EP1904088A2 - Compositions et methodes de traitement du cancer - Google Patents

Compositions et methodes de traitement du cancer

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Publication number
EP1904088A2
EP1904088A2 EP06784482A EP06784482A EP1904088A2 EP 1904088 A2 EP1904088 A2 EP 1904088A2 EP 06784482 A EP06784482 A EP 06784482A EP 06784482 A EP06784482 A EP 06784482A EP 1904088 A2 EP1904088 A2 EP 1904088A2
Authority
EP
European Patent Office
Prior art keywords
inhibitor
fanc
pathway
cell
neoplastic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06784482A
Other languages
German (de)
English (en)
Other versions
EP1904088A4 (fr
Inventor
Alan D. D'andrea
Toshiyasu Taniguchi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dana Farber Cancer Institute Inc
Original Assignee
Dana Farber Cancer Institute Inc
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Filing date
Publication date
Application filed by Dana Farber Cancer Institute Inc filed Critical Dana Farber Cancer Institute Inc
Publication of EP1904088A2 publication Critical patent/EP1904088A2/fr
Publication of EP1904088A4 publication Critical patent/EP1904088A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention generally relates to compositions and methods for the treatment of cancer.
  • Cisplatin cis- diamminedichloroplatinum, or CDDP
  • CDDP chemotherapeutic agent
  • Chemosensitizers generally inhibit the mechanism of resistance. Examples include verapamil, reserpine, tamoxifen and cremophor, inhibitors of efflux pumps conferring multidrug resistance (MDRl, P-glycoprotein).
  • MDRl multidrug resistance
  • chemosensitizers are effective only in a subset of tumors where drug efflux is the main mechanism of resistance.
  • a number of these chemosensitizers have undesirable side effects.
  • the invention provides a method of predicting whether a subject with a neoplastic disorder or disease will respond to a genotoxic anti-neoplastic agent.
  • the method comprises obtaining a biological sample from the subject, and determining degree of ubiquitination of the Fanconi anemia complementation group D2 (FANC D2) polypeptide within the biological sample.
  • FANC D2 Fanconi anemia complementation group D2
  • a degree of ubiquitination of the FANC D2 polypeptide in the biological sample of the subject that is less than about 70% when compared with a biological sample from a control subject is indicative of a subject that will respond to a genotoxic antineoplastic agent.
  • the invention provides a method of predicting whether a subject with a neoplastic disorder or disease will respond to a genotoxic anti-neoplastic agent.
  • the method comprises obtaining a biological sample from the subject, and determining the FANC D2-containing foci within the biological sample. A difference in foci formation, wherein the sample from the subject that contains less than about 70% of the FANC D2-containing foci when compared with the biological sample from a control subject is indicative of a subject that will respond to a genotoxic anti-neoplastic agent.
  • a method of identifying an inhibitor of a non-FA DNA damage repair pathway comprises the following steps: (a) providing a control cell that is functional in the Fanconi Anemia (FA) pathway; (b) providing a test cell that is isogenic to the test cell but is defective in the FA pathway; (c) contacting the test cell and the control cell with a test compound; and, (d) comparing the sensitivity of the test cell and said control cell to the test compound. An increased sensitivity of the test cell to the test compound than the control cell is indicative of a test compound being an inhibitor of a non- FA DNA damage repair pathway.
  • FA Fanconi Anemia
  • a method of treating a neoplastic disorder in a subject in need thereof comprises administering to the subject a combination of an effective amount of: (a) an inhibitor of the FA pathway or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof, and (b) a genotoxic anti-neoplastic agent.
  • a method of treating a neoplastic disorder in a subject in need thereof comprises administering to the subject a combination of an effective amount of: (a) an inhibitor of the FA pathway or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof, and (b) an inhibitor of a non-FA DNA damage repair pathway.
  • a method of treating a neoplastic disorder in a subject in need thereof comprises administering to the subject a combination of an effective amount of: (a) an inhibitor of the FA pathway or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof, (b) an inhibitor of a non-FA DNA damage repair pathway, and (c)a genotoxic anti-neoplastic agent or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof.
  • a method of increasing the sensitivity of a neoplastic disorder to a genotoxic anti-neoplastic agent comprises administering, before, after or concurrently with a therapeutically effective dose of the agent an effective amount of an inhibitor of the FA p athway .
  • a method of increasing the sensitivity of a neoplastic disorder to a genotoxic anti-neoplastic agent comprises administering, before, after or concurrently with a therapeutically effective dose of the agent a combination of an effective amount of (a) an inhibitor of the FA pathway or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof, and (b) an inhibitor of a non-FA DNA damage repair pathway.
  • Figure 1 outlines an example of the workflow of screens for the identification of inhibitors and agonists of the FA pathway.
  • Figure 2 outlines the high-throughput scheme for identification of FA pathway agonists and antagonists using fluorescence microscopy.
  • Figure 3 is a schematic showing the protein components identified within the FA pathway of DNA damage repair
  • Figure 4 shows the schematic of eGFP-FANC D2 fusion construct, and its ubiquitination in transfected PD20F, GM6914 and HeLa cells upon exposure to ionizing radiation (IR), HU, or Mitomycin C (MMC).
  • IR ionizing radiation
  • MMC Mitomycin C
  • Figure 5 are fluorescence micrographs showing the fluorescence signal emitted by eGFP- FANC D2 transfected PD20 and GM6914 cells.
  • GM6914 cells additionally transfected with FANCA shows punctate, FANC D2-containing foci upon exposure to ionizing radiation.
  • Figure 6 outlines a method for screening for FA inhibitors by fluorescence microscopy.
  • Figure 7 are fluorescence micrographs showing inhibition of IR-mediated formation of FANC D2-containing foci upon exposure of eGFP-FANC D2 containing cells to the kinase inhibitor H-9.
  • Figure 8 shows an immunoblot analysis showing the effect of H-9 on inhibition of IR- inducible monoubiquitination of FANC D2 (top two panels), phosphorylation of ATR kinase (middle panel), Chkl phosphorylation (fourth panel), and Chkl polypeptide levels (bottom panel).
  • the graph shows the enhanced sensitivity of
  • 2008 cells to cisplatin by H-9 The 2008 cells are inherently sensitive to cisplatin because of a deficiency FANCF (Open Square). This sensitivity to cisplatin is reduced upon transfection of FANCF (2008 + F, open circle). Treatment of 2008 + F cells with H-9 re-establishes sensitivity to cisplatin (closed circle), while not affecting sensitivity to cisplatin in untranfected cells (closed square).
  • FANCF Open Square
  • Figure 9 shows the inhibition of cisplatin-dependent FANC D2 monoubiquitination by treatment with curcumin.
  • the graph (9B) shows the sensitization of FANCF- corrected 2008 cells to cisplatin, but a reduced sensitizing effect on the chemosensitization of the parental 2008 cells by curcumin treatment.
  • Figure 10 shows the effects of curcumin on FANCD2 foci, and sensitivity towards cisplatin.
  • Curcumin sensitizes human tumor cells to Cisplatin.
  • 2008 cells an ovarian tumor line which is deficient in the FANCF protein and therefore has a defect in the FA pathway
  • 2008 cells corrected with the FANCF cDNA The cells were pretreated for 24 hours with or without curcumin (20 micromolar) as indicated, and the cells were then exposed to increasing doses of cisplatin. Importantly, the corrected 2008 cells are sensitized to cisplatin by pretreatment with curcumin.
  • Pretreatment of Clone 7 cells with Curcumin prevents the assembly of FANCD2 foci.
  • Figure 11 shows an immunoblot analysis showing the effect of alsterpaullone on inhibition of IR-inducible monoubiquitination of FANC D2.
  • Alsterpaullone a CdId inhibitor, does not inhibit IR-inducible D2 phosphorylation on T691 but rather enhances it.
  • Alsterpaullone inhibits the IR-inducible monoubiquitination of FANC D2 and phosphorylation of Chkl (on Ser345) in an ovarian cancer cell line.
  • Figure 12 are micrographs showing inhibition of IR-mediated FANC D2- and BRCAl- containing foci in cells treated with alsterpaullone.
  • Figure 13 shows that treatment of cells with spermine NONOate induces phosphorylation and monoubiquitination of FANC D2, and phosphorylation of Chkl in the absence of IR.
  • the graph shows a nominal reduction in sensitivity of 2008 cells to spermine NONOate upon FANC F transfection (2008 +F), when compared with vector transfected 2008 cells (2008 + vec).
  • Figure 14 shows that treatment of cells with spermine NONOate induces phosphorylation and monoubiquitination of FANC D2, and phosphorylation of Chkl in the absence of IR.
  • Figure 15 are micrographs showing that spermine NONOate induces phosphorylation of histone H2AX in the absence and in the presence of IR treatment.
  • Figure 16 shows an immunoblot analysis showing that geldanamycin (an Hsp90 inhibitor) inhibits IR-inducible D2 monoubiquitination in HeLa cells.
  • Geldanamycin also causes decreased Chkl expression.
  • the graph shows that transfection of 2008 cells with FANC F does not alter sensitivity of cells to geldanamycin.
  • Figure 17 are micrographs showing inhibition of IR-mediated FANC D2- and BRCAl- containing foci in cells treated with geldanamycin.
  • Figure 18 shows the effects of Go6976, a PKC, Chkl inhibitor, on IR-inducible monoubiquitination of FANC D2 in HeLa cells.
  • Go6976 enhances phosphorylation of Chkl in HeLa cells.
  • transfection of 2008 cells with FANC F has no effect in sensitivity to Go6976.
  • Figure 19 are micrographs showing inhibition of IR-mediated FANC D2- containing foci formation in cells treated with Go6976.
  • Figure 20 are immunoblot panels showing the effects of AG370, a PDGFR kinase inhibitor, in inhibiting IR-inducible monoubiqiiitination of FANC D2 in HeLa cells.
  • AG370 a PDGFR kinase inhibitor
  • Figure 21 are micrographs showing inhibition of IR-mediated FANC D2- and BRCAl- containing foci in cells treated with AG370.
  • Figure 22 shows that inactivation of the FA pathway causes increased sensitivity to DNA cross-linking agents, including BCNU.
  • Isogenic PD20 (circle) and PD20 retrovirally corrected with FANCD2 (square) showed differential sensitivity to BCNU. Percentage survival was plotted on the Y-axis. Similar data were obtained using 2008 (FANCF deficient cell line) and 2008 complemented with FANF.
  • Model of FA pathway (c) BCNU induced FANCD2 monoubiquitination only in the presence of an intact FA pathway (lane 4). 2008 and 2008 retrovirally complemented with FANCF were either untreated or treated with BCNU. Whole cell lysates were fractionated on SDS-PAGE and immunoblotted with FANCD2 antisera.
