Composition for treating third generation EGFR TKI-resistant lung cancer, comprising caesalpinia sappan l extract and osimertinib

Abstract

The present invention relates to a composition for treating third-generation EGFR TKI-resistant lung cancer, the composition comprising a Caesalpinia sappan L extract and Osimertinib. Co-administration of a Caesalpinia sappan L extract and Osimertinib according to the present invention has a synergistic effect on cytotoxicity in third-generation EGFR TKI-resistant lung cancer cell lines and was found to inhibit the proliferation of tumor cells by inhibiting mitochondrial membrane potential. In addition, the co-administration regulates the expression of apoptosis-related proteins and was found to reduce tumor volume in a third-generation EGFR TKI-resistant lung cancer animal model.

Description

A composition for treating lung cancer resistant to third-generation EGFR TKIs comprising a safflower extract and osimertinib

[0001] The present invention relates to a composition for treating lung cancer resistant to third-generation EGFR TKIs, comprising a safflower extract and osimertinib.

[0002]

[0003] Lung cancer is a major cause of cancer-related deaths worldwide, accounting for 30% of all cancer-related deaths. Microscopically, lung cancer is classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) based on the size and morphology of cancer cells. However, the recurrence rate after lung cancer removal surgery is 20-50%, and NSCLC, in particular, has a high recurrence rate after treatment. Furthermore, despite its high incidence, it is known to easily develop resistance to existing chemotherapy drugs, making effective treatment difficult. Despite recent advances in cancer treatment, the 10-year survival rate for NSCLC remains extremely low, at less than 10%.

[0004] Cells in the body are eliminated through the process of apoptosis. However, cancer cells proliferate indefinitely due to continuous division. To eliminate these cancer cells, apoptosis must be induced. PARP (Poly ADP-Ribose Polymerase) plays a role in repairing DNA damage. However, it ceases to function when apoptosis occurs. Therefore, the amount of PARP is shown to decrease in cells undergoing apoptosis. Reactive oxygen species (ROS) are one of the factors that induce apoptosis in cancer cells. Staining with DCF-DA allows us to detect the level of ROS generation, and an increase in ROS indicates an increase in cancer cell death.

[0005] Meanwhile, although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) have been shown to be effective in non-small cell lung cancer (NSCLC) that is positive for the EGFR mutation, it has been reported that resistance to EGFR-TKIs is enhanced due to EGFR mutations even during targeted therapy using EGFR-TKIs, resulting in disease progression. It is known that about 50% of cancers resistant to first-generation EGFR-TKIs, gefitinib and erlotinib, and second-generation EGFR-TKIs, afatinib, have the T790M mutation in the EGFR gene. Osimertinib, a third-generation EGFR-TKI, is known to be an effective drug for NSCLC that has been confirmed to be positive for the EGFR T790M mutation. However, no effective treatment has yet been approved for NSCLC resistant to osimertinib. Furthermore, no effective treatment has been approved for NSCLC resistant to EGFR-TKIs and negative for the EGFR T790M mutation.

[0006] Accordingly, the inventors of the present invention confirmed that the combination of a pine extract and osimertinib exhibited an anticancer effect in third-generation EGFR-TKI-resistant lung cancer, thereby completing the present invention.

[0007]

[0008] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, comprising an extract of Caesapinia sappan L.

[0009] Another object of the present invention is to provide an anticancer adjuvant for the prevention or treatment of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI)-resistant lung cancer, comprising an extract of Caesapinia sappan L.

[0010] Another object of the present invention is to provide a food composition for preventing or improving epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, comprising an extract of Caesapinia sappan L.

[0011] Another object of the present invention is to provide a method for preventing or treating epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, comprising a step of administering to a subject a composition containing an extract of Caesapinia sappanL.

[0012]

[0013] In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, comprising an extract of Caesapinia sappan L.

[0014] In addition, the present invention provides an anticancer adjuvant for the prevention or treatment of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI)-resistant lung cancer, comprising an extract of Caesapinia sappan L.

[0015] In addition, the present invention provides a food composition for preventing or improving epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, which comprises an extract of Caesapinia sappan L.

[0016] In addition, the present invention provides a method for preventing or treating epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, comprising a step of administering to a subject a composition containing an extract of Caesapinia sappanL.

