Therapeutic combination comprising 4-(4-methyl-piperazin-1-YL)-n-{6-[2-(4-trifluoromethyl-benzyloxy)-ethoxy]-1h-indazol-3-YL}-benzamide and an antineoplastic agent

Abstract

The present invention provides a therapeutic combination comprising (a) a compound of formula (I) or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof and (b) one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL2 family inhibitors, immune checkpoint inhibitors and anthracyclines. The combinations are described for use in the treatment of cancer.

Description

[0001]NMS 123 THERAPEUTIC COMBINATION COMPRISING 4-(4-METHYL-PIPERAZIN-1-YL)-N-{6-[2-(4- TRIFLUOROMETHYL-BENZYLOXY)-ETHOXY]-1H-INDAZOL-3-YL}-BENZAMIDE AND AN ANTINEOPLASTIC AGENT Field of the Invention The present invention relates in general to the field of cancer treatment and, more particularly, provides an anti-tumor combination comprising 4-(4-methyl-piperazin-1-yl)-N-{6-[2-(4-trifluoromethyl-benzyloxy)-ethoxy]-1H-indazol-3-yl}- benzamide or a pharmaceutically acceptable salt thereof and a demethylating agent or a BCL2 family inhibitor or an antimetabolite agent or an immune checkpoint inhibitor or an anthracycline. Background of the invention The compound 4-(4-methyl-piperazin-1-yl)-N-{6-[2-(4-trifluoromethyl-benzyloxy)-ethoxy]-1H-indazol-3-yl}- benzamide of formula (I) N (I) is described in Valuable pharmacological properties are attributed to this compound; it can be used as a protein kinase inhibitor useful in the therapy of diseases which respond to inhibition of protein kinase activity. In particular, this compound can be used to treat diseases associated with FMS-like tyrosine kinase 3 (FLT3) and / or c-KIT and / or colony stimulating factor 1 receptor (CSF-1R) mutated activity or overexpressed protein kinases. FLT3, c-KIT and CSF-1R are all members of the PDGFR family class III receptor tyrosine kinases. FLT3 mutations can be detected in 30% of acute myeloid leukemia (AML) patients (Nakao M, et al. Leukemia.1996 Dec; 10(12): 1911-8) and also in 5-10% of patients with myelodisplastic syndrome (Horiike S, et al. Leukemia.1997 Sep; 11(9): 1442-6). There are two frequent types of somatic FLT3 genetic mutations: internal tandem duplications (ITDs) in the juxtamembrane domain and point mutations in the activation loop of the tyrosine kinase domain (TKD). Both types of FLT3 mutation cause ligand-independent activation of the receptor and activation of downstream signaling pathways. Mutant FLT3 provides survival advantage to leukemic cells because it causes activation of three major intracellular signalling pathways: PI3K / AKT; RAS / RAF / MAPK and JAK / STAT (Masson K, Rönnstrand L. Cell Signal.2009 Dec;21(12):1717-26). Also c-KIT overexpression or mutations can lead to cancer. Mutation of c-KIT have been identified in GIST (Antonescu CR. J Pathol.2011; 223(2): 251-6, melanoma (Curtin JA, JCO, 2006, 24 (26): 4340-4346), and primary adenoid cystic carcinoma of the salivary gland (Vila L, Liu H, Al-Quran SZ, Coco DP, Dong HJ, Liu C, Mod Pathol. 2009; 22(10): 1296-302), and also in acute myeloid leukemia (Malaise M, Steinbach D, Corbacioglu S, Curr Hematol Malig Rep.2009, 4(2): 77-82). Overexpression is reported also in thymic carcinoma (Ströbel P, Hohenberger P, Marx A, J Thorac Oncol.2010; 5 (10 Suppl 4): S286-90), glioma (Morris PG, Abrey LE.Target Oncol.2010; 5(3):193-200), testicular seminoma (Nikolaou M. et al. Anticancer Res.2007; 27(3B): 1685-8), and small cell lung cancers (SCLC) (Micke P, et al. Clin Cancer Res.2003; 9(1): 188-94). CSF-1R (M-CSF receptor, c-FMS kinase or CD115) and its ligands, CSF-1 and interleukin 34 (IL-34), regulate the function and survival of tumor-associated macrophages, which are involved in tumorigenesis and in the suppression of antitumor immunity (Cannarile MA, Weisser M, Jacob W, Jegg AM, Ries CH, Ruttinger D, J Immunother o Cancer 2017, 5:53). High CSF-1 or CSF-1R expression levels in the tumor or peritumoural tissue have been associated with poor patient survival in different malignancies, such as Hodgkin’s lymphoma, breast cancer, and hepatocellular carcinoma [Mantovani A, et al. Nature Reviews Clin Oncol 2017; 14, 399–416.]