IDH mutations as biomarkers for zotiracillin therapy

Abstract

Provided are methods of using isocitrate dehydrogenase (IDH1 and IDH2) mutations to select cancer patients and treat with zotiracillin (TG02) therapies, e.g., to treat cancer, such as glioma, myeloblastoma, soft osteosarcoma, cholangioma, AML, astrocytoma, and the like, as a single agent in combination with other chemotherapeutic agents.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to PCT / CN2022 / 111327, filed on August 10, 2022, which is incorporated by reference in its entirety. Technical Field

[0003] Embodiments of the present disclosure relate to the use of isocitrate dehydrogenase (IDH1 and IDH2) mutations to select cancer patients for zotiracillin (TG02) therapy, such as for the treatment of cancers such as glioma, medulloblastoma, chondrosarcoma, cholangiocarcinoma, AML, astrocytoma, etc. Background Art

[0004] Zotiraciclib (TG02) is a selective kinase inhibitor for the treatment of cancer (William et al., Journal of Medicinal Chem. 55: 169-196, 2012). It is an inhibitor of cyclin-dependent kinases (CDKs), Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3), and is being evaluated in various clinical trials (see, e.g., Wu et al., Clin Cancer Res. 27: 3298–3306, 2021).

[0005] However, there is a need in the art to better predict the anti-cancer therapeutic efficacy of zotiracillin and thereby identify patients who will most benefit from treatment with this chemotherapeutic agent. Summary of the invention

[0006] Embodiments of the present disclosure include methods for treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, the method comprising administering to the subject a composition comprising zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof, thereby treating the cancer comprising the IDH1 or IDH2 mutation. In specific embodiments, the cancer is a glioma. Certain methods comprise (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; and (b) administering to the subject a composition comprising zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof if the tissue sample comprises the IDH1 or IDH2 mutation.

[0007] In some embodiments, the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, wherein X is selected from any amino acid except R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T, or R140Q.

[0008] In some embodiments, step (a) comprises determining the IDH1 or IDH2 mutation status in the tissue sample by DNA or RNA sequencing, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), whole exome sequencing (WES), single nucleotide polymorphism (SNP) array, next generation sequencing (NGS) or comparative genomic hybridization (CGH) on human IDH1 or IDH2 protein or gene.

[0009] Certain embodiments include obtaining a tissue sample from the subject. In some embodiments, the tissue sample is a liquid biopsy, optionally a blood sample, a surgical sample, or other biopsy sample obtained from the subject, optionally wherein the tissue sample is a cancer tissue sample.

[0010] In some embodiments, the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), astrocytoma, sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL), and glioblastoma (optionally secondary glioblastoma).

[0011] Certain embodiments comprise administering to the subject an oral composition of zotiracillin or an analog, derivative, or pharmaceutically acceptable salt thereof. In some embodiments, the composition comprising zotiracillin is administered in combination with radiation therapy and / or one or more additional agents, the one or more additional agents being optionally selected from chemotherapeutic agents, hormone therapeutic agents, and / or kinase inhibitors. In some embodiments, the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), enasidenib, or AG-221.

[0012] Certain embodiments include the use of a diagnostic kit for treating cancer in a human subject in need thereof with zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, the diagnostic kit comprising a means for determining the isocitrate dehydrogenase (IDH1 or IDH2) mutation status in a tissue sample from the subject.

[0013] In some embodiments, the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, wherein X is selected from any amino acid except R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T, or R140Q.

[0014] In some embodiments, the device for determining the IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay selected from one or more of the following: DNA or RNA sequencing, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), whole exome sequencing (WES), single nucleotide polymorphism (SNP) array, next generation sequencing (NGS), or comparative genomic hybridization (CGH) on human IDH1 or IDH2 protein or gene.

[0015] In some embodiments, the tissue sample is a liquid biopsy, optionally a blood sample, a surgical sample or other biopsy sample obtained from the subject, optionally a biopsy of a prostate cancer tissue. In some embodiments, the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL), astrocytoma and glioblastoma (optionally secondary glioblastoma). In some embodiments, the diagnostic / therapeutic kit comprises a composition comprising zotiracillin or its analog, derivative or pharmaceutically acceptable salt, optionally an oral composition of zotiracillin. In some embodiments, the diagnostic / therapeutic kit comprises one or more additional agents, and the one or more additional agents are optionally selected from chemotherapeutic agents, hormone therapeutic agents and / or kinase inhibitors. In some embodiments, the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), etanercept or AG-221.

[0016] Also included is a patient care kit comprising: (a) a device for determining the mutation status of isocitrate dehydrogenase (IDH1 or IDH2) in a tissue sample from a human subject suffering from cancer; and (b) a composition comprising zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof.

[0017] In some embodiments, the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, wherein X is selected from any amino acid except R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T, or R140Q.

[0018] In some embodiments, the device for determining the mutation status of IDH1 or IDH2 in a tissue sample comprises reagents for performing one or more diagnostic assays selected from the following: DNA or RNA sequencing, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), whole exome sequencing (WES), single nucleotide polymorphism (SNP) array, next generation sequencing (NGS) or comparative genomic hybridization (CGH) on human IDH1 or IDH2 protein or gene. In some embodiments, the tissue sample is a liquid biopsy, optionally a blood sample, a surgical sample, or other biopsy sample obtained from the subject, optionally a biopsy of prostate cancer tissue.

[0019] In some embodiments, the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), astrocytoma, sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL), and glioblastoma (optionally secondary glioblastoma).

[0020] In some embodiments, (b) comprises an oral composition of zotiracillin or an analog, derivative or pharmaceutically acceptable salt thereof. In some embodiments, the patient care kit comprises one or more additional agents, the one or more additional agents being optionally selected from chemotherapeutic agents, hormone therapeutic agents and / or kinase inhibitors. In some embodiments, the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), etanercept or AG-221.

[0021] Certain embodiments include a pharmaceutical composition for use in a method of treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, the pharmaceutical composition comprising zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof. In some embodiments, the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, wherein X is selected from any amino acid except R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q.

[0022] In some embodiments, the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), astrocytoma, sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL) and glioblastoma (optionally secondary glioblastoma). Certain embodiments include the use of an oral composition of zotiracillin or an analog, derivative or pharmaceutically acceptable salt thereof. Certain embodiments include the use of one or more additional agents, which are optionally selected from chemotherapeutic agents, hormone therapeutic agents and / or kinase inhibitors. In some embodiments, the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), etanercept or AG-221. In some embodiments, the method comprises (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; and (b) if the tissue sample comprises the IDH1 or IDH2 mutation, administering to the subject the composition comprising zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof.

[0023] Also included is a use of a composition for preparing a medicament for treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, and the composition comprises zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof. In some embodiments, the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the tumor metabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, wherein X is selected from any amino acid other than R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q. In some embodiments, the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), astrocytoma, sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL) and glioblastoma (optionally secondary glioblastoma). Certain uses comprise an oral composition of zotiracillin or an analog, derivative or pharmaceutically acceptable salt thereof. Particular uses comprise one or more additional agents, the one or more additional agents being optionally selected from chemotherapeutic agents, hormonal therapeutic agents and / or kinase inhibitors. In some embodiments, the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), etanercept or AG-221. Certain uses comprise the steps of: (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; and (b) if the tissue sample comprises the IDH1 or IDH2 mutation, administering to the subject the composition comprising zotiracillin (TG02) or an analog, derivative or pharmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The 2-dimensional chemical structure of Zoltiracillin is shown.

[0025] Figure 2A-2B It is shown that IDH1 mutant chondrosarcoma (HT-1080) and cholangiocarcinoma (RBE, Hcc-9810) cells are more sensitive to TG02 than IDH1-WT (Hucct-1) cells. Cells were treated with 0.2 μM TG02 for the indicated times. Cell apoptosis was determined by FACS assay.

