Cancer combination therapy comprising BCL-2 inhibitor

Abstract

The present invention relates inter alia to a combination of (i) a BCL-2 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 a BCL-2 inhibitor and (ii) a dosage form comprising SEL24 / MEN1703; and a dosage form comprising (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703, as well as a pharmaceutical composition comprising the same.

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 BCL-2 inhibitor and (ii) SEL24 / MEN1703 on one hand, which is used as a medicine. On the other hand, the present invention relates to a combination of (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703 for treating patients with cancer. On another hand, the present invention relates to a kit of dosage forms, which comprises (i) a dosage form comprising a BCL-2 inhibitor and (ii) a dosage form comprising SEL24 / MEN1703. On another hand, the present invention relates to a dosage form, which comprises (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703. Background Art

[0002] The protein B-cell lymphoma 2 (BCL-2) was originally discovered in B-cell lymphomas, and BCL-2 was subsequently found to be upregulated in other hematological malignancies, including acute myeloid leukemia (AML). BCL-2 inhibits pro-apoptotic proteins, thereby preventing apoptosis of AML cells. Venetoclax was developed as a BCL-2 inhibitor with the goal of promoting cell death by apoptosis because the inhibition of pro-apoptotic 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 be safe with modest anti-leukemic activity. Due to this rather modest activity when used as a monotherapy, studies have been initiated to combine Venetoclax with other active agents (see Samra et al., 2020).

[0003] The tested venetoclax combinations particularly relate to venetoclax-hypomethylating agent combinations, venetoclax-cytarabine combinations (wherein cytarabine is administered at low doses), venetoclax-intensive chemotherapy combinations (e.g., cytarabine and anthracyclines), venetoclax-IDH inhibitor combinations, venetoclax-FLT3 inhibitor combinations, and venetoclax-p53 restoring compound combinations (see Samra et al., 2020). When referring to the venetoclax-hypomethylating agent combinations and venetoclax-cytarabine (low-dose cytarabine) combinations, currently venetoclax products are available for specific patient populations with AML, namely, the European product. and American products

[0004] Unfortunately, as noted by Samra et al., 2020, despite the impressive response rates and improved survival of the venetoclax combination, one-third of patients appear to be refractory, with the most recognized mechanism of resistance to venetoclax being upregulation of BCL-2 family anti-apoptotic proteins (e.g., BCL-XL and MCL-1) leading to leukemic cell survival. Resistance to venetoclax and hypomethylating agents in AML was also mentioned and reviewed by Saliba et al., 2021, who noted that despite the success of venetoclax in combination with the hypomethylating agents (HMAs) decitabine or azacitidine in inducing remissions in elderly, previously untreated AML patients, resistance—primary or secondary—still poses a significant obstacle in the quest to prolong the duration of response. Zhang et al., 2022 also discussed patients who did not respond to venetoclax plus azacitidine, in which the FLT3 inhibitor gilteritinib was subsequently administered with venetoclax and azacitidine in one patient (induction therapy, where maintenance therapy was venetoclax and gilteritinib) and the FLT3 inhibitor gilteritinib was administered with venetoclax in the other patient (induction therapy, where maintenance therapy was gilteritinib).

[0005] Despite progress in single case studies, such as the two cases discussed above in Zhang et al., 2022, there remains a general need to overcome resistance to venetoclax and venetoclax combination therapy. This applies not only to venetoclax but also to BCL-2 inhibitors in general.

[0006] Object and content of the invention

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

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

[0009] 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.

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

[0011] In a preferred embodiment, 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

[0014] In an embodiment, S65487 is administered at a therapeutically effective dose. Preferably, S65487 is administered once daily. Further preferably, S65487 is administered intravenously.

[0015] APG-2575 as a BCL-2 inhibitor

[0016] In an 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. Further preferably, APG-2575 is administered orally.

[0017] LOXO-338 as a BCL-2 inhibitor

[0018] In an embodiment, LOXO-338 is administered at a therapeutically effective dose. Preferably, LOXO-338 is administered once daily. Further preferably, LOXO-338 is administered orally.

[0019] ZN-d5 as a BCL-2 inhibitor

[0020] In an embodiment, ZN-d5 is administered at a therapeutically effective dose. Preferably, ZN-d5 is administered once daily. Further preferably, ZN-d5 is administered orally.

[0021] BGB-11417 as a BCL-2 inhibitor

[0022] In an embodiment, BGB-11417 is administered at a therapeutically effective dose. Preferably, BGB-11417 is administered once daily. Further preferably, BGB-11417 is administered orally.

[0023] AZD0466 as a BCL-2 inhibitor

[0024] In an embodiment, AZD0466 is administered at a therapeutically effective dose. Preferably, AZD0466 is administered once daily. Further preferably, AZD0466 is administered intravenously.

[0025] AZD4320 as a BCL-2 inhibitor

[0026] In an embodiment, AZD4320 is administered at a therapeutically effective dose. Preferably, AZD4320 is administered once daily. Further preferably, AZD4320 is administered intravenously.

[0027] Venetoclax as a BCL-2 inhibitor

[0028] In embodiments, 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 the first day of a treatment cycle, then at a daily dose of about 200 mg on the second day, and then at a daily dose of about 400 mg on the third day and thereafter, or alternatively, then at a daily dose of about 600 mg on the fourth day, and then also at about 600 mg after the fourth day. Further preferably, venetoclax is administered orally.

[0029] In an embodiment, particularly if the BCL-2 inhibitor is venetoclax, the combination of use of the first aspect further comprises (iii) a hypomethylating agent or cytarabine. Preferably, the hypomethylating agent is selected from azacitidine, decitabine, CC-486 and ASTX727. More preferably, the hypomethylating agent is azacitidine or decitabine, most preferably azacitidine.

[0030] In embodiments, particularly if the BCL-2 inhibitor is venetoclax, the hypomethylating agent is administered at about 10 mg / m 2 About 100 mg / m 2 (The unit used in this article is mg / m 2 Refers to body surface area [BSA] in mg / m 2 Preferably, the hypomethylating agent is administered at a daily dose of about 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 If the hypomethylating agent is azacitidine, 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 If the hypomethylating agent is decitabine, 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 hypomethylating agent is administered once a day. Further preferably, the hypomethylating agent is administered intravenously, subcutaneously or orally, wherein azacitidine is preferably administered intravenously or subcutaneously, and decitabine is preferably administered intravenously.

