Compositions and methods for treating cancer

Abstract

Disclosed are novel uses of estrogen-related receptor gamma (ERRgamma) inhibitors in enhancing cancer treatment and pharmaceutical compositions for inhibiting cancer resistance to tyrosine kinase inhibitors and enhancing anti-cancer effects. The pharmaceutical composition contains an ERRgamma inhibitor as an active ingredient. The pharmaceutical composition is used for treating tyrosine kinase inhibitor resistance advanced cancers. The composition may be administered in combination with a tyrosine kinase inhibitor. A method for determining whether a cancer has tyrosine kinase resistance is also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 348,185, filed on June 2, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the use of an estrogen-related receptor gamma (ERRγ) inhibitor or a composition comprising the inhibitor as an active ingredient in enhancing cancer treatment. The present disclosure also relates to a method for treating cancer or improving cancer treatment. The present disclosure also relates to a method and composition for treating a tyrosine kinase inhibitor-resistant proliferative disease. Background Art

[0004] According to the World Health Organization (WHO), cancer is the leading cause of death worldwide, causing nearly 10 million deaths in 2020. The most common cancers in 2020 (in terms of new cancer cases) were breast, lung, colorectal, prostate, skin, and stomach cancers, and the most common causes of cancer deaths in 2020 were lung, colorectal, liver, stomach, and breast cancers.

[0005] The traditional approach to fighting cancer is chemotherapy using cytotoxic agents. However, these cytotoxic agents are often poorly selective for cancer cells, resulting in damage to rapidly dividing normal cells. Other treatment options include radiation therapy, targeted therapy, immunotherapy, hormone therapy, and surgery. The choice of treatment depends on a variety of factors, including the type of cancer, the severity of the tumor, the stage of the disease, and the patient's general condition.

[0006] Targeted therapeutic agents are generally prepared to exert efficacy on patients by targeting dysfunctional proteins specific to cancer cells. An example is a tyrosine kinase inhibitor (TKI). Tyrosine kinase inhibitors (TKI) are targeted specific inhibitors of abnormal protein tyrosine kinases (PTK). TKI has significant advantages over traditional chemotherapeutics, including high efficiency, low toxicity and high specificity. At present, TKI is widely used in the treatment of leukemia, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), gastrointestinal stromal tumors (GIST), breast cancer and hepatocellular carcinoma (HCC), etc., and their clinical application is growing rapidly. The use of tyrosine kinase inhibitors (TKI) produces the reason for effective therapeutic response in patients. At present, drug resistance is the main reason for limiting the efficacy of TKI in treating cancer.

[0007] Therefore, there is a need for therapeutic methods and pharmaceutical compositions that can treat malignancies in patients resistant to TKI chemotherapy and / or enhance cancer therapy (such as TKI therapy) and / or prevent the development of TKI resistance. Summary of the invention

[0008] One aspect of the present disclosure provides a pharmaceutical composition for preventing or enhancing treatment of tyrosine kinase inhibitor (TKI)-resistant cancer, comprising an ERRγ inhibitor as an active ingredient.

[0009] One aspect of the present disclosure provides a pharmaceutical composition for suppressing cancer TKI resistance, comprising an ERRγ inhibitor as an active ingredient.

[0010] Another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating cancer, comprising an ERRγ inhibitor as an active ingredient, wherein the pharmaceutical composition is administered in combination with a TKI.

[0011] Another aspect of the present disclosure provides a method for screening a material that enhances TKI-resistant cancer therapy, comprising treating TKI-resistant cancer cells with a candidate material; and evaluating the activity or expression of ERRγ in the cells.

[0012] Still another aspect of the present disclosure provides a kit for diagnosing TKI-resistant cancer, comprising an agent for measuring the level of mRNA of an estrogen-related receptor γ (ERRγ) gene or a protein expressed thereby.

[0013] Another aspect of the present disclosure provides a method for providing information for diagnosing TKI-resistant cancer, comprising the following steps:

[0014] (a) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a first cancer patient to examine whether the cancer exhibits resistance to a RAF inhibitor;

[0015] (b) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a second patient with cancer that is not resistant to TKI or a subject without cancer; and

[0016] (c) when the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (a) is higher than the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (b), determining the first cancer patient in step (a) as a TKI-resistant cancer patient.

[0017] Yet another aspect of the present disclosure provides a method for providing information required for determining a treatment method for a first cancer patient, comprising the following steps:

[0018] (a) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a first cancer patient;

[0019] (b) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a second patient with cancer that is not resistant to RAF inhibitors or a subject without cancer; and

[0020] (c) when the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (a) is higher than the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (b), determining the first cancer patient in step (a) as a TKI-resistant cancer patient.

[0021] Another aspect of the present disclosure provides a method for diagnosing or treating TKI-resistant cancer, comprising the following steps:

[0022] (a) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a first cancer patient;

[0023] (b) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a second patient with cancer that is not resistant to TKI or a subject that does not have cancer; and

[0024] (c) when the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (a) is higher than the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (b), determining that the first cancer patient in step (a) is a TKI-resistant cancer patient and applying another cancer therapeutic agent other than TKI.

[0025] Yet another aspect of the present disclosure provides a method of preventing or enhancing treatment of TKI-resistant cancer, comprising administering an ERRγ inhibitor to a patient suffering from TKI-resistant cancer.

[0026] Yet another aspect of the present disclosure provides a method of inhibiting cancer TKI resistance, comprising administering an ERRγ inhibitor to a patient having TKI-resistant cancer.

[0027] Yet another aspect of the present disclosure provides a method for preventing or treating cancer, comprising administering an ERRγ inhibitor and a TKI to a cancer patient. In one embodiment, the cancer patient (or a first patient with cancer) may have previously received TKI treatment.

[0028] Another aspect of the present disclosure provides a combination, comprising: a first composition comprising an ERRγ inhibitor; and a second composition comprising a TKI, the combination being used to treat cancer. In one embodiment, the cancer may be resistant to TKI.

[0029] Another aspect of the present disclosure provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of a composition comprising an ERRγ inhibitor, wherein the subject has received, is receiving, or will receive TKI treatment. The method may further comprise administering TKIs to the subject sequentially or simultaneously.

[0030] In the above-mentioned pharmaceutical compositions, kits, screening methods, treatment methods and diagnostic methods, TKI includes but is not limited to inhibitors of VEGFR (vascular endothelial growth factor receptor), FGFR (fibroblast growth factor receptor), PEGFR (platelet-derived growth factor receptor), EGFR (epidermal growth factor receptor), ABL (Abelson kinase), TIE (tyrosine kinase with immunoglobulin-like and EGF-like domains), Src kinase, JAK (Janus kinase), etc. TKI may be selective for multiple targets or act on multiple targets. TKI may be a combination of different inhibitors for the same or different tyrosine kinase targets.

[0031] In the above-mentioned pharmaceutical compositions, kits, screening methods, treatment methods and diagnostic methods, the cancer can be breast cancer, prostate cancer, melanoma, colorectal cancer, glioma (including recurrent glioblastoma), lung cancer, liver cancer, kidney cancer, ovarian cancer, sarcoma, desmoid tumor (aggressive fibromatosis) or thyroid cancer.

[0032] In the above pharmaceutical compositions, kits, screening methods, treatment methods and diagnostic methods, TKI resistance can be intrinsic resistance or acquired resistance. Therefore, in some embodiments, the cancer patient may have previously been treated with a TKI inhibitor and developed resistance to the TKI inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figures 1A to 1C The figure shows the results of constructing liver cancer cell lines showing resistance to sorafenib (Huh7: liver cancer cell line; Huh7-R: sorafenib-resistant liver cancer cell line; SK-Hep: liver cancer cell line; SK-Hep-R: sorafenib-resistant liver cancer cell line).

[0034] Figure 2A and Figure 2B Sorafenib resistance in the liver cancer cell line Huh7-R ( Figure 2A ) and SK-Hep-R( Figure 2B ) shows an increase in the orphan nuclear receptor ERRγ.

[0035] Figure 3A and Figure 3B The present invention shows that the compound of formula 1 (DN200434) (an inverse agonist against ERRγ) is used to treat the sorafenib-resistant liver cancer cell line Huh7-R ( Figure 3A) and SK-Hep-R( Figure 3B ), indicating that reactive oxygen species (ROS) were increased in each sorafenib-resistant liver cancer cell line.

[0036] Figure 4A and 4B Sorafenib-resistant liver cancer cell line Huh7-R ( Figure 4A ) and SK-Hep-R( Figure 4B ), a graph showing the reduction in cell proliferation when treated simultaneously with the compound of formula 1 (DN200434) and sorafenib.

[0037] Figure 5A are images showing that when the compound of Formula 1 (DN200434) was administered in combination with sorafenib, the tumor size in an animal model (xenograft) derived from the sorafenib-resistant liver cancer cell line Huh7-R was significantly reduced compared to the control group (the group administered with sorafenib alone).

[0038] Figure 5B : is a graph showing that when the compound of Formula 1 (DN200434) was administered in combination with sorafenib, the tumor weight in the animal model derived from the sorafenib-resistant liver cancer cell line Huh7-R was significantly reduced compared with the control group (the group administered with sorafenib alone).

[0039] Figure 5C are images showing that when the compound of Formula 1 (DN200434) was administered in combination with sorafenib, the tumor volume in the animal model derived from the sorafenib-resistant liver cancer cell line Huh7-R was significantly reduced compared with the control group (Huh7-R-Con; a group administered with sorafenib alone).

[0040] Figure 5D It is a graph showing that there was no change in body weight among the groups as a result of an experiment using an animal model derived from the sorafenib-resistant liver cancer cell line Huh7-R.

