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WO2024086533A2 - Procédés de traitement ou de prévention de la formation d'une tumeur neuroendocrine à l'aide d'inhibiteurs de cdc7 - Google Patents

Procédés de traitement ou de prévention de la formation d'une tumeur neuroendocrine à l'aide d'inhibiteurs de cdc7 Download PDF

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WO2024086533A2
WO2024086533A2 PCT/US2023/076996 US2023076996W WO2024086533A2 WO 2024086533 A2 WO2024086533 A2 WO 2024086533A2 US 2023076996 W US2023076996 W US 2023076996W WO 2024086533 A2 WO2024086533 A2 WO 2024086533A2
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cdc7
inhibitor
adenocarcinoma
patient
sclc
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WO2024086533A8 (fr
WO2024086533A3 (fr
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Álvaro D. QUINTANAL VILLALONGA
Charles Michael Rudin
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof

Definitions

  • the present disclosure provides methods for treating or preventing neuroendocrine tumor formation in subjects diagnosed with TP53 and RBI deficient adenocarcinomas (e.g., lung or prostate adenocarcinomas) using CDC7 inhibitors. Also disclosed herein are methods for preventing neuroendocrine tumor formation in subjects diagnosed with adenocarcinomas (e.g., TP53 and RBI deficient lung or prostate adenocarcinomas) using CDC7 inhibitors in combination with androgen receptor (AR) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, or chemotherapeutic drugs.
  • adenocarcinomas e.g., TP53 and RBI deficient lung or prostate adenocarcinomas
  • AR androgen receptor
  • EGFR epidermal growth factor receptor
  • Lineage plasticity the capacity of cells to transition from one committed identity to that of a distinct developmental lineage, can promote survival of cancer cells under unfavorable conditions such as oncogenic driver-targeted therapy.
  • AD adenocarcinoma
  • NE neuroendocrine
  • the present disclosure provides a method for treating or preventing neuroendocrine tumor formation in a patient diagnosed with adenocarcinoma comprising administering to the patient an effective amount of a CDC7 inhibitor, wherein the adenocarcinoma exhibits reduced expression and/or activity of TP53 and RBI.
  • the adenocarcinoma comprises (a) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in TP53 and (b) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in RBI.
  • the present disclosure provides a method for preventing neuroendocrine tumor formation in a patient diagnosed with adenocarcinoma comprising administering to the patient an effective amount of a CDC7 inhibitor and an effective amount of at least one chemotherapeutic drug, wherein the adenocarcinoma exhibits reduced expression and/or activity of TP53 and RBI.
  • a method for enhancing responsiveness of a patient with neuroendocrine tumors to systemic chemotherapy comprising administering to the patient an effective amount of a CDC7 inhibitor and an effective amount of at least one chemotherapeutic drug, wherein the neuroendocrine tumors exhibit reduced expression and/or activity of TP53 and RBI.
  • the adenocarcinoma or the neuroendocrine tumors comprise(s) (a) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in TP53 and (b) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in RB 1.
  • the CDC7 inhibitor and the at least one chemotherapeutic drug are administered sequentially, simultaneously, or separately. Additionally or alternatively, in some embodiments, the at least one chemotherapeutic drug is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, or subcutaneously, intratumorally, or topically.
  • the at least one chemotherapeutic drug may be an alkylating agent, a platinum agent, a taxane, a vinca agent, an aromatase inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine/hormonal agent, or a bisphosphonate therapy agent.
  • chemotherapeutic drugs include, but are not limited to cyclophosphamide, fluorouracil (or 5 -fluorouracil or 5-FU), methotrexate, edatrexate (10- ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, proteinbound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, ty
  • the present disclosure provides a method for preventing neuroendocrine tumor formation in a patient diagnosed with adenocarcinoma comprising administering to the patient an effective amount of a CDC7 inhibitor and an effective amount of an androgen receptor (AR) inhibitor.
  • the adenocarcinoma may exhibit reduced expression and/or activity of TP53 and RBI.
  • the adenocarcinoma comprises (a) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in TP53 and (b) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in RBI.
  • the CDC7 inhibitor and the AR inhibitor are administered sequentially, simultaneously, or separately. Additionally or alternatively, in some embodiments, the AR inhibitor is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, or subcutaneously, intratumorally, or topically.
  • AR inhibitors include, but are not limited to apalutamide, bicalutamide, darolutamide, enzalutamide, flutamide, abiraterone acetate, ARN- 509, and nilutamide.
  • the present disclosure provides a method for preventing neuroendocrine tumor formation in a patient diagnosed with adenocarcinoma comprising administering to the patient an effective amount of a CDC7 inhibitor and an effective amount of an epidermal growth factor receptor (EGFR) inhibitor (EGFRi) (e.g., EGFR tyrosine kinase inhibitor (TKI), anti-EGFR antibodies).
  • EGFR epidermal growth factor receptor
  • EGFRi epidermal growth factor receptor
  • TKI EGFR tyrosine kinase inhibitor
  • the adenocarcinoma comprises (a) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in TP53 and (b) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in RB 1.
  • the CDC7 inhibitor and the EGFRi are administered sequentially, simultaneously, or separately.
  • the EGFRi is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, or subcutaneously, intratumorally, or topically.
  • Examples of EGFRis include, but are not limited to, osimertinib, afatinib, erlotinib, gefitinib, icotinib, dacomitinib, rociletinib, olmutinib, cetuximab, panitumumab, nimotuzumab, and necitumumab.
  • the patient is diagnosed with TP537" and RB17" mutant adenocarcinoma.
  • the TP53" I' and RBI 7" mutant adenocarcinoma is lung adenocarcinoma or prostate adenocarcinoma.