  • the present invention is based in part on a series of discoveries showing the critical role played by the FA pathway in the sensitivity of cancers to anti-neoplastic agents.
  • the role of the FA pathway in modulating the sensitivity of neoplastic disorders and/or cancer cells to antineoplastic agents has been demonstrated using cell lines deficient in FA pathway components, and using inhibitors of the FA pathway. Therefore, in one embodiment, a method for treating a subject with a neoplastic disorder is provided.
  • One such method comprises administering an effective dose of an FA pathway inhibitor in combination with a genotoxic anti-neoplastic agent.
  • Another method comprises administering an effective dose of an FA pathway inhibitor in combination with an inhibitor of a non-FA DNA damage repair pathway.
  • compositions useful for the treatment of neoplastic disorders comprising an FA pathway inhibitor in combination with a genotoxic anti-neoplastic agent and/or an inhibitor of a non-FA DNA damage repair pathway.
  • pharmaceutical compositions, as well as kits thereof, comprising FA pathway inhibitors and anti-neoplastic agents and/or an inhibitor of a non-FA DNA damage repair pathway.
  • neoplasm As used herein, the terms “neoplasm”, “neoplastic disorder”, “neoplasia” “cancer,” “tumor” and “proliferative disorder” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth which generally forms a distinct mass that show partial or total lack of structural organization and functional coordination with normal tissue.
  • the terms are meant to encompass hematopoietic neoplasms (e.g. lymphomas or leukemias) as well as solid neoplasms (e.g.
  • Hematopoietic neoplasms are malignant tumors affecting hematopoietic structures (structures pertaining to the formation of blood cells) and components of the immune system, including leukemias (related to leukocytes (white blood cells) and their precursors in the blood and bone marrow) arising from myeloid, lymphoid or erythroid lineages, and lymphomas (relates to lymphocytes).
  • Solid neoplasms include sarcomas, which are malignant neoplasms that originate from connective tissues such as muscle, cartilage, blood vessels, fibrous tissue, fat or bone.
  • Solid neoplasms also include carcinomas, which are malignant neoplasms arising from epithelial structures (including external epithelia (e.g., skin and linings of the gastrointestinal tract, lungs, and cervix), and internal epithelia that line various glands (e.g., breast, pancreas, thyroid).
  • leukemia and hepatocellular cancers
  • sarcoma vascular endothelial cancers
  • breast cancers e.g. astrocytoma, gliosarcoma, neuroblastoma, oligode
  • a “genotoxic agent” or “genotoxin” refers to any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents can be chemical or radioactive.
  • a genotoxic agent is one for which a primary biological activity of the chemical (or a metabolite) is alteration of the information encoded in the DNA.
  • Genotoxic agents can vary in their mechanism of action, and can include: alkylating agents such as ethylmethane sulfonate (EMS), nitrosoguanine and vinyl chloride; bulky addition products such as benzo(a)pyrene and aflatoxin Bl; reactive oxygen species such as superoxide, hydroxyl radical; base analogs such as 5-bromouracil; intercalating agents such as acridine orange and ethidium bromide.
  • alkylating agents such as ethylmethane sulfonate (EMS), nitrosoguanine and vinyl chloride
  • bulky addition products such as benzo(a)pyrene and aflatoxin Bl
  • reactive oxygen species such as superoxide, hydroxyl radical
  • base analogs such as 5-bromouracil
  • intercalating agents such as acridine orange and ethidium bromide.
  • a “genotoxic anti-neoplastic agent”, as used herein, is a genotoxic agent used for chemotherapy, for example, to treat cancer.
  • “genotoxic anti-neoplastic agents” encompass agents, both chemical or otherwise, which cause damage to DNA. These agents include DNA alkylating agents, intercalating agents, and the like.
  • Non-limiting examples of “genotoxic anti-neoplastic agents” include l,3-Bis(2-Chloroethyl)-l-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.
  • “Genotoxic anti-neoplastic agents” also include radiation, in particular the types used in radiation therapy for the treatment of cancer, in a dosages sufficient to cause damage to DNA in a subject.
  • DNA damage refers to chemical and/or physical modification of the DNA in a cell, including methylation, alkylation double-stranded breaks, cross-linking, thymidine dimers caused by ultraviolet light, and oxidative lesions formed by oxygen radical binding to DNA bases.
  • a "chemosensitizer” and “chemosensitizing agent” refer to a compound which, when administered in a therapeutically effective amount in a subject, increases the sensitivity to chemotherapy compounds, and/or increases the therapeutic efficacy of the compounds, for example, in the treatment of a disease, such as neoplastic diseases, benign and malignant tumors, and cancerous cells.
  • a disease such as neoplastic diseases, benign and malignant tumors, and cancerous cells.
  • An increase in sensitivity to chemotherapy compounds, including genotoxic anti-neoplastic agents can be measured, for example, by measuring the decrease in LD 50 of a cell towards a compound in the presence of the chemosensitizer.
  • a “radiosensitizer” and “radiosensitizing agent”, as used herein, refer to a compound which, when administered in a therapeutically effective amount in a subject, increases the sensitivity to radiation therapy (treatment by electromagnetic radiation), and/or increases the therapeutic efficacy of radiation therapy, for example, in the treatment of a disease, such as neoplastic diseases, benign and malignant tumors and cancer cells. Also contemplated are electromagnetic radiation treatment of other diseases not listed herein.
  • sample or “biological sample” is meant any cell or tissue, or cell or tissue- containing composition or isolate derived from the subject.
  • the sample may be derived from heart, brain, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus, small intestine, or colon.
  • Another type of biological sample maybe a preparation containing white blood cells, e.g., peripheral blood, sputum, saliva, urine, etc., for use in detecting the presence or absence of DNA damage in a subject that has been exposed to a genotoxic agent, such as radiation, chemicals, etc.
  • degree of ubiquitination of the FANC D2 polypeptide refers generally to the level of activation of the FA pathway, as measured by the degree of monoubiquitination of the FANC D2 polypeptide within a subject or biological sample therefrom.
  • the "degree of ubiquitination" of the FANC D2 polypeptide can encompass the proportion of total FANC D2 polypeptide within a sample that is monoubiquitinated, and can be expressed on a fractional or percentage basis.
  • the "degree of ubiquitination" of the FANC D2 polypeptide can also be measured using any substitute methods of detecting activation of the FA pathway, including the degree of foci formation.
  • degree of foci formation refers to the total number or the rate of formation of FANC D2-containing foci in a sample.
  • FANC D2-containing foci are nuclear protein complexes formed in response to the activation of the FA pathway, for example by exposure to a genotoxic agent.
  • FANC D2-containing foci can be detected, for example, by immunofluorescence microscopy using a labeled antibody directed against the FANC D2 polypeptide, as further described herein.
  • FANC D2-containing foci can also be detected in cells expressing a functional fusion protein comprising GFP and the FANC D2 polypeptide. In these cells, FANC D2-containing foci can be detected using fluorescence microscopy without the use of anti-FANC D2 antibodies.
  • the degree of foci formation can be normalized from one sample to another, for example, to total number of cells, total number of intact nuclei, total sample volume, or total sample mass.
  • difference in foci formation is meant a difference, whether higher or lower, in the number, size or persistence of FANC D2-containing foci, when comparing a test sample with either a control sample or reference sample.
  • a difference includes an increase or decrease that is 2-fold or more, or less, for example 5, 10, 20, 100, 1000-fold or more as compared to a control or reference sample.
  • a difference also includes an increase or decrease that is 5% more or less, for example, 10%, 20%, 30%, 50%, 75%, 100%, as compared to a control or reference sample.
  • Modulate formation of FANC D2-containing foci refers to a change or an alteration in the formation of FANC D2-containing foci in a biological sample. Modulation may be an increase or a decrease in foci number, size or persistence within a biological sample, and includes an increase or decrease that is 2-fold or more, or less, for example 5, 10, 20, 100, 1000-fold or more as compared to a control or reference sample. Modulation may also be an increase or decrease that is 5% more or less, for example, 10%, 20%, 30%, 50%, 75%, 100%, as compared to a control or reference sample.
  • exposure to a "low level" of a genotoxic anti-neoplastic agent refers to exposure to a dose of a particular genotoxic anti-neoplastic agent which results in no more than 20% of the maximal number of FANC D2-containing foci in biological samples. Because of the multitude of genotoxic anti-neoplastic agents to which a sample may be exposed, as well as the varying sensitivities of different samples to such genotoxic antineoplastic agents, it is preferable to express the dosage relative to the formation of FANC D2-containing foci, rather than in the absolute dose of a particular genotoxic anti-neoplastic agent.
  • modulator refers to a chemical compound (naturally occurring or non- naturally occurring), such as a biological macromolecule (e.g., nucleic acid, protein, non- peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
  • Modulators are evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (e.g., agonist, partial antagonist, partial agonist, antagonist, anti-neoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation-promoting agents, and the like) by inclusion in screening assays described herein.
  • the activities (or activity) of a modulator may be known, unknown or partially-known. Such modulators can be screened using the methods described herein.
  • test modulator refers to a compound to be tested by one or more screening method(s) of the invention as a putative modulator. Usually, various predetermined concentrations are used for screening such as 0.0 l ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M, and lO.O ⁇ M, as described more fully below.
  • Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
  • an “FA pathway inhibitor” and “inhibitor of the FA pathway” refer to a chemical compound (naturally occurring or non-naturally occurring), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
  • a biological macromolecule e.g., nucleic acid, protein, non-peptide, or organic molecule
  • an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
  • An “FA pathway inhibitor” and “inhibitor of the FA pathway” refer broadly to compounds which inhibit the ability of the FA pathway to repair DNA damage.
  • Inhibition of the FA pathway by an "FA pathway inhibitor” or an “inhibitor of the FA pathway” can be assessed using the techniques described herein, including without limitation, the detection of FANC D2-containing foci and detection of monoubiquitination of the FANC D2 polypeptides.
  • the method contemplates any other method currently known or known in the future, for the detection of the inhibition of the FA pathway.
  • Inhibition may be a decrease in number, size or persistence of FANC D2-containing foci, and includes a decrease that is 2- fold or more, for example, 2, 5, 10, 20, 100, 1000-fold or more as compared to a control or reference.
  • Inhibition may also be an decrease of 5% or more, for example 5%, 10%, 20%, 30%, 50%, 75%, or up to 100%, as compared to a control or reference.
