Use of a composition comprising an estrogen-related receptor gamma inhibitor as an active ingredient for enhancing anti-cancer effects

Abstract

The present invention relates to a pharmaceutical composition comprising an estrogen-related receptor γ (ERRγ) inhibitor as an active ingredient for suppressing sorafenib resistance in liver cancer and enhancing the anticancer effect. The present invention can be useful as a pharmaceutical composition for treating sorafenib-resistant progressive liver cancer.

Description

Technical Field

[0001] The present invention relates to the use of a composition comprising an estrogen-related receptor γ (ERRγ) inhibitor as an active ingredient for enhancing anti-cancer effects. The present invention more specifically provides a pharmaceutical composition comprising an ESRγ inhibitor as an active ingredient that suppresses liver cancer resistance to sorafenib and enhances anti-cancer effects, and a method for treating sorafenib-resistant liver cancer using the same. Furthermore, the present invention provides a kit for diagnosing sorafenib-resistant liver cancer that uses ESRγ as a biomarker for diagnosing sorafenib-resistant liver cancer, and a method for providing information for diagnosing sorafenib-resistant liver cancer using the same. Background Art

[0002] One of the leading causes of death in South Korea is malignant neoplasms (cancer). According to death statistics released by the National Statistical Office, the liver cancer mortality rate was 21.5 per 100,000 people in 2016, second only to lung cancer mortality (Hepatocellular Carcinoma Diagnosis and Treatment Guidelines, Korean Liver Cancer Society, 2018).

[0003] The fundamental treatment for liver cancer is liver resection. It is the primary treatment for patients with single hepatocellular carcinoma confined to the liver without cirrhosis (Lang H.,Liver resection for hepatocellular carcinoma in non-cirrhotic liver without underlying viral hepatitis.Br J Surg.2005Feb;92(2):198-202). Even in the presence of cirrhosis, it can be given priority when the residual liver function is expected to be sufficient (Capussotti L, Muratore A, Massucco P, Ferrero A, Polastri R, Bouzari H Major liver resections for hepatocellular carcinoma on cirrhosis: early and long-term outcomes..Liver Transpl.2004Feb;10(2Suppl 1):S64-8). However, only 30-40% of patients diagnosed in the early stage can be treated surgically.

[0004] When there is extrahepatic metastasis such as to local lymph nodes and lungs or hepatic vascular invasion, sorafenib, a multikinase inhibitor, is administered. ) treatment, the clinical survival improvement is far below the expected level, and most patients relapse within 6 months. Efforts are underway to overcome this problem (Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378-390).

[0005] On the other hand, to overcome the limitations of existing anticancer drugs or targeted therapeutics, cancer metabolism research and new drug development research targeting the metabolic characteristics of cancer are carried out by studying the specific metabolic regulation unique to cancer cells that is different from normal cells (Vander Heiden MG. Targeting cancer metabolism: a therapeutic window opens. Nat Rev Drug Discov 2011; 10: 671-684).

[0006] Prior art literature

[0007] Patent Literature

[0008] (Patent Document 1) Korean Patent No. 10-1704533 Summary of the Invention

[0009] Technical issues

[0010] In response to this, the present inventors confirmed that inhibitors of estrogen-related receptor γ can suppress the resistance of liver cancer to sorafenib and effectively inhibit the proliferation of liver cancer, thereby completing the present invention.

[0011] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or enhancing treatment of sorafenib-resistant liver cancer, comprising an estrogen-related receptor γ inhibitor as an active ingredient.

[0012] Another object of the present invention is to provide a pharmaceutical composition for suppressing sorafenib resistance in liver cancer, comprising an estrogen-related receptor γ inhibitor as an active ingredient.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer, comprising an estrogen-related receptor γ inhibitor and sorafenib as active ingredients.

[0014] Another object of the present invention is to provide a method for screening a substance that enhances the treatment of sorafenib-resistant liver cancer, comprising the steps of treating sorafenib-resistant liver cancer cells with a candidate substance and evaluating the activity or expression of estrogen-related receptor γ in the cells.

[0015] Another object of the present invention is to provide a kit for diagnosing sorafenib-resistant liver cancer comprising a preparation for measuring the level of estrogen-related receptor γ (ERRγ) gene messenger RNA (mRNA) or protein expressed thereby.

[0016] Another object of the present invention is to provide an information providing method for diagnosing sorafenib-resistant liver cancer, comprising:

[0017] Step (a) measuring the expression level of estrogen-related receptor γ gene messenger RNA or protein expressed thereby in a biological sample isolated from a liver cancer patient to be confirmed to be resistant to sorafenib;

[0018] Step (b), measuring the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a patient with common liver cancer other than sorafenib-resistant liver cancer; and

[0019] Step (c), when the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (a) is higher than the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (b), the liver cancer patient in the above step (a) is judged as a sorafenib-resistant liver cancer patient.

[0020] Another object of the present invention is to provide a method for determining information required for a treatment method for a liver cancer patient, comprising:

[0021] Step (a), measuring the expression level of estrogen-related receptor γ gene messenger RNA or protein expressed thereby in a biological sample isolated from a liver cancer patient;

[0022] Step (b), measuring the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a patient with common liver cancer other than sorafenib-resistant liver cancer; and

[0023] Step (c), when the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (a) is higher than the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (b), the liver cancer patient in the above step (a) is judged as a sorafenib-resistant liver cancer patient.

[0024] Another object of the present invention is to provide a method for diagnosing and treating sorafenib-resistant liver cancer, comprising:

[0025] Step (a), measuring the expression level of estrogen-related receptor γ gene messenger RNA or protein expressed thereby in a biological sample isolated from a liver cancer patient;

[0026] Step (b), measuring the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a patient with common liver cancer other than sorafenib-resistant liver cancer; and

[0027] Step (c), when the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (a) is higher than the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (b), the liver cancer patient in the above step (a) is judged to be a sorafenib-resistant liver cancer patient, and other liver cancer therapeutic agents other than sorafenib are applicable.

