WO2011031308A1 - Novel combinations - Google Patents

Abstract

Provided are pharmaceutical combinations that may be useful as cancer treatments. Also provided are methods relating to a cancer treatment that includes administering a CENP-E inhibitor and at least one additional compound to a human suffering from cancer.

Description

NOVEL COMBINATIONS

[0001] This application claim priority to and the benefit of U.S. Provisional Patent Applications No. 61/240,764, filed on September 9, 2009; 61/258,355, filed on November 5, 2009; and 61/260,927, filed on November 13, 2009, all of which are incorporated herein by reference.

[0002] Provided are methods of treating cancer in a subject and combinations of pharmaceutical compounds useful in such treatment. In particular, the method relates to novel combinations each comprising a CENP-E inhibitor and at least one additional compound, pharmaceutical compositions comprising the same, and methods of using such combinations and compositions in the treatment of cancer.

[0003] Centromere-associated protein E (CENP-E) is a plus end-directed microtubule motor which belongs to the kinesin-7 super family. CENP-E plays an essential role in correct positioning and stabilization of attached chromosome to spindle microtubules at the metaphase plate. Dysfunction of CENP-E in cultured human tumor cells results in a failure to pass the mitotic checkpoint resulting in cell cycle, arrest at metaphase with bipolar mitotic spindles and misaligned chromosomes and eventual cell death. CENP-E mRNA is overexpressed in various human tumors compared to normal tissues.

[0004] Combination therapy is rapidly becoming the norm in cancer treatment, rather than the exception. Oncologists are continuously looking for antineoplastic compounds which when utilized in combination provides a more effective and/or enhanced treatment to the individual suffering the effects of cancer. Typically, a successful combination therapy provides improved and even synergistic effect over monotherapy.

[0005] siPvNAs are small double-stranded pieces of RNAs (generally over 19 bp), one strand of which matches a complementary sequence in the gene to be knocked down. Upon transfection, it causes the degradation of protein-coding messenger RNA (mRNA) transcripts, thus silencing the expression of the target gene. This allows the research scientist to assess the function of the gene. siRNA expression constructs are now available as libraries that contain reagents targeting any number of genes, which means that a number of genes can be screened simultaneously for their involvement in a chosen biological phenotype. One usage of siRNA library that is gaining popularity is synthetic lethal screen, designed to identify gene targets knocking down of which can specifically reduce cell viability in the presence of otherwise sublethal concentrations of the compound of choice, or increase sensitivity of the cells to the compound (Nature, 446(7137):815-9, April 12, 2007). The results from this type of studies can help researchers understand the molecular mechanism of cancer chemoresponsiveness and identify gene inhibitors from the screen that can potentially be useful as combination therapy with the compound of choice in treating cancer.

[0006] Provided are novel combinations for the treatment of cancer and novel cancer treatment methods which include administration of 3-chloro-N-{ (lS)-2-[(N,N- dimethylglycyl)amino]- 1 -[(4- { 8-[( 1 S)- 1 -hydroxyethyl]imidazo[ 1 ,2-a]pyridin-2- yl }phenyl)methyl]ethyl }-4-[(l-methylethyl)oxy]benzamide (hereinafter "Compound A"), and/or a pharmaceutically acceptable salt thereof, in combination with at least one additional compound, and/or a pharmaceutically acceptable salt thereof.

Compound A

[0001] Compound A is a potent inhibitor of the mitotic kinesin centromere- associated protein E (CENP-E) for the treatment of cancer. It can be prepared according to the procedure described in U.S. Patent No. 7,504,413, the entire disclosure of which is hereby incorporated by reference for all purposes.

Description of Figures

[0002] Figure 1 is a table that lists genes validated as showing sensitization in an A427 cell line treated with Compound A, along with each gene's respective biological functionality.

[0003] Figure 2 is a table that lists the number of siRNA that showed sensitization for each validated gene in A427, A549 and NCI-H460 cell lines treated with Compound A. [0004] Figure 3 is a graph depicting cell death in a SW48 colon cancer cell line after 24 hour incubation with varying concentrations of Compound A, Compound C and combinations of Compound A and Compound C.

[0005] Figure 4 is a graph depicting cell death in a RKO colon cancer cell line after 24 hour incubation with varying concentrations of Compound A, Compound C and combinations of Compound A and Compound C.

[0006] Provided is a novel combination for the treatment of cancer in a subject which comprises Compound A, and/or a pharmaceutically acceptable salt thereof, in combination with at least one additional compound (hereinafter "Compound B"), and/or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments, Compound B, and/or a pharmaceutically acceptable salt thereof, comprises a sensitizer to CENP-E inhibition. "Sensitizer to CENP-E inhibition" refers to a compound, or pharmaceutically active salt thereof, that renders one or more types of cancer more sensitive to chemotherapy with Compound A and/or a pharmaceutically acceptable salt thereof, works in synergy with Compound A and/or a pharmaceutically acceptable salt thereof, to provide an improved synergistic effect, and/or acts additively to Compound A and/or a pharmaceutically acceptable salt thereof.

[0008] In some embodiments, Compound B and/or a pharmaceutically acceptable salt thereof is an inhibitor of at least one gene selected from:

ANAPC1 , CCL11, HNMT ID1, MAP2K6, PRKRA, ROCK1, SSR3, ANAPC5, APRIN, CYP4F8, FLOT1, GADD45A, GARS, GCSH, GPR155, INPP4B, KIF22, MAP2K5, MAT2B, P2RY1 1, PGAM2, PGM2L1, PIGA, PIGB, PIGT, PTPLA, RABGGTB, RaLP, RGL2, SCAP1, SHMT2, SKIP, SLC2A12, SLC6A8, SLC9A2, . SNRK, TAS2R5, TLK2, TSC1, UBE2C, UGT1A3, UGT2B10, ACTL6A, AK2, AKT1 , ARHGAP26, ASK, BRAF, C14orfl30, C7orf9, CAMK2D, CDC6, CDKN2C, CINP, CLSPN, CNKSR1, CS, CYLD, CYP24A1, CYP2A13, DMPK, DYRK3, EMR2, FALZ, FBN2, FLJ2331 1 , GALK1, GNPDA1 , HP, HSD17B3, IBSP,

KCNJ13, KIF2C, LATS2, LIPE, MAP2K1IP1, MAPK3, MCP, MGC14156, MTCH1, OXCT1, OXCT2, PCK2, PDHB, PGLS, PGM3, PIGC, PIGH, PIGS, PNLIPRP2, PPP1CA, PPP1R12B, PRKCA, PRLR, PTPRN2, RPTOR, RIPK2, RORA,

RPS6KA1, SBDS, SERPINIl, SFXN3, SLC2A1, SLC2A1 1, SSR1, TBXAS1, TTBK1, ULK1 , VASP and SLC2A8. [0009] In some embodiments, Compound B and/or a pharmaceutically acceptable salt thereof is an inhibitor of at least one gene selected from: MAPK1/MAPK3, ROCK1 , WEEl, PRKCA, SHH, MAP2K2, DNMT1, BRAF, CCL1 1 , HNMT, SLC6A8, PDK1 and P2RY1 1.

