IDH mutations as biomarkers for zotiraclib therapy

Abstract

For example, the use of isocitrate dehydrogenase (IDH1 and IDH2) mutations to select cancer patients for zotiraclib (TG02) therapy is provided in the treatment of cancers such as glioma, medulloblastoma, chondrosarcoma, cholangiocarcinoma, AML, astrocytoma, etc. Embodiments of the present disclosure include a method for treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, comprising administering to the subject a composition comprising zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof, thereby treating the cancer comprising an IDH1 or IDH2 mutation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to PCT / CN2022 / 111327, filed August 10, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] (Technical field) Embodiments of the present disclosure relate to the use of isocitrate dehydrogenase (IDH1 and IDH2) mutations to select cancer patients for zotiraclib (TG02) therapy, for example, in the treatment of cancers such as glioma, medulloblastoma, chondrosarcoma, cholangiocarcinoma, AML, and astrocytoma.

[0003] Description of Related Art Zotiraclib (TG02) is a selective kinase inhibitor for the treatment of cancer (William et al., J. 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, for example, Wu et al., Clin Cancer Res. 27: 3298-3306, 2021). However, there is a need in the art to better predict the anti-cancer therapeutic efficacy of zotiraclib, thereby identifying patients who will most likely benefit from treatment with this chemotherapy. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] William et al., J. of Medicinal Chem. 55: 169-196, 2012 [Non-patent document 2] Wu et al., Clin Cancer Res.27:3298-3306,2021 Summary of the Invention [Means for solving the problem]

[0005]

[0003] Embodiments of the present disclosure include a method for treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, comprising administering to the subject a composition comprising zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof, thereby treating the cancer comprising an IDH1 or IDH2 mutation. In certain embodiments, the cancer is glioma. Certain methods include (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject, and (b) if the tissue sample comprises an IDH1 or IDH2 mutation, administering to the subject a composition comprising zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof.

[0006] 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) compared to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, where X is selected from any amino acid other than R, and optionally the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, where X is selected from any other R of any amino acid, and optionally the IHD2 mutation is R172G, R172K, R172M, R172S, R172T, or R140Q.

[0007] 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 the human IDH1 or IDH2 protein or gene.

[0008] Certain embodiments include obtaining a tissue sample from a subject, hi some embodiments, the tissue sample is a liquid biopsy, optionally a blood sample, a surgical sample, or other biopsy sample obtained from the subject, and optionally, the tissue sample is a cancer tissue sample.

[0009] 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).

[0010] Certain embodiments include administering to a subject an oral composition of zotilaclib, or an analog, derivative, or pharmaceutically acceptable salt thereof. Some embodiments include administering a composition comprising zotilaclib in combination with radiation therapy and / or optionally one or more additional agents selected from a chemotherapeutic agent, a hormonal therapy agent, and / or a kinase inhibitor. In some embodiments, the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), enasidenib, or AG-221.

[0011] Certain embodiments include use of a diagnostic kit for treating cancer in a human subject in need thereof with zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, including means for determining isocitrate dehydrogenase (IDH1 or IDH2) mutation status in a tissue sample from the subject.

[0012] 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) compared to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, where X is selected from any amino acid other than R, and optionally the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, where X is selected from any other R of any amino acid, and optionally the IHD2 mutation is R172G, R172K, R172M, R172S, R172T, or R140Q.

[0013] In some embodiments, the means for determining IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay selected from one or more of DNA or RNA sequencing on the human IDH1 or IDH2 protein or gene, in situ hybridization (ISH), fluorescent in situ hybridization (FISH), whole exome sequencing (WES), single nucleotide polymorphism (SNP) array, next generation sequencing (NGS), or comparative genomic hybridization (CGH).

[0014] In some embodiments, the tissue sample is a liquid biopsy, surgical sample, or other biopsy sample, optionally obtained from a subject, and optionally a blood sample, and optionally a biopsy of 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 zotilaclib, or an analog, derivative, or pharmaceutically acceptable salt thereof, optionally an oral composition of zotilaclib. In some embodiments, the diagnostic / therapeutic kit comprises one or more additional agents, 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), enasidenib, or AG-221.

[0015] Also included is a patient care kit comprising: (a) a means for determining isocitrate dehydrogenase (IDH1 or IDH2) mutation status in a tissue sample from a human subject having cancer; and (b) a composition comprising zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof.

[0016] 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) compared to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, where X is selected from any amino acid other than R, and optionally the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, where X is selected from any other R of any amino acid, and optionally the IHD2 mutation is R172G, R172K, R172M, R172S, R172T, or R140Q.

[0017] In some embodiments, the means for determining IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay selected from one or more of DNA or RNA sequencing on the human IDH1 or IDH2 protein or gene, in situ hybridization (ISH), fluorescent in situ hybridization (FISH), whole exome sequencing (WES), single nucleotide polymorphism (SNP) array, next generation sequencing (NGS), or comparative genomic hybridization (CGH). In some embodiments, the tissue sample is a liquid biopsy, surgical sample, or other biopsy sample, optionally obtained from the subject, optionally a blood sample, and optionally a biopsy of prostate cancer tissue.

[0018] 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).

[0019] In some embodiments, (b) comprises an oral composition of zotiraclib, or an analog, derivative, or pharmaceutically acceptable salt thereof. In some embodiments, the patient care kit comprises one or more additional agents, optionally selected from a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor. In some embodiments, the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), enasidenib, or AG-221.

[0020] Certain embodiments include pharmaceutical compositions for use in methods of treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, including zotiraclib (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) compared to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, where X is selected from any amino acid other than R, and optionally the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, where X is selected from any other R of an amino acid; optionally, the IHD2 mutation is R172G, R172K, R172M, R172S, R172T, or R140Q.

[0021] 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 zotiraclib, or an analog, derivative, or pharmaceutically acceptable salt thereof. Certain embodiments include the use of one or more additional agents, 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), enasidenib, or AG-221. In some embodiments, the method includes (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject, and (b) administering to the subject a composition comprising zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof, if the tissue sample contains an IDH1 or IDH2 mutation.

