WO2009050143A1 - Combinations 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-pyrimidineamine - Google Patents

Abstract

The invention relates to combinations of (1 R,2R,3S,4S)-N4-(3- aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3-nnethyl-4-(4- methylpiperazin-1 -yl)]phenyl-2,4-pyrimidinediamine and/or its physiologically acceptable salts and solvates, and other cancer therapeutics, and the use of such combinations for the treatment of cancer.

Description

COMBINATIONS OF ^l

(1 R , 2R , 3S , 4S ) ^4-(3-AMINOCARBONYLBICYCLO [2 . 2 . 1]

HEPT-5-ENE-2-YL) - 5-FLUORO-N2- [ ( 3 -

METHYL-4- (4 -METHYLPIPERAZIN-1 -YL] PHENYL-2 ,

4-PYRIMIDINEAMINE

The invention relates to combinations of (1 R,2R,3S,4S)-N4-(3- aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3-nnethyl-4-(4- methylpiperazin-1 -yl)]phenyl-2,4-pyrimidinediamine (hereinafter referred to as AS703569) and/or its physiologically acceptable salts and solvates, and other cancer therapeutics, and the use of such combinations for the treatment of cancer.

Background of the invention

AS703569, processes for its preparation and its use for the treatment of cancer are disclosed in WO 05/118544. This compound is a novel selective, highly potent, adenosine triphosphate (ATP) competitive inhibitor of Aurora kinases (A, B and C), as demonstrated in a variety of cell-based assays. AS703569 was shown to exhibit potent anti-tumor activity against a broad panel of cancer cell lines. Lung carcinoma cells, breast cancer cells, pancreatic carcinoma cells, cervical carcinoma cells, histiocytic lymphoma cells and ovarian carcinoma cells are particularly sensitive to AS703569 (Maier et al., 2007). AS703569 also strongly inhibits tube formation by human umbilical vein endothelial cell (HUVEC).

The Aurora family of conserved serine/threonine kinases perform essential functions during cell division. The three mammalian paralogues are very similar in sequence, but differ significantly in their localization, function, substrates and regulatory partners. Aurora A is mainly associated with the spindle poles during mitosis, where it is required for centrosome separation and maturation (Sausville, 2004). Spindle assembly requires that targeting protein for XKLP 2 (TPX2) targets Aurora A to spindle pole microtubules through a mechanism that requires Ran- GTP (Marumoto et al., 2005). Aurora A also functions in meiosis promoting oocyte maturation, polar-body extrusion, spindle positioning and exit from metaphase I. Regulation of Aurora A occurs through phosphorylation/dephosphorylation and degradation. Protein phosphatase 1 negatively regulates Aurora and this interaction is modulated by TPX2. Aurora B is a chromosomal-passenger protein with multiple functions in mitosis. Inner centromere protein (INCENP) and survivin, two other components of the passenger complex, function as targeting and regulatory factors for the kinase (Bishop and Shumacher, 2002). Aurora B is required for phosphorylation of histone H3, targeting of condensin and normal chromosome compaction. It has also been recently shown to be essential for chromosome biohentation, kinetochore-microtubule interactions and the spindle- assembly checkpoint. Aurora B is essential for completion of cytokinesis. Myosin Il regulatory chain, vimentin, desmin and glial fibrillary acidic protein are among its cleavage furrow substrates. Aurora B phosphorylates MgcRacGAP, transforming it into an activator of RhoA in the contractile ring (Minoshima et al., 2003). Much less is known about Aurora C kinase, other than that it seems to be preferentially expressed in meiotic cells. During the cell cycle, Aurora kinases travel to their subcellular targets aided by their binding partner-substrates, INCENP, survivin and TPX2. This provides an additional level of regulation that might be essential for the choreography of mitotic events.

Aurora A and B kinases are frequently elevated in human cancers making them attractive targets for therapeutic intervention. Small molecule inhibitors of Aurora kinases have recently been reported, but their effect on cytokinesis has yet to be investigated in detail. For example a high selective and potent small-molecule inhibitor of Aurora kinases, VX-680, blocks cell-cycle progression and induces apoptosis in a diverse range of human tumor types. This compound causes profound inhibition of tumor growth in a variety of in vivo xenograft models, leading to regression of leukemia, colon and pancreatic tumors at well-tolerated doses (Harrington et al., 2004). Another novel cell cycle inhibitor, JNJ-7706621 , showed potent inhibition of several cyclin-dependent kinases (CDK) and Aurora kinases and selectively blocked proliferation of tumor cells of various origins, but was about 10-fold less effective at inhibiting normal human cell growth in vitro. In human cancer cells, treatment with JNJ-7706621 inhibited cell growth independent of p53, retinoblastoma, or P-glycoprotein status; activated apoptosis; and reduced colony formation. At low concentrations, JNJ-7706621 slowed the growth of cells and at higher concentrations induced cytotoxicity. Inhibition of CDK1 kinase activity, altered CDK1 phosphorylation status, and interference with downstream substrates such as retinoblastoma were also shown in human tumor cells following drug treatment. JNJ-7706621 delayed progression through G1 and arrested the cell cycle at the G2-M phase (Emanuel et al., 2005). Additional cellular effects due to inhibition of Aurora kinases included endoreduplication and inhibition of histone H3 phosphorylation. In a human tumor xenograft model, several intermittent dosing schedules were identified that produced significant antitumor activity.

The present invention had the objective of finding ways to further advance the pharmaceutical utility for AS703569. Therefore, combinations of AS703569 with other anticancer agents were studied in several cancer cell lines.

Surprisingly, it has been found by the inventors of the present patent application that AS703569 acts in a synergistic way when combined with other cancer therapeutics.

Figures

Different geometrical shapes (or orientations thereof) relate to different experiments conducted.

Fig. 1 : Evaluation of the effect of combining AS703569 with Erlotinib.

Fig. 1.A.1 : Cl / f_a plot of the sequential combination of AS703569 and

Erlotinib tested in cell line SQ20B. Fig. 1.A.2: Cl / f_a plot of the sequential combination of Erlotinib and

AS703569 tested in cell line SQ20B.

Fig. 1.A.3: Cl / f_a plot of the simultaneous combination of Erlotinib and

AS703569 tested in cell line SQ20B.

Fig. 1.B.1 : Cl / f_a plot of the sequential combination of AS703569 and Erlotinib tested in cell line OVCAR3.

Fig. 1.B.2: Cl / f_a plot of the sequential combination of Erlotinib and

AS703569 tested in cell line OVCAR3. Fig. 2: Evaluation of the effect of combining AS703569 with Gefitinib.

Fig. 2.A.1 : Cl / f_a plot of the sequential combination of AS703569 and Gefitinib tested in cell line SCC61. Fig. 2.A.2: Cl / f_a plot of the simultaneous combination of Erlotinib and

AS703569 tested in cell line SCC61.