[0041] Fig. 6A are images showing that when the compound of Formula 1 (DN200434) was administered in combination with sorafenib, the tumor size in an animal model (xenograft) derived from the sorafenib-resistant liver cancer cell line SK-Hep-R was significantly reduced compared to the control group (the group administered with sorafenib alone).

[0042] Figure 6B is a line graph showing that when the compound of Formula 1 (DN200434) was administered in combination with sorafenib, the tumor weight in the animal model derived from the sorafenib-resistant liver cancer cell line SK-Hep-R was significantly reduced compared with the control group (the group administered with sorafenib alone).

[0043] Figure 6C are images showing that when the compound of Formula 1 (DN200434) was administered in combination with sorafenib, the tumor volume in the animal model derived from the sorafenib-resistant liver cancer cell line SK-Hep-R was significantly reduced compared with the control group (Huh7-R-Con; a group administered with sorafenib alone).

[0044] Fig.6D This is a graph showing that there was no change in body weight among the groups as a result of an experiment using an animal model derived from the sorafenib-resistant liver cancer cell line SK-Hep-R.

[0045] Figure 7 The structures of exemplary non-limiting TKIs are shown.

[0046] Figure 8 The results showed that the combination of DN434 and bosutinib had an unexpectedly excellent synergistic anticancer effect in an in vitro experiment using A549 cells.

[0047] Fig. 9 The results showed that the combination of DN434 and gefitinib had an unexpectedly excellent synergistic anticancer effect in in vitro experiments using HCC827 cells and PC9 cells.

[0048] Fig.10 The combination of DN434 and lenvatinib was shown to have an unexpectedly excellent synergistic anticancer effect in in vitro experiments using Huh-7 cells and SK-Hep-1 cells.

[0049] Fig.11 The results showed that the combination of DN434 and ceritinib had an unexpectedly excellent synergistic anticancer effect in an in vitro experiment using Pancl cells.

[0050] Fig.12 The results showed that the combination of DN434 and ruxolitinib had an unexpectedly excellent synergistic anticancer effect in an in vitro experiment using SKOV-3 cells.

[0051] Fig.13 It was shown that DN434 has a cancer therapeutic effect on erlotinib-resistant cancer in an in vitro experiment using erlotinib-resistant HCC837 cells. DETAILED DESCRIPTION

[0052] definition

[0053] Unless expressly stated otherwise, general terms used herein have the following meanings:

[0054] In the context of describing the present disclosure (especially in the context of the following claims), the terms "a" and "an" and "the" and similar references should be interpreted as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Where the plural form is used for compounds, salts, etc., this should be considered to also mean a single compound, salt, etc.

[0055] The term "about" or "approximately" shall have the meaning within 10%, more preferably within 5% of a given value or range.

[0056] The term "IC 50 "" refers to the amount, concentration or dose of a particular test compound that achieves a maximal response, such as 50% inhibition of cell growth or proliferation, as measured by any of the in vitro or cell-based assays described herein.

[0057] Unless otherwise indicated, the terms "comprising" and "including" are used herein in their open and non-limiting sense. The terms "consisting essentially of" or "consists essentially of" and the like when applied to the methods and compositions covered by the present disclosure have the meanings ascribed thereto by U.S. patent law, and the terms are open ended, allowing for more than the listed contents, as long as the basic or novel features of the listed contents are not altered by the presence of the listed contents, but do not include embodiments of the prior art.

[0058] The term "hydrate" means a compound provided herein, or a salt thereof, that further comprises a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0059] The term "solvate" means a solvate formed by the association of one or more solvent molecules with a compound provided herein. The term "solvate" includes hydrates (eg, monohydrate, dihydrate, trihydrate, tetrahydrate, etc.).

[0060] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0061] The term "administering" refers to introducing an agent of the present disclosure into a subject or patient. One preferred route of administration of the agent is oral administration. Another preferred route is intravenous administration. However, any route of administration may be used, such as topical, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal, introduction into cerebrospinal fluid, or instillation into a body compartment.

[0062] The term "co-administration" or "combination administration" or "combination use" or "combination with..." is intended to cover the administration of two therapeutic agents (e.g., an ERRγ inhibitor provided herein and another anticancer agent) to a single subject (e.g., a patient) in need thereof, and is intended to include a treatment regimen in which the two agents are administered simultaneously, in parallel, or sequentially without specific time limits. In an embodiment, the two agents are present in a cell or in a patient's body at the same time, or exert their biological or therapeutic effects at the same time. In an embodiment, the two therapeutic agents are in the same composition or unit dosage form. In another embodiment, the two therapeutic agents are in separate compositions or unit dosage forms.

[0063] The term "combined therapeutically effective" or "combined therapeutic effect" as used herein means that the therapeutic agents can be administered separately (in a chronologically staggered manner, especially in a sequence-specific manner) at such time intervals that they are preferably in the mammal, warm-blooded animal, especially the human to be treated. Still show a (preferably synergistic) interaction (combined therapeutic effect). Whether this is the case can be determined in particular by following the blood levels, thereby indicating that both compounds are present in the blood of the human to be treated at least during a certain time interval.

[0064] As used herein, the term "synergistic effect" refers to the effect of two therapeutic agents (such as, for example, ERRγ inhibitors provided herein and bosutinib as TKI) producing, for example, slowing down the symptomatic progression of a proliferative disease (particularly cancer) or its symptoms, which is greater than the simple addition of the effect of each drug administered alone. Synergistic effect can be calculated, for example, using a suitable method such as Sigmoid-Emax equation (Holford, NHG and Scheiner, LB, Clin. Pharmacokinet. 6: 429-453 (1981)), Loewe additivity equation (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and median effect equation (Chou, TC and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation mentioned above can be applied to experimental data to obtain a corresponding chart that helps to evaluate the effect of drug combinations. The corresponding graphs associated with the equations mentioned above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

[0065] The term "anticancer agent" refers to antimetabolites (e.g., 5-fluorouracil, methotrexate, fludarabine), antimicrotubule agents (e.g., vinca alkaloids such as vincristine, vinblastine; taxanes such as paclitaxel, docetaxel), alkylating agents (e.g., cyclophosphamide, melphalan, carmustine, nitrosoureas such as bischloroethyl nitrosourea and hydroxyurea), platinum agents (e.g., cisplatin, carboplatin, oxaliplatin, JM-216 or satraplatin, CI-973), anthracyclines (e.g., doxorubicin, daunorubicin), antitumor antibiotics (e.g., mitomycin, idarubicin, doxorubicin, daunorubicin), topoisomerase inhibitors (e.g., etoposide, camptothecin), antiangiogenic agents (e.g., and bevacizumab) or any other cytotoxic agent, (estramustine phosphate, prednimustine), hormone or hormone agonist, antagonist, partial agonist or partial antagonist, kinase inhibitor, radiation therapy or therapeutic antibody.

[0066] The term "pharmaceutical composition" or "formulation" is defined herein to mean a mixture or solution containing at least one therapeutic agent for administration to a subject (e.g., a mammal or a human) to prevent or treat a particular disease or condition affecting the subject or patient (including a mammal or a human).

[0067] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" means excipients, diluents, carriers and / or adjuvants used to prepare pharmaceutical compositions, which are generally safe, non-toxic and have no biological and other adverse effects, and include excipients, diluents, carriers and adjuvants suitable for veterinary use and / or human pharmaceutical use.

[0068] The terms "treating" or "treatment" as used herein include treatment that alleviates, reduces or alleviates at least one symptom of a subject or achieves a delay in disease progression. For example, treatment may be a reduction in one or several symptoms of a condition or a complete eradication of a condition such as cancer. Within the meaning of the present disclosure, the term "treatment" also means preventing, delaying the onset of a disease (i.e., the period before the clinical manifestation of a disease) and / or reducing the risk of disease development or worsening, and / or inhibiting an increase in tumor volume or size, or reducing tumor volume or size.

[0069] As used herein, the term "enhancing treatment" refers to all types of measures to improve or beneficially alter the symptoms of TKI-resistant cancer by administering the pharmaceutical composition of the present disclosure after the cancer acquires resistance to TKI.

[0070] The terms "ameliorate" and "ameliorating" and grammatical variations thereof mean a decrease in the severity of disease symptoms in a subject.

[0071] The term "protect" is used herein to mean preventing, delaying or treating the development, persistence or worsening of a disease in a subject (eg, a mammal or human), or both, as the case may be.

[0072] As used herein, the terms "prevent", "preventing" and "prevention" include preventing at least one symptom associated with or caused by the condition, disease or disorder being prevented.

[0073] The term "effective amount" or "therapeutically effective amount" or "pharmaceutically effective amount" of an active pharmaceutical compound (APC) or a pharmaceutical composition containing an APC is an amount of Compound A or a composition sufficient to achieve a beneficial or desired result when it is administered to a subject. Effective dosage forms, modes of administration, and dosages can be determined empirically, and making such determinations is within the capabilities of those skilled in the art. It is understood by those skilled in the art that dosages will vary with the route of administration, excretion rate, duration of treatment, identification of any other drugs administered, age, size, and species of the mammal (e.g., human patient), and similar factors well known in the medical and veterinary fields. In general, a suitable dose of an agent or composition will be the amount of the lowest dose at which the agent or composition can effectively produce the desired effect. An effective dose of an agent or composition may be administered as two, three, four, five, six, or more sub-doses administered separately at appropriate intervals within a day.

[0074] As used herein, the term "subject" or "patient" includes animals that are capable of or suffer from cancer or any condition directly or indirectly related to cancer. Examples of subjects include animals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In preferred embodiments, the subject is a human, e.g., a human that suffers from, is at risk of suffering from, or is potentially capable of suffering from cancer.

