Kinase inhibitor combinations for cancer treatment

Abstract

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 and / or physiologically acceptable salts and solvates thereof with inhibitors of MEK kinase and, as an optional third inhibitor, inhibitors of EGFR, and to 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

Technical Field

[0001] The present invention relates to 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 its physiologically acceptable salts and solvates, in combination with an inhibitor of MEK kinase, and an inhibitor of the receptor tyrosine protein kinase ERBB-1, also known as EGFR (epidermal growth factor receptor), as an optional third inhibitor, and to the use of such combinations for the treatment of cancer. The present invention also relates to combinations with an EGFR inhibitor without M2698 and an inhibitor of MEK kinase.

Background Art

[0002] Background of the Invention M2698, processes for its preparation and its use for the treatment of cancer are disclosed in WO2012 / 069146 (referred to as compound A). This compound is a selective, highly potent dual inhibitor of p70S6K and Akt, as demonstrated in various cell-based assays. M2698 has been shown to exhibit potent antitumor activity against a broad panel 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 family of structurally related enzymes responsible for regulating a wide variety of signal transduction processes within cells (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book. I and II, Academic Press, San Diego, CA). Kinases may also be classified by the substrates they phosphorylate (e.g., protein tyrosine, protein serine / threonine, lipids, etc.). Sequence motifs have generally been identified corresponding to each of these kinase families (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 may also be characterized by their regulatory mechanisms. These mechanisms include, for example, autophosphorylation, transphosphorylation by other kinases, protein-protein interactions, protein-lipid interactions, and protein-polynucleotide interactions. Individual protein kinases may be regulated by more than one mechanism. Kinases, by adding phosphate groups to target proteins, regulate a wide range of cellular processes, including proliferation, differentiation, apoptosis, motility, transcription, translation, and other signaling processes. These phosphorylation events act as molecular on / off switches that can modulate or regulate the biological function of target proteins. Protein phosphorylation occurs in response to various extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.), cell cycle events, environmental or nutritional stress, etc.Appropriate protein kinases function in signaling pathways, activating or inactivating (either directly or indirectly) metabolic enzymes, regulatory proteins, receptors, cytoskeletal proteins, ion channels or pumps, or transcription factors, for example. Unregulated signaling resulting from defective control of protein phosphorylation is associated with numerous diseases, including, for example, inflammation, cancer, allergies / asthma, immune system disorders and diseases, central nervous system disorders, and angiogenesis disorders and diseases.

[0004] P70S6K inhibition The protein kinase 70S6K, also known as 70kDa ribosomal protein kinase p70S6K (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 its activation occurs through phosphorylation at numerous sites in response to countless mitogens, hormones, and growth factors. Since rapamycin acts to inhibit p70S6K activity, p70S6K activity is also regulated by a complex containing mTOR (TORC1). p70S6K is regulated by Akt and PKC, which are downstream targets of PI3K. Akt directly phosphorylates and inactivates TSC2, thereby activating mTOR. In addition, studies using mutant alleles of p70S6K that are inhibited by woltmannin but not by rapamycin suggest that p70S6K may exert effects independently of the regulation of mTOR activity. The enzyme p70S6K regulates protein synthesis by phosphorylating S6 ribosomal proteins. S6 phosphorylation correlates with increased translation of mRNAs encoding components of the translation machinery, including ribosomal proteins and translation elongation factors, whose increased expression is essential for cell growth and proliferation. These mRNAs contain oligopyrimidine tracts (called 5'TOPs) at their 5' transcription initiation, which has been shown to be essential for their regulation at the translational level.

[0005] In addition to its involvement in translation, p70S6K activation is also involved in cellular responses crucial to cell cycle regulation, neuronal cell differentiation, regulation of cell motility, tumor metastasis, immune responses, and tissue repair. Antibodies against p70S6K abolish the pro-mitotic response that drives entry into the S phase of rat fibroblasts, indicating that p70S6K function is essential for progression from G1 to S phase in the cell cycle. Furthermore, rapamycin-induced inhibition of cell cycle proliferation from G1 to S phase has been identified as a result of inhibiting the production of the hyperphosphorylated active form of p70S6K.

[0006] The role of p70S6K in tumor cell proliferation and protection against apoptosis is supported by its participation in growth factor receptor signaling, overexpression, and activation in tumor tissue. For example, Northern and Western chromatographic analyses have revealed that amplification of the PS6K gene is accompanied by increases in the corresponding mRNA and protein, respectively (Cancer Res. (1999) 59: 1408-11 - Localization of PS6K to Chromosomal Region 17q23 and Determination of Its Amplification in Breast Cancer). 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 inhibition of p70S6K activity was reported.

[0007] In response to energy stress, the tumor suppressor LKB1 activates AMPK, which phosphorylates the TSC1 / 2 complex, and enables the inactivation of the mTOR / p70S6K pathway. Mutations in LKB1 cause Peutz-Jeghers syndrome (PJS), and patients with PSJ are 15 times more likely to develop cancer than the general population. In addition, one-third of lung adenocarcinomas have inactivated LKB1 mutations. Compounds described as suitable for p70S6K inhibition are disclosed in WO03 / 064397, WO04 / 092154, WO05 / 054237, WO05 / 056014, WO05 / 033086, WO05 / 117909, WO05 / 039506, WO06 / 120573, WO06 / 136821, WO06 / 071819, WO06 / 131835, WO08 / 140947, WO10 / 093419, WO12 / 013282 and WO12 / 069146.

