COMBINATION CANCER THERAPY OF WEE1 AND MTOR INHIBITORS

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treating cellular proliferative disorders or disorders associated with WEE1 kinase and mTor activity and for inhibiting WEE1 kinase and mTor activity. BACKGROUND OF THE INVENTION

The phosphatidylinositol-3 -kinase (PI3K) signaling pathway is important for the growth and survival of cancer cells in many different types of human malignancy. See, Granville CA et al, "Handicapping the Race to Develop Inhibitors of the Phosphoinositide 4- Kinase/Akt/Mammalian Target of Rapamycin Pathway," Clin Cancer Res, 2006; 12(3) 679-89. This pathway receives upstream input from ligand-receptor interactions, such as the epidermal growth factor receptor and insulin-like growth factor receptor, and signals through downstream effectors, such as the mammalian target of rapamycin (mTOR). mTOR is a critical downstream effector molecule that regulates the production of proteins critical for cell cycle progression and many other important cellular growth processes. See, Abraham RT and Gibbons, JJ, "The mammalian target of rapamycin signaling pathway: twists and turns in the road to cancer therapy." Clin Cancer Res, 2007; 13(11) 3109-14.

Dysregulation of the PI3 kinase axis is common in human cancer due to overactive growth factor receptor signaling, activating mutations of PI3K, loss of function of the PTEN tumor suppressor, and several other mechanisms that result in activation of mTOR kinase activity. Clinically, successful pharmacological inhibition of the PI3K axis has focused on the upstream growth factor receptors and the downstream effectors of PI3 kinase, such as mTOR. There is now substantial clinical evidence showing that mTOR inhibitors can provide clinical benefit to patients with advanced malignancies.

WEE1 is an essential tyrosine kinase best recognized as a mitotic gatekeeper that phosphorylates and inactivates cyclin dependent kinase 1 (CDK1 = CDC2), the only

indispensible human cyclin dependent kinase (Malumbres, M., and Barbacid, M., Nature Reviews Cancer, 2009, 9(3): 153-166). As cells transition into mitosis, WEE1 activity is reduced, allowing CDK 1 /cyclin Bl to intiate mitotic events. WEE1 is therefore critical for properly timing cell division in unperturbed cells, and loss of WEE 1 results in chromosomal aneuploidy and accumulated DNA damage (Tominaga, Y., et al., Intl. J. Biol. Sci., 2006, 2(4): 161-170). Additionally, WEEl activity can be increased as a result of DNA damage, causing cells to arrest in G2 and allowing for repair of DNA lesions before beginning mitosis (Raleigh, J.M., and O'Connell, M.J., J. Cell Sci., 2000, 113(10): 1727-1736). Recently, WEEl has been shown to be indispensible for genomic integrity specifically as cells traverse S-phase, describing a previously unrecognized role for WEEl in maintaining fidelity of DNA replication (Beck. H., et al, J. Cell Biology, 2010, 188(5):629-638). Knockdown of WEEl by siRNA led to rapid and S-phase specific accumulation of γΗ2ΑΧ, a phosphorylated histone protein that quantitatively represents DNA damage. Interfering with WEEl has been shown to repress cancer cell proliferation and lead to greater anti-tumor effects of DNA-damaging

chemotherapeutics than either single agent alone could achieve.

Cancer therapeutics targeting single nodes in oncogenic pathways historically have had limited clinical activity. This can be attributed to numerous factors, including negative feedback loops, redundant signaling pathways, and resistance mechanisms to name a few.

Combining cancer therapeutics is one approach to circumventing some of these mechanisms with the desired outcome being improved efficacy.

SUMMARY OF THE INVENTION

The instant invention relates generally to methods for treating cancer by administering a therapeutically effective amount of the combination of a WEEl inhibitor and an mTor inhibitor. In one embodiment, the WEEl inhibitor is WEE 1-1 or a pharmaceutically acceptable salt thereof, or WEE 1-2 or a pharmaceutically acceptable salt thereof, and the mTOR inhibitor is ridaforolimus, everolimus, temsirolimus, a rapamycin-analog or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Synergistic interaction of WEEl- 1 and ridaforolimus in 39 solid tumor cell lines (lung (NCIH1650, A427, NCIH520, SKMES1, MSTO, NCIH460, NCIH2122, NCIH23), breast (KPL1, OCUBM, EFM192B, T47D, MDAMB436, ZR751), colon (DLD1, RKO, HT29, SW620, LOVO, SW837, COLO320DM, HCT116), ovarian (SKOV3, A2780, PA1, ES2, OVCAR3, OV90, UWB1289BRCA1, CAOV3, UWB1289, prostate (LNCAP, VCAP) or melanoma (A375, A2058, SKMEL30, UAC062, RPMI7951, HT144)). Observed synergy from the screen is reported for each line as vBliss, which is the volumetric difference between the surface of predicted combination effect and the surface of observed combination effect as illustrated in Figure 2. The Bliss independence (BI) model is used to define the effect of two drugs assumed to act through independent mechanisms. BI is described by the equation Ei = (EA + E _B ) - (EA X E _B ), where Ei is the predicted effect (percentage of inhibition) by the combination of drugs A and B if they were to act additively and independently, and E _A and E _B are the observed effects (percentage of inhibition) of each drug alone, respectively. When observed inhibition exceeds predicted inhibition, the two compounds are considered to act synergistically (Synergy was considered achieved when Vbliss was equal to or greater than 0.1).

Figure 2. The A2780 (Fig. 2A) and SKOV-3 (Fig. 2B) ovarian cancer cell lines were used to validate the synergy observed in the screen. Eight concentrations each of WEEl-1 and Ridaoforolimus were titrated and proliferation at 72 hours was plotted as a fraction of

DMSO treated control cells. The predicted effect on proliferation (using Bliss synergy model) is represented as the upper surface on the plot whereas the observed effect on proliferation is represented by black dots. Observed effects are connected by vertical lines to the corresponding Bliss predicted effect for those concentrations.

