Cancer combination therapy including BCL-2 inhibitors

Abstract

The present invention relates inter alia to a combination of (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703 for use as a medicine, preferably for use in treating patients suffering from cancer. The present invention also relates to a kit of dosage forms comprising (i) a dosage form comprising a BCL-2 inhibitor and (ii) a dosage form comprising SEL24 / MEN1703, and to dosage forms comprising (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703.

Description

[Technical Field]

[0001] The present invention is in the field of cancer therapy. More specifically, in one aspect, the present invention is directed to a combination of (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703 for use as a medicament. In another aspect, the present invention relates to a combination of (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703 for use in treating patients suffering from cancer. In yet another aspect, the present invention is directed to a kit of dosage forms comprising (i) a dosage form comprising a BCL-2 inhibitor and (ii) a dosage form comprising SEL24 / MEN1703. In yet another aspect, the present invention relates to a dosage form comprising (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703. [Background technology]

[0002] The protein B-cell lymphoma 2 (BCL-2) was first identified in B-cell lymphoma, and subsequently, BCL-2 was found to be upregulated in other hematologic malignancies, including acute myeloid leukemia (AML). BCL-2 inhibits proapoptotic proteins, thus preventing apoptosis in AML cells. Venetoclax was developed as a BCL-2 inhibitor to promote apoptotic cell death, in that the inhibition of proapoptotic proteins is blocked (because BCL-2 is inhibited by venetoclax). Venetoclax has been shown to be effective in the treatment of chronic lymphocytic leukemia (CLL) and has also been tested as a single agent in the treatment of AML, where it was found to have modest anti-leukemic activity and to be safe. Due to this rather modest activity when used as monotherapy, studies have been initiated to combine venetoclax with other active agents (see Samra et al., 2020).

[0003] The venetoclax combinations that have been tested include, inter alia, venetoclax-hypomethylating agent combinations, venetoclax-cytarabine combinations (where cytarabine is administered at a low dose), venetoclax-intensive chemotherapy combinations (e.g., cytarabine and anthracycline), venetoclax-IDH inhibitor combinations, venetoclax-FLT3 inhibitor combinations, and venetoclax-p53 repair compound combinations (see Samra et al., 2020). Regarding the venetoclax-hypomethylating agent combinations and venetoclax-cytarabine (low-dose cytarabine) combinations, venetoclax products are available today for specific patient populations suffering from AML, namely, the product Venclyxto® in Europe and the product VENCLEXTA® in the United States.

[0004] Unfortunately, despite the impressive response rates and improved survival with venetoclax combinations, one-third of patients appear to be refractory, as proposed by Samra et al. (2020), and the most recognized mechanism of resistance to venetoclax is the upregulation of BCL-2 family anti-apoptotic proteins, such as BCL-XL and MCL-1, which leads to leukemia cell survival. Resistance to venetoclax and hypomethylating agents in AML was also mentioned and reviewed in Saliba et al. (2021), which stated that despite the success of combining venetoclax with the hypomethylating agents (HMAs) decitabine or azacitidine in inducing remission in elderly, previously untreated patients with AML, resistance—either primary or secondary—remains a major obstacle in the pursuit of extending the duration of response. Patients who did not respond to venetoclax plus azacitidine were also discussed in Zhang et al., 2022, where the FLT3 inhibitor gilteritinib was administered to one patient with venetoclax and azacitidine (induction therapy; maintenance therapy was venetoclax and gilteritinib) and to the other patient with venetoclax (induction therapy; maintenance therapy was gilteritinib).

[0005] Although progress has been made in single case studies, such as the two cases in Zhang et al., 2022, supra, there remains a general need to overcome resistance to venetoclax and venetoclax combination treatments, and this applies not only to venetoclax but also to BCL-2 inhibitors in general. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Samra B. et al., Frontiers in Oncology, 2020, 10, Article 562558 [Non-patent document 2] Saliba A. et al., Cancer Drug Resistance, 2021, 4, 125-42 [Non-patent document 3] Zhang et al., Onco Targets and Therapy, 2022:15, 159-164 Summary of the Invention [Means for solving the problem]

[0007] The inventors of the present invention have surprisingly found that the BCL-2 inhibitor combinations of the present invention, in particular the venetoclax combination, are superior to BCL-2 inhibitor monotherapy, in particular venetoclax monotherapy, and / or existing BCL-2 inhibitor combinations, in particular venetoclax combinations, and therefore provide a very promising new treatment option for cancer patients.

[0008] In a first aspect, the present invention is directed to a combination of (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703 for use as a medicine.

[0009] In one embodiment, 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. It is further preferred that SEL24 / MEN1703 be administered orally.

[0010] In one embodiment, the BCL-2 inhibitor is selected from the group consisting of venetoclax, S65487, APG-2575, LOXO-338, ZN-d5, BGB-11417, AZD4320, and AZD0466.

[0011] In preferred embodiments, the BCL-2 inhibitor is S65487, APG-2575, or venetoclax.

[0012] In a more preferred embodiment, the BCL-2 inhibitor is venetoclax.

[0013] S65487 as a BCL-2 inhibitor In one embodiment, S65487 is administered at a therapeutically effective dose. Preferably, S65487 is administered once daily. More preferably, S65487 is administered intravenously.

[0014] APG-2575 as a BCL-2 inhibitor In one embodiment, APG-2575 is administered at a therapeutically effective dose, which can be a daily dose of about 100 mg, or about 200 mg, or about 300 mg, or about 400 mg, or about 500 mg, or about 600 mg, or about 700 mg, or about 800 mg. Preferably, APG-2575 is administered once a day. More preferably, APG-2575 is administered orally.

[0015] LOXO-338 as a BCL-2 inhibitor In one embodiment, LOXO-338 is administered at a therapeutically effective dose. Preferably, LOXO-338 is administered once daily. More preferably, LOXO-338 is administered orally.

[0016] ZN-d5 as a BCL-2 inhibitor In one embodiment, ZN-d5 is administered at a therapeutically effective dose. Preferably, ZN-d5 is administered once daily. More preferably, ZN-d5 is administered orally.

[0017] BGB-11417 as a BCL-2 inhibitor In one embodiment, BGB-11417 is administered at a therapeutically effective dose. Preferably, BGB-11417 is administered once daily. More preferably, BGB-11417 is administered orally.

[0018] AZD0466 as a BCL-2 inhibitor In one embodiment, AZD0466 is administered at a therapeutically effective dose. Preferably, AZD0466 is administered once daily. More preferably, AZD0466 is administered intravenously.

[0019] AZD4320 as a BCL-2 inhibitor In one embodiment, AZD4320 is administered at a therapeutically effective dose. Preferably, AZD4320 is administered once daily. More preferably, AZD4320 is administered intravenously.

[0020] Venetoclax as a BCL-2 inhibitor In one embodiment, venetoclax is administered at a daily dose of about 50 mg to about 600 mg. Preferably, venetoclax is administered at a daily dose of about 50 mg, about 100 mg, about 200 mg, about 300 mg, or about 400 mg, or about 500 mg, or about 600 mg. Most preferably, venetoclax is administered at a daily dose of about 350 mg, about 380 mg, or about 400 mg, or at a daily dose of about 550 mg, about 580 mg, or about 600 mg. Preferably, venetoclax is administered once daily. Venetoclax may be administered at a daily dose of about 100 mg on day 1 of a treatment cycle, followed by a daily dose of about 200 mg on day 2, followed by a daily dose of about 400 mg on days 3 and beyond; or, alternatively, on days 4 and beyond, followed by a daily dose of about 600 mg on day 4, followed by a daily dose of about 600 mg on days 5 and beyond. It is further preferred that venetoclax be administered orally.

[0021] In one embodiment, particularly when the BCL-2 inhibitor is venetoclax, the combination for use of the first aspect further comprises (iii) a methylation inhibitor or cytarabine. Preferably, the methylation inhibitor is selected from the group consisting of azacitidine, decitabine, CC-486, and ASTX727. More preferably, the methylation inhibitor is azacitidine or decitabine, and most preferably azacitidine.

