EA039621B1 - Combination of a bcl-2 inhibitor and a mcl1 inhibitor, uses and pharmaceutical compositions thereof - Google Patents

Abstract

The invention relates to a combination comprising a BCL-2 inhibitor and a MCL1 inhibitor and the use of said combination in treating cancer.

Description

Prerequisites for the creation of the invention

Apoptosis is a highly regulated cell death pathway that is initiated by a variety of cytotoxic stimuli, including oncogenic stress and chemotherapeutic agents. It has been shown that apoptosis evasion is a hallmark of malignancy and that the efficacy of many chemotherapeutic agents depends on activation of the intrinsic mitochondrial pathway. Three different subgroups of BCL-2 family proteins control the intrinsic apoptotic pathway: (i) pro-apoptotic BH3 (BCL-2 homology 3)-only proteins; (ii) pro-survival members such as BCL-2 itself, BCL-XL, Bcl-w, MCL1 and BCL-2a1; and (III) pro-apoptotic effector proteins BAX and BAK (Czabotar et al., Nature Reviews Molecular cell biology 2014 vol. 15:49-63). Overexpression of anti-apoptotic members of the BCL-2 family has been observed in many malignant neoplasms, especially in hematological malignancies such as mantle cell lymphoma (MCL), follicular lymphoma/diffuse large B-cell lymphoma (FL/D), and multiple myeloma (Adams and Cory Oncogene 2007 vol 26:1324-1337). Pharmacological inhibition of the anti-apoptotic proteins BCL-2, BCLXL and Bcl-w by recently developed BH3 mimetic drugs such as ABT-199 and ABT-263 is a therapeutic strategy to induce apoptosis and induce tumor regression in cancer (Zhang et al., Drug Resist Update 2007 Vol 10(6):207-17). However, observations and studies are ongoing regarding the mechanisms of resistance to these drugs (Choudhary GS et al., Cell Death and Disease 2015 volume 6, e1593; doi:10.1038/cddis.2014.525).

Acute myeloid leukemia (AML) is a rapidly developing fatal malignant blood disease resulting from clonal transformation of hematopoietic stem cells, which leads to paralysis of the normal functioning of the bone marrow and death due to complications from absolute pancytopenia. AML is responsible for 25% of all adult leukemias, with the highest incidence rates in the United States of America, Australia and Europe (WHO. GLOBOCAN 2012. Estimated cancer incidence, mortality and prevalence worldwide in 2012. International Agency for Research on Cancer). Worldwide, approximately 88,000 new cases are diagnosed each year. AML continues to have the lowest survival rate of all leukemias, with an estimated 5-year survival rate of only 24%. Although the standard therapy for AML (cytarabine in combination with anthracyclines) was developed over 4 decades ago, the introduction of successful targeted therapies for this disease remains an elusive goal. In addition, there remains a need for the possibility of treatment without chemotherapy for patients with AML. The concept of targeted therapy in AML has been put on hold due to the discovery that a multiclonal hierarchy is involved in this disease, with the proliferation of leukemic subclones as the main cause of drug resistance and disease recurrence (Ding L. et al., Nature 2012 481:506-10 ). Recent clinical studies have shown the effectiveness of BCL-2 inhibitors for the treatment of AML (Konopleva M et al., American Society of Hematology 2014:118). While these inhibitors are clinically active, it is likely that other members of the BCL-2 family will need to be targeted to enhance overall efficacy in AML. In addition to BCL-2, MCL1 has also been identified as an important regulator of cell survival in AML (Glaser SP et al., Genes & development 2012 26:120-5).

Multiple myeloma (MM) is a rare and incurable disease characterized by the accumulation of cloned plasma cells in the bone marrow (MM) and is responsible for 10% of all malignant diseases of the blood system. In Europe, approximately 27,800 new cases are registered every year. With the availability of new agents in recent years, including bortezomib and lenalidomide, and autologous stem cell transplantation (ASCT), survival rates have improved. However, the response to these new agents is often short-lived and the need for new treatments becomes apparent, especially for relapsed/refractory patients and patients with a poor prognosis (poor cytogenetic profile). Recent studies support the promising activity of BCL-2 inhibitors in a subgroup of patients with multiple myeloma (Touzeau C., Dousset C., Le Gouill S., et al. Leukemia. 2014; 28(1):210-212). MCL1 has also been identified as an important regulator of cell survival in multiple myeloma (Derenne S., Monia B., Dean NM, et al. Blood. 2002;100(1): 194-199; Zhang B., Gojo I, Fenton RG Blood 2002; 99(6): 1885-1893).

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Diffuse large B-cell lymphoma (DLBCL) is the most common type (25-35%) of non-Hodgkin's lymphoma, with 24,000 new patients/year. DLBCL is a heterogeneous disease with over twelve subtypes including double-hit/MYC translocation, Activated B cells (ABC) and Germ center B cells (GCB). Approximately 60% of patients with DLBCL are treated with modern immune chemotherapy (R-CHOP), but for the remaining 40% there is little treatment option and the prognosis is poor. Therefore, there is a significant medical need to improve treatment efficacy and clinical outcomes in high-risk DLBCL, such as the ABC subtype (35% of DLBCL), which exhibits constitutive activation of the survival-promoting NF-kB pathway.

Neuroblastoma (NB) is the most common extracranial solid tumor in neonates and children, accounting for 8-10% of all tumors in children, and is currently classified into low, intermediate, or high risk categories. It accounts for about 15% of all deaths associated with malignant neoplasms in pediatric patients. The frequency of NB is 10.2 cases per million children under 15 years of age, and about 500 new cases are registered annually. The median age at diagnosis is 22 months. Outcomes in patients with NB have improved steadily over the past 30 years, with 5-year survival rates rising from 52% to 74%. However, it is estimated that 50-60% of high-risk patients relapse and therefore experience only a modest reduction in mortality. The median time to relapse is 13.2 months and 73% of those who relapse were 18 months of age or older. Taken together, NB overall survival rates remain completely unacceptable (~20% at 5 years) despite more aggressive therapies (Colon and Chung, Adv Pediatr 2013 58: 297-311). The main treatments are chemotherapy, surgical resection and/or radiation therapy. However, many aggressive NBs have developed resistance to chemotherapeutic agents, keeping the likelihood of recurrence fairly high (Pinto et al., J Clin Oncol 201533: 3008-11). Standards of care for NB according to risk stratification are often carboplatin, cisplatin cyclophosphamide, doxorubicin, etoposide, cytokines (GM-CSF and IL2), and vincristine. Deterioration after initial response to chemotherapy is a major cause of treatment failure, especially at high risk of NB.

Chemoresistance may develop due to the activation of BCL2 survival-promoting proteins (eg, BCL-2 and MCL1 proteins). NB expresses high levels of BCL-2 and MCL1 and low levels of BCL-XL. Inhibition of BCL-2 sensitizes cells to death and induces NB tumor regression in vivo (Ham et al., Cancer Cell 29:159-172). BCL-2 and MCL1 antagonisms restore chemotherapy at high risk of NB (Lestini et al., Cancer Biol Ther 2009 8:1587-1595; Tanos et al., Cancer BMC 2016 16:97). Thus, there is a strong case for combining BCL-2 and MCL1 inhibitors in previously untreated or resistant patients.

The present invention provides a novel combination of a BCL-2 inhibitor and an MCL1 inhibitor. These results suggest that with the development of effective small molecule targeted BCL-2 and MCL1, highly synergistic proapoptotic activity is found in primary human AML samples (FIGS. 2A and 17) as well as in AML (FIGS. 9, 13 and 14). multiple myeloma (Example 4), lymphoma (Fig. 4 and 12), neuroblastoma (Fig. 10), T-ALL, B-ALL cell lines (Fig. 11) and small cell lung cancer cell lines (Fig. 15 (a )-(e)). We have also shown that combined in vivo BCL-2 and MCL1 targeting is effective at tolerated doses in AML and xenograft lymphoma models in mice and rats (FIGS. 2, 5, 6, 7, 8 and 16) and significantly increases the time to development relapse in AML (FIGS. 2B and 2C). In addition, in clonogenic studies, we have shown that BCL-2+MCL1 targeting is specifically toxic to leukemia cells, but not to normal hematopoietic stem cells (Fig. 3), in contrast to previous MCL1 gene targeting experiments in mice. Prior to the development of these effective and selective inhibitors, the feasibility of targeting both BCL-2 and MCL1 remains uncertain. Previous deletion patterns specific to clonal lineages indicate a potential risk for cardiac disease (Wang X et al., Genes & development. 2013; 27(12): 1351-1364; Thomas RL et al., Genes & development. 2013; 27 (12): 1365-1377), granulocyto/hematopoietic (Opferman J et al., Science's STKE. 2005; 307(5712):1101; Dzhagalov I. et al., Blood. 2007; 109(4): 1620-1626 ; Steimer DA et al., Blood. 2009; 113(12):2805-2815), thymocyte (Dunkle A. et al., Cell Death & Differentiation. 2010;17(6):994-1002), neuronal (Arbour N. et al., Journal of Neuroscience. 2008; 28(24): 6068-6078) and liver function (Hikita H. et al., Hepatology. 2009; 50(4): 1217-1226; Vick B et al. , Hepatology 2009; 49(2): 627-636), which develops as a result of long-term ablation of MCL1. Despite these potential problems, it has recently been shown that weekly, twice weekly and even daily (for 5 consecutive days) intravenous delivery of a new potential short-acting pharmacological inhibitor of MCL1 is well tolerated and active against various malignancies in vivo. including AML (Kotschy A. et al. Nature. 2016; 538(7626): 477-482; WO 2015/097123). Short

- 2 039621 The half-life of the MCL1 protein may allow sufficient time for its regeneration in critical organs, thus allowing physiological tolerance for short exposure time MCL1 inhibitors (Yang T et al., Journal of cellular physiology. 1996; 166(3) : 523-536). So far, pulsatile inhibition of BCL-2 and MCL1 mimics a drug-like effect that has not been possible using genetic engineering approaches. Studies using BCL-2 and MCL1 inhibitors in accordance with the present invention provide experimental proof of concept that intermittent exposure to these drugs may be sufficient to trigger apoptosis and clinical response for many highly susceptible diseases such as AML without concomitant toxicity to major organ systems.

The synergistic effect of targeting both BCL-2 and MCL1 in vitro and in vivo and non-toxicity on normal bone marrow function by targeting both anti-apoptotic proteins has only been shown with a combination of effective small molecule inhibitors. These aspects cannot be predicted due to the results of gene targeting experiments, which may predict that the MCL1 deletion is poorly tolerated by hematopoietic stem cells.

The essence of the invention

The present invention relates to a combination for the treatment of cancer, which contains:

(a) a BCL-2 inhibitor selected from (i) N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)3,4-dihydro-2(1H)- isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-1indolizine carboxamide or addition salts thereof with a pharmaceutically acceptable acid or base, (ii) 5-(5 -chloro-2-{[(3S)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin-2(1H)-yl]carbonyl} phenyl)-N-(5-cyano-1,2- dimethyl-1 H-pyrrole-3-ul)-N-(4-hydroxyphenyl)-1,2-dimethyl-1 H-pyrrole-3-carboxamide or an addition salt thereof with a pharmaceutically acceptable acid or base, and (iii) 4-( 4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(3-nitro-4-{[(oxan- 4-yl)methyl]amino}phenyl)sulfonyl]-2-[(1H-pyrrolo[2,3-b]pyridin-5-yl)oxy]benzamide (AWT-199); and (b) an MCL1 inhibitor selected from (iv) (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1yl)ethoxy] phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)1H -pyrazol-5-yl]methoxy}phenyl)propionic acid or an addition salt thereof with a pharmaceutically acceptable acid or base, and (v) (2R)-2-{[(5S _a )-5-{3-chloro-2- methyl-4-[2-(4-methylpiperazin-1yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[ 2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid or its addition salt with a pharmaceutically acceptable acid or base, for simultaneous, sequential or separate use.

Certain BCL-2 inhibitors, their synthesis, their use in the treatment of cancer, and their pharmaceutical preparations are described in WO 2013/110890, WO 2015/011397, WO 2015/011399 and WO 2015/011400, the contents of which are incorporated by reference.

In a preferred embodiment of the invention, the BCL-2 inhibitor is selected from the following compounds:

4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(3-nitro-4{[( oxan-4-yl)methyl]amino}phenyl)sulfonyl]-2-[(1H-pyrrolo[2,3-b]pyridin-5-yl)oxy]benzamide (venetoclax or ABT-199);

4-(4-{[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(4-{[(2R)-4 (morpholin-4-yl)-1-(phenylsulfanyl)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzenesulfonyl]benzamide (navitoclax or ABT-263);

oblimersen (G3139);

obatoclax (GX15-070);

HA14-1;

(±)-gossypol (BL-193);

(-)-gossypol (AT-101); apogossypol; TW-37;

antimycin A, ABT-737 (Oltersdorf T et al., Nature 2005 June 2;435(7042):677-81) and compounds described in WO 2013/110890, WO 2015/011397, WO 2015/011399 and WO 2015/ 011400, the contents of which are incorporated by reference.

Said MCL1 inhibitors, their synthesis, their use in the treatment of cancer, and their pharmaceutical formulations are described in WO 2015/097123, the contents of which are incorporated by reference.

In a preferred embodiment, the MCL1 inhibitor is selected from A1210477 (Cell Death and Disease 20156, e1590; doi:10.1038/cddis.2014.561) and compounds described in WO 2015/097123, WO 2016/207216, WO 2016/207217, WO 2016/ 207225, WO 2016/207226 or WO 2016/033486, the contents of which are incorporated by reference.

In one embodiment, the invention provides a combination comprising:

- 3 039621 (a) Compound 1: N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2(1H)isoquinolinyl)carbonyl] -1,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-1-indolizine carboxamide, or a pharmaceutically acceptable salt thereof, and (b) an MCL1 inhibitor as described herein for simultaneous , sequential or separate application.

In another embodiment, the invention provides a combination comprising:

(a) Compound 4: 5-(5-chloro-2-{[(3S)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin-2(1H)yl]carbonyl}phenyl)-N- (5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-1Hpyrrol-3-carboxamide or a pharmaceutically acceptable salt thereof, and (b) MCL1 inhibitor, as described here, for simultaneous, sequential or separate use.

Alternatively, the invention provides a combination comprising:

(a) BCL-2 inhibitor as described here, and (b) Compound 2: (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4-[2-(4 -methylpiperazin-1-yl)ethoxy]phenyl}-6(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[1-(2 ,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propionic acid for simultaneous, sequential or separate use.

In another embodiment, the invention provides a combination comprising:

(a) BCL-2 inhibitor as described here, and (b) Compound 3: (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4-[2-(4 -methylpiperazin-1-yl)ethoxy]phenyl}-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidine -4-yl]methoxy}phenyl)propionic acid, for simultaneous, sequential or separate use.

In another embodiment, the invention provides the use of a combination as described herein for the preparation of a medicament for the treatment of cancer.

In another embodiment, the invention provides a medicament comprising, separately or together, (a) a BCL-2 inhibitor as described herein and (b) an MCL1 inhibitor as described herein for simultaneous, sequential or separate administration, and wherein BCL- 2 inhibitor and MCL1 inhibitor are provided in effective amounts for the treatment of cancer.