Figure 3. In vivo combination benefit of WEEl-1 (Weeli) and ridaforolimus

(mTORi) in A2780 ovarian cancer xenograft model. A2780 tumor bearing mice were treated with a single dose of ridaforolimus (1 mg/kg), WEEl-1 (60 mg/kg), or both. Tumors were processed at either 4 hours or 24 hours post dose for Western blotting against phosphotyrosine 15 of Cdc2 (pCdc2-Y15), total Cdc2, phosphoserine 235/236 of ribosomal protein S6 (pS6rp), total S6rp, cleaved PARP, and GAPDH as a loading control.

Figure 4. Kaplan-Meier plot for survival of A2780 tumor bearing mice in the study. Mice were treated until tumors reached > 1,000 mm3 at which time they were removed from the study. Mice treated with vehicle are illustrated as lines with dots. Mice treated with ridaforolimus are illustrated as lines with squares. Mice treated with WEEl-1 are illustrated as lines with triangles pointing up. Mice treated with WEEl-1 and ridaforolimus are illustrated as lines with triangles pointing down.

Figure 5. Efficacy was tested for the monotherapies of WEEl-1 and ridaforolimus (mTOR-1) at their MTD as well as the drug combination in two xenograft models of ovarian cancer, A2780 (Figure 5A) and SKOV-3 (Figure 5B).

Figure 6. Mean change in body weight loss for each treatment group in the mice described in Figure 5 A, indicating that the combination was tolerated (mean body weight loss did not exceed 15%). DETAILED DESCRIPTION OF THE INVENTION

Applicants have unexpectedly found that synergistic anticancer activity can be achieved by using a WEEl inhibitor with an mTor inhibitor, specifically wherein the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof, or WEE 1-2 or a

pharmaceutically acceptable salt thereof, and the mTOR inhibitor is ridaforolimus or a pharmaceutically acceptable salt thereof. This result is unexpected from the known mechanisms of action of both compounds. WEEl is a central regulator of CDKl/2 and prevents premature CDK activation in unperturbed DNA replication as well as in the presence of DNA damaging agents. In contrast, mTOR is a central mediator of the PI3K pathway and has roles in controlling cell growth and proliferation.

Accordingly, the instant invention relates to methods for treating

cancer by administering a therapeutically effective amount of the combination of a WEEl inhibitor and an mTOR inhibitor. Alternatively, the invention also provides the combination of a WEEl inhibitor and an mTOR inhibitor for the treatment of cancer. In one embodiment, the WEEl inhibitor is WEE 1-1 or a pharmaceutically acceptable salt thereof, or WEE 1-2 or a pharmaceutically acceptable salt thereof, and the mTOR inhibitor is ridaforolimus, everolimus, temsirolimus, a rapamycin-analog or a pharmaceutically acceptable salt thereof.

The instant invention also relates to methods for treating cellular proliferative disorders or disorders associated with WEEl kinase and mTor activity by administering a therapeutically effective amount of the combination of a WEEl inhibitor and an mTOR inhibitor. Alternatively, the invention also provides the combination of a WEEl inhibitor and an mTOR inhibitor for the treatment of cellular proliferative disorders or disorders associated with WEEl kinase and mTor activity.

The instant invention also relates to methods for modulating the activity of WEEl kinase and mTor in a patient by administering a therapeutically effective amount of the combination of a WEEl inhibitor and an mTOR inhibitor. Alternatively, the invention also provides the combination of a WEEl inhibitor and an mTOR inhibitor for modulating the activity of WEEl kinase and mTor.

In an embodiment of the invention, the WEEl inhibitor is WEEl-1 or a pharmaceutically acceptable salt thereof.

In another embodiment of the invention, the mTOR inhibitor is ridaforolimus or a pharmaceutically acceptable salt thereof.

In another embodiment of the invention, the WEEl inhibitor is administered in a dose between 100 mg per day and 250 mg per day. In an embodiment of the invention, the WEE1 inhibitors may be dosed twice a day (BID) over the course of two and a half days (for a total of 5 doses) or once a day (QD) over the course of two days (for a total of 2 doses).

In another embodiment of the invention, the WEE1 inhibitor is administered in a dose between 200 mg per day and 400 mg per day, and preferably 250 -350 mg per day. In an embodiment of the invention, the WEE1 inhibitors may be dosed once a day (QD) over the course of five days.

In another embodiment of the invention, the mTOR inhibitor is administered in a dose between 10 mg and 40 mg. In one embodiment, the ridaforolimus is administered five times a week.

The WEE1 inhibitor and the mTOR inhibitor can be prepared for simultaneous, separate or successive administration.

Reference to the embodiments set forth above is meant to include all combinations of particular groups unless stated otherwise. The meanings of the terms used in this description are described below, and the invention is described in more detail hereinunder.

The term "simultaneous" as referred to in this description means that the pharmaceutical preparations of the invention are administered simultaneously in time.

The term "separate" as referred to in this description means that the pharmaceutical preparations of the invention are administered at different times during the course of a common treatment schedule.

The term "successive" as referred to in this description means that administration of one pharmaceutical preparation is followed by administration of the other pharmaceutical preparation; after administration of one pharmaceutical preparation, the second pharmaceutical preparation can be administered substantially immediately after the first pharmaceutical preparation, or the second pharmaceutical preparation can be administered after an effective time period after the first pharmaceutical preparation; and the effective time period is the amount of time given for realization of maximum benefit from the administration of the first

pharmaceutical preparation.

The term "cancer" as referred to in this description includes various sarcoma and carcinoma and includes solid cancer and hematological malignancy. The solid cancer as referred to herein includes, for example, brain cancer, cervicocerebral cancer, esophageal cancer, thyroid cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, endometrial cancer, lung cancer, stomach cancer, gallbladder/bile duct cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, choriocarcinoma, uterus body cancer, uterocervical cancer, renal pelvis/ureter cancer, bladder cancer, prostate cancer, penis cancer, testicles cancer, fetal cancer, Wilms' tumor, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor, and soft part sarcoma. On the other hand, the hematological cancer includes, for example, acute leukemia, chronic lymphatic leukemia, chronic myelocytic leukemia, polycythemia vera, malignant lymphoma, multiple myeloma, Hodgkin's lymphoma, and non- Hodgkin's lymphoma.

