WO2010085462A1

WO2010085462A1 - Method for treating triple negative breast cancer

Info

Publication number: WO2010085462A1
Application number: PCT/US2010/021455
Authority: WO
Inventors: Angela Romanelli; Edward T. Spooner; Xiaohong Liu
Original assignee: Merck Patent Gmbh
Priority date: 2009-01-22
Filing date: 2010-01-20
Publication date: 2010-07-29

Abstract

The present invention relates to a method for the prevention or treatment of triple negative breast cancer comprising administering to a patient in need thereof a therapeutically effective amount of (1R,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3-methyl-4-(4-methylpiperazin-1-yl)]phenyl-2,4-pyrimidinediamine.

Description

METHOD FOR TREATING TRIPLE NEGATIVE BREAST CANCER

Field of the Invention

The present invention relates to a method for the prevention or treatment of triple negative breast cancer comprising administering to a patient in need thereof a therapeutically effective amount of (l R,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5- fluoro-N2-[(3-methyl-4-(4-methylpiperazin-l-yl)]phenyl-2,4-pyrimidinediamine.

Background of the Invention

Proliferative diseases are characterized by uncontrolled cellular growth. Pathways that regulate or modify communication and/or division in these cells are altered or nonfunctional, and their uncontrolled growth and proliferation eventually result in a cellular mass or rumor. Such tumors can be benign or malignant. When malignant, the cell population comprises abnormal cells that may become locally invasive or may metastasize to distant sites within the body as the tumor grows and evolves.

Malignant tumors comprise a heterogeneous population of cells, making their treatment and destruction difficult. Common treatments for cancerous tumors include subjecting them to ionizing radiation and to cytotoxic agents in order to damage their DNA and chromosomal structure, eventually leading to cellular death. An alternative path to inhibiting or preventing proliferative diseases is to disrupt the mitotic phase of the cell cycle. Members of the Aurora kinase family of protein kinases are known to be intimately involved with mitosis, and this family of kinases is overexpressed in tumors and malignant transformations (Li et al., Clin Cancer Res, 2003;9:991-7; Katayama et a\., J Natl Cancer Inst, 1999, 91 : 160- 2; Gritskpo et al., Clin Cancer Res, 2003 ;9: 1420-6; Sakakura et al., BrJ Cancer, 2001 ;84:824-31 ; Fraizer et al., M J Oncol 2004;25: 1631-9; Jeng et al., Clin Cancer Res 2004; 10:2065-71; and Tong et al., Clin Cancer Res, 2004; 10:7304-10). The overexpression of Aurora kinase A in particular has been associated specifically with poor prognosis in cancers, especially breast cancers (Tchatchou et al., Cancer Letters, 2007, 247:266-272). Breast cancers comprise a group of increasingly common malignant tumors. They are identified by their gene expression patterns for the hormonal receptor binding of estrogen, progesterone and HER2/neu. Identification of hormonal receptor activity is important both for the categorization and treatment of various kinds of cancers. For example, a cancer that is HER2/neu receptor positive will, in many cases, respond to treatment with the cytotoxic agent Trastuzumab^®, while one that is estrogen receptor positive will, in many cases, respond to treatment with doxorubicin (Adriamycin^®), cyclophosphamide (Cytoxan^®), and/or tamoxifen. However, use of these agents, with the exception of Traztuzumab^®, as well as radiation treatments are non-selective in nature. Thus, many perfectly normal cells are destroyed along with the cancer cells, and the patient may become severely debilitated, sometimes with long- lasting effects.

Triple negative breast cancer is recognized as a specific, basal-subtype cancer in which the offending tumor is estrogen receptor negative, progesterone receptor negative, and epidermal growth factor receptor 2 (HER2 or HER2/neu) negative / low. Neve et al. (Cancer Cell 19, 515-527, 2006) give an overview over the different breast cancer types, including the basal-like subtype and its phenotypes. Triple negative breast cancer lacks receptors for all three hormones that normally are used to identify and categorize breast cancers, thereby determining the kind of treatment that should be applied. Since known oncology treatment regimen depend upon the presence of one of the three hormonal receptors, triple negative breast cancer is especially difficult to treat (Schneider B. P. , Clinical Cancer Research 14(24), pp 8010-8018, 2008); Soni A., MoI Cancer Ther 7 (7), p. 1782-8, 2008; Finn RS, Clin Cancer Res 1 1 (No. 24, Pt. 2 Suppl. S), p. Abs A 233, 2005; Cleator S., Lancet Oncology 8(3), pp 235-244, 2007; Yehiely F., Trends in Molecular Medicine 12(1 1), pp 537-544, 2006.

Even the very recent literature reports that no proven targeted therapy is currently available for the treatment of triple-negative breast cancer (Corkery B., Annals of Oncology 20(5), 862-7, 2009).

Based upon the stage of cancerous progression, triple negative breast cancer often is particularly aggressive, and associated with poor prognosis compared to other forms of breast cancer (Bouchalova K., Biomedical Papers of the Medical Faculty of Palacky University in Olomouc, Czech Republic. 153(1): 13-7, 2009), and strikes women at younger ages, usually in their early 30s.

Thus, there is an urgent and compelling need to find a treatment or cure for this devastating disease. Summary of the Invention

This invention comprises a new and unexpected use for (lR,2R,3S,4S)-N4-(3- aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3-methyl-4-(4-methylpiperazin- l-yl)]phenyl-2,4-pyrimidinediamine, as previously disclosed in WO 05/1 18544 (which is hereby incorporated by reference in its entirety), for the treatment of triple negative breast cancer.