In another embodiment, the invention provides a method for treating cancer comprising administering a jointly therapeutically effective amount of:

(a) a BCL-2 inhibitor as described herein, and (b) an MCL1 inhibitor as described herein to a subject in need thereof.

In a particularly preferred embodiment, the BCL-2 inhibitor is N-(4hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2(1 H)- isoquinolinyl)carbonyl]-1,3benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-1-indolizine carboxamide hydrochloride (compound 1, HCl).

In a particularly preferred embodiment, the BCL-2 inhibitor is 5-(5chloro-2-{[(3S)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin-2(1H)-yl]carbonyl}phenyl )-(5-cyano-1,2dimethyl-1H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-N-pyrrole-3-carboxamide hydrochloride (compound 4, HCl).

In another embodiment, the BCL-2 inhibitor is ABT-199.

In another embodiment, the MCL1 inhibitor is (2R)-2-{[(5S _a )-5-{3-chloro-2methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6 -(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)-1H-pyrazole-5- yl]methoxy}phenyl)propionic acid (compound 2).

In another embodiment, the MCL1 inhibitor is (2R)-2-{[(5S _a )-5-{3-chloro-2methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6 -(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid (compound 3).

Brief description of the figures

Fig. 1. Expression of BCL-2 and MCL1 is predominant in AML. Samples of 7 AML cell lines and 13 primary AML with >70% blasts were immunoblotted to detect these proteins, and BCL-2 and MCL1 proteins were shown to be dominantly expressed in contrast to BCL-XL, which was expressed in fewer samples.

Fig. 2. Combined BCL-2/MCL1 targeting has synergistic activity in AML in vitro and in vivo. (A) 54 primary AML samples were incubated at 6-log concentration range of Compound 1 (HCl salt), Compound 2 or 1:1 concentration in RPMI/15% FCS for 48 h and LC ₅₀ determined. (B) Four groups of NSG mice were inoculated MV4; 11 cells expressing luciferase. Tumor engraftment was verified on day 10 (baseline) and then compound 1, HCl 100 mg/day po on weekdays (expressed as free base) or compound

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25 mg/kg IV twice a week for 4 weeks. The effect of Compound 2 and the combination with Compound 1 was evidenced by a decrease in the amount of luciferase mass on days 14 and 28 after the start of therapy and an increase in overall survival (C).

Fig. 3. Determination of the toxicity of combined BCL-2/MCL1 targeting normal CD34+ cells from normal donors or leukemic blast cells. Sorted normal CD34+ or leukemic blast cells were seeded and treated with Compound 1, HCl and Compound 2 at a 1:1 ratio at the indicated concentrations. The combination of Compound 1 + Compound 2 is toxic to leukemic but not normal CD34+ progenitors.

Fig. 4. Matrices of cell growth inhibitory effect and combination synergy for cell growth inhibition (left) and Loewe excess inhibition (right) provided by Compound 3 in combination with Compound 1, HCl in DB cells (A) and Toledo cells (B). The values in the influence matrix range from 0 (no inhibition) to 100 (total inhibition). The values in the synergism matrix represent the degree of growth inhibition above the theoretical additivity calculated from the activity of Compound 3 and Compound 1 alone, HCl at the concentrations tested. Synergistic effects are manifested for a wide range of concentrations of individual agents.

Fig. 5. Antitumor effects of Compound 1, HCl, Compound 3 and the combination of Compound 1, HCl + Compound 3 in the Karpass422 lymphoma xenograft model in rats.

Fig. 6. Body weight changes in animals treated with Compound 1, HCl, Compound 3 and the combination of Compound 1, HCl + Compound 3 in the Karpass422 lymphoma xenograft model in rats.

Fig. 7. Antitumor effects of Compound 1, HCl, Compound 3 and the combination of Compound 1, HCl + Compound 3 on the DLBCL Toledo mouse xenograft model.

Fig. 8. Body weight changes in animals treated with Compound 1, HCl, Compound 3 and the combination of Compound 1, HCl + Compound 3 in the DLBCL Toledo mouse xenograft model.

Fig. 9. Cell Growth Inhibitory Effect and Combination Synergy Matrices for Cell Growth Inhibition (left) and Loewe Excess Inhibition (right) provided by Compound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in an AML cell line OCIAML3 in two independent experiments.

The values in the influence matrix range from 0 (no inhibition) to 100 (total inhibition). The values in the synergism matrix represent the degree of growth inhibition above the theoretical additivity calculated from the activity of Compound 3 and Compound 1 alone, HCl at the concentrations tested. Synergistic effects are manifested for a wide range of concentrations of individual agents.

Fig. 10. Cell Growth Inhibitory Effect and Combination Synergy Matrices for Cell Growth Inhibition (left) and Loewe Excess Inhibition (right) provided by Compound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in an NB cell line LAN-6 in two independent experiments (N1: top panel; N2: bottom panel). The values in the influence matrix range from 0 (no inhibition) to 100 (total inhibition). The values in the synergism matrix represent the degree of growth inhibition above the theoretical additivity calculated from the activity of Compound 3 and Compound 1 alone, HCl at the concentrations tested.

Fig. 11. Cell Growth Inhibitory Effect and Combination Synergy Matrices for Cell Growth Inhibition (left) and Loewe Excess Inhibition (right) provided by Compound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in B-ALL cell line NALM-6 in two independent experiments (N1: upper panel; N2: lower panel).

Fig. 12. Cell Growth Inhibitory Effect and Combination Synergy Matrices for Cell Growth Inhibition (left) and Loewe Excess Inhibition (right) provided by Compound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in an MCL cell line Z138.

Fig. 13. Matrices of cell growth inhibitory effect and combination synergy for cell growth inhibition (left) and Loewe excess inhibition (right) provided by Compound 3 (MCL1 inhibitor) in combination with ABT-199 (BCL-2 inhibitor) in the AML cell line OCI -AML3 in two independent experiments (N1: upper panel; N2: lower panel).

Fig. 14. Representative matrices of cell growth inhibitory effect and combination synergism for cell growth inhibition (left) and Loewe excess inhibition (right) provided by Compound 3 (MCL1 inhibitor) in combination with Compound 4, HCl (BCL-2 inhibitor) in AML cellular lines (ML-2 cells in A and OCI-AML-3 in B).

Fig. 15(a)-(e). Dose matrices for inhibition (left), Loewe excess inhibition (middle), and growth inhibition provided by Compound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in a panel of SCLC cell lines.

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Fig. 16(a)-(b). Antitumor effects of Compound 1, HCl, ABT-199, Compound 3 and the combination of Compound 1, HCl or ABT-199 + Compound 3 in a patient-derived primary AML mouse model HAMLX5343.

Fig. 17. Heat map comparison of AML susceptibility (LC ₅₀ ) to BH3-mimetic monotherapy, or drug combinations (tested at 1:1 ratio), versus chemotherapy (idarubicin) after 48 h exposure. Cell viability of each primary AML sample after 48 h is shown. h in DMSO.

Detailed description of the invention

The invention thus provides, in Embodiment E1, a combination for the treatment of cancer comprising:

(a) a BCL-2 inhibitor selected from (i) N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2(1H) -isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-1-indolizine carboxamide or addition salts thereof with a pharmaceutically acceptable acid or base, (ii) 5 -(5-chloro-2-{[(3S)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin-2(1H)-yl]carbonyl}phenyl)N-(5-cyano-1, 2-dimethyl-1 H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-1 H-pyrrol-3-carboxamide or an addition salt thereof with a pharmaceutically acceptable acid or base, and (iii) 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(3-nitro-4{[( oxan-4-yl)methyl]amino}phenyl)sulfonyl]-2-[(1H-pyrrolo[2,3-b]pyridin-5-yl)oxy]benzamide (ABT199);

and (b) an MCL1 inhibitor selected from (iv) (2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy ]phenyl}-6-(5-fluorofuran2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)-1H -pyrazol-5-yl]methoxy}phenyl)propionic acid or an addition salt thereof with a pharmaceutically acceptable acid or base, and (v) (2R)-2-{[(5S _a )-5-{3-chloro-2- methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-( 2-{[2-(2-Methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid or its addition salt with a pharmaceutically acceptable acid or base for simultaneous, sequential or separate use.

Additional numbered embodiments (E) of the invention are described in this application. It should be appreciated that the features indicated in each embodiment may be combined with other specified features to provide additional embodiments of the present invention.

E2. The combination according to embodiment E1, wherein the BCL-2 inhibitor is N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2( 1H)-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-1-indolizine carboxamide.

E3. A combination according to embodiment E1 wherein the BCL-2 inhibitor is 5-(5-chloro-2-{[(3S)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinoline-2(1 H)-yl]carbonyl} phenyl)-N (5-cyano-1,2-dimethyl-1 H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-1 H-pyrrole -3-carboxamide.

E4. A combination according to embodiment E2 wherein N(4-hydroxyphenyl)-3-{6[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2(1H)-isoquinolinyl)carbonyl ]-1,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-1-indolizine carboxamide is present in the form of the hydrochloride salt.

E5. A combination according to embodiment E3 wherein 5-(5-chloro-2-{[(3S)-3(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin-2(1H)-yl]carbonyl}phenyl )-N-(5-cyano-1,2-dimethyl-1Hpyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-1H-pyrrole-3-carboxamide is in the form of the hydrochloride salt.

E6. The combination according to embodiment E1, wherein the BCL-2 inhibitor is ABT-199.

E7. A combination according to embodiment E1 wherein the MCL1 inhibitor is (2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl) ethoxy]phenyl}-6-(5-fluorofuran-2yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)- 1H-pyrazol-5-yl]methoxy}phenyl)propionic acid.

E8. A combination according to embodiment E1 wherein the MCL1 inhibitor is (2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl) ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy }phenyl)propionic acid.

E9. A combination according to any embodiment of E1-E8 which further comprises one or more excipients.

E10. The use of a combination according to any embodiment of E1-E8, for the preparation of a medicament for the treatment of cancer.

E11. Use according to embodiment E10, wherein the malignancy is leukemia.

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E12. Use according to embodiment E11, wherein the malignancy is acute myeloid leukemia, T-ALL or B-ALL.

E13. Use according to embodiment E10, wherein the malignancy is a myelodysplastic syndrome or a myeloproliferative disease.

E14. Use according to embodiment E10, wherein the malignancy is lymphoma.

E15. Use according to embodiment E14, wherein the lymphoma is non-Hodgkin's lymphoma.

E16. Use according to embodiment E15, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma or mantle cell lymphoma.

E17. Use according to embodiment E10, wherein the malignancy is multiple myeloma.

E18. Use according to embodiment E10, wherein the malignancy is a neuroblastoma.

E19. Use according to embodiment E10, wherein the malignant neoplasm is small cell lung cancer.

E20. A medicament comprising, separately or together, (a) a BCL-2 inhibitor as defined in Embodiment E1, and (b) an MCL1 inhibitor as defined in Embodiment E1, for simultaneous, sequential or separate administration, and where BCL- 2 inhibitor and MCL1 inhibitor are provided in effective amounts for the treatment of cancer.

E21. The use of a therapeutically effective amount of (a) a BCL-2 inhibitor as defined in Embodiment E1 and (b) an MCL1 inhibitor as defined in Embodiment E1 for the preparation of a medicament for the treatment of cancer.

E22. The use of a synergistically effective amount of (a) a BCL-2 inhibitor as defined in Embodiment E1 and (b) an MCL1 inhibitor as defined in Embodiment E1 for the preparation of a medicament for the treatment of cancer.

E23. Use according to embodiment E22, wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in synergistically effective amounts that are capable of reducing the dose required of each compound for the treatment of cancer, while providing effective treatment of cancer, with a reduction in the eventual side effects. effects.

E24. Use according to Embodiment E21 wherein the BCL-2 inhibitor is N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2( 1H)-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-1-indolizine carboxamide, and the BCL-2 inhibitor is used in combination treatment at a dose of from 50 to 1500 mg.

E25. Use according to embodiment E21 wherein the BCL-2 inhibitor is administered once a week.

E26. Use according to Embodiment E21 wherein the BCL-2 inhibitor is N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2( 1H)-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl}-K-phenyl-5,6,7,8-tetrahydro-1-indolizine carboxamide, moreover, the BCL-2 inhibitor is administered once during the combined treatment day.

E27. Use according to embodiment E21 wherein the BCL-2 inhibitor and the MCL1 inhibitor are orally administered.

E28. Use according to embodiment E21 wherein the BCL-2 inhibitor is administered orally and the MCL1 inhibitor is administered intravenously.

E29. Use according to embodiment E21 wherein the BCL-2 inhibitor and the MCL1 inhibitor are administered intravenously.

Combination refers to either a fixed dose combination in a single unit dosage form (e.g., a capsule, tablet, or sachet), a non-fixed dose combination, or a combination administration kit, wherein a compound of the present invention and one or more components from the combination (e.g., another drug, as explained below, also referred to as a therapeutic agent or co-agent) can be administered independently at the same time or separately over time intervals, especially if these time intervals allow the components of the combination to exhibit joint, for example, synergistic Effect.

The terms co-administration or combined administration, or the like, as used herein, encompass the administration of a selected combination component to a single subject in need (e.g., a patient) and include treatment regimens in which the agents are not necessarily administered by the same route. administration or at the same time.

The term fixed dose combination means that the active ingredients, for example a compound of formula (I) and one or more combination components, are both administered to the patient simultaneously in the form of a single unit or dosage.

The term "non-fixed dose combination" means that the active ingredients, e.g., a compound of the present invention and one or more components of the combination, are both administered to the patient as separate components, either simultaneously or sequentially, without specific time limits, where such administration provides therapeutically effective levels of the two compounds. in the patient's body.

The latter is also used for cocktail therapy, for example, the administration of three or more active ingredients.

Malignancy refers to a class of diseases in which a group of cells exhibits uncontrolled growth. Types of malignancies include hematologic malignancies (lymphoma and leukemia) and solid tumors including carcinoma, sarcoma, or blastoma. In particular, malignancy refers to leukemia, lymphoma, or multiple myeloma, and more specifically to acute myeloid leukemia.

The term co-therapeutically effective means that the therapeutic agents can be administered separately (in a chronological order, in particular sequence-specific manner) at such time intervals that they can preferentially, in a warm-blooded animal, especially a human, treat while still exhibiting ( preferably synergistic) interaction (joint therapeutic effect). In any case, this can be established, in particular, by determining subsequent levels in the blood, indicating that both compounds are present in the blood of the person being treated, at least for certain time intervals.

Synergistically effective or synergistic means that the therapeutic effect observed after the administration of two or more agents is greater than the sum of the therapeutic effects observed after the administration of each single agent.

As used herein, the term treat, treat, or treat any disease or disorder refers, in one embodiment, to alleviating the disease or disorder (i.e., alleviating or stopping or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, to treat, treatment or therapy refers to the reduction or alleviation of at least one physical parameter, including those that may not be felt by the patient. In yet another embodiment, treating, treating, or therapy refers to modulating a disease or disorder, either physically (eg, stabilizing a discernible symptom), physiologically (eg, stabilizing a physical parameter), or both.

