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
  • A61K31/395—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
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • 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
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
• • 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
  • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
• • 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
  • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
• • 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
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • 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
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
• • 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
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• Cancer cell quiescence effectively a cell in a state of sleep, has been recognized recently as a major mechanism of the resistance of cancer cells to treatments and for providing a pathway for disease recurrence.
• This quiescence alternatively called cellular dormancy, is due to arrest at Go phase of the cell cycle.
• a cell enters a cell cycle from gap phase 1 (Gi), as shown in FIG. 1.
• Gi gap phase 1
• S synthesis phase
• G 2 short premitotic interval
• the cell divides by mitosis (M) followed by a return to Gi.
• Gi mitosis
• a cell can enter cellular dormancy or quiescence, designated as the Go phase.
• Cancer cells can either enter an irreversible state before undergoing terminal differentiation, termed senescence, or enter a reversible, true quiescent Go state from which they could resume cycling, like quiescent fibroblasts (Coller HA, Sang L, and Roberts JM (2006) A new description of cellular quiescence, PLoS Biology 4, e83).
• a population of cells naturally may be in a quiescent state at any given time and remain quiescent for an indeterminate period until receipt of a signal to enter the cell division cycle.
• the proportion of cancer cells in quiescent state within a population in a tumor may be increased by environmental factors, such as lack of nutrients, hypoxia, high concentration of reactive oxygen species, etc.
• Cells may also be induced into the quiescent state by the action of a drug substance, as in pharmacological quiescence.
• a quiescent cancer cell is resistant to treatments that affect one of more cellular proliferation processes by means of damaging exposed DNA, interfering with DNA replication or repair, interfering with mitosis, or other mechanisms.
• the present invention provides methods and combinations for the treatment of neoplasms that features the targeting of quiescent cancers cells by small molecules, particularly molecules effective against quiescent cancer cells, in combination with treatments known to be effective against certain neoplastic cells.
• the present invention provides compositions and methods for the treatment of neoplasms, in particular, by targeting of quiescent cancer cells with therapeutic agents in combination with other treatments effective against certain neoplastic conditions, in particular, anti-cancer treatment with therapeutic agents that are inhibitors of mitosis (a mitotic inhibitor).
• the invention features a method of treating a neoplasm comprising:
• the neoplasm is a cancer or a population of cancer cells in vitro or in vivo.
• the subject receiving the treatment is diagnosed with cancer (e.g., metastatic or pre-metastatic).
• the subject has been previously treated with a first-line therapy against cancer.
• the subject is treated, or has been treated, with two or more inhibitors of mitosis sequentially or concomitantly.
• the combined treatment may result in improved outcomes, such as increased survival, reduction of severity, delay or elimination of recurrence, or reduced side effects of the primary treatments (i.e., the inhibitor of mitosis).
• the second agent is administered at lower dose and/or for a shorter duration when administered as part of the combination as compared to a treatment with the agent alone.
• the EC50 value of the inhibitor of mitosis is at least 20% lower in the combination treatment when compared to the same treatment with the inhibitor of mitosis alone, as determined, for example, in cell-based assays.
• the combination treatment increases fraction of apoptotic cells in a treated population as compared to either agent alone, by at least by 2-fold as determined, for example, by fraction of sub-Go cells by FACS assay.
• the therapeutic agent effective against quiescent cancer cells is a DYRKl inhibitor.
• the DYRKl inhibitor is a compound that inhibits activity of a DYRKl kinase, either DYRKIA or DYRKIB (in vitro or in vivo), for example, with an IC50 of 100 nM or lower in biochemical assays.
• the DYRKl inhibitor reduces the fraction of quiescent cancer cells (in vitro or in vivo) that would otherwise be found in the absence of such inhibitor, for example, by at least 10%.
• the DYRKl inhibitor inhibits both DYRKIA and DYRKIB.
• the DYRKl inhibitor is selective for DYRKIA or DYRKIB.
• the therapeutic agent effective against quiescent cancer cells is a DYRKl inhibitor.
• the DYRKl inhibitor is a compound of formula I:
• Ri is a substituted or unsubstituted Ci-s alkyl, a substituted or unsubstituted phenyl, or a substituted or unsubstituted benzyl;
• R2 is phenyl, optionally substituted with up to four groups independently selected from halo, CN, NO2, NHC(0)Ci- 4 alkyl, Ci- 4 alkyl, OH, OCi- 4 alkyl, wherein two adjacent groups and their intervening carbon atoms may form a 5- to 6-membered ring containing one or more heteroatoms selected from N, O, or S.
• the compound of formula I is selected from:
• the methods of the invention further provide (c)
• administering to the subject another cancer therapy, for example, radiation therapy or other cancer treatment.
• another cancer therapy for example, radiation therapy or other cancer treatment.
• the methods of the invention comprise: administering to a subject in need thereof a therapeutically effective amount of (a) a therapeutic agent of formula I; (b) an inhibitor of mitosis; and (c) radiation therapy; each therapy being administered sequentially or concomitantly.
• the subject is first treated with radiation therapy, whereupon the subject is administered a therapeutic agent of Formula I, alone or in combination with the inhibitor of mitosis.
• the subject is co-administered (a) the therapeutic agent effective against quiescent cancer cells, (b) the inhibitor of mitosis and, optionally, (c) the radiation therapy.
• the inhibitor of mitosis is an inhibitor of mitosis effective to treat or prevent a neoplasm, including but not limited to, all such compounds approved for the treatment of cancer and compounds that otherwise demonstrate efficacy in treating cancer in mammalian subject (e.g., mice, rats, dogs, monkeys, humans), and compounds that demonstrate efficacy against neoplastic cells in vitro. Many such compounds are known.
• the inhibitor of mitosis is a taxane.
• the taxane inhibitor of mitosis is, for example, BMS- 188796, BMS- 188797, cabazitaxel, DEP cabazitaxel, docetaxel, larotaxel (XRP9881, RPR109881), paclitaxel, taxoprexin (DHA- paclitaxel), and tesetaxel (DJ-927).
• the inhibitor of mitosis is a vinca alkaloid.
• the vinca alkaloid inhibitor of mitosis is, for example, vinblastine, vincristine, vindesine, vinflunine, and vinorelbine.
• the vinca alkaloid inhibitor of mitosis is vintafolide.
• the inhibitor of mitosis is a PLK1 inhibitor.
• the PLK1 inhibitor of mitosis is, for example, BI-2536, GSK 461364,
• the inhibitor of mitosis is BI-2536 or
• the neoplasm being treated is a cancer, for example, biliary cancer, brain cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, kidney cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer (e.g. melanoma), testicular cancer, thyroid cancer, or uterine cancer.
