Kinase inhibitor combinations for cancer treatment

Abstract

FIELD: medicine.SUBSTANCE: kit for treating cancer is disclosed, comprising separate packages of an effective amount of 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide and an effective amount of a MEK inhibitor, which is pimasertib. Also a method for treating cancer is disclosed comprising orally administering 20 or 25 mg / kg of 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide and 15 or 20 mg / kg of pima-sertib.EFFECT: synergistic effect of 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid and pima-sertib in the treatment of cancer.7 cl, 10 dwg, 4 tbl, 6 ex

Description

[0001] The present invention relates to combinations of 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)ethylamino]quinazoline-8-carboxylic acid amide (hereinafter referred to as M2698) and / or physiologically acceptable salts and solvates thereof, and a MEK kinase inhibitor, and as an optional third inhibitor, with an inhibitor of the tyrosine-protein kinase receptor ERBB-1, also known as EGFR (epidermal growth factor receptor), and to the use of such combinations for the treatment of cancer. The invention also relates to combinations of M2698 with an EGFR inhibitor without a MEK kinase inhibitor.

[0002] Prerequisites for the creation of the invention

[0003] M2698, methods for its production, and its use in the treatment of cancer are disclosed in WO 2012 / 069146 (referred to as Compound A). This compound is a selective, potent dual inhibitor of p70S6K and Akt, which has been demonstrated in various cellular assays. M2698 has been shown to exhibit potent antitumor activity against a wide range of cancer cell lines. Breast cancer cells, glioblastoma multiforme (GBM) cells, endometrial cancer cells, and ovarian cancer cells have been found to be particularly sensitive to M2698. M2698 penetrates the blood-brain barrier in vivo.

[0004] Protein kinases constitute a large family of structurally related enzymes responsible for the control of a wide range of signaling processes within the cell (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book. I and II, Academic Press, San Diego, CA). Kinases can be divided into families based on the substrates they phosphorylate (e.g., protein tyrosine, protein serine / threonine, lipids, etc.). Sequence motifs that generally correspond to each of these kinase families have been identified (e.g., Hanks, SK, Hunter, T., FASEB J., 9:576–596 (1995); Knighton, et al., Science, 253:407–414 (1991); Hiles, et al., Cell, 70:419–429 (1992); Kunz, et al., Cell, 73:585–596 (1993); Garcia-Bustos, et al., EMBO J., 13:2352–2361 (1994)). Protein kinases can be characterized by their mechanisms of regulation.

[0005] These mechanisms include, for example, autophosphorylation, transphosphorylation by other kinases, protein-protein interactions, protein-lipid interactions, and protein-polynucleotide interactions. An individual protein kinase can be regulated by more than one mechanism. Kinases regulate a variety of cellular processes, including, but not limited to, proliferation, differentiation, apoptosis, motility, transcription, translation, and other signaling processes by adding phosphate groups to target proteins. These phosphorylation events act as molecular switches that can modulate or regulate the biological function of the target protein. Phosphorylation of target proteins occurs in response to various extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.), cell cycle events, environmental or nutritional stresses, etc.The corresponding protein kinase functions in signaling pathways to activate or inactivate (directly or indirectly), for example, a metabolic enzyme, a regulatory protein, a receptor, a cytoskeletal protein, an ion channel or pump, or a transcription factor. Uncontrolled signaling due to defective control of protein phosphorylation is associated with a number of diseases, including, for example, inflammation, cancer, allergies / asthma, immune system diseases and conditions, central nervous system diseases and conditions, and angiogenesis.

[0006] P70S6K inhibition

[0007] Protein kinase 70S6K, 70 kDa ribosomal protein kinase p70S6K (also known as SK6, p70 / p85 S6 kinase, p70 / p85 ribosomal S6 kinase, and pp70S6K) is a member of the AGC subfamily of protein kinases. p70S6K is a serine-threonine kinase that is a component of the phosphatidylinositol-3 kinase (PI3K) / AKT pathway. p70S6K is downstream of PI3K and is activated through phosphorylation at a number of sites in response to numerous mitogens, hormones, and growth factors. p70S6K activity is also under the control of the mTOR-containing complex (TORC1), as rapamycin inhibits p70S6K activity. p70S6K is regulated by the downstream PDK targets Akt and PKC. Akt directly phosphorylates and inactivates TSC2, thereby activating mTOR. Furthermore, studies of mutant p70S6K alleles, which are inhibited by wortmannin but not rapamycin, suggest that the PI3K pathway may influence Hap70S6K independently of its regulation of mTOR activity.The p70S6K enzyme modulates protein synthesis by phosphorylating ribosomal protein S6. S6 phosphorylation correlates with increased translation of mRNAs encoding components of the translational apparatus, including ribosomal proteins and translation elongation factors, the increased expression of which is necessary for cell growth and proliferation. These mRNAs contain an oligopyrimidine tract at the beginning of their 5' transcription (called 5'TOP), which has been shown to be essential for their regulation at the translational level.

