EA040162B1 - COMBINATION OF MCL-1 INHIBITOR AND TAXANE COMPOUND, THEIR APPLICATIONS AND PHARMACEUTICAL COMPOSITIONS - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to a combination of an MCL-1 inhibitor and a taxane compound. The invention also relates to the use of said combination for the treatment of cancer, especially breast, lung, ovarian, bladder and prostate cancers, more preferably breast and lung cancers. Pharmaceutical formulations suitable for administering such combinations are also provided.

Prerequisites for the creation of the invention

A malignant neoplasm is characterized by uncontrolled cell proliferation. Antimitotic agents and antimicrotubulin agents are being investigated for cancer therapy due to their important effect on cell division. Inhibition of the mitotic mechanism leads to various outcomes, mainly leading to cell cycle arrest and cell death. Antimicrotubulin agents such as taxanes are currently used in clinical protocols. For example, paclitaxel and docetaxel have a similar spectrum of clinical activity, including malignant neoplasms of the ovaries, lung, breast, bladder, and prostate. Taxanes are anti-mitotic agents that bind to tubulin and inhibit microtubule depolymerization, thus disrupting the normal balance involved in microtubule assembly and disassembly and hence disrupting microtubule function. Microtubules are extremely important for cell division, and cells exposed to taxanes may be unable to divide. Cell cycle arrest after taxane treatment may eventually lead to cell death due to failed mitosis. Despite the benefits in cancer treatment, there is a long unmet need for more effective therapies with limited toxicities. In fact, the toxicities associated with paclitaxel and docetaxel include neutropenia as the main do-limiting toxic effect, as well as significant peripheral neuropathy. In fact, dose reductions often occur in severe previously treated patients to mitigate the severity of these toxicities. In addition, the development of resistance to taxanes also limits their clinical use.

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, MCL-1 and BCL-2A1; and (III) pro-apoptotic effector proteins BAX and BAK (Czabotar et al., Nature Reviews Molecular Cell Biology 2014, 15, 49-63). Overexpression of anti-apoptotic members of the BCL-2 family has been observed in many cancers (Adams and Cory, Oncogene 2007, 26, 1324-1337) and pharmacological inhibition of anti-apoptotic proteins by BH3-mimetic drugs such as ABT-199 (venetoclax), ABT- 263 (navitoclax) and S63845 are considered as a therapeutic strategy to induce apoptosis and induce tumor regression in malignancy (Zhang et al., Drug Resist. Updat. 2007, 10, 207-217; Kotschy et al., Nature 2016, 538, 477-482). However, mechanisms of resistance to BH3 mimetics have been observed (Choudhary et al., Cell Death and Disease 2015, 6, e1593) and combination therapies may improve efficacy and slow or even halt the development of resistance.

Breast cancer is a heterogeneous disease and can be divided into at least five major subgroups based on gene expression profile: Luminal A, Luminal B (estrogen positive, ER+), HEK2-amplified, basal-like (predominantly triple negative breast cancer or TNBC) and normal-like (Curtis et al., Nature 2012, 486, 346-352; Perou et al., Nature 2000, 406, 747-752). These subtypes typically predict clinical behavior with respect to response and resistance to therapy, the nature of metastases, and overall survival. Various mechanisms contribute to tumor progression and resistance to cancer therapy, including evasion of cell death due to dysregulation of the balance between anti- and pro-apoptotic members of the BCL2 family (Merino et al., Oncogene 2016, 35, 1877-1887; Beroukim et al., Nature 2010, 463, 899-905; Wertz et al., Nature 2011, 471, 110-114; Goodwin et al., Cell Death Differ. 2015). In the triple-negative breast cancer subset, remaining disease after neoadjuvant therapy is associated with a higher risk of metastatic recurrence compared with patients who achieve an abnormal complete response (Liedtke et al., J. Clin. Oncol. 2008, 26(8) , 1275-1281). Approximately 70% of patients with TNBC do not achieve a complete response after neoadjuvant chemotherapy (such as paclitaxel or docetaxel) and suffer from a significantly poorer outcome, with a greater likelihood of metastatic recurrence and a 3-year overall survival of only 60-70%.

Lung cancer is classified into non-small cell lung cancer (NSCLC), small cell lung cancer

- 1 040162 ci (SCLC) and carcinoid. Approximately 80% of all lung cancers correspond to NSCLC, which can be further classified into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Lung cancer is the most common cause of cancer-related death in men and the second most common cause in women after breast cancer. Although lung cancer therapy has improved significantly since the advent of targeted therapies, there is still an urgent need to develop new therapies to improve treatment efficacy, reduce side effects, and avoid resistance (Rothschild Cancers 2015, 7(2), 930-949).

The present invention provides a novel combination of an MCL-1 inhibitor and a taxane compound. The results show that the association of effective small molecules targeting MCL1 with antimicrotubulin agents is extremely synergistic on breast and lung cancer cell lines (FIGS. 1-7; Tables 1 and 4). We have also shown that the combined disruption of microtubule function and MCL-1 targeting in vivo is effective at tolerated doses in the PDX breast cancer model (Patient Derived Xenograft) (Figs. 8 and 9) and in various doses in female athymic rats bearing MDA-MB-231 xenograft, triple negative breast cancer model (FIGS. 10-14). A positive combination was also observed in various PDX models of TNBC using various administration schedules (FIG. 15). A synergistic effect of targeted MCL-1 and disruption of microtubule function in vitro and in vivo at tolerated doses has been shown with a combination of effective small molecule inhibitors. In conclusion, residual tumor analysis after neoadjuvant treatment is a major and poorly understood area for chemoresistance research and thus we have performed experiments in a chemotherapy resistant PDX model indicating that the combination of an MCL-1 inhibitor and a taxane compound is effective. (example 9).

The essence of the invention

The invention relates to a combination for the treatment of cancer, containing: (a) MCL-1 inhibitor, which is (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-(2,2,2trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propanoic acid or (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)propanoic acid, or addition salts thereof with a pharmaceutically acceptable acid or base, and (b) a taxane compound which is paclitaxel or docetaxel, for simultaneous, sequential or separate use.

Said compounds, their synthesis, their uses in the treatment of cancer and their pharmaceutical preparations are described in WO 2015/097123, WO 2016/207216, WO 2016/207217, WO 2016/207225, WO 2016/207226 and WO 2017/125224, content which are incorporated by reference.

In certain embodiments, the taxane compound is selected from paclitaxel, docetaxel, cabazitaxel, tezetaxel, Opaxio® (paclitaxel polyglumex), Abraxane® (nab-paclitaxel) larotaxel, taxoprexin, BMS-184476, hongdoushan A, hongdoushan B, and hongdoushan C and others.

In another embodiment, the invention provides a combination comprising:

(a) compound 1: (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)propanoic acid, or a pharmaceutically acceptable salt thereof, and (b) a taxane compound selected from paclitaxel or docetaxel, for simultaneous, sequential or separate use.

