The combination of TRX-e-002-1 with a BCL-2 inhibitor or a hypomethylating agent in the treatment of acute myeloid leukemia

Abstract

(3R, 4S)-3-(4-hydroxy-3,5-dimethoxyphenyl)-4-(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2H- chromen-7-ol (TRX-E-002-1) for use in a method of treatment for a hematological cancer being acute myeloid leukemia or multiple myeloma in a subject in need thereof, the method comprising administering to the subject an effective amount of TRX-E-002-1. The method may further comprise administering to the subject an effective amount of a second compound selected from the group consisting of (i) a BCL-2 inhibitor such as venetoclax, navitoclax or obatoclax, (ii) a deoxycytidine analogue chemotherapeutic such as cytarabine or gemcitabine, (iii) a hypomethylating agent such as azacitidine or decitabine, (iv) an anthracycline chemotherapeutic such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin or mitoxantrone, (v) a proteasome inhibitor such as carfilzomib, bortezomib, and ixazomib, (vi) an immunomodulatory drug such as pomalidomide, lenalidomide, and thalidomide and(vii) a corticosteroid drug such as dexamethasone or prednisone, and (viii) an alkylator such as bendamustine, cyclophosphamide, melphalan, and melflufen. Corresponding combination pharmaceutical compositions.

Description

[0001] SYNERGISTIC TRX-E-002-1 CANCER THERAPIES

[0002] TECHNICAL FIELD

[0003] The present invention relates to the treatment of cancer using TRX-E-002-1, particularly in a combination therapy with one or more additional anti-cancer agents having synergistic effect with TRX-E-002-1.

[0004] BACKGROUND TO THE INVENTION

[0005] Cancer remains a formidable health challenge globally and a leading cause of death worldwide. One of the most significant challenges in combating cancer is its complexity. There are hundreds of different types of cancer, each with its own unique characteristics and behaviors. This diversity makes it difficult to develop a one-size-fits-all approach to treatment and prevention.

[0006] Combination therapies for cancer represent a promising frontier in oncology. In principle, combination therapies can be tailored to the specific characteristics of a patient's cancer (or at least the specific subtype of cancer), allowing for a more personalized treatment plan. This tailored approach can lead to better outcomes and fewer side effects, as treatments can be chosen to complement each other and minimize toxicity. However, there are challenges to be addressed, including the need to discover the optimal combinations, especially for relapsing / refractory forms of cancer.

[0007] TRX-E-002-1 is a third generation benzopyran designed to target a broad spectrum of cancer cells, including those resistant to chemotherapy, which are often responsible for disease relapse (W02015 / 117202; Stevenson et al. Scientific Reports (2018) 8:5144 DOI:10.1038 / s41598-018-22882-w). TRX-E-002-1 has shown promise in preclinical studies and has completed a Phase I clinical trial in ovarian cancer, establishing clinical proof of concept (Saif et al. Cancer Chemother Pharmacol. 2017 Feb;79(2):303-314. doi: 10.1007 / s00280-016-3224-2. Epub 2016 Dec 24; Coward et al. Cancers (Basel). 2021 Jun 26;13(13):3196. doi: 10.3390 / cancersl313319).

[0008] A second generation benzopyran ME-344 has been reported to show synergy with venetoclax (a BCL-2 inhibitor) in acute myeloid leukemia (AML) by suppressing oxidative phosphorylation (OXPHOS) and / or purine biosynthesis (Hurrish et al. Biochemical Pharmacology. Volume 220 (2024), 115981 https: / / doi.Org / 10.1016 / j.bcp.2023.115981).

[0009] An object of the present invention is the provision of new and / or improved therapies utilizing TRX-E-002-1 as an active ingredient, for one or more types of cancer.

[0010] DEFINITIONS

[0011] Acute Myeloid Leukemia (AML) is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. AML is the most common acute leukemia affecting adults, and its incidence increases with age. The ICD-10-CM (International Classification of Diseases, Tenth Revision, Clinical Modification) code for Acute Myeloid Leukemia (AML) is C92.0. The specific code C92.00 is used for acute myeloblastic leukemia, not having achieved remission, while C92.01 indicates acute myeloblastic leukemia, in remission, and C92.02 denotes acute myeloblastic leukemia, in relapse. AML is also known as acute myelogenous leukemia or acute nonlymphocytic leukemia (ANLL).

[0012] Multiple Myeloma (MM) is a cancer of plasma cells that normally produce antibodies. The ICD-10-CM (International Classification of Diseases, Tenth Revision, Clinical Modification) code for Multiple Myeloma (MM) is C90.0. The specific code C90.00 is used for MM, not having achieved remission, while C90.01 indicates MM, in remission, and C90.02 denotes MM, in relapse. MM is also known as Kahler's disease, medullary plasmacytoma, myelomatosis and plasma cell myeloma.

[0013] The term TRX-E-002-1 refers to the compound according to Formula I:

[0014]

[0015] (3R, 4S)-3-(4-hydroxy-3,5-dimethoxyphenyl)-4-(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2 / 7- chromen-7-ol (CAS No.: 2135511-22-5). Synthesis of the compound was initially disclosed in W02015 / 117202. Stereoisomer separation is discussed in US2017 / 0281588 Al, with certain forms being more pharmacologically active than others with (3 / ?, 45) being the most active and being currently developed as a clinical candidate. It belongs to the third generation of benzopyran tubulin inhibitors with a broad and potent anti-cancer activity.

[0016] The terms Trilexium and TRX-E-009-1 refer to a reference compound according to Formula II:

[0017] (3 / ?, 4S)-3-(4-hydroxy-3,5-dimethoxyphenyl)-4-(3-fluoro-4-hydroxyphenyl)-8-methyl-3,4- dihydro-2 / 7-chromen-7-ol (CAS No.: 1983180-82-0). Trilexium / TRX-E-009-1 has shown potent and broad anti-cancer activity and is structurally a close analogue to TRX-E-002-1. Similarly to TRX-E-002-1, it also targets tubulin. The term ME-344 refers to a reference compound according to Formula III:

[0018] (3R, 4S)-3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2 / 7-chromen-7-ol (CAS No.: 1374524-68-1). ME-344 is second-generation benzopyran which has shown potent activity on a broad range of cancers. Structurally it is a less close analogue of TRX-E-002-1 compared to TRX-E-009-l / Trilexium. Its mode of action has been demonstrated to be predominantly through the inhibition of mitochondrial oxidative phosphorylation (OXPHOS) which depletes cancer cells of ATP therefore, causing cell death.

[0019] B-cell lymphoma 2 (BCL2 or BCL-2) is an important protein regulator of apoptosis. It is highly expressed in many hematological malignancies, where it contributes to evasion of programmed cell death, which is a hallmark of cancer. Inhibition of BCL-2 may trigger apoptosis in cancer cells and it therefore represents a therapeutic target.

[0020] BCL-2 inhibitors are a class of anti-cancer drugs defined functionally by being inhibitors of BCL-2. BH3-mimetics represent a subclass of BCL-2 inhibitors. Venetoclax is the only currently clinically approved BCL-2 inhibitor, but other compounds with similar activity such as navitoclax and obatoclax have progressed to late clinical development.

[0021] Venetoclax is a BH3-mimetic small molecule that selectively inhibits BCL-2, an anti- apoptotic protein, restoring programmed cell death in cancer cells. It is currently an approved medication for AML, CLL (chronic lymphocytic leukemia) and SLL (small lymphocytic lymphoma).

[0022] The term deoxycytidine analogue chemotherapeutics are a subclass of pyrimidine nucleoside analogs that mimic the natural nucleoside cytidine. They interfere with DNA synthesis and repair, ultimately inducing cell death and are thus useful in the treatment of cancer. Examples include cytarabine or gemcitabine. Cytarabine (also known as Ara-C or cytosine arabinose) is a deoxycytidine analogue chemotherapeutic that inhibits DNA synthesis by incorporation into DNA during the S-phase of the cell cycle causing chain termination and concomitant cell death. It is primarily used in the treatment of AML, ALL (acute lymphoblastic leukemia), CML (chronic myelogenous leukemia), and non-Hodgkin's lymphoma.

[0023] The term hypomethylating agents refers to anti-cancer drugs that inhibit DNA methyltransferases, leading to the reactivation of silenced tumor suppressor genes and altering gene expression. Examples include azacitidine and decitabine.

[0024] Azacitidine (also known as 5-azacitidine) is a DNA methyltransferase inhibitor (hypomethylating agent) that incorporates into RNA and DNA, leading to inhibition of DNA methylation, reactivation of tumor suppressor genes and disruption of RNA metabolism. It is currently approved medication for MDS (myelodysplastic syndromes), chronic lymphoma (i.e. juvenile myelomonocytic leukemia) and myeloid leukemia (i.e. AML, CML).

[0025] The term anthracycline chemotherapeutic refers to a class of classic chemotherapeutic drugs, that act mainly by disrupting DNA synthesis by intercalation and by inhibiting DNA topoisomerases, enzymes critical for unwinding DNA for cell replication. The class originated in the 1950's with the identification of daunorubicin from the soil bacterium Streptomyces peucetius. Examples include daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin and mitoxantrone.

[0026] Daunorubicin (also known as daunomycin) is an anthracycline aminoglycoside antibiotic compound that damages the DNA by intercalating between base pairs and forms a stable daunorubicin-DNA-topoisomerase II ternary complex. This topoisomerase-ll-mediated DNA damage causes cellular growth arrest and ultimately initiates programmed cell death. This drug is currently approved for AML, ALL, CML and Kaposi's sarcoma.

[0027] Proteasome inhibitors are a class of drugs that block the proteasome's function which prevents the degradation of proteins within the cell. This leads to the accumulation of damaged proteins, inducing apoptosis in rapidly dividing cancer cells. Examples include bortezomib, carfilzomib, ixazomib.

[0028] Bortezomib is a dipeptidyl boronic acid and the first-in-class proteasome inhibitor currently approved in MM and Mantle Cell Lymphoma (MCL). Carfilzomib is a tetrapeptide epoxyketone and a selective, irreversible proteasome inhibitor.

[0029] It is approved for MM.

[0030] Immunomodulatory drugs (IMiDs) are small molecules that modulate the immune system, enhancing T-cell and NK-cell activity, inhibiting angiogenesis and directly inducing cancer cell death. Examples include lenalidomide, pomalidomide, and thalidomide.

[0031] Pomalidomide is a third-generation immunomodulatory small molecule drug that has direct activity against myeloma cells, affecting gene expression and promoting apoptosis. It is known to enhance T-cell- and natural killer (NK) cell-mediated immunity and also inhibits angiogenesis. It is currently approved for MM and AIDS-related Kaposi sarcoma.

[0032] Corticosteroids are anti-inflammatory drugs that suppress the immune system and induce apoptosis in lymphoid cells. They are often used in hematological malignancies to reduce tumor burden and manage inflammation. Examples are dexamethasone and prednisone.

[0033] Dexamethasone is a synthetic corticosteroid with potent anti-inflammatory and immunosuppressive effects. It binds to glucocorticoid receptors which leads to apoptosis of lymphoid cells. It is currently approved as part of combination regimens for MM, lymphomas and leukemias, enhancing the efficacy of other anti-cancer agents.