[0010] In some embodiments, Compound B and/or a pharmaceutically acceptable salt thereof is an inhibitor of the PDK/AKT/mTOR pathway, e.g., Compound B and/or a pharmaceutically acceptable salt thereof is an inhibitor of one or more of PI3K, AKT and mTOR. Non-limiting examples of inhibitors of the PI3K/AKT/mTOR pathway include: sirolimus (rapamycin), temsirolimus, everolimus, ridaforolimus, SF1 126, PX-866, GDC- 0941 , NVP-BEZ235, XL147, XL765, D-87503, D106669, GSK615, and CAL101.

[0011] In some embodiments, Compound B and/or a pharmaceutically acceptable salt thereof is an inhibitor of the Raf/MEK/ERK or MAPK/ERK pathway, e.g., Compound B and/or a pharmaceutically acceptable salt thereof is an inhibitor of one or more of Raf, MEK, ERK and MAPK. Non-limiting examples of inhibitors of the Raf/MEK/ERK or

MAPK/ERK pathway include: ARRY-438162, AS703026, AZD6244 (selumetinib), AZD8330 , CI-1040, FR180204, GDC-0973, GSK1 120212 (Compound C), PD0325901 , PD169316, PD98059, RDEA1 19, R04987655, SB202190, SB203580, SL327, TAK-733, and U0126. In some embodiments, Compound B and/or a pharmaceutically acceptable salt thereof comprises Compound C and/or a pharmaceutically acceptable salt thereof.

[0012] Also provided is a method of treating cancer in a subject (e.g., a human) in need thereof, comprising administering to the subject a therapeutically effective amount of any of the above combinations, i.e., Compound A, and/or a pharmaceutically acceptable salt thereof, and Compound B, and/or a pharmaceutically acceptable salt thereof, wherein Compound B and/or a pharmaceutically acceptable salt thereof may be a sensitizer to CENP- E inhibition. Cancers amenable to such treatment include, for example, primary and metastatic forms of head and neck cancer, breast cancer, lung cancer, colon cancer, ovary cancer, prostate cancer and neuroblastoma.

[0013] In some embodiments, the subject's cancer cells bear one or more specific genetic mutations. For example, in some embodiments, the cancer cells have mutations in one or more genes selected from KRAS, PIK3CA, BRAF and PTEN. In some embodiments, the cancer is colon or pancreatic cancer and the cancer cells have mutations in one or more genes selected from KRAS, PIK3CA and BRAF. In some embodiments, the cancer is breast or lung cancer and the cancer cells have mutations in one or more genes selected from PIK3CA and PTEN. In some embodiments, the cancer may be wild type for any or all of the genes selected from KRAS, PIK3CA, BRAF and PTEN.

[0014] Also provided is a method comprising determining whether a subject's cancer cells contain one or more specific genetic mutations, then administering a

therapeutically effective amount of any of the above combinations, i.e., Compound A, and/or a pharmaceutically acceptable salt thereof, and Compound B and/or a pharmaceutically acceptable salt thereof, wherein Compound B and/or a pharmaceutically acceptable salt thereof may be a sensitizer to CENP-E inhibition. In some embodiments, the specific genetic mutations may occur in one or more of the genes selected from KRAS, PIK3CA, BRAF and PTEN.

[0015] Also provided is a pharmaceutical composition comprising any of the above combinations with a pharmaceutically acceptable carrier.

[0016] As used herein the term "neoplasm" refers to an abnormal growth of cells or tissue and is understood to include benign, i.e., non-cancerous growths, and malignant, i.e., cancerous growths. The term "neoplastic" means of or related to a neoplasm.

[0017] As used herein the term "agent" is understood to mean a substance that produces a desired effect in a tissue, system, animal, mammal, human, or other subject. Accordingly, the term "anti-neoplastic agent" is understood to mean a substance producing an anti-neoplastic effect in a tissue, system, animal, mammal, human, or other subject. It is also to be understood that an "agent" may be a single compound or a combination or composition of two or more compounds.

[0018] As used herein, the term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

[0019] As used herein, the term "inhibitor of a gene" means that a compound inhibits at least one function of the protein product encoded by the gene. [0020] The present compounds may have the ability to crystallize in more than one form or "polymorph", and it is understood that all such polymorphs are within the scope of Compound A, Compound B, and Compound C. Polymorphism generally can occur as a response to changes in temperature or pressure or both and can also result from variations in the crystallization process. Polymorphs can be distinguished by various physical characteristics known in the art such as x-ray diffraction patterns, solubility, and melting point.

[0021] Compound A and/or Compound B and/or Compound C includes solvates of Compound A and/or Compound B and/or Compound C, respectively. As used herein, the term "solvate" refers to a complex of variable stoichiometry formed by a solute (e.g.,, Compound A and/or Compound B and/or Compound C and/or a salt of Compound A and/or Compound B and/or Compound C) and a solvent. Such solvents generally do not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol and acetic acid. In someembodiments, the solvent is water (i.e., Compound A and/or Compound B and/or Compound C is a hydrate). Hydrates include complexes containing any ratio of compound to water, e.g., 1 : 1 (monohydrate), 1 :2 (dihydrate), 2: 1 (hemi-hydrate) and the like.

[0022] Compound A and/or Compound B may exist as a pharmaceutically acceptable salt. "Pharmaceutically acceptable salts" include, but are not limited to, salts with an organic or inorganic acid, or salts with an organic or inorganic base. "Pharmaceutically acceptable salts" also include, but are not limited to, solvates of pharmaceutically acceptable salts. Examples of pharmaceutically acceptable anions include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, 1 ,2-ethanedisulfonate, lauryl sulfate,

ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, p- glycollamidophenylarsonate, hexylresorcinate, N,N'-di(dehydroabietyl)ethylenediamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, 2-hydroxyethanesulfonate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate, diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, 8- chlorotheophyllinate, and triethiodide. Similarly, examples of pharmaceutically acceptable cations include aluminum, benzathine, calcium, chloroprocaine, choline, diethanolamine, ethylenediamine, lithium, magnesium, meglumine, potassium, procaine, sodium and zinc. See, e.g., Berge et al., J. Pharm. Sc , 66(1 ) 1- 19 (1977).