Alternatively, the invention provides a combination comprising:

(a) 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-pyrazole-5 -yl]methoxy}phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof, and (b) a taxane compound selected from paclitaxel or docetaxel, for simultaneous, sequential or separate use.

In another embodiment, the invention provides a combination as described herein for use in the treatment of breast cancer or lung cancer.

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

In another embodiment, the invention provides a medicinal product containing, separately or together:

(a) an MCL-1 inhibitor as described herein, and (b) a taxane compound as described herein, or (a) an MCL-1 inhibitor as described herein, and

- 2 040162 (b) a taxane compound as described herein for simultaneous, sequential or separate administration, and wherein the MCL-1 inhibitor and the taxane compound are provided in effective amounts for the treatment of breast cancer or lung cancer.

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

(a) an MCL-1 inhibitor of formula (I) as described herein, and (b) a taxane compound as described herein, or (a) an MCL-1 inhibitor of formula (II) as described herein, and (b) a taxane compound as described herein to a subject in need thereof.

In another embodiment, the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3-chloro2-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)propanoic acid (compound 1).

In another embodiment, the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3-chloro2-methyl-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)propanoic acid (compound 2).

In another embodiment, the taxane compound is paclitaxel.

In another embodiment, the taxane compound is docetaxel.

Brief description of the figures

In FIG. 1 illustrates matrices for inhibition, Loewe excess inhibition and growth inhibition for combinations of paclitaxel with Compound 1 in a representative breast cancer cell line MDA-MB-453;

in fig. 2 Matrices for inhibition, Loewe excess inhibition and growth inhibition for combinations of paclitaxel with Compound 1 on a representative breast cancer cell line MDA-MB-468;

in fig. 3 shows matrices for inhibition, Loewe excess inhibition, and growth inhibition for combinations of paclitaxel with Compound 1 in a representative lung cancer H522 cell line;

in fig. 4 is a typical cell growth inhibition effect and synergistic combination matrices for cell growth inhibition (left) and Loewe excess inhibition (right) provided by Compound 2 in combination with paclitaxel on the MDA-MB-453 breast cancer cell line in two independent experiments. Values in the effect matrix range from 0 (no inhibition) to 100 (complete inhibition). The values in the synergism matrix represent the degree of growth inhibition in excess of the theoretical additivity calculated from the activity of the individual agents of compound 2 and paclitaxel at the concentrations tested;

in fig. 5 is a typical cell growth inhibitory effect and synergistic combination matrices for cell growth inhibition (left) and Loewe excess inhibition (right) provided by Compound 2 in combination with paclitaxel on the H522 lung cancer cell line in two independent experiments. Values in the effect matrix range from 0 (no inhibition) to 100 (complete inhibition). The values in the synergism matrix represent the degree of growth inhibition in excess of the theoretical additivity calculated from the activity of the individual agents of compound 2 and paclitaxel at the concentrations tested;

in fig. 6 - synergistic effect of compound 2 with docetaxel. SK-BR-3 cells were treated with docetaxel (2 nM), or left untreated in the presence of compound 2 (30 nM), with or without QVD (10 μM) for 72 h, after which viability was analyzed by propidium iodide (PI) staining. Results are presented as percentage of untreated cells and represent 3-5 independent experiments;

in fig. 7 - synergistic effect of compound 2 with docetaxel. SK-BR-3 cells were treated with increasing concentrations of compound 2 and docetaxel for 72 h, after which viability was analyzed using Cell Titer Glo followed by BLISS analysis. BLISS synergy values are >0.0 on the vertical axis;

in fig. 8 - Efficacy of compound 2 with docetaxel in improving animal survival in the TNBC PDX model. Kaplan-Meier survival curves of mice carrying 110T (n=10-12 mice/arm), 838T (n=12 mice/arm), or PDX OD-BRE-0589 (8 mice/arm) treated with vehicle alone (black line), docetaxel (10 mg/kg ip on days 0 and 21) plus vehicle for compound 2 (dark gray line), compound 2 (25 mg/kg iv once a week for 6 weeks) plus vehicle for docetaxel (light gray line), or combined docetaxel and Compound 2 (dashed line). Right panels: Individual tumor volume curves. The PDX 838T model was terminated after 120 days due to age-related disease, regardless of treatment. Log-rank (Mantel-Cox) p value is presented for combination therapy relative to docetaxel alone;

in fig. 9 - maintaining a normal body weight during the implementation of therapy. Knockout mice were treated with NOD SCID IL2 γ receptor using docetaxel (15 mg/kg, once ip) and compound 2 at 25 or 50 mg/kg (3 mice per group, iv injection, one once a week for 3 weeks). Their body weight was monitored three times a week for 3 weeks. Therapy with Compound 2 alone or in combination with docetaxel was well tolerated;

in fig. 10 Efficacy of paclitaxel and Compound 1 (designated Compound A in the figure) alone and in combination on female nude rats bearing MDA-MB-231 xenografts, TNBC model. Tumors were inoculated into female nude rats by subcutaneous inoculation of human TNBC MDA-MB-231 cells (1x107 cells/200 μl HBSS/Matrigel 1:1 v/v). Animals with suitable tumor sizes were randomized into groups (n=7-8) with a mean tumor volume of approximately 400 mm ³ . Tumor volumes were assessed using the two largest diameters according to (LxW ² xn/6) and body weight was measured 2-3 times a week. Data are presented as mean±SPS or as individual tumor volumes. * p < 0.05, compared with the vehicle group (one-way ANOVA with Dunnett's post hoc test);

in fig. 11 - tolerability of paclitaxel and compound 1 (designated compound A in the figure) alone and in combination in female nude rats bearing MDA-MB-231 xenografts, TNBC model. Tumors were inoculated into female nude rats by subcutaneous inoculation of human TNBC MDA-MB-231 cells (1x10 ⁷ cells/200 μl HBSS/Matrigel 1:1 v/v). Animals with suitable tumor sizes were randomized into groups (n=7-8) with a mean tumor volume of approximately 400 mm ³ . Tumor volumes were assessed using the two largest diameters according to (LxWxn/6) and body weight was measured 2-3 times a week;

in fig. 12 Efficacy of paclitaxel and Compound 1 (designated Compound A in the figure) alone and in combination in various dosing schedules in female nude rats bearing MDA-MB-231 xenografts, TNBC model. Tumors were inoculated into female nude rats by subcutaneous inoculation of human TNBC MDA-MB-231 cells (1x10 ⁷ cells/200 μl HBSS/Matrigel 1:1 v/v). Animals with suitable tumor sizes were randomized into groups (n=7-8) with a mean tumor volume of approximately 400 mm ³ . Tumor volumes were assessed using the two largest diameters according to (LxWxn/6) and body weight was measured 2-3 times a week. Data are presented as mean±SPS or as individual tumor volumes. * p < 0.05, compared with the vehicle group (one-way ANOVA with Dunnett's post hoc test);