[0026] Figure 3A-3B It was shown that increased TG02 sensitivity can be conferred to IDH1-WT (Hucct-1) cells by transfection of mutant IDH1. Figure 3A Hucct-1 cells were transfected with IDH1 R132H plasmid and treated with 0.1 μM TG02 for 72 h. Cell apoptosis was determined by FACS assay. Figure 3B In the present study, Hucct-1 cells were transfected with IDH1 R132H plasmid, and the level of D-2-hydroxyglutarate (D-2HG) was measured in Hucct-1 WT cells, Hucct-1 cells transfected with IDH1 R132H plasmid, and RBE (cholangiocarcinoma cell line with IDH1 R132S mutation) cells. D-2HG is a metabolic biomarker for gain-of-function mutations in IDH1 and / or IDH2 genes.

[0027] Figure 4 It is shown that TG02-induced apoptosis of mutant IDH1 (Hccc-9810) cells can be rescued by adding excess exogenous substrate α-ketoglutarate (α-KG). Mutant IDH1 / 2 converts α-KG to the tumor metabolite D-2HG, which then inhibits a class of α-KG-dependent enzymes involved in epigenetic regulation. Cells were pretreated with α-KG for 24 hours and then treated with TG02 for 72 hours. Apoptosis was determined by FACS assay.

[0028] Figure 5A-5B It is shown that TG02-induced DNA damage in mutant IDH1 (HT-1080) cells can be rescued by adding exogenous α-KG or AG-120. AG-120 is an inhibitor of mutant IDH1. Figure 5A Cells were treated with the indicated drugs for 48 h, and DNA damage was determined by the neutral comet assay. Figure 5B Statistical analysis of the data in 5A is shown (left panel: tail moment. right panel: positive percentage of cells with DNA damage).

[0029] Figure 6 Shown is the rescue of TG02-induced DNA damage in mutant IDH1 cells (Hccc-9810) only by the addition of exogenous AG-120. Cells were treated with the indicated drugs for 48 hours. pH2AX, a marker of DNA damage, was determined by WB.

[0030] Figure 7A-7B It was shown that cells with IDH1 mutations are more sensitive to TG02-induced DNA damage. Fig. 7AIDH1 mutant Hucct1 cells (Hucct1 R132H / + ). Hucct1, Hucct1 R132H / + HT-1080 cells were treated with 0.2 μM TG02 or 0.1% DMSO for 48 h and analyzed by comet assay. Quantification of tail moment in neutral comet assay is presented. ns: no significant difference; ***: P < 0.001, ****: P < 0.0001 compared with NC; #: P < 0.05 compared with Hucct1 cells treated with TG02. Figure 7B Hucct1 cells and Hucct1 were treated with 2 μM AG120, 0.2 μM TG02, or 0.01 μM AZD4573 (CDK9 inhibitor) as indicated. R132H / + Cells. Western blot analysis of phosphorylated γH2AX (a marker of DSBs) after 16 hours of treatment is shown. β-Actin was used as a loading control.

[0031] Figures 8A-8C TG02 in combination with AG120 is shown to induce apoptosis in cells with mutant IDH1. Mutant IDH1 cells HT-1080 (8A), RBE (8B) and Hccc-9810 (8C) were treated with TG02 (0.1 μM) and AG120 (2 μM) for 48 hours as indicated, and apoptosis was measured by Annexin V / PI staining and analyzed by flow cytometry. DETAILED DESCRIPTION

[0032] Embodiments of the present disclosure relate to the surprising discovery that cancers containing isocitrate dehydrogenase (IDH1 and IDH2) mutations show significantly higher sensitivity to zotiracillin (TG02) therapy. Therefore, IDH1 / 2 mutation status and its associated gain-of-function phenotype can be used as a biomarker or companion diagnostic to select patients for optimized zotiracillin cancer therapy.

[0033] Unless otherwise defined, all technical and scientific terms used in this article have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. Although any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used to practice or test the subject matter of the present disclosure, preferred methods and materials are described. All publications and references, including, but not limited to, patents and patent applications cited in this specification, are incorporated herein by reference in their entirety, as if clearly and individually indicating that each individual publication or reference is incorporated herein by reference for complete elaboration. Any patent application for which the present application claims priority is also incorporated herein by reference in its entirety in the manner described above with respect to publications and references.

[0034] For the purposes of this disclosure, the following terms are defined below.

[0035] The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0036] "Antagonist" or "inhibitor" refers to a biological structure or chemical agent (e.g., compound) that interferes with or otherwise reduces the physiological effects of another molecule (e.g., protein). In some cases, an antagonist or inhibitor specifically binds to other molecules and / or functional ligands of other molecules. In some cases, an antagonist or inhibitor downregulates the expression of other molecules. Both complete and partial antagonists are included.

[0037] "Agonist" or "activator" refers to a biological structure or chemical agent that increases or enhances the physiological effect of another agent or molecule. In some cases, agonists specifically bind to other agents or molecules. Both full and partial agonists are included.

[0038] “About” means that an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length varies by up to 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0039] The term "binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges).

[0040] Throughout this disclosure, unless the context requires otherwise, the words “comprise”, “comprises”, or “comprising” will be understood to imply the inclusion of a stated step or element, or group of steps or elements, but not the exclusion of any other step or element, or group of steps or elements.

[0041] "Consisting of" means including and limited to the object following the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action detailed in this disclosure with respect to the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the listed elements.

[0042] The term "half maximal effective concentration" or "EC 50 "EC" refers to the concentration of an agent (e.g., a compound) as described herein that induces a response between baseline and maximum after some specified exposure time; thus, the EC of a graded dose-response curve is 50 EC represents the concentration of an agent at which 50% of its maximum effect is observed. 50 It also refers to the plasma concentration required to achieve 50% of the maximal effect in vivo. Similarly, “EC 90 ” refers to the concentration of an agent or composition at which 90% of its maximum effect is observed. “EC 90 ” can be based on “EC 50 " and Hill slope, or can be determined directly from the data using routine knowledge in the art. In some embodiments, the EC of an agent is 50 In some embodiments, the EC of the agent is less than about 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, or 500 nM. 50 The value was about 1 nM or less.

[0043] The “half maximal inhibitory concentration” (or “IC 50 IC (inhibitor) is a measure of the potency of an agent that inhibits a specific biological or biochemical function. This quantitative measure indicates how much of a specific agent (inhibitor) is needed to inhibit a given biological process (or component of a process, i.e., an enzyme, cell, cell receptor, or microorganism) by half. The value is usually expressed as a molar concentration. Concentration is often used as a measure of antagonist drug potency in pharmacological studies. In some cases, IC 50 Represents the concentration of an agent required for 50% inhibition in vitro. The IC of an agent can be determined by constructing a dose-response curve and examining the effects of varying concentrations of the agent on the desired activity, e.g., inhibition of tumor cell proliferation, tumor cell killing. 50 .

[0044] The "half-life" of a drug refers to the time it takes for a drug to lose half of its pharmacological, physiological or other activity relative to such activity when administered to the serum or tissue of an organism, or relative to any other defined time point. "Half-life" can also refer to the time it takes for the amount or concentration of a drug to reduce half of the initial amount administered to the serum or tissue of an organism relative to such amount or concentration when administered to the serum or tissue of an organism, or relative to any other defined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0045] The terms "modulate" and "alter" include "increase," "enhance," or "stimulate," as well as "decrease" or "reduction," typically by a statistically significant or physiologically significant amount or degree relative to a control. An "increased," "stimulated," or "enhanced" amount is typically a "statistically significant" amount, and can include an amount that is about or at least about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 times or more the amount produced by no composition or a control composition (e.g., no agent or a different agent). An “increased,” “stimulated,” or “enhanced” amount can also include an amount that is about or at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000% or more of the amount produced by no composition or a control composition. A "reduced" or "decreased" amount is typically a "statistically significant" amount, and can include an amount that is about or at most about 1 / 1.1, 1 / 1.2, 1 / 1.5, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 2000, 1 / 3000, 1 / 4000, or 1 / 5000 of the amount produced by no composition or a control composition."Reduced" or "decreased" amounts can also include less than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000% or 5000% of the amount produced by no composition or a control composition. Examples of comparisons and "statistically significant" amounts are described herein.