[0022] Also included is the use of the composition in 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, including zotiraclib (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) compared to homozygous wild-type IDH1 or IDH2. In some embodiments, the IHD1 mutation is R132X, where X is selected from any amino acid other than R, and optionally the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, where X is selected from any other R of any amino acid; optionally, 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). Particular uses include oral compositions of zotiraclib, or analogs, derivatives, or pharmaceutically acceptable salts thereof. Particular uses include one or more additional agents 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), enasidenib, or AG-221. A particular use includes (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject, and (b) if the tissue sample contains an IDH1 or IDH2 mutation, administering to the subject a composition comprising zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the two-dimensional chemical structure of zotiraclib. [Figure 2A] Figures 2A-2B show 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. Apoptosis was determined by FACS assay. [Figure 2B] Figures 2A-2B show 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. Apoptosis was determined by FACS assay. [Figure 3A] Figures 3A-3B show that increased TGO2 sensitivity can be conferred to IDH1-WT (Hucct-1) cells by transfection with mutant IDH1. In Figure 3A, Hucct-1 cells were transfected with the IDH1 R132H plasmid and treated with 0.1 μM TGO2 for 72 hours. Apoptosis was determined by FACS assay. [Figure 3B] In Figure 3B, Hucct-1 cells were transfected with the IDH1 R132H plasmid, and the levels of D-2-hydroxyglutarate (D-2HG) were measured in Hucct-1 WT, Hucct-1 transfected with the IDH1 R132H plasmid, and RBE (a cholangiocarcinoma cell line harboring the IDH1 R132S mutation) cells. D-2HG is a metabolic biomarker of gain-of-function mutations in the IDH1 and / or IDH2 genes. [Figure 4]Figure 4 shows that TG02-induced apoptosis of mutant IDH1 (Hccc-9810) cells can be rescued by the addition of excess exogenous substrate α-ketoglutarate (α-KG). Mutant IDH1 / 2 converts α-KG to the oncometabolite 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. [Figure 5A] Figures 5A-5B show that TG02-induced DNA damage in mutant IDH1 (HT-1080) cells can be rescued by the addition of exogenous α-KG or AG-120. AG-120 is an inhibitor of mutant IDH1. In Figure 5A, cells were treated with the indicated drugs for 48 h, and DNA damage was determined by the neutral comet assay. [Figure 5B] FIG. 5B shows the statistical analysis of the data in 5A (left panel: tail moments; right panel: positive percentage of cells with DNA damage). [Figure 6] Figure 6 shows that TGO2-induced DNA damage was rescued only in mutant IDH1 cells (Hccc-9810) by the addition of exogenous AG-120. Cells were treated with the indicated drugs for 48 hours. pH2AX, a marker of DNA damage, was assayed by WB. [Figure 7A] Figures 7A-7B show that cells with IDH1 mutations are more sensitive to DNA damage induced by TG02. In Figure 7A, IDH1 mutant Hucct1 cells (Hucct1R132H / +) were generated by transduction with lentivirus-expressing IDH1 R132H. Hucct1, Hucct1R132H / +, and 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 the neutral comet assay is presented. ns: no significant difference; ***: P<0.001, ****: P<0.0001 compared to NC; #: P<0.05 compared to Hucct1 cells treated with TG02. [Figure 7B] In Figure 7B, Hucct1 and Hucct1R132H / + cells were treated with 2 μM AG120, 0.2 μM TG02, or 0.01 μM AZD4573 (a CDK9 inhibitor) as indicated. Western blot analysis of phosphorylated γH2AX (a marker for DSBs) after 16 hours of treatment is shown. β-actin was used as a loading control. [Figure 8A] Figures 8A-8C show that TGO2 in combination with AG120 induced cell apoptosis in cells harboring mutant IDH1. Mutant IDH1 cells, HT-1080 (8A), RBE (8B), and Hccc-9810 (8C), were treated with TGO2 (0.1 μM) and AG120 (2 μM) as indicated for 48 hours, and apoptosis was measured by Annexin V / PI staining and flow cytometric analysis. [Figure 8B] Figures 8A-8C show that TGO2 in combination with AG120 induced cell apoptosis in cells harboring mutant IDH1. Mutant IDH1 cells, HT-1080 (8A), RBE (8B), and Hccc-9810 (8C), were treated with TGO2 (0.1 μM) and AG120 (2 μM) as indicated for 48 hours, and apoptosis was measured by Annexin V / PI staining and flow cytometric analysis. [Figure 8C] Figures 8A-8C show that TGO2 in combination with AG120 induced cell apoptosis in cells harboring mutant IDH1. Mutant IDH1 cells, HT-1080 (8A), RBE (8B), and Hccc-9810 (8C), were treated with TGO2 (0.1 μM) and AG120 (2 μM) as indicated for 48 hours, and apoptosis was measured by Annexin V / PI staining and flow cytometric analysis. DETAILED DESCRIPTION OF THE INVENTION

[0024] Embodiments of the present disclosure relate to the surprising discovery that cancers harboring isocitrate dehydrogenase (IDH1 and IDH2) mutations exhibit significantly greater sensitivity to zotiraclib (TG02) therapy. Thus, IDH1 / 2 mutation status and its associated gain-of-function phenotype can be used as biomarkers or companion diagnostics to select patients for optimized zotiraclib cancer therapy.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used to implement or test the subject matter of this disclosure, preferred methods and materials are described. All publications and references cited herein, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated herein by reference as if fully set forth. Any patent application to which this application claims priority is also incorporated herein by reference in its entirety in the manner described above for publications and references.

[0026] For purposes of this disclosure, the following terms are defined below.

[0027] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of an article. By way of example, "an element" means one element or more than one element.

[0028] "Antagonist" or "inhibitor" refers to a biological structure or chemical agent (e.g., a compound) that prevents or otherwise reduces the physiological action of another molecule, such as a protein. In some cases, an antagonist or inhibitor specifically binds to the other molecule and / or a functional ligand of the other molecule. In some cases, an antagonist or inhibitor downregulates the expression of the other molecule. Included are full antagonists and partial antagonists.

[0029] "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 instances, agonists specifically bind to other agents or molecules. Full agonists and partial agonists are included.

[0030] "About" means an amount, level, value, frequency, percentage, dimension, size, amount, weight, or length that varies by about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reference amount, level, value, frequency, percentage, dimension, size, amount, weight, or length.

[0031] The term "bond" refers to a direct association between two molecules, for example, through covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges.

[0032] Throughout this disclosure, unless the context requires otherwise, the terms "comprise", "comprises", and "comprising" are understood to imply the inclusion of stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements.

[0033] "Consisting of" means including, but not limited to, everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that other elements may not 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 conduct specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they materially affect the activity or action of the listed elements.

[0034] "Median Effective Concentration" or "EC 50 The term "EC" refers to the concentration of an agent (e.g., compound) described herein that elicits a response between baseline and maximum after some specified exposure time, and is therefore the EC of a graded dose-response curve. 50 represents the concentration of a drug at which 50% of its maximal effect is observed. 50 The EC2 also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. 90 " refers to the concentration of an agent or composition at which 90% of its maximum effect is observed. 90 " is "EC 50 and the Hill slope, or can be determined directly from the data using routine knowledge in the art. In some embodiments, the EC 50 is less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 nM. 50 It has a value.