[0075] As used herein, the term "inverse agonist" or "antagonist" refers to a molecule that can directly or indirectly reduce the biological activity of a receptor, and includes molecules that can reduce the effect of a ligand when used in conjunction with a ligand of a receptor, but the present disclosure is not limited thereto.

[0076] As used herein, the terms "miRNA", "siRNA" and "shRNA" refer to nucleic acid molecules that primarily bind to mRNA transcribed from a target gene to mediate RNA interference or gene silencing, thereby inhibiting mRNA translation. Since miRNA, siRNA and shRNA can inhibit the expression of target genes at the translation level, they can be used in effective gene knockout methods or gene therapy methods.

[0077] As used herein, the term "antisense oligonucleotide" refers to a DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a specific mRNA sequence. In this case, the antisense oligonucleotide can bind to a complementary sequence in mRNA, thereby inhibiting the translation of mRNA into protein.

[0078] The term "antibody" as used herein refers to a protein molecule that can specifically bind to an antigenic site of a protein or peptide molecule. In this case, the protein encoded by the marker gene can be obtained by cloning each gene into an expression vector according to conventional methods, and such antibodies can be prepared from the obtained protein using conventional methods.

[0079] The term "aptamer" as used herein refers to a nucleic acid molecule that has binding activity to a predetermined target molecule. An aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and may be in a linear or cyclic form. Generally speaking, the shorter the nucleotide sequence constituting the aptamer, the easier it may be to chemically synthesize and mass-produce, and it is known that aptamers have excellent advantages in terms of cost, are easy to chemically modify, and exhibit excellent in vivo stability and low toxicity.

[0080] ERRγ Inhibitors and Agents for Inhibiting ERRγ Gene Expression In pharmaceutical compositions, combinations, kits, screening methods, therapeutic methods and diagnostic methods, the ERRγ inverse agonist (or inhibitor or antagonist) can be a compound of the following formula A:

[0081]

[0082] in

[0083] L is (C6-C20)arylene, (C3-C20)heteroarylene or (C3-C20)fused heterocyclic ring;

[0084] R 1 is (C3-C20)heterocycloalkyl, (C3-C20)heteroaryl, -O-(CH2) m -R 11 、-(CH2) m -R 12 、-NH-(CH2) m -R 13 、-NHCO-(CH2) n -R 14 Or-SiR 16 R 17 -(CH2) m -R 15 ;

[0085] R 11 To R 15 are independently (C3-C20)heterocycloalkyl;

[0086] R 16 and R 17 are independently (C1-C20)alkyl;

[0087] m is an integer from 1 to 3; and

[0088] n is an integer of 0 or 1;

[0089] Ar is (C6-C20) aryl or (C3-C20) heteroaryl, wherein the aryl or heteroaryl of Ar may be further substituted by one or more selected from the group consisting of: hydroxyl, halogen, (C1-C20) alkyl, halogenated (C1-C20) alkyl, (C1-C20) alkoxy, nitro, cyano, -NR 21 R 22 , (C1-C20) alkylcarbonyloxy, (C1-C20) alkylcarbonylamino, guanidino, -SO2-R 23 and-OSO2-R 24 ;

[0090] R 21 and R 22 are independently hydrogen, (C1-C20)alkylsulfonyl or (C3-C20)cycloalkylsulfonyl;

[0091] R 23 and R 24 are independently (C1-C20)alkyl, halogenated (C1-C20)alkyl or (C3-C20)cycloalkyl;

[0092] R 2 is hydroxy, halogen, (C1-C20)alkylcarbonyloxy or (C1-C20)alkylsulfonyloxy;

[0093] R 1 The heterocycloalkyl or heteroaryl group and R 11 To R 15 The heterocycloalkyl group may be further substituted by one or more selected from the group consisting of: (C1-C20)alkyl, (C3-C20)cycloalkyl, (C2-C20)alkenyl, amidino, (C1-C20)alkoxycarbonyl, hydroxy, hydroxy(C1-C20)alkyl and di(C1-C20)alkylamino(C1-C20)alkyl; and

[0094] The heterocycloalkyl group and the heteroaryl group contain one or more heteroatoms selected from the group consisting of N, O and S, and the heterocycloalkyl group is a saturated or unsaturated monocyclic, bicyclic or spirocyclic ring having a carbon atom or a nitrogen atom as a binding site within the ring, an isomer, or a pharmaceutically acceptable salt thereof, or a solvate including a hydrate.

[0095] Compounds of formula A and methods for their preparation are disclosed in US Application No. 16 / 677,596 or US Pat. No. 10,934,303, the contents of which are incorporated herein by reference in their entirety.

[0096] According to one embodiment of the present disclosure, the inverse agonist for ERRγ may be a compound of the following formula 1a-1d, a pharmaceutically acceptable salt thereof, or an isomer or solvate (including a hydrate) thereof, but is not limited to the compound, as long as it is an inverse agonist for ERRγ that exhibits an effect equivalent to that of the compound:

[0097]

[0098] In certain embodiments, the ERRγ inverse agonist may be (E)-5-(4-hydroxyphenyl)-5-(4-(4-isopropylpiperazin-1-yl)phenyl)-4-phenylpent-4-en-1-ol (DN434 or DN200434) of Formula 1a, or a pharmaceutically acceptable salt thereof, an isomer thereof, or a solvate thereof.

[0099] The present inventors unexpectedly discovered that the specific estrogen-related receptor gamma (ERRγ) inhibitors described herein have potent anticancer effects against TKI-resistant cancers (e.g., Examples 4 and Fig.13 Moreover, the combination of ERRγ inhibitor and TKI showed significant synergistic anticancer effect compared with ERRγ inhibitor alone and TKI alone in in vitro experiments (e.g., Examples 5 and Figures 8 to 12 Without being bound by a particular theory, the potent anticancer effects and significant synergistic effects in TKI-resistant cancers may be attributed to the effects of ERRγ inhibitors in enhancing sensitivity to anticancer drugs TKIs, inhibiting proliferation of TKI-resistant cancer cells, and significantly reducing cancer size.

[0100] Therefore, ERRγ inhibitors may be effective alone or in combination with TKIs for the treatment of advanced TKI-resistant cancers.

[0101] The compounds of the present disclosure may be used in the form of pharmaceutically acceptable salts. In this case, an acid addition salt formed from a pharmaceutically acceptable free acid may be used as the salt.

[0102] Acid addition salts can be obtained from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, nitrous or phosphorous acids and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, and nontoxic organic acids such as aromatic acids, aliphatic and aromatic sulfonic acids and the like.

[0103] Such pharmaceutically nontoxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, decanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexane benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or glycolate.

[0104] The acid addition salts of the present disclosure can be prepared by conventional methods, for example, by dissolving the compound of formula A in an aqueous acid solution and precipitating the salt in an organic solvent miscible with water, such as methanol, ethanol, acetone or acetonitrile. In addition, the acid addition salt can be prepared by evaporating the solvent or excess acid from the mixture and drying the mixture, or by filtering the precipitated salt with suction.

[0105] In addition, pharmaceutically acceptable metal salts can be prepared using alkali. Alkali metal or alkaline earth metal salts can be, for example, prepared by dissolving the compound of formula A in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating the filtrate to dryness. In this case, it may be suitable to prepare sodium salts, potassium salts or calcium salts as metal salts pharmaceutically. The silver salt corresponding to the metal salt is obtained by reacting an alkali metal salt or an alkaline earth metal salt with an appropriate silver salt (e.g., silver nitrate).

[0106] All isomers, hydrates, solvates and pharmaceutically acceptable salts thereof which can be prepared by conventional methods are included within the scope of the compounds disclosed herein.

[0107] In an embodiment, the agent for inhibiting the activity of ERRγ protein may include an inverse agonist or antagonist for ERRγ, or an antibody or aptamer that can specifically bind to ERRγ, but the present disclosure is not limited thereto. According to another embodiment of the present disclosure, the agent for inhibiting the expression of ERRγ gene may be selected from the group consisting of miRNA, siRNA, shRNA and antisense oligonucleotides, all of which can specifically bind to the mRNA of the gene, but the present disclosure is not limited thereto.