[0022] In one embodiment, the methylation inhibitor is about 10 mg / m, particularly when the BCL-2 inhibitor is venetoclax. 2 ~about 100mg / m 2 (units used herein mg / m 2 is mg / m 2 (Refers to body surface area [BSA]). Methylation inhibitors are approximately 10 mg / m 2 , about 20mg / m 2 , about 30mg / m 2 , about 40mg / m 2 , about 50mg / m 2 , about 60mg / m 2 , about 70mg / m 2 , about 80mg / m 2 , about 90mg / m 2 , or about 100 mg / m 2 When the methylation inhibitor is azacitidine, the azacitidine is preferably administered at a daily dose of about 60 mg / m 2 , about 65mg / m 2 , about 70mg / m 2 , or about 75 mg / m 2 When the methylation inhibitor is decitabine, the decitabine is preferably administered at a daily dose of about 10 mg / m 2 , about 15mg / m 2 , or about 20 mg / m 2 Preferably, the methylation inhibitor is administered once a day. Even more preferably, the methylation inhibitor is administered intravenously, subcutaneously, or orally, with azacitidine preferably administered intravenously or subcutaneously and decitabine preferably administered intravenously.

[0023] In one embodiment, particularly when the BCL-2 inhibitor is venetoclax, the methylation inhibitor is administered (a) for 7 consecutive days on days 1 to 7 of a 28-day cycle, or (b) for 5 consecutive days on days 1 to 5 of a 28-day cycle, wherein the methylation inhibitor in (a) is preferably azacitidine and the methylation inhibitor in (b) is preferably decitabine.

[0024] In one embodiment, particularly when the BCL-2 inhibitor is venetoclax, cytarabine is administered at about 5 mg / m 2 , about 10mg / m 2 , about 15mg / m 2 , or about 20 mg / m 2 Cytarabine is administered at a daily dose of approximately 20 mg / m 2 Preferably, cytarabine is administered at a daily dose of about 600 mg. More preferably, cytarabine is administered once daily. Even more preferably, cytarabine is administered subcutaneously. In one embodiment, cytarabine is administered for 10 consecutive days on days 1-10 of a 28-day cycle. When cytarabine is used in combination with venetoclax and SEL24 / MEN1703, venetoclax may be administered at a daily dose of about 600 mg starting on day 4 (i.e., on day 4 and thereafter).

[0025] In another embodiment, the combination for use of the first aspect does not include an additional administration of chemotherapy.

[0026] In one embodiment of the combination for pharmaceutical use, (i) and (ii) (and optionally (iii)) are administered as separate dosage forms. In this embodiment, administration may be simultaneous or sequential. In yet another embodiment of the combination for pharmaceutical use, (i) and (ii) (and optionally (iii)) are administered together in one dosage form.

[0027] In a second aspect, the present invention is directed to a combination of (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703 for use in treating a patient suffering from cancer.

[0028] In a preferred embodiment, the cancer is a blood cancer. In an even more preferred embodiment, the cancer is leukemia. In a most preferred embodiment, the cancer is AML. In one embodiment, the AML may be newly diagnosed AML, particularly when the BCL-2 inhibitor is venetoclax. In one embodiment, the combination of the present invention may be used to treat patients with AML who are ineligible for intensive chemotherapy, preferably newly diagnosed adult patients with AML who are ineligible for intensive chemotherapy, particularly when the BCL-2 inhibitor is venetoclax. In another embodiment, the combination of the present invention may be used to treat patients 75 years of age or older, particularly when the BCL-2 inhibitor is venetoclax. In one embodiment, the combination of the present invention may be used to treat patients with comorbidities that prevent the use of intensive induction chemotherapy, particularly when the BCL-2 inhibitor is venetoclax.

[0029] If a patient is suffering from AML, the patient may exhibit a FLT3 mutation that results in overactivation of FLT3 signaling. A mutation in FLT3 may even result in constitutively active FLT3 signaling (in the sense that the signaling activity of FLT3 is constitutively active). A FLT3 mutation is caused by at least one base mutation in the FLT3 gene, which results in the above-mentioned FLT3 mutation at the protein level, which results in overactivation of FLT3 signaling. Such mutations have been known in the art 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). The FLT3 mutation may be a FLT3-ITD mutation, a FLT3-TKD mutation, or a combination of a FLT3-ITD mutation and a FLT3-TKD mutation. Additionally or alternatively, patients with cancer, including patients with AML, may exhibit at least one IDH1 and / or IDH2 mutation, preferably at least two IDH1 and / or IDH2 mutations. Alternatively, if the patient has AML, the patient with AML may exhibit FLT3 wt status.

[0030] In one embodiment, 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. It is further preferred that SEL24 / MEN1703 be administered orally.

[0031] In one embodiment, the BCL-2 inhibitor is selected from the group consisting of venetoclax, S65487, APG-2575, LOXO-338, ZN-d5, BGB-11417, AZD4320, and AZD0466.

[0032] In a preferred embodiment, the BCL-2 inhibitor is S65487, APG-2575, or venetoclax.

[0033] In a more preferred embodiment, the BCL-2 inhibitor is venetoclax.

[0034] S65487 as a BCL-2 inhibitor In one embodiment, S65487 is administered at a therapeutically effective dose. Preferably, S65487 is administered once daily. More preferably, S65487 is administered intravenously.

[0035] APG-2575 as a BCL-2 inhibitor In one embodiment, APG-2575 is administered at a therapeutically effective dose, which can be a daily dose of about 100 mg, or about 200 mg, or about 300 mg, or about 400 mg, or about 500 mg, or about 600 mg, or about 700 mg, or about 800 mg. Preferably, APG-2575 is administered once a day. More preferably, APG-2575 is administered orally.

[0036] LOXO-338 as a BCL-2 inhibitor In one embodiment, LOXO-338 is administered at a therapeutically effective dose. Preferably, LOXO-338 is administered once daily. More preferably, LOXO-338 is administered orally.

[0037] ZN-d5 as a BCL-2 inhibitor In one embodiment, ZN-d5 is administered at a therapeutically effective dose. Preferably, ZN-d5 is administered once daily. More preferably, ZN-d5 is administered orally.

[0038] BGB-11417 as a BCL-2 inhibitor In one embodiment, BGB-11417 is administered at a therapeutically effective dose. Preferably, BGB-11417 is administered once daily. More preferably, BGB-11417 is administered orally.

[0039] AZD0466 as a BCL-2 inhibitor In one embodiment, AZD0466 is administered at a therapeutically effective dose. Preferably, AZD0466 is administered once daily. More preferably, AZD0466 is administered intravenously.

[0040] AZD4320 as a BCL-2 inhibitor In one embodiment, AZD4320 is administered at a therapeutically effective dose. Preferably, AZD4320 is administered once daily. More preferably, AZD4320 is administered intravenously.

[0041] Venetoclax as a BCL-2 inhibitor In one embodiment, venetoclax is administered at a daily dose of about 50 mg to about 600 mg. Preferably, venetoclax is administered at a daily dose of about 50 mg, about 100 mg, about 200 mg, about 300 mg, or about 400 mg, or about 500 mg, or about 600 mg. Most preferably, venetoclax is administered at a daily dose of about 350 mg, about 380 mg, or about 400 mg, or at a daily dose of about 550 mg, about 580 mg, or about 600 mg. Preferably, venetoclax is administered once daily. Venetoclax may be administered at a daily dose of about 100 mg on day 1 of a treatment cycle, followed by a daily dose of about 200 mg on day 2, followed by a daily dose of about 400 mg on days 3 and beyond; or, alternatively, on days 4 and beyond, followed by a daily dose of about 600 mg on day 4, followed by a daily dose of about 600 mg on days 5 and beyond. It is further preferred that venetoclax be administered orally.

[0042] In one embodiment, particularly when the BCL-2 inhibitor is venetoclax, the combination for use of the second aspect further comprises (iii) a methylation inhibitor or cytarabine. Preferably, the methylation inhibitor is selected from the group consisting of azacitidine, decitabine, CC-486, and ASTX727. More preferably, the methylation inhibitor is azacitidine or decitabine, and most preferably azacitidine.