The terms "triple negative breast cancer", "basal-like breast cancer", "basal-subtype breast cancer'Or just "basal breast cancer" are used synonymously in the literaure (Tan et al., Breast Cancer Research & Treatment 1 15(3), 453-95, 2009; Huang et al., Cancer Research 67(5), 2226-38, 2007; Schneider et al., Clinical Cancer Research 14(24), pp 8010-8018, 2008).

Therefore, the present invention relates to a method for the prevention or treatment of breast cancer, comprising administering to a patient (lR,2R,3S,4S)-N4-(3-aminocarbonyl bicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3-methyl-4-(4-methylpiperazin-l-yl)]phenyl- 2,4-pyrimidinediamine, or its pharmaceutically acceptable salts and solvates, wherein the breast cancer is chosen from the group consisting of triple negative breast cancer, basal-like breast cancer, basal-subtype breast cancer and basal breast cancer.

(l R,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3- methyl-4-(4-methylpiperazin-l-yl)]phenyl-2,4-pyrimidinediamine (also known as AS703965, R945763, R763 or MSC 1992371, and hereinafter referred to as Compound A) has been shown to be highly potent adenosine triphosphate (ATP) competitive inhibitors of Aurora kinases, based on their activity in cell-based assays and in vivo xenograft models. The compound exhibited anti-tumor activity against various cell lines including cell lines derived from lung, breast, pancreatic, cervical and ovarian cells (Maier et al., 98^th AACR Annual Meeting, Los Angeles, CA; April 14-18, 2007; Sarno et al., 49^th Annual ASH Meeting, Atlanta, GA. December 8-1 1 , 2007). In an effort to elucidate further the oncolytic effect of the compound on breast cancer cell models and patient-derived tumor cell lines, the inventors employed both in vitro and in vivo models. While anticipating cellular inhibition in most cell lines, to their astonishment, Compound A exhibited high levels of inhibition for triple negative breast cancer cells. This surprising and unexpected result was confirmed by additional clonogenic, proliferation and in vivo assays. Brief Description of the Drawings

FIG. 1 is a bar graph depicting cellular apoptosis as cleaved caspase-3 levels in ZR75-1 cells. FIG. 2 is a bar graph depicting cellular apoptosis as cleaved caspase-3 levels in BT747 cells. FIG. 3 is a bar graph depicting cellular apoptosis as cleaved caspase-3 levels in MDA-

MB-231 cells. FIG. 4 is a bar graph depicting cellular apoptosis as cleaved caspase-3 levels in MDA-

MB-468 cells. FIG. 5 is a bar graph illustrating potent anti-tumor activity in "triple negative" cell line models in clonogenic assays.

FIG. 6 is a bar graph depicting cell cycle modulation in ZR75-1 cells. FIG. 7 is a bar graph depicting cell cycle modulation in BT-474 cells. FIG. 8 is a bar graph depicting cell cycle modulation in MDA-MB-231 cells. FIG. 9 is a bar graph depicting cell cycle modulation in MDA-MB-468 cells. FIG. 10 is a line graph illustrating potent in vivo anti-tumor effect in the MDA-MB-231 xenograft model. FIG. 1 1 is a line graph illustrating in vivo anti-rumor effect in the primary patient derived

HBCx-BC 146 (HBCx-10) xenograft. FIG. 12 is a line graph illustrating in vivo anti-tumor effect in the primary patient derived

HBCx- 12b xenograft. FIG. 13 is a line graph illustrating in vivo anti-tumor effect in the primary patient derived

HBCx-8 xenograft. FIG. 14 is a line graph illustrating in vivo anti-tumor effect in the primary patient derived

HBCx- 17 xenograft. FIG. 15 is a line graph illustrating in vivo anti-tumor effect in the primary patient derived

HBCx- 15 xenograft. Detailed Description of the Invention

Compound A and related compounds have been disclosed in WO 05/1 18544 for use in the treatment of proliferative diseases. In 2007, its structure was published together with the code names R763 and AS703569 (kinasepro wordpress com, 2007/02/13/R763).

While it is known from these publications that Compound A can be used to treat cancer in general, no one would have expected that it can be used even for the treatment of triple negative breast cancer.

The finding was particularly surprising based upon problems encountered by the medical community in arriving at a treatment method for triple negative breast cancer tumor cells that lack estrogen, progesterone, and HER2/neu receptors on which treatment regimen are based, and for which cancer even today no proven targeted therapy is available (Corkery B., Annals of Oncology 20(5), 862-7, 2009). This surprising and unexpected result was confirmed by additional clonogenic, proliferation and in vivo assays, as shown below.

As used herein, a description of the compounds of the invention in every case includes a pharmaceutically acceptable salt, solvate, hydrate, prodrug, tautomer, enantiomer, stereoisomer, analog or derivative thereof, including mixtures thereof in any ratios.

As used herein, the term "proliferative disease" or "proliferative disorder'" refers to a disease or disorder that is marked by aberrant and usually rapid cellular growth. This proliferation generally is caused by a malfunction in cell cycle mechanism(s) or the loss of inhibitory signaling.

The term "treatment" as used herein refers both to prevention of a particular disease or treatment of a pre-existing condition.

