Combination therapy for cancer comprising FLT3 inhibitors

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 the treatment of patients with cancer. The invention also relates to a kit of dosage forms comprising (i) a dosage form comprising an FLT3 inhibitor and (ii) a dosage form comprising SEL24 / MEN1703; and a dosage form comprising (i) an FLT3 inhibitor, and (ii) SEL24 / MEN1703.

Description

Technical Field

[0001] The present invention belongs to the field of cancer therapy. More specifically, the present invention relates to a combination of (i) a FLT3 inhibitor and (ii) SEL24 / MEN1703 on the one hand, which is used as a medicine. On the other hand, the present invention relates to a combination of (i) a FLT3 inhibitor and (ii) SEL24 / MEN1703, for treating patients with cancer. On the other hand, the present invention relates to a kit of dosage form, which comprises (i) a dosage form comprising a FLT3 inhibitor and (ii) a dosage form comprising SEL24 / MEN1703. On the other hand, the present invention relates to a dosage form, which comprises (i) a FLT3 inhibitor and (ii) SEL24 / MEN1703. Background Art

[0002] FLT3 mutations in AML patients have been known for more than 25 years now, with the most common FLT3-ITD ("internal tandem duplication") mutation first reported in 1996 (see Nakao M et al., 1996). Such mutations result in constitutively active FLT3, which has kinase activity. Therefore, FLT3-inhibitors designed to treat diseases were subsequently developed, and clinical trials of FTL3 inhibitors in AML recruited the first patients in 2002, as summarized by Levis and Perl, 2020. Kennedy and Smith, 2020's review summarizes the understanding of FLT3 mutations in AML, and in particular provides an overview of confirmed FTL3 inhibitors (see Table 1 therein, in which first and second generation inhibitors are given and in which the types of inhibition are given) and confirmed FLT3 inhibitors in newly diagnosed AML clinical trials (see Table 2 therein). Kennedy and Smith, 2020 further summarizes clinical trials of confirmed FLT3 inhibitors in combination therapy (see Table 4 therein) or clinical trials of novel FLT3 inhibitors including inhibitors with dual inhibitory activity ("dual agents", see Table 5 therein). The review by Yuan et al., 2019 focuses on such dual FLT3 inhibitors.

[0003] The first approved FLT3 inhibitor was midostaurin, which is approved for patients with AML that has a FLT3 mutation ( Approved in the United States and Europe in 2017). Further FLT3 inhibitors were tested, including quizartinib. The use of quizartinib has shown limitations that can arise during treatment, namely resistance due to on-target kinase-activating FLT3 mutations, which usually occurs within two to four months of starting quizartinib.

[0004] Thus, there remains a need for improvements in initial or "first generation" FLT3 inhibitors.

[0005] Taking into account the results of the initial FLT3 inhibitors, further inhibitors were developed or are being developed, including gilteritinib. Clinical studies with gilteritinib have shown that gilteritinib therapy is more effective and less toxic than standard chemotherapy. Subsequently, products containing gilteritinib have received marketing authorization for patients with AML with FLT3 mutations, particularly in the United States and Europe. Notably, on the basis of demonstrated superiority over existing therapies, It has been approved by the FDA for use in the corresponding patient population. Administered as monotherapy.

[0006] However, since resistance to gilteritinib still exists, and since responses to gilteritinib monotherapy may therefore be short-lived, there is still a need for improved therapy, and the first clinical trials investigating gilteritinib, for example in combination with intensive induction chemotherapy or less toxic agents, have been initiated and have shown promising results (see Levis and Perl, 2020). A further example of such a combination is the combination of CUDC-907 (a dual inhibitor of PI3K and histone deacetylase) with gilteritinib, which has shown promising anti-leukemic activity against FLT3-ITD AML in vitro and in vivo (see Qiao et al., 2021). In addition, gilteritinib has been studied as an additional active added to different frameworks, for example for patients who do not respond to venetoclax plus azacitidine (see Zhang et al., 2022), where gilteritinib is administered to such unresponsive patients together with venetoclax and azacitidine (induction therapy, where maintenance therapy is venetoclax and gilteritinib), with promising results.

[0007] Therefore, there remains a need for improvements in more recent or “second generation” FLT3 inhibitors, particularly gilteritinib.

[0008] Purpose and content of the invention

[0009] The inventors of the present invention have surprisingly found that the combination of a FLT3 inhibitor and SEL24 / MEN1703 acts synergistically and therefore corresponds to a very promising novel combination therapy, in particular in patients with acute myeloid leukemia (AML).

[0010] In a first aspect, the present invention relates to a combination of (i) a 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 midostaurin, gilteritinib, lestaurtinib and crenolanib.

[0012] In another preferred embodiment, the FLT3 inhibitor is a type II FLT3 inhibitor, which can be selected from quizartinib and sorafenib.

[0013] In a preferred embodiment, the FLT3 inhibitor is selected from gilteritinib, quizartinib, midostaurin and a combination thereof. Preferably, the FLT3 inhibitor is gilteritinib.

[0014] In embodiments, SEL24 / MEN1703 is administered at a daily dose of about 50 mg to about 150 mg. Preferably, 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. Preferably, SEL24 / MEN1703 is administered once daily. Further preferably, SEL24 / MEN1703 is administered orally.

[0015] In an embodiment, gilteritinib is administered at a daily dose of about 40 mg to about 400 mg. Preferably, 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. Preferably, gilteritinib is administered once daily. Further preferably, gilteritinib is administered orally.

[0016] In an embodiment, quizartinib is administered at a daily dose of about 10 mg to about 50 mg. Preferably, quizartinib 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, quizartinib is administered at a daily dose of about 15 mg, about 17 mg, about 20 mg, or about 25 mg. Preferably, quizartinib is administered once daily. Further preferably, quizartinib is administered orally.