[0017]

[0018] The combined administration of the present invention's safflower extract and osimertinib demonstrated a synergistic cytotoxic effect in a third-generation EGFR TKI-resistant lung cancer cell line, and suppressed tumor cell proliferation by inhibiting mitochondrial membrane potential. Furthermore, it modulated the expression of proteins associated with apoptosis and reduced tumor size in a third-generation EGFR TKI-resistant lung cancer animal model, making it a useful treatment for chemotherapy-resistant lung cancer.

[0019]

[0020] Figure 1 is a diagram confirming cytotoxicity in a third-generation EGFR-TKI resistant cell line following treatment with the extract of the present invention and osimertinib alone.

[0021] Figures 2 and 3 are diagrams confirming the synergistic effect of cell death by combined use of the extract of the present invention and osimertinib at the 24-hour culture time point.

[0022] Figures 4 and 5 are diagrams confirming the synergistic effect of cell death by combined use of the extract of the present invention and osimertinib at 48 hours of culture.

[0023] Figures 6 and 7 are diagrams confirming the synergistic effect of cell death by combined use of the extract of the present invention and osimertinib at the 72-hour culture time point.

[0024] Figure 8 is a diagram showing the inhibition of mitochondrial membrane potential by the combined use of the extract of the present invention and osimertinib, confirmed by JC-1 analysis.

[0025] Figure 9 is a diagram showing the expression of apoptosis-related proteins following the combined use of the extract of the present invention and osimertinib at 24 hours of culture.

[0026] Figure 10 is a diagram showing the expression of apoptosis-related proteins after 48 hours of culture following the combined use of the extract of the present invention and osimertinib.

[0027] Figure 11 is a diagram showing the expression of apoptosis-related proteins at 72 hours of culture following the combined use of the extract of the present invention and osimertinib.

[0028] Figure 12 is a diagram quantifying tumor size and body weight in a third-generation EGFR-TKI animal model following combined administration of the present invention's sapwood extract and osimertinib.

[0029] Figure 13 is a diagram quantifying tumor size and body weight in a third-generation EGFR-TKI animal model according to the combination of protosapanin B and osimertinib.

[0030]

[0031] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0032] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0033] The present invention provides a pharmaceutical composition for preventing or treating epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, comprising an extract of Caesapinia sappan L. as an active ingredient.

[0034] The term “prevention” as used in the present invention means any act of suppressing symptoms or delaying progression of a specific disease by administering the composition of the present invention.

[0035] The term "treatment" as used in the present invention means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

[0036] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to enhance its effectiveness.

[0037] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0038] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0039] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0040] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0041] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0042] According to one embodiment of the present invention, the sapwood extract may be extracted with a solvent selected from the group consisting of water, lower alcohols of C1 to C4, lower alcohol aqueous solutions, hexane, acetone, chloroform and ethyl acetate, preferably 100% ethanol but not limited thereto.

[0043] According to one embodiment of the present invention, the safflower extract may include protosappanin B.

[0044] According to one embodiment of the present invention, the composition may further comprise osimertinib, and osimertinib may be represented by the following chemical formula 1.

[0045]

[0046] According to one embodiment of the present invention, the osimertinib may be an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI).

[0047] "Osimertinib" of the present invention is a drug used to treat non-small cell lung cancer with a specific mutation, is a third-generation EGFR-TKI, and is a compound with CAS number 1421373-65-0.

[0048] The "epidermal growth factor receptor (EGFR)" of the present invention is the first growth factor receptor discovered among receptor tyrosine kinases, and is known to be overexpressed in more than 70% of non-small cell lung cancers, and the overexpression of EGFR is associated with a poor prognosis of non-small cell lung cancers. When EGFR binds to a ligand, autophosphorylation of the activation loop of the intracellular tyrosine kinase domain is induced after receptor dimerization, and the core signaling system of oncogenes such as cell proliferation, cell survival, angiogenesis, and metastasis is activated through pathways such as mitogen activated protein kinase (MAPK) and protein kinase B (Akt).

[0049] The "epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI)" of the present invention refers to a small molecule that competitively interferes with binding to ATP during the phosphorylation process of the EGFR tyrosine kinase domain within a cell.

[0050] According to one embodiment of the present invention, the osimertinib may be mixed with the safflower extract at a weight ratio of 1:1 to 3.

[0051] According to one embodiment of the present invention, the composition may inhibit tumor cell proliferation, and the inhibition of tumor cell proliferation may be by reducing the mitochondrial membrane potential (MMP) of the tumor cell.

