Kinase inhibitor combinations for cancer treatment

Abstract

The present invention relates to 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide and / or physiologically acceptable salts and solvates thereof, and inhibitors of MEK kinases, and combinations with inhibitors of EGFR as optional third inhibitors, and the use of such combinations for the treatment of cancer, and to combinations of 4-[(S)-2-azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide and / or physiologically acceptable salts and solvates thereof with inhibitors of EGFR.

Description

[0001] This invention relates to 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide (hereinafter referred to as M2698) and / or its physiologically acceptable salts and solvates, and inhibitors of MEK kinases, as an optional third inhibitor, in combination with an inhibitor of receptor tyrosine protein kinase ERBB-1 (also known as EGFR (epidermal growth factor receptor)), and the use of such combinations for the treatment of cancer. The invention also relates to combinations of M2698 with EGFR inhibitors in the absence of MEK kinase inhibitors. Background Technology

[0002] M2698, its preparation method, and its use in the treatment of cancer are disclosed in WO 2012 / 069146 (referred to as Compound A). This compound is a selective and highly potent dual inhibitor of p70S6K and Akt, as confirmed in various cell-based assays. M2698 has been shown to exhibit potent antitumor activity against a broad 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 crosses the blood-brain barrier in vivo.

[0003] Protein kinases constitute a large class of structurally related enzymes that control a wide variety of intracellular signal transduction processes (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book. I and II, Academic Press, San Diego, CA). Kinases can be classified into families based on their phosphorylated substrates (e.g., protein-tyrosine, protein-serine / threonine, lipids, etc.). Sequence motifs typically corresponding 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 regulatory mechanisms. These mechanisms include, for example, autophosphorylation, transphosphorylation via other kinases, protein-protein interactions, protein-lipid interactions, and protein-polynucleotide interactions. Individual protein kinases can be regulated by more than one mechanism. Kinases regulate a wide range 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 on / off switches that regulate or modulate the biological functions of target proteins. Target protein phosphorylation occurs in response to various extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.), cell cycle events, environmental or nutritional stresses, and so on. Appropriate protein kinases function in signaling pathways to (directly or indirectly) activate or inactivate, for example, metabolic enzymes, regulatory proteins, receptors, cytoskeletal proteins, ion channels or pumps, or transcription factors. Uncontrolled signaling due to defects in protein phosphorylation control has been implicated in many diseases, including, for example, inflammation, cancer, allergies / asthma, immune system disorders and conditions, central nervous system disorders and conditions, and angiogenesis.

[0004] P70S6K suppression

[0005] Protein kinase 70S6K, a 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 protein kinase subfamily. p70S6K is a serine-threonine kinase and a component of the phosphatidylinositol 3-kinase (PI3K) / AKT pathway. p70S6K is downstream of PI3K and its activation responds to numerous mitogens, hormones, and growth factors, occurring via phosphorylation at multiple sites. p70S6K activity is also controlled by the mTOR-containing complex (TORC1), as rapamycin acts to inhibit p70S6K activity. p70S6K is regulated by PI3K downstream targets Akt and PKC. Akt directly phosphorylates and inactivates TSC2, thereby activating mTOR. Furthermore, studies on the p70S6K mutant alleles inhibited by Wortmannin but not by rapamycin showed that the P13K pathway exhibits an effect on p70S6K that is independent of mTOR activity regulation. The enzyme p70S6K regulates protein synthesis through S6 ribosomal protein phosphorylation. S6 phosphorylation is associated with increased translation of mRNAs encoding components of the translation apparatus, including ribosomal proteins and translation elongation factors, whose increased expression is crucial for cell growth and proliferation. These mRNAs contain an oligopyrimidine fragment (referred to as 5'TOP) at their 5' transcription start site, which has been shown to be essential for their regulation at the translational level.

[0006] Beyond its role in translation, p70S6K activation has been implicated in cell cycle control, neuronal differentiation, cell motility regulation, and crucial cellular responses in tumor metastasis, immune responses, and tissue repair. Antibodies against p70S6K eliminated mitotic response-driven entry of rat fibroblasts into S phase, indicating that p70S6K function is essential for cell cycle progression from G1 to S phase. Furthermore, rapamycin's inhibition of cell cycle proliferation during the G1-S phase has been identified as a result of suppressing the hyperphosphorylation and activation of p70S6K.

[0007] The role of p70S6K in tumor cell proliferation and protection against apoptosis is supported by its involvement in growth factor receptor signaling transduction, overexpression, and activation in tumor tissues. For example, RNA and protein imprinting analyses have revealed that PS6K gene amplification is accompanied by corresponding increases in mRNA and protein expression (Cancer Res. (1999) 59:1408-11-Localization of PS6K to Chromosomal Region 17q23 and Determination of Its Amplification in Breast Cancer).

[0008] Clinical inhibition of p70S6K activation was observed in renal cell carcinoma patients treated with CCI-779 (rapamycin ester), an inhibitor of the upstream kinase mTOR. A significant linear association between disease progression and p70S6K activity inhibition was reported.

