WO2007136615A2 - Combination cancer therapy - Google Patents

Abstract

The present invention relates to a method of treating cancer in a subject in need thereof, by administering to a subject in need thereof a first amount of an Aurora kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof, in a first treatment procedure, and a second amount of an anti-cancer agent in a second treatment procedure. The first and second amounts together comprise a therapeutically effective amount. The effect of the Aurora kinase inhibitor and the anti-cancer agent may be additive or synergistic.

Description

TITLE OF THE INVENTION COMBINATION CANCER THERAPY

BACKGROUND OF THE INVENTION

Cancer is a disorder in which a population of cells has become, in varying degrees, "unresponsive to the control mechanisms that normally govern proliferation and differentiation.

Therapeutic agents used in clinical cancer therapy can be categorized into six groups: alkylating agents, antibiotic agents, antimetabolic agents, biologic agents, hormonal agents, and plant-derived agents.

Current tumor therapies are known which consist of the combinatorial treatment of patients with more than one anti-tumor therapeutic reagent. Examples are the combined use of irradiation treatment together with chemotherapeutic and/or cytotoxic reagents and more recently the combination of irradiation treatment with immunological therapies such as the use of tumor cell specific therapeutic antibodies. However, the possibility to combine individual treatments with each other in order to identify such combinations which are more effective than the individual approaches alone, requires extensive pre-clmical and clinical testing, and it is not possible without such experimentation to predict which combinations show an additive or even synergistic effect. Besides the aim to increase the therapeutic efficacy, another purpose of combination treatment is the potential decrease of the doses of the individual components in the resulting combinations in order to decrease unwanted or harmful side effects caused by higher doses of the individual components.

There is an urgent need to discover suitable methods for the treatment of cancer, including combination treatments that result in decreased side effects and that are effective at treating and controlling malignancies.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that Aurora kinase inhibitors, for example VX-680 (Compound I), can be used in combination with one or more anti-cancer agents, to provide therapeutically effective anticancer effects.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIG. 1: VX-680 exhibits synergistic effects in combination with a topoisomerase I inhibitor, camptothecin. In this example, HeLa (human cervical carcinoma) cells were treated in a sequential manner: 200 nM VX-680 for 24 hrs — > wash-out -> 50 nM camptothecin for 72 hrs -» measured by flow cytometry (FACS).

FIG. 2: VX-680 exhibits synergistic effects in combination with a topoisomerase

II inhibitor, doxorubicin. In this example, HeLa (human cervical carcinoma) cells were treated in a sequential manner: 200 nM VX-680 for 24 hrs — > wash-out — > 50 nM doxorubicin for 72 hrs → measured by flow cytometry (FACS).

FIG. 3: VX-680 exhibits synergistic effects in combination with a topoisomerase

II inhibitor, idarubicin. In this example, Molt-4 (human T-lymphoma) cells were treated in a sequential manner: VX-680 for 24 hrs —> wash-out -> idarubicin for 72 hrs — >^■ measured by Cell Titer GIo (3A) and analyzed by the Bliss additivism model (3B).

FIG. 4: VX-680 exhibits synergistic effects in combination with a DNA cross- linking agent, cisplatin. In this example, HeLa (human cervical carcinoma) cells were treated in a sequential manner: 200 nM VX-680 for 24 hrs —> wash-out -» 4 uM cisplatin for 72 hrs -» measured by flow cytometry (FACS).

FIG. 5: VX-680 exhibits synergistic effects in combination with a microtubule- stabilizing agent, taxol. In this example, HeLa (human cervical carcinoma) cells were treated in a sequential manner: 100 nM taxol for 24 hrs -» wash-out — » 200 nM VX-680 for 72 hrs -» measured by flow cytometry (FACS).

FIG. 6: VX-680 exhibits synergistic effects in combination with a microtubule- stabilizing agent, taxotere. In this example, MiaPaCa-2 cells (human pancreatic cancer) cells were treated in a sequential manner: dose-range titration of VX-680 for 24 hrs — > wash-out — > dose-range titration of taxotere for 72 hrs — > measured by Cell Titer GIo (6A) and analyzed with the Bliss additivism model (6B).

ElG. 7: VX-680 exhibits synergistic effects in combination with a microtubule- destabilizing agent, vincristine. In this example, HeLa (human cervical carcinoma) cells were co-treated with either 100 or 200 nM VX-680 and 6 nM vincristine and measured by flow cytometry (FACS).

FlG. 8: VX-680 exhibits synergistic effects in combination with an EGFR inhibitor, tarceva. In this example, NSCLC (non-small cell lung cancer) cells were co- treated with a dose-range titration of VX-680 and a dose-range titration of tarceva for 72 hrs, measured by Cell Titer GIo (8A) and analyzed with the Bliss additivism model (8B).

FlG. 9: VX-680 exhibits synergistic effects in combination with a MEK inhibitor, UOl 26. In this example, HeLa (human cervical carcinoma) cells were co-treated with a dose-range titration of VX-680 and a dose-range titration of U0126 for 72 hrs, measured by flow cytometry (9A) and analyzed with the Bliss additivism model (9B).

FlG. 10: VX-680 exhibits synergistic effects in combination with a KSP inhibitor "KSPi", over a concentration range that corresponds to less than the IC50 for single agent activity of VX-680. In this example, HeLa (human cervical carcinoma) cells were co-treated with a dose-range titration of VX-680 and a dose-range titration of KSPi for 72 hrs and measured in a WST-8 viability assay.

FlG. 11 : VX-680 exhibits synergistic effects in combination with a histone deacetylase inhibitor, vorinostat. In this example, Hl 703 (human non-small cell lung cancer) cells were co-treated with a dose-range titration of VX-680 and a dose-range titration of vorinostat for 72 hrs and measured either by a Vialight assay (1 IA; top panel) or by an activated Capase 3/7 assay (1 IB; bottom panel).

Figure 12: VX-680 exhibits synergistic effects in combination with the active metabolite of irinotecan (Camptosar®, CPT-11), a topoisomerase I inhibitor. In this example, DLDl (human colon carcinoma) cells were treated in a sequential manner with a dose-range titration of VX-680 for 24 hrs — > wash-out — » dose-range titration of SN-38 for 72 hrs and measured by an activated Capase 3/7 assay.

Figure 13: VX-680 exhibits synergistic effects in combination with carboplatin, a DNA-cross-linking agent. In this example, HeLa (human cervix tumor) cells were treated in a sequential manner with a dose-range titration of VX-680 for 24 hrs — > washout — > dose-range titration of carboplatin for 72 hrs and measured by flow cytometry (FACS, Fig. 13A) and analyzed with the Bliss additivism model (Fig. 13B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of treating cancer in a subject in need thereof, by administering to a subject in need thereof a first amount of an Aurora kinase inhibitor or a pharmaceutically acceptable salt or hydrate thereof, in a first treatment procedure, and a second amount of an anti-cancer agent in a second treatment procedure, wherein the first and second amounts together comprise a therapeutically effective amount. The effect of the Aurora kinase inhibitor and the anti-cancer agent may be additive or synergistic.

The present invention also relates to a method of treating cancer in a subject in need thereof, by administering to a subject in need thereof a first amount of Compound I or a pharmaceutically acceptable salt or hydrate thereof, in a first treatment procedure, and a second amount of an anti-cancer agent in a second treatment procedure, wherein the first and second amounts together comprise a therapeutically effective amount. The effect of Compound I and the anti-cancer agent may be additive or synergistic.

The term "treating" in its various grammatical forms in relation to the present invention refers to preventing (i.e. chemoprevention), curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or viruses) or other abnormal condition. For example, treatment may involve alleviating a symptom (i.e., not necessary all symptoms) of a disease or attenuating the progression of a disease.

Treatment of cancer, as used herein, refers to partially or totally inhibiting, delaying or preventing the progression of cancer including cancer metastasis; inhibiting, delaying or preventing the recurrence of cancer including cancer metastasis; or preventing the onset or development of cancer (chemoprevention) in a mammal, for example a human. In addition, the method of the present invention is intended for the treatment or chemoprevention of human patients with cancer. However, it is also likely that the method would be effective in the treatment of cancer in other mammals.

As used herein, the term "therapeutically effective amount" is intended to qualify the combined amount of the first and second treatments in the combination therapy. The combined amount will achieve the desired biological response. In the present invention, the desired biological response is partial or total inhibition, delay or prevention of the progression of cancer including cancer metastasis; inhibition, delay or prevention of the recurrence of cancer including cancer metastasis; or the prevention of the onset or development of cancer (chemoprevention) in a mammal, for example a human. As used herein, the terms "combination treatment", "combination therapy",

"combined treatment" or "combinatorial treatment", used interchangeably, refer to a treatment of an individual with at least two different therapeutic agents. According to the invention, the individual is treated with a first therapeutic agent, preferably Compound I or another Aurora kinase inhibitor as described herein. The second therapeutic agent may be another Aurora kinase inhibitor, or may be any clinically established anti-cancer agent as defined herein. A combinatorial treatment may include a third or even further therapeutic agents.

The method comprises administering to a patient in need thereof a first amount of an Aurora kinase inhibitor, e.g., Compound I, in a first treatment procedure, and a second amount of an anti-cancer agent in a second treatment procedure. The first and second treatments together comprise a therapeutically effective amount.