[0046] "Prodrug" is intended to indicate an agent (e.g., compound) that can be converted into a bioactive compound described herein under physiological conditions or by solvolysis. Therefore, the term "prodrug" refers to a metabolic precursor of a pharmaceutically acceptable compound. Prodrugs may be inactive when administered to a subject in need, but are converted into active compounds in vivo. Prodrugs can be rapidly converted in vivo to produce parent compounds, such as by hydrolysis in blood. Prodrug compounds generally provide advantages of solubility, tissue compatibility, or delayed release in mammalian organisms (see Bundgard, H., "Design of Prodrugs" (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). Discussions of prodrugs are provided in Higuchi, T. et al., ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press, 1987. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohols or amide derivatives of amine functional groups in the compounds of the present disclosure, and the like.

[0047] The term "prodrug" is also intended to include any covalently bonded carriers that release the active compound in vivo when such prodrugs are administered to a subject. Prodrugs of a compound can be prepared by modifying the functional groups present in the compound in a manner that allows these modifications to be cleaved into the parent compound in conventional manipulations or in vivo. Prodrugs include compounds in which a hydroxyl, an amino or a sulfhydryl group is bonded to any group that is cleaved to form a free hydroxyl, a free amino or a free sulfhydryl group when the prodrug of the compound is administered to a subject.

[0048] "Pharmaceutically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier, for example, which has been approved by the United States Food and Drug Administration for use in humans or livestock.

[0049] "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts.

[0050] "Pharmaceutically acceptable acid addition salts" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexylsulfonic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy Ethylsulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptanoic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0051] "Pharmaceutically acceptable base addition salts" refers to salts which retain the biological effectiveness and properties of the free acid, which salts are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, denanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenethylbenzylamine, benzathine penicillin, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0052] As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the agents (e.g., compounds) described herein and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be a biologically inert organic solvent. Thus, the compounds described herein may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and corresponding solvated forms. The compounds of the present disclosure may be true solvates, while in other cases, the compounds may retain only exogenous water or a mixture of water plus some exogenous solvents.

[0053] "Pharmaceutical composition" refers to a formulation of the zotiracillin (TG02) compound described herein and a medium generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). Such a medium includes all pharmaceutically acceptable carriers, diluents, and excipients.

[0054] The zotiracillin compounds described herein, or pharmaceutically acceptable salts thereof, may contain one or more asymmetric centers and may therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms, which may be defined in terms of absolute stereochemistry as (R)- or (S)- or (D)- or (L)- of an amino acid. The present disclosure is intended to include all such possible isomers and racemic and optically pure forms thereof. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques (e.g., chromatography and fractional crystallization). Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise indicated, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0055] In certain embodiments, the "purity" of any given agent in a composition can be defined. For example, certain compositions can include an agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, weight-by-weight, as measured, for example, but not limited to, by high performance liquid chromatography (HPLC), a well-known form of column chromatography commonly used in biochemistry and analytical chemistry to separate, identify, and quantify agents or compounds.

[0056] The term "solubility" refers to the property that the medicament provided herein dissolves the liquid solvent and forms a uniform solution. Solubility is usually expressed as concentration, which is the mass of the solute per unit volume of solvent (grams of solute per kilogram of solvent, g / dL (100mL), mg / ml, etc.), molar concentration, mass molar concentration, molar fraction or other similar concentration descriptions. The maximum equilibrium amount of the solute that can dissolve a unit amount of solvent is the solubility of the solute in the solvent under the specified conditions including temperature, pressure, pH and solvent properties. In certain embodiments, at physiological pH or other pH, for example, pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8 or pH 8.0 (for example, about pH 5-8) solubility is measured. In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaPO4). In specific embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO4). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C) or about body temperature (37°C). In certain embodiments, the solubility of the agent at room temperature or 37°C is at least about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml g / ml, 10mg / ml, 11mg / ml, 12mg / ml, 13mg / ml, 14mg / ml, 15mg / ml, 16mg / ml, 17mg / ml, 18mg / ml, 19mg / m l, 20mg / ml, 25mg / ml, 30mg / ml, 40mg / ml, 50mg / ml, 60mg / ml, 70mg / ml, 80mg / ml, 90mg / ml or 100mg / ml.

[0057] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and formulation into a therapeutic agent.

[0058] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA and DNA, including genomic DNA. The term generally refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single-stranded and double-stranded forms of DNA.

[0059] "Gene" means a hereditary unit consisting of a DNA sequence that occupies a specific position on a chromosome and encodes a functional molecule or protein. The structure of a gene is composed of many elements, of which the actual protein coding sequence is usually only a small part. These elements include untranscribed DNA regions and non-translated regions of RNA. In addition, a gene may have a regulatory region that changes expression and is located thousands of bases upstream or downstream of the coding sequence. The information in a gene may also be represented by (or found in) the sequence of RNA or encoded protein.

[0060] "Subject" or "subject in need thereof" includes mammalian subjects such as human subjects.

[0061] "Statistically significant" means that the results could not have occurred by chance. Statistical significance can be determined by any method known in the art. Common measures of significance include the p-value, which is the frequency or probability of the observed event occurring if the null hypothesis is true. If the p-value obtained is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0062] "Substantially" or "substantially" means almost completely or thoroughly, for example, 95%, 96%, 97%, 98%, 99% or more of a given reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, length, etc.

[0063] "Stereoisomers" refer to compounds composed of the same atoms joined by the same bonds but with different three-dimensional structures, which are not interchangeable. The present disclosure includes various stereoisomers and mixtures thereof and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.

[0064] "Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any of the described compounds.

[0065] A "therapeutic response" refers to an improvement in symptoms (whether sustained or not) based on administration of a therapeutic response.

[0066] As used herein, the terms "therapeutically effective amount," "therapeutic dose," "prophylactically effective amount," or "diagnostically effective amount" are the amount of an agent required to elicit a desired biological response following administration.

[0067] As used herein, "treatment" of a subject (e.g., a mammal such as a human) or a cell is any type of intervention used to attempt to alter the natural course of a subject or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and may be performed prophylactically, or after the onset of a pathological event or contact with a pathogen. Also included are "preventive" treatments, which may be intended to reduce the rate of progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of a disease or condition or its associated symptoms.

[0068] The term "wild-type" refers to a gene or gene product (eg, a polypeptide) that is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form of a gene.

[0069] Every embodiment in this specification is applicable to every other embodiment unless explicitly stated otherwise.

[0070] Certain embodiments include a method for treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, the method comprising administering to the subject a pharmaceutical composition comprising zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof, thereby treating the cancer comprising the IDH1 or IDH2 mutation.

[0071] Some embodiments for treating cancers with IDH1 or IDH2 mutations include:

[0072] (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; and

[0073] (b) if the tissue sample comprises the IDH1 or IDH2 mutation, administering to the subject a pharmaceutical composition comprising zotiracillin (TG02) or an analog, derivative or pharmaceutically acceptable salt thereof.

[0074] Also included is a method for predicting a human subject with cancer's response to treatment with zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof, the method comprising:

[0075] (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; and

[0076] (b)(i) if the tissue sample comprises the IDH1 or IDH2 mutation, characterizing the subject as responsive to zotiracillin (TG02) therapy; or

[0077] (ii) if the tissue sample does not comprise an IDH1 or IDH2 mutation, e.g., if the sample comprises homozygous wild-type IDH1 and IDH2, characterizing the subject as non-responsive to zotiracillin (TG02) therapy,

[0078] The treatment response of subjects with cancer to zotiracillin (TG02) is thereby predicted.