[0009] In response to energy stress, the tumor suppressor LKB1 activates AMPK, which phosphorylates the TSC1 / 2 complex and enables 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. Additionally, one-third of lung adenocarcinomas carry inactivated LKB1 mutations.

[0010] Compounds described as suitable for p70S6K inhibition are disclosed 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, WO06 / 136821, WO 06 / 071819, WO 06 / 131835, WO 08 / 140947, WO 10 / 093419, WO 12 / 013282 and WO 12 / 069146.

[0011] It has been shown that M2698, which inhibits not only p70S6K but also isoforms 1 and 3 of kinase Akt (upstream of p70S6K in the PI3K pathway), provides more effective PI3K pathway shutdown (Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J. Proc. Natl Acad Sci US A. 2008 Nov 11; 105(45):17414-9.), and allows capture of any Akt feedback loop activation (Tamburini et al., Blood 2008; 111:379–82).

[0012] Inhibition of the PI3K / Akt / mTOR pathway (PAM) is blocked by subsequent upregulation 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 downstream signaling in the Akt feedback loop; therefore, M2698 improves clinical efficacy compared to single-node PAM pathway inhibitors such as mTOR inhibitors. M2698 reduced tumor growth and prolonged survival in an orthotopic transplantation model of human GBM (Machl A et al., Am J Cancer Res. 2016; 6(4):806-818).

[0013] The present invention aims to find ways to further enhance the pharmaceutical applicability of M2698. In this context, in vivo studies were conducted on combinations of M2698 with an inhibitor of MEK kinase as a dual combination, combinations with an inhibitor of EGFR as a triple combination, and combinations of M2698 with an inhibitor of EGFR as a dual combination.

[0014] MEK inhibition

[0015] The mitogen-activated protein kinase (MAPK) signaling pathway plays a crucial role in regulating various 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 one-third of all malignant tumors. The classical MAPK pathway consists of Ras (a family of related proteins expressed in all animal cell lineages and organs), Raf (a family of three serine / threonine-specific protein kinases associated with retroviral oncogenes), MEK (mitogen-activated protein kinase kinase), and ERK (extracellular signal-regulated kinase), which sequentially transduce proliferation signals generated at cell surface receptors to the nucleus via cytoplasmic signal transduction. MEK inhibitors target the Ras / Raf / MEK / ERK signaling pathway, inhibiting cell proliferation and inducing apoptosis. Therefore, it has the potential for clinical use in cancer treatment, especially for cancers induced by RAS / RAF dysfunction (Leonard JT et al., J. Hematol. Oncol. 2016; 9:31. doi:10.1186 / s13045-016-0258-1).

[0016] Due to the widespread activation of this pathway in numerous vegetations, MEK inhibitors are under development and investigation in various clinical settings as a class of monotherapy or in combination with other targeted and cytotoxic drugs. Recently, their use in combination with immune checkpoint inhibitors has become an effective treatment for some cancers, expanding the efficacy of this class of drugs (Thompson N. et al., Curr. Opin. Pharmacol. 2005; 5:350–356).

[0017] Examples of MEK inhibitors in clinical development and / or approved by regulatory authorities include trametinib, cobimetinib, selumetinib, refatinib, and pimasertib. Pimasertib is N-(2,3-dihydroxypropyl-3-(2-fluoro-4-iodophenylamino)-isonicotinamide and has been described in WO2006045514 (Example 115).

[0018] Surprisingly, the inventors of this patent application have discovered that M2698 works synergistically when combined with MEK inhibitors.

[0019] EGFR inhibition

[0020] Epidermal growth factor receptor (EGFR) is a member of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). Mutations affecting EGFR expression or activity can lead to cancer in many cancer types (Zhang H et al., The Journal of Clinical Investigation. 117(8):2051–8). Epidermal growth factor and its receptor were discovered by Stanley Cohen of Vanderbilt University. Cohen and Rita Levi-Montalcini shared the 1986 Nobel Prize in Medicine for their discovery of growth factor. Defects in the signaling of EGFR and other receptor tyrosine kinases in humans are associated with diseases such as Alzheimer's disease, while overexpression is associated with the development of a wide variety of tumors. By blocking the EGFR binding site on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, the growth of EGFR-expressing tumors can be prevented and the condition of patients can be improved.

[0021] 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).

[0022] Surprisingly, the inventors of this patent application have discovered that M2698 works in a synergistic manner when combined with MEK inhibitors and optionally other EGFR inhibitors, or when combined with EGFR inhibitors alone. Attached Figure Description

[0023] Figure 1: In vitro analysis in GSC lines. (A) Western blot analysis of protein expression; (B) IC50 of M2698; (C) IC50 of pimasterb; and (D) apoptotic GSC11, GSC7-2, and GSC17 cells after treatment with M2698, pimasterb, or a combination of M2698 and pimasterb. Single agents were tested at their IC50; half of the IC50 of each compound was used for combinations.

