WO2009032213A1 - Lutte contre des cellules malignes par inhibition de kinase - Google Patents

Lutte contre des cellules malignes par inhibition de kinase Download PDF

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Publication number
WO2009032213A1
WO2009032213A1 PCT/US2008/010275 US2008010275W WO2009032213A1 WO 2009032213 A1 WO2009032213 A1 WO 2009032213A1 US 2008010275 W US2008010275 W US 2008010275W WO 2009032213 A1 WO2009032213 A1 WO 2009032213A1
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Prior art keywords
cells
dmat
ck2α
meg
selective inhibitor
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PCT/US2008/010275
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English (en)
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Michael Kalafatis
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Cleveland State University
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Priority claimed from PCT/US2007/019676 external-priority patent/WO2008033308A2/fr
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Publication of WO2009032213A1 publication Critical patent/WO2009032213A1/fr

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    • 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
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4192—1,2,3-Triazoles

Definitions

  • the presently disclosed embodiments relate to the control of malignant cells by inhibiting certain kinases. More specifically, the embodiments are directed to methods and compositions involving the use of particular casein kinase 2 inhibitors in the treatment of cancer.
  • Cancer cells are characterized by increased proliferation and loss of the cells' normal phenotype and function. Many types of cancer are caused by defects in signaling pathways including deregulation of a process known as apoptosis. Apoptosis is a genetically programmed and evolutionary conserved mechanism through which the normal development and tissue homeostasis are maintained. [0004] Cancer development generally requires that tumor cells achieve certain characteristics, including increased replicative potentials, anchorage and growth- factor independency, departure from apoptosis, angiogenesis and metastasis. Many of these processes involve the actions of protein kinases, which have emerged as key regulators of many aspects of abnormal and uncontrolled cell growth.
  • Disrupted protein kinase activity is repeatedly found to be associated with human malignancies, making these proteins attractive targets for anti-cancer therapy.
  • the reciprocal chromosomal translocation t(9;22), known as the Philadelphia positive chromosome (Ph+) is associated with diseases like chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), acute non- lymphocytic leukemia (ANLL) and acute lymphocytic leukemia (ALL).
  • CML chronic myelogenous leukemia
  • AML acute myelogenous leukemia
  • ANLL acute non- lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • the BCR/ABL tyrosine kinase In chronic myelogenous leukemia, the BCR/ABL tyrosine kinase is constitutively activated. Different intracellular pathways are transformed by the oncoprotein BCR/ABL, resulting in uncontrolled hematopoietic proliferation.
  • blast crisis The late phase of chronic myelogenous leukemia, named blast crisis (or blastic phase), is characterized by extreme overproliferation of stem cells and their progeny in bone marrow.
  • blast crisis a major complication is thrombosis due to high platelet counts.
  • myeloproliferative disorders like chronic myelogenous leukemia, the platelet counts and function are abnormal due to overproliferation of malignant megakaryoblasts.
  • casein kinase 2 a tyrosine kinase named casein kinase 2 (CK2) was found to be constitutively activated, elevated and serving as an oncoprotein.
  • CK2 is a pleiotropic, ubiquitous Ser/Thr kinase.
  • the protein is a heterotetramer with two catalytic subunits, ⁇ and ⁇ ', and two regulatory ⁇ subunits. Each subunit was shown to be able to execute specific functions by itself or in the holoenzyme form, the ⁇ ' ⁇ 2 tetramer.
  • CK2 ⁇ The up regulation and hyperactivity of CK2 has an anti-apoptotic effect which is associated with decreased platelet counts and function in leukemias, such as acute myelogenous leukemia and chronic myelogenous leukemia.
  • CK2 ⁇ was found to be a substrate for the ABL domain of BCR/ABL and forms a specific complex with the BCR domain of BCR/ABL. It was hypothesized that CK2 ⁇ sterically impedes the binding of the ABL SH2 domain to BCR. This results in proliferation abnormalities in Philadelphia positive cells. Therefore CK2 ⁇ was shown to be a possible arbitrator of BCR/ABL function. Other functions of CK2 ⁇ downstream of the BCR/ABL interaction yield an overall oncogenic response in Philadelphia positive cells.
  • CK2 ⁇ protein kinase inhibitors have been developed and studied, such as Emodin; 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB); 4,5,6,7- Tetrabromobenzotriazole (TBB); 2-Dimethylamino-4,5,6,7-tetrabromo-1 H- benzimidazole (DMAT); and ellagic acid.
  • DRB 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole
  • TAB 4,5,6,7- Tetrabromobenzotriazole
  • DMAT 2-Dimethylamino-4,5,6,7-tetrabromo-1 H- benzimidazole
  • ellagic acid 2-Dimethylamino-4,5,6,7-tetrabromo-1 H- benzimidazole
  • Inhibition of CK2 in various cancer cell lines produced apoptosis and proliferation arrest
  • CK2 can serve an anti-apoptic role by protecting regulatory proteins from caspase-mediated degradation, the exact mechanisms are not well understood.
  • protein kinase activity has been linked with many forms of human cancers, specific treatment methodologies using CK2 protein kinases are still needed. Accordingly, a need exists for a strategy by which abnormal cell proliferation can be arrested by controlling CK2 protein kinases. More desirably, it would be beneficial to identify a method of inducing arrest of cell proliferation using CK2 protein kinases while maintaining steady cell numbers. And, it would also be beneficial to provide such a method without attendant problems of cell necrosis.
  • the present invention provides a method for treating a disease characterized by over-proliferation of malignant cells.
  • diseases are (i) breast cancer, (ii) colon cancer, (iii) skin cancer, (iv) chronic myelogenous leukemia, (v) renal cell carcinoma, (vi) bladder cancer, and (vii) glioblastoma.
  • the present invention provides a method for treating a disease characterized by over-proliferation of malignant cells.
  • diseases include (i) breast cancer, (ii) colon cancer, (iii) skin cancer, (iv) chronic myelogenous leukemia, v) renal cell carcinoma, (vi) bladder cancer, and (vii) glioblastoma.
  • the method comprises administering an effective amount of a CK2 ⁇ inhibitor to a patient in need of treatment.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a CK2 ⁇ selective inhibitor selected from the group consisting of (i) 4,5,6, 7-Tetrabromobenzotriazole (TBBt), (ii) 2-Dimethylamino- 4,5,6,7-tetrabromo-i H-benzimidazole (DMAT), and combinations of (i) and (ii).
  • the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.
  • Fig. 1 includes photographs of MEG-01 cells prior to and after treatment with a preferred embodiment inhibitor.
  • Fig. 2 includes photographs of MEG-01 cells undergoing thrombocytopoiesis, induced by a preferred inhibitor.
  • Figs. 3-5 are photographs of MEG-01 cells producing platelets after inducement with a preferred inhibitor.
  • Figs. 6-7 are photographs of activated platelet-like particles from MEG-01 cells, the cells having been treated with a preferred inhibitor.
  • Fig. 8 is a graph further illustrating the effect of preferred embodiment inhibitors upon MEG-01 cells.
  • Fig. 9 is a photograph of a control untreated colony of MEG-01 cells.
  • Fig. 10 is a photograph of a colony of MEG-01 cells treated with a preferred embodiment inhibitor.
  • Fig. 11 is a graph comparing colony areas of a control to a sample treated in accordance with a preferred embodiment inhibitor.
  • Fig. 12 is a graph of a DNA content assay referring to the preferred embodiment inhibitors against a control.
  • Fig. 13 is a graph further illustrating the preferred embodiment inhibitors against a control.
  • Fig. 14 is a DNA content analysis of MEG-01 cell lines treated with a preferred embodiment inhibitor.
  • Figs. 15 and 16 are graphs showing apoptosis and phenotype change of a control and MEG-01 cell lines treated with a preferred inhibitor, respectively.
  • Fig. 17 is a graph illustrating the effect of preferred inhibitors on MEG-01 cell lines after 24 hours.
  • Fig. 18 is a graph illustrating the effect of preferred inhibitors on MEG-01 cell lines after 48 hours.
  • Fig. 19 is a graph illustrating the effect of preferred inhibitors on MEG-01 cell lines after 72 hours.
  • Fig. 20 is a graph illustrating the effect of preferred inhibitors on MEG-01 cell lines after 96 hours.
  • Fig. 21 is a photograph of untreated control MEG-01 cells after 96 hours.
  • Fig. 22 is a photograph of cells treated with a preferred inhibitor after 96 hours.
  • Fig. 23 is a photograph of proplatelets formation following treatment with a preferred inhibitor between 72 and 96 hours.
  • Fig. 24 is a SEM micrograph of MEG-01 cells.
  • Fig. 25 is a photograph of proplatelets formation following treatment with a preferred embodiment inhibitor.
  • Fig. 26 is a photograph of proplatelets bearing MEG-01 megakaryocytes following treatment with a preferred inhibitor after 72 to 96 hours.
  • Fig. 27 is a photograph of platelet-like particles following treatment with a preferred inhibitor after 72 to 96 hours.
  • Fig. 28 is a graph illustrating activation of platelets from MEG-01 cells obtained by use of a preferred inhibitor, compared to a control.
  • Fig. 29 is a graph illustrating activation of platelets from MEG-01 cells obtained by use of a preferred inhibitor, compared to a control.
  • Fig. 30 is a graph illustrating activation of platelets from MEG-01 cells obtained by use of a preferred inhibitor, compared to a control.
  • Fig. 31 is a graph illustrating activation of platelets from MEG-01 cells obtained by use of a preferred inhibitor, compared to a control.
  • Fig. 32 is a photograph of a fibrin clot formed from platelets derived from use of a preferred inhibitor.
  • Fig. 33 is another photograph of a fibrin clot formed from platelets derived from use of a preferred inhibitor.
  • Fig. 34 is yet another photograph of a fibrin clot formed from platelets derived from use of a preferred inhibitor.
  • Fig. 35 is a graph illustrating changes in tumor volume of MEG-01 cells treated with a preferred inhibitor compared to a control.
  • Fig. 36 is a graph illustrating changes in tumor volume of MEG-01 cells treated with a preferred inhibitor compared to a control.
  • Fig. 37 is a graph of platelet counts of a MEG-01 xenograft treated with a preferred inhibitor as compared to a control.