[0034] Alkylators (or alkylating agents) are chemotherapeutic agents that damage DNA by adding alkyl groups to DNA bases, leading to cross-linking and strand breaks. This inhibits the DNA replication and trigger apoptosis (cell death). Examples include cyclophosphamide, melphalan, melflufen, and bendamustine.

[0035] Cyclophosphamide (also known as CP or cytophosphane) is a chemotherapy that acts by alkylating cellular macromolecules such as DNA, resulting in DNA fragmentation and cell death. It is also an immunosuppressant. In a cancer setting, it is used to treat a broad range of diseases such as lymphoma, MM, leukemia, ovarian cancer, breast cancer, small cell lung cancer, neuroblastoma, and sarcoma.

[0036] Bendamustine is a chemotherapy acting by alkylating base DNA and ultimately causing cell death. It is used to treat CLL, MM, non-Hodgkin's lymphoma.

[0037] Synergistically effective refers herein to the combined administration of two or more therapeutic agents resulting in a therapeutic effect that is greater than the sum of the individual therapeutic effects of each agent administered alone. A synergistic effect of a drug combination can be demonstrated e.g. in vitro by a positive Bliss score, wherein the observed combined inhibitory effect exceeds the expected additive effect under the Bliss independence model, calculated as the product of the independent fractional effects of each drug. For example, Bliss-scores between 15-30 may indicate intermediate synergy; scores between 10-14 may indicate minor synergy and scores below 10 may indicate no significant synergy. A synergistic effect can manifest clinically as:

[0038] • Improved Disease Outcome: Such as an increased survival rate, greater tumor / tumor cell reduction, decreased metastatic proliferation, or higher rates of complete or partial response.

[0039] • Reduced Toxicity or Improved Tolerability: Achieving a desired therapeutic outcome with lower effective doses of the individual agents, thereby reducing associated side effects.

[0040] • Overcoming Resistance: Efficacy in treating a disease or condition that is refractory or non-responsive to one or both agents when administered alone.

[0041] The term "comprising" is to be interpreted as including, but not being limited to.

[0042] Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0043] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of "from 2 to 10" is inclusive of the endpoints, 2 and 10, and all the intermediate values).

[0044] The term "about" can be used to include any numerical value that can vary without changing the basic function of that value. When used with a range, "about" also discloses the range defined by the absolute values of the two endpoints, e.g. "about 2 to about 4" also discloses the range "from 2 to 4." The term "about" may refer to plus or minus 10% of the indicated number. BRIEF DESCRIPTION OF THE FIGURES

[0045] Fig 1. TRX-E-002-1 has anti-cancer activity against patient-derived AML and MM cells with IC50s in the nanomolar range (38-76 nM). Data are presented as mean ± SD from three independent experiments performed in triplicate. AML: Acute myeloid leukemia, MM: Multiple myeloma.

[0046] Fig 2. TRX-E-002-1 has anti-cancer activity against patient-derived AML cell-lines. TRX-E-002- 1 dose-response curves (top panel) and derived IC50s (bottom panel) for KASUMI-3, HL-60 and SKNO-1 cell-lines. The relapse / resistant cell-line SKNO-1 showed the highest sensitivity to TRX-E-002-1. Data represent mean ± SD from three independent experiments performed in triplicate. Statistical analysis was performed using one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001).

[0047] Fig 3. TRX-E-002-1 has anti-cancer activity against patient-derived MM cell-lines. TRX-E-002- 1 dose-response curves (top panel) and derived IC50s (bottom panel) for XG2 and XG11 celllines. Both MM cell-lines showed a similar sensitivity to TRX-E-002-1. Data represent mean ± SD from three independent experiments performed in triplicate. Statistical analysis was performed using one-way ANOVA (*P < 0.05).

[0048] Fig 4. TRX-E-002-1 synergizes with daunorubicin, cytarabine, azacitidine and venetoclax on AML cell-lines HL-60 and MOLM-13. Data represents the maximum synergistic scores obtained for TRX-E-002-1 and each combination treatment per cell-line. Synergy scores were calculated using the Bliss Model. Scores between 15-30 indicate intermediate synergy; scores between 10-14 indicate minor synergy and scores below 10 indicate no synergy. (R): Relapse / resistant.

[0049] Fig 5. TRX-E-002-1 synergizes with daunorubicin. MOLM-13 and HL-60 cell-lines were treated with the indicated concentrations of TRX-E-002-1 and daunorubicin simultaneously for three days. Synergy scores were calculated using the Bliss Model. Scores between 15-30 indicate intermediate synergy; scores between 10-14 indicate minor synergy and scores below 10 indicate no synergy.

[0050] Fig 6. TRX-E-002-1 synergizes with cytarabine. MOLM-13 and HL-60 cell-lines were treated with the indicated concentrations of TRX-E-002-1 and cytarabine simultaneously for three days. Synergy scores were calculated using the Bliss Model. Scores between 15-30 indicate intermediate synergy; scores between 10-14 indicate minor synergy and scores below 10 indicate no synergy.

[0051] Fig 7. TRX-E-002-1 synergizes with azacitidine. MOLM-13 and HL-60 cell-lines were treated with the indicated concentrations of TRX-E-002-1 and azacitidine simultaneously for three days. Synergy scores were calculated using the Bliss Model. Scores between 15-30 indicate intermediate synergy; scores between 10-14 indicate minor synergy and scores below 10 indicate no synergy.

[0052] Fig 8. TRX-E-002-1 synergizes with venetoclax. MOLM-13 and HL-60 cell-lines were treated with the indicated concentrations of TRX-E-002-1 and venetoclax simultaneously for three days. Synergy scores were calculated using the Bliss Model. Scores between 15-30 indicate intermediate synergy; scores between 10-14 indicate minor synergy and scores below 10 indicate no synergy.

[0053] Fig 9. TRX-E-002-l / venetoclax combination treatment displays a higher anti-cancer effect than TRX-E-009-l / venetoclax and ME-344 / venetoclax on AML cell-lines. MOLM-13 and HL- 60 cell-lines were treated with the indicated concentrations of TRX-E-002-1, TRX-E-009-1, ME-344 and venetoclax as single treatments or in combination simultaneously for three days. TRX-E-002-1 demonstrated the highest cytotoxicity in combination with venetoclax at the indicated concentrations. Data represent mean ± SD from one independent experiment performed in triplicate. Statistical analysis was performed using one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0054] Fig 10. TRX-E-002-l / azacitidine combination treatment displays a higher anti-cancer effect than TRX-E-009-l / azacitidine and ME-344 / azacitidine on AML cell-lines. MOLM-13 and HL- 60 cell-lines were treated with the indicated concentrations of TRX-E-002-1, TRX-E-009-1, ME-344 and azacitidine as single treatments or in combination simultaneously for three days. TRX-E-002-1 demonstrated the highest cytotoxicity in combination with azacitidine at the indicated concentrations. Data represent mean ± SD from one independent experiment performed in triplicate. Statistical analysis was performed using one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0055] Fig 11. TRX-E-002-l / cytarabine and ME-344 / cytarabine combination treatments display a higher anti-cancer effect than TRX-E-009-l / cytarabine on AML cell-lines. MOLM-13 and HL- 60 cell-lines were treated with the indicated concentrations of TRX-E-002-1, TRX-E-009-1, ME-344 and cytarabine as single treatments or in combination simultaneously for three days. TRX-E-002-1 demonstrated the highest cytotoxicity in combination with cytarabine on MOLM-13. TRX-E-002-1 also demonstrated the highest cytotoxicity in combination with cytarabine on HL-60, together with ME-344 / cytarabine. Data represent mean ± SD from one independent experiment performed in triplicate. Statistical analysis was performed using one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0056] Fig 12. AML cell-lines MOLM-13 and HL-60 display higher sensitivity for TRX-E-002-1 as compared to TRX-E-009-1 and ME-344. Dose-response curves for TRX-E-002-1, TRX-E-009-1 and ME-344 on MOLM-13 (top panel) and HL-60 (bottom panel). Data represent mean ± SD from one independent experiment performed in triplicate.

[0057] Fig 13. AML cell-lines SKNO-1 and KASUMI-3 display higher sensitivity for TRX-E-002-1 as compared to TRX-E-009-1 and ME-344. Dose-response curves for TRX-E-002-1, TRX-E-009-1 and ME-344 on SKNO-1 (top panel) and KASUMI-3 (bottom panel). Data represent mean ± SD from one independent experiment performed in triplicate.

[0058] Fig 14. MM relapse / resistant cell-lines XG21 and XG26 display higher sensitivity for TRX-E- 002-1 as compared to TRX-E-009-1 and ME-344. Dose-response curves for TRX-E-002-1, TRX- E-009-1 and ME-344 on XG21 (top panel) and XG26 (bottom panel). Data represent mean ± SD from one independent experiment performed in triplicate.

[0059] Fig 15. MM relapse / resistant cell-lines XG28 and XG30 display higher sensitivity for TRX-E- 002-1 as compared to TRX-E-009-1 and ME-344. Dose-response curves for TRX-E-002-1, TRX- E-009-1 and ME-344 on XG28 (top panel) and XG30 (bottom panel). Data represent mean ± SD from one independent experiment performed in triplicate.

[0060] Fig 16. TRX-E-002-1 reduces tumor growth in an animal model of AML. Mice engrafted with MOLM-13 cells expressing luciferase were treated with vehicle control (saline) or TRX-E-002- 1 at 50, 100 or 150 mg / kg intravenously for 14 days consecutively (Week 1, 2). Doseresponse curves represent the bioluminescent signal observed across the treatment groups over 5 weeks (n = 8 per group). Mean values ± standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA (*P < 0.05; **P < 0.01; ***p < 0.001). Fig 17. TRX-E-002-1 increases survival time in an animal model of AML. Mice engrafted with MOLM-13 cells expressing luciferase were treated with vehicle control (saline) or TRX-E-002- 1 at 50, 100 or 150 mg / kg intravenously for 14 days consecutively (Day 1-14). Kaplan-Meier survival curves for each TRX-E-002-1 treatment group as compared to control are depicted separately for visual clarity (n = 8 per group). Statistical analysis was performed using the log-rank (Mantel-Cox) test (*P < 0.05; **P < 0.01; ***P < 0.001).

[0061] Fig 18. TRX-E-002-1 in combination with venetoclax and azacitidine improves survival time in an animal model of AML. Mice engrafted with MOLM-13 cells expressing luciferase were treated with venetoclax (30 mg / kg / d, PO, Day 1-14) and azacitidine (1 mg / kg / d, IP, Day 1-5) either alone or in combination with TRX-E-002-1 (100 mg / kg / d, IV, Day 1-14). Kaplan-Meier survival curves for the standard-of-care group (venetoclax and azacitidine) and the triple combination group (TRX-E-002-1, venetoclax and azacitidine) are depicted in grey and black, respectively (n = 10 per group). Statistical analysis was performed using the log-rank (Mantel-Cox) test (*P < 0.05; **P < 0.01; ***p < 0.001). Aza: Azacitidine, Ven: Venetoclax.