[0108] The ERRγ gene may comprise or consist of a nucleotide sequence encoding ERRγ, reported as NP_001127757.1 (nucleotide sequence NM_001134285.2 [P62508-2]), NP_001230434.1 ((nucleotide sequence NM_001243505.1), NP_001230435.1 (nucleotide sequence NM_001243506.1), NP_001230436.1 (nucleotide sequence NM_001243507.1 [P62508-4]), NP_001230438.1 (nucleotide sequence NM_001243509.1 [P62508-2]), NP_001230439.1 (nucleotide sequence NM_001243510.1 [P62508-3]). 30439.1 (nucleotide sequence NM_001243510.2 [P62508-2]), NP_001230440.1 (nucleotide sequence NM_001243511.2 [P62508-2]), NP_001230441.1 (nucleotide sequence NM_001243512.1 [P62508-2]), NP_001230442.1 (nucleotide sequence NM_001243513.1 [P62508-2]), NP_001230443.1 (nucleotide sequence NM_001243514.1 [P62508-2]), and NP_001230444.1 (nucleotide sequence NM_001243515.1 [P62508-2]). 1243515.1 [P62508-2]), NP_001230447.1 (nucleotide sequence NM_001243518.1 [P62508-5]), NP_001230448.1 (nucleotide sequence NM_001243519.1 [P62508-2]), NP_001429.2 (nucleotide sequence NM_001438.3 [P62508-1]), NP_996317.1 (nucleotide sequence NM_206594.2 [P62508-2]), NP_996318.1 (nucleotide sequence NM_206595.2 [P62508-2]), XP_011507569.1 ( Nucleotide sequence XM_011509267.1 [P62508-5]), XP_011507570.1 (nucleotide sequence XM_011509268.2 [P62508-5]), XP_011507571.1 (nucleotide sequence XM_011509269.2 [P62508-5]), XP_011507576.1 (nucleotide sequence XM_011509274.1 [P62508-3]), XP_011507577.1 (nucleotide sequence XM_011509275.1 [P62508-3]), XP_011507578.1 (nucleotide sequence XM_011509276.1 [P62508-3]), XP_011507579.1 (nucleotide sequence XM_011509277.1 [P62508-3]), XP_011507580.1 (nucleotide sequence XM_011509278.1 [P62508-3]), XP_011507581.1 (nucleotide sequence XM_011509279.1 [P62508-3]), XP_011507582.1 (nucleotide sequence XM_011509280.2 [P62508-3]), XP_016856120.1 (nucleotide sequence XM_017000631.1 [P62508-3]), XP_016 856121.1 (nucleotide sequence XM_017000632.1 [P62508-3]), XP_016856122.1 (nucleotide sequence XM_017000633.1 [P62508-3]), XP_016856123.1 (nucleotide sequence XM_017000634.1 [P62508-3]), XP_016856124.1 (nucleotide sequence XM_017000635.1 [P62508-3]), XP_016856125.1 (nucleotide sequence XM_017000636.1 [P62508-3]), XP_016856126.1 (nucleotide sequence XM_017000637.1 [P62508-3]). 000637.1 [P62508-3]), XP_016856127.1 (nucleotide sequence XM_017000638.1 [P62508-2]), XP_016856128.1 (nucleotide sequence XM_017000639.1 [P62508-2]), XP_016856129.1 (nucleotide sequence XM_017000640.1), XP_016856130.1 (nucleotide sequence XM_017000641.1 [P62508-2]), XP_016856131.1 (nucleotide sequence XM_017000642.1), XP_016856132.1 (nucleotide sequence XM_017000643.1), XP_016856133.1 (nucleotide sequence XM_017000644.1), XP_016856134.1 (nucleotide sequence XM_017000645.1), XP_016856135.1 (nucleotide sequence XM_017000646.1), XP_016856136.1 (nucleotide sequence XM_017000647.1), XP_016856137.1 (nucleotide sequence XM_017000648.1), XP_016856138.1 (nucleotide sequence XM_017000649.1), XP_016856139.1 (nucleotide sequence XM_017000650.1), XP_016856139.1 (nucleotide sequence XM_017000651 XM_017000643.1), XP_016856133.1 (nucleotide sequence XM_017000644.1 [P62508-2]), XP_016856134.1 (nucleotide sequence XM_017000645.1 [P62508-2]), XP_016856135.1 (nucleotide sequence XM_017000646.1), XP_016856136.1 (nucleotide sequence XM_017000647.1), XP_016856137.1 (nucleotide sequence XM_017000648.1), XP_016856138.1 (nucleotide sequence XM_017000649.1), or its natural variant VAR_019229 (T50M), but is not limited thereto. .

[0109] Agents capable of inhibiting ERRγ protein activity or ERRγ gene expression can be used to suppress resistance to TKI in the prevention or treatment of cancer.

[0110] According to one embodiment of the present disclosure, the composition can enhance the drug sensitivity of cancer to TKI, or can enhance the anti-cancer effect of TKI on cancer, but the present disclosure is not limited thereto.

[0111] Therefore, according to another embodiment of the present invention, the composition comprising an ERRγ inhibitor or an agent that inhibits ERRγ gene expression can be administered simultaneously, separately or sequentially with TKI, but the present disclosure is not limited thereto. In addition, the method for treating cancer or preventing TKI resistance in cancer comprises administering a composition comprising an ERRγ inhibitor or an agent that inhibits ERRγ gene expression, and further comprises administering TKI simultaneously, separately or sequentially.

[0112] The nature of proliferative diseases is multifactorial, and drugs with different mechanisms of action may be used in combination.For example, the pharmaceutical composition may be administered alone or in combination with known chemotherapy, radiotherapy, cancer immunotherapy or symptomatic treatment. In an embodiment, chemotherapy may include, but is not limited to, bosutinib (src tyrosine kinase inhibitor), gefitinib (EGFR tyrosine kinase inhibitor), lenvatinib (multi-kinase inhibitor targeting VEGFR1, VEGFR2, VEGFR 3, FGFR1, FGFR2, FGFR3, etc.), ceritinib (ALK inhibitor), ruxolitinib (JAK inhibitor), erlotinib, sunitinib, pazopanib, vandetanib, axitinib, cediranib, vatal anib, motesanib, RAF265, sorafenib, afatinib, osimertinib, lazertinib, SB590885, PLX8394, PLX 4720, PLX 4032, GDC-0879, ZM 336372, dabrafenib, encorafenib, CCT3833 / BAL3833, LY3009120, BGB-283 (lifirafenib), belvarafenib, TAK-580, trametinib, RO5126766, trametiglue, nazart inib, mavelertinib, azertinib, naquotinib, olmu tinib, rociletinib or a combination thereof. Another treatment may be a therapeutic antibody, wherein the therapeutic antibody is cetuximab, panitumumab, nimotuzumab or necitumumab. Another treatment may be immunotherapy, which includes but is not limited to a therapeutic antibody, wherein the therapeutic antibody is cetuximab, panitumumab, nimotuzumab, or necitumumab.

[0113] Tyrosine kinase inhibitors and resistance

[0114] Tyrosine kinase inhibitors (TKIs) are targeted specific inhibitors of abnormal protein tyrosine kinases (PTKs). TKIs have significant advantages over traditional chemotherapeutic agents, including high efficacy, low toxicity, and high specificity.

[0115] TKIs include, but are not limited to, inhibitors of VEGFR (vascular endothelial growth factor receptor), FGFR (fibroblast growth factor receptor), PEGFR (platelet-derived growth factor receptor), EGFR (epidermal growth factor receptor), ABL (Abelson kinase), TIE (tyrosine kinase with immunoglobulin-like and EGF-like domains), Src kinase, JAK (Janus kinase), etc. TKIs may be selective for or act on multiple targets. TKIs may be a combination of different inhibitors targeting the same or different tyrosine kinase targets.

[0116] Non-limiting exemplary TKI inhibitors may include, but are not limited to, bosutinib (src tyrosine kinase inhibitor), gefitinib (EGFR tyrosine kinase inhibitor), lenvatinib (multi-kinase inhibitor targeting VEGFR1, VEGFR2, VEGFR3, FGFR1, FGFR2, FGFR3, etc.), ceritinib (ALK inhibitor), ruxolitinib (JAK inhibitor), erlotinib, sunitinib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, motesanib, RAF265, sorafenib, afatinib, osimertinib, lazertinib, SB590885, PLX8394, PLX 4720, PLX4032, GDC-0879, ZM 336372, dabrafenib, encorafenib, CCT3833 / BAL3833, LY3009120, BGB-283 (lifirafenib), belvarafenib, TAK-580, trametinib, RO5126766, trametiglue, nazartinib, mavelertinib, azertinib, naquotinib, olmutinib, rociletinib, or a combination thereof.

[0117] TKI resistance may be congenital or acquired. Therefore, in embodiments of the present disclosure, the cancer may be characterized by resistance to at least one tyrosine kinase inhibitor (TKI) due to the following reasons: (a) at least one mutation in a gene encoding a protein that is a target of at least one TKI; or (b) at least one other gene in a wild-type or mutant state, the product encoded by which is resistant to the therapeutic effect of at least one TKI. Patients may have congenital or acquired mutations or polymorphisms that affect the apoptotic response to TKI. In some embodiments, the polymorphisms that affect the apoptotic response to TKI include, but are not necessarily limited to, polymorphisms of the gene BCL2L11 (also referred to as BM, which encodes a protein containing only the BH3 domain as a member of the BCL-2 family). In some embodiments, the mutation may include EGFR L858R, EGFR L858R / T790M, EGFR L858R / C797S, L858R / T790M / C797S, EGFR Del19, EGFR Del19 / T790M, EGFR Del19 / C797S, or EGFR Del19 / T790M / C797S.

[0118] According to one aspect, the cancer can be breast cancer, prostate cancer, melanoma, colorectal cancer, glioma (including recurrent glioblastoma), lung cancer, liver cancer, kidney cancer, ovarian cancer, sarcoma, pancreatic cancer, desmoid tumor (aggressive fibromatosis) or thyroid cancer.

[0119] In another aspect, the cancer can include pseudomyxoma, intrahepatic bile duct cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphocytic leukemia, basal cell carcinoma, epithelial ovarian cancer, ovarian seminoma, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, bile duct epithelial carcinoma, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, nasal and paranasal cavity cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, childhood brain cancer, childhood lymphoma, childhood leukemia, small Bowel cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureter cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoid, gastrointestinal stromal tumor, Wilms tumor, breast cancer, sarcoma, penis cancer, pharyngeal cancer, gestational choriocarcinoma, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid, vaginal cancer, spinal cancer, vestibular schwannoma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, squamous cell lung cancer, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, and thymic cancer. Cancer can have mutations.

[0120] Pharmaceutical compositions and methods of treatment

[0121] One aspect of the present disclosure also relates to a pharmaceutical combination comprising: (a) an ERRγ inhibitor or a pharmaceutically acceptable salt thereof, and (b) one or more TKIs or a pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable carrier, for preparing a pharmaceutical composition or medicament for treating or preventing a proliferative disease in a subject in need thereof.