Fig. 2.B.1 : Cl / f_a plot of the sequential combination of AS703569 and Gefitinib tested in cell line OVCAR3.

Fig. 3: Evaluation of the effect of combining AS703569 with Cetuximab. Cl / f_a plot of the sequential combination of AS703569 and Cetuximab tested in cell line SQ20B.

Fig. 4: Evaluation of the effect of combining AS703569 with Sorafenib. Fig. 4.A.1 : Cl / f_a plot of the sequential combination of Sorafenib and

AS703569 tested in cell line OVCAR3.

Fig. 4.B.1 : Cl / f_a plot of the sequential combination of Sorafenib and AS703569 tested in cell line Miapaca2.

Fig. 5: Evaluation of the effect of combining AS703569 with Imatinib.

Cl / f_a plot of the sequential combination of AS703569 and lmatinibtested in cell Nne SCC61.

Detailed description of the invention

The present invention relates to a method for prophylaxis and/or treatment of cancer comprising administering to a subject AS703569 and/or its physiologically acceptable salts and solvates, and one or more further cancer therapeutics. AS703569 and/or its physiologically acceptable salts and solvates, and the other cancer therapeutic or therapeutics can be administered simultaneously or sequentially. When administered simultaneously, AS703569 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.

In a preferred embodiment, the method according to the invention comprises the use of AS703569 and/or its physiologically acceptable salts and solvates, and one other cancer therapeutic.

The present invention relates in particular to a method for prophylaxis and/or treatment of tumors selected from the group consisting of pancreatic tumor, breast tumor, lung tumor (such as adenocarcinoma, small-cell and non-small-cell lung carcinomas), colorectal and head and neck cancers, acute myelogenous leukemia and chronic myelogenous leukemia. However, the treatment method also relates to brain tumor (such as glioblastoma), tumor of the urogenital tract, tumor of the lymphatic system, stomach tumor, laryngeal tumor, renal cell carcinoma, endometrial carcinoma, multiple myeloma and prostate cancer.

In a preferred embodiment the methods according to the present invention relate to the treatment of cancer and, in particular, to the tumors described hereinabove and below.

Moreover, the present invention relates to a mixture of compounds, comprising AS703569 and one or more other cancer therapeutics and physiologically acceptable salts and solvates of each such compound.

Suitable acid-addition salts are inorganic or organic salts of all physiologically or pharmacologically acceptable acids, for example halides, in particular hydrochlorides or hydrobromides, lactates, sulfates, citrates, tartrates, maleates, fumarates, oxalates, acetates, phosphates, methylsulfonates, benzoates or p-toluenesulfonates.

Solvates of the compounds of the formula I are taken to mean adductions of inert solvent molecules onto the compounds of the formula I 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.

A preferred salt form of AS703569 is mono benzoate, particularly preferred is anhydrous mono benzoate.

The expression "effective amount" denotes the amount of a medicament or of a pharmaceutical active ingredient which causes in a tissue, system, animal or human a biological or medical response which is sought or desired, for example, by a researcher or physician.

In addition, the expression "therapeutically effective amount" denotes an amount which, compared with a corresponding subject who has not received this amount, has the following consequence: improved treatment, healing, prevention or elimination of a disease, syndrome, condition, complaint, disorder or prevention of side effects or also reduction in the progress of a disease, condition or disorder. The term "therapeutically effective amount" also encompasses the amounts which are effective for increasing normal physiological function.

The compound mixtures according to the invention include mixtures of two compounds, for example in the ratio 1 :1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :10, 1 :100 or 1 :1000.

Furthermore, the present invention relates to the use of the said compound mixture for the preparation of a medicament for the treatment of cancer.

The compounds and compound mixtures here can be converted into suitable pharmaceutical preparations or dosage forms together with at least one solid, liquid and/or semi-liquid excipient or adjuvant. Therefore, the invention also relates to a pharmaceutical preparation comprising the said compound mixture according to the invention and the said excipients and/or adjuvants. The invention also relates to a set (kit) consisting of separate packs of

(a) an effective amount of AS703569 and/or physiologically acceptable salts and solvates thereof,

(b) an effective amount of another cancer therapeutic and/or physiologically acceptable salts and solvates thereof and, optionally,

(c) an effective amount of a third cancer therapeutic and/or physiologically acceptable salts and solvates thereof.

The set comprises suitable containers, such as boxes, individual bottles, bags or ampoules. The set may, for example, comprise separate ampoules, each containing an effective amount of AS703569 and/or pharmaceutically usable salts and solvates thereof, and an effective amount of another cancer therapeutic in dissolved or lyophilised form.

Cancer therapeutics that can be combined with AS703569 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.

Alkylating agents include altretamine, AMD-473, AP-5280, apaziquone, bendamustine, brostallicin, busulfan, carboquone, carmustine (BCNU), chlorambucil, VNP 40101 M, cyclophosphamide, decarbazine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine (CCNU), mafosfamide, melphalan, mitobronitol, mitolactol, nimustine, nitrogen mustard N-oxide, ranimustine, temozolomide, thiotepa, treosulfan, trofosfamide and the like.

Angiogenesis inhibitors include endothelial-specific receptor tyrosine kinase (Tie- 2) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, insulin growth factor-1 receptor (IGF-1 R-) inhibitors, matrix metalloproteinase-2 (MMP-2) inhibitors, matrix metalloproteinase-9 (MMP-9) inhibitors, platelet-derived growth factor receptor (PDGFR) inhibitors, thrombospondin analogs vascular endothelial growth factor receptor tyrosine kinase (VEGFR) inhibitors and the like.

Bcr-Abl kinase inhibitors include Dasatanib, imatinib and the like.

CDK inhibitors include AZD-5438, BMM 040, BMS-032, BMS-387, CVT-2584, flavopyridol, GPC-286199, MCS-5A, PD0332991 , PHA-690509, seliciclib (CYC- 202, R-roscovitine), ZK-304709 and the like.

COX-2 inhibitors include ABT-963, etohcoxib, valdecoxib, BMS347070, celecoxib, COX-189 (lumiracoxib), CT-3, deracoxib, JTE-522, 4-methyl-2-(3,4- dimethylphenyl)-1 -(4-sulfamoylphenyl-1 H-pyrrole), MK-663 (etoricoxib), NS-398, parecoxib, RS-57067, SC-58125, SD-8381 , SVT-2016, S-2474, T-614, rofecoxib and the like.

EGFR inhibitors include ABX-EGF, anti-EGFr immunoliposomes, EGF-vaccine, matuzumab, cetuximab, HR3, IgA antibodies, gefitinib, erlotinib (OSI-774), TP-38, EGFR fusion protein, lapatinib and the like.