in fig. 13 - Tolerance of paclitaxel and Compound 1 (designated Compound A in the figure) alone and in combination in various dosing schedules in female nude rats bearing MDA-MB-231 xenografts, TNBC model. Tumors were inoculated into female nude rats by subcutaneous inoculation of human TNBC MDA-MB-231 cells (1x10 ⁷ cells/200 μl HBSS/Matrigel 1:1 v/v). Animals with suitable tumor sizes were randomized into groups (n=7-8) with a mean tumor volume of approximately 400 mm ³ . Tumor volumes were assessed using the two largest diameters according to (LxW ² xn/6) and body weight was measured 2-3 times a week;

in fig. 14 - synergistic effect of compound 1 with docetaxel;

in fig. 15 Antitumor activity of docetaxel and Compound 1 administered iv alone and in combination to female SCID mice carrying a patient-derived TNBC model. Docetaxel was administered first, followed by Compound 1 either 30 minutes or 72 hours later.

Detailed description of the invention

Thus, the invention provides in Embodiment E1 a combination for the treatment of cancer comprising: (a) an MCL-1 inhibitor which is (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)propanoic acid or (2R)-2-{[(5Sa)-5-{3- chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-( 2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid, or addition salts thereof with a pharmaceutically acceptable acid or base, and (b) a taxane compound which is paclitaxel or docetaxel, for simultaneous, sequential or separate application.

Further 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 specific features to provide further embodiments of the present invention.

E2. Combination according to E1 wherein the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy ]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)-1H -pyrazol-5-yl]methoxy}phenyl)propanoic acid.

E3. Combination according to E1 wherein the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy ]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl) propanoic acid.

E4. A combination according to E1 wherein the taxane compound is paclitaxel.

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E5. A combination according to E1 wherein the taxane compound is docetaxel.

E6. Combination according to E1 wherein the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy ]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl) propanoic acid; and the taxane compound is paclitaxel.

E7. Combination according to E1 wherein the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy ]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin4-yl]oxy}-3-(2-{[1 -(2,2,2-trifluoroethyl)-1H -pyrazol-5-yl]methoxy}phenyl)propanoic acid; and the taxane compound is paclitaxel.

E8. Combination according to E1 wherein the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy ]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl) propanoic acid; and the taxane compound is docetaxel.

E9. Combination according to E1 wherein the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy ]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)-1H -pyrazol-5-yl]methoxy}phenyl)propanoic acid; and the taxane compound is docetaxel.

E10. Combination according to E3, where the dose is (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4-[2-(4methylpiperazin-1-yl)ethoxy]phenyl}-6 -(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid during combined treatment ranges from 25 mg to 1500 mg.

E11. Combination according to E3 or E10, where (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4-[2-(4methylpiperazin-1-yl)ethoxy]phenyl}- 6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid is introduced into time of combined treatment once a week.

E12. A combination according to any of E1-E11 wherein the MCL-1 inhibitor is administered orally and the taxane compound is administered intravenously.

E13. A combination according to any of E1-E11 wherein the MCL-1 inhibitor and the taxane compound are administered intravenously.

E14. A combination according to any of E1-E13, for use in the treatment of breast cancer or lung cancer.

E15. A combination for use according to E14 wherein the MCL-1 inhibitor and the taxane compound are provided in amounts which are therapeutically effective together for the treatment of breast cancer or lung cancer.

E16. A combination for use according to E14 wherein the MCL-1 inhibitor and the taxane compound are provided in amounts that are synergistically effective in the treatment of breast cancer or lung cancer.

E17. Combination for use according to E14 wherein the MCL-1 inhibitor and the taxane compound are provided in synergistically effective amounts, allowing for a reduction in the dose required of each compound for the treatment of breast or lung cancer while still providing an effective treatment for cancer with a significant reduction in side effects.

E18. A combination for use according to any of E14-E17 wherein the malignant neoplasm is breast cancer.

E19. Combination for use according to E18, wherein the malignancy is triple negative breast cancer, especially chemoresistant triple negative breast cancer, more preferably triple negative breast cancer resistant to taxane therapy.

E20. A combination for use according to any of E14-E17 wherein the cancer is lung cancer, especially non-small cell lung cancer or small cell lung cancer.

E21. A combination according to any of E1-E13, additionally containing one or more excipients.

E22. Pharmaceutical compositions containing a combination according to any of E1-E13, in combination with one or more pharmaceutically acceptable excipients.

E23. Pharmaceutical compositions according to E22, for use in the treatment of breast cancer or lung cancer.

E24. Pharmaceutical compositions for use according to E23, wherein the malignant neoplasm is breast cancer.

E25. Pharmaceutical compositions for use according to E24 wherein the cancer is triple negative breast cancer, especially chemoresistant triple negative breast cancer, more preferably taxane-resistant triple negative breast cancer.

E26. Pharmaceutical compositions for use according to E23 wherein the cancer is lung cancer, especially non-small cell lung cancer or small cell lung cancer.

E27. The use of a combination according to any of E1-E13 for the preparation of a medicament for the treatment of breast cancer or lung cancer.

E28. Use according to E27, wherein the malignant neoplasm is breast cancer.

E29. Use according to E28, wherein the malignancy is triple negative breast cancer, especially chemoresistant triple negative breast cancer, more preferably triple negative breast cancer resistant to taxane therapy.

E30. Use according to E27, wherein the malignancy is lung cancer, in particular non-small cell lung cancer or small cell lung cancer.

E31. Medicinal product containing, separately or together:

(a) an MCL-1 inhibitor as defined in E1 and (b) a taxane compound as defined in E1 for simultaneous, sequential or separate administration, and wherein the MCL-1 inhibitor and the taxane compound are provided in effective amounts for the treatment of cancer .

E32. A method for treating a malignant neoplasm, comprising administering a jointly therapeutically effective amount of:

(a) an MCL-1 inhibitor as defined in E1, and (b) a taxane compound as defined in E1, to a subject in need thereof.

E33. A method of sensitizing a patient that (I) is refractory to at least one chemotherapy treatment, or (II) has relapsed after treatment with chemotherapy, or in both cases (I) and (II), wherein the method comprises administering a jointly therapeutically effective amount of MCL -1 inhibitor, as defined in E1, in combination with a taxane compound, as described in this application, the specified patient.

Combination refers to either a fixed dose combination in a single single 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 therapeutic agents or co-agent) may 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 co-existence, for example, synergistic effect.

The terms co-administration or combined administration or the like, in the context of this application, encompass the administration of a selected component of the combination to a single subject in need (for example, a patient), and includes 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 components of the combination, 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 disease in which a group of cells exhibits uncontrolled growth. Types of malignancies include solid tumors, including carcinoma, sarcoma, or blastoma. In particular, malignant neoplasm refers to breast and lung cancer.

