Cancer combination therapy comprising an FLT3 inhibitor

Abstract

The present invention relates, inter alia, to a combination of (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703 for use as a medicament, preferably for use in the treatment of patients suffering from cancer. The present invention also relates to a kit of dosage forms comprising a dosage form containing (i) an FLT3 inhibitor and a dosage form containing (ii) SEL24 / MEN1703, and to dosage forms containing (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703.

Description

Technical Field

[0001] The present invention is in the field of cancer treatment. More specifically, in one aspect, the present invention is directed to a combination of (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703 for use as a medicament. In another aspect, the present invention relates to a combination of (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703 for use in the treatment of a patient suffering from cancer. In yet another aspect, the present invention is directed to a kit of dosage forms comprising (i) a dosage form containing an FLT3 inhibitor and (ii) a dosage form containing SEL24 / MEN1703. In yet another aspect, the present invention relates to a dosage form comprising (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703.

Background Art

[0002] FLT3 mutations in AML patients have been known for over 25 years now, and the most common FLT3-ITD ("internal tandem duplication") mutation was first reported in 1996 (see Nakao M et al., 1996). Such mutations result in constitutively active FLT3 with kinase activity. Subsequently, therefore, as outlined by Levis and Perl, 2020, FLT3 inhibitors for the treatment of the disease were developed, and in 2002, the first patient was enrolled in a clinical trial of an FTL3 inhibitor in AML. The review by Kennedy and Smith, 2020, summarizes the understanding of FLT3 mutations in AML, and in particular, provides an overview of established FTL3 inhibitors (see Table 1 in the literature, where first- and second-generation inhibitors are given, and the type of inhibition is given), as well as clinical trials of established FLT3 inhibitors in newly diagnosed AML (see Table 2 in the literature). Kennedy and Smith, 2020, further summarize clinical trials of established FLT3 inhibitors in combination therapy (see Table 4 in the literature) or clinical trials of novel FLT3 inhibitors that have dual inhibitory activity ("dual agents", see Table 5 in the literature). The review by Yuan et al., 2019, focuses on such dual FLT3 inhibitors.

[0003] The first approved FLT3 inhibitor was midostaurin, which was approved in patients with FLT3-mutated AML (Rydapt®, approved in the United States and Europe in 2017). Further FLT3 inhibitors, including quizartinib, were tested. The use of quizartinib demonstrated limitations that can occur during treatment, namely, resistance due to on-target kinase-activated FLT3 mutations that often occur within 2 to 4 months from the start of quizartinib.

[0004] Therefore, the need to improve early or "first-generation" FLT3 inhibitors still exists.

[0005] Taking into account the results of the initial FLT3 inhibitors, further inhibitors, including gilteritinib, have been developed in the past and are still being developed. Clinical trials using gilteritinib have shown that the therapy with gilteritinib is more effective and less toxic than standard chemotherapy. Subsequently, gilteritinib-containing products have received marketing approvals for AML patients with FLT3 mutations, especially in the United States and Europe (Xospata®). It is noteworthy that Xospata® has proven its superiority over existing therapies and has been approved by the FDA for each patient population. Gilteritinib is administered as Xospata® as monotherapy.

[0006] However, resistance to gilteritinib still exists, and thus there is still a need to improve this therapy because the response to gilteritinib monotherapy may be short-lived. For example, the first clinical trials investigating combinations of gilteritinib with intensive induction chemotherapy or low-toxicity agents have been initiated and show promising results (see Levis and Perl, 2020). Further examples of such combinations are the combination of CUDC-907, a dual inhibitor of PI3K and histone deacetylase, with gilteritinib, which shows promising in vitro and in vivo anti-leukemic activity against FLT3-ITD AML (see Qiao et al., 2021). In addition, gilteritinib has been studied as an additional active substance added to different skeletons (main therapies) in patients who do not respond to, for example, venetoclax + azacitidine (see Zhang et al., 2022), and gilteritinib has been administered together with venetoclax and azacitidine to such non-responsive patients (induction therapy. Maintenance therapy was venetoclax and gilteritinib) and showed promising results.

Prior Art Documents

Non-Patent Documents

[0007]

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Summary of the Invention

Problems to be Solved by the Invention

[0008] Therefore, there is still a need to improve more recent or "second-generation" FLT3 inhibitors, particularly gilteritinib.

Means for Solving the Problems

[0009] The inventors of the present invention have surprisingly found that the combination of an FLT3 inhibitor and SEL24 / MEN1703 acts synergistically and thus corresponds to a very promising new combination therapy, particularly in patients suffering from acute myeloid leukemia (AML).

[0010] In a first aspect, the present invention is directed to a combination of (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703 for use as a medicament.

[0011] In a preferred embodiment, the FLT3 inhibitor is a type I FLT3 inhibitor. The type I FLT3 inhibitor may be selected from the group consisting of midostaurin, gilteritinib, lestaurtinib, and crenolanib.

[0012] In another preferred embodiment, the FLT3 inhibitor is a type II FLT3 inhibitor. The type II FLT3 inhibitor may be selected from the group consisting of quizartinib and sorafenib.

[0013] In a preferred embodiment, the FLT3 inhibitor is selected from the group consisting of gilteritinib, quizartinib, midostaurin, and combinations thereof. In some cases, it may be preferred that the FTL3 inhibitor is gilteritinib.

[0014] In one embodiment, SEL24 / MEN1703 is administered at a daily dose of about 50 mg to about 150 mg. It is preferred that SEL24 / MEN1703 is administered at a daily dose of about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, or about 150 mg. Most preferably, SEL24 / MEN1703 is administered at a daily dose of about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, or about 125 mg. It is preferred that SEL24 / MEN1703 is administered once daily. More preferably, SEL24 / MEN1703 is administered orally.

[0015] In one embodiment, gilteritinib is administered at a daily dose of about 40 mg to about 400 mg. It is preferred that gilteritinib is administered at a daily dose of about 40 mg, about 80 mg, about 120 mg, or about 240 mg. Most preferably, gilteritinib is administered at a daily dose of about 100 mg, about 105 mg, about 110 mg, about 115 mg or about 120 mg. It is preferred that gilteritinib is administered once a day. More preferably, gilteritinib is administered orally.