The term "pharmaceutically acceptable salt" includes a salt of the active compound that is prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compound described herein. When compound of the present invention contains few acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et ai, J. Pharma. Science 1977, 66: 1 -19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

The compound according to the invention can be used in its final non-salt form. On the other hand, the present invention also encompasses the use of these compounds in the form of their pharmaceutically acceptable salts, which can be derived from various organic and inorganic acids and bases by procedures known in the art. Pharmaceutically acceptable salt forms of the compound are for the most part prepared by conventional methods. For example, if the compound contains a carboxyl group, one of its suitable salts can be formed by reacting the compound with a suitable base to give the corresponding base-addition salt. Such bases are, for example, alkali metal hydroxides, including potassium hydroxide, sodium hydroxide and lithium hydroxide; alkaline earth metal hydroxides, such as barium hydroxide and calcium hydroxide; alkali metal alkoxides, for example potassium ethoxide and sodium propoxide; and various organic bases, such as piperidine, diethanolamine and N methylglutamine.

Furthermore, the base salts of the compound according to the invention include aluminium, ammonium, calcium, copper, iron(III), iron(II), lithium, magnesium, manganese(III), manganese(II), potassium, sodium and zinc salts, but this is not intended to represent a restriction.

As used herein, the term "prodrug" means a form of the compound that readily undergoes one or more chemical changes under physiological conditions to provide an active form of the compound of the present invention. For instance, typical prodrugs include carboxylic acid ester forms of the compounds of the invention. In an exemplary embodiment, the prodrug is suitable for treatment /prevention of those diseases and conditions that require the drug molecule to cross the blood brain or other membrane barrier. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

The compounds and compound mixtures according to the invention can be adapted for administration via any desired suitable method, for example by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) methods.

Combinations of Compound A and further cancer therapeutics have been described in WO 09/050143. Another object of the present invention is, therefore, a method for prevention or treatment of triple negative breast cancer, basal-like breast cancer, basal- subtype breast cancer or basal breast cancer, comprising administering to a patient Compound A and/or its pharmaceutically acceptable salts and solvates, and at least one further cancer therapeutics.

Cancer therapeutics that can be combined with Compound A according to the invention, include alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, kinase inhibitors (such as other aurora kinase inhibitors, cyclin-dependent kinase inhibitors, Bcr-Abl kinase inhibitors, polo-like kinase inhibitors, receptor tyrosine kinase inhibitors), biologic response modifiers, cell cycle inhibitors, cyclooxygenase-2 inhibitors, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors inhibitors, hormonal therapies, immunologicals, intercalating antibiotics, mammalian target of rapamycin inhibitors, mTOR inhibitors, platinum chemotherapeutics, VEGFR inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, retinoids/deltoids plant alkaloids, topoisomerase inhibitors, signalling inhibitors and the like. Preferred cancer therapeutics for combination with Compound A include gefitinib, cetuximab, erlotinib, imatinib, sorafenib and gemcitabine.

Compound A and/or its physiologically acceptable salts and solvates, and the other cancer therapeutic or therapeutics can be administered simultaneously or sequentially. When administered simultaneously, Compound A and/or its physiologically acceptable salts and solvates, and the other cancer therapeutic or therapeutics may be administered as one pharmaceutical composition or as separate pharmaceutical compositions. Solvates of Compound A are taken to mean adductions of inert solvent molecules onto Compound A which form owing to their mutual attractive force. Solvate are, for example, hydrates, such as monohydrates or dihydrates, or alcoholates, i.e. addition compounds with alcohols, such as, for example, with methanol or ethanol.

The term "patient" as used herein may be any mammalian species, for example a primate species, particularly humans; rodents; rabbits; horses, cows, sheep, dogs, cats, etc. Animal models are of interest for veterinary treatment and for experimental investigations, providing a model for treatment of human disease.

"Therapeutically effective amount" of Compound A means the amount of the compound that, upon administration, provides the desired beneficial result in a patient in need thereof. This amount depends on a number of factors, including, for example, the age and weight of the patient, the precise condition that requires treatment and its severity, the nature of the formulation, and the method of administration, and is ultimately determined by the a physician or veterinarian. An effective amount of Compound A for the treatment of neoplastic growth, for example, is generally in the range from 0.1 to 100 mg/kg/day of body weight of the patient.

Any drug dosage depends on the specific disease, patient status, etc. A therapeutic dose typically is considered sufficient at the level at which it reduces the undesired cell . population in the target tissue while the viability of the patient is maintained. The treatment is generally continued until a reduction in cell population has occurred, for example, minimally about 50% reduction in cell burden, and may be continued until essentially no more undesired cells are detected in the body.

A convenient synthesis for Compound A is described in WO 2005/1 18554 (p. 71, example 7.1 1.3).

Biologic Assays

Clonogenic Assay (FIG. 5)

Compound A

For the cumulative tests in tumor xenografts, two batches of Compound A were used. Compound R945763, also termed Batch 1 of compound A, Batch No. 857-067, was provided by Rigel Pharmaceuticals, Inc., South San Fransisco in a single shipment. AS703569/1 or Batch 2 of Compound A was provided by Serono Research Institute, Boston, in a single shipment. Compounds were stored at +4°C. Table 1 shows the name of all test articles and the designation for in vitro data processing at Oncotest (Freiburg, Germany):

Table 1 : Designation of test article

Vehicles and Dose Levels

The compound was tested at concentrations ranging from 0.0001 to 10.0 μg/mL. Stock solutions were prepared in DMSO at 3 mg/mL and small aliquots were stored at -20⁰C in the dark. Final dilutions were prepared in IMDM (Iscove's Modified Dubelcco's Medium) immediately prior to use.

Chemicals and Enzymes

Other chemicals and enzymes needed to perform the clonogenic assay are detailed in

Table 2.