The term jointly 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.

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Synergistically effective or synergism 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 individual agent.

In the context of this application, the term to 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 development of the disease or at least one of its clinical symptoms). In another embodiment, treating, treating, or therapy refers to alleviating or alleviating at least one physical parameter, including those that may not be perceived by the patient. In still 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.

In the context of the present application, a subject is in need of treatment if such subject would benefit biologically, medically, or quality of life from such treatment.

In another aspect, there is provided a method of sensitizing a human that is (I) refractory to at least one chemotherapy treatment, or (II) has relapsed after treatment with chemotherapy, or in both cases (I) and (II), wherein the method comprises administering An MCL-1 inhibitor, in combination with a taxane compound as described herein, to a patient. A sensitized patient is a patient who responds to treatment involving the administration of an MCL-1 inhibitor of formula (I) or formula (II) in combination with a taxane compound as described herein, or who has not developed resistance to such treatment.

Drug means a pharmaceutical composition or a combination of several pharmaceutical compositions that contain one or more active ingredients in the presence of one or more excipients.

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 route, more specifically tablets, dragees, sublingual tablets, hard gelatin capsules, tablets for slow dissolution under the tongue, capsules, lozenges, injectables, aerosols, eye or nose drops, suppositories, creams, ointments, skin gels, etc.

Pharmaceutical compositions in accordance with the invention may contain one or more pharmaceutically acceptable excipients or carriers selected from diluents, lubricants, binders, disintegrants, stabilizers, preservatives, absorbents, colors, sweeteners, flavors, and others.

As a non-limiting example, you can specify:

as diluents: lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycerin, as lubricants: silicon dioxide, talc, stearic acid and its magnesium and calcium salts, polyethylene glycol, as binders: magnesium aluminosilicate, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone, as disintegrants: agar, alginic acid and its sodium salt, effervescent mixtures.

The combination compounds may be administered simultaneously or sequentially. The route of administration is preferably intravenous infusion or injection, 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.

Dosing regimens used vary depending on the sex, age and weight of the patient, route of administration, nature of the malignancy, and any concomitant treatments, and range from 25 to 1500 mg of the MCL-1 inhibitor per week, more preferably from 50 to 1400 mg per week. The dose of the taxane compound will be the same as used for its self-administration.

pharmacological data.

Example 1 Effect on in vitro proliferation of the combination of an MCL-1 inhibitor (compound 1) with paclitaxel on breast and lung cancer cell lines.

The effect on proliferation of the combination of an MCL-1 inhibitor (compound 1) with paclitaxel was evaluated in a panel of 19 breast cancer cell lines (BT-20, BT-474, BT-549, Cal-148, HCC1143, HCC1395, HCC1500, HCC1937, HCC1954, HCC38, HCC70, Hs 578T, MCF7, MDA-MB-157, MDA-MB-231, MDA-MB-436, MDA-MB-453, MDA-MB-468 and SK-BR-3) and 3 cancer cell lines lungs (H522, H23 and A549).

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Materials and methods

Compounds were dissolved in 100% DMSO (Sigma, catalog # D2438-50ML) at a stock concentration of 10 mM and stored at -20° C. until use. Compounds were dispensed into 2 ml 96 well plates (Greiner bio-one, catalog number 780271) serially diluted 3-fold. Compound 1 was used in the concentration range 0.0 - 10.0 μM. Paclitaxel was used in the concentration range of 0.0-1.0 μM in breast cancer cells and in the concentration range of 0.0-2.0 μM in lung cancer cells.

All cell lines were obtained from the American Type Culture Collection and cultured according to the manufacturer's recommendations. All lines were supplemented with 10% FBS (GIBCO, Cat. No. 10099-141). All cell lines were determined to be free of mycoplasma contamination by detection PCR assay performed at Idexx Radil (Columbia, MO, USA) and authenticated by SNP assay. Cells were thawed from frozen stocks, carried through > 1 passage and grown at 37° C. in 5% CO ₂ . Cells were expanded into T-75 flasks and their viability was assessed in a Beckman-Coulter ViCell counter prior to seeding. For digestion and propagation of cell lines, cells were detached from the flasks using 0.25% Trypsin-EDTA (Corning Costar, Cat # 25-053-CL).

Cell proliferation was measured in 72 hour CellTiter-Glo™ (CTG) assays and all results shown are at least triplicate measurements. Cells were dispersed into 96-well tissue culture-treated plates (Costar, catalog number 3904) with a final volume of 80 μl of medium and at a density of 3000 cells per well. 16-24 hours after inoculation, 20 μl of each of the serial dilutions of the compound was transferred to plates containing cells, obtaining concentrations of compounds in the above ranges and a final concentration of DMSO of 0.16%. Additionally, the day 0 plate was examined at this time using the CellTiter-Glo® fluorescent cell viability assay as described below. 72 hours after compound treatment, the effects of the compounds on cell proliferation were determined using the CellTiter-Glo™ fluorescent cell viability assay (Promega, cat. no. G7573). It is a homogeneous method for determining the number of viable cells in culture based on the amount of ATP present, which signals the presence of metabolically active cells. The method is described in more detail in Technical Bulletin, TB288 Promega. Briefly, 100 μl of CTG reagent was added to the plates and the plates were incubated for 20-30 minutes on an orbital shaker. The plates were then analyzed on a Perkin Elmer Victor™ X4 plate reader.

Percent growth inhibition, excess inhibition and growth inhibition were calculated using the Combo Module software using the Loewe synergy model (as described in Lehar et al., Nature Biotechnology 2009, 27(7), 659-66) which determines the effect on growth higher than would be expected if the two drugs behave in a dose-additive fashion. Positive numbers represent areas of increasing synergy. The percentage of growth inhibition relative to DMSO is shown in the panel labeled Inhibition. The amount of inhibition in excess of the expected amount is shown in the panel labeled Loewe Inhibition Excess. The amount of inhibition normalized to day 0 is shown in the panel labeled Growth Inhibition. Compound 1 concentrations are presented in the bottom row from left to right and increasing concentrations of paclitaxel along the leftmost column from bottom to top. All remaining points on the grids are the results for the combination of the two inhibitors, which correspond to the concentrations of the individual agents represented on the two axes. The absolute IC ₅₀ was determined by finding the compound concentration at which the calculated curve crosses the 50% activity mark. The IC ₅₀ and synergy score were calculated using the Combo module software as described in Lehar et al., 2009.