[0028] Another object of the present invention is to provide a method for preventing or enhancing treatment of sorafenib-resistant liver cancer, comprising the step of administering an estrogen-related receptor γ inhibitor to a patient with sorafenib-resistant liver cancer.

[0029] Another object of the present invention is to provide a method for suppressing sorafenib resistance in liver cancer, comprising the step of administering an estrogen-related receptor γ inhibitor to a patient with sorafenib-resistant liver cancer.

[0030] Another object of the present invention is to provide a method for preventing or treating liver cancer, comprising the step of administering an estrogen-related receptor γ inhibitor and sorafenib to a liver cancer patient.

[0031] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and ordinary technicians in the technical field to which the present invention belongs can clearly understand other problems not mentioned from the following description.

[0032] Solutions to the Problem

[0033] To achieve the purpose of the present invention, the present invention provides a preparation capable of inhibiting the activity of estrogen-related receptor γ (ERRγ) protein or the expression of estrogen-related receptor γ gene for the prevention or treatment enhancement of sorafenib-resistant liver cancer.

[0034] In this regard, the present invention provides a pharmaceutical composition for preventing or enhancing the treatment of sorafenib-resistant liver cancer, comprising the above-mentioned estrogen-related receptor γ inhibitor as an active ingredient.

[0035] Furthermore, the present invention provides a method for preventing or enhancing treatment of sorafenib-resistant liver cancer, comprising the step of administering the above-mentioned estrogen-related receptor γ inhibitor to an individual.

[0036] Furthermore, the present invention provides the use of the above-mentioned estrogen-related receptor γ inhibitor for preventing, improving or enhancing the treatment of sorafenib-resistant liver cancer.

[0037] In one embodiment of the present invention, the agent that inhibits the activity of the estrogen-related receptor γ protein can be an inverse agonist or antagonist of estrogen-related receptor γ, or an antibody or aptamer that can specifically bind to estrogen-related receptor γ, but is not limited thereto.

[0038] In one embodiment of the present invention, the inverse agonist for the estrogen-related receptor γ can be a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, but is not limited to the above-mentioned compound as long as it is an estrogen-related receptor γ inverse agonist that exhibits the same effect as the above-mentioned compound.

[0039] Chemical formula 1

[0040]

[0041] In another example of the present invention, the preparation for inhibiting the expression of the above-mentioned estrogen-related receptor γ gene can be selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides that specifically bind to the messenger RNA of the above-mentioned gene, but is not limited thereto.

[0042] Furthermore, the present invention provides a preparation capable of inhibiting the activity of the estrogen-related receptor γ protein or the expression of the estrogen-related receptor γ gene for suppressing sorafenib resistance in preventing or treating liver cancer.

[0043] In this regard, the present invention provides a pharmaceutical composition for suppressing sorafenib resistance in liver cancer, comprising the above-mentioned estrogen-related receptor γ inhibitor as an active ingredient.

[0044] Furthermore, the present invention provides a method for suppressing sorafenib resistance in liver cancer, comprising the step of administering the above-mentioned estrogen-related receptor γ inhibitor to an individual.

[0045] Furthermore, the present invention provides the use of the estrogen-related receptor γ inhibitor to inhibit sorafenib resistance in liver cancer.

[0046] Furthermore, the present invention provides a composition comprising an agent capable of inhibiting the activity of estrogen-related receptor γ protein or the expression of estrogen-related receptor γ gene and sorafenib for preventing or treating liver cancer.

[0047] In this regard, the present invention provides a pharmaceutical composition for preventing or treating liver cancer, comprising the above-mentioned estrogen-related receptor γ inhibitor and sorafenib as active ingredients.

[0048] Furthermore, the present invention provides a method for preventing or treating liver cancer, comprising the step of administering a composition comprising an estrogen-related receptor γ inhibitor and sorafenib as active ingredients to an individual.

[0049] Furthermore, the present invention provides a composition comprising an estrogen-related receptor γ inhibitor and sorafenib as active ingredients for use in preventing, improving or treating liver cancer.

[0050] In one embodiment of the present invention, the composition can enhance the susceptibility of liver cancer to sorafenib or enhance the anti-cancer effect of sorafenib on liver cancer, but is not limited thereto.

[0051] In another embodiment of the present invention, the above composition can be administered simultaneously, separately or sequentially with sorafenib, but is not limited thereto.

[0052] Furthermore, the present invention provides a method for screening a substance that enhances the treatment of sorafenib-resistant liver cancer, comprising: treating sorafenib-resistant liver cancer cells with a candidate substance; and evaluating the activity or expression of estrogen-related receptor γ in the cells.

[0053] The present invention also provides a kit for diagnosing sorafenib-resistant liver cancer, comprising a preparation for measuring the level of estrogen-related receptor γ (ERRγ) gene messenger RNA or protein expressed thereby.

[0054] In one embodiment of the present invention, the preparation for measuring the messenger RNA level of the above-mentioned gene can include a primer pair, a probe or an antisense nucleotide that specifically binds to the above-mentioned gene, but is not limited thereto.

[0055] In another embodiment of the present invention, the preparation for measuring the level of the above protein can contain an antibody or aptamer specific to the above protein, but is not limited thereto.

[0056] In another example of the present invention, the above-mentioned kit can be a reverse transcription-polymerase chain reaction (RT-PCR) kit, a competitive reverse transcription-polymerase chain reaction kit, a real-time reverse transcription-polymerase chain reaction kit, a deoxyribonucleic acid (DNA) chip kit or a protein chip kit, but is not limited thereto.

[0057] Furthermore, the present invention provides an information providing method for diagnosing sorafenib-resistant liver cancer, comprising:

[0058] Step (a) measuring the expression level of estrogen-related receptor γ gene messenger RNA or protein expressed thereby in a biological sample isolated from a liver cancer patient to be confirmed to be resistant to sorafenib;

[0059] Step (b), measuring the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a patient with common liver cancer other than sorafenib-resistant liver cancer; and

[0060] Step (c), when the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (a) is higher than the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (b), the liver cancer patient in the above step (a) is judged as a sorafenib-resistant liver cancer patient.

