A composition for the prevention, amelioration or treatment of cancer comprising narciclasine or a pharmaceutically or foodstably acceptable salt thereof

Abstract

This invention relates to a composition for the prevention, improvement, or treatment of epidermal growth factor receptor (EGFR) mutant cancer comprising narcissine or a pharmaceutically or food-acceptable salt thereof; or a composition for the prevention, improvement, or treatment of tyrosine kinase inhibitor (TKI) resistant cancer. The composition can promote the degradation of EGFR via proteasome or lysosomal degradation pathways in tyrosine kinase inhibitor (TKI) resistant cancer and EGFR mutant cancer, thereby inhibiting or reducing the activity or expression of EGFR, thus significantly inhibiting or suppressing cancer cell growth. Therefore, it can be effectively applied to the prevention and treatment of cancer.

Description

Technical Field

[0001] This invention relates to a composition for the prevention, improvement, or treatment of epidermal growth factor receptor (EGFR) mutant cancer comprising narcissin or a pharmaceutically or food-acceptable salt thereof; or a composition for the prevention, improvement, or treatment of tyrosine kinase inhibitor (TKI) resistant cancer. Background Technology

[0002] The epidermal growth factor receptor (EGFR), also known as the HER or ErbB family of tyrosine kinase receptors, is a key gene regulating cell growth, cell survival, and migration. Overexpression or mutation of EGFR is known to enhance tyrosine kinase activity, thus being associated with the progression of various solid tumors such as breast cancer and lung cancer. In particular, EGFR mutations are found in non-squamous cell carcinomas of non-small cell lung cancer (NCSCLC), especially adenocarcinomas. The detection rate of EGFR-mutated non-squamous cell carcinomas is approximately 50% in Asian populations and approximately 20% in European and American populations. Therefore, EGFR mutations are most common in young children, women, Asian populations, and non-smokers with adenocarcinoma.

[0003] Epidermal growth factor receptor (EGFR) mutations occur in the tyrosine kinase domain and continuously activate the cell signaling system by increasing the activity of EGFR kinase, thereby enabling cells to continue differentiating and proliferating. The most common EGFR mutations include exon 19 deletion and the L858R mutation, which is caused by a point mutation. These two types of mutations account for approximately 90% of all EGFR mutations and are also known as sensitive EGFR mutations because they respond well to treatment with first- and second-generation EGFR tyrosine kinase inhibitors (TKIs) (Erminia Massarelli et al., Lung Cancer (review) 2013 Jun;80(3):235-41). However, lung cancer cells with mutations at codon 790, namely the T790M mutation, are resistant to existing tyrosine kinase inhibitors (TKIs) (gefitinib), making them difficult to treat with existing anticancer agents.

[0004] Therefore, this invention discovered that a single compound derived from the bulb extract of Narcissus simonii, namely Narciclasine, has a strong anti-cancer effect against epidermal growth factor receptor (EGFR) mutant cancers, thus completing this invention. Summary of the Invention

[0005] The purpose of this invention is to solve the problems described above and other related problems.

[0006] The purpose of this invention is to provide a pharmaceutical composition for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancers comprising narcissine or a pharmaceutically acceptable salt thereof.

[0007] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of tyrosine kinase inhibitor (TKI) resistant cancers comprising narcissin or a pharmaceutically acceptable salt thereof.

[0008] Another object of the present invention is to provide a food composition for the prevention or improvement of epidermal growth factor receptor (EGFR) mutant cancers comprising narcissin or a food-grade acceptable salt thereof.

[0009] Another object of the present invention is to provide a food composition for the prevention or improvement of tyrosine kinase inhibitor (TKI) resistant cancers containing narcissin or a food-grade acceptable salt thereof.

[0010] Another object of the present invention is to provide an anticancer adjuvant comprising narcissin or a pharmaceutically acceptable salt thereof.

[0011] Another object of the present invention is to provide the use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancers.

[0012] Another object of the present invention is to provide the use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for the prevention or treatment of tyrosine kinase inhibitor-resistant cancers.

[0013] Another object of the present invention is to provide the use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for manufacturing a medicament for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancers.

[0014] Another object of the present invention is to provide the use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of tyrosine kinase inhibitor-resistant cancers.

[0015] Another object of the present invention is to provide a method for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancer, including the step of administering a composition comprising narcissin or a pharmaceutically acceptable salt thereof to a subject.