Table 2: Chemicals and enzymes

Buffers and Solvents

Buffers and solvents needed to perform the clonogenic assay are given in detail in

Table 3.

Table 3: Buffers, media and solvents

Tumor Models used in Colony-Assay

The origin of the xenograft models was described previously (Fiebig et al., Contrib. Oncol. Basel. Karger, 1999, 54:29-50; and Fiebig et al., Contrib. Oncol. Basel, Karger 1992, 42:321 -351).

Tumor-Colony-Assay

Preparation of single cell suspensions from human tumor xenografts: solid human tumor xenografts growing subcutaneously in serial passages in thymus aplastic nude mice (NMRI nu/nu strain, obtained from Oncotest's breeding facility) were removed under sterile conditions, mechanically disaggregated and subsequently incubated with an enzyme cocktail consisting of collagenase type IV (41 U/ml), DNase I (125 U/ml), hyaluronidase type III (100 U/ml) and dispase II (1.0 U/ml) in RPMI 1640-Medium at 37°C for 45 minutes. Cells were passed through sieves of 200 μm and 50 μm mesh size and washed twice with sterile PBS- buffer. The percentage of viable cells was determined in a Neubauer-hemocytometer using trypan blue exclusion.

Culture Methods of Cells from Human Tumor Xenografts

The clonogenic assay was performed in a 24-well format according to a modified two- layer soft agar assay introduced by Hamburger & Salmon (Primary bioassay of human tumor stem cells. Science 197, 461-463 (1977)). The bottom layer consisted of 0.2 ml/well IMDM (supplemented with 20% (v/v) fetal calf serum, 0.01% (w/v) gentamicin) and 0.75% (w/v) agar. 1.5- 10⁴ to 4 10⁴ cells were added to 0.2 ml of the same culture medium supplemented with 0.4% (w/v) agar and plated in 24-multiwell dishes onto the bottom layer. The test compounds were applied by continuous exposure (drug overlay) in 0.2 ml culture medium. Every dish included six untreated control wells and drug-treated groups in triplicate at 6 concentrations. Cultures were incubated at 37⁰C and 7.5% CO₂ in a humidified atmosphere for 7-20 days and monitored closely for colony growth using an inverted microscope. Within this period, in vitro tumor growth led to the formation of colonies with a diameter of > 50 μm. At the time of maximum colony formation, counts were performed with an automatic image analysis system (OMNICON 3600, Biosys GmbH). 24 hours prior to evaluation, vital colonies were stained with a sterile aqueous solution of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5- phenyltetrazolium chloride (1 mg/ml, lOO μl/well).

Data Evaluation

An assay was considered fully evaluable, if the following quality control criteria were fulfilled: mean number of colonies in the control wells of 24-multiwell plates > 20 colonies with a colony diameter of > 50 μm; coefficient of variation in the control wells of each plate < 50%; the positive reference compound 5-fluorouracil (5-FU, at the cytotoxic concentration of 1.0 mg/ml) must effect a reduction of colony number to < 30% of the controls; or initial plate counts on days 0 or 2 were < 30% of the final control count.

Drug effects were expressed in terms of the percentage of colony formation, obtained by comparison of the mean number of colonies in the treated wells with the mean colony count of the untreated controls (relative colony count expressed by the test-versus-control- group value, T/C-value %):

T ₌ colony count_{UealBd group} ^ _%

C colony count_{∞ntrol group}

IC₅₀- and IC7o-values, the drug concentrations necessary to inhibit colony formation by 50% (T/C = 50%) and 70% (T/C = 30%), respectively, were determined by plotting compound concentration versus relative colony count. Mean IC50- and ICγo-values were calculated according to the formula:

where "x" is the specific tumor model, and "n" is the total number of tumor models studied. If an IC₅₀- or IC ₇₀- value could not be determined within the examined dose range because a compound was either too active or too inactive, the lowest or highest concentration studied was used for the calculation.

Cell Culture conditions and compound treatment for ATPlite, cell cycle and apoptosis (Cleaved Caspase-3 assays, FIGS. 1-4):

All cell lines were maintained according to the complete growth medium conditions outlined by the American Type Culture Collection (ATCC) (Manassas, VA) with the exception of MCF-7 (obtained from Barbara Ann Karmanos Cancer Institute), which was grown in DMEM/F12 with 12mg/ml sodium bicarbonate (Invitrogen, Grand Island, NY), 10% Fetal Bovine Serum (FB S)(In vitrogen), 1OmM Hepes (Invitrogen), lOug/ml Insulin (Invitrogen) and 10 ^~n estradiol (Sigma Chemicals). Compound A dilutions were made in dimethylsulfoxide (DMSO) prior to addition to cells. The final DMSO concentration did not exceed 0.4%.

Determination of cell viability using ΛTP-lite:

Cells were seeded on white, flat bottom tissue culture plates (Corning, USA) at predetermined optimal cell densities and grown in the presence of inhibitor for 96 hours. Intracellular ATP concentrations were determined using the ATP-lite Luminescence ATP Detection Assay System (Perkin Elmer, Beaconsfield, UK) After 96h in culture, the growth medium was aspirated and replaced with lOOul phosphate buffered saline, with calcium chloride, with magnesium chloride (PBS +/+) (Invitrogen). 50ul of cell lysis buffer was added to each well and mixed for 5 minutes on a Titer Plate Shaker (Fisher Scientific, Pittsburgh, PA). 50ul of ATP-lite reagent was then added to each well and mixed for 5 minutes. After incubating for 10 minutes in the dark, plates were measured for luminescence on a Victor-5 1428 Multilabel HTS counter (Perkin Elmer). Results were displayed as relative light units.

