AST-3424 combined with other therapies for leukemia and lymphoma

Abstract

Treatment method, using drugs containing AST-3424, AST-3423 or AST-2870 and their salts, esters, solvates, isotopomers in combination with drugs containing cytarabine or daunorubicin and their salts, esters, solvates, isotopomers to treat patients with leukemia and lymphoma. When the concentration of daunorubicin or cytarabine is much lower than its clinical dosage, it is not necessary to limit the patient to have a high (overexpressed, excessive) AKR1C3 protein or RNA expression. The combination of AST-3424 and cytarabine or daunorubicin still has a significant effect on the in vitro proliferation inhibition of leukemia and lymphoma cells. The combination has a good in vitro proliferation inhibition effect on cell lines with different AKR1C3 protein or RNA expression levels. For this reason, the above-mentioned combination regimen is expected to be developed as a new treatment regimen for the treatment of leukemia and lymphoma without limiting the patient's AKR1C3 enzyme expression level.

Description

AST-3424 combination therapy for leukemia and lymphoma Technical Field

[0001] The present invention relates to a method for treating tumors, in particular to the combined use of AST-3424 and drugs to treat leukemia and lymphoma, belonging to the field of tumor treatment. Background Art

[0002] AST-3424 (WO2016145092, WO2017087428), a DNA alkylating agent prodrug targeting overexpressed aldehyde-keto reductase 1C3 (AKR1C3), has a CAS number of 2097713-69-2 and its structure is as follows:

[0003] AST-3424 (also known as OBI-3424, TH-3424) enters cancer cells and is activated by the AKR1C3 enzyme overexpressed by cancer cells to release the metabolite AST-2660 (also known as AST-2660). AST-3424 itself has low toxicity to cancer cells. In its animal models and in vitro pharmacological experiments, its pharmacological effects are related to the expression of AKR1C3 enzyme: the prodrug AST-3424 is metabolized to AST-2660 under the action of AKR1C3 enzyme and NADPH, and the expression level of the enzyme is positively correlated with the efficacy (Meng F, Li WF, Jung D, et al. A novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad anti-tumor activity. Am J Cancer Res. 2021; 11(7): 3645-3659; Evans K, Duan J, Pritchard T, et al. OBI-3424, a Novel AKR1C3-Activated Prodrug, Exhibits Potent Efficacy against Preclinical Models of T-ALL. Clin Cancer Res.2019;25(14):4493-4503.doi:10.1158 / 1078-0432.CCR-19-0551;WangY,LiuY,Zhou C,et al.An AKR1C3-specific prodrug with potent anti-tumor activities against T-ALL.Leuk Lymphoma.2020;61(7):1660-1668.doi:10.1080 / 10428194.2020.1728746; He P,Wang C,WangY,et al.A Novel AKR1C3 Specific Prodrug TH3424 With Potent Antitumor Activity in Liver Cancer [retracted in: Clin Pharmacol Ther.2021 Jul;110(1):262].Clin Pharmacol Ther. 2021, 110(1):229-237.doi:10.1002 / cpt.2171).

[0004] Clinical trials (US NCT03592264, castration-resistant prostate cancer and liver cancer; US NCT04315324, T-ALL T-lymphocytic acute leukemia; China CTR20191399, various solid tumors; CTR20201915, T-lymphocytic acute leukemia and B-lymphocytic acute leukemia) all include exploring the relationship between AKR1C3 and the efficacy of AST-3424. Furthermore, in the published Phase I clinical trial results (Safety, pharmacokinetics, and clinical activity of OBI-3424, an AKR1C3-activated prodrug, in patients with advanced or metastatic solid tumors: A phase 1 dose-escalation study. J Clin Oncol 40, 2022 (suppl 16; abstr 3030), DOI: 10.1200 / JCO.2022.40.16_suppl.3030) showed that patients would only respond to the drug when the AKR1C3 expression score (H-score) detected by IHC method was higher than a certain value, and it was decided to select patients with an H-score greater than or equal to 135 for inclusion in subsequent clinical trials.

[0005] Similar conclusions were repeated in the combination experiments, that is, AST-3424 combined with abiraterone had better efficacy than single drugs in the CDX model of VCap cells with high expression of AKR1C3, in combination with prednisolone for the CDX model of VCap cells with high expression of AKR1C3, in combination with 5-fluorouracil for the CDX model of SNU-16 cells with high expression of AKR1C3, in combination with sunitinib for the CDX model of A498 cells with high expression of AKR1C3, and in combination with gemcitabine for the CDX model of A498 cells with high expression of AKR1C3 (Meng F, Li WF, Jung D, et al. A novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad anti-tumor activity. Am J Cancer Res. 2021; 11(7): 3645-3659).

[0006] In particular, the latest research has found that AST-3424 has excellent inhibitory effects on HCC cell lines HepG2 and PLC / PRF / 5, which have high AKR1C3 expression. The inhibitory effect is even stronger when combined with oxaliplatin (OXA) or 5-fluorouracil (5-FU), showing a synergistic effect. Through various experimental methods, it was found that AST-3424 monotherapy combined with OXA or 5-FU can exert a synergistic anti-tumor effect on liver cancer cells in vitro by inhibiting cell proliferation, inducing S-phase cell arrest, promoting cell apoptosis induction, and inhibiting cell metastasis.

[0007] West-bolt test showed that its mechanism may be related to the upregulation of P21 protein, but may not be related to the expression of β-catenin.