[0024] Figure 2: (A) Tumor volume and (B) median survival in an orthotopic GSC xenograft model after treatment with mediator, M2698, pimasertib, or M2698+pimasertib combination.

[0025] Figure 3 : Expression of (A) pS6, (B) pERK and (C) Ki-67 in GSC17 and GSC7-2 xenograft models after treatment with mediator control, M2698, pimasertib or M2698+pimasertib combination.

[0026] Figure 4 NSCLC brain metastasis PDX model

[0027] Figure 5 Her2+ / HR- Breast Cancer PDX Model

[0028] Figure 6 The combination of M2698 and Pimasertib in 12 PDX models of cholangiocarcinoma

[0029] Figure 7: M2698, Pimasertib, and cetuximab (single-drug group) in 75 PDX models of CRC

[0030] Figure 8: M2698, Pimasertib, and cetuximab (dual combination) in 75 PDX models of CRC

[0031] Figure 9: M2698, Pimasertib, and cetuximab (triple combination) in 75 PDX models of CRC

[0032] Figure 10 M2698 and cetuximab (dual combination) in 38 PDX models in SCCHN Detailed Implementation

[0033] This 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, MEK inhibitors, and optionally EGFR inhibitors.

[0034] M2698 and / or its physiologically acceptable salts and solvates and (one or more) other active ingredients may be administered concurrently or sequentially. When administered concurrently, M2698 and / or its physiologically acceptable salts and solvates and MEK inhibitors may be administered as a mixture of compounds in a pharmaceutical composition or as separate pharmaceutical compositions.

[0035] In a preferred embodiment, the method according to the invention comprises sequential administration of M2698 and / or its physiologically acceptable salts and solvates, a MEK inhibitor, and optionally an EGFR inhibitor.

[0036] This invention relates in particular to methods for the prevention and / or treatment of tumors selected from colorectal cancer, breast cancer (especially Her2+ / HR- type), cholangiocarcinoma, GBM, SCCHN and NSCLC (especially brain metastases of NSCLC).

[0037] Furthermore, the present invention relates to pharmaceutical compositions comprising an active pharmaceutical ingredient (API) M2698 and its physiologically acceptable salts and solvates, as well as a mixture of MEK inhibitors and physiologically acceptable salts and solvates.

[0038] Suitable acid addition salts are inorganic or organic salts of all physiologically or pharmaceutically acceptable acids, such as halides, especially hydrochlorides or hydrobroms, lactates, sulfates, citrates, tartrates, maleates, fumarates, oxalates, acetates, phosphates, methanesulfonates, benzoates, or p-toluenesulfonates.

[0039] Solvates of M2698 and MEK inhibitors are considered to be adducts of inert solvent molecules onto M2698, formed due to their mutual attraction. Solvates are, for example, hydrates, such as monohydrates or dihydrates, or alcohols, i.e., addition compounds with alcohols (e.g., with methanol or ethanol).

[0040] The preferred salt form of M2698 is its free base. Also preferred are its hydrochloride, dihydrochloride, methanesulfonate, succinate, or malonate.

[0041] The expression "effective amount" refers to the amount of a drug or pharmaceutical active ingredient that elicits a biological or medical response in an tissue, system, animal, or human, such response as is sought or desired by an investigator or physician.

[0042] Additionally, the expression "therapeutic effective amount" refers to an amount that, compared to a corresponding subject who did not receive that amount, has the following results: improvement of disease, syndrome, condition, discomfort, symptom treatment, cure, prevention or elimination, or prevention of side effects, or also reduction of the progression of disease, condition or symptom. The term "therapeutic effective amount" also encompasses amounts that are effective in improving normal physiological function.

[0043] The pharmaceutical composition according to the invention comprises a mixture 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.

[0044] The pharmaceutical composition further comprises at least one solid, liquid, and / or semi-liquid excipient or adjuvant. Therefore, the present invention also relates to pharmaceutical compositions comprising the API mixture according to the invention and the excipient and / or adjuvant.

[0045] Furthermore, the present invention relates to the use of the pharmaceutical composition in the preparation of a medicament for treating cancer.

[0046] The present invention also relates to a kit (reagent kit) consisting of the following separately packaged components:

[0047] (a) A pharmaceutical composition containing an effective amount of M2698,

[0048] (b) A pharmaceutical composition comprising an effective amount of a MEK inhibitor, and optionally,

[0049] (c) A pharmaceutical composition containing an effective amount of an EGFR inhibitor.

[0050] The present invention also relates to a kit (reagent kit) consisting of the following separately packaged components:

[0051] (a) A pharmaceutical composition comprising an effective amount of M2698, and

[0052] (b) A pharmaceutical composition containing an effective amount of an EGFR inhibitor.