  • an "FA pathway inhibitor” and “inhibitor of the FA pathway” encompass the pharmaceutically acceptable salts, solvates, esters, derivatives or prodrugs.
  • non-FA DNA damage repair pathway refers to any of the DNA damage repair pathways selected from the group consisting of the direct reversal, non- homologous end joining (NHEJ), base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MR) pathways.
  • NHEJ non- homologous end joining
  • BER base excision repair
  • NER nucleotide excision repair
  • MR mismatch repair
  • compositions of the present invention can be administered using any amount and any route of administration effective for increasing the therapeutic efficacy of drugs.
  • therapeutically effective amount when used in combination with a chemosensitizer or radiosensitizer, refers to a sufficient amount of the chemosensitizing agent to provide the desired effect against target cells or tissues. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject; the particular chemosensitizing agent; its mode of administration; and the like.
  • 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.
  • the term specifically excludes cell culture medium.
  • pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
  • suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents.
  • Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
  • a "therapeutically effective dose” refers to that amount of protein or its antibodies, antagonists, or inhibitors which prevent or ameliorate the symptoms or conditions, for example, a neoplastic disorder.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD 5 o/ED 5 o.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animals studies is used in formulating a range of dosage for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration.
  • the exact dosage is chosen by the individual physician or veterinarian in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state; age, weight and gender of the subject; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on a half-life and clearance rate of the particular formulation.
  • salt refers to both acid addition salts and base addition salts.
  • the nature of the salt is not critical, provided that it is pharmaceutically acceptable.
  • Exemplary acid addition salts include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, phosphoric, formic, acetic, citric, tartaric, succinic, oxalic, malic, glutamic, propionic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, ⁇ - hydroxybutyric, malonic, galactaric, galacturonic acid and the like.
  • Suitable pharmaceutically acceptable base addition salts include, without limitation, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N 3 N' -dibenzylethylenediarnine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, procaine and the like. Additional examples of pharmaceutically acceptable salts are listed in Journal of Pharmaceutical Sciences (1977) 66:2. All of these salts may be prepared by conventional means from a modulator of FANC D2-containing foci by treating the compound with the appropriate acid or base.
  • the cellular response to DNA damage is a complex interacting network of pathways that mediate cell cycle checkpoints, DNA repair, and apoptosis.
  • a model lesion for the investigation of these pathways has been DNA double-strand breaks, which rapidly induce cell cycle checkpoints and are repaired by a number of different pathways.
  • both homologous recombination and nonhomologous recombination pathways are utilized.
  • Extensive studies in mammalian cells have shown that complexes of DNA repair and cell cycle checkpoint proteins rapidly localize to sites of double-strand breaks induced by ionizing radiation. These proteins create foci that can be detected by immunofluorescent analyses.
  • Fanconi anemia complementation group D2 is a component of a protein complex involved in chromosome stability and repair.
  • Fanconi anemia FA
  • Fanconi anemia FA
  • BRCAl Breast Cancer, Type 1 polypeptide
  • Activation of FANC D2 occurs by phosphorylation of a serine 222 residue by the Ataxia-Telangiectasia Mutated (ATM) kinase.
  • ATM Ataxia-Telangiectasia Mutated
  • activation via the FA pathway occurs via monoubiquitination of FANC D2 at lysine 561.
  • FANC D2 In its unmodified form, FANC D2 is diffusely located throughout the nucleus. When ubiquitinated, FANC D2 forms dots, or foci, in the nucleus. The ubiquitination of FANC D2 and subsequent formation of nuclear foci occurs in response to DNA damage.
  • NBSl Nijmegen Breakage Syndrome 1
  • Rad51 foci which contain the tumor suppressor proteins BRCAl and BRCA2, also appear during S phase in the absence of exogenous induction of DNA damage.
  • Mrel 1-Rad5 ⁇ -NBS1 foci can be detected as early as 10 min after irradiation and are clearly present at sites of DNA breaks, while DNA repair is ongoing. These foci also colocalize with the BRCAl protein, which has been shown to be required for their formation, possibly through its physical interaction with human Rad50 (hRad50).
  • coimmuiioprecipitation experiments performed with BRCAl have indicated the presence of a large number of additional proteins in this complex (referred to as the BRCAl -associated surveillance complex). These include the mismatch repair proteins Msh2, Msh6, and Mlhl, the checkpoint kinase ATM, the product of the Bloom's syndrome gene BLM, and replication factor C.
  • BRCAl, NBSl, and hMrel 1 have all been shown to be substrates of the ATM kinase and to become phosphorylated in response to the presence of DNA breaks.
  • the present invention is related to the discovery that cells exposed to genotoxic anti- neoplastic agents form FANC D2-containing foci.
  • Multiple DNA damage response proteins have now been identified which form nuclear foci, also called IRIFs (Ionizing-Radiation Inducible foci) in response to DNA damage.
  • IRIFs Ionizing-Radiation Inducible foci
  • Methods of detecting FANC D2-containing foci, as well as detecting and quantitating the relative amount of ubiquitinated FANC D2 polypeptide are described in U.S. App. No. 10/165,099 and U.S. App. No. 60/540,380, the contents of which are incorporated in their entirety herein by reference.
  • the total cellular level of FANC D2 protein does not significantly change in response to DNA damage. Rather, DNA damage results in monoubiquitination of FANC D2, as well as recruitment into FANC D2-containing foci. It will be appreciated by one skilled in the art that an alternative to measuring the presence of FANC D2-containing foci is to use a ligand which specifically binds the monoubiquitinated, but not the unubiquitinated form of FANC D2. To detect the presence of monoubiquitinated FANC D2, the ligand is preferably associated with a detectable label as described above.
  • the main advantage of using such a ligand is that, due to the typically low basal level of monoubiquitinated FANC D2 in cells with undamaged DNA, the level of FANC D2- containing foci can be measured in a sample taken from a living subject using the level of monoubiquitinated FANC D2 as a surrogate marker.
  • An antibody which specifically recognizes the monoubiquitinated form of FANC D2 has considerable utility as a rapid diagnostic. For instance, this antibody could be used for:
  • IH Immunohistochemistry
  • Peripheral blood lymphocytes could be screened with this antibody.
  • a positive signal suggests the presence of activated FANC D2, consistent with a recent exposure of an individual to IR. or toxin.
  • this antibody is a useful extension of the radiation dosimeter assay described in this application.
  • the new monoclonal antibody will be a useful reagent for end product detection. Additional methods of measuring FANC D2-containing foci using a ligand which specifically recognizes monoubiquitinated FANC D2 include immunoblot analysis, or Enzyme linked immunosorbant assays (ELISA) using extracts of samples collected from living subjects, or FACS analysis (Harlow et al, 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY).
  • ELISA Enzyme linked immunosorbant assays
  • a sensitive measure of IR exposure is the increased monoubiquitination of FANC D2.
  • the ratio of FANC D2-L (monoubiquitinated isoform) to FANC D2-S (unubiquitinated isoform) is approximately 0.5-0.6. This ratio (L/S) is readily calculated by comparing the density of the L band to the S band on a western blot.
  • a sensitive indicator of increased FANC D2 monoubiquitination and IR exposure is the conversion of the L/S ratio to 1.0 or greater.
  • FANC D2 activation and foci formation An alternative approach for the detection of FANC D2 activation and foci formation is the use of a FANC D2 protein fused with a fluorescent protein, for example, GFP.
  • a functional fusion protein of FANC D2 and GFP is able to form foci upon exposure to genotoxic anti-neoplastic agents. These foci are then visible by fluorescence microscopy. Therefore, 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-neoplastic agents.
  • the present invention encompasses methods and compositions useful for the treatment of neoplastic diseases using inhibitors of the FA pathway.
  • Inhibitors of the FA pathway can be identified by methods described herein, and also methods previously described, for example, in U.S. App. No. 10/165,099 and U.S. App. No. 60/540,380, the contents of which are incorporated herein by reference.
  • inhibitors of the FA pathway can be identified systematically using a three-tiered approach, as summarized in Figure 1.
  • the first tier of screening comprises a high-throughput method to identify agents which alter the formation of FANC D2-containing foci.
  • Detection of FANC D2-containing foci for example by using a FANC D2 ligand such as anti-FANC D2 antibodies or cell lines expressing a functional eGFP-FANC D2 fusion protein, are described in U.S. App. No. 10/165,099 and U.S. App. No. 60/540,380, the contents of which are incorporated herein by reference.
  • the method comprises contacting cells or a biological sample with a test compound simultaneously with, before or after exposure to a genotoxic anti-neoplastic agent, for example ionizing radiation (IR), mitomycin C or cisplatin, at a dosage which induces formation of FANC D2-containing foci.
  • a genotoxic anti-neoplastic agent for example ionizing radiation (IR), mitomycin C or cisplatin
  • IR ionizing radiation
  • mitomycin C ionizing radiation
  • Potential agonists and inhibitors thus identified can be further tested to determine whether they exert their effects directly on the FA pathway, or act indirectly, for example, by directly causing damage to DNA (in the case of potential agonists of the FA pathway), or by reducing the effect of the genotoxic anti-neoplastic agent that was used in the screen.
  • the second tier of screening involves the detection of ubiquitinated FANC D2 polypeptides. As previously described, activation of the FA pathway results in monoubiquitination of the FANC D2 polypeptide. Activation of the FA pathway can therefore be measured by detecting the relative amount of ubiquitinated FANC D2 compared with unubiquitinated FANC D2 polypeptide.
  • the ubiquitination of FANC D2 can be detected by performing immunoblot analysis of protein extracts.
  • Ubiquitinated FANC D2 migrates at a higher molecular weight band on immunoblot analyses, and can be detected using a labeled FANC D2 ligand, for example an anti-FANC D2 antibody. Therefore, the second tier of the screening comprises The method comprises contacting cells or a biological sample with a test compound simultaneously with, before or after exposure to a genotoxic anti-neoplastic agent, for example ionizing radiation (IR), mitomycin C or cisplatin, at a dosage which induces formation of FANC D2-containing foci.
  • a genotoxic anti-neoplastic agent for example ionizing radiation (IR), mitomycin C or cisplatin
  • the amount of ubiquitinated FANC D2 polypeptide relative to unubiquitinated FANC D2 polypeptide is detected, and compared with samples from control cells or biological samples which were not contacted with the test compound. A difference in the relative amount of ubiquitinated FANC D2 relative to control cells indicates that the test compound is a modulator of the FA pathway.
  • An increase in the relative amount of ubiquitinated FANC D2 polypeptide compared with control cells or biological samples is indicative of an agonist of the FA pathway, whereas a decrease in the relative amount of ubiquitinated FANC D2 polypeptide compared with control cells or biological samples is indicative of an inhibitor of the FA pathway.