[0061] Furthermore, the present invention provides a method for providing information required for determining a treatment method for a liver cancer patient, comprising:

[0062] Step (a), measuring the expression level of estrogen-related receptor γ gene messenger RNA or protein expressed thereby in a biological sample isolated from a liver cancer patient;

[0063] Step (b), measuring the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a patient with common liver cancer other than sorafenib-resistant liver cancer; and

[0064] Step (c), when the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (a) is higher than the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (b), the liver cancer patient in the above step (a) is judged as a sorafenib-resistant liver cancer patient.

[0065] Furthermore, the present invention provides a method for diagnosing and treating sorafenib-resistant liver cancer, which comprises:

[0066] Step (a), measuring the expression level of estrogen-related receptor γ gene messenger RNA or protein expressed thereby in a biological sample isolated from a liver cancer patient;

[0067] Step (b), measuring the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a patient with common liver cancer other than sorafenib-resistant liver cancer; and

[0068] Step (c), when the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (a) is higher than the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (b), the liver cancer patient in the above step (a) is judged to be a sorafenib-resistant liver cancer patient, and other liver cancer therapeutic agents other than sorafenib are applicable.

[0069] In one embodiment of the present invention, the biological sample can be liver tissue, liver cells, whole blood, plasma, serum or blood, but is not limited thereto.

[0070] Effects of the Invention

[0071] According to the present invention, estrogen-related receptor γ (ERRγ, Estrogen-related receptor γ) inhibitors have the effect of increasing the anticancer sensitivity to sorafenib, inhibiting the proliferation of sorafenib-resistant liver cancer cells, and significantly reducing the size of liver cancer. Therefore, it is expected that the present invention can be usefully used as a pharmaceutical composition for treating sorafenib-resistant progressive liver cancer. In addition, by providing information for diagnosing sorafenib-resistant liver cancer of the present invention, the therapeutic responsiveness of liver cancer patients to sorafenib can be predicted, and treatment strategies can be formulated for different patients based on the prediction results to effectively treat liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figures 1a to 1cGraph showing the results of constructing sorafenib-resistant liver cancer cell lines (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).

[0073] Figure 2a and Figure 2b To show that Huh7-R ( Figure 2a ) and SK-Hep-R( Figure 2b ) shows an increase in the orphan nuclear receptor estrogen-related receptor gamma.

[0074] Figure 3a and Figure 3b The compound of Chemical Formula 1 (DN200434), an estrogen-related receptor γ inverse agonist, was treated with Huh7-R ( Figure 3a ) and SK-Hep-R( Figure 3b ), and the results show that reactive oxygen species (ROS) increased in various sorafenib-resistant liver cancer cell lines.

[0075] Figure 4a and Figure 4b To show that Sorafenib alone has no anticancer effect on drug-resistant liver cancer cells Huh7-R ( Figure 4a ) and SK-Hep-R( Figure 4b ) A diagram showing reduction in cell proliferation by simultaneous treatment with the compound of Chemical Formula 1 (DN200434) and sorafenib.

[0076] Figure 5a The photographs show that the compound of Chemical Formula 1 (DN200434) and sorafenib were administered in combination to an animal model (xenograft) derived from the sorafenib-resistant liver cancer cell line Huh7-R. The results show that the tumor size was significantly reduced compared to the control group (sorafenib alone administration group).

[0077] Figure 5b This is a graph showing that the compound of Chemical Formula 1 (DN200434) and sorafenib were administered in combination to an animal model derived from the sorafenib-resistant liver cancer cell line Huh7-R. The results showed that the tumor weight was significantly reduced compared to the control group (sorafenib alone administration group).

[0078] Figure 5cThis graph shows that the compound of Chemical Formula 1 (DN200434) and sorafenib were administered in combination to an animal model derived from the sorafenib-resistant liver cancer cell line Huh7-R. The results showed that the tumor volume was significantly reduced compared to the control group (Huh7-R-Con, sorafenib alone administration group).

[0079] Figure 5d This graph shows the results of an experiment using an animal model derived from the sorafenib-resistant liver cancer cell line Huh7-R, without showing changes in body weight between groups.

[0080] Figure 6a The photograph shows that the compound of Chemical Formula 1 (DN200434) and sorafenib were administered in combination to an animal model (xenograft) derived from the sorafenib-resistant liver cancer cell line SK-Hep-R. The results showed that the tumor size was significantly reduced compared to the control group (sorafenib alone administration group).

[0081] Figure 6b This is a graph showing that the compound of Chemical Formula 1 (DN200434) and sorafenib were administered in combination to an animal model derived from the sorafenib-resistant liver cancer cell line SK-Hep-R. The results showed that the tumor weight was significantly reduced compared to the control group (sorafenib alone administration group).

[0082] Figure 6c This graph shows that the compound of Chemical Formula 1 (DN200434) and sorafenib were administered in combination to an animal model derived from the sorafenib-resistant liver cancer cell line SK-Hep-R. The results showed that the tumor volume was significantly reduced compared to the control group (Huh7-R-Con, sorafenib alone administration group).

[0083] Figure 6d This graph shows the results of an experiment using an animal model derived from the sorafenib-resistant hepatocellular carcinoma cell line SK-Hep-R, without showing changes in body weight between groups. DETAILED DESCRIPTION

[0084] Best Mode for Carrying Out the Invention

[0085] The present invention provides a preparation for preventing or enhancing the treatment of sorafenib-resistant liver cancer, which can inhibit the activity of estrogen-related receptor γ (ERRγ) protein or the expression of estrogen-related receptor γ gene.

[0086] In this regard, the present invention provides a pharmaceutical composition for preventing or enhancing the treatment of sorafenib-resistant liver cancer, comprising as an active ingredient an agent that can inhibit the activity of estrogen-related receptor γ (ERRγ) protein or the expression of estrogen-related receptor γ gene.

[0087] As another embodiment of the present invention, the present invention provides a preparation capable of inhibiting the activity of the estrogen-related receptor γ protein or the expression of the estrogen-related receptor γ gene for suppressing sorafenib resistance in preventing or treating liver cancer.