The term "treatment of cancer" as referred to in this description means that an anticancer agent is administered to a cancer patient so as to inhibit the growth of the cancer cells in the patient. In an embodiment, the treatment results in cancer growth regression and/or a reduction in the size of a detectable tumor. In an embodiment, the treatment results in complete disappearance of cancerous tumor(s). mTOR Inhibitors

The mTOR inhibitors in current clinical development are structural analogs of rapamycin. The mTOR inhibitors of the instant invention include ridaforolimus, temsirolimus, everolimus, a rapamycin-analog and combinations thereof.

Ridaforolimus, also known as AP 23573 and deforolimus, is a unique, non- prodrug analog of rapamycin that has antiproliferative activity in a broad range of human tumor cell lines in vitro and in murine tumor xenograft models utilizing human tumor cell lines.

Ridaforolimus has been administered to patients with advanced cancer. Thus far, these trials have demonstrated that ridaforolimus is generally well-tolerated with a predictable and manageable adverse event profile, and possesses anti-tumor activity in a broad range of cancers.

A description and preparation of ridaforolimus is described in U.S. Patent No. 7,091,213 to

Ariad Gene Therapeutics, Inc., which is hereby incorporated by reference in its entirety.

Temsirolimus, also known as Torisel®, is currently marketed for the treatment of renal cell carcinoma. A description and preparation of temsirolimus is described in U.S. Patent

No. 5,362,718 to American Home Products Corporation, which is hereby incorporated by reference in its entirety.

Everolimus, also known as Certican® or RAD001, marketed by Novartis, has greater stability and enhanced solubility in organic solvents, as well as more favorable pharmokinetics with fewer side effects, than rapamycin (sirolimus). Everolimus has been used in conjunction with microemulsion cyclosporin (Neoral®, Novartis) to increase the efficacy of the immunosuppressive regime.

WEE1 Inhibitors In an embodiment of the invention, the WEE1 inhibitor used in the methods of the instant invention is -1, the structure of which is as shown below.

WEEl-1

WEEl-1 is a WEE1 inhibitor which may be useful for the treatment of cancer.

WEEl-1 is also known as 2-allyl-l-[6-(l-hydroxy-l-methylethyl)pyridin-2-yl]-6-{[4-(4 - methylpiperazin- 1 -yl)phenyl] amino} - 1 ,2-dihydro-3H-pyrazolo[3 ,4-d]pyrimidin-3-one. WEE 1-1 has been described in U.S. Patent No.7, 834,019, and in PCT International Publications

WO2007/126122, WO 2007/126128 and WO2008/153207, which are incorporated by reference herein in their entirety. Crystalline forms of WEEl-1 are described in US Publication US2010- 0124544 and PCT International Publication WO2011/034743, which are incorporated by reference herein in their entirety.

In an embodiment of the invention, the WEE1 inhibitor of the instant invention is WEE 1-2, the structure of which is as shown below.

WEE 1-2

WEE 1-2 is a WEE1 inhibitor which may be useful for the treatment of cancer. WEE1-2 is also known as 3-(2,6-dichlorophenyl)-4-imino-7-[(2'-methyl-2',3'-dihydro- H- spiro[cyclopropane-l,4'-isoquinolin]-7'-yl)amino]-3,4-dihydr opyrimido[4,5-d]pyrimidin-2(lH)- one. WEE 1-2 has been described in PCT International Publication WO2008/153207 and US Publication US2011-0135601, which are incorporated by reference herein in their entirety.

Crystalline forms of WEE 1-2 are described in International Publication WO2009/151997 and US Publication US2011-0092520, which are incorporated by reference herein in their entirety. The compounds used in the methods of the instant invention may also exist as various crystalline forms, amorphous substances, pharmaceutically acceptable salts, hydrates and solvates. Further, the compounds may be provided as prodrugs. In general, such prodrugs are functional derivatives of the inhibitors used in the methods of the instant invention that can be readily converted into compounds that are needed by living bodies. Accordingly, in the methods of treatment of various cancers of the invention, the term "administration" includes not only the administration of a specific compound but also the administration of a compound which, after administered to patients, can be converted into the specific compound in the living body.

Conventional methods for selection and production of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985, which is referred to herein and is entirely incorporated herein as a part of the present description. Metabolites of the compound may include active compounds that are produced by putting the compound in a biological environment, and are within the scope of the compounds described in the invention.

The compounds used in the methods of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119- 1190), and may occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, all such stereoisomers being included in the present invention. In addition, the compounds disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted.

In the compounds described in the present invention, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include use of all suitable isotopic variations of the compounds disclosed herein. For example, different isotopic forms of hydrogen (H) include protium (1H) and deuterium (2H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. Iso topically-enriched compounds disclosed herein can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and/or intermediates. Dosing and Routes of Administration

With regard to the mTOR inhibitors and WEE1 inhibitors used in the methods of the invention, various preparation forms can be selected, and examples thereof include oral preparations such as tablets, capsules, powders, granules or liquids, or sterilized liquid parenteral preparations such as solutions or suspensions, suppositories, ointments and the like. The mTOR inhibitors and WEE1 inhibitors described in the present invention are prepared with

pharmaceutically acceptable carriers or diluents.

The term "pharmaceutically acceptable salt" as referred to in this description means a conventional, well-known pharmaceutically acceptable salt. For example, when the compound has a hydroxy 1 group, or an acidic group such as a carboxyl group then it may form a base-addition salt at the hydroxyl group or the acidic group; or when the compound has an amino group or a basic heterocyclic group, then it may form an acid-addition salt at the amino group or the basic heterocyclic group.

The base-addition salts include, for example, alkali metal salts such as sodium salts, potassium salts; alkaline earth metal salts such as calcium salts, magnesium salts;

ammonium salts; and organic amine salts such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, procaine salts, and N,N'-dibenzylethylenediamine salts.