[0008] M2698, which inhibits not only p70S6K but also isoforms 1 and 3 of the kinase Atk (upstream of p70S6K in the PI3K pathway), has been shown to provide more efficient deactivation of the PI3K pathway (Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J. Proc. Natl Acad Sci US A. 2008 Nov 11;105(45):17414-9.), and enables the capture of activation of either Akt feedback loop (Tamburini et al. Blood 2008;111:379-82). Inhibition of the PI3K / Akt / mTOR pathway (PAM) is hindered by subsequent upmodulation 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 may improve clinical efficacy compared to single-node inhibitors of the PAM pathway, such as mTOR inhibitors. M2698 reduces tumor growth and prolongs survival in an orthotopic transplant model of human GBM (Machl A et al. Am J Cancer Res. 2016; 6(4):806-818).

[0009] The present invention aimed to find a method for further advancing the pharmaceutical practicality of M2698. In this regard, the combination of M2698 with a MEK kinase inhibitor as a double combination, the combination of M2698 with an EGFR inhibitor as a triple combination, and the combination of M2698 with an EGFR inhibitor as a double combination were studied in vivo.

[0010] MEK inhibition The mitogen-activated protein kinase (MAPK) signaling pathway plays a crucial role in regulating 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 one-third of all malignancies. 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 route proliferative signals generated at cell surface receptors to the nucleus via cytoplasmic signaling. MEK inhibitors target the Ras / Raf / MEK / ERK signaling pathway to inhibit cell proliferation and induce apoptosis. Therefore, it shows promise in clinical use for cancer treatment, particularly for cancers induced by RAS / RAF dysfunction (Leonard JT et al., J. Hematol. Oncol. 2016;9:31. doi: 10.1186 / s13045-016-0258-1).

[0011] Due to the widespread activation of this pathway in countless neoplasms, MEK inhibitors are in the process of development and research in various clinical settings as monotherapy, other targeted therapies, and combination therapies with cytotoxic agents. More recently, combinations with the use of immune checkpoint inhibitors have emerged as an effective treatment for some cancers, extending the efficacy of this class of agents (Thompson N. et al., Curr. Opin. Pharmacol. 2005;5:350-356). Examples of MEK inhibitors under clinical development and / or approved by regulatory authorities include trametinib, cobimetinib, selumetinib, refametinib, and pimacertib. Pimasertib is N-(2,3-dihydroxypropyl)-3-(2-fluoro-4-iodophenylamino)-isonicotinamide, described in particular in WO2006045514 (Example 115). Surprisingly, the inventors of this patent application discovered that M2698 works synergistically when combined with a MEK inhibitor.

[0012] EGFR inhibition The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptors, 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 types of cancer (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 shared the 1986 Nobel Prize in Physiology or Medicine with Rita Levi-Montalcini for their discovery of the growth factor. In humans, EGFR and other receptor tyrosine kinase-deficient signaling is associated with diseases such as Alzheimer's disease, while overexpression is associated with the development of a wide variety of tumors. Disrupting EGFR signaling, either by blocking the EGFR binding site on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, can inhibit the growth of EGFR-expressing tumors and improve the patient's condition.

[0013] Examples of EGFR inhibitors under clinical development and / or approved by regulatory authorities include gefitinib, erlotinib, afatinib, brigatinib, icotinib, osimertinib, and cetuximab (a chimeric (mouse / human) monoclonal antibody used for the treatment of metastatic colorectal cancer and head and neck cancer). Surprisingly, the inventors of this patent application have found that M2698 acts synergistically when combined with a MEK inhibitor, and optionally with an EGFR inhibitor, or with an EGFR inhibitor alone. [Brief explanation of the drawing]

[0014] figure [Figure 1]Figure 1: In vitro analysis of GSC strains. (A) Western blot analysis of protein expression; (B) IC50 of M2698; (C) IC50 of pimacertib; and (D) apoptotic GSC11, GSC7-2, and GSC17 cells after treatment with M2698, pimacertib, or a combination of M2698 + pimacertib. Monochemicals were tested with their IC50; for combinations, half the IC50 of each compound was used.

[0015] [Figure 2-1] Figure 2: (A) Tumor volume and (B) Median survival in orthotopic GSC xenograft models after treatment with vehicle, M2698, pimacerutib, or a combination of M2698 and pimacerutib. [Figure 2-2] Figure 2: (A) Tumor volume and (B) Median survival in orthotopic GSC xenograft models after treatment with vehicle, M2698, pimacerutib, or a combination of M2698 and pimacerutib. [Figure 2-3] Figure 2: (A) Tumor volume and (B) Median survival in orthotopic GSC xenograft models after treatment with vehicle, M2698, pimacerutib, or a combination of M2698 and pimacerutib. [Figure 2-4] Figure 2: (A) Tumor volume and (B) Median survival in orthotopic GSC xenograft models after treatment with vehicle, M2698, pimacerutib, or a combination of M2698 and pimacerutib.