[0016] In one embodiment, crizotinib is administered at a daily dose of about 10 mg to about 50 mg. It is preferred that crizotinib is administered at a daily dose of about 10 mg, about 20 mg, about 30 mg, about 40 mg, or about 50 mg. Most preferably, crizotinib is administered at a daily dose of about 15 mg, about 17 mg, about 20 mg, or about 25 mg. It is preferred that crizotinib is administered once a day. More preferably, crizotinib is administered orally.

[0017] In one embodiment, midostaurin is administered at a daily dose of about 20 mg to about 80 mg. It is preferred that midostaurin is administered at a daily dose of about 20 mg, about 30 mg, about 40 mg, about 50 mg, or about 60 mg. Most preferably, midostaurin is administered at a daily dose of about 35 mg, about 40 mg, about 45 mg, or about 50 mg. It is preferred that midostaurin is administered twice a day. More preferably, midostaurin is administered orally.

[0018] In one embodiment, the combination for use in the first aspect does not include additional administration of chemotherapy.

[0019] In one embodiment regarding the combination for use as a medicament, (i) and (ii) are administered as separate dosage forms. In this embodiment, the administration may be carried out simultaneously or sequentially. In yet another embodiment regarding the combination for use as a medicament, (i) and (ii) are administered together in one dosage form.

[0020] In a second aspect, the present invention is directed to a combination of (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703 for use in the treatment of a patient suffering from cancer.

[0021] In a preferred embodiment, (i) the FLT3 inhibitor is a type I FLT3 inhibitor. The type I FLT3 inhibitor may be selected from the group consisting of midostaurin, gilteritinib, lestaurtinib, and quizartinib.

[0022] In another preferred embodiment, (i) the FLT3 inhibitor is a type II FLT3 inhibitor. The type II FLT3 inhibitor may be selected from the group consisting of quizartinib and sorafenib.

[0023] In a preferred embodiment, the FLT3 inhibitor is selected from the group consisting of gilteritinib, quizartinib, midostaurin, and combinations thereof. In some cases, it may be preferred that the FTL3 inhibitor is gilteritinib.

[0024] In a preferred embodiment, the cancer is a hematological cancer. In an even more preferred embodiment, the cancer is leukemia. In a most preferred embodiment, the cancer is AML. The combination for use in the second aspect can be a first-line treatment for AML. However, the combination for use in the second aspect may alternatively be used to treat patients who progress beyond previous treatment lines. In one embodiment, the combination of the present invention may be used to treat relapsed or refractory AML.

[0025] When a patient has AML, it is preferred that the patient with AML shows an FLT3 mutation that results in overactivation of FLT3 signaling. The mutation in FLT3 may even result in constitutively active FLT3 signaling (in the sense that the signaling activity of FLT3 is constitutively active). The FLT3 mutation is caused by at least one base mutation in the FLT3 gene, and this base mutation results in the above-mentioned FLT3 mutation at the protein level that causes overactivation of FLT3 signaling. Such mutations are known in the art as described in the background art section above. Preferably, the FLT3 mutation is an FLT3-ITD mutation, an FLT3-TKD mutation, or a combination of an FLT3-ITD mutation and an FLT3-TKD mutation. Additionally or alternatively, a patient with cancer, including a patient with AML, may show at least one IDH1 and / or IDH2 mutation, preferably at least two IDH1 and / or IDH2 mutations. Additionally or alternatively, a patient with cancer may be unsuitable for chemotherapy.

[0026] When a patient has AML, however, the patient may show the FLT3 wild-type sequence. Additionally or alternatively, a patient with cancer may be unsuitable for chemotherapy.

[0027] In one embodiment, SEL24 / MEN1703 is administered at a daily dose of about 50 mg to about 150 mg. It is preferred that SEL24 / MEN1703 is administered at a daily dose of about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, or about 150 mg. It is most preferred that SEL24 / MEN1703 is administered at a daily dose of about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg or about 125 mg. It is preferred that SEL24 / MEN1703 is administered once a day. It is more preferred that SEL24 / MEN1703 is administered orally.

[0028] In one embodiment, gilteritinib is administered at a daily dose of about 40 mg to about 400 mg. It is preferred that gilteritinib is administered at a daily dose of about 40 mg, about 80 mg, about 120 mg, or about 240 mg. Most preferably, gilteritinib is administered at a daily dose of about 100 mg, about 105 mg, about 110 mg, about 115 mg or about 120 mg. It is preferred that gilteritinib is administered once daily. More preferably, gilteritinib is administered orally.

[0029] In one embodiment, crizotinib is administered at a daily dose of about 10 mg to about 50 mg. It is preferred that crizotinib is administered at a daily dose of about 10 mg, about 20 mg, about 30 mg, about 40 mg, or about 50 mg. Most preferably, crizotinib is administered at a daily dose of about 15 mg, about 17 mg, about 20 mg, or about 25 mg. It is preferred that crizotinib is administered once daily. More preferably, crizotinib is administered orally.

[0030] In one embodiment, midostaurin is administered at a daily dose of about 20 mg to about 80 mg. It is preferred that midostaurin is administered at a daily dose of about 20 mg, about 30 mg, about 40 mg, about 50 mg, or about 60 mg. Most preferably, midostaurin is administered at a daily dose of about 35 mg, about 40 mg, about 45 mg, or about 50 mg. It is preferred that midostaurin is administered twice daily. More preferably, midostaurin is administered orally.

[0031] In one embodiment, the combination for use in the second aspect, particularly the combination for use in the treatment of AML, does not include additional administration of chemotherapy.

[0032] In one embodiment regarding a combination for use in the treatment of a patient suffering from cancer, (i) and (ii) are administered as separate dosage forms. In this embodiment, the administration may be carried out simultaneously or sequentially. In yet another embodiment regarding a combination for use in the treatment of a patient suffering from cancer, (i) and (ii) are administered together in one dosage form.

[0033] In a preferred embodiment, the FLT3 inhibitor is gilteritinib, the cancer is AML, SEL24 / MEN1703 is administered at a daily dose of about 80 mg to about 120 mg, preferably orally, and gilteritinib is administered at a daily dose of about 100 mg to about 115 mg, preferably orally. In this embodiment, it may be preferred that the patient suffering from AML exhibits an FLT3 mutation and optionally also exhibits at least one IDH1 and / or IDH2 mutation.