[0088] In this regard, the present invention provides a pharmaceutical composition for suppressing sorafenib resistance in liver cancer, comprising as an active ingredient an agent capable of inhibiting the activity of estrogen-related receptor γ protein or the expression of estrogen-related receptor γ gene.

[0089] As another embodiment of the present invention, the present invention provides a composition comprising an agent capable of inhibiting the activity of estrogen-related receptor γ protein or the expression of estrogen-related receptor γ gene for preventing or treating liver cancer and sorafenib.

[0090] In this regard, the present invention provides a pharmaceutical composition for preventing or treating liver cancer, comprising as active ingredients an agent capable of inhibiting the activity of estrogen-related receptor γ protein or the expression of estrogen-related receptor γ gene and sorafenib.

[0091] The term "liver cancer" used in this specification refers to a primary malignant tumor that first occurs in the liver. Pathologically (tissue-based), primary liver cancer includes hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, and many other types, among which hepatocellular carcinoma and cholangiocarcinoma account for the majority.

[0092] In the present invention, the liver cancer may be hepatocellular carcinoma (HCC), but is not limited thereto.

[0093] The term "therapy enhancement" used in this specification refers to all behaviors in which, after liver cancer acquires resistance to sorafenib, the symptoms of sorafenib-resistant liver cancer are improved or favorably changed by administering the pharmaceutical composition of the present invention.

[0094] The term "treatment" used in this specification refers to all actions that improve or favorably modify the symptoms caused by liver cancer by administering the pharmaceutical composition of the present invention before or after liver cancer acquires resistance to sorafenib.

[0095] In the present invention, the pharmaceutical composition of the present invention can enhance the drug sensitivity (susceptibility) to sorafenib, or enhance the anti-cancer effect of sorafenib on liver cancer, but is not limited thereto.

[0096] In one embodiment of the present invention, Huh7-SR or SK-Hep-R cell lines were constructed as sorafenib-resistant liver cancer cell lines, and it was confirmed that the orphan nuclear receptor estrogen-related receptor γ (ERRγ) was significantly increased in the liver cancer cell lines that acquired the above-mentioned sorafenib resistance (see Example 1).

[0097] Furthermore, in one embodiment of the present invention, the compound represented by Chemical Formula 1 (DN200434), which is an inverse agonist of estrogen-related receptor γ, was treated with sorafenib-resistant liver cancer cells. As a result, an increase in ROS was confirmed. It was confirmed that the resistant liver cancer cells that were ineffective when treated with sorafenib alone had reduced cell proliferation due to the combined treatment with the compound represented by Chemical Formula 1 and sorafenib. Thus, it was confirmed that the compound represented by Chemical Formula 1 increased sensitivity to sorafenib by inhibiting the activity of estrogen-related receptor γ, thereby exhibiting an effect of overcoming drug resistance (see Example 2).

[0098] Furthermore, in one embodiment of the present invention, after sorafenib and the compound represented by the above-mentioned Chemical Formula 1 were administered in combination to a sorafenib-resistant liver cancer animal model, the size change of the formed tumor was measured. As a result, it was confirmed that the size of the liver cancer was significantly reduced compared with the control group (sorafenib alone treatment group) (see Example 3).

[0099] In the present invention, the agent that inhibits the activity of the estrogen-related receptor γ protein can be an inverse agonist or antagonist of estrogen-related receptor γ, or an antibody or aptamer that can specifically bind to estrogen-related receptor γ, but is not limited thereto.

[0100] The term "inverse agonist" or "antagonist" used in this specification refers to a molecule that can directly or indirectly reduce the biological activity of a receptor. When used together with a receptor ligand, it includes molecules that can reduce the effect of the above ligand, but is not limited to this.

[0101] The term "antibody" as used in this specification refers to a protein molecule that can specifically bind to the antigenic site of a protein or peptide molecule. Such an antibody can be prepared by conventional methods by cloning each gene into an expression vector to obtain the protein encoded by the above-mentioned marker gene.

[0102] As used herein, the term "aptamer" refers to a nucleic acid molecule that has binding activity against a specified target molecule. Aptamers can be ribonucleic acid (RNA), DNA, modified nucleic acids, or mixtures thereof, and can be linear or cyclic. It is generally known that shorter nucleotide sequences constituting aptamers facilitate chemical synthesis and mass production, resulting in greater cost advantages, easier chemical modification, excellent in vivo stability, and lower toxicity.

[0103] The aforementioned inverse agonists and antagonists may be compounds.

[0104] In the present invention, the inverse agonist for the estrogen-related receptor γ may be a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, but is not limited thereto.

[0105] Chemical formula 1

[0106]

[0107] The term "pharmaceutically acceptable" as used in this specification refers to compounds or compositions that are free of excessive toxicity, irritation, allergic reaction or other problems or complications, have a reasonable benefit / risk ratio, are suitable for use in contact with the tissues of a subject (e.g., human), and are within the scope of sound medical judgment.

[0108] The compound of the present invention may be in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed from a pharmaceutically acceptable free acid may be useful.

[0109] Acid addition salts can be derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, nitrous or phosphorous acids, and nontoxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, and aliphatic and aromatic sulfonic acids.

[0110] 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, propionates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexane benzoate, chlorobenzoate, methyl benzoate, 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 mandelate.

[0111] The acid addition salts of the present invention can be prepared by conventional methods, for example, by dissolving the compound of Chemical Formula 1 in an aqueous acid solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone, or acetonitrile. Alternatively, the acid addition salts can be prepared by evaporating the solvent or excess acid from the mixture and then drying or filtering the precipitated salt by suction.

[0112] In addition, a pharmaceutically acceptable metal salt can be prepared using a base. For example, an alkali metal or alkaline earth metal salt is prepared by dissolving the compound of the above chemical formula 1 in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-dissolved compound salt, evaporating the filtrate, and drying it. In this case, as a metal salt, a sodium, potassium or calcium salt is prepared, which may be suitable for pharmaceutical use. The corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (such as silver nitrate).