As used herein, a subject is in need of treatment if such subject would benefit biologically, medically, or quality of life from such treatment.

In another aspect, a method is provided for sensitizing a human that is (I) refractory to at least one chemotherapy treatment, or (II) relapsed after treatment with chemotherapy, or both (I) and (II), wherein the method comprises administering BCL-2 inhibitor in combination with an MCL1 inhibitor as described herein to a patient. A patient who is sensitized is a patient who responds to treatment involving the administration of a BCL-2 inhibitor in combination with an MCL1 inhibitor as described herein or who has not developed resistance to such treatment.

Drug means a pharmaceutical composition, or a combination of several pharmaceutical compositions, which contains one or more active ingredients in the presence of one or more excipients.

AML stands for acute myeloid leukemia.

'T-ALL' and 'B-ALL' refer to T-cell acute lymphoblastic leukemia and B-cell acute lymphoblastic leukemia.

Free base refers to a compound when it is not in the form of a salt.

In pharmaceutical compositions according to the invention, the proportion of active ingredients by weight (weight of active ingredients in relation to the total weight of the composition) is 5-50%.

As pharmaceutical compositions according to the invention, more specifically those suitable for administration by the oral, parenteral and especially intravenous, transdermal or transdermal, nasal, rectal, sublingual, ocular or respiratory routes are used, more specifically tablets, dragees, sublingual tablets. , hard gelatin capsules, tablets for slow dissolution under the tongue, capsules, lozenges, injectables, aerosols, eye drops or nasal drops, suppositories, creams, ointments, skin gels, etc.

Pharmaceutical compositions according to the invention may contain one or more excipients or carriers selected from diluents, lubricants, binders, disintegrators, stabilizers, preservatives, absorbents, colorants, sweeteners, flavoring agents, and others.

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As a non-limiting example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycerin can be mentioned as diluents, silicon dioxide, talc, stearic acid and its magnesium and calcium salts as lubricants, polyethylene glycol, as binders magnesium aluminosilicate, starch , gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone, agar as disintegrators, alginic acid and its sodium salt, effervescent mixtures.

The combination compounds may be administered simultaneously or sequentially. The route of administration is preferably the oral route and the corresponding pharmaceutical compositions may allow immediate or delayed release of the active ingredients. The combination compounds may also be administered in the form of two separate pharmaceutical compositions each containing one of the active ingredients, or in the form of a single pharmaceutical composition in which the active ingredients are present in admixture. Pharmaceutical compositions in the form of tablets are preferred.

Pharmaceutical composition of the compound 1 HCl salt, which is a film-coated tablet containing 50 and 100 mg of the medicinal substance ______________

Quantity (mg) Function Tablet 50 mg dosage 100 mg dosage Compound 1 HC1 salt 52.58 105.16 medicinal substance Equivalently, not a base fifty 100 Lactose monohydrate 178.51 357.02 Diluent Corn starch 66.6 133.2 disintegrant Povidone 23.31 46.62 Binder magnesium stearate 3.33 6.66 Lubricant Silica, colloidal anhydrous 0.67 1.34 Flow Agent Sodium Starch Glycolate (Type A) ten 20 disintegrant For uncoated tablets with weight 335 670 film sheath Glycerol 0.507 1.014 plasticizing material hypromellose 8.419 16.838 Film former Macrogol 6000 0.538 1.076 Smoothing agent magnesium stearate 0.507 1.014 Lubricant Titanium dioxide 1.621 3.242 Pigment Transition fillers Purified water qs. qs. Diluent For a film-coated tablet with a mass 346.6 693.2

Pharmacological data Materials and methods for examples 1-3

Primary AML samples from patients:

Bone marrow or peripheral blood samples from AML patients were collected after informed consent was obtained according to requirements approved by the Alfred Hospital Human Research Ethics Committee. Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation (GE Healthcare, VIC, Aus), followed by erythrocyte depletion in ammonium chloride (NH4Cl) lysis buffer at 37°C for 10 min. The cells were then resuspended in phosphate buffered saline containing 2% fetal bovine serum (Sigma, NSW, Aus). Thereafter, mononuclear cells were suspended in RPMI-1640 (GIBCO VIC, Aus) medium containing penicillin and streptomycin (GIBCO) and heat inactivated fetal bovine serum 15% (Sigma).

Cell lines, cell cultures and development of luciferase reporter cell lines:

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Cell lines MV4; 11, OCI-AML3, HL-60, HEL, K562, KG-1 and EOL-1 were maintained at 37°C, 5%

CO ₂ in RPMI-1640 (GIBCO) supplemented with 10% (v/v) fetal bovine serum (Sigma) and penicillin and streptomycin (GIBCO). MV4; 11 luciferase cell lines were generated by lentiviral transduction.

Antibodies: Primary antibodies used for western blotting were MCL1, BCL-2, Bax, Bak, Bim, BCL-XL (in-house developed by WEHI) and tubulin (T-9026, Sigma).

Cell Viability: Freshly purified mononuclear cells from patient AML samples were adjusted to a concentration of 2.5 x 105/ml and 100 μl cell aliquots per well were plated in 96 well plates (Sigma). Cells were then treated with Compound 1, HCl, Compound 2, ABT199 (Active Biochem, NJ, USA), or Idarubicin (Sigma), in a 6-log range of 1 nM to 10 μM for 48 h. agents were added in a 1:1 ratio from 1 nM to 10 μM and incubated at 37°C 5% CO2. Thereafter, cells were stained with sytox blue polynucleotide dye (Invitrogen, VIC, Aus) and fluorescence was measured by flow cytometric analysis using an LSR-II Fortessa (Becton Dickinson, NSW, Aus). FACSDiva software was used for data collection, and FlowJo software for analysis. Blast cells were adjusted using the properties of forward and side light scattering. Viable cells, excluding sytox blue, were determined at 6 concentrations for each drug and the 50% lethal concentration (LC ₅₀ , in wm) was determined.

Definition of LC ₅₀ and Synergy:

Graphpad Prism was used to calculate LC ₅₀ using non-linear regression. Synergism was determined by Additivity Index (AI based on the Chou Talalay method as described (Chou Cancer Res; 70(2) January 15, 2010).

Colony research:

Colony-forming studies were performed on freshly prepared and frozen mononuclear fractions from AML patients. Primary cells were cultured in duplicate in 35 mm dishes (Griener-bio, Germany) at concentrations from 1x10 ⁴ to 1x105. Cells were seeded in 0.6% agar (Difco NSW, Aus): AIMDM 2x (IMDM powder-Invitrogen) supplemented with NaHCO ₃ , dextran, Pen/Strept, B mercaptoethanol and asparagine): Fetal bovine serum (Sigma) at a ratio of 2: 1:1. For optimal growth conditions, all plates containing GM-CSF (100 ng per plate), IL-3 (100 ng/plate R&D Systems, USA) SCF (100 ng/plate R&D Systems) and EPO (4 U/plate) (Growth occurred within 2-3 weeks in the presence and absence of drug at 37°C at 5% CO2 in a high humidity incubator After incubation, the plates were fixed with 2.5% glutaraldehyde in saline and evaluated using GelCount from Oxford Optronix ( Abingdon, United Kingdom).

Western blotting:

Lysates were prepared in NP40 lysis buffer (10 mM Tris-HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1% NP40) supplemented with a protease inhibitor cocktail (Roche, Dee Why, NSW, Australia). Protein samples were boiled at reduced dye loading prior to separation on 4-12% Bis-Tris polyacrylamide gels (Invitrogen, Mulgrave, VIC, Australia), and transferred to Hybond C nitrocellulose membrane (GE, Rydalmere, NSW, Australia) for incubation with specific antibodies . All membrane blocking and antibody dilution steps were performed using 5% (v/v) skimmed milk in PBS containing 0.1% (v/v) Tween-20 phosphate buffered saline (PBST) or TRIS- buffered saline, and washing steps were performed with PBST or TBST. Western blots were visualized using enhanced chemiluminescence (GE).

In Vivo AML Inoculation Experiments: Animal studies were performed according to the rules approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee, MV4; 11 cells transduced with a luciferase reporter (pLUC2) were intravenously injected at 1x105 cells into irradiated (100 rad) non-obese diabetic/severe combined immunodeficient (NOD/SCID/IL2rynull) mice as previously described (Jin et al., Cell Stem Cell July 2, 2009, Volume 5, Edition 1, pp. 31-42). Adherent cells were measured on day 7 by quantifying hCD45+ cells in RV using flow cytometry and IVIS imaging of MV4 bioluminescence; 11 cells. On day 10, mice received daily oral force feeding Compound 1, HCl (200 μl 100 mg/kg - dosage expressed as free base) dissolved in PEG400 (Sigma), absolute ethanol (Sigma) and distilled H2O 40:10:60 or Compound 2 (200 µl 25 mg/kg) twice a week, dissolved in 50% 2hydroxypropyl)-in-cyclodextrin (Sigma) and 50% 50 mM HCl, or drug or vehicle combination, for 4 weeks. Blood counts were performed using a hematology analyzer (BioRad, Gladesville, NSW).

IVIS imaging: Bioluminescence imaging was performed using the Caliper IVIS Lumina III XR imaging system. Mice were anesthetized with isofleurin and injected intraperitoneally with 100 μl of 125 mg/kg luciferin (Perkin Elmer, Springvale, VIC).

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Materials and Methods for Example 4

Cell lines: Human myeloma cell lines (HMCL) were derived from primary myeloma cells cultured in RPMI 1640 medium supplemented with 5% fetal calf serum and 3 ng/ml recombinant IL-6 for IL-6 dependent cell lines. The HMCLs were representative of phenotypic and genomic heterogeneity and variability in patient response to therapy.

MTT study:

Cell viability was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric survival assay. Cells were incubated with compounds in 96 well plates containing a final volume of 100 μl/well over time. (2R)-2-{[(5S _a )5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-fluorofuran-2 -yl)thieno[2,3d]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propionic acid (compound 2) was used in 9 different concentrations according to sensitivity to one agent. N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2(1H)isoquinolinyl)carbonyl]-1,3-benzodioxol-5- yl}-N-phenyl-5,6,7,8-tetrahydro-1-indolizine carboxamide hydrochloride (compound 1, HCl) was used at a fixed dose of 1 μM. After the end of each treatment, the cells were incubated with 1 mg/ml MTT (50 µl of 2.5 mg/ml MTT solution for each well) at 37° C. for 3 hours, allowing MTT to be metabolized. Lyse buffer (100 μl Lyse buffer: DMF (2:3)/SDS (1:3)) was added to each well to dissolve the formazan crystals, and after incubation for 18 hours, the viable cell absorbance at 570 nm was measured using a spectrophotometer. As a control, cells were incubated with medium alone and with medium containing 0.1% DMSO. As a control for the growth of myeloma cells, the absorption of myeloma cells was recorded every day (DO, D1, D2, D3 and D4). All experiments were repeated 3 times and each experimental condition was repeated in at least three wells in each experiment.

The inhibitory effect was calculated according to the following formula:

Inhibitory effect (%) = (1-Absorption value of treated cells/Absorption value of control cells)x100

Example 1 BCL-2 and MCL1 are dominant survival proteins expressed in AML

Samples of 7 AML cell lines and 13 primary AML with >70% blasts were immunoblotted for protein detection as indicated in FIG. 1. As illustrated in FIG. 1, a proteomic overview of the expression of members of the BCL-2 family in AML indicates that, in addition to BCL-2, most primary AML and AML cell line samples co-express the survival-promoting protein MCL1. BCL-XL is less frequently expressed in AML.

Example 2 Combined BCL-2 and MCL1 Targeting Shows Synergistic Killing in AML

AML samples from patients were incubated at a 6-log concentration range of Compound 1 (HCl salt), Compound 2 or 1:1 concentration in RPMI/15% FCS for 48 h and LC ₅₀ was determined (FIG. 2A).

Approximately 20% of primary AML samples were extremely sensitive to either Compound 1 or Compound 2, with a lethal drug concentration required to kill 50% of primary AML blasts after 48 h (LC ₅₀ ) in the lower nanomolar range (LC ₅₀ <10 nM) ( Fig. 2A). In contrast, if Compound 1 and Compound 2 were combined, the proportion of AML samples that were sensitive increased significantly to 70%, indicating synergistic activity while targeting BCL-2 and MCL1 (FIG. 2A). Some of the results are shown in Fig. 17.

To verify the activity of this approach in vivo, MV4; 11 AML cells expressing luciferase were inoculated into NSG mice and treated with Compound 1 (HCl salt) or Compound 2 alone or in combination and tumor burden was assessed after 14 and 21 days of therapy (FIG. 2B). After completion of 28 days of therapy, mice were observed to determine survival (FIG. 2C). These experiments indicate that the combination of Compound 1 and Compound 2 is highly effective in vivo, confirming the extremely good activity observed when using primary AML cells in vitro.

The data shown in FIG. 2A-2C in the present application indicate the synergistic activity of the combination between Compound 1, HCl and Compound 2 on AML.

Example 3 Combined BCL-2 and MCL1 inhibition targeting the function of leukemic progenitors but not the function of normal progenitors

To evaluate the toxicity of BCL-2 inhibition in combination with MCL1 inhibition on the function of normal human CD34+ cells or ficollated blast cells from AML patients, clonogenic potential was assessed after 2 weeks of exposure to combination therapies. Colonies were grown on agar supplemented with 10% FCS, IL3, SCF, GM-CSF and EPO for 14 days and colonies were counted using an automated Gelcount® analyzer. Research for samples

- 11,039,621 primary AMLs were performed in duplicate and averaged. Uncertainties for CD34+ are the mean ± SD of 2 independent normal donor samples. The results were normalized to the number of colonies counted in the DMSO control. The indicated concentrations of drugs were placed in the plate in D1. It should be noted that Compound 1 + Compound 2 inhibits the colony-forming activity of AML without affecting the growth functions of normal CD34+ colonies.

Taken together, Examples 2 and 3 demonstrate that dual pharmacological inhibition of BCL-2 and MCL1 represents a novel approach for the treatment of AML without the need for additional chemotherapy and with an acceptable therapeutic safety window.

Example 4 In Vitro Evaluation of Multiple Myeloma Cell Survival Response to an MCL1 Inhibitor Alone or in Combination with a BCL-2 Inhibitor

The sensitivity of 27 human multiple myeloma cell lines to Compound 1, Compound 2, or Compound 2 in the presence of 1 μM Compound 1 was analyzed using an MTT cell viability assay. 50% inhibitory concentrations were determined (1C ₅ o, in nM).

