Combination of methionine aminopeptidase 2 inhibitor and VEGFR / VEGF inhibitor (cancer drug combination)

Abstract

The present invention relates to a combination of (S)-3-hydroxy-1-(1H-indol-5-yl)-2-oxo-pyrrolidine-3-carboxylic acid 3,5-difluoro-benzylamide with a VEGFR / VEGF inhibitor, or a physiologically acceptable salt thereof, and to the use of such a combination for the prevention or treatment of cancer.

Description

[Technical Field]

[0001] Technical Field The present invention relates to a combination of (S)-3-hydroxy-1-(1H-indol-5-yl)-2-oxo-pyrrolidine-3-carboxylic acid 3,5-difluoro-benzylamide (hereinafter referred to as Compound A) with one or more inhibitors of VEGFR1, VEGFR2, VEGFR3 and VEGF (hereinafter referred to as VEGFR / VEGF inhibitors), or physiologically acceptable salts thereof, and to the use of such a combination for the prevention or treatment of cancer. [Background technology]

[0002] background In 1990, Judah Folkman's laboratory reported the antiangiogenic activity of a natural compound named fumagillin, isolated in 1951 from the fungus Aspergillus fumigatus (Ingber et al., Nature 1990;348(6301):555-7). The molecular target of fumagillin and its derivatives was identified in 1997 by Craig Crews' laboratory as methionine aminopeptidase 2 (MetAP2) (Sin et al., Proc Natl Acad Sci USA 1997;94:6099-103). Methionine aminopeptidase is an enzyme that removes the amino-terminal amino acid methionine from proteins during or after the translation process. Methionine is cleaved from some, but not all, cellular proteins, especially those with small amino acids at the second position. As part of protein maturation, cleavage is thought to be involved in protein stability (N-end rule) and proper function (protein folding, N-terminal myristoylation). Three MetAPs can be found in humans: MetAP1, MetAP2, and MAP1D. MetAP2 is widely expressed in all human tissues, although there are differences in expression levels based on mRNA expression data. Complete knockout of MetAP2 in mice is embryonic lethal with a significant block at the gastrulation stage. Tissue-restricted knockout of the vascular endothelial cell compartment is embryonic lethal and shows significant defects in vascular development (Yeh et al., Proc Natl Acad Sci USA 2006;103(27):10379-84), indicating that MetAP2 is important during development and required for the formation of the vasculature.

[0003] Pharmacological inhibition of MetAP2 using different classes of inhibitors has been shown to block vascular endothelial cell proliferation, at least partially explaining its antiangiogenic mechanism of action (Wang et al., Proc Natl Acad Sci USA 2008;105(6):1838-43; Bernier et al., J Cell Biochem 2005;95(6):1191-203). Subsequently, antiproliferative activity against tumor cell lines was demonstrated, indicating potential direct antitumor effects (Wang et al., Cancer Res 2003;63(22):7861-9; Wang et al., Proc Natl Acad Sci USA 2008;105(6):1838-43). The discovery of fumagillin, which has potent antiangiogenic and antiproliferative activities, has prompted the development of MetAP2 antagonists as a new class of anticancer agents. MetAP2 plays a key role in the development of various types of cancer. MetAP2 inhibition leads to delayed cell cycle progression in a subset of endothelial cells and tumor cells. Consequently, MetAP2 inhibitors block angiogenesis both in vitro and in vivo and exhibit potent antitumor effects in mouse models of various human-derived tumor types.

[0004] In clinical trials, fumagillin proved unsuitable as an anticancer drug due to its pronounced neurotoxicity. Compound A is a potent, reversible, noncovalent, and orally bioavailable inhibitor of MetAP2 (Heinrich et al., J Med Chem. 2019; 62(24): 11119-11134. See also doi:10.1021 / acs.jmedchem.9b01070). Compound A is chemically unrelated to fumagillin and has not shown signs of neurotoxicity in toxicology studies. Compound A was found to inhibit the growth of endothelial cells, mouse and human tumor cells, and patient-derived tumors. Compound A demonstrated antiangiogenic and antitumor activity in mouse models. It has been shown that compound A can be used to effectively inhibit MetAP2, as evidenced by the accumulation of the unprocessed MetAP2 substrate, methionylated elongation factor 1α, Met-EF1α. Compared to irreversible MetAP2 inhibitors, Compound A is expected to have an improved safety and tolerability profile, which is beneficial for long-term administration. Compound A, its preparation method, and its use in the treatment of cancer are disclosed in WO 2013 / 149704 A1 (Compound A is referred to as "B8" in WO 2013 / 149704 A1); see also WO 2012 / 048775 A1. In addition, an asymmetric synthesis of Compound A is disclosed in Heinrich et al. J Med Chem. 2019;62(24):11119-11134. doi:10.1021 / acs.jmedchem.9b01070.

[0005] The object of the present invention was to find ways to further improve the pharmaceutical utility of Compound A. In this context, the combination of Compound A with a VEGFR / VEGF inhibitor was investigated. Known VEGFR / VEGF inhibitors include aflibercept, apatinib, axitinib, bevacizumab, brivanib alaninate, cabozantinib, cediranib, lenvatinib, linifanib, pazopanib, ponatinib, ramucirumab, regorafenib, sorafenib, sunitinib, vandetanib, and the anti-VEGFR2 antibody 33C3.

[0006] Cabozantinib (S)-malate is a tyrosine kinase inhibitor initially approved by the Food and Drug Administration (FDA) as second-line therapy for patients with metastatic renal cell carcinoma (mRCC) who have developed resistance to first-line treatments; more recently, cabozantinib (S)-malate has been approved as first-line therapy. Cabozantinib targets VEGF receptors (VEGFR) 1–3, among other kinases. The recommended dosage for cabozantinib (S)-malate in mRCC is 60 mg once daily without food until the patient no longer experiences clinical benefit or experiences unacceptable toxicity (e.g., see the label for the commercial cabozantinib (S)-malate product, Cabometyx, from January 2020, which is published on the FDA website: https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2020 / 208692s007lbl.pdf). Cabozantinib is commercially available, for example, in the form of the (S)-malate salt of cabozantinib (ie, cabozantinib (S)-malate).

[0007] Axitinib is another tyrosine kinase inhibitor. Information about axitinib can be found, for example, in the June 2020 labeling for the commercial axitinib product INLYTA, which is published on the FDA website (https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2020 / 202324s011lbl.pdf): Axitinib has been found to inhibit receptor tyrosine kinases, including VEGFR-1, VEGFR-2, and VEGFR-3, at therapeutic plasma concentrations. VEGF-mediated endothelial cell proliferation and survival were inhibited by axitinib in vitro and in mouse models. Axitinib was shown to inhibit tumor growth and VEGFR-2 phosphorylation in tumor xenograft mouse models. Axitinib has been approved as a single agent for the treatment of advanced renal cell carcinoma (RCC) after failure of one prior systemic therapy. In combination with avelumab, axitinib is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC). In combination with pembrolizumab, axitinib is indicated for the first-line treatment of patients with advanced renal cell carcinoma. Axitinib is commercially available, for example, in the form of the free base.

[0008] For the first-line treatment of advanced RCC, the recommended axitinib dose is 5 mg orally twice daily (12 hours apart) with or without food, combined with avelumab 800 mg administered as a 60-minute intravenous infusion every 2 weeks, continued until disease progression or unacceptable toxicity. When axitinib is combined with avelumab, dose escalation beyond the initial 5 mg dose of axitinib may be considered at intervals of 2 weeks or longer. The recommended axitinib dose is 5 mg orally twice daily (12 hours apart) with or without food, combined with pembrolizumab 200 mg administered as a 30-minute intravenous infusion every 3 weeks or 400 mg administered as a 30-minute intravenous infusion every 6 weeks, continued until disease progression or unacceptable toxicity. When axitinib is used in combination with pembrolizumab, dose escalation beyond the initial 5 mg dose of axitinib may be considered at intervals of 6 weeks or longer. When used as a single agent in the second-line treatment of advanced RCC, the recommended starting oral dose of axitinib is 5 mg twice daily. In this context, axitinib doses should be administered approximately 12 hours apart, with or without food.

