WO2014029016A1 - Composition comprenant une glycylcycline et un inhibiteur de tyrosine kinase pour le traitement du cancer - Google Patents

Composition comprenant une glycylcycline et un inhibiteur de tyrosine kinase pour le traitement du cancer Download PDF

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WO2014029016A1
WO2014029016A1 PCT/CA2013/000742 CA2013000742W WO2014029016A1 WO 2014029016 A1 WO2014029016 A1 WO 2014029016A1 CA 2013000742 W CA2013000742 W CA 2013000742W WO 2014029016 A1 WO2014029016 A1 WO 2014029016A1
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glycylcycline
tki
cancer
tigecycline
combination
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PCT/CA2013/000742
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English (en)
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Aaron David Schimmer
Wei Zheng
John Mckew
Catherine Chen
Noel Southall
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University Health Network
The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2014029016A1 publication Critical patent/WO2014029016A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • 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
    • A61K31/65—Tetracyclines
    • 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

Definitions

  • the disclosure relates to methods and compositions comprising a glycylcycline such as tigecycline and a tyrosine kinase inhibitor for the treatment of cancer and particularly to methods and compositions comprising a glycylcycline such as tigecycline and a tyrosine kinase inhibitor for the treatment of hematological malignancies, particularly leukemia such as acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • AML acute myeloid leukemia
  • Tyrosine kinase inhibitors are used to treat a variety of cancers.
  • Glycylcyclines such as tigecycline have been described in WO201 1/109899, for treating cancer.
  • An aspect includes a method of inducing cytotoxicity in a cancer cell comprising contacting the cancer cell with a glycylcycline compound and a tyrosine kinase inhibitor (TKI), wherein the cell is in vitro or in vivo.
  • the method comprises contacting the cell with an amount of the glycylcycline compound and TKI that demonstrates and/or produces synergistic cell toxicity.
  • Another aspect includes a method for treating a cancer wherein the cancer cell is in a subject and the subject is administered a glycylcycline compound and a TKI.
  • the subject is administered an amount of glycylcycline and
  • TKI that demonstrates and/or produces synergistic cell toxicity.
  • a further aspect includes a use of a glycylcycline compound and a TKI for treating a cancer.
  • Another aspect includes a combination comprising a glycylcycline compound and a TKI.
  • the glycylcycline compound and TKI are in a single composition.
  • the glycylcycline compound and TKI are in separate compositions.
  • the combination demonstrates and/or produces synergistic cell toxicity
  • TKI are for use in the treatment of a cancer.
  • Another aspect includes a method of treating a cancer comprising: a) obtaining cancer cells from a test sample from a subject; b) determining a mitochondrial DNA copy number and/or a mitochondrial mass of the test sample cancer cells; c) comparing the mitochondrial DNA copy number and/or mitochondrial mass of the test sample cancer cells to a mitochondrial DNA copy number and/or a mitochondrial mass of a control, and d) administering a glycylcycline compound such as tigecycline and a TKI to the subject when the mitochondrial DNA copy number and/or the mitochondrial mass of the test sample cancer cells is at least 2 fold increased compared to the mitochondrial DNA copy number and/or the mitochondrial mass of the control.
  • a glycylcycline compound such as tigecycline and a TKI
  • the use is for treating a cancer with an at least 2 fold increased mitochondrial DNA copy number and/or mitochondrial mass compared to a control.
  • the glycylcycline compound is a glycylcycline that inhibits mitochondrial ribosome activity.
  • the glycylcycline compound comprises a 9-tert-butyl- glycylamido tetracycline backbone derivative.
  • the glycylcycline comprises a 9-substituted minocycline, tetracycline, 6-demethyl-6-deoxytetracycline, flurocycline or doxycycline derivative.
  • the glycylcycline compound is tigecycline.
  • the cancer is a hematological cancer or the cancer cell is a hematological cancer cell or wherein the cancer is a solid cancer or the cancer cell is a solid cancer cell.
  • the hematological cancer is a leukemia, a lymphoma or myeloma or the cancer cell is a leukemia cell, a lymphoma cell or a myeloma cell.
  • the leukemia is AML, ALL, CLL or CML or the leukemia cell is an AML cell, an ALL cell, a CLL cell or a CML cell.
  • the solid tumour cancer is a lung, ovarian or prostate cancer or the cancer cell is a lung cancer cell, an ovarian cancer cell or a prostate cancer cell.
  • the glycylcycline such as tigecycline
  • the TKI is comprised in a TKI composition and/or the glycylcycline and the TKI are comprised in a combined (e.g. single) composition.
  • one or more of the compositions comprises one or more suitable excipients, diluents, buffers carriers or vehicles.
  • one or more of the compositions optionally the glycylcycline composition, comprises one or more of a carbohydrate, an acid, a buffer, a chelator, pyruvic acid or a salt or ester thereof, and ascorbic acid.
  • the glycylcycline and/or TKI and/or the one or more of the compositions comprising the glycylcycline and/or TKI is in a dosage form selected from a solid dosage form and a liquid dosage form.
  • the glycylcycline and/or TKI and/or the one or more of the compositions comprising the glycylcycline and/or TKI is administered and/or is for administration by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration.
  • one or more of the compositions is formulated as an injectable dosage form.
  • the glycylcycline compound and TKI and/or the one or more of the compositions is administered and/or is for administration by intratumoural injection, intravenous injection or intratumour vasculature injection.
  • combination, composition, the dosage contacted, administered or for administration and/or each unit dosage form contacted, administered or for administration comprises an amount of the TKI and glycylcycline that produces a synergistic cell toxicity.
  • combination, composition, the dosage contacted, administered or for administration and/or each unit dosage form contacted, administered or for administration comprises from about 50 mg to about 2000 mg, from about 50 mg to about 1500 mg, from about 50 mg to about 1000 mg, from about 50 mg to about 700 mg, from about 50 mg to about 500 mg, from about 50 mg to about 350 mg, from about 50 mg to about 300 mg or from about 50 mg to about 250 mg of a glycylcycline, for example tigecycline.
  • the dosage contacted, administered or for administration and/or each unit dosage form contacted, administered or for administration comprises about 20 to about 100 mg of an glycylcycline/kg body weight, about 30 to about 100 mg of an glycylcycline/kg body weight, about 40 to about 100 mg of an glycylcycline/kg body weight, or about 50 to about 100 mg of an glycylcycline/kg body weight of a subject in need of such treatment formulated into a solid oral dosage form or a liquid oral dosage form, optionally an injectable dosage form.
  • the dosage contacted, administered or for administration and/or each unit dosage form contacted, administered or for administration comprises about 1 mg to about 40 mg of an glycylcycline/kg body weight, about 5 mg to about 40 mg of an glycylcycline/kg body weight, about 10 to about 40 mg of an glycylcycline/kg body weight, or about 20 to about 40 mg of an glycylcycline/kg body weight of a subject in need of such treatment formulated into a solid oral dosage form or a liquid oral dosage form, optionally an injectable dosage form.
  • the dosage contacted, administered or for administration and/or each unit dosage form contacted, administered or for administration comprises about 1 mg to about 10 mg of an glycylcycline/kg body weight, about 2 mg to about 10 mg of an glycylcycline/kg body weight, about 2 to about 5 mg of an glycylcycline/kg body weight of a subject in need of such treatment formulated into a solid oral dosage form or a liquid oral dosage form, optionally an injectable dosage form.
  • the TKI is a TKI that increases ROS production and/or
  • the TKI is a growth factor receptor tyrosine kinase, such as an EGF receptor tyrosine kinase.
  • the TKI is selected from erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, and bosutinib as well as sorafenib (NexavarTM), cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), lestaurtinib, canertinib, zactima, vatalanib, leflunomide, ARQ197 and mixtures thereof as well as derivatives thereof that maintain comparable or have increased kinase inhibitory activity compared to the parent TKI.
  • the TKI is or comprises erlotinib (TarcevaTM).
  • the TKI is or comprises sunitinib (SutentTM).
  • the TKI is or comprises dasatinib (SprycelTM).
  • the TKI is or comprises vandetanib.
  • the TKI is or comprises gefitinib.
  • the TKI is bosutinib.
  • the dosage contacted, administered or for administration and/or each unit dosage form contacted, administered or for administration comprises from about 50 mg to about 2000 mg, from about 50 mg to about 1500 mg, from about 50 mg to about 1000 mg, from about 50 mg to about 700 mg, from about 50 mg to about 500 mg, from about 50 mg to about 350 mg, from about 50 mg to about 300 mg or from about 50 mg to about 250 mg of a TKI, for example erlotinib.
  • a further aspect includes a method of identifying a subject likely to benefit from administration of a glycylcycline such as tigecycline and a TKI comprising:
  • the subject is identified as likely to benefit from administration of a glycylcycline such as tigecycline and a TKI when the cancer cells have an at least 2 fold increased mitochondrial DNA copy number and/or mitochondrial mass compared to the control.
  • a glycylcycline such as tigecycline and a TKI when the cancer cells have an at least 2 fold increased mitochondrial DNA copy number and/or mitochondrial mass compared to the control.
  • kits comprising a glycylcycline compound and optionally a TKI and instructions for use with a TKI and/or packaging materials for use in a method, or use described herein.
  • kits comprising a TKI and optionally a glycylcycline and instructions for use with a glycylcycline and/or packaging materials for use in a method, or use described herein.
  • the kit comprises a glycylcycline compound that is a glycylcycline that inhibits mitochondrial ribosome activity/protein synthesis such as tigecycline.
  • the kit comprises a TKI and glycylcycline that in combination can produce synergistic cell toxicity in a cancer cell.
  • the TKI is a TKI that increases NOX4 protein expression in a cancer cell when the cell is contacted with the TKI in combination with tigecycline.
  • the TKI included in the kit is selected from eriotinib
  • FIG. 1 TEX leukemia cells plated into 384 well plates and were treated with increasing concentrations of an in-house library of known on-patent and off-patent drugs with and without tigecycline at its IC20 (1.5 ⁇ ). Seventy two hours after incubation, cell growth and viability was measured by the Cell Titre Glo assay. For each compound, the IC50 was calculated in the presence or absence of tigecycline. Drugs whose IC50 were lowered greater than 4-fold by the addition of tigecycline were considered hits in the screen.
  • FIG. 4A Normal cord blood mononuclear cells were treated with increasing concentrations of tigecycline and eriotinib. Cell viability was measured by Annexin V and PI staining.
  • FIG. 4B Primary AML and normal hematopoietic cells were treated with increasing concentrations eriotinib with and without tigecycline (5uM) for 48 hours. After incubation, cell viability was measured by Annexin V and PI staining. Hatched lines are PBSC and solid lines are patient samples.
  • FIG. 5 TEX cells were treated with tigecycline and eriotinib for 8 hours. After incubation, cells were harvested, lysed, and the intracellular levels of tigecycline were determined by HPLC-UV. Data represent mean + SD from a representative experiment.