  • Fig. 38 is a graph of percentage abnormal cells of a control, normal mice, and an inhibitor-treated xenograph.
  • Fig. 39 is a graph of tail bleeding times in an inhibitor-treated MEG-01 xenograft mice compared to a control and normal mice.
  • Fig. 40 is a graph of spleen size in an inhibitor-treated MEG-01 xenograft mice compared to a control and normal mice.
  • Fig. 41 is a graph of apoptotic-necrotic areas in MEG-01 cells and a control.
  • Fig. 42 is a graph of angiogenesis areas in MEG-01 cells and a control.
  • Fig. 43 is a graph of cell counts in vitro of MCF-7 cells and a control.
  • Figs. 44-49 are photographs of MCF-7 cells (controls and inhibitor treated) after 24 hours.
  • Figs. 50-52 are graphs showing apoptosis and phenotype change in a control and MCF-7 cell lines treated with a preferred inhibitor.
  • Fig. 53 is a graph comparing percentages of apoptotic cells in the samples depicted in Figs. 50-52.
  • Figs. 54-56 are photographs and a graph illustrating anchorage independence of a MCF-7 cell line.
  • Fig. 57 is a graph illustrating changes in tumor size in MCF-7 cells and a control.
  • Fig. 58 is a graph of apoptotic-necrotic areas in MCF-7 cells and a control.
  • Fig. 59 is a graph of angiogenesis areas in MCF-7 cells and a control.
  • Fig. 60 is a graph of cell counts in vitro of SW-480 cells and a control.
  • Figs. 61-66 are photographs of SW-480 cells (controls and inhibitor treated) after 24 hours.
  • Figs. 67-69 are graphs showing apoptosis and phenotype change in a control and SW-480 cell lines treated with a preferred inhibitor.
  • Fig. 70 is a graph comparing percentages of apoptotic cells in the samples depicted in Figs. 67-69.
  • Figs. 71-73 are photographs and a graph illustrating anchorage independence of a SW-480 cell line.
  • Fig. 74 is a graph illustrating changes in tumor size in SW-480 cells and a control.
  • Fig. 75 is a graph of apoptotic-necrotic areas in SW-480 cells and a control.
  • Fig. 76 is a graph of angiogenesis areas in SW-480 cells and a control.
  • Fig. 77 is a graph of cell counts in vitro of WM-164 cells and a control.
  • Figs. 78-83 are photographs of WM-164 cells (controls and inhibitor treated) after 24 hours.
  • Figs. 84-86 are graphs showing apoptosis and phenotype change in a control and WM-164 cell lines treated with a preferred inhibitor.
  • Fig. 87 is a graph comparing percentages of apoptotic cells in the samples shown in Figs. 84-86.
  • Figs. 88-90 are photographs and a graph illustrating anchorage independence of a WM-164 cell line.
  • Fig. 91 is a graph illustrating changes in tumor size in WM-164 cells and a control.
  • Fig. 92 is a graph of apoptotic-necrotic areas in WM-164 cells and a control.
  • Fig. 93 is a graph of angiogenesis areas in WM-164 cells and a control.
  • Fig. 94 is a graph illustrating changes in tumor size in another cell line,
  • Fig. 95 is a graph illustrating changes in tumor size in another cell line
  • Fig. 96 is a graph illustrating changes in tumor size in another cell line
  • Cancer is so widespread and lethal that it can be considered the biggest health problem of this century.
  • Various unknown causes, diverse genetic and protein abnormalities, as well as different and complex molecular mechanisms make cancer drug development a real challenge. Harmful side-effects are another problem that needs to be overcome in the search for possible cancer therapies.
  • Harmful side-effects are another problem that needs to be overcome in the search for possible cancer therapies.
  • cells are growing abnormally and uncontrollably.
  • Casein kinase 2 (CK2) was found to be up-regulated and over-expressed in tumor tissue and may be responsible for cancer growth and sustainability.
  • CK2 inhibitors antisense nucleotides or ATP analogues
  • MEG-01 cells are malignant megakaryoblasts isolated from a patient with chronic myelogenous leukemia in blast crisis.
  • MCF-7 cells are breast cancer cells, estrogen dependent with a highly abnormal proliferation rate.
  • SW-480 cells are malignant colon cells (epithelial cancer cells).
  • WM-164 cells are very aggressive melanoma cells. Such cells are resistant to apoptosis and manifest anchorage independence by growing colonies in soft agar.
  • a specific CK2 inhibitor induced proliferation arrest and apoptosis in all these cell lines when tested in vitro (in cell culture) and in vivo (with mice xenografts). All treated tumors showed necrosis, apoptosis and reduced angiogenesis versus untreated tumors.
  • the liver, brain, kidney, and muscle tissue of all mice treated with the inhibitor appeared to be normal following histological analysis.
  • CK2 is localized in the cytoplasm of normal cells; CK2 is translocated in the nucleus of malignant cells and phosphorylates a protein or a group of proteins responsible for normal cell growth. Phosphorylation of these proteins will severely impair their normal function resulting in uncontrollable cell growth.
  • specific CK2 inhibitors could be used as anti-cancer drugs and will stop abnormal cell proliferation independently of the type of cancer. These inhibitors will have no side effect because they have no effect on normal cells.
  • CK2 has common targets in the nucleus of malignant cells. Inhibition of phosphorylation of these target proteins can be used as a starting point for the development of a potential cancer therapy. Basis for Treatment Strategy
  • CK2 is localized in the cytoplasma of normal cells; CK2 is translocated in the nucleus of malignant cells and phosphorylates a protein or a group of proteins responsible for normal cell growth. Phosphorylation of these proteins will severely impair their normal function resulting in uncontrollable cell growth.
  • specific CK2 inhibitors could be used as anti-cancer drugs and will stop abnormal cell proliferation independently of the type of cancer. These inhibitors have no side effect because they have no effect on normal cells.
  • CK2 ⁇ and CK2 ⁇ ' exhibit approximately 90% identity which is consistent with the fact that they display similar enzymic properties (including turnover rates and substrate specificity) in vitro.
  • C-terminal domains of CK2 ⁇ and CK2 ⁇ ' are completely unrelated. Very little is currently known about CK2 ⁇ ", which was identified only recently.
  • Megakaryocytes are polyploid cells, originating from hematopoietic stem cells in the bone marrow.
  • Thrombocytopoiesis refers to the production of blood platelets or thrombocytes. More specifically, thrombocytopoiesis is the process of producing of anucleated cells or platelets, from megakaryocytes.
  • Megakaryoblasts are precursors of platelets that first differentiate to the stage of megakaryocytes. Mature megakaryocytes form pseudopodia and give rise to platelets. More specifically, megakaryoblasts undergo endomitosis and maturation to the stage of megakaryocytes, through a process called megakaryocytopoiesis.
  • Platelets (thrombocytes) are vital for maintaining normal hemostasis and for the response of the body to trauma.
  • the process of platelet formation is complex and at present, not well understood.
  • the thrombocytopoiesis process has been linked to the constitutive apoptosis of megakaryocytes.
  • Caspase activation in megakaryocytes has also been connected with platelets production. Pro-apoptotic and pro-survival balance are shifted towards apoptosis during megakaryocytopoiesis and thrombocytopoiesis.
  • MEG-01 cells were previously isolated from a patient with CML, Ph+, in blast crisis, with high peripheral blast counts and thrombocytosis (high platelets counts). The cells were characterized as being megakaryoblasts in an early stage of differentiation in the megakaryocyte lineage. The cells expressed the integrin ⁇ n b ⁇ 3 on their surface and were positive for platelet peroxidase.
  • MEG-01 cells expressed the p210 BCR/ABL tyrosine kinase. MEG-01 cells were found to be cytokine independent and capable of differentiating in vitro in response to PMA, nitric oxide (NO), aphidicolin, nocodazole and staurosporine. MEG-01 cells were found to release platelet-like particles following all of these treatments. Inhibition of caspases in a MEG-01 cell line was shown to result in impaired proplatelet formation and platelets release.
  • casein kinase 2 alpha subunit (CK2 ⁇ ) inhibition with specific preferred embodiment inhibitors was studied in a megakaryoblastic cell line from a CML patient in blast crisis (MEG-01 ). It was surprisingly discovered that the preferred embodiment casein kinase 2 inhibitors induce proliferation arrest while maintaining a steady cell number for an extended time period, such as one week. Treated cells grew at a significantly lower rate than non-treated cells. Apoptosis of MEG-01 was induced by the preferred embodiment CK2 inhibitors, and the apoptosis was dose and time dependent. No necrosis was detected in the presence of the inhibitors, demonstrating that the preferred compounds are not cytotoxic.
  • MEG-01 megakaryoblastic cell line from a CML patient in blast crisis
  • CK2 inhibitors In the presence of the preferred embodiment CK2 inhibitors, megakaryocytes matured to a pro-platelets bearing stage. Platelets were subsequently released through rupture, following cytoplasmic fragmentation and nuclear extrusion. Thrombocytopoiesis due to the use of the preferred embodiment CK2 inhibitors occurred both in suspension and with MEG-01 cells grown on a fibronectin matrix. Platelets obtained following these treatments were found to undergo shape change in response to various agonists. The platelets obtained in culture, following CK2 ⁇ inhibition with specific kinase inhibitors were functional. These platelets formed a clot visible with the eye (a normal fibrin clot as seen by SEM), when exposed to agonists. Thus, by using the preferred embodiment CK2 inhibitors, the abnormal proliferation of a transformed cell line was successfully stopped and its path reversed towards its normal function.
  • CK2 ⁇ inhibition studies with the preferred inhibitors demonstrate a key role of CK2 in oncogenic development as well as in the megakaryocytopoiesis and thrombocytopoiesis processes. These significant advances suggest a wide array of potential applications and CK2 targeted drug design for patients with cytokine and BCR/ABL inhibitors resistance. Furthermore, due to the importance of protein kinases in malignant processes, the present invention has significant future therapeutic interest.
  • CK2 ⁇ inhibitors are DMAT and TBB.
  • DMAT is 2-dimethylamino-4,5,6,7-tetrabromo-1 H- benzimidazole and has the following structural formula (1 ):
  • TBB (or sometimes referred to as TBBt) is 4,5,6,7-tetrabromobenzotriazole and has the following structural formula (2):
  • the present invention includes the use of either or both of the inhibitors DMAT and TBB, and/or their pharmaceutically acceptable salts.