[0035] "Half-maximal inhibitory concentration" (or "IC 50 ") is a measure of the potency of a drug to inhibit a specific biological or biochemical function. This quantitative measure indicates how much of a particular drug (inhibitor) is required to half-inhibit a given biological process (or component of the process, i.e., enzyme, cell, cell receptor, or microorganism). The value is typically expressed as molar concentration. Concentration is commonly used as a measure of drug potency for antagonists in pharmacological studies. In some cases, the IC 50 represents the concentration of drug required for 50% inhibition in vitro. IC 50 can be determined by constructing a dose-response curve and examining the effect of different concentrations of the agent on the desired activity, eg, inhibition of tumor cell growth, tumor cell killing.

[0036] 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, compared to such activity upon administration to the serum or tissues of an organism, or compared 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 decrease by half of the starting amount administered to the serum or tissues of an organism, compared to such amount or concentration upon administration to the serum or tissues of an organism, or compared to any other defined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0037] The terms "modulate" and "alter" include "increase," "enhancement," or "stimulation," as well as "reduction" or "decreasement," typically by a statistically significant or physiologically significant amount or degree compared to a control. An "increase," "stimulation," or "enhancement" amount is typically a "statistically significant" amount and can include amounts that are 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 the amount produced by no composition or a control composition (e.g., the absence of an agent or a different agent). An "increasing," "stimulating," or "enhancing" 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 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, or 5000 times less than the amount produced by the absence of the composition or a control composition.A "reduced" or "decreased" amount can also include 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 the absence of the composition or a control composition. Examples of comparative and "statistically significant" amounts are described herein.

[0038] "Prodrug" is meant to refer to an agent (e.g., a compound) that can be converted under physiological conditions or by solvolysis into a biologically active compound as described herein. Thus, the term "prodrug" refers to a metabolic precursor of a pharmaceutically acceptable compound. A prodrug may be inactive when administered to a subject in need thereof, but is converted to an active compound in vivo. A prodrug can be rapidly transformed in vivo to yield the parent compound, for example, by hydrolysis in blood. Prodrug compounds often offer 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)). For a discussion of prodrugs, see Higuchi, T., et al., ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987). Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol or amide derivatives of amine functional groups in the compounds of the present disclosure, and the like.

[0039] The term "prodrug" is also intended to include any covalently bonded carrier that releases an active compound in vivo when the prodrug is administered to a subject. Prodrugs of a compound may be prepared by modifying functional groups present in the compound such that the modifications are cleaved to the parent compound, either by routine manipulation or in vivo. Prodrugs include compounds in which a hydroxy group, an amino group, or a mercapto group is bonded to any group that is cleaved to form a free hydroxy group, a free amino group, or a free mercapto group, respectively, when the prodrug of the compound is administered to a subject.

[0040] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0041] "Pharmaceutically acceptable salt" includes both acid and base addition salts.

[0042] "Pharmaceutically acceptable acid addition salts" refers to salts that retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and include those derived from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; 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, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, and the like. , gentisic acid, glucoheptonic 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, and the like.

[0043] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and are not biologically 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, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, isopropylamine such as ammonia, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0044] As used herein, the term "solvate" refers to an aggregate containing one or more molecules of an agent (e.g., a compound) described herein with one or more molecules of a solvent. 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., as well as corresponding solvate forms. The compounds of the present disclosure may be true solvates, while in other cases, the compounds may simply retain extraneous water or may be a mixture of water and some extraneous solvent.

[0045] "Pharmaceutical composition" refers to a formulation of a zotiraclib (TG02) compound described herein and a medium generally accepted in the art for the delivery of biologically active compounds to a mammal, e.g., a human. Such a medium includes all pharmaceutically acceptable carriers, diluents, and excipients.

[0046] The zotiraclib compounds described herein, or pharmaceutically acceptable salts thereof, may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined in terms of absolute stereochemistry as (R)- or (S)- for amino acids, or as (D)- or (L)-. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-) isomers, (R) and (S) isomers, or (D) and (L) isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of the racemate (or racemate of a salt or derivative), using, for example, chiral high-pressure liquid chromatography (HPLC). When the compounds described herein contain an olefinic double bond or other center of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0047] In certain embodiments, the "purity" of any given agent in a composition may be defined. For example, a particular composition may contain an agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure by weight, including, but not limited to, all decimal points and ranges therebetween, as measured, for example, by high performance liquid chromatography (HPLC), a well-known form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify agents or compounds.

[0048] The term "solubility" refers to the ability of a drug provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as the concentration of solute per unit volume of solvent (e.g., g of solute per kg of solvent, g per dL (100 mL), mg / mL, etc.), molarity, molar concentration, molar fraction, or other similar descriptions of concentration. The maximum equilibrium amount of solute that can be dissolved per volume of solvent is the solubility of the solute in that solvent under specific conditions, including temperature, pressure, pH, and solvent properties. In certain embodiments, solubility is measured at physiological pH or other pHs, such as pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In certain embodiments, solubility is measured in water or a physiological buffer solution, such as PBS or NaCl, with or without NaPO4. In certain embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and a relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO). 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, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain embodiments, the agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg / ml at room temperature or 37°C.

[0049] "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.

[0050] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA, including genomic DNA. The terms refer to polymeric forms of nucleotides, typically at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The terms include single- and double-stranded DNA.

[0051] "Gene" refers to a hereditary unit that occupies a specific location on a chromosome and consists of a sequence of DNA that codes for a functional molecule or protein. The structure of a gene consists of many elements, and the actual protein-coding sequence is often only a small portion. These elements include untranscribed DNA regions as well as untranslated RNA regions. In addition, genes may have expression-altering regulatory regions many kilobases upstream or downstream of the coding sequence. The information in a gene may also be represented by (or found in) the sequence of the RNA or the encoded protein.

[0052] A "subject" or "subject in need thereof" includes a mammalian subject, such as a human subject.

[0053] "Statistically significant" means that the result is unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used significance measures include p-values, which are the frequency or probability of an observed event occurring if the null hypothesis is true. If the obtained p-value 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.

[0054] "Substantially" or "essentially" means, for example, nearly completely or completely, some given reference amount, level, value, number, frequency, proportion, dimension, size, quantity, weight, length, or other reference amount, 95%, 96%, 97%, 98%, 99%, or more.

[0055] "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 one another.

[0056] "Tautomer" refers to a proton migration from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any such compounds.

[0057] "Therapeutic response" refers to an improvement in symptoms (whether sustained or not) upon administration of the therapeutic agent.

