WO2015181628A1

WO2015181628A1 - Treatment of acute myeloid leukemia with an hck inhibitor

Info

Publication number: WO2015181628A1
Application number: PCT/IB2015/001192
Authority: WO
Inventors: Yusuke Narita; Masahiro Matsuki; Kimiyo Tabata
Original assignee: Eisai R&D Management Co., Ltd.
Priority date: 2014-05-27
Filing date: 2015-05-26
Publication date: 2015-12-03

Abstract

Treatment of acute myeloid leukemia (AML) patients with conventional chemotherapy and radiation results in complete remission but it is frequently followed by a recurrence of the disease that is resistant to chemotherapy. To target the leukemic stem cell population responsible for this relapse, a small molecule compound is described that inhibits HCK tyrosine kinase activity and eliminates the emergence of a highly aggressive AML leukemic stem cell proliferation that is believed to contribute to the relapse of AML in patients that have received chemotherapy treatment.

Description

TREATMENT OF ACUTE MYELOID LEUKEMIA WITH AN HCK INHIBITOR

FIELD OF THE INVENTION

[001] The present invention relates to the treatment of acute myeloid leukemia (AML) with an HCK kinase inhibitor. BACKGROUND

[002] According to the National Cancer Institute, approximately 60% to 70% of adults with acute myeloid leukemia (AML) can be expected to attain complete remission status following appropriate induction therapy. About 45 % of those who attain complete remission can be expected to survive 3 or more years and may be cured. Data however suggests that once attained, duration of remission may be shorter in older patients. Thus, in a significant number of patients, AML will ultimately relapse in a form that is resistant to chemotherapy treatment.

[003] In recent years, research has focused on the survival of acute myeloid leukemic stem cells as the principal reason why AML is able to elude chemotherapy treatment.

[004] In 1997, using transplantation experiments in SCID mice, Bonnet and Dick first demonstrated that AML cell populations comprise a small cell population of leukemic stem cells or LSCs that exhibit enhanced self renewal and tumor initiating potential (Bonnet D, Dick JE. 1997. Human AML is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 3:730-37). Subsequent cellular radiolabeling with tritiated thymidine in human AML patients confirmed the inherent heterogeneity of leukemic cell populations and that the vast majority of leukemic blasts are post-mitotic and continuously replenished from a small fraction of leukemic stem cells (~5 %) that cycled rapidly (reviewed by Dick, J. Nature Biotechnology 27, 44 - 46 (2009); Dalerba et al. Cancer stem cells: models and concepts. Ann. Rev. Med. 2007; 58:267-84).

[005] AML cancer stem cell survival is believed to proceed through a Darwinian evolutionary process in which cells are able to survive by adapting and ultimately overcoming the selective pressures imposed by radiotherapy and chemotherapy (Gerlinger and Swanton, 2010 How Darwinian models inform therapeutic failure initiated by clonal heterogeneity in cancer medicine. Br. J. Cancer 103, 1139-1143). [006] A multi- kinase inhibitor, RK-20449, was recently shown to significantly reduce human leukemic stem cell and non-stem AML burden in non-obese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg^nu11 mice engrafted with highly aggressive therapy resistant AML (Saito et al. A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo. Sci Transl Med 2013: Vol. 5, Issue 181, p. 181). This study highlights the importance of discovering novel targeted small molecule drugs for the treatment of AML patients who relapse.

[007] Thus, there is an unmet and urgent need for therapies that target chemotherapy resistant AML. SUMMARY OF THE INVENTION

[008] The present invention addresses the need in the art for small molecule drugs that can inhibit the proliferation of chemotherapy resistant acute myeloid leukemia stem cells.

[009] In one embodiment, a method for treating a subject with acute myeloid leukemia (AML) is disclosed that comprises administering a therapeutically effective amount of a kinase inhibitor compound to a subject with acute myeloid leukemia, wherein the compound inhibits HCK tyrosine kinase activity in the subject's leukemic stem cells.

[010] In another embodiment, the compound also inhibits Syk tyrosine kinase activity or at least one of FLT3, MEK1, MEK2, MEKK1 and LCK in the subject's leukemic cells.

[011] In another embodiment, the compound inhibits the proliferation of acute myeloid leukemic stem cells.

[012] In another embodiment, the acute myeloid leukemic stem cells have enhanced aldehyde dehydrogenase activity.

[013] In yet another embodiment, the acute myeloid leukemic stem cells fail to undergo apoptosis in response to chemotherapy and/or radiation treatment.