. Moreover, the CSF-1R / CSF-1 axis has been implicated in the pathogenesis of pigmented villonodular synovitis (PVNS), a benign tumor of the synovium (Brahmi M, Vinceneux A, Cassier PA. Curr Treat Options Oncol.2016 Feb;17(2):10). CSF-1R has been recently reported also as a novel therapeutic target of AML acting through a mechanism of paracrine cytokine / growth factor signaling in this disease (Edwards DK, et al. Blood.2019 Feb 7;133(6):588-599). CSF-1R has also been found to be highly expressed in blast samples from chronic myelomonocytic leukemia (CMML), a clonal hematopoietic stem cell disorder with poor survival rates post- blast transformation, with no standard targeted therapy. Based on the collection of data, CSF-1R kinase activity appears to be relevant in supporting the growth of many malignancies, both hematological and solid cancer diseases, thereby suggesting that it could represent a good therapeutic target for the treatment of these pathologies. There is a continuous need of anticancer drugs in order to optimise the therapeutic treatment. The present invention provides new combinations of a FLT3 / c-KIT / CSF1R inhibitor with known pharmaceutical agents that are particularly suitable for the treatment of proliferative disorders, especially leukemia. In particular, the combinations of the present invention display a therapeutic effect even when each combination partner is administered at a dose that would be non-effective if administered alone. The patient will thus benefit from a mitigation of the drawbacks associated with the antitumor drugs, i.e. reduced toxicity and milder side effects. Moreover, the combinations of the present invention provide for a clear synergistic effect. Summary of the invention The present invention provides, in a first aspect, a therapeutic combination comprising (a) a compound of formula (I): N (I) or a pharmaceutically form thereof and (b) one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL2 family inhibitors, immune checkpoint inhibitors and anthracyclines. The present invention also provides a combined preparation for simultaneous, separate or sequential use of the combination as described above. In a further aspect the invention provides a combination comprising a compound of formula (I) as defined above and one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL2 family inhibitors, immune checkpoint inhibitors and anthracyclines, for use in a method of treating cancer in a subject, wherein said compound of formula (I) and the antineoplastic agents can be administered simultaneously, sequentially or separately. In a still further aspect, the invention provides a pharmaceutical composition comprising a combination according to the invention admixed with a pharmaceutically acceptable carrier, diluent or excipient. A still further aspect relates to the use of a compound of formula (I) as defined above in the preparation of a medicament for the treatment of cancer, wherein said treatment comprises simultaneously, sequentially or separately administering to a subject in need thereof a compound of formula (I) as defined above and one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL-2 family inhibitors, immune checkpoint inhibitors and anthracyclines. A still further aspect of the invention relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined above, in combination with a therapeutically effective amount of one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL2 family inhibitors, immune checkpoint inhibitors and anthracyclines wherein the compound of formula (I) and the antineoplastic agents can be administered simultaneously, sequentially or separately to a subject in need thereof. The compound of formula (I) has the chemical name 4-(4-methyl-piperazin-1-yl)-N-{6-[2-(4-trifluoromethyl- benzyloxy)-ethoxy]-1H-indazol-3-yl}-benzamide. It can be prepared as described in WO2012 / 152763, is endowed with protein kinase inhibitory activity and is thus useful in therapy as antitumor agent. According to a preferred embodiment the pharmaceutically acceptable salts of compound of formula (I) are selected from the group consisting of hydrochloride, hydrobromide, benzene sulphonate (besylate), naphthalene-2-sulphonate (napsylate) p-toluene sulphonate (tosylate), L-malate, hydrogensulfate and hemioxalate or any hydrate, any crystal form thereof. According to a more preferred embodiment the salts are selected from the group consisting of hydrochloride, benzene sulphonate (besylate) and hemioxalate or any hydrate, any crystal form thereof. According to a preferred embodiment of the invention, the demethylating agent is selected from the group consisting of cytidine analogs such as 5-azacytidine (azacitidine) and 5-azadeoxycytidine (decitabine) According to a more preferred embodiment of the invention, the demethylating agent is 5-azacytidine.5-azacytidine can be administered, e.g., in the form as it is marketed, e.g. under the trademark Vidaza. According to a preferred embodiment of the invention the antimetabolite agent is selected from the group consisting of cytarabine, 5-fluorouracil, capecitabine, gemcitabine, pemetrexed and methotrexate. According to a more preferred embodiment of the invention, the antimetabolite agent is cytarabine. Cytarabine can be administered, e.g., in the form as it is marketed, e.g. under the trademark Cytosar. According to another more preferred