  • the potential agonists and inhibitors thus identified can be further tested to determine whether they exert their effects directly on the FA pathway, or act indirectly, for example, by directly causing damage to DNA (in the case of potential agonists of the FA pathway), or by reducing the effect of the genotoxic anti-neoplastic agent that was used in the screen.
  • the third tier of screening comprises in vitro testing of compounds for sensitivity to genotoxic anti-neoplastic agents.
  • Contacting cells or biological samples with inhibitors of the FA pathway would be expected to increase the sensitivity of the samples/cells to genotoxic anti-neoplastic agents.
  • Specific inhibition of the FA pathway by a test agent is expected to increase the sensitivity to a degree comparable to, for example, a cell line with a specific 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 is deficient in FANCF.
  • the three tiers of screening described above provide a stream-lined approach to rapidly identifying and characterizing potential modulators of the FA pathway. It should be understood that methods to identify modulators are not limited to the particular embodiments of the invention described above, and variations of the embodiments can be made and still fall within the scope of the invention. In addition, the terms used herein are for the purpose of describing the particular embodiments and are not intended to be limiting.
  • An inhibitor of the FA pathway includes any compound which results in the inhibition of formation of FANC D2-containing foci, when administered before, after or concomitantly with a genotoxic anti-neoplastic agent(s) which normally cause formation of FANC D2- containing foci.
  • genotoxic anti-neoplastic agents which induce 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 amounts of ubiquitinated and unubiquitinated FANC D2 polypeptide of samples subjected to an agent which normally induces ubiquitination.
  • Detection of FANC D2-containing foci using, for example, microscopic detection means, as well as determination of the relative ubiquitination state of the FANC D2 polypeptide is described in U.S. Serial No. 10/165099, filed June 6, 2002, and U.S. Serial No. 60/540380, filed January 30, 2004, the contents of which are incorporated herein by reference.
  • FANC D2-containing foci can be detected using immunofluorescence microscopy, using anti-FANC D2 antibodies.
  • a fluorescent protein-tagged version of FANC D2 can be transfected into the cells of interest, and formation of FANC D2-containing foci measured microscopically be detecting fluorescent 'foci', again, as described in U.S. Serial No. 60/540,380.
  • Compounds which inhibit the FA pathway such as wortmannin and
  • Trichostatin A have previously been disclosed, for example in U.S. Serial No. 60/540380, filed January 30, 2004.
  • the present invention describes additional examples of inhibitors of the FA pathway, including curcumin, H-9 and alsterpaullone, which were identified using the screening methods described herein.
  • H-9 kinase inhibitor also known as N-2-Aminoethyl-5-Isoquinolinesulfonamide (formula I) is a known inhibitor of several kinases, including PKA, PKG, PKC, Calcium/Calmodulin dependent protein kinase, and myosin light chain kinase (Inagaki et al., (1985) J Biol. Chem. 260(5):2922-5; Ito et al, (1988) Int. JImmunopharmacol. 10:211-216)
  • Alsterpaullone (formula II), is known to inhibit Cdkl/cycline B, Gsk-3B, and Cdk5
  • Curcumin (Turmeric yellow, also known as 1,7-bis (4'-hydroxy-3'-methoxyphenyl)- l,6-heptadiene-3,5-dione, diferuloylmethane), a low molecular weight polyphenol derived from the spice, turmeric, is associated with regression of some solid tumors in humans (Cheng et al. (2001) Anticancer Res. 21 :2895-2900). Curcumin is safe in human trials at doses as high as 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 which binds to Hs ⁇ 90 (Heat Shock Protein 90) and alters its function.
  • the present invention encompasses compositions and methods comprising geldanamycin and its analogs. Analogs of geldanamycin include 17-(Allylaniino)-17-demethoxy- geldanamycin (Schnur et al., (1995) J Med. Chem. 38:3806-12; Dunn (2002) J. Natl. Cancer Inst 94, 1194-5).
  • compositions and methods comprising other inhibitors of HSP90, in particular benzoquinone ansamycin inhibitors of HSP90, coumarin derivatives (described, for example, in WO 00/53169).
  • Other compounds which can inhibit the FA pathway include those compounds listed within Table 2.
  • the inhibitor of the FA pathway can be selected from the group consisting of Alsterpaullone, (+-)13-F£ODE, nifedipine, penitrem A, Geladanamycin, Go6976, leukotriene B3, Trichostatin-A, AG-370, Mitomycin C, Amanitin (alpha-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.
  • cancers have a defect in at least one of the six major DNA damage repair pathways.
  • disruption of any of these DNA repair mechanisms can lead to increased sensitivity to genotoxic anti-neoplastic agents. Therefore, these cancers have increased dependence on one of the other five DNA damage repair pathways for survival.
  • disruption of a second, non-FA DNA damage repair pathway in these neoplastic disorders for example by a small molecule inhibitor may result in selective cancer cell death.
  • many cancers may turn out to have a dominant (primary) DNA damage repair pathway. Since one DNA damage repair pathway is already abolished or significantly reduced in the cancer, an extra burden is placed on the dominant pathway in order to maintain the high proliferation rate and to prevent DNA damage of these cells.
  • Disruption of the dominant pathway in a cancer cell in which a major DNA damage repair pathway is abolished or diminished, by means of an exogenous inhibitor, may therefore have a profound cytotoxic effect on the tumor cells but a relatively small cytotoxic effect on the surrounding normal cells.
  • BER Base Excision Repair
  • the present invention also contemplates the use of inhibitors of various other DNA damage repair pathways.
  • DNA damage repair includes non-homologous end joining (NHEJ), base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MR).
  • NHEJ non-homologous end joining
  • BER base excision repair
  • NER nucleotide excision repair
  • MR mismatch repair
  • NHEJ Non-Homologous End Joining
  • DNA double strand breaks can be caused by any number of environmental or other factors, including reactive oxygen species, ionizing radiation (IR) and certain antineoplastic drugs like bleomycin. Failure to repair DSBs can lead to a number of consequences, including mutations, chromosomal aberrations, and eventually cell death.
  • NHEJ Non-homologous end-joining
  • Some members of the NHEJ pathway are shown in Table 1.
  • DNA-dependent protein kinase consists of the catalytic subunit (DNA-PKcs) and the regulatory subunit (the Ku70/Ku80 heterodimer).
  • the DNA-PKcs subunit is a serine/threonine kinase which belongs to the phosphatidyl inositol-3 kinase family.
  • the Ku80/Ku70 heterodimer (Ku) exhibits sequence-independent affinity for double- stranded termini and, upon binding to DNA, recruits and activates the DNA-PKcs catalytic subunit.
  • viridins Hanson, J. R. Nat. Prod.
  • SSBs Single Strand DNA breaks
  • BER Base Excision Repair
  • AP apurinic/apyrimidinic
  • PARP Poly(ADP-ribose) polymerase
  • PJ-34 N-(6-oxo-5,6-dihydiOphenanthridin-2-yl)-N, N- dimethylacetamide.Hcl, INHBP 5-iodo-6-amino-l,2-benzopyrone, 3-Aminobenzamide, Benzamide, 4-Amino-l,8-naphthalimide, 6(5H)-Phenanthridinone, 5-Aminoisoquinolinone (5-AIQ).
  • NER Nucleotide excision repair
  • pyrimidine dimers which are induced by ultraviolet light (UV).
  • UV ultraviolet light
  • XP xeroderma pigmentosum
  • Eukaryotic NER includes two major branches, transcription-coupled repair (TCR) and global genome repair (GGR) (de Laat et al. (1999) Genes Dev. 13:768-85, Tornaletti & Hanawalt (1999) Biochimie. 81:139-46) .
  • GGR is a slow random process of inspecting the entire genome for injuries, while TCR is highly specific and efficient and concentrates on damage-blocking RNA polymerase II. The two mechanisms differ in substrate specificity and recognition.
  • the XPC-HR23B complex recognizes damage located in nontranscribed regions (Sugasawa et al. (2001) Genes Dev.
  • RNAPII RNA polymerase II
  • Mismatch repair removes both nucleotides mispaired by DNA polymerases and insertion/deletion loops caused by slippage during replication of repetitive sequences (Harfe & Jinks-Robertson (2000) Annu Rev Genet 34: 359-399). Initially, the heterodimeric MSH complex recognizes the nucleotide mismatch, subsequently followed by interaction with MLH1/PMS2 and MLH1/MLH3 complexes. Several proteins participate in process of the nucleotide excision and resynthesis. Tumor cells deficient in mismatch repair have much higher mutation frequencies than normal cells (Parsons et al. (1993) Cell 75: 1227-1236, Bhattacharyya et al.
  • the DNA damage repair pathways of the cell can be partially redundant. This presents difficulties in identifying agents which specifically block one pathway. Inhibitors identified using cell-based methods wherein the cells have functional DNA damage repair pathways may therefore have multiple targets, including in a plurality of DNA damage repair pathways. Therefore, use of cell lines deficient in one or more DNA damage repair pathways may greatly accelerate the identification of novel, specific inhibitors. Therefore, according to one aspect, a method of identifying agents which inhibit a non-FA DNA damage repair pathway is provided. The method employs cells which have a lesion in the FA pathway. The method comprises contacting cells with an agent, and testing for sensitivity to a genotoxic anti-neoplastic agent.
  • test and control cells are isogenic, except that the test cell contains a lesion in at least one component of the FA/BRCA pathway, for example, in FANCA, FANCB, FANCC, FANCD FANCE FANCF FANCG FANCL, and the ATR protein kinase, among others.
  • the method comprises comparing the sensitivities to genotoxic anti-neoplastic agents of two isogenic cell lines which differ in the functionality of the FA pathway.
  • two isogenic ovarian tumor lines a parental 2008 and the 2008 cells complemented with the FANCF cDNA, are employed.
  • the parental 2008 cells fail to express FANCF, these cells have a disruption of the FA pathway, and they are hypersensitive to cisplatin. Complementation of the 2008 cells with the FAN CF cDNA restores FA pathway in these cells. Therefore, these control cells therefore serve as a basis for comparison.
  • This isogenic pair of cells is subjected to a high throughput chemical screen with a library of compounds, for example kinase inhibitors.
  • Agents which selectively kill 2008 cells lacking the FA/BRCA pathway) but which do not kill the corrected 2008 plus FANCF control cells are candidate inhibitors of a non-FA DNA damage repair pathway.
  • genes affecting the viability of the parental but not the control cells are tested by systematic, mass inhibition using an siRNA library.
  • a bar-coded siRNA library can be used to for stable transfection of the two cell lines.