  • the CDC7 inhibitor is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, or subcutaneously, intratumorally, or topically.
  • Examples of CDC7 inhibitors include, but are not limited to simurosertib (TAK-931), PHA-767491, carvedilol, dequalinium chloride, ticagrelor, and clofoctol.
  • the patient is human. Additionally or alternatively, in some embodiments, the patient is non-responsive to at least one prior line of cancer therapy such as chemotherapy.
  • FIGs. 1A-1J demonstrate that CDC7 is highly expressed in SCLC and exert pro- oncogenic effects in this setting.
  • FIG. 1A CDC7 mRNA expression in cell lines derived from different tumor types. The data was obtained from CCLE through UCSC Xenabrowser portal (https /xenabrowser. net/) in December 2020. Lines indicate the median CDC7 mRNA expression in SCLC cell lines.
  • FIG. 1A CDC7 mRNA expression in cell lines derived from different tumor types. The data was obtained from CCLE through UCSC Xenabrowser portal (https /xenabrowser. net/) in December 2020. Lines indicate the median CDC7 mRNA expression in SCLC cell lines.
  • FIG. IB CDC
  • FIG. 1C Western blot showing CDC7 KO in H82 (SCLC-N) and H146 (SCLC-A) SCLC cell lines. Proliferation (FIG. ID) and soft agar colony formation (FIG. IE) assays in isogenic H82 and H146 cell lines with CDC7 KO. In FIG. ID, lower line graphs correspond to CDC7 KO (sgCDC7).
  • FIG. IF Ectopic overexpression of CDC7 in H69 (SCLC-A) and DMS114 (SCLC-Y) SCLC cell lines. Proliferation (FIG. 1G) and soft agar colony formation (FIG. 1H) assays in isogenic H69 and DMS114 cell lines with ectopic CDC7 overexpression.
  • FIG. 1G top line graphs correspond to CDC7 overexpression, p-value legend: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • FIG. II Bivariate correlation of CDC7 mRNA and protein expression with simurosertib or LY3143921 sensitivity (assessed as growth inhibition) in an array of SCLC cell lines. Correlation was calculated with Spearman’s test.
  • 1J Plot showing viability of control and CDC7 CRISPR-Cas9 KO cell lines using two different sgRNAs (sgl and sg2) after treatment with simurosertib GI50 for 5 days. Viability is normalized to the untreated condition for each experimental condition (control, sgl and sg2). Student’s t-test was performed to assess statistical significance (two-tailed, assuming heterogeneous value distribution) p-value legend: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • FIGs. 2A-2H demonstrate that CDC7 inhibition strongly sensitizes SCLC to chemotherapy.
  • FIG. 2B Proliferation assays of untreated and cisplatin- treated (GI20 concentration) H82 and H146 cell lines with endogenous CDC7 expression versus CDC7 KO.
  • FIG. 2B Proliferation assays of untreated and cisplatin- treated (GI20 concentration) H82 and H146 cell lines with endogenous CDC7 expression versus CDC7 KO.
  • FIG. 2C Synergy plots showing the occurrence of synergy (red), addition (white) or antagonism (green) of the different combinations of simurosertib and cisplatin or irinotecan, calculated with the HSA method using the SynergyFinder web application (2.0).
  • FIG. 2D CDC7 protein expression, shown as H-score, assessed by IHC in an array of SCLC PDXs derived from chemotherapy-naive and -treated tumors.
  • FIG. 2E Tumor growth curves of chemotherapy-naive SCLC PDXs with high (Lxl231), intermediate (Lx33) and low (Lx276) CDC7 protein expression treated with cisplatin, etoposide, simurosertib or their combinations.
  • FIG. 2F Tumor growth curves of SCLC PDXs derived from pre-treated tumors with high (Lx761c), intermediate (Lx674c) and low (Lx95) CDC7 protein expression treated with irinotecan, simurosertib or their combinations.
  • FIG. 2G Representative experiment for apoptosis (Annexin V/PI) assay of H82 and H146 SCLC cell lines treated with cisplatin (cis), simurosertib (simu) or their combination (combo).
  • FIG. 2H Mouse weight measurements of in vivo treatments shown in FIG. 3G.
  • FIGs. 3A-3J demonstrate that CDC7 is upregulated during NE transformation in prostate and lung tumors and its inhibition sensitizes NE-transformed tumors to chemotherapy.
  • CDC7 mRNA (FIG. 3A) and protein (FIG. 3B) expression in lung tumor clinical specimens categorized as control never transformed adenocarcinomas (LU AD), transforming adenocarcinomas (T-LUAD) and small cell carcinomas (T-SCLC) and control de novo small cell carcinomas (SCLC).
  • LU AD transforming adenocarcinomas
  • T-SCLC small cell carcinomas
  • SCLC control de novo small cell carcinomas
  • FIG. 3C H-score medians and standard deviation are shown.
  • FIG. 3C CDC7 mRNA expression in PRAD tumors with or without NE features. Data from Abida et al., PNAS 2019.
  • FIG. 3C CDC7 mRNA expression in PRAD tumors with or without NE features. Data from Abida e
  • FIG. 3D CDC7 protein expression in PRAD and NEPC clinical specimens, as assessed by IHC. H-score medians and standard deviation and representative images (FIG. 3E) are shown.
  • FIG. 3F In vitro synergy assays in Lx 1042 (T-SCLC) and H660 (NEPC) cell lines of the combination of simurosertib and cisplatin with average synergy score displayed, as assessed by ZIP and calculated using the SynergyFinder web application (2.0).