[0058] As used herein, the term "therapeutically effective amount," "therapeutic dose," "prophylactically effective amount," or "diagnostically effective amount" is the amount of an agent required to elicit a desired biological response following administration.

[0059] As used herein, "treatment" of a subject (e.g., a mammal, such as a human) or cell is any type of intervention used in an attempt to change the natural course of the subject or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and can be performed either prophylactically or after the onset of a pathological event or contact with a pathogenic agent. Also included is "prophylactic" treatment, which can be aimed at reducing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0060] The term "wild-type" refers to a gene or gene product (e.g., a polypeptide) that is most frequently observed in a population and is thus, optionally, the designated "normal" or "wild" form of the gene.

[0061] Each embodiment herein is intended to apply to all other embodiments unless expressly stated otherwise.

[0062] 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, comprising administering to the subject a pharmaceutical composition comprising zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof, thereby treating the cancer comprising the IDH1 or IDH2 mutation.

[0063] Some embodiments for treating IDH1 or IDH2 mutant cancer include: (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; (b) if the tissue sample contains an IDH1 or IDH2 mutation, administering to the subject a pharmaceutical composition comprising zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof.

[0064] Also included is a method for predicting therapeutic response to zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof, in a human subject having cancer, the method comprising: (a) determining the IDH1 or IDH2 mutation status in a tissue sample from the subject; (b)(i) characterizing the subject as responsive to zotiraclib (TG02) therapy if the tissue sample contains an IDH1 or IDH2 mutation; or (ii) characterizing the subject as non-responsive to zotiraclib (TG02) therapy if the tissue sample does not contain an IDH1 or IDH2 mutation, e.g., if the sample contains homozygous wild-type IDH1 and IDH2; and and thereby predicting the therapeutic response to zotiraclib (TG02) in a subject with cancer.

[0065] Some embodiments include administering zotilaclib to the subject if the subject is characterized as responsive to zotilaclib therapy. Some examples include administering a chemotherapeutic agent other than zotilaclib to the subject if the subject is characterized as non-responsive to zotilaclib therapy.

[0066] "Zofuracilliclib" or "TG02" has the IUPAC name (16E)-14-methyl-20-oxa-5,7,14,27-tetrazatetracyclo[19.3.1.1 2,6 .1 8,12 ] refers to a small molecule having the formula heptacosa-1(25),2(27),3,5,8,10,12(26),16,21,23-decaene, PubChem CID: 16739650, and CAS Number: 1204918-72-8, including pharmaceutically acceptable salts and acids thereof. Also included are biologically active or equivalent analogs and / or derivatives of zotiraclib, including prodrugs and pharmaceutically acceptable salts thereof.

[0067] The term "isocitrate dehydrogenase" or "IDH" refers to the enzyme (and the IDH gene encoding it) 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 beta-carboxylation 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 is composed of three subunits and catalyzes the third step of the citric acid cycle in mitochondria, converting NAD+ to NADH. The IDH1 and IDH2 isoforms catalyze the same reaction outside the context of the citric acid cycle, using NADP+ as a cofactor instead of NAD+.

[0068] As described above, certain embodiments include administering zotiraclib to a subject when a tissue sample contains an IDH1 or IDH2 mutation compared 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, where X is selected from any amino acid except R (arginine). In certain embodiments, the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S. In some embodiments, the IHD2 mutation is R172X or R140X, where X is selected from any amino acid other than R (arginine). In certain 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) compared 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 D-2HG oncometabolite in a tissue sample, and administering zotiraclib to the subject if the level of D-2HG oncometabolite is present or increased in the tissue sample compared to a reference or standard (e.g., D-2HG levels in a homozygous wild-type IDH1 or IDH2 tissue sample or cell).

[0069] The IDH1 or IDH2 mutation status in a tissue sample can be determined by any of a variety of methods. For example, in some embodiments, the IDH1 or IDH2 mutation status is determined directly by, for example, DNA or RNA sequencing on the human IDH1 or IDH2 protein or gene, in situ hybridization (ISH), fluorescent in situ hybridization (FISH), whole exome sequencing (WES), single nucleotide polymorphism (SNP) array, next-generation sequencing (NGS), or comparative genomic hybridization (CGH). CGH refers to a molecular cytogenetic method for analyzing copy number variations (CNVs) in the DNA of a test sample compared to a reference sample, without the need for cell culture. This technique allows for rapid and efficient comparison between two genomic DNA samples, often from two closely related sources, suspected of containing differences in terms of gain or loss of either entire chromosomes or subchromosomal regions (portions of entire chromosomes). This technology was originally developed to assess differences between the chromosomal complement of solid tumors and normal tissues (see, e.g., Kallioniemi et al., Science. 258 (5083): 818-821, 1992). The use of DNA microarrays in conjunction with CGH technology has led to the development of a more specific form of array CGH (aCGH), which allows for locus-by-locus measurement of CNVs with increased resolution of as little as 100 kilobases (see, e.g., Pinkel, Annu Rev Genom Hum Genet. 6: 331-354, 2005). In situ hybridization (ISH) and fluorescence in situ hybridization (FISH) refer to types of hybridization that use labeled complementary DNA, RNA, or modified nucleic acid strands (i.e., probes) to localize specific DNA or RNA sequences in a portion or section of tissue (in situ) (see, e.g., Parra & Windle, Nature Genetics. 5:17-21, 1993, and Gall & Pardue, PNAS USA. 63:378-383, 1969).Thus, determining IDH1 or IDH2 status can be performed according to routine techniques in the art, for example, to identify a subject's IDH1 or IDH2 mutation (or absence thereof). In some cases, the methods and kits described herein use and / or include reagents for performing any one or more of the foregoing techniques.

[0070] In some embodiments, the IDH1 or IDH2 mutation status is determined indirectly, for example, by determining the D-2HG level in a tissue sample. The D-2HG level can be determined or measured according to various techniques in the art, including biochemical detection (e.g., colorimetric assay), biosensor, gas or liquid chromatography-mass spectrometry (GC- or LC-MS), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), and others (see, for example, Xiao et al., Nat Commun 12: 7108, 2021, D-2HG assay in Example 1; Longuespee et al., Acta Neuropathol Commun. 6: 21, 2018). Thus, determining the D-2HG level can be performed according to routine techniques in the art, for example, to indirectly determine the IDH1 or IDH2 mutation status. In some cases, the methods and kits described herein use any one or more of the aforementioned techniques and / or include reagents for performing them.