Synergy assessment

SS ~ 0 —> Additive Dose

SS >2 —► Synergy

SS >1 —► Poor synergy

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Table 1. Absolute IC ₅₀ values of the single agent for each compound and synergy score measurements for the combination of compound 1 and paclitaxel are indicated. Interactions are considered synergistic if a score > 2.0 is observed

cell line Origin Compound 1 Paclitaxel Combination Abs 1C ₅₀ (nM) Abs 1C ₅₀ (nM) Synergy score (SS) VT-20 Breast 653 5.97 7.93 VT-474 Breast 1262.5 1.7 4.7 BT-549 Breast >10000 8.82 9.01 Sa1-148 Breast 1330 4.25 13.8 NSS1143 Breast >10000 9.43 2.58 NSS1395 Breast 9915 8.03 1.52 NSS1500 Breast 257.5 >1000 6.38 NSS1937 Breast 137 I,6 9.4 NSS1954 Breast 158.5 6.5 3.69 NSS38 Breast 3385 4.33 5.19 HSS70 Breast 704 8.39 I,9 Hs 578T Breast 519.5 0.363 8.49 MCF7 Breast 1292.5 10.4 3.82 MDA-MB-157 Breast >10000 >1000 0.389 MDA-MB-231 Breast >10000 17.5 4.86 MDA-MB-436 Breast >10000 14.7 1.41 MDA-MB-453 Breast >10000 4.2 23.9 MDA-MB-468 Breast 9917.5 3.07 from SK-BR-3 Breast 301 2.66 4.52 NCI-H522 Lungs 713 0.48 14.9 NCI-H23 Lungs 160 0.22 6.97 A549 Lungs >10000 0.163 4.82

Results.

Compound 1 as the only agent inhibited the growth of 7/19 breast cancer cell lines and 2/3 lung cancer cell lines, with an IC ₅₀ below 1000 nM (Table 1).

Paclitaxel alone inhibits the growth of 17/19 breast cancer cell lines and 3/3 lung cancer cell lines, with an IC ₅₀ below 1000 nM.

In combination, treatment with Compound 1 and paclitaxel induces synergistic growth inhibition (i.e., synergism score greater than 2 (Lehar et al., 2009)) in 16/19 breast cancer cell lines and 3/3 lung cancer cell lines (Table . 1). In 11 cell lines, the synergistic effect is notable, with synergism scores above 6. Importantly, the synergism is independent of the antiproliferative effects of the individual agent, and synergistic effects are observed over a wide range of individual agent concentrations (Figures 1-3), which should be useful in vivo in terms of flexibility for levels and dosing regimens.

Example 2 In vitro effect on proliferation of the combination of an MCL-1 inhibitor (compound 2) with paclitaxel in breast and lung cancer cell lines.

The effect on proliferation of the combination of an MCL-1 inhibitor (compound 2) with paclitaxel was evaluated in a panel of 2 breast cancer cell lines (MDA-MB-453 and MDA-MB-468) and one lung cancer cell line (H522).

Materials and methods.

Cell lines were generated and maintained in basal medium supplemented with fetal bovine serum as indicated in Table 1. 2. Additionally, all culture media contained penicillin (100

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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 distributed in T-150 flasks. In all cases, cells were thawed from frozen stocks, passed through > 1 passage using appropriate dilutions, counted and assessed for viability using a CASY cell counter before seeding at 150 µl/well at the densities shown in Table 1. 2, in 96 well plates.

All cell lines were determined to be free of mycoplasmic contamination using proprietary methods.

Prepared stock solutions of compounds 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 processed in seven or eight 3.16-fold serial dilutions for each dispersed compound, either individually or in optional staggered permutations, directly in cell assay plates, as indicated in Fig. 5 and 6. The effects of single agents, as well as their checkerboard combinations, on cell viability were assessed after incubation for 3 days at 37°C/5% CO ₂ by quantifying cellular ATP levels using CellTiterGlo in an amount of 75 μl of reagent /well. Carried out at least two independent experiments, each of them was carried out in duplicate. Luminescence was quantified on a multipurpose plate reader.

The IC ₅₀ was calculated for the individual funds taken using standard four-parameter curve fitting. Potential synergistic interactions between compound combinations were assessed using a 2D inhibition excess matrix according to the Loewe additivity model and presented as Synergy Scores (Lehar et al. 2009). All calculations were performed using the Clalice™ Bioinformatics software available from the Horizon website.

The doubling time indicated in the table. 2 is the average doubling time obtained in different passages (in T-150 flasks) from thawed cells when seeded in 96-well plates.

Synergy assessment

SS ~ 0 —► Additive

SS >1 —> Weak synergy

SS >2 —► Synergy

Table 2. Identification and assay conditions for cell lines used in combined experiments

cell line Origin Wednesday %FBS Source Doubling time (hours) Number of seeded cells/well MDA-MB-453 Breast L-15 10 ATCC 41.4 15000 MDA-MB-468 Breast L-15 20 ATCC 34.3 30000 H522 Lungs RPMI 10 ATCC 67.2 30000

Table 3. IC ₅₀ values are given for compound 2 and paclitaxel alone. Compounds were incubated with cells for 3 days

cell line Compound 2 Paclitaxel Ref. CONC. [µM] 1С ₅₀ [µM] Ref. CONC. [µM] 1С ₅₀ [µM] MDA-MB-453 2.00 > 2 1.0 > 1 MDA-MB-468 2.00 > 2 1.0 0.0009 H522 2.00 0.140 0.01 0.0002

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Table 4 Synergy scores for the combination of compound 2 and paclitaxel are indicated.

Interactions are considered synergistic if a score > 2.0 is observed. Baseline compound concentrations, mean maximum inhibition, and standard deviation (sd) of synergism scores are indicated. Compounds were incubated with cells for 3 days

cell line Compound 2 Paclitaxel Combination Ref. conc. [µM] Average Max. Ing. Ref. conc. [µM] Average Max. Ing. Average Synergy scores Synergy estimation error [%] [%] (SS) (co) MDA-MB-453 2.0 21.0 1.00 37.0 16.9 0.5 MDA-MB-468 2.0 24.0 1.00 75.0 6.5 0.2 H522 2.0 92.0 0.01 61.0 3.7 0.6

Results.

Compound 2 as the only agent inhibits the growth of 1/3 of the cell lines tested, with an IC ₅₀ of 140 nM for the H522 cell line (Table 3).

Paclitaxel alone inhibited the growth of 2/3 of the tested lines, with an IC ₅₀ below 1 nM.

In combination, Compound 2 and paclitaxel treatment produced synergistic growth inhibition (ie synergism score greater than 2 (Lehar et al., 2009)) in the three cell lines tested (Table 4). In 2 cell lines, the synergistic effect is notable, with synergy scores of 6.5 and 16.9. Importantly, synergism is independent of the antiproliferative effects of a single agent, and synergistic effects are observed over a wide range of single agent concentrations (FIGS. 4 and 5), which should be useful in vivo in terms of flexibility for levels and dosing schedules.

Example 3 Synergy between an MCL-1 inhibitor and docetaxel in vitro.

The inventors investigated whether an MCL-1 inhibitor (Compound 2) would exhibit synergistic effects with agents currently used to treat TNBC. Compound 2 was combined with docetaxel in SK-BR-3 cells.