[0008] Flow cytometry analysis of the percentage of cells in different phases indicated that AST-3424 monotherapy and combination therapy with OXA or 5-Fu could block cells in the S phase. Flow cytometry analysis of the induction of apoptosis after AST-3424 monotherapy and combination therapy with OXA or 5-Fu indicated that AST-3424 monotherapy and combination therapy with OXA or 5-Fu may induce apoptosis by upregulating Bax and Caspase3 proteins and downregulating PARP protein.

[0009] Transwell cell migration and invasion assays showed that AST-3424 monotherapy and combined treatment with OXA or 5-Fu inhibited tumor cell migration and invasion, and the mechanism may be related to the downregulation of MMP-9 protein;

[0010] By studying the effects of AST-3424 monotherapy and OXA or 5-Fu combination therapy on the TGF-β signaling pathway, the experimental results showed that AST-3424 monotherapy and OXA or 5-Fu combination therapy can reduce the proliferation effect by inhibiting the TGF-β signaling pathway, and the inhibitory effect is significant only when AST-3424 and OXA are used in combination.

[0011] Summary of the Invention

[0012] Existing studies have shown that the efficacy of AST-3424 is correlated with AKR1C3 enzyme expression, with patients or animal models with high AKR1C3 enzyme expression exhibiting better efficacy. Published studies of drug combinations (abiraterone, prednisolone, 5-fluorouracil, sunitinib, oxaliplatin) in vitro cell proliferation experiments and CDX animal models also correlate with AKR1C3 enzyme expression. These studies have shown that AST-3424, whether used as a single agent or in combination therapy, requires patients to have a high level of AKR1C3 enzyme expression (as detected by kits, such as IHC / q-PCR).

[0013] The inventors found in combination studies that the in vitro proliferation inhibition effect of AST-3424 in combination with cytarabine or daunorubicin on leukemia and lymphoma cells was independent of the AKR1C3 expression level in the cell lines. The combination had a significant in vitro proliferation inhibitory effect on cell lines with varying expression levels. Therefore, this combination regimen is expected to be developed as a new treatment option for leukemia and lymphoma, without the need to restrict patients to overexpress or high expression of the AKR1C3 enzyme (often corresponding to higher RNA quantification values ​​and higher IHC H-scores), as previously suggested in research literature.

[0014] The new treatment regimen has significantly lowered the threshold for medication use for patients who benefit. If the test result is positive and there is expression, this combination regimen can be used, as long as medical professionals determine that the application of this regimen for treatment is beneficial or may benefit the patient.

[0015] Treatment method, using a drug containing AST-3424, AST-3423 or AST-2870 and their salts, esters, solvates, or isotopomers in combination with a drug containing cytarabine or daunorubicin and their salts, esters, solvates, or isotopomers to treat patients with leukemia or lymphoma. "AST-3424, AST-3423 or AST-2870 and their salts, esters, solvates, or isotopomers" is a list of seven options, namely AST-3424, AST-3423, AST-2870, salts of AST-3424, AST-3423 or AST-2870, esters of AST-3424, AST-3423 or AST-2870, solvates of AST-3424, AST-3423 or AST-2870, and isotopomers of AST-3424, AST-3423 or AST-2870. "Cytarabine or daunorubicin and their salts, esters, solvates, and isotopic isomers" are 6 parallel options, namely cytarabine, daunorubicin, salts of cytarabine or daunorubicin, esters of cytarabine or daunorubicin, solvates of cytarabine or daunorubicin, and isotopic isomers of cytarabine or daunorubicin.

[0016] The pharmaceutical use of AST-3424, AST-3423 or AST-2870 and their salts, esters, solvates and isotope isomers is used to prepare a drug for treating leukemia or lymphoma in combination with a drug containing daunorubicin or cytarabine and their salts, esters, solvates and isotope isomers.

[0017] According to the data disclosed in patents WO2016145092 and WO2017087428, AST-3423 is the enantiomer of AST-3424, AST-3424 is the S configuration, AST-3423 is the R configuration, and AST-2870 (TH2870) is a racemate. The three have similar AKR1C3 enzyme activation properties and are all catalyzed by NADPH and AKR1C3 to release the cytotoxic substance AST-2660 that exerts its pharmacological effect.

[0018] The drug described herein refers to a medicine or preparation, wherein the prepared medicine contains an active ingredient or its salt or solvate within a specific dosage range, and / or the prepared medicine is in a specific dosage form and is administered by a specific administration method.

[0019] The prepared medicine, drug, preparation may also include pharmaceutically acceptable adjuvants or excipients. The medicine may be in any dosage form for clinical use, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small needles for injection, freeze-dried powder for injection, or large infusions. Depending on the specific dosage form and mode of administration, the pharmaceutically acceptable adjuvants or excipients in the medicine may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, adhesives, fillers, flavorings, sweeteners, antioxidants, surfactants, preservatives, wrappers, and pigments.

[0020] Monotherapy refers to single-drug therapy. Combination therapy refers to combined drug therapy. Monotherapy refers to the use of only one anticancer drug in a single course of treatment. Combination therapy refers to the use of two or more anticancer drugs simultaneously or sequentially in a single course of treatment.

[0021] Generally speaking, combination therapy requires exploring different dosages and dosing cycles based on the characteristics of the disease and the types of combined drugs. Only based on the above conditions can the combination drug treatment plan explored achieve better therapeutic effects than single drug treatment.

[0022] The drug dosage, administration cycle and administration regimen of monotherapy and combination therapy are explored through clinical trials.