The results are presented in the following table:____________________________

Cell lines Compound 1, HC1 (1C ₅₀ nM) Compound 2 (1C ₅₀ nM) 1C ₅ o Compound 2 in the presence of 1 μM Compound 1, HC1 (nM) AMO1 8610.3 0.5 0.2 ANBL6 1905.0 79.5 20.8 BCN 22217.0 1111.4 59.3 LMZ >30000 56.3 25.9 JJN3 2692.0 15.6 2.4 KMM1 23926.3 57.8 8.6 KMS11 10486.7 44.1 3.9 KMS12BM 1393.7 44.1 0.03 L363 7581.3 7.6 3.4 LP1 9770.0 158.2 2.9 MM1S 21407.0 138.5 23.0 ΝΑΝΙ 6659.0 5.7 1.4 ΝΑΝ3 8241.3 1.5 1.2 ΝΑΝ6 4074.8 7.1 1.8 ΝΑΝ8 9096.3 75.4 32.5 ΝΑΝ9 23157.6 9.7 1.1 NCI-H929 15688.3 2.3 1.0 ORM2 6460.7 9.4 1.2 RPMI8226 3204.0 27.4 h, o SBN 21273.7 221.1 14.6 U266 >30000 170.1 14.9 XG1 9779.7 5.9 0.2 XG11 7912.0 374.7 8.3 XG2 15297.7 6.4 2.7 XG3 7224.7 6.1 1.3 XG6 8544.3 19.2 0.5 XG7 18121.7 16.3 8.0

Strong synergistic activity has been shown when Compound 1 and Compound 2 are combined in most cell lines compared to compounds alone.

Example 5 In Vitro Effect on Proliferation of Combining an MCL1 Inhibitor with a BCL-2 Inhibitor in a Panel of 17 Diffuse Large B Cell Lymphoma (DLBCL) Cell Lines

Materials and methods

Cell lines were sourced and maintained in basic medium supplemented with FCS (Fetal calf serum) as indicated in Table 1. one.

Additionally, all among contained penicillin (100 IU/ml), streptomycin (100 μg/ml) and L-glutamine (2 mm). Unless otherwise noted, culture media and supplements were obtained from Amimed/Bioconcept (Allschwil, Switzerland).

Cell lines were cultured at 37° C. in a humidified atmosphere containing 5% CO ₂ and maintained in T-75 flasks. In all cases, cells were thawed from frozen stock solutions, >1 passage using appropriate dilutions, counting and viability using a CASY cell counter (Omni Life Science, Bremen, Germany) before seeding at 25 µl/well at densities indicated in Table. 1 in 384 well plates (Coming). All cell lines were determined to be free of mycoplasmic contamination by PCR analysis performed on Idexx Radii (Columbia, MO, USA) and misidentification was ruled out using a small nucleotide polymorphism (SNP) panel 48 at Asuragen (Austin, TX, USA).

USA) or independently.

Compound stock solutions were prepared at a concentration of 10 mM in DMSO (Sigma) and stored at -20°C. If necessary, to obtain a complete dose-response curve, the mother liquors were preliminarily diluted in DMSO to 1000-fold the desired initial concentration (see Table 2). The day after cell seeding, eight 2.5-fold serial dilutions of each compound were measured, either individually or in all possible staggered permutations, directly into cell assay plates using a 300D Digital Dispenser (TECAN, Mannedorf, Switzerland). ), as shown in Fig. 4. The final concentration of DMSO was 0.2% in all wells.

The effects of single agents as well as their checkerboard combinations on cell viability were assessed after incubation for 2 days at 37°C/5% CO ₂ by quantifying cellular ATP levels using CellTiterGlo (Promega, Madison, WI, USA) at 25 µl reagent/well and n=2 plate repeats per condition. Luminescence was quantitatively measured on a M1000 multifunctional plate reader (TECAN, Mannedorf, Switzerland). The number/viability of cells at the time of compound addition was also evaluated and used to estimate the order of doubling time of a population of a particular cell line.

The IC ₅ o for individual agents was calculated using four parameter curve fitting. Potential synergistic interactions between compound combinations were assessed using a 2D excess inhibition matrix according to the Loewe additivity model and described as a Synergy Score (Lehar et al., Nat Biotechnol. July 2009; 27(7): 659- 666). All calculations were performed using the combination analysis module with its own software. The IC ₅₀ was defined as the compound concentration at which the CTG signal is reduced by 50% relative to that measured with the vehicle control (DMSO).

Interpretation of the synergy score (Synergy Score) was carried out as follows:

SS~0^ Additive

SS>1 ^Weak synergy

SS>2^Synergism

Table 1. Identity and study conditions for 17 diffuse large B-cell lymphoma cell lines used in combined experiments

cell line Wednesday (source) %FCS Doubling time (hours) Number of seeded cells/well D.B. RPMI (ATCC) ten 31.7 500 DOHH-2 RPMI (DSMZ) ten 25.3 500 NT RPMI (ATCC) ten 34.3 2000 JM1 MEM Pskov* (ATCC) ten 22.8 500 KARPAS-422 RPMI (DSMZ) ten 26.5 500 NU-DHL-1 RPMI (DSMZ) 20 28.0 500 OCI-LY-19 MEM alpha (DSMZ) 20 25.8 500 Pfeiffer RPMI (ATCC) ten 46.2 2000 RL RPMI (ATCC) ten 28.9 500 SU-DHL-10 RPMI (DSMZ) 20 105.7 1000 SU-DHL-4 RPMI (DSMZ) ten 25.2 500 SU-DHL-5 RPMI (DSMZ) 20 25.9 500 SU-DHL-6 RPMI (DSMZ) 20 30.1 500 SU-DHL-8 RPMI (DSMZ) 20 23.6 500 Toledo RPMI (ATCC) ten 49.6 2000 U-937 RPMI (ATCC) ten 28.7 500 WSU-DLCL2 RPMI (DSMZ) ten 26.1 500

*This medium was additionally supplemented with 50 μM 2-mercaptoethanol.

The doubling time was calculated based on the difference in ATP levels after the end of the experiment compared to the start of incubation with the compound.

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Table 2. IC50 values of single agents for Compound 3 and Compound 1 are presented.

HCl, as well as synergy scores for their combination. Interactions are interpreted ___________ as synergistic if the observed scores are >2.0.__________

cell line Compound 3 Compound 1, HC1 Combination Beginning CONC. [µM] Abs 1C ₅₀ [µM] Max Ing [%] Beginning CONC. [µM] Abs IC50 [µM] Max Ing [%] synergism assessment Synergism estimation error D.B. one 0.0129 99.2 ten 2.76 95.7 17.3 0.18 DOHH-2 0.1 0.00122 98.3 ten 0.156 101.9 2.90 oh p NT one 0.00638 99.3 ten >10 37.2 2.35 0.06 JM1 one 0.0588 99.7 ten 0.697 99.1 5.83 0.25 KARPAS422 one 0.00214 98.3 ten 2.18 90.9 9.74 0.32 NU-DHL-1 one 0.0579 98.1 ten 0.0515 102.1 4.97 0.12 OCI-LY-19 one 0.0604 98.5 ten 0.0895 100.1 3.92 0.07 Pfeiffer one 0.0426 82.7 ten 4.50 92.0 3.44 0.19 RL ten 3.03 95.4 ten 0.281 99.0 12.7 0.38 SU-DHL-10 one 0.00384 98.3 ten >10 43.9 2.44 0.26 SU-DHL-4 one 0.0178 99.5 ten 0.86 96.9 10.8 0.28 SU-DHL-5 0.1 0.00094 98.2 ten >10 49.6 1.45 0.14 SU-DHL-6 one 0.00213 99.1 ten 0.614 101.0 4.57 0.21 SU-DHL-8 ten 0.305 95.6 ten >10 28.7 9.88 0.20 Toledo one >1 45.8 ten 0.137 101.9 P,1 0.51 U-937 one 0.00832 97.1 ten 6.83 62.1 5.63 0.20 WSU-DLCL2 one 0.00792 99.0 ten 1.02 99.3 8.67 0.13

Beginning conc. denotes the initial concentration.

Abs IC50 stands for Absolute IC50

Max Ing denotes maximum inhibition.

results

The effect on proliferation of combining MCL1 inhibitor Compound 3 with BCL-2 inhibitor Compound 1, HCl was evaluated in a panel of 17 diffuse large B cell lymphoma (DLBCL) cell lines. Compound 3 as the only agent strongly inhibited the growth of most of the 17 DLBCL lines tested (Table 1). Thus, 14 cell lines showed IC ₅₀ values below 100 nM and an additional 1 cell line showed IC ₅₀ values ranging from 100 nM to µM. Only 2 cell lines showed IC ₅₀ above 1 μm.

Compound 1, HCl alone also inhibited the growth of most of the 17 DLBCL lines tested, although slightly less effectively (Table 2). Thus, 2 cell lines showed IC50 values below 100 nM and 6 cell lines showed IC50 values between 100 nM and 1 μM. Nine cell lines exhibited IC50s greater than 1 μM (four of which were greater than 10 μM).

In combination, treatment with Compound 3 and Compound 1, HCl causes synergistic growth inhibition (i.e., Synergism Score greater than 2 - Lehar et al., Nat Biotechnol. July 2009; 27(7): 659-666) at 16 of 17 DLBCL cell lines tested (Table 2). In 5 cell lines, there is a marked synergistic effect, with synergy scores between 5 and 10. In 4 cell lines, the synergistic effect is extraordinary, reaching synergy scores between 10 and 17.3. It is extremely important that the synergism is independent of the antiproliferative effects of a single agent, and in fact, is particularly strong at concentrations of Compound 3 and Compound 1, which do not exhibit an antiproliferative effect on their own. For example, in DB cells, Compound 3 and Compound 1 at the second lowest concentration tested exhibited only 1 and 2% growth inhibition, respectively, while the respective combination of the two compounds provided 96% growth inhibition (Fig. 4A, left panel) , thus being 91% higher than the additivity calculated from the activity of the individual agents (FIG. 4A, right panel). As a further example, on Toledo cells, in which Compound 3 is less potent and provides only partial growth inhibition (46%) at the highest concentration tested, combination with the second lowest concentration of Compound 1 results in 98% synergistic growth inhibition (Fig. 4B, left panel), thus being 52% higher than the additivity calculated from the activity of individual agents (Fig. 4B, right panel).

In addition, it should be noted that synergistic effects appear for a wide range of concentrations of individual agents, which should be useful in vivo in relation to flexibility regarding levels and dosing regimens.

In conclusion, the combination of Compound 3 and Compound 1 provides strong to extraordinary synergistic growth inhibition in most DLBCL cell lines tested.

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Example 6 In Vivo Efficacy on Karpas422 Xenografts Using the Combination

MCL1 inhibitor (compound 3) and BCL-2 inhibitor (compound 1)

Materials and methods

Tumor cell culture and cell inoculation

The cell line Karpas 422 B-cell non-Hodgkin's lymphoma human (NHL) was obtained from the pleural exudate of a patient with chemotherapy resistant NHL. Cells were obtained from a DSMZ cell bank and cultured in RPMI-1640 medium (BioConcept Ltd. Amimed) supplemented with 10% FCS (BioConcept Ltd. Amimed), 2 mM L-glutamine (BioConcept Ltd. Amimed), 1 mM sodium pyruvate (BioConcept Ltd. . Amimed) and 10 mM HEPES (Gibco) at 37° C. under 5% CO ₂ in air. Cells were maintained between 0.5 and 1.5x10 ⁶ cells/ml. To establish Karpas 422 xenografts, cells were harvested and resuspended in HBSS (Gibco) and mixed with Matrigel (BD Bioscience) (1:1 v/v) before injecting 200 µl containing 1 x 10 ⁷ cells subcutaneously into the right paw of the animals, which were anesthetized with isoflurane. 24 h prior to cell inoculation, all animals were irradiated with 5 Gy for 2 min using a γ-emitter.

tumor growth

Tumor growth was monitored regularly after cell inoculation and animals were randomized to treatment groups (n=5) when tumor size reached an appropriate volume. During the treatment period, tumor volume was measured approximately twice a week using calipers. Tumor size, in mm ³ , was calculated based on the formula: (LxW2xk/6). Where W = width and L = length of the tumor.

Treatment

Tumor-bearing animals (rats) were divided into treatment groups (n=5) when their tumors reached an appropriate size to form a group with an average tumor volume of approximately 450 mm ³ . The treated groups are presented in table. 3. Vehicle for Compound 1, HC1 or Compound 1, HC1 was administered by oral (po) forced administration 1 hour before vehicle for Compound 3 or Compound 3, which was administered as a 15 minute IV infusion. For iv infusion, animals were anesthetized with isoflurane/O ₂ and vehicle or Compound 3 was cannulated into the tail vein. Animals were weighed on dosing day(s) and dose adjusted for body weight, dosed volume was 10 ml/kg for both compounds.

Body mass

Animals were weighed at least 2 times per week and examined more frequently for signs of any side effects.

Data analysis and statistical evaluation

Tumor data were statistically analyzed using GraphPad Prism 7.00 (GraphPad Software). If the variances in the data are normally distributed, then the data were analyzed by one-way ANOVA with Dunnett's post hoc test to compare treatments relative to the control group.

Tukey's test with a posteriori test was used for intergroup comparisons. In other cases, the Kruskal-Wallis test with Dunn's post hoc test was used. If applicable, data were presented as mean±SPS.

As a measure of efficacy, the %T/C (treatment/control) value after the end of the experiment was calculated according to (Tumor volume ^treated /Tumor volume ^control )*100

Tumor regression was calculated according to /a g treated/g treated at the start of treatments & ι n (A tumor volume/tumor volume)*100 where A tumor volume is the mean tumor volume on the day of assessment minus the mean tumor volume at the start of the experiment.

- 15 039621

Table 3. Treatment groups for determining the effectiveness of combinations in rats,

bearing Kar pass422 xenograft Groups Treatment Dose (expressed as free base) Scheme Number of rats one Compound 1 vehicle, HC1 (PEG400/EtOH/Phosal 50 PG (30/10/60)), 1 hour before Compound 3 vehicle, 15 minutes IV infusion 10 ml/kg 10 ml/kg +10 ml/kg RN, 1 hour before + RN, iv infusion 5 2 Filler for Compound 1, HC1 + Compound 3 0 mg/kg + 20 mg/kg RN, 1 hour before + RN, iv infusion 5 3 Compound 1, HC1 + Filler for Compound 3 150 mg/kg + 0 mg/kg RN, 1 hour before + RN, iv infusion 5 4 Compound 1, HC1 + Compound 3 150 mg/kg + 20 mg/kg RN, 1 hour before + RN, iv infusion 5

Treatment was started when the average tumor volume was approximately 450 mm ³ . Compound 1, HCl was prepared in PEG400/EtOH/Phosal 50 PG (30/10/60) and compound 3 was placed in solution. PH stands for once a week.

results

Treatment with Compound 1 free base combination at 150 mg/kg 1 hour prior to Compound 3 at 20 mg/kg iv infusion induces complete regression in all Karpas422 tumors 30 days from the start of treatment (Figure 5). All animals in the treated group were in remission without tumor after treatment, which stopped developing from day 35 until day 90. A beneficial effect of the combination was observed in the combination group compared to the active single agent. At day 34, the tumor response in the Compound 3 single agent group and the combination group were significantly different from the vehicle group (p<0.05). Treatment with the combination was well tolerated based on the change in body weight (FIG. 6).