  • the preferred inhibitors can be incorporated into a wide array of compositions, formulations, and pharmaceuticals.
  • the pharmaceutical compositions may include an inhibitor by itself, or in combination and optionally including one or more suitable diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants/flavoring, carriers, excipients, buffers, stabilizers, solubilizers, other materials well known in the art and combinations thereof.
  • suitable diluents fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants/flavoring, carriers, excipients, buffers, stabilizers, solubilizers, other materials well known in the art and combinations thereof.
  • Any pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media may be used.
  • Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma, methyl- and propylhydroxybenzoate, talc, alginates, carbohydrates, especially mannitol, alpha-lactose, anhydrous lactose, cellulose, sucrose, dextrose, sorbitol, modified dextrans, gum acacia, and starch.
  • Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Erncompress and Avicell.
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the inhibitor compounds, see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. pp. 1435- 1712 (1990).
  • Pharmaceutically acceptable fillers can include, for example, lactose, microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, calcium sulfate, dextrose, mannitol, and/or sucrose.
  • Inorganic salts including calcium triphosphate, magnesium carbonate, and sodium chloride may also be used as fillers in the pharmaceutical compositions.
  • Amino acids may be used such as used in a buffer formulation of the pharmaceutical compositions.
  • Disintegrants may be included in solid dosage formulations of the inhibitors of the present invention.
  • Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Additional examples include, but are not limited to sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange 1 peel, acid carboxymethylcellulose, natural sponge and bentonite may all be used as disintegrants in the pharmaceutical compositions.
  • Other disintegrants include insoluble cationic exchange resins. Powdered gums including powdered gums such as agar, Karaya or tragacanth may be used as disintegrants and as binders.
  • Binders may be used to hold the composition, formulation, or pharmaceutical together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) can both be used in alcoholic solutions to facilitate granulation of the therapeutic ingredient.
  • An antifrictional agent may be included in the composition, formulation, or pharmaceutical to prevent sticking during the formulation process.
  • Lubricants may be used as a layer between the ingredients and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. [00102] Glidants that might improve the flow properties of the composition, formulation, or pharmaceutical during formulation and to aid rearrangement during compression might be added. Suitable glidants include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • a surfactant might be added as a wetting agent.
  • Natural or synthetic surfactants may be used.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate.
  • Cationic detergents such as benzalkonium chloride and benzethonium chloride may be used.
  • Nonionic detergents that can be used in the pharmaceutical formulations include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants can be present in the pharmaceutical compositions of the invention either alone or as a mixture in different ratios. [00104] Controlled release formulations may be desirable.
  • the inhibitors of the invention can be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums.
  • Slowly degenerating matrices may also be incorporated into the pharmaceutical formulations, e.g., alginates, polysaccharides.
  • Another form of controlled release is a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push the inhibitor compound out through a single small opening due to osmotic effects.
  • Some enteric coatings also have a delayed release effect.
  • Colorants and flavoring agents may also be included in the pharmaceutical compositions.
  • the inhibitors of the invention may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a beverage containing colorants and flavoring agents.
  • the therapeutic agent can also be given in a film coated tablet.
  • Nonenteric materials for use in coating the pharmaceutical compositions include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy- methyl cellulose, povidone and polyethylene glycols.
  • Enteric materials for use in coating the pharmaceutical compositions include esters of phthalic acid. A mix of materials might be used to provide the optimum film coating. Film coating manufacturing may be carried out in a pan coater, in a fluidized bed, or by compression coating.
  • compositions can be administered in solid, semi-solid, liquid or gaseous form, or may be in dried powder, such as lyophilized form.
  • the pharmaceutical compositions can be packaged in forms convenient for delivery, including, for example, capsules, sachets, cachets, gelatins, papers, tablets, capsules, suppositories, pellets, pills, troches, lozenges or other forms known in the art.
  • the type of packaging will generally depend on the desired route of administration.
  • Implantable sustained release formulations are also contemplated, as are transdermal formulations. The Preferred Methods of Treatment
  • the inhibitor compounds may be administered by various routes.
  • pharmaceutical compositions may be for injection, or for oral, nasal, transdermal or other forms of administration, including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., aerosolized drugs) or subcutaneous injection (including depot administration for long term release e.g., embedded under the splenic capsule, brain, or in the cornea); by sublingual, anal, vaginal, or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • the methods of the invention involve administering effective amounts of an inhibitor of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers, as described above.
  • the invention provides methods for oral administration of a pharmaceutical composition of the invention.
  • Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, supra at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, and cachets or pellets.
  • liposomal or proteinoid encapsulation may be used to formulate the compositions as for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673.
  • Liposomal encapsulation may include liposomes that are derivatized with various polymers, e.g., U.S. Pat. No. 5,013,556.
  • the formulation will include a compound of the invention and inert ingredients which protect against degradation in the stomach and which permit release of the biologically active material in the intestine.
  • the inhibitors can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
  • the capsules could be prepared by compression.
  • the preferred embodiment inhibitors DMAT and TBB can be used and administered in a variety of forms, vehicles, and concentrations. Generally, the preferred embodiment inhibitors are used in conjunction with a vehicle such as DMSO, however a wide array of other vehicles may be employed.
  • the inhibitor DMAT can be used so as to achieve in vivo or ex vivo concentrations in the vicinity of the cells of interest, ranging from as low as 0.1 ⁇ M to as high as 1 ,000 ⁇ M or more, however a preferred concentration range is from about 1 ⁇ M to about 100 ⁇ M and more preferably, from about 10 ⁇ M to about 50 ⁇ M.
  • the inhibitor TBB can be used so as to achieve in vivo or ex vivo concentrations in the vicinity of the cells of interest, ranging from as low as 0.1 ⁇ M to as high as 1 ,000 ⁇ M or more, however a preferred concentration range is from about 1 ⁇ M to about 150 ⁇ M and more preferably, from about 15 ⁇ M to about 75 ⁇ M. Generally, these concentrations are designated as effective amounts.
  • the instant pharmaceutical composition will generally contain a per dosage unit (e.g., tablet, capsule, powder, injection, teaspoonful and the like) from about 0.001 to about 100 mg/kg.
  • the instant pharmaceutical composition contains a per dosage unit of from about 0.01 to about 50 mg/kg of compound, and preferably from about 0.05 to about 20 mg/kg.
  • Methods are known in the art for determining therapeutically effective doses for the instant pharmaceutical composition.
  • the therapeutically effective amount for administering the pharmaceutical composition to a human for example, can be determined mathematically from the results of animal studies.
  • the present invention provides methods for treating a wide array of diseases, and preferably various types of cancers. Most preferably, the present invention methods can be utilized to treat diseases characterized by over- proliferation of malignant cells, and most notably, chronic myelogenous leukemia, breast cancer, colon cancer, and skin cancer. Indications for treating other types of cancers are described later herein, in a preferred treatment method, an effective amount of one or more preferred CK2a inhibitors) is administered to a subject in need of treatment for a duration sufficient to induce proliferation arrest while maintaining a steady cell number. Preferably, the duration ranges from about 1 to about 14 days, and more preferably from about 3 to about 7 days.
  • the one or more preferred inhibitor(s) can be administered multiple times per day so as to produce a preferred effective amount. In addition, it is preferred that prolonged treatment strategies can be defined in accordance with the present invention.
  • the present invention provides methods for treating a wide array of myeloproliferative disorders, and in particular, for treating chronic myelogenous leukemia.
  • the present invention also provides methods for treating various hematological malignancies, and in particular, for inhibiting hematological malignancies, inducing maturation of malignant megakaryoblasts, inducing thrombocytosis, reducing platelet production otherwise occurring from malignant megakaryoblasts, and methods for inducing thrombocytopoiesis.
  • the present invention provides strategies for treating cancers such as breast cancer, colon cancer, and skin cancer.
  • the present invention also provides methods for treating renal cell carcinoma, bladder cancer, and glioblastoma.
  • an effective amount of one or more preferred CK2a inhibitors) is administered to a subject for a duration sufficient to induce thrombocytopoiesis.
  • the duration ranges from about 1 to about 14 days, and more preferably from about 3 to about 7 days.
  • the one or more preferred inhibitors) can be administered multiple times per day so as to produce a preferred effective amount.
  • prolonged treatment strategies can be defined in accordance with the present invention.
  • the inhibitor compositions may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug product.
  • Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual to be treated. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration.
  • the optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage, see, for example, Remington's Pharmaceutical Sciences, pp. 1435-1712. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents.
  • a suitable dose may be calculated according to body weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein, as well as the pharmacokinetic data observed in human clinical trials. Appropriate dosages may be ascertained by using established assays for determining blood level dosages in conjunction with an appropriate physician considering various factors which modify the action of drugs, e.g., the drug's specific activity, the severity of the indication, and the responsiveness of the individual, the age, condition, body weight, sex and diet of the individual, the time of administration and other clinical factors.
  • the term "effective amount" means a dosage sufficient to produce a desired or stated effect.
  • leukemia generally refers to cancers that are characterized by an uncontrolled increase in the number of at least one leukocyte and/or leukocyte precursor in the blood and/or bone marrow.
  • Leukemias including but not limited to acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); and, hairy cell leukemia are contemplated.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • hairy cell leukemia hairy cell leukemia
  • the methods of the invention may be applied to cell populations in vivo or ex vivo.
  • "In vivo" means within a living individual, as within an animal or human.
  • the methods of the invention may be used therapeutically in an individual, as described herein.
  • Ex vivo means outside of a living individual.
  • ex vivo cell populations include in vitro cell cultures and biological samples including but not limited to fluid or tissue samples obtained from individuals. Such samples may be obtained by methods well known in the art.
  • Exemplary biological fluid samples include blood, cerebrospinal fluid, urine, saliva.
  • Exemplary tissue samples include tumors and biopsies thereof.
  • the invention may be used for a variety of purposes, including therapeutic and experimental purposes. Information gleaned from such use may be used for experimental purposes or in the clinic to set protocols for in vivo treatment. Other ex vivo uses for which the invention may be suited are described below or will become apparent to those skilled in the art.
  • Ex vivo applications include in vitro applications, studies, and investigations.
