EP3463319A1 - Méthodes et compositions permettant d'éradiquer des cellules leucémiques - Google Patents

Méthodes et compositions permettant d'éradiquer des cellules leucémiques

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Publication number
EP3463319A1
EP3463319A1 EP17807621.2A EP17807621A EP3463319A1 EP 3463319 A1 EP3463319 A1 EP 3463319A1 EP 17807621 A EP17807621 A EP 17807621A EP 3463319 A1 EP3463319 A1 EP 3463319A1
Authority
EP
European Patent Office
Prior art keywords
cells
subject
leukemic
population
agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP17807621.2A
Other languages
German (de)
English (en)
Other versions
EP3463319A4 (fr
Inventor
Amir SCHAJNOVITZ
David T. Scadden
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harvard College
General Hospital Corp
Original Assignee
Harvard College
General Hospital Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harvard College, General Hospital Corp filed Critical Harvard College
Publication of EP3463319A1 publication Critical patent/EP3463319A1/fr
Publication of EP3463319A4 publication Critical patent/EP3463319A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
    • A61K31/77—Polymers containing oxygen of oxiranes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • Acute myeloid leukemia is a genetically heterogeneous disease of blood stem and myeloid progenitor cells, characterized by the accumulation of malignant blasts in the bone marrow that severely impairs normal blood formation.
  • AML is one of the best characterized malignancies from a genetic viewpoint. Numerous genetic transformation events leading to leukemia have been characterized (Marcucci, et al., J.
  • the inventions disclosed herein related to methods of eradicating leukemic cells in a population of cells, the method comprising contacting the population of cells with an effective amount of an agent (e.g., carbenoxolone or an analog or derivative thereof), thereby eradicating leukemic cells in the cell population.
  • an agent e.g., carbenoxolone or an analog or derivative thereof
  • disclosed herein are methods of treating acute myeloid leukemia in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent (e.g., a glycyrrhetinic acid derivative, carbenoxolone or an analog or derivative thereof), thereby treating acute myeloid leukemia in the subject.
  • an agent e.g., a glycyrrhetinic acid derivative, carbenoxolone or an analog or derivative thereof
  • the inventions disclosed herein relate to methods of promoting the differentiation of a leukemic cell into a non-leukemic cell, the method comprising contacting the leukemic cell with an effective amount of an agent (e.g., 18 - ⁇ -glycyrrhetinic acid, carbenoxolone or an analog or derivative thereof), thereby promoting the differentiation of the leukemic cell into a non-leukemic cell.
  • an agent e.g., 18 - ⁇ -glycyrrhetinic acid, carbenoxolone or an analog or derivative thereof
  • the agents for use in accordance with the inventions disclosed herein comprise a glycyrrhetinic acid derivative.
  • exemplary glycyrrhetinic acid derivatives include glycyrrhizine, glycyrrhizinic acid, 18- -glycyrrhetinic acid,
  • the agent is or comprises 18- -glycyrrhetinic acid or a derivative thereof.
  • the agent is or comprises carbenoxolone or an analog or derivative thereof.
  • the carbenoxolone derivative is a pegylated carbenoxolone.
  • the agent is or comprises a gap junction blocker.
  • the agent is or comprises a hemichannel blocker.
  • the agent is or comprises a blocker or inhibitor of one or more of connexins, pannexins and/or
  • hydroxysteroid dehydrogenase also disclosed are methods of selectively eradicating leukemic cells in a population of cells, the methods comprising contacting the population of cells with an effective amount of an agent (e.g., carbenoxolone or an analog or derivative thereof, carbenoxolone or an analog or derivative thereof) that selectively induces oxidative stress in the leukemic cell, thereby eradicating the leukemic cells in the cell population.
  • the oxidative stress is detected by determining the total levels of reactive oxygen species (ROS) in the population of cells.
  • ROS reactive oxygen species
  • oxidative stress is detected by determining the total levels of mitochondrial reactive oxygen species (ROS) in the population of cells.
  • the present inventions are directed to methods of selectively eradicating leukemic cells in a population of cells, the methods comprising contacting the population of cells with an effective amount of an agent (e.g., a glycyrrhetinic acid derivative, carbenoxolone or an analog or derivative thereof) that selectively induces lipid damage in the leukemic cell, thereby eradicating the leukemic cells in the cell population.
  • an agent e.g., a glycyrrhetinic acid derivative, carbenoxolone or an analog or derivative thereof
  • lipid damage is detected by determining the ratio of oxidized to non-oxidized lipids in the population of cells.
  • lipid damage is detected by detecting lipid peroxidation by flow cytometry.
  • methods of selectively eradicating leukemic cells in a population of cells comprising contacting the population of cells with an effective amount of an agent (e.g., carbenoxolone or analog or derivative thereof) that selectively induces DNA damage in the leukemic cell, thereby eradicating the leukemic cells in the cell population.
  • an agent e.g., carbenoxolone or analog or derivative thereof
  • DNA damage is detected by determining yH2AX phosphorylation in the population of cells. .
  • yH2AX phosphorylation is detected by flow cytometry (e.g., FACS).
  • the agent is or comprises a glycyrrhetinic acid derivative selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, 18- ⁇ - glycyrrhetinic acid, carbenoxolone or 2-hydroxyethyl-18 -glycyrrhetinic acid amide.
  • the glycyrrhetinic acid derivative comprises carbenoxolone or a derivative or analog thereof.
  • the methods of eradicating leukemic cells further comprise inducing the differentiation of the leukemic cells into granulocytes.
  • the granulocytes comprise neutrophils.
  • the neutrophils comprise CD66b+/CD 14- neutrophils.
  • eradicating leukemic cells further comprises disrupting intercellular communications involving the leukemic cells that promote leukemia cell survival.
  • disrupting intercellular communications involving leukemic cells comprises interfering with heterotypic interactions between leukemic cells and stromal cells.
  • disrupting intercellular communications involving leukemic cells comprises interfering with homotypic interactions between leukemic cells.
  • the methods disclosed herein cause the selective eradication of leukemic cells while inducing proliferation of normal leukocytes in the population of cells.
  • the leukemic cells are selectively eradicated without eradicating normal leukocytes cells in the population of cells. In certain aspects, at least about 20%, about 50%, about 60%, about 75%, about 80%, about 90%, about 95%, about 99% or about 100% of the leukemic cells in the population of cells are eradicated.
  • the leukemic cells eradicated in accordance with the methods disclosed herein comprise an acute myeloid leukemia cell line selected from the group consisting of MLL-AF9 cells, KG-1 cells, KG- la cells, U937 cells, HL60 cells, NB-4 cells, and THP1 cells.
  • the population of cells comprises primary leukocytes selected from the group consisting of bone marrow leukocytes and peripheral blood leukocytes.
  • an effective amount of the agent comprises a concentration in the range of 50 ⁇ to 400 ⁇ in vitro or 10 mg/kg to 100 mg/kg in vivo.
  • the leukemic cells are contacted with the agent in vitro or ex vivo. In some embodiments of the methods disclosed herein, the leukemic cells are contacted with an agent in vivo (e.g., contact is in a subject).
  • the subject is a mouse. In certain aspects, the subject is a human. In certain aspects, the subject suffers from leukemia (e.g., acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL)). In some embodiments, the methods disclosed herein prolong survival of the subject. In some embodiments, the methods and agents disclosed are coadministered with a chemotherapeutic agent.
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • the disclosure provides a method of eradicating leukemic cells in a population of cells, the method comprising contacting the population of cells with an effective amount of a gap junction blocker, thereby eradicating leukemic cells in the cell population.
  • the disclosure provides a method of eradicating leukemic cells in a population of cells, the method comprising contacting the population of cells with an effective amount of a hemichannel blocker, thereby eradicating leukemic cells in the cell population.
  • the agent or gap junction blocker comprises an inhibitor of 1 ⁇ -hydroxysteroid dehydrogenase (1 ⁇ -HSD).
  • the agent or gap junction blocker is selected from the group consisting of the following formulas I to
  • Xy Y and Z each independently represent halog en, in particular, F, CI, I or Br, Ci_C 3 ⁇ 4 alkyl, C5-C 15 aryl or C 1 -C5 alkoxy, n represents an integer from 1 to 10, in particular, from 1 to 4, L represents an amide, amine, sulfonamide, ester, thioester or keto group, T, U, V and W each independently represent an oxo, thio, ketone, thioketone, Ci-C 6 alkyl or Ci-C 6 alkanol group, Ar represents ring system,
  • A represents a C 1 -C 10 ester (C 1 -C 10 alkyl- CO-O-), a C 1 -C 10 amide (C ⁇ do alkyl-CO-NH-), a C 1 -C 10 ether or a C 1 -C 10 ketone (C 1 -C 10 alkyl-CO-) group
  • B and C each independently represent an oxo group, a keto group, a Ci-C 6 alkanol group or a Ci-C 6 alkyl group
  • m is an integer from 1 to 10, in particular, from 1 to 4,
  • D is a group selected from COOR 1 or CONR z R J , wherein R , R z and R J each
  • E represents an OH, a C 1 -C 10 ester (Ci-
  • C 10 alkyl-CO-O- a C 1 -C 10 amide (C 1 -C 10 alkyl-CO-NH-), a C 1 -C 10 ether (C 1 -C 10 -O-) or a Ci- C 10 ketone (C 1 -C 10 alkyl-CO-) group
  • F represents an oxo group, keto group, a Ci-C 6 alkanol group or a Ci- C 6 alkyl group
  • G is a group selected from COOR 1 or CONR 2 R 3 , wherein R 1 , R 2 and R 3 each independently represent H or a Ci-C 2 o hydrocarbon group, in particular, a Ci-C 6 alkyl group.
  • the disclosure provides a method of eradicating leukemic cells in a population of cells, the method comprising contacting the population of cells with an effective amount of an agent or gap junction blocker, thereby eradicating leukemic cells in the cell population.
  • the agent or gap junction blocker is 18- ⁇ - glycyrrhetinic acid or a derivative thereof.
  • the agent or gap junction blocker is a derivative of 18- -glycyrrhetinic acid is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, carbenoxolone or 2-hydroxyethyl-18 -glycyrrhetinic acid amide.
  • the agent or gap junction blocker comprises carbenoxolone or an analog or derivative thereof. In some embodiments, the agent or gap junction blocker is not 18- -glycyrrhetinic acid.
  • the agent or gap junction blocker is selected from the group consisting of heptanol octanol, anadamide, fenamate, retinoic acid, oleamide, spermine, aminosulphates, halothane, enflurane, isoflurane, propofol, thiopental, glycyrrhetinic acid, quinine, 2-aminoethoxydiphenyl borate or a pharmaceutically acceptable derivatives thereof, and any combination thereof.
  • the agent or gap junction blocker is selected from the group consisting of heptanol octanol, anadamide, fenamate, retinoic acid, oleamide, spermine, aminosulphates, halothane, enflurane, isoflurane, propofol, thiopental, glycyrrhetinic acid, quinine, 2-aminoethoxydiphenyl borate or
  • pharmaceutically acceptable derivatives comprise: a pharmaceutically acceptable derivative of heptanol selected from the group consisting of 1 -heptanol, 2-heptanol, 3 -heptanol, 4- heptanol, and combinations thereof; a pharmaceutically acceptable derivative of fenamate selected from the group consisting of meclofenamic acid, niflumic acid, flufenamic acid, and combinations thereof; a pharmaceutically acceptable derivative of glycyrrhetinic acid selected from the group consisting of hydrogen esters of glycyrrhetinic acid, salts of hydrogen esters of glycyrrhetinic acid, carbenoxolone, and combinations thereof; and a pharmaceutically acceptable derivative of quinine selected from the group consisting of quinidine, mefloquine, and combinations thereof.
  • eradicating leukemic cells comprises inducing the differentiation of the leukemic cells into granulocytes.
  • the granulocytes comprise neutrophils.
  • the neutrophils comprise
  • eradicating leukemic cells comprises disrupting intercellular communications involving the leukemic cells that promote leukemia cell survival.
  • disrupting intercellular communications involving leukemic cells comprises interfering with heterotypic interactions between leukemic cells and stromal cells (e.g., mesenchymal stromal cells).
  • disrupting intercellular communications involving leukemic cells comprises interfering with heterotypic interactions between leukemic cells and any other cell types (e.g., osteolineage cells, endothelial cells, pericytes, mesenchymal cells or other hematopoietic cells).
  • disrupting intercellular communications involving leukemic cells comprises interfering with homotypic interactions between leukemic cells.
  • the leukemic cells are selectively eradicated while inducing proliferation of normal leukocytes in the population of cells.
  • the leukemic cells are selectively eradicated without eradicating normal leukocytes cells in the population of cells.
  • at least 20% of the leukemic cells in the population of cells are eradicated.
  • at least 50% of the leukemic cells in the population of cells are eradicated.
  • at least 70% of the leukemic cells in the population of cells are eradicated.
  • all of the leukemic cells in the population of cells are eradicated.
  • the leukemic cells are selectively eradicated, while minimally eradicating normal leukocytes cells in the population of cells.
  • the leukemic cells are selectively eradicated by carbenoxolone or an analog or derivative thereof (e.g., at a concentration of carbenoxolone of about 5 ⁇ , 10 ⁇ , 25 ⁇ , 50 ⁇ , ⁇ , 200 ⁇ , 250 ⁇ or 500 ⁇ ), while minimally eradicating normal leukocytes cells in the population of cells.
  • the leukemic cells are selectively eradicated (e.g., at a concentration of carbenoxolone or an analog or derivative thereof of about 50 ⁇ ), while less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001% or less of the normal cells are eradicated.
  • carbenoxolone or an analog or derivative thereof selectively eradicates stem cells (e.g., leukemic stem cells) in the population of cells, relative non-stem cells in the population of cells.
  • the leukemic cells comprise an acute myeloid leukemia cell line selected from the group consisting of MLL-AF9 cells, MLL-ENL cells, Nup98-HoxA9 cells, AML1-ET09A cells, KG-1 cells, KG- la cells, U937 cells, HL60 cells, NB-4 cells, HoxA9/Meis 1 cells, and THP1 cells.
  • an acute myeloid leukemia cell line selected from the group consisting of MLL-AF9 cells, MLL-ENL cells, Nup98-HoxA9 cells, AML1-ET09A cells, KG-1 cells, KG- la cells, U937 cells, HL60 cells, NB-4 cells, HoxA9/Meis 1 cells, and THP1 cells.
  • the population of cells comprises primary leukocytes selected from the group consisting of bone marrow leukocytes and peripheral blood leukocytes.
  • the effective amount comprises a concentration in the range of 50 ⁇ to 400 ⁇ in vitro or 10 mg/kg to 100 mg/kg in vivo.
  • the contacting occurs in vitro or ex vivo. In some embodiments, the contacting occurs in vivo. In some embodiments, the in vivo contact is in a subject. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human. In some embodiments, the subject suffers from leukemia. In some embodiments, the subject suffers from acute myeloid leukemia.
  • the disclosure provides a method of promoting the differentiation of a leukemic cell into a non-leukemic cell, the method comprising contacting the leukemic cell with an effective amount of an agent or a gap junction blocker, thereby promoting the differentiation of the leukemic cell into a non-leukemic cell.
  • the leukemic cell comprises a leukemic stem or progenitor cell.
  • the leukemic stem or progenitor cell comprises an acute myeloid leukemia cell.
  • the acute myeloid leukemia comprises a cell line selected from the group consisting of MLL-AF9 cells, MLL-ENL cells, Nup98- HoxA9 cells, AML1-ET09A cells, KG-1 cells, KG- la cells, U937 cells, HL60 cells, NB-4 cells, HoxA9/Meisl cells, and THP1 cells.
  • the non-leukemic cell comprises a mature or terminally differentiated cell. In some embodiments, the non-leukemic cell comprises a granulocyte. In some embodiments, the granulocyte comprises a short-lived granulocyte. In some embodiments, the non-leukemic cell comprises a neutrophil. In some embodiments, the neutrophil comprises a CD66b+/CD14- neutrophil.
  • the agent or gap junction blocker comprises an inhibitor of 1 ⁇ -hydroxysteroid dehydrogenase (1 ⁇ -HSD).