[0008] In addition to its role in translation, p70S6K activation is also involved in cell cycle control, neuronal differentiation, regulation of cell motility, and cellular responses, which are important in tumor metastasis, immune response, and tissue repair. Antibodies to p70S6K abolish the mitogenic response induced by rat fibroblast entry into S phase, indicating that p70S6K function is necessary for the G1-S phase transition in the cell cycle. Furthermore, inhibition of cell cycle proliferation in the G1-S phases of the cell cycle by rapamycin was identified as a consequence of inhibition of the production of the hyperphosphorylated activated form of p70S6K.

[0009] The role of p70S6K in tumor cell proliferation and cell protection from apoptosis is supported by its involvement in growth factor receptor signal transduction, overexpression, and activation in tumor tissues. For example, Northern and Western analyses showed that PS6K gene amplification was accompanied by a corresponding increase in mRNA and protein expression, respectively (Cancer Res. (1999) 59: 1408-11—Localization of PS6K to Chromosomal Region 17q23 and Determination of Its Amplification in Breast Cancer).

[0010] Clinical inhibition of p70S6K activation was observed in patients with renal cell carcinoma treated with CCI-779 (rapamycin ester), an mTOR upstream kinase inhibitor. A significant linear relationship between disease progression and inhibition of p70S6K activity was reported.

[0011] In response to energy stress, the tumor suppressor LKB1 activates AMPK, which phosphorylates the TSC1 / 2 complex and allows it to inactivate the mTOR / p70S6K pathway. Mutations in LKB1 cause Peutz-Jeghers syndrome (PJS), in which patients with PJS are 15 times more likely to develop cancer than the general population. Furthermore, one-third of lung adenocarcinomas contain inactivating mutations in LKB1.

[0012] Compounds described as suitable for inhibiting p70S6K are described in WO 03 / 064397, WO 04 / 092154, WO 05 / 054237, WO 05 / 056014, WO 05 / 033086, WO 05 / 117909, WO 05 / 039506, WO 06 / 120573, WO 06 / 136821, WO 06 / 071819, WO 06 / 131835, WO 08 / 140947, WO 10 / 093419, WO 12 / 013282 and WO 12 / 069146.

[0013] M2698, which not only inhibits p70S6K but also inhibits Akt kinase isoforms 1 and 3 (upstream of p70S6K in the PI3K pathway), has been shown to provide more efficient PI3K pathway shutdown (Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J. Proc. Natl Acad Sci USA. 2008 Nov 11; 105(45): 17414-9.), and allow any activation of the Akt feedback loop to be captured (Tamburini et al. Blood 2008; 111: 379 82).

[0014] Inhibition of the PAM / Akt / mTOR (PAM) pathway is antagonized by subsequent activation of the Akt feedback loop (O'Reilly KE et al. Cancer Res. 2006; 66(3): 1500-1508). M2698, a selective dual inhibitor of p70S6K and Akt1 / 3, blocks signaling downstream of the Akt feedback loop; therefore, M2698 may improve clinical efficacy compared with single-node PAM pathway inhibitors such as mTOR inhibitors. M2698 reduces tumor growth and prolongs survival in orthotopically implanted human glioblastoma multiforme (GBM) models (Machl A et al. Am J Cancer Res. 2016; 6(4): 806-818).

[0015] The present invention aimed to find ways to further improve the pharmaceutical utility of M2698. In this context, combinations of M2698 with a MEK kinase inhibitor as a dual combination and, in addition, with an EGFR inhibitor as a triple combination, as well as combinations of M2698 with an EGFR inhibitor as a dual combination were studied in vivo.

[0016] MEK inhibition

[0017] The mitogen-activated protein kinase (MAPK) signaling pathway plays a crucial role in the regulation of diverse cellular activities, including cell proliferation, survival, differentiation, and motility (Karin LCM Nature. 2001; 410:37-40). Dysregulation of the MAPK pathway occurs in more than a third of all malignancies. The classical MAPK pathway consists of Ras (a family of related proteins that are expressed in all animal cell lines and organs), Raf (a family of three serine / threonine-specific protein kinases related to retroviral oncogenes), MEK (mitogen-activated protein kinase kinase), and ERK (extracellular signal-regulated kinases), which sequentially transmit proliferative signals generated by cell surface receptors to the nucleus via cytoplasmic signaling. The MEK inhibitor targets the Ras / Raf / MEK / ERK signaling pathway, inhibiting cell proliferation and inducing apoptosis.Therefore, it has potential for clinical application in cancer treatment, especially for those cancers that are caused by RAS / RAF dysfunction (Leonard JT et al., J. Hematol. Oncol. 2016;9:31. doi: 10.1186 / sl3045-016-0258-l).

[0018] Due to the widespread activation of this pathway in numerous malignancies, MEK inhibitors are under development and study as monotherapy or combination therapy with other targeted and cytotoxic agents in various clinical settings. More recently, combination therapy with immune checkpoint inhibitors has emerged as an effective treatment for certain cancers, expanding the efficacy of this class of agents (Thompson N et al., Curr. Opin. Pharmacol. 2005; 5:350-356).

[0019] Examples of MEK inhibitors in clinical development and / or approved by regulatory authorities include trametinib, cobimetinib, selumetinib, refametinib, and pimasertib. Pimasertib is N-(2,3-dihydroxypropyl)-3-(2-fluoro-4-iodophenylamino)isonicotinamide and has been described, among other things, in WO 2006045514 (Example 115).