ErbB2 receptor inhibitors include CP-724-714, CM 033 (canertinib), trastuzumab, lapatinib, 2C4 (petuzumab), TAK-165, GW-572016 (ionafamib), GW-282974, EKB- 569, PM 66, dHER2 (HER2 vaccine), APC-8024 (HER-2 vaccine), anti-HER/2neu bispecific antibody, B7.her21gG3, AS HER2 trifunctional bispecific antibodies, mAb AR-209, mAb 2B-1 and the like. Histone deacetylase inhibitors include depsipeptide, LAQ-824, MS-275, trapoxin, suberoylanilide hydroxamic acid (SAHA), TSA, valproic acid and the like.

HSP-90 inhibitors include 17-AAG-nab, 17-AAG, CNF-101 , CNF-1010, CNF-2024, 17-DMAG, geldanamycin, IPI-504, KOS-953, NCS-683664, PU24FC1 , PU-3, radicicol, SNX-2112, STA-9090 VER49009 and the like.

MEK inhibitors include ARRAY-142886, ARRAY-438162 PD-325901 , PD-98059, XL-518, RDEA119, the compounds disclosed in WO 06/045514 and the like.

mTOR inhibitors include AP-23573, CCI-779, everolimus, RAD-001 , rapamycin, temsirolimus and the like. In a preferred embodiment AS703569 is combined with mTOR inhibitors. Rapamycin is a preferred mTOR inhibitor according to the invention.

Polo-like kinase inhibitors include BI-2536 and the like.

Thrombospondin analogs include ABT-510, ABT-567, ABT-898, TSP- 1 and the like.

VEGFR inhibitors include bevacizumab, ABT-869, AEE-788, axitinib (AG-13736), AZD-2171 , CP-547,632, IM-862, Macugen (pegaptamib), sorafenib (BAY43-9006), pazopanib (GW-786034), (PTK-787, ZK-222584), sunitinib (SU-11248), VEGF trap, vatalanib, vandetanib (ZD-6474) and the like. Antimetabolites include premetrexed disodium, LY231514, MTA), 5-azacitidine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, cytarabine ocfosfate, cytosine arabinoside, decitabine, deferoxamine, doxifluhdine, eflornithine, EICAR, enocitabine, ethnylcytidine, fludarabine, hydroxyurea, 5-fluorouracil (5-FU) alone or in combination with leucovorin, gemcitabine, hydroxyurea, melphalan, mercaptopurine, 6-mercaptopurine riboside, methotrexate, mycophenolic acid, nelarabine, nolatrexed, ocfosate, pelitrexol, pentostatin, raltitrexed, Ribavirin, triapine, trimetrexate, S-1 , tiazofurin, tegafur, TS-1 , vidarabine, UFT and the like. Antibiotics include intercalating antibiotics aclarubicin, actinomycin D, amrubicin, annamycin, adriamycin, bleomycin, daunorubicin, doxorubicin, elsamitrucin, epirbucin, glarbuicin, idarubicin, mitomycin C, nemorubicin, neocarzinostatin, peplomycin, pirarubicin, rebeccamycin, stimalamer, streptozocin, valrubicin, zinostatin and the like.

Topoisomerase inhibitors include aclarubicin, 9-aminocamptothecin, amonafide, amsacrine, becatecarin, belotecan, BN-80915, irinotecan hydrochloride, camptothecin, dexrazoxine, diflomotecan, edotecarin, epirubicin, etoposide, exatecan, 10-hydroxycamptothecin, gimatecan, lurtotecan, mitoxantrone, orathecin, pirarbucin, pixantrone, rubitecan, sobuzoxane, SN-38, tafluposide, topotecan and the like.

Antibodies include bevacizumab, CD40-specific antibodies, chTNT-1/B, denosumab, cetuximab, zanolimumab, IGF1 R-specific antibodies, lintuzumab, edrecolomab, WX G250, htuximab, ticilimumab, trastuzimab, cetuximab and the like.

Hormonal therapies include anastrozole, exemestane, arzoxifene, bicalutamide, cetrorelix, degarelix, deslorelin, trilostane, dexamethasone, flutamide, raloxifene, fadrozole, toremifene, fulvestrant, letrozole, formestane, glucocorticoids, doxercalciferol, lasofoxifene, leuprolide acetate, megesterol, mifepristone, nilutamide, tamoxifen citrate, abarelix, predisone, finasteride, rilostane, buserelin, luteinizing hormone releasing hormone (LHRH)), vantas, trilostane (modrastane), fosrelin, goserelin and the like.

Deltoids and retinoids include seocalcitol (EB1089, CB1093), lexacalcitrol (KH1060), fenretinide, aliretinoin, liposomal tretinoin, bexarotene, LGD-1550 and the like.

Plant alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, vinorelbine and the like. Proteasome inhibitors include bortezomib, MG132, NPI-0052, PR-171 and the like.

Examples of immunologicals include interferons and other immune-enhancing agents. Interferons include interferon alpha, interferon alpha-2a, interferon alpha- 2b, interferon beta, interferon gamma-l a, interferon gamma-i b, or interferon gamma-ni , combinations thereof and the like. Other agents include BAM-002, tasonermin, tositumomab, alemtuzumab, CTLA4 (cytotoxic lymphocyte antigen 4), decarbazine, denileukin, epratuzumab, lenograstim, lentinan, leukocyte alpha interferon, imiquimod, MDX-010, melanoma vaccine, mitumomab, molgramostim, gemtuzumab ozogamicin, filgrastim, OncoVAC-CL, oregovomab, pemtumomab (Y- muHMFGI ), sargaramostim, sizofilan, teceleukin, ubenimex, Z 100, WF-10, aldesleukin, thymalfasin, daclizumab, 90Y-lbritumomab tiuxetan and the like. Biological response modifiers are agents that modify defense mechanisms of living organisms or biological responses, such as survival, growth, or differentiation of tissue cells to direct them to have anti-tumor activity and include krestin, lentinan, sizofuran, picibanil PF-3512676 (CpG-8954), ubenimex and the like.

Pyrimidine analogs include cytarabine (ara C), cytosine arabinoside, doxifluridine, fludarabine, 5-FU (5-fluorouracil), floxuridine, gemcitabine, ratitrexed, triacetyluridine troxacitabine and the like.

Purine analogs include thioguanine and mercaptopuhne.

Antimitotic agents include batabulin, epothilone D (KOS-862), N-(2-((4- hydroxyphenyl)amino)pyridin-3-yl)-4-methoxybenzenesulfonamide, ixabepilone (BMS 247550), paclitaxel, docetaxel, PNU100940 (109881 ), patupilone, XRP- 9881 , vinflunine, ZK-EPO and the like.