  • the agent or gap junction blocker is selected from the group consisting of the following formulas I to
  • X x Y and Z each independently represent halogen, in particular, F, CI, I or Br, Ci_C 6 alkyl, C 5 -Ci 5 aryl or Ci-C 6 alkoxy
  • n represents an integer from 1 to 10, in particular, from 1 to 4
  • L represents an amide, amine, sulfonamide, ester, thioester or keto group
  • T, U, V and W each independently represent an oxo, thio, ketone, thioketone, Ci-C 6 alkyl or Ci-C 6 alkanol group
  • Ar represents ents a cyclic ring system
  • B and C each independently represent an oxo group, a keto group, a Ci-C 6 alkanol group or a Ci-C 6 alkyl group
  • m is an integer from 1 to 10, in particular, from 1 to 4
  • D is a group selected from COOR 1 or CONR 2 R 3 , wherein R 1 , R 2 and R 3 each
  • Ci-C 6 alkyl group independently represent H or a Ci-C 6 alkyl group, wherein E represents an OH, a Ci-Ci 0 ester (Ci-
  • Cio alkyl-CO-O- a Ci-Ci 0 amide (Ci-Ci 0 alkyl-CO-NH-), a Ci-Ci 0 ether (Ci-Cio-O-) or a Ci- Cio ketone (Ci-Cio alkyl-CO-) group
  • F represents an oxo group, keto group, a Ci-C 6 alkanol group or a Ci- C 6 alkyl group
  • G is a group selected from COOR 1 or CONR 2 R 3 , wherein R 1 , R 2 and R 3 each independently represent H or a Ci-C 2 o hydrocarbon group, in particular, a Ci-C ⁇ 5 alkyl group.
  • the agent or gap junction blocker is 18- ⁇ - glycyrrhetinic acid or a derivative thereof. In some embodiments, the agent or gap junction blocker is a derivative of 18- -glycyrrhetinic acid is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, carbenoxolone or 2 ⁇ hydroxyethyl-18 -glycyrrhetinic acid amide. In some embodiments, the agent of gap junction blocker comprises carbenoxolone or an analog thereof.
  • the agent or gap junction blocker is selected from the group consisting of heptanol, octanol, anadamide, fenamate, retinoic acid, oleamide, spermine, aminosulphates, halothane, enflurane, isoflurane, propofol, thiopental,
  • glycyrrhetinic acid quinine, 2-aminoethoxydiphenyl borate or a pharmaceutically acceptable derivatives thereof, and any combination thereof.
  • the pharmaceutically acceptable derivatives comprise: a pharmaceutically acceptable derivative of heptanol selected from the group consisting of 1- heptanol, 2-heptanol, 3 -heptanol, 4-heptanol, and combinations thereof; a pharmaceutically acceptable derivative of fenamate selected from the group consisting of meclofenamic acid, niflumic acid, flufenamic acid, and combinations thereof; a pharmaceutically acceptable derivative of glycyrrhetinic acid selected from the group consisting of hydrogen esters of glycyrrhetinic acid, salts of hydrogen esters of glycyrrhetinic acid, carbenoxolone, and combinations thereof; and a pharmaceutically acceptable derivative of quinine selected from the group consisting of quinidine, mefloquine, and combinations thereof.
  • the disclosure provides a method of treating acute myeloid leukemia in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent or gap junction blocker, thereby treating acute myeloid leukemia in the subject.
  • the agent or gap junction blocker comprises an inhibitor of 1 ⁇ -hydroxysteroid dehydrogenase (1 ⁇ -HSD). In some embodiments, the agent or gap junction blocker comprises carbenoxolone or an analog thereof. In some
  • the agent or gap junction blocker is not 18- -glycyrrhetinic acid. In some embodiments, the agent or gap junction blocker selectively eradicates leukemic cells in the subject without eradicating normal leukocytes in the subject. In some embodiments, the agent or gap junction blocker selectively eradicates leukemic cells in the subject with minimal eradication of normal leukocytes in the subject. In some embodiments, the agent or gap junction blocker selectively eradicates leukemic cells in the subject while inducing proliferation of normal leukocytes in the subject.
  • the method further includes administering an induction chemotherapy treatment regimen to the subject.
  • the induction chemotherapy comprises administering an antimetabolite agent and an
  • the antimetabolite agent comprises cytarabine.
  • the anthracycline agent comprises doxorubicin.
  • the induction chemotherapy comprises administering cytarabine and doxorubicin to the patient for a period of 5 days. In some embodiments, the induction chemotherapy comprises administering cytarabine and doxorubicin to the patient for a period of 3 days, followed by administering cytarabine alone to the patient for a period of 2 days.
  • the agent or gap junction blocker is administered to the subject for at least a day before administering the induction chemotherapy treatment regimen to the subject. In some embodiments, the agent or gap junction blocker is administered to the subject for at least a day before administering the induction chemotherapy treatment regimen to the subject concomitantly with the agent or gap junction blocker.
  • the methods of the invention further comprise subjecting the subject to hyperbaric oxygen therapy (HBOT).
  • HBOT hyperbaric oxygen therapy
  • the subject is administered a glycyrrhetinic acid derivative (e.g., carbenoxolone or an analog or derivative thereof) and subjected to HBOT.
  • a glycyrrhetinic acid derivative e.g., carbenoxolone or an analog or derivative thereof
  • HBOT can be initiated prior to, during, or after administration of a glycyrrhetinic acid derivative (e.g., carbenoxolone or an analog or derivative thereof) as long as the subject exhibits elevated oxygen levels in the blood and/or tissues for at least a portion of time when the glycyrrhetinic acid derivative (e.g., carbenoxolone or an analog or derivative thereof) is present in the subject.
  • HBOT is performed at a pressure from about 1.0 ATA to 3.0 ATA, in another embodiment about 1.5 ATA to 2.75 ATA, and in another embodiment about 2.0 ATA to 2.5 ATA.
  • the time per treatment may vary from about 1 to 120 minutes or longer.
  • the subject is suffering from refractory or relapsed acute myeloid leukemia.
  • the method further includes evaluating the subject to determine if the subject has refractory or relapsed acute myeloid leukemia.
  • the subject is a subject who relapses from complete remission of acute myeloid leukemia after induction chemotherapy.
  • treating acute myeloid leukemia comprises inducing complete remission of acute myeloid leukemia in the subject.
  • treating acute myeloid leukemia comprises inducing complete remission of acute myeloid leukemia in the subject in the absence of a relapse risk due to residual leukemic cells in the subject's bone marrow or peripheral blood.
  • the disclosure provides a method of promoting survival of a subject suffering from acute myeloid leukemia, the method comprising administering to the subject an effective amount of an agent or gap junction blocker, thereby promoting survival of the subject.
  • the agent or gap junction blocker comprises an inhibitor of 1 ⁇ -hydroxysteroid dehydrogenase (1 ⁇ -HSD). In some embodiments, the agent or gap junction blocker comprises carbenoxolone or an analog thereof. In some
  • the method further includes administering an induction chemotherapy treatment regimen to the subject.
  • the induction chemotherapy comprises administering an antimetabolite agent and an anthracycline agent to the subject.
  • the antimetabolite agent comprises cytarabine.
  • the anthracycline agent comprises doxorubicin.
  • the induction chemotherapy comprises administering cytarabine and doxorubicin to the patient for a period of 5 days.
  • the induction chemotherapy comprises administering cytarabine and doxorubicin to the patient for a period of 3 days, followed by administering cytarabine alone to the patient for a period of 2 days.
  • the agent or gap junction blocker is administered to the subject for at least a day before administering the induction chemotherapy treatment regimen to the subject. In some embodiments, the agent or gap junction blocker is administered to the subject for at least a day before administering the induction chemotherapy treatment regimen to the subject concomitantly with the agent or gap junction blocker.
  • the method further includes selecting a subject suffering from or exhibiting a terminal state of acute myeloid leukemia.
  • the subject has advanced tumor metastasis.
  • the subject has a high tumor burden.
  • the agent or gap junction blocker increases the subject's length of survival compared to the subject's length of survival in the absence of receiving the agent or gap junction blocker. In some embodiments, the agent or gap junction blocker increases the subject's likelihood of survival compared to the subject's likelihood of survival in the absence of receiving the agent or gap junction blocker.
  • the disclosure provides a method of inducing complete remission in a subject having relapsed or refractory acute myeloid leukemia by selectively eradicating leukemic cells in the subject, the method comprising: (a) evaluating the subject to determine if the subject has relapsed or refractory acute myeloid leukemia; (b) administering to the subject an agent or gap junction blocker at least a day before administering an induction chemotherapy treatment regimen to the subject; and (c) administering to the subject an induction chemotherapy treatment regimen comprising an antimetabolite agent and an anthracycline agent for proscribed periods of time, thereby inducing complete remission in the subject by selectively eradicating leukemic cells in the subject.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of an agent or gap junction blocker, an effective amount of at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises an antimetabolite agent. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises cytarabine. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises an anthracycline agent. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises doxorubicin. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises an antimetabolite agent and anthracycline agent. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises cytarabine and doxorubicin.
  • the agent or gap junction blocker comprises carbenoxolone or an analog thereof. In some embodiments, the agent or gap junction blocker is not 18- -glycyrrhetinic acid.
  • the disclosure provides a kit comprising an agent or gap junction blocker, at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia, and instructions for administering the agent or gap junction blocker and the at least one chemotherapeutic agent to a subject suffering from acute myeloid leukemia.
  • the kit further comprises a prophylactic treatment to be administered with the agent or gap junction blocker and/or the at least one chemotherapy agent, and instructions for administering the prophylactic treatment with the agent or gap junction blocker and/or the at least one chemotherapy agent.
  • the prophylactic treatment comprises a pharmaceutically active agent described herein for treating or preventing hypertension, hypokalemia, and edemas.
  • the instructions further comprise directions for administering the at least one chemotherapeutic agent as part of an induction chemotherapy treatment regimen for the subject.
  • the instructions further comprise directions for administering the agent or gap junction blocker, and the at least one therapeutic agent to induce complete remission of acute myeloid leukemia in the subject.
  • the instructions further comprise directions for administering the agent or gap junction blocker, and the at least one therapeutic agent to induce complete remission of acute myeloid leukemia in the subject, without risk of relapse by completely eradicating leukemic cells in the subject. In some embodiments, the instructions further comprise directions for administering the agent or gap junction blocker, and the at least one therapeutic agent to induce complete remission of acute myeloid leukemia in the subject by completely eradicating leukemic cells in the subject by inducing the leukemic cells to differentiate from proliferating, immortalized leukemic cells into shortlived, non-leukemic cells.
  • the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises an antimetabolite agent. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises cytarabine. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises an anthracycline agent. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises doxorubicin. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises an antimetabolite agent and an anthracycline agent. In some embodiments, the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises cytarabine and the anthracycline agent comprises doxorubicin.
  • the agent or gap junction blocker comprises carbenoxolone or an analog thereof.
  • identifying a candidate agent that selectively eradicates leukemic cells in a population of cells comprise: (a) contacting a population of cells comprising leukemic cells and normal cells with a test agent; and (b) detecting induction of oxidative stress in the population of cells; wherein a test agent that selectively induces oxidative stress in the leukemic cells is a candidate agent.
  • the oxidative stress is detected by determining the total levels of reactive oxygen species (ROS) in the population of cells.
  • ROS reactive oxygen species
  • oxidative stress is detected by determining the total levels of mitochondrial reactive oxygen species (ROS) in the population of cells.
  • lipid damage is detected by determining the ratio of oxidized to non-oxidized lipids in the population of cells. In some embodiments, lipid damage is detected by detecting lipid peroxidation by flow cytometry.
  • a candidate agent that selectively eradicates leukemic cells in a population of cells comprising: (a) contacting a population of cells comprising leukemic cells and normal cells with a test agent; and (b) detecting induction of DNA damage in the population of cells; wherein a test agent that selectively induces DNA damage in the leukemic cells is a candidate agent.
  • DNA damage is detected by determining yH2AX
  • yH2AX phosphorylation in the population of cells.
  • yH2AX phosphorylation is detected by flow cytometry (e.g., FACS).
  • the methods and assays disclosed herein are useful for identifying whether a test agent comprising a glycyrrhetinic acid derivative is capable of selectively eradicating leukemic cells from a population of cells.
  • the glycyrrhetinic acid derivative is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, 18 - ⁇ -glycyrrhetinic acid, carbenoxolone or 2-hydroxyethyl-18 - glycyrrhetinic acid amide.
  • the glycyrrhetinic acid derivative comprises carbenoxolone or an analog or derivative thereof.
  • the leukemic cells useful in such assays and methods comprise an acute myeloid leukemia cell line selected from the group consisting of MLL-AF9 cells, KG-1 cells, KG- la cells, U937 cells, HL60 cells, NB-4 cells, and THP1 cells.
  • an acute myeloid leukemia cell line selected from the group consisting of MLL-AF9 cells, KG-1 cells, KG- la cells, U937 cells, HL60 cells, NB-4 cells, and THP1 cells.
  • leukemia e.g., acute myeloid leukemia
  • the efficacy of the compositions and methods disclosed herein are not limited to leukemia cells, but rather extend broadly to all cancer cell types (e.g., solid and non-solid tumors).
  • the inventions disclosed herein relate to methods of eradicating cancer cells in a population of cells, the methods comprising contacting the population of cells with an effective amount of a glycyrrhetinic acid derivative, thereby eradicating the cancer cells (e.g., leukemic cells) in the cell population.
  • the inventions disclosed herein are directed to methods of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a glycyrrhetinic acid derivative, thereby treating cancer in the subject.
  • the glycyrrhetinic acid derivative is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, 18- -glycyrrhetinic acid, carbenoxolone, or 2-hydroxyethyl-18 -glycyrrhetinic acid amide.
  • the glycyrrhetinic acid derivative comprises carbenoxolone or an analog or derivative thereof.
  • the cancer cells are selectively eradicated (or the cancer is treated) by the compositions and methods disclosed herein without eradicating non-cancer cells in the population of cells.
  • the non-cancer cells in the population of cells comprise primary leukocytes selected from the group consisting of bone marrow leukocytes and peripheral blood leukocytes.
  • an effective amount of the glycyrrhetinic acid derivative comprises a concentration in the range of 50 ⁇ to 400 ⁇ in vitro or 10 mg/kg to 100 mg/kg in vivo.
  • the contacting of the cancer cells occurs in vitro or ex vivo. In certain aspects, the contacting occurs in vivo (e.g., wherein the in vivo contact is in a subject, such as a human subject suffering from acute myeloid leukemia).
  • the cancer cells comprise leukemia cells. In some embodiments, the cancer cells comprise breast cancer cells. In yet other embodiments, the cancer cells comprise melanoma cells. In still other embodiments, the cancer cells comprise prostate cancer cells. In still other embodiments, the cancer cells comprise cervix carcinoma cells. In certain aspects, the cancer cells comprise lung cancer cells.
  • the methods disclosed herein further comprise contacting the population of cells with a chemotherapeutic agent, for example, by co-administering the glycyrrhetinic acid derivative and the chemotherapeutic to a subject in need thereof.
  • the agents disclosed herein e.g., glycyrrhetinic acid derivatives, such as carbenoxolone or an analog or derivative thereof
  • the agents disclosed herein do not induce an increase in superoxide levels in non-cancer cells.
  • Some embodiments of the invention are directed to a method of eradicating cancer cells in a population of cells, the method comprising contacting the population of cells with an effective amount of a glycyrrhetinic acid derivative, thereby eradicating cancer cells in the cell population.
  • the glycyrrhetinic acid derivative is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, 18- -glycyrrhetinic acid,
  • carbenoxolone a carbenoxolone derivative, a carbenoxolone analog, or 2-hydroxyethyl-18 - glycyrrhetinic acid amide.
  • the carbenoxolone derivative is a pegylated carbenoxolone.
  • the cancer cells are selectively eradicated. In some aspects, the at least 20% of the cancer cells in the population of cells are eradicated. In some aspects, the at least 50% of the cancer cells in the population of cells are eradicated. In some aspects, the at least 70% of the cancer cells in the population of cells are eradicated. In some aspects, the all of the cancer cells in the population of cells are eradicated.
  • the effective amount of the glycyrrhetinic acid derivative comprises a concentration in the range of 50 ⁇ to 400 ⁇ in vitro or 10 mg/kg to 100 mg/kg in vivo.
  • the contacting occurs in vitro or ex vivo (e.g., in a subject).
  • the subject is a mouse or a human.
  • the subject suffers from leukemia (e.g., acute leukemia, acute myeloid leukemia).
  • the subject suffers from a solid tumor.
  • the subject suffers from a cancer selected from the group consisting of melanoma, prostate cancer, cervical cancer, breast cancer, prostate cancer, and lung cancer.
  • Some aspects of the methods of the invention further comprise measuring calcium flux in the population of cells after contacting with the glycyrrhetinic acid derivative. In some aspects measuring calcium flux is performed within about 1, 2, 3, 4, 5, or 10 minutes after contact with the glycyrrhetinic acid derivative. In some embodiments, calcium flux is measured in a population of cells from a subject refractory to the methods of treatment disclosed herein. In some embodiments, calcium flux is measured in a population of cells from a subject that has not been subjected to a method of treatment disclosed herein to aid in determining whether such treatment is likely to be effective.
  • Some aspects of the methods of the invention further comprise detecting lipid damage in the population of cells after contacting with the glycyrrhetinic acid derivative.