  • T-SCLC Lx 1042
  • NEPC H660
  • 3G In vivo treatment of Lxl042 (T-SCLC) and LuCAP49 (NEPC) PDXs was conducted to compare the efficacy of the combination of cisplatin and simurosertib versus that of cisplatin and etoposide. Student’s t-test was performed to assess statistical significance (two-tailed, assuming heterogeneous value distribution) p-value legend: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • FIG. 3H CDC7 mRNA expression in adenocarcinoma clinical specimens, categorized by their TP53/RB1 status.
  • FIG. 31 DNA accessibility ATACseq data from isogenic control and TP53/RBl-loss of function Hl 563 and 22PC isogenic cell lines. The transcription start site for the CDC7 gene is highlighted.
  • FIG. 3J Plot showing a representative biological replicate of an experiment assessing viability of control and TP53/RBl- ⁇ oss Hl 563 and 22PC cells treated with 0.5 pM simurosertib for 5 days. Student’s t-test was performed to assess statistical significance (two-tailed, assuming heterogeneous value distribution), p-value legend: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • FIGs. 4A-4C demonstrate that CDC7 upregulation after loss of TP53/RB 1 function induces sensitivity to simurosertib.
  • FIG. 4A CDC7 mRNA expression in adenocarcinoma clinical specimens, categorized by their TP53/RB1 status. Data obtained from LU AD TCGA (PanCancer), LU AD OncoSG (OncoSG, Nat Genetics 2020) and PRAD TCGA (PanCancer).
  • FIG. 4B Western blot showing CDC7 protein levels in isogenic Hl 563 (LU AD) and 22PC (PRAD) cell lines with or without induced loss of function of TP53 and/or RBI (see methods).
  • FIG. 4A CDC7 mRNA expression in adenocarcinoma clinical specimens, categorized by their TP53/RB1 status. Data obtained from LU AD TCGA (PanCancer), LU AD OncoSG (OncoSG, Nat Genetics 2020) and PRAD TCGA (PanCan
  • 4C Plot showing a representative biological replicate of an experiment assessing viability of control and TP53/RBl- ⁇ oss Hl 563, 22PC and LnCap (PRAD) cells treated with 0.5 pM simurosertib. Student’s t-test was performed to assess statistical significance (two-tailed, assuming heterogeneous value distribution), p-value legend: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • FIGs. 5A-5E demonstrate that CDC7 inhibition attenuated NE transformation in the prostate and lung.
  • FIG. 5A In vivo treatment of cell line xenografts for TP53/RB1- inactivated LnCap and 22PC cells with enzalutamide, simurosertib or their combination.
  • FIG. 5B Bar plot showing percentage of tumor classified as PRAD or NEPC, divided by treatment category, from (FIG. 5A) collected and endpoint for each of the groups. Average percentages and SEM is shown, per histology, per treatment group. Plots showing average and SEM protein expression, quantified as H-score, of NE markers (FIG. 5C) or AR (FIG.
  • FIG. 5D In vivo treatment of the EGFF-mutant combined NSCLC/SCLC MSK_Lxl51 PDX with osimertinib, simurosertib or their combination. Student’s t-test was performed to assess statistical significance (two-tailed, assuming heterogeneous value distribution) p-value legend: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • FIG. 5E In vivo treatment of the EGFF-mutant combined NSCLC/SCLC MSK_Lxl51 PDX with osimertinib, simurosertib or their combination. Student’s t-test was performed to assess statistical significance (two-tailed, assuming heterogeneous value distribution) p-value legend: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • FIG. 5E In vivo treatment of the EGFF-mutant combined NSCLC/SCLC MSK_Lxl51 PDX with osimertin
  • 5E In vivo treatment of the MSK_Lxl51 PDX with osimertinib, simurosertib or their combination. Student’s t-test was performed to assess statistical significance (two-tailed, assuming heterogeneous value distribution) p-value legend: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • FIGs. 6A-6E demonstrate that CDC7 inhibition attenuated NE transformation in the prostate and lung by activating the proteasome and inducing MYC degradation.
  • FIG. 6A Pathway enrichment analyses on differentially expressed genes between the combo- and enzalutamide-treated tumors, collected at an intermediate time point in the for TP53/RB1- inactivated LnCap and 22PC experiments shown in FIG. 5.
  • FIG. 6A Pathway enrichment analyses on differentially expressed genes between the combo- and enzalutamide-treated tumors, collected at an intermediate time point in the for TP53/RB1- inactivated LnCap and 22PC experiments shown in FIG. 5.
  • FIG. 6B Western blot showing MYC upregulation induced by targeted therapy (either enzalutamide or osimertinib), and MYC downregulation by simurosertib or the combination of targeted therapy and simurosertib in tumors collected at an intermediate time point from preclinical models treated in FIG. 5A and FIG. 5E.
  • FIG. 6C MYC mRNA levels in the tumors collected at endpoint in the prostate transformation models treated in FIG. 5A.
  • FIG 6D Barplot showing proteasome activity in control and CDC7-inhibited (either pharmacologically by simurosertib, or genetically by CDC7 CRISPR KO) prostate transformation models.
  • 6E Western blots showing neuroendocrine marker expression in prostate models of transformation, when treated with enzalutamide, simurosertib or their combination. Specifically, for each treatment condition, isogenic control and MYC T58A -expressing cells were analyzed.
  • the present disclosure demonstrates that (1) CDC7 inhibition delays NE transformation in lung and prostate adenocarcinoma at high risk of transformation when treated with targeted therapy, and (2) CDC7 inhibition robustly sensitizes NE-transformed lung and prostate tumors to chemotherapy.
  • the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • the term “adenocarcinoma” refers to cancer that forms in the glandular tissue, which lines certain internal organs and makes and releases substances in the body, such as mucus, digestive juices, and other fluids. Most cancers of the breast, lung, esophagus, stomach, colon, rectum, pancreas, prostate, and uterus are adenocarcinomas.