[0016] Another object of the present invention is to provide a method for the prevention or treatment of tyrosine kinase inhibitor-resistant cancer, comprising the step of administering to a subject a composition comprising narcissin or a pharmaceutically acceptable salt thereof.

[0017] The technical problems to be solved by the technical concept of the present invention disclosed in this specification are not limited to solving the problems mentioned above. Those skilled in the art will be able to further understand other problems not mentioned through the following description.

[0018] The present invention will now be described in detail. Furthermore, the various descriptions and embodiments disclosed in this application are also applicable to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this application are included within the scope of this application. Moreover, it should not be construed that the scope of this application is limited to the specific descriptions set forth below.

[0019] The terminology used in this invention has been selected as widely used general terms as possible with regard to its function in this invention, but this may change depending on the intention of those skilled in the art or the emergence of new technologies.

[0020] Furthermore, in specific cases, arbitrarily chosen terms may be used, and as described above, their meanings will be detailed in the description section of the corresponding embodiments. Therefore, the terms used in this invention should not be defined based on their mere names, but rather on their meanings and the overall content of the invention.

[0021] In this invention, when it is described as "including" a certain constituent element or a certain step, unless otherwise expressly stated to the contrary, it does not mean that other constituent elements or other steps are excluded, but rather that other constituent elements or other steps may also be included.

[0022] As one embodiment of achieving the stated purpose, the present invention provides a pharmaceutical composition for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancer comprising narcissine or a pharmaceutically acceptable salt thereof.

[0023] In this invention, the "narcissic cyclin" can be a compound represented by the following chemical formula 1, and can be isolated from the bulb extract of Narcissus simonii. Furthermore, the narcissic cyclin of this invention can be chemically synthesized using methods known in the art, or using commercially available substances.

[0024]

[0025]

[0026] In this invention, the pharmaceutically acceptable salt of the narcissin can be an acid addition salt formed from a pharmaceutically acceptable free acid or a metal salt formed from a base. In one example, inorganic and organic acids can be used as the free acid; inorganic acids can include hydrochloric acid, sulfuric acid, hydrobromic acid, sulfurous acid, or phosphoric acid, while organic acids can include citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, and methanesulfonic acid. Furthermore, alkali metal salts or alkaline earth metal salts, such as sodium, potassium, or calcium salts, can be used as the metal salt. However, this is not a limitation.

[0027] In this invention, "cancer" can refer to epidermal growth factor receptor (EGFR) mutant solid tumors, and the solid tumors can be selected from the group consisting of lung cancer, breast cancer, colorectal cancer, colon cancer, pancreatic cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, prostate cancer, skin cancer, liver cancer, thyroid cancer, gallbladder cancer, bile duct cancer, esophageal cancer, bladder cancer, and oral cancer. Preferably, the solid tumor can be lung cancer, but it is not limited to this.

[0028] Furthermore, the lung cancer may be selected from the group consisting of non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell lung cancer, but is not limited thereto.

[0029] In this invention, the “epidermal growth factor receptor (EGFR) mutation” may include one or more mutations selected from the group consisting of the deletion of exon 19 of epidermal growth factor receptor (EGFR), L858R mutation, T790M mutation and C797S mutation. Preferably, it may include one or more mutations selected from the group consisting of L858R mutation and T790M mutation.

[0030] The L858R mutation refers to a mutation in which amino acid position 858 of the epidermal growth factor receptor (EGFR) protein is replaced by R (arginine) instead of L (leucine). The T790M mutation refers to a mutation in which amino acid position 790 of the epidermal growth factor receptor (EGFR) protein is replaced by M (methionine) instead of T (threonine). The sequence of the epidermal growth factor receptor (EGFR) protein can be found in UniProt (P00533) and other publications.

[0031] In this invention, the composition can inhibit or reduce the activity or expression of epidermal growth factor receptor (EGFR), and can promote the degradation of epidermal growth factor receptor (EGFR) through the proteasome degradation pathway or the lysosome degradation pathway.

[0032] This study has confirmed that compositions containing narcissin can target cancers with L858R mutations, T790M mutations, or L858R / T790M double mutations of the epidermal growth factor receptor (EGFR) by promoting the degradation of EGFR through proteasome or lysosomal degradation pathways, thereby reducing the activity or expression of EGFR, and thus have significant effects on the prevention or treatment of cancer, especially lung cancer.