[0009] The proangiogenic signaling molecule VEGF and its receptors VEGFR1, VEGFR2, and VEGFR3 play important roles in tumor development. These receptors are involved in pathological angiogenesis, tumor growth, and cancer progression. Induction of angiogenesis is increasingly recognized as a critical step in tumor progression and is one of the hallmarks of cancerous growth. For example, angiogenesis is a key factor in the development and progression of renal cell carcinoma (RCC). RCC is a heterogeneous group of cancers originating in the kidney. The most common histological variant of RCC is clear cell RCC, which accounts for approximately 70% of RCC cases and has the highest metastatic potential. Clear cell RCC is characterized by inactivation of the von-Hippel Lindau (VHL) tumor suppressor gene, which leads to increased activity of hypoxia-inducible factor α (HIFα). This leads to the production of angiogenic factors such as VEGF and platelet-derived growth factor. The activity of these factors is associated with the oncogenesis, growth, and metastatic potential of RCC.

[0010] Medical treatment for metastatic RCC (mRCC) has expanded considerably from nonspecific immune approaches to targeted therapy with VEGFR and mammalian target of rapamycin (mTOR). Today, salvage approaches have evolved to include immune checkpoint blockade (ICB) targeting programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) (Salgia et al., Curr Treat Options Oncol 2019;20(5):41). VEGFR TKIs alone, ICB, or a combination of VEGFR TKIs and ICB are first-line treatment options for mRCC. Cabozantinib, nivolumab, and axitinib are subsequent treatment options. Therefore, the tyrosine kinase inhibitors cabozantinib and axitinib are among the standard-of-care (SoC) agents for the treatment of RCC. Nevertheless, there remains a need for new treatment options for cancer, including RCC. The present invention addresses this and other needs in the art. DISCLOSURE OF THE INVENTION

[0011] Summary of the Invention Surprisingly, the inventors of the present patent application have found that Compound A achieves significant combination benefits over monotherapy when combined with cabozantinib or axitinib in the treatment of RCC patient-derived xenografts (PDXs). The inventors' findings suggest that Compound A can be used in combination with VEGFR / VEGF inhibitors, such as axitinib or cabozantinib, to successfully treat cancers, including RCC. The present invention relates to the combination of compound A with a VEGFR / VEGF inhibitor, such as cabozantinib or axitinib, or a physiologically acceptable salt thereof, and to the use of such a combination for the prevention or treatment of cancer, including RCC. The present invention relates to the embodiments set forth in the claims. The combinations and their uses according to the present invention, as summarized above and further described below, provide surprisingly significant combination benefits in cancer treatment, including the treatment of RCC, as illustrated in the examples below. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1: Treatment with vehicle or Compound A and / or cabozantinib: Progression-free survival of mice bearing RCC patient-derived xenografts (PDX), represented as Kaplan-Meier curves. Days required to reach 173% relative tumor volume (RTV) (censored at latest assessment if endpoint not reached). CompA, Compound A. [Figure 2] Figure 2: Treatment with vehicle or Compound A and / or cabozantinib: Overall survival of mice bearing RCC patient-derived xenografts (PDX), represented as Kaplan-Meier curves. Days required to reach 1000% RTV (censored at latest assessment if endpoint not reached). CompA, Compound A. [Figure 3] Figure 3: Treatment with vehicle or Compound A and / or cabozantinib: Body weight change in mice bearing RCC patient-derived xenografts (PDX); one exemplary PDX model is shown for each treatment group.

[0013] [Figure 4] Figure 4: Treatment with vehicle or Compound A and / or axitinib: Progression-free survival of mice bearing RCC patient-derived xenografts (PDX), represented as Kaplan-Meier curves. Days required to reach 173% RTV (censored at latest assessment if endpoint not reached). CompA, Compound A. [Figure 5] Figure 5: Treatment with vehicle or Compound A and / or axitinib: Overall survival of mice bearing RCC patient-derived xenografts (PDX), represented as Kaplan-Meier curves. Days required to reach 1000% RTV (censored at latest assessment if endpoint not reached). CompA, Compound A. [Figure 6]Figure 6: Treatment with vehicle or Compound A and / or axitinib: Body weight change in mice bearing RCC patient-derived xenografts (PDX); one exemplary PDX model is shown for each treatment group.

[0014] Detailed Description of the Invention The present invention relates to combinations of Compound A with VEGFR / VEGF inhibitors or physiologically acceptable salts thereof, and to the use of such combinations for the prevention or treatment of cancers as described herein above and below.

[0015] The present invention relates to a compound mixture comprising the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; b) A VEGFR / VEGF inhibitor or a physiologically acceptable salt thereof. In some embodiments of the present invention, the VEGFR / VEGF inhibitor is selected from the group consisting of aflibercept, apatinib, axitinib, bevacizumab, brivanib alaninate, cabozantinib, cediranib, lenvatinib, linifanib, pazopanib, ponatinib, ramucirumab, regorafenib, sorafenib, sunitinib, vandetanib, and 33C3. In some embodiments of the present invention, the VEGFR / VEGF inhibitor is cabozantinib or axitinib. In some specific embodiments, the VEGFR / VEGF inhibitor is cabozantinib, for example, cabozantinib (S)-malate. In other specific embodiments, the VEGFR / VEGF inhibitor is axitinib. Cabozantinib is also known as N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]phenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide. Cabozantinib (S)-malate is also known as N-{4-[(6,7-dimethoxyquinolin-4-yl)oxy]phenyl}-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide, (2S)-hydroxybutanedioate.

[0016] Axitinib is also known as N-methyl-2-({3-[(1E)-2-(pyridin-2-yl)ethenyl]-1H-indazol-6-yl}sulfanyl)benzamide. In some embodiments, the present invention thus relates to a compound mixture comprising the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; b) Cabozantinib or axitinib, or a physiologically acceptable salt thereof. In some embodiments, the present invention relates to a compound mixture comprising the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; b) Cabozantinib or a physiologically acceptable salt thereof. In some embodiments, the present invention relates to a compound mixture comprising the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; b) Cabozantinib (S)-malate.

[0017] In some embodiments, the present invention relates to a compound mixture comprising the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; b) Axitinib or a physiologically acceptable salt thereof. The present invention further relates to a pharmaceutical composition comprising a compound mixture of Compound A as an active pharmaceutical ingredient (API) and a VEGFR / VEGF inhibitor or a physiologically acceptable salt thereof, optionally further comprising one or more excipients and / or adjuvants. In some embodiments of the present invention, the VEGFR / VEGF inhibitor contained in the pharmaceutical composition is selected from the group consisting of aflibercept, apatinib, axitinib, bevacizumab, brivanib alaninate, cabozantinib, cediranib, lenvatinib, linifanib, pazopanib, ponatinib, ramucirumab, regorafenib, sorafenib, sunitinib, vandetanib, and 33C3. In some embodiments of the present invention, the VEGFR / VEGF inhibitor is cabozantinib or axitinib. In some specific embodiments, the VEGFR / VEGF inhibitor is cabozantinib, e.g., cabozantinib (S)-malate. In other specific embodiments, the VEGFR / VEGF inhibitor is axitinib.

[0018] The present invention relates to pharmaceutical compositions comprising any one of the compound mixtures according to the invention, optionally further comprising one or more excipients and / or adjuvants. For some compounds, suitable acid addition salts are all physiologically or pharmacologically acceptable inorganic or organic salts of acids, such as halides, especially hydrochlorides or hydrobromides, lactates, sulfates, citrates, tartrates, maleates, fumarates, oxalates, acetates, phosphates, methylsulfonates, benzoates or p-toluenesulfonates. The preferred form of Compound A is its free base. Salt forms of Compound A include its sodium, potassium, calcium, or magnesium salts. A pharmaceutical composition according to the present invention comprises a mixture of two APIs in a ratio of, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:100 or 1:1000.

[0019] The pharmaceutical composition may further comprise at least one solid, liquid and / or semi-liquid excipient and / or adjuvant.Accordingly, the present invention also relates to a pharmaceutical composition comprising said API mixture according to the present invention and said excipient and / or adjuvant. The present invention also relates to a set (also called a kit) comprising separate packs of the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; b) A VEGFR / VEGF inhibitor or a physiologically acceptable salt thereof.

[0020] For example, the present invention relates to a set (also called a kit) comprising separate packs of the following components: a) a pharmaceutical composition comprising Compound A or a physiologically acceptable salt thereof; b) a pharmaceutical composition comprising a VEGFR / VEGF inhibitor or a physiologically acceptable salt thereof, and optionally c) A pharmaceutical composition comprising a third cancer therapeutic agent. In some embodiments of the present invention, the VEGFR / VEGF inhibitor in the set (kit) is selected from the group consisting of aflibercept, apatinib, axitinib, bevacizumab, brivanib alaninate, cabozantinib, cediranib, lenvatinib, linifanib, pazopanib, ponatinib, ramucirumab, regorafenib, sorafenib, sunitinib, vandetanib, and 33C3. In some embodiments of the present invention, the VEGFR / VEGF inhibitor is cabozantinib or axitinib. In some specific embodiments, the VEGFR / VEGF inhibitor is cabozantinib, for example, cabozantinib (S)-malate. In other specific embodiments, the VEGFR / VEGF inhibitor is axitinib.