[0031] In embodiments, particularly if the BCL-2 inhibitor is venetoclax, the hypomethylating agent is administered for (a) seven consecutive days from day 1 to day 7 of a 28-day cycle, or (b) five consecutive days from day 1 to day 5 of a 28-day cycle, wherein the hypomethylating agent in (a) is preferably azacitidine and the hypomethylating agent in (b) is preferably decitabine.

[0032] In embodiments, particularly if 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 Preferably, cytarabine is administered at a daily dose of about 20 mg / m 2 Preferably, cytarabine is administered once daily. Preferably, cytarabine is administered subcutaneously. In an embodiment, cytarabine is administered for 10 consecutive days from day 1 to day 10 of a 28-day cycle. If cytarabine is used in combination with venetoclax and SEL24 / MEN1703, venetoclax can be administered starting from day 4 (i.e., day 4 and thereafter) at a daily dose of about 600 mg.

[0033] In an alternative embodiment, the combination used in the first aspect does not comprise the administration of an additional chemotherapy.

[0034] In an embodiment involving a combination for use as a medicament, (i) and (ii) (and optionally (iii)) are administered in separate dosage forms. In this embodiment, administration may be concomitant or sequential. In yet another alternative embodiment involving a combination for use as a medicament, (i) and (ii) (and optionally (iii)) are administered together in a dosage form.

[0035] In a second aspect, the present invention relates to a combination of (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703 for use in treating a patient suffering from cancer.

[0036] In a preferred embodiment, the cancer is a hematological cancer. In an even more preferred embodiment, the cancer is a leukemia. In a most preferred embodiment, the cancer is AML. In one embodiment, particularly if the BCL-2 inhibitor is venetoclax, the AML may be newly diagnosed AML. In one embodiment, particularly if the BCL-2 inhibitor is venetoclax, the combination of the present invention can be used to treat patients with AML who are not suitable for intensive chemotherapy, preferably adult patients with newly diagnosed AML who are not suitable for intensive chemotherapy. In another embodiment, particularly if the BCL-2 inhibitor is venetoclax, the combination of the present invention can be used to treat patients aged 75 years or older. In one embodiment, particularly if the BCL-2 inhibitor is venetoclax, the combination of the present invention can be used to treat patients with comorbidities that prevent the use of intensive induction chemotherapy.

[0037] When the patient suffers from AML, the patient with AML can show the FLT3 mutation causing the over-activation of FLT3 signal transduction.The mutation in FLT3 can even cause the FLT3 signal transduction of constitutive activity (this means that the signal transduction activity of FLT3 is constitutive activity).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 over-activation of FLT3 signal transduction.This type of mutation is known in the art for more than 25 years, and the most common FLT3-ITD ("internal tandem duplication") mutation was first reported in 1996 (see Nakao M et al., 1996).The FLT3 mutation can be 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) can show at least one IDH1 and / or IDH2 mutation, preferably at least two IDH1 and / or IDH2 mutations. Alternatively, when the patient has AML, the patient with AML may exhibit FLT3 wt status.

[0038] 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.

[0039] In an embodiment, the BCL-2 inhibitor is selected from venetoclax, S65487, APG-2575, LOXO-338, ZN-d5, BGB-11417, AZD4320, and AZD0466.

[0040] In a preferred embodiment, the BCL-2 inhibitor is S65487, APG-2575 or venetoclax.

[0041] In a more preferred embodiment, the BCL-2 inhibitor is venetoclax.

[0042] S65487 as a BCL-2 inhibitor

[0043] In an embodiment, S65487 is administered at a therapeutically effective dose. Preferably, S65487 is administered once daily. Further preferably, S65487 is administered intravenously.

[0044] APG-2575 as a BCL-2 inhibitor

[0045] In an 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. Further preferably, APG-2575 is administered orally.

[0046] LOXO-338 as a BCL-2 inhibitor

[0047] In an embodiment, LOXO-338 is administered at a therapeutically effective dose. Preferably, LOXO-338 is administered once daily. Further preferably, LOXO-338 is administered orally.

[0048] ZN-d5 as a BCL-2 inhibitor

[0049] In an embodiment, ZN-d5 is administered at a therapeutically effective dose. Preferably, ZN-d5 is administered once daily. Further preferably, ZN-d5 is administered orally.

[0050] BGB-11417 as a BCL-2 inhibitor

[0051] In an embodiment, BGB-11417 is administered at a therapeutically effective dose. Preferably, BGB-11417 is administered once daily. Further preferably, BGB-11417 is administered orally.

[0052] AZD0466 as a BCL-2 inhibitor

[0053] In an embodiment, AZD0466 is administered at a therapeutically effective dose. Preferably, AZD0466 is administered once daily. Further preferably, AZD0466 is administered intravenously.

[0054] AZD4320 as a BCL-2 inhibitor

[0055] In an embodiment, AZD4320 is administered at a therapeutically effective dose. Preferably, AZD4320 is administered once daily. Further preferably, AZD4320 is administered intravenously.

[0056] Venetoclax as a BCL-2 inhibitor

[0057] In embodiments, 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 the first day of a treatment cycle, then at a daily dose of about 200 mg on the second day, and then at a daily dose of about 400 mg on the third day and thereafter, or alternatively, then at a daily dose of about 600 mg on the fourth day, and then also at a daily dose of about 600 mg after the fourth day. Further preferably, venetoclax is administered orally.

[0058] In an embodiment, particularly if the BCL-2 inhibitor is venetoclax, the combination of use of the second aspect further comprises (iii) a hypomethylating agent or cytarabine. Preferably, the hypomethylating agent is selected from azacitidine, decitabine, CC-486 and ASTX727. More preferably, the hypomethylating agent is azacitidine or decitabine, most preferably, azacitidine.