[0033] In this invention, "prevention" refers to all actions that suppress or delay cancer symptoms through administration of the compositions of this invention.

[0034] In this invention, "treatment" refers to all actions that result in improvement or beneficial changes in cancer symptoms through administration of the compositions of this invention.

[0035] As another embodiment for achieving the stated purpose, the present invention provides a pharmaceutical composition for the prevention or treatment of tyrosine kinase inhibitor (TKI) resistant cancers comprising narcissin or a pharmaceutically acceptable salt thereof.

[0036] The terms "narcissin", "pharmacologically acceptable salt", "cancer", "prevention", "treatment" and "composition" are as previously described.

[0037] In this invention, the term "tyrosine kinase inhibitor" refers to a substance that inhibits tyrosine kinase, an enzyme responsible for the activation of various proteins through a signal transduction cascade. Proteins regulated by tyrosine kinase are activated by the addition of a phosphate group, and the tyrosine kinase inhibitor works by inhibiting this phosphorylation process.

[0038] In this invention, the tyrosine kinase inhibitor, as an anticancer agent, may be gefitinib, erlotinib, or afatinib, but is not limited to these.

[0039] In this invention, "drug resistance" or "resistance" refers to the absence of a significant cellular or biological response to a specific drug. Specifically, it means that the rate of apoptosis or cell apoptosis in cancer cells does not change or decreases in response to treatment with the drug.

[0040] It has been confirmed that the narcissin-containing compositions of the present invention significantly inhibit or suppress the growth of cancer cells in epidermal growth factor receptor (EGFR) mutant cancers and tyrosine kinase inhibitor (TKI) resistant cancers (especially lung cancer), and have a stronger anticancer effect even at lower concentrations compared to the therapeutic concentrations for EFGF wild-type cancers.

[0041] The pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable carrier. In this invention, the term "pharmaceutically acceptable" means exhibiting properties that are non-toxic to cells or the human body exposed to the composition. As a pharmaceutically acceptable carrier, for example, an oral or non-oral delivery carrier may be additionally included. As an oral delivery carrier, lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., may be included.

[0042] In addition, as a non-oral drug delivery carrier, it may contain water, suitable oils, physiological saline, aqueous glucose, and glycols. Furthermore, it may contain additional stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propylparaben, and chlorobutanol.

[0043] The pharmaceutical compositions of the present invention can be administered to mammals, including humans, in any manner. For example, they can be administered orally or non-orally, and as non-oral administration methods, they can be intravenously, intramuscularly, intra-arterially, centrally, intramedullaryly, intradurally, intracardiacly, percutaneously, subcutaneously, intraperitoneally, intranasally, intestinally, locally, sublingually, or rectally, but are not limited thereto.

[0044] The pharmaceutical compositions of the present invention can be formulated into oral or non-oral formulations according to the routes of administration described above. During formulation, one or more buffers (e.g., physiological saline or phosphate-buffered saline (PBS), antioxidants, antibacterial agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrants or surfactants, diluents or excipients may be used for preparation.

[0045] Solid dosage forms for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, suspensions, or capsules. These solid dosage forms can be formulated by mixing the pharmaceutical composition of the present invention with at least one excipient (e.g., starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, glucose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose or gelatin, etc.). For example, the active ingredient can be pulverized after being combined with the solid excipient, and then processed into a granular mixture after adding suitable excipients to obtain tablets or sugar-coated tablets.

[0046] In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. As oral liquid formulations, including suspensions, internal solutions, emulsions, or syrups, they may contain various excipients (such as wetting agents, sweeteners, flavorings, or preservatives) in addition to the commonly used simple diluents, i.e., water or liquid paraffin. Furthermore, depending on the specific circumstances, croscarmellose, agar, alginate, or sodium alginate may be added as disintegrants, and additional anti-caking agents, lubricants, wetting agents, flavorings, emulsifiers, and preservatives may also be included.

[0047] For non-oral administration, the pharmaceutical compositions of the present invention can be formulated into injections, transdermal administrations, and nasal inhalers in accordance with methods known in the art, together with suitable non-oral carriers. In the case of injections, sterilization is necessary, and they must be protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), mixtures thereof, and / or solvents or dispersion media containing vegetable oils, but are not limited thereto. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate-buffered saline (PBS) containing triethanolamine, or sterile water for injection, as well as isotonic solutions of 10% ethanol, 40% propylene glycol, and 5% glucose. To protect the injections from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal can be additionally included. In addition, the injectable may, in most cases, contain additional exfoliating agents such as sugar or sodium chloride.