The acid-addition salts include, for example, inorganic acid salts such as hydrochlorides, sulfates, nitrates, phosphates, and perchlorates; organic acid salts such as maleates, fumarates, tartrates, citrates, ascorbates, and trifluoroacetates; and sulfonates such as methanesulfonates, isethionates, benzenesulfonates, and p-toluenesulfonates.

The term "pharmaceutically acceptable carrier or diluent" refers to excipients [e.g., fats, beeswax, semi-solid and liquid polyols, natural or hydrogenated oils, etc.]; water (e.g., distilled water, particularly distilled water for injection, etc.), physiological saline, alcohol (e.g., ethanol), glycerol, polyols, aqueous glucose solution, mannitol, plant oils, etc.); additives [e.g., extending agent, disintegrating agent, binder, lubricant, wetting agent, stabilizer, emulsifier, dispersant, preservative, sweetener, colorant, seasoning agent or aromatizer, concentrating agent, diluent, buffer substance, solvent or solubilizing agent, chemical for achieving storage effect, salt for modifying osmotic pressure, coating agent or antioxidant], and the like.

Solid preparations can be prepared in the forms of tablet, capsule, granule and powder without any additives, or prepared using appropriate carriers (additives). Examples of such carriers (additives) may include saccharides such as lactose or glucose; starch of corn, wheat or rice; fatty acids such as stearic acid; inorganic salts such as magnesium metasilicate aluminate or anhydrous calcium phosphate; synthetic polymers such as polyvinylpyrrolidone or polyalkylene glycol; alcohols such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives such as methylcellulose, carboxymethylcellulose, ethylcellulose or

hydroxypropylmethylcellulose; and other conventionally used additives such as gelatin, talc, plant oil and gum arabic.

These solid preparations such as tablets, capsules, granules and powders may generally contain, for example, 0.1 to 100% by weight, and preferably 5 to 98%> by weight, of the inhibitor, based on the total weight of each preparation.

Liquid preparations are produced in the forms of suspension, syrup, injection and drip infusion (intravenous fluid) using appropriate additives that are conventionally used in liquid preparations, such as water, alcohol or a plant-derived oil such as soybean oil, peanut oil and sesame oil.

In particular, when the preparation is administered parenterally in a form of intramuscular injection, intravenous injection or subcutaneous injection, appropriate solvent or diluent may be exemplified by distilled water for injection, an aqueous solution of lidocaine hydrochloride (for intramuscular injection), physiological saline, aqueous glucose solution, ethanol, polyethylene glycol, propylene glycol, liquid for intravenous injection (e.g., an aqueous solution of citric acid, sodium citrate and the like) or an electrolytic solution (for intravenous drip infusion and intravenous injection), or a mixed solution thereof.

Such injection may be in a form of a preliminarily dissolved solution, or in a form of powder per se or powder associated with a suitable carrier (additive) which is dissolved at the time of use. The injection liquid may contain, for example, 0.1 to 10% by weight of an active ingredient based on the total weight of each preparation.

Liquid preparations such as suspension or syrup for oral administration may contain, for example, 0.1 to 10% by weight of an active ingredient based on the total weight of each preparation.

Each preparation in the invention can be prepared by a person having ordinary skill in the art according to conventional methods or common techniques. For example, a preparation can be carried out, if the preparation is an oral preparation, for example, by mixing an appropriate amount of the compound of the invention with an appropriate amount of lactose and filling this mixture into hard gelatin capsules which are suitable for oral administration. On the other hand, preparation can be carried out, if the preparation containing the compound of the invention is an injection, for example, by mixing an appropriate amount of the compound of the invention with an appropriate amount of 0.9% physiological saline and filling this mixture in vials for injection.

The components of this invention may be administered to mammals, including humans, either alone or in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The components can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.

Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved. Further information about suitable dosages is provided below.

The term "administration" and variants thereof (e.g., "administering" a compound) in reference to a component of the invention means introducing the component or a prodrug of the component into the system of the animal in need of treatment. When a component of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., the mTOR inhibitor), "administration" and its variants are each understood to include concurrent and sequential introduction of the component or prodrug thereof and other agents.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The term "therapeutically effective amount" as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

A suitable amount of an mTOR inhibitor is administered to a patient undergoing treatment for cancer. In an embodiment, the mTOR inhibitor is administered in doses from about 10 mg - 40 mg per day. In an embodiment of the invention, the mTOR inhibitor is administered in a dose of 10 mg per day. In another embodiment of the invention, the mTOR inhibitor is administered in a dose of 20 mg per day. In another embodiment of the invention, the mTOR inhibitor is administered in a dose of 30 mg per day. In another embodiment of the invention, the mTOR inhibitor is administered in a dose of 40 mg per day.

In an embodiment of the invention, the mTOR inhibitor can be administered 5 times per week. For example, ridaforolimus is started on Day 1, and continued at the specified dosing level for five consecutive days, followed by two days of no ridaforolimus treatment. Ridaforolimus is then continued on this daily X 5 schedule each week.

A suitable amount of a WEEl inhibitor is administered to a patient undergoing treatment for cancer. In one embodiment of the invention, the WEEl inhibitor is administered in a dose between 100 mg per day and 250 mg per day. In an embodiment of the invention, the WEEl inhibitors may be dosed twice a day (BID) over the course of two and a half days (for a total of 5 doses) or once a day (QD) over the course of two days (for a total of 2 doses).

In another embodiment of the invention, the WEEl inhibitor is administered in a dose between 200 mg per day and 400 mg per day, and preferably 250 -350 mg per day. In an embodiment of the invention, the WEEl inhibitors may be dosed once a day (QD) over the course of five days.

The combination therapeutic comprising the WEEl inhibitors and mTOR inhibitors of the invention are administered to a human patient, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.