  • the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), intratumorally, or topically. Administration includes self-administration and the administration by another.
  • control is an alternative sample used in an experiment for comparison purpose.
  • a control can be "positive” or “negative.”
  • a positive control a compound or composition known to exhibit the desired therapeutic effect
  • a negative control a subject or a sample that does not receive the therapy or receives a placebo
  • the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein.
  • the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the therapeutic compositions may be administered to a subject having one or more signs or symptoms of lung or prostate adenocarcinomas.
  • a “therapeutically effective amount” of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.
  • expression includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • the terms “individual”, “patient”, or “subject” are used interchangeably and refer to an individual organism, a vertebrate, a mammal, or a human. In certain embodiments, the individual, patient or subject is a human.
  • prevention refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • a “sample” or “biological sample” refers to a body fluid or a tissue sample isolated from a subject.
  • a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like.
  • sample may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids.
  • Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art.
  • a blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.
  • the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
  • sequential therapeutic use refers to administration of at least two active ingredients at different times. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
  • the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
  • the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.
  • Treating”, “treat”, or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.
  • treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
  • the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.
  • the treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
  • the present disclosure provides a method for treating or preventing neuroendocrine tumor formation in a patient diagnosed with adenocarcinoma comprising administering to the patient an effective amount of a CDC7 inhibitor, wherein the adenocarcinoma exhibits reduced expression and/or activity of TP53 and RBI.
  • the adenocarcinoma comprises (a) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in TP53 and (b) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in RBI.
  • the present disclosure provides a method for preventing neuroendocrine tumor formation in a patient diagnosed with adenocarcinoma comprising administering to the patient an effective amount of a CDC7 inhibitor and an effective amount of at least one chemotherapeutic drug, wherein the adenocarcinoma exhibits reduced expression and/or activity of TP53 and RBI. Also provided herein is a method for enhancing responsiveness of a patient with neuroendocrine tumors to systemic chemotherapy comprising administering to the patient an effective amount of a CDC7 inhibitor and an effective amount of at least one chemotherapeutic drug, wherein the neuroendocrine tumors exhibit reduced expression and/or activity of TP53 and RBI.
  • the adenocarcinoma or the neuroendocrine tumors comprise(s) (a) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in TP53 and (b) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in RB 1.
  • the CDC7 inhibitor and the at least one chemotherapeutic drug are administered sequentially, simultaneously, or separately.
  • the at least one chemotherapeutic drug is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intratumorally, topically, by inhalation spray, buccally, or via an implanted reservoir.
  • the at least one chemotherapeutic drug may be an alkylating agent, a platinum agent, a taxane, a vinca agent, an aromatase inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine/hormonal agent, or a bisphosphonate therapy agent.
  • chemotherapeutic drugs include, but are not limited to cyclophosphamide, fluorouracil (or 5- fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, tykerb
  • the present disclosure provides a method for preventing neuroendocrine tumor formation in a patient diagnosed with adenocarcinoma comprising administering to the patient an effective amount of a CDC7 inhibitor and an effective amount of an androgen receptor (AR) inhibitor.
  • the adenocarcinoma may exhibit reduced expression and/or activity of TP53 and RBI.
  • the adenocarcinoma comprises (a) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in TP53 and (b) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in RBI.
  • the CDC7 inhibitor and the AR inhibitor are administered sequentially, simultaneously, or separately. Additionally or alternatively, in some embodiments, the AR inhibitor is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intratumorally, topically, by inhalation spray, buccally, or via an implanted reservoir.
  • AR inhibitors include, but are not limited to apalutamide, bicalutamide, darolutamide, enzalutamide, flutamide, abiraterone acetate, ARN-509, and nilutamide.
  • the present disclosure provides a method for preventing neuroendocrine tumor formation in a patient diagnosed with adenocarcinoma comprising administering to the patient an effective amount of a CDC7 inhibitor and an effective amount of an epidermal growth factor receptor (EGFR) inhibitor (EGFRi) (e.g., EGFR tyrosine kinase inhibitor (TKI), anti-EGFR antibodies).
  • EGFR epidermal growth factor receptor
  • EGFRi epidermal growth factor receptor
  • TKI EGFR tyrosine kinase inhibitor
  • the adenocarcinoma comprises (a) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in TP53 and (b) a genetic mutation, an indel, a copy number alteration or epigenetic downregulation in RB 1.
  • the CDC7 inhibitor and the EGFRi are administered sequentially, simultaneously, or separately.
  • the EGFRi is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intratumorally, topically, by inhalation spray, buccally, or via an implanted reservoir.
  • Examples of EGFRi s include, but are not limited to, osimertinib, afatinib, erlotinib, gefitinib, icotinib, dacomitinib, rociletinib, olmutinib, cetuximab, panitumumab, nimotuzumab, and necitumumab.
  • the patient is diagnosed with TP537’ and RB17" mutant adenocarcinoma.
  • the TP53' /' and RB17" mutant adenocarcinoma is lung adenocarcinoma or prostate adenocarcinoma.
  • the CDC7 inhibitor is administered orally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intratumorally, topically, by inhalation spray, buccally, or via an implanted reservoir.
  • Examples of CDC7 inhibitors include, but are not limited to simurosertib (TAK- 931), PHA-767491, carvedilol, dequalinium chloride, ticagrelor, and clofoctol.
  • the patient is human. Additionally or alternatively, in some embodiments, the patient is non-responsive to at least one prior line of cancer therapy such as chemotherapy.
  • the CDC7 inhibitor can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the AR inhibitor, EGFRi, or chemotherapeutic drug to the patient.