[0020] Unexpectedly, the inventors of the present patent application found that M2698 acts synergistically when combined with a MEK inhibitor.

[0021] EGFR Inhibition

[0022] The epidermal growth factor receptor is a member of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). In many types of cancer, mutations affecting the expression or activity of EGFR can lead to cancer (Zhang H et al., The Journal of Clinical Investigation. 117 (S): 2051–2058). Epidermal growth factor and its receptor were discovered by Stanley Cohen of Vanderbilt University. Cohen shared the 1986 Nobel Prize in Medicine with Rita Levi-Montalcini for their discovery of growth factors. Deficient signaling of EGFR and other receptor tyrosine kinases in humans is associated with diseases such as Alzheimer's disease, while overexpression is associated with the development of a wide range of tumors.Interruption of EGFR signaling either by blocking EGFR binding sites on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity can prevent the growth of EGFR-expressing tumors and improve patient outcome.

[0023] Examples of EGFR inhibitors in clinical development and / or approved by regulatory authorities include gefitinib, erlotinib, afatinib, brigatinib, icotinib, osimertinib, and cetuximab (a chimeric (mouse / human) monoclonal antibody used to treat metastatic colorectal and head and neck cancer).

[0024] Unexpectedly, the inventors of the present patent application have found that M2698 acts synergistically in combination with a MEK inhibitor and, optionally, additionally, with an EGFR inhibitor, or in combination with an EGFR inhibitor alone.

[0025] Figures

[0026] Fig. 1: In vitro assays in GSC lines. (A) Western blot analysis of protein expression; (B) IC 50 M2698; (C) IC 50 pimasertib; and (D) apoptotic GSC11, GSC7-2, and GSC17 cells after treatment with M2698, pimasertib, or the combination of M2698 and pimasertib. Individual agents were tested at their IC 50 ; half of the IC was used for the combination 50 each connection.

[0027] Fig. 2: (A) Tumor volume and (B) median survival in orthotopic GSC xenograft models after treatment with vehicle, M2698, pimasertib, or the combination of M2698 + pimasertib.

[0028] Fig. 3: (A) pS6 expression, (B) pERK expression, and (C) Ki-67 expression in GSC17 and GSC7-2 xenograft models after treatment with vehicle control, M2698, pimasertib, or M2698+pimasertib combination.

[0029] Fig. 4: PDX models of NSCLC brain metastasis.

[0030] Fig. 5: PDX models of Her2+ / HR- breast cancer.

[0031] Fig. 6: Combination of M2698 and pimacertib in 12 PDX models of cholangiocarcinoma.

[0032] Fig. 7: M2698, pimasertib and cetuximab in 75 PDX CRC models (individual agent groups).

[0033] Fig. 8: M2698, pimacertib and cetuximab in 75 PDX CRC models (dual combinations).

[0034] Fig. 9: M2698, pimacertib and cetuximab in 75 PDX CRC models (triple combination).

[0035] Fig. 10: M2698 and cetuximab in 38 PDX models of PCKGS (double combination).

[0036] Detailed description of the invention

[0037] The present invention relates to a method for the prevention and / or treatment of cancer, comprising administering to a subject M2698 and / or its physiologically acceptable salts and solvates, a MEK inhibitor and, optionally, an EGFR inhibitor.

[0038] M2698 and / or its physiologically acceptable salts and solvates, as well as other active ingredients, can be administered simultaneously or sequentially. When administered simultaneously, M2698 and / or its physiologically acceptable salts and solvates and the MEK inhibitor can be administered as a mixture of the compounds in a single pharmaceutical composition or as separate pharmaceutical compositions.

[0039] In a preferred embodiment, the method according to the invention comprises the use of M2698 and / or physiologically acceptable salts and solvates thereof, a MEK inhibitor and, optionally, an EGFR inhibitor, which are administered sequentially.

[0040] The present invention relates, in particular, to a method for the prevention and / or treatment of tumors selected from the group consisting of colorectal cancer, breast cancer (in particular, the Her2+ / HR- type), cholangiocarcinoma, GBM, SCCHN and NSCLC (in particular, NSCLC brain metastases).

[0041] Furthermore, the present invention relates to a pharmaceutical composition comprising a mixture of active pharmaceutical ingredients (API) M2698 and physiologically acceptable salts and solvates thereof, as well as a MEK inhibitor and physiologically acceptable salts and solvates.

[0042] Suitable acid addition salts are inorganic or organic salts of all physiologically or pharmacologically acceptable acids, such as halides, in particular hydrochlorides or hydrobromides, lactates, sulfates, citrates, tartrates, maleates, fumarates, oxalates, acetates, phosphates, methylsulfonates, benzoates or p-toluenesulfonates.

[0043] Solvates of M2698 and MEK inhibitors refer to the adduction of inert solvent molecules onto M2698, resulting from their mutual attraction. Solvates include, for example, hydrates, such as monohydrates or dihydrates, or alcoholates, i.e., addition compounds with alcohols, such as methanol or ethanol.

[0044] The preferred salt form of M2698 is its free base. Also preferred are its hydrochloride, dihydrochloride, mesylate, succinate, or malonate salts.