In the combination therapy regimen, the compositions of the present invention are administered in a therapeutically effective or synergistic amount. As used herein, a

therapeutically effective amount is such that co-administration of a WEEl inhibitor and an mTor inhibitor, or administration of a composition of the present invention, results in reduction or inhibition of the targeting disease or condition. A therapeutically synergistic amount is that amount of WEEl inhibitor and mTOR inhibitor necessary to synergistically or significantly reduce or eliminate conditions or symptoms associated with a particular disease.

In a broad embodiment, the treatment of the present invention involves the combined administration of a WEEl inhibitor and an mTOR inhibitor. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.

Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). The mTOR inhibitor may precede or follow administration of the WEE1 inhibitor or may be given simultaneously therewith. The clinical dosing of therapeutic combination of the present invention could be affected by the extent of adverse reactions.

Additional anti-cancer agents

An mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be useful in combination with additional therapeutic, chemotherapeutic and anti-cancer agents. Further combinations of an mTOR inhibitor and WEE1 inhibitor combination of the instant invention with therapeutic, chemotherapeutic and anti-cancer agents are within the scope of the invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita and S. Hellman (editors), 6 ^th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Such additional agents include the following: estrogen receptor modulators, programmed cell death protein 1 (PD-1) inhibitors, programmed death-ligand 1 (PD- Ll) inhibitors, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, γ-secretase inhibitors, agents that interfere with receptor tyrosine kinases (RTKs) and agents that interfere with cell cycle checkpoints. The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may be particularly useful when co- administered with radiation therapy.

PD-1 inhibitors include pembrolizumab (lambrolizumab), nivolumab and

MPDL3280A.

"Estrogen receptor modulators" refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381,

LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-l-oxopropoxy-4-methyl-2-[4-[2-(l- piperidinyl)ethoxy]phenyl]-2H-l-benzopyran-3-yl]-phenyl-2,2- dimethylpropanoate, 4,4'- dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646. "Androgen receptor modulators" refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5a-reductase inhibitors, nilutamide, fiutamide, bicalutamide, liarozole, and abiraterone acetate.

"Retinoid receptor modulators" refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, a- difluoromethylornithine, ILX23-7553, trans-N-(4'-hydroxyphenyl) retinamide, and N-4- carboxyphenyl retinamide.

"Cytotoxic/cytostatic agents" refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signal transduction pathways, antimetabolites, biological response modifiers, hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and aurora kinase inhibitors.

Examples of cytotoxic/cytostatic agents include, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2- methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu- (hexane-l,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(ch loro)platinum (II)]tetrachloride, diarizidinylspermine, arsenic trioxide, 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7- dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3'-deamino-3'-morpholino-13-deoxo-10- hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN 10755, 4-demethoxy-3-deamino- 3-aziridinyl-4-methylsulphonyl-daunorubicin (see WO 00/50032), Raf kinase inhibitors (such as Bay43-9006) and additional mTOR inhibitors.

An example of a hypoxia activatable compound is tirapazamine. Examples of proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade).

Examples of microtubule inhibitors/microtubule-stabilising agents include paclitaxel, vindesine sulfate, 3',4'-didehydro-4'-deoxy-8'-norvincaleukoblastine, docetaxol, rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881, BMS 184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L- proline-t-butylamide, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS 188797. In an embodiment the epothilones are not included in the genus of microtubule inhibitors/microtubule-stabilising agents.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3',4'-0-exo-benzylidene-chartreusin, 9-methoxy-N,N- dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, l-amino-9-ethyl-5-fluoro-2,3- dihydro-9-hydroxy-4-methyl-lH,12H-benzo[de]pyrano[3',4' :b,7]-indolizino[l,2b]quinoline- 10, 13(9H, 15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP 1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2'- dimethylamino-2'-deoxy-etoposide, GL331 , N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6- dimethyl-6H-pyrido[4,3-b]carbazole-l-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2- (dimethylamino)ethyl] -N-methylamino] ethyl]-5 - [4-hydro0xy-3 ,5 -dimethoxyphenyl] - 5,5a,6,8,8a,9-hexohydrofuro(3',4':6,7)naphtho(2,3-d)-l,3-dio xol-6-one, 2,3-(methylenedioxy)-5- methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2- aminoethyl)amino]benzo[g]isoguinoline-5,10-dione, 5-(3-aminopropylamino)-7,10-dihydroxy-2- (2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5 , 1 -de]acridin-6-one, N-[ 1 - [2(diethylamino)ethylamino] -7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl] formamide, N-(2- (dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy- 7H-indeno[2,l-c] quinolin-7-one, and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kinesin KSP, are described in PCT International Publications WO03/039460, WO03/050064, WO03/050122, WO03/049527, WO03/049679, WO03/049678, WO04/039774, WO03/079973, WO03/099211, WO03/105855, WO03/106417, WO04/037171, WO04/058148, WO04/058700, WO04/126699, WO05/018638, WO05/019206, WO05/019205, WO05/018547, WO05/017190, and US Publication No. 2005/0176776. In an embodiment inhibitors of mitotic kinesins include, but are not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK and inhibitors of Rab6-KIFL. Examples of "histone deacetylase inhibitors" include, but are not limited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Further reference to other histone deacetylase inhibitors may be found in the following manuscript; Miller, T.A. et al. J. Med. Chem. 46(24):5097-5116 (2003).

"Inhibitors of kinases involved in mitotic progression" include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub- 1 and inhibitors of bub-Rl . An example of an "aurora kinase inhibitor" is VX-680.

"Antiproliferative agents" includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231 , and INX3001 , and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylidenecytidine, 2'- fluoromethylene-2 ' -deoxycytidine, N- [5 -(2,3 -dihydro-benzofuryl)sulfonyl] -N ' -(3 ,4- dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L -glycero- B-L-manno-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo- 4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][l,4]thiazin-6-yl-(S) -ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-flurouracil, alanosine, l l-acetyl-8-(carbamoyloxymethyl)-4-formyl-6- methoxy-14-oxa-l,l l-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl-l-B-D- arabino furanosyl cytosine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab.

Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar.