embodiment of the invention, the antimetabolite agent is gemcitabine. Gemcitabine can be administered, e.g., in the form as it is marketed, e.g. under the trademark Gemzar. According to a preferred embodiment of the invention, the BCL-2 family inhibitor is venetoclax. Venetoclax can be administered, e.g., in the form as it is marketed, e.g. under the trademark Venclexta or Venclyxto. According to a preferred embodiment of the invention the immune checkpoint inhibitor is selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3 (T cell immunoglobulin and mucin-3), OX-40 and its ligand OX40L, LAG-3 (lymphocyte activation gene-3), KIR (Killer-cell Immunoglobulin like Receptor), VISTA (V-domain Ig-containing suppressor of T cell activation), ID01 (Indoleamine 2,3-dioxygenase), TIGIT (T cell immunoglobulin and ITIM domain), BTLA (B and T lymphocyte attenuator), A2AR (Adenosine receptor A2), SIGLEC7 (Sialic acid-binding immunoglobulin-type lectin 7), GITR (Glucocorticoid-Induced TNFR family Related gene), ICOS (Inducible T-cell costimulator), NOX-2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), Arginase I, CD276 (Cluster of Differentiation 276, also referred to as B7H4), CD27 (Cluster of Differentiation 27) and its ligand CD27 (Cluster of Differentiation 27), CD160 (Cluster of Differentiation 160) and CD39 (Cluster of Differentiation 39). According to a preferred embodiment of the invention the immune checkpoint inhibitor is selected from the group consisting of PD-1 inhibitor and PD-L1 inhibitor. According to a more preferred embodiment of the invention, the PD-1 inhibitor is an anti-PD-1 antibody. More preferably the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, tislelizumab, cemiplimab, dostarlimab and retifanlimab. According to another more preferred embodiment the PD-L1 inhibitor is an anti-PD-L1 antibody. More preferably the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab and durvalumab. According to a most preferred embodiment of the invention, the immune checkpoint inhibitor is nivolumab. Nivolumab can be administered, e.g., in the form as it is marketed, e.g. under the trademark Opdivo. According to a preferred embodiment the anthracycline is selected from the group consisting of daunorubicin, doxorubicin, epirubicin and idarubicin. According to a more preferred embodiment of the invention, anthracycline is doxorubicin. Doxorubicin can be administered, e.g., in the form as it is marketed, e.g. under the trademark Adriamycin. In the present invention, each of the active ingredient of the combination is in amount effective to produce a synergic antineoplastic effect. The present invention also provides a method for lowering the side effects caused by antineoplastic therapy with an antineoplastic agent in mammals, including humans, in need thereof, the method comprising administering to said mammal a combined preparation comprising the compound of formula (I) as defined above and one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL-2 family inhibitors, immune checkpoint inhibitors and anthracyclines, in amounts effective to produce a synergic antineoplastic effect. By the term “synergic antineoplastic effect” as used herein is meant the inhibition of the growth tumor, preferably the complete regression of the tumor, by administering an effective amount of the combination of the compound of formula (I) as defined above and a demethylating agent, an antimetabolite agent, a BCL-2 family inhibitor, an immune checkpoint inhibitor or an anthracycline to mammals, including human. The term “combined preparation” as used herein defines especially a “ kit of parts” in the sense that the combination partners (a) and (b) as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b), i.e. simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. Very preferably, the time intervals are chosen such that the therapeutic effect of the combination of the invention on the treated disease is greater than the effect which would be obtained by use of only any one of the combination partners (a) and (b). The ratio of the total amounts of the combination partner (a) to the combination partner (b) to be administered in the combined preparation can be varied, e.g. in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to the particular disease, age, sex, body weight, etc. of the patients. By the term “administered“ or “administering” as used herein is meant parenteral and / or oral administration. By “parenteral” is meant intravenous, subcutaneous and intramuscolar administration. In the method of the subject invention, for the administration of the compound of formula (I), the course of therapy generally employed is in the range from 20 mg / m2 to 300 mg / m2 of body surface area. More preferably, the course therapy employed