[0043] In one embodiment, the methylation inhibitor is about 10 mg / m, particularly when the BCL-2 inhibitor is venetoclax. 2 ~about 100mg / m 2 (units used herein mg / m 2 is mg / m 2 (Refers to body surface area [BSA]). Methylation inhibitors are approximately 10 mg / m 2 , about 20mg / m 2 , about 30mg / m 2 , about 40mg / m 2 , about 50mg / m 2 , about 60mg / m 2 , about 70mg / m 2 , about 80mg / m 2 , about 90mg / m 2 , or about 100 mg / m 2 When the methylation inhibitor is azacitidine, the azacitidine is preferably administered at a daily dose of about 60 mg / m 2 , about 65mg / m 2 , about 70mg / m 2 , or about 75 mg / m 2 When the methylation inhibitor is decitabine, the decitabine is preferably administered at a daily dose of about 10 mg / m 2 , about 15mg / m 2 , or about 20 mg / m 2 Preferably, the methylation inhibitor is administered once a day. Even more preferably, the methylation inhibitor is administered intravenously, subcutaneously, or orally, with azacitidine preferably administered intravenously or subcutaneously and decitabine preferably administered intravenously.

[0044] In one embodiment, particularly when the BCL-2 inhibitor is venetoclax, the methylation inhibitor is administered (a) for 7 consecutive days on days 1 to 7 of a 28-day cycle, or (b) for 5 consecutive days on days 1 to 5 of a 28-day cycle, wherein the methylation inhibitor in (a) is preferably azacitidine and the methylation inhibitor in (b) is preferably decitabine.

[0045] In one embodiment, particularly when the BCL-2 inhibitor is venetoclax, cytarabine is administered at about 5 mg / m 2 , about 10mg / m 2 , about 15mg / m 2 , or about 20 mg / m 2 Cytarabine is administered at a daily dose of approximately 20 mg / m 2 Preferably, cytarabine is administered at a daily dose of about 600 mg. More preferably, cytarabine is administered once daily. Even more preferably, cytarabine is administered subcutaneously. In one embodiment, cytarabine is administered for 10 consecutive days on days 1-10 of a 28-day cycle. When cytarabine is used in combination with venetoclax and SEL24 / MEN1703, venetoclax may be administered at a daily dose of about 600 mg starting on day 4 (i.e., on day 4 and thereafter).

[0046] In one embodiment, the combination for use of the second aspect does not include an additional administration of chemotherapy.

[0047] In one embodiment of the combination for pharmaceutical use, (i) and (ii) (and optionally (iii)) are administered as separate dosage forms. In this embodiment, administration may be simultaneous or sequential. In yet another embodiment of the combination for pharmaceutical use, (i) and (ii) (and optionally (iii)) are administered together in one dosage form.

[0048] In a preferred embodiment of the second aspect, the combination comprises (i) venetoclax as the BCL-2 inhibitor and (ii) SEL24 / MEN1703 as the sole active agents, wherein the cancer is AML, and wherein venetoclax is preferably administered orally at a daily dose of about 400 mg (starting on day 3) (after an initial titration starting from about 100 mg on day 1 to about 200 mg on day 2), and SEL24 / MEN1703 is preferably administered orally at a daily dose of about 80 mg to about 120 mg. In this embodiment, it may be preferred that the patient with AML is a newly diagnosed adult patient with AML who may be ineligible for intensive chemotherapy.

[0049] In yet another preferred embodiment, the combination comprises (i) venetoclax as a BCL-2 inhibitor, (ii) SEL24 / MEN1703, and (iii) a methylation inhibitor, preferably azacitidine or decitabine, wherein the cancer is AML, and wherein venetoclax is administered preferably orally at a daily dose of about 400 mg (starting on day 3) (after an initial titration starting from about 100 mg on day 1 to about 200 mg on day 2), SEL24 / MEN1703 is administered preferably orally at a daily dose of about 80 mg to about 120 mg, and the methylation inhibitor is administered at a dose of about 20 mg / m 2 ~about 75mg / m 2 (Azacitidine is approximately 75 mg / m 2 is preferred, and for decitabine it is about 20 mg / m 2 is preferably administered intravenously at a daily dose of 100 mg / kg / day (preferably 100 mg / kg / day). In this embodiment, it may be preferred that the patient suffering from AML is an adult patient with newly diagnosed AML who may not be eligible for intensive chemotherapy.

[0050] In yet another preferred embodiment, the combination comprises (i) venetoclax as a BCL-2 inhibitor, (ii) SEL24 / MEN1703, and (iii) cytarabine, wherein the cancer is AML, and wherein venetoclax is preferably administered orally at a daily dose of about 600 mg (starting on day 4) (after an initial titration starting from about 100 mg on day 1 to about 200 mg on day 2 and about 400 mg on day 3), SEL24 / MEN1703 is preferably administered orally at a daily dose of about 80 mg to about 120 mg, and cytarabine is administered at a dose of about 20 mg / m 2 and preferably subcutaneously. In this embodiment, it may be preferred that the AML is newly diagnosed and the patient is 75 years of age or older. In one embodiment, the combination may be used to treat patients (who may be newly diagnosed with AML) who have comorbidities that prevent the use of intensive induction chemotherapy.

[0051] In a third aspect, the present invention is directed to a kit of dosage forms comprising (i) a dosage form comprising a BCL-2 inhibitor and (ii) a dosage form comprising SEL24 / MEN1703.

[0052] In one embodiment, the dosage form comprising a BCL-2 inhibitor comprises a BCL-2 inhibitor selected from the group consisting of venetoclax, S65487, APG-2575, LOXO-338, ZN-d5, BGB-11417, AZD4320, and AZD0466.

[0053] In a preferred embodiment, the dosage form comprising a BCL-2 inhibitor comprises a BCL-2 inhibitor selected from the group consisting of S65487, APG-2575, and venetoclax.

[0054] In a more preferred embodiment, the dosage form comprising a BCL-2 inhibitor comprises venetoclax as the BCL2 inhibitor.

[0055] In one embodiment, the dosage form comprising the BCL-2 inhibitor comprises venetoclax in an amount of about 50 mg to about 600 mg, preferably about 50 mg, about 100 mg, about 200 mg, about 300 mg, or about 400 mg, more preferably about 350 mg, about 380 mg, or about 400 mg, or about 550 mg, about 580 mg, or about 600 mg. Preferably, the dosage form comprising venetoclax is a once-daily dosage form. More preferably, the dosage form comprising venetoclax is an oral dosage form.

[0056] In one embodiment, the dosage form containing SEL24 / MEN1703 contains 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 containing SEL24 / MEN1703 is a once-daily dosage form. It is further preferred that the dosage form comprising SEL24 / MEN1703 is an oral dosage form.

[0057] Each dosage form typically contains at least one pharmaceutically acceptable excipient as defined in Section 2 of the Detailed Description below.

[0058] In particular, when venetoclax is included in the kit as the BCL-2 inhibitor, the kit may, in one embodiment, further comprise a dosage form comprising a methylation inhibitor, preferably azacitidine or decitabine, or a dosage form comprising cytarabine, each dosage form being administered at the dosage levels outlined above in the second aspect (as outlined above, in mg / m 2The kit further comprises a dosage form containing azacitidine, along with instructions on how to prepare the corresponding intravenous or subcutaneous dosage form.

[0059] In one embodiment, the kit of the third aspect further comprises a leaflet with instructions on how to use and administer the dosage form.

[0060] In a fourth aspect, the present invention is directed to a dosage form comprising (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703.

[0061] In one embodiment, the dosage form comprises a BCL-2 inhibitor selected from the group consisting of venetoclax, S65487, APG-2575, LOXO-338, ZN-d5, BGB-11417, AZD4320, and AZD0466.

[0062] In a preferred embodiment, the dosage form comprises a BCL-2 inhibitor selected from the group consisting of S65487, APG-2575, and venetoclax.

[0063] In a more preferred embodiment, the dosage form comprises venetoclax as the BCL2 inhibitor.

[0064] In one embodiment, the dosage form comprises venetoclax as a BCL-2 inhibitor in an amount of about 50 mg to about 600 mg, preferably about 50 mg, about 100 mg, about 200 mg, about 300 mg, or about 400 mg, more preferably about 350 mg, about 380 mg, or about 400 mg, or about 550 mg, about 580 mg, or about 600 mg. Preferably, the dosage form comprising venetoclax is a once-daily dosage form. More preferably, the dosage form comprising venetoclax is an oral dosage form.

[0065] In one embodiment, the dosage form 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.