[0023] Regarding the compounds described herein, their chemical structures contain organic amine structures and P=O double bond structures, and therefore, the compounds may also be administered in the form of salts. That is, the present invention provides pharmaceutically acceptable salts of the compounds, which may be basic salts, including salts formed with inorganic bases (e.g., alkali metal hydroxides, alkaline earth metal hydroxides, etc.) or with organic bases (e.g., monoethanolamine, diethanolamine, or triethanolamine, etc.). Alternatively, the salts may be acidic salts, including salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid, or phosphoric acid, etc.) or with organic acids (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, citric acid, etc.). Similarly, they may react with certain acids or alcohols to form esters, and therefore, the compounds may also be administered in the form of esters. For various reasons, these compounds may also form solvates with certain solvents, such as hydrates or alcoholates, and thus the compounds may also be administered in the form of solvates. The selection and preparation of acceptable salts, esters and solvates of compounds are well known in the art.

[0024] The term "isotopic variant" refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound. In certain embodiments, an "isotopic variant" of a compound contains unnatural proportions of one or more isotopes, including but not limited to hydrogen ( 1 H), deuterium ( 2 H), tritium ( 3 H), carbon-11 ( 11 C), carbon-12 ( 12 C), carbon-13 ( 13 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), oxygen-16 ( 16 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O), fluorine-17 ( 17 F), fluorine-18 ( 18 F), phosphorus-31 ( 31 P), phosphorus-32 ( 32 P), phosphorus-33 ( 33 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-35 ( 35S), sulfur-36 ( 36 S), chlorine-35 ( 35 Cl), chlorine-36 ( 36 Cl), chlorine-37( 37 Cl), bromine-79( 79 Br), bromine-81( 81 Br), iodine-123 ( 123 I), iodine-125( 125 I), iodine-127( 127 I), iodine-129( 129 I) and iodine-131( 131 I). In certain embodiments, an "isotopic variant" of a compound is a stable form, i.e., non-radioactive. In certain embodiments, an "isotopic variant" of a compound contains unnatural proportions of one or more isotopes, including but not limited to hydrogen ( 1 H), deuterium ( 2 H), carbon-12 ( 12 C), carbon-13 ( 13 C), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-16 ( 16 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O), fluorine-17 ( 17 F), phosphorus-31 ( 31 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-36 ( 36 S), chlorine-35 ( 35 Cl), chlorine-37( 37 Cl), bromine-79( 79 Br), bromine-81( 81 Br) and iodine-127( 127 I). In certain embodiments, an "isotopic variant" of a compound is an unstable form, i.e., radioactive. In certain embodiments, an "isotopic variant" of a compound contains unnatural proportions of one or more isotopes, including but not limited to tritium ( 3 H), carbon-11 ( 11 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), fluorine-18 ( 18 F), phosphorus-32 ( 32 P), phosphorus-33 ( 33P), sulfur-35( 35 S), chlorine-36 ( 36 Cl), iodine-123 ( 123 I), iodine-125( 125 I), iodine-129( 129 I) and iodine-131( 131 I) It is understood that in the compounds provided herein, any hydrogen may be replaced by, for example, 2 H is D, or any carbon can be, for example 13 C, or any nitrogen may be e.g. 15 N, and any oxygen may be 18 O. In certain embodiments, an "isotopic variant" of a compound contains unnatural proportions of deuterium (D).

[0025] Leukemia, also known as blood cancer, is a malignant tumor of the hematopoietic system. The cause of the disease is the abnormal functioning of the hematopoietic tissue in the bone marrow due to mutations in the deoxyribonucleic acid in the cells. There are many types of leukemia, and the types of leukemia are mainly distinguished by the types of abnormal blood cells in the blood. Academically, there are many classification methods, the commonly used classification methods are the FAB classification method and the new WHO classification method promoted by the World Health Organization. These classification methods can provide guidance for patient prognosis and treatment. Clinically, it is generally divided into acute leukemia and chronic leukemia, including:

[0026] Acute lymphoblastic leukemia (ALL) can be diagnosed and divided into different subtypes using immunological techniques based on the different differentiation antigens on the surface of leukemia cells. They are generally divided into T and B cell lineages, namely acute T lymphoblastic leukemia (T-ALL) and acute B lymphoblastic leukemia (B-ALL).

[0027] Acute myeloid leukemia, also known as acute myeloid leukemia, Acute myeloid leukemia, referred to as AML, is a blood cancer characterized by abnormal proliferation of bone marrow white blood cells (rather than lymphocyte white blood cells);

[0028] Chronic lymphocytic leukemia, also known as CLL, mainly includes chronic B-lymphocytic leukemia (B-CLL) and chronic T-lymphocytic leukemia (T-CLL), but it is rare.

[0029] Chronic myeloid leukemia (commonly known as "CML"), also known as chronic myeloid leukemia, Chronic myeloid leukemia, referred to as CML;

[0030] Lymphoma, a malignant tumor originating from lymph nodes or other lymphoid tissues, can be divided into two major categories: Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL). Non-Hodgkin's lymphoma is divided into two major categories: B-cell lymphoma and T-cell and NK-cell lymphoma.

[0031] B-cell lymphoma is divided into two major categories: precursor B-cell lymphoma and mature B-cell lymphoma. Precursor B-cell lymphoma refers to precursor B-lymphoblastic lymphoma, while mature B-cell lymphomas are more common clinically, including small cell lymphoma, plasma cell myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse B-cell lymphoma, Burkitt lymphoma, etc.

[0032] The classification of T-cell and NK-cell lymphomas also includes precursor T-cell lymphoma and mature T-cell lymphoma. Precursor T-cell lymphoma mainly refers to precursor T-lymphoblastic lymphoma. Common mature T-cell lymphomas include T-cell prolymphocytic leukemia, adult T-cell lymphoma, extranodal NK-T lymphoma, anaplastic large cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, etc.

[0033] Particularly, the leukemia includes lymphocytic leukemia (including acute lymphocytic leukemia and chronic lymphocytic leukemia), and the lymphoma includes lymphoblastic lymphoma.