[0122] Another aspect of the present disclosure also relates to a drug combination comprising: (a) an ERRγ inhibitor of Formula 1a, 1b, 1c or 1d or a pharmaceutically acceptable salt thereof, and (b) a TKI selected from the group consisting of bosutinib, gefitinib, lenvatinib, ceritinib, ruxolitinib, erlotinib, sunitinib, pazopanib, vandetanib, axitinib, cediranib, varanib, motesanib, RAF265, sorafenib, afatinib, osimertinib, lazertinib, SB590885, PLX8394, PLX4720, PLX4032, GDC-0879, ZM 336372, dabrafenib, conafenib, CCT3833 / BAL3833, LY3009120, BGB-283 (lifirafenib), belvarafenib, TAK-580, trametinib, RO5126766, trametiglue, nazartinib, mavelertinib, azertinib, nacotinib, omotinib, loxitinib or a combination thereof, and optionally at least one pharmaceutically acceptable carrier, for treating or preventing a proliferative disease in a subject in need thereof.

[0123] One aspect of the present disclosure also provides a commercial package comprising a combination as a therapeutic agent, the combination comprising: (a) an ERRγ inhibitor or a pharmaceutically acceptable salt thereof, and (b) one or more TKIs or pharmaceutically acceptable salts thereof, and optionally at least one pharmaceutically acceptable carrier for preparing a pharmaceutical composition, and instructions for simultaneous, separate or sequential administration for delaying progression or treating a proliferative disease.

[0124] Also provided are the above combinations for simultaneous, separate or sequential administration, in particular for use in the treatment or prevention of a proliferative disease.

[0125] Combinations of two compounds according to various aspects, which optionally contain another chemotherapeutic agent, can be used to treat proliferative diseases or cancer. The nature of proliferative diseases is multifactorial, and drugs with different mechanisms of action can be used in combination. However, merely considering any combination of therapeutic agents with different modes of action does not necessarily produce a combination with favorable effects. The use of the drug combination of the present disclosure can not only produce beneficial effects, such as synergistic therapeutic effects, such as related to relieving symptoms, delaying the progression of symptoms or inhibiting symptoms, but also produce other unexpected beneficial effects, such as fewer side effects, improved quality of life or reduced incidence compared to a monotherapy of a pharmaceutical active ingredient used in the combination of the present disclosure. A further benefit is that a lower dose of the therapeutic agent of the combination of the present disclosure can be used, for example, the dose can be smaller, the drug application frequency is lower, or it can be used to reduce the incidence of side effects observed when one of the combination partners is used alone.

[0126] The active ingredient compounds, i.e., ERRγ inhibitors and TKIs according to the embodiments are preferably formulated or used to have a combined (preventive or particularly therapeutic) activity. And, for example, the compounds can be administered separately or sequentially (in a chronic staggered manner, particularly in a sequence-specific manner) at such time intervals, they are preferably in the subject to be treated and still show (preferably synergistic) interactions (combined therapeutic effects). The combined therapeutic effect can be determined, in particular, by tracking blood levels, indicating that both compounds are present in the blood of the person to be treated at least at certain time intervals, but this does not exclude the situation where the compounds are not present in the blood at the same time but they have a combined activity.

[0127] Suitable pharmaceutical compositions or formulations include, for example, tablets, capsules, suppositories, solutions, in particular injection (iv, sc, im) and infusion solutions, syrups, elixirs, emulsions or dispersible powders. The content of the pharmaceutically active compound may be in the range of from 0.1% to 90% by weight, preferably 0.5% to 50% by weight, of the total weight or volume of the composition.

[0128] The composition of the pharmaceutical combination may be administered in a single formulation or unit dosage form, simultaneously but optionally separately, or sequentially by any suitable route. The unit dosage form may also be a fixed combination.

[0129] Suitable tablets can be obtained, for example, by mixing one or more active substances with known excipients, for example inert diluents such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as magnesium stearate or talc and / or release-delaying agents such as carboxymethylcellulose, cellulose acetate phthalate or polyvinyl acetate. Tablets may also contain several layers.

[0130] Coated tablets can be prepared accordingly by coating cores prepared similarly to the tablets with substances commonly used for tablet coatings, such as collodion or shellac, gum arabic, talc, titanium dioxide or sugar. The cores can also consist of multiple layers in order to achieve delayed release or to prevent incompatibilities. Similarly, the tablet coating can consist of multiple layers to achieve delayed release, possibly using the excipients mentioned above for tablets.

[0131] Syrups or elixirs containing the active substances of the invention or their combination may additionally contain sweeteners (such as saccharin, sugar substitutes, glycerol or sugar) and flavor enhancers (e.g. flavoring agents such as vanillin or orange extract). They may also contain suspending adjuvants or thickeners (such as sodium carboxymethylcellulose), wetting agents (such as, for example, condensation products of fatty alcohols with ethylene oxide) or preservatives (such as parabens).

[0132] Injection and infusion solutions are prepared in the usual manner, for example by adding isotonic agents, preservatives (such as parabens) or stabilizers (such as alkali metal salts of ethylenediaminetetraacetic acid), optionally using emulsifiers and / or dispersants, and if water is used as a diluent, for example, an organic solvent may optionally be used as a solubilizing agent or solubilizing aid, and transferred to injection vials or ampoules or infusion bottles.

[0133] Capsules containing one or more active substances or a combination of active substances can be prepared, for example, by mixing the active substances with inert carriers such as lactose or sorbitol and packing them into gelatin capsules.

[0134] Excipients that can be used include, for example, water, pharmaceutically acceptable organic solvents such as paraffins (e.g., petroleum fractions), vegetable oils (e.g., peanut oil or sesame oil), monofunctional or polyfunctional alcohols (e.g., ethanol or glycerol), carriers such as, for example, natural mineral powders (e.g., kaolin, clay, talc, chalk), synthetic mineral powders (e.g., highly dispersed silicic acid and silicates), sugars (e.g., sucrose, lactose and glucose), emulsifiers (e.g., lignin, sulfite waste liquor, methylcellulose, starch and polyvinyl pyrrolidone) and lubricants (e.g., magnesium stearate, talc, stearic acid and sodium lauryl sulfate).

[0135] The composition is administered by conventional methods, preferably by oral or transdermal routes, most preferably by oral routes. For oral administration, the tablets may contain additives, such as sodium citrate, calcium carbonate and dicalcium phosphate, and various additives, such as starch, preferably potato starch, gelatin, etc., in addition to the above-mentioned carriers. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate and talc may be used simultaneously in the tableting process. For aqueous suspensions, in addition to the above-mentioned excipients, the active substance may be used in combination with various flavor enhancers or coloring agents.

[0136] For parenteral use, solutions of the active ingredient with suitable liquid carriers may be used.

[0137] The dosage of the pharmaceutical composition is the amount that can effectively treat or prevent an individual, subject or patient, and can be administered orally or parenterally as needed. When administered orally, 0.01 to 1000 mg / kg body weight per day, more specifically 0.1 to 1000 mg, based on the active ingredient during parenteral administration need to be administered in an amount of 0.01 to 100 mg / kg body weight / day, more specifically 0.1 to 50 mg based on the active ingredient. It can be administered in divided doses from 1 to several times. The dosage of a specific individual, subject or patient should be determined according to several relevant factors such as the patient's weight, age, sex, health status, diet, administration time, administration method, severity of the disease, and can be appropriately increased or decreased by an expert. It should be understood that such dosages are not intended to limit the scope of the present disclosure in any way. A doctor or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a doctor or veterinarian can use the dosage of the compound of the present disclosure used in the pharmaceutical composition, starting from a level lower than that required to achieve the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved.

[0138] Diagnostic methods and kits

[0139] According to still another aspect of the present disclosure, provided is a kit for diagnosing TKI-resistant cancer, comprising an agent for measuring the level of mRNA of ERRγ gene or protein expressed thereby.

[0140] According to one embodiment of the present disclosure, the agent for measuring the mRNA level of a gene may include a pair of primers, probes or antisense nucleotides that specifically bind to the gene, but the present invention is not limited thereto.

[0141] According to still another embodiment of the present invention, the agent for measuring a protein level may include an antibody or an aptamer specific to the protein, but the present disclosure is not limited thereto.

[0142] According to yet another embodiment of the present disclosure, the kit may include an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip kit, or a protein chip kit, but the present disclosure is not limited thereto.

[0143] According to another aspect of the present disclosure, there is provided a method for providing TKI-resistant cancer diagnosis information for a first patient suffering from cancer (first cancer patient), comprising the following steps:

[0144] (a) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a first cancer patient to examine whether the cancer exhibits resistance to TKI;

[0145] (b) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a second patient with cancer that is not resistant to TKI or a subject without cancer; and

[0146] (c) when the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (a) is higher than the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (b), determining the first cancer patient in step (a) as a TKI-resistant cancer patient.

[0147] According to another aspect of the present disclosure, a method for providing information required for determining a treatment method for a first patient suffering from cancer (first cancer patient) is provided, comprising the following steps:

[0148] (a) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a first cancer patient;

[0149] (b) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a second patient with cancer that is not resistant to TKI or a subject without cancer; and

[0150] (c) when the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (a) is higher than the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (b), the first cancer patient in step (a) is determined to be a TKI-resistant cancer patient.

[0151] According to yet another aspect of the present disclosure, there is provided a method of diagnosing and treating TKI-resistant cancer in a first patient, comprising the following steps:

[0152] (a) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a first patient;

[0153] (b) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a second patient with cancer that is not resistant to TKI or a subject without cancer; and

[0154] (c) when the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (a) is higher than the expression level of mRNA of the ERRγ gene or the protein expressed thereby measured in step (b), determining that the first cancer patient in step (a) is a TKI-resistant cancer patient and applying another cancer therapeutic agent other than TKI.