  • the CDC7 inhibitor and the AR inhibitor or EGFRi or chemotherapeutic drug are administered to a patient, for example, a mammal, such as a human, in a sequence and within a time interval such that the inhibitor or drug that is administered first acts together with the inhibitor or drug that is administered second to provide greater benefit than if each inhibitor were administered alone.
  • the CDC7 inhibitor and the AR inhibitor or EGFRi or chemotherapeutic drug can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, the CDC7 inhibitor and the AR inhibitor or EGFRi or chemotherapeutic drug are administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect of the combination of the two inhibitors.
  • the CDC7 inhibitor and the AR inhibitor or EGFRi or chemotherapeutic drug exert their effects at times which overlap.
  • the CDC7 inhibitor and the AR inhibitor or EGFRi or chemotherapeutic drug are each administered as separate dosage forms, in any appropriate form and by any suitable route.
  • the CDC7 inhibitor and the AR inhibitor or EGFRi or chemotherapeutic drug are administered simultaneously in a single dosage form.
  • any of these therapeutic agents can be administered once or more than once over a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 20 days, about 28 days, about a week, about 2 weeks, about 3 weeks, about 4 weeks, about a month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, about every year, about every 2 years, about every 3 years, about every 4 years, or about every 5 years.
  • the CDC7 inhibitor or the AR inhibitor or EGFRi or chemotherapeutic drug may be administered daily, weekly, biweekly, or monthly for a particular period of time.
  • the CDC7 inhibitor or the AR inhibitor or EGFRi or chemotherapeutic drug may be dosed daily over a 14 day time period, or twice daily over a seven day time period.
  • the CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug may be administered daily for 7 days.
  • a CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug may be administered daily, weekly, biweekly, or monthly for a particular period of time followed by a particular period of non-treatment.
  • the CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug can be administered daily for 14 days followed by seven days of non-treatment, and repeated for two more cycles of daily administration for 14 days followed by seven days of non-treatment.
  • the CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug can be administered twice daily for seven days followed by 14 days of non-treatment, which may be repeated for one or two more cycles of twice daily administration for seven days followed by 14 days of non-treatment.
  • the CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug is administered daily over a period of 14 days. In another embodiment, the CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug is administered daily over a period of 12 days, or 11 days, or 10 days, or nine days, or eight days. In another embodiment, the CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug is administered daily over a period of seven days. In another embodiment, the CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug is administered daily over a period of six days, or five days, or four days, or three days.
  • individual doses of the CDC7 inhibitor and the AR inhibitor or EGFRi or chemotherapeutic drug are administered within a time interval such that the two therapeutic agents can work together (e.g., within 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 1 week, or 2 weeks).
  • the treatment period during which the therapeutic agents are administered is then followed by a non-treatment period of a particular time duration, during which the therapeutic agents are not administered to the patient. This non-treatment period can then be followed by a series of subsequent treatment and non-treatment periods of the same or different frequencies for the same or different lengths of time.
  • the treatment and non-treatment periods are alternated. It will be understood that the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the treatment may be stopped. Alternatively, the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the period of treatment may continue for a particular number of cycles. In some embodiments, the length of the period of treatment may be a particular number of cycles, regardless of patient response. In some other embodiments, the length of the period of treatment may continue until the patient relapses.
  • the CDC7 inhibitor and the AR inhibitor or EGFRi or chemotherapeutic drug are each administered at a dose and schedule typically used for that agent during monotherapy.
  • one or both of the agents can advantageously be administered at a lower dose than typically administered when the agent is used during monotherapy, such that the dose falls below the threshold that an adverse side effect is elicited.
  • the therapeutically effective amounts or suitable dosages of the CDC7 inhibitor and the AR inhibitor or EGFRi or chemotherapeutic drug in combination depends upon a number of factors, including the nature of the severity of the condition to be treated, the particular inhibitor, the route of administration and the age, weight, general health, and response of the individual patient.
  • the suitable dose level is one that achieves a therapeutic response as measured by tumor regression or other standard measures of disease progression, progression free survival, or overall survival. In other embodiments, the suitable dose level is one that achieves this therapeutic response and also minimizes any side effects associated with the administration of the therapeutic agent.
  • Suitable daily dosages of AR inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of AR inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, the suitable dosages of AR inhibitors are from about 25% to about 90% of the maximum tolerated dose as a single agent. In some embodiments, the suitable dosages of AR inhibitors are from about 30% to about 80% of the maximum tolerated dose as a single agent. In other embodiments, the suitable dosages of AR inhibitors are from about 40% to about 75% of the maximum tolerated dose as a single agent.
  • the suitable dosages of AR inhibitors are from about 45% to about 60% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of AR inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
  • Suitable daily dosages of EGFRis can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of EGFRis are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, the suitable dosages of EGFRis are from about 25% to about 90% of the maximum tolerated dose as a single agent. In some embodiments, the suitable dosages of EGFRis are from about 30% to about 80% of the maximum tolerated dose as a single agent. In other embodiments, the suitable dosages of EGFRis are from about 40% to about 75% of the maximum tolerated dose as a single agent.
  • the suitable dosages of EGFRis are from about 45% to about 60% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of EGFRis are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
  • Suitable daily dosages of chemotherapeutic drugs can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of chemotherapeutic drugs are from about 20% to about 100% of the maximum tolerated dose as a single agent. In other embodiments, the suitable dosages of chemotherapeutic drugs are from about 25% to about 90% of the maximum tolerated dose as a single agent. In some embodiments, the suitable dosages of chemotherapeutic drugs are from about 30% to about 80% of the maximum tolerated dose as a single agent. In other embodiments, the suitable dosages of chemotherapeutic drugs are from about 40% to about 75% of the maximum tolerated dose as a single agent.