[0023] The present compounds may contain one or more chiral atoms, or may otherwise be capable of existing as two enantiomers or two or more diastereomers.

Accordingly, the compounds described herein include mixtures of enantiomers or disastereomers as well as purified enantiomers, diastereomers, or enantiomerically or diastereomerically enriched mixtures. Also, it is understood that all tautomers and mixtures of tautomers are included within the scope of Compound A and Compound B.

[0024] In some embodiments, Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof (or pharmaceutical compositions comprising Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof) are administered sequentially. In some instances, Compound A and/or a pharmaceutically acceptable salt thereof is administered to the subject after administration with Compound B and/or a pharmaceutically acceptable salt thereof has ended. The administration of

Compound A and/or a pharmaceutically acceptable salt thereof may begin immediately following termination of treatment with Compound B and/or a pharmaceutically acceptable salt thereof, or there may be a time interval (e.g., one day, one week, one month, six months, one year, etc.) between the end of treatment with Compound B and/or a pharmaceutically acceptable salt thereof and the beginning of treatment with Compound A and/or a pharmaceutically acceptable salt thereof. In other instances, the Compound B and/or a pharmaceutically acceptable salt thereof is administered to the subject after treatment with Compound A and/or a pharmaceutically acceptable salt thereof has ended. The

administration of Compound B and/or a pharmaceutically acceptable salt thereof may begin immediately following termination of the administration of Compound A and/or a pharmaceutically acceptable salt thereof, or there may be a time interval (e.g., one day, one week, one month, six months, one year, etc.) between the end of treatment with Compound A and/or a pharmaceutically acceptable salt thereof and the beginning of treatment with Compound B and/or a pharmaceutically acceptable salt thereof. In each instance, alternate administration may be repeated during a single treatment protocol. The determination of the order of administration and the number of repetitions of administration of each therapy during a treatment protocol is within the knowledge of the skilled physician after evaluation of the condition of the patient.

[0025] In some embodiments, Compound A and/or a pharmaceutically acceptable salt thereof is administered to the subject concurrently with Compound B and/or a pharmaceutically acceptable salt thereof, i.e., Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof are administered simultaneously, essentially simultaneously or within the same treatment protocol. In some embodiments, administration of Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof begin and end at the same time (i.e., on the same day or within the same treatment protocol). In some embodiments, only one of Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof is administered for a first period of time, followed by co-administration of Compound A and/or a

pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof for a second period of time. For example, the subject may receive Compound B and/or a pharmaceutically acceptable salt thereof for a first period of time, then receive both Compound B and/or a pharmaceutically acceptable salt thereof and Compound A and/or a pharmaceutically acceptable salt thereof for a second period of time.

Administration of either Compound A and/or a pharmaceutically acceptable salt thereof or Compound B and/or a pharmaceutically acceptable salt thereof may then continue for a third period of time. In some embodiments, the subject may receive Compound A and/or a pharmaceutically acceptable salt thereof for a first period of time, then receive both

Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof for a second period of time. Administration of either Compound A and/or a pharmaceutically acceptable salt thereof or Compound B and/or a pharmaceutically acceptable salt thereof may then continue for a third period of time. In some embodiments, Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof are co-administered for a first period of time, followed by administration of only one of Compound A and/or a

pharmaceutically acceptable salt thereof or Compound B and/or a pharmaceutically acceptable salt thereof for a second period of time. For example, the subject may receive both Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof for a first period of time, then receive Compound B and/or a pharmaceutically acceptable salt thereof for a second period of time. In another example, the subject may receive both Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof for a first period of time, then receive Compound A and/or a pharmaceutically acceptable salt thereof for a second period of time. In some embodiments, alternate administration may be repeated during a single treatment protocol. The determination of the order of administration and the number of repetitions of administration of each therapy during a treatment protocol is within the knowledge of the skilled physician after evaluation of the condition of the patient.

[0026] In some embodiments, Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof are administered during a single day. Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof may be administered within about 24 hours of each other, or within about 12 hours of each other, or within about 1 1 hours of each other, or within about 10 hours of each other, or within about 9 hours of each other, or within about 8 hours of each other, or within about 7 hours of each other, or within about 6 hours of each other, or within about 5 hours of each other, or within about 4 hours of each other, or within about 3 hours of each other, or within about 2 hours of each other, or within about 1 hour of each other.

[0027] Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof may be administered for a specified period of time during a treatment protocol. In some embodiments, one or both of Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof are administered within a specified period for at least one day, or for at least 3 consecutive days, or for at least 5 consecutive days, or for at least 7 consecutive days, or for at least 14 consecutive days, or for at least 30 consecutive days. Administration of one or both of Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof may not occur on every day of the specified period of time during the treatment protocol. For example, Compound A and/or a pharmaceutically acceptable salt thereof and/or Compound B and/or a pharmaceutically acceptable salt thereof may be administered every other day, every third day, every fifth day, or once a week during the treatment protocol. During a treatment protocol, Compound A and/or a pharmaceutically acceptable salt thereof may be administered on the same or different schedule as Compound B and/or a pharmaceutically acceptable salt thereof.

[0028] While it is possible that, for use in therapy, therapeutically effective amounts of Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof as well as pharmaceutically acceptable salts thereof, may be administered as the raw chemical, it is possible to include Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or pharmaceutically acceptable salt thereof as active ingredients of one or more pharmaceutical compositions. Accordingly, provided is a pharmaceutical composition comprising Compound A, and/or a pharmaceutically acceptable salt thereof, and Compound B and/or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carrier, diluent, or excipient. The carrier(s), diluent(s) or excipient(s) may be acceptable in the sense of being compatible with the other ingredients of the formulation, capable of pharmaceutical formulation, and not deleterious to the recipient thereof. Suitable pharmaceutical excipients are well known in the art, and may be used in a variety of formulations. See, e.g.,

Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, Editor, Mack Publishing Company (1990); Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Editor, Lippincott Williams & Wilkins (2005); Handbook of Pharmaceutical Excipients, 3rd Edition, A. H. Kibbe, Editor, American Pharmaceutical Association, and Pharmaceutical Press (2000); Handbook of Pharmaceutical Additives, compiled by Michael and Irene Ash, Gower (1995).