[014] In another embodiment, the AML has relapsed after chemotherapy treatment.

[015] In one embodiment, the compound has the structure of:

or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[016] Figure 1 depicts kinase inhibition profiling of E6201 and reference compounds, AC220 (a FLT3 inhibitor), RK-20449 (a HCK inhibitor) and Dasatinib (a multi- BCR/Abl and Src family tyrosine kinase inhibitor).

[017] Figure 2 depicts the effect of E6201 on a leukemic stem cell population in AML FLT3-ITD cells.

DETAILED DESCRIPTION [018] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions disclosed herein; as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[019] Furthermore, the practice of the invention employs, unless otherwise indicated, conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements. I. DEFINITIONS

[020] A "subject" includes a mammal. The mammal can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or a pig. Preferably the subject is a human. When the subject is a human, the subject may be referred to herein as a "patient". The terms "treat," "treating" or "treatment" of a disease of a subject refers to any improvement in one or more clinical symptoms of the disease. [021] The term "cancer stem cell" is used in accordance with its plain ordinary meaning within cancer biology and cell biology and refers to cancer cells with characteristics associated with non-cancerous stem cells, including for example the ability of self-renewal and the ability to differentiate into all cell types that make up a cancer sample (e.g. cancer cells). In certain embodiments, cancer stem cells may be identified by the presence (or absence) of cell markers (e.g. CD133 (PROM1), CD44 (PGP1), CD24 (HSA), EpCAM (epithelial cell adhesion molecule, ESA (epithelial specific antigen)), THY1 (CD90), ATP- binding cassette B5 (ABCB5), Hoechst33342, CD34, and/or ALDH1 (aldehyde

dehydrogenase).

[022] "Leukemic stem cells" or LSCs refer to AML cancer stem cells derived from white blood cells. LSCs are functionally defined to possess the property to generate more acute myeloid leukemia stem cells (self renewal) and non-stem cell acute myeloid leukemia cells. Additionally, these cells are characterized by the expression of certain cell surface markers, which include but are not limited to CD34, CD123, and CD117. Examples of cell surface markers that may be used are disclosed in US20120070450, which is incorporated herein in its entirety.

[023] As used herein, "treating" or "treat" describes the management and care of a patient for the purpose of combating AML and includes the administration of a compound of the present invention, or a pharmaceutically acceptable salt, pro-drug, metabolite, polymorph or solvate thereof, that results to any indicia of success in the treatment or amelioration of the cancer, including any objective or subjective parameter such as abatement; remission;

diminishing of symptoms or making the pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation.

[024] As used herein, "preventing" or "prevent" describes reducing or eliminating the onset of the symptoms or complications of AML.

[025] As used herein, the term "compounds of the invention" is used interchangeably with "pharmaceutical agent" or "pharmaceutical compound" or "pharmaceutical composition."

[026] The term "agent" is used herein to denote a chemical compound, a small molecule, a mixture of chemical compounds and/or a biological macromolecule (such as a nucleic acid, an antibody, an antibody fragment, a protein or a peptide). Agents may be identified as having a particular activity by screening assays described herein below. The activity of such agents may render them suitable as an "anti-cancer agent" which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically to treat AML in a subject.

[027] As defined herein, the term "inhibition", "inhibit", "inhibiting" and the like in reference to a protein-inhibitor (e.g. antagonist) interaction means negatively affecting (e.g. decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein.

[028] As used herein, the term "administering" includes oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[029] "Therapy resistant" acute myeloid leukemia, refer to AML that has become resistant to one or more cancer therapies including, but not limited to, an anti-cancer agent, a chemotherapy (e.g. a chemotherapeutic), a radiotherapy, an immunotherapy, and/or combinations thereof.

II. COMPOUNDS OF THE INVENTION

[030] Compounds useful in the methods described herein inhibit the proliferation of acute myeloid leukemic stem cells (LSC). In one embodiment, the compounds of the invention are kinase inhibitors. In another embodiment, the compounds of the invention are inhibitors of one or more kinases in the Src tyrosine kinase family, which includes cSrc, FYN, Lyn, LCK, YES, Fgr, HCK and BLK. Src family tyrosine kinases have been implicated as intermediates in signaling pathways that regulate cellular functions such as proliferation, differentiation, survival, and migration in various cell lineages. Of these, Lyn, Fgr, and HCK are expressed in myeloid lineages and are involved in normal myelopoiesis.