[0053] The kit includes suitable containers, such as boxes, individual bottles, bags, or ampoules. The kit may, for example, contain separate ampoules, each containing a pharmaceutical composition in dissolved or lyophilized form containing an effective amount of M2698 and / or its pharmaceutically usable salts, a pharmaceutical composition containing an effective amount of a MEK inhibitor and / or its pharmaceutically usable salts, and optionally a pharmaceutical composition containing an effective amount of an EGFR inhibitor.

[0054] The preferred brain-penetrating MEK inhibitor that binds to M2698 is pimasertib. The preferred EGFR inhibitor that binds to M2698 (with or without a MEK inhibitor) is cetuximab. The preferred triplet combination is M2698 + pimasertib + cetuximab.

[0055] The compounds and mixtures thereof according to the invention are adaptable for administration by any desired and suitable method, such as oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) methods. Such pharmaceutical products can be prepared using all methods known in the pharmaceutical field by, for example, combining the active ingredient with one or more excipients or adjuvants.

[0056] Compounds and mixtures of compounds suitable for oral administration may be administered as separate units, such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foamed foods; or oil-in-water or water-in-oil liquid emulsions.

[0057] Therefore, for example, in the case of oral administration in tablet or capsule form, the compound or mixture of compounds can be combined with an inert excipient that is orally non-toxic and pharmaceutically acceptable, such as ethanol, glycerin, water, etc. The powder is prepared by pulverizing the compound to a suitable fine size and mixing it with a pharmaceutical excipient pulverized in a similar manner, such as an edible carbohydrate, like starch or mannitol. Similarly, flavoring agents, preservatives, dispersants, and dyes may be present.

[0058] The capsules are produced by preparing the powder mixture as described above and filling it into a gelatin shell. Prior to the filling operation, glidants and lubricants, such as highly dispersed silica, talc, magnesium stearate, calcium stearate, or polyethylene glycol in solid form, may be added to the powder mixture. Similarly, disintegrants or solubilizers, such as agar, calcium carbonate, or sodium carbonate, may be added to improve the usability of the compound or compound mixture after the capsule is ingested.

[0059] Additionally, suitable binders, lubricants, disintegrants, and dyes may be incorporated into the mixture if desired or necessary. Suitable binders include starch; gelatin; natural sugars such as glucose or β-lactose; sweeteners made from corn; natural and synthetic rubbers such as gum arabic, tragacanth, or sodium alginate; carboxymethyl cellulose; polyethylene glycol; waxes, etc. Lubricants used in this dosage form include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc. Tablets are formulated, for example, by preparing a powder mixture, granulating or dry-compressing the mixture, adding lubricants and disintegrants, and pressing the entire mixture to obtain tablets. The powder mixture is prepared by mixing a compound, pulverized in a suitable manner, with a diluent or alkali, and optionally with a binder, such as carboxymethyl cellulose, alginate, gelatin, or polyvinylpyrrolidone; a dissolution delay agent, such as paraffin; an absorption accelerator, such as quaternary salt; and / or an absorbent, such as bentonite, kaolin, or dicalcium phosphate. The powder mixture can be granulated by wetting it with a binder, such as syrup, starch paste, acadia mucilage, or a solution of cellulose or polymeric material, and then extruding it through a sieve. Alternatively, the powder mixture can be run through a tablet press to obtain irregularly shaped lumps, which are then broken into granules. The granules can be lubricated by adding stearic acid, stearate, talc, or mineral oil to prevent them from sticking to the tablet mold. The lubricated mixture is then pressed to obtain tablets. The compounds and compound mixtures according to the invention can also be combined with free-flowing inert excipients and then directly pressed to obtain tablets without granulation or dry pressing steps. A transparent or opaque protective layer may be present, consisting of a shellac sealing layer, a sugar or polymer material layer, and a glossy wax layer. Dyes may be added to these coatings to allow for differentiation between different dosage units.

[0060] Oral liquids, such as solutions, syrups, and elixirs, can be prepared in dosage units, such that a given quantity contains a pre-specified amount of compound. Syrups are prepared by dissolving compounds and mixtures of compounds in an aqueous solution with a suitable flavor, while elixirs are prepared using non-toxic alcoholic mediators. Suspensions are formulated by dispersing compounds in a non-toxic mediator. Solubilizers and emulsifiers, such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitan ether; preservatives; flavor additives, such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, can be added similarly.

[0061] If desired, dosage units for oral administration can be encapsulated in microcapsules. Formulations can also be prepared in ways that prolong or delay release, for example, by coating or embedding particulate materials in polymers, waxes, etc.

[0062] The compounds and mixtures thereof, as well as their salts and solvates, according to the invention can also be administered in the form of liposome delivery systems, such as small monolayer vesicles, large monolayer vesicles, and multilayer vesicles. Liposomes can be formed from various phospholipids, such as cholesterol, stearamine, or phosphatidylcholine.