  • the treatment methods of the invention are useful in the fields of human medicine and veterinary medicine.
  • the individual to be treated may be a mammal, preferably human, or other animals.
  • individuals include but are not limited to farm animals including cows, sheep, pigs, horses, and goats; companion animals such as dogs and cats; exotic and/or zoo animals; laboratory animals including mice, rats, rabbits, guinea pigs, and hamsters; and poultry such as chickens, turkeys, ducks, and geese.
  • “Pharmaceutically acceptable salts” means any salts that are physiologically acceptable insofar as they are compatible with other ingredients of the formulation and not deleterious to the recipient thereof. Some specific preferred examples are: acetate, trifluoroacetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, oxalate.
  • prodrug refers to compounds that are rapidly transformed in vivo to a more pharmacologically active compound. Prodrug design is discussed generally in Hardma et al. (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th ed., pp. 11-16 (1996). A thorough discussion is provided in Higuchi et al., Prodrugs as Novel Delivery Systems, Vol. 14, ASCD Symposium Series, and in Roche (ed.), Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).
  • the inhibitors of the invention may be covalently or noncovalently associated with a carrier molecule including but not limited to a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see U.S. Pat. Nos. 4,289,872 and 5,229,490; PCT Publication No. WO 93/21259), a lipid, a cholesterol group (such as a steroid), or a carbohydrate or oligosaccharide.
  • a carrier molecule including but not limited to a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see U.S. Pat. Nos. 4,289,872 and 5,229,490; PCT Publication No. WO 93/21259), a lipid, a cholesterol group (such as a steroid), or a carbohydrate or oligos
  • carriers for use in the pharmaceutical compositions of the invention include carbohydrate-based polymers such as trehalose, mannitol, xylitol, sucrose, lactose, sorbitol, dextrans such as cyclodextran, cellulose, and cellulose derivatives. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
  • Other carriers include one or more water soluble polymer attachments such as polyoxyethylene glycol, or polypropylene glycol as described U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301 ,144, 4,670,417, 4,791 ,192 and 4,179,337.
  • Still other useful carrier polymers known in the art include monomethoxy-polyethylene glycol, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers.
  • kits for disease diagnosis, prognosis, risk assessment, and/or treatment efficacy determination are useful in a clinical setting for use in diagnosing a patient for a disease, monitoring the disease progression, testing patient's samples (e.g. biopsied), for example, to determine or predict if the patient's disease (e.g., cancer) will be resistant or sensitive to a given treatment or therapy with a drug, compound, chemotherapy agent, or biological treatment agent.
  • hypotheses presented herein were evaluated by a two pronged approach: 1 ) studies using direct inhibition of malignant cells growth in culture and xenografts in mice; and 2) studies to identify the molecular mechanism by which CK2 inhibition induces arrest of proliferation and apoptosis only in malignant cells.
  • Cell biology experiments aimed to identify nuclear proteins responsible for cell growth were employed.
  • Four cell lines presented herein were used plus several other cell lines (renal, bladder, and brain).
  • MEG-01 megakaryoblastic cell line was selected and characterized as being early stage megakaryoblasts with Philadelphia positive chromosome, isolated from a patient with CML, in blast crisis. These cells are extremely malignant with an increased proliferation rate.
  • MEG-01 megakaryoblastic cell line was a generous gift from Dr. P. B. Tracy, (Department of Biochemistry, University of Vermont, College of Medicine, Burlington, VT, USA). MEG-01 cells were also purchased from American Tissue Culture Collection (Manassas, VA).
  • Cells were maintained in an incubator, with a humidified atmosphere of CO 2 5%, and at 37 0 C.
  • Cell culture media was RPMI 1640 1 X with L-Glutamine (2mM) from Central Cell Services, Media Lab, (Lerner Research Institute, Cleveland Clinic, Cleveland, USA), and adjusted to contain 10 mM HEPES (Invitrogen, Carlsbad, CA, USA), 1.0 mM Sodium Pyruvate (Invitrogen, Carlsbad, CA, USA), 10 % heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA), and Penicillin/Streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were seeded at 2 X 10 5 cells/ml, media was renewed and cell number adjusted two times per week.
  • CK2 ⁇ inhibitors 4,5,6, 7-Tetrabromobenzotriazole (TBB), and 2-Dimethylamino- 4,5,6,7-tetrabromo-1H-benzimidazole (DMAT).
  • TBB dimethylsulfoxide
  • PMA Phorbol 12-myristate 13-acetate
  • Apoptosis and viability assays were performed to choose the non-cytotoxic concentrations of TBB and DMAT that have a significant effect. At the beginning of each treatment cells were counted with a hemacytometer and were split to approximately 2X10 5 cells/ml. To assess the effect of CK2 inhibitors, the treatment lengths went up to 4 days without splitting.
  • CD62P anti human P-Selectin monoclonal antibody conjugated with FITC
  • CD41a anti human ⁇ n b ⁇ 3 monoclonal antibody conjugated with RPE
  • PAC-1 antibody conjugated and Annexin V conjugated with FITC and Pl (Annexin V-FITC and Pl Apoptosis Kit I)
  • Human thrombin was purchased from Haematologic Technologies Inc, Essex Junction, VT, USA.
  • Flow cytometric analysis Cells were analyzed using a FACSCalibur flow cytofluorometer (Becton-Dickinson), with CellQuestPro ver.3.3 software. The data was further analyzed with FlowJo ver. 6.2, and WinMDI 2.8 software. Proper gating was performed to characterize each cell population (MEG-01 cells and platelets). The population that corresponds to platelets (small, granulated particles), is Pl negative because thrombocytes are anucleated cells (only the viable cells were considered) and were distinguished by their capacity to become activated, undergoing shape change in response to agonist and to show phosphatidylserine (PS) exposure when activated.
  • PS phosphatidylserine
  • Platelets were further separated from the megakaryocyte cells, by differential centrifugation, considering the size difference between these cell populations (1-5 ⁇ m for platelets and 35-150 ⁇ m for megakaryocyte cell line MEG-01 ) and analyzed separately. Voltage and channels settings were adjusted accordingly. Analyzed values were obtained with WinMDI ver.2.8.
  • Pl Flow Cytometry with Annexin V-FITC and propidium iodide
  • the staurosporine treated cells were stained as follows: control 1 with Pl, control 2 with Annexin V-FITC and control 3 both Pl and Annexin V-FITC in order to have the brightest controls for compensation and proper collection of the flow cytometry data.
  • Cell necrosis was induced by heat shock (65°C for 30 minutes).
  • Annexin V-FITC corresponds to FL1 H channel, and Pl to FL3H or FL2H channels.
  • Annexin V-FITC and Pl apoptosis assay staining protocol.
  • Annexin V-FITC and Pl kit from BD Biosciences, CA, USA, was used. 10 5 - 10 6 cells were stained with 50 ⁇ g/ml Pl and with 0.5 ⁇ g/ml FITC-labeled Annexin V using the staining protocol provided by the supplier. Samples were then analyzed immediately by flow cytometry.
  • Flow cytometric DNA content assessment assay using PI/RNAse A Cells were serum starved in order to synchronize them in the GO phase. Treatment with DMAT 10 ⁇ M for a period of 4 days was performed and then cells were collected for further processing. Cells were fixed in 80% cold Ethanol/PBS added drop-wise. Before Pl staining cells were washed with sterile, RNAse, DNAse free, PBS buffer. Fixed cells were incubated with 50 ⁇ g/ml Pl and 100 ⁇ g/ml RNAse A-I in hypotonic citrate staining buffer for 30 minutes in dark, at room temperature. [00135] RPE-CD41a immunophenotyping of MEG-01 cells.
  • CD41a is the antigen for ⁇ n b ⁇ 3 complex and it is found on platelets and platelet precursors, including MEG-01 cell line.
  • ⁇ n b ⁇ 3 complex is a marker of differentiation for megakaryocytes.
  • the staining protocol provided by BD Biosciences was used. Briefly, cells were washed and resuspended in 1 X PBS with 0.1 % FBS, 0.01 % NaISb, and 0.22 ⁇ m filtered buffer. Cells were counted and adjusted to 10 6 cells/ml and 20 ⁇ l of RPE-CD41a was used for 180 ⁇ l cell suspension. RPE-CD41a stained cells were collected on FL2H channel and gating was performed on FSC and SSC logarithmic modes.
  • the unstained control signal was subtracted from the stained cell signal, in order to measure the staining of the cells without background noise.
  • Platelets isolation from culture Platelets were separated from the megakaryocytic cells, by differential centrifugation and analyzed separately. Suspension cells were centrifuged first at 100 g - 150 g for 15 minutes, and then the platelets rich supernatant was kept and centrifuged again at 800 g for 15 minutes. For impeding artefactual aggregation of platelets in the control, EDTA and RGD or RGDS were added in the cell suspension from the beginning of the centrifugations, and with each centrifugation step.
  • PAC-I-FITC binding due to platelet activation flow cytometric assay Monoclonal antibody PAC-1 recognizes an epitope on the glycoprotein ⁇ n b ⁇ 3 of activated platelets. PAC-1 binds only to the activated platelets. PAC-1 will not bind EDTA and RGD or RGDS treated platelets. 20 ⁇ l PAC-1 -FITC were used for 5 ⁇ l fresh platelets suspension, in Tyrode's buffer with CaCI 2 , Activation of platelets with 1 ⁇ g/ml TRAP was performed for 10 minutes. The protocol provided by BD Biosciences was used for staining.
  • CD62P-FITC exposure due to platelet activation flow cytometric assay CD62P is a monoclonal antibody that recognizes an epitope on P-Selectin. P-Selectin is exposed as response to agonist and is a specific sign of platelet activation. 20 ⁇ l CD62P-FITC were used for 5 ⁇ l fresh platelets suspension, in Tyrode's buffer with CaCI 2 . Activation of platelets with 1 ⁇ g/ml TRAP was performed for 10 minutes. Incubation was performed in the dark at room temperature for 30 minutes, as recommended by BD Biosciences. [00139] Fibrinogen-Alexa Fluor 488 binding to platelets flow cytometric assay.
  • Viability (proliferation) assay Trypan blue exclusion. Cells were counted using a Neubauer hemacytometer. Trypan blue dye was used according to the manufacturer (Sigma-Aldrich). DMSO, which is the vehicle for TBB, DMAT and PMA, was used as a mock control, considering the highest amount that was used as a vehicle for TBB and DMAT.