[0017] In an embodiment, midostaurin is administered at a daily dose of about 20 mg to about 80 mg. Preferably, 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. Preferably, midostaurin is administered twice daily. Further preferably, midostaurin is administered orally.

[0018] In an embodiment, the combination for use of the first aspect does not comprise the administration of an additional chemotherapy.

[0019] In an embodiment involving a combination for use as a medicament, (i) and (ii) are administered in separate dosage forms. In this embodiment, administration may be concomitant or sequential. In yet an alternative embodiment involving a combination for use as a medicament, (i) and (ii) are administered together in a dosage form.

[0020] In a second aspect, the invention relates to a combination of (i) a FLT3 inhibitor and (ii) SEL24 / MEN1703 for use in treating 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 midostaurin, gilteritinib, lestaurtinib and cleranib.

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

[0023] In a preferred embodiment, the FLT3 inhibitor is selected from gilteritinib, quizartinib, midostaurin and a combination thereof. Preferably, the FLT3 inhibitor is gilteritinib.

[0024] In preferred embodiments, the cancer is a hematological cancer. In even more preferred embodiments, the cancer is a leukemia. In a most preferred embodiment, the cancer is AML. The combination for the purposes of the second aspect can be a first-line treatment for AML. However, the combination for the purposes of the second aspect can alternatively be used to treat patients who have progressed in a previous line of treatment. In one embodiment, the combination of the present invention can be used to treat recurrent or recurrent AML, or refractory AML.

[0025] When the patient suffers from AML, preferably, the patient with AML shows an overactivated FLT3 mutation causing FLT3 signal transduction. The mutation in FLT3 can even cause constitutively active FLT3 signal transduction (this means that the signal transduction activity of FLT3 is constitutively active). The FLT3 mutation is caused by at least one base mutation in the FLT3 gene, resulting in the above-mentioned FLT3 mutation at the protein level, which causes the overactivation of FLT3 signal transduction. Such mutations are known in the art, as mentioned in the technical background section above. Preferably, the FLT3 mutation is a combination of FLT3-ITD mutation, FLT3-TKD mutation or FLT3-ITD mutation and FLT3-TKD mutation. Additionally or alternatively, the patient with cancer (including the 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, the patient with cancer may not be suitable for chemotherapy.

[0026] However, when the patient has AML, the patient may also exhibit a FLT3 wild-type sequence. Additionally or alternatively, the patient with cancer may not be suitable for chemotherapy.

[0027] In embodiments, SEL24 / MEN1703 is administered at a daily dose of about 50 mg to about 150 mg. Preferably, 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. Preferably, SEL24 / MEN1703 is administered once daily. Further preferably, SEL24 / MEN1703 is administered orally.

[0028] In an embodiment, gilteritinib is administered at a daily dose of about 40 mg to about 400 mg. Preferably, 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. Preferably, gilteritinib is administered once daily. Further preferably, gilteritinib is administered orally.

[0029] In an embodiment, quizartinib is administered at a daily dose of about 10 mg to about 50 mg. Preferably, quizartinib 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, quizartinib is administered at a daily dose of about 15 mg, about 17 mg, about 20 mg, or about 25 mg. Preferably, quizartinib is administered once daily. Further preferably, quizartinib is administered orally.

[0030] In an embodiment, midostaurin is administered at a daily dose of about 20 mg to about 80 mg. Preferably, 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. Preferably, midostaurin is administered twice daily. Further preferably, midostaurin is administered orally.

[0031] In an embodiment, the combination for use in the second aspect, in particular for use in the treatment of AML, does not comprise the administration of additional chemotherapy.

[0032] In an embodiment involving a combination for treating a patient suffering from cancer, (i) and (ii) are administered as separate dosage forms. In this embodiment, administration may be performed concomitantly or sequentially. In yet an alternative embodiment involving a combination for treating a patient suffering from cancer, (i) and (ii) are administered together in a 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, preferably, the patient with AML exhibits a FLT3 mutation, and optionally exhibits at least one IDH1 and / or IDH2 mutation.

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

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

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

[0037] In a preferred embodiment, the FLT3 inhibitor is selected from gilteritinib, quizartinib, midostaurin and a combination thereof. Preferably, the FLT3 inhibitor is gilteritinib.

[0038] In an embodiment, a dosage form comprising SEL24 / MEN1703 comprises SEL24 / MEN1703 in an amount of about 50 mg to about 150 mg, preferably 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, and most preferably about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, or about 125 mg. Preferably, the dosage form comprising SEL24 / MEN1703 is a once-daily dosage form. Further preferably, the dosage form comprising SEL24 / MEN1703 is an oral dosage form.

[0039] In an embodiment, the dosage form comprising gilteritinib as a FLT3 inhibitor comprises gilteritinib in an amount of about 40 mg to about 400 mg, preferably about 40 mg, about 80 mg, about 120 mg or about 240 mg, most preferably about 100 mg, about 105 mg, about 110 mg, about 115 mg or about 120 mg. Preferably, the dosage form comprising gilteritinib as a FLT3 inhibitor is a once-daily dosage form. Further preferably, the dosage form comprising gilteritinib as a FLT3 inhibitor is an oral dosage form.

[0040] In an embodiment, the dosage form comprising quizartinib as a FLT3 inhibitor comprises quizartinib in an amount of about 10 mg to about 50 mg, preferably about 10 mg, about 20 mg, about 30 mg, about 40 mg or about 50 mg, most preferably about 15 mg, about 17 mg, about 20 mg or about 25 mg. Preferably, the dosage form comprising quizartinib as a FLT3 inhibitor is a once-daily dosage form. Further preferably, the dosage form comprising quizartinib as a FLT3 inhibitor is an oral dosage form.