  • the suitable dosages of chemotherapeutic drugs are from about 45% to about 60% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of chemotherapeutic drugs are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
  • Suitable daily dosages of CDC7 inhibitors can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages of CDC7 inhibitors are from about 20% to about 100% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of CDC7 inhibitors are from about 25% to about 90% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of CDC7 inhibitors are from about 30% to about 80% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages of CDC7 inhibitors are from about 40% to about 75% of the maximum tolerated dose as a single agent.
  • the suitable dosages of CDC7 inhibitors are from about 45% to about 60% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages of CDC7 inhibitors are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
  • Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 z.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • an effective amount of the CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug may range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks.
  • a single dosage of CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.
  • An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • a therapeutically effective amount of a CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug may be defined as a concentration of the CDC7 inhibitor or AR inhibitor or EGFRi or chemotherapeutic drug at the target tissue of 10" 12 to 10' 6 molar, e.g., approximately 10' 7 molar.
  • This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area.
  • the schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).
  • the skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
  • the mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human.
  • compositions of the present technology can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others.
  • Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions.
  • Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • the compositions disclosed herein are formulated for administration to a mammal, such as a human.
  • Formulations including any CDC7 inhibitor disclosed herein may be designed to be shortacting, fast-releasing, or long-acting.
  • compounds can be administered in a local rather than systemic means, such as administration (e.g., by injection) at a tumor site.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.
  • the rate of compound release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and g
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner.
  • Examples of embedding compositions that can be used include polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form with one or more excipients as noted above.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents.
  • opacifying agents may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions include polymeric substances and waxes.
  • kits comprising one or more CDC7 inhibitors disclosed herein, and instructions for treating or preventing neuroendocrine tumor formation.
  • the kit may comprise a CDC7 inhibitor and an AR inhibitor or EGFRi or chemotherapeutic drug that has been formulated into a single pharmaceutical composition such as a tablet, or as separate pharmaceutical compositions.
  • the kit may comprise a CDC7 inhibitor and an AR inhibitor or EGFRi or chemotherapeutic drug that has been formulated as separate pharmaceutical compositions either in a single package, or in separate packages.
  • kits further comprise at least one AR inhibitor that are useful for treating or preventing neuroendocrine tumor formation.
  • AR inhibitors include, but are not limited to apalutamide, bicalutamide, darolutamide, enzalutamide, flutamide, abiraterone acetate, ARN-509, and nilutamide.
  • kits further comprise at least one EGFRi that are useful for treating treating or preventing neuroendocrine tumor formation.
  • EGFRis include, but are not limited to, osimertinib, afatinib, erlotinib, gefitinib, icotinib, dacomitinib, rociletinib, olmutinib, cetuximab, panitumumab, nimotuzumab, and necitumumab.
  • kits further comprise at least one chemotherapeutic agent that are useful for treating or preventing neuroendocrine tumor formation.
  • chemotherapeutic drugs include, but are not limited to cyclophosphamide, fluorouracil (or 5 -fluorouracil or 5-FU), methotrexate, edatrexate (10- ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, proteinbound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane
  • kits may further comprise pharm ceutically acceptable excipients, diluents, or carriers that are compatible with one or more kit components described herein.
  • the above described components of the kits of the present technology are packed in suitable containers and labeled for the treatment or prevention of neuroendocrine tumors.
  • the kits may optionally include instructions customarily included in commercial packages of therapeutic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • H1563 (CRL-5875), H660 (CRL-5813), H69 (HTB-119), H82 (HTB-175), SHP-
  • Cells were similarly spin-transduced as described in 17 with lentiviral particles made out of lentiviral LV03 vectors expressing sgRNAs for CDC7 (#HSPD0000047627 and HSPD0000047628, Sigma) or the respective control vector expressing a safe targeting sgRNA BFP (#HSCONTROL_AAVS1 on LV03, Sigma), or the Lvl51 vector overexpressing CDC7 (#EX-M0793-Lvl51 Genecopoeia).
  • 7P5.3/7787 -deficient LU AD cell lines were generated by lentiviral transduction of a construct expressing a dominant negative TP53 isoform and a short hairpin RNA against RBI, produced from the FU-CYW vector that was previously described 18 and kindly shared by Dr. Owen Witte.
  • Cytotoxic assays were performed as described in 19 . A total of 1,500 cells/well were seeded in 96-well plates and treated with the drugs/doses described for 96 hours. Viability was assessed with the CellTiter-Glo 2.0 Assay (Promega, G9242) as indicated by manufacturer.
  • Synergy assays [0086] Cells were seeded in 96-well plates (1500 cells/well) and treated with the interval of concentrations of cisplatin or simurosertib for 5 days. Then, cell viability was assessed with CellTiter-Glo 2.0 Assay (Promega, G9242) and normalized to the untreated wells. Synergy was calculated using the ZIP method using the SynergyFinder web application (2.O) 20 .
  • Protein extraction and western blot were performed as previously described 21 .
  • Antibodies for CDC7 (#3603, Cell Signaling Technology), MYC ( #5605, Cell Signaling Technology), synaptophysin (#36406, Cell Signaling Technology), CD56 (#99746, Cell Signaling Technology), AR (#5153, Cell Signaling Technology vinculin (#13901, Cell Signaling Technology), tubulin (#3873, Cell Signaling Technology) and actin (#3700, Cell Signaling Technology). Quantifications were performed with the Image Studio software (Version 3.1, Li-Cor).
  • Parental cells were treated with cisplatin (GI20), selinexor (GI20) or their combination for 3 days.
  • control and XP01 CRISPR-KO cells were treated with cisplatin (GI20) for 3 days.