[0079] Some embodiments include administering zotiracillin to the subject if the subject is characterized as responsive to zotiracillin therapy. In some cases, including administering a chemotherapeutic agent other than zotiracillin to the subject if the subject is characterized as non-responsive to zotiracillin therapy.

[0080] "Zoltiracillin" or "TG02" refers to the compound having the IUPAC name: (16E)-14-methyl-20-oxa-5,7,14,27-tetraazatetracyclo[19.3.1.1 2,6 .1 8,12 ] Heptadecan-1(25),2(27),3,5,8,10,12(26),16,21,23-decane, PubChem CID: 16739650 and CAS number: 1204918-72-8, and includes pharmaceutically acceptable salts and acids thereof. Also included are biologically active or equivalent analogs and / or derivatives of zotiracillin, including prodrugs and pharmaceutically acceptable salts thereof.

[0081] The term "isocitrate dehydrogenase" or "IDH" refers to an enzyme (and the IDH gene encoding) that catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate and CO2. The two-step process involves the oxidation of isocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followed by β-decarboxylation of the carboxyl group to the ketone, forming α-ketoglutarate. In humans, IDH exists in three isoforms: IDH1 (Uniprot: O75874), IDH2 (Uniprot: P48735), and IDH3 (Uniprot: P50213, O43837, and P51553). The IDH3 isoform consists of three subunits and catalyzes the third step of the citric acid cycle, while converting NAD+ to NADH in the mitochondria. IDH1 and IDH2 isoforms catalyze the same reaction outside the context of the citric acid cycle and use NADP+ instead of NAD+ as a cofactor.

[0082] As described above, certain embodiments include administering zotiracillin to a subject if the tissue sample comprises an IDH1 or IDH2 mutation relative to wild-type IDH1 or IDH2. Exemplary IDH1 and IDH2 mutations in cancer are described in the art (see, e.g., Pirozzi and Yan, Nature Reviews Clinical Oncology. 18:645–661, 2021; and Persico et al., Cancers (Basel). 14(5); 1125, 2022, doi: 10.3390 / cancers14051125). In certain embodiments, the IHD1 mutation is R132X, wherein X is selected from any amino acid except R (arginine). In specific embodiments, the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R (arginine). In specific embodiments, the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q. In certain embodiments, the IDH1 or IDH2 mutation is a "gain of function mutation" characterized by increased conversion of α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2 (see, e.g., Chowdhury et al., EMBO Rep. 12(5):463-9, 2011). Thus, certain embodiments include, for example, determining the level, presence or absence of a D-2HG oncometabolite in a tissue sample, and administering zotiracillin to a subject if the D-2HG oncometabolite is present or increased in the tissue sample relative to a reference or standard (e.g., D-2HG levels in a homozygous wild-type IDH1 or IDH2 tissue sample or cell).

[0083] The IDH1 or IDH2 mutation status in tissue samples can be determined by a variety of methods. For example, in some embodiments, the IDH1 or IDH2 mutation status is directly determined by DNA or RNA sequencing, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), whole exon sequencing (WES), single nucleotide polymorphism (SNP) array, next generation sequencing (NGS) or comparative genomic hybridization (CGH) on human IDH1 or IDH2 protein or gene. CGH refers to a molecular cytogenetic method, which is used to analyze the copy number variation (CNV) of the ploidy level in the DNA of the test sample compared with the reference sample, without the need to culture cells. This technology allows fast and efficient comparison between two genomic DNA samples derived from the two most commonly closely related sources, because they are suspected of containing differences in terms of the acquisition or loss of the entire chromosome or subchromosomal region (a part of the entire chromosome). The technology was originally developed to evaluate the difference between the chromosome complement of solid tumors and normal tissues (see, for example, Kallioniemi et al., Science.258 (5083): 818–821, 1992). The use of DNA microarrays combined with CGH technology has led to the development of a more specific form of array CGH (aCGH), allowing for locus-by-locus measurement of CNVs with increased resolution down to 100 kilobases (see, e.g., Pinkel, Annu Rev Genom Hum Genet. 6:331–354, 2005). In situ hybridization (ISH) and fluorescent in situ hybridization (FISH) represent a class of hybridizations that use labeled complementary DNA, RNA, or modified nucleic acid chains (i.e., probes) to locate specific DNA or RNA sequences in a portion or slice (in situ) of a tissue (see, e.g., Parra and Windle, Nature Genetics. 5:17-21, 1993; and Gall and Pardue, PNAS USA. 63:378–383, 1969). Therefore, the step of determining IDH1 or IDH2 status, for example to identify an IDH1 or IDH2 mutation of interest (or its absence) can be performed according to conventional techniques in the art. In some cases, the methods and kits described herein employ any one or more of the aforementioned techniques, and / or comprise reagents for performing the techniques.

[0084] In some embodiments, IDH1 or IDH2 mutation status is indirectly determined, for example, by determining the D-2HG level in a tissue sample. D-2HG levels can be determined or measured according to a variety of techniques in the art, including biochemical detection (e.g., colorimetric determination), biosensors, gas or liquid chromatography-mass spectrometry (GC- or LC-MS), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), etc. (see, for example, Xiao et al., "Natural Communications (Nat Commun)" 12:7108, 2021; D-2HG determination in Example 1; Longuespée et al., "Acta Neuropathol Commun" .6:21, 2018). Therefore, determining D-2HG levels, such as indirectly determining the step of IDH1 or IDH2 mutation status can be performed according to the conventional techniques in the art. In some cases, the methods and kits described herein use any one or more of the aforementioned techniques, and / or include reagents for performing the techniques.

[0085] Examples of "references" include values, amounts, sequences, or other properties obtained from a database, such as a homozygous "wild-type" IDH1 or IDH2 sequence (see, e.g., NCBI gene 3417 and RefSeq NM_005896 for IDH1, and NCBI gene 3418 and RefSeq NM_002168 for IDH2). "References" also include values, amounts, sequences, or other properties obtained from one or more control tissues, such as wild-type IDH1 / 2 homozygous tissues (cancerous or non-cancerous) from one or more controls, such as one or more control subjects (e.g., a control subject population). As with cancerous tissues, the IDH1 / 2 mutation status from a control can be determined by various methods, including, e.g., ISH, FISH, WES, SNP arrays, NGS, or CGH on human IDH1 / 2 proteins or genes. As also described above, D-2HG levels from a control can be determined by a variety of methods, including, for example, by biochemical detection (eg, colorimetric assay), biosensor, GC- or LC-MS, MALDI-TOF, etc. (supra).

[0086] In some embodiments, the tissue sample is a liquid biopsy (e.g., a blood sample), a surgical sample, or other biopsy samples obtained from a subject. In a specific embodiment, the tissue sample is a cancer tissue sample. Certain embodiments include, for example, a step of obtaining a tissue sample from a subject before determining IDH1 or IDH2 mutation status and / or D-2HG levels. In certain embodiments, the subject is a human subject.

[0087] The methods provided herein can be performed on various cancer types. In certain embodiments, the cancer is selected from glioma (including low-grade glioma and high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), astrocytoma, sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL) and glioblastoma (optionally secondary glioblastoma).

[0088] Certain embodiments include combination therapies for treating cancer, including a method of treating a subject in need of amelioration of symptoms of cancer or inhibition of cancer progression, the method comprising administering to the subject zotiracillin in combination with at least one additional agent, such as an immunotherapeutic agent (e.g., a checkpoint inhibitor), a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor. In some embodiments, administration of zotiracillin enhances the susceptibility of cancer to the additional agent (e.g., an immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor) by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more relative to the additional agent alone.

[0089] Certain combination therapies employ one or more chemotherapeutic agents, e.g., small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include IDH1 and IDH2 inhibitors, alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), anti-microtubule agents, and the like.