[0041] In an embodiment, a dosage form comprising midostaurin as a FLT3 inhibitor comprises 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, most preferably about 35 mg, about 40 mg, about 45 mg or about 50 mg. Preferably, the dosage form comprising midostaurin is a twice-daily dosage form. Further preferably, the dosage form comprising midostaurin is an oral dosage form.

[0042] Each dosage form generally contains at least one pharmaceutically acceptable excipient, as defined in Section 2 of the Detailed Description below.

[0043] In an embodiment, the kit of the third aspect further comprises a booklet listing instructions on how to use and administer the dosage form.

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

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

[0046] In another preferred embodiment, the FLT3 inhibitor contained in the dosage form is a type II FLT3 inhibitor, and the type II FLT3 inhibitor can be selected from quizartinib and sorafenib.

[0047] In a preferred embodiment, the FLT3 inhibitor is selected from gilteritinib, quizartinib, midostaurin and a combination thereof. Preferably, the FLT3 inhibitor is gilteritinib.

[0048] In an embodiment, a dosage form comprising SEL24 / MEN1703 comprises 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, and 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] If the dosage form comprises gilteritinib as the FLT3 inhibitor, in an embodiment, the dosage form comprises gilteritinib in an amount of about 40 mg to about 400 mg, preferably about 40 mg, about 80 mg, about 120 mg or about 240 mg, most preferably about 100 mg, about 105 mg, about 110 mg, about 115 mg or about 120 mg.

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

[0051] If the dosage form comprises midostaurin as the FLT3 inhibitor, the dosage form comprises 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, most preferably about 35 mg, about 40 mg, about 45 mg or about 50 mg.

[0052] In an embodiment, the dosage form is a once-daily or twice-daily dosage form. Further preferably, the dosage form is an oral dosage form.

[0053] The dosage form generally contains at least one pharmaceutically acceptable excipient, as defined in Section 2 of the Detailed Description below.

[0054] In an embodiment, the dosage form of the fourth aspect is accompanied by a leaflet setting out instructions on how to use and administer the dosage form.

[0055] In a fifth aspect, the invention relates to a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of (i) a 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] Figure 1 : Statistical analysis of cytotoxicity data at IC75 concentrations in MV4-11 cells ("men" = MEN, "gilt" = Gilt, "men+gilt" = "MEN+Gilt"). The cytotoxicity induced by the MEN+Gilt combination was significantly different from that induced by gilteritinib (Student's t-test, p = 0.0331).

[0058] Figure 2: In vivo studies in AML MV4-11 cell line xenografts. Tumor cells were injected sc into SCID mice on day 0 and treatment with MEN ("MEN1703"), Gilentinib, MEN+Gilt combination or control (vehicle only) was started on day 19. The first arrow under the x-axis shows MEN administration (from day 19 to day 32), while the second arrow under the x-axis shows Gilt administration (from day 19 to day 40). Statistical analysis was performed according to the Mann-Whitney rank test, with evaluations performed on days 33 and 40. Tumor growth inhibition with the combination of MEN+Gilt was significantly different from that of the monotherapy and control groups. (A) shows tumor volume over time for monotherapy as well as combination treatment, while (B) shows tumor volume at day 33.

[0059] Fig. 3: in vivo study in AML MOLM-13 cell line xenograft.Tumor cells were injected sc into SCID mice at day 0, and drugs were administered using MEN ("MEN1703"), Gileritinib, MEN+Gilt combination or control (vehicle only) starting at day 15.The first arrow under the x-axis shows MEN administration (from day 15 to day 28), while the second arrow under the x-axis shows Gilt administration (from day 15 to day 28).Statistical analysis was performed according to the Mann-Whitney rank test and evaluated at day 29.The tumor growth inhibition with the combination of MEN+Gilt was significantly different from the control group.(A) shows the tumor volume of monotherapy and combination therapy over time, while (B) is shown in the tumor volume at day 29.

[0060] Fig. 4: (A) Statistical analysis of cytotoxicity data in MOLM13 cell line at IC50 concentration and (B) IC75 concentration ("Quiz" = Quizartinib, "MEN1703 + Quiz" = "MEN1703 + Quizartinib"). The cytotoxicity induced by the MEN1703 + Quiz combination was significantly different from that induced by Quizartinib (Tukey multiple comparisons one-way ANOVA analysis; p < 0.05 *).

[0061] Figure 5: Statistical analysis of cytotoxicity data in FLT3 wt KG1 cell line at (A) MEN1703 IC50 and gilteritinib IC75 concentrations and (B) MEN1703 IC25 and gilteritinib IC75 concentrations. The cytotoxicity induced by the combination of MEN1703 IC50 + gilteritinib IC75 and MEN1703 IC25 + gilteritinib IC75 was significantly different from that of MEN1703 as a single agent (Tukey's multiple comparison one-way ANOVA test; p = 0.0158*, p = 0.0005***, respectively). DETAILED DESCRIPTION OF THE INVENTION

[0063] Before describing the present invention in more detail, the following definitions are introduced.

[0064] 1. definition

[0065] As used in the specification and claims, the singular forms "a", "an" and "the" include the corresponding plural forms as well, unless the context clearly dictates otherwise.

[0066] The term "about" in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand as still ensuring the technical effect of the feature in question. The term generally denotes a deviation of ±10% from the indicated numerical value, and preferably ±5%.

[0067] It should be understood 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". If a group is defined below as comprising at least a certain number of embodiments, this also means including a group that preferably consists only of these embodiments.