[0113] The scope of the compounds of the present invention fully encompasses all isomers, hydrates and solvates that can be prepared by conventional methods, in addition to pharmaceutically acceptable salts.

[0114] In the present invention, the agent for inhibiting the expression of the estrogen-related receptor γ gene can be selected from the group consisting of microRNA, small interfering RNA, short hairpin RNA and antisense oligonucleotide that specifically bind to the messenger RNA of the gene, but is not limited thereto.

[0115] As used herein, the terms "microRNA, small interfering RNA, and short hairpin RNA" refer to nucleic acid molecules that primarily bind to messenger RNA transcribed from a target gene to inhibit translation of the messenger RNA, mediating RNA interference or gene silencing. These microRNAs, small interfering RNAs, and short hairpin RNAs can inhibit target gene expression at the translational level and are therefore useful in effective gene knockdown or gene therapy approaches.

[0116] The term "antisense oligonucleotide" used in this specification refers to DNA or RNA or their derivatives containing a nucleic acid sequence complementary to the sequence of a specific messenger RNA, which can inhibit the translation of proteins into messenger RNA by binding to the complementary sequence within the messenger RNA.

[0117] On the other hand, the pharmaceutical compositions of the present invention may contain, in addition to the active ingredients, appropriate carriers, excipients, and / or diluents commonly used in the preparation of pharmaceutical compositions. Furthermore, the pharmaceutical compositions of the present invention may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, topical preparations, suppositories, and sterile injectable solutions according to conventional methods.

[0118] The carriers, excipients and diluents that may be included in the above composition include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. When the above composition is formulated, it can be formulated with commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants.

[0119] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective amount level can be determined based on factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, the time of administration, route of administration and excretion rate, duration of treatment, concomitant medications, and other factors well known in the medical field.

[0120] In the present invention, the pharmaceutical composition of the present invention can be administered simultaneously, separately or sequentially with sorafenib, and can be administered once or multiple times.

[0121] It is important to administer the drug in an amount that minimizes the amount required to achieve the maximum effect without side effects, taking all of the above factors into consideration. This can be determined by one of ordinary skill in the art. Specifically, the effective amount of the pharmaceutical composition of the present invention can vary depending on the patient's age, sex, condition, weight, absorption, inactivity, and excretion rate of the active ingredient in the body, the type of disease, and concomitant medications.

[0122] The pharmaceutical compositions of the present invention can be administered to a subject in a variety of ways. For example, they can be administered orally, intranasally, intrabronchially, intraarterially, intravenously, subcutaneously, intramuscularly, or intraperitoneally. The daily dosage can be administered once or several times daily.

[0123] As another embodiment of the present invention, the present invention provides a method for preventing or enhancing treatment of sorafenib-resistant liver cancer, comprising the step of administering an estrogen-related receptor γ inhibitor to an individual.

[0124] Specifically, provided is a method for preventing or enhancing treatment of sorafenib-resistant liver cancer, comprising the step of administering an estrogen-related receptor γ inhibitor to a patient with sorafenib-resistant liver cancer.

[0125] Furthermore, as another embodiment of the present invention, the present invention provides a method for suppressing sorafenib resistance in liver cancer, comprising the step of administering an estrogen-related receptor γ inhibitor to an individual.

[0126] Specifically, a method for suppressing sorafenib resistance in liver cancer is provided, comprising the step of administering an estrogen-related receptor γ inhibitor to a patient with sorafenib-resistant liver cancer.

[0127] Furthermore, as another embodiment of the present invention, the present invention provides a method for preventing or treating liver cancer, comprising the step of administering a composition comprising an estrogen-related receptor γ inhibitor and sorafenib as active ingredients to an individual.

[0128] Specifically, the present invention provides a method for preventing or treating liver cancer, comprising administering an estrogen-related receptor γ inhibitor and sorafenib to a liver cancer patient.

[0129] In the method of the present invention, the liver cancer may be sorafenib-resistant liver cancer, but is not limited thereto.

[0130] The term "subject" as used herein refers to a subject for whom disease prevention, treatment, enhanced treatment, or suppression of drug resistance is desired. For example, the subject may be a human or a non-human primate mammal, including mice, dogs, cats, horses, sheep, and cattle.

[0131] In another embodiment, the present invention provides a method for screening for substances that enhance the treatment of sorafenib-resistant liver cancer, comprising: step (a) treating sorafenib-resistant liver cancer cells with a candidate substance; and step (b) evaluating the activity or expression of estrogen-related receptor γ in the cells. Using the above method, substances that inhibit the activity or expression of estrogen-related receptor γ can be screened in sorafenib-resistant liver cancer cells and used as enhancers or even adjuvants for the treatment of sorafenib-resistant liver cancer.

[0132] As another embodiment of the present invention, the present invention provides a kit for diagnosing sorafenib-resistant liver cancer.

[0133] Specifically, the kit for diagnosing sorafenib-resistant liver cancer comprises a preparation for measuring the level of messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby.

[0134] In the present invention, the term "diagnosis" refers to confirming the presence or characteristics of a pathological condition. The diagnosis in the present invention confirms the presence or development of sorafenib-resistant liver cancer by measuring the level of estrogen-related receptor γ gene messenger RNA or protein expressed thereby.

[0135] In the present invention, the term "preparation for determining the expression level of estrogen-related receptor γ messenger RNA or its protein" refers to a molecule that can be used to confirm the expression level of the estrogen-related receptor γ gene of the present invention or the protein encoded by these genes, preferably, it can be a primer pair, probe or antisense nucleotide that specifically binds to the estrogen-related receptor γ gene, or an antibody or aptamer specific for the protein encoded by the estrogen-related receptor γ gene.

[0136] In the present invention, the term "primer" refers to a nucleic acid sequence with a short free 3'-terminal hydroxyl group that can form a base pair with a complementary template and serves as a starting point for template replication. In the present invention, PCR amplification using sense and antisense primers for the marker polynucleotide of the present invention can be used to screen patients for sorafenib-resistant liver cancer based on the production of a desired product. PCR conditions and the lengths of the sense and antisense primers can vary based on what is known in the art.