is from about 60 mg / m2 / day to about 240 mg / m2 / day of body surface area or, as flat dose, from about 100 mg / day to about 400 mg / day, for up to 28 consecutive days. The compound of formula (I) can be administered in a variety of dosage forms, e.g., orally, in the form of tablets, capsules, sugar or film coated tablets, liquid solutions or suspensions; rectally in the form of suppositories; parenterally, e.g., intramuscularly, or through intravenous and / or intrathecal and / or intraspinal injection or infusion. In the method of the subject invention, for the administration of a demethylating agent, preferably 5-azacytidine, the course of therapy generally employed is from 10 mg / m2 to 500 mg / m2 daily. More preferably, the course of therapy generally employed is from about 50 mg / m2 to 150 mg / m2 daily for up to 7 consecutive days in a 28-day cycle. For the administration of an antimetabolite, preferably cytarabine, the course of therapy generally employed is from 20 mg / m2 / day to 1500 mg / m2 / day for up to 7 consecutive days every 2-4 weeks. More preferably, the course of therapy generally employed is from about 100 mg / m2 to 400 mg / m2 for up to 7 consecutive days, once every 2 -4 weeks. For the administration of a BCL-2 family inhibitor, preferably venetoclax, the course of therapy generally employed is from 10 mg to 600 mg daily by oral administration. More preferably, the course of therapy generally employed is from about 50 mg to 400 mg daily by oral administration for 28 days in a 28-day cycle. For the administration of an immune checkpoint inhibitor, preferably nivolumab, the course of therapy generally employed is from 2 mg / kg to 20 mg / kg daily by IV infusion weekly or every 2-4 weeks. More preferably, the course of therapy generally employed is from about 200 mg to 800 mg flat dose by infusion every 2 or 4 weeks. For the administration of an anthracycline, preferably doxorubicin., the course of therapy generally employed is from 10 mg / m2 to 120 mg / m2 daily by IV infusion every 3-4 weeks. More preferably, the course of therapy generally employed is from about 30 mg / m2 to 100 mg / m2 by infusion every 3 or 4 weeks. The antineoplastic therapy of the present invention is in particular suitable for treating all form of cancer including, but not limited to: carcinoma such as bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, oesophagus, gall- bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma; haematopoietic tumors of lymphoid lineage including leukaemia, acute lymphocytic leukaemia, acute lymphoblastic leukaemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; haematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukaemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; mesothelioma; tumors of the central and peripheral nervous system, including astrocytoma neuroblastoma, glioma and schwannomas, including brain metastases; other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratocanthoma, thyroid follicular cancer and Kaposi's sarcoma. As stated above, the effect of the combination of the invention is significantly increased without a parallel increased toxicity. In other words, the combined therapy of the present invention enhances the antitumoral effects of the partner (a) and / or of partner (b) of the combination of the invention and thus yields the most effective and less toxic treatment for tumors. The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, diluent, adjuvant, vehicle or excipient that does not adversely affect the pharmacological activity of the compound with which it is formulated, and which is also safe for human use. Pharmaceutically acceptable carriers that may be used in the compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (e.g., microcrystalline cellulose, hydroxypropyl methylcellulose and sodium carboxymethylcellulose), lactose monohydrate, sodium lauryl sulfate, polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, pregelatinized starch, and wool fat. The present invention further provides a commercial package comprising, in a suitable container mean, (a) a compound of formula (I) as defined above, and (b) one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL-2 family inhibitors, immune checkpoint inhibitors and anthracyclines, wherein the active ingredients are present in each case in free form or in the form of a pharmaceutically acceptable salt or any hydrate thereof, together with instructions for simultaneous, separate or sequential use thereof. In a package according to the invention each of partner (a) and (b) are present within a single container mean or within distinct container means. Another embodiment of the present invention is a commercial package comprising a pharmaceutical composition or product as described above. The activities of the combination of the present invention are shown for instance by the following in vitro and in vivo tests, which are intended to illustrate but not to limit the present invention. Experimental part Example 1. In vitro cytotoxic activity of the combination with gemcitabine Exponentially growing NKM-1 cells were seeded in 96-well plates at a density of 40,000 cells per ml in 200 µ l of appropriate medium (RPMI1640.