Example 7 In Vivo Efficacy on DLBCL Toledo Xenograft Using a Combination of an MCL1 Inhibitor (Compound 3) and a BCL-2 Inhibitor (Compound 1, HCl)

Materials and methods Cell implantation

A xenograft model was established by direct subcutaneous (SC) implantation of 3 million suspension of Toledo cells with 50% Matrigel into the subcutaneous region of SCID/natural killer cell-deficient mice. All procedures were performed using aseptic techniques. Mice were anesthetized for the entire duration of the procedure. Overall, a total of 6 animals per group were included in the efficacy study. Animals were dosed by oral gavage (po) for compound 1 and intravenously (iv) via the tail vein for compound 3 to study the effects of a single agent and combination. Compound 1, HCl was prepared as a solution in PEG300/EtOH/water (40 /10/50), and compound 3 was placed in solution. When tumors reached approximately 220 mm on day 26 post-cell implantation, tumor-bearing mice were randomized to treatment groups.

The study design, including dosing regimen, for all treated groups is summarized in Table 1. below. Animals were weighed on the day(s) of dosing and the dose adjusted for body weight, the dosed volume was 10 ml/kg. Tumor sizes and body weights were collected at the time of randomization and twice a week thereafter during the study. After each day of data collection, the following data were collected: mortality rate, individual and group mean body weights, and individual and group mean tumor volumes.

- 16 039621

Groups Treatment Dose (expressed as free base) Scheme Number of mice one Filler REOZOO/EUN/water (40/10/50) RN, according to 6 2 Compound 1, HC1 100 mg/kg RN, according to 6 3 Compound 3 25 mg/kg RN, cc 6 4 Compound 1, HC1 + Compound 3 100 mg/kg 25 mg/kg RN, according to RN, cc 6

For the Toledo model study, treatments started on day 26 post cell implantation, when the mean tumor volume was ~218-228 mm ³ . PH stands for opse-weekly. Body weight (BW) % change in body weight was calculated according to the formula (BW _teK in _Shai - BW _initial )/(BW _initial ) x 100.

Data are presented as a percentage change in body weight from the day the treatment was started.

Tumor volume and percentage of mice remaining in the study

Percentage treatment/control (T/C) values were calculated using the following formula:

% T/S = 100 x LT/LS if ΔΤ >0 % regression = 100 χ ΔΤ/Τ ₀ if ΔΤ <0 where

T = mean tumor volume of the drug-treated group on the end day of the study;

Δ? = mean tumor volume of the drug-treated group on the end day of the study - mean tumor volume of the drug-treated group on the starting day of dosing;

T ₀ = mean tumor volume of the drug-treated group on the day of grouping;

C = mean tumor volume of the control group on the final day of the study; and

ΔC = mean tumor volume of the control group on the end day of the study - mean tumor volume of the control group on the starting day of dosing.

Percentage of mice remaining in the study = 6 - Number of mice reaching the endpoint/6 x 100

Statistical analysis

All data were expressed as mean ± standard error of the mean (SEM). The difference (delta) of tumor volume and percent change in body weight was used for statistical analysis. Comparisons between groups were performed by one-way ANOVA followed by Tukey's post hoc test. For all statistical evaluations, the significance level was set at p < 0.05. Describe the reliability compared to the control group with the filler, unless specifically indicated otherwise.

results

Treatment T/C% per day 42 Filler 100 Compound 1, HC1 37 Compound 3 102 Compound 1, HC1 + Compound 3 3

In the Toledo model, Compound 1 as a free base at 100 mg/kg produced statistically significant antitumor effects with 37% T/C. Compound 3 at 25 mg/kg resulted in no antitumor effects with 102% T/C (FIG. 7). The combination of Compound 1 + Compound 3 resulted in tumoristasis with 3% T/C, which is statistically significant compared to groups administered with Vehicle, Compound 1, and Compound 3 (p<0.05, by one-way ANOVA test). Thus, the combined inhibition of BCL-2 and MCL1 in DLBCL may provide a therapeutic benefit in the clinic. Additionally, the average body weight change for Toledo is shown in FIG. 8. Treatment of mice with Compound 1, HCl, and Compound 3 showed an increase in body weight (1.081 and 2.3%, respectively). The group treated with the combination showed a slight loss of body weight (-3.2%). No other signs of side effects were observed in this

- 17 039621 research. All 6 animals survived during the study.

Taken together, Examples 2, 6, and 7 showed that the combination of an MCL1 inhibitor and a BCL-2 inhibitor is effective at tolerated doses in mice and rats bearing xenograft cell lines derived from acute myeloid leukemia and human lymphoma, indicating that with this combination achieves a suitable therapeutic window for these diseases.

Example 8 In vitro effect on proliferation of a combination of an MCL1 inhibitor with a BCL-2 inhibitor in a panel of 13 acute myeloid leukemia (AML) cell lines.

Materials and methods

Cell lines were sourced and maintained in basic medium supplemented with FBS (Fetal Bovine Serum) as indicated in Table 1. 1. Additionally, all media contained penicillin (100 IU/ml), streptomycin (100 μg/ml) and L-glutamine (2 mm).

Cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO ₂ and grown in T-150 flasks. In all cases, cells were thawed from frozen materials, passed through >1 passage using appropriate dilutions, counted and analyzed for viability using a CASY cell counter before seeding at 150 µl/well at the densities shown in Table 1. 1, in 96 well plates. All cell lines were defined as free of mycoplasmic contaminants on their own basis.

Compound stock solutions were prepared at a concentration of 5 mM in DMSO and stored at -20°C.

To assay the activity of compounds as the only means, cells were seeded and treated with nine 2-fold serial dilutions of each compound, individually dispersed, directly into cell assay plates. The effects of compounds on cell viability were assessed after 3 days of incubation at 37° C./5% CO 2 by quantifying cellular ATP levels using CellTiterGlo at a concentration of 75 μl reagent/well. All experiments were carried out in triplicate. Luminescence was quantified on a multifunctional plate reader. The IC ₅₀ for a single agent was calculated using a standard four-parameter curve fitting. The IC ₅₀ was defined as the compound concentration at which the CTG signal is reduced by 50% relative to that measured with the vehicle control (DMSO).

To assay the activity of compounds in combination, cells were seeded and treated with seven or eight 3.16-fold serial dilutions of each compound distributed, either individually or in all possible staggered combinations, directly into cell assay plates as shown in FIG. 9. The effects of agents as monotherapy as well as their staggered combinations on cell viability were assessed after 3 days of incubation at 37°C/5% CO2 by quantifying cellular ATP levels using CellTiterGlo at a concentration of 75 μl of reagent/well. Carried out two independent experiments, each of them was carried out in duplicate. Luminescence was quantified on a multifunctional tablet reader.

Potential synergistic interactions between compound combinations were assessed using a 2D overinhibition matrix according to the Loewe additivity model and presented as a Synergy Score (Lehar et al., Nat Biotechnol. July 2009; 27(7): 659-666). All calculations were performed using Clalice™ Bioinformatics Software.

The doubling time indicated in the table. 3 is the mean doubling time obtained from different passages (in T-150 flasks) made from thawed cells when seeded in 96-well plates.

The Synergy Score was interpreted as follows:

SS ~ 0^Additive

SS > 1 ^Weak synergy

SS > 2^Synergism

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Table 3 Identity and study conditions for 13 acute myeloid leukemia (AML) cell lines, incl.

cell line Wednesday (source) %FBS Doubling time (hours) Number of seeded cells/well MV4;11 RPMI (ATCC) ten 31.0 56520 MOLM-13 RPMI (DSMZ) ten 32.4 56520 PL-21 RPMI (DSMZ) ten 32.4 56520 ML-2 RPMI (DSMZ) ten 31.6 56520 Nomo-1 RPMI (DSMZ) ten 43.5 56520 TNR-1 RPMI (ATCC) ten 49.6 56520 HL-60 IMDM (ATCC) 20 34.8 56520 Kasumi-1 RPMI (ATCC) 20 59.4 56520 OCI-AML3 MEM alpha (DSMZ) 20 25.7 56520 EOL-1 RPMI (DSMZ) ten 37.6 113040 GDM-1 RPMI (ATCC) ten 31.6 56520 KG1 IMDM (ATCC) 20 45.7 56520 KGla IMDM (ATCC) 20 36.5 56520

Table 4a. Monotherapy IC ₅₀ values for Compound 3, Compound 1, HCl and ABT-199 in 13

AML cell lines are presented. Compounds were incubated with cells for 3 days

cell line Compound 3 Compound 1, HCl AVT-199 Beginning CONC. [µM] IC _5O [µM] Beginning CONC. [µM] 1С ₅₀ [µM] Beginning CONC. [µM] 1С ₅₀ [µM] MV4;11 0.01 0.001 0.1 0.03 N.d. N.d. MOLM-13 0.01 0.002 0.1 0.04 N.d. N.d. PL-21 0.10 0.065 30.0 2.78 15.0 3,300 ML-2 0.10 0.005 2.0 0.04 n.a. n.a. Nomo-1 0.05 0.013 30.0 7.45 15.0 5,000 THP-1 0.10 0.017 30.0 0.75 2.0 0.900 HL-60 0.10 0.025 30.0 1.42 15.0 2,100 Kasumi-1 2.00 0.033 30.0 0.77 n.a. n.a. OCI-AML3 2.00 0.146 30.0 8.09 15.0 8,500 EOL-1 0.10 0.001 2.0 0.04 0.2 0.004 GDM-1 0.10 0.008 2.0 0.06 n.a. n.a. KG1 30.00 0.390 30.0 4.70 15.0 3,400 KGla 30.00 2,000 30.0 1.75 15.0 0.900

Table 4b. Monotherapy IC ₅₀ values for Compound 4, HCl in 5 AML cell lines are shown. The compound was incubated with cells for 3 days.

cell line Compound 4, HCl Beginning CONC. [µM] IC50 [µM] MV4;11 0.5 0.01 MOLM-13 θ.5 0.012 ML-2 0.5 0.01 OCI-AML3 fifteen 5.41 GDM-1 0.5 0.002

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Table 5a. Synergy scores for Compound 3 and Compound 1 and combination in 13 AML cell lines are presented. Interactions are interpreted as synergistic if observed scores are >2.0. Initial compound concentrations, mean max, inhibition, and standard deviation (sd) of synergism scores are presented. Compounds were incubated with cells for 3 days.

cell line Compound 3 Compound 1, HC1 Combination(s) Beginning CONC. [µM] Average Max Ing [%] Beginning CONC. [µM] Average Max Ing [%] Average Synergism Score Synergy estimation error (co) MV4;11 ο,ι 100.0 0.3 99.5 4.3 0.7 MOLM-13 0.1 99.5 0.3 90.0 8.2 1.3 PL-21 0.3 91.5 5.0 75.0 17.9 2.7 ML-2 0.1 99.5 oh, s 88.0 10.9 1.8 Nomo-1 0.3 97.0 5.0 31.0 11.8 1.0 TNR-1 0.3 99.0 5.0 55.5 13.2 0.1 HL-60 0.3 97.5 5.0 61.5 12.9 1.7 Kasumi-1 0.3 84.5 2.0 52.5 10.5 0.5 OCI- AML3 2.0 100.0 5.0 53.5 19.8 0.2 EOL-1 0.1 100.0 1.0 100.0 5.8 0.8 GDM-1 0.1 99.0 oh, s 87.0 I,1 1.4 KG1 2.0 80.5 5.0 53.0 14.6 1.7 KGla 2.0 28.5 5.0 73.0 13.0 0.9

Table 5b. Synergy scores for Compound 3 and ABT-199 combination in 8 AML cell lines are presented. Interactions are interpreted as synergistic if observed scores are >2.0. Initial compound concentrations, mean max, inhibition, and standard deviation (sd) of synergism scores are presented. Compounds were incubated with cells for 3 days.

cell line PL-21 Nomo-1 ТНР-1 Connect Beginning CONC. [µM] oh, s 0.3 0.3 3 Average Max Ing [%] 89.0 95.5 97.0 ΑΒΊ Beginning CONC. [µM] 2.0 2.0 o.s G-199 Average Max Ing [%] 41.5 45.5 47.0 Combi! Mean Synergy scores 19.7 10.7 12.4 data (b) Synergy estimation error (co) 2.7 1.6 0.7 HL-60 0.3 97.5 2.0 56.0 12.9 1.6 OCI- AML3 2.0 100.0 2.0 58.5 16.4 0.5 EOL-1 0.1 100.0 0.1 97.5 4.0 oh, s KG1 2.0 89.0 2.0 54.5 12.2 0.8 KGla 2.0 57.5 2.0 73.0 17.6 0.1

Table 5c. Synergy scores for Compound 3 and Compound 4, HCl combination with 5 AML cell lines are presented. Interactions are interpreted as synergistic if observed scores are >2.0. Initial compound concentrations, mean max, inhibition, and standard deviation (sd) of synergism scores are presented. Compounds were incubated with cells for 3 days.

cell line Compound 3 Compound 4, HC1 Combination (with) Beginning CONC. [µM] Average Max Ing [%] Beginning CONC. [µM] Average Max Ing [%] Mean value of the synergism score Synergy estimation error (co) MV4;11 0.01 100.0 0.03 70 3.37 0.75 MOLM-13 0.1 100 0.1 99 3.84 0.02 ML-2 0.1 100 0.1 99 7.09 0.96 OCI- AML3 2.0 100.0 5.0 53.5 16.53 1.62 GDM-1 0.1 100 0.1 99 7.03 0.52

results

Combination (a). The effect on proliferation of combining MCL1 inhibitor Compound 3 with BCL-2 inhibitor Compound 1 was evaluated in a panel of 13 acute myeloid leukemia (AML) cell lines. Compound 3 as the only agent strongly inhibited the growth of most of the 13 AML lines tested (Table 4a). Thus, 10 cell lines showed an IC ₅₀ below 100 nM, and

- 20 039621 additional 2 cell lines showed IC ₅₀ between 100 nm and 1 μm. Only 1 cell line showed IC ₅₀ above 1 μm.

Compound 1, HCl alone also inhibited the growth of several AML test lines, although slightly less effectively (Table 4a). Thus, 5 cell lines showed IC ₅₀ below 100 nM and 2 cell lines showed IC ₅₀ between 100 nM and 1 μM. Six cell lines showed IC ₅₀ above 1 μm.

In combination, Compound 3 and Compound 1 HCl treatment produced synergistic growth inhibition (ie synergism score greater than 2) in all 13 cell lines tested (Table 5a). In 2 cell lines, there is a marked synergistic effect with synergy scores between 5 and 10. In 10 cell lines, the synergistic effect is extraordinary, reaching synergy scores between 10 and 19.8. It is extremely important that the synergism is independent of the antiproliferative effects of a single agent, and in fact, is particularly strong at concentrations of Compound 3 and Compound 1, which themselves do not have an antiproliferative effect. For example, on OCI-AML3 cells, Compound 3 and Compound 1 at the third lowest concentration tested exhibited 5% and 1% growth inhibition, respectively, while the respective combination of the two compounds provided 84% growth inhibition (Fig. 9A, upper left panel), thus being 79% higher than the additivity calculated from the activity of individual agents (Fig. 9A, upper right panel).

In addition, it should be noted that Synergistic effects appear for a wide range of concentrations of individual agents, which should be useful in vivo in relation to flexibility regarding levels and dosing regimens.