In a preferred embodiment AS703569 is combined with signalling inhibitors. The term "signalling inhibitor" as used herein refers to signalling inhibitors or analogues of signalling inhibitors as described herein, including the ionic, salt, solvate, isomers, tautomers, N-oxides, ester, prodrugs, isotopes and protected forms thereof (preferably the salts or tautomers or isomers or N-oxides or solvates thereof, and more preferably, the salts or tautomers or N-oxides or solvates thereof), as described above.

A malignant tumour is the product of uncontrolled cell proliferation. Cell growth is controlled by a delicate balance between growth-promoting and growth-inhibiting factors. In normal tissue the production and activity of these factors results in differentiated cells growing in a controlled and regulated manner that maintains the normal integrity and functioning of the organ. The malignant cell has evaded this control; the natural balance is disturbed (via a variety of mechanisms) and unregulated, aberrant cell growth occurs.

One driver for growth is the epidermal growth factor (EGF), and the receptor for EGF (EGFR) has been implicated in the development and progression of a number of human solid tumours including those of the lung, breast, prostate, colon, ovary, head and neck. EGFR is a member of a family of four receptors, namely EGFR (HERI or ErbBI), ErbB2 (HER2lneu), ErbB3 (HER3), and ErbB4 (HER4). These receptors are large proteins that reside in the cell membrane, each having a specific external ligand binding domain, a transmembrane domain and an internal domain which has tyrosine kinase enzyme activity. When EGF attaches to EGFR, it activates the tyrosine kinase, triggering reactions that cause the cells to grow and multiply. EGFR is found at abnormally high levels on the surface of many types of cancer cells, which may divide excessively in the presence of EGF. Activating mutations of EGFR that lead to constitutive activity in the absence of ligand have also been identified in certain types of cancers such as Non-Small Cell Lung Cancer. Inhibition of EGFR activity has therefore been a target for molecularly targeted therapeutic research in the treatment of cancer. Such inhibition can be effected by direct interference with the target EGFR on the cell surface, for example by the use of antibodies, or by inhibiting the subsequent tyrosine kinase activity. Examples of antibodies which target EGFRs are the monoclonal antibodies trastuzumab and cetuximab.

Amplification of the human epidermal growth factor receptor 2 protein (HER 2) in primary breast carcinomas has been shown to correlate with a poor clinical prognosis for certain patients. Trastuzumab is a highly purified recombinant DNA- derived humanized monoclonal IgGI kappa antibody that binds with high affinity and specificity to the extracellular domain of the HER2 receptor. In vitro and in vivo preclinical studies have shown that administration of trastuzumab alone or in combination with paclitaxel or carboplatin significantly inhibits the growth of breast tumour-derived cell lines that over-express the HER2 gene product. In clinical studies trastuzumab has been shown to have clinical activity in the treatment of breast cancer. The most common adverse effects of trastuzumab are fever and chills, pain, asthenia, nausea, vomiting, diarrhea, headache, dyspnea, rhinitis, and insomnia. Trastuzumab has been approved for the treatment of metastatic breast cancer involving over-expression of the HER2 protein in patients who have received one or more chemotherapy regimes.

Cetuximab has been used for the treatment of irotecan-refractory colorectal cancer. It is also being evaluated both as a single agent and in combination with other agents for use in the treatment of a variety of other cancers for example head and neck cancer, metastatic pancreatic carcinoma, and non-small-cell lung cancer. Examples of agents which target EGFR tyrosine kinase activity include the tyrosine kinase inhibitors gefitinib and erlotinib. Gefitinib which has the chemical name N- (3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4- amine, is used for the treatment of non-small-cell lung cancer, and is also under development for other solid tumours that over-express EGF receptors such as breast and colorectal cancer. It has been found that patients receiving gefitinib may develop interstitial lung disease that causes inflammation within the lung. Eye irritation has also been observed in patients receiving gefitinib. Erlotinib, which has the chemical name N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4- amine has also been used for the treatment of non-small-cell lung cancer, and is being developed for the treatment of various other solid tumours such as pancreatic cancer, the most common side effects being rash, loss of appetite and fatigue; a more serious side effect which has been reported is interstitial lung disease.

Another growth factor which has received attention as a target for anticancer research is the vascular endothelial growth factor (VEGF). VEGF is a key regulator of vasculogenesis during angiogenic processes including wound healing, retinopathy, psoriasis, inflammatory disorders, tumour growth and metastasis. Studies have shown that over-expression of VEGF is strongly associated with invasion and metastasis in human malignant disease.

An example of an antibody that targets the VEGF antigen on the surface of a cell is the monoclonal antibody bevacizumab which is a recombinant humanised monoclonal IgGI antibody that binds to and inhibits VEGF.

Bevacizumab has been used for the treatment of colorectal cancer, for example in combination with 5-fluorouracil. Bevacizumab also being developed as a potential treatment for other solid tumours such as metastatic breast cancer, metastatic non- small-cell lung cancer and renal cell carcinoma. The most serious adverse events associated with bevacizumab include gastrointestinal perforations, hypertensive crises, nephrotic syndrome and congestive heart failure. Other therapeutic agents in development which target the action of VEGF at alternate points in the signal transduction cascade intiated by this growth factor include sunitinib which inhibits the kinase activity of the VEGF receptor.

Another growth factor of importance in tumour development is the platelet-derived growth factor (PDGF) that comprises a family of peptide growth factors that signal through cell surface tyrosine kinase receptors (PDGFR) and stimulate various cellular functions including growth, proliferation, and differentiation. PDGF expression has been demonstrated in a number of different solid tumours including glioblastomas and prostate carcinomas. The tyrosine kinase inhibitor imatinib, which has the chemical name 4-[(4- methylpiperazin-1 -yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]- phenyl]-benzamide, blocks activity of the Bcr-Abl oncoprotein and the cell surface tyrosine kinase receptor c-Kit, and as such is approved for the treatment on chronic myeloid leukemia and gastrointestinal stromal tumours. Imatinib mesylate is also a potent inhibitor of PDGFR kinase and is currently being evaluated for the treatment of chronic myelomonocytic leukemia and glioblastoma multiforme, based upon evidence in these diseases of activating mutations in PDGFR. The most frequently reported drug-related adverse events were edema, nausea, vomiting, cramps and musculoskeletal pain.

A further kinase target for cancer chemotherapy is inhibition of Raf which is a key enzyme in the chain reaction of the body's chemistry that triggers cell growth. Abnormal activation of this pathway is a common factor in the development of most cancers, including two-thirds of melanomas. By blocking the action of Raf kinase, it may be possible to reverse the progression of these tumours. One such inhibitor is sorafenib (BAY 43-9006) which has the chemical name 4-[4-[[4-chloro-3- (trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2- carboxamide. Sorafenib targets both the Raf signalling pathway to inhibit cell proliferation and the VEGFR/PDGFR signalling cascades to inhibit tumour angiogenesis. Raf kinase is a specific enzyme in the Ras pathway. Mutations in the Ras gene occur in approximately 20 percent of all human cancers, including 90 percent of pancreatic cancers, 50 percent of colon cancers and 30 percent of non- small cell lung cancers.