[031] mRNA for HCK (hematopoietic cell kinase) is overrepresented in primary human acute myeloid leukemic stem cells relative to normal hematopoietic stem cells. Accordingly, in another embodiment, the compounds of the invention are inhibitors of HCK.

[032] Signal transducer and activator of transcription 5 (STAT5) is constitutively activated by BCR/ABL, the oncogenic tyrosine kinase responsible for chronic myelogenous leukemia. BCR/ABL SH3 and SH2 domains interact with HCK, leading to the stimulation of HCK catalytic activity. Accordingly, in another embodiment, the compounds of the invention are inhibitors of BCR/ABL.

[033] In one embodiment, the compound of the invention is compound (1):

[034] Compound (1), and methods of making and using compound (1) are disclosed in United States Patent Nos. 7,799,827 and 8,350,059, which are incorporated herein in their entirety. Compound (1) has been found to inhibit HCK (see Pxample 1 and Figure 1).

E6201 ((3S,4R,5Z,8S,9S,l lE)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,10- tetrahydro-lH-benzo[c][l]oxacyclotetradecine-l,7(8H)-dione) is compound (1).

[035] In certain embodiments, the compounds of the invention are selected from the compounds of Table I, Table II, and pharmaceutically acceptable salts thereof.

Table I

Table II

[036] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the compounds of the present invention wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxy naphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurring amine acids, e.g. , glycine, alanine, phenylalanine, arginine, etc.

[037] Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-l-carboxylic acid, 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The present invention also encompasses salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

[038] It should be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) as defined herein, of the same salt.

[039] The compounds of the present invention can also be prepared as prodrugs, for example, pharmaceutically acceptable prodrugs. The terms "pro-drug" and "prodrug" are used interchangeably herein and refer to any compound which releases an active parent drug in vivo. Since prodrugs are known to enhance numerous desirable qualities of

pharmaceuticals (e.g. , solubility, bioavailability, manufacturing, etc.), the compounds of the present invention can be delivered in prodrug form. Thus, the present invention is intended to cover prodrugs of the presently claimed compounds, methods of delivering the same and compositions containing the same. "Prodrugs" are intended to include any covalently bonded carriers that release an active parent drug of the present invention in vivo when such prodrug is administered to a subject. Prodrugs in the present invention are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include compounds of the present invention wherein a hydroxy, amino, sulfhydryl, carboxy or carbonyl group is bonded to any group that may be cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl, free carboxy or free carbonyl group, respectively.

[040] Examples of prodrugs include, but are not limited to, esters (e.g. , acetate, dialkylaminoacetates, formates, phosphates, sulfates and benzoate derivatives) and carbamates (e.g. , N,N-dimethylaminocarbonyl) of hydroxy functional groups, esters (e.g. , ethyl esters, morpholinoethanol esters) of carboxyl functional groups, N-acyl derivatives (e.g. , N-acetyl) N-Mannich bases, Schiff bases and enaminones of amino functional groups, oximes, acetals, ketals and enol esters of ketone and aldehyde functional groups in compounds of the invention, and the like, See Bundegaard, H., Design of Prodrugs, pl-92, Elesevier, New York-Oxford (1985).

III. DOSAGE AND FORMULATION

[041] An appropriate composition comprising the compound of the invention to be administered can be prepared in a physiologically acceptable vehicle or carrier and optional adjuvants and preservatives.

[042] Any route of administration is compatible with the present invention including, but not limited to, oral, peroral, internal, pulmonary, lingual, intravenous, intraarterial, transdermal and subcutaneous formulations. The compound of the invention may be administered to a patient as granules, tablets, syrups, pills, capsules, powders, aerosols, skin patches, parenterals, and oral liquids, include oil aqueous suspensions, solutions, and emulsions as well as in sustained release (long acting) formulations and devices. [043] The composition may include additional ingredient including, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; creams, ointments, lotions, oils, pastes, suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. (1980)).

[044] A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.

[045] In certain embodiments, compositions comprising the compounds of the invention can be administered separately. In a preferred embodiment, compositions comprise the compound of the invention at a designated dosage in a single compound tablet.

[046] A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients.

Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include potato starch and sodium starch glycollate. Known surface active agents include sodium lauryl sulfate. Known diluents include calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include corn starch and alginic acid. Known binding agents include gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include magnesium stearate, stearic acid, silica, and talc.

[047] Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in, e.g., U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.

[048] Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

[049] Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[050] Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.