[0137] In the present invention, the term "probe" refers to a nucleic acid fragment equivalent to RNA or DNA, which is as short as a few bases to as long as hundreds of bases and can achieve specific binding with messenger RNA. Due to being labeled, the presence or absence of specific messenger RNA can be confirmed. The probe can be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. In the present invention, hybridization is performed using a probe complementary to the estrogen-related receptor γ polynucleotide of the present invention, and sorafenib-resistant liver cancer patients can be screened by the degree of hybridization. The selection of appropriate probes and hybridization conditions can be modified based on what is known in the art.

[0138] Primer of the present invention or probe can use phosphoramidite solid support method or other widely known method chemosynthesis.This nucleotide sequence can also utilize known multiple means deformation in this field.The non-limitative example of this deformation has methylation, capping, natural nucleotide to the replacement of more than one homologue and the deformation between nucleotide, for example, to the deformation of uncharged linker (such as methyl phosphate, phosphotriester, phosphoramidate, carbamate etc.) or charged linker (such as thiophosphate, phosphorodithioate etc.).

[0139] When considering variations with biologically equivalent activity, the base sequence of the agent used in the present invention for measuring the expression level of the estrogen-related receptor γ gene is interpreted as also including sequences that exhibit substantial identity with the sequence that specifically binds to the estrogen-related receptor γ gene. The term "substantial identity" refers to alignment of a specific sequence with any other sequence to maximize correspondence. When the aligned sequences are analyzed using an algorithm commonly used in the art, it refers to sequences that exhibit at least 60% identity, more specifically 70% identity, further specifically 80% identity, and most specifically 90% identity.

[0140] In the kit of the present invention, the terms "antibody" and "aptamer" are the same as those described in the above-mentioned pharmaceutical composition, and thus their description is omitted.

[0141] The kit of the present invention can be used to diagnose sorafenib-resistant liver cancer or predict the therapeutic response to sorafenib by confirming the expression level of messenger RNA of estrogen-related receptor γ gene or the expression level of protein encoded by these genes.

[0142] The kit of the present invention can be used to predict the therapeutic response to sorafenib, and in this regard, can be used to predict the prognosis of liver cancer patients.

[0143] In the present invention, the term "prognosis prediction" refers to the pre-estimation of medical trends, and for the purpose of the present invention, refers to the pre-estimation of the drug resistance of liver cancer patients to sorafenib.

[0144] According to one example of the present invention, the above-mentioned kit can be a reverse transcription-polymerase chain reaction kit, a competitive reverse transcription-polymerase chain reaction kit, a real-time reverse transcription-polymerase chain reaction kit, a DNA chip kit or a protein chip kit, but is not limited thereto.

[0145] According to one embodiment of the present invention, a kit for determining the expression level of messenger RNA of the estrogen-related receptor γ gene can be a kit comprising the necessary elements for performing a reverse transcription-polymerase chain reaction. In addition to primer pairs specific for the marker gene, the reverse transcription-polymerase chain reaction kit can also include test tubes or other appropriate containers, reaction buffer, deoxyribonucleic acid (dNTPs), Taq polymerase and reverse transcriptase, deoxyribonuclease (DNAse), ribonuclease (RNase) inhibitor, DEPC-water, sterile water, etc.

[0146] The kit of the present invention 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.

[0147] According to another embodiment of the present invention, the kit of the present invention can be a kit for detecting estrogen-related receptor γ, including the necessary factors for performing DNA chip analysis. The DNA chip kit can include a substrate to which a gene or a cDNA equivalent to a fragment thereof is attached via a probe, and the substrate contains a quantitative control gene or a cDNA equivalent to a fragment thereof.

[0148] According to another embodiment of the present invention, a kit for measuring the expression level of a protein encoded by estrogen-related receptor γ may include a substrate, an appropriate buffer solution, a chromogenic enzyme, or a secondary antibody labeled with a fluorescent substance, and a chromogenic substrate, etc., for immunological detection of antibodies. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from a polyethylene resin, a 96-well plate synthesized from a polystyrene resin, or a glass slide, etc.; the chromogenic enzyme may be peroxidase or alkaline phosphatase; the fluorescent substance may be FITC or RITC, etc.; and the chromogenic substrate solution may be ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), TMB (tetramethylbenzidine), etc.

[0149] The kit of the present invention may further include a composition, solution or device having one or more other structural components suitable for the analytical method.

[0150] As another embodiment of the present invention, a method for providing information for diagnosing sorafenib-resistant liver cancer is provided.

[0151] Specifically, the information providing method for diagnosing sorafenib-resistant liver cancer includes: step (a), determining the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a liver cancer patient who is confirmed to be resistant to sorafenib; step (b), determining the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a common liver cancer patient who is not sorafenib-resistant liver cancer; and step (c), when the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (a) is higher than the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby measured in the above step (b), judging the liver cancer patient in the above step (a) as a sorafenib-resistant liver cancer patient.

[0152] In the present invention, the term "biological sample" includes tissues (liver tissue), cells (hepatocytes), whole blood, plasma, serum, blood, saliva, synovial fluid, urine, sputum, lymph fluid, cerebrospinal fluid, autopsy tissue samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, or disrupted eukaryotic cells that show differences in the expression and / or activity levels of estrogen-related receptor γ, a marker for sorafenib-resistant liver cancer. This includes samples derived from primary cancer lesions as well as metastatic lesions. The activity or expression level of estrogen-related receptor γ can be confirmed with or without manipulation of these biological samples.

[0153] In the information-providing method for diagnosing sorafenib-resistant liver cancer of the present invention, the biological sample of step (a) can be obtained using specific methods known to those skilled in the art. For example, the biological sample can be obtained from a vertebrate, particularly a mammal, and preferably from a liver cancer patient whose sorafenib resistance is to be determined. In this case, the liver cancer patient is a human. Tissue biopsy is often used to obtain representative fragments of tumor tissue. Alternatively, tumor cells can be obtained indirectly using tissue or fluid known to contain tumor cells of interest.