[0090] Examples of IDH1 or IDH2 inhibitors include ivosidenib (AG-120), etanercept, and AG-221 (see, e.g., Zarei et al., Cancer Treat Rev. 103:102334, 2022. doi:10.1016 / j.ctrv.2021.102334; Hansen et al., Blood. 124(21):3734, 2014; Quivoron et al., Blood. 124(21):3735, 2014).

[0091] Examples of alkylating agents include nitrogen mustards (e.g., dichloromethane, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine, carbazine, mitozolomide and temozolomide), aziridines (e.g., thiotepa, mytomycin and diaziquone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin) and atypical alkylating agents (optionally procarbazine and hexamethylmelamine).

[0092] Examples of antimetabolites include: antifolates (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine);

[0093] Examples of cytotoxic antibiotics include: anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include: camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin.

[0094] Examples of anti-microtubule agents include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

[0095] Some combination therapies use at least one hormone therapy agent. General examples of hormone therapy agents include hormone agonists and hormone antagonists. Specific examples of hormone agonists include: progesterone (progesterone), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, androgen, estrogen and somatostatin analogs. Examples of hormone antagonists include hormone synthesis inhibitors, such as aromatase inhibitors and gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), including analogs thereof. Also included are hormone receptor antagonists, such as selective estrogen receptor modulators (SERMs, e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0096] Also included are hormone pathway inhibitors, such as antibodies against hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1), such as cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, and robatumumab; inhibitors of vascular endothelial growth factor receptors 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as alacizumab, pegol), bevacizumab, icrucumab, ramucirumab; inhibitors of TGF-β receptors R1, R2, and R3, such as fresolimumab and metelimumab; inhibitors of c-Met, such as naxitamab; inhibitors of EGF receptors, such as cetuximab, depatuxizumab, and sutuximab. Therapeutic agents include mafodotin, futuximab, imgatuzumab, laprituximabemtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomucotuximab, and zalutumumab; inhibitors of FGF receptors, such as aprutumab ixadotin and bemarituzumab; and inhibitors of PDGF receptors, such as olaratumab or tovetumab.

[0097] Certain combination therapies employ at least one kinase inhibitor, including a tyrosine kinase inhibitor. Examples of kinase inhibitors include, but are not limited to, adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, tinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemuafenib.

[0098] In some embodiments, the methods and pharmaceutical compositions described herein increase the median survival time of the subject by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks or longer. In some embodiments, the methods and compositions described herein increase the progression-free survival, overall survival and / or post-progression survival of the subject in need by, for example, about or at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months or 24 months or longer, or increase by about or at least about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years or 10 years or longer. In certain embodiments, the methods and compositions described herein are sufficient to stabilize the disease.

[0099] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor (e.g., a decrease in tumor mass of at least 10%, 20%, 30%, 40%, 50% or more) or by a change in scan size (e.g., a statistically significant decrease). In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause disease stabilization.

[0100] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to result in a clinically relevant reduction in symptoms for a specific disease indication known to the skilled clinician.

[0101] The method for treating cancer can be combined with other treatment modes. For example, cancer therapy as described herein can be applied to subjects before, during or after other therapeutic interventions, including symptomatic care, radiotherapy (radiationtherapy / radiotherapy), surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy or any combination thereof. Symptomatic care includes the use of corticosteroids to reduce brain edema, headache, cognitive dysfunction and vomiting, and the use of anticonvulsants to reduce epileptic seizures. Radiotherapy includes whole brain irradiation, fractionated radiotherapy and radiosurgery such as stereotactic radiosurgery, which can also be combined with traditional surgery. In some embodiments, radiation therapy comprises administering a total radiation dose of about 1 Gray (Gy) to about 70 Gy, or about 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 65 Gy, or 70 Gy.

[0102] Methods for identifying subjects suffering from one or more of the diseases or conditions described herein are known in the art.

[0103] For in vivo use, eg, for treatment of human disease or testing, the agents described herein are typically incorporated into one or more therapeutic or pharmaceutical compositions prior to administration.

[0104] In order to prepare a therapeutic composition or pharmaceutical composition, an effective amount or desired amount of one or more agents is usually mixed with any pharmaceutical carrier or excipient, which is known to those skilled in the art to be suitable for a particular agent and / or mode of administration. The pharmaceutical carrier can be liquid, semi-liquid or solid. Solutions or suspensions for parenteral, intradermal, subcutaneous or topical application can include, for example, sterile diluents (such as water), saline solutions (e.g., phosphate buffered saline; PBS), fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents (such as benzyl alcohol and methyl paraben); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates). If administered intravenously (e.g., by intravenous infusion), suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickeners and solubilizers (such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof).

[0105] Administration of the agents described herein in pure form or in the form of suitable therapeutic or pharmaceutical compositions can be carried out by any accepted mode of administration of pharmaceutical agents for serving similar purposes. Therapeutic or pharmaceutical compositions can be prepared by combining the composition containing the agent with a suitable physiologically acceptable carrier, diluent or excipient, and can be formulated into solid, semisolid, liquid or gaseous preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. In addition, other pharmaceutically active ingredients (including other small molecules as described elsewhere herein) and / or suitable excipients such as salts, buffers and stabilizers may be present in the composition but need not be.

[0106] Administration can be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous or topical. The preferred mode of administration depends on the nature of the condition to be treated or prevented. Specific embodiments include administration by intravenous infusion.

[0107] The carrier may include, for example, a pharmaceutically or physiologically acceptable carrier, excipient or stabilizer that is nontoxic to the cells or mammals exposed to it at the dosage and concentration employed. Physiologically acceptable carriers are often aqueous pH buffered solutions. Examples of physiologically acceptable carriers include: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN TM ) Polyethylene glycol (PEG) and poloxamer (PLURONICS TM )wait.

[0108] In certain embodiments, one or more agents may be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., ed., (1980). The particles or liposomes may further contain other therapeutic or diagnostic agents.

[0109] The exact dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known test protocols, or by testing the composition in a model system known in the art and inferring therefrom. Controlled clinical trials can also be conducted. The dosage can also vary with the severity of the condition to be alleviated. Pharmaceutical compositions are usually formulated and administered to exert therapeutically useful effects while minimizing undesirable side effects. The composition can be administered in a one-time application, or can be divided into a number of smaller doses to be administered at intervals. For any particular subject, a specific dosage regimen can be adjusted over time according to individual needs.

[0110] Therefore, typical routes of administering these and related therapeutic compositions or pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhaled, parenteral, sublingual, buccal, rectal, vaginal and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. The therapeutic composition or pharmaceutical composition according to certain embodiments of the present disclosure is formulated so as to allow the active ingredients contained therein to be bioavailable after the composition is administered to a subject or patient. The composition to be administered to a subject or patient can take the form of one or more dosage units, wherein, for example, a tablet can be a single dose unit, and a container of an agent described herein in the form of an aerosol can accommodate multiple dosage units. The actual method of preparing such dosage forms is known or obvious to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will typically contain a therapeutically effective amount of an agent described herein for treating the disease or condition of interest.

[0111] The therapeutic composition or pharmaceutical composition can be in the form of a solid or liquid. In one embodiment, the carrier is a microparticle so that the composition is in the form of, for example, a tablet or powder. The carrier can be a liquid, and the composition is, for example, an oral oil, an injectable liquid or an aerosol, which can be used, for example, for inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably in the form of a solid or liquid, wherein semi-solid, semi-liquid, suspension and gel forms are included in the forms considered as solid or liquid in this article. Certain embodiments include sterile, injectable solutions.

[0112] As a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gum, glutinous rice paper capsules, etc. Such solid compositions will generally contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrin, disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as mint, methyl salicylate or orange flavor; and coloring agents. When the pharmaceutical composition is in capsule form (e.g., gelatin capsules), it may also contain liquid carriers such as polyethylene glycol or oil in addition to the above-mentioned types of materials.