[0045] The expression "effective amount" means the amount of a drug or pharmaceutically active ingredient that produces in a tissue, system, animal or human being the biological or medical response sought or desired, for example, by a researcher or physician.

[0046] Furthermore, the term "therapeutically effective amount" means an amount that, compared to a corresponding subject who has not received this amount, has the following effect: improving the treatment, healing, prevention, or elimination of a disease, syndrome, condition, complaint, or disorder, or preventing side effects, or also slowing the progression of a disease, condition, or disorder. The term "therapeutically effective amount" also encompasses amounts effective in enhancing normal physiological function.

[0047] The pharmaceutical composition of the present invention comprises mixtures of two APIs, for example, in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:100 or 1:1000.

[0048] Furthermore, the pharmaceutical composition comprises at least one solid, liquid and / or semi-liquid excipient or adjuvant. Thus, this invention also relates to a pharmaceutical composition comprising the said API mixture of this invention and the said excipients and / or adjuvants.

[0049] Furthermore, the present invention relates to the use of said pharmaceutical composition for the preparation of a medicament for the treatment of cancer.

[0050] The present invention also relates to a set (kit) consisting of individual packs

[0051] (a) a pharmaceutical composition containing an effective amount of M2698,

[0052] (b) a pharmaceutical composition comprising an effective amount of a MEK inhibitor and, optionally,

[0053] (c) a pharmaceutical composition comprising an effective amount of an EGFR inhibitor.

[0054] The present invention also relates to a set (kit) consisting of individual packs

[0055] (a) a pharmaceutical composition containing an effective amount of M2698, and

[0056] (b) a pharmaceutical composition comprising an effective amount of an EGFR inhibitor.

[0057] The kit includes suitable containers, such as boxes, individual vials, bags, or ampoules. The kit may, for example, include individual ampoules, each containing a pharmaceutical composition containing an effective amount of M2698 and / or its pharmaceutically acceptable salts, a pharmaceutical composition containing an effective amount of a MEK inhibitor and / or its pharmaceutically acceptable salts, and, optionally, a pharmaceutical composition containing an effective amount of an EGFR inhibitor in dissolved or lyophilized form.

[0058] A particularly preferred brain-penetrating MEK inhibitor for combination with M2698 is pimasertib. A particularly preferred EGFR inhibitor for combination with M2698, with or without a MEK inhibitor, is cetuximab. A particularly preferred triple combination is M2698 + pimasertib + cetuximab.

[0059] The compounds and mixtures of compounds of the present invention may be adapted for administration by any desired suitable route, for example, 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 may be prepared using all methods known in the pharmaceutical art, for example, by combining the active ingredient with auxiliary substance(s) or adjuvant(s).

[0060] The compounds and mixtures of compounds intended for oral administration may be administered as discrete units such as, for example, capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foam products; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0061] For example, for oral administration in tablet or capsule form, the compound or mixture of compounds can be combined with an orally acceptable, non-toxic, and pharmaceutically acceptable inert excipient, such as ethanol, glycerol, water, and the like. Powders are obtained by grinding the compound to a suitable fine size and mixing it with a similarly ground pharmaceutical excipient, such as a food carbohydrate such as starch or mannitol. Flavoring, preservatives, dispersants, and coloring agents may also be present.

[0062] Capsules are produced by preparing a powder mixture as described above and filling it into molded gelatin shells. Glidants and lubricants, such as highly dispersed silicic acid, talc, magnesium stearate, calcium stearate, or solid polyethyleneglycol, may be added to the powder mixture before filling. A disintegrating agent or solubilizer, such as agar-agar, calcium carbonate, or sodium carbonate, may also be added to improve the availability of the compound or compound mixtures after ingestion.

[0063] Furthermore, if desired or necessary, suitable binders, lubricants, and disintegrating agents, as well as coloring agents, can also be introduced into the mixture. Suitable binders include starch, gelatin, natural sugars such as, for example, glucose or beta-lactose, sweeteners made from corn, natural and synthetic rubber such as, for example, acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrating agents include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.Tablets are prepared, for example, by preparing a powder mixture, granulating or dry-pressing the mixture, adding a lubricant and disintegrant, and compressing the entire mixture to obtain tablets. The powder mixture is prepared by mixing a suitably ground compound with a diluent or base as described above and, optionally, with a binder such as, for example, carboxymethylcellulose, alginate, gelatin, or polyvinylpyrrolidone, a dissolution retarder such as, for example, paraffin, an absorption accelerator such as, for example, a quaternary salt, and / or an absorbent 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, Acadian mucilage, or solutions of cellulose or polymeric materials, and pressing it through a sieve.As an alternative to granulation, the powder mixture can be passed through a tableting machine, producing irregularly shaped lumps that are broken into granules. The granules can be lubricated with stearic acid, a stearate salt, talc, or mineral oil to prevent sticking to tablet molds. The lubricated mixture is then compressed to form tablets. The compounds and mixtures of compounds of this invention can also be combined with a free-flowing inert filler and then directly compressed to form tablets, without granulation or dry pressing steps. A transparent or opaque protective layer may be present, consisting of a sealing layer of shellac, a layer of sugar or polymeric material, and a glossy wax layer. Dyes can be added to these coatings to differentiate the different dosage units.