  • Genes that are required for viability of the 2008 cells, but not for the corrected cells are expected to have the result that siRNA knockdown of such a gene will be lethal in the parental 2008 cells, but not in the control 2008 cells which have been transfected with the FANCF cDNA.
  • Agents thus identified which can kill a cell in which one or more DNA damage repair pathways is disrupted but do not kill an isogenic cell line in which the disruption is restored can be used in the treatment of cancer. Disruption of two or more of the six major DNA damage repair pathways can result in cell death. Since many cancers already have the one pathway knocked out or repressed, a relatively non-toxic inhibitor of the second pathway, for example the BER pathway, maybe sufficient to cause cytoreduction of the cancer, even in the absence of a chemotherapeutic agent.
  • a pro-drug strategy to enhance uptake of these agents by cancer cells provide the necessary therapeutic index.
  • Anti-neoplastic agents which are particularly useful include, but are not limited to, agents which cause damage to the DNA. These agents include DNA alkylating agents, intercalating agents, and the like.
  • DNA- damaging chemotherapeutic compounds including, but 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.
  • methods described herein can also employ radiotherapeutic methods of treating neoplastic disorders.
  • the genotoxic anti-neoplastic agents do not inhibit DNA damage repair at the concentrations administered.
  • the present invention is based on the surprising discovery that the efficacy of the FA pathway of a cell strongly correlates with its sensitivity to chemotherapeutic agents. Therefore, in one aspect, the invention provides a method of predicting whether a subject with a neoplastic disorder or disease will respond to a genotoxic anti-neoplastic agent.
  • the method comprises obtaining a biological sample from the subject, and determining degree of ubiquitination of the Fanconi anemia complementation group D2 (FANC D2) polypeptide within the biological sample.
  • FANC D2 Fanconi anemia complementation group D2
  • a degree of ubiquitination of the FANC D2 polypeptide in the biological sample of the subject that is less than about 70% when compared with a biological sample from a control subject is indicative of a subject that will respond to a genotoxic antineoplastic agent.
  • the invention provides a method of predicting whether a subject with a neoplastic disorder or disease will respond to a genotoxic anti-neoplastic agent.
  • the method comprises obtaining a biological sample from the subject, and determining the FANC D2-containing foci within the biological sample. A difference in foci formation, wherein the sample from the subject that contains less than about 70% of the FANC D2-containing foci when compared with the biological sample from a control subject is indicative of a subject that will respond to a genotoxic anti-neoplastic agent.
  • the neoplastic disorder is selected from the group consisting of leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphatic leukemia, myelodysplasia, multiple myeloma, Hodgkin's disease or non-Hodgkin's lymphoma, small or non-small cell lung carcinoma, gastric, intestinal or colorectal cancer, prostate, ovarian or breast cancer, head, brain or neck cancer, cancer in the urinary tract, kidney or bladder cancer, malignant melanoma, liver cancer, uterine or pancreatic cancer.
  • the ability of a biological sample to activate the FA pathway is determined to identify responders to chemotherapeutic agents, particularly genotoxic anti- neoplastic agents.
  • the anti-neoplastic agents can be any which are used for the treatment of cancer, and in one embodiment, anti-neoplastic agents' mechanism of action is through the damage of DNA.
  • 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, Mnotecan, Lomustine, Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin, Temozolomide, and Topotecan and ionizing radiation.
  • BCNU 1,3-Bis(2-Chloroethyl)-1- NitrosoUrea
  • Busulfan Carboplatin
  • Carmustine Chlorambucil
  • Cisplatin Cisplatin
  • Cyclophosphamide dacarbazine
  • Daunorubicin Daunorubicin
  • the patient or, alternatively, the biological sample obtained from the patient can be exposed to the anti-neoplastic agent prior to determining the degree of ubiquitination of the FANC D2 polypeptide.
  • the patient or biological sample obtained from the subject is exposed at a dose that is less than or equal to the therapeutically effective dose. In another embodiment, the exposure is at 50% or less of the therapeutically effective dose of the anti-neoplastic agent.
  • the degree of ubiquitination of the FANC D2 polypeptide can be compared with that of a control subject.
  • a control subject can be a single subject that has previously been determined to be normal with respect to response to anti-neoplastic agents, or a number of normal subjects.
  • Biological samples from either a single control subject or a number of control subjects can be used.
  • a subject is deemed to be a responder to an anti-neoplastic agent if the percentage of FANC D2 ubiquitination is less than about 70% when compared with a sample from a subject, 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, when compared with a sample from a subject that has received the same or equivalent dose of anti-neoplastic agent as the test sample.
  • control samples can be prepared prior to preparation of the test samples, or prepared simultaneously to preparation of the test samples.
  • the subject or alternatively the biological sample taken from the subject, can be treated with a genotoxic anti-neoplastic agent prior to measurement of the efficacy of the FA pathway.
  • the dosage of the anti-neoplastic agent would be that necessary to induce the FA pathway in a normal subject.
  • the dosage of the anti-neoplastic agent would be from between about 5% to 100% of the typical therapeutically effective dose, more typically between 20% to 100%, and most typically between about 35% - 100%.
  • the degree of ubiquitination of the FANC D2 polypeptide can be measured using inimunoblot analysis as previously described.
  • Subjects are considered responders if the formation of ubiquitinated FANC D2 polypeptide is about 70% or less when compared with normal subjects, for example 70% or less, 65% or less, 60% or less, 50% or less, 40% or less, 30% or less than in normal subjects.
  • a subject or patient is administered with a therapeutically effective dose of a genotoxic anti-neoplastic agent, simultaneously, before or after administration with an inhibitor of a non-FA DNA damage repair pathway, for example the FA pathway.
  • Therapeutically effective dosages of many anti-neoplastic agents are well- established, and can be found, for example, in Cancer Chemotherapy and Biotherapy: A Reference Guide Edition Number: 2 Tenenbaum, ed. Saunders & CO (1994) which is incorporated herein by reference.
  • the method comprises administering to the subject an effective amount of an inhibitor of the FA pathway and a geno toxic anti-neoplastic agent.
  • the antineoplastic agent can be selected from the group consisting of 1,3-Bis(2-Chloroethyl)-1- Nitrosourea (BCNU), Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, dacarbazine, Daunorubicin, Doxorubicin, Epirubicin, Etoposide, Idarubicin, Ifosfamide, Mnotecan, Lomustine, Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin, Temozolomide, and Topotecan and ionizing radiation.
  • BCNU 1,3-Bis(2-Chloroethyl)-1- Nitrosourea
  • Busulfan Carboplatin
  • Carmustine Chlorambucil
  • a method of treating a neoplastic disorder in a subject in need thereof comprises administering to the subject an effective amount of an inhibitor of the FA pathway and an inhibitor of a non-FA DNA damage repair pathway.
  • the inhibitor of a non-FA DNA damage repair pathway can be selected which inhibits any of the repair pathways, and can be selected from the group consisting of PARP inhibitors, DNA- PK inhibitors, mTOR inhibitors, ERCCl inhibitors ERCC3 inhibitors, ERCC6 inhibitors, ATM inhibitors, XRCC4 inhibitors, Ku80 inhibitors, Ku70 inhibitors, XPA inhibitors, CHKl inhibitors, CHK2 inhibitors, or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof.
  • the inhibitor of the FA pathway can be administered before, simultaneously with, or after administration of the inhibitor of the non-FA DNA damage repair pathway.
  • the inhibitors can be administered parenterally, orally or directly into the tumor.
  • the inhibitor of the FA pathway as well as inhibitor of a non-FA DNA damage repair pathway, can act to increase the sensitivity of a neoplastic disorder to a genotoxic antineoplastic agent. Therefore, in another aspect, a method of increasing the sensitivity of a neoplastic disorder to a genotoxic anti-neoplastic agent is provided.
  • the method comprises administering before, after or concurrently with a therapeutically effective dose of the agent a combination of an effective amount of an inhibitor of the FA pathway and an inhibitor of a non-FA DNA damage repair pathway.
  • the method can be useful for the treatment of many types of neoplastic disorders, and can be selected from the group consisting of leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphatic leukemia, myelodysplasia, multiple myeloma, Hodgkin's disease or non-Hodgkin's lymphoma, small or non-small cell lung carcinoma, gastric, intestinal or colorectal cancer, prostate, ovarian or breast cancer, head, brain or neck cancer, cancer in the urinary tract, kidney or bladder cancer, malignant melanoma, liver cancer, uterine or pancreatic cancer.
  • leukemia acute myeloid leukemia, chronic myeloid leukemia, chronic lymphatic leukemia, myelodysplasia, multiple myeloma, Hodgkin's disease or non-Hodgkin's lymphoma, small or non-small cell lung carcinoma, gastric, intestinal or colorectal cancer, prostate, ovarian or
  • the invention provides a method of increasing the sensitivity of a neoplastic disorder to a genotoxic anti-neoplastic agent.
  • the method comprises administering before, after or concurrently with a therapeutically effective dose of an genotoxic anti-neoplastic agent, an effective amount of an inhibitor of the FA pathway.
  • the inhibitor of the FA pathway can be administered before, simultaneously with, or after administration of the inhibitor of the non-FA DNA damage repair pathway, and can be administered parenterally, orally or directly into the tumor.
  • the method further comprises administering an inhibitor of a non-FA DNA damage repair pathway, in addition to the FA inhibitor and genotoxic anti-neoplastic agent.
  • the inhibitor of the non-FA DNA damage repair pathway can be administered before, after, or concurrently with a therapeutically effective dose of the FA pathway inhibitor and genotoxic anti-neoplastic agent.
  • compositions disclosed herein in preventing or treating neoplastic disorders can be tested, for example, in animal models of specific neoplastic disorders.
  • animal models are well known to those skilled in the art, and are disclosed, 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 to Oncology, VoI 51) (Karger, 1996), the contents of which are incorporated herein in their entirety.
  • test Compounds According to the Invention Whether in an in vitro or in vivo system, the invention encompasses methods by which to screen compositions which can inhibit the formation of FANC D2-containing foci, as well as compositions which inhibit DNA damage repair pathways other than the FA pathway.
  • Candidate modulator compounds from large libraries of synthetic or natural compounds can be screened. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. 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, WI). Combinatorial libraries are available and can be prepared. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible by methods well known in the art. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
  • Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, including small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 Daltons, preferably less than about 750, more preferably less than about 350 Daltons. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents.
  • peptide agents are identified, they maybe modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g., for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.
  • an unnatural amino acid such as a D-amino acid, particularly D-alanine
  • Candidate modulators which may be screened according to the methods of the invention include receptors, enzymes, ligands, regulatory factors, and structural proteins.