The invention further relates to pharmaceutical combinations useful for the treatment of cancer. The pharmaceutical combination comprises a first amount of an Aurora kinase inhibitor, e.g., Compound I and a second amount of an anti-cancer agent. The first and second amount together comprises a therapeutically effective amount. The invention further relates to the use of a first amount of an Aurora kinase inhibitor and a second amount of an anti -cancer agent for the manufacture of a medicament for treating cancer. hi particular embodiments of this invention, the combination of the Aurora kinase inhibitor and anti-cancer agent is additive, i.e. the combination treatment regimen produces a result that is the additive effect of each constituent when it is administered alone. In accordance with this embodiment, the amount of the Aurora kinase inhibitor and the amount of the anticancer agent together constitute an effective amount to treat cancer. In another particular embodiment of this invention, the combination of the Aurora kinase inhibitor and anti-cancer agent is considered therapeutically synergistic when the combination treatment regimen produces a significantly better anticancer result (e.g., cell growth arrest, apoptosis, induction of differentiation, cell death) than the additive effects of each constituent when it is administered alone at a therapeutic dose. Standard statistical analysis can be employed to determine when the results are significantly better. For example, a Mann- Whitney Test or some other generally accepted statistical analysis can be employed.

The treatment procedures can take place sequentially in any order, simultaneously or a combination thereof. For example, the first treatment procedure, administration of an Aurora kinase inhibitor, can take place prior to the second treatment procedure, i.e., the anticancer agent, after the second treatment with the anticancer agent, at the same time as the second treatment with the anticancer agent, or a combination thereof. For example, a total treatment period can be decided for the Aurora kinase inhibitor. The anti-cancer agent can be administered prior to onset of treatment with the Aurora kinase inhibitor or following treatment with the Aurora kinase inhibitor. In addition, treatment with the anti-cancer agent can be administered during the period of Aurora kinase inhibitor administration but does not need to occur over the entire Aurora kinase inhibitor treatment period. In another embodiment, the treatment regimen includes pre-treatment with one agent, either the Aurora kinase inhibitor or the anti-cancer agent, followed by the addition of the second agent. The methods of the present invention are useful in the treatment in a wide variety of cancers, including but not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma,' lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, colorectal, rectal ; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerrninoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma.

Cancers that may be treated by the methods of the invention include, but are not limited to: breast, prostate, colon, colorectal, lung, brain, testicular, stomach, ovarian, pancrease, skin, small intestine, large intestine, throat, head and neck, oral, bone, liver, bladder, kidney, thyroid and blood.

The methods of the present invention are useful in the treatment of cancers, including: breast, prostate, colon, ovarian, colorectal and lung. The methods of the present invention are useful in the treatment of cancers, including: breast, colon, (colorectal) and lung.

The methods of the present invention are useful in the treatment of cancers, including: lymphoma and leukemia. The methods of the present invention are useful in the treatment of CML. The methods of the present invention are useful in the treatment of CML in patients with the T315IAbl mutation.

The methods of the present invention are useful in the treatment of cervical carcinoma, T-lymphoma, pancreatic cancer and non-small cell lung cancer.

In one particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with an additional Aurora kinase inhibitor. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with an alkylating agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with an antibiotic agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with an antimetabolic agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with a hormonal agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with a plant-derived agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with an anti-angiogenic agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with a differentiation inducing agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with a cell growth arrest inducing agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with an apoptosis inducing agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with a cytotoxic agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with a biologic agent. In another particular embodiment of the present invention, the Aurora kinase inhibitor can be administered in combination with any combination of an additional Aurora kinase inhibitor, an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant-derived agent, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent or a biologic agent.

In one particular embodiment of the present invention, the Aurora kinase inhibitor is Compound I, which can be administered in combination with any one or more of another Aurora kinase inhibitor, an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant-derived agent, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a biologic agent, a gene therapy agent, or any combination thereof.

In another particular embodiment of the present invention, the Aurora kinase inhibitor is Compound I, which can be administered in combination with the following anticancer agents: abarelix (Plenaxis depot®); aldesleukin (Prokine®); Aldesleukin (Proleukin®); Alemtuzumabb (Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole (Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine (Vidaza®); bevacuzimab (Avastin®); bexarotene capsules (Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); busulfan intravenous (Busulfex®); busulfan oral (Myleran®); calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®); carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®);'dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®); Darbepoetin alfa (Aranesp®); daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®); docetaxel (Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal (Doxil®); dromostanolone propionate (dromostanolone®); dromostanolone propionate (masterone injection®); Elliott's B Solution (Elliott's B Solution®); epirubicin (Ellence®); Epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine (Emcyt®); etoposide phosphate (Etopophos®); etoposide, VP- 16 (Vepesid®); exemestane (Aromasin®); Filgrastim (Neupogen®); floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU (Adrucil®); folvestrant (Faslodex®); gefitinib (Iressa®); gemcitabine (Gemzar®); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate (Zoladex Implant®); goserelin acetate (Zoladex®); histrelin acetate (Histrelin implant®); hydroxyurea (Hydrea®); Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide (IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a (Roferon A®); Interferon alfa-2b (Intron A®); irinotecan (Camptosar®); lenalidomide (Revlϊmid®); letrozole (Femara®); leucovorin (Wellcovorin®,

Leucovorin®); Leuprolide Acetate (Eligard®); levamisole (Ergamisol®); lomustine- CCNU

(CeeBU®); meclorethamine, nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnex tabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); mitomycin C

(Mutamycin®); mitotane (Lysodren®); mitoxantrone (Novantrone®); nandrolone phenpropionate (Durabolin-50®); nelarabine (Arranon®); Nofetumomab (Verluma®);

Oprelvekin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®); pegademase (Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®);

Pegfilgrastim (Neulasta®); pemetrexed disodium (Alimta®); pentostatin (Nipent®); pipobroman

(Vercyte®); plicamycin, mithramycin (Mithracin®); porfimer sodium (Photofrin®); procarbazine (Matulane®); quinacrine (Atabrine®); Rasburicase (Elitek®); Rituximab

(Rituxan®); sargramostim (Leukine®); Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen

(Nolvadex®); temozolomide (Temodar®); teniposide, VM-26 (Vumon®); testolactone

(Teslac®); thioguanine, 6-TG (Thioguanine®); thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab (Bexxar®); Tositumomab/I-131 tositumomab (Bexxar®);

Trastuzumab (Herceptin®); tretinoin, ATRA (Vesanoid®); Uracil Mustard (Uracil Mustard Capsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine (Oncovin®); vinorelbine

(Navelbine®); and zoledronate (Zometa®).

In another embodiment, Compound I may also be useful for treating cancer in combination with Gleevec® or dasatinib or nilotinib.

In another particular embodiment of the present invention, Compound I can be administered in combination with camptothecin.

In another particular embodiment of the present invention, Compound I can be administered in combination with doxorubicin.

In another particular embodiment of the present invention, Compound I can be administered in combination with idarubicin. In another particular embodiment of the present invention, Compound I can be administered in combination with cisplatin. In another particular embodiment of the present invention, Compound I can be administered in combination with taxol.

In another particular embodiment of the present invention, Compound I can be administered in combination with taxotere. In another particular embodiment of the present invention, Compound I can be administered in combination with vincristine.

In another particular embodiment of the present invention, Compound I can be administered in combination with tarceva.

In another particular embodiment of the present invention, Compound I can be administered in combination with a MEK inhibitor.

In another particular embodiment of the present invention, Compound I can be administered in combination with a KSP inhibitor.

In another particular embodiment of the present invention, Compound I can be administered in combination with vorinostat. In another particular embodiment of the present invention, Compound I can be administered in combination with SN-38.

In another particular embodiment of the present invention, Compound I can be administered in combination with carboplatin.

The combination therapy can provide a therapeutic advantage in view of the differential toxicity associated with the two treatment modalities. For example, treatment with Aurora kinase inhibitors can lead to a particular toxicity that is not seen with the anti-cancer agent, and vice versa. As such, this differential toxicity can permit each treatment to be administered at a dose at which said toxicities do not exist or are minimal, such that together the combination therapy provides a therapeutic dose while avoiding the toxicities of each of the constituents of the combination agents. Furthermore, when the therapeutic effects achieved as a result of the combination treatment are enhanced or synergistic, for example, significantly better than additive therapeutic effects, the doses of each of the agents can be reduced even further, thus lowering the associated toxicities to an even greater extent. Aurora Kinase Inhibitors Aurora kinase inhibitors include, but are not limited to: Hesperadin, ZM447439,

AV445, AT-9283, AX39459, MKC1693, MLN-8054, MP-235, SNS314, AZD-1152, R763, CYC-116 and

In a further embodiment, Aurora kinase inhibitors are: Hesperadin, ZM447439, AV445, AT-9283, AX39459, MKC1693, MLN-8054, MP-235, SNS314, AZD-1152, R763, CYC-116 and Compound I. Alkylating Agents

Alkylating agents react with nucleophilic residues, such as the chemical entities on the nucleotide precursors for DNA production. They affect the process of cell division by alkylating these nucleotides and preventing their assembly into DNA.