[0064] Liquids for oral administration, such as solutions, syrups, and elixirs, can be prepared in unit dosage form, each containing a predetermined amount of the compound. Syrups can be prepared by dissolving the compounds and mixtures of compounds in a suitably flavored aqueous solution, while elixirs can be prepared using a non-toxic alcoholic carrier. Suspensions can be prepared by dispersing the compound in a non-toxic carrier. Solubilizers and emulsifiers, such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitan ethers, preservatives, flavoring agents such as peppermint oil, or natural sweeteners, or saccharin, or other artificial sweeteners, and the like, can also be added.

[0065] Standard dosage formulations for oral administration can, if desired, be encapsulated in microcapsules. The formulation can also be formulated to provide extended or delayed release, for example, by coating or incorporating the particulate material into polymers, wax, or the like.

[0066] The compounds and mixtures of the compounds of this invention, as well as their salts and solvates, can also be administered in the form of liposomal 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.

[0067] The compounds and mixtures of the compounds of this invention can also be delivered using monoclonal antibodies as individual carriers to which the compound molecules are linked. The compounds and mixtures of the compounds can also be linked to soluble polymers as targeted drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidophenol, polyhydroxyethylaspartamidophenol, or polylysine polyethylene oxide substituted with palmitoyl radicals. In addition, the compounds can be linked to a class of biodegradable polymers suitable for achieving controlled drug release, such as polylactic acid, polyepsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates, and cross-linked or amphipathic hydrogel block copolymers.

[0068] Compounds and mixtures of compounds intended for transdermal administration can be administered as stand-alone patches for prolonged, intimate contact with the recipient's epidermis. For example, the active ingredient can be delivered from the patch using iontophoresis, as described generally in Pharmaceutical Research, 3(6):318, 1986.

[0069] The compounds and mixtures of compounds intended for topical use may be prepared as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0070] For treating the eyes or other external tissues, such as the oral cavity and skin, the compositions are preferably applied as an ointment or cream for topical application. For ointment formulations, the compounds or mixtures of compounds can be used with either a paraffin or water-miscible cream base. Alternatively, the compounds or mixtures of compounds can be formulated to form a cream using an oil-in-water cream base or a water-in-oil base.

[0071] Compounds and mixtures of compounds intended for topical application to the eye include eye drops in which the active ingredient is dissolved or suspended in a suitable vehicle, in particular an aqueous solvent.

[0072] Compounds and mixtures of compounds intended for topical use in the mouth include lozenges, pastilles and mouthwashes.

[0073] Compounds and mixtures of compounds intended for rectal administration may be administered in the form of suppositories or enemas.

[0074] Compounds and mixtures of compounds intended for nasal administration, in which the carrier substance is a solid, comprise a coarse powder with a particle size of, for example, 20-500 microns, which is administered by nasal inhalation, i.e., by rapidly inhaling through the nasal passages from a container containing the powder held to the nose. Suitable formulations for administration as a nasal spray or nasal drops with a liquid as the carrier substance include solutions of the active ingredient in water or oil.

[0075] Compounds and mixtures of compounds intended for administration by inhalation include fine dusts or aerosols which may be generated by various types of pressurized aerosol dispensers, nebulizers or insufflators.

[0076] Compounds and mixtures of compounds intended for vaginal administration may be administered as pessaries, tampons, creams, gels, pastes, foams or sprays.

[0077] Compounds and mixtures of compounds intended for parenteral administration include aqueous and non-aqueous sterile injection solutions containing antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the recipient being treated; and aqueous and non-aqueous sterile suspensions, which may contain a suspending medium and thickening agents. The formulations may be administered in single-dose or multi-dose containers, such as sealed ampoules and vials, and stored in a freeze-dried (lyophilized) state so that only the addition of a sterile carrier liquid, such as water for injection, is necessary immediately before use. Prescribed injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0078] It goes without saying that, in addition to the above-mentioned specifically mentioned components, the medicinal products of the present invention may also include other agents conventional in the art with respect to the particular type of pharmaceutical composition; thus, for example, compounds or mixtures of compounds suitable for oral administration may contain flavoring agents.

[0079] The therapeutically effective amount of a compound or mixture of compounds of the present invention depends on a number of factors, including, for example, the age and weight of the recipient, the specific condition requiring treatment and its severity, the nature of the preparation and the route of administration, and is ultimately determined by the attending physician or veterinarian. However, the effective amount of the API for the treatment of diseases according to the present invention is typically in the range of 0.1 to 100 mg / kg of the recipient's (mammal's) body weight per day, and is particularly typically in the range of 1 to 10 mg / kg of body weight per day. Thus, the actual amount per day for an adult mammal weighing 70 kg is typically 70 to 700 mg, where this amount can be administered as an individual dose per day or more often as a series of partial doses (e.g., 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 a physiologically functional derivative thereof can be defined as a proportion of the effective amount of the compounds and mixtures of compounds of the invention per se.