[0052] According to one embodiment of the present invention, the inhibition of tumor cell proliferation may be by regulating the expression or phosphorylation of a cell death-associated protein, and the cell death-associated protein may be a protein selected from the group consisting of cleaved-Poly ADP-Ribose Polymerase (c-PARP), pro-caspase9, Cyclin D1, extracellular signal-regulated kinases 1 / 2 (ERK 1 / 2), phosphorylated EKR 1 / 2 (p-ERK 1 / 2), protein kinase B (Akt), phosphorylated Akt (pAkt), and phosphorylated S6 kinase (p-S6).

[0053] According to one embodiment of the present invention, regulating the expression or phosphorylation of the cell death-associated protein may be increasing the expression of c-PRAP, and inhibiting the expression or phosphorylation of a protein selected from the group consisting of pro-caspase9, Cyclin D1, ERK 1 / 2, p-ERK 1 / 2, Akt, pAkt, and p-S6.

[0054] According to one embodiment of the present invention, the composition may reduce tumor volume.

[0055] According to one embodiment of the present invention, the lung cancer may be non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC), and the lung cancer may be EGFR-TKI resistant lung cancer.

[0056] The above non-small cell lung cancer, also called non-small cell lung cancer, is a type of epithelial cancer (carcinoma) and refers to all epithelial lung cancers other than small cell lung cancer, and accounts for approximately 85% to 90% of all lung cancers. Non-small cell lung cancer is relatively less sensitive to chemotherapy compared to small cell lung cancer, and the cancer is staged based on the TNM classification: the size of the tumor, the extent of cancer spread to regional lymph nodes, and the presence or absence of cancer metastasis. In the treatment of non-small cell lung cancer, surgery is generally performed together with adjuvant chemotherapy involving cisplatin, a platinum-containing drug, because early, non-metastatic non-small cell lung cancer has very low sensitivity to chemotherapy and radiation. On the other hand, for metastatic non-small cell lung cancer (NSCLC) that has progressed beyond the early stages, various chemotherapy regimens are used. Symptoms of NSCLC include persistent cough, chest pain, weight loss, nail damage, joint pain, and shortness of breath. However, because NSCLC typically progresses slowly, symptoms are rarely apparent in the early stages.

[0057] The "EGFR-TKI resistance" of the present invention is a resistance mechanism that occurs after treatment with first-generation and second-generation EGFR TKIs, and the expression of T790M, a novel mutation in exon 20 of EGFR, amplification of the MET oncogene, activation of AXL, amplification of HER2, activation of the PIK3CA signaling pathway, and histological changes to small cell carcinoma are known to be the main causes of EGFR-TKI resistance. In particular, the T790M mutation is a new EGFR mutation that occurs when threonine is substituted with methionine at position 790 of exon 20, and has been reported to account for approximately 50% of the causes of resistance to first-generation and second-generation EGFR-TKI treatments. Among the various resistance mechanisms, the development of drugs targeting the T790M mutation, which accounts for the largest proportion, is continuing, and osimertinib is currently being used as a third-generation EGFR-TKI. However, if resistance develops despite treatment with osimertinib, and if non-small cell lung cancer worsens even after treatment with the third-generation EGFR-TKIs developed to date, resistance that makes additional EGFR-TKI treatment impossible will appear.

[0058] In addition, the present invention provides an anticancer adjuvant for the prevention or treatment of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI)-resistant lung cancer, which comprises an extract of Caesapinia sappan L. as an active ingredient.

[0059] The "anticancer adjuvant" of the present invention is a formulation capable of improving, enhancing, or augmenting the anticancer effect of an anticancer agent. Although it does not exhibit anticancer activity on its own, it may be a formulation capable of improving, enhancing, or augmenting the anticancer effect of the anticancer agent when used in combination with the anticancer agent. In addition, a formulation that exhibits concentration-dependent anticancer activity may be a formulation capable of improving, enhancing, or augmenting the anticancer effect of the anticancer agent when used in combination with the anticancer agent at a level that does not exhibit anticancer activity on its own.

[0060] The above anticancer adjuvant may be administered via any conventional route as long as it can reach the target tissue. The anticancer adjuvant of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonary, or rectally, depending on the intended use, but is not limited thereto. Furthermore, the anticancer adjuvant may be administered via any device capable of transporting the active substance to target cells.

[0061] According to one embodiment of the present invention, the adjuvant may increase the efficacy of osimertinib in the treatment of EGFR-TKI-resistant lung cancer.