Analysis of cell cycle and apoptosis:

Cells were plated on 6-well tissue culture dishes (BD Falcon, Franklin Lakes, NJ) and allowed to grow overnight to a density of 40-50%. They were then treated with various concentrations of inhibitor and harvested after 48, 72 and 96 hours. At the time of harvest, cells were washed once with phosphate buffered saline, without calcium chloride, without magnesium chloride, (PBS -/-) (Invitrogen) and incubated with 0.5% Trypsin/EDTA (Invitrogen) for 5 minutes to allow cells to detach. Cells were harvested in ImI of complete medium and pelleted for 5 minutes at 600 x g in centrifuge. Cells next were fixed and permabolized with the addition of 100% ice cold methanol (Fisher Scientific) and incubated on ice for 30 minutes. Then the cells were pelleted and washed twice in 1% bovine serum albumin (BSA)(Fisher Scientific)/PBS and split into two tubes, one to measure cell cycle by Propidium Iodide (PI) staining and the other to measure apoptosis by cleaved caspase-3 staining. Cells to be analyzed for cell cycle were resuspended in PI/RNAse (Becton Dickenson) and incubated for 15 minutes at room temperature. Samples were then washed in PBS followed by analysis by flow cytometry on a Guava EasyCyte instrument (Guava, Hayward, CA). Cells to be analyzed for the presence of cleaved caspase-3 were stained with Alexa-488 conjugated cleaved caspase-3 (Asp 175) antibody (Cell Signaling Technologies, Beverly, MA) followed by flow cytometric analysis on a Guava EasyCyte instrument (Guava).

Primary patient derived breast cancer xenografts

Tumor material was cut into 3 x 3 mm pieces. Swiss nu/nu (nude) female mice, 7- 9 weeks old, were used as xenograft recipients. Tumor fragments were implanted subcutaneously and maintained by serial transplantations. Mice originated from Charles River Laboratories France (Les Arbresles) and maintained under specific pathogen-free conditions. Tumor-bearing mice were randomly distributed into groups of 12 mice assigned to controls or treatments. All treatments started at day one as the tumors reached a volume comprised between 60 mm³ (minimum) and 200 mm³. In cases of heterogenous tumor take and growth, inclusion of mice was delayed until tumors reached the initial optimal volume (60 - 200 mm³). Mice of the same group (control or treated) were randomly distributed into 2 cages at least, to avoid any cage-dependent effects. Compound A was suspended in sterile NaCl 0.9% solution at the right dosages and administered by gavage. Administration was repeated weekly. The compound was given at a dose of 50mg/kg/once a week during 6 weeks consecutively

Evaluation of xenograft growth

Tumor growth was assessed by measuring two perpendicular diameters with a caliper, every 2 or 3 days. Tumor volumes (V) were calculated as V = a² x b/2 where a is the width of the tumor in millimeters and b is the length of the tumor in millimeters

References for cell lines:

General

A. F. Gazdar, V. Kurvari. A. Virmani, L. Gollahon, M. Sakaguchi, M. Westerfield, D. Kodagoda, V. Stasny, H. T. Cunningham, I. I. Wistuba, G. Tomlinson, V. Tonk, R. Ashfaq, A.M. Leitch, J.D. Minna and J.W.Shay (1998). Characterization of paired tumor and non- rumor cell lines established from patients with breast cancer. Int. J. Cancer 78 : 766-774.

MDA-MB-361

Her-2 expression: Pierceall, W.E., Woodard, A.S., Morrow, J.S., Rimm, D., Fearon, E.R. Frequent alterations in E-cadherin and alpha- and beta-catenin expression in human breast cancer cell lines. Oncogene, U: 1319-1326, 1995. Progesterone Receptor: Sutherland, R.L., Hall, R.E., Pang, G. Y., Musgrove, E.A., Clarke, CL. Effect of medroxyprogesterone acetate on proliferation and cell cycle kinetics of human mammary carcinoma cells. Cancer Res, 48: 5084-5091, 1988.

Estrogen Receptor: Hall, R.E., Lee, C.S., Alexander, I.E., Shine, J., Clarke, C.L., Sutherland, R.L. Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorticoid receptor messenger RNA levels. Int J Cancer, 46: 1081-1087, 1990.

MDA-MB-231

Estrogen Receptor: Cailleau, R., Olive, M., Crueiger, Q.V.J. Long term human breast carcinoma cell lines of metastatic origin: preliminary characterization. In Vitro, 14: 91 1-915, 1978.

Progesterone Receptor: Sutherland, R.L., Hall, R.E., Pang, G. Y., Musgrove, E.A., Clarke, CL. Effect of medroxyprogesterone acetate on proliferation and cell cycle kinetics of human mammary carcinoma cells. Cancer Res, 48: 5084-5091, 1988 Her2: Hollywood, D. P., Hurst, H.C. A novel transcription factor, OB2-1 , is required for overexpression of the proto-oncogne c-ebB-2 in mammary tumour lines. EMBO J., 12: 2369- 2375, 1993

BT-474

Estrogen Receptor: Hall, R.E., Lee, CS., Alexander, I.E., Shine, J., Clarke, C. L., Sutherland, R.L. Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorticoid receptor messenger RNA levels. Int J Cancer, 46: 1081-1087, 1990.

Progesterone Receptor: Sutherland, R.L., Hall, R.E., Pang, G. Y., Musgrove, E.A., Clarke, CL. Effect of medroxyprogesterone acetate on proliferation and cell cycle kinetics of human mammary carcinoma cells. Cancer Res, 48: 5084-5091, 1988.