• the neoplasm being treated is a bladder cancer, breast cancer, colorectal cancer, non- small cell lung cancer, small cell lung cancer, ovarian cancer, and prostate cancer.
• the cancer is primary or metastatic.
• the cancer is of the type represented by the cell line types shown in the Examples.
• the embodiments are not meant to be limiting with regard to additional combination components, especially therapeutic agents and inhibitors that are part of existing treatment combinations, such as, for example, TPF wherein T stands for Taxotere ® , that is docetaxel, or PCV wherein V stands for vincristine sulfate.
• TPF Taxotere ®
• PCV vincristine sulfate
• the embodiments are not meant to be limiting with regard to routes and order of administration or with regard to patient type (previously untreated or previously treated, absence or presence of co-morbid conditions, sex, etc.) or stage of patient's disease, type of inhibitor of mitosis, etc.
• Figure 1 shows a schematic diagram of a mitotic cycle of a eukaryotic cell.
• Figure 2 shows a schematic diagram of a mitotic cycle of a eukaryotic cancer cell annotated to indicate the stages of the cell cycle upon which some of the available anti-cancer therapeutic agents are believed act.
• Figure 3 shows effect of combination of paclitaxel and Compound 1-5 (0, 2, and 4 ⁇ ) on the growth of SW620 cells.
• Figure 4 shows effect of combination of paclitaxel and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of DMS273 cells.
• Figure 5 shows effect of combination of paclitaxel and Compound 1-5 (0, 2, and 4 ⁇ ) on the growth of LNCap cells.
• Figure 6 shows effect of combination of paclitaxel and Compound 1-7 (0, 3, and 6 ⁇ ) on the growth of HCC827 cells.
• Figure 7 shows effect of combination of paclitaxel and Compound 1-7 (0, 3, and 6 ⁇ ) on the growth of A549 cells.
• Figure 8 shows effect of combination of paclitaxel and Compound 1-7 (0, 2, 4, 8, and 10 ⁇ ) on the growth of SK-OV-3 cells.
• Figure 9 shows effect of combination of paclitaxel and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of OVCAR3 cells.
• Figure 10 shows effect of combination of docetaxel and Compound 1-5 (0, 2, and 4 ⁇ ) on the growth of SW620 cells.
• Figure 11 shows effect of combination of docetaxel and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of DMS273 cells.
• Figure 12 shows effect of combination of docetaxel and Compound 1-7 (0, 3, and 6 ⁇ ) on the growth of HCC827 cells.
• Figure 13 shows effect of combination of docetaxel and Compound 1-7 (0, 3, and 6 ⁇ ) on the growth of A549 cells.
• Figure 14 shows effect of combination of docetaxel and Compound 1-7 (0, 4, and 8 ⁇ ) on the growth of SK-OV-3 cells.
• Figure 15 shows effect of combination of docetaxel and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of OVCAR3 cells.
• Figure 16 effect of combination of vincristine and Compound 1-5 (0, 2, and 4 ⁇ ) on the growth of DMS273 cells.
• Figure 17 shows effect of combination of vincristine and Compound 1-5 (0, 2, and 4 ⁇ ) on the growth of HI 975 cells.
• Figure 18 shows effect of combination of vincristine and Compound 1-7 (0, 4, and 8 ⁇ ) on the growth of SK-OV-3 cells.
• Figure 19 shows effect of combination of vincristine and Compound 1-7 (0, 1, and 3 ⁇ ) on the growth of OVCAR3 cells.
• Figure 20 shows effect of combination of vinorelbine and Compound 1-5 (0, 2, and 4 ⁇ ) on the growth of DMS273 cells.
• Figure 21 shows effect of combination of vinorelbine and Compound 1-5 (0, 2, and 4 ⁇ ) on the growth of HI 975 cells.
• Figure 22 shows effect of combination of vinorelbine and Compound 1-7 (0, 1, and 3 ⁇ ) on the growth of OVCAR3 cells.
• Figure 23 shows effect of combination of vincristine and Compound 1-7 (0, 4, and 8 ⁇ ) on the growth of SK-OV-3 cells.
• Figure 24 shows effect of combination of vincristine and Compound 1-7 (0, 3, and 6 ⁇ ) on the growth of A549 cells.
• Figure 25 shows effect of combination of BI2536 and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of HI 975 cells.
• Figure 26 shows effect of combination of BI2536 and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of PANC1 cells.
• Figure 27 shows effect of combination of BI2536 and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of DMS273 cells.
• Figure 28 shows effect of combination of BI2536 and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of A549 cells.
• Figure 29 shows effect of combination of GSK461364 and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of HI 975 cells.
• Figure 30 shows effect of combination of GSK461364 and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of PANC1 cells.
• Figure 31 shows effect of combination of GSK461364 and Compound 1-7 (0, 2, and 4 ⁇ ) on the growth of DMS273 cells.
• Figure 32 shows effect of combination of GSK461364 and Compound 1-7 (0, 3, and 6 ⁇ ) on the growth of A549 cells.
• Figure 33 shows FACS analyses of cell cycle distribution of DMS273 cells incubated for 24 hours in Panel A: FBS+ media; Panel B: FBS- media; Panel C: FBS+ media with 4 ⁇ Compound 1-7; Panel D: FBS+ media with 2.7 nM paclitaxel; Panel E: FBS+ media with 5 ⁇ Compound 1-7 and 2 nM paclitaxel.
• Figure 34 shows FACS analyses of cell cycle distribution of SW620 cells incubated for 24 hours in Panel A: FBS+ media; Panel B: FBS- media; Panel C: FBS+ media with 4 ⁇ Compound 1-7; Panel D: FBS+ media with 2 nM paclitaxel; Panel E: FBS+ media with 4 ⁇ Compound 1-7 and 2 nM paclitaxel.
• Figure 35 shows FACS analyses of cell cycle distribution of DMS273 cells incubated for 24 hours in Panel A: FBS+ media; Panel B: FBS- media; Panel C: FBS+ media with 4 ⁇ Compound 1-7; Panel D: FBS+ media with 1.1 nM vincristine; Panel E: FBS+ media with 4 ⁇ Compound 1-7 and 1.1 nM vincristine.
• Figure 36 shows FACS analyses of cell cycle distribution of DMS273 cells incubated for 24 hours in Panel A: FBS+ media; Panel B: FBS- media; Panel C: FBS+ media with 4 ⁇ Compound 1-7; Panel D: FBS+ media with 8.1 nM vinorelbine; Panel E: FBS+ media with 4 ⁇ Compound 1-7 and 8.1 nM vinorelbine.