[0029] Also provided is a process for the preparation of a pharmaceutical formulation including admixing Compound A, and/or a pharmaceutically acceptable salt thereof, and Compound B, and/or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents or excipients.

[0030] Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof may be administered to the subject in separate pharmaceutical compositions or they may be formulated together in one pharmaceutical composition. In some embodiments, the method comprises administering to the subject a first pharmaceutical composition comprising Compound A, and/or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients and a second pharmaceutical composition comprising Compound B, and/or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0031] In some embodiments, the method comprises administering to the subject a single pharmaceutical composition comprising Compound A, and/or a pharmaceutically acceptable salt thereof, Compound B, and/or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Also provided is such a pharmaceutical composition comprising Compound A, and/or a pharmaceutically acceptable salt thereof, Compound B, and/or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. One example of a pharmaceutical composition comprises 3-chloro-N-{ (l S)-2-[(N,N-dimethylglycyl)amino]-l - [(4- { 8-[( 1 S)- 1 -hydroxyethyl]imidazo[ 1 ,2-a]pyridin-2-yl } phenyl)methyl]ethyl } -4-[( 1 - methylethyl)oxy]benzamide (20 mg), N-[3-[3-cyclopropyl-5-[(2-fluoro-4- iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin- l(2h)-yl]phenyl]acetamide (20 mg), microcrystalline cellulose (30 mg), sucrose (4 mg), starch (2 mg), talc ( 1 mg) and stearic acid (0.5 mg).

[0032] Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. As is known to those skilled in the art, the amount of active ingredient per dose will depend on the condition being treated, the route of administration and the age, weight and condition of the patient, or the pharmaceutical formulations may be presented in unit dose forms containing a

predetermined amount of active ingredient per unit dose. In some embodiments, the unit dosage formulations are those containing a daily dose or sub-dose, or an appropriate fraction thereof, of an active ingredient. Such pharmaceutical formulations may be prepared by any of the methods known in the pharmacy art.

[0033] Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof may be administered by any appropriate route. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intraveneous, intradermal, intrathecal, and epidural). In some embodiments, the route may vary with, for example, the condition of the subject and the cancer to be treated. Each of the agents administered may be administered by the same or different routes and that Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof may be compounded together in a pharmaceutical composition/formulation.

[0034] Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0035] For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing and coloring agent can also be present.

[0036] Capsules are made by preparing a powder mixture as described above, and filling the powder mixture into formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.

[0037] When desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be added. Suitable binders include, for example, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Suitable lubricants include, for example, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

[0038] Tablets may be formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then compressed into tablets. The present compounds can also be combined with free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.

[0039] Oral fluids such as solution, syrups and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound.

Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like can also be added.

[0040] Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax or the like.

[0041] The present agents can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

[0042] The present agents may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer,

polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans,

polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

[0043] Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical

Research, 3(6), 318 (1986).

[0044] Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0045] For treatments of the eye or other external tissues, for example mouth and skin, the formulations may be applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water- miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical formulations adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, such as an aqueous solvent.

[0046] Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

[0047] Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas.

[0048] Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

[0049] Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists that may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators: [0050] Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0051] Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0052] It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral

administration may include flavoring agents.

[0053] In some embodiments, the pharmaceutical combination includes

Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a pharmaceutically acceptable salt thereof, and optionally at least one additional antineoplastic agent.

[0054] In some embodiments, therapeutically effective amounts of Compound A and/or a pharmaceutically acceptable salt thereof and Compound B and/or a

pharmaceutically acceptable salt thereof, and optionally additional anti-neoplastic therapies, are administered to a mammal. Typically, the therapeutically effective amount of one of the administered agents will depend upon a number of factors including, for example, the age and weight of the mammal, the precise condition requiring treatment, the severity of the condition, the nature of the formulation, and the route of administration. Ultimately, the therapeutically effective amount will be at the discretion of the attendant physician or veterinarian.

[0055] Examples of cancers that are suitable for treatment by the methods described herein include, but are not limited to, both primary and metastatic forms of head and neck cancer, breast cancer, lung cancer, colon cancer, ovary cancer, prostate cancer and neuroblastoma. [0056] The following examples are presented for illustrative purposes and in no way serve to limit the true scope of this disclosure.

Examples

Example 1: High Throughput Screen of Druggable Genome

[0057] A high-throughput screen (HTS) was performed to identify genes that are sensitizers to CENP-E inhibition with 3-chloro-N-{ (l S)-2-[(N,N-dimefhylglycyl)amino]-l- [(4- { 8-[( 1 S)- 1 -hydroxyethyl]imidazo[ 1 ,2-a]pyridin-2-yl } phenyl)methyl]ethyl } -4-[( 1 - methylethyl)oxy]benzamide (Compound A). The Human Druggable Genome v2.0 siRNA library, which consists of approximately 7,000 genes, was purchased from Qiagen. The HTS was performed in cell line A427 (a non-small cell lung cancer cell line) and used 2 siRNAs per gene. Experiments were run in triplicate with five concentrations of Compound A and one vehicle (DMSO) controls applied. The concentrations of Compound A were 0.0650μΜ, 0.1025μΜ, Ο.ΠΟΟμΜ, 0.2900μΜ, and 8.0000μΜ.

[0058] The library siRNAs were printed onto white, solid bottom, 384-well plates (1 μί volume with the final concentration of 13 nM) and stored frozen at -80°C. On the day before screening, the plates were removed from the freezer and allowed to thaw overnight in a 4°C refrigerator. The selected transfection reagent (Lipofectamine RNAiMAX

(Invitrogen)) was diluted in complexing media and 20 μί was added into each well of the plate containing siRNA. The plates were then incubated at room temperature for approximately 30 minutes to allow for complexing of the siRNA and the lipid reagent. A suspension of A427 cells was prepared and 20 μ]_, of the solution was then added to each well to achieve a seeding density of 500 cells/well. The plates were then placed in a humidified incubator at 37°C with 5% C0₂. Twenty-four (24) hours after the seeding of the cells, 10 pL of diluted drug or diluted vehicle were added to the plates. The cells were incubated at 37°C with 5% C0₂. After 72 hours of drug treatment, the cell viability was determined for each well by the addition of CellTiter-Glo reagent. All plates were assured a one-hour incubation time of CellTiter-Glo (CTG) before data capture on a multilabel plate reader.

[0059] The HTS identified 703 genes as potential sensitizers for enhanced efficacy of Compound A.