[0048] For transdermal drug delivery systems, these include ointments, creams, lotions, gels, topical liquids, pastes, liniments, and aerosols. "Transdermal delivery" refers to the local administration of a pharmaceutical composition onto the skin, thereby delivering an effective dose of the active ingredient contained in the composition into the skin.

[0049] In the case of inhaled administration, the compositions according to the invention can be conveniently delivered in the form of an aerosol spray using a suitable propellant (e.g., dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases) via a pressurized canister or nebulizer. In the case of pressurized aerosols, the dosage unit can be determined by equipping a valve for delivering a prescribed dose. For example, gelatin capsules and cartridges used in inhalers or blowpipes can be formulated into powder mixtures containing the compound and a suitable powder matrix such as lactose or starch. For non-oral dosage forms, descriptions are found in the universally recognized formulary in all pharmaceutical chemistry fields (Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

[0050] The pharmaceutical compositions of the present invention are administered at pharmaceutically effective doses. A pharmaceutically effective dose is a dose sufficient to treat a disease without inducing side effects, based on a reasonable benefit / risk ratio applicable to medical treatment. The effective dose level can be determined based on factors including the patient's health status, the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the method of administration, the timing of administration, the route of administration and the excretion rate, the duration of treatment, concurrent or combined medications, and other factors known in the medical field. The dosage and frequency of administration are in no way a limitation on the scope of the invention.

[0051] The pharmaceutical compositions of the present invention can be used alone or in combination with methods such as surgery, radiation therapy, hormone therapy, chemotherapy, or the use of biological response modulators.

[0052] As another embodiment for achieving the stated purpose, the present invention provides a food composition for the prevention or improvement of epidermal growth factor receptor (EGFR) mutant cancer comprising narcissin or a food-grade acceptable salt thereof.

[0053] The terms "narcissin", "epidermal growth factor receptor (EGFR) mutation", "cancer", "prevention" and "composition" are as previously described.

[0054] As another embodiment for achieving the stated purpose, the present invention provides a food composition for the prevention or improvement of tyrosine kinase inhibitor (TKI) resistant cancers comprising narcissin or a food-grade acceptable salt thereof.

[0055] The terms "narcissin", "tyrosine kinase inhibitor", "cancer", "prevention" and "composition" are as previously described.

[0056] In this invention, "improvement" refers to all behaviors that result in an improvement in cancer symptoms through the application of the compositions of this invention.

[0057] In this invention, the food-acceptable salt of the narcissin can be an acid addition salt formed from a food-acceptable free acid or a metal salt formed from a base. In one example, inorganic and organic acids can be used as the free acid. Inorganic acids can include hydrochloric acid, sulfuric acid, hydrobromic acid, sulfurous acid, or phosphoric acid, while organic acids can include citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, and methanesulfonic acid. Furthermore, alkali metal salts or alkaline earth metal salts, such as sodium, potassium, or calcium salts, can be used as the metal salt. However, this is not a limitation.

[0058] The food compositions of the present invention can cover all foods in the conventional sense, and can be used interchangeably with terms known in the industry such as functional foods and health functional foods.

[0059] In this invention, the term "functional food" refers to food manufactured and processed using raw materials or ingredients that have functions beneficial to the human body, as specified in Law No. 6727 concerning functional foods. "Functional" refers to the intake of food for health purposes, such as obtaining beneficial effects related to regulating nutrients or improving physiological functions in order to target the structure and function of the human body.

[0060] The term "health functional food" in this invention refers to food made by using specific ingredients as raw materials or by extracting, concentrating, purifying, or mixing specific ingredients contained in food raw materials for the purpose of health assistance. It is a food that can be designed and processed to fully exert the biological regulatory functions of the organism, such as the body's defense, regulation of biological rhythms, prevention and recovery from diseases, by means of the ingredients. The composition of the health food can play a role in the prevention and recovery of diseases.