[0059] In embodiments, particularly if the BCL-2 inhibitor is venetoclax, the hypomethylating agent is administered at about 10 mg / m 2 About 100 mg / m 2 (The unit used in this article is mg / m 2 Refers to body surface area [BSA] in mg / m 2 Preferably, the hypomethylating agent is administered at a daily dose of about 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 If the hypomethylating agent is azacitidine, azacitidine is preferably administered at a daily dose of about 60 mg / m 2 , about 65mg / m 2 , about 70mg / m 2or about 75 mg / m 2 If the hypomethylating agent is decitabine, 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 hypomethylating agent is administered once a day. Further preferably, the hypomethylating agent is administered intravenously, subcutaneously or orally, wherein azacitidine is preferably administered intravenously or subcutaneously, and decitabine is preferably administered intravenously.

[0060] In embodiments, particularly if the BCL-2 inhibitor is venetoclax, administration of the hypomethylating agent continues (a) for seven consecutive days from day 1 to day 7 of a 28-day cycle, or (b) for five consecutive days from day 1 to day 5 of a 28-day cycle, wherein the hypomethylating agent in (a) is preferably azacitidine and the hypomethylating agent in (b) is preferably decitabine.

[0061] In embodiments, particularly if 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 Preferably, cytarabine is administered at a daily dose of about 20 mg / m 2 Preferably, cytarabine is administered once daily. Preferably, cytarabine is administered subcutaneously. In an embodiment, cytarabine is administered for 10 consecutive days from day 1 to day 10 of a 28-day cycle. If cytarabine is used in combination with venetoclax and SEL24 / MEN1703, venetoclax can be administered starting from day 4 (i.e., day 4 and thereafter) at a daily dose of about 600 mg.

[0062] In an embodiment, the combination for use of the second aspect does not comprise the administration of an additional chemotherapy.

[0063] In an embodiment involving a combination for use as a medicament, (i) and (ii) (and optionally (iii)) are administered in separate dosage forms. In this embodiment, administration may be concomitant or sequential. In yet another alternative embodiment involving a combination for use as a medicament, (i) and (ii) (and optionally (iii)) are administered together in a dosage form.

[0064] In a preferred embodiment of the second aspect, the combination comprises (i) venetoclax as a BCL-2 inhibitor and (ii) SEL24 / MEN1703 as the sole active agent; the cancer is AML; venetoclax is administered at a daily dose of about 400 mg (starting on day 3) (after initial titration, starting from about 100 mg on day 1 to about 200 mg on day 2), preferably orally; SEL24 / MEN1703 is administered at a daily dose of about 80 mg to about 120 mg, preferably orally. Preferably, in this embodiment, the patient with AML is an adult patient newly diagnosed with AML who may not be suitable for intensive chemotherapy.

[0065] In yet another preferred embodiment, the combination comprises (i) venetoclax as a BCL-2 inhibitor, (ii) SEL24 / MEN1703, and (iii) a hypomethylating agent, preferably azacitidine or decitabine; the cancer is AML; venetoclax is administered 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), preferably orally; SEL24 / MEN1703 is administered at a daily dose of about 80 mg to about 120 mg, preferably orally; the hypomethylating agent is administered at a daily dose of about 20 mg / m 2 Up to about 75 mg / m 2 A daily dose of about 75 mg / m 2 The decitabine is preferably about 20 mg / m 2 ), preferably intravenously. Preferably, in this embodiment, the patient with AML is an adult patient newly diagnosed with AML who may not be suitable for intensive chemotherapy.

[0066] In yet another preferred embodiment, the combination comprises (i) venetoclax as a BCL-2 inhibitor, (ii) SEL24 / MEN1703 and (iii) cytarabine; the cancer is AML; venetoclax is administered 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), preferably orally; SEL24 / MEN1703 is administered at a daily dose of about 80 mg to about 120 mg, preferably orally; cytarabine is administered at about 20 mg / m 2 The combination is administered at a daily dose of, preferably subcutaneously. Preferably, in this embodiment, the AML is newly diagnosed AML and the patient is 75 years of age or older. In one embodiment, the combination can be used to treat patients (optionally newly diagnosed AML) with comorbidities that preclude the use of intensive induction chemotherapy.

[0067] In a third aspect, the present invention 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.

[0068] In an embodiment, the dosage form comprising a BCL-2 inhibitor comprises a BCL-2 inhibitor selected from venetoclax, S65487, APG-2575, LOXO-338, ZN-d5, BGB-11417, AZD4320, and AZD0466.

[0069] In a preferred embodiment, the dosage form comprising a BCL-2 inhibitor comprises a BCL-2 inhibitor selected from S65487, APG-2575, and venetoclax.

[0070] In a more preferred embodiment, the dosage form comprising a BCL-2 inhibitor comprises venetoclax as the BCL2 inhibitor.

[0071] In an embodiment, the dosage form comprising a BCL-2 inhibitor comprises venetoclax, and 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. Further preferably, the dosage form comprising venetoclax is an oral dosage form.

[0072] 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, 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.

[0073] Each dosage form generally contains at least one pharmaceutically acceptable excipient, as defined in Section 2 of the detailed description below.

[0074] Specifically, if venetoclax is included in the kit as a BCL-2 inhibitor, in an embodiment, the kit may further comprise: a dosage form comprising a hypomethylating agent (preferably azacitidine or decitabine), or a dosage form comprising cytarabine, wherein each dosage form comprises an amount of cytarabine to achieve the administration level (mg / m 2 The kit further comprises a dosage form comprising azacitidine.

[0075] In an embodiment, the kit of the third aspect further comprises a booklet listing instructions on how to use and administer the dosage form.

[0076] In a fourth aspect, the present invention relates to a dosage form comprising (i) a BCL-2 inhibitor and (ii) SEL24 / MEN1703.

[0077] In an embodiment, the dosage form comprises a BCL-2 inhibitor selected from venetoclax, S65487, APG-2575, LOXO-338, ZN-d5, BGB-11417, AZD4320, and AZD0466.

[0078] In a preferred embodiment, the dosage form comprises a BCL-2 inhibitor selected from S65487, APG-2575, and venetoclax.