[0063] The compounds and mixtures thereof according to the invention can also be delivered using monoclonal antibodies as separate carriers conjugated to the compound molecules. The compounds and mixtures thereof can also be conjugated to soluble polymers as targeted drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropyl methacryloyl-aminophenol, polyhydroxyethyl asparagine, or polyethylene oxide polylysine, substituted with palmitoyl radicals. Furthermore, the compounds can be conjugated to a class of biodegradable polymers suitable for controlled drug release, such as polylactic acid, poly-ε-caprolactone, polyhydroxybutyrate, polyorthoesters, polyacetals, polydihydroxypyran, polycyanoacrylates, and crosslinked or amphiphilic block copolymers of hydrogels.

[0064] Compounds and mixtures of compounds suitable for transdermal application can be applied as standalone patches for prolonged close contact with the recipient's epidermis. Thus, for example, active ingredients can be delivered from patches via iontophoresis, as described in the general section of Pharmaceutical Research, 3(6):318, 1986.

[0065] Compounds and mixtures of compounds suitable for topical application can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0066] For the treatment of the eyes or other external tissues, such as the mouth and skin, the formulation is preferably applied as a topical ointment or cream. When formulating to obtain an ointment, the compound or mixture of compounds may be used together with a paraffinic or water-miscible cream base. Alternatively, the compound or mixture of compounds may be formulated to obtain a cream having an oil-in-water or water-in-oil base.

[0067] Compounds and mixtures of compounds suitable for topical application to the eye include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent.

[0068] Compounds and compound mixtures suitable for topical application to the mouth include lozenges, tablets, and mouthwashes.

[0069] Compounds and mixtures of compounds suitable for rectal administration may be administered in suppository or enema form.

[0070] Compounds and mixtures of compounds suitable for nasal application, wherein the carrier is a solid, comprise coarse powder having a particle size, for example, in the range of 20-500 micrometers, which are applied by inhalation of nasal smoke, i.e., by rapid inhalation through the nasal passages from a container containing the powder placed near the nose. Suitable formulations having a liquid as a carrier for application as nasal sprays or drops comprise a solution of the active ingredient in water or oil.

[0071] Suitable for compounds and compound mixtures administered by inhalation, encompassing fine particulate powders or mists, which can be produced by various types of pressurized dispensers with aerosols, nebulizers, or blowpipes.

[0072] Compounds and mixtures thereof suitable for vaginal application may be used as vaginal suppositories, sanitary napkins, creams, gels, pastes, foams or sprays.

[0073] Compounds and mixtures of compounds suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions containing antioxidants, buffers, antibacterial agents, and solutes, which make the formulation isotonic with the blood of the recipient to be treated; and aqueous and non-aqueous sterile suspensions, which may contain a suspension medium and a thickener. The formulations are administered in single-dose or multi-dose containers, such as sealed ampoules and vials, and stored in a lyophilized (freeze-dried) state, so that a sterile carrier liquid, such as water for injection, only needs to be added immediately before use. Injectable solutions and suspensions formulated according to the specifications can be prepared from sterile powders, granules, and tablets.

[0074] It goes without saying that, in addition to the components specifically mentioned above, the pharmaceutical products according to the present invention may also contain other pharmaceutical agents that are common in the art for a particular type of pharmaceutical preparation; thus, for example, compounds or mixtures of compounds suitable for oral administration may contain flavoring agents.

[0075] The therapeutically effective amount of the compounds or mixtures of compounds of the present invention depends on many factors, including, for example, the recipient's age and weight, the exact condition requiring treatment and its severity, the nature of the formulation, and the method of administration, and is ultimately determined by the treating physician or veterinarian. However, the effective amount of the API used to treat disease according to the present invention is generally in the range of 0.1-100 mg / kg recipient (mammal) body weight / day, and particularly generally in the range of 1-10 mg / kg body weight / day. Therefore, for an adult mammal weighing 70 kg, the actual daily dose is often between 70-700 mg, which may be administered as a single daily dose, or more often as a series of partial daily doses (e.g., two, three, four, five, or six) to ensure a consistent total daily dose. The effective amount of the salt or solvate, or its physiologically functional derivatives, can be determined as a fraction of the effective amount of the compound or mixture of compounds according to the present invention itself.

[0076] Pharmaceutical formulations according to the invention can be used as medicines for humans and veterinarians. Suitable excipients are organic or inorganic substances suitable for oral (e.g., oral), parenteral, or topical application and that do not react with the novel compound, such as water, vegetable oil, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc, or petrolatum. Tablets, coated tablets, capsules, syrups, juices, drops, or suppositories are particularly suitable for oral administration; solutions, preferably oil-based or aqueous solutions, are suitable for parenteral application; suspensions, emulsions, or implants are also possible; and ointments, creams, or powders are suitable for topical application. The compounds and mixtures of compounds may also be lyophilized, and the resulting lyophilized formulations are used, for example, in the preparation of injectable formulations.

[0077] The formulations may be sterilized and / or contain excipients such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for altering osmotic pressure, buffering substances, dyes, flavoring agents, and / or aromatic substances. If desired, they may also contain one or more further active ingredients, such as one or more vitamins.