Sorafenib is being investigated for the treatment of a number of cancers including liver and kidney cancer. The most common side effects of sorafenib are pain, swelling, redness of the hands and/or feet, and also rash, fatigue and diarrhea.

The signalling inhibitors used in combination with AS according to the invention are specific inhibitors of cell signalling proteins as described above and have activity against various cancers. These combinations are beneficial in the treatment of many types of cancer.

Combinations with a molecularly targeted agent such as a signalling inhibitor (e.g. gefitinib, bevacizumab, trastuzumab, or imatinib) would find particular application in relation to cancers which express or have activated the relevant molecular target such as EGF receptor, VEGF receptor, ErbB2, BCRabl, c-kit, PDGF. Diagnosis of such tumours could be performed using techniques known to a person skilled in the art and as described herein such as RTPCR, immunohistochemistry and FISH.

Preferred signalling inhibitors for use in accordance with the invention include antibodies targeting EGFR such as monoclonal antibodies trastuzumab and cetuximab, EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib, VEGF targeting antibody is bevacizumab, PDGFR inhibitor such as imatinib mesylate and Raf inhibitor such as sorafenib referred to herein.

Preferred antibodies targeting EGFR include the monoclonal antibodies trastuzumab and cetuximab. Especially preferred is cetuximab.

Trastuzumab is commercially available from Genentech lnc under the trade name Herceptin, or may be obtained as described in US 5,821 ,337. Cetuximab is commercially available from Merck Serono S.A. or Bristol-Myers Squibb Corporation under the trade name Erbitux, or may be obtained as described in WO 96/40210.

Preferred EGFR tyrosine kinase inhibitors include gefitinib and erlotinib. Gefitinib is commercially available from AstraZeneca pic under the trade name Iressa, or may be obtained as described in WO 96/33980. Erlotinib is commercially available from Pfizer lnc under the trade name Tarceva, or may be obtained as described in WO 96/30347. A preferred antibody targeting VEGF is bevacizumab which is commercially available from Genentech lnc under the trade name Avastin, or may be obtained as described in WO 94/10202.

A preferred PDGFR inhibitor is imatinib which is commercially available (mesylate salt) from Novartis AG under the trade name Gleevec (also referred to as Glivec), or may be obtained as described in EP 564409 A1.

A preferred Raf inhibitor is sorafenib which is available from Bayer AG, or may be obtained as described in WO 00/42012.

Especially preferred signalling inhibitors for use in combination with AS703569 according to the invention, are gefitinib, cetuximab, erlotinib, imatinib and sorafenib.

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. Such medicaments can be prepared using all processes known in the pharmaceutical art by, for example, combining the active ingredient with the excipient(s) or adjuvant(s).

Compounds and compound mixtures adapted for oral administration can be administered as separate units, such as, for example, capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foam foods; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

Thus, for example, in the case of oral administration in the form of a tablet or capsule, the compound or compound mixtures can be combined with an oral, nontoxic and pharmaceutically acceptable inert excipient, such as, for example, ethanol, glycerol, water and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing it with a pharmaceutical excipient comminuted in a similar manner, such as, for example, an edible carbohydrate, such as, for example, starch or mannitol. A flavour, preservative, dispersant and dye may likewise be present.

Capsules are produced by preparing a powder mixture as described above and filling shaped gelatine shells therewith. Glidants and lubricants, such as, for example, highly disperse silicic acid, talc, magnesium stearate, calcium stearate or polyethylene glycol in solid form, can be added to the powder mixture before the filling operation. A disintegrant or solubiliser, such as, for example, agar-agar, calcium carbonate or sodium carbonate, may likewise be added in order to improve the availability of the compound or compound mixtures after the capsule has been taken.

In addition, if desired or necessary, suitable binders, lubricants and disintegrants as well as dyes can likewise be incorporated into the mixture. Suitable binders include starch, gelatine, natural sugars, such as, for example, glucose or beta- lactose, sweeteners made from maize, natural and synthetic rubber, such as, for example, acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. The lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. The disintegrants include, without being restricted thereto, starch, methylcellulose, agar, bentonite, xanthan gum and the like. The tablets are formulated by, for example, preparing a powder mixture, granulating or dry-pressing the mixture, adding a lubricant and a disintegrant and pressing the entire mixture to give tablets. A powder mixture is prepared by mixing the compound comminuted in a suitable manner with a diluent or a base, as described above, and optionally with a binder, such as, for example, carboxymethylcellulose, an alginate, gelatine or polyvinylpyrrolidone, a dissolution retardant, such as, for example, paraffin, an absorption accelerator, such as, for example, a quaternary salt, and/or an absorbant, such as, for example, bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting it with a binder, such as, for example, syrup, starch paste, acadia mucilage or solutions of cellulose or polymer materials and pressing it through a sieve. As an alternative to granulation, the powder mixture can be run through a tableting machine, giving lumps of non-uniform shape which are broken up to form granules. The granules can be lubricated by addition of stearic acid, a stearate salt, talc or mineral oil in order to prevent sticking to the tablet casting moulds. The lubricated mixture is then pressed to give tablets. The compounds and compound mixtures according to the invention can also be combined with a free-flowing inert excipient and then pressed directly to give tablets without carrying out the granulation or dry- pressing steps. A transparent or opaque protective layer consisting of a shellac sealing layer, a layer of sugar or polymer material and a gloss layer of wax may be present. Dyes can be added to these coatings in order to be able to differentiate between different dosage units.

Oral liquids, such as, for example, solution, syrups and elixirs, can be prepared in the form of dosage units so that a given quantity comprises a prespecified amount of the compound. Syrups can be prepared by dissolving the compounds and compound mixtures in an aqueous solution with a suitable flavour, while elixirs are prepared using a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersion of the compound in a non-toxic vehicle. Solubilisers and emulsifiers, such as, for example, ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavour additives, such as, for example, peppermint oil, or natural sweeteners or saccharin or other artificial sweeteners, and the like, can likewise be added.

The dosage unit formulations for oral administration can, if desired, be encapsulated in microcapsules. The formulation can also be prepared in such a way that the release is extended or retarded, such as, for example, by coating or embedding of particulate material in polymers, wax and the like. The compounds and compound mixtures according to the invention and salts and solvates thereof can also be administered in the form of liposome delivery systems, such as, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from various phospholipids, such as, for example, cholesterol, stearylamine or phosphatidylcholines.