Examples of alkylating agents include, but are not limited to, bischloroethylamines (nitrogen mustards, e.g., chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard), aziridines (e.g., thiotepa), alkyl alkone sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, streptozocin), nonclassic alkylating agents (altretamine, dacarbazine, and procarbazine), platinum compounds (carboplastin and cisplatin). These compounds react with phosphate, amino, hydroxyl, sulfihydryl, carboxyl, and imidazole groups.

Under physiological conditions, these drugs ionize and produce positively charged ion that attach to susceptible nucleic acids and proteins, leading to cell cycle arrest and/or cell death. The alkylating agents are cell cycle phase nonspecific agents because they exert their activity independently of the specific phase of the cell cycle. The nitrogen mustards and alkyl alkone sulfonates are most effective against cells in the Gl or M phase. Nitrosoureas, nitrogen mustards, and aziridines impair progression from the Gl and S phases to the M phases. Chabner and Collins eds. (1990) "Cancer Chemotherapy: Principles and Practice", Philadelphia: JB Lippincott.

The alkylating agents are active against wide variety of neoplastic diseases, with significant activity in the treatment of leukemias and lymphomas as well as solid tumors.

Clinically this group of drugs is routinely used in the treatment of acute and chronic leukemias; Hodgkin's disease; non-Hodgkin's lymphoma; multiple myeloma; primary brain tumors; carcinomas of the breast, ovaries, testes, lungs, bladder, cervix, head and neck, and malignant melanoma.

The major toxicity common to all of the alkylating agents is myelosuppression.

Additionally, gastrointestinal adverse effects of variable severity occur commonly and various organ toxicities are associated with specific compounds. Black and Livingston (1990) Drugs 39:

489-501; and 39: 652-673.

Antibiotics

Antibiotics (e.g., cytotoxic antibiotics) act by directly inhibiting DNA or RNA synthesis and are effective throughout the cell cycle. Examples of antibiotic agents include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin and anthracenedione), mitomycin C, bleomycin, dactinomycin, plicatomycin. These antibiotic agents interfere with cell growth by targeting different cellular components. For example, anthracyclines are generally believed to interfere with the action of DNA topoisomerase II in the regions of transcriptionally active DNA, which leads to DNA strand scissions. Bleomycin is generally believed to chelate iron and forms an activated complex, which then binds to bases of DNA, causing strand scissions and cell death.

The antibiotic agents have been used as therapeutics across a range of neoplastic diseases, including carcinomas of the breast, lung, stomach and thyroids, lymphomas, myelogenous leukemias, myelomas, and sarcomas. The primary toxicity of the anthracyclines within this group is myelosuppression, especially granulocytopenia. Mucositis often accompanies the granulocytopenia and the severity correlates with the degree of myelosuppression. There is also significant cardiac toxicity associated with high dosage administration of the anthracyclines.

Antimetabolic Agents Antimetabolic agents (i.e., antimetabolites) are a group of drugs that interfere with metabolic processes vital to the physiology and proliferation of cancer cells. Actively proliferating cancer cells require continuous synthesis of large quantities of nucleic acids, proteins, lipids, and other vital cellular constituents.

Many of the antimetabolites inhibit the synthesis of purine or pyrimidine nucleosides or inhibit the enzymes of DNA replication. Some antimetabolites also interfere with the synthesis of ribonucleosides and RNA and/or amino acid metabolism and protein synthesis as well. By interfering with the synthesis of vital cellular constituents, antimetabolites can delay or arrest the growth of cancer cells. Examples of antimetabolic agents include, but are not limited to, fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatiπ, fludarabine phosphate, cladribine (2- CDA), asparaginase, and gemcitabine.

Antimetabolic agents have widely used to treat several common forms of cancer including carcinomas of colon, rectum, breast, liver, stomach and pancreas, malignant melanoma, acute and chronic leukemia and hair cell leukemia. Many of the adverse effects of antimetabolite treatment result from suppression of cellular proliferation in mitotically active tissues, such as the bone marrow or gastrointestinal mucosa. Patients treated with these agents commonly experience bone marrow suppression, stomatitis, diarrhea, and hair loss. Chen and Grem (1992) Curr. Opin. Oncol 4: 1089-1098. Hormonal Agents

The hormonal agents are a group of drug that regulate the growth and development of their target organs. Most of the hormonal agents are sex steroids and their derivatives and analogs thereof, such as estrogens, progestogens, anti-estrogens, androgens, anti- androgens and progestins. These hormonal agents may serve as antagonists of receptors for the sex steroids to down regulate receptor expression and transcription of vital genes. Examples of such hormonal agents are synthetic estrogens (e.g., diethylstibestrol), antiestrogens (e.g., tamoxifen, toremifene, fluoxymesterol and raloxifene), antiandrogens (bicalutamide, nilutamide, flutamide), aromatase inhibitors (e.g., aminoglutethimide, anastrozole and tetrazole), luteinizing hormone release hormone (LHRH) analogues, ketoconazole, goserelin acetate, leuprolide, megestrol acetate and mifepristone.

Hormonal agents are used to treat breast cancer, prostate cancer, melanoma and meningioma. Because the major action of hormones is mediated through steroid receptors, 60% receptor-positive breast cancer responded to first-line hormonal therapy; and less than 10% of receptor-negative tumors responded. The main side effect associated with hormonal agents is flare. The frequent manifestations are an abrupt increase of bony pain, erythema around skin lesions, and induced hypercalcemia.

Specifically, progestogens are used to treat endometrial cancers, since these cancers occur in women that are exposed to high levels of oestrogen unopposed by progestogen. Antiandrogens are used primarily for the treatment of prostate cancer, which is hormone dependent. They are used to decrease levels of testosterone, and thereby inhibit growth of the tumor. Hormonal treatment of breast cancer involves reducing the level of oestrogen- dependent activation of oestrogen receptors in neoplastic breast cells. Anti-oestrogens act by binding to oestrogen receptors and prevent the recruitment of coactivators, thus inhibiting the oestrogen signal. LHRH analogues are used in the treatment of prostate cancer to decrease levels of testosterone and so decrease the growth of the tumor.

Aromatase inhibitors act by inhibiting the enzyme required for hormone synthesis. In post-menopausal women, the main source of oestrogen is through the conversion of androstenedione by aromatase. Plant-derived Agents

Plant-derived agents are a group of drugs that are derived from plants or modified based on the molecular structure of the agents. They inhibit cell replication by preventing the assembly of the cell's components that are essential to cell division.

Examples of plant derived agents include vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinzolidine and vinorelbine), podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)), taxanes (e.g., paclitaxel and docetaxel). These plant-derived agents generally act as antimitotic agents that bind to tubulin and inhibit mitosis. Podophyllotoxins such as etoposide are believed to interfere with DNA synthesis by interacting with topoisomerase II, leading to DNA strand scission. Plant-derived agents are used to treat many forms of cancer. For example, vincristine is used in the treatment of the leukemias, Hodgkin's and non-Hodgkin's lymphoma, and the childhood tumors neuroblastoma, rhabdomyosarcoma, and Wilms' tumor. Vinblastine is used against the lymphomas, testicular cancer, renal cell carcinoma, mycosis fungoides, and Kaposi's sarcoma. Doxetaxel has shown promising activity against advanced breast cancer, non- small cell lung cancer (NSCLC), and ovarian cancer.

Etoposide is active against a wide range of neoplasms, of which small cell lung cancer, testicular cancer, and NSCLC are most responsive.

The plant-derived agents cause significant side effects on patients being treated. The vinca alkaloids display different spectrum of clinical toxicity. Side effects of vinca alkaloids include neurotoxicity, altered platelet function, myelosuppression, and leukopenia. Paclitaxel causes dose-limiting neutropenia with relative sparing of the other hematopoietic cell lines. The major toxicity of the epipophyllotoxins is hematologic (neutropenia and thrombocytopenia). Other side effects include transient hepatic enzyme abnormalities, alopecia, allergic reactions, and peripheral neuropathy. Biologic Agents

Biologic agents are a group of biomolecules that elicit cancer/tumor regression when used alone or in combination with chemotherapy and/or radiotherapy. Examples of biologic agents include immuno-modulating proteins such as cytokines, monoclonal antibodies against tumor antigens, tumor suppressor genes, and cancer vaccines.

Cytokines possess profound immunomodulatory activity. Some cytokines such as interleukin-2 (IL-2, aldesleukin) and interferon-a (IFN-a) demonstrated antitumor activity and have been approved for the treatment of patients with metastatic renal cell carcinoma and metastatic malignant melanoma. IL-2 is a T-cell growth factor that is central to T-cell-mediated immune responses. The selective antitumor effects of IL-2 on some patients are believed to be the result of a cell-mediated immune response that discriminate between self and nonself.

Ihterferon-α includes more than 23 related subtypes with overlapping activities. IFN-a has demonstrated activity against many solid and hematologic malignancies, the later appearing to be particularly sensitive.

Examples of interferons include, interferon-α, interferon-β (fibroblast interferon) and interferon-γ (fibroblast interferon). Examples of other cytokines include erythropoietin (epoietin- α), granulocyte-CSF (filgrastϊn), and granulocyte, macrophage-CSF (sargramostim). Other immuno-modulating agents other than cytokines include bacillus Calmette-Guerin, levamisole, and octreotide, a long-acting octapeptide that mimics the effects of the naturally occuring hormone somatostatin.