  • FIG. 7 Human leukemia (OCI-AML2) cells were injected subcutaneously into the flank of SCID mice. Seven days after injection, when tumors were palpable, mice were treated with tigecycline (50 mg/kg daily by i.p. injection) and/or erlotinib (100 mg/kg by i.p.
  • OCI-AML2 Human leukemia
  • mice were sacrificed, tumors excised, and the volume and mass of the tumors were measured and mean values determined. The tumor volume and mass are shown. * p ⁇ 0.05, ** p ⁇ 0.01 , as determined by t-test. Error bars represent mean ⁇ SD.
  • FIG. 8 A) TEX leukemia cells were treated with increasing concentrations of erlotinib (erl) or the DMSO control (DMSO) for 48 hours. After incubation, ROS production was measured by flow cytometry and staining with Carboxy-H 2 DCFDA. Cells were treated with H202 as a control. Data represent the mean fold increase in ROS production + SD compared to the control treated cells.
  • B) TEX leukemia cells were treated with increasing concentrations of tigecycline (tig) or buffer control for 48 hours. After incubation, ROS production was measured by flow cytometry and staining with Carboxy-H 2 DCFDA. Cells were treated with H202 as a control.
  • FIG. 9 Tex cells were treated with Erlotinib (3 ⁇ or 6 ⁇ ) for 48 hours, and then analyzed by immunoblotting for Nox4 and Actin.
  • glycylcycline compound as used herein means any glycyl derivative of any tetracycline backbone, for example a tert-butyl-glycylamido derivative, and includes any salt forms, such as any pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, prodrug or mixtures thereof.
  • the glycyl derivative is a tetracycline, minocycline, 6-demethyl-6-deoxytetracycline, fluorocycline (e.g. 7-fluoro) or doxycycline derivative wherein a glycyl group is attached at the 9 position of the tetracyclic structure.
  • the glycylcycline compound is a tetracycline-based compound having, at the 9-position, a dimethylglycylamido moiety.
  • the 9-glycyl moiety is 9-tert-butyl-glycylamido.
  • the glycylcycline compound is tigecycline.
  • the glycylcycline has a structure shown below, eg.
  • tetracycline backbone means a family of related substances that that contain the same four ring backbone system, including for example, but not limited to tetracycline, 6-demethyl-6-deoxytetracycline, fluorocycline comprising a fluorine atom at position C7, chlortetracycline, oxytetracycline declomycin, demeclocycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, and rolitetracycline.
  • glycol as used herein means a group of the formula:
  • R' and R" are independently selected from the group H, Ci. 20 alkyl, C 6 -ioaryl and C 3 . 10 cycloalkyl, or R' and R" are joined to form, together with the nitrogen to which they are attached, a 3 to 10 membered ring.
  • one of R' and R" is H and the other of R' and R" is C ⁇ alkyl (branched or unbranched).
  • R' and R" are each independently selected C1 -6alkyl (branched or unbranched), to form a moiety such as ⁇ , ⁇ -dimethylglycylamido (DMG).
  • tigecycline as used herein means a compound having the structure:
  • Tigecycline can be produced according to methods known in the art for example as described in U.S. Patent Publication Nos.: 2006-0247181 , titled “Tigecycline compositions and methods of preparation”; and 2007-0026080, titled “Manufacturing process for tigecycline".
  • tyrosine kinase inhibitors or "TKIs” as used herein means a compound that inhibits the tyrosine kinase activity of one or more tyrosine kinases, for example by at least 50%, 60%, 70%, 80%, 90% or 95% and includes for example, erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, and bosutinib as well as sorafenib (NexavarTM), cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), lestaurtinib, canertinib, zactima, vatalanib, leflunomide, ARQ197 and mixtures thereof as well as derivatives thereof that maintain comparable or have increased kinase inhibitory activity compared to the parent TKI, and optionally TKIs that increase ROS
  • Acceptable salts include for example, erlotinib hydrochloride (brand name TarcevaTM (available from Hoffman La Roche)) which is a reversible tyrosine kinase inhibitor approved used to treat for example non-small cell lung cancer and pancreatic cancer.
  • unitinib as used herein means a compound having the structure:
  • Sunitinib (trade name Sutent (available from Pfizer), and previously known as SU1 1248) is a multi-targeted receptor tyrosine kinase (RTK) inhibitor approved for the treatment of for example renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumours.
  • RTK receptor tyrosine kinase
  • diatsanib as used herein means a compound having the structure:
  • Datsanib brand name SprycelTM (available from Bristol Myers Squib)
  • CML chronic myelogenous leukemia
  • Ph+ ALL Philadelphia chromosome-positive acute lymphoblastic leukemia
  • vandetanib as used herein means a compound having the structure:
  • Vandetanib brand name Caprelsa (available from AstraZeneca)
  • ZD6474 is approved for treating medullary thyroid cancer.
  • Gefitinib as used herein means a compound having the structure: or pharmaceutically acceptable salts, solvates or prodrugs thereof as well as mixtures thereof.
  • Gefitinib brand name Iressa (available from AstraZeneca and Teva)
  • bisutinib as used herein means a compound having the structure:
  • Bosutinib is a third generation tyrosine kinase inhibitor that is useful in patients whose leukemia is resistant to both first and second generation tyrosine kinase inhibitors.
  • mixture means a composition comprising two or more compounds.
  • a mixture is a mixture of two or more distinct compounds.
  • a compound in a mixture can also exist as a mixture of forms.
  • a compound may exist as a hydrate of a salt or as a hydrate of a salt of a prodrug of the compound. All forms of the compounds disclosed herein are within the scope of the present disclosure.
  • cancer means a metastatic and/or a non- metastatic cancer, and includes primary and secondary cancers.
  • Reference to cancer includes reference to cancer cells.
  • cancer includes although is not limited to hematological cancers and solid cancers.
  • hematological cancer refers to cancers of blood and bone marrow, such as myeloproliferative disorders, leukemia, multiple myeloma and lymphoma and includes primary and secondary cancers.
  • Reference to hematological cancer includes reference to hematological cancer cells.
  • leukemia means any disease involving the progressive proliferation of abnormal leukocytes found in hematopoietic tissues, other organs and usually in the blood in increased numbers.
  • Leukemia includes, but is not limited to, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML).
  • AML acute myeloid leukemia
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • myeloma and/or “multiple myeloma” as used herein means any tumor or cancer composed of cells derived from the hematopoietic tissues of the bone marrow. Multiple myeloma is also known as MM and/or plasma cell myeloma.
  • myeloproliferative disorders such as “myeloproliferative diseases” and “myeloproliferative neoplasms (MPN)", most notably polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF).
  • PV polycythemia vera
  • ET essential thrombocythemia
  • PMF primary myelofibrosis
  • MDS myelodysplastic syndromes
  • AML acute myelogenous leukemia
  • lymphoma means any disease involving the progressive proliferation of abnormal lymphoid cells.
  • lymphoma includes mantle cell lymphoma, Non-Hodgkin's lymphoma, and Hodgkin's lymphoma.
  • Non-Hodgkin's lymphoma would include indolent and aggressive Non-Hodgkin's lymphoma.
  • Aggressive Non-Hodgkin's lymphoma would include intermediate and high grade lymphoma.
  • Indolent Non-Hodgkin's lymphoma would include low grade lymphomas.
  • solid tumour cancer refers to a cancer resulting in one or more solid tumours composed of cancer cells and includes, for example, lung cancer, such as non-small cell lung cancer, brain (glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma, ependymomas), liver, thyroid, bone, adrenal, spleen, kidney (such as renal cell carcinoma), lymph node, small intestine (including for example gastrointestinal stromal tumours), pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, and esophagus.
  • lung cancer such as non-small cell lung cancer
  • brain glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma, ependymomas
  • liver thyroid
  • bone adrenal
  • adrenal spleen
  • kidney such as renal cell carcinoma
  • lymph node small intestine (including
  • composition is defined herein to refer to a mixture or solution containing at least one therapeutic agent to be administered to a subject, e.g., a mammal or human, in order to prevent or treat a particular disease or condition affecting the mammal.
  • pharmaceutically acceptable means compatible with the treatment of animals, in particular humans.
  • pharmaceutically acceptable salt means an acid addition salt or basic addition salt which is suitable for, or compatible with, the treatment of patients.
  • pharmaceutically acceptable acid addition salt means any non-toxic organic or inorganic salt of any basic compound.
  • Basic compounds that form an acid addition salt include, for example, compounds comprising an amine group.
  • Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen, orthophosphate and potassium hydrogen sulfate.
  • Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids.
  • Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form.
  • acid addition salts are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art.
  • compositions that form a basic addition salt include, for example, compounds comprising a carboxylic acid group.
  • Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide.
  • Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline, alkylammonias or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.
  • a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
  • prodrug refers to a derivative of an active form of a known compound or composition which derivative, when administered to a subject, is gradually converted to the active form to produce a better therapeutic response and/or a reduced toxicity level.
  • prodrugs will be functional derivatives of the compounds disclosed herein which are readily convertible in vivo into the compound from which it is notionally derived.
  • Prodrugs include, without limitation, acyl esters, carbonates, phosphates, and urethanes. These groups are exemplary and not exhaustive, and one skilled in the art could prepare other known varieties of prodrugs.
  • Prodrugs may be, for example, formed with available hydroxy, thiol, amino or carboxyl groups.
  • the available OH and/or NH 2 in the compounds of the disclosure may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine).
  • Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C,-C 2 4) esters, acyloxymethyl esters, carbamates and amino acid esters.
  • the prodrugs of the compounds of the disclosure are those in which the hydroxy and/or amino groups in the compounds is masked as groups which can be converted to hydroxy and/or amino groups in vivo. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985.
  • the compounds according to the disclosure possess one or more than one asymmetric centres, they may exist as "stereoisomers", such as enantiomers and diastereomers. It is to be understood that all such stereoisomers and mixtures thereof in any proportion are encompassed within the scope of the present disclosure. It is to be understood that, while the stereochemistry of the compounds of the disclosure may be as provided for in any given compound shown herein, such compounds may also contain certain amounts (e.g. less than 20%, less than 10%, less than 5%) of compounds having alternate stereochemistry.
  • solvate as used herein means a compound or its pharmaceutically acceptable salt, wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered.
  • suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate".
  • solvates will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
  • subject includes all members of the animal kingdom including mammals, and suitably refers to humans.
  • inducing cytotoxicity in a cell means causing cell damage that results in cell death.
  • cell death includes all forms of cell death including necrosis and apoptosis.
  • treating means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
  • Treating and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Treating” and “treatment” as used herein also include prophylactic treatment.
  • a subject with early stage leukemia can be treated to prevent progression or metastases, or alternatively a subject in remission can be treated with a compound or composition described herein to prevent recurrence.
  • Treatment methods comprise administering to a subject a therapeutically effective amount of a compound described herein and optionally consists of a single administration, or alternatively comprises a series of applications.
  • the compounds described herein may be administered at least once a week.
  • the compounds may be administered to the subject from about one time per three weeks, or about one time per week to about once daily for a given treatment.