[0034] Furthermore, the leukemia is acute lymphocytic leukemia, preferably acute T-lymphocytic leukemia, and the lymphoma is T-lymphoblastic lymphoma.

[0035] In particular, leukemia includes acute lymphoblastic leukemia, acute lymphoblastic T-cell leukemia (T-ALL), T-cell leukemia, and acute lymphoblastic leukemia; lymphoma includes T lymphoblastoma, specifically T lymphoblastic lymphoma and T cell lymphoblastic lymphoma.

[0036] The dosage of daunorubicin and cytarabine in combination therapy is lower than the clinical dosage of their single drugs.

[0037] Daunorubicin, clinically used is its hydrochloride, i.e., daunorubicin hydrochloride, in the form of sterile powder for injection. Before use, it is dissolved in normal saline for injection and then administered intravenously or by drip. It is mainly used clinically to treat:

[0038] (1) Acute myeloid leukemia. Whether used alone or in combination with other anti-tumor drugs, daunorubicin is suitable for the treatment of all stages of the disease. It is also used to treat promyelocytic leukemia.

[0039] (2) Acute lymphocytic leukemia: Daunorubicin is used to treat this disease, with a high remission rate. However, due to its significant side effects and the availability of other effective treatments, daunorubicin is only suitable for patients who have developed resistance to other drugs.

[0040] (3) Other tumors: Daunorubicin has been observed to have a good therapeutic effect on neuroblastoma and rhabdomyosarcoma.

[0041] Cytarabine is clinically used as cytarabine for injection (using either cytarabine or cytarabine hydrochloride). It is administered as a lyophilized powder. Before use, benzyl alcohol and water are added to prepare a benzyl alcohol injection (generally sold together with the lyophilized powder) for intravenous or subcutaneous injection. It is primarily indicated for the induction and maintenance treatment of acute non-lymphocytic leukemia in adults and children. It also has therapeutic effects on other types of leukemia, such as acute lymphocytic leukemia and chronic myeloid leukemia (blast crisis). It can be used alone or in combination with other anti-tumor drugs, with combined therapy offering enhanced efficacy.

[0042] The AKR1C3 corresponding RNA test in the blood sample of the leukemia patient is positive, and the AKR1C3 protein test in the tumor tissue sample of the lymphoma patient is positive;

[0043] or

[0044] The leukemia patient was detected as positive for RNA corresponding to AKR1C3, and the lymphoma patient was detected as positive for AKR1C3 protein.

[0045] Previous studies and experimental papers have shown that AST-3424 monotherapy or combination therapy is limited to animal models or patients with excessive or overexpressed AKR1C3. However, this treatment regimen / pharmaceutical application involves the combination of AST-3424 and daunorubicin / cytarabine for the treatment of leukemia / lymphoma, which only requires a positive AKR1C3 protein or RNA test, i.e., expression, and still has significant therapeutic effects in the positive case.

[0046] Overexpression means that the expression level of cancer cells is higher than that of normal cells. This situation is called overexpression. Alternatively, based on statistics, a threshold is set artificially, and overexpression means exceeding this threshold. For information on the overexpression or overexpression of AKR1C3 in different tumors (solid tumors or blood cancers), please refer to Evans K, Duan J, Pritchard T, et al. OBI-3424, a Novel AKR1C3-Activated Prodrug, Exhibits Potent Efficacy against Preclinical Models of T-ALL. Clin Cancer Res. 2019; 25(14): 4493-4503. doi: 10.1158 / 1078-0432. CCR-19-0551 (disclosing the expression of AKR1C3 in acute lymphoblastic leukemia) and Guise CP, Abbattista MR, Singleton RS, et al. The bioreductive prodrug PR-104A is activated under aerobic conditions by human aldo-keto reductase 1C3. Cancer Res. 2010; 70(4): 1573-1584. doi: 10.1158 / 0008-5472.CAN-09-3237 (discloses the expression of AKR1C3 in different solid tumors).

[0047] Previous AKR1C3 enzyme-activated anticancer prodrugs often required testing AKR1C3 enzyme expression levels before medication and clarifying that the treatment plan was only effective for patients with high or overexpression of AKR1C3. However, the new combination drug combination provided this time has almost no correlation with AKR1C3 in its drug effect. Patients only need to have AKR1C3 expression, that is, a positive test result, to be effective.

[0048] Furthermore, the AKR1C3 corresponding RNA detection Log2FPKM quantitative value of the blood sample of the leukemia patient is greater than or equal to 0.71, and the AKR1C3 protein quantitative value of the tumor tissue sample of the lymphoma patient is the ratio of the AKR1C3 protein band to the reference protein band in the protein band detected by western blot (WB), which is greater than or equal to 0.84;

[0049] or

[0050] The leukemia patient was detected to have RNA corresponding to AKR1C3, and the Log2FPKM quantitative value was greater than or equal to 0.71. The lymphoma patient was detected to have AKR1C3 protein quantitatively, which was the ratio of the AKR1C3 protein band to the reference protein band in the protein band detected by western blot (WB), and the value was greater than or equal to 0.84.

[0051] If the above conditions are met, the effect may be better.