+ 10% FCS) After 24 hours, compound DMSO solutions, alone or in combination matrices, were directly administered to cells using a D300e digital dispenser (Tecan, Männedorf, Switzerland). Cell viability was determined after 72 hours of treatment by ATP determination in each test well with a luciferase- based detection system (CellTiterGlo, Promega, Madison, WI, USA). The intensity of emitted light was measured using an EnVision reader (PerkinElmer, Waltham, MA USA) and expressed as RLU. The data were expressed as Fractional Survival (FS) versus DMSO treated controls (CTR): FS = Treated Sample / CTR and analyzed according to the Bliss independence model [Eugene Demidenko, Todd W. Miller. Statistical determination of synergy based on Bliss definition of drugs independence. PLOS ONE, Published: November 25, 2019. https: / / doi.org / 10.1371 / journal.pone.0224137]. Assuming that compound of formula (I) (compound A) and gemcitabine (compound B) act independently, this model predicts the effect of a combination to be equal to the product of the effect of its constituents, according to the equation: FUAB= FUA* FUB;; where FUAand FUBare the individual Fraction Unaffected of the two agents (A and B). The combination indices were calculated as ratio between experimentally measured survival values (FS) and predicted combination values. Bliss C.I. = FSAB / FUAB Combination indices (C.I.) ≤0.8 indicate synergism; 0.8-1.2 additivity; ≥1.2 antagonism. The resulted C.I. at different combined concentrations are reported in Table 1. Table 1. In vitro combination with gemcitabine: combination indices Gemcitabine (nM) Compound of formula (I) (nM) 4.6 5.5 6.2 20 0.65 0.60 0.74 34 0.57 0.49 0.65 50 0.50 0.47 0.50 74 0.50 0.40 0.50 110 0.47 0.34 0.37 170 0.51 0.33 0.22 The results show that on human tumor cells compound of formula (I) can be effectively combined with the antimetabolite agent gemcitabine, producing a synergic effect, since all C.I.s are lower than 0.8. Example 2. In vivo antitumor efficacy in combination with azacytidine Scid female mice, from Charles River, Italy, were maintained in cages with paper filter covers, provided with food and acidified drinking water ad libitum and sterilized bedding. MOLM-13 human acute myeloid leukemia (from American Type Culture Collection) cell line was maintained in vitro. For the experiment, 5x106cells were injected into the tail vein of the mice. This tumor model was selected because it was representative of FLT3 mutant patients, bearing the FLT3-ITD mutation. The treatments started at day 2. Both compounds were prepared immediately before treatment. Compound of formula (I) was administered by oral route in a volume of 10 ml / kg at the dose of 5 mg / kg daily for 10 consecutive days. Azacytidine was administered by intraperitoneal route in a volume of 10 ml / kg at the dose of 1 mg / kg for 5 consecutive days. Mice were monitored daily for mortality and clinical signs. The body weights were evaluated every three days. Antitumor efficacy was evaluated in terms of survival increase respect to control and of number of long-term survival mice (mice found tumor-free at the end of the experiment). For each group Median Survival Time (MST) was calculated and T / C (%) value was reported: T / C (%) = (MSTmicetreated / MST mice control group) x 100 on the basis of the body weight reduction. are in table 2 Table 2. In vivo efficacy in combination with azacytidine Treatment T / C (%) Tumor- free mice Toxicity Compound of formula (I) 165 0 / 10 0 / 10 Azacytidine 163 0 / 10 0 / 10 Compound of formula (I) 228 2 / 10 0 / 10 + azacytidine The compound of formula (I) combined with azacytidine produced a clear synergistic effect. Compound of formula (I), administered by oral route in a volume of 10 ml / kg at the dose of 5 mg / kg daily for 10 consecutive days, was active producing an increase in the survival time (MST= 38 days vs 23 days of control mice, T / C= 165 %). Azacytidine, administered at 1 mg / kg for 5 consecutive days, produced an increase of survival time (MST= 37.5 days; T / C: 163%). In the combination group the MST was 52.5 days with a T / C of 228%. At the end of the experiment (120 days) 2 out of 10 mice were cured. No cured mice were found in the groups treated with compound as single agent. No toxicity was observed in any of the