[0062] SUMMARY OF THE INVENTION

[0063] The present invention is based on the unexpected synergistic effect discovered between TRX-E-002-1 and standard-of-care (SOC) anti-cancer medications for hematological cancers, providing a basis for new combination therapies using TRX-E-002-1 as one of the active substances.

[0064] Briefly, TRX-E-002-1 was tested against a panel of several different cell-lines representing the hematological malignancies acute myeloid leukemia (AML) and multiple myeloma (MM) (Example 1). TRX-E-002-1 had a strong and similar cytotoxic effect against all cell-lines, regardless of their glycolytic score, which demonstrates no dependency on metabolic processes such as OXPHOS or glycolysis.

[0065] TRX-E-002-1 was also tested in two AML cell-lines, alone and in combination with standard- of-care treatments including daunorubicin, cytarabine, azacitidine and venetoclax (Example 2). Synergistic cytotoxic effects were observed in both cell-lines. In comparison to two structurally related compounds TRX-E-009-l / Trilexium and ME-344, it was shown that TRX-E-002-1 exhibited superior anti-cancer efficacy (Examples 3 and 4).

[0066] TRX-E-002-1 alone or when added to the standard-of-care anti-cancer drugs venetoclax and azacitidine improved outcomes in a disseminated mouse model of AML (Examples 5 and 6).

[0067] The present invention relates to the following items. The subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims.

[0068] 1. (3 / ?, 4S)-3-(4-hydroxy-3,5-dimethoxyphenyl)-4-(4-hydroxyphenyl)-8-methyl-3,4- dihydro-2 / 7-chromen-7-ol (TRX-E-002-1) for use in a method of treatment for a hematological cancer being acute myeloid leukemia (AML) or multiple myeloma (MM) in a subject in need thereof, the method comprising administering to the subject an effective amount of TRX-E-002-1.

[0069] 2. TRX-E-002-1 for use according to item 1, wherein the method further comprises administering to the subject an effective amount of a second compound selected from the group consisting of: (i) a hypomethylating agent such as azacitidine or decitabine (ii) a deoxycytidine analogue chemotherapeutic such as cytarabine or gemcitabine, (iii) a BCL-2 inhibitor such as venetoclax, navitoclax or obatoclax, and (iv) an anthracycline chemotherapeutic such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin or mitoxantrone.

[0070] 3. TRX-E-002-1 for use according to item 2, wherein the TRX-E-002-1 and the second compound are administered in a synergistically effective manner and dose for the treatment of the hematological cancer.

[0071] 4. TRX-E-002-1 for use according any of items 1-3, wherein the cancer is AML.

[0072] 5. TRX-E-002-1 for use according to item 4, wherein the second compound is a BCL-2 inhibitor.

[0073] 6. TRX-E-002-1 for use according to item 5, wherein the second compound is venetoclax, navitoclax, obatoclax, lisaftoclax, pelcitoclax, sonrotoclax, asaretoclax (ZN-d5), lacutoclax, oblimersen, S55746 (BCL201, CAS No. 1448584-12-0), S65487 (VOB560, prodrug of S55746, CAS Nos. 1644600-79-2 , 1644543-95-2, 2416937-01-2 depending on salt form), AZD4320 (CAS No. 1357576-48-7, including its PEGylated / poly-lysine conjugated version AZD0466), R-(-)-enantiomer of gossypol (AT 101, CAS No. 866541-93-7 for acetic acid salt, CAS No. 90141-22-3 for free base), LOXO-338 (FCN-338, LY3847429, CAS No. 2248046-39-9) or BM-1197 (UBX1967, CAS No. 1391107-89-3).

[0074] 7. TRX-E-002-1 for use according to item 6, wherein the second compound is venetoclax, navitoclax or obatoclax.

[0075] 8. TRX-E-002-1 for use according to item 7, wherein the second compound is venetoclax.

[0076] 9. TRX-E-002-1 for use according to item 4, wherein the second compound is a deoxycytidine analogue chemotherapeutic.

[0077] 10. TRX-E-002-1 for use according to item 9, wherein the second compound is cytarabine or gemcitabine.

[0078] 11. TRX-E-002-1 for use according to item 10, wherein the second compound is cytarabine.

[0079] 12. TRX-E-002-1 for use according to item 4, wherein the second compound is a hypomethylating agent.

[0080] 13. TRX-E-002-1 for use according to item 12, wherein the second compound is azacitidine, decitabine, guadecitabine (SG 1-110, CAS No. 929904-85-8), 5-Fluoro-2'- deoxycytidine (FdCyd, CAS No. 10356-76-0), nanaomycin A (CAS No. 52934-83-5), 4- Deoxyuridine (zebularine, CAS No. 3690-10-6), N-Phthalyl-L-tryptophan (RG108, CAS No. 48208-26-0), 3-Bromo-3-nitroflavanone (CAS No. 6513-51-5), SGI-1027 (CAS No. 1020149-73-8), MC3343 (CAS No. 1535187-91-7), or 5-aza-4'-thio-2'-deoxycytidine (NTX-301, CAS No. 169514-76-5).

[0081] 14. TRX-E-002-1 for use according to item 13, wherein the second compound is azacitidine or decitabine.

[0082] 15. TRX-E-002-1 for use according to item 14, wherein the second compound is azacitidine.

[0083] 16. TRX-E-002-1 for use according to item 4, wherein the second compound is an anthracycline chemotherapeutic.

[0084] 17. TRX-E-002-1 for use according to item 16, wherein the second compound is daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin or mitoxantrone. 18. TRX-E-002-1 for use according to item 17, wherein the second compound is daunorubicin.

[0085] 19. TRX-E-002-1 for use according to item 4 or any item dependent thereon, wherein the AML is an AML subtype selected from the list consisting of: (i) AML with defining genetic abnormalities (e.g., RUNX1::RUNX1T1 fusion, CBFB::MYH11 fusion, PML::RARA fusion, KMT2A rearrangement, DEK::NUP214 fusion, MECOM rearrangement, RBM15::MRTFA fusion, BCR::ABL1 fusion, NUP98 rearrangement, NPMl mutation, CEBPA mutation or other defined genetic alteration), (ii) AML defined by differentiation (e.g., AML with minimal differentiation, AML without maturation, AML with maturation, acute myelomonocytic leukemia, acute monoblastic / monocytic leukemia, pure erythroid leukemia, acute megakaryoblastic leukemia, acute basophilic leukemia), (iii) AML with myelodysplasia-related changes (AML-MR), (iv) therapy-related AML (e.g. FLT3 mutation, IDH1 / 2 mutation), and (v) AML not otherwise specified (NOS) and (vi) myeloid sarcoma.

[0086] 20. TRX-E-002-1 for use according to item 4 or any item dependent thereon, wherein the AML is an AML with a FAB classification of MO, Ml, M2, M3, M4, M5, M6 or M7.

[0087] 21. TRX-E-002-1 for use according to item 4, wherein the cancer is an AML subtype selected from Table 1 and the second compound is as indicated in Table 1 for said AML subtype.

[0088] 22. TRX-E-002-1 for use according to item 6 or any item dependent thereon, wherein the method comprises administering an effective amount of a third compound being a hypomethylating agent.

[0089] 23. TRX-E-002-1 for use according to item 22, wherein the TRX-E-002-1, the second compound and the third compound are administered in a synergistically effective manner and dose for the treatment of the AML.

[0090] 24. TRX-E-002-1 for use according to item 22 or 23, wherein the third compound is selected from the list consisting of: azacitidine, decitabine, guadecitabine (SG 1-110, CAS No. 929904-85-8), 5-Fluoro-2'-deoxycytidine (FdCyd, CAS No. 10356-76-0), nanaomycin A (CAS No. 52934-83-5), 4-Deoxyuridine (zebularine, CAS No. 3690-10- 6), N-Phthalyl-L-tryptophan (RG108, CAS No. 48208-26-0), 3-Bromo-3-nitroflavanone (CAS No. 6513-51-5), SGI-1027 (CAS No. 1020149-73-8), MC3343 (CAS No. 1535187- 91-7), and 5-aza-4'-thio-2'-deoxycytidine (NTX-301, CAS No. 169514-76-5).

[0091] 25. TRX-E-002-1 for use according to item 24, wherein the third compound is azacitidine or decitabine.

[0092] 26. TRX-E-002-1 for use according to item 25, wherein the third compound is azacitidine.

[0093] 27. TRX-E-002-1 for use according to any of the preceding items, wherein the method comprises administering TRX-E-002-1 to a human subject in need thereof a dose being from 1 mg / kg to 25 mg / kg per administration.

[0094] 28. TRX-E-002-1 for use according to any of the preceding items, wherein the method comprises administering at least two doses of TRX-E-002-1 separated by one to seven days a human subject in need thereof.

[0095] 29. TRX-E-002-1 for use according to any of the preceding items, wherein the method comprises administering TRX-E-002-1 intravenously to a human subject in need thereof.

[0096] 30. TRX-E-002-1 for use according to item 1, wherein the cancer is MM.

[0097] 31. TRX-E-002-1 for use according to item 30, wherein the cancer is a MM subtype selected from Table 2 and the method further comprises administering to the subject an effective amount of a second compound as indicated in Table 2 for said MM subtype.

[0098] 32. TRX-E-002-1 for use according to item 31, wherein the method further comprises administering to the subject an effective amount of a second compound selected from the group consisting of (v) a proteasome inhibitor such as carfilzomib, bortezomib, and ixazomib, (vi) an immunomodulatory drug (I Mi D) such as pomalidomide, lenalidomide, and thalidomide, (vii) a corticosteroid drug such as dexamethasone, prednisolone or prednisone, and (viii) an alkylator such as bendamustine, cyclophosphamide, melphalan, and melflufen.

[0099] 33. TRX-E-002-1 for use according to item 32, wherein the second compound is a proteasome inhibitor.

[0100] 34. TRX-E-002-1 for use according to item 33, wherein the second compound is carfilzomib, bortezomib or ixazomib, preferably carfilzomib.

[0101] 35. TRX-E-002-1 for use according to item 32, wherein the second compound is an I Mi D. 36. TRX-E-002-1 for use according to item 35, wherein the second compound is pomalidomide, lenalidomide or thalidomide, preferably pomalidomide.

[0102] 37. TRX-E-002-1 for use according to item 32, wherein the second compound is a corticosteroid drug.

[0103] 38. TRX-E-002-1 for use according to item 37, wherein the second compound is dexamethasone, prednisolone or prednisone, preferably dexamethasone.

[0104] 39. TRX-E-002-1 for use according to item 32, wherein the second compound is an alkylator.

[0105] 40. TRX-E-002-1 for use according to item 39, wherein the second compound is bendamustine, cyclophosphamide, melphalan or melflufen, preferably cyclophosphamide.

[0106] 41. Venetoclax for use in a method of treatment for a hematological cancer being AML in a subject in need thereof, the method comprising administering to the subject effective amounts of venetoclax and TRX-E-002-1.

[0107] 42. Cytarabine for use in a method of treatment for a hematological cancer being AML in a subject in need thereof, the method comprising administering to the subject effective amounts of cytarabine and TRX-E-002-1.