[0021] The set comprises suitable containers, such as boxes, individual bottles, bags, ampoules, etc. The set may, for example, comprise separate ampoules each containing: a pharmaceutical composition comprising an effective amount of Compound A and / or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising an effective amount of a VEGFR / VEGF inhibitor (e.g., cabozantinib or axitinib) and / or a pharmaceutically acceptable salt thereof, and, optionally, a pharmaceutical composition comprising an effective amount of a third cancer therapeutic agent in dissolved or lyophilized form.

[0022] Cancer therapeutic agents that can be combined with Compound A according to the present invention and a VEGFR / VEGF inhibitor (e.g., cabozantinib or axitinib) as a "third cancer therapeutic agent" can include one or more, preferably one, of the following agents: - alkylating agents, such as altretamine, bendamustine, busulfan, carmustine, chlorambucil, chlormethine, cyclophosphamide, dacarbazine, ifosfamide, improsulfan tosylate, lomustine, melphalan, mitobronitol, mitolactol, nimustine, ranimustine, temozolomide, thiotepa, treosulfan, mechlorethamine, carboquone, apaziquone, fotemustine, glufosfamide, palifosfamide, pipobroman, trofosfamide, uramustine, etc.; - platinum compounds, such as carboplatin, cisplatin, eptaplatin, miriplatin hydrate, oxaliplatin, lobaplatin, nedaplatin, picoplatin, satraplatin, etc.; DNA-modifying agents, such as amrubicin, bisantrene, decitabine, mitoxantrone, procarbazine, trabectedin, clofarabine, amsacrine, brostallicin, pixantrone, laromustine, etc.;

[0023] -Topoisomerase inhibitors, such as etoposide, irinotecan, razoxane, sobuzoxane, teniposide, topotecan, amonafide, belotecan, elliptinium acetate, voreloxin, etc.; -microtubule modifiers, such as cabazitaxel, docetaxel, eribulin, ixabepilone, paclitaxel, vinblastine, vincristine, vinorelbine, vindesine, vinflunine, fosbretabine, tesetaxel, etc.; -Antimetabolites, such as asparaginase, azacitidine, levofolinate calcium, capecitabine, cladribine, cytarabine, enocitabine, floxuridine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, nelarabine, pemetrexed, pralatrexate, azathioprine, thioguanine, carmofur, doxifluridine, elacitarabine, raltitrexed, sapacitabine, tegafur, trimetrexate, etc.; -Anti-cancer antibiotics, such as bleomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, levamisole, miltefosine, mitomycin C, romidepsin, streptozocin, valrubicin, zinostatin, zorubicin, daunorubicin, plicamycin, aclarubicin, peplomycin, pirarubicin, etc.;

[0024] - Hormones / antagonists, such as abarelix, abiraterone, bicalutamide, buserelin, calusterone, chlorotrianisene, degarelix, dexamethasone, estradiol, fluocortolone, fluoxymesterone, flutamide, fulvestrant, goserelin, histrelin, leuprorelin, megestrol, mitotane, nafarelin, nandrolone, nilutamide, octreotide, prednisolone, raloxifene, tamoxifen, thyrotropin alfa, toremifene, trilostane, triptorelin, diethylstilbestrol, acolbifene, danazol, deslorelin, epithiostanol, orteronel, enzalutamide, etc.; - Aromatase inhibitors, such as aminoglutethimide, anastrozole, exemestane, fadrozole, letrozole, testolactone, formestane, etc.; small molecule kinase inhibitors, such as crizotinib, dasatinib, erlotinib, imatinib, lapatinib, nilotinib, pazopanib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, bosutinib, gefitinib, afatinib, alisertib, dabrafenib, dacomitinib, dinaciclib, dovitinib, enzastaurin, Nintedanib, lenvatinib, linifanib, linsitinib, masitinib, midostaurin, motesanib, neratinib, orantinib, perifosine, ponatinib, radotinib, rigosertib, tipifarnib, tivantinib, tivozanib, trametinib, pimasertib, brivanib alaninate, cediranib, apatinib, carfilzomib, ibrutinib, icotinib, etc.;

[0025] -Photosensitizers, such as methoxsalen, porfimer sodium, talaporfin, temoporfin, etc.; - Cytokines, such as aldesleukin, interferon alpha, interferon alpha 2a, interferon alpha 2b, tasonermin, teceleukin, oprelvekin, etc.; -drug conjugates, such as denileukin diftitox, ibritumomab tiuxetan, iobenguane I123, prednimustine, trastuzumab emtansine, estramustine, gemtuzumab ozogamicin, aflibercept, syntredekin besudotox, edotreotide, inotuzumab ozogamicin, naptumomab estafenatox, oportuzumab monatox, technitium (99mTc) arcitumomab, vintafolide, etc.; - Vaccines, such as sipuleucel, vitespen, emepepimt-S, oncoVAX, rindopepimt, troVax, stimuvax, etc.;

[0026] -Other medications, such as alitretinoin, bexarotene, bortezomib, everolimus, ibandronate, imiquimod, lenalidomide, lentinan, metyrosine, mifamurtide, pamidronate, pegaspargase, pentostatin, sipuleucel, sizofiran, tamibarotene, temsirolimus, thalidomide, tretinoin, vismodegib, zoledronic acid, thalidomide, vorinostat, celecoxib, cilengitide, entinostat, etani Dazole, ganetespib, idronoxyl, iniparib, ixazomib, lonidamine, nimorazole, panobinostat, peretinoin, plitidepsin, pomalidomide, procodazole, ridaforolimus, tasquinimod, telotristat, simalfasin, tirapazamine, tosedostat, travedelsen, ubenimex, valspodar, gendicine, picibanil, leolysin, letaspimycin hydrochloride, trebananib, bilirudin, etc.

[0027] The compounds and compound mixtures according to the present invention can be adapted for administration by any desired suitable method, for example, oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) methods. Such pharmaceutical compositions (also called pharmaceutical formulations) can be prepared using any method known in the pharmaceutical art, for example, by combining the active ingredient with excipient(s) and / or adjuvant(s). Compounds and compound mixtures adapted for oral administration can be administered as discrete units, such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foam foods; or oil-in-water or water-in-oil liquid emulsions.

[0028] Thus, for example, for oral administration in the form of a tablet or capsule, the compound or compound mixture can be combined with an oral, non-toxic, and pharmaceutically acceptable inert excipient, such as ethanol, glycerol, water, etc. Powders are prepared by comminuting the compound to a suitable fine size and mixing it with a similarly comminuted pharmaceutical excipient, such as an edible carbohydrate, for example, starch or mannitol. Flavoring agents, preservatives, dispersing agents, and dyes may also be present. Capsules can be produced by preparing a powder mixture as described above and filling it into shaped gelatin shells.Glidants and lubricants, such as highly dispersible silicic acid in solid form, talc, magnesium stearate, calcium stearate or polyethylene glycol, can be added to the powder mixture before filling.Disintegrants or solubilizers, such as agar, calcium carbonate or sodium carbonate, can also be added to improve the availability of compound and compound mixture after taking the capsule.

[0029] Furthermore, if desired or necessary, suitable binders, lubricants, disintegrants, and dyes can also be included in the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, sweeteners made from corn, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or dry-pressing the mixture, adding a lubricant and disintegrant, and compressing the entire mixture to obtain tablets. Powder mixtures are prepared by mixing the compound, milled in a suitable manner, with a diluent or base, as described above, and optionally with a binder, such as carboxymethylcellulose, alginate, gelatin, or polyvinylpyrrolidone; a solution retarder, such as paraffin; an absorption accelerator, such as a 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, acacia mucilage, or a cellulose solution or polymeric material, and forcing it through a sieve. As an alternative to granulation, the powder mixture can be passed through a tablet press to obtain lumps of non-uniform shape, which are then broken down to form granules. The granules can be lubricated by adding stearic acid, a stearate salt, talc, or mineral oil to prevent them from sticking to the tablet casting mold. The lubricated mixture is then compressed to form tablets. The compounds and compound mixtures of the present invention can also be combined with a free-flowing inert excipient and then compressed directly to give tablets without the need for granulation or dry-pressing. A transparent or opaque protective layer consisting of a shellac sealing layer, a layer of sugar or polymeric material and a gloss layer of wax may be present.Dyes can be added to these coatings to make it possible to distinguish between different dosage units.