"HMG-CoA reductase inhibitors" refers to inhibitors of 3-hydroxy-3- methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that may be used include, but are not limited to, lovastatin (MEVACOR®; see U.S. Patent Nos. 4,231,938,

4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Patent Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S. Patent Nos. 4,346,227, 4,537,859,

4,410,629, 5,030,447 and 5,180,589), fiuvastatin (LESCOL®; see U.S. Patent Nos. 5,354,772,

4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896), atorvastatin (LIPITOR®; see U.S. Patent Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin and BAYCHOL®; see US Patent No. 5,177,080). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 85-89 (5 February 1996) and US Patent Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefore the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.

"Prenyl-protein transferase inhibitor" refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and

geranylgeranyl-protein transferase type-II (GGPTase-II, also called Rab GGPTase).

Examples of prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Patent No. 5,420,245, U.S. Patent No. 5,523,430, U.S. Patent No. 5,532,359, U.S. Patent No. 5,510,510, U.S. Patent No. 5,589,485, U.S. Patent No. 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ. 0 696 593, WO 94/19357, WO

95/08542, WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Patent No.

5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Patent No. 5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO

96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477,

WO 96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436, and U.S. Patent No. 5,532,359. For an example of the role of a prenyl-protein transferase inhibitor on angiogenesis see, European J. of Cancer, Vol. 35, No. 9, pp.1394-1401 (1999).

"Angiogenesis inhibitors" refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-l/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-α, interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992);

JNCI, Vol. 69, p. 475 (1982); Arch. Opthalmol, Vol. 108, p.573 (1990); Anat. Rec, Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p.107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-0-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin- 1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. 105: 141-145 (1985)), and antibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp.963-968 (October 1999); Kim et al, Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis and may also be used in combination with the compounds of the instant invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see Thromb. Haemost. 80:10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101 :329-354 (2001)).

"Agents that interfere with cell cycle checkpoints" refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents. Such agents include inhibitors of ATR, ATM, the CHK11 and CHK12 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7- hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

"Agents that interfere with receptor tyrosine kinases (RTKs)" refer to compounds that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor progression. Such agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors of RTKs as described by Bume- Jensen and Hunter, Nature, 411 :355-365, 2001.

"Inhibitors of cell proliferation and survival signaling pathway" refer to compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such agents include inhibitors of serine/threonine kinases (including but not limited to, inhibitors of Akt such as described in the following patents and publications: WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO

2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US 2005/43361), inhibitors of Raf kinase (for example BAY-43-9006 ), inhibitors of MEK (for example CI-1040 and PD-098059), inhibitors of mTOR (for example Wyeth CCI-779), and inhibitors of PI3K (for example LY294002).

Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]- l-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-l-[[3,5-dichloro-4- (4-chlorobenzoyl)phenyl]methyl] - 1 H- 1 ,2,3 -triazole-4-carboxamide,CM 101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl- bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyr role]-carbonylimino]-bis-(l,3- naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).

As used above, "integrin blockers" refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the α _ν β3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the ανβ5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the α _ν β3 integrin and the α _ν β5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the α _ν β6 _? α _ν β8 _? α-ΐβΐ, α2βΐ, 5βΐ, α6βΐ and α6β4 integrins. The term also refers to antagonists of any combination of α _ν β3, α _ν β5, α _ν β6> α _ν β8> ^α ΐβΐ> ^α 2βΐ, < 5βΐ, α6βΐ and α6β4 integrins.

Some specific examples of tyrosine kinase inhibitors include N- (trifluoromethylphenyl)-5 -methylisoxazol-4-carboxamide, 3 - [(2,4-dimethylpyrrol-5 - yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro-4- fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]qu inazoline, N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11,12- hexahydro- 10-(hydroxymethyl)- 10-hydroxy-9-methyl-9, 12-epoxy- lH-diindolo[ 1 ,2,3 -fg: 3 ' ,2', 1 '- kl]pyrrolo[3,4-i][l,6]benzodiazocin-l-one, SH268, genistein, STI571, CEP2563, 4-(3- chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinem ethane sulfonate, 4-(3-bromo- 4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4'-hydroxyphenyl)amino-6,7- dimethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-l- phthalazinamine, and EMD121974. Combinations with compounds other than anti-cancer compounds are also encompassed in the instant methods. For example, combinations of the mTOR inhibitor and WEE1 inhibitor combination of the instant invention with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists may be useful in the treatment of certain malingnancies. PPAR-γ and PPAR-δ are the nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol. 1998; 31 :909-913; J. Biol. Chem.

1999;274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41 :2309-2317). More recently, PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice. {Arch. Ophthamol. 2001; 119:709-717). Examples of PPAR-γ agonists and PPAR- γ/α agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR- H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-l,2- benzisoxazol-6-yl)oxy]-2-methylpropionic acid (disclosed in USSN 09/782,856), and 2(R)-7-(3- (2-chloro-4-(4-fluorophenoxy) phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid.

Another embodiment of the instant invention is the use of the presently disclosed compounds in combination with gene therapy for the treatment of cancer. For an overview of genetic strategies to treating cancer, see Hall et al {Am. J. Hum. Genet. 61 :785-789, 1997) and Kufe et al (Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. Patent No. 6,069,134, for example), a uPA/uPAR antagonist ("Adenovirus-Mediated Delivery of a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice," Gene Therapy, August 1998;5(8): 1105-13), and interferon gamma (J. Immunol.

2000;164:217-222).

The compounds used in the methods of the instant invention may also be administered in combination with an inhibitor of inherent multidrug resistance (MDR), in particular MDR associated with high levels of expression of transporter proteins. Such MDR inhibitors include inhibitors of p-glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar). The compounds described in the present invention may be employed in conjunction with anti-emetic agents to treat nausea or emesis, including acute, delayed, late- phase, and anticipatory emesis, which may result from the use of a compound of the present invention, alone or with radiation therapy. For the prevention or treatment of emesis, a compound described in the present invention may be used in conjunction with other anti-emetic agents, especially neurokinin- 1 receptor antagonists, 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as disclosed in U.S.Patent Nos. 2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, an antidopaminergic, such as the phenothiazines (for example prochlorperazine, fluphenazine, thioridazine and

mesoridazine), metoclopramide or dronabinol. In another embodiment, conjunctive therapy with an anti-emesis agent selected from a neurokinin- 1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is disclosed for the treatment or prevention of emesis that may result upon administration of the instant compounds.