  • SEM Scanning electron microscopy
  • mice were used, provided and housed by Dr. Lindner, DJ from Taussig Cancer Center, Cleveland Clinic and Case Western Reserve University. The mice were checked every day. The mice were housed in filtered air flow cabinets with autoclaved bedding at a density of 5 mice/cage. They were fed autoclaved Purina Lab Rodent Chow® 5010 and HCI-acidified distilled water ad libitum and were placed in rooms with controlled temperature, humidity and 12-hr light-dark cycles.
  • MEG-01 myeloid blast crisis cells (10*10 6 cells/100 ⁇ l in cell culture media (RPMI 1640 1X, 10% FBS) were injected subcutaneously into the lower flanks of mice (left and right). Cells were counted with a hemacytometer using Trypan blue (only live cells were counted).
  • mice After 10 days the mice developed tumors large enough to start the treatment. Tumors were visible after 6-7 days from inoculation. The average weight of the mice typically ranged 30-35 g.
  • Treatment with DMAT was started when tumors were at least 100-200 mm 3 volumes (prolate spheroid). Tumor volumes were calculated as prolate spheroid (4/3 * ⁇ * (a) 2* (b), where "2a” is the minor axis and "2b” is the major axis of the prolate spheroid. "2a” and “2b” were measured with a caliper (mm). Animals were treated with DMAT for approximately 2 weeks. When tumor volumes reached a size unacceptable with the IACUC protocols, animals were sacrificed in a CO 2 euthanasia chamber. Tumors were collected for further histological analysis.
  • mice were sacrificed and tumors and organs were collected. Samples were fixed in formalin for further processing.
  • mice were supposed to necropsy. Tumors were collected from under the skin from both flanks and were measured for the last time and then fixed in formalin fixative. Spleen, liver, kidney, brain, lungs and legs were collected and fixed. Spleens were also measured (as length, mm). Fresh tissue was immersed immediately into liquid nitrogen and kept frozen at -8O 0 C. Fixed tissue was embedded in paraffin and next processed for H&E (the basic dye hematoxylin, and the alcohol-based synthetic material, eosin) by the Cleveland Clinic Histology Core facility.
  • H&E the basic dye hematoxylin, and the alcohol-based synthetic material, eosin
  • mice blood collection and blood counts Blood counts are dependent upon the method and time of blood collection. Whole blood was collected from the retro-orbital sinus (under the eye) of anesthesiated mice (both DMSO treated and DMAT treated batch). EDTA and prostaglandin E1 (PGE1 ) were used at collection to prevent clotting during blood collection. 500 ⁇ l mice whole blood with 100 ⁇ l anticoagulant were used for counting (a 1 :5 dilution). A hematological analyzer was used for this. Samples were compared (gated) with normal mice (C57BL strain).
  • Tail-bleeding assay Tail-bleeding assay. Tail-bleeding times are important to investigate whether the platelets could establish hemostasis in vivo. Platelet aggregation and clot retraction in response to physiologic agonists adenosine diphosphate (ADP), epinephrine, and thrombin will affect tail-bleeding times.
  • ADP adenosine diphosphate
  • epinephrine epinephrine
  • thrombin thrombin
  • Normal tail-bleeding times are an average of 1.5-2 minutes in C57BL mouse strain.
  • Pre-warm tubes of saline (PBS) at 37 0 C and maintained at this temperature during the measurements were used. Inhalation of isoflurane vapor or, alternatively, intraperitoneal injection of avertin was used to induce general anesthesia. Using a sharp new razor or scalpel blade, tails were cut exactly 0.5 cm of the distal tip of the tail of the adult mouse and immediately inserted into the pre- warmed tube of saline. A stopclock was started at this time. The tail was hold gently, near its base, to avoid a "torniquet effect.” Venous blood flowing into the tube can be observed and can it can be detected when this bleeding stops. The stopclock provides an accurate bleeding time.
  • Cells in cell culture media were injected.
  • Cells (4*10 6 cells/100 ⁇ l MCF-7 cells, 2 X 10 6 cells/100 ⁇ l SW-480 and 3 X 10 6 cells/100 ⁇ l WM- 164) in cell culture media (DMEM F12, 10% FBS) were injected subcutaneously into the lower flanks of mice (left and right). Cells were counted with a hemacytometer using Trypan blue (only live cells were counted).
  • Tumors were visible after 6-7 days from inoculation. The average weight of the mice used in these sets of experiments was 30-35 g for male mice and 20-25 g for female mice. Female mice must be used for MCF-7 xenografts, because require hormone supplementation (estradiol). This hormone was provided in drinking water with glucose to be more paleatable.
  • Treatment with DMAT was started when tumors were at least 100-200 mm 3 volumes (prolate spheroid). Tumor volumes were calculated as prolate spheroid (4/3* ⁇ r*(a)2*(b), where "2a" is the minor axis and "2b" is the major axis of the prolate spheroid.
  • Tumor diameters were measured using a caliper and tumor volume was calculated using the prolate-spheroid formula.
  • DMAT in DMSO as well as just DMSO as a control were administered by injection subcutaneous in the neck (exogenous from the tumor). Tumor measurements will show if DMAT induces tumor ablation in MCF-7 xenografts.
  • DMAT in DMSO as well as just DMSO as a control were administered by injection subcutaneous in the neck (exogenous from the tumor). Tumor measurements will show if DMAT induces tumor ablation in SW-480 xenografts.
  • mice were supposed to necropsy. Tumors were collected from under the skin from both flanks and were measured for the last time and then fixed in formalin fixative. Spleen, liver, kidney, brain, lungs and legs were collected and fixed. Spleens were also measured (as length, mm). Fresh tissue was immersed immediately into liquid nitrogen and kept frozen at -8O 0 C. Fixed tissue was embedded in paraffin and next processed for H&E (the basic dye hematoxylin, and the alcohol-based synthetic material, eosin) by the Cleveland Clinic Histology Core facility.
  • H&E the basic dye hematoxylin, and the alcohol-based synthetic material, eosin
  • Sections (4- ⁇ m thick) were stained with hematoxylin and eosin and evaluated for pathologic changes in a blinded fashion. H&E staining gives morphological information (vascularization, normal proliferating tissue, necrosis and apoptosis of the tissue).
  • CK2 ⁇ the inhibition of CK2 ⁇ was investigated. Specifically, inhibition of CK2 ⁇ with the preferred inhibitors induced thrombocytopoiesis, forming proplatelets from a demarcation membrane system.
  • Fig. 1 the photograph on the left illustrates a MEG-01 cell prior to thrombocytopoiesis.
  • the photograph on the right illustrates a MEG-01 cell undergoing thrombocytopoiesis, at 20,000 X magnification, and 60 kV.
  • the MEG-01 cells in the photographs of Fig. 1 were treated with DMAT 10 ⁇ M for 4 days.
  • Fig. 2 includes photographs of MEG-01 cells undergoing thrombocytopoiesis, at 20,000 X magnification, and 60 kV. The cells were treated with DMAT 10 ⁇ M for 4 days.
  • Figs. 3-5 illustrate MEG-01 cells producing platelets after treatment with a preferred inhibitor.
  • Fig. 3 shows MEG-01 cells producing platelets, the image obtained from confocal microscopy (Phalloidin-Alexa Fluor 488 in DAPI mounting media) at 63 X magnification.
  • Fig. 4 shows MEG-01 cells producing platelets, with Phalloidin only, at 63X.
  • Fig. 5 shows MEG-01 cells producing platelets, with Phalloidin only, at 63X and 8X digital zoom.
  • Figs. 6 and 7 illustrate that platelets produced from the MEG-01 cell line, treated with the preferred embodiment inhibitor DMAT, respond to thrombin.
  • Fig. 6 is a photograph of a resting platelet-particle from MEG-01 cells, at 10,000 X and 60 kV.
  • Fig. 7 is a photograph of an activated platelet-particle from MEG-01 cells, at 20,000 X and 60 kV.
  • CK2 inhibitors DMAT and TBB induces proliferation arrest and decreases the tumorogenicity (anchorage independence) of these malignant megakaryoblasts.
  • Fig. 8 illustrates cell proliferation (viability) assay, with Trypan Blue exclusion of five days of treatment of MEG-01 cells. Open squares represent control untreated, open triangles 50 ⁇ M TBB, filled triangles 100 ⁇ M TBB, open circles 25 ⁇ M DMAT, filled circles 50 ⁇ M DMAT and open diamonds PMA 5 nM. Each day quadruplicate measurements were taken and triplicate sets of experiments were considered for the measurements.
  • CK2 ⁇ inhibitors TBB and DMAT
  • TBB and DMAT Trypan blue proliferation assay
  • FIG. 9 illustrates control untreated colony formation in soft agar by MEG-01 cells, magnification X20, phase-contrast micrograph.
  • Fig. 10 shows DMAT treated (25 ⁇ M) colony formation in soft agar by MEG-01 cells, magnification X20, phase-contrast micrograph.
  • Fig. 11 illustrates anchorage independence assay in soft agar. Comparison of colonies areas (pixels) between control untreated and DMAT treated MEG-01 cells. Anchorage independence of growth in soft agar assay is strongly connected with tumorogenicity and invasiveness.
  • RPECD41 a ( ⁇ n b ⁇ 3 integrin expression) flow cytometric immunophenotyping for DMAT, TBB and PMA treatments versus control untreated is indicated as follows. Histogram shows results from one set of treatments (total relative fluorescence). Control untreated unstained - plot A, control untreated stained - plot B, 10 ⁇ M DMAT - plot C, 25 ⁇ M TBB - plot D, 1 nM PMA treatments - plot E. Fig. 12 shows that 10 ⁇ M DMAT is sufficient to obtain significant maturation levels compared to the control untreated cells. A further increase in the concentration of DMAT (up to 20 ⁇ M) induces a slight increase in the maturation level of MEG-01 cells. In Fig.
  • the graph represents total relative fluorescence percentages for RPE-CD41a immunophenotyping, conform analysis of data in WinMDI ver 2.8, from triplicate experiments.
  • Fig. 13 also shows that 20 ⁇ M DMAT induced a similar maturation level as 1 nM PMA and 25 ⁇ M TBB.