[0016] [Figure 3] Figure 3: (A) pS6 expression, (B) pERK expression, and (C) Ki-67 expression in GSC17 and GSC7-2 xenograft models following treatment with vehicle control, M2698, pimacertib, or a combination of M2698 and pimacertib. [Figure 4] Figure 4: PDX model of NSCLC brain metastases.

[0017] [Figure 5] Figure 5: Her2+ / HR- breast cancer PDX model. [Figure 6] Figure 6: Combination of M2698 and pimasertib in the PDX model of cholangiocarcinoma of 12.

[0018] [Figure 7-1] Figure 7: M2698, pimasertib, and cetuximab (single-agent group) in the PDX model of CRC of 75. [Figure 7-2] Figure 7: M2698, pimasertib, and cetuximab (single-agent group) in the PDX model of CRC of 75. [Figure 7-3] Figure 7: M2698, pimasertib, and cetuximab (single-agent group) in the PDX model of CRC of 75.

[0019] [Figure 8-1] Figure 8: M2698, pimasertib, and cetuximab (dual combination) in the PDX model of CRC of 75. [Figure 8-2] Figure 8: M2698, pimasertib, and cetuximab (dual combination) in the PDX model of CRC of 75. [Figure 8-3] Figure 8: M2698, pimasertib, and cetuximab (dual combination) in the PDX model of CRC of 75.

[0020] [Figure 9-1] Figure 9: M2698, pimasertib, and cetuximab (triple combination) in the PDX model of CRC of 75. [Figure 9-2] Figure 9: M2698, pimasertib, and cetuximab (triple combination) in the PDX model of CRC of 75. [Figure 10] Figure 10: M2698 and cetuximab (dual combination) in the PDX model of SCCHN of 38.

[0021] Detailed description of the present invention The present invention relates to a method for the prevention and / or treatment of cancer, comprising administering M2698 and / or its physiologically acceptable salts and solvates, a MEK inhibitor, and optionally an EGFR inhibitor to a subject. M2698 and / or its physiologically acceptable salts and solvates, and other active ingredients (one or more) may be administered simultaneously or sequentially. When administered simultaneously, M2698 and / or its physiologically acceptable salts and solvates, and the MEK inhibitor may be administered as a compound mixture in a single pharmaceutical composition or as separate pharmaceutical compositions. In a preferred embodiment, a method according to the present invention comprises the use of M2698 and / or its physiologically acceptable salts and solvates, wherein a MEK inhibitor and, optionally, an EGFR inhibitor are administered sequentially.

[0022] 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 Her2+ / HR- type), cholangiocarcinoma, GBM, SCCHN, and NSCLC (in particular from brain metastases of NSCLC). Furthermore, the present invention relates to a compound mixture of the active pharmaceutical ingredient (API) M2698, its physiologically acceptable salts and solvates, and a pharmaceutical composition comprising a MEK inhibitor and its physiologically acceptable salts and solvates.

[0023] Suitable acid addition salts are inorganic or organic salts of all physiologically or pharmacologically acceptable acids, such as halides, particularly hydrochlorides or hydrobroms, lactates, sulfates, citrates, tartrates, maleates, fumarates, oxalates, acetates, phosphates, sulfons, benzoates, or p-toluenesulfons. The solvates of M2698 and MEK inhibitors are understood to mean adducts on M2698 formed due to their mutual attraction. Solvates are adducts with hydrates such as monohydrates or dihydrates, or with alcohols, i.e., alcohols such as methanol or ethanol. The preferred salt form of M2698 is its free base. Other preferred forms are its hydrochloride, dihydrochloride, mesylate, succinate, or malonate.

[0024] The term "effective dose" refers to the amount of a drug or pharmaceutically active ingredient that elicits, for example, a biological or medical response sought or desired by a researcher or physician in a tissue, system, animal or human. In addition, the expression "therapeutably effective dose" means a dose that, compared to a corresponding subject not receiving that dose, results in: improved treatment, cure, prevention or elimination of a disease, syndrome, illness, complaint, or disorder, or prevention of side effects or reduction in the progression of the disease, illness, or disorder. The term "therapeutably effective dose" also encompasses doses that are effective in increasing normal physiological function.

[0025] A pharmaceutical composition according to the present invention comprises, for example, a mixture of two APIs in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:100, or 1:1000. The pharmaceutical composition further comprises at least one solid, liquid, and / or semi-liquid excipient or adjuvant. Accordingly, the present invention also relates to a pharmaceutical composition comprising the API mixture and the excipient and / or adjuvant according to the present invention.

[0026] Furthermore, the present invention relates to the use of the pharmaceutical composition for the preparation of a pharmacopoeia for the treatment of cancer. The present invention also, (a) A pharmaceutical composition containing an effective amount of M2698, (b) A pharmaceutical composition comprising an effective amount of MEK inhibitor, and optionally, (c) A pharmaceutical biologic containing an effective amount of EGFR inhibitor, This refers to a set (kit) consisting of separate packs. The present invention also, (a) A pharmaceutical composition containing an effective amount of M2698, and (b) A pharmaceutical composition comprising an effective amount of EGFR inhibitor, This refers to a set (kit) consisting of separate packs.