[051] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl-methylcellulose. Known dispersing or wetting agents include naturally- occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include lecithin and acacia. Known preservatives include methyl, ethyl, or n-propyl- para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[052] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[053] Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations. [054] A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[055] Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

[056] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen- free water) prior to parenteral administration of the reconstituted composition. [057] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a nontoxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other parentally administrable formulations that are useful include those, which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[058] For intravenous administration, compounds disclosed herein may be formulated as a sterile solution of the active ingredient, either in its free or salt form, in physiological buffer or sterile water. Sugar-containing carrier liquids (such as Ringer's lactate, or other glucose or dextrose solutions) can be used if desired, provided that the total sugar content does not cause undesired levels of lactic acidosis. Intravenous administration can be either through bolus injection (preferably several times per day), or through continuous infusion over a sustained period of time. Total preferred dosages for bolus injection or infusion may vary substantially, depending on a patient's physical condition; in general, they will usually range from about 25 mg/kg to about 250 mg/kg.

[059] Other modes of administration of a pharmaceutical composition can be found in U.S. Patent No. 7,276,514, which is incorporated herein in its entirety.

IV. METHODS OF TREATING ACUTE MYELOID LEUKEMIA

[060] Disclosed herein are novel therapeutic methods of treating acute myeloid leukemia that include administering to a subject, a therapeutically effective amount of a compound that inhibits the tyrosine kinase activity of HCK (an HCK kinase inhibitor). A subject in need thereof may include, for example, a subject who has been diagnosed with AML, or a subject who had been previously treated for AML, including subjects who have been treated with chemotherapy. In some embodiments, the instant invention relates to methods of preventing a relapse of AML in a subject who had previously been treated with chemotherapy.

[061] The term "therapeutically effective amount", as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent AML, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.

[062] For any compound, the "therapeutically effective amount" can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[063] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and/or administer doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[064] The term "prophylactic ally effective amount" refers to an amount of an active compound of the invention that inhibits or delays the onset of AML in a subject.

[065] The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[066] Generally, the dose should be sufficient to result in slowing, and preferably regressing, the growth of the AML and also preferably causing complete regression of the cancer. Dosages can range from about 0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects, dosages can range from about 1 mg/kg per day to about 1000 mg/kg per day. In an aspect, the dose will be in the range of about 0.1 mg/day to about 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day to about 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about 1 g/day, in single, divided, or continuous doses (which dose may be adjusted for the patient's weight in kg, body surface area in m², and age in years). An effective amount of a pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. For example, regression of AML in a patient may be measured with reference to the number of leukemic cells in the blood circulation or the morphology of cells found in a bone aspirate or a decrease in the number of leukemic stem cells.

[067] Treating AML with the compound of the invention can result in a decrease in number of metastatic lesions in other tissues or organs. Preferably, after treatment, the number of metastatic lesions is reduced by 5 % or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. The number of metastatic lesions may be measured by any reproducible means of measurement. The number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified

magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.

[068] Treating AML with the compound of the invention can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[069] Treating AML with the compound of the invention can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof.

Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[070] Treating AML with the compound of the invention can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating AML can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof. Preferably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.

[071] Treating or preventing AML can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement. An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope. An abnormal cellular morphology can take the form of nuclear pleiomorphism.

[072] Treating AML can result in cell death, and preferably, cell death results in a decrease of at least 10% in number of cells in a population. More preferably, cell death means a decrease of at least 20%; more preferably, a decrease of at least 30%; more preferably, a decrease of at least 40%; more preferably, a decrease of at least 50%; most preferably, a decrease of at least 75%. Number of cells in a population may be measured by any reproducible means. A number of cells in a population can be measured by fluorescence activated cell sorting (FACS), immunofluorescence microscopy and light microscopy.

Methods of measuring cell death are as shown in Li et al., Proc Natl Acad Sci U S A. 100(5): 2674-8, 2003. In an aspect, cell death occurs by apoptosis.

[073] In certain embodiments, the efficacy of treatment of AML in a patient is determined by measuring the ability of the treatment regimen to reduce and/or eliminate the number of acute myeloid leukemic stem cells (LSCs) in the patient's blood or bone marrow.

[074] Acute myeloid leukemic stem cells can be identified by FACS analysis using cell- specific surface markers. In one embodiment, LSCs are identified as a CD34⁺ fraction. In other embodiments, the LSC fraction is CD34⁺ CD38^". Additional methods to isolate and characterize LSCs can be found, for example, in US20120070450, which is incorporated herein in its entirety.