Her2: Hollywood, D.P., Hurst, H.C. A novel transcription factor, OB2-1 , is required for overexpression of the proto-oncogne c-ebB-2 in mammary tumour lines. EMBO J., 12: 2369-2375, 1993. MDA-MB-468

Estrogen Receptor: Sheikh, M.S., Shao, Z.M., Hussain, A., Fontana, J. The p53- binding protein MDM2 gene is differentially expressed in human breast carcinoma. Cancer Res, 53: 3226-3228, 1993.

Progesterone Receptor: Maemura, M., Akiyama, S. K., Woods, V.L.J., Dickson, R.B. Expression and ligand binding of alpha 2 beta 1 integrin on breast carcinoma cells. Clin Exp Metastasis, 13: 223-235, 1995.

Her2: Pierceall, W.E., Woodard, A.S., Morrow, J.S., Rimm, D., Fearon, E.R. Frequent alterations in E-cadherin and alpha- and beta-catenin expression in human breast cancer cell lines. Oncogene, 11: 1319-1326, 1995.

SK-BR-3

Estrogen Receptor: Thompson, E.W., Paik, S., Brunner, N., Sommers, C. L., Zugmaier, G., Clarke, R., Shima, T.B., Torri, J., Donahue, S., Lippman, M. E. Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines. J Cell Physiol, 150: 534-544, 1992.

Progesterone Receptor: Tong, D., Czerwenka, K., Sedlak, J., Schneeberger, C, Schiebel, I., Concin, N., Leodolter, S., Zeillinger, R. Association of in vitro invasiveness and gene expression of estrogen receptor, progesterone receptor, pS2 and plasminogen activator inhibitor- 1 in human breast cancer cell lines [In Process Citation]. Breast Cancer Res Treat, 56: 91 -97, 1999.

Her2: Roetger, A., Merschjann, A., Dittmar, T., Jackisch, C, Barnekow, A., Brandt, B. Selection of potentially metastatic subpopulations expressing c-erbB-2 from breast cancer tissue by use of an extravasation model. Am J Pathol, 153: 1797-1806, 1998.

ZR-75-1

Estrogen Receptor: Fabbro, D., Kung, W., Roos, W., Regazzi, R., Eppenberger, U. Epidermal growth factor binding and protein kinase C activities in human breast cancer cell lines: possible quantitative relationship. Cancer Res, 46: 2720-2725, 1986. Progesterone Receptor: Sutherland, R.L., Hall, R.E., Pang, G. Y., Musgrove, E.A., Clarke, CL. Effect of medroxyprogesterone acetate on proliferation and cell cycle kinetics of human mammary carcinoma cells. Cancer Res, 48: 5084-5091, 1988.

Her2: Hollywood, D.P., Hurst, H.C. A novel transcription factor, OB2-1, is required for overexpression of the proto-oncogne c-ebB-2 in mammary tumour lines. EMBOJ., 12: 2369-2375, 1993.

MCF- 7

Estrogen Receptor/Progesterone Receptor: Fabbro, D., Kung, W., Roos, W., Regazzi, R., Eppenberger, U. Epidermal growth factor binding and protein kinase C activities in human breast cancer cell lines: possible quantitative relationship. Cancer Res, 46: 2720-2725, 1986.

Examples

Example 1

Table 4: Antiproliferative activity of Compound A in relation to HER2, estrogen and progesterone receptor status in a panel of breast cancer cell lines.

Triple negatιve/Her2 "~ ER+/PR+ or HER2++

MDA- MDA- HCC HCC HCC HCC HCC SKBFU ZR75-1 MB-468 MB-231 1143 1395 38 1419 2218 MI&I ^{BT474 MC}"

Sub-type BaA BaS BaA unkn BaB unkn unkn Lum Lum Lum Lum Lum

ER neg neg neg neg neg neg neg pos pos pos neg pos

PR neg neg neg neg neg neg neg pos pos pos neg pos

HER2' neg ^' neg neg neg low pos pos pos pos low pos pos

IC₅₀ (μM) 0.004 0.240 0.694 0.277 0.572 7.37 6.85 2.96 3.60 4.08 2.52 5.31 AS70356S.

Compound A exhibited significant anti-proliferative activity against breast cancer cell lines, with an average IC50 of 2873 nM. Cell lines that responded with an IC50 lower than the average were considered to be more "sensitive" to the compound. These include: HCC-1 143, HCC-38, HCC- 1395, MDA-MB-231 and MDA-MB-468 Table 4 below illustrates that these cell lines are characterized as showing lower expression levels of estrogen (ER), progesterone (PR) receptors and HER2 Cell lines considered more resistant with an IC50 above 2000 nM exhibited higher levels of ER, PR and HER2 Trend towards sensitivity correlated with basal- like A or B phenotype (BaA or BaB) whereas Luminal (Lum) breast cancer lines were more resistant (designations based on Neve et al , CANCER CELL 10, 515-527, DECEMBER 2006) Receptor status was confirmed by lmmunostaining, and HER2 expression also was confirmed by Western blot

ER, PR and HER2 cell lines were obtained from different sources including ATCC references as given below