[0027] The set includes suitable containers such as boxes, individual bottles, bags, or ampoules. The set may also include separate ampoules containing, for example, an effective amount of a pharmaceutical composition comprising M2698 and / or pharmaceutically usable salts thereof, an effective amount of a pharmaceutical composition comprising MEK inhibitor and / or pharmaceutically usable salts thereof, and optionally, an effective amount of a pharmaceutical composition comprising EGFR inhibitor, each in a dissolved or lyophilized form. The particularly preferred brain-penetrating MEK inhibitor to be combined with M2698 is pimacertib. The particularly preferred EGFR inhibitor to be combined with M2698, with or without a MEK inhibitor, is cetuximab. The particularly preferred triple combination is M2698 + pimacertib + cetuximab.

[0028] Compounds and compound mixtures according to the present invention may be adapted for administration by any preferred method, for example, oral (including oral or sublingual), rectal, nasal, topical (including oral, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal). Such pharmaceuticals may be prepared using any process known in the field of pharmacy, for example, by combining the active ingredient with one or more excipients or adjuvants. Compounds and compound mixtures adapted 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 foam or foam food; or oil-in-water or water-in-oil liquid emulsions.

[0029] Therefore, for example, in the case of oral administration in the form of tablets or capsules, the compound or compound mixture may be combined with an orally, non-toxic, and pharmaceutically acceptable inert excipient such as ethanol, glycerol, or water. Powders are prepared by grinding the compound to a suitable fine size and mixing it with a similarly ground pharmaceutical excipient, such as an edible carbohydrate like starch or mannitol. Flavors, preservatives, dispersants, and colorants may also be present. Capsules are produced by preparing the powder mixture as described above and filling it into a molded gelatin shell. Lubricants or additives, such as highly dispersible silicic acid, talc, magnesium stearate, calcium stearate, or polyethylene glycol in solid form, may even be added to the powder mixture before the filling operation. Disintegrants or solubilizers, such as agar, calcium carbonate, or sodium carbonate, may also be added to improve the availability of the compound or compound mixture after the capsule has been ingested.

[0030] In addition, suitable binders, lubricants, and disintegrants, as well as pigments, may also be incorporated into the mixture if desired or necessary. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, sweeteners made from corn, natural and synthetic rubbers such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, wax, etc. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. Tablets are formulated, for example, by preparing a powder mixture, granulating or dry-pressing the mixture, adding lubricants and disintegrants, and compressing the whole mixture to give tablets. The powder mixture is prepared by mixing a compound, finely ground in a suitable manner, with a diluent or base as described above, and optionally with a binder such as carboxymethylcellulose, alginic acid, gelatin, or polyvinylpyrrolidone, a dissolution inhibitor such as paraffin, an absorption enhancer such as a quaternary salt, and / or an absorbent such as bentonite, kaolin, or calcium diphosphate. The powder mixture can be granulated by moistening it with a binder such as syrup, starch paste, acadia mucilage, or a solution of cellulose or polymer material, and compressing it through a sieve. As an alternative to granulation, the powder mixture can be put through a tablet press to give it a non-uniformly shaped mass, which is then crushed to form granules. The granules can be lubricated by adding stearic acid, stearates, talc, or mineral oil to prevent them from sticking to the tablet mold. The lubricated mixture is then compressed to give tablets. Compounds and compound mixtures according to the present invention may also be combined with a highly fluid, inert excipient, and then directly compressed to obtain tablets without performing a granulation or dry pressing step.A transparent or opaque protective layer may be present, consisting of a shellac seal layer, a layer of sugar or polymer material, and a glossy layer of wax. Dyes may be added to these coatings so that they can be distinguished between different dosage units.

[0031] For example, oral liquids such as solutions, syrups, and elixirs may be prepared in the form of dosage units such that a given volume contains a predetermined amount of the compound. Syrups may be prepared by dissolving the compound and compound mixtures in an aqueous solution with a suitable flavor, while elixirs are prepared using a non-toxic alcoholic vehicle. Suspensions may be formulated by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether, preservatives, flavor additives such as peppermint oil, or natural sweeteners or saccharin or other artificial sweeteners may also be added. Dosage units for oral administration may be encapsulated in microcapsules if desired. The formulations may also be prepared in a manner that extends or delays release, for example, by coating or embedding particulate materials in polymers, waxes, etc.

[0032] Compounds and compound mixtures thereof, as well as their salts and solvates according to the present 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, stearylamine, or phosphatidylcholine.

[0033] Compounds and compound mixtures according to the present invention may also be delivered using monoclonal antibodies as individual carriers to which compound molecules are bound. Compounds and compound mixtures may also be bound to soluble polymers as targeted pharmaceutical carriers. Such polymers may encompass polyvinylpyrrolidone, pyran copolymers, polyhydroxypropyl methacryl-amidophenol, polyhydroxyethyl aspartamidophenol, or polyethylene oxide polylysine, substituted with palmitoyl radicals. Compounds may also be bound to a class of biodegradable polymers suitable for achieving controlled release of pharmaceuticals, such as crosslinked or amphiphilic block copolymers of polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyran, polycyanoacrylates, and hydrogels.