[0071] Examples of a "reference" include values, amounts, sequences, or other features obtained from a database, such as homozygous "wild-type" IDH1 or IDH2 sequences (e.g., for IDH1, see NCBI Gene 3417 and RefSeq NM_005896; for IDH2, see NCBI Gene 3418 and RefSeq NM_002168). A "reference" also includes values, amounts, sequences, or other features obtained from one or more control tissues, such as wild-type IDH1 / 2 homozygous tissues (cancerous or non-cancerous) from one or more control subjects (e.g., a control subject population). As with cancer tissues, IDH1 / 2 mutation status from controls can be determined by any of a variety of methods, including, for example, ISH, FISH, WES, SNP arrays, NGS, or CGH on human IDH1 / 2 proteins or genes. Also, as noted above, D-2HG levels from a control can be determined by any of a variety of methods, including, for example, by biochemical detection (e.g., colorimetric assays), biosensors, GC- or LC-MS, MALDI-TOF, and others (as described above).

[0072] In some embodiments, the tissue sample is a liquid biopsy (e.g., a blood sample), a surgical sample, or other biopsy sample obtained from a subject. In certain embodiments, the tissue sample is a cancer tissue sample. Certain embodiments include, for example, obtaining a tissue sample from a subject before determining the IDH1 or IDH2 mutation status and / or D-2HG level. In some embodiments, the subject is a human subject.

[0073] 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).

[0074] Certain embodiments include combination therapies for treating cancer, including methods of ameliorating symptoms of or inhibiting the progression of cancer in a subject in need thereof, comprising administering zotilaclib to the subject in combination with at least one additional agent, such as, for example, an immunotherapeutic agent (e.g., a checkpoint inhibitor), a chemotherapeutic agent, a hormonal therapy, and / or a kinase inhibitor. In some embodiments, administering zotilaclib enhances the sensitivity of the cancer to the additional agent (e.g., the immunotherapeutic agent, the chemotherapeutic agent, the hormonal therapy, and / or the 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, compared to the additional agent alone.

[0075] Certain combination therapies employ one or more chemotherapeutic agents, e.g., small molecule chemotherapeutic agents, non-limiting examples of which include IDH1 and IDH2 inhibitors, alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), and anti-microtubule agents, among others.

[0076] Examples of IDH1 or IDH2 inhibitors include ivosidenib (AG-120), enasidenib, 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).

[0077] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mustine, 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, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine).

[0078] 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).

[0079] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin.

[0080] Examples of anti-microtubule agents include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastone, vincristine, vindesine, vinorelbine).

[0081] Certain combination therapies employ at least one hormonal therapeutic agent. Common examples of hormonal therapeutic agents include hormone agonists and hormone antagonists. Examples of hormonal agonists include progestogens (progestins), corticosteroids (e.g., prednisolone, methylprednisolone, or dexamethasone), insulin-like growth factors, VEGF-induced angiogenesis and lymphangiogenesis factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factors (FGFs), galectins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgens, estrogens, 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, and hormone receptor antagonists, such as selective estrogen receptor modulators (SERMs, e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0082] Also included are hormone pathway inhibitors, such as antibodies directed against hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1), such as cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, and lobatumumab, 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-beta receptors R1, R2, and R3, such as fresolimumab and methimumab; inhibitors of the EGF receptor, such as cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomzotuximab, and zalutumumab; inhibitors of the FGF receptor, such as aprituximab ixadotin and bemarituzumab; and inhibitors of the PDGF receptor, such as olaratumab and tobetumab.

[0083] Certain combination therapies employ at least one kinase inhibitor, including a tyrosine kinase inhibitor. Examples of kinase inhibitors include, but are not limited to, adavocetib, afantinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and bemafenib.

[0084] In some embodiments, the methods and pharmaceutical compositions described herein increase the median survival time of subjects 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 more.In some embodiments, the methods and compositions described herein increase the progression-free survival, overall survival, and / or post-progression survival time of subjects in need thereof by, for example, about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months or more, or about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or more.In certain embodiments, the methods and compositions described are sufficient to cause disease stabilization.

[0085] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to result in a statistically significant reduction in viable tumor burden, e.g., a reduction in tumor mass of at least 10%, 20%, 30%, 40%, 50%, or more, or tumor regression as indicated by a changed (e.g., statistically significantly reduced) scan dimension. In certain embodiments, the methods and therapeutic compositions described herein are sufficient to result in stable disease.

[0086] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to result in a clinically meaningful reduction in the symptoms of a particular disease indication known to one of skill in the art.

[0087] The methods for treating cancer may be combined with other therapeutic modalities. For example, the cancer therapies described herein may be administered to a subject before, during, or after other therapeutic interventions, including symptomatic care, radiation therapy (radiation therapy), surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptom care includes the administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment, and vomiting, and the administration of anticonvulsants to reduce seizures. Radiation therapy includes radiosurgery, such as whole-brain irradiation, fractionated radiation therapy, and stereotactic radiosurgery, which may be further combined with conventional surgery. In some embodiments, radiation therapy includes administering a total radiation dose of about 1 Gray (Gy) to about 70 Gy, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 Gy.

[0088] Methods for identifying a subject having one or more of the diseases or conditions described herein are known in the art.

[0089] For in vivo use, eg, for the treatment or testing of human disease, the agents described herein are generally incorporated into one or more therapeutic or pharmaceutical compositions prior to administration.

[0090] To prepare a therapeutic or pharmaceutical composition, an effective or desired amount of one or more agents is typically mixed with any pharmaceutical carrier or excipient known to those skilled in the art to be suitable for the particular agent and / or administration method. Pharmaceutical carriers may be liquid, semi-liquid, or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical applications may contain, for example, sterile diluents (such as water), saline (e.g., phosphate-buffered saline, PBS), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents (such as benzyl alcohol and methylparabens), antioxidants (such as ascorbic acid and sodium bisulfite), and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates, and phosphates). For intravenous administration (e.g., by intravenous infusion), suitable carriers include saline or phosphate-buffered saline (PBS), as well as solutions containing thickeners and solubilizers, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0091] The agents described herein can be administered in pure form or in suitable therapeutic or pharmaceutical compositions via any of the accepted methods for administering drugs to serve the same purpose. Therapeutic or pharmaceutical compositions can be prepared by combining the drug-containing composition with suitable physiologically acceptable carriers, diluents, or excipients, and can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, 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 described elsewhere herein) and / or suitable excipients such as salts, buffers, and stabilizers can be present in the composition, but need not be.

[0092] 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 will depend on the nature of the condition being treated or prevented. Certain embodiments include administration by IV infusion.

[0093] Carriers may include, for example, pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. Physiologically acceptable carriers are often aqueous pH buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, 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 dextrin; 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™), polyethylene glycol (PEG), poloxamer (PLURONICS™), and the like.