  • the compound is administered twice daily.
  • the length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the patient, the concentration, the activity of the compounds described herein, and/or a combination thereof. It will also be appreciated that the effective dosage of the compound used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compounds are administered to the subject in an amount and for a duration sufficient to treat the patient.
  • the term "dosage form” refers to the physical form of a compound or composition for example comprising a compound and/or mixture of compounds of the disclosure, and includes without limitation liquid and solid dosage forms including, for example tablets, including enteric coated tablets, caplets, gelcaps, capsules, ingestible tablets, buccal tablets, troches, elixirs, suspensions, syrups, wafers, resuspendable powders, liquids, solutions as well as injectable dosage forms, including, for example, sterile solutions and sterile powders for reconstitution, and the like, that are suitably formulated for injection.
  • liquid and solid dosage forms including, for example tablets, including enteric coated tablets, caplets, gelcaps, capsules, ingestible tablets, buccal tablets, troches, elixirs, suspensions, syrups, wafers, resuspendable powders, liquids, solutions as well as injectable dosage forms, including, for example, sterile solutions and sterile powders for reconstitution, and the like
  • dosage means an amount or quantity of a compound or composition contacted with a cell, administered to a subject or for administration to a subject.
  • an effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • an effective amount e.g. of a glycylcycline compound and a TKI
  • an amount e.g. of the combination
  • Effective amounts may vary according to factors such as the disease state, age, sex, weight of the subject.
  • the amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • a mitochondrial translated polypeptide refers to a polypeptide that is exclusively translated by a ribosome located in a mitochondria.
  • Mitochondrial mass refers to the overall number and/or weight of mitochondria in a cell or number of cells. Mitochondrial mass may be determined or characterized, for example, by incubating cells with Mitotracker Green FM dye, subsequently performing flow cytometry, and determining the median fluorescence intensity of the cells. Mitochondrial mass may also be determined or characterized by incubating cells with Mitotracker Green FM dye, subsequently performing confocal scanning laser microscopy, and quantifying the fluorescence levels using an image software, for example ImageJ (see for example Agnello et al. A method for measuring mitochondrial mass and activity. Cytotechnology Vol 56(3): 145-1 9).
  • ImageJ see for example Agnello et al. A method for measuring mitochondrial mass and activity. Cytotechnology Vol 56(3): 145-1 9).
  • the mitochondrial mass of a cell or average mitochondrial mass of a number of cells, for example, in a sample taken from a subject with a cancer, can be compared to a mitochondrial mass of a control cell or number of cells in a sample taken for example from a control subject.
  • DNA copy number and can be determined by quantitating the level of a mitochondrial gene such as an ND gene, for example ND1 , relative to a non-mitochondrial gene such as beta- globin.
  • ND1 , ND2 is a family of mitochondrial ⁇ encoded NADH dehydrogenases, wherein ND1 , ND2, ND2, ND3, ND4, ND4L, ND5 are ND6 are specific family members.
  • Mouse ND1 has Accession number NP_904328 and Human ND1 for example has accession Ensembl: ENSG00000198888, UniProtKB: P03886. the sequences therein herein incorporated by reference. Human ND1 can be detected for example using Sigma Aldrich Anti MT-ND1 antibody.
  • control refers to a suitable comparator subject, sample, cell or cells such as non-cancerous subject, blood sample, cell or cells from such a subject, for comparison to a cancer subject, sample (e.g. test sample) cell or cells from a cancer subject; or an untreated subject, cell or cells, for comparison to a treated subject, cell or cells, according to the context.
  • a control for comparing mitochondrial mass includes for example non-cancerous cells such as normal CD34+ hematopoietic cells, for example in a blood sample taken from a control subject free of cancer and/or cancer cells known to have low and/or about normal mitochondrial mass.
  • Control can also refer to a value representative of a control subject, cell and/or cells and/or a population of subjects, for example representative of a normal mitochondrial mass.
  • sample refers to any biological fluid comprising a cell, a cell or tissue sample from a subject including a sample from a test subject, i.e. a test sample, such as from a subject whose mitochondrial mass is being tested, for example, a subject with a cancer, wherein the test sample comprises cancer cells, and a control sample from a control subject, e.g., a subject without a cancer, whose mitochondrial mass is being tested.
  • the sample can comprise a blood sample, for example a peripheral blood sample, a fractionated blood sample, a bone marrow sample, a biopsy, a frozen tissue sample, a fresh tissue specimen, a cell sample, and/or a paraffin embedded section.
  • the sample comprises mononuclear cells.
  • the term "inhibiting a mammalian mitochondrial ribosome in a cell” as used herein means to reduce compared to an untreated cell, interfering with mitochondrial polypeptide translation of mRNA, reflected for example in the steady state level or the amount of translation product produced over a period of time.
  • Mitochondrial ribosome translated polypeptides include for example ND1 , ND2, ND3, ND4, ND4L, ND5, ND6, Cyt B, Cox-2, Cox-3, ATP6, and ATP8.
  • ND1 , ND2, ND3, ND4, ND4L, ND5 and ND6 are mitochondrially encoded NADH dehydrogenases
  • Cyt B is cytochrome B
  • Cox-2 and Cox-3 are cytochrome oxidase subunits 2 and 3 respectively
  • ATP6 and ATP8 are ATP synthase F0 subunit 6 and 8 respectively.
  • NOX4 or "NADPH oxidase 4" as used herein means NADPH oxidase enzyme encoded by the NOX4 gene, in the human for example having amino acid and nucleic acid sequences with accession numbers: UniProt Q9NPH5, Entrez Gene ID: 50507 and RefSeq NP_001 37308, (in the mouse having for example accession numbers: UniPort Q9JHI8, Entrez 50490 and RefSeq NP 056575), which sequences identified thereby are specifically incorporated herein by reference, and as well as all naturally occurring variants. NOX4 is also known for example as KOX; KOX-1 ; and RENOX.
  • chelator as used herein is a compound that binds for example mono- and/or divalent cations, removing them from solution. Suitable chelators for use in the compositions of the application include without limitation EDTA and derivatives thereof, for example, disodium edetate, trisodium edetate, tetrasodium edetate, disodium calcium edentate, and the like, EGTA, penicillamine, dimercapto-propane sulfonate citrates, and tartrates.
  • pyruvic acid, or an ester or salt thereof as used herein means a compound having the structure
  • R'" is selected from H, a cation, Ci. 24 alkyl, C 6 . 10 aryl and C 3 .i 0 cycloalkyl, or pharmaceutically acceptable solvates or prodrugs thereof, as well as mixtures thereof.
  • the carboxylate (COOH) anion of pyruvic acid, CH 3 COCOO " is pyruvate, which can be for example sodium pyruvate, calcium pyruvate, potassium pyruvate, magnesium pyruvate, or dihydroxyacetone pyruvate, or any other suitable salt.
  • corbic acid as used herein means a compound having the structure
  • cyclodextrin refers to a family of compounds that are made up of sugar molecules bound together in a ring.
  • Non-limiting examples include 2- hydroxylpropyl-beta-cyclodextrin (2-HP-beta-CD) alpha-cyclodextrin, beta-cyclodextrin and gam ma-cyclodextrin .
  • the term "administered contemporaneously" as used herein means that two substances are administered to a subject such that they are both biologically active in the subject at the same time and provide joint therapeutic effect.
  • the TKI and glycyclcycline can be may be given separately (in a chronologically staggered manner, for example a sequence-specific manner) in such time intervals that they prefer, and which still show a (preferably synergistic) interaction (joint therapeutic effect). Whether this is the case can, for example be determined by following the blood levels, showing that both compounds are present in the blood of the subject to be treated at least during certain time intervals.
  • two substances will be administered substantially simultaneously, i.e. within minutes of each other, or in a single composition that comprises both substances.
  • synergistic effect refers to action of two therapeutic agents such as, for example, a glycylcycline, and a TKI, producing an effect, for example, slowing the symptomatic progression of a cancer, or symptoms thereof, which is greater than the additive effect of each drug alone.
  • a synergistic effect can be calculated, for example, using combination index analysis applied to experimental data to generate a combination index graph to assess the effects of the drug combination as is shown in Fig. 1 and/or by calculating Excess Bliss over additivism scores as was done in Fig 6E.
  • a synergistic effect determined by one or more of these methods can identify the combination as synergistic.
  • a composition containing "a compound” includes a mixture of two or more compounds.
  • the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
  • a TKI can be one or more than one different TKIs and a glycylcycline compound can be one or more than one glycylcyclines.
  • compositions comprising an "additional” or “second” component
  • the second component as used herein is chemically different from the other components or first component.
  • a “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.
  • composition or “pharmaceutical combination” is defined herein to refer to either a fixed combination in one dosage unit form, a non- fixed combination or a kit of parts for the combined administration where COMPOUND A or a pharmaceutically acceptable salt thereof, and at least one TKI may be administered simultaneously, independently at the same time or separately within time intervals that allow the combination partners to show a cooperative, i.e., synergistic, effect.
  • fixed combination means that the active ingredients, e.g. a glycylcycline and a TKI combination partner, are both administered to a subject simultaneously in the form of a single entity or dosage.
  • non-fixed combination means that the active ingredients, e.g. a glycylcycline and a TKI combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the subject.
  • cocktail therapy e.g. the administration of three or more active ingredients.
  • Tigecycline which is for example sold under the brand name Tygacil®, is presently used for the treatment of certain infections. It was demonstrated in WO201 1/109899 (Application Number PCT/CA2011/000258) filed March 10, 201 1 , herein incorporated by reference in its entirety, that pharmacologically achievable concentrations of tigecycline can be used for treating cancer and particularly leukemia. Minocycline and tetracycline were also tested and did not show efficacy.
  • tyrosine kinase inhibitors erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, gefitnib (IressaTM) and bosutinib synergize with tigecyline (FIGs. 1 and 3).
  • tigecycline reduced the IC50 of erlotinib, sunitinib, dasatinib, vandetanib and bosutinib toxicity to leukemia cells by at least 4 fold (e.g. see Examplel ).
  • the TKI, gefitinib was also shown to synergize with tigecycline
  • an aspect of the present disclosure includes a method of treating a cancer comprising administering to a subject in need thereof a glycylcycline compound such as tigecycline and a TKI.
  • the disclosure includes use of a glycylcycline compound such as tigecycline and a TKI for treating a cancer.
  • Another aspect includes use of a glycylcycline compound such as tigecycline and a TKI for the manufacture of a medicament for the treatment of a cancer.
  • the disclosure includes a combination comprising a glycylcycline compound such as tigecycline and a TKI. In an embodiment, combination is for use in the treatment of cancer.
  • tigecycline induced cytotoxicity correlates with increasing mitochondrial mass in AML samples.
  • AML cells with an increased mitochondrial mass compared to normal cells were more sensitive to tigecycline compared to cells with a decreased mitochondrial mass.