[0061] The types of food for which the compositions of the present invention can be used are not limited in any way. Furthermore, the compositions of the present invention can be prepared by mixing with other suitable auxiliary ingredients and known additives that may be included in food, as chosen by those skilled in the art. Types of food to which they can be added include meat, sausage, bread, chocolate, confectionery, snacks, pastries, pizza, ramen, other noodle products, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and multivitamin preparations, etc., which can be manufactured by adding them to juices, teas, jellies, and fruit juices made with extracts and components according to the present invention as main ingredients.

[0062] Furthermore, the foods applicable to this invention may include special nutritional foods (such as formula milk, infant complementary foods, etc.), meat products, aquatic products, tofu, jelly, noodle products (such as ramen, noodles, etc.), health supplements, condiments (such as soy sauce, soybean paste, chili sauce, compound sauce, etc.), sauces, pastries (such as snacks), dairy products (such as fermented milk, cheese, etc.), other processed foods, pickles, preserved foods (such as various pickles, preserved vegetables, etc.), beverages (such as fruit and vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (such as ramen soup base, etc.), and all other foods.

[0063] When the health functional food composition of the present invention is used in the form of a beverage, it can contain various sweeteners, flavoring agents, or natural carbohydrates as an additive, similar to ordinary beverages. In addition, the health functional food composition of the present invention may also contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, it may contain fruit pulp used in the manufacture of natural fruit juices, fruit juice beverages, and vegetable beverages.

[0064] As another embodiment for achieving the stated purpose, the present invention provides an anticancer adjuvant comprising narcissin or a pharmaceutically acceptable salt thereof.

[0065] The terms "narcissin", "pharmacologically acceptable salt", and "cancer" are as previously stated.

[0066] The anticancer adjuvant can be administered in combination with anticancer agents.

[0067] The aforementioned anticancer adjuvants refer to all forms used to enhance the anticancer effect of anticancer agents or to inhibit or improve the side effects of anticancer agents. The anticancer adjuvants of the present invention can be administered in combination with various types of anticancer agents or other adjuvants. When administered in combination, even at a lower dose compared to typical anticancer agent dosages, equivalent anticancer therapeutic effects can be achieved, thus allowing for safer implementation of anticancer treatment.

[0068] The administration route of the anticancer adjuvant can be any general route that can reach the target tissue. The anticancer adjuvant of the present invention can be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intrapulmonaryly, and rectally, depending on the purpose, but is not limited thereto. Furthermore, the anticancer adjuvant can be administered through any device that allows the active substance to move to the target cells.

[0069] The anticancer adjuvant of the present invention may, in addition to the active ingredient, contain one or more pharmaceutically acceptable carriers for administration purposes, and is preferably formulated into an anticancer adjuvant. Carriers, excipients, or diluents that may be included in the anticancer adjuvant of the present invention include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, but are not limited thereto.

[0070] The anticancer adjuvant of the present invention can be a formulation for non-oral administration, and for descriptions related to the formulation, please refer to the description of the formulation of the pharmaceutical composition.

[0071] Furthermore, all anticancer adjuvants disclosed in this technical field can be applied to this invention without special limitations.

[0072] As one embodiment of achieving the stated purpose, the present invention provides the use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancers.

[0073] The terms “epidermal growth factor receptor (EGFR) mutation”, “cancer”, “prevention”, “treatment”, “narcissin”, and “composition” are as previously described.

[0074] As one embodiment of achieving the stated purpose, the present invention provides the use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for the prevention or treatment of tyrosine kinase inhibitor-resistant cancers.

[0075] The terms "tyrosine kinase inhibitor", "drug resistance", "cancer", "prevention", "treatment", "narcissin", and "composition" are as previously described.

[0076] As one embodiment of achieving the stated purpose, the present invention provides the use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for manufacturing a medicament for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancer.

[0077] The terms “epidermal growth factor receptor (EGFR) mutation”, “cancer”, “prevention”, “treatment”, “narcissin”, and “composition” are as previously described.

[0078] As one embodiment of achieving the stated purpose, the present invention provides the use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for manufacturing a medicament for the prevention or treatment of tyrosine kinase inhibitor-resistant cancer.

[0079] The terms "tyrosine kinase inhibitor", "drug resistance", "cancer", "prevention", "treatment", "narcissin", and "composition" are as previously described.

[0080] As one embodiment of achieving the stated purpose, the present invention provides a method for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancer, comprising the step of administering to a subject a composition comprising narcissin or a pharmaceutically acceptable salt thereof.