• Figure 37 FACS analyses by DNA content of cell cycle distribution of SW620 cells. The cells were incubated in Panel A: 24 hours in FBS- media; Panel B: 24 hours in FBS- media with 2.5 ⁇ AZ191; Panel C: 24 hours in FBS- media with 5 ⁇ AZ191; Panel D: 24 hours in FBS- media with 10 ⁇ AZ191.
• Figure 38 shows FACS analyses by DNA content of cell cycle distribution of SW620 cells.
• the cells were incubated in Panel A: 24 hours in FBS- media with DMSO control; Panel B: 24 hours in FBS- media with 1.25 ⁇ Compound 1-7; Panel C: 24 hours in FBS- media 2.5 ⁇ Compound 1-7; Panel D: 24 hours in FBS- media with 5 ⁇ Compound 1-7.
• Panel A 24 hours in FBS- media with DMSO control
• Panel B 24 hours in FBS- media with 1.25 ⁇ Compound 1-7
• Panel C 24 hours in FBS- media 2.5 ⁇ Compound 1-7
• Panel D 24 hours in FBS- media with 5 ⁇ Compound 1-7.
• an "alkyl” group is a saturated, straight or branched, hydrocarbon group, comprising from 1 to 8 carbon atoms (C 1-8 alkyl group), in particular from 1 to 6, or from 1 to 4 carbons atoms, unless otherwise indicated.
• alkyl groups having from 1 to 6 carbon atoms inclusive are methyl, ethyl, propyl (e.g., n-propyl, iso-propyl), butyl (e.g., tert-butyl, sec-butyl, n-butyl), pentyl (e.g., neo-pentyl), hexyl (e.g., n- hexyl), 2-methylbutyl, 2-methylpentyl and the other isomeric forms thereof.
• propyl e.g., n-propyl, iso-propyl
• butyl e.g., tert-butyl, sec-butyl, n-butyl
• pentyl e.g., neo-pentyl
• hexyl e.g., n- hexyl
• 2-methylbutyl 2-methylpentyl and the other isomeric
• Alkyl groups may be unsubstituted or substituted by at least one group chosen from halogen atoms, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxyl, alkenyl, alkynyl, CN, nitro, and amino groups.
• an "alkenyl” group is a straight or branched hydrocarbon group comprising at least one double carbon-carbon bond, comprising from 2 to 8 carbon atoms (unless otherwise indicated).
• alkenyl groups containing from 2 to 6 carbon atoms are vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1- pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5- hexenyl and the isomeric forms thereof.
• Alkenyl groups may be unsubstituted, or substituted by at least one group chosen from halogen atoms, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxyl, alkenyl, alkynyl, CN, nitro, and amino groups.
• an "alkynyl” group is a straight or branched hydrocarbon group comprising at least one triple carbon-carbon bond, comprising from 2 to 8 carbon atoms.
• Alkynyl groups may be substituted by at least one group chosen from halogen atoms, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxyl, alkenyl, alkynyl, CN, nitro, and amino groups.
• an "aryl” group is an aromatic hydrocarbon cycle, comprising from 5 to 14 carbon atoms. Most preferred aryl groups are mono- or bi-cyclic and comprises from 6 to 14 carbon atoms, such as phenyl, alpha-naphtyl, 3-naphtyl, antracenyl, preferably phenyl. "Aryl” groups also include bicycles or tricycles comprising an aryl cycle fused to at least another aryl, heteroaryl, cycloalkyl or heterocycloalkyl group, such as benzodioxolane, benzodioxane, dihydrobenzofurane or benzimidazole.
• Aryl groups may be unsubstituted, or substituted by at least one (e.g. 1, 2 or 3) group chosen from halogen atoms, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxyl, alkenyl, alkynyl, CN, nitro, and amino groups.
• aryl groups may be substituted by adjacent substituents which can, taken together with the carbon atom to which they are attached, form a 5- to 6- membered ring which may contain one or more heteroatom(s) selected from N, O, and S.
• halogen atom or "halo” is a CI, Br, F, or I atom.
• an "alkoxyl” group is an alkyl group linked to the rest of the molecule through an oxygen atom, of the formula O-alkyl.
• an "amino" group is a NH 2 , NH-alkyl, or N(alkyl) 2 group.
• a "heteroaryl” group is an aryl group whose cycle is interrupted by at least at least one heteroatom, for example a N, O, or S, atom, such as thiophene or pyridine.
• Heteroaryl groups may be unsubstituted, or substituted by at least one (e.g. 1, 2 or 3) group chosen from halogen atoms, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxyl, alkenyl, alkynyl, CN, nitro and amino groups.
• heteroaryl groups may be substituted by adjacent substituents which can, taken together with the carbon atom to which they are attached, form a 5- to 6-membered ring which may contain one or more heteroatom(s) selected from N, O, and S.
• a "cycloalkyl” denotes a saturated alkyl group that forms one cycle having preferably from 3 to 14 carbon atoms, and more preferably 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
• Cycloalkyl groups may be unsubstituted or substituted by at least one (e.g. 1, 2 or 3) group chosen from halogen atoms, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxyl, alkenyl, alkynyl, CN, nitro, and amino groups.
• cycloalkyl groups may be substituted by adjacent substituents which can, taken together with the carbon atom to which they are attached, form a 5- to 6-membered ring which may contain one or more
• heteroatom(s) selected from N, O, and S.
• a "heterocycloalkyl” group is a cycloalkyl group comprising at least one heteroatom, such as pyrrolidine, tetrahydrothiophene, tetrahydrofuran, piperidine, pyran, dioxin, morpholine or piperazine.
• a heterocycloalkyl group may in particular comprise from four to fourteen carbon atoms, such as morpholinyl, piperidinyl, pyrrolidinyl, tetrahydropyranyl, dithiolanyl.
• Heterocycloalkyl groups may be unsubstituted, or substituted by at least one group chosen from halogen atoms, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxyl, alkenyl, alkynyl, CN, nitro and amino groups.
• heterocycloalkyl groups may be substituted by adjacent substituents which can, taken together with the carbon atom to which they are attached, form a 5- to 6-membered ring which may contain one or more heteroatom(s) selected from N, O, and S.
• a neoplasm means an abnormal mass of tissue that results from neoplasia.
• “Neoplasia” means a process of an abnormal proliferation of cells.
• a neoplasm is a solid cancer, or alternately a hematopoietic cancer.
• the neoplasia may be benign, pre-malignant, or malignant.
• neoplasm encompasses mammalian cancers, in some embodiments, human cancers, and carcinomas, sarcomas, blastomas of any tissue (for example adenocarcinomas, squamous cell carcinoma, osteosarcomas, etc.), germ cell tumors, glial cell tumors, lymphomas, leukemias, including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, rectal, pancreatic, stomach, brain, head and neck, skin, uterine, cervical, testicular, esophagus, thyroid, biliary cancer, liver cancer, and cancer of the bone and cartilaginous tissue, including non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia, multiple myeloma, and myelodysplastic syndrome.