[0080] The pharmaceutical preparations of the present invention can be used as medicinal products in human and veterinary medicine. Suitable excipients are organic or inorganic substances suitable for enteral (e.g. oral), parenteral or topical administration and not reacting with the new compounds, such as, for example, water, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc or petrolatum. For enteral administration, tablets, coated tablets, capsules, syrups, juices, drops or suppositories are particularly suitable; for parenteral administration, solutions, preferably oil-based or aqueous solutions, furthermore suspensions, emulsions or implants are suitable; and for topical application, ointments, creams or powders are suitable.Compounds and mixtures of compounds can also be lyophilized, and the resulting lyophilisates can be used, for example, to prepare injection preparations.

[0081] These preparations may be sterilized and / or contain adjuvants such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for modifying osmotic pressure, buffering agents, colorants, flavorings, and / or aromatic substances. If desired, they may also contain one or more additional active ingredients, such as one or more vitamins.

[0082] The present invention also relates to a method for the prevention and / or treatment of cancer, especially head and neck squamous cell carcinoma (HNSCC), comprising administering to a subject M2698 and / or its physiologically acceptable salts and solvates and an EGFR inhibitor, especially cetuximab, as well as corresponding compound mixtures, pharmaceutical compositions, formulations and methods of administration, as described above.

[0083] In some embodiments, the invention relates to such as:

[0084] 1. A mixture of compounds containing 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide or a physiologically acceptable salt thereof and a MEK inhibitor or a physiologically acceptable salt thereof.

[0085] 2. A mixture of compounds as described above in this list of embodiments, wherein the MEK inhibitor is pimasertib.

[0086] 3. A mixture of compounds as described above in this list of embodiments, further comprising an EGFR inhibitor.

[0087] 4. A pharmaceutical composition comprising a mixture of compounds as described above in this list and optionally excipients and / or adjuvants.

[0088] 5. A set (kit) containing individual packages

[0089] (a) an effective amount of 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide or a physiologically acceptable salt thereof and

[0090] (b) an effective amount of a MEK inhibitor, or a physiologically acceptable salt thereof.

[0091] 6. A kit (set) as described above in this list of embodiments, further comprising a separate package

[0092] (c) an effective amount of an EGFR inhibitor or a physiologically acceptable salt thereof.

[0093] 7. A kit (set) as described above in this list of embodiments, wherein the EGFR inhibitor is cetuximab.

[0094] 8. A method for preventing or treating cancer, comprising administering to a subject 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide or a physiologically acceptable salt thereof and a MEK inhibitor or a physiologically acceptable salt thereof.

[0095] 9. The method as described above in this list of embodiments, wherein the MEK inhibitor is pimasertib.

[0096] 10. The method as described above in this list of embodiments, further comprising administering to the subject an EGFR inhibitor or a physiologically acceptable salt thereof.

[0097] 11. The method as described above in this list of embodiments, wherein the EGFR inhibitor is cetuximab.

[0098] 12. The method as described above in this list of embodiments, wherein 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide, a MEK inhibitor and, optionally, an EGFR inhibitor are administered simultaneously.

[0099] 13. The method as described above in this list of embodiments, wherein 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide, a MEK inhibitor and optionally an EGFR inhibitor are administered sequentially.

[0100] 14. A mixture of compounds containing 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide or a physiologically acceptable salt thereof and an EGFR inhibitor, preferably cetuximab.

[0101] 15. A set (kit) containing individual packages

[0102] (a) an effective amount of 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide or a physiologically acceptable salt thereof and

[0103] (b) an effective amount of an EGFR inhibitor, preferably cetuximab.

[0104] 16. A method for preventing or treating cancer, comprising administering to a subject 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide or a physiologically acceptable salt thereof and an EGFR inhibitor, preferably cetuximab, wherein the two agents are administered either simultaneously or sequentially.

[0105] 17. In preferred embodiments, the cancer is colorectal cancer, breast cancer, cholangiocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, and non-small cell lung cancer.

[0106] Examples

[0107] Example 1: Combination of M2698 and pimacertib in patient-derived xenograft (PDX) models of glioblastoma (GBM)

[0108] GSC Lines: In vitro susceptibility to M2698 and pimacertib

[0109] Although the effects of M2698 in some GBM models were significant, the responses were relatively modest. Because both the PAM and MAPK pathways are involved in GBM, M2698 was combined with the brain-infiltrating cell / glioblastoma stem cell (GSC) model of the MEK inhibitor pima-sertib 19 in vivo and in vitro.

[0110] IC Data 50 in vitro were used to select GSC lines for in vivo studies; with IC values 50<2 μM cells were considered sensitive, and at values ​​>2 μM cells were considered resistant. All GSC lines expressed Akt. Relatively high pAkt expression in GSC17, GSC7-2, GSC231, GSC11, GSC20, GSC6-27, and GSC8-11 was comparable to GSC272 and GSC267 (Fig. 1A). Cell lines with relatively higher pAkt expression were sensitive to M2698, except for GSC20, indicating that constitutive activation of Akt increased cell sensitivity to M2698 (Fig. 1B). Fewer cell lines (GSC17, GSC11, and GSC231) were sensitive to pimasertib (IC 50 <2 μM), while GSC20, GSC272, GSC6-27, and GSC7-2 were resistant (Fig. 1C).