Furthermore, the anti-cancer treatment can comprise treatment by immunotherapy with antibodies and reagents used in tumor vaccination approaches. The primary drugs in this therapy class are antibodies, alone or carrying e.g. toxins or chemotherapeutics/cytotoxics to cancer cells. Monoclonal antibodies against tumor antigens are antibodies elicited against antigens expressed by tumors, preferably tumor-specific antigens. For example, monoclonal antibody HERCEPTIN® (trastuzumab) is raised against human epidermal growth factor receptor2 (HER2) that is overexpressed in some breast tumors including metastatic breast cancer. Overexpression of HER2 protein is associated with more aggressive disease and poorer prognosis in the clinic. HERCEPTIN® is used as a single agent for the treatment of patients with metastatic breast cancer whose tumors over express the HER2 protein.

Another example of monoclonal antibodies against tumor antigens is RITUXAN® (rituximab) that is raised against CD20 on lymphoma cells and selectively deplete normal and malignant CD20+ pre-B and mature B cells.

RITUXAN is used as single agent for the treatment of patients with relapsed or refractory low-grade or follicular, CD20+, B cell non-Hodgkin's lymphoma. MYELOTARG® (gemtuzumab ozogamϊcin) and CAMPATH® (alemtuzumab) are further examples of monoclonal antibodies against tumor antigens that may be used.

Tumor suppressor genes are genes that function to inhibit the cell growth and division cycles, thus preventing the development of neoplasia. Mutations in tumor suppressor genes cause the cell to ignore one or more of the components of the network of inhibitory signals, overcoming the cell cycle checkpoints and resulting in a higher rate of controlled cell growth- cancer. Examples of the tumor suppressor genes include Duc-4, NF-I, NF-2, RB, p53, WTl, BRCAl and BRCA2.

DPC4 is involved in pancreatic cancer and participates in a cytoplasmic pathway that inhibits cell division. NF-I codes for a protein that inhibits Ras, a cytoplasmic inhibitory protein. NF-I is involved in neurofibroma and pheochromocytomas of the nervous system and myeloid leukemia. NF-2 encodes a nuclear protein that is involved in meningioma, schwanoma, and ependymoma of the nervous system. RB codes for the pRB protein, a nuclear protein that is a major inhibitor of cell cycle. RB is involved in retinoblastoma as well as bone, bladder, small cell lung and breast cancer. P53 codes for p53 protein that regulates cell division and can induce apoptosis. Mutation and/or inaction of p53 is found in a wide ranges of cancers. WTI is involved in Wilms' tumor of the kidneys. BRCAl is involved in breast and ovarian cancer, and BRCA2 is involved in breast cancer. The tumor suppressor gene can be transferred into the tumor cells where it exerts its tumor suppressing functions.

Cancer vaccines are a group of agents that induce the body's specific immune response to tumors. Most of cancer vaccines under research and development and clinical trials are tumor-associated antigens (TAAs). TAAs are structures (i.e., proteins, enzymes or carbohydrates) that are present on tumor cells and relatively absent or diminished on normal cells. By virtue of being fairly unique to the tumor cell, TAAs provide targets for the immune system to recognize and cause their destruction. Examples of TAAs include gangliosides (GM2), prostate specific antigen (PSA), α-fetoprotein (AFP), carcinoembryonic antigen (CEA)

(produced by colon cancers and other adenocarcinomas, e.g., breast, lung, gastric, and pancreatic cancers), melanoma-associated antigens (MART-I, gap 100, MAGE 1,3 tyrosinase), papillomavirus E6 and E7 fragments, whole cells or portions/lysates of autologous tumor cells and allogeneic tumor cells.

Other Therapies

Recent developments have introduced, in addition to the traditional cytotoxic and hormonal therapies used to treat cancer, additional therapies for the treatment of cancer.

For example, many forms of gene therapy are undergoing preclinical or clinical trials. hi addition, approaches are currently under development that are based on the inhibition of tumor vascularization (angio genesis). The aim of this concept is to cut off the tumor from nutrition and oxygen supply provided by a newly built tumor vascular system. hi addition, cancer therapy is also being attempted by the induction of terminal differentiation of the neoplastic cells. Suitable differentiation agents include the compounds disclosed in any one or more of the following references, the contents of which are incorporated by reference herein. a) Polar compounds Friend, C, Scher, W., Holland, J. W., and Sato, T. (1971)

Proc. Natl. Acad. ScL (USA) 68: 378-382; Tanaka, M., Levy, J., Terada, M., Breslow, R.,

Rifkind, R. A., and Marks, P. A. (1975) Proc. Natl. Acad. ScL (USA) 72: 1003-1006; Reuben, R.

C, Wife, R. L-, Breslow, R., Rifkind, R. A., and Marks, P. A. (1976) Proc. Natl. Acad. ScL

(USA) 73: 862-866); b) Derivatives of vitamin D and retinoic acid (Abe, E., Miyaura, C, Sakagami, H.,

Takeda, M., Konno, K., Yamazaki, T., Yoshika, S., and Suda, T. (1981) Proc. Natl. Acad. ScL

(XJSA) 78: 4990-4994; Schwartz, E. L., Snoddy, J. R., Kreutter, D., Rasmussen, H., and

Sartorelli, A. C. (1983) Proc. Am, Assoc. Cancer Res. 24: 18; Tanenaga, K., Hozumi, M., and

Sakagami, Y. (1980) Cancer Res. 40: 914-919); c) Steroid hormones (Lotem, J. and Sachs, L. (1975) Int. J. Cancer 15: 731 -740); d) Growth factors (Sachs, L. (1978) Nature (Lond.) 274: 535, Metcalf, D. (1985) Science, 229: 16-22); e) Proteases (Scher, W., Scher, B. M., and Waxman, S. (1983) Exp. Hematol. 11: 490-498; Scher, W., Scher, B. M., and Waxman, S. (1982) Biochem. & Biophys. Res. Comm. 109: 348-354); f) Tumor promoters (Huberman, E. and Callaham, M. F. (1979) Proc. Natl. Acad. ScL (USA) 76: 1293-1297; Lottem, J. and Sachs, L. (1979) Proc. Natl. Acad. ScL (USA) 76: 5158-5162); and g) Inhibitors of DNA or RNA synthesis (Schwartz, E. L. and Sartorelli, A. C. (1982) Cancer Res. 42: 2651-2655, Terada, M., Epner, E., Nudel, U., Salmon, J., Fibach, E., Rifkind, R. A., and Marks, P. A. (1978) Proc. Natl. Acad. ScL (USA) 75: 2795-2799; Morin, M. J. and Sartorelli- A. C. (1984) Cancer Res. 44: 2807-2812; Schwartz, E. L., Brown, B. J., Nierenberg, M., Marsh, J. C, and Sartorelli, A. C. (1983) Cancer Res. 43: 2725-2730; Sugano, H., Furusawa, M., Kawaguchi, T., and Ikawa, Y. (1973) Bibl. Hematol 39: 943-954; Ebert, P. S., Wars, L, and Buell, D. N. (1976) Cancer Res. 36: 1809-1813; Hayashi, M., Okabe, J., and Hozumi, M. (1979) Gann 70: 235-238),

The use of all of these approaches in combination with Aurora kinase inhibitors, e.g. Compound I, is within the scope of the present invention. Modes and Doses of Administration

The methods of the present invention comprise administering to a patient in need thereof a first amount of an Aurora kinase inhibitor, e.g., Compound I, in a first treatment procedure, and a second amount of an anti-cancer agent in a second treatment procedure. The first and second treatments together comprise a therapeutically effective amount.

"Patient" as that term is used herein, refers to the recipient of the treatment. Mammalian and non-mammalian patients are included. In a specific embodiment, the patient is a mammal, such as a human, canine, murine, feline, bovine, ovine, swine or caprine. In a particular embodiment, the patient is a human. Administration of the Aurora Kinase Inhibitor Routes of Administration

The Aurora kinase inhibitor (e.g. Compound I), can be administered by any known administration method known to a person skilled in the art. Examples of routes of administration include but are not limited to oral, parenteral, intraperitoneal, intravenous (IV), intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, liposomal, via inhalation, vaginal, intraoccular, via local delivery by catheter or stent, subcutaneous, intraadiposal, intraarticular, intrathecal, or in a slow release dosage form.

For example, the Aurora kinase inhibitors of the invention can be administered in such oral forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Likewise, the Aurora kinase inhibitors can be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. In one embodiment, administration of the Aurora kinase inhibitor is oral administration. In another embodiment, administration of the Aurora kinase inhibitor is intravenous administration.

The Aurora kinase inhibitors can also be administered in the form of a depot injection or implant preparation, which may be formulated in such a manner as to permit a sustained release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants. Implants may employ inert materials such as biodegradable polymers or synthetic silicones, for example, Silastic, silicone rubber or other polymers manufactured by the Dow-Corning Corporation. The Aurora kinase inhibitor 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.

The Aurora kinase inhibitors can also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.