[0094] In some embodiments, one or more agents may be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), for example, in microcapsules prepared by coacervation techniques or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively). Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particles or liposomes may further comprise other therapeutic or diagnostic agents.

[0095] The exact dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known testing protocols or by testing the composition in a model system known in the art and extrapolating from there.Comparative clinical trials can also be conducted.Dosage can also vary depending on the severity of the condition being alleviated.Pharmaceutical compositions are generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects.The composition can be administered once or divided into several smaller doses that are administered at regular intervals.For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs.

[0096] Thus, typical routes of administration of these and related therapeutic or pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intrasternal injection, or infusion techniques. Therapeutic or pharmaceutical compositions according to certain embodiments of the present disclosure are formulated so that the active ingredients contained therein are bioavailable upon administration of the composition to a subject or patient. The composition administered to a subject or patient may take the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of a drug described herein in aerosol form may hold multiple dosage units. Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The administered composition typically contains a therapeutically effective amount of a drug described herein for treating the disease or condition of interest.

[0097] The therapeutic composition or pharmaceutical composition may be in solid or liquid form.In one embodiment, the carrier is particulate, so that the composition is, for example, in tablet or powder form.The carrier may also be liquid, and the composition is, for example, oral oil, injectable liquid, or aerosol, which is useful for, for example, inhalation administration.When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, and semi-solid, semi-liquid, suspension, and gel form are included in the form considered herein as either solid or liquid.Certain embodiments include sterile injectable solutions.

[0098] As a solid composition for oral administration, the pharmaceutical composition may be formulated into powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavors such as peppermint, methyl salicylate, or orange flavoring; and colorings. When the pharmaceutical composition is in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to the above-mentioned materials, a liquid carrier such as polyethylene glycol or oil.

[0099] Therapeutic or pharmaceutical compositions may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the compound, one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. In compositions intended to be administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included.

[0100] Liquid therapeutic or pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following adjuvants: sterile diluents such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, etc.; fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can serve as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparabens; 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 dextrose. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. Physiological saline is a preferred adjuvant. Pharmaceutical compositions for injection are preferably sterilized.

[0101] Liquid therapeutic or pharmaceutical compositions intended for either parenteral or oral administration should contain an amount of drug to ensure an appropriate dosage. Typically, this amount is at least 0.01% of the drug of interest in the composition. When intended for oral administration, this amount may vary from 0.1 to about 70% by weight of the composition. Particular oral therapeutic or pharmaceutical compositions contain from about 4% to about 75% of the drug of interest. In certain embodiments, therapeutic or pharmaceutical compositions and preparations according to the invention are prepared so that a parenteral dosage unit contains from 0.01 to 10% by weight of the drug of interest before dilution.

[0102] Therapeutic or pharmaceutical compositions may contain various materials that modify the physical form of solid or liquid dosage units. For example, the composition may contain a material that forms a coating shell around the active ingredient. The material that forms the coating shell is typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. Therapeutic or pharmaceutical compositions in solid or liquid form may contain a component that binds to the drug and thereby assists in the delivery of the compound. Suitable components that can act in this capacity include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0103] The compositions described herein can be prepared with carriers that protect the agent against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include, but are not limited to, controlled-release formulations such as implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and others known to those skilled in the art.

[0104] Therapeutic or pharmaceutical compositions may be prepared by methods well known in the pharmaceutical arts. For example, therapeutic or pharmaceutical compositions intended for administration 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 a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with drugs to promote the dissolution or homogeneous suspension of the drug in an aqueous delivery system.

[0105] Certain embodiments include the use of a diagnostic kit for treating cancer in a human subject in need thereof with zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof, comprising a means for determining isocitrate dehydrogenase (IDH1 or IDH2) mutation status in a tissue sample from the subject. Also included are patient care kits comprising: (a) a means for determining isocitrate dehydrogenase (IDH1 or IDH2) mutation status in a tissue sample from a human subject with cancer; and (b) a composition comprising zotiraclib (TG02), or an analog, derivative, or pharmaceutically acceptable salt thereof.

[0106] In some embodiments, the means for (directly) determining IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay selected from one or more of DNA or RNA sequencing on the human IDH1 or IDH2 protein or gene, ISH, FISH, WES, SNP array, NGS, or CGH. In certain embodiments, the means for (indirectly) determining IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay for determining D-2HG levels in the tissue sample, e.g., selected from biochemical detection (e.g., colorimetric), biosensor, GC- or LC-MS, and MALDI-TOF.

[0107] Some diagnostic 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 instructions for determining or measuring, for example, the IDH1 / 2 mutation status and / or the level, presence, or absence of D-2HG in tissue samples from subjects and / or controls.

[0108] Certain diagnostic or patient care kits include one or more additional agents, such as, for example, immunotherapeutic agents, chemotherapeutic agents, hormonal therapeutic agents, and / or kinase inhibitors, as described herein.

[0109] In some embodiments, a diagnostic kit or patient care kit contains separate containers, dividers, or compartments for the composition and informational material. For example, the composition or reagent may be contained in a bottle, vial, or syringe, and the informational material may be contained in association with the container. In some embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition or reagent is contained in a bottle, vial, or syringe to which the informational material in the form of a label is attached. In some embodiments, the kit contains multiple (e.g., packs) of individual containers, each containing one or more compositions, reagents, and / or unit dosage forms of zotiraclib. For example, the kit contains multiple syringes, ampoules, foil packets, or blister packs, each containing a reagent or a single unit dose of zotiraclib. The containers of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.

[0110] The patient care kit optionally includes a device suitable for administering the medication, e.g., a syringe, inhaler, dropper (e.g., eye dropper), swab (e.g., cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses a metered amount of the medication. Also included are methods of providing the kit, e.g., by combining the components described herein.

[0111] In certain aspects, the diagnostic or therapeutic response test or method described herein is carried out in a diagnostic laboratory, and the results are then provided to the subject or to a doctor or other healthcare provider involved in the subject's health care and cancer treatment.Therefore, certain embodiments include a method for providing the results of response test to a subject in need thereof, or to a doctor or other healthcare provider.These results or data may be in the form of a hard copy or paper copy, or in electronic form, such as a computer-readable medium.

[0112] 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.

[0113] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made without departing from the spirit or scope of the description or the appended claims. The following examples are provided for illustrative purposes only, and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.

[0114] [Example]

[0115] [Example 1] Experiments were conducted to test the activity of zotiraclib (TG02) against IDH1 mutant cancer cells and to evaluate the mechanism of such activity.

[0116] material and method Cell culture. The human chondrosarcoma cell line HT-1080 was cultured in MEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gemini, USA). The human cholangiocarcinoma cell lines RBE, Hccc-9810, and Hucct-1 were cultured in RPMI 1640 (Hyclone, USA) supplemented with 10% FBS. Cells were incubated at 37°C in 5% CO2.