treatment groups. Example 3. In vivo antitumor efficacy in combination with cytarabine Scid female mice, from Charles River, Italy, were maintained in cages with paper filter covers, provided with food and acidified drinking water ad libitum and sterilized bedding. MOLM-13 human acute myeloid leukemia (from American Type Culture Collection) cell line was maintained in vitro. For the experiment, 5x106cells were injected into the tail vein of the mice. This tumor model was selected because it was representative of FLT3 mutant patients, bearing the FLT3-ITD mutation. The treatments started at day 2. Both compounds were prepared immediately before treatment. Compound of formula (I) was administered by oral route in a volume of 10 ml / kg at the dose of 15 mg / kg daily for 10 consecutive days. Cytarabine was administered by intraperitoneal route in a volume of 10 ml / kg at the dose of 50 mg / kg daily for 2 cycles of 5 consecutive days. Mice were monitored daily for mortality and clinical signs. The body weights were evaluated every three days. Antitumor efficacy was evaluated in terms of survival increase respect to control and of number of long-term survival mice (mice found tumor-free at the end of the experiment). For each group Median Survival Time (MST) was calculated and T / C (%) value was reported: T / C (%) = (MSTmicetreated / MST mice control group) x 100 on the basis of the body weight reduction. are in table 3 Table 3. In vivo efficacy in combination with cytarabine Treatment T / C (%) Tumor- free mice Toxicity Compound of formula (I) 281 1 / 6 0 / 6 Cytarabine 102 0 / 6 0 / 6 Compound of formula (I) >571 3 / 6 0 / 6 + cytarabine The compound of formula (I) combined with cytarabine produced a clear synergistic effect. Compound of formula (I), administered by oral route in a volume of 10 ml / kg at the dose of 15 mg / kg daily for 10 consecutive days, was active producing an increase in the survival time (MST= 59 days vs 21 days of control mice, T / C= 281 %). Cytarabine, administered at 50 mg / kg for 2 cycles of 5 consecutive days, was not active (T / C: 102%). In the combination group the MST was >120 days with a T / C higher than 571%. At the end of experiment (120 days) 3 out of 6 mice were cured. No toxicity was observed in any of the treatment groups. Example 4. In vivo antitumor efficacy in combination with venetoclax Scid female mice, from Charles River, Italy, were maintained in cages with paper filter covers, provided with food and acidified drinking water ad libitum and sterilized bedding. MOLM-13 human acute myeloid leukemia (from American Type Culture Collection) cell line was maintained in vitro. For the experiment, 5x106cells were injected into the tail vein of the mice. This tumor model was selected because it was representative of FLT3 mutant patients, bearing the FLT3-ITD mutation. The treatments started at day 2. Both compounds were prepared immediately before treatment. Compound of formula (I) was administered by oral route in a volume of 10 ml / kg at the dose of 10 mg / kg daily for 10 consecutive days. Venetoclax was administered by oral route in a volume of 10 ml / kg at the dose of 100 mg / kg for 10 consecutive days. Mice were monitored daily for mortality and clinical signs. The body weights were evaluated every three days. Antitumor efficacy was evaluated in terms of survival increase respect to control and of number of long-term survival mice (mice found tumor-free at the end of the experiment). For each group Median Survival Time (MST) was calculated and T / C (%) value was reported: T / C (%) = (MSTmicetreated / MSTmice control group) x 100 on the basis of the body weight reduction. are in table 4 Table 4. In vivo efficacy in combination with venetoclax Treatment T / C (%) Tumor- free mice Toxicity Compound of formula (I) 212 3 / 10 0 / 10 Venetoclax 112 0 / 10 0 / 10 Compound of formula (I) >590 8 / 10 0 / 10 + venetoclax The compound of formula (I) combined with venetoclax produced a clear synergistic effect. Compound of formula (I), administered by oral route in a volume of 10 ml / kg at the dose of 10 mg / kg daily for 10 consecutive days, was active producing an increase in the survival time (MST= 44.5 days vs 21 days of control mice, T / C= 212%, with 3 out of 10 mice found tumor free). Venetoclax, orally administered at 100 mg / kg for 10 consecutive days, was not active (T / C: 112%). In the combination group the MST was >124 days with a T / C higher than 590%. At the end of experiment (124 days) 8 out of 10 mice were cured. No toxicity was observed in any of the treatment groups. Example 5. In vivo antitumor efficacy in combination with anti-PD-1 antibody C57BL / 6J female mice, from Shanghai Lingchang Bio-Technology Co. Ltd (LC, Shanghai, China), were maintained in ventilated