[0068] As used herein, the term "combination" or "combined with ... " is not intended to imply that therapy or active agents (i) and (ii) must be administered simultaneously and / or formulated for delivery together, although such therapies and preparations are within the scope of the present invention. The active agents in the combination can be administered simultaneously, before or after each other and even one or more other additional therapies or active agents. The active agent or treatment regimen can be administered in any order. Typically, each active agent will be administered with a dosage and / or time schedule determined for the active agent. In addition, typically, it is contemplated that the active agent used in the combination is administered with a dosage that does not exceed the dosage used alone. In some embodiments, the dosage used in the combination will be lower than the dosage used alone. In some embodiments, one of the two active agents is administered with a therapeutic dose or less than a therapeutic dose, for example, a FLT3 inhibitor is administered with a dose less than a therapeutic dose or SEL24 / MEN1703 is administered with a dose less than a therapeutic dose (wherein "less than a therapeutic dose" is derived from a comparison of the therapeutic dose of a single active agent in a monotherapy). A subtherapeutic dose may 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.

[0069] 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 tyrosine kinase class III.

[0070] As used herein, the term "FLT3 inhibitor" refers to a compound capable of inhibiting the kinase FLT3, particularly a mutant version of FLT3, which results in overactivation of FLT3 signaling, such as constitutively active FLT3. Such compounds are known in the art, particularly when it comes to AML treatment (see Levis and Perl, 2020). Typically, such compounds are small molecules that strongly (and, depending on the situation, may [i.e., not necessarily] selectively) bind to FLT3 so that FLT3 is inhibited. FLT3 inhibitors can be subdivided according to how they interact with the intracellular kinase domain of the FLT3 receptor. In normal physiology, the FLT3 ligand binds to the extracellular domain, resulting in FLT3 receptor dimerization, presenting an enzymatically active conformation and subsequently activating downstream signaling. Type I FLT3 inhibitors bind to receptors in an active conformation, thereby inhibiting FLT3-TKD and FLT3-ITD mutant receptors. Type II FLT3 inhibitors bind to the region near the ATP binding pocket and only inhibit receptors in an inactive conformation. Type II inhibitors are ineffective against most FLT3-TKD mutations because these mutations favor the active kinase conformation of FLT3 (see also Kennedy and Smith, 2020, paragraph spanning pages 3 and 4).

[0071] As used herein, the term "FLT3-ITD mutation" refers to a FLT3 "internal tandem duplication, ITD" mutation. As used herein, the term "FLT3-TKD mutation" refers to a FLT3 "tyrosine kinase domain" mutation.

[0072] As used herein, the term "SEL24 / MEN1703" (alternatively referred to herein as "MEN," "Men," "men," or "Men1703") refers to the compound 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazole, in the form of a free base or a pharmaceutically acceptable salt thereof (e.g., an HCl salt). The CAS number for the free base form is 1616359-00-2, while the CAS number for the HCl salt form is 2769008-22-0. The compound is a dual pan-PIM / FLT3 inhibitor that has been shown, inter alia, to inhibit the growth of a broad group of AML cell lines in xenograft models. The rationale for developing this dual inhibitor is that PIM kinases are believed to be the main driver of resistance to FLT3 inhibitors. SEL24 / MEN1703 is characterized in more detail, e.g., in 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 fails to disclose the combination of SEL24 / MEN1703 with a FLT3 inhibitor, let alone the combination of any compound within the scope of WO 2014 / 096388 with a FLT3 inhibitor.

[0073] As used herein, the term "gilteritinib" refers to the FLT3 inhibitor gilteritinib, marketed under the trade name Gilteritinib is marketed and is approved for the treatment of AML. More details about Gilteritinib can be found in particular in the product brochure or in the regulatory dossier.

[0074] As used herein, the term "quizartinib" refers to the FLT3 inhibitor quizartinib, which is manufactured by Daiichi Sankyo Europe GmbH under the name A marketing authorization for quizartinib was applied for in Europe. The EMA issued an opinion in 2019 recommending the refusal of a marketing authorization because, in the EMA's opinion, the benefits of quizartinib treatment did not outweigh its risks.

[0075] As used herein, the term "midostaurin" refers to the FLT3 inhibitor midostaurin, available under the trade name marketed, it is approved for the treatment of AML, where at the start of treatment, It must always be used together with chemotherapy. More details about midostaurin can be found in particular in the product leaflet or in the regulatory dossier.

[0076] As used herein, the term "overactivation" of FLT3 means that FLT3 is more active than the wild-type state, particularly more active in downstream activation and signaling, thereby leading to cancer cell growth.

[0077] As used herein, the term "small molecule" refers to a small organic compound with a low molecular weight. The small molecule in the context of the present invention preferably has 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 a particularly preferred embodiment, the small molecule in the context of the present invention has a molecular weight of less than 800 daltons. In another preferred embodiment, the small molecule in the context of the present invention has a molecular weight of 50 to 3000 daltons, preferably 100 to 2000 daltons, more preferably 100 to 1500 daltons, and even more preferably 100 to 1000 daltons.

[0078] As used herein, the term "treatment" refers to clinical intervention performed in order to cure or ameliorate a disease, prevent recurrence of a disease, alleviate symptoms of a disease, reduce any direct or indirect pathological consequences of a disease, achieve a stable state of disease (i.e., not worsening), prevent metastasis, reduce the rate of disease progression, and / or prolong survival compared to expected survival if not receiving treatment.

[0079] As used herein, the term "relapsed or relapsed AML" refers to AML that reappears after treatment (with a different drug, optionally a combination of drugs, such as the combination of the present invention) and remission.