In conclusion, the combination of Compound 3 and Compound 1 provided synergistic growth inhibition in all 13 AML cell lines tested. Critically, an extraordinary synergistic growth inhibition was observed in most AML cell lines tested (10/13).

Combination (b). The effect on proliferation of combining the MCL1 inhibitor Compound 3 with the BCL-2 inhibitor ABT-199 was evaluated in a panel of 8 acute myeloid leukemia (AML) cell lines.

Compound 3 as the only agent strongly inhibited the growth of most of the 8 AML lines tested (Table 4a). Thus, 5 cell lines showed an IC ₅₀ below 100 nM and an additional 2 cell lines showed an IC ₅₀ between 100 nM and 1 μM. Only 1 cell line showed an IC50 above 1 μM.

AVT-199 as the only agent also inhibited the growth of AML lines, although with less efficiency (Table 4a). Thus, only one cell line showed an IC50 below 100 nM and 2 cell lines showed an IC ₅₀ between 100 nM and 1 μM. Five cell lines showed IC ₅₀ above 1 μm.

In combination, treatment with an MCL1 inhibitor and ABT-199 produced synergistic growth inhibition (ie, a synergism score greater than 2) across a panel of 8 cell lines tested (Table 5b). In most cell lines, the synergistic effect is extraordinary, reaching synergy scores between 10 and 17.6. It is extremely important that the synergism is independent of the antiproliferative effects of a single agent, and in fact, is particularly strong at concentrations of the MCL1 inhibitor and ABT-199, which alone have no antiproliferative effect. For example, in OCI-AML3 cells, MCL1 and ABT-199 at the third lowest concentration tested exhibited 26% and 18% growth inhibition, respectively, while the corresponding combination of the two compounds provided 91% growth inhibition (Fig. 13, upper left panel). In addition, it should be noted that synergistic effects appear for a wide range of concentrations of individual agents, which should be useful in vivo in relation to flexibility regarding levels and dosing regimens.

In conclusion, the combination of Compound 3 and ABT-199 provided synergistic growth inhibition in all 8 AML cell lines tested. Critically, an extraordinary synergistic growth inhibition was observed in most AML cell lines tested (7/8).

Combination (s). The effect on proliferation of combining MCL1 inhibitor Compound 3 with BCL-2 inhibitor Compound 4 was evaluated in a panel of 5 acute myeloid leukemia (AML) cell lines.

Compound 3 as the only agent strongly inhibited the growth of the 5 AML test lines (Table 4b). Thus, all cell lines showed IC50 below 200 nM. Compound 4, HCl as the only agent also inhibited the growth of 4 out of 5 cell lines tested with an IC50 less than or equal to 40 nM, one cell line is resistant to compound 4 with an IC50 of 10 μM. In combination, treatment with Compound 3 and Compound 4, HCl produced synergistic growth inhibition (ie synergism score greater than 2) in all 5 cell lines tested (Table 5c). In 2 cell lines, there is a marked synergistic effect, with synergy scores between 5 and 10. In 1 cell line, the synergistic effect is extraordinary, providing a synergy score of 16.5. Extremely

- 21 039621 it is important that the synergism is independent of the antiproliferative effects of a single agent and in fact is particularly strong at concentrations of compound 4, HCl and compound 3, which alone have no or low antiproliferative effect. For example, in OCI-AML3 cells, Compound 4, HCl, and Compound 3 at the third lowest concentration tested exhibited growth inhibition of 1 and 40%, respectively, while the corresponding combination of the two compounds provided 98% growth inhibition (Fig. 1A, left panel; representative of two independent experiments), thus being 53% higher than the additivity calculated from the activity of the individual agents (Fig. 14A, right panel). In ML-2, Compound 4, HCl, and Compound 3 at the fifth lowest concentration tested exhibited 18% and 26% growth inhibition, respectively, while the respective combination of the two compounds provided 100% growth inhibition (Fig. 14B, left panel). ; characteristic of two independent experiments), thus being 51% higher than the additivity calculated from the activity of individual agents (Fig. 15, right panel).

In conclusion, the combination of Compound 4 and Compound 3 provided synergistic growth inhibition in all 5 AML cell lines tested.

Example 9 In Vitro Proliferation Effect of Combining an MCL1 Inhibitor with a BCL-2 Inhibitor in a Panel of 12 Neuroblastoma (NB) Cell Lines

Materials and methods

Cell lines were sourced and maintained in basic medium supplemented with FBS as indicated in Table 1. 1. Additionally, all media contained penicillin (100 IU/ml), streptomycin (100 μg/ml) and L-glutamine (2 mM). Cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO ₂ and grown in T-150 flasks. In all cases, cells were thawed from frozen materials, passed through >1 passage using appropriate dilutions, counted and analyzed for viability using a CASY cell counter before seeding at 150 µl/well at the densities shown in Table 1. 6 in 96 well plates. All cell lines were defined as free of mycoplasmic contaminants on their own basis.

Compound stock solutions were prepared at a concentration of 5 mM in DMSO and stored at -20°C. To assay the activity of compounds as the only means, cells were seeded and treated using nine 3.16-fold serial dilutions of each compound, individually dispersed, directly into cell assay plates. The effects of compounds on cell viability were assessed after 2 or 3 days of incubation (as indicated in Table 6) at 37°C/5% CO2 by quantifying cellular ATP levels using CellTiterGlo at 150 µl of reagent/well.

Conducted two independent experiments, each of them was carried out in duplicate. All experiments were carried out in triplicate. Luminescence was quantified on a multifunctional plate reader. The IC ₅₀ for individual agents as monotherapy was calculated using a standard four-parameter curve fitting. The IC ₅₀ was defined as the compound concentration at which the CTG signal is reduced by 50% relative to that measured with the vehicle control (DMSO).

Carried out identical experiments to evaluate potential synergistic interactions between combinations of compounds. Synergy was evaluated using a 2D excess inhibition matrix according to the Loewe additivity model (Lehar et al., Nat Biotechnol. July 2009; 27(7): 659-666). All calculations were performed using Chalice TM Bioinformatics software.

The doubling time indicated in the table. 6 is the mean doubling time obtained from different passages (in T-150 flasks) made from thawed cells when seeded in 96-well plates.

Interpretation of the synergy score (Synergy Score) was carried out as follows:

SS~0^ Additive

SS>1 ^Weak synergy

SS>2^Synergy

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Table 6. Identity and study conditions for 12 neuroblastoma (NB) cell lines used in combined experiments

cell line Wednesday (source) %FBS Doubling time (hours) Number of seeded cells/well Days of incubation with Comm. SK-N-AS DMEM (ATCC) ten 33 9375 3 SK-N-BE EMEM/Ham F12 (ATCC) ten fifty 37500 3 SK-N-DZ DMEM (ATCC) ten 42 37500 3 LAN-6 DMEM (DSMZ) 20 100 9375 NBL-S MDM Pskov (DSMZ) ten 46 18750 3 SIMA RPMI (DSMZ) ten 60 18750 3 KELLY RPMI (ECACC) ten 34 3750 2 IMR-32 EMEM (ATCC) ten 55 28125 2 SH-SY-5Y 1/2 EMEM No Glutamine + 1/2 Ham F12 + 2mM Glutamine + NEAA (ECACC) fifteen 35 3750 2 SK-N-SH EMEM (ATCC) ten 65 3750 2 NB-1 RPMI (JCRB) ten 35 15000 2 SK-N-FI DMEM (ATCC) ten 60 7500 2

Table 7. Monotherapy IC ₅₀ values for Compound 3 and Compound 1, HCl, are shown.

Compounds were incubated with cells for 2 or 3 days._____________________

cell line Compound 3 Compound 1, HCl IC ₅₀ [µM] IC ₅₀ [µM] SK-N-AS 0.26 > 1 SK-N-BE >2 >2 SK-N-DZ >2 >2 LAN-6 >2 >2 NBL-S >2 >2 SIMA >2 >2 NB1 0.123 >3 SK-N-SH >3 >3 SH-SY5Y >3 >3 Kelly 0.031 >3 SK-N-FI >3 >3

Table 8. Synergism scores are shown for combination with Compound 3 and Compound 1, HCl. Interactions are interpreted as synergistic if observed scores are >2.0. Compounds were incubated with cells for 2 or 3 days.

cell line Compound 3 Compound 1, HC1 Combination Beginning CONC. [µM] Average Max Ing [%1 Beginning CONC. [µM] Max Ing [%] Synergy assessment Synergism estimation error SK-N-AS 2 84 one nine 2.78 0.46 SK-N-BE 2 27 2 27 10.72 0.78 SK-N-DZ 2 2.5 2 ten 0.34 0.06 LAN-6 2 17.5 2 26 10.51 0.39 NBL-S 2 thirteen 2 ten 17.81 3.7 SIMA 2 0 2 48.5 2.41 0.75 NB1 3 99 3 And 10.72 4.33 SK-N-SH 3 40 3 fifteen 4.07 0.23 SH-SY5Y 3 24 3 ten 10.21 0.54 Kelly 3 99 3 27 9.62 0.48 SK-N-FI 3 33 3 6 4.35 0.91

results

The effect on proliferation of combining MCL1 inhibitor Compound 3 with BCL-2 inhibitor Compound 1 was evaluated in a panel of 12 neuroblastoma cell lines. Three of the 12 cell lines tested are sensitive to compound 3 as the only agent (Table 7). One cell line showed an IC ₅₀ below 100 nM and an additional 2 cell lines showed an IC ₅₀ between 100 nM and 1 μM.

All cell lines are resistant to Compound 1, HCl as the only agent where all cell lines tested show an IC ₅₀ greater than 1 μM. In combination, treatment with Compound 3 and Compound 1 produces synergistic growth inhibition (i.e., score

- 23 039621 synergy above 2 - Lehar et al., Nat Biotechnol. July 2009; 27(7): 659-666) in 11 of the 12 NB cell lines tested (Table 8). In 5 cell lines, the synergistic effect is extraordinary, reaching synergy scores between 10 and 17.81. It is extremely important that the synergism is independent of the antiproliferative effects of a single agent, and in fact, is particularly strong at concentrations of Compound 3 and Compound 1, HCl, which alone have no antiproliferative effect. For example, in LAN-6 cells, Compound 3 and Compound 1, HCl at 630 nM exhibited only 12% and 0% growth inhibition, respectively, while the respective combination of the two compounds provided 95% growth inhibition (Fig. 10, upper left panel), thus being 76% higher than the additivity calculated from the activity of individual agents (Fig. 10, upper right panel). In conclusion, the combination of Compound 3 and Compound 1 provides strong to extraordinary synergistic growth inhibition in most neuroblastoma cell lines tested.

Example 10 In vitro effect on proliferation of combining an MCL1 inhibitor with a BCL-2 inhibitor in a panel of 8 B-cell acute lymphoblastic leukemia (B-ALL) cell lines and 10 T-cell acute lymphoblastic leukemia (T-ALL) cell lines.

Materials and methods

Cell lines were sourced and maintained in basic medium supplemented with FBS as indicated in Table 1. 1. Additionally, all media contained penicillin (100 IU/ml), streptomycin (100 μg/ml) and L-glutamine (2 mm). Cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO ₂ and grown in T-150 flasks. In all cases, cells were thawed from frozen materials, passed through >1 passage using appropriate dilutions, counted and analyzed for viability using a CASY cell counter before seeding at 150 µl/well at the densities shown in Table 1. 9 in 96 well plates. All cell lines were defined as free of mycoplasmic contaminants on their own basis.

Compound stock solutions were prepared at a concentration of 5 mM in DMSO and stored at 20°C. To assay the activity of compounds as the only means, cells were seeded and treated with nine 2-fold serial dilutions of each compound, individually dispersed, directly into cell assay plates. The effects of compounds on cell viability were assessed after 3 days of incubation at 37° C./5% CO 2 by quantifying cellular ATP levels using CellTiterGlo at a concentration of 75 μl reagent/well. All conditions were tested in triplicate. Luminescence was quantified on a multifunctional plate reader. IC ₅₀ was calculated for single agents as monotherapy using standard four-parameter curve fitting. The IC ₅₀ was defined as the compound concentration at which the CTG signal is reduced by 50% relative to that measured with the vehicle control (DMSO).

To assay the activity of compounds in combination, cells were seeded and treated with seven or eight 3.16-fold serial dilutions of each compound distributed, either individually or in all possible staggered combinations, directly into cell assay plates as shown in FIG. 1. The effects of agents in the form of monotherapy, as well as their combinations in a checkerboard pattern on cell viability was assessed after 3 days of incubation at 37°C/5% CO ₂ by quantifying cellular ATP levels using CellTiterGlo at a concentration of 75 μl of reagent/well. For B-ALL cell lines, two independent experiments were carried out, each of them was carried out in duplicate. For T-ALL cell lines, one performed experiment was performed in triplicate. Luminescence was quantified on a multifunctional plate reader.

Potential synergistic interactions between compound combinations were assessed using a 2D overinhibition matrix according to the Loewe additivity model and presented as a Synergy Score (Lehar et al., Nat Biotechnol. July 2009; 27(7): 659-666). All calculations were performed using the Chalice™ Bioinformatics software available from the Horizon website.

The doubling time indicated in the table. 9 is the mean doubling time obtained from different passages (in T-150 flasks) made from thawed cells when seeded in 96-well plates.