[0034] In a third aspect, the invention is directed to a kit of dosage forms comprising (i) a dosage form comprising an FLT3 inhibitor and (ii) a dosage form comprising SEL24 / MEN1703.

[0035] In a preferred embodiment, the FLT3 inhibitor comprised in dosage form (i) is a type I FLT3 inhibitor. The type I FLT3 inhibitor may be selected from the group consisting of midostaurin, gilteritinib, lestaurtinib, and quizartinib.

[0036] In another preferred embodiment, the FLT3 inhibitor comprised in dosage form (i) is a type II FLT3 inhibitor. The type II FLT3 inhibitor may be selected from the group consisting of quizartinib and sorafenib.

[0037] In a preferred embodiment, the FLT3 inhibitor is selected from the group consisting of gilteritinib, quizartinib, midostaurin, and combinations thereof. It may be preferred that the FTL3 inhibitor is gilteritinib.

[0038] In one embodiment, a dosage form containing SEL24 / MEN1703 contains SEL24 / MEN1703 in an amount of about 50 mg to about 150 mg, preferably in an amount of about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, or about 150 mg, most preferably in an amount of about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg or about 125 mg. It is preferred that the dosage form containing SEL24 / MEN1703 is a once-daily dosage form. More preferably, the dosage form containing SEL24 / MEN1703 is an oral dosage form.

[0039] In one embodiment, a dosage form containing gilteritinib as an FLT3 inhibitor contains gilteritinib in an amount of about 40 mg to about 400 mg, preferably in an amount of about 40 mg, about 80 mg, about 120 mg, or about 240 mg, most preferably in an amount of about 100 mg, about 105 mg, about 110 mg, about 115 mg or about 120 mg. It is preferred that the dosage form containing gilteritinib as an FLT3 inhibitor is a once-daily dosage form. More preferably, the dosage form containing gilteritinib as an FLT3 inhibitor is an oral dosage form.

[0040] In one embodiment, a dosage form containing quizartinib as an FLT3 inhibitor contains quizartinib in an amount of about 10 mg to about 50 mg, preferably in an amount of about 10 mg, about 20 mg, about 30 mg, about 40 mg, or about 50 mg, most preferably in an amount of about 15 mg, about 17 mg, about 20 mg, or about 25 mg. It is preferred that the dosage form containing quizartinib as an FLT3 inhibitor is a once-daily dosage form. More preferably, the dosage form containing quizartinib as an FLT3 inhibitor is an oral dosage form.

[0041] In one embodiment, a dosage form containing midostaurin as an FLT3 inhibitor contains midostaurin in an amount of about 20 mg to about 80 mg, preferably about 20 mg, about 30 mg, about 40 mg, about 50 mg, or about 60 mg, and most preferably about 35 mg, about 40 mg, about 45 mg, or about 50 mg. The dosage form containing midostaurin is preferably a twice-daily dosage form. More preferably, the dosage form containing midostaurin is an oral dosage form.

[0042] Each dosage form typically contains at least one pharmaceutically acceptable excipient as defined in Section 2 of the Formulation section for carrying out the following invention.

[0043] In one embodiment, the kit of the third aspect further includes a leaflet describing how to use and administer the dosage form.

[0044] In the fourth aspect, the present invention is directed to a dosage form containing (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703.

[0045] In a preferred embodiment, the FLT3 inhibitor contained in the above dosage form is a type I FLT3 inhibitor. The type I FLT3 inhibitor may be selected from the group consisting of midostaurin, gilteritinib, lestaurtinib, and quizartinib.

[0046] In another preferred embodiment, the FLT3 inhibitor contained in the above dosage form is a type II FLT3 inhibitor. The type II FLT3 inhibitor may be selected from the group consisting of quizartinib and sorafenib.

[0047] In a preferred embodiment, the FLT3 inhibitor is selected from the group consisting of gilteritinib, quizartinib, midostaurin, and combinations thereof. In some cases, it is preferred that the FTL3 inhibitor is gilteritinib.

[0048] In one embodiment, the dosage form containing SEL24 / MEN1703 contains SEL24 / MEN1703 in an amount of about 50 mg to about 150 mg, preferably in an amount of about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, or about 150 mg, most preferably in an amount of about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg or about 125 mg.

[0049] When the dosage form contains gilteritinib as an FLT3 inhibitor, in one embodiment, the dosage form contains gilteritinib in an amount of about 40 mg to about 400 mg, preferably in an amount of about 40 mg, about 80 mg, about 120 mg, or about 240 mg, most preferably in an amount of about 100 mg, about 105 mg, about 110 mg, about 115 mg or about 120 mg.

[0050] When the dosage form contains quizartinib as an FLT3 inhibitor, the dosage form contains quizartinib in an amount of about 10 mg to about 50 mg, preferably in an amount of about 10 mg, about 20 mg, about 30 mg, about 40 mg, or about 50 mg, most preferably in an amount of about 15 mg, about 17 mg, about 20 mg, or about 25 mg.

[0051] When the dosage form contains midostaurin as an FLT3 inhibitor, the dosage form contains midostaurin in an amount of about 20 mg to about 80 mg, preferably in an amount of about 20 mg, about 30 mg, about 40 mg, about 50 mg, or about 60 mg, most preferably in an amount of about 35 mg, about 40 mg, about 45 mg, or about 50 mg.

[0052] In one embodiment, the dosage form is a once-daily or twice-daily dosage form. It is more preferred that the dosage form is an oral dosage form.

[0053] The dosage form typically contains at least one pharmaceutically acceptable excipient as defined in Section 2 of the Forms for Carrying Out the Following Invention.

[0054] In one embodiment, the dosage form of the fourth aspect comprises a leaflet describing how to use and administer the dosage form.

[0055] In a fifth aspect, the present invention is directed to a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of (i) an FLT3 inhibitor and an effective amount of (ii) SEL24 / MEN1703.

[0056] All embodiments outlined above for the second aspect are equally applicable to the fifth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0057]

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Figure 5A

Figure 5B

BEST MODE FOR CARRYING OUT THE INVENTION

[0058] Before explaining the present invention in more detail, the following definitions are introduced.

[0059] 1. Definitions As used in this specification and the claims, the singular forms "a" and "an" include the corresponding plural forms unless the context clearly contradicts.