  • AML samples have increased mitochondrial DNA copy number of ND1 (also known as mitochondrially encoded NADH dehydrogenase 1 ) relative to human globulin gene DNA (HGB).
  • the disclosure includes a method of identifying a subject likely to benefit from administration of a glycylcycline compound and a TKI comprising: a) obtaining cancer cells from a test sample from the subject; b) determining a mitochondrial DNA copy number and/or a mitochondrial mass of the test sample cancer cells; c) comparing the mitochondrial DNA copy number and/or the mitochondrial mass of the test sample cancer cells to the mitochondrial DNA copy number and/or the mitochondrial mass of a control, wherein the subject is identified likely to benefit from administration of a glycylcycline compound and a TKI when the test sample cancer cells have an at least 2 fold increased mitochondrial DNA copy number and/or mitochondrial mass compared to the control.
  • the disclosure includes a method of treating a cancer comprising: a) obtaining cancer cells from a test sample from a subject; b) determining a mitochondrial DNA copy number and/or a mitochondrial mass of the test sample cancer cells; c) comparing the mitochondrial DNA copy number and/or mitochondrial mass of the test sample cancer cells to a mitochondrial DNA copy number and/or mitochondrial mass of a control, and d) administering a glycylcycline compound and a TKI to the subject when the mitochondrial DNA copy number and/or mitochondrial mass of the test sample cancer cells is at least 2 fold increased compared to the mitochondrial DNA copy number and/or mitochondrial mass of the control.
  • the mitochondrial DNA copy number of a test sample is determined by quantitating a DNA level of a mitochondrial gene such as ND1 and a DNA level of a non-mitochondrial gene (i.e. a nuclear gene) such as human globulin (HGB), which serves as an internal control, and comparing a ratio of the DNA levels of the mitochondrial gene to the non-mitochondrial gene in the test sample to a control.
  • the method comprises using PCR for example real-time PCR.
  • a DNA level of any of the genes encoded by the mitochondrial genome can be used.
  • the non-mitochondrial gene can be any suitable nuclear gene such as but not limited to beta-globin (i.e. human globulin), 18S and GAPDH.
  • a further aspect includes a method of treating a cancer with an at least 2 fold increased mitochondrial DNA copy number and/or mitochondrial mass compared to a control comprising administering to the subject in need thereof, a glycylcycline compound such as tigecycline and a TKI.
  • a glycylcycline compound such as tigecycline and a TKI.
  • a further aspect includes use of a glycylcycline compound such as tigecyline and a TKI for treating a cancer with an at least 2 fold increased mitochondrial DNA copy number and/or mitochondrial mass compared to a control.
  • Another aspect includes use of a glycylcycline compound such as tigecycline and a TKI for the manufacture of a medicament for treating a cancer with an at least 2 fold increased mitochondrial DNA copy number and/or mitochondrial mass compared to a control.
  • Yet another aspect includes a combination comprising a glycylcycline compound such as tigecycline and a TKI for use in treating a cancer with an at least 2 fold increased mitochondrial DNA copy number and/or mitochondrial mass compared to a control.
  • a cancer or cell mitochondrial mass is assessed by taking a biopsy sample e.g. test sample from a subject and determining the mitochondrial mass of the test sample cancer cells using for example a method described herein, in WO2011/109899 and/or known in the art.
  • the cancer and/or cancer cells have at least a 3 fold increase, at least a 4 fold increase and/or at least a 5 fold increase in mitochondrial DNA copy number and/or mitochondrial mass compared to a control.
  • the disclosure includes a method of inducing cytotoxicity in a cancer cell comprising contacting the cancer cell with or administering to the cancer cell a glycylcycline compound such as tigecycline and a TKI.
  • a glycylcycline compound such as tigecycline and a TKI.
  • the contact is for example under a suitable length of time and under suitable conditions to induce cytotoxicity in the cell.
  • the disclosure provides use of a glycylcycline compound such as tigecycline and a TKI for inducing cytotoxicity in a cancer cell.
  • Another aspect of the disclosure includes use of a glycylcycline compound such as tigecycline and a TKI for the manufacture of a medicament for inducing cytotoxicity in a cancer cell.
  • a further aspect provides a combination comprising a glycylcycline compound and a TKI for inducing cytotoxicity in a cancer cell.
  • the cancer cell is in vitro.
  • the cancer cell is in vivo.
  • the cancer cell is located in a human subject.
  • Synergy is demonstrated in several cancer cell models. Synergy was demonstrated for tigecycline TKi combinations in leukemia, myeloma and lung cell lines.
  • a glycylcycline and a TKI may result not only in a beneficial effect, e.g. a synergistic therapeutic effect, e.g. with regard to alleviating, delaying progression of or inhibiting the symptoms, but also in further surprising beneficial effects, e.g. fewer side-effects, an improved quality of life or a decreased morbidity, compared with a monotherapy applying only one of the pharmaceutically therapeutic agents used in the combination
  • the disclosure includes a method of treating a cancer wherein the cancer cell is in a subject and the subject is administered a glycylcycline compound such as tigecycline and a TKI.
  • the cancer cell is a hematological cancer cell. In another embodiment, the cancer cell is a solid cancer cell. In a further embodiment, the cancer cell is a cancer stem cell.
  • the disclosure includes a method of inhibiting a mammalian mitochondrial ribosome in a cell comprising contacting the cell with or administering to the cancer cell a composition comprising a glycylcycline compound such as tigecycline and a TKI, for example for a suitable time and under suitable conditions.
  • a suitable time includes for example a time sufficient to decrease mitochondrial ribosome activity by at least 50%, 60%, 70%, 80%, 90%, 95% or more.
  • Suitable conditions include for example appropriate cell culture conditions for the cell if in vitro and/or appropriate formulation if in vivo.
  • the method is for inhibiting a mammalian mitochondrial ribosome in a cell, in the absence of producing increased radical oxygen production.
  • Another aspect of the disclosure includes use of a glycylcycline compound such as tigecycline compound and a TKI for inhibiting a mammalian mitochondrial ribosome in a cell.
  • the disclosure includes use of a glycylcycline compound such as tigecycline and a TKI for the manufacture of a medicament for inhibiting a mammalian mitochondrial ribosome in a cell.
  • a further aspect includes a combination comprising a glycylcycline compound and a TKI for use in inhibiting a mammalian mitochondrial ribosome in a cell.
  • inhibition of the mammalian mitochondrial ribosome is assessed by determining the level of a mitochondrial translated polypeptide.
  • the mitochondrial translated polypeptide is Cox-1.
  • the mitochondrial translated polypeptide is Cox-2.
  • any protein that is translated by mitochondrial ribosomes, preferably exclusively by mitochondrial ribosomes, can be assayed to assess inhibition of a mitochondrial ribosome. Without wishing to be bound to any particular theory, it is predicted that tigecycline, induces cell death by inhibiting mitochondrial ribosomal protein synthesis that thereby blocks oxidative phosphorylation and cellular metabolism and/or leads to disruption of the mitochondria.
  • the mitochondrial ribosome translated polypeptide is selected from ND1 , ND2, ND3, ND4, ND4L, ND5, ND6, Cyt B, Cox-2, Cox-3, ATP6, and ATP8.
  • ND1 , ND2, ND3, ND4, ND4L, ND5 and ND6 are mitochondrially encoded NADH dehydrogenase
  • Cyt B is cytochrome B
  • Cox-2 and Cox-3 are cytochrome oxidase subunits 2 and 3 respectively
  • ATP6 and ATP8 are ATP synthase F0 subunit 6 and 8 respectively.
  • the glycylcycline compound is a glycylcycline that inhibits mitochondrial ribosome activity for example as assessed by measuring protein synthesis.
  • the glycylcycline compound comprises tigecycline.
  • the glycylcycline compound is tigecycline.
  • the TKI is a TKI that increases ROS production and/or NOX4 protein expression in a cancer cell when he cell is contacted with the TKI in combination with tigecycline.
  • the protein expression level of NOX4 can be detected by a plurality of methods including antibody based methods such as western blot, immunohistochemistry, immunoprecipitation and the like or imputed from increases in NOX4 mRNA, for example by PCR based methods such as RT-PCR or probe based methods.
  • NOX4 antibodies are available for example from GE Health, Santa Cruz and Abeam.
  • Mitochondrial ROS can be detected for example using fluorescent probes, electron-spin resonance approaches, and immunoassays including for example HPLC superoxide detection, fluorescent boronate-containing probes, use of cell-targeted hydroxylamine spin probes, and immunospin trapping.
  • the TKI is selected from erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, bosutinib, sorafenib (nexavar), cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), lestaurtinib, canertinib, zactima, vatalanib, leflunomide and ARQ197 and mixtures thereof.
  • the TKI is selected from erlotinib, sunitinib, dasatinib, vandetanib and bosutinib and mixtures thereof and/or derivatives thereof that maintain comparable or have increased kinase inhibitory activity compared to the parent TKI.
  • the TKI is a TKI that demonstrates synergistic cell toxicity (e.g. decreased cell viability) with tigecycline, in a cancer cell such an H1573 cell or other cell shown in Fig. 3 or 6, wherein the synergy is determined for example by calculating the combination index and/or Excess Bliss over additivism score.
  • the glycylcycline compound is a glycylcycline compound that demonstrates synergistic cell toxicity (e.g. decreased cell viability) with erlotinib in a cancer cell such an H1573 cell or other cell shown in Fig 3 or 6, wherein the synergy is determined for example by calculating the combination index and/or Excess Bliss over additivism score.
  • the TKI and glycyclcycline are comprised in a combination kit or composition, administered or for administration in a synergistic ratio having a combination index of less than 1.
  • the combination index less than 1 , less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4 or less than O.3..
  • the TKI and glycyclcycline are comprised in a combination kit or composition, administered or for administration in a synergistic ratio having an Excess Bliss over additivism score of at least 20.
  • the Excess Bliss over additivism score is at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 or at least 31.
  • the TKI and glycyclcycline are comprised in a combination kit or composition, administered or for administration, in respective proportions to provide a synergistic cell toxicity effect.
  • the ratio of TKI to glycylcycline amount or concentration or glycylcycline to TKI amount or concentration is from about 10: 1 to about 1 : 10, about 6 1 to about 1 :6, for example about 1 : 1 , about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1 : 10 or about 2:3, about 3:4, about 2: 1 , about 3: 1 , about 3:2, or about 4: 1.
  • the ratio is a molar ratio of TKI to glycylcycline or glycylcycline to TKI.
  • the molar ratio of TKI to glycylcycline is about 10; 1 to about 1 : 10, about 8: 1 to about 1 :8, about 6:1 to about 1 :6 about 4: 1 to about 1 :4 about 2: 1 to about 1 :2 or; at least 1 :4, at least 1 :2 at least 1 : 1 ; at least 2:1 at least 4:1 or; at least 6: 1..