[0080] As used herein, the term "refractory AML" refers to a leukemia that has not responded to previous treatment (using different drugs, optionally a combination of drugs, such as the combination of the present invention).

[0081] As used herein, the term "IDH1 and / or IDH2 mutation" refers to a mutation in the "isocitrate dehydrogenase 1" gene or the "isocitrate dehydrogenase 2" gene encoding the corresponding isocitrate dehydrogenase.

[0082] 2. Pharmaceutical composition

[0083] "FLT3 inhibitors" and "SEL24 / MEN1703" are "pharmaceutically active agents" or "active agents" for the purposes of the present invention. As described above, they may be present in separate dosage forms or contained in a single dosage form.

[0084] As used herein, a "pharmaceutically active agent" refers to a compound that effectively modulates a response in a patient (i.e., a human or animal). As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient commonly included in a pharmaceutical dosage form or composition known to those skilled in the art. Examples of such excipients are as follows. In view of the definition of "pharmaceutically active agent" given above, a pharmaceutically acceptable excipient can be defined as pharmaceutically inactive.

[0085] If a marketed FLT3 inhibitor is used in combination with SEL24 / MEN1703, it is generally preferred that administration occurs via a different dosage form, and the FLT3 inhibitor is in the dosage form and administered via an approved route of administration. SEL24 / MEN1703 can be administered in a dosage form as listed below or in a dosage form currently undergoing clinical trials.

[0086] The dosage form used according to the present invention can 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 form of the present invention can also be specified as a preparation or a pharmaceutical composition.

[0087] In general, the pharmaceutical composition according to the present invention may include various pharmaceutically acceptable excipients, which are selected according to the functionality to be achieved by the composition. "Pharmaceutically acceptable excipient" in the meaning of the present invention can be any substance used to prepare a pharmaceutical dosage form, including coating materials, film-forming materials, fillers, disintegrants, release-modifying materials, carrier materials, diluents, adhesives and other adjuvants. Typical pharmaceutically acceptable excipients include substances such as sucrose, mannitol, sorbitol, starch and starch derivatives, lactose and lubricants (e.g., magnesium stearate), disintegrants and buffers.

[0088] The term "carrier" refers to a pharmaceutically acceptable organic or inorganic carrier material with which the active ingredient is combined to facilitate application. Suitable pharmaceutically acceptable carriers include, for example, water, saline solutions, alcohols, oils, preferably vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, surfactants, essential oils, fatty acid monoglycerides and diglycerides, petroleum ether fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical composition can be sterilized and, if necessary, can be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavoring agents and / or aromatic substances, etc., which do not react adversely with the active compound.

[0089] If liquid dosage forms are contemplated by the present invention, these may 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 imparting volume, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners / flavoring agents.

[0090] For parenteral use, particularly suitable vehicles are composed of solutions, preferably oily or aqueous solutions, and suspensions, emulsions or implants. Pharmaceutical preparations for parenteral administration are particularly preferred, and include aqueous solutions of water-soluble forms. In addition, suspensions can 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 can contain materials that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

[0091] For injectable preparations, sterile injectable aqueous or oily suspensions can be prepared, for example, using suitable dispersants, wetting agents and / or suspending agents according to known techniques. Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenteral acceptable diluents or solvents. Acceptable vehicles and solvents that can be used are water and isotonic sodium chloride solutions. Sterile oils are also commonly used as solvents or suspension media.

[0092] Suppositories for rectal administration of the pharmaceutical compositions of this invention can be prepared, for example, by mixing the compound with suitable non-irritating excipients such as cocoa butter, synthetic triglycerides and polyethylene glycols which are solid at room temperature but liquid at the rectal temperature so that they melt in the rectum and release the active agent from the suppository.

[0093] For administration by inhalation, the pharmaceutical compositions according to the invention may conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant, for example 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 containing a powder mix of the compound and a suitable powder base, for example lactose or starch.

[0094] Oral dosage forms can be liquid or solid, and include, for example, tablets, lozenges, pills, capsules, powders, effervescent preparations, dragees and granules. Pharmaceutical preparations for oral use can be obtained as solid excipients, optionally grinding the resulting mixture, and processing the granular mixture after adding suitable adjuvants (if necessary) to obtain tablets or dragee cores. Suitable excipients are particularly fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or its salt, such as sodium alginate, can be added. Oral dosage forms can be formulated to ensure the immediate release of the active agent or the sustained release of the active agent. 3. Example

[0095] The following examples are merely illustrative and will further describe the present invention, but these examples should not be construed as limiting the present invention thereto.

[0096] Example 1: In vitro studies in cell lines

[0097] In a standard in vitro cytotoxicity assay, two AML cell lines (MV4-11 [FLT3 ITD] and MOLM-13 [FLT3ITD]) were treated with MEN and Gilt (type I inhibitors) as individual agents or in combination for 72 hours. The synergistic effect of the combination was measured according to the combination index calculation (Chou TC, 2006). At all concentration levels, the combination of MEN and Gilt was slightly synergistic in MV4-11 cells (see Table 1). The cytotoxicity induced by the combination of MEN and Gilt at the IC75 concentration was significantly different from the cytotoxicity induced by Gilter in MV4-11 cells (Student's t test, p = 0.0331; see Figure 1 The combination of MEN and Gilt did show synergy at IC25 and IC10 concentration levels in MOLM-13 cells (see Table 2).

[0098] Table 1: In vitro combination index in MV4-11 cells

[0099]

[0100] The combination of MEN+Gilt was moderately synergistic (CI≤1) at different concentration levels. IC75 and IC50 are clinically relevant concentrations (grey rows).