[0060] In the context of the present invention, the term "about" indicates an interval of precision that a person skilled in the art understands to still guarantee the technical effect of the feature being discussed. This term typically indicates a deviation of ±10%, preferably ±5%, from the indicated numerical value.

[0061] It is necessary to understand that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". In the following, when a group is defined as including at least a specific number of embodiments, this also preferably means including a group consisting of only these embodiments.

[0062] As used herein, the terms "combination" or "in combination with" do not imply that the therapies or active agents (i) and (ii) must be administered simultaneously and / or formulated for co-delivery. However, such therapies and formulations are within the scope of the invention. The active agents in a combination can be administered to each other simultaneously, before each other, or after each other, and further, simultaneously with, before, or after one or more additional therapies or active agents. The active agents or treatment protocols can be administered in any order. Generally, each active agent is administered at a dosage and / or time schedule determined for that active agent. Further, generally, the active agents used in combination are expected to be used at dosages that do not exceed the dosages at which they are used individually. In some embodiments, the dosages used in combination are less than the dosages used individually. In some embodiments, one of the two active agents is administered at a therapeutic dosage or less than a therapeutic dosage, for example, the FLT3 inhibitor is administered at less than a therapeutic dosage, or SEL24 / MEN1703 is administered at less than a therapeutic dosage ( "less than a therapeutic dosage" is derived from a comparison with the therapeutic dosage of the single active agent in monotherapy). A dosage less than a therapeutic dosage (a dosage lower than the therapeutic dosage) can be, for example, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% lower than the corresponding monotherapy.

[0063] As used herein, the term "FLT3" refers to the receptor-type tyrosine-protein kinase FLT3 (also known as "Cluster of differentiation antigen 135", "CD135", "fms-like tyrosine kinase 3", "fetal liver kinase-2" or "Flk2"), which is a protein encoded by the FLT3 gene in humans. FLT3 is a cytokine receptor belonging to the receptor-type tyrosine kinase class III.

[0064] As used herein, the term "FLT3 inhibitor" means a compound capable of inhibiting the kinase FLT3, particularly a compound capable of inhibiting the overactivation of FLT3 signaling, such as a mutant form of FLT3 that results in constitutively active FLT3. Such compounds are known in the art, particularly with respect to the treatment of AML (see Levis and Perl, 2020). Typically, such compounds are small molecules that bind strongly (and optionally [i.e., not necessarily] selectively) to FLT3 such that FLT3 is inhibited. FLT3 inhibitors can be subclassified based on how they interact with the intracellular kinase domain of the FLT3 receptor. In normal physiological conditions, the FLT3 ligand binds to the extracellular domain, causing the FLT3 receptor to dimerize, adopt an enzymatically active conformation, and subsequently activate downstream signaling. Type I FLT3 inhibitors bind to the active conformation of the receptor and thus inhibit both FLT3-TKD and FLT3-ITD mutant receptors. Type II FLT3 inhibitors bind to a region adjacent to the ATP-binding pocket and inhibit only the inactive conformation of the receptor. Type II inhibitors are inactive against most FLT3-TKD mutations because these mutations bias the conformation of the active kinase of FLT3 (see also the description spanning pages 3-4 of Kennedy and Smith, 2020).

[0065] As used herein, the term "FLT3-ITD mutation" means a FLT3 "intragenic tandem duplication, ITD" mutation. As used herein, the term "FLT3-TKD mutation" means a FLT3 "tyrosine kinase domain" mutation.

[0066] As used herein, the term "SEL24 / MEN1703" (alternatively referred to herein as "MEN", "Men", "men", or "Men1703") means the compound 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole in the form of the free base or a pharmaceutically acceptable salt thereof (such as the HCl salt). The free base form has the CAS number 1616359-00-2 and the HCl salt form has the CAS number 2769008-22-0. This compound is a dual pan-PIM / FLT3 inhibitor and has been shown to inhibit the growth of a broad panel of AML cell lines, particularly in xenograft models. The rationale for the development of this dual inhibitor was that the PIM kinase was thought to be a major driver of resistance to FLT3 inhibitors. SEL24 / MEN1703 has been characterized in more detail, for example, by Czardybon et al., 2018. WO 2014 / 096388 discloses SEL24 / MEN1703 as compound 26A therein and characterizes SEL24 / MEN1703 as a dual pan-PIM / FLT3 inhibitor (see Table 1A of WO 2014 / 096388). WO 2014 / 096388 does not disclose a combination of SEL24 / MEN1703 with an FLT3 inhibitor, nor does it disclose a combination of any compound within the scope of WO 2014 / 096388 with an FLT3 inhibitor.

[0067] As used herein, the term "gilteritinib" means gilteritinib, an FLT3 inhibitor approved for the treatment of AML and commercially available under the trade name Xospata®. Further details regarding gilteritinib can be found, inter alia, in the product leaflet or regulatory dossier.

[0068] As used herein, the term "quizartinib" means quizartinib, an FLT3 inhibitor, for which Daiichi Sankyo Europe GmbH has applied for marketing authorization in Europe under the name "Vanflyta®". The EMA issued an opinion in 2019 recommending refusal of marketing authorization because the benefits of quizartinib treatment did not outweigh its risks in the EMA's opinion.

[0069] As used herein, the term "midostaurin" means midostaurin, an FLT3 inhibitor sold under the trade name Rydapt®, which is approved for the treatment of AML, and at the start of treatment, Rydapt® must always be used in combination with chemotherapy. Further details regarding midostaurin can be found, inter alia, in the product leaflet or regulatory dossier.

[0070] As used herein, the term "hyperactivation" of FLT3 means that FLT3 is more active compared to the wild-type situation, particularly with respect to downstream activation and signal transduction, and thus results in cancer cell proliferation.

[0071] As used herein, the term "small molecule" refers to a small organic compound having a low molecular weight. Small molecules in the context of the present invention preferably have a molecular weight of less than 5000 daltons, more preferably less than 4000 daltons, more preferably less than 3000 daltons, more preferably less than 2000 daltons, or even more preferably less than 1000 daltons. In particularly preferred embodiments, small molecules in the context of the present invention have a molecular weight of less than 800 daltons. In another preferred embodiment, small molecules in the context of the present invention have a molecular weight of 50 - 3000 daltons, preferably 100 - 2000 daltons, more preferably 100 - 1500 daltons, and even more preferably 100 - 1000 daltons.