ZR-75-1 ATCC Disease ductal carcinoma

Organ mammary gland, breast

Tissue Duct HCC2218 ATCC

Disease ductal carcinoma Organ mammary gland, breast

Tissue Duct

SK-BR-3 ATCC Tumor stage TMN stage IHA, grade 3

Organ mammary gland, breast Disease primary ductal carcinoma

Disease Adenocarcinoma

HCC1419 ATCC

MDA-MB-468 ATCC Organ mammary gland, breast Organ mammary gland, breast Tumor stage TMN stage HIA, grade 3 Disease Adenocarcinoma Disease primary ductal carcinoma

MDA-MB-361 ATCC HCC38 ATCC Organ mammary gland, breast Organ mammary gland, breast Disease Adenocarcinoma Tumor stage TMN stage HB, grade 3 MDA-MB-231 ATCC Disease primary ductal carcinoma Organ mammary gland, breast Disease Adenocarcinoma HCC 1395 ATCC Organ mammary gland, breast

BT-474 ATCC Tumor stage TMN stage I, grade 3

Organ mammary gland, breast Disease primary ductal carcinoma

Tissue Duct MCF-7: Barbara Ann Karmanos Cancer

HCCl 143: ATCC Institute, Wayne State University Organ: mammary gland, breast Organ: mammary gland, breast Tumor stage: TMN stage HA, grade 3 Disease: adenocarcinoma Disease: primary ductal carcinoma

Example 2

Table 5: Cell cycle profile of breast cancer cell lines treated with Compound A.

Distribution of cells throughout the cell cycle was evaluated for up to 96 hrs. Observations are summarized in Table 5. Most cell lines with the exception of MDA-MB- 468 exhibited G2/M and/or endoreduplication, phenotypes well documented to be associated with aurora kinase inhibitors. All cell lines also undergo apoptosis to some extent. Cells lines characterized as devoid of expression or expressing relatively lower levels of ER, PR and HER2 undergo apoptosis to a larger extent at lower doses of Compound A that were found to be effective in the cell lines expressing higher levels of ER, PR and HER2. These observations were further confirmed by the cleaved caspase-3 assay described in the following figures. Example 3

tire 1 : Cleaved caspase-3 staining in ZR-75-1 cells.

Cleaved Caspase-3 : ZR75-1

A dose dependent increase in apoptosis is seen at all time points as determined by an increase in cleaved caspase-level. Highest levels are observed at 96 hrs. Graph bars from left to right represent results of treatment of ZR-75-1 cells with DMSO, 0.1 μM Compound A, and 1.0 μM Compound A.

Example 4

Figure 2: Cleaved caspase-3 staining in BT474 cells.

Cleaved Caspase-3 : BT474

In the BT-474 cells, cleaved caspase-3 did not reach appreciable levels compared to DMSO controls even after 96 hrs of treatment. Therefore, the BT-474 cell line was relatively more resistant to apoptosis compared to the ZR-75-1 cell line. Graph bars from left to right represent results of treatment of BT-474 cells with DMSO, 0.1 μM Compound A, and 1.0 μM Compound A.

Example 5

Figure 3: Cell cycle analysis and Cleaved Caspase-3 staining in MDA-MB-231 cells

A significant increase in cleaved caspase-3 levels was observed after 72 hrs and 96 hrs of treatment with Compound A compared to those cells treated with DMSO. Higher levels of apoptosis were reached in the MDA-MB-231 cell line with lower concentrations of Compound A compared to the other cell lines described above. This cell line is therefore more susceptible to undergoing apoptosis in response to being subjected to Compound A. Graph bars from left to right represent results of treatment of MDA-MB-231 cells with DMSO, 0.01 μM Compound A, and 0.1 μM Compound A. and 1.0 μM Compound A. Example 6

Figure 4: Cleaved Caspase-3 staining in MDA-MB-468 cells.

A significant increase in cleaved caspase-3 levels was observed as early as 48 hrs post- treatment with low doses of Compound A. This cell line was the most sensitive to Compound A in terms of an apoptotic response as measured by cleaved caspase-3 staining. Graph bars from left to right represent results of treatment of MDA-MB-468 cells with DMSO, 0.005 μM Compound A, 0.0075 μM Compound A, 0.01 μM Compound A, and 0.1 μM Compound A.

Example 7

Figure 5- Clonogenic Assay Data

IC70 of Compound A in colony forming assays using Breast cancer cell lines

1000 2000 3000 4000 5000 6000 IC70 concentration (nM)

In a clonogenic assay that examines the effect of Compound A on tumor model growth in an anchorage independent culture system, the IC70 for the two triple negative cell lines models were in the nanomolar range ( MDA-MB-231 : 83nM; MX1/12: 12 nM), whereas the non-triple negative cell line was in the micromolar range (MCF-7: 5541 nM).

Example 8

Figure 6-Cell cycle analysis in the ZR-75-1 cell line, "middle responder". Cell incubated with indicated doses of Compound A or vehicle DMSO for 96 hours:

Each bar in the graph above comprises 4 cell cycle phases given as distinct blocks from bottom to top: sub Gl, G0/G1, S and G2M. The uppermost block represents >4n DNA cell content. Blocks representing certain phases may be negligible and so barely visible, or non-existent.

Example 9

Figure 7-Cell cycle analysis in BT-474 cell line, "non responder". Cell incubated with indicated doses of Compound A or vehicle DMSO for 96 hours:

Example 10

Figure 8 Cell cycle analysis in MDA-MB-231 cell line, "responder". Cell incubated with indicated doses of Compound A or vehicle DMSO for 96 hours:

Example 1 1

Figure 9- Cell cycle analysis in MDA-MB-468 cell line, "most sensitive responder". Cell incubated with indicated doses of Compound A or vehicle DMSO for 96 hours:

₍₃₎ V_{l j}mo_ro ₃mmum_i

It is understood that in light of the teachings of this invention to one of ordinary skill in the art that certain changes and modifications may be made thereto without departing from the spirit and scope of the invention.