[0066] In particular, when venetoclax is included in the dosage form as a BCL-2 inhibitor, the dosage form, in one embodiment, due to oral administration, further comprises a methylation inhibitor, preferably CC-486 or ASTX727.

[0067] In one embodiment, the dosage form is a once-daily dosage form. More preferably, the dosage form is an oral dosage form.

[0068] Dosage forms typically contain at least one pharmaceutically acceptable excipient as defined in Section 2 of the Detailed Description below.

[0069] In one embodiment, the dosage form of the fourth aspect comprises a leaflet with instructions on how to use and administer the dosage form.

[0070] In a fifth aspect, the present invention is directed to a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of (i) a BCL-2 inhibitor and an effective amount of (ii) SEL24 / MEN1703.

[0071] All embodiments outlined above for the second aspect apply equally to the fifth aspect. [Brief explanation of the drawings]

[0072] [Figure 1]Statistical analysis of cytotoxicity data at IC75 concentrations in KG1 cells. When the triple combination Men+Aza+Ven (mav) was compared with Men+Ven, Ven+Aza, Men+Aza, and single-agent treatments, significant differences were found for Men+Aza+Ven treatment versus Men+Ven and single-agent treatments (Student's t-test, p<0.01**, p<0.1*). The Ven+Men combination showed significant differences compared to Ven single-agent treatment. [Figure 2] Statistical analysis of cytotoxicity data at IC75 concentrations in MV4-11 cells. When the triple combination Men+Aza+Ven (mav) was compared with Men+Ven, Ven+Aza, Men+Aza, and single-agent treatments, significant differences were found for Men+Aza+Ven treatment versus Men alone and for Men+Ven combination treatment versus Ven as a single-agent treatment (Student's t-test, p<0.01**, p<0.1*). [Figure 3A] In vivo study in AML MOLM-16 cell line xenografts. Antitumor activity of MEN1703, 5-azacytidine, and venetoclax as single agents (left graph) and as double and triple drug combinations (right graph) in the MOLM-16 xenograft tumor model. Tumor cells were injected sc (subcutaneously) into SCID mice on day 0, and drug administration began on day 29. The first arrow below the x-axis represents 5-azacytidine administration, the second arrow below the x-axis represents MEN1703 administration, and the third arrow below the x-axis represents venetoclax administration. (A) Shows tumor volume for single-agent treatment (left graph) and combination treatment (right graph) over the total days of each administration. [Figure 3B] In vivo study in AML MOLM-16 cell line xenografts. Antitumor activity of MEN1703, 5-azacytidine, and venetoclax as single agents (left graph) and as double and triple drug combinations (right graph) in the MOLM-16 xenograft tumor model. Tumor cells were injected sc into SCID mice on day 0, and drug administration began on day 29. (B) Tumor volume on day 41. [Figure 4A]In vivo study in AML MV4-11 cell line xenograft. Antitumor activity of MEN1703, 5-azacytidine, and venetoclax as single agents (left graph) and as double and triple drug combinations (right graph) in the MV4-11 xenograft tumor model. Tumor cells were injected sc into SCID mice on day 0, and drug administration began on day 21. The first arrow under the x-axis represents 5-azacytidine administration, the second arrow under the x-axis represents MEN1703 administration, and the third arrow under the x-axis represents venetoclax administration. (A) Shows tumor volumes for single-agent treatment (left graph) and combination treatment (right graph) over the total days of each administration. [Figure 4B] In vivo study in AML MV4-11 cell line xenograft. Antitumor activity of MEN1703, 5-azacytidine, and venetoclax as single agents (left graph) and as double and triple drug combinations (right graph) in the MV4-11 xenograft tumor model. Tumor cells were injected sc into SCID mice on day 0, and drug administration began on day 21. (B) Tumor volume at day 45. [Figure 5] Engraftment of human hematopoietic cells (CD45 positive) in the peripheral blood of NSG mice monitored at the indicated time points. [Figure 6] Histograms represent the percentage of human CD45+ AML cell engraftment in mice 110 days after transplantation. Results are expressed as mean ± SD (**p<0.01; ***p<0.001; ****p<0.0001). [Figure 7] Kaplan-Meier curves comparing overall survival in PDX AML mouse models and statistical results of treatment group comparisons. [Figure 8A]Statistical analysis of cytotoxicity data at IC50 concentrations in MOLM-13 cells ("MEN1703" = MEN, "MEN1703+S65487" = "MEN+S65487"). The combination of MEN1703+S65487 induced cytotoxicity that was significantly different from MEN1703-induced cytotoxicity and S65487-induced cytotoxicity (Tukey's multiple comparison one-way ANOVA test; p<0.05*, p<0.01**). [Figure 8B] Statistical analysis of cytotoxicity data at IC50 concentrations in MV4-11 cells ("MEN1703" = MEN, "MEN1703+S65487" = "MEN+S65487"). The combination of MEN1703+S65487 induced cytotoxicity that was significantly different from MEN1703-induced cytotoxicity and S65487-induced cytotoxicity (Tukey's multiple comparison one-way ANOVA test; p<0.05*, p<0.001***). [Figure 9A] Statistical analysis of cytotoxicity data at IC50 concentrations in MOLM-13 cells ("MEN1703" = MEN, "MEN1703 + AZD4320" = "MEN + AZD4320"). The combination of MEN1703 + AZD4320 induced cytotoxicity that was significantly different from MEN1703-induced cytotoxicity and AZD4320-induced cytotoxicity (Tukey's multiple comparison one-way ANOVA test; p<0.05*). [Figure 9B] Statistical analysis of cytotoxicity data at IC50 concentrations in MV4-11 cells ("MEN1703" = MEN, "MEN1703+AZD4320" = "MEN+AZD4320"). The combination of MEN1703+AZD4320 induced cytotoxicity that was significantly different from MEN1703-induced cytotoxicity and AZD4320-induced cytotoxicity (Tukey's multiple comparison one-way ANOVA test; p<0.05*). DETAILED DESCRIPTION OF THE INVENTION

[0073] Before describing the present invention in more detail, the following definitions are introduced.

[0074] 1. Definition As used in this specification and claims, the singular forms "a" and "an" include the corresponding plural forms unless the context clearly dictates otherwise.

[0075] The term "about" in the context of the present invention indicates an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature being discussed. This term typically indicates a deviation of ±10%, preferably ±5%, from the indicated numerical value.

[0076] 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". In the following, when a group is defined as comprising at least a certain number of embodiments, this also means to encompass a group that preferably consists only of these embodiments.

[0077] As used herein, the terms "combination" or "in combination with" do not imply that the therapies or active agents (i) and (ii) (and optionally (iii)) must be administered simultaneously and / or formulated for delivery together. However, such therapies and formulations are within the scope of the present invention. Active agents in a combination can be administered simultaneously with each other, before each other, or after each other, as well as simultaneously with, before, or after one or more other additional therapies or active agents. Active agents or treatment protocols can be administered in any order. Generally, each active agent will be administered at a dose and / or at a time schedule determined for that active agent. Furthermore, it is generally expected that active agents used in combination will be used in doses that do not exceed the doses used when they are used individually. In some embodiments, the doses used in combination are lower than the doses used individually. In some embodiments, one of the two active agents is administered at a therapeutic or subtherapeutic dose, e.g., venetoclax is administered at a subtherapeutic dose or SEL24 / MEN1703 is administered at a subtherapeutic dose (where "subtherapeutic" is derived relative to the therapeutic dose as the sole active agent in a monotherapy). A subtherapeutic dose (a dose lower than a therapeutic dose) 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.

[0078] As used herein, the term "venetoclax" refers to the BCL-2 inhibitor venetoclax, which is marketed under the trade names Venclyxto® (EMA) and Venclexta® (FDA), both of which are approved, among other things, for the treatment of AML. Further details regarding venetoclax can be found, among other things, in the product leaflet or regulatory dossier.