The compounds and compound mixtures according to the invention can also be delivered using monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds and compound mixtures can also be coupled to soluble polymers as targeted medicament carriers. Such polymers may encompass polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidophenol, polyhydroxyethylaspartamidophenol or polyethylene oxide polylysine, substituted by palmitoyl radicals. The compounds may furthermore be coupled to a class of biodegradable polymers which are suitable for achieving controlled release of a medicament, for example polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

Compounds and compound mixtures adapted for transdermal administration can be administered as independent plasters for extended, close contact with the epidermis of the recipient. Thus, for example, the active ingredient can be delivered from the plaster by iontophoresis, as described in general terms in Pharmaceutical Research, 3(6):318, 1986.

Compounds and compound mixtures adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

For the treatment of the eye or other external tissue, for example mouth and skin, the formulations are preferably applied as topical ointment or cream. In the case of formulation to give an ointment, the compounds or compound mixtures can be employed either with a paraffinic or a water-miscible cream base. Alternatively, the compounds or compound mixtures can be formulated to give a cream with an oil-in- water cream base or a water-in-oil base.

Compounds and compound mixtures adapted for topical application to the eye include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, in particular an aqueous solvent.

Compounds and compound mixtures adapted for topical application in the mouth encompass lozenges, pastilles and mouthwashes.

Compounds and compound mixtures adapted for rectal administration can be administered in the form of suppositories or enemas.

Compounds and compound mixtures adapted for nasal administration in which the carrier substance is a solid comprise a coarse powder having a particle size, for example, in the range 20-500 microns, which is administered in the manner in which snuff is taken, i.e. by rapid inhalation via the nasal passages from a container containing the powder held close to the nose. Suitable formulations for administration as nasal spray or nose drops with a liquid as carrier substance encompass active-ingredient solutions in water or oil.

Compounds and compound mixtures adapted for administration by inhalation encompass finely particulate dusts or mists, which can be generated by various types of pressurised dispensers with aerosols, nebulisers or insufflators.

Compounds and compound mixtures adapted for vaginal administration can be administered as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Compounds and compound mixtures adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions comprising antioxidants, buffers, bacteriostatics and solutes, by means of which the formulation is rendered isotonic with the blood of the recipient to be treated; and aqueous and nonaqueous sterile suspensions, which may comprise suspension media and thickeners. The formulations can be administered in single-dose or multidose containers, for example sealed ampoules and vials, and stored in freeze-dried (lyophilised) state, so that only the addition of the sterile carrier liquid, for example water for injection purposes, immediately before use is necessary. Injection solutions and suspensions prepared in accordance with the recipe can be prepared from sterile powders, granules and tablets.

It goes without saying that, in addition to the above particularly mentioned constituents, the medicaments according to the invention may also comprise other agents usual in the art with respect to the particular type of pharmaceutical formulation; thus, for example, compounds or compound mixtures which are suitable for oral administration may comprise flavours.

A therapeutically effective amount of a compound or compound mixture of the present invention depends on a number of factors, including, for example, the age and weight of the recipient, 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 treating doctor or vet. However, an effective amount of a compound of the formula I for the treatment of the diseases according to the invention is generally in the range from 0.1 to 100 mg/kg of body weight of the recipient (mammal) per day and particularly typically in the range from 1 to 10 mg/kg of body weight per day. Thus, the actual amount per day for an adult mammal weighing 70 kg is usually between 70 and 700 mg, where this amount can be administered as an individual dose per day or more usually in a series of part- doses (such as, for example, two, three, four, five or six) per day, so that the total daily dose is the same. An effective amount of a salt or solvate or of a physiologically functional derivative thereof can be determined as a fraction of the effective amount of the compounds and compound mixtures according to the invention per se. The pharmaceutical preparations according to the invention can be employed as medicaments in human and veterinary medicine. Suitable excipients are organic or inorganic substances which are suitable for enteral (for example oral), parenteral or topical administration and do not react with the novel compounds, for example water, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates, such as lactose or starch, magnesium stearate, talc or Vaseline. Suitable for enteral administration are, in particular, tablets, coated tablets, capsules, syrups, juices, drops or suppositories, suitable for parenteral administration are solutions, preferably oil-based or aqueous solutions, furthermore suspensions, emulsions or implants, and suitable for topical application are ointments, creams or powders. The compounds and compound mixtures may also be lyophilised and the resultant lyophilisates used, for example, for the preparation of injection preparations.

The preparations indicated may be sterilised and/or comprise adjuvants, such as lubricants, preservatives, stabilisers and/or wetting agents, emulsifiers, salts for modifying the osmotic pressure, buffer substances, dyes, flavours and/or aroma substances. They can, if desired, also comprise one or more further active ingredients, for example one or more vitamins.

Examples

The examples below relate to pharmaceutical preparations:

Example A1 : Injection vials

A solution of 100 g of a compound or a compound mixture according to the invention and 5 g of disodium hydrogenphosphate in 3 I of bidistilled water is adjusted to pH 6.5 using 2N hydrochloric acid, sterile filtered, transferred into injection vials, lyophilised and sealed under sterile conditions. Each injection vial contains 5 mg of active ingredients.

Example A2: Suppositories 20 g of a compound or a compound mixture according to the invention is melted with 100 g of soya lecithin and 1400 g of cocoa butter, poured into moulds and allowed to cool. Each suppository contains 20 mg of active ingredients.

Example A3: Solution

A solution is prepared from 1 g of a compound or a compound mixture according to the invention, 9.38 g of NaH₂PO₄ x 2 H₂O, 28.48 g of NaH₂PO₄ x 12 H₂O and 0.1 g of benzalkonium chloride in 940 ml of bidistilled water. The pH is adjusted to 6.8, and the solution is made up to 1 I and sterilised by irradiation. This solution can be used in the form of eye drops.

Example A4: Ointment

500 mg of a compound or a compound mixture according to the invention are mixed with 99.5 g of Vaseline under aseptic conditions.

Example A5: Tablets

1 kg of a compound or a compound mixture according to the invention, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is pressed to give tablets in a conventional manner in such a way that each tablet contains 10 mg of active ingredients.

Example A6: Coated tablets

Tablets are pressed analogously to Example E and subsequently coated in a conventional manner with a coating of sucrose, potato starch, talc, tragacanth and dye.

Example A7: Capsules

2 kg of a compound or a compound mixture according to the invention are introduced into hard gelatine capsules in a conventional manner in such a way that each capsule contains 20 mg of the active ingredients. Example A8: Ampoules

A solution of 1 kg of a compound or a compound mixture according to the invention in 60 I of bidistilled water is transferred into ampoules, lyophilised under aseptic conditions and sealed under sterile conditions. Each ampoule contains 10 mg of active ingredients.