• non-Hodgkin's lymphomas
• the terms “treat,” “treating,” or “treatment,” mean to counteract a medical condition (e.g. , cancer) to the extent that the medical condition is improved according to a clinically-acceptable standard. Improvement in cancer can include: 1) reduced rate of tumor growth (tumor growth inhibition), 2) tumor shrinkage (regression), 3) remission, whether partial or total, 4) reduction in metastases, 5) prolonging progression free survival, and 6) delay or elimination of recurrence.
• treating includes achieving, partially or substantially, one or more of the following results: partially or totally reducing the cancer mass, or volume, or the malignant cell count;
• Treatment also can mean prolonging survival compared to expected survival without treatment or compared to standard of care treatment.
• Treating includes prophylactic or preventative treatment.
• “Prophylactic treatment” refers to treatment before appearance or re-appearance of clinical symptoms of a target disorder to prevent, inhibit, or reduce its occurrence, severity, or progression.
• an "effective amount” refers to an amount of a therapeutic agent or a combination of therapeutic agents that is therapeutically or prophylactic ally sufficient to affect the desired improvement in the targeted disorder.
• effective amounts typically range from about 0.0001 mg/ kg of body weight to about 500 mg/kg of body weight per single administered dose, such doses being administered once or over a period of time.
• An example range is from about 0.0001 mg/ kg of body weight to about 5 mg/kg per dose. In other examples, the range can be from about 0.0001 mg/kg to about 5 mg/kg per single administered dose.
• effective amounts range from about 0.01 mg/ kg of body weight to 50 mg/kg of body weight per single administered dose, or from 0.01 mg/kg of body weight to 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, or 40 mg/kg of body weight per single administered dose.
• an example of an effective dose is that amount approved of by a regulatory agency for treatment of an indication.
• the term "subject” refers to a mammal, for example a human, but can also mean an animal in need of veterinary treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like), and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
• companion animals e.g., dogs, cats, and the like
• farm animals e.g., cows, sheep, pigs, horses, and the like
• laboratory animals e.g., rats, mice, guinea pigs, and the like.
• the term "therapeutic agent” means any chemical molecule used or contemplated for use or investigated for use in cancer treatment, including cytotoxic, cytostatic, or targeted agents, whether small molecules, or peptides, or antibodies, or oligonucleotides, irrespective of mechanism of action.
• cytotoxic, cytostatic, or targeted agents whether small molecules, or peptides, or antibodies, or oligonucleotides, irrespective of mechanism of action.
• terapéutica refers to either the active pharmaceutical ingredient (API) or its pharmaceutically acceptable salt or hydrate (solvate), or a drug product containing the therapeutic agent, however formulated, and whether API is amorphous or crystalline and of whatever polymorphic form.
• Formulation means a combination of an active pharmaceutical ingredient (API, drug substance) or ingredients (APIs) combined with excipients and/or delivery vehicle to make an administrable dosage form (drug product).
• paclitaxel includes Taxol ® , Abraxane ® , Lipusu ® , and any other drug product with paclitaxel as the active ingredient.
• the therapeutic agents of the invention can be administered alone, but are generally administered with a pharmaceutically acceptable carrier, with respect to standard
• compositions defined herein and pharmaceutically acceptable carriers.
• inhibitor means any composition that reduces the activity of an enzyme.
• An example of an inhibitor is a chemical molecule.
• a measure of the potency of an inhibitor is its "50% inhibitory concentration" (IC50).
• IC50 concentration or IC50 value is the concentration of an inhibitor at which 50% of the enzymatic activity is inhibited by the inhibitor.
• Methods for the determination of IC50 values, for example, of kinase inhibitors are known to persons of ordinary skill in the art and include direct and indirect functional assays, such as the HotSpotTM kinase assay technology (Reaction Biology Corporation, Malvern, PA, www.reactionbiology.com) or competition binding assays, such as KINOMEscan ®
• EC 50 50% effective concentration
• EC50 concentration or EC50 value is the concentration of a drug that produces half-maximal response, such as, for example, 50% growth inhibition or 50% reduction in cell viability.
• the term "quiescence” or “quiescent state” refers to the Go state of the cell cycle, as understood by the practitioners of the art.
• the term "therapeutic agent effective against quiescent cancer cells” refers to a molecule that either reduces the fraction of quiescent cancer cells in a cell population or prevents, completely or substantially, an increase in fraction of quiescent cancer cells in a cell population under conditions that otherwise would lead to such an increase.
• a “quiescent neoplastic cell”, alternately referred to as a “quiescent cancer cell” means a cancer cell that exists in the quiescent, or Go, state of the cell cycle.
• a “fraction of quiescent neoplastic cells” or “fraction of quiescent cancer cells”, as used herein, means the portion of a cancer cell population that exists in the Go state of the cell cycle. Determining the fraction of quiescent neoplastic cells includes characterizing a cell population by distribution of its constituent cells within the stages of the cell cycle. The fraction of cells in the Go state (i.e., quiescent neoplastic cells) is quantified relative to the total cell population. The fraction may be expressed as a percentage of the total cell population (i.e.
• Characterization of the cell population by distribution of its constituent cells within the stages of the cell cycle may be achieved by techniques known to persons of ordinary skill in the art, and may include analysis by DNA and/or RNA content using flow cytometry methods, for example, fluorescence-activated cell sorting (FACS). Detailed description
• the present invention provides compositions and methods for the treatment of neoplasms, in particular, by targeting of quiescent cancers cells with therapeutic agents in combination with other treatments effective against certain neoplastic conditions, in particular, anti-cancer treatment with therapeutic agents which are inhibitors of mitosis.
• the invention features a method of treating a neoplasm comprising:
• the neoplasm is a cancer or a population of cancer cells in vitro or in vivo.
• the subject receiving the treatment is diagnosed with cancer (e.g., metastatic or pre-metastatic).
• the subject has been treated previously with a first-line therapy against cancer.
• the subject has been treated previously with second-line and/or other therapies.
• the subject is treated, or has been treated, with radiation therapy.
• the subject was treated with surgery, for example to resect or de-bulk a tumor.
• the subject's neoplasm has recurred.
• the subject is treated, or has been treated, with two or more inhibitor of mitosis sequentially or concomitantly.
• the combined treatment may result in improved outcomes, such as increased survival, reduction of severity, delay or elimination of recurrence, or reduced side effects of the primary treatments (i.e., the inhibitor of mitosis).