[0154] In the present invention, the term "mRNA expression level measurement" refers to the process of confirming the presence and expression level of mRNA of the estrogen-related receptor γ gene in a biological sample, which can be determined by measuring the amount of mRNA. Analysis methods used for this purpose include, but are not limited to, reverse transcription-polymerase chain reaction, competitive RT-PCR, real-time RT-PCR, RNase protection assay, northern blotting, or DNA chip technology.

[0155] In the present invention, the term "protein expression level determination" refers to the process of confirming the presence and expression level of a protein expressed from the estrogen-related receptor γ gene in a biological sample. The amount of the protein can be confirmed using an antibody that specifically binds to the protein expressed from the gene. Analytical methods used for this purpose include, but are not limited to, western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, bidirectional immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, fluorescence-activated cell sorter (FACS) analysis, and protein chip technology.

[0156] In the information providing method for diagnosing sorafenib-resistant liver cancer of the present invention, common liver cancer patients other than sorafenib-resistant liver cancer may be hepatocellular carcinoma (HCC) or hepatic adenocarcinoma patients who do not exhibit sorafenib-resistant liver cancer, but are not limited thereto.

[0157] In the information-providing method for diagnosing sorafenib-resistant liver cancer of the present invention, the expression level of the estrogen-related receptor γ gene can be measured at the messenger RNA (mRNA) level or the protein level. The mRNA or protein can be isolated from a biological sample using known procedures. The analytical methods for measuring mRNA levels and protein levels are described above.

[0158] The information providing method for diagnosing sorafenib-resistant liver cancer of the present invention can be used to predict the therapeutic responsiveness of liver cancer patients to sorafenib, and treatment strategies can be formulated for different patients based on the prediction results to effectively treat liver cancer.

[0159] In this regard, as another embodiment of the present invention, a method for providing information required for determining a treatment method for a liver cancer patient is provided, which includes measuring the expression level of estrogen-related receptor γ gene messenger RNA or protein expressed thereby in a biological sample isolated from a liver cancer patient.

[0160] Specifically, the method for providing information required to determine a treatment method for a liver cancer patient includes: step (a), determining the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a liver cancer patient; step (b), determining the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a common liver cancer patient who is not a sorafenib-resistant liver cancer patient; and step (c), when the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby determined in the above step (a) is higher than the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby determined in the above step (b), judging the liver cancer patient in the above step (a) as a sorafenib-resistant liver cancer patient.

[0161] When sorafenib resistance is confirmed in a liver cancer patient using the method for providing information necessary for determining a treatment method for the liver cancer patient, other known liver cancer therapeutic agents other than sorafenib can be used to induce a liver cancer therapeutic effect.

[0162] Thus, in an additional aspect, the present invention provides methods for diagnosing and treating sorafenib-resistant liver cancer.

[0163] Specifically, the method for diagnosing and treating sorafenib-resistant liver cancer of the present invention includes: step (a), determining the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a liver cancer patient; step (b), determining the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby in a biological sample isolated from a patient with ordinary liver cancer who does not have sorafenib-resistant liver cancer; and step (c), when the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby determined in the above step (a) is higher than the expression level of the messenger RNA of the estrogen-related receptor γ gene or the protein expressed thereby determined in the above step (b), the liver cancer patient in the above step (a) is judged to be a sorafenib-resistant liver cancer patient, and other liver cancer therapeutic agents other than sorafenib are applicable.

[0164] The details of the method for providing information required to determine a treatment method for a liver cancer patient and / or the diagnosis and treatment method for sorafenib-resistant liver cancer of the present invention are the same as those of the above-mentioned method for providing information for diagnosing sorafenib-resistant liver cancer, and thus description thereof is omitted.

[0165] The terms or words used in this specification and the scope of protection of the invention should not be interpreted limited to the usual or dictionary meanings. In order to explain their own invention in the best way, the inventor should interpret them as meanings and concepts that are consistent with the technical ideas of the present invention based on the principle of appropriately defining the concepts of terms.

[0166] In order to facilitate understanding of the present invention, preferred embodiments are provided below. However, the following embodiments are only intended to facilitate understanding of the present invention, and the present invention is not limited to the following embodiments.

[0167] Modes for Carrying Out the Invention

[0168] Experimental example

[0169] Conventional experimental methods

[0170] The present inventors used animal models (xenografts) derived from sorafenib-resistant liver cancer cell lines (Huh7-SR cell line, SK-Hep-R cell line) and liver cancer cell lines to perform experiments. In order to confirm the effect of the synthetic compound DN200434, an estrogen-related receptor γ inverse agonist, on the proliferation and ROS production of liver cancer cells, ROS were measured using cell counts and DCF-DA (2',7'-dichlorofluorescin diacetate). Finally, after sorafenib-resistant liver cancer cell lines were injected into mice to induce the formation of sorafenib-resistant liver cancer, changes in the size of liver cancer were confirmed in the groups injected with estrogen-related receptor γ inverse agonist DN200434 and sorafenib and the groups injected with control drugs.

[0171] Example 1

[0172] Construction of a sorafenib-resistant hepatocellular carcinoma cell line and confirmation of increased expression of estrogen-related receptor γ

[0173] Hepatocellular carcinoma cell lines [Huh7 cells (cell) (Korean Cell Line Bank KCLB No. 60104), SK-Hep cells ( HTB-52 TM )] were continuously exposed to sorafenib (gradually increasing to 10 μM) to construct sorafenib-resistant liver cancer cell lines (Huh7-SR cell line, SK-Hep-R cell line). First, in order to evaluate cancer cell death using FACS, the cancer cell lines were cultured in FITC-conjugated annexin (Annexin) and propidium iodide (PI) for 15 minutes, and then the binding of annexin and PI was measured by flow cytometry. The data were obtained using a BD accuriC6 flow cytometer (BD Biosciences) and analyzed using the Accuri C6 analysis program (BD Biosciences) / FlowJo software (FlowJo, LLC.). Cleaved caspase-3 antibody (Cell Signaling Technology) was used to evaluate cancer cell death, as shown in Figure 2. Figures 1a to 1c As shown, it was confirmed that sorafenib did not induce an increase in apoptosis in the Huh7-SR cell line and the SK-Hep-R cell line, which are sorafenib-resistant liver cancer cell lines.