Materials and methods.

Cell lines. The SK-BR-3 breast cancer cell line was maintained in RPMI-1640 plus GlutaMAX-1 (Gibco) supplemented with 10% fetal bovine serum (FCS) and 10 μg/ml insulin. For viability testing, cells were plated at 2 x 10 ⁵ cells/ml in 96 well plates, in RPMI-1640 medium (Gibco) supplemented with 10% FCS and 10 μg/ml insulin, and treated with increasing concentrations of Compound 2.

cell viability. Cell viability was assessed using the Cell Titer Glo (Promega) luminescence assay according to the manufacturer's instructions. The broad spectrum caspase inhibitor QVD-OPh hydrate (Sigma-Aldrich) was used at a concentration of 10 μM. Displacement of propidium iodide (5 μg/ml) was analyzed by flow cytometry. To study cells in vitro to determine synergy between different drugs, the effects of combinations were determined using the independent Bliss method (Prichard et al., Antimicrobial Agents and Chemotherapy 1991, 35, 1060-5).

Results.

Docetaxel and MCL-1 inhibitors exhibit marked synergy at very low concentrations of both components. In particular, docetaxel and Compound 2 exhibit marked synergy at very low concentrations of docetaxel (2 nM) and Compound 2 (31 nM) (FIGS. 6 and 7). Caspase inhibition with the pan-caspase inhibitor QVD-OPH effectively blocks cell death, suggesting that cell death is mediated by apoptosis (FIG. 6).

Example 4 MCL-1 inhibition sensitizes tumor PDX to taxane treatment in vivo.

Since breast cancer cell lines were found to be sensitive to compound 2 in combination therapy in in vitro assays, in the next step we determined their therapeutic effect in vivo in three PDX models representing three TNBCs (110T, 838T and PDX OD- BRE-0589).

Materials and methods.

Human breast cancer tissues were obtained from consenting patients at the Royal Melbourne Hospital Tissue Bank, Victorian Cancer Biobank and Georges-Francois Leclerc Center with relevant biomedical ethics commission clearance. Ethics and Clinical Research Permission has been obtained from the Human Research Ethics Committee of the Walter and Eliza Hall Institute (WEHI) and

- 11 040162 from the Georges-Francois Leclerc Center Human Research Ethics Committee. NOD SCID IL2 gamma receptor knockout mice or SCID mice were bred and maintained according to institutional guidelines. All animal experiments have been approved by the WEHI Animal Ethics Committee and the Servier Research Institute (IdRS).

Compound 2 (25 mg/kg) or vehicle was injected intravenously weekly for six weeks. Compound 2 was dissolved in 20% (2-hydroxypropyl)-in-cyclodextrin and 25 mm hydrochloric acid. Docetaxel (10 mg/kg ip) or vehicle was prepared as previously described (Oakes et al., Proceedings of the National Academy of Sciences of the USA 2012, 109, 2766-71) and injected ip weekly for one day prior to compound 2 administration. Mice were monitored for tumor development three times a week and tumor size was measured using electronic calipers. Tumor volume was assessed by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter) ² (maximum diameter)/2.

After tumor development, mice were randomized into treatment groups. Treatment was started when the tumor volume reached 80-120 mm ³ . Randomization and measurement of tumors was performed using Study Director software (v 3.0, studylog). Mice were sacrificed at the first measurement at which the tumor volume exceeded 600 mm ³ or an acute deterioration in the health of the animal, which was not due to disease progression or drug toxicity (test mice).

Results.

Compound 2 alone is ineffective in inhibiting tumor growth. However, we observed significantly improved activity in combination with docetaxel compared to docetaxel administered alone, resulting in significantly improved animal survival in the three PDX models (FIG. 8).

These results indicate that MCL-1 inhibitors in combination with taxane compounds are likely to significantly enhance tumor response to treatment and clinical outcome.

Example 5 The MCL-1 inhibitor in combination with docetaxel is well tolerated in vivo.

NOD SCID IL2 gamma receptor knockout mice were treated with docetaxel (15 mg/kg, once ip) and compound 2 at 25 or 50 mg/kg (3 mice per group, iv injection, once a week within 3 weeks). Their body weight was monitored three times a week for 3 weeks.

Compound 2 in combination with docetaxel was well tolerated and did not induce significant body weight loss (FIG. 9).

Example 6 Efficacy of compound 1 in combination with paclitaxel in the MDA-MB-231 mammary xenograft model in nude rats.

Methods.

This study evaluated the antitumor activity and tolerability of Compound 1 in combination with paclitaxel in a triple negative breast cancer (TNBC) model, MDAMB-231, in female NTac:NIH-Whn nude rats (Taconic).

Compound 1 (free base) and paclitaxel (Sandoz) were used in these studies. Paclitaxel was diluted according to the manufacturer's instructions with sterile 5% (w/v) glucose solution to 1.5 mg/ml to administer 7.5 mg/kg in a dose volume of 5 ml/kg [final concentration of ethanol and Cremophor EL was 10 and 15%, respectively, in 5% (w/v) glucose solution]. Compound 1 was formulated in a liposomal formulation (Novartis) at 5 mg/ml for a 50 mg/kg dose in a dose volume of 10 ml/kg.

MDA-MB-231, a triple negative breast cancer cell line, was obtained from the ATCC cell bank. Cells were cultured at 37°C under 5% CO2 in air in high glucose DMEM medium (BioConcept Ltd. Amimed) supplemented with 10% FCS (BioConcept Ltd. Amimed, no. 2-01F36-I) and 4 mM L-glutamine (BioConcept Ltd. Amimed, #5-10K00-H). To obtain MDA-MB-231 xenografts, cells were harvested and resuspended in HBSS (Gibco, #14175) and mixed with Matrigel (BD Bioscience, #354234) (1:1 v/v) prior to injection of 200 µl containing 1x10 ⁷ cells subcutaneously into the right paws of animals anesthetized with isoflurane. 24 h before cell inoculation, all animals were irradiated at a dose of 5 Gy for 2 min using a γ-emitter. Tumor growth was monitored regularly after cell inoculation and animals were randomized into treatment groups (n=7-8) with a mean tumor volume of approximately 400 mm ³ .

The groups were treated with:

1) vehicle used to prepare paclitaxel, IV plus liposomal vehicle IV; or

2) 7.5 mg/kg IV bolus of paclitaxel plus IV liposomal vehicle; or

3) the vehicle used to prepare the IV paclitaxel preparation plus Compound 1 at a dose of 50 mg/kg IV; or

4) 7.5 mg/kg IV paclitaxel plus 50 mg/kg IV Compound 1.