[0052] In other words, this combination regimen broadens the population of patients who can benefit from AST-3424 treatment. Previous technical literature (Evans K, Duan J, Pritchard T, et al. OBI-3424, a Novel AKR1C3-Activated Prodrug, Exhibits Potent Efficacy against Preclinical Models of T-ALL. Clin Cancer Res. 2019; 25(14): 4493-4503. doi: 10.1158 / 1078-0432. CCR-19-0551; Guise CP, Abbattista MR, Singleton RS, et al. The biore induced prodrug PR-104A is activated under aerobic conditions by human aldo-keto reductase 1C3. Cancer Res.2010;70(4):1573-1584.doi:10.1158 / 0008-5472.CAN-09-3237;Safety,pharma cokinetics,and clinical activity of OBI-3424,an AKR1C3-activated prodrug,in patients with advanced or metastatic solid tumors:A phase 1 dose-escalation study.J Clin Oncol 40,2022(suppl 16;abstr 3030), DOI: 10.1200 / JCO.2022.40.16_suppl.3030), revealed that the population benefiting from AST-3424 in the treatment of leukemia or solid tumors is strictly limited to patients with excessive or underexpressed AKR1C3 protein or RNA (often corresponding to higher RNA quantification values ​​and higher IHC H-score scores). However, the new treatment regimen significantly lowers the threshold for patients to receive medication. Before administering this combination therapy to patients for the treatment of leukemia / lymphoma, the AKR1C3 assay result does not need to be overexpressed / overexpressed as previously defined in the literature. If the test result is positive and there is expression, the combination regimen can be administered, as long as medical professionals determine that the administration of this regimen will benefit or may benefit the patient.

[0053] The present application also provides a pharmaceutical combination comprising the following substances as active ingredients:

[0054] AST-3424, AST-3423 or AST-2870 and their salts, esters, solvates, isotopic isomers, and

[0055] Daunorubicin and its salts, esters, solvates, isotopic isomers, or

[0056] Cytarabine and its salts, esters, solvates, and isotopic isomers;

[0057] The active ingredients are formulated together or separately for combined use, simultaneous use or separate use.

[0058] The drug combination of the present application includes at least two active ingredients, such as AST-3424 and daunorubicin, or AST-3424 and cytarabine, and may also include pharmaceutically acceptable excipients.

[0059] Typically, the two active ingredients that characterize the pharmaceutical combinations described herein may be formulated either together (single dosage unit) or separately (kit).

[0060] The two active ingredients, formulated together or separately, may generally be administered simultaneously; or, given the nature of their respective formulations, separately with a time interval which allows for the optimization of the synergistic effect of the two active ingredients.

[0061] The drug combination can be a compound drug comprising one active ingredient being AST-3424, AST-3423, AST-2870, or their salts, esters, solvates, or isotopomers, and another active ingredient being daunorubicin, cytarabine, or their salts, esters, solvates, or isotopomers. In this case, the active ingredients are co-formulated and administered simultaneously.

[0062] A drug combination can also be a packaged product, similar to Pfizer's COVID-19 drug Paxlovid, that can be sold together or separately. In this case, the active ingredients are formulated separately, but the two or more separately formulated drugs can be administered simultaneously or at intervals, depending on the properties of their respective formulations.

[0063] As a specific embodiment of the present application, when formulated separately, one drug contains the active ingredients AST-3424, AST-3423, AST-2870 or their salts, esters, solvates, isotopomers, and pharmaceutically acceptable excipients, and the other drug contains the active ingredients daunorubicin and cytarabine or their salts, esters, solvates, isotopomers, and pharmaceutically acceptable excipients. The drug combination consisting of the above two drugs can be administered simultaneously or separately at a certain time interval depending on the properties of each formulation.

[0064] Among the drug combinations, the combined dose of daunorubicin and cytarabine is lower than the clinical dose of their single drugs.

[0065] The present application also provides a pharmaceutical preparation, which is prepared from the active ingredients of the above-mentioned drug combination and pharmaceutically acceptable excipients.

[0066] As a specific embodiment of the present application, the dosage form of the above-mentioned pharmaceutical preparation is selected from granules, tablets, pills, capsules or injections.

[0067] The present application also provides the use of the above-mentioned drug combination and / or the above-mentioned drug preparation in the preparation of anti-tumor drugs.

[0068] As a specific embodiment of the present application, the above-mentioned tumor is selected from leukemia and lymphoma.

[0069] As a preference, leukemia includes lymphocytic leukemia, and lymphoma includes lymphoblastic lymphoma; preferably, the leukemia is acute lymphocytic leukemia, more preferably acute T-lymphocytic leukemia, and the lymphoma is T-lymphoblastic lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 shows the proliferation inhibition curves of five cell lines by three drugs;

[0071] FIG2 shows the experimental results of the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on the proliferation of SUP-T1 cells;

[0072] FIG3 shows the experimental results of the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on CCRF-CEM cell proliferation;

[0073] FIG4 shows the experimental results of the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on the proliferation of PF-382 cells;

[0074] FIG5 shows the experimental results of the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on the proliferation of Jurkat and CloneE6-1 cells;

[0075] FIG6 shows the experimental results of the effects of AST-3424, daunorubicin, and cytarabine alone or in combination on the proliferation of MOLT-4 cells.

[0076] In Figure 2, Ara-c represents cytarabine, and Daunorubicin represents daunorubicin. The 4th, 5th, and 6th bar graphs from left to right represent the cell survival percentages of 0.1nM, 20nM, and 80nM daunorubicin alone, respectively. The 7th, 8th, and 9th bar graphs to the right correspond to the cell survival percentages of 0.1nM, 20nM, and 80nM daunorubicin combined with 0.13nM AST-3424, respectively. The 10th, 11th, and 12th bar graphs to the right correspond to the cell survival percentages of 0.1nM, 20nM, and 80nM daunorubicin combined with 1nM AST-3424, respectively. The contents of the remaining Figures 2 to 6 are similar. Example

[0077] 1. In vitro cell experiments

[0078] 1.1 Single-drug in vitro cell proliferation inhibition experiment

[0079] Steps

[0080] On the first day, cells were seeded in 96-well cell culture plates (180 μL / well).