• the second agent is administered at lower dose and/or for a shorter duration when administered as part of the combination as compared to a treatment with the agent alone.
• the EC50 value of the inhibitor of mitosis is at least 20%, 25%, 30%, 40%, 50%, 100%, 3-fold, 5-fold, 10-fold lower in the combination treatment when compared to the same treatment with the first agent, as determined, for example, in cell-based assays.
• the combination treatment increases fraction of apoptotic cells in a treated population as compared to either agent alone, by at least by 2-fold, 3-fold, 4-fold, 5-fold as determined, for example, by fraction of sub-Go phase cells in a FACS assay.
• the therapeutic agent effective against quiescent cancer cells is a DYRK1 inhibitor.
• the DYRK1 inhibitor is a compound that inhibits activity of a DYRKl kinase, either DYRK1A or DYRK1B (in vitro or in vivo), for example, with an IC50 value of ⁇ 100 nM, ⁇ 90 nM, ⁇ 80 nM, ⁇ 70 nM, ⁇ 60 nM, ⁇ 50 nM, ⁇ 40 nM, ⁇ 30 nM, ⁇ 20 nM, ⁇ 10 nM, ⁇ 5 nM or lower in biochemical assays.
• the DYRKl inhibitor reduces the fraction of quiescent cancer cells (in vitro or in vivo) in a population or a tumor that would otherwise be found in the absence of such inhibitor, for example, by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more.
• the DYRKl inhibitor inhibits both DYRKl A and DYRK1B. In some embodiments, the DYRKl inhibitor is selective for DYRK1A, with ratio of
• the DYRKl inhibitor is selective for DYRK1B, with ratio of DYRKl A IC50 to DYRK1B IC50 of 1000, 100, 50, 25, 10, or 5 to 1.
• the DYRKl inhibitor is selective for DYRKl by at least 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold as compared to DYRK2 and/or DYRK3 and/or DYRK4, as determined by ratios of IC50 values.
• the DYRKl inhibitor is selective for DYRKl by at least 4-fold, 5-fold, 10- fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold as compared to cyclin dependent kinases (CDKs) such, as for example, CDK2, as determined by ratios of IC50 values.
• CDKs cyclin dependent kinases
• DYRKl inhibitors examples include AZ191, DYRKi, harmine, ID-8, leucettine L41, NCGC00185981, INDY, ProINDY, TC-S 7004, and TG003.
• At least one known DYRKl inhibitor, TC-S 7004, (US20120184562) is reported to be effective against quiescent cancer cells in vitro (Ewton DZ, Hu J, Vilenchik M, Deng X, Luk KC, Polonskaia A, Hoffman AF, Zipf K, Boylan JF, and Friedman EA. (2011) Inactivation of
• MIRK/DYRKIB kinase targets quiescent pancreatic cancer cells.
• the DYRKl inhibitor is a compound of formula I:
• Ri is a substituted or unsubstituted Ci-s alkyl, a substituted or unsubstituted phenyl, or a substituted or unsubstituted benzyl;
• R 2 is phenyl, optionally substituted with up to four groups independently selected from halo, CN, N0 2 , NHC(0)Ci- 4 alkyl, Ci- 4 alkyl, OH, OCi- 4 alkyl, wherein two adjacent groups and their intervening carbon atoms may form a 5- to 6-membered ring containing one or more heteroatoms selected from N, O, or S.
• the compound of formula I is selected from:
• the methods of the invention further provide (c)
• administering to the subject another cancer therapy, for example, radiation therapy or other cancer treatment.
• another cancer therapy for example, radiation therapy or other cancer treatment.
• the methods of the invention comprise: administering to a subject in need thereof a therapeutically effective amount of (a) a therapeutic agent of formula I; (b) an inhibitor of mitosis; and (c) radiation therapy; each therapy being administered
• the subject is first treated with radiation therapy, whereupon the subject is administered a therapeutic agent of Formula I, alone or in combination with the inhibitor of mitosis.
• the subject is co-administered (a) the therapeutic agent effective against quiescent cancer cells, (b) the inhibitor of mitosis, and, optionally (c) the radiation therapy.
• the inhibitor of mitosis is an inhibitor of mitosis effective to treat or prevent a neoplasm, including but not limited to, all such compounds approved for the treatment of cancer, compounds in clinical trials for the treatment of cancer, compounds that otherwise demonstrate efficacy in treating cancer in mammalian subject (e.g., mouse, rats, monkeys, humans), and compounds that demonstrate efficacy against neoplastic cells in vitro. Many such compounds are known.
• the inhibitor of mitosis is a taxane.
• Taxanes useful for the methods of the invention include BMS- 188796, BMS- 188797, cabazitaxel, DEP cabazitaxel, docetaxel, larotaxel (XRP9881, RPR109881), paclitaxel, taxoprexin (DHA-paclitaxel), and tesetaxel (DJ-927).
• the inhibitor of mitosis is a vinca alkaloid.
• Vinca alkaloids useful for the methods of the invention include vinblastine, vincristine, vindesine, vinflunine, and vinorelbine.
• the vinca alkaloid inhibitor of mitosis is vintafolide.
• the inhibitor of mitosis is a PLK1 inhibitor.
• PLK1 inhibitors useful for the methods of the invention include BI-2536, GSK461364, GW843682X, HMN- 214 and HMN-176, MLN-0905, NMS-P937, rigosertib, Ro3280, SBE 13, and volasertib.
• the inhibitor of mitosis is BI-2536 or GSK461364.
• a neoplasm is biliary cancer, brain cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer (e.g. melanoma), testicular cancer, thyroid cancer, or uterine cancer.
• the neoplasm is selected from bladder cancer, breast cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, and prostate cancer.
• the cancer is primary or metastatic.
• the cancer is of the type represented by the cell line types shown in the Examples.
• the embodiments are not meant to be limiting with regard to additional combination components, especially compounds that are part of existing treatment combinations, such as, for example, TPF wherein T stands for Taxotere ® , that is docetaxel, or PCV wherein V stands for vincristine sulfate.
• TPF Taxotere ®
• PCV vincristine sulfate
• the embodiments described here are illustrative and are not meant to be limiting with regard to routes and order of administration, patient type (previously untreated or previously treated, absence or presence of co-morbid conditions, age, sex, etc.), or stage of patient's disease, type of inhibitor of mitosis, etc.
• mitosis Targeting mitosis in cancer: emerging strategies, Molecular Cell 60, 524-536).
• mitosis As used herein, the terms “inhibitor of mitosis” and “mitotic inhibitor” are equivalent and may be used interchangeably.