  • lipid damage is detected by detecting lipid peroxidation with flow cytometry (e.g., fluorescent activated cell sorting (FACS)).
  • flow cytometry e.g., fluorescent activated cell sorting (FACS)
  • lipid damage is detected by the ratio of oxidized to non-oxidized lipids in the population of cells.
  • lipid damage is detected by detecting lipid peroxidation by flow cytometry (e.g., FACS).
  • Some aspects of the methods of the invention further comprise detecting DNA damage in the population of cells after contacting with the glycyrrhetinic acid derivative.
  • DNA damage is detected by detecting double -stranded DNA breaks.
  • DNA damage is detected by determining yH2AX phosphorylation in the population of cells.
  • Some aspects of the methods of the invention further comprise detecting induction of oxidative stress in the population of cells after contacting with the glycyrrhetinic acid derivative.
  • oxidative stress is detected by detecting the total levels of reactive oxygen species (ROS) in the population of cells.
  • oxidative stress is detected by detecting the total levels of mitochondrial reactive oxygen species (ROS) in the population of cells.
  • Some aspects of the methods of the invention further comprise detecting a metabolic product after contacting with the glycyrrhetinic acid derivative.
  • the metabolic product is hypoxanthine.
  • Some aspects of the methods of the invention further comprise contacting the population of cells with one or more chemotherapeutics as described herein.
  • the one or more chemotherapeutics comprises an antimetabolite agent and/or an anthracycline agent.
  • the one or more chemotherapeutics comprise one or more demethylating agents.
  • the one or more chemotherapeutics comprise cytarabine and doxorubicin.
  • the one or more chemotherapeutics comprise a chemotherapeutic subject to resistance by the cancer cells.
  • FIGS. 1A, IB, 1C and ID demonstrate the results of kinetic studies of a 5 day induction chemotherapy regimen administered in a mouse model of acute myeloid leukemia (AML).
  • FIG. 1A is a schematic illustration shows the experimental design for the mouse model of AML.
  • FIG. IB is an example of IVIS imaging of mice at day 14, showing luciferase activity in the bones.
  • FIGS. 1C and ID are bar graphs showing the results of FACS analysis of GFP-positive MLL-AF9 cells from blood (FIG. 1C) and bone marrow (BM) (FIG. ID) samples.
  • FIGS. 2A, 2B, 2C and 2D demonstrate that gap junctions activity plays a key role in maintaining leukemic cell drug resistance.
  • 100,000 MLL-AF9 cells were incubated for 16-hours with or w/o chemotherapy (50nM Cytarabine + 20nM Doxorubicin) and with or w/o sub-confluent MS-5 layer (FIG. 2A).
  • FIG. 2D shows whole body bioluminescence imaging (IVIS) 1 week after indicated treatment.
  • White arrows indicate minimal residual AML cells.
  • FIGS. 3A and 3B demonstrate that in vivo administration of Carbenoxolone alone or in combination with chemotherapy, to mice transplanted with MLL-AF9 leukemia (established mouse model of human leukemia), results in prolonged survival.
  • FIG 3A shows survival curves of mice left untreated (red line), or mice that received treatment on Day 27, upon detection of Leukemic cells in the bones (green, blue, purple or black lines, as indicated).
  • Chemo cytarabine (lOOmg/kg) and doxorubicin (3 mg/kg) to the subject for a period of 3 days, followed by administering cytarabine alone (100 mg/kg) to the subject for a period of 2 days.
  • CBX Carbenoxolone (20mg/kg) to the subject for a period of 3 or 6 days, with or w/o chemotherapy.
  • FIG. 3B shows survival curves of terminally ill mice that left untreated (red line), or that received treatment on Day 68 (blue line), a terminal state of the disease, with leukemic cells spread all over the body and high tumor burden.
  • CBX Carbenoxolone (20mg/kg) to the subject for a period of 3 or 6 days, with or w/o chemotherapy.
  • FIG. 3B shows survival curves of terminally ill mice that left untreated (red line), or that received treatment on Day 68 (blue line), a terminal state of the disease, with leukemic cells spread all over the body and high tumor burden.
  • CBX Carbenoxolone (20mg/kg) to the subject for a period of 3 or 6 days, with or w/o chemotherapy.
  • FIG. 3B shows survival curves of terminally ill mice that left untreated
  • FIGS. 4A, 4B, 4C and 4D demonstrate the selective eradication of different AML cell types by gap junction blockade in vitro.
  • Carbenoxolone (CBX) at the indicated concentrations, was added to either 100,000 MLL-AF9, primary bone marrow leukocytes or primary peripheral blood leukocytes for 16 hours (FIG. 4A).
  • Either 100,000 MLL-AF9 (clone A) cells (FIG. 4B), 100,000 MLL-AF9 (clone B) cells (FIG. 4C), or 100,000 HoxA9/Meisl cells (FIG. 4D) were mixed with 100,000 primary BM leukocytes and exposed to CBX, as indicated, for 16 hours.
  • Cells were distinguished by Cd45.1/CD45.2 expression and cell viability was determined by % of 7AAD-negative cells, in FACS analysis. (*p ⁇ 0.01).
  • FIGS. 5A, 5B, 5C and 5D demonstrate that carbenoxolone selectively eradicates different murine AML cells (blue bars) without affecting non-leukemic normal counterparts (red bars).
  • FIG. 5 A is a bar graph showing the results of MLL-AF9 (clone A) cells mixed 1 : 1 with freshly isolated primary blood leukocytes and exposed to carbenoxolone, as indicated, for 16 hours. MLL-AF9 (clone A) cells (FIG. 5B), MLL-AF9 (clone B) cells (FIG. 5C), and HoxA9/Meis 1 cells (FIG.
  • FIGS. 6A, 6B, 6C, 6D, 6E, and 6F demonstrate that carbenoxolone treatment eradicates murine leukemic cancer stem cells while inducing proliferation of normal stem cells.
  • Colony formation assays were performed on MLL-AF9 cells (FIG. 6A), bone marrow primary leukocytes (FIG. 6B), and a 1 : 1 mixture of both MLL-AF9 cells and bone marrow primary leukocytes (FIG. 6C) exposed to increasing concentrations of carbenoxolone, as indicated, and the colony forming units in culture CFU-C per 10,000 cells was determined at each concentration.
  • 6D, 6E and 6F are images showing formation of leukemic colonies (green) and normal colonies (blue) upon exposure to 0 ⁇ (FIG. 6D), 50 ⁇ (FIG. 6E), and 200 ⁇ or 100 ⁇ (FIG. 6F) of carbenoxolone.
  • FIGS. 7A, 7B, and 7C demonstrate that a gap-junction blockade promotes differentiation of AML cells into short-lived granulocytes in vitro.
  • Primary MLL-AF9 cells were exposed to increasing concentrations of carbenoxolone, as indicated, for 16 hours and then analyzed by FACS analysis for the expression of Grl (FIG. 7A), Macl (FIG. 7B), or Grl/Mac l (FIG. 7C). Cell viability was determined by % of 7AAD-negative cells, in FACS analysis. (*p ⁇ 0.01).
  • FIGS. 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, 81, 8 J, 8K, and 8L demonstrate that carbenoxolone induces rapid (within 16 hours) neutrophil differentiation (CD66b+/CD 14-) in human AML cell line U937.
  • Human AML U937 cells were exposed to increasing concentrations of carbenoxolone, as indicated, for 16 hours and then analyzed by FACS analysis for the expression of live cells (FIG. 8A), CD 14+ (FIG. 8B), CD66b+ (FIG. 8C), Mac l+ (FIG. 8D), CD66b+/Macl- (FIG. 8E), CD66b+/Macl+ (FIG.
  • CD66b-/Mac l+ (FIG. 8G), CD66b-/Macl- (FIG. 8H), CD66+/CD 14- (FIG. 81), CD66b+/CD 14+ (FIG. 8J), CD66b-/CD14+ (FIG. 8K), and CD66b-/CD14- (FIG. 8L).
  • Cell viability was determined by % of 7AAD-negative cells, in FACS analysis. (*p ⁇ 0.01).
  • FIGS. 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, 91, 9J, 9K, and 9L demonstrate that carbenoxolone induces rapid (within 16 hours) neutrophil differentiation (CD66b+/CD14-) in human AML cell line HL60.
  • Human AML HL60 cells were exposed to increasing concentrations of carbenoxolone, as indicated, for 16 hours and then analyzed by FACS analysis for the expression of live cells (FIG. 9A), CD 14+ (FIG. 9B), CD66b+ (FIG. 9C), Macl+ (FIG. 9D), CD66b+/Macl- (FIG. 9E), CD66b+/Macl+ (FIG.
  • FIGS. 10A, 10B, IOC, 10D, 10E, 10F, 10G, 10H, 101, 10 J, 1 OK and 10L demonstrate that carbenoxolone does not induce neutrophil differentiation (CD66b+/CD14-) in freshly isolated primary human leukocytes.
  • Primary human leukocytes were freshly isolated and exposed to increasing concentrations of carbenoxolone, as indicated, for 16 hours and then analyzed by FACS analysis for the expression of live cells (FIG. 10A), CD14+ (FIG. 10B), CD66b+ (FIG. IOC), Macl+ (FIG. 10D), CD66b+/Macl- (FIG. 10E), CD66b+/Macl+ (FIG.
  • FIGS. 11A, 1 IB, 11C and 1 ID demonstrate that Carbenoxolone treatment eradicates human leukemic cancer stem and progenitor cells (1 lA-11C) while inducing proliferation of normal human stem and progenitor cells (1 ID).
  • Colony formation assays were performed on THP1 cells (FIG. 11A), HL60 (FIG. 1 IB), U937 (FIG. 11C) and freshly isolated primary non-leukemic normal human leukocytes (FIG. 1 ID) exposed to increasing concentrations of Carbenoxolone, as indicated, and the colony forming units in culture CFU- C per 2,000 cells was determined at each concentration.
  • FIGS. 12A, 12B, 12C, 12D, 12E, 12F, 12G, 12H, 121, 12J, 12K, 12L, 12M, 12N, 120, 12P, and 12Q are gene expression profiles from published arrays demonstrating that 1 I HSD, and members of the Connexin gap junction family are over-expressed in human AML.
  • FIG. 12A is an expression profile showing 1 I HSD expression in malignant and normal blood.
  • FIG. 12B is an expression profile showing Cx40.1 expression in malignant and normal blood.
  • FIG. 12C is an expression profile showing Cx30.2 expression in malignant and normal blood.
  • FIG. 12D is an expression profile showing Cx31.1 expression in malignant and normal blood.
  • FIG. 12A, 12B, 12C, 12D, 12E, 12F, 12G, 12H, 121, 12J, 12K, 12L, 12M, 12N, 120, 12P, and 12Q are gene expression profiles from published arrays demonstrating that 1 I HSD, and
  • FIG. 12E is an expression profile showing Cx36 expression in malignant and normal blood.
  • FIG. 12F is an expression profile showing Cx45 expression in malignant and normal blood.
  • FIG. 12G is an expression profile showing Cx47 expression in malignant and normal blood.
  • FIG. 12H is an expression profile showing Cx32 expression in malignant and normal blood.
  • FIG. 121 is an expression profile showing Cx50 expression in malignant and normal blood.
  • FIG. 12J is an expression profile showing Cx30.3 expression in malignant and normal blood.
  • FIG. 12K is an expression profile showing Cx31 expression in malignant and normal blood.
  • FIG. 12L is an expression profile showing Cx26 expression in malignant and normal blood.
  • FIG. 12M is an expression profile showing Cx40 expression in malignant and normal blood.
  • FIG. 12E is an expression profile showing Cx36 expression in malignant and normal blood.
  • FIG. 12F is an expression profile showing Cx45 expression in malignant and normal blood.
  • FIG. 12G is an expression profile showing Cx
  • FIG. 12N is an expression profile showing Cx37 expression in malignant and normal blood.
  • FIG. 120 is an expression profile showing Cx46 expression in malignant and normal blood.
  • FIG. 12P is an expression profile showing Cx43 expression in malignant and normal blood.
  • FIG. 12Q is an expression profile showing Cx30 expression in malignant and normal blood. Every dot in each of the expression profiles depicted in FIGS. 12A-12Q represents a different independent study.
  • FIG. 13 demonstrates an experimental design for the real time analysis of leukemic cell interactions and communication during induction chemotherapy, which enables intravital microscopy, monitoring of disease progression, synchronization of treatment, monitoring of relapse, valid comparison of different oncogenes, minimization of use of viruses, in vitro live video microscopy, and in vitro screens.
  • FIGS. 14A, 14B and 14C demonstrate that CBX overcomes stroma-mediated drug resistance. Results obtained using 25 nM cytarabine and 10 nM doxorubicin as chemo. Asterisks indicate results obtained after 16 hours of incubation. Viability was detected via GFP expression and 7AAD exclusion. Results in FIG. 14C achieved with transwell insert separation.
  • FIG. 15 demonstrates that in vitro exposure to CBX (at greater than or equal to 100 ⁇ ) leads to the eradication of MLL-AF9 leukemia, but not the eradication of normal blood cells. Results obtained via flow cytometry viability assays after 16 hours of incubation with the amounts of CBX indicated with viability detection by GFP expression and 7AAD exclusion.
  • FIGS. 16A, 16B, 16C and 16D demonstrate that CBX selectively eradicates different murine AML cells (blue bars) without affecting non-leukemic normal counterparts (red bars) - in 1 : 1 cell mixtures.
  • FIG. 16A shows results of CBX treatment at the concentrations indicated of MLL-AF9 (bulk A) cells mixed 1 : 1 with freshly isolated blood leukocytes.
  • FIG. 16B shows results of CBX treatment at the concentrations indicated of MLL-AF9 (bulk A) cells mixed 1 : 1 with freshly isolated bone marrow leukocytes.
  • FIG. 16A shows results of CBX treatment at the concentrations indicated of MLL-AF9 (bulk A) cells mixed 1 : 1 with freshly isolated bone marrow leukocytes.
  • FIG. 16C shows results of CBX treatment at the concentrations indicated of MLL-AF9 (bulk B) cells mixed 1 : 1 with freshly isolated bone marrow leukocytes.
  • FIG. 16D shows results of CBX treatment at the concentrations indicated of HoxA9/Meis 1 cells mixed 1 : 1 with freshly isolated bone marrow leukocytes.
  • FIGS. 17A, 17B, 17C, and 17D demonstrates the differential effect of CBX (at greater than or equal to 50 ⁇ ) on malignant vs. normal immature stem and progenitor cells (CFU-C assay).
  • FIG. 17A is a bar graph showing results of CBX treatment at the concentrations indicated on MLL-AF9 leukemic cells alone.
  • FIG. 17B is a bar graph showing results of CBX treatment at the concentrations indicated on normal bone marrow primary leukocytes alone.
  • FIG. 17C is a bar graph showing results of CBX treatment at the concentrations indicated on a 1 : 1 mixture of MLL-AF9 cells and normal bone marrow primary leukocytes.
  • FIG. 17D shows images of the results of CBX treatment on the cell mixture described in FIG. 17C.
  • FIGS. 18A, 18B, 18C and 18D demonstrate that the human APL cell line HL60 is sensitive to CBX.
  • FIG. 18A is a bar graph showing the viability of HL60 cells 16 hours post exposure to CBX at the concentrations indicated, as assessed by flow cytometry.
  • FIG. 18B is a bar graph showing the viability of freshly isolated human peripheral blood leukocytes 16 hours post exposure to CBX at the concentrations indicated, as assessed by flow cytometry.
  • FIG. 18C is a bar graph showing colony formation of HL60 cells 7 days post exposure to CBX at the concentrations indicated, as assessed by CFU-C assay for a functional quantification of cancer and normal stem and progenitor cells.
  • FIG. 18D is a bar graph showing colony formation of freshly isolated human peripheral blood leukocytes 7 days post exposure to CBX at the concentrations indicated, as assessed by CFU-C assay for a functional quantification of cancer and normal stem and progenitor cells.
  • FIGS. 19A, 19B, 19C and 19D demonstrate that CBX treatment (100 ⁇ ) eradicates human leukemic progenitors but induces proliferation of normal progenitors.
  • FIG. 19A is a bar graph showing proliferation of THP1 cells treated with CBX at the
  • FIG. 19B is a bar graph showing proliferation of HL60 cells treated with CBX at the concentrations indicated, as assessed by CFU-C assay.
  • FIG. 19C is a bar graph showing proliferation of U937 cells treated with CBX at the concentrations indicated, as assessed by CFU-C assay.
  • FIG. 19D is a bar graph showing proliferation of primary human progenitor cells treated with CBX at the
  • FIGS. 20A, 20B and 20C demonstrate that CBX rapidly induces apoptosis of MLL-AF9 leukemic cells.