  • cells were collected and stained with the APC Annexin V Apoptosis Detection Kit with PI (#640932, Biolegend) and apoptosis was analyzed by flow cytometry as previously described 22
  • CDC7 mRNA expression data from Cancer Cell Line Encyclopedia (CCLE) 11 was downloaded from UCSC Xenabrowser portal (https://xenabrowser. et/) in December 2020.
  • mice Female 6-week-old athymic nude mice (cell line xenografts) were engrafted per treatment arm and until tumors reached 100-150 mm 3 . At that point, mice were randomized into groups and treated with either vehicle, cisplatin (2 mg/kg i.p. once/week), etoposide (3 mg/kg i.p. QDx3), simurosertib (40 mg/kg p.o. QDx3), enzalutamide (10 mg/kg p.o. QDx5), osimertinib (25 mg/kg p.o.
  • Transcript abundances were quantified using RNA-seq reads by Salmon vl .1 ,0 23 Raw reads of RNA-seq were mapped to 25 mer indexed hg38 genome.
  • mapping validation validatemappings
  • bootstrapping with 30 re-samplings — numBootstraps
  • sequence specific biases correction — seqBias
  • coverage biases correction — posBias
  • GC biases correction — gcBias
  • RNAseq expression distribution of XPO1, SOX2 and CDC7 were presented in box plots for the above four groups of samples. RNAseq expression values were downloaded through cBioPortal.
  • ⁇ data type 1 The expression levels for LUSD (OncoSG, Nat Genet 2020) are in RSEM (RNAseq by Expectation-Maximization) that have been normalized using DESeq2 v.1.16.1 followed by log transformation while that for PRAD (TCGA, PanCancer) are in batch normalized RSEM then followed by log transformation.
  • RSEM z-score> Log-transformed mRNA expression z-scores compared to the expression distribution of all samples were downloaded for both LUSD (OncoSG, Nat Genet 2020) 26 and PRAD (TCGA, PanCancer) 27 . The pairwise comparisons of mean expressions were conducted among previously mentioned four groups and evaluated by Wilcoxon test.
  • RNAseq DEG Using traditional RNAseq DEG approach to evaluate DE p value by limma pipeline: linear modelling was applied on the normalized and log transformed RSEM values which are assumed to be normally distributed using limma (v3.28.14) 28 . The coefficients and standard errors were then estimated for each pair of contrast from the linear model. Empirical Bayes Statistics for differential expressions were carried out to evaluate the significance level.
  • XPO1 and SOX2 were correlated in scatter plots for previously mention seven cohorts. RNAseq expression values were downloaded through cBioPortal.
  • ⁇ data type 1 RSEM> The expression levels are in RSEM (RNAseq by Expectation-Maximization) that have been using DESeq2 v.1.16.1 normalization, LUSD (OncoSG, Nat Genet 2020) 26 , or batch normalized followed by log transformation.
  • RSEM z-score> Log-transformed mRNA expression z-scores compared to the expression distribution of all samples were downloaded. The expression correlations were evaluated by Pearson (Spearman).
  • GSEA 29 Gene set enrichment analysis 29 was conducted on the full sets of differential gene expression output from the previously mentioned comparisons. Genes were ranked by p value scores computed as -loglO(p value)*(sign of beta). The annotations of gene set were taken from Molecular Signatures Database (MSigDB v7.O.l) 29 ’ 30 of gene set enrichment was evaluated using permutation test and the p value was adjusted by Benjamini- Hochberg procedure. Any enriched gene sets with adjusted p value ⁇ 0.1 were regarded as significant. This analysis was conducted using ClusterProfiler R package v3.18.1 31 . Some enriched gene sets of interests were selected and their pathway annotations were concatenated manually to remove redundancy and achieve high level generality.
  • Enriched regions in individual samples were called using MACS2 34 and then filtered against genomic ‘blacklisted’ regions (http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/hg38- human/hg38.blacklist.bed.gz).
  • the filtered peaks within 500 bp were merged to create an union of peak atlas.
  • Raw read counts were tabulated over this peak atlas using featureCounts vl.6.0 35 .
  • the read counts were then normalized with DESeq2.
  • the read density profile in the format of bigwig file for each sample was created using the BEDTools suite (htp s : /7b edtool s . readth edocs .io) with the normalization factor from DESeq2 36 .
  • All bigwig genome tracks on XPO1 gene region were generated using pyGenomeTracks v3.5 37 .
  • Proteasome activity was measured with the Proteasome 20S Activity Assay Kit (#MAK172, Sigma), following the manufacturer’s instructions.
  • SCLCs are highly proliferative tumors exhibiting high dependency for cell cycle and DNA repair genes. Genes in these pathways are even upregulated during the process of adenocarcinoma to SCLC transformation 1,2 , highlighting their importance in the SCLC setting, and consistently, targeting genes in these pathways such as CDK7 3 or Chkl 4 has yielded promising preclinical efficacy against SCLC tumors.
  • the MCM complex involved in the initiation of DNA replication before cell division, has been previously involved in chemotherapy resistance in the SCLC setting 5 . This complex is activated by CDC7 6 , a gene previously involved in tumorigenesis in different tumor settings 7 .
  • CDC7 expression is induced by inactivation of TP53 and RBI -hallmarks of SCLC- in different tumor types, including lung cancer 8,9 . These results were suggestive of a potential pro-oncogenic role for this gene in the SCLC setting. Additionally, the recent development of potent and clinically safe inhibitors for CDC7 7,10 made it an attractive therapeutic target candidate for SCLC, and thus the role of CDC7 in this setting was evaluated.