• An inhibitor of mitosis interrupts cell cycling during the M phase or at a checkpoint entering (the G 2 /M checkpoint) or leaving (the post-mitotic checkpoint) the M phase. In M phase, the chromosomes and cytoplasm are divided into two daughter cells (cytokinesis). Mitosis proceeds in five phases: prophase, prometaphase, metaphase, anaphase, and telophase and an inhibitor of mitosis can interrupt any of these phases.
• inhibitors of mitosis include taxanes, vinca alkaloids, and PLK1 inhibitors.
• taxanes include BMS- 188796, BMS- 188797, cabazitaxel, DEP cabazitaxel, docetaxel, larotaxel (XRP9881, RPR109881), paclitaxel, taxoprexin (DHA- paclitaxel), and tesetaxel (DJ-927);
• vinca alkaloids include vincristine, vinblastine, vindesine, vinflunine, and vinorelbine;
• PLK1 inhibitors include BI-2536, GSK461364, GW843682X, HMN-214 and HMN-176, MLN-0905, NMS-P937, rigosertib, Ro3280, SBE 13, and volasertib.
• DYRK1 kinases such as DYRK1A and DYRKIB, may be important for the maintenance of cancer cells in Go state (quiescent state).
• DYRKIB/Mirk is a member of the Minibrain/DYRK family of kinases which mediates survival and differentiation in certain normal tissues.
• Dyrk a dual specificity protein kinase with unique structural features whose activity is dependent on tyrosine residues between subdomains VII and VIII, Journal of Biological Chemistry 271, 3488-3495; Becker W, Weber Y, Wetzel K, Eirmbter K, Tejedor FJ, and Joost HG (1998) Sequence characteristics, subcellular localization, and substrate specificity of DYRK-related kinases, a novel family of dual specificity protein kinases, Journal of Biological Chemistry 273, 25893-25902).
• DYRKIB is expressed at detectable levels in skeletal muscle cells and testes. Knockout of DYRKIB caused no evident abnormal phenotype in mice even in developing muscle, suggesting that DYRKIB is not an essential gene for normal development. Supporting this interpretation, normal fibroblasts exhibited no alteration in survival after 20-fold depletion of DYRKIB kinase levels. Thus, DYRKIB does not appear to be an essential gene for survival of normal cells yet there is evidence that it is upregulated in certain malignant cancer cells in which DYRKIB is believed to mediate survival by retaining cancer cells in quiescent state. These unusual characteristics suggest that DYRKIB may be an attractive target for therapeutic intervention and in particular for anti-cancer therapy directly against quiescent cancer cells.
• the disclosed combinations and methods may permit reduction in doses and/or frequency of administration of therapeutic agents and radiation to achieve the same improvements as a result of treatment relative to what is possible using individual components or existing single and combination treatments.
• quiescent cancer cells are inherently less susceptible to anticancer therapeutics, including mitotic inhibitors, and even a small fraction of quiescent cells that survives post treatment can lead to recurrence. Consequently, eradicating the resistant, quiescent cell populations in a neoplasm may or may not yield a synergistic reduction in EC50 values yet may yield a significant improvement in cancer recurrence and appearance of metastatic neoplasms.
• combinations may well vary depending on the neoplasm treated, extent of progression of the neoplasm, exact combination selected, age, sex, and physical condition of the subject, and other factors.
• Administration regimen may include multiple doses per period of time, the treatments administered concurrently or sequentially, etc.
• the combinations may be administered to subjects who are naive to treatment (have not been treated), or subjects who underwent previous treatments, or have undergone surgical resection or debulking of a solid tumor, or subjects whose cancers relapsed.
• therapeutic agent effective against quiescent cancer cells may be administered before the inhibitor of mitosis.
• the therapeutic agent effective against quiescent cancer cells may be administered 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, one week before the inhibitor of mitosis.
• the therapeutic agent effective against quiescent cancer cells may be administered at the same time (concomitantly) as the inhibitor of mitosis.
• the therapeutic agent effective against quiescent cancer cells may be administered 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours after the inhibitor of mitosis.
• the therapeutic agent effective against quiescent cancer cells and/or the inhibitor of mitosis may be administered before, after, or concomitantly with radiation or other therapy.
• the therapeutic agent effective against quiescent cancer cells may be administered daily, every two days, every three days, every four days, once weekly, once every two weeks, once per month by oral, intravenous (IV), intraperitoneal (IP), subcutaneous (SC), intratumoral (IT), intrathecal, or other routes of administration.
• IV intravenous
• IP intraperitoneal
• SC subcutaneous
• I intratumoral
• intrathecal or other routes of administration.
• the combinations may be administered to subjects who are naive to treatment (have not been treated), or subjects who underwent previous treatments with first-line, second-line, third-line, or other therapies, radiation treatments, or have undergone surgical resection or debulking of a solid tumor, or subjects whose cancers relapsed, or subjects whose cancers are non-metastatic or metastatic.
• Example 1 Determination of fraction of quiescent cancer cells within a population
• the following cell lines were obtained from ATCC and cultured according to the ATCC recommendations: DMS273 - small cell lung cancer cell line; HI 975 - non-small cell lung cancer cell line harboring L858R and T790M mutations in EGFR TK; A549 - a non- small cell lung cancer cell line with wild type EGFR; LNCap - prostate cancer cell line; SW620 - colon cancer cell line; MiaPaCa2 - pancreatic cancer cell line; PANC 1 - pancreatic cancer cell line; OVCAR3 - ovarian cancer cell line; SK-OV-3 - ovarian cancer cell line.
• Cell cultures were seeded into 6-well plates at 3xl0 5 - 6xl0 5 cells/well; the plated number of cells depended on cell size and rate of proliferation, aiming for approximately 50% confluency. After seeding, the cells were allowed to attach for 24 hours while incubated at 37 °C in a humidified 5% C0 2 atmosphere, and then treated with compounds for desired amount of time (usually 24 hours) incubating under same conditions. Then the cells were harvested by trypsinization, pooled with the floating cells, washed in PBS, and fixed in 70% ice-cold ethanol overnight.
• cells were seeded into 96- well plates at 2xl0 3 - 6xl0 3 cells/well; the plated number of cells depended on cell size and rate of proliferation aiming for approximately 50% confluency. After seeding, the cells were allowed to attach for 24 hours incubated at 37 °C in a humidified 5% C0 2 atmosphere.
• the treatments were performed using at least 6 different concentrations of a compound in 1:3 serial dilutions in DMSO such that the DMSO concentration in the cell medium was ⁇ 1%.
• the cell cultures were incubated for an additional 96 hours in 5% CO2 incubator at 37 °C. Treatments were performed in triplicate. Results were analyzed by CellTiter-GloTM Luminescent Cell Viability Assay (Promega, cat. # G7571) according to the manufacturer's instructions using Spectra MAX Gemini Spectrophotometer (Molecular Devices).