[0101] Table 2: In vitro combination index in MOLM-13 cells

[0102]

[0103] The combination of MEN+Gilt was synergistic (CI≤1) at concentration levels IC10 and IC25.

[0104] Materials and methods

[0105] Human cell lines

[0106] Human acute myeloid leukemia cell lines MV4-11 and MOLM-13 are obtained from DSMZ, Braunschweig, Germany (ACC102 and ACC554). MV4-11 cells are cultivated in RPMI 1640 culture medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, SaintLouis, CA, USA). MOLM-13 cells are grown in RPMI with 20% FBS. All cells are incubated at 37°C, 5% CO2 and 80% relative humidity.

[0107] drug

[0108] MEN1703 (alternatively referred to as "Men", "MEN" or "SEL24 / MEN1703"), in HCl salt form (CAS No. 2769008-22-), whose IUPAC name is 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazol-4-amine hydrochloride (lot A / 2201 / 24 / 1), synthesized at Aptuit (VR), and gilteritinib (alternatively referred to as "Gilt", MedChem Express, Monmouth Junction, NJ, USA) were dissolved in DMSO, aliquoted and stored at -80°C until use. The stock solution was added to the culture medium at the appropriate concentration before addition to the cells.

[0109] Cytotoxicity

[0110] For cytotoxicity studies, cells were seeded at an appropriate density (50,000 cells / well) before adding MEN and Gilt on day 0. The concentration range at the same time point was previously determined in a single-agent cytotoxicity assay so that the IC10, IC25, IC50, and IC75 values ​​of each drug and each AML cell line were included in the combination experiment (see Tables 1 to 2). After 72 hours, CellTiter96Aqueous One Solution Reagent (MTS) (Promega, Madison.WI, USA) was added to assess cell viability. After 4h, fluorescence was measured using Tecan Infinite M200 (Tecan Trading AG, Switzerland), and the absorbance at 490nm was recorded. CompuSyn (ComboSyn, Inc. Paramus, NJ, USA) was used to assess the quantitative measurement of synergy / antagonism using the combination index (CI) of the affected fraction (Fa) (Chou TC, 2006).

[0111] Example 2: In vivo xenograft studies

[0112] In an in vivo xenograft study with MV4-11 (FLT3 ITD) cells, MEN and gilteritinib as single agent treatment induced significant antitumor activity at day 33, showing 57.2% and 84.1% tumor volume inhibition (TVI), respectively, while the combination treatment showed 99.2% TVI at day 33 (see Figure 2A and Table 3). In the combination treatment group, the combination treatment ended on day 33, and thereafter long-term treatment with gilteritinib alone for an additional 8 days induced durable antitumor activity until day 47. After day 47, regrowth of tumor masses was observed in the combination treatment group. On day 40, when gilteritinib treatment alone ended, single agent treatments of Men and gilteritinib showed 53.1% and 87.6% TVI, respectively, while the combination treatment showed 100% TVI (see Figure 2A and Table 3).

[0113] Table 3: In vivo tumor volume inhibition (TVI) at day 33 and day 40, respectively.

[0114]

[0115] At the end of each treatment, tumor growth inhibition was improved with the combination of MEN + gilteritinib compared to single agent treatment.

[0116] According to the Mann Whitney test evaluated on day 33, the reduction in TV was statistically significant when the single-agent treatment groups and the combination group were compared to the vehicle group (p value = 0.007; p value = 0.001 and p value = 0.002, respectively), and the combination treatment group was statistically significant when compared to both single-agent treatment groups (p value = 0.001, p value = 0.01, see Figure 2B ).

[0117] In an in vivo xenograft study with MOLM-13 (FLT3ITD) cells, MEN and gilteritinib as single-agent treatment induced significant antitumor activity at day 29, showing 38.8% and 60.1% tumor volume inhibition (TVI), respectively, while the combination treatment showed 78.3% TVI at day 29 (see Figure 3A and Table 4).

[0118] Table 4: In vivo tumor volume inhibition (TVI) at day 29.

[0119]

[0120] At the end of each treatment, tumor growth inhibition was improved with the combination of MEN+Gilt compared to single agent treatment.

[0121] The reduction in TV was statistically significant when gilteritinib alone and combination treatments were compared to the vehicle group according to the Mann Whitney test assessed on day 29 (p value = 0.04 and p value = 0.002, respectively, see Figure 3B ).

[0122] Materials and methods

[0123] Human cell lines

[0124] MV4-11 and MOLM-13 cells were obtained and cultured as described in Example 1.

[0125] drug

[0126] MEN1703 (alternatively referred to as "Men", "MEN" or "SEL24 / MEN1703"), in the form of a hydrochloride salt (CAS No. 2769008-22-), 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 at MENARINI RICERCHE SpA Pisa) was dissolved in sterile water. Gilteritinib (MedChem Express, Monmouth Junction, NJ, USA) was dissolved in a solution of carboxymethylcellulose (0.5%).

[0127] MV4-11 Model

[0128] For AML xenograft models, 10x10 6 MV4-11 cells were resuspended in 0.2 ml BME type III (Trevigen) at 5.6 mg / ml + DPBS (1:1) and then injected subcutaneously into the right flank of 6-8 week old female SCID mice (Charles River, Calco, Italy).

[0129] After injection, mice were maintained in microisolator cages under continuously monitored environmental conditions. Drinking water and a specific sterile diet (VRF1, Charles River) were provided ad libitum. Environmental conditions and procedures for housing and handling animals 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 assessed and recorded twice a week.