[075] In other embodiments, the acute myeloid leukemic stem cell fraction is identified on the basis of their aldehyde dehydrogenase (ALDH) activity using an Aldefluor assay (see Examples) or Hoechst 33342 staining of the side population (SP) cell fraction. The isolation and characterization of leukemic stem cells is described, for example in US 8,153,388, which is incorporated herein in its entirety.

[076] Any patent, patent application, publication, or other disclosure material identified in the specification is hereby incorporated by reference herein in its entirety. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material.

EXAMPLES

[0103] Examples have been set forth below for the purpose of illustration and to describe certain specific embodiments of the invention. However, the scope of the claims is not to be in any way limited by the examples set forth herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art.

EXAMPLE 1 : INHIBITION OF TYROSINE KINASE ACTIVITY

Kinase assay

[077] Off-chip mobility shift Assay of HCK was performed. The 5 mL of 4x compound solution, 5 mL of 4x Substrate/ ATP/Metal solution, and 10 mL of 2x kinase solution were prepared with assay buffer (20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, pH7.5) and mixed and incubated in a well of polypropylene 384 well microplate for 1 hour at room temperature.

[078] The kinase reaction was evaluated by the product ratio calculated from peak heights of product(P) and substrate(S) peptides (P/(P+S)) (see Figure 1).

HCK kinase assay [079] As the result of kinase assay, compound (1) was found to inhibit HCK activity in a dose dependent manner. The IC50 value was 58nM. This activity is almost same as other kinase inhibition activity. These results suggest that E6201 have the activity of HCK inhibition. EXAMPLE 2: TREATMENT OF LEUKEMIC CELL LINE WITH THE HCK

KINASE INHIBITOR, E6201

Cell lines and cell cultures

[080] The human acute myeloid leukemia cell lines MV4-11 was obtained from the American Type Culture in 2007. This cell line was cultured in IMDM containing 10% FBS.

Aldefluor assay

[081] The assay kit (ST-01700) was purchased from STEM CELL TECHNOLOGIES. The concentration of the cells was adjusted to 1 x 10⁶ cells/mL using the ALDEFLUOR™ assay buffer. 5 of ALDEFLUOR™ DEAB reagent was then added to 1.0 mL of the adjusted cell suspension. DEAB was added to a half of the sample. After a 45min incubation at 37°C, all tubes were centrifuged for 10 minutes at 250 x g and the supernatant was removed. Cell pellets were then resuspended in 0.5 mL of ALDEFLUOR™ assay buffer and the cells were stored at 4°C. Data acquisition was performed by flow cytometer. Gate was created at the place of ALDH bright population. Results

[082] Recent data have shown that the AML CD34⁺CD38^" fraction with high levels of aldehyde dehydrogenase activity population ALDH^br is highly enriched for leukemic stem cells (LSC).

[083] The percentage of ALDH^br was evaluated in MV4-11 using ALDFLUOR assay system to test the anti-LSC activity of E6201. As shown in Figure 2, E6201 reduced the number of cells in the ALDH^br cell fraction in a dose dependent manner. The ALDH^br population decreased from 1.1% to 0.4% in the presence of 30nM E6201 and to 0% in the presence of ΙΟΟηΜ E6201. These data indicate that E6201 inhibits the proliferation of the leukemic stem cell population.

Claims

CLAIMS What is claimed is:

1. A method for treating a subject with acute myeloid leukemia, the method comprising:

administering a therapeutically effective amount of a kinase inhibitor compound to a subject with acute myeloid leukemia,

wherein said compound inhibits HCK tyrosine kinase activity in the subject' s leukemic stem cells.

2. The method of claim 1, wherein said compound also inhibits Syk tyrosine kinase activity in the subject's leukemic stem cells.

3. The method of claim 1 , wherein said compound inhibits the proliferation of leukemic stem cells.

4. The method of claim 3, wherein said leukemic stem cells have enhanced aldehyde dehydrogenase activity.

5. The method of claim 1, wherein said leukemic stem cells fail to undergo apoptosis in response to chemotherapy and/or radiation treatment.

6. The method of claim 1 , wherein the leukemia has relapsed after chemotherapy

treatment.

7. The method of claim 1, wherein said compound further inhibits the tyrosine kinase activity of at least one of FLT3, MEK1, MEK2, MEKK1 and LCK in the subject's leukemic stem cells.

8 The method of claim 1 , wherein said compound has the structure of:

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