubiquitination of the FANC D2 polypeptide in the cell for the test compound identified as the inhibitor in step (c); wherein the degree of monoubiquitination of the FANC D2 polypeptide is When lower in the control cell, the test compound is further identified as an inhibitor of the Fanconi anemia DNA repair pathway;
(E) for a test compound further identified as an inhibitor in step (d), contacting a test cell having a functional Fanconi anemia pathway with said test compound and said genotoxic anti-tumor agent;
(F) determining the sensitivity of the test cell to a genotoxic anti-tumor agent; and (g) comparing the sensitivity of the test cell to the drug with that of the second control cell; wherein the second control cell Is syngeneic with the test cell but lacks the Fanconi anemia pathway; where the test compound is of the Fanconi anemia DNA repair pathway when the sensitivity of the test cell is comparable to that of the second control cell A method comprising further being identified as an inhibitor.   16. The method of claim 15, wherein in step (c), the number of FANC D2-containing focus is determined.   16. The method according to claim 15, wherein in step (c) the size of the FANC D2 containing focus is determined.   16. The method of claim 15, wherein in step (c), an FANC D2-containing focus is determined using an antibody specific for monoubiquitinated FNAC D2.   16. The method of claim 15, wherein step (c) is performed in a high throughput format.   16. The method of claim 15, wherein the degree of monoubiquitination of the FANC D2 polypeptide in step (d) is determined by immunoblot analysis.   16. The method of claim 15, wherein the sensitivity of the test cell and the second control cell to the anti-tumor agent is determined by measuring cell survival when using one or more concentrations of the anti-tumor agent.   16. The test cell according to claim 15, wherein the test cell has a disorder in a protein selected from the group consisting of FANC A, FANC B, FANC C, FANC D, FANC E, FANC F, FANC G, FANC L and ATR protein kinase. Method.   16. The method of claim 15, wherein the second control cell is prepared by transfecting a test cell with a cDNA that restores impairment of the Fanconi anemia pathway.   16. The method of claim 15, wherein the test cell and the second control cell are human cells.   16. The method of claim 15, wherein the test cell and the second control cell are ovarian cells.   26. The method of claim 25, wherein the test cell is an ovarian cell that does not express FANC F, and the second control cell is an ovarian cell transfected with a cDNA encoding FANC F.   Genotoxic antitumor agents include 1,3-bis (2-chloroethyl) -1-nitrosourea, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin 16. The method of claim 15, selected from the group consisting of: ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide, topotecan and ionizing radiation. A method of identifying an inhibitor of a non-Fanconi anemia DNA repair pathway comprising:
(A) contacting a test cell having a functional Fanconi anemia pathway with a test compound and a genotoxic antitumor agent;
(B) measuring the sensitivity of the test cell to genotoxic anti-tumor agent; and (c) comparing the sensitivity of the test cell to the drug with that of the control cell,
Here, the control cell is syngeneic with the test cell but lacks the Fanconi anemia pathway; when the test cell is more sensitive than the control cell, the test compound is an inhibitor of the non-Fanconi anemia DNA repair pathway. Identified;
Said method.   29. The method of claim 28, wherein the sensitivity of the test and control cells to the anti-tumor agent is determined by measuring cell survival when using one or more concentrations of the anti-tumor agent.   29. The test cell of claim 28, wherein the test cell has a disorder in a protein selected from the group consisting of FANC A, FANC B, FANC C, FANC D, FANC E, FANC F, FANC G, FANC L, and ATR protein kinase. Method.   30. The method of claim 28, wherein the control cells are prepared by transfecting test cells with cDNA that restores impairment of the Fanconi anemia pathway.   30. The method of claim 28, wherein the test compound does not inhibit the Fanconi anemia pathway using the method of claim 15.   30. The method of claim 28, wherein the test cell and control cell are human cells.   30. The method of claim 28, wherein the test cell and control cell are ovarian cells.   35. The method of claim 34, wherein the test cell is an ovarian cell that does not express FANC F and the control cell is an ovarian cell transfected with a cDNA encoding FANC F. To a subject in need of treatment for a neoplastic disease:
Administering a combination of (a) an inhibitor of the Fanconi anemia pathway; and (b) an effective amount of a genotoxic anti-tumor agent.   Genotoxic antitumor agents include 1,3-bis (2-chloroethyl) -1-nitrosourea, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin 37. The method of claim 36, selected from the group consisting of: ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide, topotecan and ionizing radiation.   Inhibitors of Fanconi anemia pathway are Alster Paulon, (+-) 13-HODE, Nifedipine, Penitrem A, Geldanamycin, Go6976, Leukotriene B3, Trichostatin-A, AG-370, Mitomycin C, Amanitin (α-Amanitin) ), HNMPA- (AM) 3, propidium iodide, DRB, ochratoxin, Ca-074-Me, K252c, wortmannin, actinomycin D, AG213, BAPTA-AM, curcumin, puromycin, bumetanide, methyladenine (3- 37. The method of claim 36, selected from the group consisting of (methyladenine), H9, TPEN, and spermine NONOate.   40. The method of claim 36, wherein the Fanconi anemia pathway inhibitor is selected from the group consisting of Ulster Paulon, Curcumin and H9.   37. The method of claim 36, wherein the Fanconi anemia pathway inhibitor is curcumin and the genotoxic anti-tumor agent is carboplatin.   40. The method of claim 36, wherein the combination is administered parenterally, orally or directly to the tumor tissue. To a subject in need of treatment for a neoplastic disease:
(A) an inhibitor of the Fanconi anemia pathway; and (b) an inhibitor of the non-Fanconi anemia DNA damage repair pathway;
Administering a combination of effective amounts of:   Inhibitors of Fanconi anemia pathway are Alster Paulon, (+-) 13-HODE, Nifedipine, Penitrem A, Geldanamycin, Go6976, Leukotriene B3, Trichostatin-A, AG-370, Mitomycin C, Amanitin (α-Amanitin) ), HNMPA- (AM) 3, propidium iodide, DRB, ochratoxin, Ca-074-Me, K252c, wortmannin, actinomycin D, AG213, BAPTA-AM, curcumin, puromycin, bumetanide, methyladenine (3- 43. The method of claim 42, wherein the method is selected from the group consisting of (methyladenine), H9, TPEN, and spermine NONOate.   43. The method of claim 42, wherein the Fanconi anemia pathway inhibitor is selected from the group consisting of Ulster Paulon, Curcumin and H9.   Non-fanconi anemia DNA damage repair pathway inhibitors include PARP inhibitors, DNA-PK inhibitors, mTOR inhibitors, ERCC1 inhibitors, ERCC3 inhibitors, ERCC6 inhibitors, ATM inhibitors, XRCC4 inhibitors, Ku80 inhibitors, 43. The method of claim 42, selected from the group consisting of a Ku70 inhibitor, an XPA inhibitor, a CHK1 inhibitor, and a CHK2 inhibitor.   43. The method of claim 42, wherein the combination is administered parenterally, orally or directly to the tumor tissue. To a subject in need of treatment for a neoplastic disease:
(A) an inhibitor of the Fanconi anemia pathway;
(B) an inhibitor of a non-Fanconi anemia DNA damage repair pathway; and (c) a genotoxic antitumor agent;
Administering a combination of effective amounts of:   Inhibitors of Fanconi anemia pathway are Alster Paulon, (+-) 13-HODE, Nifedipine, Penitrem A, Geldanamycin, Go6976, Leukotriene B3, Trichostatin-A, AG-370, Mitomycin C, Amanitin (α-Amanitin) ), HNMPA- (AM) 3, propidium iodide, DRB, ochratoxin, Ca-074-Me, K252c, wortmannin, actinomycin D, AG213, BAPTA-AM, curcumin, puromycin, bumetanide, methyladenine (3- 48. The method of claim 47, selected from the group consisting of (methyladenine), H9, TPEN, and spermine NONOate.   48. The method of claim 47, wherein the Fanconi anemia pathway inhibitor is selected from the group consisting of Ulster Paulon, Curcumin and H9.   Non-fanconi anemia DNA damage repair pathway inhibitors include PARP inhibitors, DNA-PK inhibitors, mTOR inhibitors, ERCC1 inhibitors, ERCC3 inhibitors, ERCC6 inhibitors, ATM inhibitors, XRCC4 inhibitors, Ku80 inhibitors, 48. The method of claim 47, wherein the method is selected from the group consisting of a Ku70 inhibitor, an XPA inhibitor, a CHK1 inhibitor, and a CHK2 inhibitor.   