[0108] 43. Azacitidine for use in a method of treatment for a hematological cancer being AML in a subject in need thereof, the method comprising administering to the subject effective amounts of azacitidine and TRX-E-002-1.

[0109] 44. Daunorubicin for use in a method of treatment for a hematological cancer being AML in a subject in need thereof, the method comprising administering to the subject effective amounts of daunorubicin and TRX-E-002-1.

[0110] 45. Carfilzomib for use in a method of treatment for a hematological cancer being MM in a subject in need thereof, the method comprising administering to the subject effective amounts of carfilzomib and TRX-E-002-1.

[0111] 46. Pomalidomide for use in a method of treatment for a hematological cancer being MM in a subject in need thereof, the method comprising administering to the subject effective amounts of pomalidomide and TRX-E-002-1. Dexamethasone for use in a method of treatment for a hematological cancer being MM in a subject in need thereof, the method comprising administering to the subject effective amounts of dexamethasone and TRX-E-002-1. Cyclophosphamide for use in a method of treatment for a hematological cancer being MM in a subject in need thereof, the method comprising administering to the subject effective amounts of cyclophosphamide and TRX-E-002-1. A pharmaceutical composition, comprising TRX-E-002-1 and a second compound selected from the group consisting of (i) a BCL-2 inhibitor such as venetoclax, navitoclax or obatoclax, (ii) a deoxycytidine analogue chemotherapeutic such as cytarabine or gemcitabine, (iii) a hypomethylating agent such as azacitidine or decitabine, (iv) an anthracycline chemotherapeutic such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin or mitoxantrone, (v) a proteasome inhibitor such as carfilzomib, bortezomib, and ixazomib, (vi) an immunomodulatory drug (I MiD) such as pomalidomide, lenalidomide, and thalidomide, (vii) a corticosteroid drug such as dexamethasone or prednisone, and (viii) an alkylator such as bendamustine, cyclophosphamide, melphalan, and melflufen. The pharmaceutical composition according to item 49, wherein the second compound is a BCL-2 inhibitor. The pharmaceutical composition according to item 49, wherein the second compound is venetoclax, navitoclax or obatoclax. The pharmaceutical composition according to item 49, wherein the second compound is venetoclax. The pharmaceutical composition according to item 49, wherein the second compound is a deoxycytidine analogue chemotherapeutic. The pharmaceutical composition according to item 49, wherein the second compound is cytarabine or gemcitabine. The pharmaceutical composition according to item 49, wherein the second compound is cytarabine. The pharmaceutical composition according to item 49, wherein the second compound is a hypomethylating agent. 57. The pharmaceutical composition according to item 49, wherein the second compound is azacitidine or decitabine.

[0112] 58. The pharmaceutical composition according to item 49, wherein the second compound is azacitidine.

[0113] 59. The pharmaceutical composition according to item 49, wherein the second compound is an anthracycline chemotherapeutic.

[0114] 60. The pharmaceutical composition according to item 49, wherein the second compound is daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin or mitoxantrone.

[0115] 61. The pharmaceutical composition according to item 49, wherein the second compound is daunorubicin.

[0116] 62. The pharmaceutical composition according to item 49, wherein the second compound is a proteasome inhibitor.

[0117] 63. The pharmaceutical composition according to item 49, wherein the second compound is carfilzomib, bortezomib or ixazomib, preferably carfilzomib.

[0118] 64. The pharmaceutical composition according to item 49, wherein the second compound is an I MiD.

[0119] 65. The pharmaceutical composition according to item 49, wherein the second compound is pomalidomide, lenalidomide or thalidomide, preferably pomalidomide.

[0120] 66. The pharmaceutical composition according to item 49, wherein the second compound is a corticosteroid drug.

[0121] 67. The pharmaceutical composition according to item 49, wherein the second compound is dexamethasone, prednisolone or prednisone, preferably dexamethasone.

[0122] 68. The pharmaceutical composition according to item 49, wherein the second compound is an alkylator.

[0123] 69. The pharmaceutical composition according to item 49, wherein the second compound is bendamustine, cyclophosphamide, melphalan or melflufen, preferably cyclophosphamide.

[0124] 70. The pharmaceutical composition according to any one of items 49-69, further comprising a vehicle, an excipient, a solubilizer and / or a stabilizer. 71. The pharmaceutical composition according to any one of items 49-70, being formulated for parenteral administration.

[0125] The arrangement of the present disclosure into sections with headings and subheadings is merely to improve legibility and is not to be interpreted limiting in any way, in particular, the division does not in any way preclude or limit combining features under different headings and subheadings with each other. All references are hereby incorporated by reference.

[0126] DETAILED DESCRIPTION

[0127] Single therapy and combination therapy for AML and MM

[0128] In a first aspect, the present invention provides (3 / ?, 4S)-3-(4-hydroxy-3,5-dimethoxyphenyl)- 4-(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2 / 7-chromen-7-ol (TRX-E-002-1) for use in a method of treatment for a hematological cancer being acute myeloid leukemia (AML) or multiple myeloma (MM) in a subject in need thereof, the method comprising administering to the subject an effective amount of TRX-E-002-1.

[0129] For the treatment of AML, the method may further comprise administering to the subject an effective amount of a second compound selected from the group consisting of (i) a BCL-2 inhibitor such as venetoclax, navitoclax or obatoclax, (ii) a deoxycytidine analogue chemotherapeutic such as cytarabine or gemcitabine, (iii) a hypomethylating agent such as azacitidine or decitabine and (iv) an anthracycline chemotherapeutic such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin or mitoxantrone.

[0130] The TRX-E-002-1 and the second compound are preferably administered in a synergistically effective manner and dose for the treatment of the AML.

[0131] In certain embodiments, both the TRX-E-002-1 and the second compound (and optionally one or more additional compounds, such as a third compound, a fourth compound, or further compounds) are administered at dosages and in a manner where each compound is individually effective, but the combination achieves a greater therapeutic effect, such as enhanced efficacy, improved duration of response, or increased patient response rate, when administered together. In other embodiments, the AML may be refractory or non-responsive to the second or additional compound(s) alone (and / or refractory or non-responsive to the TRX-E-002-1 alone) at the dosage(s) used, but responds to the combination treatment, resulting in a greater therapeutic effect when the compounds are administered together. For example, the combination may overcome one or more resistance mechanisms in AML (such as upregulation of alternative signaling pathways, alterations in microRNAs, epigenetic changes, efflux pump activity, presence of dormant leukemia stem cells or mutational escape), leading to a degree of disease regression, symptom alleviation and / or disease stabilization that is not observed with monotherapy.

[0132] In yet other embodiments, the combination of the TRX-E-002-1 and the second compound (and optionally one or more additional compounds) permits the use of lower dosages of one or both compounds, wherein such lower dosages are not individually effective (or are sub- therapeutic) when administered alone but are effective when administered as a combination treatment. This dosage reduction advantageously minimizes side effects, improves tolerability, reduces toxicity (such as myelosuppression, neuropathy, gastrointestinal adverse events, or organ-specific damage associated with higher doses), and enhances patient compliance. In specific embodiments, the dosage of the TRX-E-002-1 is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to its monotherapy dosage, while maintaining or exceeding the efficacy of the monotherapy. Similarly, the dosage of the second (and optionally one or more of further compounds) may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, while maintaining or exceeding the efficacy of monotherapy with the second (or further) compound.

[0133] In certain embodiments, the second compound is a BCL-2 inhibitor. The BCL-2 inhibitor may be venetoclax, navitoclax or obatoclax. In certain embodiments, the second compound is venetoclax. Additional alternatives that have reached clinical development include lisaftoclax, pelcitoclax, sonrotoclax, asaretoclax (ZN-d5), lacutoclax, oblimersen, S55746 (BCL201, CAS No. 1448584-12-0), S65487 (VOB560, prodrug of S55746, CAS Nos. 1644600- 79-2 , 1644543-95-2, 2416937-01-2 depending on salt form), AZD4320 (CAS No. 1357576- 48-7, including its PEGylated / poly-lysine conjugated version AZD0466), R-(-)-enantiomer of gossypol (AT 101, CAS No. 866541-93-7 for acetic acid salt, CAS No. 90141-22-3 for free base), LOXO-338 (FCN-338, LY3847429, CAS No. 2248046-39-9), BM-1197 (UBX1967, CAS No. 1391107-89-3).

[0134] In certain embodiments, the second compound is a deoxycytidine analogue chemotherapeutic. The deoxycytidine analogue chemotherapeutic may be cytarabine or gemcitabine. In certain embodiments, the second compound is cytarabine.

[0135] In certain embodiments, the second compound is a hypomethylating agent. The hypomethylating agent may be azacitidine or decitabine. In certain embodiments, the second compound is azacitidine. Additional alternatives that have reached clinical development include guadecitabine (SG 1-110, CAS No. 929904-85-8), 5-Fluoro-2'- deoxycytidine (FdCyd, CAS No. 10356-76-0), nanaomycin A (CAS No. 52934-83-5), 4- Deoxyuridine (zebularine, CAS No. 3690-10-6), N-Phthalyl-L-tryptophan (RG108, CAS No. 48208-26-0), 3-Bromo-3-nitroflavanone (CAS No. 6513-51-5), SGI-1027 (CAS No. 1020149- 73-8), MC3343 (CAS No. 1535187-91-7), 5-aza-4'-thio-2'-deoxycytidine (NTX-301, CAS No. 169514-76-5).

[0136] In certain embodiments, the second compound is an anthracycline chemotherapeutic. The anthracycline chemotherapeutic may be daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin or mitoxantrone. In certain embodiments, the second compound is daunorubicin.

[0137] For the treatment of MM, the method may further comprise administering to the subject an effective amount of a second compound selected from the group consisting of (v) a proteasome inhibitor such as carfilzomib, bortezomib, and ixazomib, (vi) an immunomodulatory drug (I Mi D) such as pomalidomide, lenalidomide, and thalidomide, (vii) a corticosteroid drug such as dexamethasone or prednisone, and (viii) an alkylator such as bendamustine, cyclophosphamide, melphalan, and melflufen.

[0138] In certain embodiments, the second compound is a proteasome inhibitor. The proteasome inhibitor may be carfilzomib, bortezomib or ixazomib, preferably carfilzomib.

[0139] In certain embodiments, the second compound is an immunomodulatory drug (I Mi D) . The I MiD may be pomalidomide, lenalidomide, and thalidomide, preferably pomalidomide.

[0140] In certain embodiments, the second compound is a corticosteroid drug. The corticosteroid may be dexamethasone, prednisolone or prednisone, preferably dexamethasone. In certain embodiments, the second compound is an alkylator. The alkylator may be bendamustine, cyclophosphamide, melphalan or melflufen, preferably cyclophosphamide.

[0141] Indications for single and combination therapy

[0142] Preferably, the hematological cancer of the first aspect is AML. In other embodiments, the hematological cancer of the first aspect is MM. The hematological cancer may be a relapsing and / or refractory form of the cancer. In certain embodiments, the AML may be newly diagnosed.