The Aurora kinase inhibitors can also be prepared with soluble polymers as targetable drug carriers. Such polymers can include polyvinlypyrrolidone, pyran copolymer, polyhydroxy-propyl-methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the Aurora kinase inhibitors can be prepared with biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels. Dosafies and Dosage Schedules

The dosage regimen utilizing the Aurora kinase inhibitors can be selected in accordance with a variety of factors including type, species, age, weight, sex and the type of cancer being treated; the severity (i.e., stage) of the cancer to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to treat, for example, to prevent, inhibit (fully or partially) or arrest the progress of the disease. For example, Compound I or any one of the Aurora kinase inhibitors can be administered in a total daily dose of up to 800 mg, The Aurora kinase inhibitor can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID). The Aurora kinase inhibitor can be administered at a total daily dosage of up to 800 mg, e.g., 200 mg, 300 mg, 400 mg, 600 mg or 800 mg, which can be administered in one daily dose or can be divided into multiple daily doses as described above. In one embodiment, the administration is oral.

In addition, the administration can be continuous, i.e., every day, or intermittently. The terms "intermittent" or "intermittently" as used herein means stopping and starting at either regular or irregular intervals. For example, intermittent administration of an Aurora kinase inhibitor may be administration one to six days per week or it may mean administration in cycles (e.g. daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days.

Compound I or any of the Aurora kinase inhibitors may be administered to the patient at a total daily dosage of between 25-4000 mg/m². In one embodiment, the treatment protocol comprises continuous administration (i.e., every day), once, twice or three times daily at a total daily dose in the range of about 200 mg to about 600 mg.

In another embodiment, the treatment protocol comprises intermittent administration of between three to five days a week, once, twice or three times daily at a total daily dose in the range of about 200 mg to about 600 mg.

In another particular embodiment, the Aurora kinase inhibitor is administered continuously once daily at a dose of 400 mg or twice daily at a dose of 200 mg.

In another particular embodiment, the Aurora kinase inhibitor is administered intermittently three days a week, once daily at a dose of 400 mg or twice daily at a dose of 200 mg.

In another particular embodiment, the Aurora kinase inhibitor is administered intermittently four days a week, once daily at a dose of 400 mg or twice daily at a dose of 200 mg.

In another particular embodiment, the Aurora kinase inhibitor is administered intermittently five days a week, once daily at a dose of 400 mg or twice daily at a dose of 200 mg. In another particular embodiment, the Aurora kinase inhibitor is administered continuously once daily at a dose of 600 mg, twice daily at a dose of 300 mg, or three times daily at a dose of 200 mg.

In another particular embodiment, the Aurora kinase inhibitor is administered intermittently three days a week, once daily at a dose of 600 mg, twice daily at a dose of 300 mg, or three times daily at a dose of 200 mg.

In another particular embodiment, the Aurora kinase inhibitor is administered intermittently four days a week, once daily at a dose of 600 mg, twice daily at a dose of 300 mg, or three times daily at a dose of 200 mg. In another particular embodiment, the Aurora kinase inhibitor is administered intermittently five days a week, once daily at a dose of 600 mg, twice daily at a dose of 300 mg, or three times daily at a dose of 200 mg.

In addition, the Aurora kinase inhibitor may be administered according to any of the schedules described above, consecutively for a few weeks, followed by a rest period. For example, the Aurora kinase inhibitor may be administered according to any one of the schedules described above from two to eight weeks, followed by a rest period of one week, or twice daily at a dose of 300 mg for three to five days a week. In another particular embodiment, the Aurora kinase inhibitor is administered three times daily for two consecutive weeks, followed by one week of rest. Intravenously or subcutaneously, the patient would receive the Aurora kinase inhibitor in quantities sufficient to deliver between about 3-1500 mg/m² per day, for example, about 3, 30, 60, 90, 180, 300, 600, 900, 1200 or 1500 mg/m² per day. Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of Aurora kinase inhibitor during one extended period of time or several times a day. The quantities can be administered for one or more consecutive days, intermittent days or a combination thereof per week (7 day period). Alternatively, low volumes of high concentrations of Aurora kinase inhibitor during a short period of time, e.g. once a day for one or more days either consecutively, intermittently or a combination thereof per week (7 day period). For example, a dose of 300 mg/m² per day can be administered for 5 consecutive days for a total of 1500 mg/m² per treatment. In another dosing regimen, the number of consecutive days can also be 5, with treatment lasting for 2 or 3 consecutive weeks for a total of 3000 mg/m² and 4500 mg/m² total treatment. In an embodiment, Compound I can be administered intravenously for a 5-day continuous infusion at 24-64 mg/m²/hr with a cycle duration every 21-28 days. In another embodiment, Compound I can be administered intravenously for a 5-day continuous infusion at 6-12 mg/m²/hr with a cycle duration every 21-28 days. In another embodiment, Compound I can be administered intravenously for a 5-day continuous infusion at 8-10 mg/m /hr with a cycle duration every 21-28 days. In another embodiment, Compound I can be administered intravenously for a 24 hr infusion every 21 days at 32-200 mg/mVhr. In another embodiment, Compound I can be administered intravenously for a 24 hr infusion every 21 days at 32-64 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 48 hr infusion every 21-28 days at 8-12 mg/m²/hr.

In another embodiment, Compound I can be administered intravenously for a 5-day continuous infusion at 24-64 mg/m²/hτ with a cycle duration every 14-28 days. In another embodiment, Compound I can be administered intravenously for a 5-day continuous infusion at 6-12 rng/m²/hr with a cycle duration every 14-28 days. In another embodiment, Compound I can be administered intravenously for a 5-day continuous infusion at 8-10 mg/m²/hr with a cycle duration every 14-28 days. In another embodiment, Compound I can be administered intravenously for a 24 hr infusion every 14-21 days at 32-200 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 24 hr infusion every 14-21 days at 32-64 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 48 hr infusion every 21-28 days at 8-12 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 6 hr infusion every 14-21 days at 32-200 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 6 hr infusion every 14-21 days at 32-64 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 3 hr infusion every 14-21 days at 32-200 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 3 hr infusion every 14-21 days at 32-64 mg/m²/hr.

In another embodiment, Compound I can be administered intravenously for a 5- day continuous infusion at 24-64 mg/m²/hr with a cycle duration every 14-28 days. In another embodiment, Compound I can be administered intravenously for a 5-day continuous infusion at 8-10 mg/m²/hr with a cycle duration every 21 days. In another embodiment, Compound I can be administered intravenously for a 24 hr infusion every 21 days at 64-96 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 24 hr infusion every 21 days at 32-64 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 6 hr infusion every 14-21 days at 32-200 mg/m²/hr. In another embodiment, Compound I can be administered intravenously for a 3 hr infusion every 14-21 days at 32-200 mg/m²/hr. Typically, an intravenous formulation may be prepared which contains a concentration of Aurora kinase inhibitor of between about 1.0 mg/mL to about 10 mg/mL, e.g. 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL and 10 mg/mL and administered in amounts to achieve the doses described above. In one example, a sufficient volume of intravenous formulation can be administered to a patient in a day such that the total dose for the day is between about 300 and about 1500 mg/m².

Subcutaneous formulations, preferably prepared according to procedures well known in the art at a pH in the range between about 5 and about 12, also include suitable buffers and isotonicity agents, as described below. They can be formulated to deliver a daily dose of Aurora kinase inhibitor in one or more daily subcutaneous administrations, e.g., one, two or three times each day.

The Aurora kinase inhibitors can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime.

It should be apparent to a person skilled in the art that the various modes of administration, dosages and dosing schedules described herein merely set forth specific embodiments and should not be construed as limiting the broad scope of the invention. Any permutations, variations and combinations of the dosages and dosing schedules are included within the scope of the present invention. Administration of Anti-Cancer Agent

Any one or more of the specific dosages and dosage schedules of the Aurora kinase inhibitors, is also applicable to any one or more of the anti-cancer agents to be used in the combination treatment.

Moreover, the specific dosage and dosage schedule of the anti-cancer agent can further vary, and the optimal dose, dosing schedule and route of administration will be determined based upon the specific anti-cancer agent that is being used.

Of course, the route of administration of Compound I or any one of the other Aurora kinase inhibitors is independent of the route of administration of the anti-cancer agent. In an embodiment, the administration for Compound I is oral administration. In another embodiment, the administration for Compound I is intravenous administration. Thus, in accordance with these embodiments, Compound I is administered orally or intravenously, and the second agent (anti-cancer agent) can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally. or in a slow release dosage form.

In addition, the Aurora kinase inhibitor and anti-cancer agent may be administered by the same mode of administration, i.e. both agents administered e.g. orally, by IV.

However, it is also within the scope of the present invention to administer the Aurora kinase inhibitor by one mode of administration, e.g. oral, and to administer the anti-cancer agent by another mode of administration, e.g. IV or any other ones of the administration modes described hereinabove.