[0072] As used herein, the term "treatment" refers to a clinical intervention for curing or ameliorating a disease, preventing recurrence of the disease, alleviating symptoms of the disease, reducing any direct or indirect pathological consequences of the disease, achieving a stabilized (i.e., non-worsening) state of the disease, preventing metastasis, reducing the rate of disease progression, and / or extending survival as compared to expected survival without treatment.

[0073] As used herein, the term "relapsed or relapsing AML" means that AML has recurred after treatment and remission (using drugs different from the combinations of the present invention, optionally in combination with drugs).

[0074] As used herein, the term "refractory AML" means that leukemia has not responded to previous treatment (using drugs different from the combinations of the present invention, optionally in combination with drugs).

[0075] As used herein, the term "IDH1 and / or IDH2 mutation" means a mutation in the "isocitrate dehydrogenase 1" gene or "isocitrate dehydrogenase 2" gene encoding the corresponding isocitrate dehydrogenase.

[0076] 2. Pharmaceutical Composition "FLT3 inhibitor" and "SEL24 / MEN1703" are "pharmaceutically active agents" or "active agents" for the purposes of the present invention. As described above, they may exist in separate dosage forms or may be included in a single dosage form.

[0077] As used herein, "pharmaceutically active agent" means that a compound has the efficacy to modulate a response in a patient, i.e., a human or animal in vivo. As used herein, the term "pharmaceutically acceptable excipient" refers to excipients commonly included in a pharmaceutical dosage form or pharmaceutical composition, which are known to those skilled in the art. Such excipients are exemplified below. In view of the definition of the above "pharmaceutically active agent", pharmaceutically acceptable excipients can be defined as being pharmaceutically inert.

[0078] When a commercially available FLT3 inhibitor is used in combination with SEL24 / MEN1703, typically, the administration is carried out via separate dosage forms, and the FLT3 inhibitor is preferably administered via the approved administration route in the dosage form. SEL24 / MEN1703 may be administered in the dosage forms shown below or in the dosage forms currently undergoing clinical trials.

[0079] The dosage forms for the use according to the present invention may be formulated for oral, buccal, nasal, rectal, topical, transdermal or parenteral application. Oral application is particularly preferred. Parenteral application includes intravenous, intramuscular or subcutaneous administration. The dosage forms of the present invention may sometimes also be referred to as formulations or pharmaceutical compositions.

[0080] Generally, the pharmaceutical compositions according to the present invention can include various pharmaceutically acceptable excipients selected according to what functionality is to be achieved for the composition. "Pharmaceutically acceptable excipients" in the meaning of the present invention can be any substance used in the preparation of pharmaceutical dosage forms, including coating materials, film-forming materials, fillers, disintegrants, release-regulating materials, carrier materials, diluents, binders and other adjuvants. Typical pharmaceutically acceptable excipients include substances such as sucrose, mannitol, sorbitol, starch and starch derivatives, lactose, etc., as well as lubricants such as magnesium stearate, disintegrants and buffering agents.

[0081] The term "carrier" means a pharmaceutically acceptable organic or inorganic carrier substance which is combined with the active ingredient to facilitate its application (administration). Suitable pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohols, oils, preferably vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, surfactants, perfumes, monoglycerides and diglycerides of fatty acids, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and the like. The pharmaceutical composition can be sterilized and, if desired, can be mixed with auxiliaries such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for influencing the osmotic pressure, buffers, colorants, flavoring agents and / or aromatic substances which do not react detrimentally with the active compound.

[0082] When liquid dosage forms are contemplated for the present invention, these can include pharmaceutically acceptable emulsions, solutions, suspensions and syrups containing inert diluents commonly used in the art such as water. These dosage forms may contain, for example, microcrystalline cellulose for bulking, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweetening / flavoring agents.

[0083] For parenteral application, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Pharmaceutical formulations for parenteral administration are particularly preferred and include aqueous solutions in water-soluble form. Furthermore, suspensions may be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0084] In the case of injectable preparations, sterile aqueous or oily suspensions can be formulated according to known techniques, for example, using suitable dispersing agents, wetting agents and / or suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that can be used include water and isotonic sodium chloride solution. Sterile oils have also been conventionally used as solvents or suspending media.

[0085] For rectal administration of the pharmaceutical composition of the present invention, suppositories can be prepared, for example, by mixing the compound with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and melts in the rectum to release the active agent from the suppository, such as cocoa butter, synthetic triglycerides and polyethylene glycol.

[0086] For administration by inhalation, the pharmaceutical composition according to the present invention may be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, for use in an inhaler or insufflator may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0087] The oral dosage form may be liquid or solid, and examples thereof include tablets, troches, pills, capsules, powders, effervescent preparations, sugar-coated tablets, and granules. Pharmaceutical preparations for oral use can be obtained with a solid excipient, and optionally, the resulting mixture is ground, appropriate auxiliaries are added as necessary, and then the mixture of granules is processed to obtain a core of a tablet or a sugar-coated tablet. Suitable excipients include, in particular, sugars such as lactose, sucrose, mannitol, or sorbitol, starches such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, cellulose preparations such as methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or fillers such as polyvinylpyrrolidone (PVP). Optionally, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts such as sodium alginate may be added. The oral dosage form may be formulated to ensure immediate release or sustained release of the active agent.

Example

[0088] 3. Example The following examples are merely illustrative and are intended to further illustrate the present invention. These examples should not be construed as limiting the present invention to those examples.

[0089] Example 1: In Vitro Study in Cell Lines In a standard in vitro cytotoxicity experiment, two AML cell lines (MV4-11 [FLT3 ITD] and MOLM-13 [FLT3 ITD]) were treated with MEN and Gilt (type I inhibitor) as single agents or in combination for 72 hours. The synergistic effect of the combination was measured according to the calculation of the combination index (Chou TC, 2006). The combination of MEN and Gilt was slightly synergistic in MV4-11 cells at all concentration levels (see Table 1). The combination of MEN and Gilt at the IC75 concentration induced cytotoxicity significantly different from that of gilteritinib-induced cytotoxicity in MV4-11 cells (Student's t-test, p = 0.0331; see Figure 1). The combination of MEN and Gilt in MOLM-13 cells seemed to be synergistic at the IC25 and IC10 concentration levels (see Table 2).