[0081] The terms "narcissin", "epidermal growth factor receptor (EGFR) mutation", "cancer", "prevention", "treatment" and "composition" are as previously described.

[0082] As one embodiment of achieving the stated purpose, the present invention provides a method for the prevention or treatment of tyrosine kinase inhibitor-resistant cancer, comprising the step of administering to a subject a composition comprising narcissin or a pharmaceutically acceptable salt thereof.

[0083] The terms "narcissin", "tyrosine kinase inhibitor", "drug resistance", "cancer", "prevention", "treatment" and "composition" are as described above.

[0084] The compositions of the present invention can promote the degradation of epidermal growth factor receptor (EGFR) through the proteasome degradation pathway or lysosomal degradation pathway in tyrosine kinase inhibitor (TKI) resistant cancers and epidermal growth factor receptor (EGFR) mutant cancers, thereby inhibiting or reducing the activity or expression of epidermal growth factor receptor (EGFR), thus significantly inhibiting or suppressing the growth of cancer cells, and therefore can be effectively applied to the prevention and treatment of cancer. Attached Figure Description

[0085] Figure 1 shows the A549 cell line (epidermal growth factor receptor wild-type) treated with various concentrations (25 nM, 50 nM, 100 nM, 500 nM, 1 μM, 5 μM and 10 μM) of narcissin (NCS). Figure 1a H1299 cell line (epidermal growth factor receptor wild-type (EGFR wild-type)). Figure 1b ) and the H1975 cell line (epidermal growth factor receptor mutant (EGFR mutant) T790M / L858R, Figure 1c The results were then obtained by measuring the cell growth curve using a live cell imaging device.

[0086] Figure 2 The results are based on simulation models that predict the binding of narcissin (NCS) to wild-type or mutant (T790M / L858R) epidermal growth factor receptor (EGFR).

[0087] Figure 3 shows the results of treatment with 50 nM gefitinib (Gef) or 50 nM narcissin (NCS) on wild-type epidermal growth factor receptor (EGFR) kinase. Figure 3a ) or epidermal growth factor receptor (EGFR) mutant (T790M / L858R) kinase ( Figure 3b The amount of adenine triphosphate (ATP) consumed afterward was measured, thereby determining the kinase activity.

[0088] Figure 4 This is a confirmation of cell viability after treatment with narcissin (NCS) on epidermal growth factor receptor (EGFR) mutant PC-9 cell lines and gefitinib-resistant PC-9 (PC-9-GR) cell lines.

[0089] Figure 5a The results were confirmed by Western blotting of the expression levels of phosphorylated epidermal growth factor receptor (p-EGFR) and total epidermal growth factor receptor (Total EGFR) in H1975 cell lines treated with 50 nM narcissin (NCS) for 4, 8 and 24 hours.

[0090] Figure 5bThe results were obtained by confirming the expression sites of lysosome-associated membrane protein 1 (LAMP1, green) and epidermal growth factor receptor (EGFR, red), markers of lysosome degradation of intracellular substances, in H1975 cell lines treated with narcissin (NCS) using immunofluorescence staining.

[0091] Figure 5c The results compare the expression levels of epidermal growth factor receptor (EGFR) after treatment with the protein synthesis inhibitor cycloheximide (CHX) and before treatment with narcissin (NCS) or without treatment.

[0092] Figure 5d The results compare the expression levels of epidermal growth factor receptor (EGFR) after treatment with the proteasome inhibitor MG132 and the lysosomal inhibitor bafilomycin A1 (Baf A1) followed by treatment with narcissin (NCS) and the untreated / untreated results.

[0093] Figure 6 (A) shows the results of comparing the formation of the multivulva phenotype in the epidermal growth factor receptor (EGFR) mutant (L858R / T790M) Caenorhabditis elegans cancer model with that in wild-type Caenorhabditis elegans; (B) shows the results of confirming the formation of the multivulva phenotype after treatment with narcissin (NCS); and (C) shows the results of comparing the relative degree of formation of the multivulva phenotype after treatment with gefitinib or narcissin (NCS). Detailed Implementation

[0094] The present invention will now be described in more detail through embodiments. These embodiments are merely illustrative of the invention, and the scope of the invention is not limited to these embodiments.