The following examples relate to combination studies using AS703569 and other cancer therapeutics:

Example B: Combination of AS703569 and further cancer therapeutics in human cancer cell lines

In the following examples the anhydrous mono benzoate salt form of AS703569 was used.

Cell lines: The cell lines OVCAR3, Miapaca2, SCC61 and SQ20B were obtained from the ATCC (Rockville, MD, USA). Cells were grown as monolayers in RPMI medium supplemented with 10% fetal calf serum (InVitrogen, Cergy Pontoise, France), 2 mM glutamine, 100 units/mL penicillin and 100 g/mL streptomycin. All cells were split twice a week using trypsin/EDTA (0.25% and 0.02%, respectively; InVitrogen, CergyPontoise, France) and seeded at a concentration of 2.5 x 10⁴ cells/mL. All cell lines were tested regularly for Mycoplasma contamination by PCR using a Stratagene kit (La JoIIa, CA, USA).

In vitro growth inhibition assay (MTT assay): The MTT assay was carried out as described previously (Hansen et al., 1989). In brief, cells were seeded in 96well tissue culture plates at a density of 2 x 10³ cells/well. Cell viability was determined after 120 h incubation by the colorimetric conversion of yellow, water soluble tetrazolium MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma, SaintQuentin Fallavier, France), into purple, water insoluble formazan. This reaction is catalyzed by mitochondrial dehydrogenases and is used to estimate the relative number of viable cells (Mosmann, 1983). Cells were incubated with 0.4 mg/mL MTT for 4 h at 37°C. After incubation, the supernatant was discarded, the cell pellet was resuspended in 0.1 ml_ of DMSO and the absorbance was measured at 560 nm by use of a microplate reader (Dynatech, Michigan, USA). Wells with untreated cells or with drug-containing medium without cells were used as positive and negative controls respectively. Growth inhibition curves were plotted as a percentage of untreated control cells.

Single agent study: The cells were seeded at 2 x 10³ cells/well in 96well plates and treated 24 h later with increasing concentrations of AS703569. After 4 h, 24 h or 48 h incubation the cells were washed and postincubated in drugfree medium for 72 h. Growth inhibition were then determined by the MTT assay.

Combination study: The effects of exposure of the cells to AS703569 combined with other cancer therapeutics were studied in SCC61 , Miapaca2, OVCAR3 and SQ20B cell lines using combination indices (Cl) that represent the ratio of the concentrations necessary for a given cell death rate, as previously described by Chou and Talalay (1984). Calculation of a combination index (Cl) < 1 indicates synergy, the value of 1 indicates additive effects, and values >1 indicate antagonism.

Statistical analysis and determination of synergistic activity: Effects of drug combinations were evaluated using the Chou and Talalay method which is based on the median-effect principle. This involves plotting dose-effect curves for each drug and for multiple diluted, fixed-ratio combinations, using the equation: f_a / f_u = (C / C_m)^m, where C is the drug concentration, IC₅₀ the concentration required for a halfmaximal effect (i.e., 50% inhibition of cell growth), f_a the cell fraction affected by the drug concentration C (e.g., 0.9 if cell growth is inhibited by 90%), f_u the unaffected fraction, and m the sigmoidicity coefficient of the concentration-effect curve. On the basis of the slope of the curve for each drug in a combination, it can be determined whether the drugs have mutually nonexclusive effects (e.g., independent or interactive modes of action). The combination index (Cl) is then determined by the equation:

Cl = [(C)₁Z(Cx)₁]+ [(C)₂ / (Cx)₂] + [α (C)₁ (C)₂ / (Cx)₁ (Cx)₂], where (Cx)₁ is the concentration of drug 1 required to produce an x percent effect of that drug alone, and (C)₁, the concentration of drug 1 required to produce the same x percent effect in combination with (C)₂. If the mode of action of the drugs is mutually exclusive or nonexclusive, then α is 0 or 1 , respectively. Cl values will be calculated by solving the equation for different values of f_a (i.e., for different degrees of cell growth inhibition). Data were analyzed on an IBM-PC computer using concentrationeffect analysis for microcomputer software (Biosoft, Cambridge, UK). For statistical analysis and graphs we used lnstat and Prism software (GraphPad, San Diego, USA). The dose-effect relationships for the drugs tested, alone or in paired combinations, were subjected to median-effect plot analysis to determine their relative potency (IC₅₀), shape (m), and conformity (r) in each selected cell line. As stated above, the IC₅O and m values were respectively used to calculate synergism and antagonism on the basis of the Cl equation. Results were expressed as the mean ± standard deviation of at least 3 experiments performed in duplicate. In each experiment, cells were exposed to the paired combinations for 48h as described above. Means and standard deviations were compared using Student's t-test (two-sided p value).

Example B1 : Combination of AS703569 and Erlotinib

1.A) Cell line SQ20B

1.A.1 ) The cells were seeded at 2 x 10³ cells/well in 96well plates and allowed to grow for 24 h. Cells were then exposed to various concentrations of AS703569 (corresponding to the IC₂O, IC₄₀ or IC₆O values) for 24 h, the drug was removed, the cells were washed and various concentrations of Erlotinib (corresponding to the IC₂O, IC₄₀ or IC₆O values) were added. After an exposure for 24 h or 48 h, Erlotinib was removed, the cells were washed and post-incubated in drug-free medium for 72 h. Growth inhibition was then determined by the MTT assay. As can be seen in Figure 1.A.1 , the sequential combination of the two cancer therapeutics produces a synergistic effect.

1.A.2) The cells were cultured as described in Example 1.A.1. Cells were then exposed to various concentrations of Erlotinib (corresponding to the IC₂O, IC₄₀ or IC₆O values for 24 h or 48 h. The drug was removed, the cells were washed and various concentrations of AS703569 (corresponding to the IC₂O, IC₄₀ or IC₆O values were added. After an exposure for 24 h, AS703569 was removed, the cells were washed and post-incubated in drug-free medium for 72 h. Growth inhibition was then determined by the MTT assay.

As can be seen in Figure 1.A.2, the sequential combination of the two cancer therapeutics produces a synergistic effect.

1 A3) The cells were seeded at 2 x 10³ cells/well in 96well plates and treated 24 hours later with various concentrations of AS703569 and Erlotinib, corresponding to the IC₂O, IC₄₀ or IC₆O values, simultaneously for 24 h or 48 h. Both drugs were then removed, cells were washed and further inclubated in drug-free medium for an additional 72h, after which the growth inhibitory effects were measured by the MTT assay. As can be seen in Figure 1 A3, the simultaneous combination of the two cancer therapeutics produces a synergistic effect.

1.B) Cell line OVCAR3

1.B.1 ) It was proceeded as described in Example 1.A.1.

As can be seen in Figure 1.B.1 , the sequential combination of the two cancer therapeutics produces a synergistic effect.