  • the molar ratio of TKI selected from sunitinib (SutentTM), dasatinib, vandetanib, bosutinib, sorafenib, cedivanib, gefitinib, imatinib, lestaurtinib, canertinib, zactima, vatalanib, leflunomide and ARQ197 and mixtures thereof to tigecycline is about 10; 1 to about 1 : 10, about 8: 1 to about 1 :8, about 6: 1 to about 1 :6 about 4: 1 to about 1 :4 about 2: 1 to about 1 :2 or; at least 1 :4, at least 1 :2 at least 1 : 1 ; at least 2: 1 at least 4: 1 or; at least 6: 1.
  • the amount or concentrations corresponding to the molar ratios can be calculated for different combinations of TKI and glycylcyclines.
  • the molecular weight of tigecycline is about 586 Daltons and the molecular weight of eriotinib is about 430 Daltons.
  • the TKI composition and/or combination composition comprises about 0.001 to about 30 weight percent, about 0.01 to about 30 weight percent, about 1 to about 30 weight percent, about 0.001 to about 20 weight percent, about 0.01 to about 20 weight percent, about 1 to about 20 weight percent, about 0.001 to about 10 weight percent, about 0.01 to about 0 weight percent, or about 1 to about 10 weight percent of the TKI.
  • the glycylcycline composition and or combination composition comprises about 0.001 to about 30 weight percent, about 0.01 to about 30 weight percent, about 1 to about 30 weight percent, about 0.001 to about 20 weight percent, about 0.01 to about 20 weight percent, about 1 to about 20 weight percent, about 0.001 to about 10 weight percent, about 0.01 to about 10 weight percent, or about 1 to about 10 weight percent of the glycylcycline.
  • the TKI is selected from eriotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib and bosutinib.
  • the TKI is a TKI that inhibits one or more of the kinases inhibited by eriotinib, sunitinib, dasatinib, vandetanib, gefitinib and/or bosutinib.
  • the TKI is eriotinib (TarcevaTM).
  • the TKI is sunitinib (SutentTM).
  • the TKI is dasatinib (SprycelTM)
  • the TKI is vandetanib.
  • the TKI is gefitinib.
  • the TKI is bosutinib.
  • the combination comprises tigecycline and one or more of eriotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, gefitinib and bosutinib and/or a TKI that inhibits one or more of the kinases inhibited by eriotinib, sunitinib, dasatinib, vandetanib, gefitinib and/or bosutinib.
  • the combination comprises tigecycline and eriotinib (TarcevaTM).
  • the combination comprises tigecycline and sunitinib (SutentTM).
  • the combination comprises tigecycline and dasatinib (SprycelTM).
  • the combination comprises tigecycline and vandetanib.
  • the combination comprises tigecycline and gefitinib.
  • the combination comprises tigecycline and bosutinib.
  • the method or use comprises administration or use of tigecycline and eriotinib (TarcevaTM).
  • the method or use comprises administration or use of tigecycline and sunitinib (SutentTM).
  • the method or use comprises administration or use of tigecycline and dasatinib (SprycelTM).
  • the method or use comprises administration or use of tigecycline and vandetanib.
  • the method or use comprises administration or use of tigecycline and gefitinib.
  • the method or use comprises administration or use of tigecycline and bosutinib.
  • Cancers and cancer cells that can be treated include, but are not limited to, hematological cancers, including leukemia, lymphoma and myeloma, and solid cancers, including for example tumors of the brain (glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma, ependymomas), lung, liver, thyroid, bone, adrenal, spleen, kidney (such as renal cell cancer), lymph node, small intestine (including gastrointestinal stromal tumours), pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, and esophagus. More generally, it is contemplated that the present drug combination will be useful to treat those cancers that are responsive to tigecycline therapy, at the very least. The list of treatable cancers may of course be expanded beyond these, when combination therapy including the TKI is exploited.
  • the cancer is a hematological cancer.
  • the hematological cancer is a leukemia.
  • the hematological cancer is a myeloma.
  • the hematological cancer is a lymphoma.
  • the leukemia is selected from acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML).
  • AML acute myeloid leukemia
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • the leukemia is AML.
  • the leukemia is ALL.
  • the leukemia is CLL.
  • the leukemia is CML.
  • the cancer cell is a leukemic cell, for example, but not limited to, an AML cell, an ALL cell, a CLL cell or a CML cell.
  • the hematological cancer is a myeloma.
  • the hematological cancer cell is a myeloma cell.
  • the hematological cancer is a lymphoma.
  • the hematological cancer cell is a lymphoma cell.
  • the cancer is a solid tumour cancer.
  • the solid tumour cancer is selected from ovarian cancer, prostate cancer and lung cancer.
  • the cancer cell is an ovarian cancer cell, a prostate cancer cell or a lung cancer cell.
  • the glycylcycline compound can be formulated with no excipients.
  • tigecycline can be formulated as a powder with no excipients.
  • the drug is reconstituted in a vial at a concentration of 10 mg/mL in saline, 5% dextrose, or Ringer's lactate. In this form, the drug is stable for only 6 hours at room temperature.
  • Tigecycline can be further diluted to a concentration of 1 mg/mL in the above solutions. At this concentration, the drug is stable for 24 hours at room temperature (19).
  • the glycylcycline compound can be comprised in a composition and/or TKI can also be comprised in a composition.
  • the glycylcycline compound and the TKI can also be formulated in a single composition.
  • the glycylcycline compound is comprised in a composition (e.g. glycylcycline composition) with one or more suitable excipients, diluents, buffers, carriers or vehicles.
  • the composition comprises tigecycline with one or more suitable excipients, diluents, buffers, carriers or vehicles.
  • the TKI e.g.
  • TKI composition is comprised in a composition with one or more suitable excipients, diluents, buffers carriers or vehicles.
  • the composition comprises a glycylcycline, a TKI and one or more suitable excipients, diluents, buffers carriers or vehicles.
  • a suitable excipient, excipient, diluent, buffer, carrier or vehicle can increase compound stability.
  • US Patent Serial No. 7,879,828 issued February 1 , 201 1 to Wyeth relates to compositions of tigecycline with improved stability comprising a suitable carbohydrate, such as lactose, an acid and a buffer.
  • a suitable carbohydrate such as lactose, an acid and a buffer.
  • tigecycline is supplied as a vial containing 50 mg of lyophilized powder with 100 mg lactose monohydrate, hydrochloric acid and sodium hydroxide (20).
  • US patent application publication no. 2008/0014256 describes pharmaceutical compositions comprising tigecycline for oral administration.
  • the composition can comprise a glycylcycline compound such as tigecycline having at least one enteric coating.
  • the composition for example comprising a glycylcycline compound, optionally tigecycline, and/or a TKI further comprises a suitable carbohydrate, such as lactose, an acid and a buffer.
  • a suitable carbohydrate such as lactose, an acid and a buffer.
  • lactose is lactose monohydrate.
  • suitable carbohydrates are disclosed in US Patent No. 7,879,828, herein incorporated by reference.
  • one or more of the compositions comprises lactose monohydrate, hydrochloric acid and/or sodium hydroxide.
  • composition for use in a method or use described herein comprises tigecycline and, one or more suitable excipients, diluents, buffers, carriers or vehicles.
  • the composition comprises tigecycline with improved stability and which retains anti-leukemic activity and anti-bacterial activity.
  • a formulation of tigecycline in the presence of EDTA, sodium pyruvate, and 2-hydroxylpropyl- beta-cyclodextrin (2-HP-beta-CD) remained stable and active when stored at a concentration of 1 mg/mL for up to 7 days.
  • the addition of ascorbic acid can permit increased concentrations of tigecycline in the solution.
  • compositions comprising tigecycline at drug concentrations of up to 5 mg/mL were stable and retained activity when stored at room temperature for 7 days.
  • the composition for example a composition comprising a glycylcycline compound such as tigecycline and/or a composition comprising a TKI and/or a composition comprising a glycylcycline such as tigecycline and a TKI such as one or more of erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, gefitinib and bosutinib or other TKI herein referred to, also comprises one or more of a chelator, pyruvic acid, or a salt or ester thereof and ascorbic acid and optionally one or more excipients, diluents or buffers.
  • one or more of the compositions further comprises a cyclodextrin.
  • tigecycline for contacting a cell, for administering or for administration is comprised in a composition
  • a composition comprising, consisting of, or consisting essentially of, a glycylcycline compound such as tigecycline and/or a TKI such one or more of erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, gefitinib and bosutinib, and one or more of a chelator; pyruvic acid or a salt or ester thereof and ascorbic acid and optionally one or more excipients, diluents or buffers.
  • the composition further comprises a cyclodextrin.
  • the composition comprises a glycylcycline and a TKI.
  • the composition comprises tigecycline and erlotinib (TarcevaTM).
  • the composition comprises tigecycline and sunitinib (SutentTM).
  • composition comprises tigecycline and dasatinib (SprycelTM).
  • the composition comprises tigecycline and vandetanib.
  • the composition comprises tigecycline and gefitinib.
  • the composition comprises tigecycline and bosutinib.
  • the pyruvic acid, or salt or ester of pyruvic acid is present in the composition in an amount of about 0.06% to about 30% (w/v), or any 0.01 % increment in between. In an embodiment, the pyruvic acid, or salt or ester of pyruvic acid, is present in the composition in an amount of about 1 % to about 25% (w/v). In an embodiment, the pyruvic acid, or salt or ester of pyruvic acid is present in the composition in an amount of at least about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% (w/v).
  • pyruvic acid, or salt or ester of pyruvic acid is present in the composition in an amount of at most about 7%, about 8%, about 9%, about 10%, about 1 1 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29% or about 30% (w/v).
  • the chelator is present in the composition in an amount of about 0.006% to about 3% (w/v), or any 0.001 % increment in between. In an embodiment, the chelator is present in the composition in an amount of about 0.1 % to about 2.5% (w/v). In an embodiment, the chelator is present in the composition in an amount of at least about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9% or about 1 % (w/v).
  • the chelator is present in the composition in an amount of at most about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1 .1 %, about 1 .2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1 .8%, about 1.9%, about 2.0%, about 2.1 %, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9% or about 3.0% (w/v).
  • cyclodextrin is present in the composition in an amount of about 0.05% to about 25% (w/v), or any 0.01 % increment in between. In an embodiment, the cyclodextrin is present in the composition in an amount of about 1 % to about 25% (w/v). In an embodiment, cyclodextrin is present in the composition in an amount of at least about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% (w/v).
  • the cyclodextrin is present in the composition in an amount of at most about 7%, about 8%, about 9%, about 10%, about 1 1 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24% or about 25% (w/v).
  • ascorbic acid is present in the composition in an amount of about 0.03% to about 15% (w/v), or any 0.01 % increment in between. In an embodiment, the ascorbic acid is present in the composition in an amount of about 1 % to about 15% (w/v). In an embodiment, cyclodextrin is present in the composition in an amount of at least about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% (w/v).
  • the cyclodextrin is present in the composition in an amount of at most about 7%, about 8%, about 9%, about 10%, about 1 1 %, about 12%, about 13%, about 14% or about 15% (w/v).