[0095] Example 1. Cancer cell growth inhibition effect using narcissine An experiment was conducted to confirm whether narcisine (NCS) has an inhibitory effect on cancer cell growth.

[0096] A549 cell line (EGFR wild-type), H1299 cell line (EGFR wild-type), and H1975 cell line (EGFR mutant T790M / L858R) were cultured at 4 × 10⁻⁶ cells per cell line. 3 Cells were seeded at a density of 1 / 2 wells in 96-well plates and treated with various concentrations (25 nM, 50 nM, 100 nM, 500 nM, 1 μM, 5 μM, and 10 μM) of narcissin (NCS). The NCS-treated cells were then cultured in a Live Cell Imager, and cell growth curves were plotted by measuring cell growth every 3 hours over 48 hours. The IC50 value was then derived by calculating the survival rate at each concentration.

[0097] The results, shown in Figure 1, confirmed that narcissin (NCS) significantly inhibited or blocked cell growth in three types of lung cancer cell lines: wild-type (EGFR) and EGFR mutant (T790M / L858R) cell lines in a concentration-dependent manner. Specifically, in the wild-type (EGFR) A549 and H1299 cell lines, the IC50 value of narcissin (NCS) that inhibited cell growth by 50% was 0.11 μM (…). Figure 1a ) and 0.08μM ( Figure 1b Conversely, it can be confirmed that in the H1975 cell line with the epidermal growth factor receptor (EGFR) mutant (T790M / L858R), the IC50 value is 0.022 μM (…). Figure 1c This confirms that narcissin (NCS) has a significant cell growth inhibitory effect on the epidermal growth factor receptor (EGFR) mutant (T790M / L858R) cancer cell line, even at lower concentrations.

[0098] The results confirm that the narcissin (NCS) of the present invention has a significant effect in inhibiting or blocking the growth of epidermal growth factor receptor (EGFR) mutant (T790M / L858R) cancer cells.

[0099] Example 2. Three-dimensional binding structure analysis of narcissin (NCS) and epidermal growth factor receptor (EGFR) The binding structure of narcissin (NCS) to wild-type or mutant epidermal growth factor receptor (EGFR) (T790M / L858R) was predicted using structural biology methods based on a binding simulation model.

[0100] The result is as follows Figure 2 As shown, it can be confirmed that in the binding structure of narcissin (NCS) to the mutant epidermal growth factor receptor (EGFR) (T790M / L858R), there are more residues involved in hydrophobic interaction compared to the binding structure of the wild type of epidermal growth factor receptor (EGFR).

[0101] The results confirm that, compared with the wild-type epidermal growth factor receptor (EGFR), the narcissin (NCS) of the present invention can form a stronger binding structure with the mutant epidermal growth factor receptor (EGFR) (T790M / L858R).

[0102] Example 3. The effect of reducing kinase activity in vitro using narcissin (NCS) To confirm whether narcissin (NCS) has an inhibitory or antagonistic effect on the activity of wild-type and mutant epidermal growth factor receptor (EGFR) (T790M / L858R), an in vitro kinase assay was performed.

[0103] Purified wild-type epidermal growth factor receptor (EGFR) kinase and mutant EGFR kinase (T790M / L858R) kinase were added to microcentrifuge tubes along with reaction buffer, substrate, and adenine triphosphate (ATP). After treatment with 50 nM gefitinib or 50 nM narcissin (NCS), the amount of ATP consumed was measured by incubation at 30°C, thereby verifying kinase activity.

[0104] The result is as follows Figure 3aAs shown, it can be confirmed that in the epidermal growth factor receptor (EGFR) wild-type (WT) kinase response, when treated with the positive control group (50 nM gefitinib), the relative kinase activity significantly decreased from approximately 8 to approximately 3, but no significant decrease was observed when treated with 50 nM narcissin (NCS). Conversely, as... Figure 3b As shown, it can be confirmed that in the epidermal growth factor receptor (EGFR) mutant (T790M / L858R) kinase response, there was no significant change in kinase activity when treated with 50 nM gefitinib, but the relative kinase activity was significantly reduced when treated with 50 nM narcissin (NCS).

[0105] The results confirm that the narcissin (NCS) of the present invention can significantly reduce the activity of epidermal growth factor receptor (EGFR) kinase in EGFR mutant (T790M / L858R) cancer cells, thereby inhibiting or suppressing the growth of cancer cells.