Neurokinin- 1 receptor antagonists of use in conjunction with the compounds of the present invention are fully described, for example, in U.S. Patent Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147;

European Patent Publication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430 771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0 512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517 589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0 545 478, 0 558 156, 0 577 394, 0 585 913,0 590 152, 0 599 538, 0 610 793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0 707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776 893; PCT International Patent Publication Nos. WO 90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181, 93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304, 96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2 302 689. The preparation of such compounds is fully described in the aforementioned patents and publications, which are incorporated herein by reference.

In an embodiment, the neurokinin- 1 receptor antagonist for use in conjunction with the compounds of the present invention is selected from: 2-(R)-(l-(R)-(3,5- bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4- (3-(5-oxo-lH,4H-l,2,4- triazolo)methyl)morpholine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Patent No. 5,719,147.

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be administered with an agent useful in the treatment of anemia. Such an anemia treatment agent is, for example, a continuous eythropoiesis receptor activator (such as epoetin alfa).

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be administered with an agent useful in the treatment of neutropenia. Such a neutropenia treatment agent is, for example, a hematopoietic growth factor which regulates the production and function of neutrophils such as a human granulocyte colony stimulating factor, (G-CSF). Examples of a G-CSF include filgrastim.

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be administered with an immunologic-enhancing drug, such as levamisole, isoprinosine and Zadaxin.

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be useful for treating or preventing cancer, including bone cancer, in combination with bisphosphonates (understood to include bisphosphonates, diphosphonates, bisphosphonic acids and diphosphonic acids). Examples of bisphosphonates include, but are not limited to: etidronate (Didronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate, EB-1053, minodronate, neridronate, piridronate and tiludronate, including any and all pharmaceutically acceptable salts, derivatives, hydrates and mixtures thereof.

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be useful for treating or preventing breast cancer in combination with aromatase inhibitors. Examples of aromatase inhibitors include but are not limited to: anastrozole, letrozole and exemestane.

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be useful for treating or preventing cancer in combination with RNA interference molecules (e.g., siRNA).

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be administered in combination with γ-secretase inhibitors and/or inhibitors of NOTCH signaling. Such inhibitors include compounds described in, but not limited to the following pulications: WO 01/90084, WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO 03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO

2004/039370, WO 2005/030731, WO 2005/014553, USSN 10/957,251, WO 2004/089911, WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031137, WO 2004/031139, WO

2004/031138, WO 2004/101538, WO 2004/101539 and WO 02/47671 (including LY-450139).

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be useful for treating or preventing cancer in combination with inhibitors of Akt. Such inhibitors include compounds described in, but not limited to, the following publications: WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004- 0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, and US 2005/43361.

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be useful for treating or preventing cancer in combination with PARP inhibitors.

Radiation therapy itself means an ordinary method in the field of treatment of cancer. For radiation therapy, employable are various radiations such as X-ray, γ-ray, neutron ray, electron beam, proton beam; and radiation sources. In a most popular radiation therapy, a linear accelerator is used for irradiation with external radiations to produce γ-ray.

The mTOR inhibitor and WEE1 inhibitor combination of the instant invention may also be useful for treating cancer in further combination with the following therapeutic agents: pembrolizumab (Keytruda®), abarelix (Plenaxis depot®); prokine (Leukine®);

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®); fulvestrant (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 (Revlimid®); 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®).

Indications

The WEE1 inhibitor and mTOR inhibitor combination may be useful for the treatment of the following cancers: 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, dysgerminoma, 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); hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplasia syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; and skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma. Thus, the term "cancerous cell" as provided herein, includes a cell afflicted by any one of the above-identified conditions.

Further included within the scope of the invention is a method of treating or preventing a disease in which angiogenesis is implicated, which is comprised of administering to a mammal in need of such treatment a therapeutically effective amount of the combination of the present invention. Ocular neovascular diseases are an example of conditions where much of the resulting tissue damage can be attributed to aberrant infiltration of blood vessels in the eye (see WO 2000/30651, published 2 June 2000). The undesirable infiltration can be triggered by ischemic retinopathy, such as that resulting from diabetic retinopathy, retinopathy of prematurity, retinal vein occlusions, etc., or by degenerative diseases, such as the choroidal

neovascularization observed in age-related macular degeneration. Inhibiting the growth of blood vessels by administration of the present compounds would therefore prevent the infiltration of blood vessels and prevent or treat diseases where angiogenesis is implicated, such as ocular diseases like retinal vascularization, diabetic retinopathy, age-related macular degeneration, and the like.

Further included within the scope of the invention is a method of treating or preventing a non-malignant disease in which angiogenesis is implicated, including but not limited to: ocular diseases (such as, retinal vascularization, diabetic retinopathy and age-related macular degeneration), atherosclerosis, arthritis, psoriasis, obesity and Alzheimer's disease (Dredge, et al., Expert Opin. Biol. Ther., 2002, 2(8):953-966). In another embodiment, a method of treating or preventing a disease in which angiogenesis is implicated includes: ocular diseases (such as, retinal vascularization, diabetic retinopathy and age-related macular degeneration), atherosclerosis, arthritis and psoriasis.

The specific anticancer agents illustrated in the examples are not, however, to be construed as forming the only genus that is considered as the invention. The illustrative

Examples below, therefore, are not limited by the anticancer agents listed or by any particular substituents employed for illustrative purposes.

EXAMPLES

EXAMPLE 1

In vitro cell viability

Cells were plated in 96 well plates at 3500 cells/well. Cells were then treated with an eight by eight matrix of concentrations of WEEl-1 inhibitor and ridaforolimus. 96 hours later, cell viability was measured using cell titer glo (Promega).