[0079] In a more preferred embodiment, the dosage form comprises venetoclax as the BCL2 inhibitor.

[0080] In an embodiment, the dosage form comprises venetoclax as a BCL-2 inhibitor and 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. Further preferably, the dosage form comprising venetoclax is an oral dosage form.

[0081] In an 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.

[0082] In particular if venetoclax as a BCL-2 inhibitor is included in the dosage form, in an embodiment the dosage form further comprises a hypomethylating agent, preferably CC-486 or ASTX727, as they are administered orally.

[0083] In an embodiment, the dosage form is a once-daily dosage form. Further preferably, the dosage form is an oral dosage form.

[0084] The dosage form generally contains at least one pharmaceutically acceptable excipient, as defined in Section 2 of the detailed description below.

[0085] 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.

[0086] 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 BCL-2 inhibitor and an effective amount of (ii) SEL24 / MEN1703.

[0087] All embodiments outlined above for the second aspect are equally applicable to the fifth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 : Statistical analysis of cytotoxicity data at IC75 concentrations in KG1 cells. When the triple combination Men+Aza+Ven (mav) was compared to Men+Ven, Ven+Aza, Men+Aza and single agent treatments, it was found that the Men+Aza+Ven treatment was significantly different relative to Men+Ven and relative to single treatments (Student's t test, p<0.01**, p<0.1*). The Ven+Men combination showed significant differences relative to the Ven single treatment.

[0089] Figure 2: Statistical analysis of cytotoxicity data at IC75 concentrations in MV4-11 cells. When the triple combination Men+Aza+Ven (mav) was compared to Men+Ven, Ven+Aza, Men+Aza, and single agent treatments, it was found that the Men+Aza+Ven treatment was significantly different relative to Men alone, and the Men+Ven combination treatment was significantly different relative to Ven as a single agent treatment (Student's t test, p<0.01**, p<0.1*).

[0090] Fig. 3: in vivo study in AML MOLM-16 cell line xenograft.MEN1703, 5-azacytidine and Venetoclax as single agent (left figure) and as double drug and three-drug combination (right figure) anti-tumor activity in MOLM-16 xenograft tumor model.Tumor cells were injected sc into SCID mice at day 0, and drug administration was started at day 29.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 the tumor volume of single treatment (left figure) and combination treatment (right figure) in all days of each administration, and (B) is shown in the tumor volume at day 41.

[0091] Figure 4: In vivo studies in AML MV4-11 cell line xenografts. Antitumor activity of MEN1703, 5-azacytidine and venetoclax as single agents (left panel) and as dual and triple drug combinations (right panel) 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 the tumor volume for monotherapy (left panel) and combination therapy (right panel) on all days of respective administration, while (B) shows the tumor volume on day 45.

[0092] Figure 5 : Engraftment of human hematopoietic cells (CD45 positive) in the peripheral blood of NSG mice was monitored at the indicated time points.

[0093] Figure 6 : Histograms represent the percentage of human CD45+ AML cell engraftment in mice 110 days after transplantation. Results are presented as mean ± SD (**p<0.01; ***p<0.001; ****p<0.0001).

[0094] Figure 7: Kaplan-Meier curves comparing overall survival of PDX AML mouse models and statistical results of treatment group comparisons.

[0095] Fig. 8: (A) MOLM-13 cells and (B) MV4-11 cells at IC50 concentrations of cytotoxicity data statistical analysis ("MEN1703" = MEN, "MEN1703 + S65487" = "MEN + S65487"). The cytotoxicity induced by the MEN1703 + S65487 combination was significantly different from the cytotoxicity induced by MEN1703 and the cytotoxicity induced by S65487 (Tukey's multiple comparisons one-way ANOVA test; p < 0.05 *, p < 0.01 **, p < 0.001 ***).

[0096] Fig. 9: (A) MOLM-13 cells and (B) MV4-11 cells at IC50 concentrations of cytotoxicity data statistical analysis ("MEN1703" = MEN, "MEN1703 + AZD4320" = "MEN + AZD4320"). The cytotoxicity induced by the MEN1703 + AZD4320 combination was significantly different from the cytotoxicity induced by MEN1703 and the cytotoxicity induced by AZD4320 (Tukey multiple comparisons one-way ANOVA test; p < 0.05 *). DETAILED DESCRIPTION OF THE INVENTION

[0098] Before describing the present invention in more detail, the following definitions are introduced.

[0099] 1. definition

[0100] 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.

[0101] 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%.

[0102] 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.

[0103] As used herein, the term "combination" or "combination with..." is not intended to imply that therapies or active agents (i) and (ii) (and optionally (iii)) must be administered simultaneously and / or formulated for delivery together, although such therapies and formulations are within the scope of the present invention. The active agents in the combination can be administered simultaneously with each other and even one or more other additional therapies or active agents, before them, or after them. The active agents or treatment regimens 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 at a therapeutic dose or below the therapeutic dose, for example, venetoclax is administered at a dose below the therapeutic dose or SEL24 / MEN1703 is administered at a dose below the therapeutic dose (wherein "below the therapeutic dose" is derived from a comparison with 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.

[0104] As used herein, the term "venetoclax" refers to the BCL-2 inhibitor venetoclax, sold under the trade name and Both are approved, inter alia, for the treatment of AML. Further details about venetoclax can be found, inter alia, in the product leaflet or in the regulatory dossier.

[0105] 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, 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 apparent from the data of WO 2014 / 096388 that the different compounds synthesized and tested therein do not necessarily have the same mechanism of action (for example, when compound 26A is compared with other compounds (e.g., compound 1A, which differs structurally from compound 26A in terms of ring substituents and alkyl substituents), with respect to their activities against different PIM kinases, see Table 1A of WO 2014 / 096388, it can be concluded that SEL24 / MEN1703 is a pan-PIM inhibitor, whereas other compounds (e.g., compound 1A) are quite specific for certain PIM kinases).