[0143] There are several systems for classifying AML into its subtypes, each with its own focus and methodology. World Health Organization (WHO) Classification (Latest Edition: 5th edition of 2022) combines morphology, immunophenotyping, cytogenetics, and molecular genetics. Major categories include: AML with defining genetic abnormalities (e.g., RUNX1::RUNX1T1 fusion, CBFB::MYH11 fusion, PML::RARA fusion, KMT2A rearrangement, DEK::NUP214 fusion, MECOM rearrangement, RBM15::MRTFA fusion, BCR::ABL1 fusion, NUP98 rearrangement, NPMl mutation, CEBPA mutation or other defined genetic alteration), AML defined by differentiation (e.g., AML with minimal differentiation, AML without maturation, AML with maturation, acute myelomonocytic leukemia, acute monoblastic / monocytic leukemia, pure erythroid leukemia, acute megakaryoblastic leukemia, acute basophilic leukemia), AML with myelodysplasia-related changes (AML-MR), therapy-related AML (e.g. FLT3 mutation, IDH1 / 2 mutation), AML not otherwise specified (NOS) and myeloid sarcoma (a tumor mass of myeloid blasts that occurs at an anatomical site other than the bone marrow). Furthermore, patients with Down syndrome have a higher risk of developing specific types of myeloid leukemia, which are classified separately. International Consensus Classification (ICC) of 2022 is similar to WHO but with some distinctions in genetic criteria and blast thresholds (e.g. AML with mutated TP53).

[0144] French-American-British (FAB) classification is an older system introduced in 1976. It is based on the morphology and cytochemistry of leukemic cells. FAB is somewhat outdated in the sense that it does not incorporate genetic or molecular data, which are today essential for prognosis and treatment decisions. Under the FAB, AML can be categorized into subtypes M0-M7:

[0145] M0: Undifferentiated acute myeloblastic leukemia • Ml: Acute myeloblastic leukemia with minimal maturation

[0146] • M2: Acute myeloblastic leukemia with maturation

[0147] • M3: Acute promyelocytic leukemia (APL)

[0148] • M4: Acute myelomonocytic leukemia

[0149] • M4Eo: M4 with eosinophilia

[0150] • M5: Acute monocytic leukemia

[0151] • M6: Acute erythroid leukemia

[0152] • M7: Acute megakaryoblastic leukemia

[0153] Some subtypes are further divided based on the degree of cell differentiation (e.g. M5a (poorly differentiated, M5b (well differentiated), M6a, and M6b).

[0154] European LeukemiaNet (ELN) guidelines provide yet another classification system for AML, focusing on risk stratification (adverse / intermediate / favorable) and disease management prioritizing patient fitness evaluation (see e.g. Lachowiez et al. Blood Adv. 2023 May 9;7(9):1899-1909).

[0155] The AML being treated according to the present invention encompasses all subtypes according to any of WHO, ICC, FAB or ENL classification. Both primary and relapsing / recurrent forms are also encompassed.

[0156] In the Tables 1 and 2, treatment combinations for TRX-E-002-1 and a second compound for AML, MM and specific subtypes thereof are disclosed.

[0157] Table 1: Treatment combinations for AML and its subtypes, where TRX-E-002-1 is used in combination with a second compound as indicated in the table.

[0158]

[0159] Table 2: Treatment combinations for MM and its subtypes, where TRX-E-002-1 is used in combination with a second compound as indicated in the table.

[0160]

[0161] Table 3: Certain preferred BCL-2 inhibitors for the treatment of AML, in combination with

[0162] TRX-E-002-1 Table 4: Certain preferred hypomethylating agents for the treatment of AML, in combination with TRX-E-002-1.

[0163] The present invention also provides further particularly preferred three-compound (TRX-E- 002-1 plus two additional compounds) combination embodiments of the combination therapy of the first aspect, listed in Table 5.

[0164] The contents of Table 5 are to be read as follows. Each line in Table 5 corresponds to an embodiment. From the left, the first column denoted "#" shows the embodiment identification number. The second column from the left denoted "A" shows the AML subtype number for that embodiment, with reference to Table 1. The third column from the left denoted "B" shows the second compound used in that embodiment, with reference to Table 3. The fourth column from the left denoted "C" shows the third compound for that embodiment, with reference to Table 4.

[0165] To facilitate understanding of how Table 5 is to be read, the following example is provided: Embodiment 53 in Table 5 corresponds to the following values: Column A = 5, Column B = 2, Column C = 2.

[0166] From Table 1 it can thus be deduced that embodiment 53 relates to treatment of AML or R / R AML of the myeloblastic (MO, Ml, or M2) type (#5 in Table 1).

[0167] Second and third compounds can be found in Tables 3 and 4, and resolve to lisaftoclax (#2 in table 3) and decitabine (#2 in Table 4).

[0168] Taken together, embodiment 53 corresponds to: TRX-E-002-1 combined with lisaftoclax (#2 in Table 3) and decitabine (#2 in Table 4) for the treatment of AML or R / R AML of the myeloblastic (MO, Ml, or M2) type (#5 in Table 1).

[0169] Table 5: Preferred combinations for AML treatment

[0170] With regard to AML, Venclyxto (venetoclax) has been approved in Europe (EMA), as follows: Venclyxto in combination with a hypomethylating agent is indicated for the treatment of adult patients with newly diagnosed acute myeloid leukaemia (AML) who are ineligible for intensive chemotherapy. The corresponding US (FDA) Venclexta approval is: In combination with azacitidine, or decitabine, or low-dose cytarabine for the treatment of newly diagnosed acute myeloid leukemia (AML) in adults 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy.

[0171] In preferred embodiments, the combination treatment method of the invention involves administering an effective amount of TRX-E-002-1 in combination with the aforementioned approved therapies and / or indications for venetoclax.

[0172] With regard to AML, azacitidine products are indicated in Europe (EMA) for:

[0173] • the treatment of adult patients who are not eligible for haematopoietic stem cell transplantation (HSCT) with:

[0174] • acute myeloid leukaemia (AML) with 20-30 % blasts and multi-lineage dysplasia, according to World Health Organisation (WHO) classification,

[0175] • AML with >30% marrow blasts according to the WHO classification

[0176] The corresponding US (FDA) approval for azacitidine is indicated for continued treatment of adult patients with acute myeloid leukemia who achieved first complete remission (CR) or complete remission with incomplete blood count recovery (CRi) following intensive induction chemotherapy and are not able to complete intensive curative therapy.

[0177] In preferred embodiments, the combination treatment method of the invention involves administering an effective amount of TRX-E-002-1 in combination with the aforementioned approved therapies and / or indications for azacitidine.

[0178] Further therapeutic agents in a combination treatment

[0179] In further embodiments, the therapeutic agent used in combination with TRX-E-002-1 as a second compound for AML may be selected from the following list of drugs: Arsenic trioxide, azacitidine, cyclophosphamide, cytarabine, daunorubicin, decitabine, dexamethasone, doxorubicin, enasidenib, epirubicin, gemtuzumab ozogamicin, gilteritinib, glasdegib, idarubicin, ivosidenib, midostaurin, mitoxantrone, olutasidenib, pemigatinib, prednisolone, prednisone, quizartinib, thioguanine, venetoclax, vincristine.

[0180] In further embodiments, the therapeutic agent used in combination with TRX-E-002-1 as a second compound for MM may be selected from the following list of drugs: Belantamab mafodotin, bendamustine, bortezomib, carfilzomib, carmustine, cyclophosphamide, daratumumab, dexamethasone, doxorubicin, elotuzumab, elranatamab, etoposide, isatuximab, ixazomib, lenalidomide, melphalan, pomalidomide, prednisolone, prednisone, selinexor, talquetamab, teclistamab, thalidomide.

[0181] In certain embodiments, the therapeutic agent used in combination with TRX-E-002-1 as a second compound for AML may be selected from the following list: venetoclax, navitoclax, obatoclax, cytarabine, gemcitabine, azacitidine, decitabine, daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin or mitoxantrone.

[0182] In other embodiments, the therapeutic agent used in combination with TRX-E-002-1 as a second compound for MM may be selected from the following list: bendamustine, bortezomib, carfilzomib, cyclophosphamide, daratumumab, dexamethasone, isatuximab, lenalidomide, melphalan, pomalidomide, thalidomide.

[0183] In certain embodiments of the first aspect, the second compound may be a BCL-2 inhibitor as described above, and the method further comprises administering an effective amount of a third compound being a hypomethylating agent, such as azacitidine or decitabine, preferably azacitidine. Additional alternatives for third compound include guadecitabine (SGI-110, CAS No. 929904-85-8), 5-Fluoro-2'-deoxycytidine (FdCyd, CAS No. 10356-76-0), nanaomycin A (CAS No. 52934-83-5), 4-Deoxyuridine (zebularine, CAS No. 3690-10-6), N- Phthalyl-L-tryptophan (RG108, CAS No. 48208-26-0), 3-Bromo-3-nitroflavanone (CAS No. 6513-51-5), SGI-1027 (CAS No. 1020149-73-8), MC3343 (CAS No. 1535187-91-7), 5-aza-4'- thio-2'-deoxycytidine (NTX-301, CAS No. 169514-76-5). In certain embodiments of the first aspect, the cancer is AML, and the method comprises administering TRX-E-002-1, venetoclax and azacitidine to the subject.

[0184] In certain embodiments of the first aspect, the cancer is AML, and the method comprises administering TRX-E-002-1, venetoclax and cytarabine to the subject. In certain embodiments of the first aspect, the cancer is AML, and the method comprises administering TRX-E-002-1, venetoclax and decitabine to the subject.

[0185] In certain embodiments of the first aspect, the cancer is AML, and the method comprises administering TRX-E-002-1, daunorubicin and cytarabine to the subject.

[0186] In certain embodiments of the first aspect, the cancer is MM, and the method comprises administering TRX-E-002-1, Carfilzomib and dexamethasone to the subject.

[0187] In certain embodiments of the first aspect, the cancer is MM, and the method comprises administering TRX-E-002-1, Pomalidomide and dexamethasone to the subject.

[0188] Further medical uses

[0189] In a second aspect, the present invention provides venetoclax for use in a method of treatment for a hematological cancer being AML in a subject in need thereof, the method comprising administering to the subject an effective amount of venetoclax and TRX-E-002-1.

[0190] In a third aspect, the present invention provides cytarabine for use in a method of treatment for a hematological cancer being AML in a subject in need thereof, the method comprising administering to the subject an effective amount of cytarabine and TRX-E-002-1.

[0191] In a fourth aspect, the present invention provides azacitidine for use in a method of treatment for a hematological cancer being AML in a subject in need thereof, the method comprising administering to the subject an effective amount of azacitidine and TRX-E-002-1.

[0192] In a fifth aspect, the present invention provides daunorubicin for use in a method of treatment for a hematological cancer being AML in a subject in need thereof, the method comprising administering to the subject an effective amount of daunorubicin and TRX-E- 002-1.

[0193] In a sixth aspect, the present invention provides Carfilzomib for use in a method of treatment for a hematological cancer being MM in a subject in need thereof, the method comprising administering to the subject effective amounts of carfilzomib and TRX-E-002-1.