Example 2: Confirmation of HTS Hits in A427 Cell Line [0060] In a manner similar to that described in Example 1 , experiments were run in duplicate with six concentrations of Compound A and two vehicle (DMSO) controls applied. The concentrations of Compound A were 0.0750μΜ, 0.102μΜ, 0.125μΜ, Ο. ΠΟΟμΜ, 0.3000μΜ, and 9.000μΜ. Only genes with a minimum of two sequences that demonstrated Compound A sensitization in both experimental replicates were considered a confirmed HTS hit. A total of 484 confirmed hits (siRNA sequences) targeting 160 genes were identified during the confirmation step. The confirmed HTS hits are summarized in Table 1.

Table 1

Example 3: Validation of HTS Hits in Three Lung Cancer Cell Lines

[0061] 160 genes were identified and confirmed as potential sensitizers for Compound A from the HTS. An additional 80 genes were selected through knowledge mining based on biological relevance towards CENP-E. These additional 80 genes are shown in Table 2.

Table 2

[0062] These 80 additional gene targets would have been considered as confirmed hits if relaxed hit selection criteria were used during analysis. Therefore, a total of 240 genes with four sequences per target were used in the validation screen.

[0063] These genes were tested in the primary cell line (A427, more resistant to

Compound A), and also in A549 and NCI-H460 cell lines (two non-small cell lines that are more sensitive to Compound A). Four siRNA sequences were tested for each gene (two more than were tested in the HTS and confirmation steps). For each cell line, six concentrations of Compound A and two vehicle (DMSO) controls were applied. The doses of Compound A applied in each cell line are summarized in Table 3. Table 3

[0064] The data from the screen was analyzed in the same manner as the HTS and confirmation steps, using the same QC analysis assessing transfection efficiency and robustness of the experiments. Each siRNA sequence was then examined for any potential sensitizing effects with Compound A. In the validation process, a biological replicate of the experiment was performed in each cell line. In order for a gene target to be considered a validated hit, at least 2 sequences were required to show sensitization to Compound A in each biological replicate.

[0065] From the 240 genes selected, the number of validated genes was determined for each respective cell line. In the A427 cell line, 110 genes were validated as sensitizers to Compound A. The list of the validated genes, along with their respective biological functionality, is summarized in Figure 1. In addition, there were 48 genes validated in the A549 line and 95 genes validated in the NCI-H460 cell line. Figure 2 is a summary of the number of siRNA that showed sensitization for each gene in each cell line. Example 4: Synergy with Inhibitors of the PI3K/AKT/mTOR Pathway [0066] The PI3 K/AKT/mTOR pathway consists PI3-kinase, AKT, mTOR and other proteins and is a cancer pathway. Multiple genes that encode proteins along this pathway were identified as potential sensitizers for Compound A in the siRNA library screen, including AKT1, RPTOR (raptor) and a downstream gene PRKCA. Two mTOR inhibitors, rapamycin and temsirolimus, were tested to determine if they showed synergy with Compound A in chemical combination studies.

Rapamycin

[0067] Combination studies with Compound A and rapamycin were conducted using a panel of cell lines from human colon cancers (n = 23) and pancreatic cancer (n = 1) obtained from American Type Culture Collection (ATCC). Certain cell lines contained mutations in one or more of the genes KRAS, PIK3CA and BRAF, as summarized in Table 4.

Table 4

Cell Line Type KRAS PIK3CA BRAF

HCT8 Colon c.38G>A c. l633G>A WT

RKO Colon WT c.3140A>G c.l799T>A

NCIH747 Colon c.38G>A WT WT

LS 1034 Colon c.436G>A WT WT

SW948 Colon c.l 82A>T c. l624G>A WT

LS 174T Colon c.35G>A c.3140A>G WT

HT29 Colon WT c. l345C>A c.l799T>A

SW480 Colon c.35G>T WT WT

HCT15 Colon c.38G>A c. l633G>A WT

HCT1 16 Colon c.38G>A c.3140A>G WT

BxPC3 Pancreas WT WT WT

[0068] Cell lines were grown in RPMI-1640 supplemented with 2mM glutamine, ImM sodium pyruvate and 10% fetal bovine serum and maintained at 37 °C and 5% C0₂ in a humid incubator.

[0069] First, the test compounds were prepared as 5 mM stocks in 100% DMSO. Further dilutions were made with DMSO. The first test compound (designated Compound 1) was diluted horizontally in a 96 well microtiter plate in rows B-E using a 2-fold dilution series for 10 dilution points. The second test compound (designated Compound 2) was diluted horizontally in a separate 96 well microtiter plate in rows D-G using a 2-fold dilution series for 10 dilution points. The two compounds were combined using equal volumes from each drug plate into cell culture media, resulting in a 1 :50 dilution of the drugs in the cell culture media. Compound 1 is individually titrated in rows B and C, while only Compound 2 is dosed in rows F and G of the plate. An additional 1 :10 dilution of the drugs is performed in cell culture media prior to addition to the cells. Drug addition to the cells results in a further 1 :2 dilution of the drugs, resulting in a total dilution of the drug plate to the cells of 1 : 1000. The final dosing concentration range for Compound A was 1-500 nM, and the final dosing concentration range for rapamycin was 0.019-10 nM. The positive control consisted of culture media with 0.1 % DMSO and cells. The negative control consisted of culture media with 0.1 % DMSO. Assays were performed in 384 well microtiter plates with appropriate seeding densities estimated from previous studies of each cell line. Following dosing, the cell lines were incubated at 37 °C and 5% C0₂ in humid air for 72 hours. Cell proliferation was measured using the CellTiter Glo reagent (Promega

Corporation, Madison, WI) according to manufacturer's protocol. The plates were treated with CellTiter Glo solution and were analyzed for RLU (relative light units) using a

SpectraMax M5 plate reader ( Molecular Devices, Sunnyvale, CA).

[0070] Three independent metrics were used to analyze the combinatorial effects on growth inhibition of Compound A and rapamycin:

[0071] 1. Excess over Highest Single Agent ( EOHSA ). One standard criterion for measuring drug combinatorial effects is analyzing the effects on cell growth inhibition in absolute terms. In this case, the combination of drugs is compared to the more responsive of the two individual treatments (single agent). For each combination experiment, the percent effect relative to the highest single agent for each dose along the curve is generated. This measure of "Excess of Highest Single Agent (EOHSA)" is one of the criteria used for evaluating synergy of drug combinations. (Borisy AA Elliott PJ, Hurst NW, Lee MS, Lehar J, Price ER, Serbedzija G, Zimmermann GR, Foley MA, Stockwell BR, Keith CT.