  • Blue bars and red bars show viability (as assessed by the frequency of 7AAD-negative cells) and apoptosis (as assessed by frequency of 7AAD- negative, annexin-V positive cells) of MLL-AF9 leukemic cells after treatment with CBX at the concentrations indicated compared to control for 6 hours (FIG. 20A), 12 hours (FIG. 20B), and 20 hours (FIG. 20C).
  • FIG. 21 demonstrates that CBX treatment induces apoptosis of leukemic cells and enhances the replenishment of normal healthy cells.
  • FACS dot plots show the results of CBX treatment compared to control (PBS) after 4h on a 1 : 1 mixture of iRFP+ MLL-AF9 leukemic cells with normal bone marrow mononuclear cells (BM-MNC).
  • FIG. 22 demonstrates that CBX treatment induces apoptosis of leukemic cells and enhances the replenishment of normal healthy cells.
  • Blue bars and red bars show apoptotic mononuclear cells (MNC) and apoptotic leukemic cells, respectively, after treatment with CBX at the concentrations indicated compared to the PBS control.
  • MNC mononuclear cells
  • FIG. 23 demonstrates that MLL-AF9 leukemic cells are double-positive for Macl, Grl and cKit. FACS dot plots indicate that normal myoblasts are the most appropriate counterparts for comparison.
  • FIG. 24 shows FACTS dot plots showing the results of CBX treatment compared to control (PBS) after 4h on a 1 : 1 mixture of iRFP+ MLL-AF9 leukemic cells with normal myeloblasts.
  • FIG. 25 demonstrates that CBX-induced leukemia apoptosis is not cell cycle dependent.
  • FIG. 25 is a bar graph showing results of 4h of CBX treatment on a 1 : 1 mixture of iRFP+ MLL-AF9 leukemic cells (blue bars) with highly proliferating normal myeloblasts (black bars) or expanded lineage -/cKit+/Scal+ (LKS) cells (black bars).
  • Rate of proliferation for cell populations used is as follows: MLL-AF9: 3 divisions/24 hours; myeloblasts: 3 divisions/24 hours, eLKS: 7 divisions/24 hours.
  • FIGS. 26A, 26B, 26C and 26D are bar graphs showing the viability (7AAD-negative, iRFP +/-; FIG. 26A) and apoptosis (7AAD-negative, iRFP+, annexin-V-positive; FIG. 26B) of a 1 : 1 mixture of MLL-AF9 iRFP+ leukemia and normal myeloblast cells after 4h treatment with 200 ⁇ of CBX or Aldosterone.
  • FIGS. 26C and 26D are bar graphs showing the viability (7AAD-negative, iRFP +/-; FIG.
  • FIG. 26C apoptosis (7AAD-negative, iRFP+, annexin-V-positive; FIG. 26D) of a 1 : 1 mixture of MLL-AF9 iRFP+ leukemia and normal myeloblast cells after 24h treatment with 200 ⁇ of CBX or Aldosterone.
  • FIGS. 27A, 27B, 27C and 27D are bar graphs showing the viability (7AAD-negative, GFP +/-; FIG. 27A) and apoptosis (7AAD-negative, GFP+/, annexin-V-positive; FIG. 27B) of a 1 : 1 mixture of MLL-AF9 GFP+ leukemia and normal myeloblast cells after 4h treatment with 200 ⁇ of CBX or Aldosterone.
  • FIGS. 27C and 27D are bar graphs showing the viability (7AAD-negative, GFP +/-; FIG. 26C) and apoptosis (7AAD-negative, GFP+/, annexin-V-positive; FIG. 27D) of a 1 : 1 mixture of MLL-AF9 GFP+ leukemia and normal myeloblast cells after 24h treatment with 200 ⁇ of CBX or
  • FIGS. 28A-N depict compounds with similar structure and/or similar systemic "steroid-like" effects, to CBX (FIG. 28N).
  • FIGS. 28A, 28B, 28C, and 28D are the structural formulas for mineralocorticoids aldosterone (FIG. 28A), spironolactone (FIG. 28B), fludrocortisone (FIG. 28C), and deoxycorticosterone (FIG. 28D).
  • FIGS. 28E, 28F, 28G, 28H, 281, 28J, 28K 28L and 28M are the structural formulas for glucocorticoids beclometasone dipropionate (FIG. 28E), Cortisol (FIG.
  • FIG. 28F cortisone
  • FIG. 28G dexamethasone
  • FIG. 28H dexamethasone
  • FIG. 281 betamethasone
  • prednisolone FIG. 28J
  • prednisone FIG. 28K
  • methylprednisolone FIG. 28L
  • triamcinolone acetonide FIG. 28M
  • FIGS. 29A and 29B are bar graph showing the viability (7AAD- negative, iRFP+/-) of a 1: 1 mixture of MLL-AF9 iRFP+ leukemia and normal myeloblast cells after 24h treatment with 200 ⁇ of CBX or the mineralocorticoid compounds indicated.
  • FIG. 29B is a Table showing the comparative steroid potencies of the compounds shown in FIGS. 28A-28N.
  • FIGS. 30A and 30B are bar graph showing the viability (7AAD- negative, iRFP+/-) of a 1: 1 mixture of MLL-AF9 iRFP+ leukemia and normal myeloblast cells after 24h treatment with 200 ⁇ of CBX or the glucocorticoid compounds indicated.
  • FIG. 30B is a Table showing the comparative steroid potencies of the compounds shown in FIGS. 28A-28N.
  • FIGS. 31A and 3 IB are bar graph showing the viability (7AAD- negative, iRFP+/-) of a 1: 1 mixture of MLL-AF9 iRFP+ leukemia and normal myeloblast cells after 24h treatment with 200 ⁇ of CBX or the glucocorticoid compounds indicated.
  • FIG. 3 IB is a Table showing the comparative steroid potencies of the compounds shown in FIGS. 28A-28N.
  • FIG. 32 demonstrates that Macl (CD1 lb, integrin a rempli ⁇ is downregulated in CBX treated mice leukemic cells undergoing apoptosis (7AAD-negative MLL-AF9 cells).
  • FIG. 33 is a Table demonstrating that human myelo-markers are different than the mouse. Macl is expressed by neutrophils, NK cells and macrophages. CD66b is expressed exclusively on granulocytes and used as a granulocyte marker. CD 14 is expressed mainly by macrophages (and at 10-times lesser extend by neutrophils). CD66b+ CD14+ marks only monocytes. As shown in FIG. 33, CD 14 is the human equivalent to Mac-1 in mice, and CD66b is the human equivalent to Gr-1 in mice.
  • FIGS. 34A, 34B, 34C and 34D demonstrate that Macl is downregulated in CBX-treated human leukemias, but not in CBX-treated normal human blood cells, as determined by flow cytometry analysis of live 7AAD human cells incubated with CBX at the concentrations indicated for 16h.
  • FIGS. 34A-34D are bar graphs quantifying myelo-markers CDl lb (FIG. 34A), CD14+ (FIG. 34B), CD66b+ (FIG. 34C), and CD66+/CD14+ (FIG. 34D) in CBX-treated primary normal human leukocytes as compared to CBX-treated human leukemic cell lines U937, HL60 and THP1.
  • FIG. 35 is a Table demonstrating results of an in vivo gene expression study of gap-junction molecules in bone marrow leukemic cells and their normal counterparts.
  • Bone marrow leukemic cells assessed comprise MLL-AF9+, GFP -positive, Grl/Macl- positive, c-Kit high cells.
  • Bone marrow normal myeloid progenitors assessed comprise B220/CD8a/CD3e/CD4/TER119-negative GFP-negative, GRl/Mac-1 -positive, c-Kit high cells.
  • FIG. 36 is a bar graph illustrating expression of connexin sorting protein Consortin relative to expression of HPRT in normal GMP compared to leukemia after using the treatments and controls indicated for the time periods specified.
  • FIGS. 37A, 37B, 37C, 37D, 37E, 37F, 37G, 37H, and 371 are bar graphs illustrating expression of gap-junction alpha molecules relative to expression of HPRT in normal GMP compared to leukemia after using the treatments and controls indicated for the time periods specified, including gap-junction alpha molecules Al (FIG. 37A), A3 VI (FIG. 37B), A3 V2 (FIG. 37C), A4 (FIG. 37D), A5 VI (FIG. 37E), A5 V2 (FIG. 37F), A6 (FIG. 37G), A8 (FIG. 37H) and A10 (FIG. 371).
  • FIGS. 38A, 38B, 38C, 38D, 38E, 38F and 38G are bar graphs illustrating expression of gap-junction beta molecules relative to expression of HPRT in normal GMP compared to leukemia after using the treatments and controls indicated for the time periods specified, including gap-junction beta molecules B l (FIG. 38A), B2 (FIG. 38B), B3 (FIG. 38C), B4 (FIG. 38D), B5 (FIG. 38E), B6 V3 (FIG. 38F) and B6 V2 (FIG. 38G).
  • FIGS. 39A, 39B and 39C are bar graphs illustrating expression of gap- junction gamma molecules relative to expression of HPRT in normal GMP compared to leukemia using the treatments and controls indicated for the time periods specified, including gap-junction gamma molecules CI (FIG. 39A), C2 (FIG. 39B) and C3 (FIG. 39C).
  • FIGS. 40A, 40B and 40C are bar graphs illustrating expression of gap- junction delta molecules relative to expression of HPRT in normal GMP compared to leukemia using the treatments and controls indicated for the time periods specified, including gap-junction delta molecules D2 (FIG. 40A), D3 (FIG. 40B) and D4 (FIG. 40C).
  • FIG. 41 is a bar graph illustrating expression of gap-junction epsilon molecules relative to expression of HPRT in normal GMP compared to leukemia after using the treatments and controls indicated for the time periods specified.
  • FIG. 42 demonstrates that intraperitoneal (IP) administration of CBX to MLL-AF9 leukemic mice results in prolonged survival. Leukemia was induced in mice by intravenously injecting 1 million live MLL-AF9 leukemic cells (expressing GFP and luciferase) into non-irradiated recipients. In the model, leukemic cells could be detected in the bone marrow -27 days post-transplantation and non-treated mice succumbed to leukemia at day -70.
  • IP intraperitoneal
  • FIG. 42 shows whole body bioluminescence signal (IVIS) on day 36 of mice not treated (left), treated with chemotherapy (middle), and treated with CBX plus chemotherapy (right).
  • IVIS whole body bioluminescence signal
  • FIGS. 43A and 43B demonstrate that intraperitoneal (IP) administration of CBX to MLL-AF9 leukemic mice results in prolonged survival.
  • IP intraperitoneal
  • FIG. 43 A treatment was given on day 27 upon detection of leukemic cells in the bones, chemo comprised cytarabine (100 mg/kg) and doxorubicin (3 mg/kg) for 3 days, followed by administering cytarabine alone (100 mg/kg) for an additional 2 days, and CBX was administered at 20 mg/kg to the subject for 6 days.
  • chemo comprised cytarabine (100 mg/kg) and doxorubicin (3 mg/kg) for 3 days
  • CBX was administered at 20 mg/kg to the subject for 6 days.
  • CBX treatment was given on day 68, at a terminal state of the disease, with leukemic cells spread all over the body, CBX was administered at 10 mg/kg for 3 days, followed by 3 days without treatment, followed by 2 days of 20 mg/kg, followed by 3 days without treatment, followed by 3 days of 30 mg/kg.
  • FIG. 44 demonstrates that daily sub-cutaneous (SC) administration of CBX for 2 weeks (at 75 mg/kg alone or at 50 mg/kg combined with chemotherapy) results in prolonged survival of leukemic mice.
  • Leukemia was induced in mice by intravenously injecting 5 million live MLL-AF9 leukemic cells (expressing infrared fluorescent protein (iRFP) into sub-lethally irradiated recipients.
  • iRFP infrared fluorescent protein
  • leukemic cells could be detected in the bone marrow - 7 days post-transplantation and non-treated mice succumbed to leukemia at day -34.
  • Day -1 sub-lethal irradiation (4.5 Gy); Day 1 : tail vein injection of 5M live iRFP+ MLL-AF9 leukemic cells; Days 7+8: PBS or CBX treatment (subcutaneous, as depicted; Days 9-1 1 : PBS or CBX treatment + chemotherapy (100 mg/kg Cytarabine + 3 mg/kg Doxorubicin) Days 12 + 13 : PBS or CBX treatment + chemotherapy ( 100 mg/kg Cytarabine); Days 14-21 : PBS or CBX treatment.
  • chronic systemic exposure of CBX continuously for 2 weeks), without prophylactic treatment, resulted in acute lethal pseudo-hyperaldosteronism in some case (e.g., hypertension and gastric edemas)
  • FIGS. 45A, 45B and 45C demonstrate the survival curve of mice treated with 25 mg/kg CBX alone, or in combination with induction chemotherapy. 40%-60% of CBX- treated mice survived, with early mortality due to acute lethal pseudo-hyperadlosteronism. Notably, there was no statistically significant difference in survival of mice treated with CBX in combination with induction chemotherapy compared to both PBS and chemotherapy controls.
  • FIGS. 46A, 46B and 46C demonstrate the survival curve of mice treated with 50 mg/kg CBX alone, or in combination with induction chemotherapy.
  • 50 mg/kg CBX alone resulted in early mortality with no statistically significant difference (20% of CBX-treated mice survived).
  • 50 mg/kg of CBX led to significant increased survival compared to both PBS and chemotherapy controls.
  • FIGS. 47A, 47B and 47C demonstrate the survival curve of mice treated with 75 mg/kg CBX alone, or in combination with induction chemotherapy.
  • 75 mg/kg alone led to significant increased survival compared to PBS controls.
  • 75 mg/kg resulted in early mortality with no statistically significant difference (20% of CBX-treated mice survived).
  • FIG. 48 demonstrates survival curves and p values of all SC prolonged administration trials described in FIGS. 45A-C, FIGS. 46A-C and FIGS. 47A-C.
  • FIGS. 49A and 49B demonstrate that prolonged administration of CBX (SC, daily, 14 days) might result in lethal pseudo-hyperadlosteronism with no sign of leukemia.
  • FIG. 49A is an autopsy image of a healthy control mice.
  • FIG. 49B is an autopsy image of a mouse that died at day 28 after 50 mg/ml CBX treatment (FIG. 49B), which exhibited gastric edema.
  • FIGS. 50A, 50B and 50C demonstrate that early mortality in CBX treated animals (SC, daily for 14 days) is most likely induced by acute pseudo-hyperaldosteronism, and not by leukemia.
  • FIG. 5 OA is an autopsy image of a mouse treated with PBS, showing clear signs of leukemia.
  • FIG. 50B is an autopsy image of a mouse treated with chemotherapy, showing signs of leukemia.
  • FIG. 50C is an autopsy image of a mouse treated with chemotherapy plus 50 mg/kg CBX, showing no signs of leukemia.
  • Black arrows indicate swollen lymph nodes. Red arrows indicate splenomegaly. Blue arrows indicate gastric edema.
  • FIG. 51 is an autopsy image of a subject mouse treated with chemotherapy, showing signs of leukemia. Black arrows indicate swollen lymph nodes. Red arrows indicate splenomegaly. Blue arrows indicate gastric edema.
  • FIG. 52 is an autopsy image of a subject mouse treated with chemotherapy plus 50 mg/kg CBX, showing no signs of leukemia.
  • Black arrows indicate swollen lymph nodes.
  • Red arrows indicate splenomegaly.
  • Blue arrows indicate gastric edema.
  • FIG. 53 demonstrates the selective leukemia cell eradication by 18 ⁇ - glycyrrhetinic acid (enoxolone) and depicts the structure thereof.
  • Primary MLL-AF9 LKS acute myeloid leukemia cells (red bars) were co-cultured with normal BM mononuclear cells (blue bars). Cells in co-culture were exposed to enoxolone for 20 hours (at 0, 5, 20, 50, 100 and 200 ⁇ ) and then cell viability was analyzed by flow cytometry (live leukemia
  • FIG. 54 shows the effects of 18 -glycyrrhetinic acid (enoxolone) on the viability of normal bone marrow cells (myeloid cells vs. non-myeloid cells). Cells in co- culture were exposed to enoxolone for 20 hours (at 0, 5, 20, 50, 100 and 200 ⁇ ). As illustrated in FIG. 54, enoxolone was toxic to normal non-myeloid cells at 200 ⁇ .
  • enoxolone 18 -glycyrrhetinic acid
  • FIG. 55 demonstrates that carbenoxolone (CBX) selectively induces oxidative stress in leukemic cells.
  • CBX carbenoxolone
  • treatment with 200 ⁇ of CBX over varying durations (1, 2, 4 or 8 hours) caused a differential effect on both total and mitochondrial reactive oxygen species (ROS) levels in leukemia cells relative to normal cells, thereby contributing to the selective eradication observed with CBX.
  • ROS mitochondrial reactive oxygen species
  • FIG. 56 demonstrates that carbenoxolone (CBX) selectively induces lipid damage and double-strand DNA breaks in leukemia cells.