[0079] 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 the free base or a pharmaceutically acceptable salt thereof (e.g., the HCl salt). The free base form has CAS number 1616359-00-2, and the HCl salt form has CAS number 2769008-22-0. This compound is a dual pan-PIM / FLT3 inhibitor that has been shown, inter alia, to inhibit the growth of a broad panel of AML cell lines in xenograft models. The rationale for the development of this dual inhibitor was that PIM kinases are believed to be a major driver of resistance to FLT3 inhibitors. SEL24 / MEN1703 has been characterized in more detail, for example, 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, which is also characterized in Czardybon et al., 2018). WO 2014 / 096388 does not disclose the combination of SEL24 / MEN1703 with a BCL-2 inhibitor. Furthermore, it is clear from the data in WO 2014 / 096388 that the different compounds synthesized and tested therein do not necessarily share the same mechanism of action (for example, when compound 26A is compared with other compounds, such as compound 1A, which is structurally different in ring and alkyl substituents from compound 26A, with respect to its activity against different PIM kinases (see Table 1A in WO 2014 / 096388), from which it can be concluded that SEL24 / MEN1703 is a pan-PIM inhibitor, while other compounds, such as compound 1A, are rather specific for certain PIM kinases).

[0080] As used herein, the term "methylation inhibitor" refers to an agent that inhibits DNA methylation, i.e., the modification of DNA nucleotides by the addition of methyl groups. Treatment with methylation inhibitors is considered epigenetic therapy. Currently available methylation inhibitors, including azacitidine (also referred to herein as "5-azacytidine") and decitabine disclosed herein, block the activity of DNA methyltransferase. Further details regarding azacitidine and decitabine can be found, inter alia, in the product leaflets or regulatory dossiers for Venclyxto® and Venclexta®, as noted above. CC-486 is also a methylation inhibitor, i.e., an oral azacitidine; see Kipp and Wei, 2021. Yet another methylation inhibitor is ASTX727, a decitabine / cedazuridine; see Kipp and Wei, 2021. Both CC-486 and ASTX727 are orally administered methylation inhibitors.

[0081] As used herein, the term "cytarabine" refers to a chemotherapy agent that is or is called "cytosine arabinoside (ara-C)." Cytarabine combines the base cytosine with an arabinose sugar, disrupting DNA synthesis. It does this by rapidly converting cytarabine to cytosine arabinoside triphosphate, which damages DNA while the cell cycle is in the S phase. Thus, rapidly dividing cells are most affected. Further details regarding cytarabine can be found, inter alia, in the Venclexta® product leaflet or regulatory dossier referenced above.

[0082] The term "S65487," as used herein, refers to the BCL-2 inhibitor S65487, alternatively referred to as "VOB560," which is commercially available, for example, from MedChemExpress, and has CAS number 1644600-79-2. S65487 is currently undergoing clinical trials to, among other things, evaluate safety, tolerability, PK, and preliminary clinical activity, and to estimate the maximum tolerated dose / recommended Phase 2 dose as a single agent administered intravenously to adult patients with refractory or relapsed AML, non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), or chronic lymphocytic leukemia (CCL) [ClinicalTrials.gov Identifier: NCT03755154].

[0083] As used herein, the term "APG-2575" refers to the BCL-2 inhibitor APG-2575, alternatively referred to as "Lisaftoclax," which is commercially available, for example, from MedChemExpress, and has CAS number 2180923-05-9. APG-2575 is currently undergoing clinical trials to evaluate, among other things, the safety and pharmacokinetics of APG-2575 alone and in combination with homoharringtonine or azacitidine in patients with relapsed / refractory AML and related myeloid malignancies [ClinicalTrials.gov Identifier: NCT04501120].

[0084] As used herein, the term "LOXO-338" refers to the BCL-2 inhibitor LOXO-338, alternatively referred to as "LY3847429." LOXO-338 is currently undergoing clinical trials to, among other things, evaluate LOXO338 as monotherapy in patients with advanced hematologic malignancies [ClinicalTrials.gov Identifier: NCT05024045].

[0085] As used herein, the term "ZN-d5" refers to the BCL-2 inhibitor ZN-d5. On April 30, 2020, Zentalis announced FDA clearance of its IND application for ZN-d5 for the treatment of hematologic malignancies.

[0086] As used herein, the term "BGB-11417" refers to the BCL-2 inhibitor BGB-11417, which is currently undergoing clinical trials to, among other things, evaluate BGB-11417 for safety and tolerability, and to define the maximum tolerated dose and recommended Phase 2 dose, as well as to evaluate the safety and tolerability of a monotherapy ramp-up dosing schedule [ClinicalTrials.gov Identifier: NCT04277637].

[0087] The term "AZD0466" as used herein refers to the BCL-2 inhibitor AZD0466, characterized inter alia in Arulananda et al., 2021. AZD0466 has been disclosed in a clinical trial to evaluate AZD0466 for safety, tolerability, maximum tolerated dose, recommended phase 2 dose, and PK in patients with solid tumors, lymphoma, and multiple myeloma at low risk for tumor lysis syndrome [ClinicalTrials.gov Identifier: NCT04214093].

[0088] As used herein, the term "AZD4320" refers to the BCL-2 inhibitor AZD4320, characterized inter alia in Balachander et al., 2020. It is commercially available, for example from MedChemExpress, and has the CAS number 1357576-48-7.

[0089] As used herein, the term "treatment" refers to a clinical intervention to cure or ameliorate a disease, prevent recurrence of a disease, alleviate the symptoms of a disease, reduce any direct or indirect pathological consequence of a disease, achieve a stabilized (i.e., not worsening) state of a disease, prevent metastasis, slow the rate of disease progression, and / or prolong survival as compared to expected survival if not receiving treatment.

[0090] 2. Pharmaceutical Compositions The BCL-2 inhibitor, SEL24 / MEN1703, methylation inhibitor, and cytarabine are "pharmaceutical active agents" or "active agents" for purposes of the present invention. As noted above, the BCL-2 inhibitor and SEL24 / MEN1703 may be present in separate dosage forms or may be included in a single dosage form.

[0091] As used herein, "pharmaceutical active agent" means that the compound is effective in modulating a response in a patient, i.e., a human or animal in vivo. As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that is typically included in a pharmaceutical dosage form or pharmaceutical composition, and is known to those skilled in the art. Examples of such excipients are listed below. In view of the above definition of "pharmaceutical active agent," a pharmaceutically acceptable excipient can be defined as pharmaceutically inactive.

[0092] Typically, the administration of the BCL-2 inhibitor and SEL24 / MEN1703 will be via separate dosage forms, with the BCL-2 inhibitor preferably being administered via the route of administration approved in that dosage form. SEL24 / MEN1703 may be administered in the dosage forms described below or in dosage forms currently undergoing clinical trials.

[0093] The dosage forms for 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 also be referred to as formulations or pharmaceutical compositions.

[0094] Generally, the pharmaceutical composition of the present invention can contain various pharmaceutically acceptable excipients, which can be selected depending on the functionality of the composition to be achieved. A "pharmaceutically acceptable excipient" in the sense 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-modifying materials, carrier materials, diluents, binders, and other adjuvants. Typical pharmaceutically acceptable excipients include substances such as sucrose, mannitol, sorbitol, starch and starch derivatives, lactose, as well as lubricants such as magnesium stearate, disintegrants, and buffers.

[0095] The term "carrier" refers to a pharmaceutically acceptable organic or inorganic carrier substance with which an active ingredient is combined to facilitate application (administration). Suitable pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohols, oils, preferably vegetable oils, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, surfactants, flavor oils, mono- and diglycerides of fatty acids, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical compositions can be sterilized and, if desired, can be mixed with auxiliary substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors, and / or aromatic substances that do not adversely react with the active compounds.

[0096] When liquid dosage forms are contemplated for this invention, they 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 to provide bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners / flavorings.

[0097] For parenteral application, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Pharmaceutical preparations for parenteral administration are particularly preferred, including 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 that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0098] For injection preparations, sterile injectable aqueous or oily suspensions can be formulated according to known techniques, for example, using suitable dispersants, wetting agents, and / or suspending agents. The sterile injectable preparations may be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Acceptable vehicles and solvents that can be used include water and isotonic sodium chloride solution. Sterile oils are also conventionally used as solvents or suspending media.

[0099] Suppositories for rectal administration of the pharmaceutical compositions of the invention can be prepared, for example, by mixing the compound with a suitable non-irritating excipient, such as cocoa butter, synthetic triglycerides and polyethylene glycols, which is solid at room temperature but liquid at rectal temperature and will melt in the rectum releasing the active agent from the suppository.