Systematic discovery of multicomponent therapeutics. Proc Natl Acad Sci U S A. 2003 Jun 24; 100(13):7977-82; FDA 21 CFR 300.50)

[0072] 2. Bliss synergy. A second criterion often used to determine combination synergy is evaluating the excess inhibition over Bliss independence or "additivity" (Bliss, C.I, Mexico, DF, The Toxicity of Poisons Applied Jointly. Annals of Applied Biology 1939, Vol 26, Issue 3, August 1939). The model assumes a combined response of the two compounds independently using the following:

E_a + E_b - (E_a * E_b)

where E_a is the effect (or percent inhibition) of compound A and E_b is the effect of compound B. The resulting effect of the combination of the two compounds is compared to their predicted additivity by Bliss and a synergy score is generated for each dose along the response curve. [0073] 3. Combination Index (CI). A third criterion for evaluation of synergy is Combination Index (CI) derived from the Chou and Talalay (Chou TC, Talalay P.

Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984; 22:27-55). The following equation is a model used for compounds that behave with different mechanisms of action (mutually nonexclusive formula).

Combination Index =

D_a in a:b/IC₅₀(a) + D_b in a:b/IC₅₀(b) + (D_a in a:b) (D_b in a:b)/ IC₅₀(a)IC₅₀(b)

[0074] The lower the CI the more synergy the combination potentially has. A CI greater than 1 suggests that the combination being studied may be antagonistic. CI scores are also generated for inhibitory concentrations of 25% (IC25) and 75% (IC75) by replacing the IC50 in the formula above for each compound with the respective inhibitory

concentration.

[0075] The percent intensity values were used in model 205 of XLfit in Microsoft Excel to calculate glCso values using a 4 parameter logistical fit. The midpoint of the growth window (the glCso) falls half way between the number of cells at the time of compound addition (T=0) and the growth of control cells treated with DMSO at 72 hrs. The number of cells at time zero (To) is divided from the intensity value at the bottom of the response curve (Ymin) to generate a measure for cell death (Y_mi_n T₀). A value below 1 for Y_mm/To indicates stronger potency with the treatment when compared to higher values.

[0076] For EOHSA and Bliss, a synergy score must be seen in both technical replications within an experiment to make an appropriate designation (synergy, modest synergy, etc). Each combination experiment contains a replicate for the two compounds as single agents as well as a technical replicate for the combination.

[0077] Synergy scores for EOHSA and Bliss, at low concentrations, (e.g., Dose 1 , dose 2) are subject to higher variation and generally excluded from the analysis.

Conversely, synergy scores at the highest concentrations (Dose 10), far outside of the therapeutic dosing range, are generally excluded from analysis since the effects observed are more susceptible to off-target events.

[0078] For EOHSA and Bliss Synergy measures, a score is generated for each dose along the response curve. Scores were categorized as being 'Antagonistic' (< -10), 'Additive' (-10 - 10), 'Modest Synergy' (10 - 20) or 'Synergistic' (> 20). These scores reflect the percentage over the higher agent or percentage greater than Bliss additivity, depending on which model is being interpreted.

[0079] For the Combination Index, the lower the CI, the more synergy the combination potentially has. Scores between 0 and 0.7 were considered to be synergistic, while scores between 0.7 and 0.9 were considered to be modest synergy. All other scores did not indicate synergy for the Combination index.

[0080] For those cell lines that never reached an inhibitory concentration of 25% for 1 of the compounds in the combination, a CI value cannot be calculated and 'NA' was listed for the CI.

[0081] EOHSA, Bliss and CI values were calculated for each cell line treated with the combination of Compound A and rapamycin. The results showed that Compound A and rapamycin exhibit interaction across colon cancer and pancreatic cancer cell lines.

Temsirolimus

[0082] In a manner similar to that described above, combination studies with Compound A and temsirolimus were conducted using a panel of cell lines from human breast cancers (n = 4) and lung cancer (n = 4) obtained from ATCC. Certain cell lines contained mutations in one or more of the genes PIK3CA and PTEN, as summarized in Table 5.

Table 5

[0083] EOHSA, Bliss and CI values were calculated for each cell line treated with the combination of Compound A and temsirolimus. The results indicate that Compound A and temsirolimus exhibit interaction across breast cancer and lung cancer cell lines.

[0084] These data indicate that inhibitors of the PI3K/AKT/mTOR pathway sensitize the cancer cells to CENP-E inhibition with Compound A. Thus, administration of a CENP-E inhibitor such as Compound A and an inhibitor of the PI3K AKT/mTOR pathway may be a useful treatment for certain cancers, such as breast or lung cancer.

Example 5: Synergy with Inhibitors of the Other Pathways

[0085] Nineteen commercially available inhibitors of various genes identified in the HTS were obtained for further investigation in A427, A549 and NCI-H460 non-small cell lung cancer cell lines. The compounds and their gene targets are provided in Table 6.

Table 6

[0086] Initial dose response profiling was performed on each compound in the three cell lines in order to determine growth inhibition 50% (GI₅₀) concentration, EC50, and the relative dose response curve. The initial drug dose response screening was performed in a 384- well format with a serial 3-fold dilution starting at 100 μΜ. Each experiment was performed in duplicate for each cell line. The GI values were used as an alternative when response curves had a drug activity of less than 50%. In most cases GI and EC values were comparable, although varying responses were observed. Results from drug dose responses were normalized and both GI₂s and GI50 concentrations were estimated. Multiple compounds alone showed insignificant dose response curves making GI₂5 and GI50

concentrations unobtainable. In such situations, the compounds were tested at 25 μΜ and 50 μΜ respectively. The highest concentration was limited to 50 μΜ in order to minimize any potential DMSO toxicity that may occur during combination with Compound A. Once the GI concentrations were determined, their synergistic effects with Compound A were tested.

[0087] The determined high and low dose of each compound was tested in combination with a dose response of Compound A across all three cell lines. A non- combination dose response was used as a control. Potential synergistic activity was assessed by the comparison of dose response curves. Favorable combinatory activity resulted in the reduction of the EC50 value of Compound A. The higher dose of each compound was applied as a general marker to ensure that the target was inhibited without losing assay sensitivity. The lower dose was applied to ensure that an effective combination would still be obtained in the event that the higher dose was too high. The two concentrations were also used to observe potential trends in the degree of synergy (EC50 shifts) measured. A significant EC50 shift was defined as an EC50 value statistically different than the control EC50 with a greater than 95% confidence interval (p < 0.05).