[0117] 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 cultured at 1 × 10 in medium with 10% FBS containing the desired concentration of TG02. 5 Cells were seeded at a density of 1000 cells / well. 48 / 72 hours after treatment, cells were harvested and tested for apoptosis by Annexin V and PI staining. Cell analysis was performed using a FACSCelesta (BD).

[0118] 2-hydroxyglutarate (D-2HG) assay. Cells (approximately 1 × 10 7 ) were rapidly homogenized in 100 μl of D-2HG assay buffer in a D2-hydroxyglutarate assay kit (Abcam, ab211070) on ice for 10 minutes. The cells were then centrifuged at 10,000 × g for 5 minutes at 4°C, and the supernatant was collected. Equal volumes of each sample were added to triplicate wells of a 96-well clear plate.

[0119] The 100 mM D-2HG standard was diluted to 100 mM (1 nmol / μl) by adding 10 μl of the 1 mM D-2HG standard to 990 μl of D-2HG assay buffer and mixed well.

[0120] Five μl of 1 mM D-2HG standard was added to one of three samples defined as follows: spiked sample (5 nmol D2 hydroxyglutaric acid + sample), sample, and sample background. The spiked sample was used as an internal standard to correct for sample interference. The final volume of all wells was adjusted to 50 μl with D-2HG assay buffer.

[0121] For each well, 50 μl of reaction mixture was prepared (see Table E1):

[0122] [Table 1]

[0123] 50 μl of the reaction mixture was added to each well containing the standards and samples and mixed well. The plate was incubated at 37° C. for 60 minutes and the OD450 nm was measured.

[0124] The sample background reading was subtracted from the paired sample reading to obtain a corrected sample reading. The amount of D-2HG in the sample well (X) was determined based on the following formula: D-2HG amount (nmol)=() / [(())-()]×5

[0125] Comet Assay. The comet assay is a common technique for measuring DNA damage in individual cells. In the electrophoretic field, damaged cellular DNA (including fragments and strand breaks) is separated from intact DNA, resulting in the classic "comet tail" shape under a microscope. DNA damage was assayed using the OxiSelect™ Comet Assay Kit (CELL BIOLABS, INC.) according to the manufacturer's instructions. Briefly, comet agarose was pipetted onto an OxiSelect™ Comet Slide to form a substratum. Cells were combined with OxiSelect™ Comet agarose at 37°C, and then the agarose / cell mixture was pipetted on top of the substratum. Cells were treated with lysis buffer and alkaline solution. Electrophoresis was performed under neutral conditions. Slides were viewed under an epifluorescence microscope using a FITC filter.

[0126] Tail moment = (100 × tail DNA strength / cellular DNA strength) × tail moment length

[0127] Western blot analysis. Hccc-9810 and Hucct-1 cells treated with TG02 or AG120 (0.1% DMSO was added as a control) for 48 hours were harvested and centrifuged at 500 g for 5 minutes to obtain cell pellets. The pellets were then dissolved in lysis buffer (Beyotime, China), which additionally contained 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 12,000 rpm for 10 minutes at 4°C to obtain the supernatant as cell lysate. The protein concentration in the cell lysate was determined using a Micro BCA™ Protein Assay Kit (ThermoFisher, USA). 4x SDS-PAGE sample loading buffer (SolarBio, China) was added to the cell lysate containing 25 μg of total protein. After boiling, the mixture was electrophoresed in a polyacrylamide gel. After electrophoresis, the proteins on the gel were transferred to a PVDF membrane, and the membrane was cut at a position close to the molecular weight of the protein whose expression was examined (pH2AX (CST, USA), GAPDH (ZSGB-BIO, China)).

[0128] Transfection. The day before transfection, cells (approximately 2 × 10 5 1000kJ / well) were seeded into a 6-well plate. The cells were cultured overnight at 37°C in a 5% CO2 incubator. The old medium was removed and 2 ml of transfection medium was added. 50 μl of Optiti-MEM was added to the tube, and then 2 μg of the plasmid DNA mix solution was mixed by vortexing. 4 μl of PEI solution was added to the 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 PEI / DNA complex to form. The PEI / DNA complex was gently added dropwise to the well containing the cells. The cells were gently mixed by swirling and incubated at 37°C in 5% CO2 for 48 hours. The cells were then used to detect drug efficacy or gene expression.

[0129] Results. As shown in Figures 2A-2B, IDH1 mutant chondrosarcoma (HT-1080) and cholangiocarcinoma (RBE, Hcc-9810) cells were more sensitive to TG02 than IDH1-WT (Hucct-1) cells. Figures 3A-3B show that increased TG02 sensitivity can be conferred to IDH1-WT (Hucct-1) cells by transfection of mutant IDH1. In Figure 3A, Hucct-1 cells were transfected with the IDH1 R132H plasmid and treated with 0.1 μM TG02 for 72 h. Apoptosis was determined by FACS assay. In Figure 3B, Hucct-1 cells were transfected with the IDH1 R132H plasmid, and the levels of D-2-hydroxyglutarate (D-2HG) were measured in Hucct-1 WT, Hucct-1 transfected with the IDH1 R132H plasmid, and RBE (a cholangiocarcinoma cell line harboring the IDH1 R132S mutation) cells. D-2HG is a metabolic biomarker of gain-of-function mutations in the IDH1 and / or IDH2 genes.

[0130] Figure 4 shows that TG02-induced apoptosis in mutant IDH1 (Hccc-9810) cells can be rescued by the addition of exogenous substrate α-ketoglutarate (α-KG). Mutant IDH1 / 2 converts α-KG into an oncometabolite (D-2HG) and then inhibits 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, Figures 5A-5B show that TG02-induced DNA damage in mutant IDH1 (HT-1080) cells can be rescued by the addition of exogenous α-KG or AG-120. AG-120 is an inhibitor of mutant IDH1. In Figure 5A, cells were treated with the indicated agents for 48 h, and DNA damage was determined by neutral comet assay. Figure 5B shows a statistical analysis of the data in 5A. (Left panel: tail moment. Right panel: positive percentage of cells with DNA damage).

[0131] Figure 6 shows that TGO2-induced DNA damage was rescued only in mutant IDH1 cells (Hccc-9810) by the addition of exogenous AG-120. Cells were treated with the indicated drugs for 48 hours. pH2AX, a marker of DNA damage, was assayed by WB.