[0081] On the next day, 20 μL of a 10-fold test concentration of the compound working solution (AST-3424) was added, the cell plate was gently shaken, and the plate was placed in a 37° C. incubator for further culture.

[0082] On the third day, 20 μL of a 10-fold test concentration of compound (cytarabine and daunorubicin) solution was added, the cell plate was gently shaken, and the plate was placed in a 37° C. incubator for further culturing for 72 hours.

[0083] After incubation, add the prepared reagent (50 μL / well) to the cell plate according to the instructions of the CellTiter Glo reagent, mix thoroughly, and incubate at room temperature in the dark for 10 minutes.

[0084] Place the cell plate into the plate reader for analysis, set to read chemiluminescence and record the data.

[0085] Data processing

[0086] The readings in each well need to be converted into cell viability. Cell viability can be calculated using the following formula:

[0087] The processed data were analyzed by nonlinear regression using GraphPad Prism 5 software to obtain the dose-effect curve, as shown in Figure 1, and the half-maximal lethal concentration (IC 50 ), as shown in Table 1 below.

[0088] Table 1: Data on the proliferation inhibition of different drugs on different cell lines

[0089] Cytarabine, daunorubicin, and the compound AST-3424 all demonstrated potent inhibitory effects in five cell lines: CCRF-CEM, PF-382, Jurkat, Clone E6-1, SUP-T1, and MOLT-4. AST-3424 demonstrated superior in vitro cytotoxicity to daunorubicin and cytarabine in all but Jurkat and Clone E6-1 cells.

[0090] 1.2 In vitro cell proliferation inhibition experiment using drug combination

[0091] Steps

[0092] On the first day, cells were seeded in 96-well cell culture plates (180 μL / well).

[0093] On the next day, 10 μL of 20-fold test concentration AST-3424 solution (two concentrations of IC 25 or IC 10 The cell plate was gently shaken and placed in a 37°C incubator for further culture.

[0094] On the third day, 10 μL of 20-fold test concentration compound (cytarabine or daunorubicin) (three concentrations of IC 50 、IC 25 and 20 times the concentration of IC5), 10 μL of culture medium containing 2% DMSO was added to the wells where the drug was used alone, the cell plate was gently shaken, and the plate was placed in a 37°C incubator for further culture for 72 hours.

[0095] After incubation, add the prepared reagent (50 μL / well) to the cell plate according to the instructions of the CellTiter Glo reagent, mix thoroughly, and incubate at room temperature in the dark for 10 minutes.

[0096] Place the cell plate into the plate reader for analysis, set to read chemiluminescence and record the data.

[0097] Data processing

[0098] The readings in each well need to be converted into cell viability, calculated according to the formula in section 1.1.

[0099] The final survival rates under different experimental conditions were plotted as bar graphs. Figures 2-6 show the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on the proliferation of five cell lines: SUP-T1, CCRF-CEM, PF-382, Jurkat, Clone E6-1, and MOLT-4.

[0100] Experimental Conclusion

[0101] Analyzing Figure 2, the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on SUP-T1 cell proliferation show that:

[0102] 1. Single-agent AST-34240.13nM (nM indicates concentration in nmol / L, the same below) and 1nM had no effect on SUP-T1 cell proliferation;

[0103] 2. All three concentrations of daunorubicin combined with 0.13 nM or 1 nM AST-3424 resulted in decreased cell viability, and the decrease was AST-3424 dose-dependent. When combined with both concentrations of AST-3424, 80 nM daunorubicin was more effective than 0.1 nM and 20 nM daunorubicin.

[0104] 3. All three concentrations of cytarabine combined with 0.13nM or 1nM AST-3424 resulted in a decrease in cell viability. Combining 0.64nM and 400nM cytarabine with AST-3424 showed a dose-dependent decrease in AST-3424.

[0105] Analyzing Figure 3, the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on CCRF-CEM cell proliferation show that:

[0106] 1. Single-agent 0.1nM and 1nM AST-3424 had no effect on CCRF-CEM cell proliferation;

[0107] 2. Compared with the 32nM daunorubicin group alone, the combination of the drug with 0.1nM or 1nM AST-3424 resulted in a decrease in cell viability, but there was no dose-dependency of AST-3424. When combined with both concentrations of AST-3424, 32nM daunorubicin was more effective than 16nM daunorubicin; there was no effect when combined with 0.03nM daunorubicin.

[0108] 3. Compared with 80nM cytarabine alone, the combination of the drug with 0.1nM or 1nM AST-3424 resulted in a decrease in cell viability, and the decrease was dose-dependent with AST-3424. When combined with two concentrations of AST-3424, 80nM cytarabine was more effective than 40nM cytarabine, and 0.6nM cytarabine had no effect.

[0109] Analyzing Figure 4, the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on PF-382 cell proliferation show that:

[0110] 1. Single-agent 0.64 and 2.5 nM AST-3424 had no significant effect on PF-382 cell proliferation;

[0111] 2. The combination of three concentrations of daunorubicin with 0.64 nM or 2.5 nM AST-3424 resulted in a decrease in cell viability, and the decrease was dose-dependent.

[0112] 3. The combination of three concentrations of cytarabine and 0.64nM or 2.5nM AST-3424 resulted in a decrease in cell viability, and the decrease was dose-dependent with AST-3424.

[0113] Analysis of Figure 5 shows the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on the proliferation of Jurkat and CloneE6-1 cells:

[0114] 1. Single-agent AST-3424 0.64nM and 16nM had no significant effect on the proliferation of Jurkat and CloneE6-1 cells;

[0115] 2. The combination of three concentrations of daunorubicin with 0.64 nM or 16 nM AST-3424 resulted in a decrease in cell viability, and the decrease was dose-dependent.