[0088] 12 of the 19 compounds tested were found to demonstrate significant reduction of the dose response of Compound A in at least one cell line. The Combination Index (CI) values for these 12 compounds were then determined when dosed with

Compound A.

[0089] In the IC experiments, four different doses of each compound were used while the concentration of Compound A remained constant. The highest concentration of each compound was serially diluted 2-fold to produce the three other concentrations. Each concentration of drug was assessed alone and in combination with Compound A. The results of these experiments are summarized in Table 7.

Table 7

[0090] These date indicate that inhibitors of one or more of the MAPK1 MAPK3, ROCKl, WEEI, PRKCA, SHH, DNMTl, BRAF, CCLl 1 and HNMT genes may sensitize cancer cells to CENP-E inhibition, and such inhibitors may be useful in the treatment of cancers when combined with a CENP-E inhibitor such as Compound A.

Example 6: Synergy Confirmation with MEK Inhibitor

[0091] The MAPK/ERK pathway includes RAF, MEK, ERK and other proteins and represents another cancer pathway. Multiple genes along MAPK/ERK pathway were identified as potential sensitizers for Compound A in the siRNA library screen, including BRAF, MAPK3, MAP2K1 IP1 (a regulator for MEK), and RPS6KA1 (a downstream gene). Combination drug tests were performed with the CENP-E inhibitor Compound A and MEK inhibitor N-[3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo- 3,4,6,7-tetrahydropyrido[4,3-d]pyri ]acetamide, Compound C.

Compound C Compound C is described in International Application Publication No. WO 2005/121 142, the entire disclosure of which is hereby incorporated by reference for all purposes.

[0092] The studies were conducted using a panel of cell lines from human colon cancers (HCT1 16, RKO, HCT15, SW48, SW620, Colo205), lung cancers (A549, Calu-6, NCI-H1299, NCI-H1792, NCI-H2030, NCI-H2170, NCI-H358, NCI-H460, NCI-H520, NCI-H1563, NCI-H2122, NCI-H2228, NCI-H23, SK-MES-1, SW900) and pancreatic cancers (Capan- 1, HUP-T4, ASPC-1 , Capan-2, HPAF-II, MiaPaCa). Cell lines were grown in RPMI-1640 supplemented with 2 raM glutamine, ImM sodium pyruvate and 10% fetal bovine serum (except for Capan-1 and HuP-T4 which were grown with 20% fetal bovine serum) and maintained at 37°C and 5% C0₂ in a humid incubator.

[0093] The test compounds were prepared as 10 mM stocks in 100% dimethyl sulfoxide (DMSO). Further dilutions of the compounds were made with DMSO. The first test compound (designated as Compound 1) was diluted horizontally in a 96 well microtiter plate in rows B-E using a 3-fold dilution series for 10 dilution points. A second test compound (designated as Compound 2) was diluted horizontally in a separate 96 well microtiter plate in rows D-G using a 3-fold dilution series for 10 dilution points. The two compounds were combined using equal volumes from each drug plate into cell culture media. This resulted in a 1 :50 dilution of the drugs in the cell culture media. Compound 1 was individually titrated in rows B and C, while only Compound 2 was dosed in rows F and G of the plate. An additional 1 : 10 dilution of the drugs was performed in cell culture media prior to addition to the cells. Drug addition to the cells resulted in a further 1 :2 dilution of drugs. The total dilution of the drug plate to the cells was 1 : 1000. The final dosing concentration range for Compound A was 0.025. - 500.0 nM and was 0.013 - 250.0 nM for Compound C (except for cell line HCT15 which was treated with 0.25 -5000nM Compound A and 0.13 - 2500nM Compound C). The positive control consisted of culture media with DMSO at 0.1 % and cells. The negative control consisted of culture media with DMSO at 0.1 % solution.

[0094] Assays were performed in 384 well microtiter plates with appropriate seeding densities estimated from previous studies of each cell line. Following dosing, the cell lines are incubated at 37°C, 5% C0₂ in humid air for 72 hours. Cell proliferation was measured using the CellTiter Glo (Promega Corporation, Madison, WI, USA) reagent according to the manufacturer's protocol. The plates are treated with CellTiter Glo solution and are analyzed for RLU (relative light units) using a Molecular Devices SpectraMax M5 (Sunnyvale, CA, USA) plate reader.

[0095] A comprehensive categorization of the degree of synergy was done for each cell line treated with the combination of the CENP-E inhibitor Compound A and MEK inhibitor Compound C. Cell lines were considered to have synergy when at least two separate metrics were scored as synergistic. Synergy was seen in 5 of the 6 colon cancer lines (83%) by this definition. For the 15 lung cancer cell lines, synergy was observed in 5 (33%). Among the 8 pancreatic lines, 6 showed synergy (75%).

[0096] These data indicate that inhibitors of the MAPK pathway sensitize the cancer cells to CENP-E inhibition with Compound A. Thus, administration of a CENP-E inhibitor such as Compound A and an inhibitor of the MAPK pathway may be a useful treatment for certain cancers, such as colon, lung and pancreatic cancer.

[0097] One colon cancer cell line tested, SW48, is wild-type for both RAS/RAF and PI3K. Another colon cancer cell line tested, RKO, has a mutant BRAF and a mutant PI3K. Each of these cell lines was treated with: DMSO alone (control), Compound A at 20nM, Compound C at lOnM, Compound A at 20nM and Compound C at lOnM, Compound A at 200nM, Compound C at ΙΟΟηΜ, or Compound A at 200nM and Compound C at lOOnM. After 24 hr incubation, apoptosis was measured by Caspase Glo (Promega). At low concentrations of either drug, only minimal cell death was observed. Administration of Compound C alone, even at lOOnM, resulted in minimal cell death induction. Administration of Compound A at a concentration of 200nM induced a certain degree of cell death. However, in the presence of both Compound A and Compound C, the cell death was significantly higher than what was observed when either drug was administered alone. (See Figures 3 and 4). This result was observed in both cell lines, each having different

RAS/RAF and PI3K genetic backgrounds.

Claims

Claims:

1. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of 3-chloro-N-{(lS)-2-[(N,N-dimethylglycyl)amino]-l-[(4- { 8-[( 1 S)- 1 -hydroxyethyl]imidazo[ 1 ,2-a]pyridin-2-yl }phenyl)methyl]ethyl } -4-[( 1 - methylethyl)oxy]benzamide, and/or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of at least one additional compound that is a sensitizer to CENP-E inhibition.