  • Candidate modulators also include nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma-associated proteins, serum proteins, viral antigens, bacterial antigens, protozoan antigens and parasitic antigens.
  • Candidate modulators additionally comprise proteins, lipoproteins, glycoproteins, pliosphoproteins and nucleic acids (e.g., RNAs such as ribozymes or antisense nucleic acids).
  • Proteins or polypeptides which can be screened using the methods of the present invention include hormones, growth factors, neurotransmitters, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, structural proteins, viral antigens, parasitic antigens, bacterial antigens and antibodies (see below).
  • Candidate modulators which may be screened according to the invention also include substances for which a test cell or organism might be deficient or that might be clinically effective in higher-than-normal concentration as well as those that are designed to eliminate the translation of unwanted proteins.
  • Nucleic acids of use according to the invention not only may encode the candidate modulators described above, but may eliminate or encode products which eliminate deleterious proteins.
  • Such nucleic acid sequences are antisense RNA and ribozymes, as well as DNA expression constructs that encode them. Note that antisense RNA molecules, ribozymes or genes encoding them may be administered to a test cell or organism by a method of nucleic acid delivery that is known in the art, as described below.
  • Inactivating nucleic acid sequences may encode a ribozyme or antisense RNA specific for the target mRNA.
  • Ribozymes of the hammerhead class are the smallest known, and lend themselves both to in vitro production and delivery to cells (summarized by Sullivan, (1994) J. Invest. Dermatol., 103: 85S-98S; Usman et al., (1996), Curr. Opin. Struct. Biol, 6: 527- 533).
  • the invention relates to methods and pharmaceutical compositions comprising an inhibitor of the FA pathway in combination with an anti-neoplastic agent and/or inhibitor of a non-FA DNA damage repair pathway, as described in the preceding section, and a pharmaceutically acceptable carrier, as described below.
  • the pharmaceutical composition comprising an inhibitor of the FA pathway is useful for treating a variety of diseases and disorders including cancer, and may be useful as protective agents against genotoxic anti-neoplastic agents.
  • the invention provides for a method of treating a neoplastic disorder in a subject in need thereof comprising administering a combination of an effective amount of: a) an inhibitor of the FA pathway or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof, and b) a genotoxic anti-neoplastic agent.
  • inhibitors of the FA pathway include H-9, alsterpaullone and curcumin.
  • additional inhibitors of the FA pathway can be identified, for example, using the methods described herein.
  • an inhibitor of the FA pathway can be a small molecule, and antibody, a ribozyme or siRNA molecule.
  • the method can be used in the treatment of various neoplastic disorders, including leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphatic leukemia, myelodysplasia, multiple myeloma, Hodgkin's disease or non-Hodgkin's lymphoma, small or non-small cell lung carcinoma, gastric, intestinal or colorectal cancer, prostate, ovarian or breast cancer, head, brain or neck cancer, cancer in the urinary tract, kidney or bladder cancer, malignant melanoma, liver cancer, uterine or pancreatic cancer.
  • the method is used to treat ovarian cancer.
  • the dosage of the inhibitor of the FA pathway depends on several factors, including solubility, bioavailability, plasma protein binding, kidney clearance, and inhibition constants. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition of the FA pathway is defined as an "effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of the inhibitor per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. Alternatively, the dosage can be administered using a functional dosage, since the activation of the FA pathway in a subject can be determined empirically using the ubiquitination of the FANC D2 polypeptide using the methods described herein.
  • an "effective dose" of an inhibitor of the FA pathway can mean a dose required to reduce the level of FANC D2 ubiquitination to about 70% or less when compared with a control sample, more typically to about 50% or less than a control sample.
  • a control sample is ideally taken from the same subject, before administration of the inhibitor.
  • the dosage of the inhibitor of the FA pathway in relation to the dosage of the genotoxic anti-neoplastic agent can be expressed as a ratio.
  • the inhibitor of the FA pathway can be administered at a ratio of between about 100:1 to about 1:100, on a molar basis, in relation to the genotoxic antineoplastic agent, for example, at 1:100, 1:50, 1:10, 1 :5, 1 :2, 1 :1, 2:1, 5:1, 10:1, 20:1, 50:1, or 100:1.
  • the genotoxic anti-neoplastic agent are agents which are used to treat neoplastic disorders, and include 1 ,3 -Bis(2-Chloroethyl)- 1 -Nitrosourea (BCNU), Busulfan, Carboplatin, Carmustme, Chlorambucil, Cisplatin, Cyclophosphamide, dacarbazine, Daunorubicin, Doxorubicin, Epirubicin, Etoposide, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin, Temozolomide, and Topotecan.
  • BCNU 1 ,3 -Bis(2-Chloroethyl)- 1 -Nitrosourea
  • Busulfan Carboplatin, Carmustme, Chlorambucil, Cisplatin, Cyclophospham
  • Dosages of the anti-neoplastic agents listed above have been well established for different types of neoplastic disorders.
  • co-administration with inhibitors of the FA pathway can increase the sensitivity of the neoplastic disorders to the anti-neoplastic agents. Therefore, it is possible that the dosage of the anti-neoplastic agents will be less than is typically administered for the given neoplastic disorder.
  • the lower dosage may have the additional advantage of reduced side effects.
  • the dosage of the antineoplastic agent is expected to be within about 20%- 100% of the typical dosage for the given neoplastic disorder, more typically between about 35% - 100%.
  • the present invention provides for a method of treating a neoplastic disorder in a subject in need thereof, comprising administering to the subject a combination of an effective amount of:
  • the inhibitor of a DNA damage repair pathway can be selected from the group consisting of PARP inhibitors, DNA-PK inhibitors, FA inhibitors, mTOR inhibitors, ERCCl inhibitors, ERCC3 inhibitors, ERCC6 inhibitors, ATM inhibitors, XRCC4 inhibitors, Ku80 inhibitors, Ku70 inhibitors, XPA inhibitors, CHKl inhibitors, CHK2 inhibitors, or pharmaceutically acceptable salts, esters, derivatives, solvates or prodrugs thereof. ,
  • the non-FA DNA damage repair pathway is a pathway other than the FA pathway.
  • the inhibitor targets a pathway selected from the group consisting of the non-homologous end joining DNA damage repair pathway, the mismatch repair pathway, and the nucleotide excision pathway.
  • the inhibitor targets the non-homologous end joining DNA damage repair pathway.
  • the inhibitor targets the direct reversal pathway.
  • the inhibitor targets the mismatch repair pathway.
  • the inhibitor targets the nucleotide excision repair pathway.
  • the inhibitor targets the base excision repair pathway.
  • Ideal dosages of the inhibitor of a DNA damage repair pathway will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of the inhibitor per- kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used.
  • the appropriate dosage can be determined empirically, inhibition of DNA damage repair pathways can be measured using biological samples taken from the subject. Therefore, an "effective dose" of an inhibitor of the DNA damage repair pathway can mean a dose required to reduce the level of the specific pathway to about 70% or less when compared with a control sample, more typically to about 50% or less than a control sample.
  • a control sample is ideally taken from the same subject, before administration of the inhibitor.
  • the present invention provides for a method of treating a neoplastic disorder in a subject in need thereof, comprising administering to said subject a combination of an effective amount of:
  • the inhibitor of the FA pathway, its dosage and method of administration are as described previously.
  • the inhibitor of a non-FA DNA damage repair pathway, as well as its dosage and method of administration are the same as previously described.
  • administration of inhibitors of the FA pathway, as well as of a non-FA DNA damage repair pathway can heighten the sensitivity to a genotoxic anti-neoplastic agent. Therefore, it is possible that the dosage of the anti-neoplastic agents will be less than is typically administered for the given neoplastic disorder.
  • the lower dosage may have the additional advantage of reduced side effects.
  • the dosage of the antineoplastic agent is expected to be within about 20%- 100% of the typical dosage for the given neoplastic disorder, more typically between about 35% - 100%.
  • the compounds of the present invention can be formulated for oral, intravenous, intramuscular, subcutaneous, topical and/or parenteral administration for the therapeutic or prophylactic treatment of diseases.
  • compounds of the present invention can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers and the like.
  • the compositions comprising a compound of this present invention will contain from about 0.1% to about 99.9%, about 1% to about 98%, about 5% to about 95%, about 10% to about 80% or about 15% to about 60% by weight of the active compound.
  • the compounds of the present invention can be administered at separate times, using separate methods of administration. For example, in certain situations, it maybe advantageous to administer the inhibitor of the FA pathway before, simultaneously with, or after administration of the genotoxic anti-neoplastic agent or other agents. Likewise, the method of administration of each compound will depend on the optimal means of administration thereof.
  • compositions of the present invention are prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent, or eliminate cancer, or to provide a protective effect against genotoxic anti-neoplastic agents such as ionizing radiation.
  • genotoxic anti-neoplastic agents such as ionizing radiation.
  • the compositions of the present invention can be delivered using controlled (e.g., capsules) or sustained release delivery systems (e.g., biodegradable matrices).
  • compositions of the present invention comprise one or more compounds of the present invention in association with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants and/or excipients, collectively referred to herein as "carrier" materials, and if desired other active ingredients.
  • compositions may contain common carriers and excipients, such as com starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid.
  • compositions may contain crosarmellose sodium, microcrystalline cellulose, sodium starch glycolate and alginic acid.
  • Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Providone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.
  • Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicon fluid, talc, waxes, oils and colloidal silica.
  • Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the like can also be used. It may also be desirable to add a coloring agent to make the dosage form more aesthetic in appearance or to help identify the product comprising a compound of the present invention.
  • the pharmaceutical compositions are in the form of, for example, a tablet, capsule, suspension or liquid.
  • 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.
  • the tablets and capsules which can contain, in addition to the active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica or talc: disintegrants, for example, potato starch, flavoring or coloring agents, or acceptable wetting agents.
  • binding agents for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth
  • fillers for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose
  • lubricants for example, magnesium stearate, polyethylene glycol, silica or talc
  • disintegrants for example
  • Oral liquid preparations generally are in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs and may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents.
  • additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl parahydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.
  • compounds of the present invention can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion.
  • Intravenous fluids include, without limitation, physiological 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 of the carriers mentioned for use in the formulations for oral administration.
  • the compounds can be dissolved in polyethylene glycol, propylene glycol, ethanol, com oil, benzyl alcohol, sodium chloride, and/or various buffers.
  • a sterile formulation of compounds of the present invention or suitable soluble salts forming the compound can be dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline or 5% glucose.
  • WFI Water-for-Injection
  • a suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.