[0100] For administration by inhalation, the pharmaceutical compositions of the present invention may be conveniently 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, such as lactose or starch.

[0101] Oral dosage forms may be liquid or solid, such as tablets, lozenges, pills, capsules, powders, effervescent preparations, sugar-coated tablets, and granules. Pharmaceutical preparations for oral use can be obtained as solid excipients, optionally by grinding the resulting mixture and, if necessary, adding suitable additives, processing the granular mixture to obtain tablets or sugar-coated cores. Suitable excipients are, in particular, sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or fillers such as polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added. Oral dosage forms may be formulated to ensure immediate release of the active agent or sustained release of the active agent. [Example]

[0102] 3. Working Example The following examples are merely illustrative and are intended to further illustrate the present invention and should not be construed as limiting the invention to those examples.

[0103] Example 1: In vitro studies in cell lines In a standard in vitro cytotoxicity assay, three AML cell lines (KG1 [FLT3 wt], MV4-11 [FLT3 ITD], and MOLM-13 [FLT3 ITD]) were treated with Ven and / or 5-Aza for 48 hours, followed by an additional 24-hour treatment with Men. The synergistic effects of the resulting combinations of Men with either Ven or 5-Aza alone (i.e., two active agents combined) or Ven + 5-Aza (i.e., three active agents combined) were measured according to the combination index calculation (Chou TC, 2006). The triple combination of Men + 5-Aza + Ven (mav) showed high synergy in most of the AML cell lines tested (see Tables 1–3 and Figures 1 and 2). In KG1 cells (FLT3 wt), the cytotoxic effect of Men+5-Aza+Ven (mav) treatment was significantly stronger than that of Men+Ven and single-agent treatments (see Figure 1), and the combination index appeared lower for the triple combination Men+5-Aza+Ven versus Men+Ven and Ven+Aza, all applied at clinically relevant concentrations (see Table 1). In MV4-11 cells (FLT3 ITD), a similar trend was observed: the CI for the triple combination was nearly equal to that for Men+Ven and significantly lower than that for Ven+Aza (see Figure 2 and Table 2). In MOLM13 cells (FLT3 ITD), the CI for the triple combination was similar to that for the Men-Ven and Ven-Aza combinations (see Table 3).

[0104] [Table 1]

[0105] The combination of Men+Ven+Aza (mav) was strongly synergistic at clinically relevant concentrations (gray rows).

[0106] [Table 2]

[0107] The combination of Men+Ven+Aza (mav) was strongly synergistic at clinically relevant concentrations (gray rows).

[0108] [Table 3]

[0109] The CI for the Men+Ven+Aza (mav) combination was similar to the CI for the Men+Ven and Ven+Aza combinations at clinically relevant concentrations (gray rows).

[0110] Example 2: In vivo xenograft studies In vivo xenograft studies using MOLM-16 (FLT3 wt) and MV4-11 (FLT3 ITD) cells confirmed the in vitro results. In MOLM-16 xenografts, concordant tumor growth inhibition was observed with the triple combination, which was the only combination that was statistically significant when compared to the control (see Figures 3A and 3B). However, it should be noted that lower concentrations of the three active agents were used in this experiment because effects could only be observed at lower concentrations (see the Materials and Methods section below). Thus, combining the active agents at higher concentrations would likely be ineffective. This explains why such low concentrations of the single agents were not effective in this experimental setting. Furthermore, high concentrations of these active agents are typically toxic when administered in combination to mice. In MV4-11 xenografts, concordant tumor growth inhibition was observed with the triple combination, which was significantly different when compared to the control, single-agent Aza and Ven treatments, and the Ven + Aza and Men + Aza combination treatments. Ven+Men was also significantly different from control and single-agent Ven treatment in this setting (see Figures 4A and 4B).

[0111] The combination treatment was also tested in an AML patient-derived xenograft (PDX) diffuse model. This model confirmed the higher efficacy of the triple combination treatment compared to the Ven+Aza combination. Statistical analysis showed significant differences in tumor burden (assessed as % of human CD45-positive cells) between the triple combination and single-agent treatments and the Men+Aza combination treatment (see Figures 5 and 6). Statistical analysis of overall survival data showed significant differences between the triple combination treatment and single-agent treatments and the Men+Aza or Ven+Aza combination treatment (see Figure 7).

[0112] Materials and Methods for Examples 1 and 2: human cell lines Human acute myeloid leukemia cell lines KG-1, MV4-11, and MOLM-13 were obtained from DSMZ, Braunschweig, Germany (ACC14, ACC102, and ACC554). KG-1 and MV4-11 cells were cultured in RPMI 1640 medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, Saint Louis, CA, USA). MOLM-13 cells were grown in RPMI containing 20% FBS. All cells were incubated at 37°C, 5% CO2, and 80% relative humidity. The human acute myeloid leukemia cell line MOLM-16 was obtained from DSMZ, Braunschweig, Germany (cell line number ACC-555). MOLM-16 cells were grown in RPMI containing 20% FBS (Sigma, St. Louis, CA, USA). Cells were incubated at 37°C, 5% CO2, and 80% relative humidity.

[0113] Active substances in cytotoxicity assays Men (alternatively referred to as "MEN1703") (batch number A / 2201 / 24 / 1), IUPAC name 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazol-4-amine hydrochloride, in its HCl salt form (CAS number 2769008-22-), synthesized by Aptuit (VR), and venetoclax (alternatively referred to as "Ven" or "ABT-199"; ChemCruz number SC-472284A) were dissolved in DMSO, aliquoted, and stored at -80°C until use. Stock solutions were added to the appropriate concentrations in culture medium before addition to cells. 5-Azacytidine (alternatively referred to as "5-Aza" or "A2385"; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in distilled pure water and used immediately after resuspension (5-azacytidine is highly unstable in aqueous solution). The stock solution was added to the culture medium at the appropriate concentration before addition to cells.

[0114] Active substances in mouse models Men (alternatively referred to as "MEN1703") in the HCl salt form (CAS number 2769008-22-) with 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) was dissolved in sterile water. 5-Azacytidine (batch BCCC8387) was purchased from Sigma-Aldrich; the powder was dissolved in sterile saline and administered within 1 hour. Venetoclax (batch B0521) was purchased from Santa Cruz Biotechnology, Inc. The powder was first dissolved in DMSO and then diluted with a solution of 20% 2-hydroxypropyl-β-cyclodextrin (HPCD) in 100 mM citrate buffer (pH 3).

[0115] Cytotoxicity assay For cytotoxicity studies, cells were seeded at the appropriate density (50,000 cells / well) on day 0, followed by the addition of Ven and 5-Aza. For degradation (Hollenbach PW, 2010 and Chang E, 2016), 5-Aza was added every 24 hours. Then, 48 hours later, Men was added to the cultures for the final 24 hours (a total 72-hour period was tested). Concentration ranges were previously identified in single-agent cytotoxicity assays at the same time points to include IC10, IC25, IC50, and IC75 values for each drug and each AML cell line in combination experiments (see Tables 1–3). All possible combinations were evaluated: Men + Ven, Ven + 5-Aza, Men + 5-Aza, and Men + 5-Aza + Ven (mav). After 72 hours, CellTiter 96 Aqueous One Solution Reagent (MTS) (Promega, Madison, WI, USA) was added to assess cell viability. Fluorescence was measured after 4 hours using a Tecan Infinite M200 (Tecan Trading AG, Switzerland) and absorbance at 490 nm was recorded. Quantitative measurements of synergy / antagonism were assessed using the combination index (CI) for fraction affected (Fa) using CompuSyn (ComboSyn, Inc., Paramus, NJ, USA) (Chou, TC, 2006).

[0116] MOLM-16 model 10 x 10 for AML xenograft models 6 MOLM-16 cells were resuspended in 0.2 ml of 5.6 mg / ml BME type III (Trevigen) + DPBS (1:1) and then injected subcutaneously into the right flank of 6- to 8-week-old female SCID mice (Charles River, Calco, Italy).

[0117] After injection, mice were maintained in microisolator cages under continuously monitored environmental conditions. Drinking water and specific sterilized diet (VRF1, Charles River) were provided ad libitum. Environmental conditions, as well as animal housing and handling procedures, conformed to 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 assessed and recorded twice weekly.