  • DNA content analysis is shown of MEG-01 cells treated with 10 ⁇ M DMAT for 4 days, as assessed by Pl and RNAase A, flow cytometric assay.
  • DNA content assay demonstrates that MEG-01 cells become polyploid (ploidy higher than 2N) in the presence of 10 ⁇ M DMAT.
  • the increase in DNA content and cell size demonstrate that MEG-01 undergo maturation in the presence of CK2 ⁇ inhibitors treatments.
  • the data demonstrate that inhibition of CK2 in MEG-01 cells results in proliferation arrest followed by maturation of the cells.
  • Apoptosis and phenotype change induced by CK2 inhibitors (DMAT and TBB) in leukemia megakaryoblasts are dose and time dependent and indicated as follows. Percentages of total apoptotic cells are the sum of (FL1 H+, FL3H-) lower right quadrant, corresponding to early apoptotic cells gate, with (FL1 H+, FL3H+) upper right quadrant, corresponding to late apoptotic cells.
  • apoptotic cells percentages and controls were plotted for each treatment set for each day.
  • quadrant gating of a control untreated MEG-01 cells after 24 hours (Annexin V- FITC and Pl flow cytometric assay). Necrotic cells correspond to the upper left quadrant gate (FL1 H-, FL3H-).
  • quadrant gating of cells treated with 20 ⁇ M DMAT following 24 hours Fig. 15 demonstrates that following 24 hours incubation in the absence of CK2 inhibitors, 8.7% of the control untreated cells are apoptotic while 0.87% are necrotic.
  • FIG. 17 A comparative summary of the results obtained following 24 hours incubation with either TBB or DMAT is provided in Fig. 17.
  • Fig. 17 the effect of TBB and DMAT treatment on MEG-01 cells apoptosis after 24 hours.
  • Fig. 18 the effect of TBB and DMAT treatment on MEG-01 cells apoptosis after 48 hours
  • Fig. 19 the effect of TBB and DMAT treatments on MEG-01 cells apoptosis after 72 hours.
  • Fig. 20 the effect of TBB and DMAT treatments on MEG-01 cells apoptosis after 96 hours.
  • Figs. 17-20 represent the average found in three independent experiments.
  • the data shown in Figs. 18-20 demonstrate that the effect is dose dependent and reaches a maximum after four days. Following 96 hours incubation the results obtained with 10 ⁇ M DMAT are similar to the results obtained with 20 ⁇ M inhibitor.
  • a direct comparison between control cells and DMAT-treated cells establish that treatment with DMAT induces significant apoptosis in MEG-01.
  • Figs. 21-27 Phenotype change in MEG-01 cells following treatment with 10 ⁇ M DMAT is shown.
  • Fig. 21 illustrates control untreated MEG-01 cells phase-contrast micrograph after 96 hours, magnification X20
  • Fig. 22 shows MEG-01 cells treated with 10 ⁇ M DMAT phase- contrast micrograph, following 96 hours of treatment same magnification.
  • Fig. 23 shows proplatelets formation, in suspension, phase-contrast micrograph, following DMAT treatment (10 ⁇ M) between 72 and 96 hours, magnification X40.
  • Fig. 21-27 Phenotype change in MEG-01 cells following treatment with 10 ⁇ M DMAT is shown.
  • Fig. 21 illustrates control untreated MEG-01 cells phase-contrast micrograph after 96 hours, magnification X20
  • Fig. 22 shows MEG-01 cells treated with 10 ⁇ M DMAT phase- contrast micrograph, following 96 hours of treatment same magnification.
  • Fig. 23 shows proplatelets formation, in
  • FIG. 24 shows scanning electron microscopy micrograph of MEG-01 cells, magnification X 7500, voltage 15 kV.
  • Fig. 25 illustrates proplatelets formation on fibronectin coat, phase-contrast micrograph, following DMAT treatment (10 ⁇ M) between 72 and 96 hours of treatment, magnification X40.
  • Fig. 26 shows DAPI staining micrograph of proplatelets bearing MEG-01 megakaryocyte following DMAT treatment (10 ⁇ M) between 72 and 96 hours of treatment, magnification X40.
  • Fig. 27 shows platelets- like particles identified as anucleated cells with DAPI staining, magnification X40 following DMAT (10 ⁇ M treatment) at 72 to 96 hours.
  • Figs. 21-27 demonstrate that CK2 ⁇ inhibition result in thrombocytopoiesis.
  • DMAT induced MEG-01 cells to form proplatelet extensions. This process was dramatically enhanced following four days of treatment (Figs. 21-26).
  • the megakaryocytes undergoing thrombocytopoiesis showed apoptotic features, DNA condensation and fragmentation (Figs. 23 and 25). Following explosive fragmentation, long filaments with beaded ends (proplatelets) are formed. Similar results were observed with MEG-01 in suspension (Fig. 25) and MEG-01 cells grown on fibronectin (Fig. 23).
  • Fig. 26 shows scanning electron microscopy (SEM) of MEG-01 cells treated with 10 ⁇ M DMAT for 4 days.
  • Figs. 28-31 platelets from MEG-01 cells obtained due to DMAT treatment, in culture, are demonstrated to be functional. MEG-01 -derived platelets following treatment with DMAT (10 ⁇ M for 72) were collected and used. Platelets were activated with TRAP peptide. Controls were treated with EDTA and RGDS to prevent any artefactual activation.
  • Fig. 28 illustrates P-Selectin exposure (CD62P - FITC) by activated platelets. Fig.
  • FIG. 29 shows PAC-1 binding (PAC-1 -FITC) by activated platelets.
  • Fig. 30 illustrates Fibrinogen-Alexa Fluor 488 binding to activated platelets.
  • Fig. 31 illustrates Annexin V-FITC binding to activated platelets (phosphatidylserine exposure).
  • the control platelets are represented by line A, while the results obtained with activated platelets are represented by the line B.
  • the platelets are capable of undergoing shape change in response to agonists (human thrombin, TRAP, ADP, and PMA).
  • Activated platelets stain positive for PAC-1 (an antibody that recognizes a specific epitope on ⁇ n b ⁇ 3 integrin, exposed only when platelets are activated) (Fig. 28).
  • FIG. 32 details of the fibrin clot magnification X 5,000, voltage 20 kV are shown.
  • Fig. 33 platelets and fibrin net detail, magnification X 7,500, voltage 20 kV are shown.
  • Fig. 34 fibrin net detail from the clot, magnification X 20,000 voltage 20 kV is shown. [00181] Fig.
  • Fig. 35 is a graph of a murine xenograft treated with DMAT, compared to a control. Specifically, mice with subcutaneous tumors of MEG-01 cells were treated with DMAT 2 mg (in DMSO) per animal per day. The daily animal weight was averaged at 32.5 g at the start of the treatment. Tumors were measured daily and tumor volumes were calculated based upon a prolate spheroid model.
  • Fig. 35 illustrates the relatively rapid growth of tumor volume of the control (squares) as compared to tumor volume of DMAT treated MEG-01 cells (triangles).
  • Fig. 35 further illustrates that DMAT induces proliferation arrest in vivo in large tumors and tumor ablation in small tumors, the arrest being dose and time dependent. [00182] Fig.
  • FIG. 36 is a graph of a murine xenograft treated with DMAT, compared to a control.
  • Mice with subcutaneous tumors of MEG-01 cells were treated with DMAT 3 mg (in DMSO) per animal per day. The daily animal weight was averaged at 32.5 g at the start of treatment. Tumors were measured daily and tumor volumes were calculated based upon a prolate spheroid model.
  • Fig. 36 illustrates the significant benefits of treatment with DMAT (triangles) as compared to the control (squares).
  • Fig. 36 further illustrates DMAT induces proliferation arrest in vivo for large tumors and tumor ablation in small tumors, such effect being dose and time dependent.
  • Figs. 37 and 38 illustrate additional aspects of the MEG-01 xenografts.
  • Fig. 37 shows an increase in platelet counts in a batch of MEG-01 mice xenografts treated with DMAT 50 ⁇ l/day of 125 mg compared to a control.
  • Fig. 38 illustrates the percentage of abnormal cells in blood counts of MEG-01 xenografts of a control, DMAT treated MEG-01 cells, and normal mice.
  • Figs. 37 and 38 demonstrate that MEG-01 xenograft mice have abnormally high platelet counts, i.e. both with regard to a DMSO control and DMAT-treated specimens.
  • Fig. 39 is a graph illustrating the results of tail bleeding times in MEG- 01 xenograft mice.
  • MEG-01 tumors produce platelets in vivo. This is similar as to what happens in acute myelogenous leukemia (CML).
  • CML acute myelogenous leukemia
  • This data illustrates that tail bleeding times in the xenograft mice (triangles) are in certain instances, longer than tail bleeding times in normal mice (circles) or control mice (squares). This is believed to be a result of DMAT being a CK2 inhibitor (such as for example, like heparin).
  • Fig. 40 is a graph illustrating comparative spleen sizes between MEG- 01 xenografts (treated with DMAT and a control) and that of normal mice. Spleen size differences between the treated and the control may be due to the fact that MEG-01 tumors produce platelets and circulating blasts in vivo and the controls have larger tumors than the treated and more abnormal cells. This same observation is also valid for the treated xenograft only. This phenomena is similar with what happens in chronic myelogenous leukemia (CML).
  • CML chronic myelogenous leukemia
  • Fig. 41 illustrates apoptotic-necrotic areas in MEG-01 tumors identified from the histological stains hematoxylin and eosin.
  • Fig. 41 shows the significantly greater areas of apoptosis and necrosis of a DMAT treated (10 ⁇ M) MEG-01 cell line as compared to a control.
  • Fig. 42 illustrates an area of angiogenesis of a DMAT treated (10 ⁇ M) MEG-01 cell line as compared to a control.
  • Angiogenesis refers to blood vessel formation which usually accompanies the growth of malignant tissue.
  • MCF-7 is a breast cancer cell line established from the mammary gland of a 69 year old woman. It is an adenocarcinoma derived from pleural effusion (metastic site). MCF- 7 are differentiated mammary epithelium cells that express estrogen receptor. MCF- 7 cells express oncogenes (WNT7B and Tx-4) and are sensitive to TNF alpha, which inhibits their growth. MCF-7 growth in vivo is hormone dependent (estradiol). MCF- 7 cells produce insulin-like growth factor binding proteins (IGFBP). IGFBP secretion from MCF-7 can be modulated by treatment with anti-estrogeni. [00189] Fig.