  • the compounds of present invention can also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and can take the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat paints.
  • suitable forms can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient.
  • DMSO dimethylsulfoxide
  • the compounds of the present invention can be presented in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.
  • the compounds of the present invention can be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or other glyceride.
  • the compound of the present invention can be in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery.
  • the unit dosage form of the compound can be a solution of the compound or a salt thereof in a suitable diluent in sterile, hermetically sealed ampoules.
  • the amount of the compound of the present invention in a unit dosage comprises a therapeutically-effective amount of at least one active compound of the present invention which may vary depending on the recipient subject, route and frequency of administration.
  • a subject refers to an animal such as an ovine or a mammal, including a human.
  • the novel compositions disclosed herein are placed in a pharmaceutically acceptable carrier and are delivered to a recipient subject (including a human subject) in accordance with known methods of drug delivery.
  • the methods of the invention for delivering the compositions of the invention in vivo utilize art-recognized protocols for delivering the agent with the only substantial procedural modification being the substitution of the compounds of the present invention for the drugs in the art-recognized protocols.
  • 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 antineoplastic agents.
  • unit dosage refers to a quantity of a therapeutically effective amount of a compound of the present invention that elicits a desired therapeutic response.
  • treating is defined as administering, to a subject, a therapeutically effective amount of at least one compound of the present invention, both to prevent the occurrence of a pre-cancer or cancer condition, or to control or eliminate pre-cancer or cancer condition.
  • the term “desired therapeutic response” refers to treating a recipient subject with a compound of the present invention such that a pre-cancer or cancer condition is reversed, arrested or prevented in a recipient subject.
  • the compounds of the present invention can be administered as a single daily dose or in multiple doses per day.
  • the treatment regime may require administration over extended periods of time, e.g., for several days or for from two to four weeks.
  • the amount per administered dose or the total amount administered will depend on such factors as the nature and severity of the disease condition, the age and general health of the recipient subject, the tolerance of the recipient subject to the compound and the type of cancer, the sensitivity of the cancer to therapeutic agents, and, if used in combination with other therapeutic agent(s), the dose and type of therapeutic agent(s) used.
  • a compound according to this invention may also be administered in the diet or feed of a patient or animal.
  • the diet for animals can be normal foodstuffs to which the compound can be added or it can be added to a premix.
  • the compounds of the present invention may be taken in combination, together or separately with any known clinically approved agent to treat a recipient subject in need of such treatment.
  • Example 1 Methods Cell lines and Cell Culture.
  • HeLa cells, PD20 (FA-D2) fibroblasts, and GM6914 (FA-A) fibroblasts were grown as previously described (Taniguchi et al. (2002) Cell. 109:459-472). Briefly, cells were grown in Dulbecco's modified eagles medium (DMEM) supplemented with 15% fetal calf serum (FCS). The FANCF-deficient ovarian tumor line (2008) and FANCF cDNA corrected 2008 cells were previously described.
  • DMEM Dulbecco's modified eagles medium
  • FCS fetal calf serum
  • the FANCF-deficient ovarian tumor line (2008) and FANCF cDNA corrected 2008 cells were previously described.
  • Breast cancer cell line MCF7 was purchased from the American Type Culture Collection (Manassas, Virginia). OVCAR5 and OVCAR8 were grown as previously described.
  • pMMP-puro (Ory et al., (1996) Proc Natl Acad. Sd USA. 93 : 11400-11406) and pMMP-puro-FANCD2 was described previously (Timmers et al., (2001) MoI Cell. 7:241-248; Garcia-Higuera et al., (2001) MoI Cell. 7:249-262).
  • pMMPpuro EGFP-F ANCD2 was constructed by adding EGFP cDNA sequence (from pEGFP-Nl (Clontech)) to the N-terminus of the FANCD2 cDNA sequence. The cDNA insert was verified by direct DNA sequencing.
  • fibroblasts Production of pMMP retroviral supernatants and infection of fibroblasts (PD20 fibroblasts) were performed as previously described (Naf et al., (1998) MoI Cell Biol, 18:5952-5960). After 48 hours, cells were trypsinized and selected in medium containing puromycin (1 ⁇ g/mL). Dead cells were removed, and surviving cells were grown under continuous selection in puromycin.
  • Subcloning of PD20 fibroblasts infected with pMMPpuroEGFP-FANCD2 was performed by limited dilution, and a clone which showed clear EGFP-FANCD2 foci formation in response to IR treatment (15Gy, 10hr) was selected (PD20F -EGFP-F ANCD2 clone 7) for the drug screening study.
  • Human cells (HeLa cells, PD20 fibroblasts, or 2008 cells, where indicated; Taniguchi et al., (2003) Nat Med. 9:568-574) were seeded onto 12-well plates at 9 x 10 4 cells/well in DMEM-15% FCS (5 ml). After cells attached for 16 to 24 h, the medium was replaced with DMEM- 15% FCS containing cisplatin (CDDP) or MMC (Sigma) at various concentrations, with or without a kinase inhibitor or curcumin (Sigma) at either variable or the same concentration. The cells were incubated at 37° C for one day.
  • clone 7 cells were plated in 384 well plates.
  • a chemical compound from a commercial library (Kau et al., (2003) Cancer
  • Example 2 Identification and characterization of potential inhibitors of FANC D2 ubiquitination and foci formation.
  • Example 3 Identification and characterization of H-9 as an inhibitor of FANC D2 ubiquitination and foci formation.
  • H-9 was identified as an inhibitor of the FA pathway using the high-throughput screen as described above. As shown by fluorescence microscopy in Figure 7, H-9 inhibited the formation of FANC D2 foci in the 50-100 ⁇ M range. As a secondary screen using immunoblot analysis to determine the relative pools of monoubiquitinated FANC D2, H-9 treatment was found to decrease the overall level of FANC D2 monoubiquitination ( Figure 8). H-9 did not affect ATM-dependent phosphorylation of FANC D2 but did inhibit ATR dependent phosphorylation of CHKl .
  • Example 4 Identification and characterization of alsterpaullone as an inhibitor of FANC D2 ubiquitination and foci formation.
  • alsterpaullone was identified as a potential inhibitor of the FA pathway using the high-throughput screen. Alsterpaullone is known to inhibit Cdkl/cycline B, Gsk- 3B, and Cdk5 (Sausville et al. (2000) Ann N Y Acad. ScI 910:207-221; Schultz et al. (1999) J Med. Chem. 42:2909-2919). Alsterpaullone inhibited the formation of FANC D2 foci at a concentration of 10 ⁇ M ( Figure 10). Like H-9, alsterpaullone inhibited FANC D2 monoubiquitination and inhibited ATR dependent phosphorylation of Chkl ( Figure 11).
  • Example 5 Identification and characterization of c ⁇ rcumin as an inhibitor of FANC D2 ubiquitination and foci formation.
  • the natural compound, curcumin also caused a dose-dependent decrease in FANC
  • curcumin caused a dbse dependent decrease in FANC D2 monoubiquitination in HeLa cells and in cisplatin-exposed HeLa cells (Figure 10). Curcumin also caused a dose-dependent decrease in ATR-mediated Chkl phosphorylation ( Figure 11), further suggesting that curcumin blocks an upstream event in the FA/BRCA pathway.
  • curcumin The chemosensitizing activity of curcumin was further tested for its specificity to cisplatin.
  • the ovarian tumor cell line, SKO V3 is sensitive to cisplatin and taxol (Yang and Page, (1995) Oncol Res. 7:619-24). Curcumin synergized with cisplatin in the killing of these cells. In contrast, curcumin had no effect on the dose dependent taxol cytotoxicity profile of these cells.
  • Example 6 Correction of cisplatin hypersensitivity in the ovarian cancer cell line 2008 by FANCF complementation.
  • Curcumin sensitized FANCF-corrected 2008 cells to cisplatin but had a lesser effect on the chemosensitization of the parental 2008 cells ( Figure 9, and Table 3). Curcumin sensitized the cells in a dose range of 3-20 micromolar, corresponding to the curcumin dose range required for inhibition of the FA/BRCA pathway. Similar results were observed when the cells were pretreated for 24 hours with curcumin before the addition of cisplatin (data not shown). Taken together, these results indicate that curcumin synergizes with cisplatin in enhancing ovarian tumor cytoxicity and that this curcumin effect correlates with its inhibition of the FA/BRCA pathway.
  • Curcumin and Alsterpaullone are soluble at up to 2.5 mM in 25% DMSO, the maximum concentration compatible with intraventricular administration. Taking into consideration that the entire mouse ventricular system consists of no more than 20 ⁇ l, the maximum concentration achieved (50 ⁇ M) exceeds that required for FA pathway inhibition. Likewise, both AMD3100 and 06 Benzylguanine are sufficiently soluble in media, allowing estimated intraventricular concentrations to exceed those required for efficacy in vitro. For each compound tested, a group of six mice are implanted with intraventricular catheters (4 mm anterior to the lambda suture, 0.7 mm lateral of midline, and 2.5 mm below the dura).
  • the catheters are connected to a subcutaneous Alzet Osmotic pump model 1007D (90 ⁇ l volume delivered at 0.5 ⁇ l/hr) containing 2.5 mM, 1.25 mM, 0.625 mM, 0.313 mM, 0.151 mM of each compound or the vehicle media.
  • the mice are monitored daily during the infusion period for neurologic deficits and sacrificed at post-operative day 21.
  • Brain sections are performed to 1) confirm catheter continuity with the lateral ventricle, and 2) assess cellular toxicity by stained with hematoxylin and eosin as well as TUNEL staining. The highest dose of drug delivered without clinical or histologic evidence of damage are selected for subsequent efficacy studies, as detailed below.
  • Example 8 In vivo tumoricidal effects of systemic BCNU administration by intravesicular administration of an FA pathway inhibitor.
  • the mouse xenograft, bioluminescence model used herein employs the U87 GMB cell line (ATCC), which has been retrovirally transfected with the coding sequence for luciferase in a pMMP vector.
  • U87-Luciferase cell lines are harvested in mid-logarithmic growth phase, resuspended as 50,000 cells in 10 ⁇ l PBS, and introduced into mice brain using stereotactic guidance (2 mm lateral and posterior to the bregma, 3 mm below dura). Mice are given D-luciferin (Xenogen, Alameda, CA) intraperitoneal injections and imaged with the rVIS imaging system (Xenogen) on post-surgery day 5 and 10.