Commonly used anti-cancer agents and daily dosages usually administered include but are not restricted to:

Antimetabolites: Methotrexate: 20-40 mg/m i.v. or 4-6 mg/m p.o.12000 mg/ra high dose therapy; 6-Mercaptopurine: 100 mg/m²; 6-Thioguanine: 1-2 x 80 mg/m² p.o.;

Pentostatin:4 mg/m² i.v.; Fludarabinphosphate: 25 mg/m² i.v.; Cladribine: 0.14 mg/kg BW i.v.;

5-Fluorouracil: 500-2600 mg/m² i.v.; Capecitabine: 1250 mg/m² p.o.; Cytarabin: 200 mg/m² i.v. or 3000 mg/m² i.v. high dose therapy; Gemcitabine: 800-1250 mg/m² i.v.; Hydroxyurea: 800-

4000 mg/m² p.o. Antibiotics: Actinomycin D: 0.6 mg/m2 i.v.; Daunorubicin: 45-60 mg/m² i.v.;

Doxorubicin: 45-60 mg/m² i.v.; Epirubicin: 60-80 mg/m² i.v.; Idarubicin: 10-12 mg/m² i.v. or 35-

50 mg/m² p.o.; Mitoxantron: 10-12 mg/m² i.v.; Bleomycin: 10-15 mg/m² Lv., i.m., s.c;

Mitomycin C: 10-20 mg/² i.v.; Mnotecan (CPT -111: 350 mg/m² i.v.; Topotecan: 1.5 mg/m² i.v.

Alkylating Agents: Mustargen: 6 mg/m² i.v.; Estramustinphosphate: 150-200 mg/m² i.v. or 480-550 mg/m² p.o.; Melphalan: 8-10 mg/m² i.v. or 15 mg/m² i.v.; Chlorambucil:

3-6 mg/m² i.v.; Prednimustine: 40-100 mg/m² p.o.; Cyclophosphamide: 750-1200 mg/m² i.v. or

50-100 mg/m² p.o.; Ifosfamide: 1500-2000 mg/m² i. v.; Trofosfamide: 25-200 mg/m² p.o.;

Busulfa: 2-6 mg/m² p.o.; Treosulfan: 5000-8000 mg/m² i.v. or 750-1500 mg/m² p.o.; Thiotepa:

12-16 mg/m² i.v.; Carmustin (BCNU): 100 mg/m² i.v.; Lomustin (CCNTJ): 100-130 mg/m² p.o.; Nimustin (ACNU): 90-100 mg/m² i.v.; Dacarbazine (QTIO: 100-375 mg/m² i.v.; Procarbazine:

100 mg/m² p.o.; Cisplatin: 20-120 mg/m² i.v.; Carboplatin: 300-400 mg/m² i.v.

Anti-mitotic agents: Vincristine: 1.5-2 mg/m² i.v.; Vinblastine: 4-8 mg/m² i.v.;

Vindesine: 2-3 mg/m² i.v.; Etoposide (VPl 6): 100-200 mg/m² i.v. or 100 mgp.o.; Teniposide rVM26): 20-30 mg/tn² i.v.; Paclitaxel (TaxoH: 175-250 mg/m² i.v.; Docetaxel (Taxotere^*): 100- 150 mg/m² i.v.

Hormones, Cytokines and Vitamins: Interferon-a : 2-10 x 10⁶ IU/m²;

Prednisone: 40-100 mg/m² p.o.; Dexamethasone: 8-24 mg p.o.; G-CSF: 5-20 μg/kg BW s.c; aIA trans Retinoic Acid: 45 mg/m²; Interleukin-2: 18 x 10⁶ IU/m²; GM-CSF: 250 mg/m²; erythropoietin: 150 IU/kg tiw. Combination Administration

The first treatment procedure, administration of an Aurora kinase inhibitor, can take place prior to the second treatment procedure, i.e., the anti-cancer agent, after the treatment with the anti-cancer agent, at the same time as the treatment with the anti-cancer agent, or a combination thereof. For example, a total treatment period can be decided for the Aurora kinase inhibitor. The anti-cancer agent can be administered prior to onset of treatment with the inhibitor or following treatment with the inhibitor. In addition, anti-cancer treatment can be administered during the period of inhibitor administration but does not need to occur over the entire inhibitor treatment period.

Compound I or any one of the Aurora kinase inhibitors can be administered in accordance with any dose and dosing schedule that, together with the effect of the anti-cancer agent, achieves a dose effective to treat cancer. Pharmaceutical Compositions As described above, the compositions comprising the Aurora kinase inhibitor and/or the anti-cancer agent can be formulated in any dosage form suitable for oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, liposomal, via inhalation, vaginal, or intraocular administration, for administration via local delivery by catheter or stent, or for subcutaneous, intraadiposal, intraarticular, intrathecal administration, or for administration in a slow release dosage form.

The Aurora kinase inhibitor and the anti-cancer agent can be formulated in the same formulation for simultaneous administration, or they can be in two separate dosage forms, which may be administered simultaneously or sequentially as described above.

The invention also encompasses pharmaceutical compositions comprising pharmaceutically acceptable salts of the Aurora kinase inhibitors and/or the anti-cancer agents.

Suitable pharmaceutically acceptable salts of the compounds described herein and suitable for use in the method of the invention, are conventional non-toxic salts and can include a salt with a base or an acid addition salt such as a salt with an inorganic base, for example, an alkali metal salt (e.g., lithium salt, sodium salt, potassium salt, etc.), an alkaline earth metal salt (e.g., calcium salt, magnesium salt, etc.), an ammonium salt; a salt with an organic base, for example, an organic amine salt (e.g., triethylatnine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.) etc.; an inorganic acid addition salt (e.g., hydrochloride, hydrobromide, sulfate, phosphate, etc.); an organic carboxylic or sulfonic acid addition salt (e.g., formate, acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc.); a salt with a basic or acidic amino acid (e.g., arginine, aspartic acid, glutamic acid, etc.) and the like.

The invention also encompasses pharmaceutical compositions comprising hydrates of the Aurora kinase inhibitors and/or the anti-cancer agents. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like.

In addition, this invention also encompasses pharmaceutical compositions comprising any solid or liquid physical form of Compound I or any of the other Aurora kinase inhibitors. For example, the Aurora kinase inhibitors can be in a crystalline form, in amorphous form, and have any particle size. The Aurora kinase inhibitor particles may be micronized, or may be agglomerated, particulate granules, powders, oils, oily suspensions or any other form of solid or liquid physical form.

A 20 mg/mL lactic acid formulation of Compound I may be prepared according to the following steps: Prepare a 20mg/mL concentration of lactic acid in water by weighing 2.Og of lactic acid (either L-lactic acid, D-lactic acid or a racemic mixture) into a 10OmL volumetric flask. Next, weigh out 200mg of Compound I into a 1OmL volumetric flask. Next, add approximately 8mL of the 20mg/mL lactic acid solution to the 1OmL volumetric flask. Next, add the appropriate amount of sugar (for example, 15mg/mL, 50mg/mL or lOOmg/mL, depending on the desired tonicity). Stir the solution until all the drug contents are dissolved. Qs' d the solution to 1OmL with the 20mg/mL lactic acid solution and adjust the pH as needed to aid in solublization.

A 20 mg/mL lactic acid formulation of Compound I (large scale manufacture) may be prepared according to the following steps: Add water for injection equal to 80 percent of batch weight to a suitable mixing vessel. Add the necessary amount of compendial lactic acid (either L-lactic acid, D-lactic acid or a racemic mixture) equaling to 20mg/mL and mix to insure homogeneity. Add Compound I equal to 20mg/mL free base to the vessel and mix to dissolve. Add the appropriate amount of sugar (for example, 15mg/mL, 50mg/mL or lOOmg/mL, depending on the desired tonicity) to the vessel and mix to dissolve. Adjust the pH as needed. Qs' d the batch to final weight with water for injection. Sterile filter and collect the filtered formulation in an appropriate sterile receiving vessel. Fill and stopper the formulation in appropriate vials using aseptic technique in a properly classified area. Cap and terminally sterilize product as required. Store the formulation at the appropriate temperature conditions.

In another embodiment, a 20mg/mL lactic acid formulation of Compound I (large scale manufacture) may be prepared according to the following steps: Add water for injection equal to 80 percent of batch weight to a suitable mixing vessel. Add the necessary amount of compendial lactic acid (either L-lactic acid, D-lactic acid or a racemic mixture) equaling to 20mg/mL and mix to insure homogeneity. Add Compound I equal to 20mg/mL free base to the vessel and mix to dissolve. Add the appropriate amount of sugar (for example, 15mg/mL, 50mg/mL or lOOmg/mL, depending on the desired tonicity) and 0.05mg/ml EDTA (edetate disodium dihydrate) to the vessel and mix to dissolve. Adjust the pH as needed. Qs'd the batch to final weight with water for injection. Sterile filter and collect the filtered formulation in an appropriate sterile receiving vessel. Fill and stopper the formulation in appropriate vials using aseptic technique in a properly classified area. Cap and terminally sterilize product as required. Store the formulation at the appropriate temperature conditions. A lyophilized powder formulation for reconstitution with sterile water for injection may be prepared according to the following steps: Place approximately 90% of the final batch weight of water for injection, USP into a tared, clean agitated vessel. Add the specified amount of mannitol, USP; agitate for at least 15 minutes to dissolve. Add the specified amount of the sulfate salt of Compound I; agitate for at least 30 minutes to dissolve. Add water for injection, USP to the final batch weight. For purposes of this examplary formulation, the final batch contains the following proportions:

Component ms/mL mg/vial

Compound I-sulfate 12.1 91.0

(as equivalent free base) (10.0) (75.0)

Mannitol 50 375

Water for Injection q.s. to q.s. to

1.O mL 7.5 mL

Cool the solution thus prepared to 22° C. and filter through a .22 μm sterilizing filter into appropriate sterile containers. Lyophilize to form a white powder.