[0106] As used herein, the term "hypomethylating agent" refers to an agent that inhibits DNA methylation, i.e., modifies DNA nucleotides by adding methyl groups. Treatment with a hypomethylating agent is considered an epigenetic therapy. Currently available hypomethylating agents include azacitidine disclosed herein (alternatively referred to herein as "5-azacitidine") and decitabine, which block the activity of DNA methyltransferases. Further details about azacitidine and decitabine can be found in particular in the above and The product leaflet or regulatory file of CC-486 is also a hypomethylating agent, i.e., oral azacitidine, see Kipp and Wei, 2021. Yet another hypomethylating agent is ASTX727, which is decitabine / cedazuridine, see Kipp and Wei, 2021. Both CC-486 and ASTX727 are orally administered hypomethylating agents.

[0107] As used herein, the term "cytarabine" refers to a chemotherapeutic agent that may alternatively be named "cytosine arabinoside (ara-C)". Cytarabine binds the cytosine base to the sugar arabinose, interfering with DNA synthesis. It does this through its rapid conversion to cytosine arabinoside triphosphate, which damages DNA when the cell cycle is held in the S phase. Therefore, rapidly dividing cells are most affected. Further details on cytarabine can be found in particular above can be found in the product leaflet or regulatory dossier.

[0108] As used herein, the term "S65487" refers to the BCL-2 inhibitor S65487, which may alternatively be referred to as "VOB560". It is commercially available, for example, from MedChemExpress and has a CAS number of 1644600-79-2. S65487 is currently undergoing clinical studies to evaluate safety, tolerability, PK and preliminary clinical activity, and to estimate the maximum tolerated dose / recommended Phase 2 dose administered intravenously as a single agent in adult patients with refractory or relapsed AML, non-Hodgkin lymphoma (NHL), multiple myeloma (MM) or chronic lymphocytic leukemia (CCL) [ClinicalTrials.gov identifier: NCT03755154].

[0109] As used herein, the term "APG-2575" refers to the BCL-2 inhibitor APG-2575, which may alternatively be referred to as "Lisaftoclax". It is commercially available from, for example, MedChemExpress and has the CAS number 2180923-05-9. APG-2575 is currently undergoing clinical studies to evaluate the safety and pharmacokinetics of APG-2575 as a single agent and in combination with homoharringtonine or azacitidine in patients with relapsed / refractory AML and related myeloid malignancies [ClinicalTrials.gov Identifier: NCT04501120].

[0110] As used herein, the term "LOXO-338" refers to the BCL-2 inhibitor LOXO-338, which may be alternatively referred to as "LY3847429." LOXO-338 is currently undergoing clinical studies, among others, to evaluate LOXO338 as a monotherapy in patients with advanced hematological malignancies [ClinicalTrials.gov Identifier: NCT05024045].

[0111] As used herein, the term "ZN-d5" refers to the BCL-2 inhibitor ZN-d5. Zentalis announced on April 30, 2020 that the FDA approved the IND application for ZN-d5 for the treatment of hematological malignancies.

[0112] As used herein, the term "BGB-11417" refers to the BCL-2 inhibitor BGB-11417. BGB-11417 is currently undergoing a clinical study [ClinicalTrials.gov Identifier: NCT04277637] to evaluate BGB-11417 with regard to safety and tolerability, and to define the maximum tolerated dose and the recommended Phase 2 dose, and to evaluate the safety and tolerability of the dose escalation schedule of monotherapy.

[0113] As used herein, the term "AZD0466" refers to the BCL-2 inhibitor AZD0466, which is characterized in particular in Arulananda et al., 2021. AZD0466 is disclosed in a clinical study to evaluate the safety, tolerability, maximum tolerated dose, recommended Phase 2 dose and PK of AZD0466 in patients with solid tumors, lymphomas and multiple myeloma at lower risk of tumor lysis syndrome [ClinicalTrials.gov Identifier: NCT04214093].

[0114] As used herein, the term "AZD4320" refers to the BCL-2 inhibitor AZD4320, which is inter alia characterized in Balachander et al., 2020. It is commercially available, for example, from MedChemExpress and has the CAS number 1357576-48-7.

[0115] As used herein, the term "treatment" refers to clinical intervention performed in order to cure or ameliorate a disease, prevent disease recurrence, alleviate disease symptoms, reduce any direct or indirect pathological consequences of the disease, achieve a stable state of the 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.

[0116] 2. Pharmaceutical composition

[0117] BCL-2 inhibitors, SEL24 / MEN1703, hypomethylating agents and cytarabine are "pharmaceutically active agents" or "active agents" for the purposes of the present invention. As described above, BCL-2 inhibitors and SEL247MEN1703 may be present in separate dosage forms or contained in a single dosage form.

[0118] 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.

[0119] It is generally preferred that the administration of the BCL-2 inhibitor and SEL24 / MEN1703 is carried out in different dosage forms, and the BCL-2 inhibitor is in the dosage form and administered by 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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

[0129] 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.

[0130] Example 1: In vitro studies in cell lines

[0131] 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 and then treated with Men for an additional 24 hours. The synergistic effect of the resulting combination of Men with Ven or 5-Aza alone (i.e., two active combinations) or with Ven + 5-Aza (i.e., three active combinations) was 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 tested AML cell lines (see Tables 1 to 3 and Figure 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 treatment (see Figure 1 ), where the combination index appeared lower in the triple combination Men+5-Aza+Ven relative to 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, where the CI of the triple combination was approximately equal to that of Men+Ven and significantly lower than that of Ven+Aza (see Figure 2 In MOLM13 cells (FLT3 ITD), the CI of the triple combination was similar to that of the Men-Ven and Ven-Aza combinations (see Table 3).

[0132] Table 1: In vitro combination index in KG1 cells

[0133]

[0134] The combination of Men+Ven+Aza(mav) has strong synergistic effects at clinically relevant concentrations (grey row).

[0135] Table 2: In vitro combination index in MV4-11 cells

[0136]

[0137] The combination of Men+Ven+Aza(mav) has strong synergistic effects at clinically relevant concentrations (grey row).

[0138] Table 3: In vitro combination index in MOLM13 cells

[0139]

[0140] The CI for the combination of Men+Ven+Aza (mav) was similar to the CIs for the combinations of Men+Ven and Ven+Aza at clinically relevant concentrations (grey row).