2. The method of claim 1, wherein the at least one additional compound is an inhibitor of at least one gene selected from: ANAPC1 CCL11 HNMT ID1, MAP2K6, PRKRA, ROCKl, SSR3, ANAPC5, APRIN, CYP4F8, FLOTl , GADD45A, GARS, GCSH, GPR155, INPP4B, KIF22, MAP2K5, MAT2B, P2RY11, PGAM2, PGM2L1, PIGA, PIGB, PIGT, PTPLA, RABGGTB, RaLP, RGL2, SCAPl, SHMT2, SKIP, SLC2A12, SLC6A8, SLC9A2, SNRK, TAS2R5, TLK2, TSC1, UBE2C, UGT1A3, UGT2B10, ACTL6A, AK2, AKT1, ARHGAP26, ASK, BRAF, C14orfl30, C7orf9, CAMK2D, CDC6, CDKN2C, CINP, CLSPN, CNKSR1, CS, CYLD, CYP24A1, CYP2A13, DMPK, DYRK3, EMR2, FALZ, FBN2, FLJ2331 1, GALK1, GNPDA1 , HP, HSD17B3, IBSP, KCNJ13, KIF2C, LATS2, LIPE, MAP2K1IP1, MAPK3, MCP, MGC 14156, MTCHl, OXCTl , OXCT2, PCK2, PDHB, PGLS, PGM3, PIGC, PIGH, PIGS, PNLIPRP2, PPP1CA, PPP1R12B, PRKCA, PRLR, PTPRN2, RPTOR, RIPK2, RORA, RPS6KA1, SBDS, SERPINI1, SFXN3, SLC2A1, SLC2A1 1 , SSR1, TBXAS1, TTBKl, ULK1, VASP and SLC2A8.

3. The method of claim 1, wherein the at least one additional compound is an inhibitor of at least one gene selected from: MAPK1/MAPK3, ROCKl , WEE1, PRKCA, SHH, MAP2K2, DNMT1, BRAF, CCL1 1, HNMT, SLC6A8, PDK1 and P2RY1 1.

4. The method of claim 1 , wherein the at least one additional compound is an inhibitor of the PBK/AKT/mTOR pathway.

5. The method of claim 4, wherein the at least one additional compound inhibits one or more of PI3K, AKT and mTOR.

6. The method of claim 5, wherein the at least one additional compound is selected from sirolimus (rapamycin), temsirolimus, everolimus, and ridaforolimus.

7. The method of claim 1 , wherein the at least one additional compound is an inhibitor of the MAPK pathway.

8. The method of claim 7, wherein the at least one additional compound inhibits one or more of RAF, MEK and ERK.

9. The method of claim 8, wherein the at least one additional compound is N-[3-[3- cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7- tetrahydropyrido[4,3-d]pyrimidin- l (2h)-yl]phenyl]acetamide, and/or a pharmaceutically acceptable salt thereof.

10. The method of claim 1, wherein the cancer is selected from primary and metastatic forms of head and neck cancer, breast cancer, lung cancer, colon cancer, ovary cancer, prostate cancer and neuroblastoma.

1 1. A pharmaceutical composition comprising a therapeutically effective amount of 3- chloro-N-{ ( 1 S)-2-[(N,N-dimethylglycyl)amino]- 1 -[(4-{ 8-[( 1 S)- 1 -hydroxyethyl]imidazo[ 1 ,2- a]pyridin-2-yl }phenyl)methyl]ethyl }-4-[(l-methylethyl)oxy]benzamide, and/or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of at least one additional that is a sensitizer to CENP-E inhibition.

12. The pharmaceutical composition of claim 1 1 , wherein the at least one additional compound is an inhibitor of at least one gene selected from: ANAPC1 CCL1 1 HNMT ID1, MAP2K6, PPvKRA, ROCK1, SSR3, ANAPC5, APRIN, CYP4F8, FLOT1, GADD45A, GARS, GCSH, GPR155, Γ ΡΡ4Β, KIF22, MAP2K5, MAT2B, P2RY1 1, PGAM2, PGM2L1, PIGA, PIGB, PIGT, PTPLA, RABGGTB, RaLP, RGL2, SCAPl , SHMT2, SKIP, SLC2A12, SLC6A8, SLC9A2, SNRK, TAS2R5, TLK2, TSC1 , UBE2C, UGT1A3, UGT2B10,

ACTL6A, AK2, AKTl, ARHGAP26, ASK, BRAF, C14orfl30, C7orf9, CAMK2D, CDC6, CDKN2C, CINP, CLSPN, CNKSR1 , CS, CYLD, CYP24A1, CYP2A13, DMPK, DYRK3, EMR2, FALZ, FBN2, FLJ2331 1, GALK1, GNPDA1, HP, HSD17B3, IBSP, KCNJ13, KIF2C, LATS2, LIPE, MAP2K1IP1, MAPK3, MCP, MGC14156, MTCH1, OXCT1, OXCT2, PCK2, PDHB, PGLS, PGM3, PIGC, PIGH, PIGS, PNLIPRP2, PPP1CA,

PPP1R12B, PRKCA, PRLR, PTPRN2, RPTOR, RIPK2, RORA, RPS6KA1 , SBDS, SERPINI1, SFXN3, SLC2A1, SLC2A1 1, SSR1, TBXAS1, TTBKl , ULK1, VASP and SLC2A8.

13. The pharmaceutical composition of claim 1 1, wherein the at least one additional compound is an inhibitor of at least one gene selected from: MAPK1/MAPK3, ROCK1 , WEE1 , PRKCA, SHH, MAP2K2, DNMT1 , BRAF, CCL1 1, HNMT, SLC6A8, PDK1 and P2RY1 1.

14. The pharmaceutical composition of claim 1 1, wherein the at least one additional compound is an inhibitor of the PI3K/AKT/mTOR pathway.

15. The pharmaceutical composition of claim 14, wherein the at least one additional compound inhibits one or more of PI3K, AKT and mTOR.

16. The pharmaceutical composition of claim 15, wherein the at least one additional compound is selected from sirolimus (rapamycin), temsirolimus, everolimus, and ridaforolimus.

17. The pharmaceutical composition of claim 1 1, wherein the at least one additional compound is an inhibitor of the MAPK pathway.

18. The pharmaceutical composition of claim 17, wherein the at least one additional compound inhibits one or more of RAF, MEK and ERK.