[0130] The outcome was assessed as follows: tumor volume was measured by caliper and tumor mass was calculated using the following formula: [length (mm) × width (mm) 2 (mm) × d] / 2, assuming that the density of tumor tissue is d = 1 mg / mm 3 (Teicher B. Totowa, 1997). When the average tumor volume reaches 200-300mm 3 (corresponding to Figure 2A The animals were randomly divided into four groups (6-7 mice / group) and the following treatments were administered starting from day 19: Group I received oral vehicle carboxymethylcellulose 0.5% (gilteritinib diluted solution) once daily for 22 days, i.e. until the end of the administration period (i.e. until day 40, see Figure 2A ), Group II received MEN at 25 mg / kg orally once daily for 14 days (i.e. until day 33, see Figure 2A ), Group III received gilteritinib at 3 mg / kg orally once daily for 22 days, i.e. until day 40, see Figure 2A ), and Group IV received a combination of MEN and gilteritinib, with the doses and schedules of the above active agents also used in the combination.

[0131] Treatment efficacy was assessed as TVI% in treated mice relative to control mice using the following formula: TVI%=(1-mean volume of treated tumor mass / mean volume of control tumor)*100.

[0132] Mice were sacrificed when tumors reached a volume of approximately 10% of total body weight or when body weight of mice decreased by more than 20% compared to control animals for 7 days or more. Animals were euthanized with carbon dioxide exposure according to standard procedures (Annex IV of Directive 2010 / 63 / EU; 2010).

[0133] MOLM-13 Model

[0134] For AML xenograft models, 10x 10 6 MOLM-13 cells were resuspended in 0.2 ml of BME type III (Trevigen) at 5.6 mg / ml + DPBS (1:1) and then injected subcutaneously into the right flank of 6-8 week old female CD-1 mice (Charles River, Calco, Italy).

[0135] After injection, mice were maintained in microisolator cages under continuously monitored environmental conditions. Drinking water and a specific sterile diet (VRF1, Charles River) were provided ad libitum. Environmental conditions and procedures for housing and handling animals 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 assessed and recorded twice a week.

[0136] The outcome was assessed as follows: tumor volume was measured by caliper and tumor mass was calculated using the following formula: [length (mm) × width (mm) 2 (mm) × d] / 2, assuming that the density of tumor tissue is d = 1 mg / mm 3 (Teicher B. Totowa, 1997). When the average tumor volume reaches 200-300mm 3 (corresponding to Figure 3AThe animals were randomly divided into four groups (6-9 mice / group) and the following treatments were administered starting on day 15: Group I received oral vehicle carboxymethylcellulose 0.5% (gilteritinib diluted solution) once daily for 14 days (i.e., until and including day 28, see Figure 3A ), Group II received MEN at 25 mg / kg orally once daily for 14 days (i.e., up to and including Day 28, see Figure 3A ), Group III received gilteritinib at 3 mg / kg orally once daily for 14 days (i.e., until and including day 28, see Figure 3A ), and Group IV received a combination of MEN and gilteritinib, with the doses and schedules of the above active agents also used in the combination.

[0137] Treatment efficacy was assessed as TVI% in treated versus control mice using the following formula: TVI%=(1-mean volume of treated tumor mass / mean volume of control tumor)*100.

[0138] Mice were sacrificed when tumors reached a volume of approximately 10% of total body weight or when body weight of mice decreased by more than 20% compared to control animals for 7 days or more. Animals were euthanized with carbon dioxide exposure according to standard procedures (Annex IV of Directive 2010 / 63 / EU; 2010).

[0139] Statistical analysis

[0140] Statistical analysis was performed using GraphPad Prism software (GraphPAD Software Inc., California). Statistical differences were considered significant at p values ​​< 0.05 using a two-tailed Mann-Whitney rank test. In vivo data (Figures 2 to 3) are presented as mean values, with values ​​for each group represented by symbols of different shapes and lines of different colors.

[0141] Example 3: In vitro studies in cell line MOLM-13

[0142] In a standard in vitro cytotoxicity assay, the AML cell line MOLM-13 was treated with MEN and quizartinib (a type II inhibitor) as single agents or in combination for 72 hours. The synergistic effect of the combined treatment was measured according to the combination index calculation (Chou TC, 2006).

[0143] The synergistic effect of the combination of MEN and quizartinib in MOLM.13 cells is shown in Table 5 (see Table 5, CI <1.0). The cytotoxicity induced by the combination of MEN and quizartinib in MOLM-13 cells at IC50 and IC75 concentrations was significantly different from that induced by quizartinib (Tukey multiple comparisons one-way ANOVA test; p <0.05*, see Figure 4A and B).

[0144] Table 5: In vitro combination index in MOLM-13 cells

[0145]

[0146] Materials and methods

[0147] Human cell lines

[0148] The human acute myeloid leukemia cell line MOLM-13 was obtained from DSMZ, Braunschweig, Germany (ACC554). MOLM-13 cells were grown in RPMI with 20% FBS and incubated at 37°C, 5% CO2, 80% relative humidity.

[0149] drug

[0150] MEN1703 (alternatively referred to as "Men", "MEN", "MEN1703" or "SEL24 / MEN1703"), in HCl salt form (CAS No. 2769008-22-), IUPAC name 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazol-4-amine hydrochloride (lot A / 2201 / 24 / 1), synthesized at Aptuit (VR), and quizartinib (alternatively referred to as "Quiz", MedChem Express, Monmouth Junction, NJ, USA, lot 20907) were dissolved in DMSO, aliquoted and stored at -80°C until use. The stock solution was added to the culture medium at the appropriate concentration prior to addition to the cells.