[0034] Compounds and compound mixtures suitable for transdermal administration may be administered as separate ointments for long-term, close contact with the recipient's epidermis. For example, the active ingredient may be delivered from the ointment by iontophoresis, as described in the general section of Pharmaceutical Research, 3(6):318, 1986. Compounds and compound mixtures suitable for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.

[0035] 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. In the case of formulations that provide an ointment, the compound or compound mixture may be used with either a paraffinic or water-miscible cream base. Alternatively, the compound or compound mixture may be formulated to provide a cream using an oil-in-water cream base or an oil-in-water base. Compounds and compound mixtures suitable for topical use in the eye include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, in particular an aqueous solvent.

[0036] Compounds and compound mixtures suitable for topical use in the mouth encompass lozenges, troches, and mouthwashes. Compounds and compound mixtures suitable for rectal administration may be administered in the form of suppositories or enemas.

[0037] Compounds and compound mixtures suitable for nasal administration where the carrier substance is solid include, for example, coarse powders having particle sizes in the range of 20 to 500 microns, which are administered by rapid inhalation through the nasal passages from a container containing the powder held close to the nose, in the manner in which snuff is ingested. Suitable formulations for administration as nasal sprays or nasal drops with a liquid as the carrier substance consist of a solution of the active ingredient in water or oil. Compounds and compound mixtures suitable for inhalation administration comprise fine particulate powders or mist, which can be generated by pressurized dispensers, nebulizers, or injectors with various types of aerosols. Compounds and compound mixtures suitable for vaginal administration may be administered as pessaries, tampons, creams, gels, pastes, foams, or spray formulations.

[0038] Compounds and compound mixtures adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions containing antioxidants, buffers, bacteriostatic agents, and solutes, which are used to make the formulation isotonic with the blood of the recipient to whom the formulation is to be treated; and aqueous and non-aqueous sterile suspensions, which may also contain suspension media and thickeners. The formulations may be administered in single-dose or multi-dose containers, e.g., sealed ampoules and vials, and are stored in a freeze-dried state so that only the addition of a sterile carrier liquid, e.g., water for injection, is intended immediately before use. Injection solutions and suspensions prepared according to the recipe may be prepared from sterile powders, granules, and tablets. In addition to the constituent substances specifically mentioned above, the pharmaceuticals according to the present invention may also include other agents that are common in the art with respect to a particular type of pharmaceutical formulation; for example, a compound or compound mixture suitable for oral administration may include a flavor.

[0039] The therapeutically effective amount of the compound or compound mixture of the present invention depends on numerous factors, including, for example, the age and weight of the recipient, the exact condition requiring treatment and its severity, the nature of the formulation and the method of administration, and ultimately, the physician or veterinarian administering the treatment. However, an effective amount of API for treating diseases according to the present invention generally ranges from 0.1 to 100 mg / kg of the recipient's (mammalian) body weight per day, and more typically from 1 to 10 mg / kg of body weight per day. Thus, the actual amount per day for an adult mammal weighing 70 kg is usually between 70 and 700 mg, where this amount can be administered as individual doses per day or more often in a series of partial doses per day (e.g., 2, 3, 4, 5, or 6 times, etc.), such that the total daily dose is the same. The effective amount of a salt or solvate, or a derivative of its physiologically functional derivative, can be determined as the effective amount of the compound and compound mixture itself according to the present invention.

[0040] Pharmaceutical preparations according to the present invention can be used as pharmaceuticals in humans and veterinary medicine. Suitable excipients are organic or inorganic substances suitable for intestinal (e.g., oral), parenteral or topical administration 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. Suitable for intestinal administration are, among other things, tablets, coated tablets, capsules, syrups, fruit juices, infusions or suppositories; suitable for parenteral administration are solutions, preferably oil-based or aqueous solutions, and furthermore, suspensions, emulsions or grafts; and suitable for topical application are ointments, creams or powders. The compound and compound mixtures may also be lyophilized, and the resulting lyophilized products can be used, for example, for the preparation of injectable preparations.

[0041] The indicated preparations may be sterilized and / or contain adjuvants such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for altering osmotic pressure, buffering substances, colorants, flavors and / or aromatic substances. They may also contain one or more additional active ingredients, such as one or more vitamins, if desired.

[0042] The present invention also relates to methods 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, and corresponding compound mixtures, pharmaceutical compositions, formulations and methods of administration as described herein.

[0043] In one embodiment, the present invention relates to the following: A compound mixture comprising 1,4-[(S)-2-azetidine-1-yl-1-(4-chloro-3-trifluoromethylphenyl)-ethylamino]-quinazoline-8-carboxylic acid amide, or a physiologically acceptable salt thereof, and an inhibitor of MEK, or a physiologically acceptable salt thereof. 2. A compound mixture as described above in this embodiment, wherein the MEK inhibitor is pimacertib. 3. A compound mixture as described above in this embodiment, further comprising an EGFR inhibitor. 4. A pharmaceutical composition comprising, in this enumeration of embodiments, a compound mixture as described above, and optionally, an excipient and / or adjuvant.