cages, provided with food and acidified drinking water ad libitum and sterilized bedding. The MC38 murine colon cancer cells will be maintained in vitro as a monolayer culture in DMEM medium supplemented with 10% fetal bovine serum at 37ºC in an atmosphere of 5% CO2in air. The tumor cells will be routinely subcultured twice weekly. The cells in an exponential growth phase will be harvested and counted for tumor inoculation. Each mouse will be inoculated subcutaneously at the right lower flank with MC38 tumor cells (1 x 106) in 0.1 ml of PBS for tumor development. The treatments will be started when the mean tumor size reaches approximately 50mm3. Both compounds were prepared immediately before treatment. Compound of formula (I) was administered by oral route in a volume of 10 ml / kg at the dose of 30 mg / kg daily for 20 consecutive days. Anti-PD-1 antibody (RMP1-14), was administered by intraperitoneal route in a volume of 10 ml / kg at the dose of 10 mg / kg twice a week for 3 consecutive weeks. Mice were monitored daily for mortality and clinical signs. The body weights were evaluated every three days. Tumor growth was assessed by caliper. The two diameters were recorded, and the tumor weight was calculated according to the following formula: length (mm) x width2 / 2. The effect of the antitumor treatment was evaluated as the delay in the onset of an exponential growth of the tumor (see for references, Anticancer drugs 7:437-60,1996). This delay (T-C value) was defined as the difference of time (in days) required for the treatment group (T) and the control group(C) tumors to reach a predetermined size (1g). Toxicity was evaluated on the basis of body weight reduction. The results are reported in table 5 Table 5. In vivo efficacy in combination with anti-PD-1 antibody Treatment T-C (days) Toxicity Compound of formula (I) 3.8 0 / 10 Anti-PD-1 10.1 0 / 10 Compound of formula (I) + 18.3 0 / 10 Anti-PD-1 The compound of formula (I) combined with the anti-PD-1 antibody produced a clear synergistic effect. Compound of formula (I), administered by oral route in a volume of 10 ml / kg at the dose of 30 mg / kg daily for 20 consecutive days, was active producing a delay in tumor growth with a T-C=3.8 days, the anti-PD-1 antibody administered at 10 mg / kg twice a week for 3 consecutive weeks shows a T-C=10.1 days. The T-C observed when compound of formula (I) was combined with anti-PD-1 antibody was 18.3 days, highly superior to the expected by the simple addition of T-C obtained by the single treatments (13.9 days). No toxicity was observed in any of the treatment groups. Example 6. In vitro cytotoxic activity of the combination with doxorubicin Exponentially growing NKM-1 cells were seeded in 96-well plates at a density of 40,000 cells per ml in 200 µ l of appropriate medium (RPMI1640 + 10% FCS) After 24 hours, compound DMSO solutions, alone or in combination matrices, were directly administered to cells using a D300e digital dispenser (Tecan, Männedorf, Switzerland). Cell viability was determined after 72 hours of treatment by ATP determination in each test well with a luciferase- based detection system (CellTiterGlo, Promega, Madison, WI, USA). The intensity of emitted light was measured using an EnVision reader (PerkinElmer, Waltham, MA USA) and expressed as RLU. The data were expressed as Fractional Survival (FS) versus DMSO treated controls (CTR): FS = Treated Sample / CTR and analyzed according to the Bliss independence model [Eugene Demidenko, Todd W. Miller. Statistical determination of synergy based on Bliss definition of drugs independence. PLOS ONE, Published: November 25, 2019. https: / / doi.org / 10.1371 / journal.pone.0224137]. Assuming that compound of formula (I) (compound A) and doxorubicin (compound B) act independently, this model predicts the effect of a combination to be equal to the product of the effect of its constituents, according to the equation: FUAB= FUA* FUB; where FUAand FUBare the individual Fraction Unaffected of the two agents (A and B). The combination indices were calculated as ratio between experimentally measured survival values (FS) and predicted combination values. Bliss C.I. = FSAB / FUAB Combination indices (C.I.) ≤0.8 indicate synergism; 0.8-1.2 additivity; ≥1.2 antagonism. The resulted C.I. at different combined concentrations, are reported in Table 6. Table 6: In vitro combination with doxorubicin: combination indices doxorubicin (nM) Compound of formula (I) (nM) 12 25 50 20 0.55 0.49 0.54 34 0.56 0.49 0.46 50 0.44 0.36 0.32 74 0.60 0.38 0.22 110 0.34 0.23 0.18 170 0.37 0.22 0.14 The results show that on human tumor cells compound of formula (I) can be effectively combined with the anthracycline doxorubicin, producing a synergic effect, since all C.I.s are lower than 0.8.