  • D-luciferin Xenogen, Alameda, CA
  • rVIS imaging system Xenogen
  • mice For each compound tested, a group of 30 mice are implanted with U87-Luciferase and surveyed on post-implant day 5 and 10, out of which 20-2.6 mice are expected to have uptake of the implanted tumor (Rubin et aL, (2003) Proc. Natl. Acad. Sd. ' USA, 100: 13513-13518). Mice with U87-Luciferase tumor uptake are then surgically implanted with an intraventricular catheter and Alzet pump 1007D. These mice are then stratified into four groups:
  • Group 2 treated with i.p. BCNU administration (15 mg/kg);
  • Group 3 treated with i.p. administration of an FA inhibitor at the maximum tolerated dose
  • Group 4 treated with i.p. BCNU administration (15 mg/kg) as well as FA inhibitor at the maximum tolerated dose.
  • the intraventricular catheter is placed contra-lateral to the tumor implant site to minimize the effect of tumor growth on stereotactic coordinates.
  • the i.p. BCNU injection is carried out 4 days after intraventricular administration to allow for sensitization.
  • the mice are imaged on day 15 and 20 after initial tumor implant. Comparison of tumor growth is determined using LIVING IMAGE software package (Xenogen).
  • Example 9 Efficacy of FA inhibitor in sensitizing ovarian tumors to anti-neoplastic agents in an animal model.
  • mice models provide useful means to test the efficacy of FA inhibitors in sensitizing ovarian tumors to anti-neoplastic agents. 6 mice are used per group. To test the efficacy of cisplatin, alone or in combination with the FA inhibitor alsterpaullone, the following groups were used:
  • Group 1 treated with control vehicle
  • Group 2 treated with cisplatin, 4 mg/kg
  • Group 3 treated with alsterpaullone at 5 mg/kg
  • Group 4 treated with cisplatin, 4 mg/kg, and alsterpaullone, 5 mg/kg. Repeat the cycle after two days.
  • mice All treatments are started a week after tumor inoculation. Mice are treated for 10 cycles in total, and sacrificed for tumor nodule counting two weeks (on day 50) after discontinuation of drug treatment. Upon sacrifice, antitumor activity in each group is evaluated by counting the number of tumor nodules in the peritoneal cavity, measuring the diameter of the tumors, measuring the volume of the ascites and qualitatively observing the color of the peritoneal wall as an indication of the degree of tumor-induced vascularization. Toxicity is evaluated by qualitative observation of the general appearance and behavior of the mice prior to sacrifice and by measuring their body weight at various intervals during the course of the treatments.
  • Curcumin is known to be safe at high doses, with LD 50 of greater than 10,000 mg/kg. Curcumin can be administered orally, intraperitoneally or intravesicularly. In one example, i.p. administration of curcumin at 100mg/kg - 300mg/kg is performed, either alone or in combination with an anti-neoplastic agent (e.g., cisplatin) is tested in mice, as described above. In another example, intravesicular administration of curcumin is performed at the highest possible dose determined using the method outlined above.
  • an anti-neoplastic agent e.g., cisplatin
  • Example 10 Efficacy of a combination of an FA inhibitor and a DNA damage repair pathway inhibitor in treating ovarian tumor.
  • the efficacy of a combination of an FA inhibitor and an inhibitor of a DNA damage repair pathway is tested essentially as described above in Example 6. Briefly, the efficacy of the FA inhibitor (for example, curcumin, H-9, or alsterpaullone) in treating ovarian tumor, is tested alone or in combination with a DNA damage repair pathway inhibitor. In one example, the following groups are tested:
  • Group 1 treated with control vehicle
  • Group 2 treated with alsterpaullone at 5 mg/kg
  • Group 3 treated with methoxyamine at 2 mg/kg
  • Group 4 treated with alsterpaullone, 5 mg/kg, and methoxyamine, 2 mg/kg. Repeat the cycle after two days.
  • Example 11 Combination of an FA inhibitor and a DNA damage repair pathway inhibitor in sensitizing ovarian tumors to anti-neoplastic agents.
  • the ability of a combination of an FA pathway inhibitor such as curcumin, H-9 or alsterpaullone, and a DNA damage repair pathway inhibitor such as methoxyamine, in sensitizing a tumor to anti-neoplastic agents is tested using an animal model, essentially as described above.
  • the dosage ' of anti-neoplastic agent administered can be varied to determine whether sensitization results in a lower overall dosage of the antineoplastic agent necessary 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; alsterpaullone, 5 mg/kg, and methoxyamine, 2 mg/kg
  • Group 3 treated with cisplatin, 1 mg/kg; alsterpaullone, 5 mg/kg, and methoxyamine, 2 mg/kg
  • Group 4 treated with cisplatin, 2 mg/kg; alsterpaullone, 5 mg/kg, and methoxyamine, 2 mg/kg
  • Example 12 Clinical Evaluation of Treatment of Recurrent MuIIerian Malignancies With Curcumin and Carboplatin.
  • a Phase I open-label, dose-escalation safety study is conducted in patients with recurrent carcinoma of mullerian origin, less than 12 months from prior platinum-based chemotherapy. Curcumin is administered orally the night before, immediately prior to, the night of, and the morning following intravenous administration of carboplatin AUC 5.
  • a treatment cycle is 28 days with carboplatin adminstration beginning on day 1 followed by a 28-day follow-up period. Decisions regarding dose escalation and Dose Limiting Toxicity determination are made at the end of the 4 week cycle. Patients who tolerate treatment without evidence of disease progression are eligible for additional cycles of curcumin/carboplatin treatment.
  • the initial dose level will be carboplatinum AUC 5 and curcumin 900mg. If none has Dose Limiting Toxicity (DLT), then the next 3 patients get dose level 2. If a DLT occurs at any dose level, three additional patients are enrolled to that dose level. If two DTLs occur at that dose level, then it is declared above the Maximum Tolerated Dose (MTD) and the MTD is defined at the previous dose level. No intrapatient dose escalations are made.
  • DLT Dose Limiting Toxicity
  • the initial dose will be carboplatin AUC 5 infused over 60 minutes and curcumin 900mg taken orally.
  • the curcumin dose will be escalated while the carboplatin dose will remain constant based on the patient's renal function.
  • a cycle is defined as an interval of 28 days and is comprised of one treatment of carboplatin on Day 1 of the cycle and one course of curcumin administered the day prior (DayO), immediately prior to carboplatin on Day 1, the night of Day 1, and the morning of Day 2 for a total of 4 doses during each cycle.
  • the doses of curcumin will be escalated for additional cohorts of patients until the DLT and MTD are determined.
  • Carboplatin is infused intravenously over 1-hour.
  • the dose of carboplatin is calculated as follows, using the Calvert formula based on creatinine clearance:
  • Total dose (mg) Target AUC (in mg/ml per min) x (Estimated GFR + 25)
  • the carboplatin dose is calculated in mg, not mg/m 2 .
  • Creatinine clearance (CrCL) can either be measured, or estimated using the Jelliffe formula.
  • Typical pre- mediations include, zofran, ativan and decadron.
  • DLT determination of DLT for purposes of assessing dose escalation is defined as follows using the NCI CTC version 3.0 criteria with consideration of known and accepted toxicities of carboplatin. Toxicities reached without pre-medication are not considered DLT.
  • Any febrile neutropenia (defined as T > 101° F) with a neutrophil count ⁇ 500 cells/ul after curcumin/carboplatin administration
  • Platelet count 10,000 cell/ul OR Grade 3 with evidence of bleeding necessitating blood product or platelet transfusion. Hemoglobin > Grade 4 toxicity with erythropoietin co-administration
  • tumors lesions are categorized as follows:
  • measurable - lesions that can be accurately measured in at least one dimension as 20mm with conventional techniques or as 10 mm with spiral CT.
  • Non-measurable disease includes the following: bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusion, abdominal masses that are not confirmed and followed by imaging techniques, and cystic lesions.
  • Imaging- based evaluation is preferred to evaluation by clinical examination when both methods have been used to assess the antitumor effect of treatment.
  • Clinically detected lesions are considered measurable when they are superficial (i.e. skin nodules and palpable lymph nodes).
  • AU skin lesions are documented with color photography, including a ruler to estimate the size of the lesion.
  • lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined, a CT is preferable.
  • CT Computed tomography
  • MRI magnetic resonance imaging
  • CT and MRI are the best available (and most reproducible) methods for measuring target lesions selected for response assessment.
  • Conventional CT and MRI are performed with contiguous cuts of 10mm or less in slice thickness.
  • Ultrasound is not used to measure tumor lesions. Ultrasound can be considered a possible alternative to clinical measurements for superficial palpable lymph nodes and subcutaneous lesions.
  • markers alone are not used to assess response. However, if markers are initially above the upper limit, they must return to normal levels for a patient to be considered in complete clinical response when all tumor lesions have disappeared.
  • the cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease and progressive disease.
  • Target lesions All measurable lesions up to a maximum of 5 lesions per organ and 10 lesions in total, representative of all involved organs, are identified as target lesions and recorded and measured at baseline. Target lesions are selected on the basis of their size (those with the longest diameter) and their suitability for accurate repeated measurements (either by imaging techniques or clinically). A sum of the longest diameter for all target lesions is calculated and reported as the baseline sum longest diameter. The baseline sum of the longest diameter is used as the reference by which to characterize the objective tumor response.
  • the criteria used to determine the objective tumor response for nontarget lesions are: complete response — the disappearance of all nontarget lesions and normalization of tumor marker level; incomplete response/stable disease — the persistence of one or more nontarget lesion(s) and/or the maintenance of tumor marker level above the normal limits; and progressive disease — the appearance of one or more new lesions and/or unequivocal progression of existing nontarget lesions.
  • CR complete response
  • PR partial response
  • SD stable disease
  • PD progressive disease.

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Abstract

L'invention concerne des méthodes et des compositions permettant de traiter le cancer. L'invention concerne, en particulier, des inhibiteurs de chemin de l'anémie de Fanconi et leurs méthodes d'utilisation. On utilise ces inhibiteurs pour inhiber la réparation d'un ADN endommagé et pour, par exemple, traiter le cancer.
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WO2012009475A1 (fr) * 2010-07-14 2012-01-19 Oregon Health & Science University Méthodes de traitement du cancer par inhibition de la déméthylase 1 spécifique de la lysine
US9821008B2 (en) 2013-11-25 2017-11-21 The University Of Toledo Inhibitors of ERCC1-XPF and methods of using the same
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CHIRNOMAS DEBORAH ET AL: "Chemosensitization to cisplatin by inhibitors of the Fanconi anemia/BRCA pathway" MOLECULAR CANCER THERAPEUTICS, vol. 5, no. 4, April 2006 (2006-04), pages 952-961, XP002550338 ISSN: 1535-7163 *
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