  • CBX carbenoxolone
  • FIG. 57 demonstrates that varying concentrations of carbenoxolone - derivative compounds that are identified as CBX-1, CBX-2 and CBX-3, also demonstrated selective anti-leukemia activity in an ex vivo assay relative to a control compound.
  • FIGS. 58A-58B demonstrate that CBX treatment induces apoptosis in leukemia cells, but failed to induce apoptosis in normal bone marrow mononuclear cells (FIG. 58 A) and normal myeloid progenitor cells (FIG. 58B).
  • FIG. 59 demonstrates that CBX induced a differential effect on leukemia initiating cells relative to normal colony forming cells in mice.
  • FIGS. 60A-60B illustrate that carbenoxolone (CBX) selectively eradicates leukemia cells and prolongs survival.
  • FIG. 60A shows the percent survival of mice treated with a vehicle control (days 9-21), induction chemotherapy (iCT, days 9- 13), CBX alone (days 9-21) or combined treatment (iCT+CBX). *p ⁇ 0.02.
  • 61A-61D illustrate that carbenoxolone (CBX) blocks mitochondrial respiration in human and mouse leukemia cells.
  • CBX carbenoxolone
  • Oxygen consumption rates before and after PBS or CBX injections in human cells (FIG. 61C) and mouse cells (FIG. 61D) are shown. Arrows indicate time of compound injection (as indicated). Similar results were documented in all human and mouse cell lines tested.
  • FIGS. 62A-62B compare mitochondrial stress and the effects of
  • CBX carbenoxolone
  • FIG. 62A normal cells
  • FIG. 62B leukemia cells
  • Arrows indicate time of automatic compound injection (operated by the seahorse software).
  • Green lines and green legends depict the Mito- Stress assay, blue lines depict PBS injection and red lines depict CBX injection. Average data of greater than 4 samples. Similar results were documented with all human and mouse leukemia cell lines tested.
  • Oligomycin ATP Synthase (complex V) inhibitor. Used for measuring how much oxygen is being consumed for ATP production as well as for measuring maximum glycolytic capacities.
  • FCCP H+ ionophore (uncoupling agent). Used for measuring the maximal mitochondrial respiration capacities.
  • Antimycin Complex III inhibitor. Used to block mitochondrial respiration.
  • FIGS. 63A-63C illustrate that carbenoxolone (CBX) treatment induced oxidative stress-mediated cell death in leukemia cells, but not in normal cells.
  • FIG. 63 A shows measurements of superoxide levels (MitoSOX Red mitochondrial superoxide indicator).
  • FIG. 63B shows measurements of peroxyl radicals levels (BODIPY 581/591 Cl l ; Lipid Peroxida on Sensor).
  • FIG. 63C depicts phosphorylation of ⁇ 2 ⁇ (double-strand DNA breaks indicator).
  • FIGS. 64A-64B demonstrate that carbenoxolone (CBX) induced Ca 2+ influx in human leukemia cells (FIG. 64B), but did not induce Ca 2+ influx in normal human PB- MNC cells (FIG. 64A).
  • Mitochondrial stress ATP synthase inhibitor, H + ionophor and complex III inhibitor
  • Arrows indicate time of compound injection.
  • Green lines and green legends depict the Mito-Stress assay, blue lines depict PBS injection and red lines depict CBX injection. Similar results were documented with all human leukemia cell lines tested
  • FIGS. 65A-65C demonstrate the selective anti-leukemia activity induced by carbenoxolone (CBX) in vivo.
  • FIG. 65 A depicts an induction chemotherapy (iCT) regimen, the current standard of care, while FIG. 65B compares iCT and CBX in leukemia cells.
  • FIG. 65 C demonstrates that CBX eradicated leukemia cells and supports replenishment of normal myeloid cells.
  • FIG. 66 demonstrates the activity of CBX in solid tumor cell lines. All solid tumor cell lines tested exhibited greater sensitivity to CBX treatment (24-hour incubation in vitro), compared to normal BM stromal cell line (HS-5).
  • CBX efficiently eradicated melanoma cells at ⁇ (A375, blue arrows). Cells were incubated with increasing concentration of CBX (0 ⁇ -400 ⁇ ) for 24 hours. End-point cell viability was scored by flow cytometry analysis.
  • the disclosure relates to the discovery that certain agents (e.g., glycyrrhetinic acid derivatives) can be used effectively to eradicate leukemic cells in a population or subject without eradicating normal cells in the subject or, in certain instances only minimally eradicating normal cells in the subject. Accordingly, the disclosure contemplates the use of one or more agents (e.g., glycyrrhetinic acid derivatives) in methods, compositions, and kits for eradicating leukemic cells, and in related methods, compositions, and kits for treating acute myeloid leukemia.
  • agents e.g., glycyrrhetinic acid derivatives
  • a method of eradicating leukemic cells in a population of cells comprises contacting the population of cells with an effective amount of an agent or a gap junction blocker, thereby eradicating leukemic cells in the cell population.
  • the leukemic cells can be eradicated by a variety of mechanisms upon exposure to or contact with an agent described herein.
  • the agent selectively induces apoptosis of leukemic cells.
  • the agents e.g., carbenoxolone and analogs or derivatives thereof
  • eradicating leukemic cells comprises inducing programmed cell death. In some embodiments, eradicating leukemic cells comprises inducing apoptosis. In some embodiments, eradicating leukemic cells comprises inducing the differentiation of the leukemic cells into non-leukemic cells as described herein. In some embodiments, eradicating leukemic cells comprises inducing the differentiation of the leukemic cells into granulocytes. In some embodiments, the granulocytes comprise neutrophils. In some embodiments, leukemic cells are eradicated upon contact with an agent or gap junction blocker described herein by inducing the differentiation of the leukemic cells (e.g., human) into CD66b+/CD 14- neutrophils.
  • an agent or gap junction blocker described herein by inducing the differentiation of the leukemic cells (e.g., human) into CD66b+/CD 14- neutrophils.
  • eradicating leukemic cells comprises disrupting intercellular communications involving the leukemic cells that promote leukemia cell survival. In certain embodiments, eradicating leukemic cells comprises disrupting intercellular communications involving the leukemic cells that confer drug resistance to the leukemic cells. In some embodiments, disrupting intercellular communications involving leukemic cells comprises interfering with homotypic interactions between leukemic cells. In some embodiments, disrupting intercellular communications involving leukemic cells comprises interfering with heterotypic interactions between leukemic cells and stromal cells.
  • disrupting intercellular communications involving leukemic cells comprises interfering with heterotypic interactions between leukemic cells and any other cell types (e.g., mesenchymal stromal cells, osteolineage cells, endothelial cells, pericytes, mesenchymal cells or other hematopoietic cells).
  • eradicating leukemic cells comprises overcoming stroma-mediated drug resistance (e.g., to cancer treatment, e.g., induction chemotherapy).
  • disrupting intercellular communications involving the leukemic cells causes the leukemic cells to differentiate into non-leukemic cells, thereby eradicating the cells.
  • leukemic cells are selectively eradicated while inducing proliferation of normal leukocytes in the population of cells. For example, contacting a population of acute myeloid leukemia cells with a gap junction blocker described herein selectively eradicates the acute myeloid leukemia cells while inducing the proliferation of normal leukocytes in the population.
  • compositions and methods described herein preferably selectively affect leukemic cells without affecting normal cells (e.g., leukocytes) in the population of cells.
  • leukemic cells are selectively eradicated without eradicating, or in certain aspects minimally eradicating, normal leukocytes in the population of cells.
  • the leukemic cells are selectively eradicated without eradicating, or in certain embodiments minimally eradicating, normal bone marrow leukocytes or normal peripheral blood leukocytes, including without limitation, stem and progenitors, bone marrow mononuclear cells, myeloblasts, neutrophils, NK cells, macrophages, granulocytes, monocytes, and lineage-/cKit+/Scal+ (LKS) cells.
  • the amount or activity of leukemic cells in a population of cells is selectively decreased without decreasing the amount or activity of normal leukocytes in the population.
  • proliferation of leukemic cells is selectively inhibited in a population of cells without inhibiting proliferation of normal leukocytes in the population.
  • the compositions and methods described herein can be used to increase the number of normal leukocytes in a population of cells by selectively reducing the number, activity, and/or proliferation of leukemic cells in the population of cells. Without wishing to be bound by theory, it is expected that the amount of leukemic cells eradicated, reduced, or inhibited in any particular population of cells is proportional to the concentration of the agent or gap junction blocker to which the population of cells has been exposed.
  • At least 20% of the leukemic cells in the population of cells are eradicated, reduced, or inhibited. In some embodiments, at least 50% of the leukemic cells in the population of cells are eradicated, reduced, or inhibited. In some embodiments, at least 70% of the leukemic cells in the population of cells are eradicated, reduced, or inhibited. In some embodiments, all of the leukemic cells in the population of cells are eradicated, reduced, or inhibited.
  • the present invention contemplates selectively eradicating any leukemic cell by contacting a population of cells with, or exposing the population of cells to, a gap junction blocker.
  • the leukemia cells comprise leukemia cells from an acute myeloid leukemia cell line.
  • Exemplary acute myeloid leukemia cell lines include, but are not limited to, MLL-AF9 cells, MLL-ENL cells, Nup98-HoxA9 cells, AML1-ET09A cells, KG-1 cells, KG- la cells, U937 cells, THP 1 cells, HL60 cells, HoxA9/Meis 1 cells, and NB-4 cells.
  • selective eradication of leukemic cells in a population of cells means that the leukemic cells in the population are eradicated without eradicating or otherwise affecting other normal cells in the population.
  • selective eradication of leukemic cells in a population of cells means that the leukemic cells in the population are eradicated with minimal eradication or with limited untoward effects on other normal cells within the population.
  • selective eradication of leukemic cells in a population of cells means that the leukemic cells in the population are eradicated, while the population of normal cells is expanded.
  • the population of cells comprises primary leukocytes, such as bone marrow leukocytes and peripheral blood leukocytes.
  • Examples of such primary leukocytes include, without limitation, stem and progenitors, mononuclear cells, myeloblasts, neutrophils, NK cells, macrophages, granulocytes, monocytes, and lineage-/cKit+/Scal+ (LKS) cells.
  • stem and progenitors mononuclear cells, myeloblasts, neutrophils, NK cells, macrophages, granulocytes, monocytes, and lineage-/cKit+/Scal+ (LKS) cells.
  • the effective amount of the agents for use in accordance with the present inventions may vary, for example, depending on the agent or gap junction blocker being used and its location of use.
  • the effective amount of the agent (e.g., a glycyrrhetinic acid derivative or a gap junction blocker) for in vitro use comprises a concentration in the range of 50 ⁇ to 400 ⁇ .
  • the effective amount of the agent or gap junction blocker for in vivo use comprises a concentration in the range of lOmg/kg to lOOmg/kg.
  • the effective amount comprises a
  • the effective amount comprises a concentration of 25 mg/kg. In some embodiments, the effective amount comprises a concentration of 50 mg/kg. In some embodiments, the effective amount comprises a concentration of 75 mg/kg.
  • the contacting occurs in vitro or ex vivo.
  • the contacting occurs in vivo.
  • the in vivo contact is in a subject as described herein.
  • the disclosure also provides methods for promoting the differentiation of leukemic cells into non-leukemic cells. Such methods can be useful for treating leukemia, for example, acute myeloid leukemia.
  • a method of promoting the differentiation of a leukemic cell into a non-leukemic cell comprises contacting a leukemic cell with an effective amount of an agent (e.g., a gap junction blocker), thereby promoting the differentiation of the leukemic cell into a non-leukemic cell.
  • an agent e.g., a gap junction blocker
  • the disclosure contemplates differentiating any leukemic cell into a non- leukemic cell in accordance with the methods described herein.
  • the leukemic cell comprises a leukemic stem or progenitor cell.
  • the leukemic stem or progenitor cell comprises an acute myeloid leukemia cell.
  • the acute myeloid leukemia comprises a cell line selected from the group consisting of MLL-AF9 cells, MLL-ENL cells, Nup98-HoxA9 cells, AML1-ET09A cells, KG-1 cells, KG- la cells, U937 cells, HL60 cells, THP1 cells, HoxA9/Meis 1 cells, and NB-4 cells.
  • the differentiated leukemic cells may differentiate into non-leukemic cells of varying stages.
  • the non- leukemic cell comprises a mature or terminally differentiated cell.
  • the non-1 eukemic cell comprises a granulocyte.
  • the granulocyte comprises a short-lived granulocyte.
  • the non-leukemic cell comprises a neutrophil.
  • the neutrophil comprises a CD66b+/CD 14- neutrophil.
  • compositions and kits comprising the gap junction blockers and/or agents described herein.
  • disclosure contemplates the treatment of any disease in which intercellular
  • the agents and/or gap junction blockers described herein can be used to treat and/or prevent such diseases.
  • the agents and/or gap junction blockers selectively eradicate malignant or neoplastic cells (e.g., blood cells) in which intercellular communication or interaction through gap junctions or hemichannels plays a role in disease resistance to treatment or therapy.
  • the agents e.g., glycerrhetinic acid derivatives, carbenoxolone or analogs or derivatives thereof
  • the disclosure provides a method of treating leukemia (e.g., acute myeloid leukemia) in a subject in need thereof, the method comprising administering to the subject an effective amount of a gap junction blocker or an agent (e.g., a glycyrrhetinic acid derivative, carbenoxolone or analogs or derivatives thereof) described herein, thereby treating leukemia (e.g., acute myeloid leukemia) in the subject.
  • a gap junction blocker or an agent e.g., a glycyrrhetinic acid derivative, carbenoxolone or analogs or derivatives thereof
  • the agent and/or gap junction blocker selectively eradicates leukemic cells without eradicating, or minimally eradicating, normal cells in the subject. In some embodiments, the agent and/or gap junction blocker selectively eradicates leukemic cells in the subject without eradicating, or minimally eradicating, normal leukocytes in the subject. In some embodiments, the agent and/or gap junction blocker selectively eradicates leukemic cells in the subject while inducing proliferation of normal leukocytes in the subject. In some embodiments, the agent and/or gap junction blocker selectively eradicates leukemic cells in the subject while inducing replenishment of normal leukocytes in the subject.
  • the agent and/or gap junction blocker can selectively eradicate leukemic cells without eradicating, or minimally eradicating, normal leukocytes while inducing the proliferation and/or replenishment of normal leukocytes in the subject.
  • the method further comprises administering an induction chemotherapy treatment regimen to the subject.
  • the disclosure contemplates administering any induction chemotherapy treatment regimen that is useful for inducing complete remission of acute myeloid leukemia in a subject.
  • the induction chemotherapy comprises administering an antimetabolite agent (e.g., cytarabine) and an anthracycline agent (e.g., doxorubicin) to the subject.
  • the antimetabolite agent comprises cytarabine.
  • the induction chemotherapy treatment regimen can be administered to the subject over a period of hours, days, or months.
  • chemotherapeutic agents used in the induction chemotherapy treatment regimen can be administered at the same time throughout the period, or administered at different intervals within the period.
  • the induction chemotherapy comprises administering cytarabine and doxorubicin to the subject for a period of 5 days.
  • the induction chemotherapy comprises administering cytarabine and doxorubicin to the subject for a period of 3 days, followed by administering cytarabine alone to the subject for a period of 2 days.
  • the agent and/or gap j unction blocker can be administered to the subj ect before the induction chemotherapy treatment regimen is administered to the subject, at the same time the induction chemotherapy treatment regimen is administered to the subject, after the induction chemotherapy treatment regimen is administered to the subject, or any combination of the above.
  • the agent and/or gap junction blocker is administered to the subject for at least a day before administering the induction chemotherapy treatment regimen to the subject.
  • the agent and/or gap junction blocker is administered to the subject for at least a day before administering the induction chemotherapy treatment regimen to the subject concomitantly with the agent and/or gap junction blocker.
  • the agent and/or gap junction blocker is administered to the subject at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or up to at least a week before administering the induction chemotherapy treatment regimen to the subject. In some embodiments, the agent and/or gap junction blocker is administered to the subject at least 8 days, at least 9 days, at least 10 days, at least 1 1 days, at least 12 days, at least 13 days, at least 2 weeks, at least 3 weeks, or at least a month before the induction chemotherapy treatment regimen is administered to the subject.
  • the agent and/or gap junction blocker is administered to the subject for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or up to at least a week before administering the induction chemotherapy treatment regimen to the subject, and then the induction chemotherapy regimen is administered to the subject concomitantly with the gap junction blocker for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 2 weeks, at least 3 weeks, or at least a month.