Paclitaxel vehicle or paclitaxel was administered once weekly (QW) as a slow

- 12040162 tail vein bolus over 0.5 h or 16 h before Compound 1 vehicle or Compound 1 itself in a liposomal formulation and administered by 15 min IV infusion into the tail vein. For bolus injections and 15 minute infusions, animals were anesthetized for approximately 5 and 25 minutes, respectively, with isoflurane/O ₂ .

Tumor volumes were measured using calipers 2-3 times a week. Tumor size, in mm ³ , was calculated according to the formula:

(LxW ² xn/6), where W is the width and L is the length of the tumor. Animals were also weighed 2-3 times per week and frequently examined for signs of any side effects. Tumor data and body weight changes were analyzed statistically using GraphPad Prism 7.00 (GraphPad software). If the discrepancies in the data were normally distributed, then the data were analyzed using one-way analysis of variance with Dunnett's post hoc test to compare the treated relative to the control group. Retrospective analysis with Tukey's test was used for intergroup comparison. In other cases, the Kruskal-Wallis rank test and the Dunn retrospective test were used. If appropriate, the results are presented as mean±SPS.

As an efficiency criterion, the %T/C values after the end of the experiment were calculated according to (A tumor volume ^treated / A tumor volume ^control ) * 100

Tumor regression was calculated according to /a g treated/g treated per target & ι pa (Tumor volume/tumor volume)* 100 where Δtumor volumes are the mean tumor volume on the day of assessment minus the mean tumor volume at the start of the experiment.

Results: efficacy and tolerability.

Compound 1 50 mg/kg dosed 0.5 h or 16 h after paclitaxel vehicle [ethanol:Cremophor EL:5% (w/v) glucose (10:15:75%)] was well tolerated.

Compound 1 (50 mg/kg QW) in a liposomal formulation was not effective in the MDA-MB-231 xenograft model after QWx7 IV infusion (FIGS. 10 and 12).

Paclitaxel 7.5 mg/kg retarded tumor growth (T/C% = 34%) and differed significantly (p < 0.05) from the vehicle treated group (Figures 10 and 12).

The combination of 7.5 mg/kg IV paclitaxel plus 50 mg/kg IV Compound 1 administered at 0.5 or 16 hour intervals produced 82% and 59% regression, respectively, on day 28 post-treatment for animal survival (3/8 in both groups) (Fig. 10). At day 46 from the start of treatment, tumor regression was 92% and 81%, with animal survival of 2/8 and 3/8, respectively. Animal tumor volume in both of these combined groups was significantly different (p < 0.05) from that in animals treated alone with paclitaxel or compound 1 on days 28 and 46 (FIG. 10).

The combination of 7.5 mg/kg IV paclitaxel plus 25 mg/kg IV Compound 1 given 16 hours later induces tumor stasis (15% regression on day 28 and T/C% values were 2% on day 49 after start of treatment) in surviving (7/7) animals (Fig. 12). This dosing regimen was well tolerated based on changes in body weight and clinical signs.

The combination of 3.75 mg/kg IV paclitaxel plus 50 mg/kg IV Compound 1 given 16 hours later induces tumor stasis up to day 35 (3% regression on day 28 and T/C% values were 20% at day 49 after the start of treatment) in surviving (7/7) animals (Fig. 12). This dosing regimen was well tolerated based on changes in body weight and clinical signs (Figures 11 and 13). These data indicate that the combination of paclitaxel and Compound 1 has a marked positive effect on antitumor activity compared to either agent alone.

Example 7 MCL-1 inhibition sensitizes tumor PDX to taxane treatment in vivo.

We investigated the in vivo therapeutic effects of Compound 1 in combination therapy, in the TNBC 110T PDX model.

Materials and methods.

Human breast cancer tissues were obtained from consenting patients at the Royal Melbourne Hospital Tissue Bank, Victorian Cancer Biobank, and Georges-Francois Leclerc Center by relevant Biomedical Ethics Commission approval. Ethics and clinical trial clearance was obtained from the Walter and Eliza Hall Institute (WEHI) Human Research Ethics Committee and the Georges-Francois Leclerc Center Human Research Ethics Committee. NOD SCID IL2 γ receptor knockout mice or SCID mice were bred and maintained according to institutional guidelines. All animal experiments have been approved by the WEHI Animal Ethics Committee and the Servier Research Institute (IdRS).

A group of 40 female NSG mice were inoculated with thawed suspensions of single cells and early passage human mammary tumors (TNBC PDX110). Briefly, 100,000 cells were resuspended in 10 µl of transplant buffer (50% fetal bovine serum, 10% 0.04% trypan blue solution, and 40% PBS) and Matrigel with reduced growth factor [BD] at

- 13 040162 at a ratio of 3:1, and injected into purified mammary fat pads in female mice

NOD-SCID-IL2Ry _c ' ^/_ at 3 or 4 weeks of age. Mice were monitored for tumor development three times per week and tumor size was measured using electronic calipers. Tumor volume was assessed by measuring the minimum and maximum tumor diameters using the formula:

(minimum diameter) ² (maximum diameter)/2.

After tumors reached 60-110 mm ^{3 ,} mice were randomized into treatment groups and treated. Docetaxel or its excipient was prepared by dissolving the stock solution (20 mg/ml) with PBS and injected ip every 21 days for two cycles of treatment. The duration of therapy is indicated by a vertical bar. Compound 1 was dissolved in 20% (2hydroxypropyl)-β-cyclodextrin and 25 mM hydrochloric acid. Compound 1 (15 mg/kg) or its excipient was injected intravenously twice a week for six weeks. Mice were sacrificed at the first measurement at which tumor volume exceeded 600 mm ³ , or if their health deteriorated rapidly due to reasons other than disease progression or drug toxicity (censored event), n=9-10 mice per treatment group.

Results.

Compound 1 alone is ineffective in inhibiting tumor growth. However, we observed significantly improved activity in combination with docetaxel compared to docetaxel administered alone, resulting in significantly improved animal survival in the PDX model (FIG. 14).

These results indicate that MCL-1 inhibitors in combination with taxane compounds are likely to significantly enhance tumor response to treatment and clinical outcome.

Example 8 Antitumor activity of docetaxel and compound 1 in female SCID mice carrying a patient-derived TNBC model.

This study evaluated the antitumor activity of Compound 1 in combination with docetaxel in TNBC PDX model OD-BRE-00589 in female SCID mice.

Methods.

Docetaxel was formulated in 5% ethanol, 5% PS80 and 90% glucose at a concentration of 0.67 mg/ml for administration of 10 mg/kg. Compound 1 was formulated in a liposomal formulation (Novartis) at a concentration of 7.5 mg/ml for administration of 70 mg/kg.

OD-BRE-00589 is a triple negative PDX breast cancer derived from the IMODI consortium. Consenting patients were obtained from the Georges-Francois Leclerc Center Human Research Ethics Committee. They were grafted onto SCID mice as 27 mm ³ fragments.