[0062] In addition, the present invention provides a food composition for preventing or improving epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, which contains an extract of Caesapinia sappan L. as an active ingredient.

[0063] The term "improvement" as used herein means any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0064] The food composition of the present invention may contain, in addition to containing the effective ingredient of the present invention, various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions.

[0065] Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the above-mentioned flavoring agent, natural flavoring agent (thaumatin), stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.) can be advantageously used. The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0066] In addition, the food composition may contain, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0067] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating lung cancer. The term "health functional food composition" in the present invention refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention may include conventional food additives, and whether it is suitable as a food additive is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Examples thereof include natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular weight pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, a health functional food in tablet form can be prepared by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, etc. to compress and molding, or directly compressing and molding the mixture. In addition, the health functional food in tablet form can contain a maturing agent, etc., if necessary. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The above soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as needed.The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can be coated with white sugar or other coating agents, or the surface can be coated with a substance such as starch or talc. The granular health functional food can be manufactured into a granular form using a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can contain a flavoring agent, a flavoring agent, etc.

[0068] In addition, the present invention provides a method for preventing or treating epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, comprising a step of administering to a subject a composition containing an extract of Caesapinia sappanL.

[0069]

[0070] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the detailed examples described below. The present invention will now be described in detail through examples. However, these examples are intended to specifically illustrate the present invention and are not intended to limit the scope of the present invention.

[0071]

[0072] <Manufacturing Example 1> Preparation of safflower extract and acquisition of osimertinib

[0073] The present invention prepared a Caesapina extract. Specifically, commercially available Caesapina (Caesapinia sappan L.) was soaked in 100% ethanol and extracted for 72 hours. The extract was then concentrated under reduced pressure to evaporate the ethanol, yielding only the Caesapina extract. The obtained Caesapina extract was freeze-dried at -110°C using a freeze dryer and then stored at -80°C.

[0074] In addition, the osimertinib of the present invention has a molecular formula C 28 H 33 It is a compound of N7O2 and molecular weight 499.62, purchased from Aladdin Scientific (USA), and the structure of osimertinib is shown in Chemical Formula 1 below.

[0075] [Chemical Formula 1]

[0076]

[0077]

[0078] <Example 1> Confirmation of cytotoxicity of safflower extract or osimertinib

[0079] In order to confirm whether the present invention's extract of the bark of the tree and osimertinib have an anticancer effect on third-generation EGFR TKI-resistant lung cancer, the cell viability according to the treatment with the bark of the bark of the tree or osimertinib was confirmed. Specifically, 50 mg of the bark of the tree extract was dissolved in 1 ml of DMSO to prepare a concentration of 50 mg / ml. The resistant cell line was a third-generation EGFR TKI-resistant lung cancer cell line, YU-1096 cell line (mutation: EGFR L858R, SCIENTIFIC REPORTS (2019) 9:19909), among EGFR mutant non-small cell lung cancer cell lines derived from patients with advanced lung adenocarcinoma. The cell viability was analyzed using the EZ-Cytox cell viability assay kit (DoGenBio; Cat no. EZ-3000, Lot no. DLS406). Specifically, the YU-1096 cell line was seeded at 1.5 10 in a 96-well plate. 4The cells were inoculated at a concentration of 0.25, 0.5, 1, 2, and 5 μg / ml and cultured for 24 hours. The cultured cell lines were then treated with the extract of Safflower at concentrations of 0.625, 1.25, 2.5, 5, and 10 μg / ml and cultured for an additional 24 hours. After the culture was completed, 10 μl of EZ-Cytox was administered and reacted for 1 hour, and the absorbance was measured at 450 nm using a microplate reader. In addition, osimertinib was treated at concentrations of 0.25, 0.5, 1, 2, and 4 μM in the same manner as above, and the cell viability was confirmed.

[0080] As a result, as shown in Fig. 1, the extract of the present invention and osimertinib showed cytotoxicity in a concentration-dependent manner in the YU-1096 cell line, a third-generation EGFR TKI-resistant lung cancer cell line, confirming that they have an anticancer effect. It was confirmed that the IC90 of the extract of the present invention was 1 μg / ml at a concentration, and the IC90 of osimertinib was 2 μM.