  • pyruvate is present in the composition in an amount of about 0.6 milligrams/mL to about 300 milligrams/mL, or any 0.1 milligram/mL increment in between.
  • EDTA is present in the composition in an amount of about 0.06 milligrams/mL to about 30 milligrams/mL or any 0.01 milligram/mL increment in between.
  • 2-hydroxylpropyl-beta-cyclodextrin (2-HP-beta-CD) is present in the composition in an amount of about 0.5 milligrams/mL to about 250 milligrams or any 0.1 milligram/mL increment in between.
  • ascorbic acid is present in the composition in an amount of about 0.3 milligrams/mL to about 150 milligrams/mL ascorbic acid or any 0.1 milligram/mL increment in between.
  • one or more of the compositions comprise from about 0.6 milligrams/mL to about 300 milligrams/mL pyruvate or any 0.1 milligram/mL increment in between, from about 0.06 milligrams/mL to about 30 milligrams/mL EDTA or any 0.01 milligram/mL increment in between, from about 0.5 milligrams/mL to about 250 milligrams of 2-hydroxylpropyl-beta-cyclodextrin (2-HP-beta-CD) or any 0.1 milligram/mL increment in between; and/or from about 0.3 milligrams/mL to about 150 milligrams/mL ascorbic acid or any 0.1 milligram/mL increment in between.
  • the molecular weight of tigecycline is 586.
  • the molecular weight of sodium pyruvate is 292, and the molecular weight of 2-hydroxylpropyl-beta-cyclodextrin is 154.
  • a person skilled in the art could determine the molar ratios of tigecycline to each of these components to for example identify the molar ratios of the ranges provided and to determine the amount of a related compound that could be used.
  • the molecular weight of sodium pyruvate to tigecycline for the ranges provided could be computed and used to determine the amount of a different pyruvate, such as a pyruvate ester that should be included in a composition.
  • the chelator is selected from EDTA, EGTA, citrates, and tartrates.
  • the chelator is penicillamine or dimercapto-propane sulfonate.
  • the salt or ester of pyruvic acid is a pyruvate, such as sodium pyruvate, calcium pyruvate, potassium pyruvate, magnesium pyruvate, or dihydroxyacetone pyruvate.
  • the cyclodextrin is selected from 2- hydroxylpropyl-beta-cyclodextrin (2-HP-beta-CD), alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin.
  • the chelator is not EDTA.
  • the one or more of the compositions for example the composition comprising a glycylcycline compound and/or the composition comprising a TKI and/or the composition comprising a glycylcycline compound and a TKI is a pharmaceutical composition.
  • the compounds are suitably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
  • compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, such that an effective quantity of the active substance or substances is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Liquid formulations can be prepared using methods known in the art. For example, liquid formulations can be prepared by resuspending and/or diluting each component of the composition in a suitable diluent and combining the resuspended and/or diluted components to provide the desired concentrations. Alternatively, the components can be blended together to provide a solid formulation comprising each of desired components of the composition and subsequently diluted with a suitable diluent such as water or saline.
  • a suitable diluent such as water or saline.
  • a typical process for preparing a powder composition comprising a glycylcycline compound such as tigecycline and/or a TKI can involve dissolving the glycylcycline compound such as tigecycline and/or a TKI and optionally and one or more of a chelator, pyruvic acid, or a salt or ester thereof and ascorbic acid; and optionally one or more excipients, diluents or buffers and/or cyclodextrin in water and lyophilizing (freeze- drying) the solution to dryness to form solid cakes of amorphous glycylcycline compound and/or TKI containing compositions.
  • the cakes Prior to being administered to patients, the cakes are reconstituted, often in 0.9% saline, to provide a desired concentration for example a tigecycline concentration of, for example, about 10 milligrams/mL, optionally 5 milligrams, optionally 1 milligram/mL. Lyophilizing may be accomplished by any pharmaceutically acceptable means.
  • compositions include, albeit not exclusively, solutions of the substances in association with one or more than one pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • the diluent is water, saline or other suitable diluents for intravenous administration.
  • Non-limiting examples of suitable diluents include saline, such as 0.9% saline, water, 5% dextrose, Hartmann's solution, Ringer's solution and Ringer's lactate solution.
  • saline such as 0.9% saline, water, 5% dextrose, Hartmann's solution, Ringer's solution and Ringer's lactate solution.
  • Other diluents suitable for intravenous administration known in the art can also be used including combinations of the foregoing.
  • the composition may include a pH modifying agent, for example to maintain the pH of the composition in a solution between about 3 and about 8, about 3 to about 7, about 4 to about 7 or about 7.
  • the pH modifying agent can include any pharmaceutically acceptable acid or buffer capable of adjusting the pH of a composition of the application to between about 3 to about 8.
  • agents that can be used include but are not limited to citric acid, acetic acid, lactic acid, hydrogenophosphoric acid, diethylamine, hydrochloric acid, including 1.0 N HCI, gentisic acid, lactic acid, and phosphoric acid.
  • compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which optionally further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient.
  • Other components that are optionally present in such compositions include, for example, water, surfactants (such as TweenTM), alcohols, polyols, glycerin and vegetable oils.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.
  • the composition can be supplied, for example, but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the subject.
  • Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition.
  • suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1 (2,3- dioleyloxy)propyl)N,N, N-trimethylammonium chloride (DOTMA), diolesyl-phosphotidyl- ethanolamine (DOPE), and liposomes.
  • DOTMA diolesyl-phosphotidyl- ethanolamine
  • liposomes Such compositions should contain a therapeutically effective amount of the compound(s), together with a suitable amount of carrier so as to provide the form for direct administration to the subject.
  • composition comprising a glycylcycline compound and a TKI and optionally one or more suitable excipients, diluents, buffers, carriers or vehicles.
  • the composition can be used in any of the methods described herein and comprise one or more suitable excipients, diluents, buffers, carriers or vehicles described herein.
  • the glycylcycline compound and/or a composition comprising the glycylcycline compound, and the TKI and/or a composition comprising the TKI are contacted, administered or administrable at different times, e.g., sequentially, or contemporaneously. Sequential administration is performed so that the two drugs are present in active amounts for at least a period that overlaps within the subject receiving treatment.
  • the composition contacted, administered or administrable comprises a glycylcycline and a TKI.
  • the glycylcycline for example tigecycline, the TKI and/or a composition comprising the glycylcycline compound and/or TKI, is comprised in a dosage or dosage form for example a dosage form described herein.
  • the glycylcycline and the TKI can for example be administered to the cell or subject and/or is for administration contemporaneously or at different times, and/or as part of a therapeutic regimen.
  • the glycylcycline and TKI can optionally be in a single composition and/or be separate compositions each optionally comprising one or more excipients, diluents, buffers, carriers and vehicles.
  • the compounds and compositions described herein are administered, for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration.
  • the compound or composition is administered by intravenous infusion.
  • the compound or composition is administered by direct intratumoral injection.
  • the compound or composition is administered by injection into tumour vasculature.
  • the dosage form may be, for example, incorporated with excipient and used in the form of enteric coated tablets, caplets, gelcaps, capsules, ingestible tablets, buccal tablets, troches, elixirs, suspensions, syrups, wafers, and the like.
  • the oral dosage form may be solid or liquid.
  • Timed-release compositions can be formulated, e.g. liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc.
  • differentially degradable coatings such as by microencapsulation, multiple coatings, etc.
  • freeze-dry the compounds described herein and use the lyophilizates obtained, for example, for the preparation of products for injection.
  • the disclosure describes a pharmaceutical composition wherein the dosage form is a solid dosage form.
  • a solid dosage form refers to individually coated tablets, capsules, granules or other non-liquid dosage forms suitable for oral administration. It is to be understood that the solid dosage form includes, but is not limited to, modified release, for example immediate release and timed-release, formulations.
  • modified-release formulations include, for example, sustained-release (SR), extended-release (ER, XR, or XL), time-release or timed-release, controlled-release (CR), or continuous-release (CR or Contin), employed, for example, in the form of a coated tablet, an osmotic delivery device, a coated capsule, a microencapsulated microsphere, an agglomerated particle, e.g. , as of molecular sieving type particles, or, a fine hollow permeable fiber bundle, or chopped hollow permeable fibers, agglomerated or held in a fibrous packet.
  • Timed-release compositions can be formulated, e.g.
  • liposomes or those wherein the active compound is protected with differentially degradable coatings such as by microencapsulation, multiple coatings, etc. It is also possible to freeze-dry the compounds described herein and use the lyophilizates obtained, for example, for the preparation of products for injection.
  • the disclosure describes a pharmaceutical composition wherein the dosage form is a liquid dosage form.
  • the dosage form is a liquid dosage form.
  • a person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20 th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • the disclosure describes a pharmaceutical composition wherein the dosage form is an injectable dosage form.
  • An injectable dosage form is to be understood to refer to liquid dosage forms suitable for, but not limited to, intravenous, subcutaneous, intramuscular, or intraperitoneal administration.
  • Solutions of compounds described herein can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • a surfactant such as hydroxypropylcellulose.
  • a sodium chloride solution for example a 0.9% sodium chloride solution or a dextrose solution for example a 5% dextrose solution.
  • the liquid dosage form or formulation including a liquid formulation prior to lyophilizing comprises one or more a chelator, pyruvic acid, or a salt or ester thereof and ascorbic acid; and optionally one or more excipients, diluents or buffers.
  • the liquid formulation further comprises a cyclodextrin, and/or optionally tigecycline.
  • the composition comprising one or more of a chelator, pyruvic acid, or a salt or ester thereof and ascorbic acid and optionally one or more excipients, diluents or buffers and/or cyclodextrin, can be provided as a liquid which is used as is to reconstitute tigecycline and/or a composition comprising a TKI.
  • the composition can further comprise tigecycline and be provided as a liquid formulation, for example for administration.
  • the composition is a powder comprising one or more a chelator, pyruvic acid, or a salt or ester thereof and ascorbic acid; and optionally one or more excipients, diluents or buffers, a cyclodextrin and/or tigecycline and/or a TKI.
  • the powder is a lyophilized formulation for reconstitution.
  • the composition can comprise a glycylcycline compound such as tigecycline and/or a TKI and one or more of a chelator, pyruvic acid, or a salt or ester thereof and ascorbic acid and optionally one or more excipients, diluents or buffers and/or cyclodextrin, wherein the composition is a powder, for example to be reconstituted with water and/or saline, or other suitable diluent.
  • a glycylcycline compound such as tigecycline and/or a TKI and one or more of a chelator, pyruvic acid, or a salt or ester thereof and ascorbic acid and optionally one or more excipients, diluents or buffers and/or cyclodextrin, wherein the composition is a powder, for example to be reconstituted with water and/or saline, or other suitable diluent.
  • the composition can comprise one or more of a chelator, pyruvic acid, or a salt or ester thereof and ascorbic acid, a glycylcycline compound and/or a TKI and optionally one or more excipients, diluents or buffers, wherein the composition is a powder, for example to be reconstituted with water or saline or other suitable diluent.