1.B.2) It was proceeded as described in Example 1.A.2. As can be seen in Figure 1.B.2, the sequential combination of the two cancer therapeutics produces a synergistic effect. Example B2: Combination of AS703569 and Gefitinib

2.A) Cell line SCC61

2.A.1 ) The procedure according to Example 1.A.1 was followed, except that Gefitinib was used as the other cancer therapeutic.

As can be seen in Figure 2.A.1 , the sequential combination of the two cancer therapeutics produces a synergistic effect.

2.A.2) The procedure according to Example 1.A.3 was followed, except that Gefitinib was used as the other cancer therapeutic.

As can be seen in Figure 2.A.2, the simultaneous combination of the two cancer therapeutics produces a synergistic effect.

2.B) Cell line OVCAR3

2.B.1 ) The procedure according to Example 1.A.1 was followed, except that Gefitinib was used as the other cancer therapeutic.

As can be seen in Figure 2.B.1 , the sequential combination of the two cancer therapeutics produces a synergistic effect.

Example B3: Combination of AS703569 and Cetuximab tested in cell line SQ20B

The procedure according to Example 1.A.1 was followed, except that Cetuximab was used as the other cancer therapeutic.

As can be seen in Figure 3, the sequential combination of the two cancer therapeutics produces a synergistic effect.

Example B4: Combination of AS703569 and Sorafenib

4.A) Cell line OVCAR3 4.A.1 ) The procedure according to Example 1.A.2 was followed, except that Sorafenib was used as the other cancer therapeutic.

As can be seen in Figure 4.A.1 , the sequential combination of the two cancer therapeutics produces a synergistic effect.

4.B) CeII line Miapaca2

4.B.1 ) The procedure according to Example 1.A.2 was followed, except that Sorafenib was used as the other cancer therapeutic.

As can be seen in Figure 4.B.1 , the sequential combination of the two cancer therapeutics produces a synergistic effect.

Example B5: Combination of AS703569 and lmatinib tested in cell line SCC61

The procedure according to Example 1.A.1 was followed, except that lmatinib was used as the other cancer therapeutic.

As can be seen in Figure 5, the sequential combination of the two cancer therapeutics produces a synergistic effect.

Literature

1 ) Maier A , Schϋler JB , Bausch N , Fiebig HH Romanelli A , Gianella- Borradori A. Preclinical evaluation of the orally available Aurora kinase inhibitor AS703569 to support the selection of tumor indications for clinical studies. 98th AACR Annual Meeting, Los Angeles, CA. April 14-18 (2007).

2) Sausville EA. Aurora kinases dawn as cancer drug targets, Nat. Med., 10, 234-235 (2004).

3) Marumoto T, Zhang D, Saya H. Aurora A - A guardian of poles, Nature, 5, 42-50 (2005). 4) Bishop JD and Shumacher JM. Phosphorylation of the Carboxyl Terminus of Inner Centromere Protein (INCENP) by the aurora B Kinase Stimulates aurora B Kinase Activity, J. Biol. Chem. 277, 27577-27580 (2002).

5) Minoshima Y, Kawashima T, Hirose K, Tonozuka Y, Kawajiri A, Bao Y, Deng X, Tatsuka M, Narumiya S, May W Phosphorylation by aurora B converts MgcRacGAP to a RhoGAP during cytokinesis. Dev. Cell 4, 549- 560 (2003).

6) Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun

AO, Nakayama T. Graham JA, Demur C, Hercend T, Diu-Hercend A, Su M, Golec JM, Miller KM VX-680, a potent and selective small-molecule inhibitor of the aurora kinases, suppresses tumor growth in vivo, Nat. Med., 10, 262-267 (2004).

7) Emanuel S, Rugg CA, Gruninger RH, Lin R, Fuentes-Pesquera A, Connolly PJ, Wetter SK, Hollister B, Kruger WW, Napier C, JoIMfTe L, Middleton SA, The in vitro and in vivo effects of JNJ-7706621 : A dual inhibitor of cyclin- dependent kinases and aurora kinases, Cancer Res., 65, 9038-9046 (2005).

8) Hansen M, Nielsen S, Berg K (1989) Reexamination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 119: 203-210.

9) Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.

10) Chou TC, Talalay P (1984) Quantitive analysis of doseeffect relationships : the combined effects of multiple drugs or enzyme inhibitors. Adv Enz Regul 22: 27-55.

Claims

Patent Claims

1. Compound mixture, comprising (1 R,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 or physiologically acceptable salts and solvates thereof and one other cancer therapeutic or physiologically acceptable salts thereof.

2. Compound mixture according to Claim 1 , wherein the other cancer therapeutic is a signalling inhibitor.

3. Compound mixture according to Claim 2, wherein the signalling inhibitor is a kinase inhibitor.

4. Compound mixture according to Claim 2, wherein the signalling inhibitor is an antibody.

5. Compound mixture according to Claim 4, wherein the antibody is a anti-EGFR antibody.

6. Compound mixture according to Claim 3, wherein the kinase inhibitor is a EGFR tyrosine kinase inhibitor.

7. Compound mixture according to Claim 2, wherein the signalling inhibitor is selected from a group consisting of gefitinib, cetuximab, erlotinib, imatinib and sorafenib.

8. Pharmaceutical preparation, comprising a compound mixture according to Claim 1 - 7 and optionally excipients and/or adjuvants.

9. Set (kit) consisting of separate packs of

(a) an effective amount of (1 R,2R,3S,4S)-N4-(3- aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro-N2-[(3-nnethyl-4-(4- methylpiperazin-1 -yl)]phenyl-2,4-pyrimidinediannine and/or physiologically acceptable salts and solvates thereof and

(b) an effective amount of a another cancer therapeutic and/or physiologically acceptable salts and solvates thereof.

10. Method for prophylaxis and/or treatment of cancer comprising administering to a subject (1 R,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 and/or its physiologically acceptable salts and solvates, and one or more further cancer therapeutics.

11. Method for prophylaxis and/or treatment of cancer comprising administering to a subject (1 R,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 and/or its physiologically acceptable salts and solvates, and one other cancer therapeutic and/or its physiologically acceptable salts and solvates.

12. Method according to claim 11 , wherein the other cancer therapeutic is a signalling inhibitor.

13. Method according to Claim 12, wherein the signalling inhibitor is a kinase inhibitor.

14. Method according to Claim 12, wherein the signalling inhibitor is an antibody.

15. Method according to Claim 14, wherein the antibody is a anti-EGFR antibody.

16. Method according to Claim 13, wherein the kinase inhibitor is a EGFR tyrosine kinase inhibitor.

17. Method according to Claim 12, wherein the signalling inhibitor is selected from a group consisting of gefitinib, cetuximab, erlotinib, imatinib and sorafenib.