[0044] 5. (a) an effective amount of 4-[(S)-2-azetidine-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide or a physiologically acceptable salt thereof, and (b) an effective amount of MEK inhibitor or a physiologically acceptable salt thereof A set (kit) including separate packs. 6. (c) An effective amount of EGFR inhibitor or a physiologically acceptable salt thereof A set (kit) as described above in this enumeration, further including separate packs of the above. 7. The EGFR inhibitor is cetuximab, as described above in this embodiment (kit).

[0045] 8. A method for the prevention or treatment of cancer, comprising administering to a subject 8.4-[(S)-2-azetidine-1-yl-1-(4-chloro-3-trifluoromethylphenyl)-ethylamino]-quinazoline-8-carboxylic acid amide, or a physiologically acceptable salt thereof, and a MEK inhibitor, or a physiologically acceptable salt thereof. 9. The MEK inhibitor is pimacertib, as described above in this embodiment. 10. The methods described above in this list of embodiments, further comprising administering an EGFR inhibitor or a physiologically acceptable salt thereof to a subject.

[0046] 11. The method described above in this embodiment, wherein the EGFR inhibitor is cetuximab. 12.4-[(S)-2-azetidine-1-yl-1-(4-chloro-3-trifluoromethylphenyl)-ethylamino]-quinazoline-8-carboxylic acid amide, a MEK inhibitor, and optionally an EGFR inhibitor, administered simultaneously, as described above in this embodiment. 13.4-[(S)-2-azetidine-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide, a MEK inhibitor, and optionally an EGFR inhibitor, administered sequentially, as described above in this embodiment.

[0047] A compound mixture comprising 14.4-[(S)-2-azetidine-1-yl-1-(4-chloro-3-trifluoromethylphenyl)-ethylamino]-quinazoline-8-carboxylic acid amide, or a physiologically acceptable salt thereof, and an inhibitor of EGFR, preferably cetuximab. 15. (a) an effective amount of 4-[(S)-2-azetidine-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide or a physiologically acceptable salt thereof, and (b) an effective amount of EGFR inhibitor, preferably cetuximab A set (kit) that includes separate packs.

[0048] 16. A method for the prevention or treatment of cancer comprising administering to a subject 16.4-[(S)-2-azetidine-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. 17. In a preferred embodiment, cancers include colorectal cancer, breast cancer, cholangiocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, and non-small cell lung cancer.

[0049] example Example 1 : Combination of M2698 and pimacertib in patients with glioblastoma (GBM) derived from a xenograft model (PDX) GSC strain: Sensitivity to M2698 and pimacertib in vitro Despite the significant effect of M2698 in several 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 pimacertib in vivo and in vitro in 19GSC (glioblastoma stem cell) cell / models. In vitro IC50 data were used to select GSC strains for in vivo studies; IC50 values ​​<2 μM were considered highly sensitive, and >2 μM were considered resistant. All GSC strains expressed Akt. Relatively high pAkt expression was observed in GSC17, GSC7-2, GSC231, GSC11, GSC20, GSC6-27, and GSC8-11 strains compared to GSC272 and GSC267 (Figure 1A). Cell lines with relatively higher pAkt expression were sensitive to M2698, with the exception of GSC20, suggesting that constitutive Akt activation sensitized the cells to M2698 (Figure 1B). Only a few cell lines (GSC17, GSC11, and GSC231) were sensitive to pimacertib (IC50 < 2 μM), while GSC20, GSC272, GSC6-27, and GSC7-2 were resistant (Figure 1C).

[0050] The induction of apoptosis by M2698, pimacertib, and combinations thereof was measured in vitro in three of five GSC strains that were sensitive to M2698. Of these, GSC11 was the most sensitive to pimacertib, GSC17 was moderately sensitive, and GSC7-2 was not sensitive to pimacertib. Both M2698 and pimacertib, administered as monotherapy to GSC11, GSC17, and GSC7-2 strains, induced apoptosis, but there was no apparent difference in the percentage of apoptosis for each compound in terms of IC50 (56%, 11%, and 13%, respectively) versus 2×IC50 (62%, 12%, and 17%, respectively). M2698 + pimacertib exhibited either an additive effect (GSC11 and GSC7-2 strains) or an apparent synergistic effect (GSC17 strain) in inducing apoptosis compared to the use of the single agent (Figure 1D).