[0194] In a seventh aspect, the present invention provides Pomalidomide for use in a method of treatment for a hematological cancer being MM in a subject in need thereof, the method comprising administering to the subject effective amounts of pomalidomide and TRX-E-002-

[0195] 1. In an eighth aspect, the present invention provides Dexamethasone for use in a method of treatment for a hematological cancer being MM in a subject in need thereof, the method comprising administering to the subject effective amounts of dexamethasone and TRX-E- 002-1.

[0196] In a ninth aspect, the present invention provides Cyclophosphamide for use in a method of treatment for a hematological cancer being MM in a subject in need thereof, the method comprising administering to the subject effective amounts of cyclophosphamide and TRX-E- 002-1.

[0197] The first to ninth aspects also encompass the use of TRX-E-002-1 for the manufacture of a medicament for the therapeutic uses and methods disclosed in said aspects.

[0198] Preferably, the therapeutic uses and methods of the invention exclude (i.e. do not involve) the therapeutic use of a compound being a glycolytic inhibitor.

[0199] Combination compositions

[0200] In a tenth aspect, the present invention provides a pharmaceutical composition, comprising TRX-E-002-1 and a second compound selected from the group consisting of (i) a BCL-2 inhibitor such as venetoclax, navitoclax or obatoclax, (ii) a deoxycytidine analogue chemotherapeutic such as cytarabine or gemcitabine, (iii) a hypomethylating agent such as azacitidine or decitabine, (iv) an anthracycline chemotherapeutic such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin or mitoxantrone, (v) a proteasome inhibitor such as carfilzomib, bortezomib, and ixazomib, (vi) an immunomodulatory drug (I MiD) such as pomalidomide, lenalidomide, and thalidomide, (vii) a corticosteroid drug such as dexamethasone or prednisone, and (viii) an alkylator such as bendamustine, cyclophosphamide, melphalan, and melflufen. Preferred compounds in each class are as set out in the foregoing aspects.

[0201] The pharmaceutical composition may further comprise a vehicle, an excipient, a solubilizer and / or a stabilizer. The pharmaceutical composition may be formulated for parenteral administration.

[0202] Formulations, administration of treatments

[0203] Usable pharmaceutical formulations of TRX-E-002-1 with cyclodextrin are known in the art (see Saif et al, Coward et al. supra), but other formulations are also contemplated for use with the present invention. The TRX-E-002-1 administration is not particularly limited by the route of administration. It may be administered parenterally e.g. intravenously (IV, bolus or infusion) or intraperitoneally (IP). Administration via subcutaneous (SC), and intramuscular (IM) injection are also contemplated.

[0204] For intravenous administration, TRX-E-002-1 can be administered as a rapid injection (bolus) over a short period (e.g., 1-15 minutes) or as a slower, more prolonged infusion (e.g., over 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours). The choice between bolus and infusion may be guided by the desire to achieve a high peak plasma concentration (Cmax) or to maintain a sustained plasma concentration above a therapeutic threshold (e.g., the in vitro IC50), respectively, particularly in view of the compound's rapid in vivo elimination.

[0205] The second compound (and optionally the third compound) may be administered in any manner known in the art to be appropriate for said compound. For already regulatorily approved compounds (e.g. venetoclax, cytarabine, azacitidine or daunorubicin) in a combination therapy, the routine routes of administration and known formulations may be used.

[0206] The TRX-E-002-1 and the second compound (and optionally the third compound) may be administered in the same pharmaceutical compositions, in separate formulations but concomitantly, or sequentially. Concomitant administration includes co-formulation in a single pharmaceutical composition or administration as separate formulations at substantially the same time (e.g., within 30 minutes of each other). Sequential administration involves administering one agent first, followed by the other after a predetermined time interval. This interval can range from minutes to hours or days. For example, TRX-E-002-1 may be administered within 1 hour, 6 hours, 12 hours, 24 hours, or 48 hours before or after the administration of the second compound.

[0207] The therapeutically effective amount of TRX-E-002-1 to be administered in the combination therapy can vary depending on factors such as the specific second compound used, the disease being treated, the patient's age and condition, and the route of administration.

[0208] Based on preclinical data in mice (IV administration at 50, 100, or 150 mg / kg daily) and initial clinical data in humans (IP administration up to 5 mg / kg once a week), a range of effective doses in humans is contemplated. In various embodiments, a therapeutically effective amount of TRX-E-002-1 for a human patient may range from about 0.1 mg / kg to about 25 mg / kg per administration. In other embodiments, the dose may range from about 0.5 mg / kg to about 15 mg / kg, from about 1 mg / kg to about 20 mg / kg or from about 1 mg / kg to about 10 mg / kg. For example, the dose may be about 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg , 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg or 25 mg / kg.

[0209] In various embodiments, a therapeutically effective amount of TRX-E-002-1 for a human patient may be indicated in terms of mg / m2body surface area and the dose may range from about 4 mg / m2to about 1000 mg / m2per administration. In other embodiments, the dose may range from about 20 mg / m2to about 500 mg / m2, from about 4 mg / m2to about 750 mg / m2or from about 40 mg / m2to about 400 mg / m2. The dose may be about 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 75, 90, 100, 110, 150, 185, 200, 220, 250, 260, 275, 280, 295, 300, 335, 370, 400, 405, 445, 480, 500, 520, 555, 590, 600, 630, 665, 700, 705, 740, 750, 925 or 1000 mg / m2body surface area.

[0210] Alternatively, the dose may be a fixed dose, for example, ranging from about 25 mg to about 2500 mg per administration. In certain embodiments, the fixed dose may be from about 25 mg to about 1500 mg, from about 50 mg to about 750 mg, or from about 100 mg to about 500 mg. For example, the fixed dose may be about 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 750 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1750 mg, 2000 mg or 2500 mg.

[0211] The dosing frequency for TRX-E-002-1 is also contemplated to be variable. In different embodiments, TRX-E-002-1 may be administered twice daily (BID), once daily (QD), every other day, twice a week, once a week (QW), every second week, or every third week. Once a week is preferred. The administration can be part of a treatment cycle, for example, a 21- day or 28-day cycle, which may be repeated as necessary based on the patient's response and tolerance. The selection of a dosing schedule may be optimized to maintain the plasma concentration of TRX-E-002-1 above a minimal effective level for a sufficient time, for instance, a level corresponding to its nanomolar IC50 value observed in vitro.

[0212] Specific, non-limiting examples of combination regimens include:

[0213] • Regimen A: TRX-E-002-1 is administered intravenously once daily for 14 consecutive days, and the second compound, such as venetoclax, is administered orally once daily concurrently.

[0214] • Regimen B: The second compound, such as azacitidine, is administered daily for a 5- day cycle. TRX-E-002-1 is administered intravenously concomitantly from the start of the azacitidine cycle, and continued for a total of 14 days.

[0215] • Regimen C: Combination of regimens A and B. TRX-E-002-1 is administered intravenously once daily for 14 consecutive days, and the second compound, such as venetoclax, is administered orally once daily concurrently for 14 days. A third compound, such as azacitidine, is concurrently administered daily for a 5-day cycle, starting at day 0.

[0216] Needless to say, additional drugs apart from TRX-E-002-1 and the second compound (and optionally the third compound) may also be administered to the patient in the same pharmaceutical compositions, in separate formulations but concomitantly, or sequentially.

[0217] EXAMPLES

[0218] The following examples are not to be regarded as limiting. For further information on the experimental details, the skilled reader is directed to a separate section titled Materials and Methods.

[0219] Example 1: Cytotoxicity effect by TRX-E-002-1 on AML and MM cell lines

[0220] The cytotoxicity effect induced by TRX-E-002-1 for 3 days of treatment was evaluated on 5 hematological cancer cell-lines. The glycolysis status of the different cell-lines was used to assess any associations with the cytotoxic effects of TRX-E-002-1.

[0221] All cell-lines tested showed a high sensitivity to TRX-E-002-1, with IC50s ranging from 38.2 nM to 75.9 nM (Fig. 1). TRX-E-002-1 exerted similar effects on cell-lines with high, intermediate and low glycolysis status, as shown in the table below. The 3 AML cell-lines showed a high sensitivity to TRX-E-002-1. The relapse / resistant AML cell-line SKNO-1 showed a significantly lower IC50 compared to HL-60 and KASUMI-3, with IC50s of 38.2 nM, 66.8 nM and 75.9 nM respectively (Fig.l, Fig. 2).

[0222] The 2 MM cell-lines (XG2 and XG11) also showed a high sensitivity to TRX-E-002-1 with IC50s of around 56.8 nM and 59.7 nM respectively (Fig.l, Fig. 3).

[0223] AML: Acute myeloid leukemia, MM: Multiple myeloma, (R): Relapse / resistant.

[0224] Example 2: Synergistic effect induced by TRX-E-002-1 in combination treatments

[0225] The cytotoxic and synergistic effect induced by TRX-E-002-1 in combination with 4 standard- of-care treatments commonly used for treating AML patients was evaluated using synergy matrices. Treatment with TRX-E-002-1 in combination with daunorubicin, cytarabine, azacitidine and venetoclax were performed for 3 days on the AML cell-lines HL-60 and MOLM-13 (relapse / resistant). These two cell-lines are widely used in AML animal models for testing the efficacy of anti-cancer agents as a single treatment and in combination.

[0226] Cytotoxic effects of the combination treatment TRX-E-002-1 and daunorubicin were observed in both AML cell-lines (HL-60, MOLM-13). Intermediate and minor synergies for this combination treatment on MOLM-13 were observed for 3 doses of TRX-E-002-1 (7.5 nM to 30 nM) and 3 doses of daunorubicin (0.31 nM to 1.25 nM). The associated Bliss scores ranged from 10.33 to 25.59. Minor synergy was observed on HL-60 for 2 doses of TRX-E- 002-1 (15 nM, 30 nM) with daunorubicin (6 nM), with Bliss scores of 12.21 and 14.11 respectively (Fig. 4, Fig. 5). Cytotoxic effects of the combination treatment TRX-E-002-1 and cytarabine were observed in both AML cell-lines (HL-60, MOLM-13). Intermediate and minor synergies for this combination treatment on MOLM-13 was observed for 3 doses of TRX-E-002-1 (7.5 nM, 30 nM, 60 nM) and 2 doses of cytarabine (18.8 nM, 75 nM). The associated Bliss scores ranged from 11.83 to 15.76. Intermediate and minor synergies were also observed on HL-60 for the highest dose of TRX-E-002-1 (60nM) with 3 doses of cytarabine (5 nM to 20 nM), with Bliss scores ranging from 11.96 to 17.52 (Fig. 4, Fig. 6).

[0227] Cytotoxic effects of the combination treatment TRX-E-002-1 and azacitidine were observed in both AML cell-lines (HL-60, MOLM-13). An intermediate synergy for this combination treatment on MOLM-13 was observed, using the highest dose of TRX-E-002-1 (60 nM) in combination with 4 azacitidine doses (25 nM to 200 nM). A minor synergy was also observed on MOLM-13 using the highest doses of TRX-E-002-1 (60 nM) and azacitidine (400 nM). The associated Bliss scores ranged from 11.53 to 21.6. Minor synergy was observed on HL-60 for TRX-E-002-1 (60 nM) and azacitidine (3 pM), with a Bliss score of 10.33 (Fig. 6, Fig. 7).