In vivo efficacy

Six to eight- week-old female athymic (CD1 nu/nu) mice from Charles River Laboratories (Wilmington, MA) were housed under pathogen-free conditions in microisolator cages with laboratory chow and water ad libitum. SK-OV-3 cells and A2780 cells at 3 x 10 ⁶ in PBS:matrigel (1 : 1) were injected s.c. into the right flank region. Tumors were allowed to reach 150 to 400 mm ³ for efficacy studies (8-10 mice per group) or 350 to 600 mm ³ for mechanism of action studies (3 mice per group) before randomization.

WEEl-1 was prepared in 0.5% Methylcellulose. It was administered p.o. at 5 per gram of body weight (5 days on/2 days off). Ridaforolimus was prepared in 10%> DMA (Ν,Ν-Dimethyl Acetamide), 10% Tween-80, 40% PEG-400 and 40% water. It was administered i.p. at 5 per gram of body weight (5 days on/2 days off. SK-OV-3 mice were treated for 28 days (4 cycles) for the tumor growth experiment while the mice received only 1 treatment for the mechanism of action study. A2780 mice were treated until individual tumor reached 1000 mm ³ for the tumor growth experiment while the mice received only 1 treatment for the mechanism of action study. After treatment, mice were sacrificed with C0 ₂ . The tumors were then removed (mechanism of action study) and frozen in liquid nitrogen for future analysis.

Twice a week xenografts were measured with a caliper. Caliper measurements were used to calculate tumor volumes using the formula V = length x width ² x 0.5. The combination of the WEE1 inhibitor, WEE 1-1, and the mTOR inhibitor, ridaforolimus, was identified in the primary screen as providing synergy in multiple cell lines (FIG.1) and indications. To validate this finding, two cell lines in the primary screen were treated with an 8 x 8 dose titration matrix of these compounds. Viability was then assessed 96 hours post treatment. The surface plot of the 8 x 8 matrix viability data (FIG.2) demonstrates synergy at multiple doses of the WEEl-1 and ridaforolimus combination.

To further explore the WEEl-1 and ridaforolimus combination, the tolerability of combining these two agents in vivo was determined. Non-tumor bearing mice were co-dosed with the maximum tolerated doses (MTD) of the monotherapies and this combination was found to be tolerated. To confirm that target engagement was achieved in tumor bearing mice when dosed with WEEl-1 and ridaforolimus, an acute study was conducted in which mice bearing A2780 xenograft tumors were given one dose of either WEEl-1 (60 mpk) or ridaforolimus (1 mpk) or the combination of the two agents. Mice were sacrificed four or twenty four hours after treatment. Levels of pS6 ribosomal protein were reduced in tumors from mice treated with ridaforolimus, both four and twenty four hours after treatment. Decreased levels of pCdc2 were observed in tumors from mice treated with WEEl-1 at 4 hours post treatment. However, pCdc2 levels were not decreased in mice treated with WEEl-1 but sacrificed 24 hours after treatment, supporting a BID dosing of WEEl-1 in mice to achieve continuous target engagement.

Surprisingly, it was found that in mice treated with the ridaforolimus and WEEl-1 combination, the tumor pS6 ribosomal protein levels were even lower than in mice treated with ridaforolimus alone. These results suggest a previously unknown interaction between the mTOR and WEEl signaling pathways. In addition, the tumor pCdc2 levels were even lower in mice treated with the combination than in mice treated with WEEl-1 monotherapy at the 4 hour timepoint.

Increased levels of cleaved PARP were observed, indicating increased levels of apoptosis in tumors from mice twenty four hours after treatment with the ridaforolimus and WEEl-1 combination compared to tumors from mice treated with vehicle or either monotherapy (FIG.3).

To investigate the in vivo activity of this combination, efficacy was tested for the monotherapies at MTD as well as the combination in two xenograft models of ovarian cancer. These models were selected based on the robust in vitro synergy of the combination in these cell lines and their response. For the A2780 xenograft model, mice were dosed for two weeks. The WEEl-1 and ridaforolimus combination was significantly better in inhibiting tumor growth than either monotherapy. After the two week dosing period, mice were monitored for an additional period of one week. At the end of this period, the percentage of mice surviving that had received combination treatment was significantly higher than the percentage of mice surviving that had received vehicle or either monotherapy treatment, 70% of combination treated mice were still alive compared to 0 and 20% of mice that received ridaforolimus or WEE 1-1 treatment respectively (FIG. 4).

Efficacy was tested for the monotherapies of WEEl-1 and ridaforolimus at their MTD as well as the drug combination in two xenograft models of ovarian cancer, A2780 (Figure 5 A) and SKOV-3 (Figure 5B). For each model, 10 tumor bearing mice were treated per cohort. Mice were treated with the WEE 1-1 inhibitor at 60 mg/kg twice daily (5 days on, 2 days off), ridaforolimus at 1 mg/kg once daily (5 days on, 2 days off), or the combination of the WEEl-1 and ridaforolimus at the same doses and schedules. The A2780 tumor bearing mice were dosed for two weeks and the SKOV-3 tumor bearing mice were dosed for three weeks. The mean tumor volume -/+ SEM was plotted. In both models, the observed tumor growth inhibition caused by the combination was greater than the tumor growth inhibition predicted for an additive effect of the two compounds and therefore the combination demonstrated synergy in both models. The predicted additive effect was determined with the Bliss independence (BI) model. BI is described by the equation Ei = (EA + EB) - (EA x EB), where Ei is the predicted effect

(percentage of inhibition) by the combination of drugs A and B if they were to act additively and independently, and EA and EB are the observed effects (percentage of inhibition) of each drug alone, respectively. When observed inhibition exceeds predicted inhibition, the two compounds are considered to act synergistically. The predicted additive effect is 24% Tumor Growth Inhibition (TGI) in the A2780 model and 87% TGI in the SKOV-3 model.

Table 1 : Tumor growth inhibition (TGI) summary for A2780 model

Table 2: Tumor growth inhibition (TGI) summary for SKOV-3 model