• SW620 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of paclitaxel used in this assay was 100 nM and the concentrations of Compound 1-5 were 2 ⁇ and 4 ⁇ , accordingly.
• the observed EC50 values of paclitaxel were 8.1 nM when Compound 1-5 was not present, 2.3 nM when Compound 1-5 was present at a concentration of 2 ⁇ , and 0.2 nM when Compound 1-5 was present at a concentration ⁇ 4 ⁇ . See FIG. 3.
• DMS273 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of paclitaxel used in this assay was 10 nM and the concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of paclitaxel were 2.7 nM when Compound 1-7 was not present, 1.9 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 0.9 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 4.
• LNCap cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of paclitaxel used in this assay was 10 nM and the concentrations of Compound 1-5 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of paclitaxel were 3.2 nM when Compound 1-5 was not present, 2.1 nM when Compound 1-5 was present at a concentration of 2 ⁇ , and 0.9 nM when Compound 1-5 was present at a concentration of 4 ⁇ . See FIG. 5.
• HCC827 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of paclitaxel used in this assay was 10 nM and the concentrations of Compound 1-7 were 3 ⁇ and 6 ⁇ .
• the observed EC50 values of paclitaxel were 4.2 nM when Compound 1-7 was not present, 2.5 nM when Compound 1-7 was present at a concentration of 3 ⁇ , and 0.6 nM when Compound 1-7 was present at a concentration of 6 ⁇ . See FIG. 6.
• A549 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of paclitaxel used in this assay was 10 nM and the concentrations of Compound 1-7 were 3 ⁇ and 6 ⁇ .
• the observed EC50 values of paclitaxel were 4.8 nM when Compound 1-7 was not present, 2.5 nM when Compound 1-7 was present at a concentration of 3 ⁇ , and 0.7 nM when Compound 1-7 was present at a concentration of 6 ⁇ . See FIG. 7.
• SK-OV-3 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of paclitaxel used in this assay was 10 nM and the concentrations of Compound 1-7 were 2 ⁇ , 4 ⁇ , 8 ⁇ , and 10 ⁇ .
• the observed EC50 values of paclitaxel were 9.7 nM when Compound 1-7 was not present, 9.4 nM when Compound 1-7 was present at a concentration of 2 ⁇ , 4.8 nM when Compound 1-7 was present at a concentration of 4 ⁇ , 4.7 nM when Compound 1-7 was present at a concentration of 8 ⁇ , and 5.2 nM when Compound 1-7 was present at a concentration of 10 ⁇ . See FIG. 8.
• OVCAR3 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of paclitaxel used in this assay was 10 nM and the concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of paclitaxel were 3.3 nM when Compound 1-7 was not present, 2.8 nM when Compound 1-7 was present at a concentration of 3 ⁇ , and 1.7 nM when Compound 1-7 was present at a concentration of 6 ⁇ . See FIG. 9.
• SW620 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of docetaxel used in this assay was 100 nM and the concentrations of Compound 1-5 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of docetaxel were 2.85 nM when Compound 1-5 was not present, 0.69 nM when Compound 1-5 was present at a concentration of 2 ⁇ , and ⁇ 0.015 nM when Compound 1-5 was present at a concentration of 4 ⁇ . See FIG. 10.
• DMS273 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of docetaxel used in this assay was 10 nM and the concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of docetaxel were 0.64 nM when Compound 1-7 was not present, 0.38 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 0.14 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 11.
• HCC827 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of docetaxel used in this assay was 10 nM and the concentrations of Compound 1-7 were 3 ⁇ and 6 ⁇ .
• the observed EC50 values of docetaxel were 1.8 nM when Compound 1-7 was not present, 0.6 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 0.03 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 12.
• A549 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of docetaxel used in this assay was 5 nM and the concentrations of Compound 1-7 were 3 ⁇ and 6 ⁇ .
• the observed EC50 values of docetaxel were 1.4 nM when Compound 1-7 was not present, 0.6 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 0.1 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 13.
• SK-OV-3 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of docetaxel used in this assay was 10 nM and the concentrations of Compound 1-7 were 4 ⁇ and 8 ⁇ .
• the observed EC50 values of docetaxel were 2.5 nM when Compound 1-7 was not present, 1.5 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 0.3 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 14.
• OVCAR3 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of docetaxel used in this assay was 10 nM and the concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of docetaxel were 0.85 nM when Compound 1-7 was not present, 0.52 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and ⁇ 0.04 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 15.
• Example 5 Combination of a molecule effective against quiescent cancer cells with vincristine DMS273 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of vincristine used in this assay was 10 nM and the concentrations of Compound 1-5 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of vincristine were 1.1 nM when Compound 1-5 was not present, 0.4 nM when Compound 1-5 was present at a concentration of 2 ⁇ , and ⁇ 0.04 nM when Compound 1-5 was present at a concentration of 4 ⁇ . See FIG. 16.
• HI 975 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of vincristine used in this assay was 10 nM and the concentrations of Compound 1-5 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of vincristine were 1.5 nM when Compound 1-5 was not present, 0.85 nM when Compound 1-5 was present at a concentration of 2 ⁇ , and 0.35 nM when Compound 1-5 was present at a concentration of 4 ⁇ . See FIG. 17.
• SK-OV-3 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of vincristine used in this assay was 20 nM and the concentrations of Compound 1-7 were 4 ⁇ and 8 ⁇ .
• the observed EC50 values of vincristine were 8.7 nM when Compound 1-7 was not present, 2.1 nM when Compound 1-7 was present at a concentration of 4 ⁇ , and 1.0 nM when Compound 1-7 was present at a concentration of 8 ⁇ . See FIG. 18.
• OVCAR3 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of vincristine used in this assay was 1 nM and the concentrations of Compound 1-7 were 1 ⁇ and 3 ⁇ .
• the observed EC50 values of vincristine were 1.2 nM when Compound 1-7 was not present, 0.8 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 0.1 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 19.
• Example 6 Combination of a molecule effective against quiescent cancer cells with vinorelbine
• DMS273 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of vinorelbine used in this assay was 10 nM and the concentrations of Compound 1-5 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of vinorelbine were 1.1 nM when Compound 1-5 was not present, 0.4 nM when Compound 1-5 was present at a concentration of 2 ⁇ , and ⁇ 0.04 nM when Compound 1-5 was present at a concentration of 4 ⁇ . See FIG. 20.
• HI 975 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of vinorelbine used in this assay was 10 nM and the concentrations of Compound 1-5 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of vinorelbine were 9.5 nM when Compound 1-5 was not present, 5.2 nM when Compound 1-5 was present at a concentration of 2 ⁇ , and 2.1 nM when Compound 1-5 was present at a concentration of 4 ⁇ . See FIG. 21.