[0141] Example 2: In vivo xenograft studies

[0142] The in vitro results were confirmed in in vivo xenograft studies with MOLM-16 (FLT3 wt) and MV4-11 (FLT3 ITD) cells. In MOLM-16 xenografts, consistent tumor growth inhibition was observed with the triple combination, which was the only statistically significant combination when compared to control (see Figure 3A and 3B). However, it is noteworthy that these three actives were used at lower concentrations in this experiment (see Materials and Methods section below), as otherwise it would not be possible to observe an effect, i.e., there would be no effect when the actives are combined at higher concentrations. This explains why such low concentrations of the single agents were not as effective in this experimental setting. In addition, high concentrations of these actives are generally toxic when administered in combination in mice. In MV4-11 xenografts, consistent tumor growth inhibition was observed with the triple combination, which was significantly different when compared to controls, single agent Aza and Ven treatments, and Ven+Aza and Men+Aza combination treatments. Ven+Men was also significantly different from controls and single agent Ven treatments in this setting (see Figure 4A and 4B ).

[0143] The combination therapy was also tested in a diffuse AML patient-derived xenograft (PDX) model. This model demonstrated the superior efficacy of the triple combination therapy relative to the Ven+Aza combination. Statistical analysis showed significant differences in tumor burden (calculated as % of human CD45-positive cells) between the triple combination therapy and the single agent treatments and the Men+Aza combination treatment (see Figure 5 and 6 Statistical analysis of the overall survival data showed that the triple combination therapy was significantly different from the single agent therapy and from the Men+Aza or Ven+Aza combination therapy (see Figure 7 ).

[0144] Materials and methods for Examples 1 and 2:

[0145] Human cell lines

[0146] Human acute myeloid leukemia cell lines KG-1, MV4-11 and MOLM-13 are all obtained from DSMZ, Braunschweig, Germany (ACC14, ACC102 and ACC554). KG-1 and MV4-11 cells are cultured in RPMI 1640 culture medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, Saint Louis, CA, USA). MOLM-13 cells are grown in RPMI containing 20% ​​FBS. All cells are incubated at 37°C, 5% CO2, 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, Saint Louis, CA, USA). Cells were incubated at 37° C., 5% CO 2 , 80% relative humidity.

[0147] Active substances in cytotoxicity assays

[0148] Men (alternatively referred to as "MEN1703", (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)) and Venetoclax (alternatively referred to as "Ven" or "ABT-199", ChemCruz No. SC-472284A) in the form of HCl salt synthesized at Aptuit (VR) were dissolved in DMSO, aliquoted and stored at -80°C until use. The stock solution was added to the culture medium at an appropriate concentration before addition to the cells. 5-Azacitidine (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 (because it is very unstable in aqueous solution). The stock solution is added to the culture medium at an appropriate concentration prior to addition to the cells.

[0149] Active substances in mouse models

[0150] Men in the form of the HCl salt (alternatively referred to as "MEN1703", (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. 5-Azacytidine (Batch BCCC8387) was purchased from SIGMA Aldrich, and the powder was dissolved in sterile saline solution and administered within one hour. Venetoclax (Batch B0521) was purchased from Santa Cruz Biotechnology, Inc., and the powder was first dissolved in DMSO and then diluted in a solution of 20% 2-hydroxypropyl-β-cyclodextrin (HPCD) in 100 mM citrate buffer (pH 3).

[0151] Cytotoxicity assay

[0152] For cytotoxicity studies, cells were seeded at an appropriate density (50,000 cells / well) before adding Ven and 5-Aza on day 0. Due to degradation, 5-Aza was added every 24 hours (Hollenbach PW, 2010 and Chang E, 2016). Then, after 48 hours, Men was added to the cultured cells for the last 24 hours (a total of 72 hours of time was tested). The concentration range at the same time point was previously determined in a single-agent cytotoxicity assay to include the IC10, IC25, IC50 and IC75 values ​​of each drug and each AML cell line in the combination experiment (see Tables 1 to 3). All possible combinations were evaluated: Men+Ven, Ven+5-Aza, Men+5-Aza and men+5-Aza+Ven (mav). After 72 hours, CellTiter96Aqueous One Solution Reagent (MTS) (Promega, Madison.WI, USA) was added to assess cell viability. Fluorescence was measured after 4 h using Tecan Infinite M200 (Tecan Trading AG, Switzerland) and absorbance was recorded at 490 nm. Quantitative measurement of synergy / antagonism was assessed using the combination index (CI) of the affected fraction (Fraction affected, Fa) using CompuSyn (ComboSyn, Inc. Paramus, NJ, USA) (Chou TC, 2006).

[0153] MOLM-16 Model

[0154] For AML xenograft models, 10x 10 6 MOLM-16 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 SCID mice (Charles River, Calco, Italy).

[0155] 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 animal housing and handling 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.

[0156] The outcome was evaluated as follows: tumor volume was measured by caliper, and tumor mass was calculated using the following formula: [length (mm) × width 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 3A Animals were randomly divided into 8 groups (5-6 mice / group) and the following treatments were administered starting on day 29: Group I received oral vehicle (Venetracla dilute solution) once daily until day 42 (single administration, see Figure 3A Left: Combined application, see Figure 3A Right), Group II received 25 mg / kg Men orally once daily q1dx5, starting from day 38 until day 42 (single administration, see Figure 3A Left), Group III received 1.25 mg / kg 5-azacytidine intraperitoneally every two days for a total of 3 doses (single administration, see Figure 3A Left: Combined application, see Figure 3A right), and Group IV received oral venetoclax (given at 100 mg / kg) once daily q1dx14 until Day 42 (single administration, see Figure 3A Left), Group V received a combination of Men (25 mg / kg once daily q1dx14 orally starting from Day 38 until Day 51) + 5-Aza (at the same dose and regimen as the single-agent group) (combination administration, see Figure 3ARight), Group VI received a combination of Men (25 mg / kg orally once daily q1dx14 starting from day 38 until day 51) + Venetoclax (100 mg / kg orally once daily q1dx23 starting from day 29 until day 51) (combination administration, see Figure 3A Right), Group VII received a combination of 5-Aza (at the same dose and schedule as the single-agent group) + venetoclax (given orally at 100 mg / kg once daily q1dx23 starting on Day 29 until Day 51) (combination administration, see Figure 3A Right, and Group VIII received the triple combination of Men+5-Aza+Ven, with all drugs administered at the same dose and schedule as the double combination group.