[0113] Therapeutic compositions or pharmaceutical compositions can be in the form of liquids, for example, elixirs, syrups, solutions, emulsions or suspensions. As two examples, liquids can be used for oral administration or for delivery by injection. When intended for oral administration, in addition to the compounds of this invention, preferred compositions also contain one or more of sweeteners, preservatives, dyes / colorants and flavor enhancers. In compositions intended to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents can be included.

[0114] Liquid therapeutic compositions or pharmaceutical compositions (whether they are solutions, suspensions or other similar forms) may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides or diglycerides (which may serve as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or glucose. Parenteral preparations may be enclosed in ampoules, disposable syringes or multiple dose bottles made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0115] Liquid therapeutic compositions or pharmaceutical compositions intended for parenteral or oral administration should contain an amount of reagent so that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the target agent in the composition. When intended for oral administration, this amount can vary between 0.1% to about 70% of the composition weight. Some oral therapeutic compositions or pharmaceutical compositions contain about 4% to about 75% of the target agent. In certain embodiments, therapeutic compositions or pharmaceutical compositions and preparations according to the present invention are prepared in such a way that the parenteral dosage unit contains 0.01 to 10% by weight of the target agent before dilution.

[0116] Therapeutic compositions or pharmaceutical compositions can include various materials that modify the physical form of solid or liquid dosage units. For example, the composition can include materials that form a coating shell around the active ingredient. The material that forms the coating shell is usually inert and can be selected from, for example, sugar, shellac and other enteric coating agents. Alternatively, the active ingredient can be wrapped in a gelatin capsule. Therapeutic compositions or pharmaceutical compositions in solid or liquid form can include such components: it is combined with an agent and thus helps the delivery of the compound. Suitable components that can work in this capacity include monoclonal antibodies or polyclonal antibodies, one or more proteins or liposomes.

[0117] The compositions described herein can be prepared with carriers that protect the agent from rapid elimination from the body, such as time-limited release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and other substances known to those of ordinary skill in the art.

[0118] Therapeutic compositions or pharmaceutical compositions can be prepared by methods well known in the pharmaceutical field. For example, therapeutic compositions or pharmaceutical compositions intended to be administered by injection may contain one or more of salts, buffers and / or stabilizers, with sterile distilled water to form a solution. Surfactants may be added to promote the formation of uniform solutions or suspensions. Surfactants are compounds that non-covalently interact with an agent, thereby promoting the dissolution or uniform suspension of an agent in an aqueous delivery system.

[0119] Certain embodiments include the use of a diagnostic kit for treating cancer in a human subject in need thereof with zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof, the diagnostic kit comprising a device for determining the mutation status of isocitrate dehydrogenase (IDH1 or IDH2) in a tissue sample from a subject. Also included is a patient care kit comprising: (a) a device for determining the mutation status of isocitrate dehydrogenase (IDH1 or IDH2) in a tissue sample from a human subject with cancer; and (b) a composition comprising zotiracillin (TG02) or an analog, derivative, or pharmaceutically acceptable salt thereof.

[0120] In some embodiments, the device for (directly) determining the IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay selected from one or more of the following: DNA or RNA sequencing, ISH, FISH, WES, SNP arrays, NGS or CGH on human IDH1 or IDH2 proteins or genes. In certain embodiments, the device for (indirectly) determining the IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay (e.g., a diagnostic assay selected from biochemical detection (e.g., colorimetric assay), biosensor, GC- or LC-MS and MALDI-TOF) to determine the level of D-2HG in a tissue sample.

[0121] Some diagnostic kits or patient care kits include an IDH1 / 2 gene reference obtained from a database or determined from a control or reference, such as a homozygous wild-type IDH1 / 2 control. The kit may also include, for example, written instructions on how to determine or measure the level, presence, absence of D-2HG in a tissue sample from a subject and / or from a control.

[0122] Certain diagnostic kits or patient care kits comprise one or more additional agents, such as an immunotherapeutic, chemotherapeutic, hormonal therapeutic, and / or kinase inhibitor as described herein.

[0123] In certain embodiments, a diagnostic or patient care kit contains separate containers, dispensers, or compartments for a composition and informational material. For example, a composition or reagent may be contained in a bottle, vial, or syringe, and the informational material may be contained in combination with the container. In certain embodiments, the separate elements of the kit are contained in a single, undivided container. For example, a composition or reagent is contained in a bottle, vial, or syringe with informational material attached thereto in the form of a label. In certain embodiments, the kit includes multiple (e.g., a pack) of separate containers each containing one or more compositions, reagents, and / or unit dosage forms of zotiracillin. For example, the kit includes multiple syringes, ampoules, foil packets, or blister packs each containing a reagent or a single unit dose of zotiracillin. The containers of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation) and / or lightproof.

[0124] The patient care kit optionally includes a device suitable for administering the reagent, such as a syringe, an inhaler, a dropper (e.g., an eye dropper), a swab (e.g., a cotton swab or a wooden swab), or any such delivery device. In certain embodiments, the device is an implantable device that dispenses a metered dose of the reagent. Also included are methods of providing a kit, for example, by combining the components described herein.

[0125] In some aspects, the diagnosis or treatment response test or method described herein is performed in a diagnostic laboratory, and the result is then provided to the subject, or a physician or other health care provider who plays a role in the health care and cancer treatment of the subject. Therefore, specific embodiments include methods for providing the result of the responsiveness test to a subject in need, or a physician or other health care provider. These results or data can be in the form of hard copy or paper copy, or in electronic form such as computer readable medium.

[0126] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0127] Although the foregoing invention has been described in more detail by way of specific illustration and examples for the purpose of clear understanding, it will be readily apparent to those of ordinary skill that certain changes and modifications may be made thereto without departing from the spirit or scope of the specification or the appended claims. The following examples are provided merely as specific illustrations and not as limitations. Those skilled in the art will readily appreciate that a variety of non-critical parameters may be changed or modified to produce substantially similar results.

[0128] Examples

[0129] Example 1

[0130] Experiments were performed to test the activity of zotiracillin (TG02) against IDH1 mutant cancer cells and to evaluate the mechanism of such activity.

[0131] Materials and methods

[0132] Cell culture. Human chondrosarcoma cell line HT-1080 was cultured in MEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gemini, USA). Human cholangiocarcinoma cell lines RBE, Hccc-9810 and Hucct-1 were cultured in RPMI1640 (Hyclone, USA) supplemented with 10% FBS. Cells were incubated at 37° C. in 5% CO 2 .

[0133] Apoptosis assay. HT-1080, RBE, Hccc-9810, and Hucct-1 cells were treated with the indicated drugs. Apoptosis was detected by Annexin V / PI staining kit (Thermo Fisher) according to the manufacturer's instructions. Briefly, cells were plated at 1×10 5 Cells were plated at a density of 10 cells / well in medium containing 10% FBS containing the desired concentration of TG02. At 48 / 72 hours after treatment, cells were harvested and tested for apoptosis by Annexin V and PI staining. Cell analysis was performed using FACSCelesta (BD).

[0134] 2-Hydroxyglutarate (D-2HG) assay. Cells (approximately 1 x 10 7 ) were quickly homogenized on ice for 10 min. The cells were then centrifuged at 10,000 × g for 5 min at 4°C, the supernatant was collected, and the same volume of each sample was added to three wells of a 96-well clear plate.

[0135] Dilute the 100 mM D-2HG standard to 1 mM (1 nmol / μl) by adding 10 μl of 100 mM D-2HG standard solution to 990 μl D-2HG assay buffer and mix well.

[0136] 5 μl of 1 mM D-2HG standard was added to one of three samples, defined as: spiked sample (5 nmol D2 hydroxyglutarate + sample); sample; and sample background. The spiked sample was used as an internal standard to correct for any sample interference. The final volume of all wells was adjusted to 50 μl with D-2HG assay buffer.

[0137] For each well, prepare 50 μl of reaction mixture (see Table E1):

[0138]

[0139] 50 μl of the reaction mixture was added to each well containing standards and samples and mixed well. The plate was incubated at 37°C for 60 minutes and the OD450 nm was measured.