[0151] Cytotoxicity

[0152] For cytotoxicity studies, cells were seeded at an appropriate density (50,000 cells / well) before adding MEN and Quiz on day 0. The concentration range was previously determined in the single-agent cytotoxicity assay at the same time point so that the IC10, IC25, IC50 and IC75 values ​​of each drug were included in the combination experiment (see Table 5). After 72 hours, CellTiter96Aqueous single solution reagent (MTS) (Promega, Madison.WI, USA) was added to assess cell viability. After 4h, fluorescence was measured using Tecan Infinite M200 (Tecan Trading AG, Switzerland), and the absorbance at 490nm was recorded. CompuSyn (ComboSyn, Inc. Paramus, NJ, USA) was used to assess the quantitative measurement of synergy / antagonism using the combination index (CI) of the affected fraction (Fa) (Chou TC, 2006).

[0153] Example 4: In vitro studies in cell line KG-1

[0154] In a standard in vitro cytotoxicity assay, the AML cell line KG-1 (FLT wild type) was 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 combination index calculation (Chou TC, 2006).

[0155] 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). In KG-1 cells, the cytotoxicity induced by the combination of MEN at IC50 concentration and Gilt at IC75 concentration was significantly different from that induced by MEN1703 (Tukey's multiple comparison one-way ANOVA test; p <0.0158*, see Figure 5A In KG-1 cells, the cytotoxicity induced by the combination of MEN at IC25 and Gilt at IC75 was significantly different from that induced by MEN1703 (Tukey's multiple comparisons one-way ANOVA test; p<0.0005***, see Figure 5B ).

[0156] Table 6: In vitro combination index in KG-1 cells

[0157]

[0158]

[0159] Materials and methods

[0160] Human cell lines

[0161] 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, 80% relative humidity.

[0162] drug

[0163] MEN1703 (alternatively referred to as "Men", "MEN" or "SEL24 / MEN1703"), in HCl salt form (CAS No. 2769008-22-), IUPAC name 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazol-4-amine hydrochloride (lot A / 2201 / 24 / 1), synthesized at Aptuit (VR), and gilteritinib (alternatively referred to as "Gilt", MedChem Express, Monmouth Junction, NJ, USA) were dissolved in DMSO, aliquoted and stored at -80°C until use. The stock solution was added to the culture medium at the appropriate concentration prior to addition to the cells.

[0164] Cytotoxicity

[0165] For cytotoxicity studies, cells were seeded at an appropriate density (50,000 cells / well) before adding MEN and Gilt on day 0. The concentration range was previously determined in a single-agent cytotoxicity assay at the same time point so that the IC10, IC25, IC50, and IC75 values ​​of each drug were included in the combination experiment (see Table 6). After 72 hours, CellTiter96Aqueous single solution reagent (MTS) (Promega, Madison.WI, USA) was added to assess cell viability. After 4h, fluorescence was measured using Tecan Infinite M200 (Tecan Trading AG, Switzerland), and the absorbance at 490nm was recorded. CompuSyn (ComboSyn, Inc. Paramus, NJ, USA) was used to assess the quantitative measurement of synergy / antagonism using the combination index (CI) of the affected fraction (Fa) (Chou TC, 2006).

[0166] 4. References:

[0167] Chou TC,Theoretical basis,experimental design,and computerizedsimulation of synergism and antagonismin drug combination studies,PharmacolRev,2006

[0168] Czardybon W.et al.,A novel,dual pan-PIM / FLT3 inhibitor SEL24exhibitsbroad therapeutic potential in acute myeloid leukemia.Oncotarget,2018,9(24),16917-16931

[0169] Directive 2010 / 63 / Eu of The European Parliament And Of The CouncilOnThe Protection Of Animals Used For Scientific Purposes,22 September 2010

[0170] Kennedy VE and Smith CC,FLT3 mutations in acute myeloid leukemia:keyconcepts and emerging controversies,Frontiers in Oncology,2020,10:612880

[0171] Levis and Perl,Gilteritinib:potent targeting of FLT3 mutations inAML,Blood advances,2020,4(6),1178-1191

[0172] Nakao M,et al.,Internal tandem duplication of the flt3 gene found inacutemyeloid leukemia.Leukemia,1996,10(12),1911-1918

[0173] Qiao et al.The combination of CUDC-907 and gilteritinib showspromisingin vitro and in vivo antileukemic activity against FLT3-ITD AML,2021,BloodCancer Journal,11:111

[0174] Teicher B.Totowa.Anticancer Drug Development Guide.New Jersey:HumanaPress,1997

[0175] Workman P.,et al.,Guidelines for The Welfare And Use Of AnimalsInCancer Research.British Journal Of Cancer,2010,102,1555-1577

[0176] Yuan et al.,Dual FLT3 inhibitors:Against the drug resistance ofacutemyeloid leukemia in recent decade.European Journal of MedicinalChemistry,2019,178,468-483

[0177] Zhang et al.,Rapid and efficient response to gilteritinib andvenetoclax-basedtherapy in two AML patients with FLT3-ITD mutationunresponsive to venetoclaxplus azacitidine,Onco Targets and Therapy,2022:15,159-164

Claims

1. A combination of (i) a 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 gilteritinib, quizartinib, midostaurin and a combination thereof.

3. A combination of (i) a FLT3 inhibitor and (ii) SEL24 / MEN1703 for use in treating 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 4, wherein the cancer is acute myeloid leukemia (AML).

6. The combination for use according to any one of the preceding claims, wherein SEL24 / MEN1703 is administered in 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 in 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 in 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 in a daily dose of about 20 mg to about 80 mg.

10. A combination for use according to any preceding claim, wherein (i) and (ii) are administered as separate dosage forms.

11. A combination for use according to any preceding claim, wherein (i) and (ii) are administered orally.

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

15. The dosage form according to claim 14, wherein the FLT3 inhibitor is selected from gilteritinib, quizartinib, midostaurin, and a combination thereof.

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