Genotoxic antitumor agents include 1,3-bis (2-chloroethyl) -1-nitrosourea, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin 48. The method of claim 47, selected from the group consisting of: ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide, topotecan and ionizing radiation.   48. The method of claim 47, wherein the combination is administered parenterally, orally or directly to the tumor tissue.   A method of sensitizing a patient to treatment with a genotoxic anti-tumor agent, wherein the method inhibits the Fanconi anemia pathway in a subject that receives a genotoxic anti-tumor agent but is resistant to said agent A method comprising administering an agent.   Inhibitors of Fanconi anemia pathway are Alster Paulon, (+-) 13-HODE, Nifedipine, Penitrem A, Geldanamycin, Go6976, Leukotriene B3, Trichostatin-A, AG-370, Mitomycin C, Amanitin (α-Amanitin) ), HNMPA- (AM) 3, propidium iodide, DRB, ochratoxin, Ca-074-Me, K252c, wortmannin, actinomycin D, AG213, BAPTA-AM, curcumin, puromycin, bumetanide, methyladenine (3- 54. The method of claim 53, selected from the group consisting of (methyladenine), H9, TPEN and spermine NONOate.   54. The method of claim 53, wherein the Fanconi anemia pathway inhibitor is selected from the group consisting of Ulster Paulon, Curcumin and H9.   Genotoxic antitumor agents include 1,3-bis (2-chloroethyl) -1-nitrosourea, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin 54. The method of claim 53, selected from the group consisting of: Ifosfamide, Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin, Temozolomide, Topotecan and Ionizing radiation.   54. The method of claim 53, wherein the genotoxic anti-tumor agent is administered concurrently with an inhibitor of the Fanconi anemia pathway. A method of sensitizing a patient to treatment with a genotoxic anti-tumor agent, the method comprising:
(A) administering an inhibitor of the Fanconi anemia pathway to a subject that receives a genotoxic anti-tumor agent but is resistant to the drug; and (b) a subject of an inhibitor of a non-Fanconi anemia DNA repair pathway. Administration.   Inhibitors of Fanconi anemia pathway are Alster Paulon, (+-) 13-HODE, Nifedipine, Penitrem A, Geldanamycin, Go6976, Leukotriene B3, Trichostatin-A, AG-370, Mitomycin C, Amanitin (α-Amanitin) ), HNMPA- (AM) 3, propidium iodide, DRB, ochratoxin, Ca-074-Me, K252c, wortmannin, actinomycin D, AG213, BAPTA-AM, curcumin, puromycin, bumetanide, methyladenine (3- 59. The method of claim 58, selected from the group consisting of (methyladenine), H9, TPEN, and spermine NONOate.   59. The method of claim 58, wherein the Fanconi anemia pathway inhibitor is selected from the group consisting of Ulster Paulon, Curcumin and H9.   Non-fanconi anemia DNA damage repair pathway inhibitors include PARP inhibitors, DNA-PK inhibitors, mTOR inhibitors, ERCC1 inhibitors, ERCC3 inhibitors, ERCC6 inhibitors, ATM inhibitors, XRCC4 inhibitors, Ku80 inhibitors, 59. The method of claim 58, wherein the method is selected from the group consisting of a Ku70 inhibitor, an XPA inhibitor, a CHK1 inhibitor, and a CHK2 inhibitor.   Genotoxic antitumor agents include 1,3-bis (2-chloroethyl) -1-nitrosourea, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin 59. The method of claim 58, selected from the group consisting of: ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide, topotecan and ionizing radiation.   59. The method of claim 58, wherein the genotoxic anti-tumor agent is administered concurrently with an inhibitor of the Fanconi anemia pathway and an inhibitor of the DNA repair pathway.   A kit for determining whether a subject with a neoplastic disease responds to a genotoxic anti-tumor agent, antibodies specific for FANC D2-L, their packaging materials, and claims A kit comprising instructions for carrying out the method according to 1.   29. A kit for identifying an inhibitor of a non-Fanconi anemia pathway, comprising a test cell and a control cell according to claim 28, and packaging material therefor.   66. The kit of claim 65, wherein the test cell and the control cell are ovarian cells.

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