[0155] According to one embodiment of the present disclosure, the biological sample may include cancer tissue, cancer cells, whole blood, plasma, serum or blood, but the present invention is not limited thereto.

[0156] According to another aspect of the present disclosure, the present disclosure also provides a method for screening materials that enhance TKI-resistant cancer treatment, comprising (a) treating TKI-resistant cancer cells with a candidate material; and (b) evaluating the activity or expression of ERRγ in cells. Substances that can inhibit the activity or expression of ERRγ in TKI-resistant cancer cells can be screened by such methods and used as enhancers or adjuvants for TKI-resistant cancer treatment.

[0157] According to still another aspect of the present disclosure, the present disclosure provides a kit for diagnosing TKI-resistant cancer.

[0158] Specifically, the kit for diagnosing TKI-resistant cancer includes an agent for measuring the level of mRNA of the ERRγ gene or a protein expressed thereby.

[0159] In the present disclosure, the term "diagnosis" means confirming the presence or characteristics of a pathological condition. In the present disclosure, diagnosis is determining the presence or occurrence of TKI-resistant cancer by measuring the level of mRNA of the ERRγ gene or the protein expressed thereby.

[0160] In the present disclosure, the term "agent for measuring the expression level of mRNA of ERRγ gene or its protein" refers to a molecule that can be used to examine the expression level of ERRγ gene or protein encoded by such gene. In this case, the agent may preferably be a pair of primers, probes or antisense nucleotides, both of which specifically bind to ERRγ gene, or may be an antibody or aptamer specific to the protein encoded by ERRγ gene.

[0161] In the present disclosure, the term "primer" refers to a short nucleic acid sequence with a free 3' hydroxyl group, i.e., a short nucleic acid sequence that can form a base pair with a complementary template and serve as a template replication origin. In the present disclosure, the sense and antisense primers of the marker polynucleotide of the present disclosure can be used for PCR amplification to screen patients with TKI-resistant cancers by the level of production of the desired product. The PCR conditions, the length of the sense and antisense primers can be modified according to what is known in the art.

[0162] As used in the present disclosure, the term "probe" refers to a nucleic acid fragment corresponding to a few bases to several hundred bases, such as RNA or DNA, which can specifically bind to mRNA and can be labeled to determine the presence / absence of a specific mRNA. The probe can be made into the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. In the present disclosure, a probe complementary to the ERRγ polynucleotide of the present disclosure can be used for hybridization to screen patients with RAF inhibitor-resistant cancers based on the degree of hybridization. The selection of suitable probes and hybridization conditions can be modified according to knowledge known in the art.

[0163] Primers or probes disclosed herein can be chemically synthesized using the phosphoramidite solid phase support method or other well-known methods. Such nucleotide sequences can also be modified using a variety of methods known in the art. Non-limiting examples of such modifications include methylation, encapsulation, replacement of one or more homologues of natural nucleotides and internucleotide modifications (such as modification to uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

[0164] Taking into account mutations with biologically equivalent activity, the nucleotide sequence of the agent for measuring the expression level of the ERRγ gene used in the present disclosure is interpreted as including sequences that show substantial identity with sequences that specifically bind to the ERRγ gene. The term "substantial identity" means that when a specific sequence is aligned to the maximum possible correspondence with any other sequence and the aligned sequence is analyzed using an algorithm commonly used in the art, the sequence shows at least 60% identity, more specifically 70% or higher identity, even more specifically 80% or higher identity, and most specifically 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher identity.

[0165] In the kit of the present disclosure, the terms "antibody" and "aptamer" are the same as those described in the pharmaceutical composition, and thus the description thereof will be omitted.

[0166] The kit of the present disclosure can be used to diagnose TKI-resistant cancer by examining the mRNA expression level of the ERRγ gene or the expression level of the protein encoded by the gene, or to predict the therapeutic response to TKI treatment.

[0167] Since the kits of the present disclosure can be used to predict the therapeutic response to TKI therapy, the kits can also be used to predict the prognosis of cancer patients.

[0168] In the present disclosure, the term "predicting ... prognosis" refers to the process of guessing a medical outcome in advance, and for the purpose of the present disclosure, means guessing a cancer patient's resistance to TKI treatment.

[0169] According to one embodiment of the present disclosure, the kit may be an RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip kit, or a protein chip kit, but the present disclosure is not limited thereto.

[0170] According to one embodiment of the present disclosure, the kit for measuring the expression level of ERRγ gene mRNA may be a kit including the basic elements necessary for RT-PCR. In addition to each pair of primers specific to the marker gene, the RT-PCR kit also includes a test tube or other suitable container, a reaction buffer, deoxynucleotides (dNTPs), TAQ TM Polymerase and reverse transcriptase, deoxyribonuclease, ribonuclease inhibitor, DEPC water, sterile water, etc.

[0171] The kit of the present disclosure may include a kit for extracting nucleic acid (eg, total RNA) from body fluids, cells or tissues, a fluorescent substance for labeling, an enzyme and culture medium for nucleic acid amplification, instructions for use, and the like.

[0172] According to another embodiment of the present disclosure, the kit of the present disclosure may be a kit for detecting ERRγ, which comprises the elements necessary for using a DNA chip. The DNA chip kit may include a cDNA corresponding to a gene or a fragment thereof as a substrate to which a probe is attached, and the substrate may include a cDNA corresponding to a quantitatively controllable gene or a fragment thereof.

[0173] According to another embodiment of the present disclosure, a kit for measuring the expression level of a protein encoded by ERRγ of the present disclosure may include a substrate for antibody immunoassay, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like. In this case, nitrocellulose membranes, 96-well plates synthesized from polyethylene resins, 96-well plates synthesized from polystyrene resins, and glass slides may be used as substrates. Peroxidase and alkaline phosphatase may be used as chromogenic enzymes. FITC, RITC, and the like may be used as fluorescent materials, and ABTS (2,2'-azo-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), TMB (tetramethylbenzidine), and the like may be used as chromogenic substrates.

[0174] The kits of the present disclosure may be configured to further include a composition, solution, or device having one or more other elements suitable for use in an analytical method.

[0175] According to yet another aspect, the present disclosure provides a method of providing information for diagnosing TKI-resistant cancer.

[0176] Specifically, the method for providing information for diagnosing TKI-resistant cancer includes: (a) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a first cancer patient to examine whether the cancer exhibits resistance to TKI; (b) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a second patient with cancer that is not resistant to RAF inhibitor treatment or a subject without cancer; and (c) when the expression level of mRNA of the ERRγ gene or a protein expressed thereby measured in step (a) is higher than the expression level of mRNA of the ERRγ gene or a protein expressed thereby measured in step (b), determining the first cancer patient in step (a) as a patient suffering from TKI-resistant cancer.

[0177] In the present disclosure, the term "biological sample" refers to tissue (cancer tissue), cell (cancer cell), whole blood, plasma, serum, blood, saliva, synovial fluid, urine, sputum, lymph, cerebrospinal fluid, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant or ruptured eukaryotic cells, ERRγ as a TKI-resistant cancer marker has different expression and / or activity levels. In addition, biological samples include samples from metastatic lesions and primary cancer lesions. In this case, the activity or expression level of ERRγ can be measured in a state where these biological samples are operated or not operated.

[0178] In the method for providing information for diagnosing cancer resistant to RAF inhibitors according to the present disclosure, the biological sample of step (a) can be obtained using a specific method known to those skilled in the art. For example, the biological sample can be obtained from a vertebrate, particularly a mammal, and can preferably be obtained from a cancer patient to examine whether the cancer exhibits resistance to RAF inhibitors. In this case, the cancer patient is a human. Tissue biopsy is typically used to obtain a representative tumor tissue block. Alternatively, tumor cells can be obtained indirectly in the form of a tissue or liquid known or believed to contain tumor cells of interest.

[0179] In the present disclosure, the term "measuring the expression level of mRNA" refers to the process of confirming the presence and expression level of mRNA of the ERRγ gene in a biological sample, and can be understood by measuring the amount of mRNA. The analytical method for the measurement includes RT-PCR, competitive RT-PCR, real-time RT-PCR, ribonuclease protection method, Northern blotting or DNA chip technology, but the present disclosure is not limited thereto.

[0180] In the present disclosure, the term "measuring the expression level of a protein" refers to the process of confirming the presence and expression level of a protein expressed by the ERRγ gene in a biological sample. In this case, the amount of the protein can be determined using an antibody that specifically binds to the protein expressed by the gene. The analytical methods for this measurement include protein blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, fluorescence-activated cell sorting analysis (FACS analysis), protein chip technology, etc., but the present disclosure is not limited thereto.

[0181] In the method for providing information for diagnosing TKI-resistant cancer according to the present disclosure, the patient with cancer that is not resistant to TKI may be a patient with breast cancer, prostate cancer, melanoma, colorectal cancer, glioma (including recurrent glioblastoma), lung cancer, kidney cancer, ovarian cancer, sarcoma, desmoid tumor (aggressive fibromatosis) or thyroid cancer who does not show TKI-resistant cancer. For example, in TKI-resistant liver cancer, the patient with cancer that is not resistant to TKI may be a patient with hepatocellular carcinoma (HCC) or hepatic adenocarcinoma who does not show TKI-resistant liver cancer, but the present disclosure is not limited thereto.