[0157] 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.

[0158] 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).

[0159] MV4-11 Model

[0160] 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).

[0161] 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.

[0162] 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 4A The animals were randomly divided into 8 groups (5-6 mice / group) and the following treatments were administered starting on day 21: Group I received vehicle (Venetracla dilute solution) orally once daily until day 42, and Group II received 25 mg / kg Men orally once daily q1dx13 starting on day 30 until day 42 (single administration, see Figure 4A Left: Combined application, see Figure 4A Right), Group III received 1.25 mg / kg of 5-azacytidine intraperitoneally every two days for a total of 3 doses (single administration, see Figure 4A Left: For combined use, see Figure 4A Right), and Group IV received oral venetoclax (given at 100 mg / kg) once daily q1dx22 until Day 42 (single administration, see Figure 4A Left: Combined application, see Figure 4A Right), groups V-VI-VII received the combination of Men+5-Aza, Men+Ven and 5-Aza+Ven, respectively, and group VIII received the triple combination of Men+5-Aza+Ven, in which the dosages and administration methods of the above-mentioned active agents were also used in the combinations.

[0163] 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.

[0164] 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).

[0165] AML PDX Models

[0166] The human AML samples used in this study have been tested and the results indicate the mutational status of IDH2, JAK2, MIR636, SRSF2, and RUNX1. Peripheral blood mononuclear cells (PBMCs) were isolated from patient samples by Ficoll separation and T-cell depletion was performed using anti-CD3 magnetic beads (Miltenyi Biotec). T-cell depletion has been previously shown to enhance the engraftment of AML cells in female non-obese diabetic severe combined immunodeficient NOD / SCID interleukin-2 receptor gamma (IL-2Rγ)-deficient (NSG) hosts by limiting polyclonal donor T-cell expansion due to xenogeneic graft-versus-host disease (Von Bonin M. et al., 2013). For PDX, experiments were performed on female NSG mice aged 6 to 8 weeks. Mice were housed under pathogen-free conditions in the animal facility of the European Institute of Oncology-Institute of Molecular Oncology, Italian Foundation for Cancer Research (IEO-IFOM, Milan, Italy). All animal experiments were performed in strict accordance with Italian legislation (DLvo26 / 2014 and supplements below) and were approved by the institutional committee.

[0167] 10 million T cell depleted cells were transplanted intraperitoneally into 6 to 8 week old male and female NSG mice conditioned with 1 Gy irradiation. Bone marrow and spleen cells were collected from one primary recipient mouse and cryopreserved in liquid nitrogen after staining with different anti-human CD45 APC, CD33 APC-Cy7, CD13 PE-Cy7, CD117 PE and anti-mouse CD45 FITC antibodies and checked for human engraftment (hCD45>80%) and patient markers by FACS analysis. For the experiments, human blasts collected from the second generation spleen of the xenograft were injected intravenously (750.000 cells / mouse) into the lateral tail vein of NSG mice 1 day before receiving low dose irradiation (1 Gy). On day 38 after transplantation, once systemic xenografting (approximately 0.05%) is confirmed, mice will be randomized to receive treatment. The percentage of human cells in peripheral blood from the tail vein was assessed by flow cytometry, once a week until day 110, evaluating anti-human CD45-APC (clone J.33, Beckman-Coulter) and anti-mouse CD45-PE (clone 30-F11, BD) to exclude mouse cell contamination. All blood samples were lysed with ammonium chloride red blood cell buffer (Qiagen) before staining. The percentage of stained cells was determined and compared with the appropriate negative control. Hepta-aminoactinomycin D (7AAD) from Sigma-Aldrich was used to count live cells, apoptotic cells, and dead cells. Cell suspensions were evaluated by a 3-laser, 10-color flow cytometer (Navios, Beckman Coulter, Brea, CA, USA) using an analytical gate designed to exclude dead cells, platelets, and debris. Implanted mice (n=5 per study arm) were treated with vehicle or with different drugs used as single agents or in combination.

[0168] In detail, mice were treated with 5-azacytidine (1.25 mg / kg) intraperitoneally (ip) for 3 days (days 1-4-7 after implantation), or with MEN1703 (25 mg / kg) for 14 consecutive days (days 10-23 after implantation), or with venetoclax (100 mg / kg) for 23 consecutive days (starting from day 1 after implantation), all by oral gavage.

[0169] Therapeutic efficacy was assessed as expression of human CD45 in the peripheral blood of mice + Animals were euthanized by carbon dioxide exposure according to standard procedures.

[0170] Statistical analysis

[0171] 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 4 to 6) are presented as mean values, with values ​​for each group represented by symbols of different shapes and lines of different colors. Analysis was performed on day 110 of the AML PDX model using Tukey's multiple comparison test.

[0172] Example 3: In vitro studies in cell lines for combination with S65487

[0173] In a standard in vitro cytotoxicity assay, 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 measured according to the combination index calculation (Chou TC, 2006).

[0174] The synergistic effect of the combination of MEN and S65487 in MOLM13 cells is shown in Table 4 (see Table 4, combination index or CI < 1.0). The cytotoxicity induced by the combination of MEN and S65487 at IC50 concentration is significantly different from the cytotoxicity induced by S65487 in MOLM-13 cells and the cytotoxicity induced by MEN1703 in MOLM-13 cells (Tukey multiple comparisons single factor ANOVA test; p < 0.05*, p < 0.01**, see Fig. 8A ).