[0090]

Table 1

[0091] The combination of MEN + Gilt was moderately synergistic (CI ≤ 1) at different concentration levels. IC75 and IC50 are clinically relevant concentrations (gray rows).

[0092]

Table 2

[0093] The combination of MEN + Gilt was synergistic (CI ≤ 1) at the concentration levels IC10 and IC25.

[0094] Materials and Methods Human Cell Lines The human acute myeloid leukemia cell lines MV4-11 and MOLM-13 were obtained from DSMZ, Braunschweig, Germany (ACC102 and ACC554). MV4-11 cells were cultured in RPMI 1640 medium (Gibco, Life Technologies, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, Saint Louis, California, USA). MOLM-13 cells were grown in RPMI containing 20% FBS. All cells were incubated at 37 °C, 5% CO2, and 80% relative humidity.

[0095] Drug MEN1703 (alternatively referred to as "Men", "MEN", or "SEL24 / MEN1703") (batch number A / 2201 / 24 / 1), in HCl salt form (CAS number 2769008-22-), of IUPAC name 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazol-4-amine hydrochloride, synthesized by Aptuit (VR), and gilteritinib (alternatively referred to as "Gilt". MedChem Express, Monmouth Junction, New Jersey, USA) were dissolved in DMSO, aliquoted, and stored at -80 °C until use. Stock solutions were added at appropriate concentrations in culture medium prior to addition to the cells.

[0096] Cytotoxicity For the cytotoxicity study, cells were seeded at an appropriate density (50,000 cells / well) on day 0, and then MEN and Gilt were added. The concentration ranges were determined in advance in the single-agent cytotoxicity assay at the same time point to include the IC10, IC25, IC50, and IC75 values for each drug and each AML cell line in the combination experiment (see Tables 1-2). After 72 hours, CellTiter 96 Aqueous One Solution Reagent (MTS) (Promega, Madison, Wisconsin, USA) was added to evaluate cell viability. Fluorescence was measured using Tecan Infinite M200 (Tecan Trading AG, Switzerland) 4 hours later, and the absorbance at 490 nm was recorded. The quantitative measurement of synergism / antagonism was evaluated using CompuSyn (ComboSyn, Inc., Paramus, New Jersey, USA) with the combination index (CI) for the affected fraction (Fa) (Chou TC, 2006).

[0097] Example 2: In Vivo Xenograft Study In the in vivo xenograft study using MV4-11 (FLT3 ITD) cells, MEN and gilteritinib as single-agent treatments induced significant antitumor activity on day 33, showing tumor volume inhibition (TVI) of 57.2% and 84.1% respectively, while the combination treatment showed 99.2% TVI on day 33 (see Figure 2A and Table 3). The combination treatment ended on day 33, and subsequent extended treatment with gilteritinib alone for another 8 days induced long-lasting antitumor activity until day 47 in this combination treatment group. After day 47, tumor mass regrowth was observed in this combination treatment group. On day 40, when the treatment with gilteritinib alone ended, the single-agent treatments with Men and gilteritinib showed TVI of 53.1% and 87.6% respectively, while the combination treatment showed 100% TVI (see Figure 2A and Table 3).

[0098] [Table 3]

[0099] Tumor growth inhibition by the combination of MEN + gilteritinib is improved compared to monotherapy at the end of each treatment.

[0100] According to the Mann-Whitney test evaluated on day 33, when the monotherapy group and the combination group were compared with the vehicle group, the reduction in TV was statistically significant (p-value = 0.007; p-value = 0.001 and p-value = 0.002, respectively), and the combination treatment group was statistically significant when compared with both monotherapy groups (p-value = 0.001, p-value = 0.01, see Figure 2B).

[0101] In an in vivo xenograft study using MOLM-13 (FLT3 ITD) cells, MEN and gilteritinib as monotherapies induced significant antitumor activity on day 29, showing tumor volume inhibition (TVI) of 38.8% and 60.1%, respectively, while the combination treatment showed 78.3% TVI on day 29 (see Figure 3A and Table 4).

[0102] [Table 4]

[0103] Tumor growth inhibition by the combination of MEN + Gilt is improved compared to monotherapy at the end of each treatment.

[0104] According to the Mann-Whitney test evaluated on day 29, when gilteritinib monotherapy and combination treatment were compared with the vehicle group, the reduction in TV was statistically significant (p-value = 0.04 and p-value = 0.002, see Figure 3B).

[0105] Materials and Methods Human Cell Lines MV4-11 and MOLM-13 cells were obtained and cultured as described in Example 1.

[0106] Drugs The HCl salt form (CAS number 2769008-22-) of MEN1703 (alternatively also called "Men", "MEN", or "SEL24 / MEN1703") having the IUPAC name 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazol-4-amine hydrochloride (batch 76608X, synthesized by MENARINI RICERCHE SpA, Pisa, Italy) was dissolved in sterile water. Gilotrifitinib (MedChem Express, Monmouth Junction, New Jersey, USA) was dissolved in a solution of carboxymethylcellulose (0.5%).

[0107] MV4-11 model For the AML xenograft model, 10×10 6 MV4-11 cells were resuspended in 0.2 ml of 5.6 mg / ml BME type III (Trevigen) + DPBS (1:1) and then subcutaneously injected into the right flank of 6- to 8-week-old female SCID mice (Charles River, Calco, Italy).

[0108] After injection, the mice were maintained in microisolator cages under environmentally controlled conditions with continuous monitoring. Drinking water and a specific sterile diet (VRF1, Charles River) were provided ad libitum. Environmental conditions, as well as animal housing and handling procedures, were in accordance with UKCCCR guidelines (Workman P et al., 2010) and the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (Directive 2010 / 63 / EU; 2010). Tumor growth and body weight were evaluated and recorded twice a week.