Interpretation of the synergy score (Synergy Score) was carried out as follows:

SS~0^ Additive

SS>1 ^Weak synergy

SS>2^Synergy

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Table 9. Identity and test conditions for 8 B-ALL and 10 T-ALL cell lines used in combined experiments_______

cell line type of cancer Wednesday (source) %FBS Doubling time (hours) Number of seeded cells/well VOLUME-1 BALL RPMI (DSMZ) 20 70.0 112500 SUP-B15 BALL McCoy (DSMZ) 20 35.0 112500 NALM-21 B-ALL RPMI (DSMZ) ten 50.0 112500 NALM-6 BALL RPMI (DSMZ) ten 27.0 56250 TANOUE B-ALL RPMI (DSMZ) ten 26.0 30000 Kasumi-2 B-ALL RPMI (DSMZ) ten 52.0 112500 RS4;11 BALL RPMI (ATCC) ten 42.0 90000 BALL-1 BALL RPMI (DSMZ) ten 38.0 112500 BE-13 T-ALL RPMI 1640 (DSMZ) ten 37.0 13875 MOLT-4 T-ALL RPMI 1640 (ATCC) ten 24.0 28125 TALL-104 T-ALL IMDM (ATCC) 20 68.0 13875 HPB-ALL T-ALL RPMI 1640 (DSMZ) 20 42.0 56250 DND-41 T-ALL RPMI 1640 (DSMZ) ten 38.0 56250 CML-T1 T-ALL RPMI 1640 (DSMZ) ten 32.0 112500 J45.01 T-ALL RPMI 1640 (ATCC) ten 25.0 56250 CCRFCEM T-ALL RPMI 1640 (ATCC) ten 24.0 56250 J.RT3 T3.5 T-ALL RPMI 1640 (ATCC) ten 24.0 56250 Loucy T-ALL RPMI 1640 (ATCC) ten 61.0 112500

Table 10 Shows monotherapy IC ₅₀ values for Compound 3 and Compound 1, HCl in 8 B-ALL and 10 T-ALL cell lines. Compounds were incubated with cells for 3 days.

cell line type of cancer Duration of treatment (h) Compound 3 Compound 1, HC1 Beginning CONC. [µM] 1С ₅₀ [µM] Beginning CONC. [µM] 1С ₅₀ [µM] VOLUME-1 B-ALL 72 0.10 0.024 0.15 0.019 SUP-B15 BALL 72 2.00 0.078 0.90 0.025 NALM-21 BALL 72 0.10 0.012 0.50 0.095 NALM-6 BALL 72 2.00 0.120 30.00 3,630 TANOUE B-ALL 72 30.00 6,540 30.00 17,000 Kasumi-2 B-ALL 72 2.00 0.030 2.00 0.209 RS4;11 BALL 72 0.90 0.079 9.00 0.020 BALL-1 BALL 72 0.25 0.063 0.10 0.019 BE-13 T-ALL 72 0.15 0.015 30.00 6,700 MOLT-4 T-ALL 72 2.00 0.026 30.00 3.290 TALL-104 T-ALL 72 2.00 0.044 30.00 15,900 HPB-ALL T-ALL 72 2.00 0.660 30.00 4,500 DND-41 T-ALL 72 30.00 7,000 30.00 9,000 CML-T1 T-ALL 72 30.00 6,000 30.00 15,000 J45.01 T-ALL 48 0.60 0.029 30.00 9,000 CCRF-CEM T-ALL 48 0.90 0.047 30.00 7,500 J.RT3 T3.5 T-ALL 48 1.88 0.063 30.00 10,000 Loucy T-ALL 48 0.90 0.064 3.75 0.231

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Table 11 Synergism scores for Compound 3 and Compound 1, HCl and combination in 8 B-ALL and 10 T-ALL cell lines are shown. Interactions are interpreted as synergistic if observed scores are >2.0. Initial compound concentrations, mean max, inhibition, and standard deviation (sd) of synergism scores are presented. Compounds were incubated with cells for 3 days.

cell line type of cancer Duration of treatment (h) Compound 3 Compound 1, HC1 Combination Beginning conc. [µM] Average Max Ing [%] Beginning conc. [µM] Average Max Ing [%] Average Synergy score Synergy Errors (co) VOLUME-1 BALL 72 0.3 98.5 0.1 90.5 4.1 0.4 SUP-B15 B-ALL 72 2.0 99.0 0.3 97.0 5.6 0.4 NALM-21 BALL 72 0.3 99.0 0.3 84.5 9.6 0.0 NALM-6 BALL 72 2.0 71.5 5.0 56.5 15.9 1.4 TANOUE BALL 72 5.0 78.5 5.0 13.0 1.0 0.6 Kasumi-2 BALL 72 0.3 99.0 2.0 82.0 10.1 1.9 RS4;11 BALL 72 0.3 87.5 0.3 98.0 7.0 1.3 BALL-1 BALL 72 0.3 96.0 0.3 100.0 6.3 0.3 BE-13 T-ALL 72 1, oh 95.0 5.0 0.0 8.8 0.4 MOLT-4 T-ALL 72 1.0 99.0 5.0 63.0 4.4 oh 1 TALL-104 T-ALL 72 1.0 99.0 2.0 29.0 15.1 0.5 HPB-ALL T-ALL 72 1.0 49.0 5.0 15.0 5.6 0.3 DND-41 T-ALL 72 2.0 17.0 5.0 24.0 10.3 0.3 CML-T1 T-ALL 72 2.0 22.3 5.0 17.0 3.3 0.2 J45.01 T-ALL 48 2.0 100.0 2.0 23.0 2.9 0.1 CCRF-CEM T-ALL 48 2.0 92.0 2.0 55.0 4.1 1, oh J.RT3 T3.5 T-ALL 48 2.0 99.0 2.0 32.0 3.8 0.1 Loucy T-ALL 48 2.0 100.0 2.0 77.0 11.3 0.6

results

The effect on proliferation of combining an MCL1 inhibitor with a BCL-2 inhibitor was evaluated in a panel of 8 B-ALL and 10 T-ALL cell lines. The MCL1 inhibitor as the only agent strongly inhibited the growth of most of the ALL cell lines tested (Table 10). Thus, 13 ALL cell lines showed an IC ₅₀ below 100 nM and an additional 2 ALL cell lines showed an IC ₅₀ between 100 nM and 1 μM. Only 3 ALL cell lines showed IC ₅₀ above 1 μm.

The BCL-2 inhibitor alone also inhibited the growth of several ALL cell lines tested, although it was less effective (Table 10). Thus, 5 cell lines showed IC ₅₀ below 100 nM and 2 cell lines showed IC ₅₀ between 100 nM and 1 μM. Eleven ALL cell lines exhibited IC50s greater than 1 μM. In combination, treatment with an MCL1 inhibitor and a BCL-2 inhibitor causes synergistic growth inhibition (i.e. synergism score greater than 2 - Lehar et al., Nat Biotechnol. July 2009; 27(7): 659-666) in all 17 subjects tested. /18 ALL cell lines (Table 11). In 6 cell lines, a noticeable synergistic effect, with synergy scores between 5 and 10. In 5 cell lines, the synergistic effect is extraordinary, reaching synergy scores between 10 and 15.9. It is extremely important that the synergism is independent of the antiproliferative effects of a single agent, and in fact, is particularly strong at concentrations of the MCL1 inhibitor and BCL-2 inhibitor, which alone have no antiproliferative effect. For example, in NALM-6 cells, an MCL1 inhibitor and a BCL-2 inhibitor at the fourth lowest concentration tested exhibited 6% and 8% growth inhibition, respectively, while the corresponding combination of the two compounds provided 61% growth inhibition (Fig. 11 , upper left panel).

In addition, it should be noted that synergistic effects appear for a wide range of concentrations of individual agents, which should be useful in vivo in relation to flexibility regarding levels and dosing regimens.

In conclusion, the combination of an MCL1 inhibitor and a BCL-2 inhibitor provides synergistic growth inhibition in most (17/18) ALL cell lines tested. Critically, extraordinary synergistic growth inhibition was observed in 5/18 of the ALL cell lines tested.

Example 11 In Vitro Effect on Proliferation of Combining an MCL1 Inhibitor with a BCL-2 Inhibitor in a Panel of 5 Mantle Zone Lymphoma (MCL) Cell Lines.

Materials and methods

Cell lines were sourced and maintained in basic medium supplemented with FBS as indicated in Table 1. 12. Additionally, all media contained penicillin (100 IU/ml), streptomycin (100 μg/ml) and L-glutamine (2 mM).

Cell lines were cultured at 37° C. in a humidified atmosphere containing 5% CO2 and grown in T-150 flasks. In all cases, cells were thawed from frozen materials, passed through >1 passage using appropriate dilutions, counted and analyzed for viability using a CASY cell counter before seeding at 150 µl/well at the densities shown in Table 1. 12 in 96 well plates. All cell lines were defined as free of mycoplasmic contaminants on their own basis.

Compound stock solutions were prepared at a concentration of 5 mM in DMSO and stored at 20°C. For activity assays of compounds alone or in combination, cells were seeded and treated with seven or eight 3.16-fold serial dilutions of each compound distributed, either individually or in all possible staggered combinations, directly into cell assay plates. The effects of agents as monotherapy as well as their staggered combinations on cell viability were assessed after incubation for 2 days at 37°C/5% CO2 by quantifying cellular ATP levels using CellTiterGlo at 150 µl of reagent/well. All conditions were tested in triplicate. Luminescence was quantified on a multifunctional plate reader. Potential synergistic interactions between compound combinations were assessed using a 2D overinhibition matrix according to the Loewe additivity model and presented as a Synergy score (Lehar et al., Nat Biotechnol. July 2009; 27(7): 659-666). All calculations were performed using the Chalice™ Bioinformatics software available from the Horizon website.

IC ₅₀ was calculated for single agents as monotherapy using standard four-parameter curve fitting. The IC ₅₀ was defined as the compound concentration at which the CTG signal is reduced by 50% relative to that measured with the vehicle control (DMSO).

The doubling time indicated in the table. 12 is the mean doubling time obtained from different passages (in T-150 flasks) made from thawed cells when seeded in 96 well plates.

Synergy assessment

SS~0^ Additive

SS>1 ^Weak synergy

SS>2^Synergy

Table 12. Identity and study conditions for 5 lymphoma cell lines from ______ mantle zone cells used in combined experiments

cell line Wednesday %FBS Source Doubling time (hours) Number of seeded cells/well Z-138 RPMI ten ATCC 22.5 37500 Jeko RPMI 20 ATCC 26.0 27000 Mino RPMI fifteen ATCC 31.1 56250 JVM-2 RPMI ten ATCC 76.0 56250 REC-1 RPMI ten ATCC 36.0 56250

Table 13 Shows monotherapy IC ₅₀ values for Compound 3 and Compound 1, HCl in 5 mantle zone lymphoma cell lines. Compounds were incubated with cells for 2 days.

cell line Compound 3 Compound 1, HC1 Beginning CONC. [µM] 1С ₅₀ [µM] Beginning CONC. [µM] 1С ₅₀ [µM] Z-138 2 0.448 5 > 5 Jeko 2 0.023 5 > 5 Mino 2 0.008 2 0.091 JVM-2 2 >2 5 > 5 REC-1 2 0.077 2 0.703

Table 14 Synergism scores for Compound 3 and Compound 1, HCl combination in 5 mantle zone lymphoma cell lines are shown. Interactions are interpreted as synergistic if observed scores are >2.0. Initial compound concentrations, maximum inhibition, and synergism scores are presented. Compounds were incubated with cells for 2 days.

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results

The effect on proliferation of combining an MCL1 inhibitor with a BCL-2 inhibitor was evaluated in a panel of 5 mantle zone lymphoma cell lines.

As the sole agent, the MCL1 inhibitors were more active than the BCL-2 inhibitor. Thus, 3 cell lines showed an IC ₅₀ below 100 nM for the MCL1 inhibitor, while only one cell line showed an IC ₅₀ below 100 nM for the BCL-2 inhibitor (Table 13).

In combination, treatment with an MCL1 inhibitor and a BCL-2 inhibitor causes synergistic growth inhibition (i.e. synergism score greater than 2 - Lehar et al., Nat Biotechnol. July 2009; 27(7): 659-666) in all subjects tested. cell lines (Table 14) as illustrated in FIG. 12. Extremely important, in 4/5 cell lines, there is a noticeable synergistic effect, with synergy scores above 5.

Example 12 In Vitro Effect on Proliferation of Combining an MCL1 Inhibitor with a BCL-2 Inhibitor in a Panel of 5 Small Cell Lung Cancer (SCLC) Cell Lines.

All cell lines were obtained from ATCC. Culture media containing RPMI1640 (Invitrogen) supplemented with 10% FBS (HyClone) was used for COR-L95, NCI-H146, NCI-H211, SHP77, SW1271, NCI-H1339, NCI-H1963 and NCI-H889. Culture media containing Waymouth MB 752/1 (Invitrogen) with 10% FBS was used for DMS-273. Culture medium containing DMEM/F12 (Invitrogen) containing 5% FBS and supplemented with 0.005 mg/ml insulin, 0.01 mg/ml transferrin, and 30 nM sodium selenite solution (Invitrogen), 10 nM hydrocortisone (Sigma), 10 nM betaestradiol (Sigma), and 2 mM L-glutamine (HyClone) were used for NCI-H1105.

Cell lines were cultured at 37°C and 5% CO ₂ incubator and grown in T-75 flasks. In all cases, cells were thawed from frozen materials, passed through >1 passage using 1:3 dilutions, counted and analyzed for viability using a ViCell counter (Beckman-Coulter) before seeding into 384-wells. To propagate and grow cell lines, cells were detached from flasks using 0.25% Trypsin-EDTA (GIBCO). All cell lines were determined to be free of mycoplasmic contamination as determined by PCR detection methodology assayed on Idexx Radii (Columbia, MO, USA) and correctly identified by SNP panel detection. Cell proliferation was measured over 72 hours in CellTiter-Glo™ (CTG) assays (Promega G7571) and all reported results were at least triplicate. For CellTiter-Glo™ assays, cells were distributed into treated tissue culture in 384 well plates (Corning 3707) with a final volume of 35 μl of medium and at a density of 5000 cells per well. 24 hours after inoculation, 5 μl of dilution series of each compound were transferred to plates containing cells, resulting in compound concentrations in the range of 0-10 μM and a final concentration of DMSO (Sigma D8418) of 0.16%. The plates were incubated for 72 hours and the effect of the compounds on cell proliferation was determined using a CellTiter-Glo™ fluorescent cell viability assay (Promega G7571) and an Envision plate reader (Perkin Elmer).

The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method for determining the number of viable cells in culture based on the quantification of ATP present, which signals the presence of metabolically active cells. The method is described in detail in the Technical Bulletin, TB288 Promega. Briefly, cells were seeded in a light-tight multiwell plate in culture medium as described above. Also prepared control wells containing medium without cells, to obtain the value of the background luminescence. Thereafter, 15 μl CellTiter-Glo® reagent was added and the contents were mixed for 10 min on an orbital shaker to induce cell lysis. After that, the luminescence was recorded using a plate reader.

Percent growth inhibition and excess inhibition were analyzed using Chalice software (CombinatoRx, Cambridge MA). The percentage of growth inhibition relative to DMSO was shown in the labeled inhibition panel, and the amount of inhibition in excess of the calculated amount in the labeled ADD excess inhibition panel (Fig. 15(a)-(e)). The concentrations of Compound 1, HCl are presented in the bottom row from left to right and the increasing concentrations of Compound 3 in the leftmost column from bottom to top. All remaining points in the dataset are results from the combination of the two inhibitors, corresponding to the concentrations of the individual agents represented on the two axes. Cell proliferation data analysis was performed using a Chalice analyzer as described in Lehar et al., Nat Biotechnol. July 2009; 27(7): 659666. Excess inhibition was calculated using the Loewe synergy model, which measures the effect on growth relative to what would be expected if two drugs behaved additively in a dose-dependent manner. Positive numbers represent areas of increasing synergy.

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Synergy assessment

SS~0^Additive dose

SS>2^ Synergism

SS>1 ^Weak synergy

results

In combination, treatment with Compound 1 and Compound 3 produces synergistic growth inhibition (ie synergism score greater than 2) in 8/10 small cell lung cancer cell lines. Extremely important, in 6 cell lines, there is a noticeable synergistic effect, with synergy scores above 6.

Example 13 Effects in vivo on patient-derived primary AML models of HAMLX5343 using a combination of an MCL1 inhibitor (Compound 3) and a BCL-2 inhibitor (Compound 1, HCl or ABT-199)

Materials and methods

materials

Animals

NOD scid gamma (NSG) female mice weighing 17-27 g (Jackson Laboratories) were allowed to acclimate with access to food and water ad libitum for 3 days prior to manipulation.

Primary tumor models

Patient-derived primary AML models HAMLX5343 carrying the KRAS mutation and wild-type FLT3 were obtained from the Dana Farber Cancer Institute.