[0030] Oral liquid, such as solution, syrup and elixir, can be prepared in the form of dosage unit, so that a predetermined amount contains a predetermined amount of the compound.Syrup can be prepared by dissolving the compound and the compound mixture in aqueous solution with suitable flavoring, while elixir is prepared by using non-toxic alcoholic vehicle.Suspension can be prepared by dispersing the compound in non-toxic vehicle.Solubilizer and emulsifier, such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservative, flavor additive, such as peppermint oil or natural sweetener or saccharin, or other artificial sweetener and similar, can also be added.

[0031] Dosage unit formulations for oral administration can, if desired, be encapsulated in microcapsules. Formulations can also be prepared in such a way that release is prolonged or delayed, such as by coating or embedding particulate materials in polymers, waxes, and the like. The compounds and compound mixtures and salts thereof according to the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from various phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

[0032] The compounds and compound mixtures according to the present invention can also be delivered using monoclonal antibodies as individual carriers to which the compound molecules are bound. The compounds and compound mixtures can also be bound to soluble polymers as targeted drug carriers. Such polymers may include palmitoyl-substituted polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartamidophenol, or polyethylene oxide polylysine. The compounds may also be bound to a group of biodegradable polymers suitable for achieving controlled drug release, such as polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates, and crosslinked or amphiphilic block copolymers of hydrogels. Compounds and compound mixtures adapted for transdermal administration can be administered as separate plasters for prolonged, intimate contact with the recipient's epidermis. Thus, for example, the active ingredient can be delivered from the plaster by iontophoresis, as generally described in Pharmaceutical Research, 3(6), 318 (1986).

[0033] Compounds and compound mixtures adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For treating eye or other external tissue, for example mouth and skin, formulation is preferably applied as topical ointment or cream.When formulating for applying ointment, compound or compound mixture can be used with either paraffinic or water-miscible cream base.Alternatively, compound or compound mixture can be formulated to obtain the cream with oil-in-water cream base or water-in-oil base. Compounds and compound mixtures adapted for topical application to the eye include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, in particular an aqueous solvent.

[0034] Compounds and mixtures of compounds adapted for topical application in the mouth encompass lozenges, pastilles and mouthwashes. Compounds and compound mixtures adapted for rectal administration can be administered in the form of suppositories or enemas. Compounds and compound mixtures adapted for intranasal administration in which the carrier material is a solid include, for example, a coarse powder having a particle size in the range of 20 to 500 microns, which are administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container containing the powder held close to the nose. Formulations suitable for administration as intranasal sprays or nasal drops, having a liquid as the carrier material, include solutions of the active ingredient dissolved in water or oil.

[0035] Compounds and compound mixtures adapted for administration by inhalation encompass fine particulate dusts or mists, which can be generated by various types of pressurized dispensers with aerosols, nebulizers, or inhalers. Compounds and compound mixtures adapted for vaginal administration can be administered as pessaries, tampons, creams, gels, pastes, foams or spray formulations. Compounds and compound mixtures suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions containing antioxidants, buffers, bacteriostatic 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 suspension media and thickeners.The formulations can be administered in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, so that they only require the addition of a sterile carrier liquid, such as water for injection, immediately before use.The injection solutions and suspensions prepared by the formulations can be prepared from sterile powders, granules, and tablets.

[0036] Of course, in addition to the components specifically mentioned above, the compound mixtures and pharmaceutical compositions according to the invention may also contain other agents that are conventional in the art for the particular type of pharmaceutical formulation, so that, for example, a compound or compound mixture suitable for oral administration may contain flavoring agents. The pharmaceutical preparations according to the present invention can be used as medicines in human and veterinary medicine.Suitable excipients are organic or inorganic substances suitable for enteral (e.g., oral), parenteral, or topical administration that do not react with the compounds of the present invention, such as water, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc, or petrolatum.Suitable for enteral administration are, in particular, tablets, dragees, capsules, syrups, juices, drops, or suppositories; suitable for parenteral administration are solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants; suitable for topical administration are ointments, creams, or powders.The compounds and compound mixtures can also be lyophilized, and the resulting lyophilized products can be used, for example, to prepare injection preparations.

[0037] The specified formulations may be sterilized and / or contain adjuvants, such as lubricants, preservatives, stabilizers and / or wetting agents, emulsifiers, salts for adjusting the osmotic pressure, buffer substances, dyes, flavors and / or aromatic substances, etc. They may also contain one or more further active ingredients, if desired, such as one or more vitamins. In some embodiments of the present invention, Compound A is formulated as a 1 mg, 5 mg, or 30 mg hard gelatin capsule for oral administration; in some of these embodiments, no other excipients are used. For example, in some embodiments of the present invention, a set (kit) includes such hard gelatin capsules as a pharmaceutical composition containing Compound A. In other embodiments of the present invention, Compound A is formulated as a tablet for oral administration.

[0038] In some embodiments of the present invention, the commercially available cabozantinib drug product (i.e., cabozantinib (S)-malate) tablet formulation (Cabometyx; Exelixis, Inc.) is used; this tablet formulation is sold in three strengths (20, 40, and 60 mg). In some embodiments of the present invention, a commercially available pharmaceutical tablet formulation of axitinib (Inlyta; Pfizer) is used. This tablet formulation is sold in two strengths (1 mg and 5 mg).

[0039] The present invention also relates to a method for preventing and / or treating cancer, comprising administering to a subject the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; and b) a VEGFR / VEGF inhibitor or a physiologically acceptable salt thereof. In some embodiments of the present invention, the VEGFR / VEGF inhibitor administered in this method is selected from the group consisting of aflibercept, apatinib, axitinib, bevacizumab, brivanib alaninate, cabozantinib, cediranib, lenvatinib, linifanib, pazopanib, ponatinib, ramucirumab, regorafenib, sorafenib, sunitinib, vandetanib, and 33C3. In some embodiments of the present invention, the VEGFR / VEGF inhibitor administered in this method is cabozantinib or axitinib. In some specific embodiments of the invention, the VEGFR / VEGF inhibitor administered in this manner is cabozantinib, e.g., cabozantinib (S)-malate. In other specific embodiments of the invention, the VEGFR / VEGF inhibitor administered in this manner is axitinib.

[0040] In the methods of the present invention, the cancer may be, for example, a cancer for which a VEGFR / VEGF inhibitor is the standard treatment option (e.g., a cancer for which cabozantinib or axitinib is the standard treatment option) and / or a cancer that is sensitive to anti-angiogenic treatment (based on high angiogenesis). In some embodiments, the cancer is selected from the group consisting of renal cell carcinoma (RCC), colorectal cancer, lung cancer (e.g., non-small cell lung cancer), head and neck cancer, gastric cancer (e.g., gastric carcinoma), gastroesophageal junction (GEJ) adenocarcinoma, gastrointestinal stromal tumor, glioblastoma, hepatocellular carcinoma, breast cancer, thyroid cancer, soft tissue sarcoma, chronic myeloid leukemia (CML), and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). In some embodiments, the cancer is RCC. In some specific embodiments, the cancer is clear cell RCC. In some embodiments, the present invention therefore relates to a method for the prevention and / or treatment of cancer, comprising administering to a subject the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; and b) cabozantinib or a physiologically acceptable salt thereof.

[0041] In some embodiments, the present invention relates to a method for the prevention and / or treatment of RCC (e.g., clear cell RCC), comprising administering to a subject the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; and b) cabozantinib or a physiologically acceptable salt thereof. In some embodiments, the present invention relates to a method for preventing and / or treating cancer, comprising administering to a subject the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; and b) cabozantinib (S)-malate. In some embodiments, the present invention relates to a method for preventing and / or treating RCC (e.g., clear cell RCC), comprising administering to a subject the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; and b) cabozantinib (S)-malate.

[0042] In some embodiments, the present invention relates to a method for preventing and / or treating cancer, comprising administering to a subject the following compounds a) and b): a) compound A or a physiologically acceptable salt thereof; and b) axitinib or a physiologically acceptable salt thereof. In some embodiments, the present invention relates to a method for the prevention and / or treatment of RCC (e.g., clear cell RCC), comprising administering to a subject the following compounds a) and b): a) Compound A or a physiologically acceptable salt thereof; and b) axitinib or a physiologically acceptable salt thereof. In the methods according to the invention, Compound A and the VEGFR / VEGF inhibitor (e.g., cabozantinib or axitinib), or a physiologically acceptable salt thereof, can be administered via any desired suitable route, for example, by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) administration.