  • IGFBP insulin-like growth factor binding proteins
  • FIG. 43 illustrates a proliferation assay in vitro of MCF-7 cells.
  • Various cell counts were performed over a period of five days.
  • the control squares
  • the MCF-7 cells treated with 20 ⁇ M DMAT exhibited the lowest cell counts.
  • the MCF-7 cells treated with 10 ⁇ M DMAT exhibited cell counts between the control and the MCF-7 cells treated with 20 ⁇ M at 24 hours (Fig. 46).
  • Figs. 44-49 are photographs illustrating the arrest of MCF-7 proliferation, in vitro.
  • Figs. 44-46 are photographs at 5 X showing a control of MCF-7 at 24 hours (Fig. 44), MCF-7 treated with DMAT 10 ⁇ M at 24 hours (Fig. 45), and MCF-7 treated with DMAT 20 ⁇ M at 24 hours (Fig. 46).
  • Figs. 47-49 are photographs at 10 X showing a control of MCF-7 at 24 hours (Fig. 47), MCF-7 treated with DMAT 10 ⁇ M at 24 hours (Fig. 48), and MCF-7 treated with DMAT 20 ⁇ M at 24 hours (Fig. 49).
  • Figs. 50-53 are graphs illustrating assessment of the effect of the preferred inhibitor DMAT on MCF-7 cells in vitro. And so, using an assay employing Annexin V, assessment of both apoptosis and necrotic levels was made.
  • Fig. 50 demonstrates that following 24 hours incubation in the absence of CK2 inhibitors, 41 % of the control untreated cells are apoptotic while 0% are necrotic (Fig. 50). Following 24 hours treatment with 10 ⁇ M DMAT, the level of apoptotic cells significantly increased to 28.4% while the level of necrotic cells remained low at 8.7% (Fig. 51 ).
  • Fig. 53 is a comparative graph illustrating the percentage of apoptotic cells in the control and the MCF-7 DMAT 10 ⁇ M sample and the MCF-7 DMAT 20 ⁇ M sample.
  • Figs. 54-55 are photographs illustrating MCF-7 anchorage independence.
  • Fig. 54 illustrates MCF-7 control on soft agar after one week, at 10 X.
  • Fig. 55 illustrates MCF-7 treated with DMAT 10 ⁇ M on soft agar after one week, at 10 X.
  • Fig. 56 is a comparative graph showing relative area of the control and the noted MCF-7 treated cells.
  • Fig. 57 is a graph of mice injected with the MCF-7 cell line, i.e. a murine xenograft model, illustrating changes in tumor volume over a period of thirteen days.
  • a control squares
  • the MCF-7 cells treated with DMAT at 10 mg/kg (in DMSO) per day exhibited remarkably stable size and nearly no increase over the 13 day period.
  • mice were injected with subcutaneous tumors of MCF-7 cells. Tumors were measured daily and volumes were calculated based on a prolate spheroid model. Animal weight was averaged at 25 g at the beginning of treatment. This investigation demonstrates that DMAT induces proliferation arrest in vivo in large tumors, and tumor ablation in small tumors, and is dose and time dependent.
  • Fig. 58 illustrates relative areas of apoptotic-necrotic MCF-7 cells treated with DMAT 10 ⁇ M.
  • Angiogenesis in MCF-7 cells treated with DMAT 10 ⁇ M is shown in Fig. 59 compared to controls. As previously explained, the cells were stained with hematoxylin and eosin, which are two known histological stains.
  • SW-480 a cell line designated as SW-480 was obtained.
  • SW-480 was established from a 50 year old male.
  • SW-480 are colon epithelial cells from colorectal adenocarcinoma, tumor stage: Dukes' type B.
  • SW-480 cells produce carcinoembryonic antigen (CEA), keratin, transforming growth factor beta.
  • SW-480 cells exhibit epithelial growth factor receptor (EGF).
  • EAF epithelial growth factor receptor
  • Fig. 60 illustrates a proliferation assay in vitro of SW-480 cells. Various cell counts were performed over a period of four days. The control (squares) exhibited the highest number of cells. The SW-480 cells treated with 20 ⁇ M DMAT exhibited the lowest cell counts. The SW-480 cells treated with 10 ⁇ M DMAT exhibited cell counts between the control and the SW-480 cells treated with 20 ⁇ M. [00197] Figs. 61-66 are photographs illustrating the arrest of SW-480 proliferation, in vitro. Figs. 61-63 are photographs at 5 X showing a control of SW- 480 at 24 hours (Fig. 61 ), SW-480 treated with DMAT 10 ⁇ M at 24 hours (Fig.
  • FIGs. 64-66 are photographs at 10 X showing a control of SW-480 at 24 hours (Fig. 64), SW-480 treated with DMAT 10 ⁇ M at 24 hours (Fig. 65), and SW-480 treated with DMAT 20 ⁇ M at 24 hours (Fig. 66).
  • Figs. 67-70 are graphs illustrating assessment of the effect of the preferred inhibitor DMAT on SW-480 cells in vitro. Using an assay employing Annexin V, assessment of both apoptosis and necrotic levels was made.
  • Fig. 67 demonstrates that the following 24 hours incubation in the absence of CK2 inhibitors, 1.9% of the control untreated cells are apoptotic while 3.5% are necrotic. Following 24 hours treatment with 10 ⁇ M DMAT, the level of apoptotic cells significantly increased to 17.5% while the level of necrotic cells remained very low at 1.9% (Fig. 68).
  • Fig. 70 is a comparative graph illustrating the percentage of apoptotic cells in the control and the SW-480 DMAT 10 ⁇ M sample and the SW-480 DMAT 20 ⁇ M sample.
  • Figs. 71-72 are photographs illustrating SW-480 anchorage independence.
  • Fig. 71 illustrates SW-480 control on soft agar after one week, at 10 X.
  • Fig. 72 illustrates SW-480 treated with DMAT 10 ⁇ M on soft agar after one week, at 10 X.
  • Fig. 73 is a comparative graph showing relative area of the control and the noted SW-480 treated cells.
  • Fig. 74 is a graph of a murine xenograft model, i.e. mice injected with the SW-480 cell line, illustrating changes in tumor volume over a period of thirteen days.
  • a control squares
  • the SW-480 cells treated with . DMAT at 40 mg/kg (in DMSO) per day exhibited only a minor increase in tumor volume.
  • mice were injected with subcutaneous tumors of SW-480 cells. Tumors were measured daily and volumes calculated based upon a prolate spheroid model. Animal weight was averaged at 35 g at the start of treatment.
  • Fig. 75 illustrates relative areas of apoptotic-necrotic SW-480 cells treated with DMAT 10 ⁇ M.
  • Fig. 76 illustrates angiogenesis in SW-480 cells treated with DMAT 10 ⁇ M as compared to a control. As previously explained, cells were stained with hematoxylin and eosin.
  • WM-164 a cell line designated as WM-164 was obtained.
  • WM-164 was established from a 21 year old male with nodular melanoma in vertical growth phase.
  • WM-164 are skin melanocytes.
  • WM-164 exhibit spontaneous metastasis into liver and lung.
  • Fig. 77 illustrates a proliferation assay in vitro of WM-164 cells.
  • Cell counts were performed over a period of four days.
  • the control squares
  • the WM-164 cells treated with 20 ⁇ M DMAT exhibited the lowest cell counts.
  • the WM-164 cells treated with 10 ⁇ M DMAT exhibited cell counts between those of the control and those of the WM-164 cells treated with 20 ⁇ M.
  • Figs. 78-83 are photographs showing the arrest of WM-164 proliferation, in vitro.
  • Figs. 78-80 are photographs at 5 X showing a control of WM- 164 at 24 hours (Fig. 78), WM-164 treated with DMAT 10 ⁇ M at 24 hours (Fig. 79), and WM-164 treated with DMAT 20 ⁇ M at 24 hours (Fig. 80).
  • Figs. 81-83 are photographs at 10 X showing a control of WM-164 at 24 hours (Fig. 81), WM-164 treated with DMAT 10 ⁇ M at 24 hours (Fig. 82) and WM-164 treated with DMAT 20 ⁇ M at 24 hours (Fig. 83).
  • Figs. 84-86 are graphs illustrating assessment of the effect of the preferred inhibitor DMAT on WM-164 cells, in vitro. Using an assay employing Annexin V, assessment of both apoptosis and necrotic levels was made.
  • Fig. 84 demonstrates that following 24 hours incubation in the absence of CK2 inhibitors, 16.9% of the control untreated cells are apoptotic while 4.8% are necrotic. Following 24 hours treatment with 10 ⁇ M DMAT, the level of apoptotic cells increased to 36.8% while the level of necrotic cells remained low at 2.0% (Fig. 85). In another sample following treatment with 20 ⁇ M DMAT, the level of apoptic cells was 26.8% while the level of necrotic cells was 4.1 %.
  • Fig. 87 is a comparative graph illustrating the percentage of apoptotic cells in the control and the WM-164 DMAT 10 ⁇ M sample and the WM-164 DMAT 20 ⁇ M sample.
  • Figs. 88-89 are photographs illustrating WM-164 anchorage independence.
  • Fig. 88 shows WM-164 control on soft agar after one week, at 10 X.
  • Fig. 89 shows WM-164 treated with DMAT 10 ⁇ M on soft agar after one week, at 10 X.
  • Fig. 90 is a comparative graph showing relative area of the control and the noted WM-164 treated cells.
  • Fig. 91 is a graph of a murine xenograft model, i.e. mice injected with the WM-164 cell line, illustrating changes in tumor volume over a period of fourteen days.
  • a control squares
  • the WM-164 cells treated with DMAT at 10 mg/kg (in DMSO) per day exhibited only a slight increase in tumor volume.
  • mice were injected with subcutaneous tumors of WM-164 cells. Tumors were measured daily and volumes calculated based upon a prolate spheroid model. Animal weight was averaged at about 35g at the beginning of the study. This study demonstrates that DMAT induces proliferation arrest in vivo for large tumors, and tumor ablation for small tumors, and is also dose and time dependent.