Example 12

Figure 10: Anti-tumor effect of Compound A in a triple negative breast cancer cell line (MDA-MB-231) derived xenograft.

0 10 20 30 40

Days

Once weekly dosing with Compound A resulted in a significant decrease in tumor growth compared to vehicle alone (84% tumor growth inhibition).

The MDA-MB-231 breast cancer cell line was purchased from the American Type Culture Collection (ATCC), (Manassas, VA). Cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA) containing 10% fetal bovine serum, (Gibco, Carlsbad, CA). Cells in exponential growth phase were collected after trypsinization and washing steps, re-suspended in PBS and held on ice until implantation. MDA-MB231 cells (10xl0⁶ in 100 ul PBS) were subcutaneously implanted in the right shoulder area of 5 to 6-week-old female SCID mice (Taconic, Hudson, NY) in lOOul of PBS. When the average tumor size reached 150-200 mm3 (approx. 3 weeks after implantation), rumor-bearing mice were sorted into groups and treated by oral gavage with saline (vehicle control), AS703569 in saline at 50mg/kg once per week. Tumor volumes and body weights were measured twice per week during the treatment period. Tumor volume was calculated based on the formula: V (tumor volume in mm3)= (width dimension2 x length dimension)/2. All animal protocols used in this study were approved by EMD Serono Research Institute Inc. (EMD SRI) Institutional Animal Care and Use Committee (IACUC).

Example 13

Figure 1 1 : Anti-tumor effect of Compound A in the primary patient derived (triple negative breast cancer) xenograft BC 146 (otherwise known as HBCx-10) .

1 6 11 16 21 26 31

By the end of treatment, mice dosed with Compound A had lower tumor volumes compared to those treated with vehicle alone.

Example 14

Figure 12: Anti-tumor effect of Compound A in the primary patient derived (triple negative breast cancer) xenograft HBCx- 12b.

Relative Tumor Volume

C 10 20 30 40 50

Days after start of treatment

By the end of treatment, mice dosed with Compound A had lower tumor volumes compared to those treated with vehicle alone. Example 15

Figure 13: Anti-tumor effect of Compound A in the primary patient derived (triple negative breast cancer) xenograft HBCx-8.

Relative Tumor Volume

10 15 20 25

Days after start of treatment

Example 16

Figure 14: Anti-tumor effect of Compound A in the primary patient derived (triple negative breast cancer) xenograft HBCx- 17.

10 20 30 40 50 60

Days after start of treatment

Example 17

Figure 15: Anti-tumor effect of Compound A in the primary patient derived (triple negative breast cancer) xenograft HBCx- 15.

10 15 20 25

Days after start of treatment

Claims

ClaimsWhat is claimed is:

1. A method for the prevention or treatment of breast cancer, comprising administering to a patient (lR,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5- fluoro-N2-[(3-methyl-4-(4-methylpiperazin-l-yl)]phenyl-2,4-pyrimidinediamine, or its pharmaceutically acceptable salts and solvates, wherein the breast cancer is chosen from a group consisting of triple negative breast cancer, basal-like breast cancer, basal-subtype breast cancer and basal breast cancer.

2. Method according to claim 1, wherein (lR,2R,3S,4S)-N4-(3- aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3-methyl-4-(4- methylpiperazin-l-yl)]phenyl-2,4-pyrimidinediamine is administered in combination with at least one further cancer therapeutic.

3. Method according to claim 2, wherein the at least one further cancer therapeutic is selected from a group consisting of gefitinib, cetuximab, erlotinib, imatinib, sorafenib and gemcitabine.

4. (lR,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3- methyl-4-(4-methylpiperazin-l-yl)]phenyl-2,4-pyrimidinediamine for use in the treatment of breast cancer, wherein the breast cancer is chosen from a group consisting of triple negative breast cancer, basal-like breast cancer, basal-subtype breast cancer and basal breast cancer.

5. (lR,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3- methyl-4-(4-methylpiperazin-l-yl)]phenyl-2,4-pyrimidinediamine according to claim

4, administered in combination with at least one further cancer therapeutic.

6. (lR,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3- methyl-4-(4-methylpiperazin-l-yl)]phenyl-2,4-pyrimidinediamine according to claim

5, wherein the at least one further cancer therapeutic is selected from a group consisting of gefitinib, cetuximab, erlotinib, imatinib, sorafenib and gemcitabine.

7. Use of (lR,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2. l]hept-5-ene-2-yl)-5-fluoro- N2- [(3-methyl-4-(4-methylpiperazin- 1 -yl)]phenyl-2,4-pyrimidinediamine for the manufacture of a medicament for the treatment of breast cancer, wherein the breast cancer is chosen from a group consisting of triple negative breast cancer, basal-like breast cancer, basal-subtype breast cancer and basal breast cancer.

8. Use according to claim 7, wherein (lR,2R,3S,4S)-N4-(3- aminocarbonylbicyclo[2.2. l]hept-5-ene-2-yl)-5-fluoro-N2-[(3-methyl-4-(4- methylpiperazin-l-yl)]phenyl-2,4-pyrimidinediamine is administered in combination with at least one further cancer therapeutic.

9. Use according to claim 8, wherein the at least one further cancer therapeutic is selected from a group consisting of gefitinib, cetuximab, erlotinib, imatinib, sorafenib and gemcitabine.