  • the agent and/or gap junction blocker is administered to the subject for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or up to at least a week before administering the induction chemotherapy treatment regimen to the subject, and then the induction chemotherapy regimen is administered to the subject concomitantly with the agent and/or gap junction blocker for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 2 weeks, at least 3 weeks, or at least a month, before ceasing administration of the induction chemotherapy regimen while continuing administration of the agent and/or gap junction blocker to the subject for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least
  • the agent and/or gap junction blocker is administered to the subject for at least 2 days before administering an induction chemotherapy treatment regimen comprising 100 mg/kg cytarabine + 3 mg/kg doxorubicin to the subject concomitantly with or without administering the agent and/or gap junction blocker for 3 days, followed by chemotherapy with 100 mg/kg cytarabine in the absence of doxorubicin concomitantly with or without the gap junction blocker for 2 days, followed by 2 weeks (14 days) of administration of the agent and/or gap junction blocker to the subject.
  • CBX is administered to the subject for at least 2 days before
  • administering an induction chemotherapy treatment regimen comprising 100 mg/kg cytarabine + 3 mg/kg doxorubicin to the subject concomitantly with or without administering CBX for 3 days, followed by chemotherapy with 100 mg/kg cytarabine in the absence of doxorubicin concomitantly with or without the CBX for 2 days, followed by 2 weeks (14 days) of administration of CBX to the subject.
  • administration of CBX or a gap junction blocker described herein comprises administering ascending and intermittent concentrations or doses of CBX or the gap junction blocker described herein over a period of time to the subject.
  • CBX or the gap-junction blocker can be administered at 10 mg/kg for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least a week, followed by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 1 week in the absence of administering CBX or the gap junction blocker, followed by administration of 20 mg/kg for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least a week, followed by the absence of administration of CBX or the gap junction blocker for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least a week, and then followed by administration of 30 mg/kg for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least a week, and
  • the concentration or dosage of CBX or the gap junction blocker administered initially and at successive intervals after intermission of treatment can vary, as well as the escalation of the concentration or dose between treatment intervals.
  • the initial dose or concentration of CBX or the gap junction blocker can be 5 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg or more
  • the escalation of the concentration or dose between intervals can be 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, or 25 mg/kg.
  • ascending and intermittent concentrations of doses of CBX or gap junction blocker can be administered over a variety of treatment intervals, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or as many as desired until the subject enters remission, to keep the subject in remission, or to further prolong survival of the patient, for example, by inducing the patient into remission or preventing the patient from relapsing from remission.
  • treatment intervals e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10
  • the treatment and intermission from treatment intervals can be more than a week, e.g., 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or a year depending on the course of the disease in the subject.
  • the aforementioned ascending and intermittent concentration or dosing schedules can be used when a subject is at a terminal state of the disease, for example, when leukemic cells are spread all over the subject's body, to prolong survival time of the subject.
  • treat when used in reference to a disease, disorder or medical condition, refers to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition.
  • treating includes reducing or alleviating at least one adverse effect or symptom of a condition.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i. e., not worsening) state of, for example, acute myeloid leukemia, delay or slowing progression of acute myeloid leukemia, and an increased lifespan as compared to that expected in the absence of treatment.
  • treating acute myeloid leukemia comprises inducing complete remission of acute myeloid leukemia in the subject.
  • the agent and/or gap junction blocker is administered to the patient for at least a day after complete remission is induced in the acute myeloid leukemia patient.
  • the agent and/or gap junction blocker is administered to the patient for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 1 1 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 1 month, at least 2 months, at least 3 months, or more after complete remission is induced in the acute myeloid leukemia patient.
  • treating acute myeloid leukemia comprises inducing complete remission of acute myeloid leukemia in the subject in the absence of a relapse risk due to residual leukemic cells in the subject's bone marrow or peripheral blood.
  • the method further comprises evaluating the subject to determine if the subject has refractory or relapsed acute myeloid leukemia.
  • the disclosure provides a method of promoting survival of a subject suffering from acute myeloid leukemia, the method comprising administering to the subject an effective amount of an agent and/or gap junction blocker, thereby promoting survival of the subject.
  • the method contemplates any gap junction blocker described herein.
  • the agent or gap junction blocker comprises an inhibitor of 1 1 ⁇ - hydroxysteroid dehydrogenase (1 ⁇ -HSD).
  • the agent or gap junction blocker comprises carbenoxolone or an analog thereof.
  • the method further comprises administering an induction chemotherapy treatment regimen to the subject.
  • the induction chemotherapy comprises administering an antimetabolite agent and an
  • the antimetabolite agent comprises cytarabine.
  • the anthracycline agent comprises doxorubicin.
  • the induction chemotherapy comprises administering cytarabine and doxorubicin to the patient for a period of 5 days.
  • the induction chemotherapy comprises administering cytarabine and doxorubicin to the patient for a period of 3 days, followed by administering cytarabine alone to the patient for a period of 2 days. It should be appreciated that any of the administration or dosing schedules and/or treatment regiments described herein can be used with the method.
  • the agent or gap junction blocker is administered to the subject for at least a day before administering the induction chemotherapy treatment regimen to the subject. In some embodiments, the agent or gap junction blocker is administered to the subject for at least a day before administering the induction chemotherapy treatment regimen to the subject concomitantly with the agent or gap junction blocker. [0177] In some embodiments, the methods of the invention further comprise subjecting the subject to hyperbaric oxygen therapy (HBOT). Hyperbaric oxygen therapy (HBOT) is the administration of 100% oxygen at elevated pressure (greater than sea level, 1 ATA).
  • HBOT increases plasma oxygen saturation, facilitating oxygen delivery to the tissue independent of hemoglobin 0 2 saturation (Gill & Bell (2004) "Hyperbaric oxygen: its uses, mechanisms of action and outcomes," QJM. 2004 Jul; 97(7):385-95).
  • the subject is administered a glycyrrhetinic acid derivative (e.g., carbenoxolone or an analog or derivative thereof) and subjected to HBOT.
  • a glycyrrhetinic acid derivative e.g., carbenoxolone or an analog or derivative thereof
  • the selective anti-cancer e.g., anti-leukemic
  • carbenoxolone is mediated by altered cellular metabolism in cancer cells (e.g., leukemia cells) with an increased oxygen consumption rate.
  • HBOT can be initiated prior to, during, or after administration of a glycyrrhetinic acid derivative (e.g., carbenoxolone or an analog or derivative thereof) as long as the subject exhibits elevated oxygen levels in the blood and/or tissues for at least a portion of time when the glycyrrhetinic acid derivative (e.g., carbenoxolone or an analog or derivative thereof) is present in the subject.
  • a glycyrrhetinic acid derivative e.g., carbenoxolone or an analog or derivative thereof
  • HBOT is performed at a pressure from about 1.0 ATA to 3.0 ATA, in another embodiment about 1.5 ATA to 2.75 ATA, and in another embodiment about 2.0 ATA to 2.5 ATA.
  • the time per treatment may vary from about 1 to 120 minutes, in another embodiment from about 30 to 110 minutes, and in another embodiment from about 45 to 100 minutes.
  • the method further comprises selecting a subject suffering from or exhibiting a terminal state of acute myeloid leukemia.
  • the subject has advanced tumor metastasis. In some embodiments, the subject has a high tumor burden.
  • “Survival” refers to the subject remaining alive, and includes overall survival as well as progression free survival. “Overall survival” refers to the subject remaining alive for a defined period of time, such as 1 year, 2 years, 3 years, 4 years, 5 years, etc. from the time of diagnosis or treatment.
  • progression free survival refers to the subject remaining alive, without the acute myeloid leukemia progressing or getting worse.
  • Treatment survival refers to enhancing one or more aspects of survival in a treated subject relative to an untreated subject (i.e., a subject not treated with a gap junction blocker, such as carbenoxolone), or relative to a subject treated with an approved
  • the gap junction blocker increases the subject's length of survival compared to the subject's length of survival in the absence of receiving the gap junction blocker. In some embodiments, the gap junction blocker increases the subject's likelihood of survival compared to the subject's likelihood of survival in the absence of receiving the gap junction blocker.
  • administration of the gap junction blocker increases the subject's overall survival time by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more relative to subject's overall survival time in the absence of administration of the gap junction blocker and/or compared to chemotherapy treatment alone.
  • administration of the gap junction blocker increases the subject's overall survival time by at least 1.1 fold, at least 1.2 fold, 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 3 fold, at least 4 fold, or, at least 5 fold or more relative to subject's overall survival time in the absence of administration of the agent or gap junction blocker and/or compared to chemotherapy treatment alone.
  • the gap junction blocker e.g., CBX
  • administration of the gap junction blocker increases the subject's survival time by 1 day, 5 days, 10 days, 30 days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, 1 year, 18 months, 2 years, 30 months, 3 years, 40 months, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 50 years, 55 years, 60 years, 65 years, 70 years, or 75 years or more relative to subject's overall survival time in the absence of administration of the agent or gap junction blocker and/or compared to chemotherapy treatment alone.
  • the gap junction blocker e.g., CBX
  • the disclosure provides a method of inducing complete remission in a subject having relapsed or refractory acute myeloid leukemia by selectively eradicating leukemic cells in the subject, the method comprising: (a) evaluating the subject to determine if the subject has relapsed or refractory acute myeloid leukemia; (b) administering to the subject an agent or gap junction blocker at least a day before administering an induction chemotherapy treatment regimen to the subject; and (c) administering to the subject an induction chemotherapy treatment regimen comprising an antimetabolite agent and an anthracycline agent for proscribed periods of time, thereby inducing complete remission in the subject by selectively eradicating leukemic cells in the subject.
  • the efficacy of the compositions and methods disclosed herein are not limited to leukemia cells, but rather extend broadly to all cancer cell types.
  • the cancer is selected from the group consisting of solid tumors and non-solid tumors.
  • the inventions disclosed herein related to methods of eradicating cancer cells in a population of cells, the methods comprising contacting the population of cells with an effective amount of a glycyrrhetinic acid derivative (e.g., carbenoxolone or analogs or derivatives thereof), thereby eradicating leukemic cells in the cell population.
  • a glycyrrhetinic acid derivative e.g., carbenoxolone or analogs or derivatives thereof
  • the cancer cells comprise leukemia cells. In some embodiments, the cancer cells comprise breast cancer cells. In yet other embodiments, the cancer cells comprise melanoma cells. In still other embodiments, the cancer cells comprise prostate cancer cells. In still other embodiments, the cancer cells comprise cervix carcinoma cells.
  • the methods and compositions disclosed herein are useful for the treatment of one or more of colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer; cancers of the oral cavity and pharynx, esophagus, stomach, small intestine, large intestine, colon, rectum, liver and biliary passages; pancreas, bone, connective tissue, skin, cervix, uterus, corpus endometrium, testis, bladder, kidney and other urinary tissues; cancers of the eye, brain, spinal cord, and meninges, including glioblastoma; cancers of the thyroid and other endocrine glands; Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma, leukemias and lymphomas; adrenocarcinoma, angiosarcoma, astrocytoma, acoustic neuroma, anaplastic astrocytoma, basal cell
  • chondrosarcoma choriocarcinoma, chordoma, craniopharyngioma, cutaneous melanoma, cystadenocarcinoma, endotheliosarcoma, embryonal carcinoma, ependymoma, Ewing's tumor, epithelial carcinoma, fibrosarcoma, gastric cancer, genitourinary tract cancers, glioblastoma multiforme, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hepatoma, Kaposi's sarcoma, large cell carcinoma, leiomyosarcoma, leukemias, liposarcoma, lymphatic system cancer, lymphomas, lymphangiosarcoma,
  • lymphangioendotheliosarcoma medullary thyroid carcinoma, medulloblastoma, meningioma mesothelioma, myelomas, myxosarcoma neuroblastoma, neurofibrosarcoma,
  • oligodendroglioma, osteogenic sarcoma, epithelial ovarian cancer papillary carcinoma, papillary adenocarcinomas, paraganglioma, parathyroid tumours, pheochromocytoma, pinealoma, plasmacytomas, retinoblastoma, rhabdomyosarcoma, sebaceous gland carcinoma, seminoma, skin cancers, melanoma, small cell lung carcinoma, non-small cell lung carcinoma, squamous cell carcinoma, sweat gland carcinoma, synovioma, thyroid cancer, uveal melanoma, and Wilm's tumor.
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, "patient” and “subject” are used interchangeably herein.
  • the subject suffers from cancer.
  • the subject suffers from acute myeloid leukemia.
  • the subject is a patient presenting with leukemia, for example, a leukemia selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL).
  • leukemia is acute myeloid leukemia.
  • Acute myeloid leukemia encompasses all forms of acute myeloid leukemia and related neoplasms according to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia, including all of the following subgroups in their relapsed or refractory state: Acute myeloid leukemia with recurrent genetic abnormalities, such as AML with t(8;21)(q22;q22); RU X 1-RU X 1T1, AML with inv(16)(p l3.1q22) or t(16; 16)(p l3.1 ;q22); CBFB-MYH1 1, AML with t(9; l I)(p22;q23); MLLT3-MLL, AML with t(6;9)(p23;q34); DEK-NUP214, AML with inv(3)(q21 q26.2) or t(3;3)(q21;q26.2); RPN1
  • the methods described herein further comprise selecting a subject diagnosed with acute myeloid leukemia, for example, based on the symptoms presented. Symptoms associated with acute myeloid leukemia are known to the skilled practitioner. For example, a patient can be diagnosed with acute myeloid leukemia if the subject presents with a myeloid neoplasm with 20% or more blasts in the peripheral blood or bone marrow. [0190] In some embodiments, the methods described herein further comprise selecting a subject at risk of developing acute myeloid leukemia. For example, a subject can be selected as at risk of developing leukemia based on a family history of leukemias.
  • a subject is selected as diagnosed with acute myeloid leukemia or at risk of developing acute myeloid leukemia based on a genetic mutation useful as a diagnostic or prognostic marker of myeloid neoplasms.
  • exemplary such markers include mutations of: JAK2, MPL, and KIT in MPN; NRAS, KRAS, NF 1, and PTPN1 1 in
  • the methods described herein further comprise selecting a subject suspected of having acute myeloid leukemia.
  • a subject suspected of having acute myeloid leukemia for example, can be selected based on family history, diagnostic testing or based on the symptoms presented or a combination thereof.
  • the methods described herein further comprise selecting a subject suffering from refractory or relapsed acute myeloid leukemia.
  • relapsed acute myeloid leukemia is defined as reappearance of leukemic blasts in the blood or greater than 5% blasts in the bone marrow after complete remission not attributable to any other cause. For subjects presenting with relapsed AML, more than 5% blasts on baseline bone marrow assessment is required.
  • refractory acute myeloid leukemia is defined as a failure to achieve a complete remission or complete remission with incomplete blood recovery after previous therapy. Any number of prior anti- leukemia schedules is allowed.
  • complete remission is defined as morphologically leukemia free state (i.e. bone marrow with less than 5% blasts by morphologic criteria and no Auer rods, no evidence of extramedullary leukemia) and absolute neutrophil count greater than or equal to 1,000/ ⁇ and platelets greater than 100,000/ ⁇ .
  • complete remission with incomplete blood recovery is defined as
  • morphologically leukemia-free state i.e. bone marrow with less than 5% blasts by morphologic criteria and no Auer rods, no evidence of extramedullary leukemia
  • neutrophil count less than 1,000/ ⁇ or platelets less than 100,000 ⁇ ⁇ in the blood.
  • the methods described herein further comprise selecting a subject who relapses from complete remission of acute myeloid leukemia after receiving an induction chemotherapy treatment regimen.
  • the methods and compositions disclosed herein are useful for the treatment of a subject with leukemia.
  • a leukemia selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL).
  • compositions comprising the gap junction blockers and/or agents described herein (e.g., at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia).
  • the disclosure provides a pharmaceutical composition comprising an effective amount of a gap junction blocker, and an effective amount of at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia as described herein.
  • a pharmaceutical composition comprises an effective amount of an agent or gap junction blocker, an effective amount of at least one
  • chemotherapeutic agent subject to resistance by acute myeloid leukemia, and a
  • the pharmaceutical composition includes an effective amount of a prophylactic treatment for hypertension, hypokalemia, and/or edemas.
  • compositions comprising the agent or gap junction blocker and the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia can be used for treating acute myeloid leukemia as described herein.
  • the composition is useful for selectively eradicating leukemic cells in a subject without eradicating normal leukocytes in the subject.
  • the composition is useful for selectively eradicating leukemic cells in a subject with minimal eradication of normal leukocytes in the subject.
  • the composition is useful for selectively eradicating leukemic cells in a subject without eradicating normal cells in the subject.