[0116] 3. The combination of three concentrations of cytarabine and 0.64nM or 16nM AST-3424 resulted in a decrease in cell viability, and the decrease was dose-dependent with AST-3424.

[0117] Analysis of Figure 6 shows the effects of AST-3424, daunorubicin, and cytarabine alone and in combination on MOLT-4 cell proliferation:

[0118] 1. Single-agent AST-3424 at 3.2nM and 6.4nM had no significant effect on MOLT-4 cell proliferation;

[0119] 2. 40 nM daunorubicin had a very significant effect on cell proliferation, and the effect of combined drug use on cell survival could not be determined;

[0120] 3. Compared with the single-agent 5nM and 20nM daunorubicin groups, the combination of daunorubicin at both concentrations and 3.2nM or 6.4nM AST-3424 resulted in decreased cell viability, and the decrease was AST-3424 dose-dependent. The combination of 20nM daunorubicin with both concentrations of AST-3424 was more effective than 5nM daunorubicin.

[0121] 4. Combination of three concentrations of cytarabine with either 3.2nM or 6.4nM AST-3424 resulted in decreased cell viability in a dose-dependent manner. Combination with 80nM and 120nM cytarabine was more effective than 3.2nM cytarabine.

[0122] II. Cell Line and Drug Description

[0123] 2.1 Cell line description

[0124] MOLT-4 and MOLT-3 were derived from a 19-year-old male patient with relapsed acute lymphoblastic leukemia who had previously received multidrug combination chemotherapy. The MOLT-4 cell line is of T-lymphocyte origin and harbors a G→A mutation at codon 248 of the p53 gene. It does not express p53, immunoglobulins, or Epstein-Barr virus. It produces high levels of terminal deoxyribosyltransferase (TdT) and expresses CD1 (49%), CD2 (35%), CD3A (26%), B (33%), C (34%), CD4 (55%), CD5 (72%), CD6 (22%), and CD7 (77%). These cells are human acute lymphoblastic leukemia cells, corresponding to human acute T-lymphoblastic leukemia (T-ALL).

[0125] 2. PF-382, human acute lymphoblastic T-cell leukemia cells, the corresponding disease is human acute lymphoblastic T-cell leukemia, namely T-ALL.

[0126] 3. Jurkat Clone E6-1 is a clone of the Jurkat-FHCRC cell line, a derivative of the Jurkat cell line. This cell line was established from the peripheral blood of a 14-year-old male patient with acute T-cell leukemia. This cell line is a human T-lymphocytic leukemia cell line, corresponding to T-ALL.

[0127] 4. SUP-T1, human T-lymphoblastoma cells, corresponding to the disease T-cell Lymphoblastic Lymphoma (TLBL)

[0128] 5. CCRF-CEM, human acute lymphoblastic leukemia T lymphocyte serum culture cells, were obtained from the peripheral blood leukocytes of a four-year-old Caucasian female patient with acute lymphoblastic leukemia in November 1964. These cells are human acute T lymphoblastic leukemia cells, corresponding to acute T lymphoblastic leukemia (T-ALL).

[0129] 2.2 Cellular AKR1C3 expression level data

[0130] The AKR1C3 enzyme expression and RNA expression levels of the above five leukemia cells and lymphoma cells were tested using the methods reported in the literature. The results are shown in Table 2 below.

[0131] Table 2: AKR1C3 protein and RNA expression data in different cell lines

[0132] AKR1C3 RNA expression was analyzed by RNA-Seq according to the method described in the literature (Meng, F., Li, WF, Jung, D., Wang, CC, Qi, T., Shia, CS, Hsu, RY, Hsieh, YC, & Duan, J. (2021). A novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad anti-tumor activity. American journal of cancer research, 11(7), 3645-3659.) and quantified using Log2 FPKM. The data in the table are the values ​​quantified by Log2 FPKM.

[0133] AKR1C3 protein expression was determined by measuring the ratio of the AKR1C3 protein band to the reference protein band in the protein bands detected by western blot (WB). In this example, the grayscale values ​​of the AKR1C3 and α-Tubulin protein bands were processed and measured using Image J software. The AKR1C3 / α-Tubulin ratio was then calculated as the relative protein expression quantitative value and compared across different cell lines.

[0134] The superscript 1 indicates that the data are the test results of AST-3424 in this experiment, see section 1.1 Single-drug in vitro cell proliferation inhibition experiment.

[0135] The superscript 2 indicates that the data are previous experimental test results of AST-3424.

[0136] 2.3 Drug Description

[0137] Daunorubicin and cytarabine are both drugs used in clinical practice to treat leukemia or other tumors. Through investigation, comparison and conversion results as shown in Table 3 below, it was found that the dosage of the combination regimen is far less than the clinical dosage.

[0138] Table 3: Clinical usage data of daunorubicin and cytarabine

[0139] Summarize

[0140] The combination of daunorubicin and cytarabine with different concentrations of AST-3424 resulted in a decrease in the survival rate of the experimental cancer cells, and the decrease was dose-dependent with AST-3424.

[0141] The combined effect was better than that of SUP-T1 and CCRF-CEM in MOLT-4, PF-382 and Jurkat Clone E6-1 cells.

[0142] In specific leukemia and lymphoma cell lines, when cell lines were detected to have different AKR1C3 expression: the AKR1C3 protein and RNA quantitative values ​​ranged from 0.84-6.08 and 0.71-5.96. Within such a large expression level range, the effectiveness of AST-3424 combined with daunorubicin or cytarabine in vitro was not related to the AKR1C3 expression level.