[0081]

[0082] <Example 2> Confirmation of the combined effect of safflower extract and osimertinib

[0083] In order to confirm whether the combined use of the present invention's Soffit extract and osimertinib has an anticancer effect against third-generation EGFR TKI-resistant lung cancer, the combined effect was confirmed at 24, 48, and 72 hours after combination treatment. Specifically, the YU-1096 cell line cultured in the same manner as Example 1 was co-administered with the Soffit extract at a concentration of 1 μg / ml and osimertinib at a concentration of 2 μM, and the cell viability was confirmed at 24, 48, and 72 hours. In addition, the synergistic effect according to the combination was confirmed by zip code using SynergeFinder+ (https: / synergyfinder.org / ).

[0084] As a result, as shown in FIGS. 2 to 7, in the YU-1096 cell line, when the IC90 concentration of the pine extract and the IC90 concentration of osimertinib were administered together, it was confirmed that there was a synergistic effect on the cell death effect compared to single administration, and it was specifically confirmed that there was a synergistic effect with a zip code of 10 or more points every 24, 48, and 72 hours.

[0085]

[0086] <Example 3> Confirmation of membrane potential according to combined use of safflower extract and osimertinib

[0087] In order to confirm whether the combined use of the present invention's extract of the pine tree and osimertinib inhibits cancer cell proliferation and growth, it was confirmed whether mitochondrial membrane potential (MMP) was inhibited. Specifically, YU-1096 cell line was seeded in a 96-well plate at a density of 1.5 X 10 4 Cells were seeded at a concentration of 1 μg / ml and cultured for 24 h. Afterwards, 1 μg / ml of safflower extract and 2 μM osimertinib were treated alone or in combination in white media and cultured for 24, 48, and 72 h. Afterwards, 100 μl of JC-1 dye was diluted to 20 mM and treated to each well, and cultured for 15 min. Afterwards, the media was removed, washed with PBS, and 100 μl of white media was added to each well. The fluorescence expression of the monomer was measured at 480 nm / 530 nm, and the fluorescence expression of the polymer (oligomer) was measured at 535 nm / 590 nm.

[0088] As a result, as shown in Fig. 8, compared to treatment with the extract of the pine tree and osimertinib alone, it was confirmed that co-administration inhibited MMP and reduced the ratio of JC-1, and thus it was confirmed that the combined use of the extract of the pine tree of the present invention and osimertinib can inhibit the growth and metastasis of cancer cells by inhibiting the mitochondrial membrane potential.

[0089]

[0090] <Example 4> Western blot analysis

[0091] We examined whether the combined use of the present invention's safflower extract and osimertinib modulates the expression of proteins associated with apoptosis. Specifically, proteins were isolated from cells using a lysis buffer (pH 7.4, 1% NP-40, 1 mM Na3VO4, 1 M EDTa, 1 mM NaF, 50 mM Tris-HCl, 0.25% sodium deoxycholic acid, 150 mM NaCl) containing a protease inhibitor cocktail. Protein quantification was performed using the Bio-Rad DC protein assay kit II, with β-actin adjusted to a constant level. The isolated proteins were then electrophoresed on a 12% SDS-PAGE gel and subjected to Western blotting, followed by blocking with 5% skim milk. The membranes onto which the proteins were transferred were then incubated with primary antibodies for 24 hours and then with mouse and rabbit secondary antibodies for 1 hour. Afterwards, protein expression was confirmed using a chemiluminescence (ECL) system, and the expression of apoptosis-related proteins such as cleaved-Poly ADP-Ribose Polymerase (c-PARP), pro-caspase9, Cyclin D1, extracellular signal-regulated kinases 1 / 2 (ERK 1 / 2), phosphorylated EKR 1 / 2 (p-ERK 1 / 2), protein kinase B (Akt), phosphorylated Akt (pAkt), and phosphorylated S6 kinase (p-S6) was confirmed.

[0092] As a result, as shown in Figures 9 to 11, when the extract of the pine tree and osimertinib were co-administered at a concentration having a synergistic effect, c-PARP increased compared to the group treated with each alone, and the expression of pro-caspase 9, Cyclin D1, ERK 1 / 2, p-ERK 1 / 2, Akt, p-AKt, and p-S6 was suppressed, confirming that the combined use of the extract of the pine tree and osimertinib regulates the expression of proteins related to apoptosis.