  • the composition is useful for selectively eradicating leukemic cells in a subject with minimal eradication of normal cells in the subject. In some embodiments, the composition is useful for selectively eradicating leukemic cells in the subject while inducing proliferation of normal leukocytes in the subject. In some embodiments, the composition is useful for inducing complete remission of leukemia in the subject. In some embodiments, the composition is useful for inducing complete remission of acute myeloid leukemia in the subject. In some embodiments, the composition is useful for inducing complete remission of acute leukemia in the subject in the absence of a relapse risk due to residual leukemic cells in the subject's bone marrow or peripheral blood. [0202] Formulation and Administration
  • the gap junction blockers and/or agents described herein can be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
  • the term "administered” refers to the placement of an agent described herein, into a subject by a method or route which results in at least partial localization of the agent at a desired site.
  • a gap junction blocker or agent described herein can be administered by any appropriate route which results in effective treatment in the subject, i.e. administration results in delivery to a desired location in the subject where at least a portion of the composition delivered.
  • Exemplary routes of administration of the gap junction blockers and/or agents described herein include, without limitation, intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the gap junction blockers and/or agents can be formulated in pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of the agent, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents, or excipients.
  • the formulations can conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques, excipients and formulations generally are found in, e.g., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1985, 17th edition, Nema et al, PDA J. Pharm. Sci. Tech. 1997 51 : 166-171.
  • the gap junction blockers and/or agents described herein can be administrated encapsulated within a nanoparticle (e.g., a lipid nanoparticle).
  • gap junction blockers and/or agents described herein can be administered encapsulated within liposomes.
  • the manufacture of such liposomes and insertion of molecules into such liposomes being well known in the art, for example, as described in US Pat. No. 4,522,81 1.
  • Liposomal suspensions (including liposomes targeted to particular cells, e.g., endothelial cells) can also be used as pharmaceutically acceptable carriers.
  • the gap junction blockers and/or agents can be administrated to a subject in combination with other pharmaceutically active agents.
  • exemplary pharmaceutically active agents include, but are not limited to, those found in Harrison 's Principles of Internal Medicine, 13 th Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; Physician's Desk Reference, 50 th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8 Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990, the complete contents of all of which are incorporated herein by reference.
  • the pharmaceutically active agent is a conventional treatment for acute myeloid leukemia.
  • the pharmaceutically active agent is a conventional treatment for acute myeloid leukemia.
  • the pharmaceutically active agent is a conventional treatment for acute myeloid leukemia.
  • the pharmaceutically active agent is a conventional treatment for acute myeloid leuk
  • the pharmaceutically active agent is a conventional treatment for an autoimmune or inflammatory condition.
  • Chronic sub-cutaneous administration of a gap junction blocker described herein e.g., CBX
  • the pharmaceutically active agent comprises a prophylactic treatment, for example, to treat or prevent hypertension, hypokalemia, edemas, and other deleterious side effects caused by administration of the gap junction blocker (e.g., CBX).
  • such pharmaceutically active agent comprises an anti-mineralocorticoid or aldosterone inhibitor, such as an aldosterone receptor antagonist, e.g., eplerenone or spironolactone.
  • such pharmaceutically active agent comprises an angiotensin-converting-enzyme (ACE) inhibitor or other diuretic drug, e.g., thiazide diuretics, such as chlorothiazide, chlorthalidone, indapamide, hydrochlorothiazide, methyclothiazide, metolazone; loop diuretics, such as bumetanide, furosemide, ethacrynate, and torsemide; potassium sparing diuretics, such as amiloride hydrochloride, spironolactone, and triamterene; carbonic anhydrase inhibitors, such as acetazolamide, methazolamide, and osmotic diuretics, such
  • the agents and the other pharmaceutically active agent can be administrated to the subject in the same pharmaceutical composition or in different pharmaceutical compositions (at the same time or at different times).
  • a gap junction blocker and at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia can be formulated in the same composition or in different compositions.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • an effective amount means an amount of the agent (e.g., gap junction blocker) which is effective to selectively eradicate a majority or all of the leukemic cells (e.g., stem or progenitor cells) in a population of cells or a subject without eradicating, or minimally eradicating, normal cells (e.g., bone marrow or peripheral blood leukocytes) in the population or subject. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • the agent e.g., gap junction blocker
  • a therapeutically effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other agents that inhibit pathological processes in the acute myeloid leukemia or autoimmune or inflammatory disorder.
  • the gap junction blockers and/or agents described herein can be provided in a kit.
  • the kit includes (a) the agent, e.g., a composition that includes the agent, and (b) informational material.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the agent for the methods described herein.
  • the informational material describes methods for administering the gap junction blockers and/or agents to a subject for treating acute myeloid leukemia.
  • the informational material can include instructions to administer the gap junction blocker and/or agents described herein in a suitable manner, e.g., in a suitable dose, dosage form, or mode of administration.
  • gap junction blockers such as CBX
  • CBX gap junction blockers
  • the disclosure contemplates administering an effective amount of a gap junction blocker (e.g., CBX) to achieve plasma levels of about 200 ⁇ .
  • the instructions recommend administering an effective amount of a gap junction blocker (e.g., CBX) to achieve plasma levels of about 200 ⁇ .
  • the instructions recommend orally administering a gap junction blocker formulated as a tablet comprising 100 mg of the gap junction blocker (e.g., CBX) 3 times per day.
  • the informational material can include instructions for selecting a suitable subject, e.g., a human, e.g., a human suffering from relapsed or refractory acute myeloid leukemia.
  • the informational material of the kits is not limited in its form.
  • the informational material e.g., instructions
  • the informational material is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet.
  • the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording.
  • the informational material of the kit is a link or contact information, e.g., a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the modulator and/or its use in the methods described herein.
  • the informational material can also be provided in any combination of formats.
  • the kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and/or a second agent for treating a condition or disorder described herein, e.g. acute myeloid leukemia.
  • the other ingredients can be included in the kit, but in different compositions or containers than the agent.
  • the kit can include instructions for admixing the agent and the other ingredients, or for using the gap junction blocker together with the other ingredients.
  • the gap junction blocker and/or agents described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the gap junction and/or agent be substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • reconstitution generally is by the addition of a suitable solvent.
  • the solvent e.g., sterile water or buffer, can optionally be provided in the kit.
  • the kit can include one or more containers for the composition containing the agent.
  • the kit contains separate containers, dividers or compartments for the agent (e.g., in a composition) and informational material.
  • the agent e.g., in a composition
  • the agent can be contained in a bottle, vial, or syringe
  • the informational material can be contained in a plastic sleeve or packet.
  • the separate elements of the kit are contained within a single, undivided container.
  • the agent (e.g., in a composition) is contained in a bottle, vial or syringe that has attached thereto the
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agent (e.g., in a composition).
  • the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of the agent.
  • the containers of the kits can be air tight and/or waterproof.
  • a kit comprises: a gap junction blocker, at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia, and instructions for administering the agent or gap junction blocker and the at least one chemotherapeutic agent to a subject suffering from acute myeloid leukemia.
  • the instructions further comprise directions for administering the at least one chemotherapeutic agent as part of an induction chemotherapy treatment regimen for the subject.
  • the instructions further comprise directions for administering the agent or gap junction blocker, and the at least one therapeutic agent to induce complete remission of acute myeloid leukemia in the subject.
  • the instructions further comprise directions for administering the agent or gap junction blocker, and the at least one therapeutic agent to induce complete remission of acute myeloid leukemia in the subject, without risk of relapse by completely eradicating leukemic cells in the subject.
  • the instructions further comprise directions for administering the agent or gap junction blocker, and the at least one therapeutic agent to induce complete remission of acute myeloid leukemia in the subject by completely eradicating leukemic cells in the subject by inducing the leukemic cells to differentiate from proliferating, immortalized leukemic cells into short-lived, non-leukemic cells.
  • the agents e.g., glycerrhetinic acid derivatives, carbenoxolone or analogs or derivatives thereof
  • carbenoxolone treatment caused a differential effect on both total and mitochondrial reactive oxygen species (ROS) levels in leukemia cells relative to normal cells, thereby contributing to the selective eradication observed following contacting of such cells with CBX.
  • ROS mitochondrial reactive oxygen species
  • carbenoxolone treatment caused a differential effect on lipid damage (e.g., as evidenced by the observed increase in the ratio of oxidized to non-oxidized lipids in leukemic cells) and caused a differential effect on DNA damage (e.g., as evidenced by the observed increase in the ratio of yH2AK phosphorylation in leukemic cells).
  • the contemplated agents include any agents that selectively induce oxidative stress in leukemic cells.
  • the contemplated agents include any agents that selectively induce lipid damage in leukemic cells.
  • the contemplated agents include any agents that selectively induce double- strand DNA breaks in leukemic cells.
  • the agent is carbenoxolone or analogs or derivatives thereof.
  • Some aspects of the methods of the invention further comprise measuring calcium flux in the population of cells after contacting with the glycyrrhetinic acid derivative. In some aspects measuring calcium flux is performed within about 1, 2, 3, 4, 5, or 10 minutes after contact with the glycyrrhetinic acid derivative.
  • Some aspects of the methods of the invention further comprise detecting lipid damage in the population of cells after contacting with the glycyrrhetinic acid derivative. In some aspects lipid damage is detected by detecting lipid peroxidation with flow cytometry (e.g., fluorescent activated cell sorting (FACS)). In some aspects lipid damage is detected by the ratio of oxidized to non-oxidized lipids in the population of cells. In some embodiments, lipid damage is detected by detecting lipid peroxidation by flow cytometry (e.g., FACS).
  • flow cytometry e.g., fluorescent activated cell sorting
  • Some aspects of the methods of the invention further comprise detecting DNA damage in the population of cells after contacting with the glycyrrhetinic acid derivative.
  • DNA damage is detected by detecting double -stranded DNA breaks.
  • DNA damage is detected by determining yH2AX phosphorylation in the population of cells.
  • Some aspects of the methods of the invention further comprise detecting induction of oxidative stress in the population of cells after contacting with the glycyrrhetinic acid derivative.
  • oxidative stress is detected by detecting the total levels of reactive oxygen species (ROS) in the population of cells.
  • oxidative stress is detected by detecting the total levels of mitochondrial reactive oxygen species (ROS) in the population of cells.
  • Some aspects of the methods of the invention further comprise detecting a metabolic product after contacting with the glycyrrhetinic acid derivative.
  • the metabolic product is hypoxanthine.
  • the agents e.g., glycyrrhetinic acid derivatives
  • the agents may eradicate leukemic cells by blocking or otherwise interfering with one or more of hemichannels and/or gap junctions, or blocking or interfering with one or more of connexins, pannexins and/or hydroxysteroid dehydrogenase, which are the building blocks of hemichannels and gap junctions.
  • glycyrrhetinic acid derivatives relate to the use of certain glycyrrhetinic acid derivatives it should be understood that the present inventions are not limited to such glycyrrhetinic acid derivatives, for example to eradicate leukemic cells. Rather, contemplated herein are any means of interfering with interactions with or between leukemic cells and thereby eradicating (e.g., selectively eradicating) leukemic cells or for blocking or interfering with hemichannels and/or gap junctions or connexins, pannexins and/or hydroxysteroid dehydrogenase.
  • eradicating e.g., selectively eradicating
  • kits and compositions disclosed herein may comprise any agents or compositions that are capable of or useful for blocking or otherwise interfering (e.g., selectively blocking or selectively interfering) with one or more of hemichannels, gap junctions, connexins, pannexins or hydroxysteroid dehydrogenase.
  • Such agents or compositions may be selected from the group consisting of gap junction and hemichannels inhibitors such as glycyrrhizic acid, 18a-glycyrrhetinic acid, carbenoxolone, carbenoxolone derivatives, carbenoxolone analogs, fenamates, flufenemic acid, flufenemic acid derivatives, flufenemic acid analogs, heptanol, octanol, arachidonic acid, quinine, quinine derivatives (including mefloquine), connexin (Cx) fragments (including fragments from the extracellular domain of a connexin such as Connexin 43 or Connexin 30), connexin mimetic peptides including but not limited to Gap26 and Gap27, connexin inhibitors, connexin antibodies, connexin expression modulators such as clustered regularly-interspaced short palindromic repeats, CRISPR/
  • the agents (e.g., carbenoxolone or analogs or derivatives thereof) disclosed herein preferentially bind to one or more of hemichannels and/or gap junctions of leukemic cells (e.g., leukemic stem cells), relative to normal cells.
  • leukemic cells e.g., leukemic stem cells
  • carbenoxolone preferentially binds to one or more of hemichannels and/or gap junctions of leukemic cells (e.g., leukemic stem cells) that is greater than 2 fold, 3 fold, 4 fold, 5 fold, six fold, 10 fold, 12 fold, 20 fold, 25 fold, 30 fold, 40 fold, 50 fold, 75 fold, 100 fold, 150 fold or greater than the binding to gap junctions or
  • carbenoxolone has a binding affinity for hemichannels and/or gap junctions of leukemic cells, that is greater than 2 fold, 3 fold, 4 fold, 5 fold, six fold, 10 fold, 12 fold, 20 fold, 25 fold, 30 fold, 40 fold, 50 fold, 75 fold, 100 fold, 150 fold or greater than the binding affinity with which it binds to the gap junctions or hemichannels of a normal cell.
  • the agents e.g., carbenoxolone or analogs or derivatives thereof
  • carbenoxolone preferentially binds to one or more of connexins, pannexins and/or hydroxysteroid dehydrogenase of leukemic cells that is greater than 2 fold, 3 fold, 4 fold, 5 fold, six fold, 10 fold, 12 fold, 20 fold, 25 fold, 30 fold, 40 fold, 50 fold, 75 fold, 100 fold, 150 fold or greater than the binding to connexins, pannexins and/or hydroxysteroid dehydrogenase of a normal cell.
  • carbenoxolone has a binding affinity for connexins, pannexins and/or hydroxysteroid dehydrogenase of leukemic cells (e.g., leukemic stem cells), that is greater than 2 fold, 3 fold, 4 fold, 5 fold, six fold, 10 fold, 12 fold, 20 fold, 25 fold, 30 fold, 40 fold, 50 fold, 75 fold, 100 fold, 150 fold or greater than the binding affinity with which it binds to the connexins, pannexins and/or hydroxysteroid dehydrogenase of a normal cell.
  • leukemic cells e.g., leukemic stem cells
  • the disclosure contemplates the use of an agent or gap junction blocker alone, or in combination together with at least one additional chemotherapeutic agent, such as a chemotherapeutic agent subject to resistance by acute myeloid leukemia, in the methods, compositions, and kits described herein.
  • chemotherapeutic agent such as a chemotherapeutic agent subject to resistance by acute myeloid leukemia
  • the disclosure contemplates the use of any agents or gap junction blocker that is capable of selectively eradicating leukemic cells in a population of cells or subject, without eradicating, or minimally eradicating, normal cells (e.g., leukocytes) in the population or subject.
  • agents that can be used as a gap junction blocker include small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivatives; peptidomimetics; nucleic acids selected from the group consisting of siR As, shR As, antisense RNAs, ribozymes, and aptamers; an extract made from biological materials selected from the group consisting of bacteria, plants, fungi, animal cells, and animal tissues; naturally occurring or synthetic compositions; and any combination thereof.
  • the gap junction blocker comprises an inhibitor of 1 ⁇ -hydroxysteroid dehydrogenase (1 ⁇ -HSD). It should be appreciated that such inhibitor can be an inhibitor of 1 ⁇ -HSDl, an inhibitor of 1 i -HSD2, or an inhibitor both 1 ⁇ -HSDl and 1 i -HSD2. In some embodiments, the gap junction blocker is selected from the group consisting of the following formulas I to III:
  • Xi Y and Z each independently represent halogen, in particular, F, CI, I or Br,
  • Ci_C 6 alkyl C 5 -Ci 5 aryl or Ci-C 6 alkoxy
  • n represents an integer from 1 to 10, in particular, from 1 to 4,
  • L represents an amide, amine, sulfonamide, ester, thioester or keto group
  • T, U, V and W each independently represent an oxo, thio, ketone, thioketone
  • Ci-C 6 alkyl or Ci-C 6 alkanol group Ci-C 6 alkyl or Ci-C 6 alkanol group
  • Ar represents an aromatic ring system
  • Cyc represents a cyclic ring system
  • A represents a Ci-Ci 0 ester (Ci-Cio alkyl-CO-O-), a Ci-Cio amide (Ci-Cio alkyl-CO-NH-), a Ci-Cio ether or a Ci-Cio ketone (Ci-Cio alkyl-CO-) group,
  • B and C each independently represent an oxo group, a keto group, a Ci-C 6 alkanol group or a Ci-C 6 alkyl group,
  • m is an integer from 1 to 10, in particular, from 1 to 4, and
  • D is a group selected from COOR 1 or CONR 2 R 3 , wherein R 1 , R 2 and R 3 each
  • E represents an OH, a Ci-Ci 0 ester (Ci-Ci 0 alkyl-CO-O-), a Ci-Ci 0 amide (Ci- Cio alkyl-CO-NH-), a Ci-Ci 0 ether (Ci-Cio-O-) or a Ci-Ci 0 ketone (Ci-Ci 0 alkyl-CO-) group,
  • F represents an oxo group, keto group, a Ci-C 6 alkanol group or a Ci- C 6 alkyl group
  • G is a group selected from COOR 1 or CONR 2 R 3 , wherein R 1 , R 2 and R 3 each independently represent H or a Ci-C 2 o hydrocarbon group, in particular, a Ci-C 6 alkyl group.