[0140] The sample background reading was subtracted from its paired sample reading to obtain the sample correction reading. The amount of D-2HG in the sample well (X) was determined based on the following equation:

[0141] D-2HG amount (nmol) = (OD sample (corrected) / [(OD (spiked sample)) - (OD sample)] x 5

[0142] Comet assay. The comet assay is a common technique used to measure DNA damage in individual cells. Under the electrophoretic field, damaged cellular DNA (containing fragments and strand breaks) is separated from intact DNA, resulting in the classic "comet tail" shape under the microscope. TM Comet Assay Kit (CELLBIOLABS, INC.) was used to measure DNA damage. Briefly, comet agarose was pipetted onto OxiSelect TM Incubate the cells with OxiSelect at 37°C TM Comet agarose combination, then pipette the agarose / cell mixture on top of the basal layer. Treat cells with lysis buffer and alkaline solution. Perform electrophoresis under neutral conditions. Observe the slides by epifluorescence microscopy using a FITC filter.

[0143] Tail moment = (100 × tail DNA strength / cell DNA strength) × tail moment length

[0144] Western blot. Hccc-9810 and Hucct-1 cells treated with TG02 or AG120 for 48 hours as indicated (0.1% DMSO was added as a control) were collected and centrifuged at 500g for 5 minutes to obtain cell pellets. The pellets were then lysed in a lysis buffer (Beyotime, China), which additionally included a protease inhibitor cocktail (Beyotime, China) and a phosphatase inhibitor cocktail (Beyotime, China). The cells were incubated on ice for 30 minutes and then centrifuged at 12000 rpm for 10 minutes at 4°C to obtain the supernatant as a cell lysate. The supernatant was purified by Micro BCA TM The concentration of protein in the cell lysate was determined by a protein assay kit (ThermoFisher, USA). 4×SDS-PAGE sample loading buffer (SolarBio, China) was added to the cell lysate containing 25 μg of total protein, and after boiling, the mixture was electrophoresed in a polyacrylamide gel. After electrophoresis, the protein on the gel was transferred to a PVDF membrane, and the membrane was cut at a position close to the molecular weight of the protein, and the expression of the protein was examined (pH2AX (CST, USA), GAPDH (ZSGB-BIO, China)).

[0145] Transfection. One day before transfection, cells (approximately 2 x 10 5 / well) were inoculated in 6-well plates. The cells were cultured overnight at 37°C in a 5% CO2 incubator. The old culture medium was removed and 2 ml of transfection medium was added. 50 μl of opti-MEM was added to the tube, and then the 2 μg of plasmid DNA mixture solution was mixed by vortexing. 4 μl of PEI solution was added to this tube, and the solution was mixed by gently pipetting up and down. The sample was incubated at room temperature for 15 minutes to allow the formation of PEI / DNA complexes. The PEI / DNA complex was gently added dropwise to the wells containing the cells. The cells were mixed by gently swirling and incubated for 48 hours at 37°C, 5% CO2. The cells were then used to detect drug efficacy or gene expression.

[0146] Results. Figure 2A-2B As shown, IDH1-mutant chondrosarcoma (HT-1080) and cholangiocarcinoma (RBE, Hcc-9810) cells were more sensitive to TG02 compared with IDH1-WT (Hucct-1) cells. Figure 3A-3BIt was shown that increased TG02 sensitivity can be conferred to IDH1-WT (Hucct-1) cells by transfection of mutant IDH1. Figure 3A Hucct-1 cells were transfected with IDH1 R132H plasmid and treated with 0.1 μM TG02 for 72 h. Cell apoptosis was determined by FACS assay. Figure 3B In the present study, Hucct-1 cells were transfected with IDH1 R132H plasmid, and the level of D-2-hydroxyglutarate (D-2HG) was measured in Hucct-1 WT cells, Hucct-1 cells transfected with IDH1 R132H plasmid, and RBE (cholangiocarcinoma cell line with IDH1 R132S mutation) cells. D-2HG is a metabolic biomarker for gain-of-function mutations in IDH1 and / or IDH2 genes.

[0147] Figure 4 It was shown that TG02-induced apoptosis of mutant IDH1 (Hccc-9810) cells can be rescued by adding the exogenous substrate α-ketoglutarate (α-KG). Mutant IDH1 / 2 converts α-KG into tumor metabolites (D-2HG), which then inhibit a class of α-KG-dependent enzymes involved in epigenetic regulation. Cells were pretreated with α-KG for 24 h and then treated with TG02 for 72 h. Apoptosis was determined by FACS assay. Similarly, Figure 5A-5B It is shown that TG02-induced DNA damage in mutant IDH1 (HT-1080) cells can be rescued by adding exogenous α-KG or AG-120. AG-120 is an inhibitor of mutant IDH1. Figure 5A Cells were treated with the indicated drugs for 48 h, and DNA damage was determined by the neutral comet assay. Figure 5B Statistical analysis of the data in 5A is shown (left panel: tail moment. right panel: positive percentage of cells with DNA damage).

[0148] Figure 6 Shown is the rescue of TG02-induced DNA damage in mutant IDH1 cells (Hccc-9810) only by the addition of exogenous AG-120. Cells were treated with the indicated drugs for 48 hours. pH2AX, a marker of DNA damage, was determined by WB.

[0149] Figure 7A-7B It was shown that cells with IDH1 mutations are more sensitive to TG02-induced DNA damage. Fig. 7A In this study, IDH1 mutant Hucct1 cells (Hucct1 R132H / +). Hucct1, Hucct1 were treated with 0.2 μM TG02 or 0.1% DMSO. R132H / + and HT-1080 cells for 48 h. Comet assay was performed with cells. Quantification of tail moment in neutral comet assay is presented. ns: no significant difference; ***: P < 0.001, ****: P < 0.0001 compared with NC; #: P < 0.05 compared with Hucct1 cells treated with TG02. Figure 7B Hucct1 cells and Hucct1 were treated with 2 μM AG120, 0.2 μM TG02, or 0.01 μM AZD4573 (CDK9 inhibitor) as indicated. R132H / + Cells. Western blot analysis of phosphorylated γH2AX (a marker of DSBs) after 16 hours of treatment is shown. β-Actin was used as a loading control.

[0150] Figures 8A-8C TG02 in combination with AG120 is shown to induce apoptosis in cells with mutant IDH1. Mutant IDH1 cells HT-1080 (8A), RBE (8B) and Hccc-9810 (8C) were treated with TG02 (0.1 μM) and AG120 (2 μM) for 48 hours as indicated, and apoptosis was measured by Annexin V / PI staining and analyzed by flow cytometry.

[0151] Thus, the evidence shows that TG02 selectively induces DNA damage and cell killing in mutant IDH1 cancer cells relative to homozygous wild-type IDH1 cancer cells. The evidence also shows that such TG02 sensitivity is associated with the 'gain of function' cancer-promoting activity of mutant IDH1 (shared by mutant IDH2) that converts α-KG to the oncometabolite D-2HG.

Claims

1. A method for treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, the method comprising administering to the subject a composition comprising zotiraciclib (TG02) or a pharmaceutically acceptable salt thereof, Said cancer comprising said IDH1 or IDH2 mutation is thereby treated.

2. The method of claim 1, wherein the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), astrocytoma, sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL) and glioblastoma (optionally secondary glioblastoma).

3. The method according to claim 1 or 2, comprising: (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; and (b) if the tissue sample comprises the IDH1 or IDH2 mutation, administering to the subject a composition comprising zotiracillin (TG02) or a pharmaceutically acceptable salt thereof.

4. The method according to any one of claims 1 to 3, wherein the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the tumor metabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2.

5. The method according to any one of claims 1 to 4, wherein the IHD1 mutation is R132X, wherein X is selected from any amino acid except R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L or R132S.