[0109] The results were evaluated as follows: The tumor volume was measured with calipers, and the tumor mass was calculated using the following formula: [length (mm) × width 2 (mm) × d] / 2, assuming a density d = 1 mg / mm 3 for the tumor tissue (Teicher B. Totowa, 1997). When the average tumor volume reached 200 - 300 mm 3 (corresponding to day 19 in Figure 2A), the animals were randomly assigned to four groups (6 - 7 mice / group) and received the following treatments starting on day 19: Group I received 0.5% vehicle carboxymethylcellulose (gefitinib diluent solution) orally once daily for 22 days, i.e., until the end of the dosing period (i.e., until day 40, see Figure 2A). Group II received 25 mg / kg of MEN orally once daily for 14 days (i.e., until day 33, see Figure 2A). Group III received 3 mg / kg of gefitinib orally once daily for 22 days (i.e., until day 40, see Figure 2A). Group IV received a combination of MEN and gefitinib. The above dosages and regimens of the active agents were used similarly in the combination.

[0110] Treatment efficacy was evaluated as TVI% in treated mice versus control mice using the following formula. TVI% = (1 - average volume of treated tumor mass / average volume of control tumor) × 100.

[0111] When the tumor reached a volume of approximately 10% of the total body weight, or when the body weight of the mice decreased by more than 20% over a period of 7 days or more compared to the control animals, the mice were sacrificed. The animals were euthanized using carbon dioxide exposure according to standard procedures (Directive 2010 / 63 / EU; Annex IV of 2010).

[0112] MOLM - 13 model For the AML xenograft model, 10 × 10 6Individual MOLM-13 cells were resuspended in 0.2 ml of BME type III (Trevigen) at 5.6 mg / ml + DPBS (1:1), and then subcutaneously injected into the right flank of 6- to 8-week-old female CD-1 mice (Charles River, Calco, Italy).

[0113] After injection, the mice were maintained in micro-isolator cages under environmental conditions that were continuously monitored. Drinking water and a specific sterilized diet (VRF1, Charles River) were provided ad libitum. Environmental conditions, as well as the procedures for animal housing and handling, were in accordance with the UKCCCR guidelines (Workman P et al., 2010) and the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (Directive 2010 / 63 / EU; 2010). Tumor growth and body weight were evaluated and recorded twice a week.

[0114] The results were evaluated as follows: Tumor volume was measured with calipers, and tumor mass was calculated using the following formula: [length (mm) × width 2 (mm) × d] / 2, assuming a density d = 1 mg / mm 3 for the tumor tissue (Teicher B. Totowa, 1997). The average tumor volume was 200 - 300 mm 3When it reached (corresponding to the 15th day in Figure 3A), the animals were randomly assigned to four groups (6 - 9 mice / group), and the following treatments were administered starting on the 15th day: Group I received 0.5% vehicle carboxymethylcellulose (gefitinib diluent solution) orally once daily for 14 days (i.e., until the 28th day (including this day), see Figure 3A), Group II received 25 mg / kg of MEN orally once daily for 14 days (i.e., until the 28th day (including this day), see Figure 3A), Group III received 3 mg / kg of gefitinib orally once daily for 14 days (i.e., until the 28th day (including this day), see Figure 3A), and Group IV received a combination of MEN and gefitinib. The above - mentioned dosages and regimens of the active agents were also used in the combination.

[0115] Treatment efficacy was evaluated as TVI% in treated mice versus control mice using the following formula. TVI%=(1 - average volume of treated tumor mass / average volume of control tumor)×100.

[0116] When the tumor reached a volume of approximately 10% of the total body weight, or when the body weight of the mouse decreased by more than 20% over 7 days or more compared to the control animals, the mice were sacrificed. In accordance with standard procedures (Directive 2010 / 63 / EU; Annex IV of 2010), the animals were euthanized using carbon dioxide exposure.

[0117] Statistical Analysis GraphPad Prism software (GraphPAD Software Inc., California) was used for statistical analysis. Statistical differences were considered significant with a p - value < 0.05 using the two - sided Mann - Whitney rank sum test. The in - vivo data (Figures 2 - 3) are presented as means, and the values for each group are represented as symbols of different shapes and lines of different colors.

[0118] Example 3: In - vitro Study in Cell Line MOLM - 13 In a standard in vitro cytotoxicity experiment, the AML cell line MOLM-13 was treated with MEN and crizotinib (type II inhibitor) as a single agent or in combination for 72 hours. The synergistic effect of the combination was measured according to the combination index calculation (Chou TC, 2006).

[0119] The synergistic effect of the combination of MEN and crizotinib in MOLM.13 cells is shown in Table 5 (see Table 5, CI < 1.0). The combination of MEN and crizotinib at the IC50 concentration and IC75 concentration induced a cytotoxicity significantly different from that of crizotinib-induced cytotoxicity in MOLM-13 cells (Tukey's multiple comparison one-way ANOVA test; p < 0.05 * , see Figures 4A and B).

[0120]

Table 5

[0121] Materials and Methods Human Cell Lines The human acute myeloid leukemia cell line MOLM-13 was obtained from DSMZ, Braunschweig, Germany (ACC554). MOLM-13 cells were grown in RPMI containing 20% FBS and incubated at 37°C, 5% CO2, and 80% relative humidity.

[0122] Drugs MEN1703 (alternatively referred to as "Men", "MEN", "MEN1703" or "SEL24 / MEN1703") (batch number A / 2201 / 24 / 1), in its HCl salt form (CAS number 2769008-22-), which is the hydrochloride salt of 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazol-4-amine synthesized by Aptuit(VR), and quizartinib (alternatively referred to as "Quiz". MedChem Express, Monmouth Junction, New Jersey, USA, batch number 20907) were dissolved in DMSO, aliquoted, and stored at -80 °C until use. The stock solutions were added at appropriate concentrations in the culture medium prior to addition to the cells.

[0123] Cytotoxicity For the cytotoxicity study, cells were seeded at an appropriate density (50,000 cells / well) on day 0, and then MEN and Quiz were added. The concentration ranges were determined beforehand in a single-agent cytotoxicity assay at the same time points in order to include the IC10, IC25, IC50, and IC75 values for each drug in the combination experiment (see Table 5). After 72 hours, CellTiter 96 Aqueous One Solution Reagent (MTS) (Promega, Madison, Wisconsin, USA) was added to evaluate cell viability. Fluorescence was measured using a Tecan Infinite M200 (Tecan Trading AG, Switzerland) 4 hours later, and the absorbance at 490 nm was recorded. The quantitative measurement of synergism / antagonism was evaluated using CompuSyn (ComboSyn, Inc., Paramus, New Jersey, USA) with the combination index (CI) for the fraction affected (Fa) (Chou TC, 2006).