Claims

Claims 1. A therapeutic combination comprising (a) a compound of formula (I): N (I) or a pharmaceuticallyform thereof and (b) one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL2 family inhibitors, immune checkpoint inhibitors and anthracyclines.

2. A combination according to claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, benzene sulphonate (besylate) and hemioxalate.

3. A combination according to claim 1 or 2, wherein the demethylating agent is selected from the group consisting of 5-azacytidine (azacitidine) and 5-azadeoxycytidine (decitabine).

4. A combination according to claim 1 or 2, wherein the antimetabolite agent is selected from the group consisting of cytarabine, 5-fluorouracil, capecitabine, gemcitabine, pemetrexed and methotrexate.

5. A combination according to claim 1 or 2, wherein the BCL-2 inhibitor is venetoclax.

6. A combination according to claim 1 or 2, wherein the anthracycline is selected from the group consisting of daunorubicin, doxorubicin, epirubicin and idarubicin.

7. A combination according to claim 1 or 2, wherein the immune checkpoint inhibitor is selected from the group consisting of PD-1 inhibitor and PD-L1 inhibitor.

8. The combination according to claim 7, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

9. The combination according to claim 7, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody.

10. The combination according to any one of claims 1 to 9, wherein the compound of formula (I) and the antineoplastic agents are administered independently intravenously or orally.

11. The combination according to any one of claims 1 to 9 which is a combined preparation for simultaneous, separate or sequential use.

12. A combination comprising a compound of formula (I) as defined in claim 1 and one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL2 family inhibitors, immune checkpoint inhibitors and anthracyclines, for use in a method of treating cancer in a subject, wherein said compound of formula (I) and the antineoplastic agents can be administered simultaneously, sequentially or separately.

13. The combination according to claim 12, wherein the cancer is selected from the group consisting of carcinomas, haematopoietic tumors of lymphoid lineage and haematopoietic tumors of myeloid lineage.

14. A pharmaceutical composition comprising a combination according to any one of claims 1 to 9 admixed with a pharmaceutically acceptable carrier, diluent or excipient.

15. Use of a compound of formula (I) as defined in claim 1 in the preparation of a medicament for the treatment of cancer, wherein said treatment comprises simultaneously, sequentially or separately administering to a subject in need thereof a compound of formula (I) as defined above and one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL2 family inhibitors, immune checkpoint inhibitors and anthracyclines.

16. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof, or any hydrate, crystalline form thereof, in combination with a therapeutically effective amount of one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL2 family inhibitors, immune checkpoint inhibitors and anthracyclines wherein the compound of formula (I) and the antineoplastic agents can be administered the simultaneously, sequentially or separately to a subject in need thereof.

17. The method according to claim 17, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, benzene sulphonate (besylate) and hemioxalate, or any solvate, hydrate, crystalline form thereof.

18. The method according to any one of claims 16-17, wherein the cancer is selected from the group consisting of carcinoamas, haematopoietic tumors of lymphoid lineage and haematopoietic tumors of myeloid lineage.

19. A commercial package comprising, in a suitable container mean, (a) a compound of formula (I) as defined in claim 1 and (b) one or more antineoplastic agents selected from the group consisting of demethylating agents, antimetabolite agents, BCL2 family inhibitors, immune checkpoint inhibitors and anthracyclines, wherein the active ingredients are present in each case in free form or in the form of a pharmaceutically acceptable salt or any hydrate thereof, together with instructions for simultaneous, separate or sequential use thereof.

Images