[0093]

[0094] <Example 5> Confirmation of tumor growth inhibition in a third-generation EGFR TKI-resistant lung cancer animal model

[0095] The combination of the present invention's extract of the bark of the tree and osimertinib was examined for its anticancer effects in an animal model transplanted with tumor tissue isolated from a third-generation EGFR TKI-resistant lung cancer patient. Specifically, the animal model was prepared by transplanting tumor tissue isolated from a third-generation EGFR TKI-resistant lung cancer patient into mice. The animal model was then administered the bark of the tree at a concentration of 50 or 100 mg / kg in combination with osimertinib (5 mg / kg). Tumor size and body weight were measured daily thereafter.

[0096] In addition, the anticancer effect was further confirmed by co-administering 20 mg / kg of protosappanin B (ChemFaces, CN), known as the main active ingredient in the extract of the sappan tree, with osimertinib. Protosappanin B was administered orally or intraperitoneally.

[0097] As a result, as shown in Fig. 12, there was no significant effect on the body weight of each group in mice transplanted with tumors derived from third-generation EGFR TKI-resistant patients, but when the extract of the safflower and osimertinib were administered together, the tumor volume was significantly reduced compared to each treatment alone, and in particular, it was confirmed that the tumor volume was reduced in a concentration-dependent manner of the extract of the safflower.

[0098] In addition, it was confirmed that when protosaphanin B, a major active ingredient in the root of the plant, was co-administered with osimertinib, the tumor volume was significantly reduced compared to each treatment alone (Fig. 13).

[0099] Therefore, the combined administration of the present invention's safflower extract and osimertinib was confirmed to have a synergistic effect on cytotoxicity in a third-generation EGFR TKI-resistant lung cancer cell line, suppress mitochondrial membrane potential, and inhibit tumor cell proliferation. In addition, it was confirmed to regulate the expression of proteins related to cell death and reduce tumor size in a third-generation EGFR TKI-resistant lung cancer animal model.

Claims

1. A pharmaceutical composition for preventing or treating epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer containing an extract of Caesapinia sappan L.

2. A composition according to claim 1, wherein the sapwood extract is extracted with a solvent selected from the group consisting of water, lower alcohols of C1 to C4, lower alcohol aqueous solutions, hexane, acetone, chloroform, and ethyl acetate.

3. A composition according to claim 1, wherein the safflower extract contains protosappanin B.

4. A composition according to claim 1, wherein the composition further comprises osimertinib.

5. A composition according to claim 4, wherein the osimertinib is represented by the following chemical formula 1. [Chemical Formula 1] 6. A composition according to claim 4, wherein the osimertinib is an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI).

7. A composition in claim 4, wherein the osimertinib is mixed with the safflower extract in a weight ratio of 1:1 to 3.

8. A composition according to claim 1, wherein the composition inhibits tumor cell proliferation.

9. A composition according to claim 8, wherein the inhibition of tumor cell proliferation is achieved by reducing the mitochondrial membrane potential (MMP) of the tumor cells.

10. A composition according to claim 8, wherein the inhibition of tumor cell proliferation is achieved by regulating the expression or phosphorylation of a cell death-associated protein.

11. A composition according to claim 10, wherein the apoptosis-associated protein is a protein selected from the group consisting of cleaved-Poly ADP-Ribose Polymerase (c-PARP), pro-caspase9, Cyclin D1, extracellular signal-regulated kinases 1 / 2 (ERK 1 / 2), phosphorylated EKR 1 / 2 (p-ERK 1 / 2), protein kinase B (Akt), phosphorylated Akt (pAkt), and phosphorylated S6 kinase (p-S6).

12. A composition according to claim 10, wherein regulating the expression or phosphorylation of the cell death-associated protein increases the expression of c-PRAP.

13. In the 10th paragraph, a composition for regulating the expression or phosphorylation of the cell death-related protein is a composition for inhibiting the expression or phosphorylation of a protein selected from the group consisting of pro-caspase9, Cyclin D1, ERK 1 / 2, p-ERK 1 / 2, Akt, pAkt, and p-S6.

14. A composition according to claim 1, wherein the composition reduces tumor volume.

15. A composition according to claim 1, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

16. An anticancer adjuvant for the prevention or treatment of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI)-resistant lung cancer containing Caesapinia sappan L. extract.

17. In claim 16, the anticancer adjuvant increases the efficacy of osimertinib in the treatment of EGFR-TKI-resistant lung cancer.

18. A food composition for preventing or improving epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer containing an extract of Caesapinia sappan L.

19. A method for preventing or treating epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant lung cancer, comprising administering to a subject a composition containing an extract of Caesapinia sappan L.

20. A method according to claim 19, wherein the composition is administered in combination with osimertinib.

Images