[0132] Figures 7A-7B show that cells with IDH1 mutations are more sensitive to DNA damage induced by TGO2. In Figure 7A, IDH1 mutant Hucct1 cells (Hucct1 R132H / + ) were generated by lentivirus expressing IDH1 R132H. Hucct1, Hucct1 R132H / + , and HT-1080 cells were treated with 0.2 μM TG02 or 0.1% DMSO for 48 hours. The cells were used for comet assay. Quantification of tail moment in the neutral comet assay is shown. ns: no significant difference; ***: P<0.001, ****: P<0.0001 compared to NC; #: P<0.05 compared to Hucct1 cells treated with TG02. In Figure 7B, Hucct1 and Hucct1 cells were treated with 0.2 μM TG02 or 0.1% DMSO for 48 hours. The cells were used for comet assay. Quantification of tail moment in the neutral comet assay is shown. ns: no significant difference; ***: P<0.001, ****: P<0.0001 compared to NC; #: P<0.05 compared to Hucct1 cells treated with TG02. R132H / + Cells were treated with 2 μM AG120, 0.2 μM TG02, or 0.01 μM AZD4573 (CDK9 inhibitor). 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.

[0133] Figures 8A-8C show that TGO2 in combination with AG120 induced cell apoptosis in cells harboring mutant IDH1. Mutant IDH1 cells, HT-1080 (8A), RBE (8B), and Hccc-9810 (8C), were treated with TGO2 (0.1 μM) and AG120 (2 μM) as indicated for 48 hours, and apoptosis was measured by Annexin V / PI staining and flow cytometric analysis.

[0134] Thus, evidence indicates that TGO2 selectively induces DNA damage and cell killing in mutant IDH1 cancer cells compared with homozygous wild-type IDH1 cancer cells. Evidence also indicates that such TGO2 sensitivity is associated with a "gain-of-function" cancer-promoting activity of mutant IDH1 (shared by mutant IDH2) that converts α-KG to the oncometabolite D-2HG.

Claims

1. 1. A method of treating cancer in a human subject in need thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation; administering to the subject a composition comprising zotiraclib (TG02) or a pharmaceutically acceptable salt thereof; thereby treating the cancer containing an IDH1 or IDH2 mutation.

2. 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. (a) determining the IDH1 or IDH2 mutation status in a tissue sample from said subject; (b) if the tissue sample contains an IDH1 or IDH2 mutation, administering to the subject a composition comprising zotiraclib (TG02) or a pharmaceutically acceptable salt thereof.

4. 4. The method of 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 oncometabolite D-2-hydroxyglutarate (D-2HG) compared to homozygous wild-type IDH1 or IDH2.

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

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

7. 7. The method of any one of claims 3 to 6, comprising: (a) 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 the human IDH1 or IDH2 protein or gene.

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

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

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

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

12. 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 zotiraclib (TG02) or a pharmaceutically acceptable salt thereof, wherein the cancer comprises an isocitrate dehydrogenase (IDH1 or IDH2) mutation, the kit comprising a means for determining isocitrate dehydrogenase (IDH1 or IDH2) mutation status in a tissue sample from the subject.

14. 14. The use of 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 oncometabolite D-2-hydroxyglutarate (D-2HG) compared to homozygous wild-type IDH1 or IDH2.

15. 15. The use of claim 13 or 14, wherein the IHD1 mutation is R132X, where X is selected from any amino acid other than R, and optionally the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S.

16. 16. The use of any one of claims 13 to 15, wherein the IHD2 mutation is R172X or R140X, where X is selected from any amino acid other than R, and optionally the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q.

17. 17. The use of any one of claims 13 to 16, wherein the means for determining IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay selected from one or more of DNA or RNA sequencing on the human IDH1 or IDH2 protein or gene, 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).

18. 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. 19. The use according to 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 zotiraclib or a pharmaceutically acceptable salt thereof, optionally an oral composition of zotiraclib.

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

23. 1. A patient care kit comprising: (a) a means for determining isocitrate dehydrogenase (IDH1 or IDH2) mutation status in a tissue sample from a human subject having cancer; (b) a composition comprising zotiraclib (TG02), or a pharmaceutically acceptable salt thereof.

24. 24. The patient care kit of 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 oncometabolite D-2-hydroxyglutarate (D-2HG) compared to homozygous wild-type IDH1 or IDH2.

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

26. 26. The patient care kit of any one of claims 23-25, wherein the IHD2 mutation is R172X or R140X, where X is selected from any amino acid other than R, and optionally the IHD2 mutation is R172G, R172K, R172M, R172S, R172T, or R140Q.

27. 17. The patient care kit of claims 13-16, wherein the means for determining IDH1 or IDH2 mutation status in a tissue sample comprises reagents for performing a diagnostic assay selected from one or more of DNA or RNA sequencing on the human IDH1 or IDH2 protein or gene, 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).

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

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

31. 31. The patient care kit of any one of claims 23 to 30, optionally comprising one or more additional agents selected from chemotherapeutic agents, hormonal therapeutic agents, and / or kinase inhibitors.

32. 12. The patient care kit of claim 11, wherein the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), enasidenib, 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, comprising zotiraclib (TG02) or a pharmaceutically acceptable salt thereof.

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

35. 35. The pharmaceutical composition for use of claim 33 or 34, wherein the IHD1 mutation is R132X, where X is selected from any amino acid other than R, and optionally the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S.

36. 36. The 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 other than R, and optionally the IHD2 mutation is R172G, R172K, R172M, R172S, R172T or R140Q.

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

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

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

41. The method comprises: (a) determining the IDH1 or IDH2 mutation status in a tissue sample from said subject; (b) if the tissue sample contains an IDH1 or IDH2 mutation, administering to the subject a composition comprising zotiraclib (TG02) or a pharmaceutically acceptable salt thereof.

42. Use of a composition in 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, including zotiraclib (TG02) or a pharmaceutically acceptable salt thereof.

43. 43. The use of 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 oncometabolite D-2-hydroxyglutarate (D-2HG) compared to homozygous wild-type IDH1 or IDH2.

44. 44. The use of claim 42 or 43, wherein the IHD1 mutation is R132X, where X is selected from any amino acid other than R, and optionally the IHD1 mutation is R132C, R132G, R132H, R132L, or R132S.

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

46. 46. The use according to 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, comprising an oral composition of zotiraclib, or a pharmaceutically acceptable salt thereof.

48. 48. The use of 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. 50. The use of claim 49, wherein the chemotherapeutic agent comprises an IDH1 or IDH2 inhibitor, optionally ivosidenib (AG-120), enasidenib, or AG-221.

50. (a) determining the IDH1 or IDH2 mutation status in a tissue sample from said subject; (b) if the tissue sample contains an IDH1 or IDH2 mutation, administering to the subject the composition comprising zotiraclib (TG02) or a pharmaceutically acceptable salt thereof.

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