The sulfate salt of Compound I (dry powder) may be prepared according to the following steps: To Compound I in solution in ethanol at 70⁰C. (7mg of free base/ml), add one equivalent of concentrated sulfuric acid. Stir the reaction mixture at this temperature 10 minutes. After cooling, collect the precipitate by filtration and dry in a vacuum oven at 50⁰C. overnight. For oral administration, the pharmaceutical compositions can be liquid or solid.

Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.

Any inert excipient that is commonly used as a carrier or diluent may be used in the formulations of the present invention, such as for example, a gum, a starch, a sugar, a cellulosic material, an acrylate, or mixtures thereof. The compositions may further comprise a disintegrating agent and a lubricant, and in addition may comprise one or more additives selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof. Furthermore, the compositions of the present invention may be in the form of controlled release or immediate release formulations. The Aurora kinase inhibitors can be administered as active ingredients in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials or "pharmaceutically acceptable carriers") suitably selected with respect to the intended form of administration. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. For liquid formulations, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solutions or suspensions can also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. In addition, the compositions may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers (e.g., tris-HCI, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), a glidant (e.g., colloidal silicon dioxide), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hyroxypropyhnethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents (e.g., peppermint, methyl salicylate, or orange flavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

The preparation of pharmaceutical compositions that contain an active component is well understood in the art, for example, by mixing, granulating, or tablet-forming processes. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the active agents are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions and the like as detailed above.

The amount of the compound administered to the patient is less than an amount that would cause toxicity in the patient. In the certain embodiments, the amount of the compound that is administered to the patient is less than the amount that causes a concentration of the compound in the patient's plasma to equal or exceed the toxic level of the compound. In an embodiment, the concentration of the compound in the patient's plasma is maintained at about 10 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 25 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 50 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 100 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 500 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 1000 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 2500 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 5000 nM. The optimal amount of the compound that should be administered to the patient in the practice of the present invention will depend on the particular compound used and the type of cancer being treated.

The percentage of the active ingredient and various excipients in the formulations may vary. For example, the composition may comprise between 20 and 90%, preferably between 50-70% by weight of the active agent. For rv administration, Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration can be used as buffers. Sodium chloride solution wherein the pH has been adjusted to the desired range with either acid or base, for example, hydrochloric acid or sodium hydroxide, can also be employed. Typically, a pH range for the intravenous formulation can be in the range of from about 5 to about 12.

Subcutaneous formulations, preferably prepared according to procedures well known in the art at a pH in the range between about 5 and about 12, also include suitable buffers and isotonicity agents. They can be formulated to deliver a daily dose of the active agent in one or more daily subcutaneous administrations. The choice of appropriate buffer and pH of a formulation, depending on solubility of the Aurora kinase inhibitor to be administered, is readily made by a person having ordinary skill in the art. Sodium chloride solution wherein the pH has been adjusted to the desired range with either acid or base, for example, hydrochloric acid or sodium hydroxide, can also be employed in the subcutaneous formulation. Typically, a pH range for the subcutaneous formulation can be in the range of from about 5 to about 12. The compositions of the present invention can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime. The present invention also provides in-vitro methods for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells, by contacting the cells with a first amount of Compound I or a pharmaceutically acceptable salt or hydrate thereof, and a second amount of an anti-cancer agent, wherein the first and second amounts together comprise an amount effective to induce terminal differentiation, cell growth arrest of apoptosis of the cells.

Although the methods of the present invention can be practiced in vitro, it is contemplated that the preferred embodiment for the methods of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells will comprise contacting the cells in vivo, i.e., by administering the compounds to a subject harboring neoplastic cells or tumor cells in need of treatment.

As such, the present invention also provides methods for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells in a subject by administering to the subject a first amount of Compound I or a pharmaceutically acceptable salt or hydrate thereof, in a first treatment procedure, and a second amount of an anti-cancer agent in a second treatment procedure, wherein the first and second amounts together comprise an amount effective to induce terminal differentiation, cell growth arrest of apoptosis of the cells.

The invention is illustrated in the Examples that follow. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to limit in any way the invention as set forth in the claims which follow thereafter.

EXAMPLE 1: Synthesis of Compound I

4,6-Dichloropyrimidine-2-methylsulfone (A)

Prepared by methods substantially similar to those set forth in Koppell et al, JOC, 26, 1961, 792, in the following manner. To a stirred solution of 4,6-dichloro-2- (methylthio)pyrirnidine (50 g, 0.26 mol) in dichloromethane (1 L) at 0⁰C was added meta- chloroperoxybenzoic acid (143.6 g, 0.64 mol) over a period of 20 minutes. The solution was allowed to warm to room temperature and was stirred for 4 hours. The mixture was diluted with dichloromethane (1.5 L) and then treated sequentially with 50% Na₂S₂O₃ / NaHCO₃ solution (2 x 200 ml), sat. NaHCO₃ solution (4 x 300 ml), and brine (200 ml) then dried (MgSO₄). The solvent was removed in vacuo to afford an off-white solid, which was redissolved in EtOAc (IL) and treated sequentially with sat. NaHCO₃ solution (3 x 300 ml), and brine (100 ml) then dried (MgSO₄). The solvent was removed in vacuo to afford the title compound (A) as a white solid (55.6 g, 96% yield). ¹H NMR CDCl₃ δ 3.40 (3H, s, CH3), 7.75 (IH. s. ArH).

Cyclopropane carboxylic acid [4-(4,6-dichloro-pyrimidin-2-ylsulphanyl)-phenyl]- amide (O

A suspension of compound A (1Og, 44.04 mmol) and cyclopropane carboxylic acid (4-mercapto~phenyl)-amide (B, 8.51 g, 44.04 mmol) in t-butanol (300 ml) was degassed by evacuation, then flushing with nitrogen. The mixture was stirred at 90° C under nitrogen atmosphere for 1 hour then the solvent was removed in vacuo. The residue was dissolved in ethyl acetate (600 ml) and washed with an aqueous solution of potassium carbonate and sodium chloride. The organic extract was dried over magnesium sulphate, concentrated to a low volume and allowed to crystallize. The product C was collected as colourless crystals, (11.15 g, 74%). ¹H-NMR DMSO-d⁶, δ 0.82-0.89 (4H, m), 1.80-1.88 (IH, m), 7.55 (2H, d), 7.70-7.76 (3H, m), 10.49 (IH, s); M+H, 340. Cyclopropane carboxylic acid {4-[4-chloro-6-(5-methyl-2H-pyrazol-3-ylamino) - pyrimidin-2-ylsulphanyl] -phenyl} amide (D)

A mixture of compound C (1.0 g, 2.94 mmol)and 3-amino-5-methylpyrazole (314 mg, 3.23 mmol) in dimethylforrnamide (6 ml) was treated with diisopropylethylamine (0.614 ml, 3.53 mmol) and sodium iodide (530 mg, 3.53 mmol). The mixture was stirred under nitrogen at 85 ° for 4 hours, cooled to room temperature and diluted with ethyl acetate. The solution was washed with water (x 4), dried over magnesium sulphate and concentrated to 5 ml to afford, upon crystallization and harvesting of colourless crystals, the title compound D (920 mg, 78%). ¹H-NMR DMSO-d⁶, δ 0.80-0.87 (4H, m), 1.77-1.85 (IH, m), 1.92 (IH, s), 5.24 (IH, br s), 6.47 (IH, br s), 7.55 (2H, d), 7.70-7.80 (2H, m), 10.24 (IH, s), 10.47 (IH, s), 11.92 (IH, s). Cyclopropane carboxylic acid {4-[4-(4-methyl-piperazin-l-yl)-6-(5-methyl-2H- ^" pyrazol-3-ylaminoVpyrimidin-2-ylsulphanyl1-ρhenyD-amide (l)

Compound D (2.373 g, 5.92 mmol) was treated with N-methylpiperazine (10 ml) and the mixture stirred at 110° for 2 hours. The excess N-methylpiperazine was removed in vacuo then the residue was dissolved in ethyl acetate, washed with aqueous sodium bicarbonate solution, dried over magnesium sulphate, and concentrated. The residue was crystallised from methanol to give colourless crystals of desired product I (1.82 g, 66%), ¹H-NMR DMSO-d⁶, δ 0.81 (4H, d), 1.79 (IH, m), 2.01 (3H, s), 2.18 (3H, s), 2.30 (4H, m), 3.35 (masked signal), 5.42 (IH, s), 6.02 (IH, br s), 7.47 (2H, d), 7.69 (2H, d), 9.22 (IH, s). 10.39 (IH, s), 11.69 (IH, s).