[0174] Furthermore, Western blotting was performed to confirm the expression pattern of the orphan nuclear receptor estrogen-related receptor γ in Huh7-SR cell line and SK-Hep-R cell line. The results showed that Figure 2a and Figure 2b As shown, it was confirmed that the expression of estrogen-related receptor γ was significantly increased in the above two sorafenib-resistant liver cancer cell lines.

[0175] Example 2

[0176] Elucidation of the effect of orphan nuclear receptor estrogen-related receptor γ on sorafenib-resistant hepatocellular carcinoma

[0177] To investigate the effect of the compound DN200434, represented by Chemical Formula 1, which is an inverse agonist of estrogen-related receptor γ, on the generation of ROS in sorafenib-resistant liver cancer cells, FACS analysis was performed using H2-DCF-DA (2',7'-dichlorohydrofluorescein diacetate; Invitrogen, USA) as a ROS probe. Drug-resistant cells were treated with sorafenib (10 μM) and DN200434 (12 μM) for 24 hours. Then, 10 μM H2-DCF-DA was added to the cells and incubated for 30 minutes. After washing with phosphate-buffered saline (PBS), the cells were analyzed using a BD Accuri TM Data were collected using a C6 flow cytometer (BD Biosciences, USA) and Accuri TM Analysis was performed using the C6 analysis program (BD Biosciences, USA). For cell count evaluation, sorafenib-resistant hepatoma cell lines were treated with sorafenib (10 μM) and DN200434 compound (12 μM) and stained with trypan blue, and then the cell count was measured using a hemocytometer.

[0178] The results, such as Figure 3a and Figure 3b As shown in Figure 2, ROS was increased in both Huh7-SR cell line and SK-Hep-R cell line, which are sorafenib-resistant liver cancer cell lines, due to the DN200434 compound. Figure 4a and Figure 4b As shown, it was confirmed that the proliferation of drug-resistant liver cancer cells that were ineffective when treated with sorafenib alone was significantly reduced by the co-administration of DN200434 represented by the above Chemical Formula 1. These results confirm that DN200434 inhibits the activity of estrogen-related receptor γ, increases sensitivity to sorafenib, and can overcome drug resistance.

[0179] Example 3

[0180] Confirmation of the anticancer effect of sorafenib in an animal model of resistant hepatocellular carcinoma

[0181] Sorafenib-resistant liver cancer cell line Huh7-R was injected into mice to create a sorafenib-resistant liver cancer xenograft model. The estrogen-related receptor γ inverse agonist, DN200434 (Chemical Formula 1), was then administered in combination with sorafenib. Changes in the size of the resulting tumors were measured. A control group treated with sorafenib alone served as a comparison.

[0182] The results, such as Figures 5a to 5c As shown, in the group where the DN200434 compound represented by the above chemical formula 1, which is an estrogen-related receptor γ inverse agonist, was administered in combination with sorafenib to an animal model derived from the sorafenib-resistant liver cancer cell line Huh7-R, it was confirmed that the size, weight, and volume of liver cancer tumors were significantly reduced compared to the control group. Figure 5d As confirmed in the literature, there was no difference in body weight changes between the experimental and control groups.

[0183] An animal model derived from the sorafenib-resistant hepatocellular carcinoma cell line SK-Hep-R was established by the same method as above, and the combined effect of the DN200434 compound represented by the above chemical formula 1, which is an estrogen-related receptor γ inverse agonist, and sorafenib was confirmed.

[0184] Likewise, Figures 6a to 6c As shown in the animal model derived from the sorafenib-resistant liver cancer cell line SK-Hep-R, the group that received the DN200434 compound and sorafenib in combination also confirmed that the size, weight, and volume of liver cancer tumors were significantly reduced compared to the control group. Figure 6d As confirmed in the literature, there was no difference in body weight changes between the experimental and control groups.

[0185] Based on the results of Examples 1 to 3 above, it was confirmed that the expression of estrogen-related receptor γ was significantly increased in sorafenib-resistant liver cancer. In cell experiments, it was confirmed that the estrogen-related receptor γ inverse agonist, DN200434, represented by Chemical Formula 1, increased intracellular ROS to inhibit the proliferation of sorafenib-resistant liver cancer cells and increase sensitivity to sorafenib. In addition, it was confirmed that in animal experiments using sorafenib-resistant liver cancer cell lines, cancer proliferation was significantly reduced compared to the control group. The above results demonstrate that estrogen-related receptor γ plays an important role in the drug resistance of liver cancer. When the estrogen-related receptor γ inverse agonist, DN200434, is used to inhibit the activity of estrogen-related receptor γ, it is effective in treating drug-resistant progressive liver cancer.

[0186] The above description of the present invention is for illustrative purposes only. Those skilled in the art will appreciate that the present invention can be easily modified in other specific forms without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims

1. Use of an inverse agonist of estrogen-related receptor γ (ERRγ) in the manufacture of a medicament for sorafenib-resistant liver cancer, characterized in that: The inverse agonist for ERRγ is a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, Chemical formula 1 2. The use according to claim 1, characterized in that The inverse agonist enhances the drug sensitivity of liver cancer to sorafenib or enhances the anticancer effect of sorafenib on liver cancer.

3. The use according to claim 1, characterized in that The above-mentioned inverse agonist and sorafenib are administered simultaneously, separately or sequentially.

4. Use of an inverse agonist of estrogen-related receptor γ (ERRγ) in the manufacture of a medicament for inhibiting sorafenib resistance in liver cancer, characterized in that: The inverse agonist for ERRγ is a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, Chemical formula 1 5. Use of the composition for the manufacture of a drug for preventing or treating sorafenib-resistant liver cancer, characterized in that: The composition comprises an inverse agonist for estrogen-related receptor γ (ERRγ) and sorafenib, wherein the inverse agonist for ERRγ is a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof: Chemical formula 1

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