[0175] The combination of MEN and S65487 was synergistic in MV4-11 cells, as can be concluded from Table 5 (see Table 5, combination index or CI < 1.0). The cytotoxicity induced by the combination of MEN and S65487 at the IC50 concentration was significantly different from the cytotoxicity induced by S65487 in MV4-11 cells and the cytotoxicity induced by MEN1703 in MV4-11 cells (Tukey's multiple comparisons one-way ANOVA test; p < 0.05*, p < 0.001***, see Figure 8B ).

[0176] Table 4: In vitro combination index in MOLM-13 cells

[0177]

[0178] Table 5: In vitro combination index in MV4-11 cells

[0179]

[0180] Example 4: In vitro studies in cell lines for combination with AZD4320

[0181] In a standard in vitro cytotoxicity assay, 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 measured according to the combination index calculation (Chou TC, 2006).

[0182] The synergistic effect of the combination of MEN and S65487 in MOLM13 cells is shown in Table 6 (see Table 6, combination index or CI < 1.0). The cytotoxicity induced by the combination of MEN and AZD4320 at IC50 concentrations was significantly different from the cytotoxicity induced by AZD4320 in MOLM-13 cells and the cytotoxicity induced by MEN1703 in MOLM-13 cells (Tukey multiple comparisons one-way ANOVA test; p < 0.05 *, see Fig. 9A ).

[0183] The combination of MEN and AZD4320 was synergistic in MV4-11 cells, as can be concluded from Table 7 (see Table 7, combination index or CI < 1.0). The cytotoxicity induced by the combination of MEN and AZD4320 at the IC50 concentration was significantly different from the cytotoxicity induced by AZD4320 in MV4-11 cells and the cytotoxicity induced by MEN1703 in MV4-11 cells (Tukey's multiple comparisons one-way ANOVA test; p < 0.05*, see Fig. 9B ).

[0184] Table 6: In vitro combination index in MOLM-13 cells

[0185]

[0186] Table 7: In vitro combination index in MV4-11 cells

[0187]

[0188]

[0189] Materials and Methods for Examples 3 and 4

[0190] Human cell lines

[0191] 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 containing 20% ​​FBS. All cells are incubated at 37°C, 5% CO2 and 80% relative humidity.

[0192] drug

[0193] MEN1703 (alternatively referred to as "Men", "MEN", "MEN1703" 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 (lot A / 2201 / 24 / 1), synthesized at Aptuit (VR); AZD4320 (from MedChem Express, Monmouth Junction, NJ, USA, lot 64354) and S65487 (from 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 and then added to the cells.

[0194] Cytotoxicity

[0195] 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 a single-agent cytotoxicity assay at the same time point to include IC10, IC25, IC50, and IC75 values ​​for each drug and each AML cell line in the combination experiment (see Tables 4 to 7). After 72 hours, CellTiter 96 Aqueous 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 combined index (CI) of the affected fraction (Fa) (Chou TC, 2006).

[0196] 4. References:

[0197] Arulananda et al.,A novel BH3-mimetic,AZD0466,targeting BCL-XL andBCL-2 is effective in pre-clinical models of malignant pleural mesothelioma,Cell Death Discovery,2021,7,122-131.

[0198] Balachander et a.,AZD4320,a dual inhibitor of Bcl-2and Bcl-x L , induce regression in hematological cancer models without dose-limiting thrombocytopenia, Clin Cancer Res, 2020, 26(24):6535-6549

[0199] Chang E.et al.,The combination of FLT3 and DNA methyltransferaseinhibition is synergistically cytotoxic to FLT3 / ITD acute myeloid leukemiacells,Leukemia,2016

[0200] Chou TC,Theoretical basis,experimental design,and computerizedsimulation of synergism and antagonismin drug combination studies,PharmacolRev,2006

[0201] Directive 2010 / 63 / Eu of The European Parliament And Of The Council OnThe Protection Of Animals Used For Scientific Purposes,22September 2010

[0202] Hollenbach PW,et al.,A comparison of azacitidine and decitabineactivities in acute myeloid leukemia cell lines,PLoS One,2010

[0203] Kipp D.and Wei A.,The path to approval for oral hypomethylatingagents in acute myeloid leukemia and myelodysplastic syndromes,FutureOncol.2021,17(20),2563-2571

[0204] Nakao M,et al.,Internal tandem duplication of the flt3 gene found inacute myeloid leukemia.Leukemia,1996,10(12),1911-1918

[0205] Saliba A.et al.,Resistance to Venetoclax and hypomethylating agentsin acute myeloid leukemia,Cancer Drug Resistance,2021,4,125-42

[0206] Samra B,et al.,Venetoclax-based combinations in Acute MyeloidLeukemia:Current Evidence and Future Directions,Frontiers in Oncology,2020,10,Article562558

[0207] Teicher B.Totowa.Anticancer Drug Development Guide.New Jersey:HumanaPress,1997

[0208] Workman P.,et al.,Guidelines for The Welfare And Use Of Animals InCancer Research.British Journal Of Cancer,2010,102,1555–1577

[0209] 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

[0210] Von Bonin M,Wermke M,Cosgun KN,et al.In vivo expansion of co-transplanted T cells impacts on tumor re-initiating activity of human acutemyeloidleukemia 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 medicament.

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 hematological cancer.

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

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

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 the preceding claims, wherein SEL24 / MEN1703 is administered in a daily dose of about 50 mg to about 150 mg.

8. A combination for use according to any one of the preceding claims, wherein the combination further comprises (iii) a hypomethylating agent or cytarabine.

9. The combination for use according to claim 8, wherein the hypomethylating agent is selected from azacitidine, decitabine, CC-486 and ASTX727.

10. The combination for use according to claim 8 or 9, wherein the hypomethylating agent is administered at about 10 mg / m 2 About 100 mg / m 2 The daily dose is administered.

11. The combination for use according to claim 10, wherein the hypomethylating agent is azacitidine and the azacitidine is administered at about 75 mg / m 2 The daily dose is administered.

12. The combination for use according to claim 10, wherein the hypomethylating agent is decitabine and the decitabine is administered at about 20 mg / m 2 The daily dose is administered.

13. The combination for use according to claim 8, wherein cytarabine is administered at about 20 mg / m 2 The daily dose is administered.

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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