Test compounds, preparations

Compound 1, HCl was prepared in 5% Ethanol, 20% Dexolve-7 as an intravenous solution or prepared in PEG300/EtOH/water (40/10/50) for oral administration. ABT-199 was prepared in PEG300/EtOH/water (40/10/50) for oral administration. All were stable for at least one week at 4°C. Compound 3 was prepared in a liposomal preparation as a solution for intravenous preparation, which is stable for three weeks at 4°C.

The filler and dosed solutions of the compounds were prepared as needed. All animals were dosed with 10 ml/kg of Compound 1 (expressed as free base) or ABT-199, or 5 ml/kg for Compound 3.

Methods

Experiment scheme

Eight treatment groups were used in the 7844HAMLX5343-XEF study, as summarized in Table 1. 15. All treatments were started when an average tumor burden (%CD-45 positive cells) was between 8% and 15%.

In this study, Compound 1 was administered by oral forced administration (po) or intravenous administration at a dose of 50 mg/kg once a week, ABT-199 was administered at a dose of 25 mg/kg by oral forced administration (po) once a week, or as a single agent or in combination with compound 3 at a dose of 12.5 mg/kg once a week, respectively, for 18 days.

Both Compound 1 (expressed as free base) and ABT-199 were administered at a dose of 10 ml/kg. Compound 3 was administered at a dose of 5 ml/kg. The dose was adjusted for body weight. Body weight was recorded twice a week and tumor burden was recorded once a week.

Table 15. Doses* and Dosing Schedules for 7844HAMLX5343-XEF

Treatment groups Number of animals Dosing schedule Filler (10 ml/kg) 4 RN Compound 1 (50 mg/kg po) 4 RN Compound 1 (50 mg/kg IV) 4 RN ABT-199 (25mg/kg po) 4 RN Compound 3 (12.5 mg/kg IV) 4 RN Connection 1 + Connection 3 (po/vv) 4 RN + RN Connection 1 + Connection 3 (iv/vv) 4 RN + RN AVT-199 + Compound 3 (po/vv) 4 RN + RN

* Doses are presented as the free base

Primary AML Model

For this experiment, 32 mice were implanted with the primary AML line HAMLX5343. Mice were injected intravenously with 2.0 million leukemic cells. When tumor burden reached 8-15%, animals were randomized into eight groups of four animals each for vehicle, Compound 1 (po), Compound 1 (cv), ABT-199, Compound 3, or combination treatment. After 18 days of treatment, the study was stopped if the tumor burden reached 99%. Tumor burden was determined using FACS analysis.

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Animal observations

The condition and behavior of the animals, including grooming and the ability to move, was monitored twice a day. The general health of the mice was monitored and mortality was recorded daily. Any dying animals were euthanized.

Tumor measurement

Mice were bled by tail incision once a week. Blood was added to an IgG control well and a CD33/CD45 well of a 96-well plate. Blood was lysed with 200 μl RBC lysis buffer twice at RT, then washed once with FACS buffer (5% FBS in PBS). After that, the samples were incubated for 10-30 min at 4C in 100 μl of blocking buffer (5% mouse Fc Block + 5% human Fc Block + 90% FACS buffer). 20 µl IgG control mix (2.5 µl mouse igG1 K isotype control -PE + 2.5 µl mouse igG1 K isotype control-APC + 15 µl FACS buffer) was added to IgG control wells and 20 µl CD33/CD45 mix (2, 5 µl mouse anti-human CD33-PE + 2.5 µl mouse anti-human CD45-APC + 15 µl FACS buffer). Samples were incubated for 30-60 min at 4°C, then washed twice before analysis. Samples were run on Canto using FACSDiva software. Analysis was performed using FloJo software. The percentage of CD45-positive, living, single cells was described as tumor burden.

Data analysis

Percentage treatment/control (T/C) was calculated using the following formula: %T/C = 100 x ΔT/AC if ΔΤ >0% Regression = 100 x LT/T _baseline if ΔT < 0 where

T = mean tumor burden of the drug-treated group on the end day of the study;

ΔΤ = mean tumor burden of the drug-treated group on the end day of the study - mean tumor burden of the drug-treated group on the starting day of dosing;

T _initial = mean tumor burden of the drug-treated group on the initial day of dosing;

C = mean tumor burden of the control group on the end day of the study and

ΔC = mean tumor burden of the control group on the end day of the study - mean tumor burden of the control group on the starting day of dosing.

All data were expressed as mean ± SPE. The difference (delta) between tumor burden and body weight was used for statistical analysis.

Comparisons were made between groups for final measurements using ANOVA with Tukey's test. Statistical analysis was performed using GraphPad Prism.

Statistical analysis

All data were expressed as mean ± standard error of the mean (SEM). The difference (delta) between tumor volume and body weight was used for statistical analysis. Comparisons were made between groups using Kruskal-Wallis ANOVA followed by Dunn's post hoc test or Tukey's test. For all statistical evaluations, the significance level was set at p < 0.05. Describe the reliability compared to the control group with the filler, unless specifically indicated otherwise.

The standard protocols used in pharmacological studies have not been previously run to demonstrate statistically significant benefits of combinations compared to treatment-matched agents as monotherapies. Statistical power is often limited by single agent response performance and/or model variability. However, p-values are presented for combinations relative to treatment with single agents as monotherapies.

results

Synergistic antitumor effect of combined MCL1 and BCL-2 inhibition

In the 7844HAMLX5343-XEF study, Compound 1, ABT-199, or Compound 3 alone showed no antitumor activity in a HAMLX5343 model carrying the KRAS mutation when administered at a dose of 50 mg/kg (po or iv), 25 mg/kg (po), or 12.5 mg/kg (iv) once a week, respectively (%T/C 98, 92, 98, or 99%, respectively, p>0.05).

When administered orally, compound 1 at a dose of 50 mg/kg or ABT-199 at a dose of 25 mg/kg in combination with compound 3 (12.5 mg/kg IV) once a week leads to tumor stasis (%T/C 3 or 6 %, respectively, p<0.05) on this model. On the other hand, the combination of intravenously administered Compound 1 with Compound 3 induces almost complete tumor regression (% regression of 100%), which is significantly different from any single agent (p<0.05) or the combination of Compound 1/Compound 3

- 30 039621 on / cc. The mean tumor burden for each treatment group was plotted over time over a treatment period of 18 days, as shown in FIG. 1. Change in tumor load, %T/C or % regression is presented in Table. 16 and in FIG. 16(a)-(c).

Table 16 Summary of antitumor effect results in the 7844HAMLX5343-XEF study

Treatment T/C % Regression % Filler 100 Compound 1 50 mg/kg b.w. 98 Compound 1 50 mg/kg body weight iv 92 AVT-199 25 mg/kg b.w. 98 Compound 3 12.5 mg/kg body weight iv 99 Compound 1 + Compound 3 (po/vv) 3* Connection 1 + Connection 3 (iv/vv) 100** AVT-199 + Compound 3 (po/vv) 6*

* p < 0.05 compared to Excipients and single agents (ANOVA, Tukey's test) ** p < 0.05 compared to po/iv combination (ANOVA, Tukey's test)

Conclusion

AML is an aggressive and heterogeneous malignant disease of the blood system caused by the transformation of hematological progenitor cells due to acquired genetic rearrangements (Patel et al., New England Journal of Medicine 2012 366: 1079-1089). The 5-year survival rate for AML is low due to the lack of effective therapies. Apoptosis bypass is a hallmark of cancer (Hanahan et al., Cell 2000 100:57-70). One of the primary means by which cancer cells avoid apoptosis is upregulation of survival-promoting BCL-2 family proteins such as BCL-2, BCL-xL and MCL1.

The MCL1 gene is the most frequently amplified gene in cancer patients (Berukhim et al., Nature 2010 463:899-905). In addition, both BCL-2 and MCL1 are highly expressed in AML. Therefore, the combination of Compound 1 (BCL-2i) and Compound 3 (MCL1) may provide synergy by enhancing pro-apoptotic signals as a primary anti-AML mechanism.

In the present application, we have shown that the BCL-2 inhibitor compound 1 or ABT-199 in combination with compound 3 (MCL1 inhibitor) have a pronounced synergistic effect in the treatment of AML in an AML xenograft model with a KRAS mutation (dt FLT3). The iv/iv combination of Compound 1/Compound 3 is superior to iv combination treatment at the same dosage level. The results obtained indicate that the combination of MCL1 inhibitors would be an effective therapy for AML.

CLAIM

Claims (31)

CLAIM 1. Combination for the treatment of malignant neoplasm, containing: (a) a BCL-2 inhibitor selected from (i) N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2(1H) -isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetra-hydro-1-indolizine carboxamide or an addition salt thereof with a pharmaceutically acceptable acid or base, (ii) 5 -(5-chloro-2-{[(3S)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin-2(1H)-yl]carbonyl}phenyl)N-(5-cyano-1, 2-dimethyl-1H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-1H-pyrrol-3-carboxamide or an addition salt thereof with a pharmaceutically acceptable acid or base and (iii) 4- (4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(3-nitro-4{[(oxan- 4-yl)methyl]amino}phenyl)sulfonyl]-2-[(1 H-pyrrolo[2,3-b]pyridin-5-yl)oxy]benzamide (AWT-199); and (b) an MCL1 inhibitor selected from (iv) (2R)-2-{[(5Sα)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl) ethoxy]phenyl} -6-(5-fluorofuran2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)- 1H-pyrazol-5-yl]methoxy}phenyl)propionic acid or an addition salt with a pharmaceutically acceptable acid or base, and (v) (2R)-2-{[(5Sa)-5-{3-chloro-2-methyl -4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-b]pyrimidin-4-yl]oxy}-3-(2-{ [2-(2-Methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid or an addition salt thereof with a pharmaceutically acceptable acid or base, for simultaneous, sequential or separate use. 2. The combination of claim 1 wherein the BCL-2 inhibitor is N-(4-hydroxyphenyl)-3-{6-[((3S)-3(4-morpholinylmethyl)-3,4-dihydro-2(1 H)-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl}-N-phenyl5,6,7,8-tetrahydro-1-indolizine carboxamide. 3. Combination according to claim 1, wherein the BCL-2 inhibitor is 5-(5-chloro-2-{[(3S)-3-(morpholin-4ylmethyl)-3,4-dihydroisoquinolin-2(1H)-yl ]carbonyl}phenyl)-N-(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)N-(4-hydroxyphenyl)-1,2-dimethyl-1H-pyrrole-3-carboxamide. 4. The combination according to claim 2, where N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro2(1H)-isoquinolinyl)carbonyl]- 1,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-1-indolizine carboxamide is present in the form of the hydrochloride salt. 5. Combination according to claim 3, wherein 5-(5-chloro-2-{[(3S)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin2(1H)-yl]carbonyl}phenyl) -N-(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl1H-pyrrole-3-carboxamide is present in the form of the hydrochloride salt. 6. The combination of claim 1 wherein the BCL-2 inhibitor is ABT-199. 7. Combination according to claim 1, wherein the MCL1 inhibitor is (2R)-2-{[(3S _a )-5-{3-chloro-2-methyl-4[2-(4-methylpiperazin-1-yl) ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2{[1-(2,2,2-trifluoroethyl) -1H-pyrazol-5-yl]methoxy}phenyl)propionic acid. 8. Combination according to claim 1, wherein the MCL1 inhibitor is (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4[2-(4-methylpiperazin-1-yl) ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2(2-methoxyphenyl)-pyrimidin-4-yl]methoxy }phenyl)propionic acid. 9. Combination according to claim 1, where The BCL-2 inhibitor is 5-(5-chloro-2-{[(3S)-3-(morpholin-4-ylmethyl)-3,4dihydroisoquinolin-2(1H)-yl]carbonyl}phenyl)-N-( 5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-1H-pyrrol-3-carboxamide and The MCL1 inhibitor is (2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1yl)ethoxy]phenyl}-6-(4-fluorophenyl )thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid. 10. Combination according to claim 1, where BCL-2 inhibitor is ABT-199 and The MCL1 inhibitor is (2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1yl)ethoxy]phenyl}-6-(4-fluorophenyl )thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propionic acid. 11. Combination according to any one of claims 1 to 10, which additionally contains one or more fillers. 12. The use of a combination according to any one of claims 1 to 10 for the preparation of a medicament for the treatment of cancer. 13. Use according to claim 12, wherein the malignancy is leukemia. 14. Use according to claim 13 wherein the leukemia is acute myeloid leukemia, T-ALL or BALL. 15. The use of claim 12 wherein the malignancy is a myelodysplastic syndrome or a myeloproliferative disorder. 16. Use according to claim 12, wherein the malignancy is lymphoma. 17. Use according to claim 16 wherein the lymphoma is non-Hodgkin's lymphoma. 18. Use according to claim 17, wherein the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma or mantle cell lymphoma. 19. Use according to claim 12, wherein the malignancy is multiple myeloma. 20. Use according to claim 12, wherein the malignancy is a neuroblastoma. 21. Use according to claim 12, wherein the malignancy is small cell lung cancer. 22. Medicinal product containing separately or together: (a) a BCL-2 inhibitor as defined in claim 1, and (b) an MCL1 inhibitor as defined in claim 1, for simultaneous, sequential or separate administration, and wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in effective amounts for the treatment of malignant neoplasm. 23. The use of a therapeutically effective amount of (a) a BCL-2 inhibitor as defined in claim 1 and (b) an MCL1 inhibitor as defined in claim 1 for the preparation of a medicament for the treatment of cancer. 24. The use of a synergistically effective amount of (a) a BCL-2 inhibitor as defined in claim 1 and (b) an MCL1 inhibitor as defined in claim 1 for the preparation of a medicament for the treatment of cancer. 25. Use according to claim 24, wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in a synergistic manner - 32 039621 effective amounts that are able to reduce the dose required for each compound for the treatment of malignant neoplasm, while providing effective treatment of malignant neoplasm, with a decrease in the end side effects. 26. Use according to claim 23, wherein the BCL-2 inhibitor is N-(4-hydroxyphenyl)-3-{6-[((3S)3-(4-morpholinylmethyl)-3,4-dihydro-2(1 H)isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl}-N-phenyl5,6,7,8-tetrahydro-1-indolizine carboxamide, moreover, the BCL-2 inhibitor is used in the combined treatment at a dose of 50 to 1500 mg. 27. Use according to claim 23, wherein the BCL-2 inhibitor is administered once a week. 28. Use according to claim 23 wherein the BCL-2 inhibitor is N-(4-hydroxyphenyl)-3-{6-[((3S)3-(4-morpholinylmethyl)-3,4-dihydro-2(1 H)-isoquinolinyl)carbonyl]-1,3-benzodioxol-5-yl}-N-phenyl5,6,7,8-tetrahydro-1-indolizine carboxamide, wherein the BCL-2 inhibitor is administered once a day in the combined treatment. 29. Use according to claim 23, wherein the BCL-2 inhibitor and the MCL1 inhibitor are orally administered. 30. Use according to claim 23, wherein the BCL-2 inhibitor is administered orally and the MCL1 inhibitor is administered intravenously. 31. Use according to claim 23, wherein the BCL-2 inhibitor and the MCL1 inhibitor are administered intravenously.