Tumor growth was monitored regularly after fragment grafting and animals were randomized 11 days post grafting to treated groups (n=8) with a mean tumor volume of approximately 200 mm ³ . The control group was not treated, while the other groups were treated with:

1) 70 mg/kg Compound 1 IV, or

2) 10 mg/kg IV docetaxel, or

3) 10 mg/kg IV docetaxel followed 30 minutes later by 70 mg/kg Compound 1, or

4) 10 mg/kg IV docetaxel followed 72 hours later by 70 mg/kg Compound 1.

The introduction was carried out once in the tail vein.

Tumor volumes were measured using calipers 2-3 times a week. Tumor volume was calculated using the formula:

lengths width ^2/2 .

Animals were also weighed 2-3 times per week and frequently examined for signs of any side effects.

results

Compound 1 alone is ineffective in inhibiting tumor growth. However, we observed significantly improved activity in combination with docetaxel compared to docetaxel administered alone, resulting in a significant improvement in antitumor activity in the PDX model (FIG. 15).

These results indicate that MCL-1 inhibitors in combination with taxane compounds are likely to significantly enhance tumor response to treatment and clinical outcome.

Example 9 Efficacy studies of compound 1 in combination with paclitaxel in a docetaxel resistant PDX model.

Methods.

Tested PDX models, the resistance of which to docetaxel was confirmed in vivo. Each treatment group included 5 female Swiss Nude mice aged 6 to 8 weeks. Treatment was started when the xenografts reached an average tumor volume of ~120-150 mm ³ . Thereafter, the groups were randomly exposed to various treatments. The number of inoculated animals depended on the homogeneity of tumor growth. Prepared in a liposomal formulation, Compound 1

- 14 040162 was administered intravenously at a dose of 70 mg/kg once a week. The drug must be prepared extemporaneously.

Paclitaxel diluted in 0.9% NaCl was administered intravenously at a dose of 25 mg/kg. Paclitaxel was administered QW 16 hours prior to the administration of compound 1. Administration schedules for various . treatments are defined as below

Group Dose (mg/kg) Scheme Control (filler) - - Compound 1 70 in / in, Q7D, 4n: D1-D8-D15-D22 Paclitaxel 25 in/b, Q7D, 4n: D1-D8-D15-D22 Compound 1 + paclitaxel Compound 1: 70 Paclitaxel: 25 Q7D, 4n Paclitaxel, i.p.: D1-D8-D15-D22 Connection 1, I/O: D1-D8-D15-D22

Tumor sizes were measured twice a week and the weight of individual mice was measured once a week. Treatments were continued until the average tumor volume of the most responding group began to re-grow. Mice were observed after stopping treatment to compare relapse times between groups. Using Statview software, tumor volume and/or relative tumor volume (RTV, ratio of volume at time t divided by baseline volume on day 1 and multiplied by 100), optimal growth inhibition (RTV ratio (x 100) in the treated group, divided by RTV in controls), growth retardation (time in days required to increase 4 times the initial tumor volume of 200 mm ³ in the treated group and control group) and change in body weight.

If relapse was observed after treatment with paclitaxel, the tumors were re-exposed to the compound 1 + paclitaxel combination. This was done either by including 10 mice in a paclitaxel-treated group and then treating relapsed mice with paclitaxel alone (5 animals) or in combination (5 animals), or by using non-randomized mice from an efficacy study. If an initial response to paclitaxel was observed, then additional animals retained after randomization were included for this study.

Results.

Compound 1 alone is ineffective in inhibiting tumor growth. However, we observed a significantly improved activity in combination with paclitaxel compared to paclitaxel administered alone, resulting in a significant improvement in antitumor activity in the taxane compound resistant PDX model.

These results indicate that MCL-1 inhibitors in combination with taxane compounds are likely to significantly enhance tumor response to treatment and clinical outcome.

Claims (18)

CLAIM 1. Combination for the treatment of malignant neoplasm, containing: (a) an MCL-1 inhibitor which is (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4-[2-(4methylpiperazin-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)propanoic acid or (2R)-2-{[(5S _a )-5-{3-chloro2-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)propanoic acid, or addition salts thereof with a pharmaceutically acceptable acid or base; and (b) a taxane compound that is paclitaxel or docetaxel, for simultaneous, sequential or separate use. 2. The combination of claim 1 wherein the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4[2-(4-methylpiperazine-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)propanoic acid. 3. The combination of claim 1 wherein the MCL-1 inhibitor is (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4[2-(4-methylpiperazine-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)propanoic acid. 4. Combination according to claim 1, additionally containing one or more fillers. 5. The use of a combination according to claim 1 for the treatment of breast cancer or lung cancer. 6. Use according to claim 5, wherein the MCL-1 inhibitor and the taxane compound are provided in amounts that are therapeutically effective together for the treatment of breast cancer or lung cancer. 7. Use according to claim 5, wherein the MCL-1 inhibitor and the taxane compound are provided in amounts that are synergistically effective in the treatment of breast cancer or lung cancer. 8. Use according to claim 5, wherein the MCL-1 inhibitor and the taxane compound are provided in synergistically effective amounts, which makes it possible to reduce the dose required for each - 15 040162 of this compound for the treatment of breast cancer or lung cancer, while at the same time providing an effective treatment of malignancy, with a reduction in side effects. 9. A pharmaceutical composition containing a combination according to any one of claims 1 to 4, in combination with one or more pharmaceutically acceptable excipients. 10. The use of a pharmaceutical composition according to claim 9 for the treatment of breast cancer or lung cancer. 11. The use of a combination according to claim 1 for the preparation of a medicament for the treatment of breast cancer or lung cancer. 12. Medicinal product containing, separately or together: (a) an MCL-1 inhibitor as defined in claim 1; and (b) a taxane compound as defined in claim 1 for simultaneous, sequential or separate administration, and wherein the MCL-1 inhibitor and the taxane compound are provided in effective amounts for the treatment of breast cancer or lung cancer. 13. A method for the treatment of breast cancer or lung cancer, including the introduction of a jointly therapeutically effective amount of: (a) an MCL-1 inhibitor as defined in claim 1; and (b) a taxane compound as defined in claim 1 to a subject in need thereof. 14. The method of claim 13, wherein the dose of the MCL-1 inhibitor during combination treatment is between 25 and 1500 mg. 15. The method of claim 13 wherein the MCL-1 inhibitor is administered during the combination treatment once a week. 16. The method of claim 13, wherein the MCL-1 inhibitor and the taxane compound are administered intravenously. 17. The method of claim 13 wherein the MCL-1 inhibitor is administered orally and the taxane compound is administered intravenously. 18. A method of sensitizing a patient that (I) is refractory to at least one chemotherapy treatment, or (II) has relapsed after chemotherapy treatment, or in both (I) and (II), wherein the method comprises administering a co-therapeutically effective quantities: (a) an MCL-1 inhibitor as defined in claim 1; and (b) a taxane compound as defined in claim 1 to the patient.