  • Fig. 92 illustrates relative areas of apoptotic-necrotic WM-164 cells treated with DMAT 10 ⁇ M.
  • Fig. 93 illustrates angiogenesis in WM-164 cells treated with DMAT 10 ⁇ M as compared to a control. As previously explained, cells were stained with hematoxylin and eosin.
  • ACTN Renal Cell Carcinoma
  • ACHN cancerous renal cells
  • Fig. 94 illustrates the effect of administration of DMAT as compared to a control over a period of 59 days.
  • Fig. 94 is a graph of a murine xenograft treated with DMAT, compared to a control. Mice with tumors of ACHN cells were treated with DMAT at effective dosage levels (in DMSO) per animal per day. The daily animal weight was averaged at 32.5 g at the start of treatment. Tumors were measured daily and tumor volumes were calculated based upon a prolate spheroid model.
  • Fig. 94 illustrates the significant benefits of treatment with DMAT (circles) as compared to the control (squares).
  • Fig. 94 further illustrates DMAT induces proliferation arrest in vivo for large tumors and tumor ablation in small tumors, such effect being dose and time dependent. The differences in tumor volume between control cells and those treated with DMAT, as described herein, are striking.
  • HT1376 are cancerous bladder cells.
  • Fig. 95 illustrates the effect of administration of DMAT s compared to a control over a time period of 36 days.
  • Fig. 95 is a graph of a murine xenograft treated with DMAT, compared to a control. Mice with tumors of HT1376 cells were treated with DMAT at effective dosage levels (in DMSO) per animal per day. The daily animal weight was averaged at 32.5 g at the start of treatment. Tumors were measured daily and tumor volumes were calculated based upon a prolate spheroid model.
  • Fig. 95 illustrates the significant benefits of treatment with DMAT (circles) as compared to the control (squares). Although the tumor size increased for HT1376 cells treated with DMAT, the tumor volume remained approximately one-half of the size as that associated with the untreated HT1376 cells.
  • U-87 are cancerous brain cells.
  • Fig. 96 illustrates the effect of administration of DMAT as compared to a control, i.e. DMSO, for a period of approximately 24 days.
  • Fig. 96 is a graph of a murine xenograft treated with DMAT, compared to a control. Mice with tumors of U- 87 cells were treated with DMAT at effective dosage levels (in DMSO) per animal per day. The daily animal weight was averaged at 32.5 g at the start of treatment. Tumors were measured daily and tumor volumes were calculated based upon a prolate spheroid model.
  • Fig. 96 illustrates the significant benefits of treatment with DMAT (squares) as compared to the control (diamonds).
  • Fig. 96 further illustrates DMAT induces proliferation arrest in vivo for large tumors and tumor ablation in small tumors, such effect being dose and time dependent. Again, the U-87 brain cells treated with DMAT exhibited significantly less cancerous growth than the untreated control cells.
  • Thrombocytopoiesis process occurred in cells bound to fibronectin matrix as well on cells in suspension.
  • the thrombocytopoiesis process observed in the present study follows the maturation and differentiation process of MEG-01 megakaryoblasts. This differentiation is similar to the effect observed with phorbol ester (PMA), however, CK2 ⁇ inhibitors are not cytotoxic, whereas PMA is a potent tumorigenic substance as well as a powerful platelet activator. It is contemplated that maturation of MEG-01 cells is a result of proliferation arrest that makes incomplete repeated cell cycle to enter into endomitosis, probably due to the action of CK2 on the cell cycle.
  • BCR/ABL was found to be involved in the malignant transformation of Ph+ cells, but its inhibition is not sufficient to suppress anchorage independence of such cells, suggesting involvement of other molecular mechanisms.
  • the results connect CK2 with apoptosis and the mechanism of thrombocytopoiesis that follow megakaryocytopoiesis. It has been previously shown that platelet shedding results from a constitutive form of apoptosis of megakaryocytes. The present inventigation shows for the first time that CK2 inhibition induces release of functional platelets from malignant megakaryoblasts.
  • CK2 inhibition In megakaryoblasts, CK2 inhibition first produces proliferation arrest, followed by differentiation to megakaryocytes that culminate with proplatelets formation, blebbing, and compartmentalized fragmentation of megakaryocytes, finalized by thrombocytes release. Platelets obtained in culture, following CK2 ⁇ inhibition are functional. These platelets form a clot visible with the eye when exposed to agonists. The present invention successfully stopped the abnormal proliferation of a transformed cell line and reversed the path towards its normal function.
  • CK2 ⁇ inhibition studies with TBB and DMAT demonstrate a key role of CK2 in oncogenic development as well as in the megakaryocytopoiesis and thrombocytopoiesis processes. This opens up the possibility of CK2 targeting drug design for patients with cytokine and BCR/ABL inhibitors resistance.
  • mice xenografts using MEG-01 , MCF-7, SW-480 and WM-164 cells
  • the in vivo results demonstrate that the best response to this treatment was obtained with MEG-01 and MCF-7 cells.
  • the data obtained with these two cell lines showed tumor ablation (Figs. 9 and 30 respectively).
  • the data shown in Figures 47 and 64 with the xenografts obtained with SW-480 cells (female mice) and WM-164 cells (male mice) demonstrate significant reduction in tumor volume. However, at the end of the treatment small tumors remained (Figs. 47 and 64). Overall, the data demonstrate that DMAT can be used as a therapy to treat tumors in vivo.
  • DMAT appears to be of utmost importance as a tool in developing a new cancer therapy both because of its efficacy and its apparent lack of toxicity.
  • a protein marker (CK2) has been identified that is translocated to the nucleus in malignant cells. Inhibition of the function of this protein by a specific inhibitor results in the arrest of cell proliferation. Therefore, phosphorylation of nuclear proteins participating in cell growth by CK2 is required for the survival of these malignant cells. Identification of the proteins responsible for abnormal cell proliferation in all these cell lines is a major contribution to the field.
  • SW-480, WM-164, ACHN, HT-1376, U-87 xenograft murine models results in proliferation arrest and tumor ablation, suggesting that small chemical compounds that inhibit kinases have strong potential in cancer treatment.
  • the preferred inhibitor DMAT can be effectively used in a variety of cancer treatment regimes, and specifically, in the treatment of chronic myelogenous leukemia, breast cancer, colon cancer, and melanoma.
  • the other preferred embodiment inhibitor TBB can also be used in corresponding treatment regimes.
  • the present invention is not limited to the use of DMAT and TBB alone or in combination, but also includes the use of other CK2 inhibitors, and particularly,

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Abstract

L'invention porte sur des inhibiteurs de la caséine kinase 2 qui se sont révélés pouvoir arrêter une prolifération cellulaire incontrôlée, suggérant par là leur utilisation dans des stratégies de traitement du cancer. Des applications spécifiques comprennent le traitement du cancer du sein, du cancer du côlon, d'un mélanome, de la leucémie myéloïde chronique, du cancer de la vessie, du cancer du rein et du cancer du cerveau. L'invention porte sur divers procédés et diverses compositions utilisant les inhibiteurs.
PCT/US2008/010275 2007-08-30 2008-08-29 Lutte contre des cellules malignes par inhibition de kinase WO2009032213A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3144014A1 (fr) * 2015-09-21 2017-03-22 Commissariat A L'energie Atomique Et Aux Energies Alternatives Combinaison de médicaments létaux synthétiques pour traiter un carcinome cellulaire rénal

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040121968A1 (en) * 2002-12-23 2004-06-24 Alexander Ljubimov Antiangiogenesis by inhibiting protein kinase CK2 activity
WO2005092866A1 (fr) * 2004-03-29 2005-10-06 Fundacja Rozwoju Diagnostyki I Teratii Nouveaux derives de 4, 5, 6, 7-tetrabromobenzimidazole et procede pour les preparer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040121968A1 (en) * 2002-12-23 2004-06-24 Alexander Ljubimov Antiangiogenesis by inhibiting protein kinase CK2 activity
WO2005092866A1 (fr) * 2004-03-29 2005-10-06 Fundacja Rozwoju Diagnostyki I Teratii Nouveaux derives de 4, 5, 6, 7-tetrabromobenzimidazole et procede pour les preparer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MISHRA: "Protein kinase CKllalpha interacts with the Bcr moiety of Bcr/Abl and mediates proliferation of Bcr/Ab-expressng cells.", ONCOGENE, vol. 22, no. 51, 2003, pages 8255 - 8262, XP055353392 *
PAGANO ET AL.: "2-Dimethylamino-4,5,6,7-tetrabromo-1 H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2.", BIOCHEM. BIOPHYS. RES COMM., vol. 321, 2004, pages 1040 - 1044, XP004523820 *
RUSSO ET AL.: "Biochemical and Functional Characterization of Protein Kinase CK2 in Ascidian Ciona intestinalis Oocytes at Fertilization", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 31, 2004, pages 33012 - 33023, XP055353395 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3144014A1 (fr) * 2015-09-21 2017-03-22 Commissariat A L'energie Atomique Et Aux Energies Alternatives Combinaison de médicaments létaux synthétiques pour traiter un carcinome cellulaire rénal
WO2017050842A1 (fr) * 2015-09-21 2017-03-30 Commissariat A L'energie Atomique Et Aux Energies Alternatives Combinaison de médicaments létaux synthétiques pour traiter le carcinome à cellules rénales
JP2018531224A (ja) * 2015-09-21 2018-10-25 コミッサリア ア レネルジ アトミック エ オー エネルジ アルターネイティブスＣｏｍｍｉｓｓａｒｉａｔ Ａ Ｌ’Ｅｎｅｒｇｉｅ Ａｔｏｍｉｑｕｅ Ｅｔ Ａｕｘ Ｅｎｅｒｇｉｅｓ Ａｌｔｅｒｎａｔｉｖｅｓ 腎細胞癌を治療するための合成致死薬の組み合わせ
US10322133B2 (en) 2015-09-21 2019-06-18 Commissariat A L'energie Atomique Et Aux Energies Alternatives Synthetic lethal drug combination for treating renal cell carcinoma
JP7138563B2 (ja) 2015-09-21 2022-09-16 コミッサリア ア レネルジ アトミック エ オー エネルジ アルターネイティブス 腎細胞癌を治療するための合成致死薬の組み合わせ

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