[0143] The concentrations of daunorubicin and cytarabine tested in the in vitro combined experiments were much lower than the clinical doses.

[0144] In summary, the experiment revealed that the new treatment regimen for leukemia / lymphoma patients does not require patients to have high (overexpression, excessive) AKR1C3 protein or RNA expression. A positive AKR1C3 protein or RNA test result is likely to benefit from this treatment regimen. In other words, the new treatment regimen significantly lowers the threshold for patients to benefit from the drug. A positive test result indicates that the combination regimen can be used, as long as medical professionals determine that the patient will benefit or may benefit from the treatment.

Claims

1. A method of treating leukemia or lymphoma using a drug containing AST-3424, AST-3423 or AST-2870 and its salts, esters, solvates, isotopomers in combination with a drug containing cytarabine or daunorubicin and its salts, esters, solvates, isotopomers.

2. The treatment method according to claim 1, wherein the leukemia includes lymphocytic leukemia, and the lymphoma includes lymphoblastic lymphoma; preferably, the leukemia is acute lymphocytic leukemia, more preferably acute T-lymphocytic leukemia, and the lymphoma is T-lymphoblastic lymphoma.

3. The method of claim 1, wherein in the combined treatment, the dosage of daunorubicin and cytarabine is lower than the clinical dosage of each of the single drugs.

4. The method according to claim 1, wherein: The AKR1C3 corresponding RNA test in the blood sample of the leukemia patient is positive, and the AKR1C3 protein test in the tumor tissue sample of the lymphoma patient is positive; or The leukemia patient was detected as positive for RNA corresponding to AKR1C3, and the lymphoma patient was detected as positive for AKR1C3 protein.

5. The method according to claim 4, wherein: The AKR1C3 corresponding RNA detection Log2FPKM quantitative value of the blood sample of the leukemia patient is greater than or equal to 0.71, The AKR1C3 protein quantification of the tumor tissue sample of the lymphoma patient is the ratio of the AKR1C3 protein band to the reference protein band in the protein band detected by western blot (WB), and the ratio is greater than or equal to 0.84; or The leukemia patient is detected to have RNA corresponding to AKR1C3, and the Log2FPKM quantitative value is greater than or equal to 0.

71. The AKR1C3 protein detected in the lymphoma patient is quantitatively determined by a western blot (WB) method, and the ratio of the AKR1C3 protein band to the reference protein band in the protein band is greater than or equal to 0.

84.

6. Pharmaceutical use of AST-3424, AST-3423 or AST-2870 and their salts, esters, solvates and isotopomers, for preparing drugs for treating leukemia or lymphoma in combination with drugs containing daunorubicin or cytarabine and their salts, esters, solvates and isotopomers.

7. The pharmaceutical use according to claim 6, wherein the leukemia includes lymphocytic leukemia, and the lymphoma includes lymphoblastic lymphoma; preferably, the leukemia is acute lymphocytic leukemia, more preferably acute T-lymphocytic leukemia, and the lymphoma is T-lymphoblastic lymphoma.

8. The pharmaceutical use according to claim 6, wherein in the combined treatment, the dosage of daunorubicin and cytarabine is lower than the clinical dosage of the single drugs.

9. The pharmaceutical use according to claim 6, characterized in that: The AKR1C3 corresponding RNA test in the blood sample of the leukemia patient is positive, and the AKR1C3 protein test in the tumor tissue sample of the lymphoma patient is positive; or The leukemia patient was detected as positive for RNA corresponding to AKR1C3, and the lymphoma patient was detected as positive for AKR1C3 protein.

10. The pharmaceutical use according to claim 9, characterized in that: The AKR1C3 corresponding RNA detection Log2FPKM quantitative value of the blood sample of the leukemia patient is greater than or equal to 0.71, The AKR1C3 protein quantification of the tumor tissue sample of the lymphoma patient is the ratio of the AKR1C3 protein band to the reference protein band in the protein band detected by western blot (WB), and the ratio is greater than or equal to 0.84; or The leukemia patient is detected to have RNA corresponding to AKR1C3, and the Log2FPKM quantitative value is greater than or equal to 0.

71. The AKR1C3 protein detected in the lymphoma patient is quantitatively determined by a western blot (WB) method, and the ratio of the AKR1C3 protein band to the reference protein band in the protein band is greater than or equal to 0.

84.

11. A drug combination, characterized in that: Contains the following substances as active ingredients: AST-3424, AST-3423 or AST-2870 and their salts, esters, solvates, isotopic isomers, and Daunorubicin and its salts, esters, solvates, isotopic isomers, or Cytarabine and its salts, esters, solvates, and isotopic isomers; The active ingredients are formulated together or separately for combined use, simultaneous use or separate use.

12. The drug combination according to claim 11, characterized in that The combined dosage of daunorubicin and cytarabine is lower than the clinical dosage of the single drugs.

13. A pharmaceutical preparation, characterized in that The drug combination is prepared from the active ingredients of the drug combination according to claim 11 and pharmaceutically acceptable excipients.

14. The pharmaceutical preparation according to claim 13, characterized in that The dosage form of the pharmaceutical preparation is selected from granules, tablets, pills, capsules or injections.

15. Use of the drug combination according to claim 11 and / or the pharmaceutical preparation according to claim 13 in the preparation of an anti-tumor drug.

16. The use according to claim 15, wherein The tumor is selected from leukemia and lymphoma.

17. The use according to claim 16, wherein The leukemia includes lymphocytic leukemia, and the lymphoma includes lymphoblastic lymphoma; preferably, the leukemia is acute lymphocytic leukemia, more preferably acute T-lymphocytic leukemia, and the lymphoma is T-lymphoblastic lymphoma.

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