[0228] Cytotoxic effects of the combination treatment TRX-E-002-1 and venetoclax were observed in both AML cell-lines (HL-60, MOLM-13). Intermediate synergies were observed on MOLM- 13 and HL-60 for the highest dose of TRX-E-002-1 (60 nM) with 2 doses of venetoclax (2 nM and 4 nM) for MOLM-13 and with all venetoclax doses tested for HL-60. The associated Bliss scores for MOLM-13 and HL-60 ranged from 17.03 to 17.78 and 17.09 to 20.77, respectively (Fig. 6, Fig. 8).

[0229] In summary, intermediate synergies with maximum Bliss scores between 15-30 were observed for TRX-E-002-1 in combination with daunorubicin, cytarabine, azacitidine and venetoclax on MOLM-13. Intermediate synergies with maximum Bliss scores between 15-30 were observed for TRX-E-002-1 in combination with cytarabine and venetoclax on the HL-60 cell-line. Lastly, minor synergies with maximum Bliss scores between 10-14 were observed for TRX-E-002-1 in combination with daunorubicin and azacitidine on the HL-60 cell-line (Fig. 4). Example 3: TRX-E-002-1 demonstrates higher cytotoxicity compared to reference compounds TRX-E-009-1 and ME-344

[0230] The cytotoxic effect induced by TRX-E-002-1, TRX-E-009-1 and ME-344 as single treatments and in combination with 3 standard-of-care treatments (cytarabine, azacitidine and venetoclax) was evaluated in the AML cell-lines HL-6O and MOLM-13.

[0231] TRX-E-002-1 demonstrated the highest cytotoxicity in combination with venetoclax, which was significantly higher than that observed for TRX-E-009-1 and ME-344 in combination with venetoclax on both AML cell-lines (Fig. 9).

[0232] Similarly, TRX-E-002-1 displayed the highest cytotoxicity in combination with azacitidine, which was significantly higher than that observed for TRX-E-009-1 and ME-344 in combination with azacitidine on both AML cell-lines (Fig. 10).

[0233] The cytotoxic effect of TRX-E-002-1 in combination with cytarabine was significantly higher than that observed for TRX-E-009-1 in combination with cytarabine on both AML cell-lines. The cytotoxic effect of TRX-E-002-1 in combination with cytarabine was also significantly higher than that observed for ME-344 on the MOLM-13 cell-line, while their cytotoxic effect when combined with cytarabine was similar on HL-60 (Fig. 11).

[0234] In summary, TRX-E-002-1 showed the highest cytotoxicity among the 3 compounds (TRX-E- 002-1, TRX-E-009-1 and ME-344) in combination with venetoclax and azacitidine in the 2 AML cell-lines tested and in combination with cytarabine on MOLM-13.

[0235] As single treatment, the cytotoxic effect was significantly higher for TRX-E-002-1 as compared to TRX-E-009-1 on both AML cell-lines and ME-344 on MOLM-13 (Fig. 9-11).

[0236] Example 4: Dose-response comparisons of cytotoxic effects of TRX-E-002-1, TRX-E-009-1 and ME-344 in AML and MM cell-lines

[0237] The cytotoxic effects of TRX-E-002-1, TRX-E-009-1 and ME-344 were further explored in dose-response assays using 10 graded doses in 4 AML cell-lines (KASUMI-3, HL-60, MOLM- 13, SKNO-1) and 4 MM cell-lines (XG21, XG26, XG28, XG30), to ascertain potential differences among the treatments.

[0238] The 4 AML cell-lines tested showed a higher sensitivity for TRX-E-002-1 as compared to TRX- E-009-1 and ME-344 treatments. The higher sensitivity to TRX-E-002-1 is first evident at the 60 nM (HL-60, MOLM-13, SKNO-1) and 120 nM doses (KASUMI-3) of the dose-response curves (Fig. 12, Fig. 13). The percentage of cell lysis observed at these two doses in the 4 AML cell-lines for all three treatments are shown in the table below:

[0239] (R): Relapse / resistant. The 4 MM cell-lines tested showed a higher sensitivity for TRX-E-002-1 as compared to TRX- E-009-1 and ME-344 treatments. The higher sensitivity to TRX-E-002-1 is first evident at the 40 nM (XG26, XG28, XG30) and 60 nM doses (XG21) of the dose-response curves (Fig. 14, Fig. 15). The percentage of cell lysis observed at these two doses in the 4 MML cell-lines for all three treatments are shown in the table below:

[0240] (R): Relapse / resistant.

[0241] Example 5: TRX-E-002-1 treatment reduces tumor growth and increases survival time in an animal model of AML

[0242] Mice were engrafted with human MOLM-13 cells expressing luciferase to establish a disseminated and relapse / resistant model of AML. This type of model is particularly useful for evaluating therapies that target systemic disease and for studying disease progression and metastasis in a more clinically relevant setting. The human MOLM-13 cell-line is widely used in AML animal models for testing the efficacy of anti-cancer agents as a single treatment and in combination.

[0243] Starting one week post-engraftment, mice were treated with vehicle control (saline) or TRX- E-002-1 at 50, 100 or 150 mg / kg intravenously for 14 days consecutively.

[0244] TRX-E-002-1 significantly reduced tumor growth in all three treatment groups by week five, as shown by a decreased bioluminescent signal as compared to vehicle control treated animals (Fig. 16). In addition, a statistically significant increase in survival time was observed for the mice treated with TRX-E-002-1 at the highest doses, 150 mg / kg and 100 mg / kg, as compared to controls (Fig. 17). Example 6: TRX-E-002-1 in combination with venetoclax and azacitidine improves survival time in an animal model of AML

[0245] Mice were engrafted with human MOLM-13 cells (R / R AML) expressing luciferase to establish a disseminated and relapse / resistant model of AML. Starting one week post- engraftment, mice received treatment with 30 mg / kg venetoclax and 1 mg / kg azacitidine, either alone or in combination with 100 mg / kg TRX-E-002-1. TRX-E-002-1 and venetoclax were administered daily for 14 days while azacitidine was administered daily for 5 consecutive days.

[0246] Mice treated with TRX-E-002-1 in combination with venetoclax and azacitidine had a statistically significant increase in survival time as compared to mice treated with venetoclax and azacitidine (Fig. 18).

[0247] MATERIALS AND METHODS

[0248] Cell-lines

[0249] Ten cell-lines derived from patients with hematological malignancies were used. Acute myeloid leukemia (AML) cell-lines included KASUMI-3, HL-60, MOLM-13, and SKNO-1. MOLM-13 and SKNO-1 were originally derived from relapse patients with mutations associated to drug resistance and will be referred to as "relapse / resistant". A non- exhaustive summary of the characteristics of the AML cell-lines used is provided below:

[0250] HL-60 KASUMI-3

[0251] SKNO-1 (relapse / resistant) 0CI-AML2 MOLM-13 (relapse / resistant)

[0252] Six multiple myeloma (MM) cell-lines were included, XG2, XG11, XG21, XG26, XG28 and XG3O, which were obtained as previously described (Moreaux et al; Haematologica. 2011;96(4):574-582). The MM cell-lines XG21, XG26, XG28 and XG30 were derived from MM relapse patients that had developed drug resistance to at least one line of treatment and will be referred to as "relapse / resistant". The MM-cell lines were classified in molecular subgroups as previously described (Zhan et al; Blood, 2006).

[0253] Cell-cultures Cell-lines were thawed and cultured in RPMI1640 or MEMa medium with 10 to 20% Fetal Calf Serum (FCS) in the presence or absence of Granulocyte-macrophage colony-stimulating factor (GM-CSF) or in the presence or absence of IL-6 (2 ng / mL), according to the supplier's instructions. All cells were maintained at 37°C in 5% CO2 and humidified atmosphere. Cells were seeded every 3-4 days. At each cell splitting, cell viability and cell density were controlled by trypan blue exclusion assay. Before starting the experiments, the cells were confirmed as being mycoplasma negative using the MycoAlert Mycoplasma Detection kit (Lonza).

[0254] Reagents

[0255] The main cell-culture reagents used are summarized in the table below.

[0256] Compounds

[0257] TRX-E-OO2-1, TRX-E-009-1 and ME-344 were solubilized in DMSO at 5mM and stored in aliquots at -80°C. Aliquots were thawed only once. Azacitidine, cytarabine, daunorubicin and venetoclax (Selleckchem) were solubilized in DMSO at 10 mM, 50 mM, 10 mM, and 10 mM, respectively, aliquoted and stored at -80°C. Aliquots were thawed maximum twice.

[0258] In vitro drug treatments

[0259] Drug dilutions were performed using the EpMotion automated pipetting system (Eppendorf). Cells were treated on 96-well plates in single or triplicate wells (1-3 independent experiments). The cell density selected for each cell line was linked to their specific cell culture growth rate. The plates were incubated at 37°C in 5% CO2 atmosphere for 3 days.

[0260] Cell viability assay

[0261] Cells were treated with different compounds for 72 hours. The luminescent cell viability assay Cell Titer-Gio (Promega) was used to determine the cells' cytotoxicity effect of treatment for each condition. Luminescence was measured using a 96-microplate luminometer (Tecan).

[0262] Treatment experimental design

[0263] Example 1:

[0264] Dose-response assays for TRX-E-002-1 were performed in 5 hematological cancer cell-lines including 3 AML cell-lines (KASUMI-3, HL-60, and SKNO-1) and 2 MM cell-lines (XG2 and XG11). Ten graded doses including untreated conditions were used (0 nM, 3.3 nM, 8.2 nM, 20.5 nM, 51.2 nM, 128 nM, 320 nM, 800 nM, 2 pM and 5 pM).

[0265] Example 2:

[0266] Dose-response assays were performed for TRX-E-002-1 in combination with daunorubicin, cytarabine, azacitidine, and venetoclax. For each treatment, the untreated control conditions, and single treatments were assessed in the matrices.

[0267] The dose-range for TRX-E-002-1 (0 nM, 3.75 nM, 7.5 nM, 15 nM, 30 nM and 60nM) was kept constant across all cell-lines, based on the results obtained in Example 1.

[0268] The optimal graded doses for azacitidine, daunorubicin, cytarabine, and venetoclax were selected for each cell-line to maximize the synergistic assay, based on the sensitivity or resistance profile of each cell line. The dose-ranges (5 concentrations) selected for the AML cell-lines HL-60 and MOLM-13 are depicted below:

[0269] Example 3:

[0270] The cytotoxic effect induced by TRX-E-002-1, TRX-E-009-1, and ME-344 in combination with venetoclax, cytarabine or azacitidine was also assessed on AML cell-lines at specific concentrations. For HL-60, the doses tested were: TRX-E-002-1 (60 nM), TRX-E-009-1 (60 nM), and ME-344 (60 nM), venetoclax (2 nM), cytarabine (20 nM) and azacitidine (3 pM). For MOLM-13, the doses tested were: TRX-E-002-1 (45 nM), TRX-E-009-1 (45 nM), and ME- 344 (45 nM), venetoclax (2 nM), cytarabine (20 nM) and azacitidine (50 nM). These doses were selected for each cell-line were based on the sensitivity or resistance profile of each cell line to the different compounds.