[0043] In the method according to the present invention, Compound A and the VEGFR / VEGF inhibitor (e.g., cabozantinib or axitinib), or a physiologically acceptable salt thereof, can be administered simultaneously or sequentially. When administered simultaneously, Compound A and the VEGFR / VEGF inhibitor (or a physiologically acceptable salt thereof) can be administered as a compound mixture in one pharmaceutical composition or as separate pharmaceutical compositions. In one embodiment, the method for preventing or treating cancer according to the present invention comprises the sequential administration of compound A and a VEGFR / VEGF inhibitor (e.g., cabozantinib or axitinib) or a physiologically acceptable salt thereof. In some embodiments of the methods of the present invention, Compound A and cabozantinib are administered once daily (i.e., QD) at doses of 60 mg of cabozantinib (e.g., cabozantinib (S)-malate) and 20 mg or 35 mg of Compound A. In some embodiments, the daily administration of Compound A and cabozantinib is oral. In some embodiments, the daily administration of Compound A and cabozantinib is performed in a 21-day cycle.

[0044] In another embodiment of the method of the present invention, Compound A and axitinib are administered at a dose of 5 mg axitinib twice daily (preferably 12 hours apart) and 20 mg or 35 mg Compound A once daily. In some embodiments, the daily and twice daily administration of Compound A and axitinib is oral administration. In some embodiments, the daily and twice daily administration of Compound A and axitinib is performed in a 21-day cycle, respectively. In embodiments of the present invention, the subject to which the compound is administered can be any mammal; in preferred embodiments, the subject is a human subject.

[0045] The present invention also relates to compound A and a VEGFR / VEGF inhibitor (or a physiologically acceptable salt thereof) for use in the method for preventing or treating cancer according to the present invention. In some embodiments of the present invention, the VEGFR / VEGF inhibitor for such use is selected from the group consisting of aflibercept, apatinib, axitinib, bevacizumab, brivanib alaninate, cabozantinib, cediranib, lenvatinib, linifanib, pazopanib, ponatinib, ramucirumab, regorafenib, sorafenib, sunitinib, vandetanib, and 33C3. In some embodiments of the present invention, the VEGFR / VEGF inhibitor for such use is cabozantinib or axitinib. In some specific embodiments, the VEGFR / VEGF inhibitor for such use is cabozantinib, for example, cabozantinib (S)-malate. In other particular embodiments, the VEGFR / VEGF inhibitor for such uses is axitinib. The present invention also relates to a compound mixture or a pharmaceutical composition according to the present invention for use in a method for preventing or treating cancer according to the present invention.

[0046] Furthermore, the present invention relates to the use of said compound mixture or pharmaceutical composition for preparing a medicament for the prevention or treatment of cancer. The phrase "effective amount" refers to an amount of a drug or active pharmaceutical ingredient that elicits the biological or medical response sought or desired, for example, by a researcher or physician, in a tissue, system, animal or human. Furthermore, the expression "therapeutically effective amount" means an amount that produces the following results compared to a corresponding subject who does not receive this amount: improved treatment, cure, prevention, or elimination of a disease, syndrome, condition, complaint, or disorder, or prevention of side effects, or attenuation of the progression of a disease, condition, or disorder. The term "therapeutically effective amount" also encompasses an amount effective to enhance normal physiological function.

[0047] The therapeutically effective amount of a compound or compound mixture of the present invention will depend 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 is ultimately determined by the treating physician or veterinarian. For example, an effective amount of API for treating a disease according to the present invention may be in the range of 0.1 to 100 mg / kg of recipient (mammal) body weight per day, e.g., 1 to 10 mg / kg of body weight per day. Thus, for an adult mammal weighing 70 kg, the actual amount per day may be in the range of 7 to 7000 mg, e.g., 70 to 700 mg, where this amount can be administered as an individual dose per day or in a series of partial doses, usually 2, 3, 4, 5, or 6 doses per day, so that the total daily dose is the same. The effective amount of these salts or physiologically functional derivatives thereof can be determined as a percentage of the effective amount of the compound or compound mixture according to the present invention itself. [Example]

[0048] The following Examples A1 to A8 relate to pharmaceutical compositions: Example A1: Injection vial A solution of 100g of the compound or compound mixture according to the present invention and 5g of disodium hydrogen phosphate in 3L of double distilled water is adjusted to pH 6.5 using 2N hydrochloric acid, sterile filtered, transferred into injection vials, lyophilized and sealed under sterile conditions.Each injection vial contains 5mg of active ingredient.

[0049] Example A2: Suppositories 20 g of a compound or compound mixture according to the invention is melted with 100 g of soy lecithin and 1400 g of cocoa butter, poured into molds and allowed to cool. Each suppository contains 20 mg of active ingredient.

[0050] Example A3: Solution A solution is prepared by dissolving 1 g of a compound or compound mixture according to the present invention, 9.38 g of NaH2PO4 x 2H2O, 28.48 g of NaH2PO4 x 12H2O, and 0.1 g of benzalkonium chloride in 940 ml of double distilled water. The pH is adjusted to 6.8, the solution is made up to 1 L, and the solution is sterilized by irradiation. This solution can be used in the form of eye drops.

[0051] Example A4: Ointment 500 mg of a compound or mixture of compounds according to the invention is mixed with 99.5 g of petrolatum under sterile conditions.

[0052] Example A5: Tablets 1 kg of a compound or compound mixture according to the invention (e.g., Compound A), 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate are compressed by conventional means to give tablets, each containing 10 mg of the active ingredient.

[0053] Example A6: Coated tablets Tablets are compressed as in Example A5 and subsequently coated in the conventional manner with a coating of sucrose, potato starch, talc, tragacanth and dye.

[0054] Example A7: Capsule 2 kg of a compound or compound mixture according to the present invention (e.g., Compound A) is conventionally introduced into hard gelatin capsules so that each capsule contains 20 mg of active ingredient(s). Alternatively, for example, Compound A is conventionally introduced into hard gelatin capsules so that each capsule contains 1 mg, 5 mg, or 30 mg of active ingredient.

[0055] Example A8: Ampoule A solution of 1 kg of a compound or compound mixture according to the invention in 60 liters of double-distilled water is transferred into ampoules, lyophilized under aseptic conditions and sealed under sterile conditions, each ampoule containing 10 mg of active ingredient. The following Examples B1 and B2 relate to combination studies using Compound A and VEGFR / VEGF inhibitors. Compound A has been found by the inventors of this patent application to achieve significant combination benefits when combined with cabozantinib or axitinib in the treatment of cancer, as reported in more detail below:

[0056] Example B1: In vivo efficacy of Compound A in combination with axitinib or cabozantinib in RCC patient-derived tumor models (PDX) 1. Overview This example characterizes the antitumor activity of Compound A in combination with axitinib or cabozantinib (SoC of RCC) in 16 patient-derived tumor xenograft models (PDXs) of RCC. While tumors treated with Compound A, axitinib, and cabozantinib progressed under monotherapy, the combination of both agents (i.e., Compound A with axitinib or Compound A with cabozantinib) demonstrated a significant combination benefit, increasing progression-free survival and overall survival compared to monotherapy. Treatment was well tolerated.

[0057] 2. List of Abbreviations aai Aqua ad iniectabilia, water for injection AC ventilation BID twice a week BW Body Weight BWL weight loss CMC Carboxymethylcellulose D5W 5% dextrose in water EDTA Ethylenediaminetetraacetic acid ELN Electronic Labor Notebook H:0 First dose of the day = h:0 H:0+12 Take twice a day at 12-hour intervals IT induction time IVC Individually Ventilated Cage Kg kilogram MetAP2 methionine aminopeptidase 2 Mg milligram ml milliliter MTD maximum capacity n / a Not applicable NMRI Naval Medical Research Institute Athymic immunodeficiency mutant in NMRI Foxn1nu NMRI background nr not reached PBS Phosphate-buffered saline PDX patient-derived xenografts po (oral, oral) QD*n Once a day for n days RXA Kidney Xenografts in Asian Ethnics RXF Kidney Xenotransplantation Freiburg RTV relative tumor volume ROP Research Operating Procedures SOC standard of care SOP Standard Operating Procedure T / C test vs. control values TV tumor volume vs.

[0058] 3. Materials and Methods 3.1 Materials 3.1.1 Test System 3.1.2 Test and Positive Control Substances 3.1.2.1 Animals All experiments and protocols were approved by animal welfare organizations and local authorities and were conducted in accordance with all applicable international, national, and local laws and guidelines. Only animals in good health were selected for study procedures. Animals (female NMRInu / nu mice (NMRI-Foxn1nu); Envigo RMS GmbH, Netherlands) were delivered at 4–6 weeks of age and used for experiments after at least 1 week of acclimatization. Animals were randomly numbered using radio frequency identification transponders during tumor implantation. Each cage was equipped with a record card listing all relevant experimental details.