[0118] The results were evaluated as follows: the tumor volume was measured with a caliper, and the tumor mass was calculated using the following formula: [length (mm) × width]. 2 (mm) × d] / 2, and the density of tumor tissue is d = 1 mg / mm 3 Assume that the average tumor volume is 200-300 mm (Teicher B. Totowa, 1997). 3When the mean age reached 18 days (corresponding to day 29 in Figure 3A), animals were randomly assigned to eight groups (5–6 mice / group) and received the following treatments starting on day 29: Group I received vehicle (venetoclax diluent solution) orally once daily until day 42 (single-agent administration, see left graph in Figure 3A; combination administration, see right graph in Figure 3A), and Group II received 25 mg / kg Men orally once daily q1d × 5 starting on day 38 until day 42. Group III received 1.25 mg / kg 5-azacytidine intraperitoneally every 2 days for a total of 3 doses (single agent administration, see left graph in Figure 3A; combination administration, see right graph in Figure 3A), Group IV received 100 mg / kg venetoclax given orally once daily q1d x 14 until day 42 (single agent administration, see left graph in Figure 3A), and Group V received 100 mg / kg venetoclax given orally once daily q1d x 14 until day 42 (single agent administration, see left graph in Figure 3A). Group VI received a combination of 25 mg / kg Men orally once daily q1d x 14 starting on day 38 until day 51 and 100 mg / kg venetoclax given orally q1d x 23 starting on day 29 until day 51 (combination dosing, see graph on the right in Figure 3A); Group VII received a combination of 5-Aza at the same dose and schedule as the single-agent groups and 100 mg / kg venetoclax given orally q1d x 23 starting on day 29 until day 51 (combination dosing, see graph on the right in Figure 3A); Group VIII received a triple combination of Men + 5-Aza + Ven (all drugs administered at the same dose and schedule as the double combination groups).

[0119] 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) x 100.

[0120] Mice were sacrificed when tumors reached a volume of approximately 10% of their total body weight or when their body weight decreased by more than 20% compared to control animals for 7 days or more. Animals were euthanized using carbon dioxide exposure according to standard procedures (Annex IV of Directive 2010 / 63 / EU; 2010).

[0121] MV4-11 model 10 x 10 for AML xenograft models 6 MV4-11 cells were resuspended in 0.2 ml of 5.6 mg / ml BME type III (Trevigen) + DPBS (1:1) and then injected subcutaneously into the right flank of 6- to 8-week-old female SCID mice (Charles River, Calco, Italy).

[0122] After injection, mice were maintained in microisolator cages under continuously monitored environmental conditions. Drinking water and specific sterilized diet (VRF1, Charles River) were provided ad libitum. Environmental conditions, as well as animal housing and handling procedures, conformed to 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 weekly.

[0123] The results were evaluated as follows: the tumor volume was measured with a caliper, and the tumor mass was calculated using the following formula: [length (mm) × width]. 2 (mm) × d] / 2, and the density of tumor tissue is d = 1 mg / mm 3 Assume that the average tumor volume is 200-300 mm (Teicher B. Totowa, 1997). 3When the mean age reached 14 days (corresponding to day 21 in Figure 4A), animals were randomly assigned to eight groups (5-6 mice / group) and received the following treatments starting on day 21: Group I received vehicle (venetoclax diluent solution) orally once daily until day 42; Group II received 25 mg / kg Men orally once daily q1d x 13 starting on day 30 until day 42 (single agent administration, see left graph in Figure 4A; combination administration, see right graph in Figure 4A); and Group III received 1.25 mg / kg Men every 2 days for a total of 3 doses. Group IV received 100 mg / kg venetoclax given orally once daily q1d x 22 until day 42 (single-agent administration, see left graph in Figure 4A; combination administration, see right graph in Figure 4A), Groups V-VI-VII received combinations of Men+5-Aza, Men+Ven, and 5-Aza+Ven, respectively, and Group VIII received the triple combination of Men+5-Aza+Ven. The above-mentioned doses and regimens of the active agents were used in the combinations as well.

[0124] 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) x 100.

[0125] Mice were sacrificed when tumors reached a volume of approximately 10% of their total body weight or when their body weight decreased by more than 20% compared to control animals for 7 days or more. Animals were euthanized using carbon dioxide exposure according to standard procedures (Annex IV of Directive 2010 / 63 / EU; 2010).

[0126] AML PDX model The human AML samples used in this study were tested, and the results indicate the mutational status of IDH2, JAK2, MIR636, SRSF2, and RUNX1. Peripheral blood mononuclear cells (PBMCs) from patient samples were isolated by Ficoll separation and subjected to T cell depletion using anti-CD3 magnetic beads (Miltenyi Biotec). T cell depletion has previously been shown to enhance AML cell engraftment in female nonobese diabetic severe combined immunodeficient (NOD / SCID) interleukin-2 receptor gamma (IL-2Rγ)-null (NSG) hosts by limiting polyclonal donor T cell proliferation due to xenograft-versus-host disease (Von Bonin et al., 2013). For PDX, experiments were performed in 6- to 8-week-old female NSG mice. Mice were maintained under pathogen-free conditions in the animal facilities of the European Institute of Oncology - Italian Foundation for Cancer Research Institute of Molecular Oncology (IEO-IFOM, Milan, Italy). All animal experiments were performed in strict accordance with Italian legislation (DLvo 26 / 2014 and subsequent amendments) and approved by the institutional committee.

[0127] Ten million T cell-depleted cells were intraperitoneally transplanted into 6-8 week-old male and female NSG mice conditioned with 1 Gy of irradiation. Bone marrow and spleen cells were collected from one primary recipient mouse and stained with different anti-human CD45 APC, CD33 APC-Cy7, CD13 PE-Cy7, CD117 PE, and anti-mouse CD45 FITC antibodies. Human engraftment (hCD45 > 80%) and patient markers were confirmed by FACS analysis, and then cryopreserved in liquid nitrogen. For the experiment, human blasts collected from the spleen of the second xenograft passage were intravenously injected into the lateral tail vein of NSG mice (750,000 cells / mouse), and one day later, the mice received low-dose irradiation (1 Gy). 38 days after transplantation, once systemic xenograft engraftment was confirmed (approximately 0.05%), mice were randomized to receive treatment. The percentage of human cells in tail vein-derived peripheral blood was assessed by flow cytometry weekly until day 110 using anti-human CD45-APC (clone J.33, Beckman-Coulter) and anti-mouse CD45-PE (clone 30-F11, BD) to exclude mouse cell contamination. Prior to staining, all blood samples were lysed with ammonium chloride red blood cell buffer (Qiagen). The percentage of stained cells was determined and compared with the appropriate negative control. Viable, apoptotic, and dead cells were counted using 7-aminoactinomycin D (7AAD) from Sigma-Aldrich. Cell suspensions were evaluated using a three-laser, 10-color flow cytometer (Navios, Beckman-Coulter, Brea, CA, USA) using analysis gates designed to exclude dead cells, platelets, and debris. Transplanted mice (n=5 per test arm) were treated with either vehicle or different drugs used as single agents or in combination.

[0128] Specifically, mice were treated intraperitoneally (ip) with 5-azacytidine (1.25 mg / kg) for 3 days (days 1-4-7 post-engraftment), with MEN1703 (25 mg / kg) for 14 consecutive days (days 10-23 post-engraftment), or with venetoclax (100 mg / kg) for 23 consecutive days (starting on day 1 post-engraftment), both by oral gavage.

[0129] The efficacy of the treatment was assessed by measuring the level of human CD45 in the peripheral blood of the mice. + The cells were then euthanized using carbon dioxide exposure according to standard procedures.

[0130] statistical analysis GraphPad Prism software (GraphPAD Software Inc., CA) was used for statistical analysis. Statistical differences were considered significant at p values <0.05 using a two-tailed Mann-Whitney rank test. In vivo data (Figures 4-6) are presented as means, with values for each group represented by differently shaped symbols and differently colored lines. For analysis of AML PDX models at day 110, Tukey's multiple comparison test was used.