6. The method of any one of claims 1 to 4, wherein the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q.

7. The method according to any one of claims 3 to 6, wherein (a) comprises determining the IDH1 or IDH2 mutation status in the tissue sample by DNA or RNA sequencing, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), whole exome sequencing (WES), single nucleotide polymorphism (SNP) array, next generation sequencing (NGS) or comparative genomic hybridization (CGH) on human IDH1 or IDH2 protein or gene.

8. The method according to any one of claims 3 to 7, comprising obtaining the tissue sample from the subject.

9. The method according to any one of claims 3 to 8, wherein the tissue sample is a liquid biopsy, optionally a blood sample, a surgical sample or other biopsy sample obtained from the subject, optionally wherein the tissue sample is a cancerous tissue sample.

10. The method of any one of claims 1 to 9, comprising administering to the subject an oral composition of zotiracillin or a pharmaceutically acceptable salt thereof.

11. The method according to any one of claims 1 to 10, comprising administering the composition comprising zotiracillin in combination with radiation therapy and / or one or more additional agents, optionally selected from chemotherapeutic agents, hormonal therapeutic agents and / or kinase inhibitors.

12. The method of claim 11, wherein the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), enasidenib, or AG-221.

13. Use of a diagnostic kit for treating cancer in a human subject in need thereof with zotiracillin (TG02) or a pharmaceutically acceptable salt thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, and the diagnostic kit comprises a device for determining the isocitrate dehydrogenase (IDH1 or IDH2) mutation status in a tissue sample from the subject.

14. The use according to claim 13, wherein the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the tumor metabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2.

15. Use according to claim 13 or 14, wherein the IHD1 mutation is R132X, wherein X is selected from any amino acid except R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L or R132S.

16. Use according to any one of claims 13 to 15, wherein the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q.

17. The use according to any one of claims 13 to 16, wherein the device for determining the IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay selected from one or more of the following: DNA or RNA sequencing, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), whole exome sequencing (WES), single nucleotide polymorphism (SNP) array, next generation sequencing (NGS) or comparative genomic hybridization (CGH) on human IDH1 or IDH2 protein or gene.

18. The use according to any one of claims 13 to 17, wherein the tissue sample is a liquid biopsy, optionally a blood sample, a surgical sample or other biopsy sample obtained from the subject, optionally a biopsy of prostate cancer tissue.

19. The method of any one of claims 13 to 18, wherein the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL), astrocytoma and glioblastoma (optionally secondary glioblastoma).

20. The use according to any one of claims 13 to 19, wherein the diagnostic kit comprises a composition comprising zotipracillin or a pharmaceutically acceptable salt thereof, optionally an oral composition of zotipracillin.

21. Use according to claim 20, wherein the diagnostic kit comprises one or more additional agents, optionally selected from chemotherapeutic agents, hormonal therapeutic agents and / or kinase inhibitors.

22. The use according to claim 21, wherein the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), etanercept or AG-221.

23. A patient care kit comprising: (a) an apparatus for determining the mutation status of isocitrate dehydrogenase (IDH1 or IDH2) in a tissue sample from a human subject suffering from cancer; and (b) A composition comprising zotiracillin (TG02) or a pharmaceutically acceptable salt thereof.

24. A patient care kit according to claim 23, wherein the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the tumor metabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2.

25. The patient care kit of claim 23 or 24, wherein the IHD1 mutation is R132X, wherein X is selected from any amino acid except R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L or R132S.

26. A patient care kit according to any one of claims 23 to 25, wherein the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q.

27. A patient care kit according to claims 13 to 16, wherein the device for determining the IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay selected from one or more of the following: DNA or RNA sequencing, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), whole exome sequencing (WES), single nucleotide polymorphism (SNP) array, next generation sequencing (NGS) or comparative genomic hybridization (CGH) on human IDH1 or IDH2 protein or gene.

28. A patient care kit according to any one of claims 23 to 27, wherein the tissue sample is a liquid biopsy, optionally a blood sample, a surgical sample or other biopsy sample obtained from the subject, optionally a biopsy of prostate cancer tissue.

29. A patient care kit according to any one of claims 23 to 28, wherein the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), astrocytoma, sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL) and glioblastoma (optionally secondary glioblastoma).

30. The patient care kit of any one of claims 23 to 29, wherein (b) comprises an oral composition of zotiracillin or a pharmaceutically acceptable salt thereof.

31. A patient care kit according to any one of claims 23 to 30, wherein the patient care kit comprises one or more additional agents, the one or more additional agents optionally selected from chemotherapeutic agents, hormonal therapeutic agents and / or kinase inhibitors.

32. The patient care kit of claim 11, wherein the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), etanercept, or AG-221.

33. A pharmaceutical composition for use in a method of treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, the pharmaceutical composition comprising zotiracillin (TG02) or a pharmaceutically acceptable salt thereof.

34. A pharmaceutical composition for use according to claim 33, wherein the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the tumor metabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2.

35. A pharmaceutical composition for use according to claim 33 or 34, wherein the IHD1 mutation is R132X, wherein X is selected from any amino acid except R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L or R132S.

36. A pharmaceutical composition for use according to any one of claims 33 to 35, wherein the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q.

37. A pharmaceutical composition for use according to any one of claims 33 to 36, wherein the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), astrocytoma, sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL) and glioblastoma (optionally secondary glioblastoma).

38. A pharmaceutical composition for use according to any one of claims 33 to 37 comprising an oral composition of zotiracillin or a pharmaceutically acceptable salt thereof.

39. A pharmaceutical composition for use according to any one of claims 33 to 35, comprising one or more additional agents, optionally selected from chemotherapeutic agents, hormonal therapeutic agents and / or kinase inhibitors.

40. The pharmaceutical composition for use of claim 39, wherein the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), etanercept, or AG-221.

41. A pharmaceutical composition for use according to any one of claims 31 to 40, wherein the method comprises: (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; and (b) if the tissue sample comprises the IDH1 or IDH2 mutation, administering to the subject the composition comprising zotiracillin (TG02) or a pharmaceutically acceptable salt thereof.

42. Use of a composition for the preparation of a medicament for treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, and the composition comprises zotiracillin (TG02) or a pharmaceutically acceptable salt thereof.

43. The use according to claim 42, wherein the IDH1 or IDH2 mutation is a gain-of-function mutation characterized by increased conversion of α-ketoglutarate (α-KG) to the tumor metabolite D-2-hydroxyglutarate (D-2HG) relative to homozygous wild-type IDH1 or IDH2.

44. Use according to claim 42 or 43, wherein the IHD1 mutation is R132X, wherein X is selected from any amino acid except R, optionally wherein the IHD1 mutation is R132C, R132G, R132H, R132L or R132S.

45. The use according to any one of claims 42 to 44, wherein the IHD2 mutation is R172X or R140X, wherein X is selected from any amino acid except R, optionally wherein the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q.

46. ​​The method of any one of claims 42 to 45, wherein the cancer is selected from glioma (optionally low-grade or high-grade glioma), medulloblastoma, chondrosarcoma, cholangiocarcinoma, acute myeloid leukemia (AML), astrocytoma, sinonasal undifferentiated carcinoma (SNUC), angioimmunoblastic T-cell lymphoma (AITL) and glioblastoma (optionally secondary glioblastoma).

47. The use according to any one of claims 42 to 45, which comprises an oral composition of zotiracillin or a pharmaceutically acceptable salt thereof.

48. The use according to any one of claims 42 to 47, comprising one or more additional agents, optionally selected from chemotherapeutic agents, hormonal therapeutic agents and / or kinase inhibitors.

49. The use according to claim 49, wherein the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), etanercept or AG-221.

50. The use according to any one of claims 31 to 40, comprising: (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; and (b) if the tissue sample comprises the IDH1 or IDH2 mutation, administering to the subject the composition comprising zotiracillin (TG02) or a pharmaceutically acceptable salt thereof.

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