[0124] Example 4: In Vitro Study in Cell Line KG-1 In a standard in vitro cytotoxicity experiment, the AML cell line KG-1 (FLT wild-type) was treated with MEN and Gilt (type I inhibitor) as a single agent or in combination for 72 hours. The synergistic effect of the combination was measured according to the combination index calculation (Chou TC, 2006).

[0125] The synergistic effect of the combination of MEN and Gilt in KG-1 cells is shown in Table 6 (see Table 6, CI < 1.0). The combination of MEN at the IC50 concentration and Gilt at the IC75 concentration induced cytotoxicity significantly different from MEN1703-induced cytotoxicity in KG-1 cells (Tukey's multiple comparison one-way ANOVA test; p < 0.0158 * see Figure 5A). The combination of MEN at the IC25 concentration and Gilt at the IC75 concentration induced cytotoxicity significantly different from MEN1703-induced cytotoxicity in KG-1 cells (Tukey's multiple comparison one-way ANOVA test; p < 0.0005 *** see Figure 5B).

[0126]

Table 6

[0127] Materials and Methods Human Cell Lines The human acute myeloid leukemia cell line KG-1 was obtained from DSMZ, Braunschweig, Germany (ACC14). KG-1 cells were grown in RPMI + 10% FBS and incubated at 37°C, 5% CO2, and 80% relative humidity.

[0128] Drugs MEN1703 (alternatively referred to as "Men", "MEN", or "SEL24 / MEN1703") (batch number A / 2201 / 24 / 1), in HCl salt form (CAS number 2769008-22-), of the IUPAC name 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazol-4-amine hydrochloride, synthesized by Aptuit(VR), and gilteritinib (alternatively also referred to as "Gilt". MedChem Express, Monmouth Junction, New Jersey, USA) were dissolved in DMSO, aliquoted, and stored at -80 °C until use. The stock solutions were added at appropriate concentrations in culture medium prior to addition to cells.

[0129] Cytotoxicity For the cytotoxicity study, cells were seeded at an appropriate density (50,000 cells / well) on day 0, and then MEN and Gilt were added. The concentration ranges were determined beforehand in a single-agent cytotoxicity assay at the same time points in order to include the IC10, IC25, IC50, and IC75 values for each drug in the combination experiment (see Table 6). After 72 hours, CellTiter 96 Aqueous One Solution Reagent (MTS) (Promega, Madison, Wisconsin, USA) was added to evaluate cell viability. Fluorescence was measured using a Tecan Infinite M200 (Tecan Trading AG, Switzerland) 4 hours later, and the absorbance at 490 nm was recorded. The quantitative measurement of synergism / antagonism was evaluated using CompuSyn (ComboSyn, Inc., Paramus, New Jersey, USA) with the combination index (CI) for the fraction affected (Fa) (Chou TC, 2006).

[0130] 4. References: Chou TC, Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies, Pharmacol Rev, 2006 Czardybon W., et al., A novel, dual pan-PIM / FLT3 inhibitor SEL24 exhibits broad therapeutic potential in acute myeloid leukemia. Oncotarget, 2018, 9(24), 16917-16931 Directive 2010 / 63 / Eu of The European Parliament And Of The Council On The Protection Of Animals Used For Scientific Purposes, September 22, 2010 Kennedy VE and Smith CC, FLT3 mutations in acute myeloid leukemia: key concepts and emerging controversies, Frontiers in Oncology, 2020, 10:612880 Levis and Perl, Gilteritinib: potent targeting of FLT3 mutations in AML, Blood advances, 2020, 4(6), 1178-1191 Nakao M., et al., Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia, 1996, 10(12), 1911-1918 Qiao et al., The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML, 2021, Blood Cancer Journal, 11:111 Teicher B. Totowa. Anticancer Drug Development Guide. New Jersey: Humana Press, 1997 Workman P. et al., Guidelines for The Welfare And Use Of Animals In Cancer Research. British Journal Of Cancer, 2010, 102, 1555-1577 Yuan et al., Dual FLT3 inhibitors: Against the drug resistance of acute myeloid leukemia in recent decade. European Journal of Medicinal Chemistry, 2019, 178, 468-483 Zhang et al., Rapid and efficient response to gilteritinib and venetoclax-based therapy in two AML patients with FLT3-ITD mutation unresponsive to venetoclax plus azacitidine, Onco Targets and Therapy, 2022:15, 159-164

Claims

1. A combination of (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703 for use as a medicament.

2. The combination for use according to claim 1, wherein the FLT3 inhibitor is selected from the group consisting of gilteritinib, quizartinib, midostaurin, and combinations thereof.

3. A combination of (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703 for use in the treatment of a patient suffering from cancer.

4. The combination for use according to claim 3, wherein the cancer is a hematological cancer.

5. The combination for use according to claim 3 or claim 4, wherein the cancer is acute myeloid leukemia (AML).

6. The combination for use according to any one of claims 1 to 5, wherein SEL24 / MEN1703 is administered at a daily dose of about 50 mg to about 150 mg.

7. The combination for use according to any one of claims 2 to 6, wherein gilteritinib is administered at a daily dose of about 40 mg to about 400 mg.

8. The combination for use according to any one of claims 2 to 7, wherein quizartinib is administered at a daily dose of about 10 mg to about 50 mg.

9. The combination for use according to any one of claims 2 to 8, wherein midostaurin is administered at a daily dose of about 20 mg to about 80 mg.

10. The combination for use according to any one of claims 1 to 9, wherein (i) and (ii) are administered as separate dosage forms.

11. The combination for use according to any one of claims 1 to 10, wherein (i) and (ii) are administered orally.

12. A kit of dosage forms comprising a dosage form containing (i) an FLT3 inhibitor and a dosage form containing (ii) SEL24 / MEN1703.

13. The kit according to claim 12, wherein the FLT3 inhibitor is selected from the group consisting of gilteritinib, quizartinib, midostaurin, and combinations thereof.

14. A dosage form comprising (i) an FLT3 inhibitor and (ii) SEL24 / MEN1703.

15. The dosage form according to claim 14, wherein the FLT3 inhibitor is selected from the group consisting of gilteritinib, quizartinib, midostaurin, and combinations thereof.

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