[0051] In vivo efficacy and disease progression (PD) effects of M2698 and pimacertib in an orthotopic GSC xenograft model. The in vivo effects of M2698 and pimacertib, both individually and in combination, were assessed in a GSC model after orthotopic transplantation into mice. The treatment period and post-transplant euthanasia time were set at 5–10 weeks. The endpoint was apoptosis, as measured by the expression of PD markers pS6 and pERK, the proliferation marker Ki67, tumor volume, and survival. All but one of the seven GSC models (GSC20) were sensitive to M2698 monotherapy, as measured by tumor volume; M2698 significantly inhibited tumor growth (GSC17, GSC6-27, GSC7-2, GSC272, GSC231; all P<0.05; Figure 2A) or tended to do so (GSC11, P=0.10). Of the six M2698-sensitive models, five responded to pimacertib monotherapy, either showing significant tumor growth inhibition (GSC6-27, GSC7-2, GSC272; P<0.05) or tending toward a significant response (GSC231, P=0.10). Tumor growth in GSC17 was unaffected by pimacertib monotherapy (compared to vehicle, P=0.34). M2698 + pimacertib significantly inhibited tumor growth in all GSC models compared to the vehicle, even in GSC20, where monotherapy treatment was ineffective (P>0.05) (all P<0.05; Figure 2A).

[0052] The median survival in the GSC models did not fully reflect the tumor volume data (Figure 1B). M2698 (GSC272 and GSC231) and the combination (GSC17 and GSC7-2) extended survival in only two models compared to the vehicle alone (P<0.05), and pimacertib had no significant effect on survival compared to the control in any model (P>0.05). In GSC6-27, mice treated with the vehicle had a significantly longer median survival than mice from the treatment group for unknown reasons (P<0.05). In contrast, both monotherapys significantly inhibited tumor growth in some models, and the combination inhibited tumor growth in all models.

[0053] The significant effect on tumor volume indicates that the compound invaded the brain and affected orthotopic tumors. Additional evidence of BBB penetration included effects on PD markers of targeted signaling pathways in tumor cells. M2698 significantly reduced pS6 in orthotopic tumors in all GSC models except GSC20 (P<0.05; Figure 3A). Similarly, no significant effect of pimacerutib treatment on pERK protein was observed in GSC20 tumors (P=0.18), while pimacerutib significantly reduced pERK in other GSC tumors, including GSC17, which did not experience a reduction in tumor volume following pimacerutib treatment (P<0.05; Figure 3B). The M2698 + pimacerutib combination reduced pS6 and pERK in all models except GSC20 (all P<0.05). The relatively high variability in tumor growth, pS6, and pERK in mice treated with the GSC20 model vehicle may have masked the statistical significance of any biologically relevant treatment effect on these endpoints. Conversely, the lack of a significant response was predicted by the insensitivity of GSC20 to both compounds in vitro (Figures 1B and 1C).

[0054] In addition to the effects of pimacerutib on pERK and M2698 on pS6 in orthotopic brain models, each M2698 could also affect opposing PD markers. Pimaserutib significantly reduced pS6 in the GSC272 and GSC231 models. M2698 reduced pERK in all GSC models except GSC20 (P<0.05; Figure 3B). Three of the GSC tumors were analyzed at more than one time point for the PD marker. Statistical analysis of responses over time was not performed, but trends in different endpoints were evaluated. 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). The reduction of pS6 cross-pathogenesis by pimacertib in the GSC272 and GSC231 models decreased until 7.5–10 weeks, and the activity of the compound alone or in combination on pERK expression decreased until 7.5–10 weeks in the GSC231 model. Further research is needed to determine whether these trends are related to the development of resistance, but they reflect heterogeneity in the models.

[0055] Ki67 was measured by immunohistochemistry in tumor sections from GSC17 and GSC7-2, two models in which the M2698 + pimacertib combination significantly extended survival (Figure 3C). In the GSC17 model, all three treatments significantly reduced growth compared to the vehicle, as did M2698 monotherapy and its combination with pimacertib in the GSC7-2 model (all P<0.05). Neither M2698, pimacerutib, nor any combination treatment had a significant effect on apoptosis in GSC7-2 xenograft versus control at 7.5 weeks. However, at 10 weeks, similar to the in vitro data, the M2698 + pimacerutib combination significantly increased the apoptotic cell count / field (172.33 ± 60.80) compared to control (8.50 ± 1.80; P=0.002), M2698 alone (52.17 ± 25.20; P=0.01), and pimacerutib alone (65.00 ± 60.80; P=0.02).

[0056] Example 2: The combination of M2698 and pimacertib in a patient-derived xenograft (PDX) model for brain metastases in non-small cell lung cancer (NSCLC). As shown in Figure 4, the combination of M2698 and pimacertib unexpectedly showed a tumor control rate (TCR) of 26%, while monotherapy showed rates of 5% (M2698) and 16% (pimasertib).

[0057] Example 3: The combination of M2698 and pimacertib in a patient-derived xenograft (PDX) model for Her2+ / HR- breast cancer. As shown in Figure 5, the combination of M2698 with pimacertib unexpectedly reduced tumor volume to almost zero over the duration of the experiment, while the monotherapy merely achieved tumor volume stagnation.

[0058] Example 4: The combination of M2698 and pimacertib in a patient-derived xenograft (PDX) model for cholangiocarcinoma. The goal of this experiment was to evaluate the antitumor activity of M2698 alone and in combination with pimacertib in 13 patient-derived xenograft models representing human cholangiocarcinoma from Chinese patients in immunodeficient mice. The Her2 status of the models was obtained via immunohistochemistry (IHC) and is shown in Figure 6.