[0078] The present invention also relates to a method for the prevention and / or treatment of cancer, particularly squamous cell carcinoma of the head and neck (SCCHN), comprising administering to a subject M2698 and / or its physiologically acceptable salts and solvates, and EGFR inhibitors, particularly cetuximab, as well as corresponding mixtures of compounds, pharmaceutical compositions, formulations, and administration methods, as described herein above.

[0079] In some embodiments, the present invention relates to:

[0080] 1. A mixture of compounds comprising 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide or a physiologically acceptable salt thereof, and an inhibitor of MEK or a physiologically acceptable salt thereof.

[0081] 2. The compound mixture as described above in the detailed description of this embodiment, wherein the MEK inhibitor is pimasertib.

[0082] 3. The compound mixture as described above in the detailed description of this embodiment further comprises an EGFR inhibitor.

[0083] 4. A pharmaceutical composition comprising a mixture of compounds as described above in this detailed description, and optionally, excipients and / or adjuvants.

[0084] 5. A kit containing separately packaged (a) an effective amount of 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxamide or a physiologically acceptable salt thereof and (b) an effective amount of a MEK inhibitor or a physiologically acceptable salt thereof.

[0085] 6. The kit (reagent kit) as described above in the detailed description of this implementation scheme further comprises (c) separately packaged effective amounts of EGFR inhibitors or physiologically acceptable salts thereof.

[0086] 7. The kit (reagent kit) as described above in the detailed implementation scheme, wherein the EGFR inhibitor is cetuximab.

[0087] 8. A method for the prevention or treatment of cancer, comprising administering to a subject 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxamide or a physiologically acceptable salt thereof, and a MEK inhibitor or a physiologically acceptable salt thereof.

[0088] 9. The method described in the detailed implementation scheme above, wherein the MEK inhibitor is pimastetib.

[0089] 10. The method described above in the detailed description of this embodiment further comprises administering an EGFR inhibitor or a physiologically acceptable salt thereof to the subject.

[0090] 11. The method described in the detailed implementation scheme above, wherein the EGFR inhibitor is cetuximab.

[0091] 12. The method described above in the detailed description of this embodiment, wherein 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide, a MEK inhibitor and an optional EGFR inhibitor are administered simultaneously.

[0092] 13. The method described above in the detailed description of this embodiment, wherein 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide, a MEK inhibitor, and optionally an EGFR inhibitor are administered sequentially.

[0093] 14. A mixture of compounds comprising 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide or a physiologically acceptable salt thereof and an inhibitor of EGFR, preferably cetuximab.

[0094] 15. A kit containing separately packaged (a) an effective amount of 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxamide or a physiologically acceptable salt thereof and (b) an effective amount of an EGFR inhibitor, preferably cetuximab.

[0095] 16. A method for the prevention or treatment of cancer, comprising administering to a subject 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide or a physiologically acceptable salt thereof, and an EGFR inhibitor, preferably cetuximab, wherein the two agents are administered simultaneously or sequentially.

[0096] 17. In a preferred embodiment, the cancer is colorectal cancer, breast cancer, cholangiocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, and non-small lung cancer.

[0097] Example

[0098] Example 1 The combination of M2698 and Pimasertib in a patient-derived glioblastoma (GBM) xenograft model (PDX)

[0099] GSC lineage: In vitro sensitivity to M2698 and Pimasertib

[0100] Although M2698 showed significant effects in some GBM models, the response was relatively mild. Since both the PAM and MAPK pathways are involved in GBM, M2698 was combined with the brain-penetrating MEK inhibitor pimastetib19 GSC (glioblastoma stem cell) cells / models in vivo and in vitro.

[0101] GSC lines were selected for in vivo studies using in vitro IC50 data; IC50 values ​​<2 μM were considered sensitive, while >2 μM were considered resistant. All GSC lines expressed Akt. Compared to GSC272 and GSC267, pAkt expression was relatively high in GSC17, GSC7-2, GSC231, GSC11, GSC20, GSC6-27, and GSC8-11 lines (Fig. 1A). Except for GSC20, cell lines with relatively higher pAkt expression were sensitive to M2698, indicating that constitutive Akt activation sensitizes cells to M2698 (Fig. 1B). Fewer cell lines (GSC17, GSC11, and GSC231) were sensitive to pimastetib (IC50 <2 μM), while GSC20, GSC272, GSC6-27, and GSC7-2 were resistant (Fig. 1C).

[0102] The induction of apoptosis by M2698, pimasterbib, and combinations was measured in three of the five GSC lines sensitive to M2698. GSC11 was the most sensitive to pimasterbib, GSC17 was moderately sensitive, and GSC7-2 was insensitive to pimasterbib. Both M2698 and pimasterbib, when administered as monotherapy in the GSC11, GSC17, and GSC7-2 lines, induced apoptosis, although there was no significant difference in the percentage of apoptosis at IC50 (56%, 11%, and 13%, respectively) versus at 2× IC50 (62%, 12%, and 17%, respectively) for each compound. Compared to the individual agents, M2698 + pimasterbib showed an additive (GSC11 and GSC7-2 lines) or a significant synergistic effect (GSC17 line) in inducing apoptosis (Figure 1D).