  • the gap junction blocker is 18- -glycyrrhetinic acid or a derivative thereof.
  • Exemplary derivatives of 18- -glycyrrhetinic acid include, but are not limited to, glycyrrhizine, glycyrrhizinic acid, carbenoxolone, and 2 ⁇ hydroxyethyl-18 - glycyrrhetinic acid amide.
  • the gap junction blocker comprises carbenoxolone or an analog thereof. In some embodiments, the agent or gap junction blocker is not 18- ⁇ - glycyrrhetinic acid. [0251] In some embodiments, the gap junction blocker is selected from the group consisting of heptanol, octanol, anadamide, fenamate, retinoic acid, oleamide, spermine, aminosulphates, halothane, enflurane, isoflurane, propofol, thiopental, glycyrrhetinic acid, quinine, 2-aminoethoxydiphenyl borate or pharmaceutically acceptable derivatives thereof, and any combination thereof.
  • Exemplary pharmaceutically acceptable derivatives of heptanol include, but are not limited to, 1-heptanol, 2-heptanol, 3-heptanol, 4-heptanol, and combinations thereof.
  • Exemplary pharmaceutically acceptable derivatives of fenamate include, but are not limited to, meclofenamic acid, niflumic acid, flufenamic acid, and combinations thereof.
  • Exemplary pharmaceutically acceptable derivatives of glycyrrhetinic acid include, but are not limited to, hydrogen esters of glycyrrhetinic acid, salts of hydrogen esters of glycyrrhetinic acid, carbenoxolone, and combinations thereof.
  • the gap junction blocker comprises carbenoxolone, carbenoxolone or analogs or derivatives thereof. In some embodiments, the agent or gap junction blocker is not 18- -glycyrrhetinic acid.
  • the gap junction blocker comprises an analog or derivative of carbenoxolone.
  • the gap junction blocker comprises the glycyrrhetinic acid derivative carbonoxolone (CBX), depicted by structure IV below.
  • CBX a putative gap junction inhibitor, influences endogenous glucocorticoids by potently inhibiting 1 ⁇ -hydroxysteroid dehydrogenase ( 1 ⁇ -HSD) and reversible catalyzes the conversion of Cortisol to the inactive metabolite cortisone.
  • CBX is highly bioavailable when administered orally and is safe and well-tolerated. Reproductive, teratogenic and carcinogenic tests in animals showed no significant adverse effects.
  • CBX side effects largely comprise fluid overload and are generally manageable.
  • the gap junction blocker comprises an inhibitor of a member of the connexin gap junction family. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx40.1. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx30.2. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx31.1. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx36. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx45. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx47. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx32.
  • the gap junction blocker comprises an inhibitor of connexin Cx50. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx30.3. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx31. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx26. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx40. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx37. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx46. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx43. In some embodiments, the gap junction blocker comprises an inhibitor of connexin Cx30. Exemplary inhibitors of the connexin gap junction family members listed above include, but are not limited to, extracellular Ca2+,
  • chemotherapeutic agent that is useful for treating cancer (e.g., leukemia).
  • chemotherapeutic agents that can be administered in combination with the gap junction blockers of the present invention include alkylating agents (e.g. cisplatin, carboplatin, oxaloplatin, mechlorethamine, cyclophosphamide, chorambucil, nitrosureas); anti-metabolites (e.g.
  • methotrexate pemetrexed, 6-mercaptopurine, dacarbazine, fludarabine, 5-fluorouracil, arabinosycytosine, capecitabine, gemcitabine, decitabine
  • plant alkaloids and terpenoids including vinca alkaloids (e.g. vincristine, vinblastine, vinorelbine),
  • podophyllotoxin e.g. etoposide, teniposide
  • taxanes e.g. paclitaxel, docetaxel
  • topoisomerase inhibitors e.g. notecan, topotecan, amasacrine, etoposide phosphate
  • antitumor antibiotics include lactinomycin, doxorubicin, epirubicin, and bleomycin; ribonucleotides reductase inhibitors; antimicrotubules agents; and retinoids.
  • compositions, methods, and kits described herein contemplate the use of at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia.
  • the at least one chemotherapeutic agent may be subject to drug resistance by acute myeloid leukemia due to any drug resistance mechanism.
  • the at least one chemotherapeutic agent is subject to stroma-mediated drug resistance.
  • stroma-mediated drug resistance refers to chemo-resistance exhibited by acute myeloid leukemia due to heterotypic interactions between the leukemic cells and stromal cells.
  • the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises an antimetabolite agent.
  • the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises cytarabine.
  • the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises an anthracycline agent.
  • the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises doxorubicin.
  • the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises an antimetabolite agent and an anthracycline agent.
  • the at least one chemotherapeutic agent subject to resistance by acute myeloid leukemia comprises cytarabine and the anthracycline agent comprises doxorubicin.
  • administration of a gap junction blocker described herein selectively eradicates leukemic cells by, in part, overcoming chemo-resistance exhibited by leukemic cells, such as stroma-mediated chemo- resistance.
  • test agent means any compound or molecule (e.g., a small molecule organic compound, an
  • the assays and methods disclosed herein are useful for identifying test agents that are candidate agents.
  • the phrase "candidate agent” means any compound or molecule (e.g., a small molecule organic compound, an oligonucleotide, a polynucleotide, a siRNA, a shRNA, a gene, a gene product, a polypeptide, an antibody or other
  • the agents or candidate agents e.g., carbenoxolone or an analog or derivative thereof
  • the agents or candidate agents may be used, for example, to treat and/or cure leukemia (e.g., AML, CLL, MDS and ALL) in a subject (e.g., a human) or to promote survival of the subject.
  • leukemia e.g., AML, CLL, MDS and ALL
  • Such treatment may improve a diagnosed condition or make it more manageable, or improve disease symptoms, or prolong survival.
  • Treatment can also include delaying or preventing the onset of remission or preventing recurrence or relapse of leukemia.
  • such methods of identifying a candidate agent that selectively eradicates leukemic cells in a population of cells comprise: (a) contacting a population of cells comprising leukemic cells and normal cells with a test agent; and (b) detecting induction of oxidative stress in the population of cells; wherein a test agent that selectively induces oxidative stress in the leukemic cells is a candidate agent.
  • the oxidative stress is detected by determining the total levels of reactive oxygen species (ROS) in the population of cells.
  • ROS reactive oxygen species
  • oxidative stress is detected by determining the total levels of mitochondrial reactive oxygen species (ROS) in the population of cells.
  • lipid damage is detected by determining the ratio of oxidized to non-oxidized lipids in the population of cells. In some embodiments, lipid damage is detected by detecting lipid peroxidation by flow cytometry.
  • a candidate agent that selectively eradicates leukemic cells in a population of cells comprising: (a) contacting a population of cells comprising leukemic cells and normal cells with a test agent; and (b) detecting induction of DNA damage in the population of cells; wherein a test agent that selectively induces DNA damage in the leukemic cells is a candidate agent.
  • DNA damage is detected by determining yH2AX
  • yH2AX phosphorylation in the population of cells.
  • yH2AX phosphorylation is detected by flow cytometry (e.g., FACS).
  • an exemplary agent or candidate agent is a glycyrrhetinic acid derivative.
  • a glycyrrhetinic acid derivative selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, 18- -glycyrrhetinic acid, carbenoxolone or 2-hydroxyethyl-18 - glycyrrhetinic acid amide.
  • the glycyrrhetinic acid derivative comprises carbenoxolone or an analog or derivative thereof.
  • the leukemic cells useful in connection with the foregoing methods and assays comprise an acute myeloid leukemia cell line selected from the group consisting of MLL-AF9 cells, KG-1 cells, KG-la cells, U937 cells, HL60 cells, NB-4 cells, and THP 1 cells.
  • compositions, methods, kits and respective component(s) thereof are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
  • compositions, methods, kits and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • Example 1 Gap junction Intercellular Communication Regulate Leukemic Cell Survival and Drug Resistance
  • fusion proteins encoded by genetic translocations in human leukemias have been reported to impart leukemia stem cell properties on committed hematopoietic progenitors (Huntly, et al., Cancer Cell 6, 587-96 (2004); Cozzio, et al, Genes & Development 17, 3029-35 (2003); and (So, et al., Cancer Cell 3, 161-71 (2003)). Furthermore, introduction of these altered alleles into normal bone marrow cells induces AML in mouse models of the disease.
  • MML mixed lineage leukemia
  • Such AML models recapitulate the human disease phenotype and display stem cell-like properties that demonstrate the ability to serially colonize in-vitro, and the ability to confer an AML phenotype that can be serially transplanted in vivo (Huntly, et al. (2004) and Krivtsov, et al., Nature 442, 818-22 (2006)).
  • MLL-AF9 is a fusion protein, encoded by the t(9; l I)(p22;q23) translocation (Krivtsov, et al. 2006 and Sykes, et al., Cell 146, 697-708 (201 1)), present in leukemic blasts derived from patients with AML and is associated with poor prognosis (Schoch, et al., Blood 102, 2395-402 (2003)).
  • MLL-AF9 leukemic cells In order to generate primary MLL-AF9 leukemic cells for our experiments, we introduced the MLL-AF9 oncogene via retroviral transduction into lineage depleted bone marrow cells from B6.SJL mice, as depicted in FIG. 1A. Sorted GFP positive cells were then intravenously injected into sub-lethally irradiated C57B16 recipients and MLL-AF9 GFP-positive leukemic cells were harvested 3 months later, at a terminal stage of disease. In order to synchronize the timing of the treatment and to visualize disease progression, we transduced primary MLL-AF9 GFP- positive cells with Luciferase lentivirus.
  • Luciferase expressing cells with antibiotics and transplanted 1 million double positive leukemic cells into non-irradiated recipients and monitored disease progression by whole body bioluminescence imaging (FIGS. 1A and IB).
  • induction chemotherapy Cytarabine combined with anthracycline
  • a similar outcome has been described in mouse model of human AML by a 5 -days treatment regimen of combined Cytarabine and Doxorubicin for 3 days, proceeded with Cytarabine alone for additional 2 days (Zuber, et al., Genes &
  • MS-5 are murine bone-marrow stromal cells that were previously shown to support hematopoietic stem and progenitor cells (Itoh, et al., Experimental Hematology 21, 145-153 (1989) and Schajnovitz, et al., Nature Immunology 12, 391-8 (2011)).
  • co-culture of MLL-AF9 cells with BM supporting MS-5 stromal cells resulted in increased resistance (-70%) to combined therapy with only -30% cell death (FIG. 2A). This suggests that direct interactions between the leukemic cells and the stromal cells facilitate drug resistance.
  • Gap junction intercellular channels which are homo- and hetero-hexamers of connexin proteins, facilitate intercellular communication between contacting cells via the passage of secondary messengers, such as calcium and cAMP 17 .
  • Carbenoxolone (CBX) is a potent broad range gap junctions inhibitor which efficiently blocks cell-cell communication at ⁇ without affecting cell viability (Schajnovitz et al. 2011). Blocking gap junctions activity in the coculture system by ⁇ CBX, 20 minutes before induction chemotherapy, reversed the resistance and almost 90% of the ML-AF9 cells were eradicated (FIG. 2B). Gap junction blockade in co-cultures separated with transwell inserts, resulted in more than 90% eradication (FIG. 2C), suggesting that homotypic intercellular communication also contributes to acquired drug resistance.
  • mice 25mg/kg CBX was administrated to sick mice, 24 hours before induction chemotherapy and throughout the 5-days treatment. 1 week after treatment, mice were imaged and then sacrificed to evaluate treatment outcome. As depicted in FIG. 2D, mice treated with chemotherapy alone entered complete remission but minimal residual cells could be detected in the bones (white arrows). Strikingly, mice that were treated with chemotherapy and a gap junction blocker were entirely leukemia free, with no detectable leukemia cells by luciferase imaging or by flow cytometry.
  • Example 2 Selective Eradication of Leukemic Cells Using Carbenoxolone
  • AML Acute myeloid leukemia
  • Current therapy is unchanged since the 1980s and fails in approximately 75% of patients despite a high rate of initial remission.
  • Genetic understanding of the basis for AML is now quite sophisticated, yet targeted therapies have disappointed to date; IDH targeted therapy may be an exception but are relevant for only 17% of patients.
  • CBX carbenoxolone
  • SDH succinate dehydrogenase
  • CBX treatment markedly reduced leukemia burden in mice bearing the well- validated model of human AML (MLL-AF9) and prolonged survival.
  • CBX-treated mice survived longer than mice treated with standard induction chemotherapy (iCT), and a synergistic effect was documented when both therapies were combined (FIG. 60A).
  • iCT standard induction chemotherapy
  • the present inventors report that CBX-mediated leukemia cell death is not restricted to a specific oncogenic karyotype or mutation, suggesting that it affects a common fundamental pathway in leukemia cells (FIG. 60B).
  • Succinate is a substrate for Succinate dehydrogenase (SDH), which participates in both the TCA cycle and the electron transport chain.
  • SDH Succinate dehydrogenase
  • SDH activity was higher in leukemia cell lysate compare to normal cell lysate.
  • Incubation of CBX in leukemia cell lysate led to a decreased SDH activity, indicating that CBX can directly bind to SDH and inhibit its activity in vitro, whereas Antimycin A had no effect.
  • the present inventors then tested the effect of specific SDH inhibitors on leukemia cell viability. Both Oxaloacetate and Atpenin A5, potent SDH inhibitors, did not induce leukemia cell death. Thus, additional stimuli are required to induce leukemia cell death.
  • CBX-treated leukemia cells represent the hallmark of a recently described regulated cell death program, termed ferroptosis.
  • the present inventors then tested if pre-treating cells with ferroptosis inhibitors could rescue CBX-mediated cell death.
  • the present inventors employed a direct approach, using an iron chelator
  • CBX activity was significantly diminished in leukemia cells that were pre-treated with the iron chelator DFO, whereas Liproxstatin-1 and a-tocopherol pre-treatment resulted in a more modest inhibition of CBX activity.
  • the present inventors next sought to determine the activity of CBX in solid tumor cell lines. Cells were incubated with increasing concentration of CBX (0 ⁇ -400 ⁇ ) for 24 hours. End-point cell viability was scored by flow cytometry analysis.
  • CBX demonstrated activity in solid tumor cell lines. All solid tumor cell lines tested exhibited greater sensitivity to CBX treatment (24-hour incubation in vitro), compared to normal BM stromal cell line (HS-5). In particular, CBX efficiently eradicated melanoma cells at ⁇ (A375, blue arrows).
  • CBX carbenoxolone
  • CBX is not limited to leukemia cells, but instead also extends to non-leukemia cancer cell types (e.g., breast cancer cells, melanoma cells, prostate cancer cells and cervix carcinoma).
  • non-leukemia cancer cell types e.g., breast cancer cells, melanoma cells, prostate cancer cells and cervix carcinoma.
  • the present inventors are currently validating a model by which leukemia cells aggregate into functional clusters to protect themselves from toxic ROS by-products, via direct intracellular conduction of specific metabolites.
  • myeloid leukemia prognostic and therapeutic implications. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 475- 86 (201 1).
  • AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias.
  • Cell 146, 697-708 (201 1).
  • Schoch, C. et al. AML with 1 Iq23/MLL abnormalities as defined by the WHO classification: incidence, partner chromosomes, FAB subtype, age distribution, and prognostic impact in an unselected series of 1897 cytogenetically analyzed AML cases. Blood 102, 2395-402 (2003).
  • Zuber, J. et al. Mouse models of human AML accurately predict chemotherapy response. Genes & development 23, 877-89 (2009). Pardee, T. S., Zuber, J. & Lowe, S. W.
  • Flt3-ITD alters chemotherapy response in vitro and in vivo in a p53-dependent manner.

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Abstract

L'invention concerne des compositions, des méthodes et des kits comprenant des dérivés d'acide glycyrrhétinique permettant d'éradiquer sélectivement des cellules cancéreuses (par exemple, des cellules leucémiques) dans une population ou un sujet, ainsi que des méthodes associées permettant de traiter un cancer (par exemple, une leucémie myéloblastique aiguë), et de favoriser la survie de patients atteints d'un cancer (par exemple, des patients atteints d'une leucémie myéloblastique aiguë). L'invention concerne également des méthodes et des dosages permettant de déterminer si un agent d'analyse est un agent candidat approprié pour éradiquer sélectivement des cellules cancéreuses (par exemple, des cellules leucémiques) dans une population de cellules.
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