[0182] In the method for providing information for diagnosing TKI-resistant cancer according to the present disclosure, the expression level of the ERRγ gene can be measured at the mRNA level or protein level, and the mRNA or protein can be isolated from the biological sample using methods known in the art. The analytical method for measuring the mRNA level and the analytical method for measuring the protein level are as described above.

[0183] By providing information for diagnosing TKI-resistant cancer according to the present disclosure, it is possible to predict the therapeutic response of cancer patients to TKI and formulate a treatment strategy for each patient based on the prediction results, thereby making it possible to effectively treat cancer.

[0184] Therefore, according to yet another aspect, the present disclosure provides a method for providing information required for determining a treatment method for a cancer patient, comprising measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a cancer patient.

[0185] Specifically, the method for providing information required to determine a treatment method for a first cancer patient may include: (a) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from the first cancer patient; (b) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a second patient suffering from cancer that is not resistant to TKI or a subject without cancer; and (c) when the expression level of mRNA of the ERRγ gene or a protein expressed thereby measured in step (a) is higher than the expression level of mRNA of the ERRγ gene or a protein expressed thereby measured in step (b), determining the first cancer patient in step (a) as a patient suffering from TKI-resistant cancer.

[0186] According to the method of providing information required for determining a therapeutic method for a cancer patient, when resistance to TKI is confirmed in a cancer patient, a cancer therapeutic effect can be induced by applying other known cancer therapeutic agents other than TKI.

[0187] Thus, according to a further aspect, the present disclosure provides a method of diagnosing and treating TKI-resistant cancer.

[0188] Specifically, the method for diagnosing and treating TKI-resistant cancer in a first patient according to the present disclosure includes: (a) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from the first cancer patient; (b) measuring the expression level of mRNA of the ERRγ gene or a protein expressed thereby in a biological sample isolated from a second patient suffering from cancer that is not resistant to TKI; and (c) when the expression level of mRNA of the ERRγ gene or a protein expressed thereby measured in step (a) is higher than the expression level of mRNA of the ERRγ gene or a protein expressed thereby measured in step (b), determining the first cancer patient in step (a) as a patient suffering from TKI-resistant cancer and applying another cancer therapeutic agent other than TKI.

[0189] Specific details of the method for providing information required to determine a treatment method for a cancer patient and / or the method for diagnosing and treating TKI-resistant cancer according to the present disclosure are as described above in the method for providing information for diagnosing TKI-resistant cancer, and thus a description thereof will be omitted.

[0190] It should be understood that the terms and words used in this specification and the appended claims should not be understood as limited to the general and dictionary meanings, but should be interpreted based on the meanings and concepts corresponding to the technical aspects of the present disclosure according to the principles that the inventors are able to appropriately define the concepts of the terms to best describe the present disclosure.

[0191] The following preferred embodiments are provided to help understand the present disclosure. However, it should be understood that the following embodiments are only used to better understand the present disclosure and are not intended to limit the scope of the present disclosure.

[0192] [Experimental Example]

[0193] General experimental methods

[0194] As an example of a TKI-resistant cancer model, the inventors used sorafenib-resistant liver cancer cell lines (Huh7-SR and SK-Hep-R cell lines) and animal models derived from liver cancer cell lines (xenografts). In order to determine the effect of the synthetic compound DN200434 (Formula 1a compound), which is an inverse agonist of ERRγ, on liver cancer cell proliferation and ROS generation, ROS were measured using cell counting and 2',7-dichlorofluorescein diacetate (DCF-DA). Finally, sorafenib-resistant liver cancer cell lines were injected into rats to induce the formation of sorafenib-resistant liver cancer, and changes in liver cancer size were confirmed in the group injected with an inverse agonist (DN434) and sorafenib for ERRγ and the group injected with the control drug.

[0195] [Example 1]

[0196] Construction of sorafenib-resistant hepatocellular carcinoma cell line and confirmation of increased ERRγ expression

[0197] Hepatocellular carcinoma cell lines [Huh7 cells (Korean Cell Line Bank (KCLB) No. 60104), SK-Hep cells ( HTB-52 TM )] were continuously exposed to sorafenib (gradually increased to 10 μM) to construct sorafenib-resistant liver cancer cell lines (Huh7-SR and SK-Hep-R cell lines). To evaluate cancer cell death by FACS, cancer cell lines were first incubated in FITC-conjugated annexin and propidium iodide (PI) for 15 min, and then annexin and PI binding were measured by flow cytometry to acquire data using a BD Accuri C6 flow cytometer (BD Biosciences), and the ACCURI TM The analysis was performed using the C6 analysis program (BD Biosciences) / FlowJo software (FlowJo, LLC.). Cancer cell line cell death was assessed using a cleaved caspase-3 antibody (Cell Signaling Technology). Figures 1A to 1C As shown, it was confirmed that in two sorafenib-resistant liver cancer cell lines (eg, Huh7-SR and SK-Hep-R cell lines), cell death caused by sorafenib was not increased.

[0198] In addition, Western blotting was performed to confirm the expression pattern of the orphan nuclear receptor ERRγ in Huh7-SR and SK-Hep-R cell lines. Thus, it was confirmed that the expression of ERRγ was significantly increased in both sorafenib-resistant hepatoma cell lines, as shown in Figure 2. Figure 2A and Figure 2B shown.

[0199] [Example 2]

[0200] Effect of orphan nuclear receptor ERRγ on sorafenib-resistant hepatocellular carcinoma

[0201] To investigate the effect of ERRγ inhibitor (compound represented by Formula 1a (DN200434)) on ROS generation in sorafenib-resistant hepatoma cells, the effect was measured by FACS using 2',7'-dichlorohydrofluorescein diacetate (H2-DCF-DA; Invitrogen, USA) as a ROS probe. Drug-resistant cells were treated with sorafenib (10 μM) and DN200434 compound (12 μM) for 24 hours, and 10 μM H2-DCF-DA was added to the cells. Then, the cells were incubated for 30 minutes, washed with PBS, and stained with BD ACCURI TM Data were collected using a C6 flow cytometer (BD Bioscience, USA) and analyzed using ACCURI TM The analysis was performed using C6 analysis program (BD Bioscience, USA). To determine the cell number, Sorafenib-resistant hepatoma cell lines were treated with DN200434 compound (12 μM) in combination with Sorafenib (10 μM), stained with trypan blue, and then the cell number was measured using a hemacytometer.

[0202] Compounds of Formula 1b to Formula 1d were tested in the same manner as described in Example 2 as ERRγ inhibitors.

[0203] Therefore, for the compound of formula 1a, Figure 3A and Figure 3B As shown in Figure 2, it was confirmed that the increase of ROS caused by the compound was observed in both Huh7-SR and SK-Hep-R cell lines (which are sorafenib-resistant liver cancer cell lines). Figure 4A and Figure 4B As shown, it was confirmed that the cell proliferation of sorafenib-resistant liver cancer cells, which was not affected when treated with sorafenib alone, was significantly reduced when the compound of formula 1a was simultaneously administered. Based on the results as described above, it was confirmed that ERRγ inhibitors can overcome drug resistance by inhibiting ERRγ activity and increasing sensitivity to sorafenib.

[0204] [Example 3]

[0205] Confirmation of anticancer effects in sorafenib-resistant hepatocellular carcinoma animal models

[0206] Sorafenib-resistant liver cancer cell line Huh7-R was injected into mice to construct a sorafenib-resistant liver cancer animal model (xenograft). Subsequently, an inverse agonist for ERRγ (e.g., DN200434 (Formula 1a)) was administered in combination with sorafenib, and the size change of the formed mass was measured. In this case, a group of sorafenib-resistant liver cancer-derived animal models treated with sorafenib alone was used as a comparative control.

[0207] Compounds of Formula 1b to Formula 1d were tested in the same manner as described in Example 3 as ERRγ inhibitors.

[0208] As a result of the compound of formula 1a, such as FIG. 5A to FIG. 5C As shown in FIG. 1 , it was confirmed that in the group to which the compound was administered in combination with sorafenib in an animal model derived from the sorafenib-resistant liver cancer cell line Huh7-R, tumor size, weight, and volume were significantly reduced compared to the control group. Figure 5D It can be seen that there was no difference in body weight change between the experimental and control groups.

[0209] An animal model derived from the sorafenib-resistant hepatoma cell line SK-Hep-R was constructed in the same manner as described above, and the effect of co-administration of an ERRγ inhibitor with sorafenib was confirmed.

[0210] Similarly, if FIG. 6A to FIG. 6C As shown in FIG. 1 , it was confirmed that even in an animal model derived from the sorafenib-resistant liver cancer cell line SK-Hep-R, the tumor size, weight, and volume of liver cancer in the group to which the ERRγ inhibitor was administered in combination with sorafenib were significantly reduced compared with the control group. Fig.6D It can be seen that there was no difference in body weight change between the experimental and control groups.

[0211] Based on the results of Examples 1 to 3, it was confirmed that the expression of ERRγ was significantly increased in TKI-resistant cancer. In addition, it was confirmed experimentally that ERRγ inhibitors increased intracellular ROS, inhibited the proliferation of TKI-resistant cancer cells, and increased sensitivity to TKI. In addition, it was confirmed that in animal experiments using TKI-resistant cancer cell lines, cancer cell proliferation was significantly reduced compared with the control group. The above results prove that ERRγ plays an important role in drug resistance of cancer, and when ERRγ activity is inhibited by an inverse agonist against ERRγ, advanced drug-resistant cancer can be effectively treated.

[0212] [Example 4]

[0213] Anticancer effects in erlotinib-resistant lung cancer cells

[0214] Erlotinib-resistant HCC827 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% antibiotics.