• OVCAR3 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of vinorelbine used in this assay was 10 nM and the concentrations of Compound 1-7 were 1 ⁇ and 3 ⁇ .
• the observed EC50 values of vinorelbine were 2.4 nM when Compound 1-7 was not present, 2.4 nM when Compound 1-7 was present at a concentration of 1 ⁇ , and 0.1 nM when Compound 1-7 was present at a concentration of 3 ⁇ . See FIG. 22.
• SK-OV-3 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of vinorelbine used in this assay was 40 nM and the concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of vinorelbine were 15.1 nM when Compound 1-7 was not present, 10.1 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 4.7 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 23.
• A549 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of vinorelbine used in this assay was 50 nM and the concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of vinorelbine were 27 nM when Compound 1-7 was not present, 16 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 6 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 24.
• Example 7 Combination of a molecule effective against quiescent cancer cells with BI2536
• HI 975 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of BI2536 used in this assay was 50 nM and the concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of BI2536 were 46.3 nM when Compound 1-7 was not present, 17.3 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and ⁇ 0.04 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 25.
• PANC1 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of BI2536 used in this assay was 50 nM and the concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of BI2536 were 7.3 nM when Compound 1-7 was not present, 4.4 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 2.5 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 26.
• DMS273 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of BI2536 used in this assay was 50 nM and the concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of BI2536 were 6.2 nM when Compound 1-7 was not present, 4.1 nM when Compound 1-7 was present at a concentration of 2 ⁇ , and 1.7 nM when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 27.
• A549 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of BI2536 used in this assay was 50 nM and the concentrations of Compound 1-7 were 3 ⁇ and 6 ⁇ .
• the observed EC50 values of BI2536 were 6.2 nM when Compound 1-7 was not present, 4.1 nM when Compound 1-7 was present at a concentration of 3 ⁇ , and 1.7 nM when Compound 1-7 was present at a concentration of 6 ⁇ . See FIG. 28.
• Example 8 Combination of a molecule effective against quiescent cancer cells with
• HI 975 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of GSK461364 used in this assay was 10 ⁇ and the
• concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of GSK461364 were 1.9 ⁇ when Compound 1-7 was not present, 0.97 ⁇ when Compound I- 7 was present at a concentration of 2 ⁇ , and 0.58 ⁇ when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 29.
• PANC1 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of GSK461364 used in this assay was 10 ⁇ and the
• concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of GSK461364 were 0.5 ⁇ when Compound 1-7 was not present, 0.3 ⁇ when Compound 1-7 was present at a concentration of 2 ⁇ , and 0.2 ⁇ when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 30.
• DMS273 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of GSK461364 used in this assay was 10 ⁇ and the
• concentrations of Compound 1-7 were 2 ⁇ and 4 ⁇ .
• the observed EC50 values of GSK461364 were 0.43 ⁇ when Compound 1-7 was not present, 0.24 ⁇ when Compound 1-7 was present at a concentration of 2 ⁇ , and 0.12 ⁇ when Compound 1-7 was present at a concentration of 4 ⁇ . See FIG. 31.
• A549 cells were cultured, treated, and analyzed as described in Examples 1 and 2.
• the highest concentration of GSK461364 used in this assay was 1 ⁇ and the concentrations of Compound 1-7 were 3 ⁇ and 6 ⁇ .
• the observed EC50 values of GSK461364 were 1.2 ⁇ when Compound 1-7 was not present, 0.5 ⁇ when Compound 1-7 was present at a concentration of 3 ⁇ , and 0.2 ⁇ when Compound 1-7 was present at a concentration of 6 ⁇ . See FIG. 32.
• Example 9 Cell cycle effects and cytotoxicity of paclitaxel and combination of a molecule effective against quiescent cancer cells with paclitaxel
• DMS273 cells were cultured, treated, and analyzed as described in Examples 1 and 2. The results when different concentrations of paclitaxel, Compound 1-7, or both paclitaxel and Compound 1-7 are present are shown in FIG. 33.
• SW620 cells were cultured, treated, and analyzed as described in Examples 1 and 2. The results when different concentrations of paclitaxel, Compound 1-7, or both paclitaxel and Compound 1-7 are present are shown in FIG. 34.
• Example 10 Cell cycle effects and cytotoxicity of vincristine and combination of a molecule effective against quiescent cancer cells with vincristine
• DMS273 cells were cultured, treated, and analyzed as described in Examples 1 and 2. The results when different concentrations of vincristine, Compound 1-7, or both vincristine and Compound 1-7 are present are shown in FIG. 35.
• Example 11 Cell cycle effects and cytotoxicity of vinorelbine and combination of a molecule effective against quiescent cancer cells with vinorelbine
• DMS273 cells were cultured, treated, and analyzed as described in Examples 1 and 2. The results when different concentrations of vinorelbine, Compound 1-7, or both vinorelbine and Compound 1-7 are present are shown in FIG. 36.
• the S W620 cells were cultured and treated as described in Example 1.
• PI propidium iodide
• manufacturer's protocol supplied with the Guava Cell Cycle Reagent for Flow Cytometry (EMD Millipore) was followed.
• the measurements were performed with Guava PCA-96 flow cytometer (EMD Millipore) using the green laser for excitation at 535 nm and monitoring emission at 617 nm.
• the S W620 cells were cultured, treated, and analyzed as described in Examples 1.
• PI propidium iodide
• manufacturer's protocol supplied with the Guava Cell Cycle Reagent for Flow Cytometry (EMD Millipore) was followed.
• the measurements were performed with Guava PCA-96 flow cytometer (EMD Millipore) using the green laser for excitation at 535 nm and monitoring emission at 617 nm.
• SW620 cells were incubated for 24 hours in FBS- media, which contained different concentrations of either AZ191 or Compound 1-7.
• exposure to AZ191 led to no decrease in fraction of cells in quiescent state (Go) based on no observed change in the fraction of cells Go+Gi phase.
• exposure to same or lower concentrations of Compound 1-7 led to a significant decrease in fraction of cells in quiescent state (Go) based on the significant decrease in the fraction of cells Go+Gi phase.
• AZ191 inhibits DYRKIB at 17 nM (Ashford AL, Oxley D, Kettle J, Hudson K, Guichard S, Cook SJ, Lochhead PA (2014) A novel DYRKIB inhibitor AZ191 demonstrates that DYRKIB acts independently of GSK3beta to phosphorylate cyclin Dl at Thr(286), not Thr(288). Biochemical Journal 457, 43-56).

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