[0059] research design [Table 1] Vehicle: Milli-Q water with 0.5% methocel / 0.25% Tween20. Tumors from three untreated mice were collected as satellite samples when the tumor volume reached between 300 and 500 mm³.

[0060] As can be deduced from Figure 6, surprisingly, tumor stabilization was achieved with M2698 and pimacertib in 10 out of 12 tumor samples (83%), while monotherapy showed 17% (2 / 12, M2698) and 8% (1 / 12, pimacertib) results. In this experiment, neither tumor growth response nor regression was observed with any of the treatments.

[0061] Example 5: Combinations of M2698, pimacertib, and cetuximab in 75 patient-derived xenograft (PDX) models for colorectal cancer. research design [Table 2] Of the 75 models, 25 were wild-type, 25 were KRas mutants, and 25 were BRaf mutants.

[0062] result As shown in Figure 7, the tumor control rates (TCR) for monotherapy were 15% (11 / 74) for M2698, 32% (24 / 74) for pimacertib, and 21% (15 / 73) for cetuximab. As shown in Figure 8, the TCRs for the dual combinations were 59% (44 / 74) for pimacertib + cetuximab, 35% (26 / 74) for M2698 + cetuximab, and, unexpectedly, 63% (45 / 72) for M2698 + pimacertib. As shown in Figure 9, surprisingly, the TCR for the triple combination is even more significantly higher than that for the double combination, at 78% (58 / 74).

[0063] Example 6: Combination of M2698 and cetuximab in a patient-derived xenograft (PDX) model of 38 SCCHN Forty-six SCCHN PDX models were run using a 1+1 scheme (one mouse per treatment group). Eight models were withdrawn due to poor growth. These withdrawn models were adjusted to complete 38 models. M2698 was administered along with the standard of care (SoC) agents cisplatin and cetuximab in all monotherapy and "1+1" screen combinations, i.e., one mouse per treatment per model. Post-transplant, the mean tumor volume for each model was approximately 200 mm². 3 Treatment was initiated when the tumor reached approximately 1200 mm in size. This is when the model animals were euthanized, and the control tumor was approximately 1200 mm in size. 3Tumor volume was measured for each model up to that point.

[0064] research design [Table 3]

[0065] result [Table 4] As shown in the table and Figure 10 above, for the combination, the TCR is 58% (22 / 38), which is an unexpected improvement over the TCR of monotherapy in this difficult-to-treat cancer type.

Claims

1. A compound mixture comprising 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 inhibitor of MEK, or a physiologically acceptable salt thereof.

2. 2. The compound mixture of claim 1, wherein the MEK inhibitor is pimasertib.

3. 3. The compound mixture of claim 1 or 2, further comprising an EGFR inhibitor.

4. A pharmaceutical composition comprising a mixture of compounds according to any one of claims 1 to 2, and optionally excipients and / or adjuvants.

5. (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 (b) an effective amount of a MEK inhibitor, or a physiologically acceptable salt thereof; A set (kit) containing separate packs of.

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

6. The set (kit) of claim 5, further comprising a separate pack of:

7. The set (kit) of claim 6, wherein the EGFR inhibitor is cetuximab.

8. The pharmaceutical composition according to claim 4, 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.

9. The pharmaceutical composition of claim 8, wherein the MEK inhibitor is pimasertib.

10. The pharmaceutical composition of claim 8 or 9, wherein the prevention or treatment of cancer further comprises administering to the subject an EGFR inhibitor, or a physiologically acceptable salt thereof.

11. 10. The pharmaceutical composition of claim 9, wherein the EGFR inhibitor is cetuximab.

12. The pharmaceutical composition of any one of claims 8 to 11, wherein the prevention or treatment of cancer is achieved by simultaneously administering 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.

13. The pharmaceutical composition of any one of claims 8 to 11, wherein the prevention or treatment of cancer is achieved by sequentially administering 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.

14. A compound mixture comprising 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 inhibitor of EGFR.

15. 15. The compound mixture of claim 14, wherein the EGFR inhibitor is cetuximab.

16. (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 (b) an effective amount of an EGFR inhibitor. A set (kit) containing separate packs of.

17. The set (kit) of claim 16, wherein the EGFR inhibitor is cetuximab.

18. A pharmaceutical composition comprising a mixture of compounds according to claim 14 or 15, and optionally an excipient and / or adjuvant, for the prevention or treatment of cancer, comprising administering 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 to a subject.

19. 19. The pharmaceutical composition of claim 18, wherein the EGFR inhibitor is cetuximab.

20. 20. The pharmaceutical composition of claim 18 or 19, wherein 4-[(S)-2-Azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide and the EGFR inhibitor are administered simultaneously.

21. 20. The pharmaceutical composition of claim 18 or 19, wherein 4-[(S)-2-Azetidin-1-yl-1-(4-chloro-3-trifluoromethyl-phenyl)-ethylamino]-quinazoline-8-carboxylic acid amide and the EGFR inhibitor are administered sequentially.

22. The pharmaceutical composition according to any one of claims 8 to 13 and 18 to 21, wherein the cancer is colon cancer, breast cancer, cholangiocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, and non-small cell lung cancer.

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