[0271] Example 4:

[0272] Dose-response assays for TRX-E-002-1, TRX-E-009-1 and ME-344 were performed in 4 AML cell lines (HL-60, MOLM13, SKNO-1 and KASUMI-3) and 4 MM resistant / relapse cell-lines (XG21, XG26, XG28, XG30). Ten graded doses including untreated conditions were used in the experiment (0 nM, 5 nM, 10 nM, 20 nM, 40 nM, 60 nM, 120 nM, 240 nM, 480 nM and 960 nM). This dose-range was selected based on the results obtained in Example 1.

[0273] Data analysis

[0274] Analyses of raw data, dose-response curves and half maximal inhibitory concentration (IC50) determinations were performed using Excel (Microsoft) and Prism Software (Graph Pad). For all conditions, the mean signal of the controls wells without treatment (Ctrl) was used to normalize the signals.

[0275] Glycolysis scores were derived from RNA sequencing data. The glycolytic status was classified using the quartile method based on the calculated glycolytic scores of the cell-lines used.

[0276] Drug synergy analyses were performed with Synergy Finder R package version 3.2.10 (R software). The degree of synergy among drug combinations was calculated using the Bliss Equation (Greco et al. Pharmacol Rev 1995;47:331-85). Briefly, the observed drug combination response was compared against the expected response, which is calculated using a reference model that assumes no interaction between drugs. Bliss model assumes a stochastic process in which two drugs exert their effects independently, and the expected combination effect can be calculated based on the probability of independent events as yBliss=yA+yB-yA*yB where yBliss is the expected fraction of cells affected by the drug combination in the case of independence in the effect, and yA and yB are the fractions of cells affected by treatment A and B, respectively. The difference between the yBliss value and the fraction of living cells in the cytotoxicity test was considered as an estimation of the interaction effect. Synergy scores were classified as follows: strong synergy >30, intermediate synergy 15-30; minor synergy 10-14, no synergy >10.

[0277] Mouse Models:

[0278] A disseminated xenograft acute myeloid leukemia (AML) model was established by intravenously injecting immunodeficient NGS mice (JAX stock #005557) with 5 x 106MOLM- 13 cells expressing luciferase. Six days post-injection, baseline bioluminescence (BLI) imaging was performed to confirm successful engraftment. Mice were then randomized based on weight and BLI intensity into different treatment groups, as stated below. BLI imaging was conducted weekly, starting from baseline, before treatment initiation to monitor disease progression. Body weight was monitored every day during treatment and three times a week thereafter. Observations for signs of toxicity or pharmacologic effects were made once daily for each study animal. Mice were observed regularly and those reaching the predetermined humane endpoints were euthanised. Survival was recorded for each group to determine the treatment's efficacy.

[0279] Compounds

[0280] TRX-E-002-1 formulated in 20% cyclodextrin was provided by Vivesto and stored at 5°C in 10 ml vials (20 mg / ml) protected from light until dosed. Venetoclax (MedChemExpress, Cat. No. HY-15531) was formulated in 10% EtOH, 30% PEG 400 and 60% Phosal. Azacitidine (Sigma-Aldrich, Cat. No. A2385) was dissolved in sterile saline before administration.

[0281] Treatment experimental design

[0282] Example 5: In the first experimental set-up, mice were randomized into four treatment groups (n = 8 per group): Vehicle control (saline); TRX-E-002-1 (50 mg / kg); TRX-E-002-1 (100 mg / kg); TRX- E-002-1 (150 mg / kg). TRX-E-002-1 was administered intravenously (IV) once a day (QD) for 14 consecutive days.

[0283] Example 6:

[0284] In the second experimental set-up, mice were randomized into two treatment groups (n =

[0285] 10 per group). Mice in the standard-of-care group were treated with venetoclax (30 mg / kg) and azacitidine (1 mg / kg) whereas mice in the triple combination group received TRX-E-002- 1 (100 mg / kg) in addition to venetoclax (30 mg / kg) and azacitidine (1 mg / kg). TRX-E-002-1 and venetoclax were administered intravenously (IV) and per oral (PO) respectively, once a day (QD) for 14 consecutive days. Azacitidine was administered intra peritonially (IP) once a day for 5 consecutive days. Due to technical issues with the BLI system, disease progression was not monitored by optical imaging in this experiment.

[0286] Statistical analysis in vitro experiments All statistical analyses were performed using Prism Software (Graph Pad). Unpaired t-tests were used for comparisons between two groups. One-way ANOVA followed by Tukey's multiple comparison test was used to assess differences between at least three groups, with all pairwise comparison evaluated. Error bars represent mean values ± standard deviation (SD). P values <0.05 were considered statistically significant.

[0287] Statistical analysis in vivo experiments

[0288] All statistical analyses were performed using Prism Software (Graph Pad). One-way ANOVA followed by Dunnett's multiple comparison test was used to assess differences between at least three groups, with each treatment group compared to the control. Survival curves were compared using the log-rank (Mantel-Cox) test. Data represent mean values ± standard error of the mean (SEM). P values <0.05 were considered statistically significant.

Claims

64CLAIMS1. (3R, 4S)-3-(4-hydroxy-3,5-dimethoxyphenyl)-4-(4-hydroxyphenyl)-8-methyl-3,4- dihydro-2 / 7-chromen-7-ol (TRX-E-002-1) for use in a method of treatment for acute myeloid leukemia (AML), wherein the method comprises administering to the subject effective amounts of TRX-E-002-1 and a second compound selected from the group consisting of (i) a BCL- 2 inhibitor such as venetoclax, navitoclax or obatoclax and (ii) a hypomethylating agent such as azacitidine or decitabine.

2. TRX-E-002-1 for use according to claim 1, wherein the second compound is a BCL-2 inhibitor.

3. TRX-E-002-1 for use according to claim 2, wherein the second compound is selected from the list consisting of: venetoclax, navitoclax, obatoclax, lisaftoclax, pelcitoclax, sonrotoclax, asaretoclax (ZN-d5), lacutoclax, oblimersen, S55746 (BCL201, CAS No. 1448584-12-0), S65487 (VOB560, prodrug of S55746, CAS Nos. 1644600-79-2 , 1644543-95-2, 2416937-01-2 depending on salt form), AZD4320 (CAS No. 1357576- 48-7, including its PEGylated / poly-lysine conjugated version AZD0466), R-(-)- enantiomer of gossypol (AT 101, CAS No. 866541-93-7 for acetic acid salt, CAS No. 90141-22-3 for free base), LOXO-338 (FCN-338, LY3847429, CAS No. 2248046-39-9) and BM-1197 (UBX1967, CAS No. 1391107-89-3).

4. TRX-E-002-1 for use according to claim 3, wherein the second compound is venetoclax, lisaftoclax, sonrotoclax or asaretoclax.

5. TRX-E-002-1 for use according to claim 4, wherein the second compound is venetoclax.

6. TRX-E-002-1 for use according to claim 1, wherein the second compound is a hypomethylating agent.

7. TRX-E-002-1 for use according to claim 6, wherein the second compound is selected from the list consisting of: azacitidine, decitabine, guadecitabine (SG 1-110, CAS No. 929904-85-8), 5-Fluoro-2'-deoxycytidine (FdCyd, CAS No. 10356-76-0), nanaomycin A (CAS No. 52934-83-5), 4-Deoxyuridine (zebularine, CAS No. 3690-10-6), N-Phtha lyl-L- tryptophan (RG108, CAS No. 48208-26-0), 3-Bromo-3-nitroflavanone (CAS No. 6513-6551-5), SGI-1027 (CAS No. 1020149-73-8), MC3343 (CAS No. 1535187-91-7), and 5- aza-4'-thio-2'-deoxycytidine (NTX-301, CAS No. 169514-76-5).

8. TRX-E-002-1 for use according to claim 7, wherein the second compound is azacitidine, decitabine or guadecitabine.

9. TRX-E-002-1 for use according to claim 8, wherein the second compound is azacitidine.

10. TRX-E-002-1 for use according to any of claims 2-5, wherein the method comprises administering an effective amount of a third compound being a hypomethylating agent.

11. TRX-E-002-1 for use according to claim 10, wherein the third compound is selected from the list consisting of: azacitidine, decitabine, guadecitabine (SG 1-110, CAS No. 929904-85-8), 5-Fluoro-2'-deoxycytidine (FdCyd, CAS No. 10356-76-0), nanaomycin A (CAS No. 52934-83-5), 4-Deoxyuridine (zebularine, CAS No. 3690-10-6), N-Phtha lyl-L- tryptophan (RG108, CAS No. 48208-26-0), 3-Bromo-3-nitroflavanone (CAS No. 6513- 51-5), SGI-1027 (CAS No. 1020149-73-8), MC3343 (CAS No. 1535187-91-7) and 5-aza- 4'-thio-2'-deoxycytidine (NTX-301, CAS No. 169514-76-5).

12. TRX-E-002-1 for use according to claim 11, wherein the third compound is azacitidine, decitabine or guadecitabine.

13. TRX-E-002-1 for use according to claim 12, wherein the third compound is azacitidine.

14. TRX-E-002-1 for use according to any of the preceding claims, wherein the AML is selected from the list consisting of: (i) AML with defining genetic abnormalities (e.g., RUNX1::RUNX1T1 fusion, CBFB::MYH11 fusion, PML::RARA fusion, KMT2A rearrangement, DEK::NUP214 fusion, MECOM rearrangement, RBM15::MRTFA fusion, BCR::ABL1 fusion, NUP98 rearrangement, NPM1 mutation, CEBPA mutation or other defined genetic alteration), (ii) AML defined by differentiation (e.g., AML with minimal differentiation, AML without maturation, AML with maturation, acute myelomonocytic leukemia, acute monoblastic / monocytic leukemia, pure erythroid leukemia, acute megakaryoblastic leukemia, acute basophilic leukemia), (iii) AML with myelodysplasia-related changes (AML-MR), (iv) therapy-related AML (e.g. FLT366 mutation, IDH1 / 2 mutation), (v) AML not otherwise specified (NOS) and (vi) myeloid sarcoma.

15. TRX-E-002-1 for use according to any of the preceding claims, wherein the AML is an AML with a FAB classification of MO, Ml, M2, M3, M4, M5, M6 or M7.

16. TRX-E-002-1 for use according to any of the preceding claims, wherein the method comprises administering TRX-E-002-1 to a human subject in need thereof a dose being from 1 mg / kg to 25 mg / kg per administration.

17. TRX-E-002-1 for use according to any of the preceding claims, wherein the method comprises administering at least two doses of TRX-E-002-1 separated by one to seven days to a human subject in need thereof.

18. TRX-E-002-1 for use according to any of the preceding claims, wherein the method comprises administering TRX-E-002-1 intravenously to a human subject in need thereof.

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