[0111] Apoptosis induction by M2698, pimasertib, and their combination was measured in three of five GSC lines that were sensitive to M2698 in vitro. Of these, GSC11 was the most sensitive to pimasertib, GSC17 was moderately sensitive, and GSC7-2 was not sensitive to pimasertib. Both M2698 and pimasertib administered as monotherapy to GSC11, GSC17, and GSC7-2 lines induced apoptosis, although there were no obvious differences in the percentage of apoptosis at IC 50 (56%, 11%, 13%, respectively) compared to 2 x IC 50 (62%, 12%, 17%, respectively) for each compound. The combination of M2698 + pimasertib had either an additive effect (GSC11 and GSC7-2 lines) or a clear synergistic effect (GSC17 line) in inducing apoptosis compared with each agent alone (Fig. 1D).

[0112] In vivo efficacy and PD effects of M2698 and pimasertib in orthotopic xenograft GSC models

[0113] The in vivo effects of M2698 and pimasertib, individually and in combination, were assessed in GSC models following orthotopic implantation in mice. Treatment duration and euthanasia time after implantation were 5 to 10 weeks. Endpoints included expression of the PD markers pS6 and pERK, the proliferation marker Ki67, apoptosis measured by TUNEL, tumor volume, and survival.

[0114] All but one (GSC20) of the seven GSC models were sensitive to M2698 monotherapy as measured by tumor volume; M2698 either significantly inhibited tumor growth (GSC17, GSC6-27, GSC7-2, GSC272, GSC231; all P<0.05; Fig. 2A) or showed a trend to do so (GSC11, P=0.10). Of the six models sensitive to M2698, five responded to pimasertib monotherapy with either significant tumor growth inhibition (GSC6-27, GSC7-2, GSC272; P<0.05) or a trend to a significant response (GSC231, P=0.10). Only GSC17 tumor growth was unaffected by pimasertib (P=0.34 vs. vehicle).

[0115] The M2698+pimasertib combination significantly inhibited tumor growth in all GSC models compared with vehicle, even in GSC20 (all P<0.05; Fig. 2A), which were unresponsive to monotherapy in tumor growth (P>0.05).

[0116] The median survival of the GSC models did not fully reflect the tumor volume data (Fig. IB). M2698 (GSC272 and GSC231) and the combination (GSC17 and GSC7-2), each of which increased survival in only two models compared with vehicle (P < 0.05) and pimasertib, did not significantly affect survival in any model compared with the control (P > 0.05). In GSC6-27, vehicle-treated mice had a significantly higher median survival than mice in the treatment groups (P < 0.05) for unknown reasons. In contrast, both monotherapies significantly inhibited tumor growth in some models, and the combination inhibited tumor growth in every model.

[0117] The significant effect on tumor volume indicates that the compounds penetrated the brain and acted on orthotopically growing tumors. Further evidence of blood–brain barrier (BBB) ​​penetration included effects on PD markers of target signaling pathways in tumor cells. M2698 significantly reduced pS6 in orthotopic tumors of all GSC models (P < 0.05; Fig. 3A) except GSC20. Similarly, no significant effects of pimasertib treatment on pERK protein were observed in GSC20 tumor (P = 0.18), whereas pimasertib significantly reduced pERK in other GSC tumors (P < 0.05; Fig. 3B), including GSC17, which did not show a reduction in tumor volume after pimasertib treatment. The combination of M2698 + pimacertib reduced pS6 and pERK in all models (all P < 0.05) except GSC20.The relatively high variability in tumor growth, pS6, and pERK in vehicle-treated mice in the GSC20 model may have masked the statistical significance of biologically relevant treatment effects on these endpoints. Conversely, the lack of a significant response was predicted by the insensitivity of GSC20 to both compounds in vitro (Fig. 1B and Fig. 1C).

[0118] In addition to the effects of pimasertib on pERK and M2698 on pS6 in orthotopic brain models, each M2698 was also able to influence the opposite PD marker. Pimasertib significantly reduced pS6 in the GSC272 and GSC231 models. M2698 reduced pERK in all GSC models except GSC20 (P < 0.05; Fig. 3B).

[0119] Three GSC tumors were analyzed at more than one time point for PD markers. Although statistical analysis of responses over time was not performed, trends across various endpoints were assessed. The percentage of p86-positive cells increased over time in GSC7-2 and GSC272 tumors, but not in GSC231 tumors, while pERK increased over time only in GSC7-2 tumors (Fig. 3). Crossover reduction of pS6 by pimasertib in the GSC272 and GSC231 models decreased from 7.5 to 10 weeks, and the potency of the compounds on pERK expression, individually or in combination, decreased from 7.5 to 10 weeks in the GSC231 model. Whether these trends are associated with the development of resistance requires further study, but they reflect the heterogeneity of the models.

[0120] Ki67 was measured by immunohistochemistry (Fig. 3C) in tumor sections from GSC17 and GSC7-2, two models in which the M2698 + pimasertib combination significantly prolonged survival. In the GSC17 model, all three treatments significantly reduced proliferation compared with vehicle, as did M2698 monotherapy and its combination with pimasertib in the GSC7-2 model (all P < 0.05).