[0059] 3.1.2.2 Accommodation conditions Animals were housed in individually ventilated cages (TECNIPLAST Sealsafe™-IVC-System, TECNIPLAST, Hohenpeissenberg, Germany) in either type III or type II long cages, depending on group size. They were kept under an artificial light cycle of 14 L:10 D. The temperature inside the cage was maintained at 25 ± 1 °C, the relative humidity was 40–70%, and the cage was air-conditioned 60–65 times per hour. Dust-free litter consisting of poplar wood chips (ABEDD®- LAB & VET Service GmbH, Vienna, Austria, product code: LTE E-001) with approximate dimensions of 5 × 5 × 1 mm and additional nesting material was used. The litter and cages containing the nesting material were changed weekly. Animals were fed autoclaved Teklad Global 19% Protein Extruded Diet (T.2019S.12) from Envigo RMS SARL and had access to sterile-filtered, acidified (pH 2.5) tap water, which was changed twice weekly. Food and water were provided ad libitum. All materials were autoclaved before use. If needed, animals were provided with a nutrient-enriched water gel (DietGel® Recovery from ClearH2O, Maine, USA), which was changed every other day.

[0060] 3.1.2.3 Test tumor Tumor xenografts were obtained from surgical specimens of human cancer patients. After surgical resection, tumor fragments were subcutaneously transplanted into immunodeficient mice; therefore, they are called patient-derived tumor xenografts (PDXs). Establishment and characterization of PDXs were performed following primary transplantation (passage 1) into immunodeficient mice. Tumor xenografts were passaged until a stable growth pattern was established. At that point, master stocks of early-passage PDXs were frozen in liquid nitrogen. Detailed information about each PDX (e.g., histology, growth characteristics, etc.) is listed in Table 1.

[0061] Table 1. Characteristics of 16 RCC patient-derived xenografts [Table 1]

[0062] Table 2. Experimental overview [Table 2]

[0063] 3.1.3 Test materials [Table 3]

[0064] Vehicle for axitinib: 0.5% carboxymethylcellulose (CMC; also used as control vehicle 1). Vehicle for cabozantinib: 30% propylene glycol, 5% Tween 80, 65% D5W (D5W means 5% dextrose in water). Vehicle for Compound A: 0.25% Methocel (Colorcon) in sterile water for injection (wfi) (sterile wfi; also used as control vehicle 2).

[0065] Axitinib: A 0.4 mg / ml dosing solution for 4 mg / kg / dose was prepared daily by dissolving 6.4 mg of dry matter in 16 ml of vehicle and stirring for at least 30 minutes. If necessary, the solution was sonicated for 2 minutes. Cabozantinib: A 1 mg / ml dosing solution for administration at 10 mg / kg / dose was prepared immediately prior to administration by slowly adding 0.2 ml of 100% propylene glycol to 12 mg of cabozantinib. This was vigorously stirred on a magnetic stirrer until a fine suspension containing clumps was formed, followed by brief vortexing for 15–20 seconds. An additional 3.4 ml of propylene glycol was added, and the stirring and vortexing process was repeated. Next, 600 μl of 100% Tween 80 was added with continuous stirring, followed by brief vortexing for 15–20 seconds. The solution was then sonicated for 10 minutes, and 7.8 ml of D5W was added with slow stirring. At this stage, the solution was clear with many air bubbles. Care was taken to remove these when the suspension was dispensed into a syringe for treatment.

[0066] Compound A: A 10 mg / ml dosing solution for administration at 100 mg / kg / dose was prepared every 3 days by dissolving 180 mg of dry matter in 18 ml of vehicle. The resulting white suspension was stirred at 50°C for 2 hours using a magnetic stirrer equipped with a hot plate (Phoenix Instruments, model RSM-10HS). The suspension was allowed to come to room temperature and mixed thoroughly before being transferred to a syringe for administration. All treatment solutions were administered at a volume of 10 ml / kg.

[0067] 3.1.4 Solution All reagents and buffers were stored according to the manufacturer's instructions and used before the batch expiration date. Compound A was developed at Merck KGaA, Darmstadt, Germany. [Table 4] The vehicle for Compound A, 0.25% Methocel® in aai, was made by dissolving 875 mg of Methocel® in 200 ml of water for injection and stirring at 1000 rpm and 65.5° C. for 6 hours until dissolved. 150 ml of water was then added and the solution was stirred for an additional 5 minutes before being sterile filtered.

[0068] 3.1.5 Equipment [Table 5]

[0069] 3.2 Study Doses 3.2.1 Test substance concentration [Table 6]

[0070] 3.2.2 Negative Control [Table 7]

[0071] 3.3 Test Procedure Tumor cell inoculation: Tumor fragments were obtained from xenografts in serial passages in nude mice. After excision from donor mice, tumors were cut into fragments (edge ​​length 3-4 mm) and placed in PBS containing 10% penicillin / streptomycin. Recipient animals were anesthetized by inhalation of isoflurane and received unilateral or bilateral tumor implants subcutaneously in the flank. For tumor xenografts with a take rate of less than 65%, one or two tumors per mouse were implanted. In the case of bilateral take rates, one of these tumors was explanted prior to randomization.

[0072] Randomization: Animals and tumor implants were monitored daily until solid tumor growth was detected in a sufficient number of animals. At randomization, the volume of growing tumors was determined. Animals that met the randomization criteria (i.e., 50–250 mm) were randomized. 3 , preferably 80 to 200 mm3 (with tumors of 1000 or more) were distributed into experimental groups to aim for comparable median and mean group tumor volumes. Animals not used in the experiment were used as satellite animals for PD sampling, if available. The day of randomization was designated as day 0 of the experiment. The time from implantation at standard tumor volume to randomization is expressed in days as the "induction time (IT)." Tumor induction times were recorded periodically, and the median IT was calculated for characterization.

[0073] Measurements and calculations: Tumor length (L) and width (W) were measured twice weekly with calipers. Tumor volume was calculated using the formula: L x W 2 The relative volume of an individual tumor on day x (individual RTV) was calculated using the absolute volume of an individual tumor on day x (T x ) was calculated as the individual absolute tumor volume (T r ) and multiplied by 100%.

[0074] Body weight: Animals were weighed twice a week, or daily if a weight loss of more than 10% was recorded. The relative weight of an individual animal was calculated as the individual body weight on day X (BW x ) on the day of randomization r ) and multiplied by 100%. Group mean relative body weights were also calculated for evaluation purposes.

[0075] Euthanasia Criteria: In accordance with animal welfare regulations and relevant SOPs, the following euthanasia criteria will be applied to each individual animal regardless of experimental status. Tumor volume >2000mm 3 (unilateral) Ulcerative, skin-penetrating tumor Weight loss of more than 30% at one time Weight loss of more than 20% lasting for more than 2 days Documented rapid weight loss of more than 20% within two days Severe impairment of general condition (sluggishness, pain, markedly reduced food and water intake, difficulty breathing, abnormal habits or behavior)

[0076] If an individual animal met the euthanasia criteria, sampling was performed prior to the scheduled time and, if possible, at the correct time interval after administration of the last applied dose. Sampling: Samples were collected under sterile conditions in accordance with all relevant animal welfare guidelines. Samples were not taken from deceased animals. Tumors were harvested immediately after euthanasia and divided into two pieces, each approximately 100 mg in weight. The weight of this fragment was recorded and then placed in a tube (Precellys) and transferred to liquid nitrogen for quick freezing. The remaining tumor pieces were fixed in formalin. Fixation was performed in 10% neutral phosphate-buffered formalin for approximately 24 hours. The fixative was then exchanged by immersing the sample in PBS, and the sample was directly infiltrated with paraffin for embedding. FFPE samples were preferred when tumor material was limited. Blood was collected by cardiac puncture under isoflurane anesthesia (final collection of mixed venous and arterial blood). Serum was prepared by incubating blood in standard serum vials at room temperature for 30 minutes, followed by centrifugation at 10,000 × g for 5 minutes. Serum samples were stored at -80°C.