  • Example 3 Inhibition of CDC7 strongly sensitizes SCLC PDXs to first- and second-line chemotherapy
  • CDC7 inhibition has shown to sensitize to an array of chemotherapeutic agents in a variety of tumor types 10 ; and the MCM complex, downstream CDC7, has been associated to chemotherapy resistance in SCLC 5 . Consistently, increased CDC7 protein expression was observed in SCLC tumors collected after prior chemotherapy treatment as compared to treatment-naive specimens (FIG. 2A). Thus, the therapeutic potential of CDC7 inhibition in SCLC was examined. Initially, the sensitivity of an array of SCLC cell lines belonging to all SCLC subtypes to two different CDC7 inhibitors, simurosertib and LY3143921, was assessed to confirm their specificity in the SCLC setting (FIG. II).
  • CDC7 KO induced a strong sensitivity to cisplatin in both cell lines (FIG. 2B), which was validated at the pharmacological level in apoptosis assays revealing increased apoptosis in cells treated with the combination of cisplatin and simurosertib than in cells treated with either inhibitor alone (FIG.
  • the treatment-naive PDXs (FIG. 2E) exhibited extraordinarily sensitivity to the combination of cisplatin and simurosertib, dramatically outperforming the combination of cisplatin and etoposide.
  • simurosertib was able to strongly sensitize either model to irinotecan, including 3/5 and 5/5 complete tumor regressions in the Lx761c and Lx674c, respectively.
  • the combination therapy showed superiority to irinotecan, including the low CDC7-expressing PDX model Lx95 (FIG.
  • Example 4 CDC7 is Unregulated during NE Transformation and CDC7 Inhibition Sensitizes NE-transformed Tumors to Chemotherapy
  • the simurosertib and cisplatin combination showed significantly superior efficacy as compared to that of cisplatin and etoposide combo, currently used in the treatment of NE-transformed lung and prostate tumors, with tumor growth inhibition (T/C) values of 35.98% versus 59.24% and 10.18% versus 50.85% for Lxl042 and LuCap49 PDX models, respectively, at control arm experimental endpoint (FIG. 3G).
  • the DKO LnCap xenografts showed resistance to enzalutamide (T/C value of 72.53% at control arm experimental endpoint, FIG. 5A). These tumors showed also limited sensitivity to simurosertib monotherapy (T/C values of 67.38% at control arm endpoint, FIG. 5A). However, the combination treatment showed dramatic efficacy, with a T/C value of 18.85% at control arm endpoint, and a significant delay in tumor relapse compared to either drug in monotherapy (31 days versus 73 days for enzalutamide- and combo-treated tumors, respectively, FIG. 5A).
  • a PDX derived from a combined /T/7’7?-mutant NSCLC/SCLC tumor retaining both NSCLC and SCLC components (MSK_Lxl51) mimicking an intermediate state of transformation was leveraged.
  • Treatment of this PDX model with osimertinib yielded limited efficacy, with a T/C value of 73.30% at control arm experimental endpoint.
  • simurosertib monotherapy showed increased efficacy (T/C of 38.86%, FIG. 5E), but the combination outperformed any other treatment condition under assay, with a T/C value of 20.49%.
  • Example 6 CPC 7 Inhibition Attenuated NE Transformation in the Prostate and Lung by Activating the Proteasome and Inducing MYC Degradation
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
  • TAK-931 a novel CDC7-selective inhibitor. Science Advances 5, (2019). Datta, A. et al. p53 gain-of-function mutations increase Cdc7-dependent replication initiation. EMBO reports 18, 2030-2050 (2017). Sasi, N. K., Bhutkar, A., Lanning, N. J., MacKeigan, J. P. & Weinreich, M. DDK Promotes Tumor Chemoresistance and Survival via Multiple Pathways. Neoplasia (United States) 19, 439-450 (2017). Iwai, K. et al.
  • a CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery. Science Advances 7, 1-15 (2021). Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603-607 (2012). Abida, W. et al. Genomic correlates of clinical outcome in advanced prostate cancer. Proc Natl Acad Sci USA 166, 11428-11436 (2019). Park, C. K., Oh, I. J. & Kim, Y. C. Is transformed small cell lung cancer (SCLC) different from de novo SCLC? Transl Cancer Res 8, 346-349 (2019). Niederst, M. J. et al.
  • SynergyFinder 2.0 Visual analytics of multi-drug combination synergies. Nucleic Acids Res 48, W488-W493 (2021). Gardner, E. E. et al. Chemosensitive Relapse in Small Cell Lung Cancer Proceeds through an EZH2-SLFN11 Axis. Cancer Cell 31, 286-299 (2017). Shrestha, C. L. et al. Cysteamine-mediated clearance of antibiotic-resistant pathogens in human cystic fibrosis macrophages. PLoS ONE 12, 1-17 (2017). Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression.

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Abstract

La présente divulgation concerne des méthodes de traitement ou de prévention de la formation d'une tumeur neuroendocrine chez des sujets diagnostiqués avec des adénocarcinomes déficients en RBI et en TP53 (par exemple, des adénocarcinomes du poumon ou de la prostate) à l'aide d'inhibiteurs de CDC7. La divulgation concerne également des procédés de prévention de la formation d'une tumeur neuroendocrine chez des sujets chez lesquels on a diagnostiqué des adénocarcinomes (par exemple, des adénocarcinomes du poumon ou de la prostate déficients en TP53 et en RBI) à l'aide d'inhibiteurs de CDC7 en combinaison avec des inhibiteurs du récepteur des androgènes (AR), des inhibiteurs du récepteur du facteur de croissance épidermique (EGFR), ou des médicaments chimiothérapeutiques.
PCT/US2023/076996 2022-10-17 2023-10-16 Procédés de traitement ou de prévention de la formation d'une tumeur neuroendocrine à l'aide d'inhibiteurs de cdc7 Ceased WO2024086533A2 (fr)

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