[0103] In vivo efficacy and PD effects of M2698 and Pimasertib in an orthotopic GSC xenograft model

[0104] Following orthotopic transplantation into mice, the in vivo effects of M2698 alone and in combination with pimastetib were evaluated in a GSC model. The duration of treatment and euthanasia after transplantation ranged from 5 to 10 weeks. Endpoints included expression of the PD markers pS6 and pERK, the proliferation marker Ki67, apoptosis as measured by TUNEL, tumor volume, and survival.

[0105] Of the seven GSC models, all but one (GSC20) were sensitive to M2698 monotherapy, as measured by tumor volume; M2698 also significantly inhibited tumor growth (GSC17, GSC6-27, GSC7-2, GSC272, GSC231; all P < 0.05; Figure 2A) or showed a trend toward such inhibition (GSC11, P = 0.10). Of the six M2698-sensitive models, five responded to pimasertib monotherapy, either with significant tumor growth inhibition (GSC6-27, GSC7-2, GSC272; P < 0.05) or with a trend toward significant response (GSC231, P = 0.10). Tumor growth in GSC17 was not affected by pimasertib alone (P = 0.34 compared to the mediator). Compared with the mediator, M2698+pimasertib significantly inhibited tumor growth in all GSC models, even in GSC20 (all P<0.05; Figure 2A), where tumor growth did not respond to monotherapy (P>0.05).

[0106] Median survival in the GSC models does not fully reflect tumor volume data (Figure 1B). Compared to the mediators, M2698 (GSC272 and GSC231) and the combination (GSC17 and GSC7-2) prolonged survival in only two models (P<0.05), while Pimasertib had no significant effect on survival relative to the control in any model (P>0.05). In GSC6-27, for unknown reasons, mice treated with the mediators had significantly longer median survival than mice from the treatment group (P<0.05). In contrast, both monotherapies significantly inhibited tumor growth in some models, while the combination inhibited tumor growth in every model.

[0107] The significant effect on tumor volume indicates that the compound enters the brain and affects orthotopically growing tumors. Further evidence of BBB penetration includes the effect of PD markers on signaling pathways targeted in tumor cells. M2698 significantly reduced pS6 in orthotopically growing tumors in all GSC models except GSC20 (P<0.05). Figure 3 A). Similarly, the significant effect of pimastetib treatment on pERK protein was not observed in GSC20 tumors (P = 0.18), however, pimastetib significantly reduced pERK in other GSC tumors (P < 0.05); Figure 3 B), including GSC17 mice that did not experience tumor volume reduction after pimastetib treatment. The M2698+pimasertib combination reduced pS6 and pERK in all models except GSC20 (all P < 0.05). In mice treated with the mediator in the GSC20 model, the relatively high changes in tumor growth, pS6, and pERK may have masked the statistical significance of the biologically relevant therapeutic effects of these endpoints. Conversely, the lack of significant response was predicted by the insensitivity of GSC20 to both compounds in vitro (Figures 1B and 1C).

[0108] In addition to the effects of pimasertib on pERK and M2698 on pS6 in the orthotopic brain model, various M2698s also affected opposite PD markers. Pimasertib significantly reduced pS6 in the GSC272 and GSC231 models. M2698 reduced pERK in all GSC models except GSC20 (P<0.05). Figure 3 B).

[0109] PD markers for three GSC tumors were analyzed at more than one time point. Although no statistical analysis of response across time was performed, trends across different endpoints were assessed. The percentage of pS6-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. Figure 3 In the GSC272 and GSC231 models, the reduction in pS6 cross-pathway activity via pimastetib decreased from 7.5–10 weeks, and in the GSC231 model, the activity of compounds against pERK expression (alone or in combination) decreased from 7.5–10 weeks. Whether these trends are related to the development of resistance requires further investigation, but they reflect the heterogeneity of the models.

[0110] Ki67 was measured by immunohistochemistry in tumor sections from GSC17 and GSC7-2 (in both models, the M2698+pimasertib combination significantly prolonged survival). Figure 3 C). In the GSC17 model, all three treatments significantly reduced proliferation compared to the mediator, as did M2698 monotherapy and its combination with pimastetib in the GSC7-2 model (all P < 0.05).

[0111] At 7.5 weeks, M2698, pimastetib, or combination therapy did not significantly affect apoptosis in GSC7-2 xenografts compared to controls. However, at 10 weeks, similar to in vitro data, the M2698+pimasertib combination significantly increased the number of apoptotic cells per field of view (172.33±60.80) compared to controls (8.50±1.80; P=0.002), M2698 alone (52.17±25.20; P=0.01), and pimastetib alone (65.00±60.80; P=0.02).