[0077] 3.4 Endpoints Preclinical response criteria were defined according to Therasse et al., J Nat Cancer Inst 2000; 92(3):205-16. Tumor progression: Tumor progression was defined as a treatment group or individual mouse reaching a 73% change in tumor volume at the end of the experiment or treatment compared to the tumor volume at the start of treatment (0% change in tumor volume at the start). (In other words, if the relative tumor volume (RTV) at the start was set at 100%, tumor progression was defined as a treatment group reaching a median tumor volume of 173%). Tumor stasis: -66% to 73% change in tumor volume by the end of treatment Tumor regression: tumor volume change ≦-66% by the end of treatment

[0078] Progression-free survival: Treatment duration extended to 70 days in all experiments. Progression-free survival is defined as when tumors reach 173% relative tumor volume (RTV) compared to the RTV at the start of treatment (=100%). Overall survival: treatment duration extended to 70 days in all experiments. Overall survival is defined as the tumor reaching 1000% relative tumor volume (RTV) compared to the RTV at the start of treatment (=100%). MTD in Repeat Dose Studies: The maximum tolerated dose (MTD) is the dose or exposure that results in no deaths, less than 20% weight loss, and no irreversible clinical or pathological findings. A dose resulting in a 20% weight change (group mean) or 10% or more drug-related deaths was considered a toxic dose. Animal weights included tumor weight.

[0079] Table 3. Groups and treatments [Table 8]

[0080] 3.5 Computer programs used and statistical evaluation [Table 9]

[0081] 4. Results The antitumor activity of Compound A in combination with the SoC drugs axitinib or cabozantinib was evaluated in 16 selected subcutaneous patient-derived xenograft models of RCC. Tumor fragments from patient-derived tumor types were grown in donor mice, harvested, and further implanted into mice. In each efficacy study, tumor-bearing animals were assigned to experimental groups (n=5) with the same mean tumor size at the start of treatment, designated day 0. Compound A was administered orally QD at a dose of 100 mg / kg for a defined maximum period of 70 days. Axitinib and cabozantinib were administered at doses of 4 mg / kg (BID) and 10 mg / kg (QD), respectively. The same dose and schedule were applied for combination treatments. Tumor and animal weights were measured twice weekly; tumor volumes (mm 3 ) is expressed as the formula [length x width 2 [The mean age of the patients was 18 years or older.] Tolerability was assessed based on the percentage of weight loss during the treatment period. Progression-free survival (PFS) and overall survival (OS) were assessed based on defined endpoints (PFS = relative tumor volume (RTV) 173%, OS = RTV > 1000%) over the entire 70-day treatment period.

[0082] Overall, monotherapy with Compound A, axitinib, and cabozantinib did not strongly inhibit tumor growth, and tumors progressed during treatment. Combining Compound A with axitinib or cabozantinib demonstrated significant combination efficacy, resulting in increased progression-free survival (Figures 1 and 4) and overall survival (Figures 2 and 5). All treatments were well tolerated (see body weights; see Figures 3 and 6 for representative examples of PDX models shown by treatment group). RECIST-like preclinical responses were assessed according to the definitions in Therasse et al. (2000).

[0083] Example B2: An open-label, phase I, dose-escalation study of the methionine aminopeptidase 2 inhibitor Compound A in subjects with advanced solid tumors title : An open-label, phase I, dose-escalation study of the methionine aminopeptidase 2 inhibitor Compound A in subjects with advanced solid tumors Trial Phase :I / Ib Methodology: This is a two-part, Phase I / Ib, first-in-human, multicenter, open-label study of Compound A administered daily. Part 1 was a dose-escalation study designed to investigate the safety, tolerability, PK and Pd profile, and clinical activity of Compound A as a single agent over a 21-day treatment cycle in subjects with advanced solid tumors who had not received surgery, radiation therapy, or available systemic anti-cancer therapy. Part 2 is designed to determine the safety, tolerability, and antitumor activity of Compound A in combination with cabozantinib in subjects with mRCC. Part 2A includes a dose-escalation (Part 2A) and expansion cohort (Part 2B) design. In Part 2A, escalating doses of Compound A will be combined with cabozantinib in subjects with mRCC who have progressed on one or more prior lines of systemic anti-cancer therapy other than treatment with cabozantinib. Part 2A of the study allows for escalation of subjects. Thus, enrollment into Part 2A may occur while Part 1 is ongoing; however, enrollment will occur only at dose levels of Compound A that are determined to be safe in Part 1 of the study. An exploratory food effect assessment will be conducted on Day 1 of Cycle 1, in which subjects will receive a single dose of Compound A as a single agent, with food. In expansion Part 2B, compound A at the expanded recommended dose (RDE) / maximum tolerated dose (MTD) defined in Part 2A will be combined with cabozantinib in subjects with mRCC who have progressed on one or two prior lines of systemic anticancer therapy other than treatment with cabozantinib. In Part 2B, subjects must have failed only one prior antiangiogenic tyrosine kinase inhibitor (TKI) for metastatic disease. Adjuvant treatment with sunitinib will be considered as one line of treatment for metastatic disease if disease progresses during treatment or within 3 months of completing treatment.

[0084] Target number of people: Part 1: 36-42 subjects. Part 2A: 6-15 subjects. Part 2B: 30-40 subjects. Part 1, Compound A monotherapy, solid tumors Primary endpoint: Dose-limiting toxicities (DLTs) during the first 21-day treatment cycle to determine the MTD based on a predefined set of adverse events (AEs). Secondary endpoint: Incidence and severity of treatment-emergent adverse events (TEAEs) and mortality, including cause of death, from screening to the end-of-treatment visit (EOT) Changes in clinical laboratory measurements, electrocardiogram (ECG) measurements, vital signs, and Eastern Cooperative Oncology Group performance status (ECOG PS) Investigator-assessed best overall response (BOR: complete response [CR], partial response [PR], stable disease [SD], or progressive disease [PD]) according to RECIST v1.1 criteria. Clinical benefit, defined as complete response, partial response, or sustained sustained disease for ≥12 weeks -PFS (progression-free survival) period. Part 2, Compound A in combination with cabozantinib, mRCC Part 2A, dose escalation Primary endpoint: The occurrence of DLTs in subjects with mRCC receiving Compound A in combination with cabozantinib during the first 22-day treatment cycle (21 days of Compound A and cabozantinib combination, followed by 1 day of Compound A alone) Occurrence of TEAEs (including TEAEs leading to death or treatment discontinuation) in subjects with mRCC receiving Compound A in combination with cabozantinib. AEs occurring or worsening between the start of the study intervention and 30 days after the end of the study intervention are considered TEAEs. -Look for clinically relevant abnormal changes in clinical laboratory measurements from baseline, vital signs, ECOG performance status, and ECG from the start of the study intervention until 30 days after the end of the study intervention Investigator-assessed objective response by RECIST v1.1 Secondary endpoint: Investigator-assessed duration of response (DoR) according to RECIST v1.1 criteria Investigator-assessed PFS by RECIST v1.1 Part 2B, expansion cohort Primary endpoint: -Investigate the occurrence of serious AEs, including death, from the first administration of the study intervention to 30 days after the end of the study intervention. -Investigate clinically relevant abnormal changes in clinical laboratory measurements from baseline, vital signs, ECOG PS, and ECG from the start of the study intervention until 30 days after the end of the study intervention - Occurrence of TEAEs (including death) from the start of study intervention to 30 days after the end of study intervention Investigator-assessed objective response by RECIST v1.1 criteria Secondary endpoint: Investigator-assessed DoR according to RECIST v1.1 criteria Investigator-assessed PFS by RECIST v1.1

[0085] Diagnosis and key inclusion and exclusion criteria: Part 1, Compound A monotherapy, solid tumors Key inclusions: Subjects must be resistant to or intolerant of existing cancer treatment(s) known to provide clinical benefit Histologically confirmed advanced solid tumors with no clear curative treatment options available after at least one prior systemic anticancer therapy The tumor is accessible for biopsy and consent is given to perform fresh tumor biopsies pre- and post-treatment. · For men or women aged 18 and over. Major Exclusions: ECOG PS ≥ 2 Extensive radiation therapy to 30% or more of bone marrow reserves or bone marrow / stem cell transplant within 5 years of study initiation Severe bone marrow, kidney, or liver damage Part 2, Compound A in combination with cabozantinib, mRCC Key inclusions: Histologic or cytologic evidence / proof of mRCC with clear cell component Previous treatment: a. Part 2A Subjects must have progressed on one or more prior lines of systemic anti-cancer therapy other than treatment with cabozantinib b. Part 2B : Subjects must have progressed on one or two prior lines of systemic anti-cancer therapy other than treatment with cabozantinib. Subjects must have failed no more than one prior TKI for metastatic disease. Adjuvant treatment with sunitinib is considered a line of treatment for metastatic disease if disease progresses during treatment or within 3 months of completing treatment. At least one lesion measurable using RECIST v1.1. Adequate organ function (i.e., liver, kidney, bone marrow) as evidenced by multiple laboratory test results within specific parameters Major Exclusions: Previous use of cabozantinib or a MetAP2 inhibitor Thromboembolic events requiring anticoagulation therapy. Concomitant anticoagulation therapy at therapeutic doses with oral anticoagulants (i.e., warfarin, direct thrombin and factor Xa inhibitors) or platelet inhibitors (i.e., clopidogrel) initiated within the last 6 months of screening. NOTE: Low-dose aspirin and low-dose low-molecular-weight heparin (LMWH) for cardioprotection are acceptable (subjects must have received a stable dose of LMWH for at least 6 weeks prior to the first dose of study treatment and have had no clinically significant bleeding complications due to anticoagulation regimens or tumors). Current significant cardiac conduction disorders, including a prolongation of the QT interval corrected for heart rate according to Fridericia (QTcF) of greater than 450 milliseconds in men and greater than 470 milliseconds in women, or cardiovascular disorders, including symptomatic congestive heart failure, unstable angina, and severe cardiac arrhythmias.