  • composition comprising the one or more of chelator, salt, ester, or anion of pyruvic acid and ascorbic acid and optionally one or more excipients, diluents or buffers, and/or cyclodextrin can be used to reconstitute a powder formulation of tigecycline.
  • compositions can be reconstituted with saline, water or other suitable diluents for intravenous administration.
  • the ranges and concentrations provided herein can refer to the ranges and concentrations of the corresponding reconstituted or liquid formulation.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20 th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.
  • the powder is reconstitutable or reconstituted in a composition comprising, consisting of, or consisting essentially of two or more a chelator, pyruvic acid or a salt or ester thereof, ascorbic acid and a cyclodextrin; and optionally one or more excipients, diluents or buffers.
  • the dosage and/or each unit dosage form comprises from about 25 mg to about 1000 mg, from about 50 mg to about 1000 mg, from about 100 mg to about 2000 mg, from about 100 mg to about 1500 mg, from about 100 mg to about 1000 mg, from about 100 mg to about 700 mg, from about 100 mg to about 500 mg, from about 100 mg to about 350 mg, from about 100 mg to about 300 mg or from about 100 mg to about 250 mg of a glycylcycline, for example of tigecycline.
  • the dosage and/or each unit dosage form comprises from about 50 mg to about 2000 mg, from about 50 mg to about 1500 mg, from about 50 mg to about 1000 mg, from about 50 mg to about 700 mg, from about 50 mg to about 500 mg, from about 50 mg to about 350 mg, from about 50 mg to about 300 mg or from about 50 mg to about 250 mg of a glycylcycline, for example of tigecycline.
  • the dosage and/or each unit dosage form comprises about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg or about 500 mg of a glycylcycline, for example of tigecycline.
  • the dosage or dosage form comprises sufficient glycylcycline compound, for example tigecycline, to produce a peak serum concentration (i.e. C max ) from about 0.5 micrograms/mL to about 100 micrograms/mL, from about 0.5 micrograms/mL to about 80 micrograms/mL, from about 0.5 micrograms/ml to about 60 micrograms/mL, from about 0.5 micrograms/mL to about 40 micrograms/mL, from about 0.5 micrograms/mL to about 20 micrograms/mL, or from about 0.5 micrograms/mL to about 10 micrograms/mL.
  • C max peak serum concentration
  • the dosage or dosage form comprises sufficient glycylcycline, for example tigecycline, to produce a peak serum concentration (i.e. C ma ) from about 1 micrograms/mL to about 100 micrograms/mL, from about 10 micrograms/mL to about 100 micrograms/mL, from about 25 micrograms/ml to about 100 micrograms/mL, from about 40 micrograms/mL to about 100 micrograms/mL, from about 60 micrograms/mL to about 100 micrograms/mL, or from about 80 micrograms/mL to about 100 micrograms/mL.
  • C ma peak serum concentration
  • mice that received a single dose of tigecycline (50 mg/kg) i.p.
  • the Cmax was found to be 27+/-4.5 pg/mL.
  • the Tmax was about 30 min and the half life was about 4.5 hours.
  • the half life in mice is significantly shorter than the half life in humans (27 hours).
  • the dosage or dosage form can alternatively comprise about 2 to about 100 mg of a glycylcycline/kg body weight, about 3 to about 100 mg of a glycylcycline/kg body weight, about 4 to about 100 mg of a glycylcycline/kg body weight, or about 5 to about 100 mg of a glycylcycline/kg body weight of a subject in need of such treatment formulated into a solid oral dosage form, a liquid oral dosage form, or an injectable dosage form.
  • the dosage or dosage form can comprise about 2 to about 90 mg of a glycylcycline/kg body weight, about 2 to about 80 mg of a glycylcycline/kg body weight, about 2 to about 70 mg of a glycylcycline/kg body weight, about 2 to about 60 mg of a glycylcycline/kg body weight, or about 2 to about 50 mg of a glycylcycline/kg body weight of a subject in need of such treatment formulated into a solid oral dosage form, a liquid oral dosage form, or an injectable dosage form.
  • the dosage or dosage form can comprise about 1 mg to about 40 mg of an glycylcycline/kg body weight, about 5 mg to about 40 mg of an glycylcycline/kg body weight, about 10 to about 40 mg of an glycylcycline/kg body weight, or about 20 to about 40 mg of an glycylcycline/kg body weight of a subject in need of such treatment formulated into a solid oral dosage form or a liquid oral dosage form, optionally an injectable dosage form.
  • the dosage or dosage form can comprise about 2 to about 10 mg of a glycylcycline/kg body weight of a subject in need of such treatment.
  • the dosage or dosage form can comprise any weight amount or range between 2 and 100 mg of a glycyclcycline/kg body weight of a subject in need of such treatment.
  • the unit doses are for example and in preferred embodiments above the level used for anti-microbial therapy.
  • the dosage or dosage form comprises tigecycline.
  • the dosage and/or each unit dosage form comprises from about 25 mg to about 1000 mg, from about 50 mg to about 1000 mg, from about 100 mg to about 2000 mg, from about 100 mg to about 1500 mg, from about 100 mg to about 1000 mg, from about 100 mg to about 700 mg, from about 100 mg to about 500 mg, from about 100 mg to about 350 mg, from about 100 mg to about 300 mg or from about 100 mg to about 250 mg of a TKI, for example erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, bosutinib, sorafenib (nexavar), cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), lestaurtinib, canertinib, zactima, vatalanib, leflunomide and ARQ197 and mixtures thereof.
  • TKI for example erlotinib (TarcevaTM),
  • the dosage and/or each unit dosage form comprises from about 50 mg to about 2000 mg, from about 50 mg to about 1500 mg, from about 50 mg to about 1000 mg, from about 50 mg to about 700 mg, from about 50 mg to about 500 mg, from about 50 mg to about 350 mg, from about 50 mg to about 300 mg or from about 50 mg to about 250 mg of a TKI, for example erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, bosutinib, sorafenib (nexavar), cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), lestaurtinib, canertinib, zactima, vatalanib, leflunomide and ARQ197 and mixtures thereof.
  • TKI for example erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (Spr
  • the dosage and/or each unit dosage form comprises about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg or about 500 mg of a TKI, for example oerlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, bosutinib, sorafenib (nexavar), cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), lestaurtinib, canertinib, zactima, vatalanib, leflunomide and ARQ197 and mixtures thereof.
  • TKI for example oerlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, bosutinib, sorafenib (nexa
  • the usual therapeutic dose of a TKI for example erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (SprycelTM), vandetanib, bosutinib, sorafenib (nexavar), cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), lestaurtinib, canertinib, zactima, vatalanib, leflunomide and ARQ197 and mixtures thereof, is contacted, administered or for administration.
  • the maximum tolerated dose (MTD) is contacted, administered and/or for administration.
  • the amount of glycylcycline and TKI administered and/or for administration is an effective amount of the combination.
  • the combination has greater efficacy than each compound alone.
  • the amount of glycylcycline compound such as tigecycline and/or TKI such as erlotinib (TarcevaTM), sunitinib (SutentTM), dasatinib (Sprycel ), vandetanib, bosutinib, sorafenib (nexavar), , cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), lestaurtinib, canertinib, zactima, vatalanib, leflunomide and ARQ197 and mixtures thereof, administered and/or for administration can be decreased compared to its individual use obtaining the same efficacy with potentially reduced side effects.
  • the amount of TKI administered is decreased by at least 4 fold, at least 3 fold, at least 2 fold or at least 1.5 fold when administered with a glycylcycline, for example when compared to without glycylcycline administration, or for example compared to usual therapeutic dose.
  • a glycylcycline such as tigecycline or a glycylcycline that has similar activity to tigecycline, to a dosing regimen can decrease the amount of TKI into clinically relevant ranges thereby increasing the repertoire of TKIs that can be used to treat cancer.
  • kits comprises instructions for carrying out a method described herein and/or comprises a compound or composition described herein.
  • the kit is used for and/or comprises instructions for use for treating a cancer, inducing cytotoxicity in a cancer cell, or inhibiting a mammalian mitochondrial ribosome in a cell.
  • the instructions for use can include for example instructions regarding how the compositions are to be reconstituted and/or administered, including for example instructions for further diluting, such as mixing in an IV bag.
  • the kit is for use in a method and/or comprises instructions for use in treating a cancer, such as a solid cancer or a hematological cancer.
  • the kit comprises a TKI and glycylcycline that in combination can produce synergistic cell toxicity in a cancer cell.
  • the kit comprises either a TKI or a glycylcycline compound and instructions for use in combination with a glycylcycline compound or TKI respectively
  • the instructions identify glycylcycline compounds or TKIs that can produce synergistic toxic effect.
  • the kit comprises a glycylcycline compound such as tigecycline.
  • the kit comprises a glycylcycline and a TKI and/or packaging materials such as product inserts and the like.
  • the kit comprises tigecycline and a TKI selected from erlotinib (TarcevaTM), sunitinib (Sutent TM ), dasatinib (SprycelTM), vandetanib, bosutinib, sorafenib (nexavar), cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), lestaurtinib, canertinib, zactima, vatalanib, leflunomide and ARQ197 and mixtures thereof and optionally instructions for use and/or packaging materials.
  • TKI selected from erlotinib (TarcevaTM), sunitinib (Sutent TM ), dasatinib (SprycelTM), vandetanib, bosutinib, sorafenib (nexavar), cedivanib, gefitinib (IressaTM), imatinib (GleevacTM), les
  • the TKI is a TKI that inhibits one or more of the kinases inhibited by erlotinib, sunitinib, dasatinib, vandetanib, gefitinib and/or bosutinib.
  • the TKI is erlotinib (TarcevaTM).
  • the TKI is sunitinib (SutentTM).
  • the TKI is dasatinib (SprycelTM).
  • the TKI is vandetanib.
  • the TKI is gefitinib.
  • the TKI is bosutinib.
  • the kit comprises a vial comprising a glycylcycline compound and a vial comprising a TKI.
  • the vials can be sterile vials.
  • the kit comprises a composition for reconstituting the glycylcycline such as tigecycline comprising one or more of a chelator, pyruvic acid, or a salt or ester thereof and ascorbic acid and optionally one or more excipients, diluents or buffers.
  • the composition for reconstituting further comprises a cyclodextrin.
  • Each component of the reconstitution composition can be provided separately, for example to be combined at time of tigecycline reconstitution. Alternatively, the components can be blended together in a single formulation.
  • the reconstitution composition can be a solid, for example a powder to be dissolved, with for example water, saline or other suitable diluent, and used to reconstitute the glycylcycline such as tigecycline and/or composition comprising the glycylcycline.
  • the dosage forms included in the kit can be dosage described herein and the amounts of the TKI and/or glycylcycline can be amounts described herein.
  • TEX and M9-ENL1 cells derived from lineage-depleted primitive human cord blood cells (Lin- CB) transduced with the oncogenes TLS-ERG or MLL-ENL, respectively, which display properties of leukemia initiating cells, including hierarchal differentiation and marrow repopulation, as shown previously by Dr. J Dick (4, 5) were used. From the screens, the anti-microbial agent tigecycline was identified as a top candidate. A lethal action was also demonstrated on 13 of 20 primary AML samples including the CD34+/CD38- subset of progenitor/stem cells with similar potency.