[0121] Neither M2698, pimasertib, nor the combination treatment had any significant effect on apoptosis in GSC7-2 xenografts compared with control at 7.5 weeks. However, at 10 weeks, consistent with in vitro data, the M2698 + pimasertib combination significantly increased the number of apoptotic cells / field (172.33±60.80) compared with control (8.50±1.80; P=0.002), M2698 alone (52.17±25.20; P=0.01), and pimasertib alone (65.00±60.80; P=0.02).

[0122] Example 2: Combination of M2698 and pimasertib in patient-derived xenograft (PDX) models of non-small cell lung cancer (NSCLC) brain metastasis.

[0123] As shown in Fig. 4, the combination of M2698 with pimasertib unexpectedly showed a tumor control rate (TCR) of 26%, while the individual agents showed 5% (M2698) and 16% (pimasertib).

[0124] Example 3 : Combination of M2698 and pimasertib in patient-derived xenograft (PDX) models of Her2+ / HR- breast cancer

[0125] As shown in Fig. 5, the combination of M2698 with pimasertib unexpectedly reduces tumor volume to almost zero over the course of the experiment, while the individual agents simply achieve a stop in tumor volume growth.

[0126] Example 4 : Combination of M2698 and pimasertib in patient-derived xenograft (PDX) models of cholangiocarcinoma

[0127] The aim of this experiment was to evaluate the antitumor activity of M2698 alone and in combination with pimasertib in a panel of 13 patient-derived xenograft models representing human cholangiocarcinoma cancers derived from Chinese patients in immunodeficient mice. The Her2 status of the models was determined using immunohistochemistry (IHC) and is shown in Fig. 6.

[0128]

[0129] * QD every day

[0130] Vehicle: 0.5% Methylcellulose / 0.25% Tween20 in Milli-Q grade water. Tumors from three untreated mice were collected as auxiliary samples when the tumor volume reached 300-500 mm 3 .

[0131] As can be seen in Fig. 6, unexpectedly, tumor growth arrest was achieved with M2698 and pimasertib in 10 of 12 tumor samples (83%), while the individual agents demonstrated 17% (2 / 12, M2698) and 8% (1 / 12, pimasertib) tumor growth in response to treatment. Tumor growth in response to treatment or even regression were not observed in this experiment with any treatment.

[0132] Example 5 : Combinations of M2698, pimasertib, and cetuximab in 75 patient-derived xenograft (PDX) models of colorectal cancer.

[0133]

[0134] Of the 75 models, 25 were wild type, 25 were KRas mutants, and 25 were BRaf mutants.

[0135] Results

[0136] As shown in Fig. 7, for individual agents, the tumor control rates (TCR) were 15% (11 / 74) for M2698, 32% (24 / 74) for pimasertib, and 21% (15 / 73) for cetuximab.

[0137] As shown in Fig. 8, for the dual combinations, the TCRs were 59% (44 / 74) for the pimacertib + cetuximab combination, 35% (26 / 74) for the M2698 + cetuximab combination, and, surprisingly, 63% (45 / 72) for the M2698 + pimacertib combination.

[0138] As shown in Fig. 9, for the triple combinations, unexpectedly, the TCR was 78% (58 / 74), which was again significantly higher than the TCR of the double combinations.

[0139] Example 6: Combinations of M2698 and cetuximab in 38 patient-derived xenograft (PDX) models of CCCH

[0140] 46 PDX HCC models were run in a 1+1 design (one mouse per treatment group). Eight models were removed because tumors in the models did not grow. Adjusting for the removed models, 38 models were run. M2698 was administered with standard of care (SoC) cisplatin and cetuximab in all monotherapies and combinations in the 1+1 screening, i.e., one mouse per treatment per model. After implantation, treatment was started when the average tumor volume reached -200 mm 3for each model. Tumor volumes were measured for each model until the control tumor reached ~1200 mm, after which all animals in this model were euthanized.

[0141]

[0142] Results

[0143] As shown in the table above and Fig. 10, the TCR for the combination was 58% (22 / 38), which was an unexpected improvement in the TCR of individual agents in this difficult-to-treat cancer type.

Claims

1. A kit for the treatment of cancer, comprising separate packages of (a) an effective amount of 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide and (b) an effective amount of a MEK inhibitor that is pimasertib.

2. The kit of claim 1, further comprising a separate package (c) of an effective amount of an EGFR inhibitor that is cetuximab.

3. A method of treating cancer, comprising orally administering to a subject 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide at a dose of 20 or 25 mg / kg and a MEK inhibitor, which is pimasertib, at a dose of 15 or 20 mg / kg.

4. The method of claim 3, further comprising administering to the subject an EGFR inhibitor that is cetuximab.

5. The method according to any one of claims 3, 4, wherein 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide, a MEK inhibitor and optionally an EGFR inhibitor are administered simultaneously.

6. The method according to any one of claims 3, 4, wherein 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide, a MEK inhibitor and optionally an EGFR inhibitor are administered sequentially.

7. The method according to any one of claims 3-6, wherein the cancer is colorectal cancer, breast cancer, cholangiocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, and non-small cell lung cancer.

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