[0086] Investigational drug: Dose / Administration method / Administration schedule: Part 1, Compound A monotherapy, solid tumors Compound A in capsule form will be administered orally QD according to the dose escalation schedule and as determined by the Safety Monitoring Committee (SMC). The starting dose for the first dose escalation cohort will be 7 mg QD. A Bayesian two-parameter logistic regression model with overdose control will be used to assist the SMC in dose selection during the dose escalation portion. The model incorporates nonclinical toxicity and DLT information observed from all previously completed dose escalation cohorts to provide dose recommendations for the next cohort. Part 2, Compound A in combination with cabozantinib, mRCC Part 2A, dose escalation Compound A in capsule form in combination with cabozantinib will be administered orally QD in the morning under fasted conditions. Three pre-specified dose levels of Compound A (20 mg, 35 mg, and 60 mg) are planned in combination with 60 mg of cabozantinib. A Bayesian 2-parameter logistic regression model with overdose control will be used to assist the SMC in dose selection for the next cohort and determine the MTD / RDE of Compound A in combination with cabozantinib 60 mg QD in Part 2B. Dose levels may be added or skipped if not pre-specified, but will not exceed the Compound A dose levels already declared safe in Part 1. Cabozantinib will be used at the approved daily dose of 60 mg free base equivalent (FBE), with dose adjustment to 40 mg FBE or 20 mg FBE as permitted for management of AEs. Part 2B, dose expansion In Part 2B, subjects will receive Compound A at the dose level specified in Part 2A and cabozantinib at 60 mg QD in 21-day cycles until disease progression, unacceptable toxicity, withdrawal of consent, or any criteria for withdrawal from the study. In Part 2B, the SMC will continue to monitor the safety of the combination of Compound A and cabozantinib and may recommend continuation of the same dose, a dose modification (not exceeding the MTD defined in Part 2A), or discontinuation of the expansion cohort. Unacceptable toxicity in the expansion (Part 2B) cohort is an AE that meets the DLT criteria. The SMC will evaluate the safety and cumulative toxicity of the combination treatment after 10 subjects who complete the DLT period or drop out and after 20 subjects. The SMC decision in the expansion cohort will also be supported by a two-parameter Bayesian logistic regression model. Subject enrollment will not be interrupted during the SMC.

[0087] Planned study and treatment duration per subject: The study duration per subject, including screening, treatment, and follow-up, is approximately 8 months in Part 1 and 12 months in Part 2. Treatment will be administered in consecutive 21-day treatment cycles. Subjects will receive the investigational drug at a pre-specified dose level until disease progression, unacceptable toxicity, withdrawal of consent, or any criterion for withdrawal from the study. Statistical methods: Part 1, Compound A monotherapy, solid tumors Analyses will be arranged by dose level. There are no formal significance levels in this study and all analyses will be considered descriptive. Dose escalation is supported by a Bayesian two-parameter logistic regression model. The SMC dedicated to dose escalation decisions receives the results of the Bayesian two-parameter logistic model along with overdose control updated with observed DLT data. Recommendations are based on a loss function. Part 2, Compound A in combination with cabozantinib, mRCC Part 2A, dose escalation A Bayesian two-parameter logistic regression model with overdose control will be used to assist the SMC in selecting the next dose of Compound A from three pre-specified dose levels (20 mg, 35 mg, and 60 mg QD). The SMC may add or skip doses as long as they do not exceed the dose level of Compound A declared safe in Part 1. An exploratory food effect assessment will compare (using a modeling approach) PK data generated for a single dose of Compound A as a single agent with food to data obtained from Part 1 of the study, in which subjects received the same dose level of Compound A alone in the fasted state. Part 2B, expansion cohort Response will be assessed using a two-stage design with an interim analysis for futility.

Claims

1. A compound mixture comprising the following compounds a) and b): a) (S)-3-hydroxy-1-(1H-indol-5-yl)-2-oxo-pyrrolidine-3-carboxylic acid 3,5-difluoro-benzylamide or a physiologically acceptable salt thereof; b) An inhibitor of one or more of VEGFR1, VEGFR2, VEGFR3 and VEGF, wherein the inhibitor is cabozantinib, or a physiologically acceptable salt thereof.

2. 2. The compound mixture of claim 1, wherein compound b) is cabozantinib (S)-malate.

3. 10. A pharmaceutical composition comprising a compound mixture according to claim 1 or 2, optionally further comprising one or more excipients and / or adjuvants.

4. A kit comprising separate packs of the following compounds a) and b): a) (S)-3-hydroxy-1-(1H-indol-5-yl)-2-oxo-pyrrolidine-3-carboxylic acid 3,5-difluoro-benzylamide or a physiologically acceptable salt thereof; b) An inhibitor of one or more of VEGFR1, VEGFR2, VEGFR3 and VEGF, wherein the inhibitor is cabozantinib, or a physiologically acceptable salt thereof.

5. The kit of claim 4, wherein compound b) is cabozantinib (S)-malate.

6. 1. A pharmaceutical composition for use in a method for the prevention or treatment of cancer, wherein said pharmaceutical composition comprises compound a), wherein the method comprises administering compounds a) and b) to a subject, and wherein compounds a) and b) are: a) (S)-3-hydroxy-1-(1H-indol-5-yl)-2-oxo-pyrrolidine-3-carboxylic acid 3,5-difluoro-benzylamide or a physiologically acceptable salt thereof; b) an inhibitor of one or more of VEGFR1, VEGFR2, VEGFR3 and VEGF, wherein the inhibitor is cabozantinib, or a physiologically acceptable salt thereof; The pharmaceutical composition,

7. 7. The pharmaceutical composition for use according to claim 6, wherein compound b) is cabozantinib (S)-malate.

8. 8. The pharmaceutical composition for use according to claim 6 or 7, wherein the cancer is a cancer for which inhibitors are the standard therapeutic treatment option and / or a cancer sensitive to anti-angiogenic treatment.

9. 9. The pharmaceutical composition for use according to any one of claims 6 to 8, wherein the cancer is selected from the group consisting of renal cell carcinoma, colorectal cancer, lung cancer, head and neck cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, glioblastoma, hepatocellular carcinoma, breast cancer, thyroid cancer, soft tissue sarcoma, chronic myeloid leukemia, and Philadelphia chromosome-positive acute lymphoblastic leukemia.

10. The pharmaceutical composition for use according to any one of claims 6 to 9, wherein the cancer is renal cell carcinoma.

11. The pharmaceutical composition for use according to claim 10, wherein the renal cell carcinoma is renal clear cell carcinoma.

12. The pharmaceutical composition for use according to any one of claims 6 to 11, wherein compounds a) and b) are administered simultaneously.

13. The pharmaceutical composition for use according to any one of claims 6 to 11, wherein compounds a) and b) are administered sequentially.

14. 1. Use of compounds a) and b) for the manufacture of a medicament for the prevention or treatment of cancer, wherein compounds a) and b) are: a) (S)-3-hydroxy-1-(1H-indol-5-yl)-2-oxo-pyrrolidine-3-carboxylic acid 3,5-difluoro-benzylamide or a physiologically acceptable salt thereof; b) an inhibitor of one or more of VEGFR1, VEGFR2, VEGFR3 and VEGF, wherein the inhibitor is cabozantinib, or a physiologically acceptable salt thereof; The use of compounds a) and b) above, wherein

15. 10. Use of a compound mixture according to claim 1 or 2 or a pharmaceutical composition according to claim 3 for the manufacture of a medicament for the prevention or treatment of cancer.

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