[0215] To start the assay, cells were plated at 1 × 10 4 Cells were seeded in 96-well plates at a density of 10 cells and incubated overnight in a 37°C CO2 incubator, and then treated with DN434 for 24 hours. After 24 hours of incubation, the culture medium was replaced with culture medium containing 0.5 mg / ml MTT, and the plates were incubated for 1 hour. Then, the culture medium was removed and the formazan crystals were dissolved in 100 μl DMSO added to each well. The absorbance at 570 nm was measured using a microplate reader, and cell viability was normalized compared to the control. Statistical significance was calculated using the Student's t-test, with significance levels set at **p<0.01, ***p<0.001, and ****p<0.0001. The half-maximal inhibitory concentration (IC 50 ).

[0216] The results are shown in Fig.13 middle. Fig.13 The ERRγ compound (DN434) was shown to inhibit the growth of erlotinib-resistant HCC827 cells with an IC 50 The results showed that DN434 could also effectively inhibit the growth of erlotinib-resistant lung cancer cells.

[0217] [Example 5]

[0218] Synergistic anticancer effects of ERRγ compounds combined with TKIs

[0219] To evaluate the anticancer effects of ERRγ compounds alone and in combination with TKIs, various cancer cells (including A549, Panc-1, Sk-Hep-1, Huh-7, and SK-Hep-1 cells) were cultured in DMEM containing 10% FBS and 1% antibiotics. In addition, HCC827, PC9, and SKOV-3 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% antibiotics.

[0220] To start the assay, cells were plated at 1 × 10 4Cells were seeded in 96-well plates at a density of 10 cells and incubated overnight in a 37°C CO2 incubator, and then treated with TKI, DN434, or a combination of the two for 24 hours. For control, an equal volume of DMSO was added. After 24 hours of incubation, the medium was replaced with medium containing 0.5 mg / ml MTT, and the plate was incubated for 1 hour. Then, the medium was removed and the formazan crystals were dissolved in 100 μl DMSO added to each well. The absorbance at 570 nm was measured using a microplate reader, and cell viability was normalized compared to the control. Statistical significance was calculated using the Student's t-test, with significance levels set at **p<0.01, ***p<0.001, and ****p<0.0001.

[0221] The results are shown in Figures 8 to 12 .based on Figure 8 As shown in the results, the growth of A549 lung cancer cell line was inhibited by 16.3%, 26.4% and 83.7% by Bosutinib (12.5 μM), DN434 (25 μM) and the combination of the two, respectively. The results indicate a synergistic effect. It was found that the combination treatment had a significant synergistic cancer cell growth inhibition effect.

[0222] like Fig. 9 As shown, it was observed that gefitinib (25nM), DN434 (25μM) and the combination of the two inhibited the growth of HCC827 lung cancer cell line by 16.6%, 3.4% and 32.2%, respectively, and the combination had a significant synergistic effect (left figure). In addition, gefitinib (12.5nM), DN434 (8μM) and the combination of the two inhibited the growth of PC9 lung cancer cell line by 25.1%, 19% and 47.8%, respectively, and the combination also had a significant synergistic effect (right figure).

[0223] like Fig.10 As shown in Figure 3b, lenvatinib (1.3 μM), DN434 (10 μM) and the combination of the two inhibited the growth of Huh-7 liver cancer cells by 16.7%, 12.8% and 34.7%, respectively, and the combination had a significant synergistic effect (left figure). Similarly, in Figure 3b, lenvatinib (15 μM), DN434 (20 μM) and the combination of the two inhibited the growth of SK-Hep-1 liver cancer cells by 16.7%, 12.8% and 34.7%, respectively, and the combination had a significant synergistic effect (right figure).

[0224] and, Fig.11 The results showed that ceritinib (2.5 μM), DN434 (25 μM) and the combination of the two inhibited the growth of Panc1 pancreatic cancer cells by 17.0%, 21.3% and 47.6%, respectively, and the combination had a significant synergistic effect.

[0225] in addition, Fig.12 The results also showed that ruxolitinib (30 μM), DN434 (25 μM) and the combination of the two inhibited the growth of SKOV-3 ovarian cancer cells by 8.8%, 23.7% and 48.1%, respectively, and the combination had a significant synergistic effect.

[0226] The above description of the present disclosure is given by way of example only, and those skilled in the art to which the present disclosure belongs should understand that various changes and modifications can be made without departing from the technical spirit and scope of the present disclosure. Therefore, it should be understood that the above embodiments are given by way of example only and are not intended to be limiting in all aspects.

Claims

1. A combination comprising: (a) a first composition comprising an agent capable of inhibiting estrogen-related receptor gamma (ERRγ) protein activity or ERRγ gene expression; and (b) a second composition comprising a tyrosine kinase inhibitor. 2 . The combination according to claim 1 , wherein the agent for inhibiting the activity of ERRγ protein is an inverse agonist or antagonist against ERRγ, or an antibody or aptamer capable of specifically binding to ERRγ.

3. The combination according to claim 2, wherein the inverse agonist or antagonist against ERRγ is a compound of the following formula A: in L is (C6-C20)arylene, (C3-C20)heteroarylene or (C3-C20)fused heterocyclic ring; R 1 is (C3-C20)heterocycloalkyl, (C3-C20)heteroaryl, -O-(CH2) m -R 11 、-(CH2) m -R 12 、-NH-(CH2) m -R 13 、-NHCO-(CH2) n -R 14 Or-SiR 16 R 17 -(CH2) m -R 15 ; R 11 To R 15 are independently (C3-C20)heterocycloalkyl; R 16 and R 17 are independently (C1-C20)alkyl; m is an integer from 1 to 3; and n is an integer of 0 or 1; Ar is (C6-C20) aryl or (C3-C20) heteroaryl, wherein the aryl or heteroaryl of Ar may be further substituted by one or more selected from the group consisting of hydroxyl, halogen, (C1-C20) alkyl, halogenated (C1-C20) alkyl, (C1-C20) alkoxy, nitro, cyano, -NR 21 R 22 , (C1-C20) alkylcarbonyloxy, (C1-C20) alkylcarbonylamino, guanidino, -SO2-R 23 and-OSO2-R 24 ; R 21 and R 22 are independently hydrogen, (C1-C20)alkylsulfonyl or (C3-C20)cycloalkylsulfonyl; R 23 and R 24 are independently (C1-C20)alkyl, halogenated (C1-C20)alkyl or (C3-C20)cycloalkyl; R 2 is hydroxy, halogen, (C1-C20)alkylcarbonyloxy or (C1-C20)alkylsulfonyloxy; R 1 The heterocycloalkyl or heteroaryl group and R 11 To R 15 The heterocycloalkyl group may be further substituted by one or more selected from the group consisting of: (C1-C20)alkyl, (C3-C20)cycloalkyl, (C2-C20)alkenyl, amidino, (C1-C20)alkoxycarbonyl, hydroxyl, hydroxyl (C1-C20)alkyl and di (C1-C20)alkylamino (C1-C20)alkyl; and The heterocycloalkyl and heteroaryl groups contain one or more heteroatoms selected from the group consisting of N, O and S, and the heterocycloalkyl group is a saturated or unsaturated monocyclic, bicyclic or spirocyclic ring having a carbon atom or a nitrogen atom as a binding site within the ring, isomers, or pharmaceutically acceptable salts or solvates including hydrates thereof.

4. The combination according to claim 3, wherein the compound of formula A is a compound of the following formula 1a to formula 1d, a pharmaceutically acceptable salt thereof, or an isomer thereof, or a solvate thereof: Or wherein the agent for inhibiting the expression of ERRγ gene is selected from the group consisting of miRNA, siRNA, shRNA and antisense oligonucleotide, all of which specifically bind to the mRNA of said gene.

5. The combination of claim 1, wherein the tyrosine kinase inhibitor is bosutinib, gefitinib, lenvatinib, ceritinib, ruxolitinib, erlotinib, sunitinib, pazotinib, vandetanib, axitinib, cediranib, vatalanib, motesanib, sorafenib, afatinib, osimertinib, lazertinib, dabrafenib, connefenib, lifirafenib, belvarafenib, trametinib, trametiglue, nazartinib, mavelertinib, azertinib, nacotinib, omotinib, loxitinib, or a combination thereof.

6. The combination of claim 1, wherein the first composition and the second composition are administered simultaneously, separately or sequentially.

7. The combination of claim 6, which is a pharmaceutical preparation for preventing or inhibiting tyrosine kinase resistance in a patient suffering from cancer, for enhancing treatment of a tyrosine kinase inhibitor-resistant cancer in a patient, or for treating cancer in a patient.

8. The combination of claim 7, wherein the patient has been treated with a tyrosine kinase inhibitor.

9. The combination of claim 7, wherein the patient has not been treated with a tyrosine kinase inhibitor.

10. The combination of claim 7, wherein the cancer is breast cancer, prostate cancer, melanoma, colorectal cancer, glioma (including recurrent glioblastoma), lung cancer, liver cancer, kidney cancer, ovarian cancer, sarcoma, pancreatic cancer, desmoid tumor (aggressive fibromatosis) or thyroid cancer.

11. The combination of claim 7, wherein the combination is administered in combination with a third composition and / or radiation therapy, wherein the third composition comprises a therapeutic antibody.

12. Use of the combination of claim 1 in the manufacture of a medicament for preventing or inhibiting tyrosine kinase resistance in a patient suffering from cancer, for enhancing treatment of a tyrosine kinase inhibitor-resistant cancer in a patient, or for treating cancer in a patient.

13. Use of the combination of claim 1 for preventing or inhibiting tyrosine kinase resistance in a patient suffering from cancer, enhancing treatment of a tyrosine kinase inhibitor-resistant cancer in a patient, or treating cancer in a patient.

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