EXAMPLE 2

Methods Flow cytometry analysis (FAGS^'):

For co-treatment of VX-680 with a second agent, cells are seeded at 5 x 10⁴ cells/well in 6-well plates, grown overnight, and treated with VX-680 in combination with a chemotherapeutic or targeted agent for 72 hrs. For sequential treatment, cells are seeded at 4 x 10⁴ cells/well, grown overnight, and treated either with VX-680 or with a chemotherapeutic/targeted agent (single agent treatment). After 24 hrs, the supernatant is removed and the cells are washed with media 2 times. The supernatant from each wash is collected, pooled, and centrifuged. The pellet from centrifugation is resuspended in DMEM and added back to the well. The cells are then cultured in the presence of the second agent (either VX-680 or the chemotherapeutic/targeted agent) for an additional 72 hrs. At the end of treatment, cells are harvested, stained with propidium idodide and analyzed by FACS. The combination effects are evaluated by the Bliss additivism model (Tallarida, RJ. Dose-response analysis. Drug synergism and dose effect data-analysis (Borisy et al. (2003) Proc. Natl. Acad. Sci. U.S. A. 100, 7977-7982). Cell Titer GIo viability assay:

Logarithmically growing cells are seeded in black clear-bottom 96-well plates (Costar) at 5000 cells / well and allowed to attach overnight in 50 uL of DMEM + 10% inactivated fetal bovine serum (iFBS). For simultaneous combinations, VX-680 and a chemotherapeutic/targeted agent are added separately at 4X final concentrations in 25 uL. For sequential combinations either VX-680 or the chemotherapeutic/targeted agent is added at 2X final concentration in 50 uL for 24 hrs after which time the cells were washed 2X in compound free media and then exposed to the second agent at IX final concentration in 100 uL. Cell viability was assessed by adding 50 uL of Cell Titer GIo (Promega) reagent to each well and measuring luminescence on a Wallac Microbeta plate reader (Millipore). ViaLieht viability assay: Logarithmically growing cells were seeded, onto white clear-bottom 96-well plates (Costar) at 4000 cells / well and allowed to attach overnight in 100 uL of RPMI complete media supplemented with 10% fetal bovine serum, ImM Na pyruvate, 1OmM HEPES, pencillin / streptomycin, 0.225% glucose and 2mM glutamax. Combinations were set up as a matrix with 5 concentrations of VX-680 (including the DMSO control) and 5 concentrations of Vorinostat (including the DMSO control). A stock solution of 10 mM Vorinostat in DMSO was first diluted to 5x (of the final concentration) in media and then 25 ul of the 5x drug solution was added to the 100 ul of media in each well. After 72 hrs of treatment, a ViaLight assay (Cambrex cat# LT07-121) for cell viability was performed. Prior to measurement, the assay reagents were warmed to room temperature and the AMR PLUS reagent was reconstituted in assay buffer and equilibrated at room temperature for 15 minutes. Cell plates were removed from the incubator and allowed to cool to room temperature for at least 5 minutes. Cells were then treated for a minimum of 10 min with Cell Lysis Reagent (50 ul / well) followed by addition of AMR PLUS for an additional 2 min. Cell viability was assessed by measuring luminescence on a Victor Spectrophotometer and %ATP content was normalized to vehicle-treated cells. Caspase 3/7 assay:

Logarithmically growing cells were seeded onto black clear-bottom 96-well plates (Costar) at 4000 cells / well and allowed to attach overnight in 100 uL of RPMI complete media supplemented with 10% fetal bovine serum, ImM Na pyruvate, 1OmM HEPES, pencillin / streptomycin, 0.225% glucose and 2mM glutamax. Combinations were set up as a matrix with 5 concentrations of VX-680 (including the DMSO control) and 5 concentrations of Vorinostat (including the DMSO control). A stock solution of 10 mM Vorinostat in DMSO was first diluted to 5x (of the final concentration) in media and then 25 ul of the 5x drug solution was added to the 100 ul of media in each well. After 72 hrs of treatment, a Caspase 3/7 assay (BD Biosciences Clonetech Apo- Alert Fluorescent Caspase 3 kit, cat. No. 630215) for apoptosis was performed. After 72 hrs of treatment, the 96-well cell plates were centrifuged at 1300 rpm for 10 min at 6⁰C. The media was gently aspirated without disturbing the cell monolayer and 50 ul of lysis buffer (Clonetech kit) was added to each well. Cells were incubated at 4⁰C for 10 min and then wrapped in plastic and incubated at -70⁰C overnight. The following day, the plates were thawed to room temperature and 50 ul of 2X reaction buffer containing 200 mM HEPES, 1 mM EDTA, 10 mM DTT, and 10 uM capase 3 substrate (Biosource International #77-934 (5 mgs) or #77-935 (25 mgs)) was added. The plates were re-wrapped in plastic and incubated overnight at 37°C. The following day, the fluorescence from the cleaved Caspase 3 substrate was measured at 400 nm excitation / 505 ran emission on a Spectramax fluorometer. Caspase 3/7 assay (method for Fig 12V.

DLD-I colorectal cells were plated at 10,000 cells per well in a clear-bottom black 96- well plates (Costar) and allowed to attach overnight in 50 ul DMEM + 10% iFBS. VX- 680 was added at 2X final concentration in 50 ul for 24 hrs following which time media was removed and cells were washed 2X in compound free media. SN-38 was then added at IX final concentration in 100 ul media. After 24 hrs media was removed and cells were washed IX in PBS. Cells were incubated in 60 ul of RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS) at 4°C for 10 minutes. Thirty ul of the lysed cell extract was added into 100 ul of caspase assay buffer: (120 mM HEPES, 12 mM EDTA, 30 mM DTT, and 12.5 ug/ml AC-DEVD-AMC (Sigma)). Reaction was left to occur overnight at room temperature. The remaining 30 ul of crude extract was analyzed for protein content with BCA reagent (Pierce). Caspase activity was measured as fluorescene with excitation at 360 nm and emission at 460 nm and corrected for protein content. WST-8 viability assay:

Cells were seeded at 1500 cells/well to 96 well plate in 100 ul of DMEM + 10% heat inactivated fetal bovine serum (FBS) and they were incubated in a humidified atmosphere of 5 % CO₂ at 37°C. Next day, equal volume of medium containing DMSO or VX-680 and/or test compound adjusted to 2-fold of final concentration was added to each wells. Final DMSO concentration of each wells are 0.5%. Cell viability was assessed by adding 20 uL of WST-8 kit (KISHIDA CHEMICAL CO., LTD) reagent to each well followed by 30-60 min incubation in a humidified atmosphere of 5% CO₂ at 37°C and measuring optical density at 450 nm and 650 nm (background) on a SpectraMax Plus384 plate reader (Molecular Devices Corporation). The background optical density was subtracted from the optical density of each well and then the average of blank was subtracted from average of DMSO control and that of each test point. Bliss Additivism Model:

Bliss additivism model was used to evaluate the combination effect. The predicted Bliss additivity (E_A+B) of two agents was calculated by the following equation: EA+B = EA + E_B(1 -E_A/100) where E_A = the inhibitory effect of VX-680 and EB = inhibitory effect of the second test agent. If the observed inhibitory effect of the combination is greater than the predicted E_A+B , the combination effect is determined synergistic. If the observed inhibitory effect of the combination is less than or equal to E_A+B , the combination therapy is shown not to enhance the effect of either single agent. Additional Reference:

Borisy AA, Elliott PJ, Hurst NW, Lee MS, Lehar J, Price ER₅ Serbedzija G, Zimmermann GR, Foley MA, Stockwell BR, & Keith CT. Systematic discovery of multicomponent therapeutics. (2003) Proc. Natl. Acad. Sci. U S A. 100(13), 7977-82.

PBS = phosphate-buffered saline

CONCLUSIONS:

Chemotherapeutics and targeted agents that have been shown to be additive/synergistic with VX-680 include topoisomerase I inhibitors (SN-38 and camptothecin), topoisomerase II inhibitors (doxorubicin and idarubicin), DNA cross-linking agents (cisplatin), anti-microtubule agents (taxol, taxotere, and vincristine), EGFR inhibitors (Iressa and Tarceva), a MEK inhibitor (UO 126), a KSP inhibitor, and a histone deacetylase inhibitor (Vorinostat). Figures 1-13 show specific examples for VX-680 in combination with chemotherapeutics and targeted agents with cancer indications.

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject in need thereof, comprising the sequential or co-administration of Compound I:

or a pharmaceutically acceptable salt or hydrate thereof, and an anti-cancer agent.

2. The method of claim 1, wherein the anti-cancer agent is camptothecin.

3. The method of claim 1, wherein the anti-cancer agent is doxorubicin.

4. The method of claim 1, wherein the anti-cancer agent is idarubicin.

5. The method of claim 1, wherein the anti-cancer agent is cisplatin.

6. The method of claim 1, wherein the anti-cancer agent is taxol.

7. The method of claim 1, wherein the anti -cancer agent is taxotere.

8. The method of claim 1, wherein the anti-cancer agent is vincristine.

9. The method of claim 1 , wherein the anti-cancer agent is tarceva.

10. The method of claim 1, wherein the anti-cancer agent is the MEK inhibitor.

11. The method of claim 1, wherein the anti-cancer agent is a KSP inhibitor.

12. The method of claim 1, wherein the anti-cancer agent is vorinostat.

13. The method of claim 1, wherein the anti-cancer agent is SN-38.

14. The method of claim 1, wherein the anti-cancer agent is carboplatin.