[0131] Example 3: In vitro studies in cell lines for combination with S65487 In a standard in vitro cytotoxicity experiment, two AML cell lines (MOLM-13 [FLT3 ITD] and MV4-11 [FLT3 ITD]) were treated with MEN and S65487 as single agents or in combination for 72 hours. The synergistic effect of the combination was determined according to the combination index calculation (Chou TC, 2006).

[0132] The synergistic effect of the combination of MEN and S65487 in MOLM-13 cells is shown in Table 4 (see Table 4, combination index or CI<1.0). The combination of MEN and S65487 at IC50 concentrations induced cytotoxicity that was significantly different from S65487-induced cytotoxicity in MOLM-13 cells and MEN1703-induced cytotoxicity in MOLM-13 cells (Tukey's multiple comparison one-way ANOVA test; p<0.05). * , p<0.01 ** , see Figure 8A).

[0133] The combination of MEN and S65487 was synergistic in MV4-11 cells (see Table 5, combination index or CI<1.0), as can be derived from Table 5. The combination of MEN and S65487 at IC50 concentrations induced cytotoxicity that was significantly different from S65487-induced cytotoxicity in MV4-11 cells and MEN1703-induced cytotoxicity in MV4-11 cells (Tukey's multiple comparison one-way ANOVA test; p<0.05). * , p<0.001 *** , see Figure 8B).

[0134] [Table 4]

[0135] [Table 5]

[0136] Example 4: In vitro studies in cell lines for combination with AZD4320 In a standard in vitro cytotoxicity experiment, two AML cell lines (MOLM-13 [FLT3 ITD] and MV4-11 [FLT3 ITD]) were treated with MEN and AZD4320 as single agents or in combination for 72 hours. The synergistic effect of the combination was determined according to the combination index calculation (Chou TC, 2006).

[0137] The synergistic effect of the combination of MEN and S65487 in MOLM-13 cells is shown in Table 6 (see Table 6, combination index or CI<1.0). The combination of MEN and AZD4320 at IC50 concentrations induced cytotoxicity that was significantly different from AZD4320-induced cytotoxicity in MOLM-13 cells and MEN1703-induced cytotoxicity in MOLM-13 cells (Tukey's multiple comparison one-way ANOVA test; p<0.05). * , see Figure 9A).

[0138] The combination of MEN and AZD4320 was synergistic in MV4-11 cells (see Table 7, combination index or CI<1.0), as can be derived from Table 7. The combination of MEN and AZD4320 at IC50 concentrations induced cytotoxicity that was significantly different from AZD4320-induced cytotoxicity in MV4-11 cells and MEN1703-induced cytotoxicity in MV4-11 cells (Tukey's multiple comparison one-way ANOVA test; p<0.05). * , see Figure 9B).

[0139] [Table 6]

[0140] [Table 7]

[0141] Materials and Methods for Examples 3 and 4 human cell lines 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, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, CA, USA). MOLM-13 cells were grown in RPMI containing 20% FBS. All cells were incubated at 37°C, 5% CO2, and 80% relative humidity.

[0142] drugs MEN1703 (alternatively referred to as "Men," "MEN," "MEN1703," or "SEL24 / MEN1703") (Batch No. A / 2201 / 24 / 1), IUPAC name 5,6-dibromo-4-nitro-2-(piperidin-4-yl)-1-(propan-2-yl)-1H-1,3-benzodiazol-4-amine hydrochloride, in its HCl salt form (CAS No. 2769008-22-), synthesized by Aptuit (VR), AZD4320 from MedChem Express (Monmouth Junction, NJ, USA, Batch No. 64354), and S65487 from MedChem Express (Monmouth Junction, NJ, USA) were dissolved in DMSO, aliquoted, and stored at -80°C until use. Stock solutions were added to the appropriate concentrations in culture medium before addition to cells.

[0143] Cytotoxicity For cytotoxicity studies, cells were seeded at the appropriate density (50,000 cells / well) on day 0, followed by the addition of MEN and Quiz. Concentration ranges were previously identified in single-agent cytotoxicity assays at the same time points to include IC10, IC25, IC50, and IC75 values for each drug and each AML cell line in combination experiments (see Tables 4–7). After 72 hours, CellTiter 96 Aqueous One Solution Reagent (MTS) (Promega, Madison, WI, USA) was added to assess cell viability. Fluorescence was measured after 4 hours using a Tecan Infinite M200 (Tecan Trading AG, Switzerland), and absorbance at 490 nm was recorded. Quantitative measurements of synergy / antagonism were assessed using the combination index (CI) for fraction affected (Fa) using CompuSyn (ComboSyn, Inc., Paramus, NJ, USA) (Chou TC, 2006).

[0144] 4. References: Arulananda et al., A novel BH3-mimetic, AZD0466, targeting BCL-XL and BCL-2 is effective in pre-clinical models of malignant pleural mesothelioma, Cell Death Discovery, 2021, 7, 122-131. Balachander et al., AZD4320, a dual inhibitor of Bcl-2 and Bcl-X L , induces tumor regression in hematological cancer models without dose-limiting thrombocytopenia, Clin Cancer Res, 2020, 26(24):6535-6549 Chang E. et al., The combination of FLT3 and DNA methyltransferase inhibition is synergistically cytotoxic to FLT3 / ITD acute myeloid leukemia cells, Leukemia, 2016 Chou TC, Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies, Pharmacol Rev, 2006 Directive 2010 / 63 / Eu of The European Parliament And Of The Council On The Protection Of Animals Used For Scientific Purposes, September 22, 2010 Hollenbach PW. et al., A comparison of azacitidine and decitabine activities in acute myeloid leukemia cell lines, PLoS One, 2010 Kipp D. and Wei A., The path to approval for oral hypomethylating agents in acute myeloid leukemia and myelodysplastic syndromes, Future Oncol. 2021, 17(20), 2563-2571 Nakao M. et al., Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia, 1996, 10(12), 1911-1918 Saliba A.ら、Resistance to Venetoclax and hypomethylating agents in acute myeloid leukemia、Cancer Drug Resistance、2021、4、125-42 Samra B.ら、Venetoclax-based combinations in Acute Myeloid Leukemia: Current Evidence and Future Directions、Frontiers in Oncology、2020、10、Article 562558 Teicher B.Totowa. Anticancer Drug Development Guide. New Jersey:Humana Press、1997 Workman P.ら、Guidelines for The Welfare And Use Of Animals In Cancer Research. British Journal Of Cancer、2010、102、1555-1577 Zhangら、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 Von Bonin M、Wermke M、Cosgun KN.ら、In vivo expansion of co-transplanted T cells impacts on tumor re-initiating activity of human acute myeloid leukemia in NSG mice. PLoS One. 2013;8(4):e60680

Claims

1. A combination of (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703 for use as a medicine.

2. A combination of (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703 for use in treating a patient suffering from cancer.

3. The combination for use according to claim 2, wherein the cancer is a blood cancer.

4. 4. The combination for use according to claim 2 or claim 3, wherein the cancer is acute myeloid leukemia (AML).

5. The combination for use according to any one of claims 1 to 4, wherein the BCL-2 inhibitor is venetoclax.

6. 6. The combination for use according to claim 5, wherein venetoclax is administered in a daily dose of about 50 mg to about 600 mg.

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

8. 8. The combination for use according to any one of claims 1 to 7, wherein the combination further comprises (iii) a methylation inhibitor or cytarabine.

9. The combination for use according to claim 8, wherein said methylation inhibitor is selected from the group consisting of azacitidine, decitabine, CC-486 and ASTX727.

10. The methylation inhibitor is about 10 mg / m ２ ~Approx. 100mg / m ２ 10. The combination for use according to claim 8 or claim 9, wherein the combination is administered in a daily dose of

11. The methylation inhibitor is azacitidine, and the azacitidine is about 75 mg / m ２ 11. The combination for use according to claim 10, wherein the combination is administered in a daily dose of

12. The methylation inhibitor is decitabine, and the decitabine is about 20 mg / m ２ 11. The combination for use according to claim 10, wherein the combination is administered in a daily dose of

13. Cytarabine is approximately 20 mg / m ２ 9. The combination for use according to claim 8, wherein the combination is administered in a daily dose of

14. A kit of dosage forms comprising (i) a dosage form comprising a BCL-2 inhibitor and (ii) a dosage form comprising SEL24 / MEN1703.

15. A dosage form comprising (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703.

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