[0112] Example 2: The combination of M2698 and Pimasertib in a patient-derived xenograft model (PDX) of brain metastases in non-small lung cancer (NSCLC).

[0113] like Figure 4 As can be seen, the combination of M2698 and pimastetib unexpectedly showed a tumor control rate (TCR) of 26%, while single-agent therapy showed 5% (M2698) and 16%.

[0114] (pimasertib).

[0115] Example 3:The combination of M2698 and Pimasertib in a patient-derived Her2+ / HR- breast cancer xenograft model (PDX)

[0116] like Figure 5 As can be seen, the combination of M2698 and pimasertib unexpectedly reduced tumor volume to almost zero during the duration of the trial, while the single agent only achieved stagnation of tumor volume.

[0117] Example 4: The combination of M2698 and Pimasertib in a patient-derived xenograft model (PDX) of cholangiocarcinoma

[0118] The aim of this study was to evaluate the antitumor activity of M2698 alone and in combination with Pimasertib in a cohort of 13 patient-derived xenograft models in immunodeficient mice, representing human cholangiocarcinoma derived from Chinese patients. Her2 status of the models was obtained via immunohistochemistry (IHC) and is shown in [the image / image / etc.]. Figure 6 middle.

[0119] Research Design

[0120]

[0121] Medium: 0.5% methylcellulose (Methocel) / 0.25% Tween 20 in Milli-Q water. When reaching a depth of 300-500 mm... 3 When the tumor volume was between that of the mice, tumors from three untreated mice were collected as supplementary samples.

[0122] As from Figure 6 Surprisingly, tumor arrest was achieved in 10 out of 12 tumor samples (83%) with M2698 and pimastetib, compared to 17% (2 / 12, M2698) and 8% (1 / 12, pimastetib) with single-agent therapy. No tumor growth response or even regression was observed with either treatment in this study.

[0123] Example 5: The combination of M2698, Pimasertib, and cetuximab in 75 patient-derived xenograft models (PDX) of colorectal cancer.

[0124] Research Design

[0125]

[0126]

[0127] Of the 75 models, 25 were wild-type, 25 were KRas mutants, and 25 were BRaf mutants.

[0128] result

[0129] As shown in Figure 7, the tumor control rate (TCR) for single agents was 15% (11 / 74) for M2698, 32% (24 / 74) for Pimasertib, and 21% (15 / 73) for cetuximab.

[0130] As shown in Figure 8, for the dual combination, TCR was 59% (44 / 74) for Pimasertib + cetuximab and 35% (26 / 74) for M2698 + cetuximab, and surprisingly, 63% (45 / 72) for M2698 + Pimasertib.

[0131] As shown in Figure 9, for the triple combination, surprisingly, the TCR was 78% (58 / 74), which was again significantly higher than the TCR of the dual combination.

[0132] Example 6: The combination of M2698 and cetuximab in xenograft models (PDX) of 38 patient-derived SCCHN

[0133] Forty-six SCCHN PDX models were run in a 1+1 regimen (one mouse / treatment group). Eight models were cancelled because they failed to grow. With adjustments made to the cancelled models, 38 models were completed. In the 1+1 selection (i.e., one mouse / treatment / model), M2698 was administered in combination with the standard-of-care (SoC) agents cisplatin and cetuximab in all monotherapy and combination treatments. After engraftment, when the average tumor volume reached ~200 mm for each model... 3 Treatment began at that time. Tumor volume was measured for each model until the control tumor reached approximately 1200 mm². 3 At this point, all the animals in the model were euthanized.

[0134] Research Design

[0135]

[0136] result

[0137]

[0138] As shown in the table above and Figure 10 The results showed that for the combination, the TCR was 58% (22 / 38), which is an unexpected improvement in TCR for a single agent in this difficult-to-treat type of cancer.

Claims

1. 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide or a physiologically acceptable salt thereof and a MEK inhibitor or a physiologically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of cancer, wherein the MEK inhibitor is pimasertib, and wherein the cancer is colorectal cancer, breast cancer, cholangiocarcinoma, glioblastoma multiforme, or non-small cell lung cancer.

2. The use according to claim 1, wherein the drug further comprises an EGFR inhibitor or a physiologically acceptable salt thereof.

3. The use according to claim 2, wherein the EGFR inhibitor is cetuximab.

4. The use according to claim 1, wherein 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide and the MEK inhibitor are administered simultaneously.

5. The use according to claim 2 or 3, wherein 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide, the MEK inhibitor and the EGFR inhibitor are administered simultaneously.

6. The use according to claim 1, wherein 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide and the MEK inhibitor are administered sequentially.

7. The use according to claim 2 or 3, wherein 4-[(S)-2-azacyclobut-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazolin-8-carboxylic acid amide, the MEK inhibitor and the EGFR inhibitor are administered sequentially.

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