  • tigecycline reduced the clonogenic growth of primary AML samples by 93 ⁇ 4% and was effective in reducing the ability of AML cells to regenerate disease in transplanted immunodeficient mice. In contrast, tigecycline had no effect on the clonogenic growth or repopulating potential of normal human hematopoietic cells (3).
  • tigecycline Using Haplo-lnsufficiency Profiling, a functional chemical genomic screen in S. cerevisiae, inhibition of mitochondrial translation was identified as the most likely mechanism of tigecycline in eurkaryotes. It was subsequently demonstrated that tigecycline inhibited mitochondrial but not cytoplasmic translation in AML cell lines and in primary AML samples. It was also demonstrated that tigecycline directly inhibited mitochondrial translation using cell-free assays with mitochondria isolated from leukemia cells. Consistent with the inhibition of mitochondrial translation, tigecycline decreased the enzyme activity of Complex I and IV, which contain mitochondrially-translated subunits, but not complex II (nuclear- encoded subunits only).
  • Tigecycline also decreased oxygen consumption and decreased mitochondrial-membrane potential in AML cell lines and primary AML samples, but not normal hematopoietic cells. Interestingly, unlike many mitochondrial inhibitors, Tigecycline did not increase ROS production in AML cells (3).
  • Tigecycline is an anti-microbial agent of the glycylcycline class that is active against a range of gram-positive and gram-negative bacteria, particularly drug-resistant pathogens 13 and FDA-approved for the treatment of complicated gram positive and gram negative infections. Tigecycline was developed synthetically as an analogue to minocycline with the addition of a tert-butyl-glycylamido side chain to the tetracycline backbone 14 . This approach was used to decrease drug resistance effects mediated by efflux pumps and improve its affinity for the ribosome.
  • tigecycline has been shown to inhibit bacterial protein synthesis 3- and 20-fold greater than minocycline and tetracycline respectively 15 .
  • tigecycline reversibly binds to the 30S subunit of the bacterial ribosome, blocking the aminoacyl-tRNA form entering the A site 16 , thereby inhibiting elongation of the peptide chain and protein synthesis.
  • Tigecycline is routinely administered as 50 mg intravenously every 12 hours without significant toxicity, but higher doses have also been used safely.
  • intravenous doses of 300 mg are well tolerated save for mild nausea and produce a Cmax of 2.82 pg/mL (5 ⁇ ) 17 , a concentration within the range required for anti-leukemic effects.
  • Toxicology studies in animals have been conducted. Rats receiving > 30 mg/kg/day x 2 weeks developed reversible anemia, thrombocytopenia, and leucopenia with a hypocellular bone marrow 18 .
  • the dose of 30 mg/kg translates to 150 mg of drug in humans based on scaling for body surface area and weight, and is within 3 times the antimicrobial dose of drug.
  • Erlotinib is an EGFR inhibitor that is approved for the treatment of non-small cell lung cancer and pancreatic cancer.
  • a recent report has reported signals of clinical activity in AML including a complete remission (6).
  • these reports of remission have been sporadic, suggesting that identifying the appropriate patients who would most likely respond to this treatment and using this drug in combination would be important development strategies.
  • concentrations of erlotinib required for synergy in the experiments described herein appears clinically relevant as steady state plasma concentrations in the range of 2 to 5 ⁇ have been achieved in previous clinical studies (7, 8).
  • TEX human leukemia cells were maintained in IMDM, 15% FBS, 1 %, penicillin-streptomycin, 20 ng/mL SCF, 2 ng/mL IL-3 and 2 mM L-glutamine.
  • the other human leukemia cell lines were maintained in RPMI 1640 medium. All cells were incubated at 37 °C in a humidified air atmosphere supplemented with 5% C02.
  • AML human acute myeloid leukemia
  • PBSC peripheral blood mononuclear cells
  • the mononuclear cells were isolated from the samples by Ficoll density centrifugation. Primary cells were cultured at 37°C in IMDM supplemented with 20% FCS, 1 mM of L-glutamine and appropriate antibiotics.
  • Cell death was measured by Annexin V fluorescein isothiocyanate (FITC) and Propidium Iodide (PI) (Biovision Research Products, Mountain View, CA) staining using flow cytometry according to the manufacturer's instructions.
  • FITC Fluorescein isothiocyanate
  • PI Propidium Iodide
  • Cell growth and viability was measured with the Cell Titre Glo® (Promega, Madison, Wl) and Sulphorhodamine B (Sigma, St. Lois, MO), assays according to manufacturer's instructions.
  • Intracellular tigecycline was measured in TEX and RTEX+TIG cells by HPLC with UV detection (350 nm). Cellular proteins were precipitated by addition of 20 pL 100%- trichloroacetic acid containing 200 ⁇ g/mL minocycline as an internal standard. Then the aqueous phase was loaded on Symmetry C18 column (3.9*150 mm, 5 prm). Tigecycline and minocycline were separated by 25:75 (v/v) acetonitrile-phosphate buffer (0.023 M, pH 3.0) containing 4 mM 1 -octanesulfonic acid.
  • Total cell lysates were prepared from cells as described previously (3). Briefly, cells were washed twice with phosphate buffered saline pH 7.4 and suspended in lysis buffer (1.5% n-dodecyl ⁇ -maltoside (Sigma Aldrich, St. Louis, MO)) containing protease inhibitor tablets (complete tablets; Roche, IN). Protein concentrations were measured by the DC Protein assay (Bio Rad, Hercules, CA) Equal amounts of protein were subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gels followed by transfer to nitrocellulose membranes.
  • lysis buffer (1.5% n-dodecyl ⁇ -maltoside (Sigma Aldrich, St. Louis, MO)
  • protease inhibitor tablets complete tablets; Roche, IN. Protein concentrations were measured by the DC Protein assay (Bio Rad, Hercules, CA) Equal amounts of protein were subjected to sodium dodecyl sulphate (SDS)-polyacryl
  • Membranes were probed with anti-EGFR (Cell Technologies, Mountain View, CA), anti-a-tubulin 1 :2000 (Sigma Aldrich, St. Louis, MO), and secondary antibodies from GE Health (IgG peroxidase linked species-specific whole antibody). Detection was performed by the enhanced chemical luminescence method (Pierce, Rockford, IL).
  • TEX leukemia cells were treated with increasing concentrations of an in-house library of known on-patent and off-patent drugs with and without tigecycline at its IC20 (1.5 ⁇ ). From the screen, the IC50 was calculated for each drug in the library. Drugs whose IC50 were lowered greater than 4-fold by the addition of tigecycline were considered hits in the screen.
  • eriotinib has also recently been reported to inhibit drug efflux pumps (20). Therefore, it was investigated whether eriotinib may be increasing intracellular levels of tigecycline and through this mechanism producing synergy. To test this potential pharmacokinetic basis for the synergy, intracellular levels of tigecycline was measured in cells treated with increasing concentrations of tigecycline with or without co- treatment of eriotinib. The addition of eriotinib did not alter intracellular levels of tigecycline ( Figure 5). Therefore, the mechanism of resistance is not simply related to altering drug uptake or efflux.
  • ROS Reactive oxygen species
  • Erlotinib and tigecycline delay tumor growth in vivo
  • NADPH oxidases are a family of enzymes associated with the plasma membrane. These enzymes catalyze the conversion of NADPH to NADP by transferring electrons to molecular oxygen with the resultant formation of reactive oxygen species(21 ).
  • a previous study in head and neck cancer demonstrated that treatment of FaDu cells with eriotinib increased expression of NOX4 and ROS production (22). Therefore, the impact of eriotinib on ROS production and NOX expression in AML cells was tested.
  • TEX cells were treated with increasing concentrations of eriotinib for 48 hours followed by the measurement of intracellular ROS production by Carboxy-H 2 DCFDA staining and flow cytometry.
  • Eriotinib increased ROS production at concentrations associated with synergy with tigecycline ( Figure 8A).
  • treatment of TEX cells with tigecycline did not increase ROS production ( Figure 8B).
  • the combination of eriotinib and tigecycline was evaluated.
  • the combination of eriotinib and tigecycline increased ROS production greater than either drug alone ( Figure 8C).
  • the combination of drugs also increased mitochondrial ROS production greater than either drug alone (Figure 8D).
  • eriotinib increased NOX4 protein expression in TEX cells ( Figure 9).
  • Tex cells were treated with Eriotinib (3 ⁇ or 6 ⁇ ) for 48 hours, then analyzed by immunoblotting for Nox4 and Actin.
  • A549 lung cancer cells were next treated with increasing concentrations of eriotinib. After 48 hours, oxygen consumption was measured. As shown in Figure 6C, increasing concentrations of eriotinib produced and increase in oxygen consumption rate (OCR) in the cells.
  • OCR oxygen consumption rate
  • KMS1 1 myeloma cells were treated with eriotinib and tigecycline and synergy was determined by the combination index analysis where CI ⁇ 1 indicates synergy. As shown in Figure 6D, KMS1 1 myeloma cells showed synergy for the combination treatment.

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Abstract

L'invention concerne des méthodes, des trousses et des compositions pour le traitement d'un cancer, la methode comprenant l'administration à un sujet en ayant besoin d'une glycylcycline, par exemple tigécycline, et un TKI, la glycylcycline et le TKI étant fournis dans un rapport synergique.
PCT/CA2013/000742 2012-08-24 2013-08-23 Composition comprenant une glycylcycline et un inhibiteur de tyrosine kinase pour le traitement du cancer WO2014029016A1 (fr)

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CN109331026A (zh) * 2018-04-21 2019-02-15 温州医科大学附属第医院 替加环素在制备抗多发性骨髓瘤药物中的应用
WO2020056064A1 (fr) * 2018-09-11 2020-03-19 Stanley Satz Thérapie par radionucléides ciblée antitumorale et imagerie moléculaire de cancers her2 + et d'autres néoplasmes, et médicament de précision
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Cited By (4)

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Publication number Priority date Publication date Assignee Title
US20220096502A1 (en) * 2017-04-21 2022-03-31 Lunella Biotech, Inc. Targeting hypoxic cancer stem cells (cscs) with doxycycline: implications for improving anti-angiogenic therapy
CN109331026A (zh) * 2018-04-21 2019-02-15 温州医科大学附属第医院 替加环素在制备抗多发性骨髓瘤药物中的应用
WO2020056064A1 (fr) * 2018-09-11 2020-03-19 Stanley Satz Thérapie par radionucléides ciblée antitumorale et imagerie moléculaire de cancers her2 + et d'autres néoplasmes, et médicament de précision
US11357874B2 (en) 2018-09-11 2022-06-14 Stanley Satz Tumor targeted radionuclide therapy and molecular imaging of HER2+ cancers and other neoplasms

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