[0106] Example 4. Effect of narcissin (NCS) on gefitinib-resistant cell lines The epidermal growth factor receptor (EGFR) mutant (exon 19 deletion) cell line, namely the PC-9 cell line, is a cell line that undergoes apoptosis induced by gefitinib. Gefitinib-resistant (GR) cell lines were prepared using the PC-9 cell line, and then treated with narcissin to confirm cell viability.

[0107] The result is as follows Figure 4 As shown, apoptosis can be confirmed in both PC-9 and PC-9-GR cell lines with the aid of narcissin (NCS). However, in the PC-9-GR cell line, which is resistant to the tyrosine kinase inhibitor gefitinib, the number of apoptotic cells is significantly increased.

[0108] Example 5. Molecular mechanism of reduced activity or expression of epidermal growth factor receptor (EGFR) via narcissin (NCS) 5.1. Confirmation of epidermal growth factor receptor (EGFR) expression changes using narcissin (NCS) treatment An experiment was performed to confirm whether narcissin (NCS) affects the activity of epidermal growth factor receptor (EGFR) in the EGFR mutant (T790M / L858R) H1975 cell line.

[0109] The expression levels of phosphorylated epidermal growth factor receptor (phospho-EGFR, p-EGFR) and total epidermal growth factor receptor (Total EGFR) in H1975 cell lines treated with 50 nM narcissin (NCS) for 4, 8, and 24 hours were confirmed using Western blotting. The results are as follows: Figure 5a As shown, it can be confirmed that the activity of epidermal growth factor receptor (EGFR) is significantly reduced according to the treatment time of narcissin (NCS), and the expression of total epidermal growth factor receptor (Total EGFR) is also significantly reduced at 24 hours.

[0110] 5.2. Confirmation of the mechanism of epidermal growth factor receptor (EGFR) expression changes by means of narcissin (NCS) treatment To confirm the cause of the decreased expression of epidermal growth factor receptor (EGFR), the expression sites of lysosome markers that degrade intracellular substances, namely lysosome-associated membrane protein 1 (LAMP1, green) and epidermal growth factor receptor (EGFR, red), were identified in H1975 cell lines treated with 100 nM narcissin (NCS) by immunofluorescence (IF).

[0111] The result is as follows Figure 5b As shown, it can be confirmed that epidermal growth factor receptor (EGFR) and lysosomal-associated membrane protein 1 (LAMP1) are simultaneously stained in cell lines treated with narcissin (NCS). This confirms that the reduced expression of epidermal growth factor receptor (EGFR) with the aid of narcissin (NCS) is achieved through signal transduction associated with lysosomal degradation.

[0112] In addition, to confirm whether the reduced expression of epidermal growth factor receptor (EGFR) is related to protein synthesis, an experiment was performed to confirm the expression level of epidermal growth factor receptor (EGFR) over time after treatment with 50 nM cycloheximide (NCS) and the protein synthesis inhibitor cycloheximide (CHX).

[0113] The result is as follows Figure 5cAs shown, it can be confirmed that in cell lines treated with narcissin (NCS), the expression of epidermal growth factor receptor (EGFR) was significantly reduced according to the treatment time with actinomycin (CHX) compared to the control group. This confirms that the reduction in EGFR expression assisted by narcissin (NCS) is achieved by promoting the degradation of the EGFR protein.

[0114] Next, an experiment was performed to confirm whether the expression morphology of epidermal growth factor receptor (EGFR) of narcissin (NCS) changed when treated with MG132, an inhibitor of the proteasome and lysosome that degrade intracellular proteins, and bafilomycin A1 (Baf A1). The results are as follows. Figure 5d As shown, it can be confirmed that the reduced expression of epidermal growth factor receptor (EGFR) by means of narcissin (NCS) can be restored by treatment with proteasome inhibitors or lysosomal inhibitors.

[0115] The results confirm that the narcissin (NCS) of the present invention can reduce the expression of epidermal growth factor receptor (EGFR) by promoting the degradation of EGFR through the proteasome and lysosome pathways.

[0116] Example 6. In vivo anticancer effects of narcissin (NCS) To confirm the in vivo anticancer effect of narcissin (NCS), epidermal growth factor receptor (EGFR) mutant (L858R / T790M) Caenorhabditis elegans was used. Caenorhabditis elegans, C. elegans Experiments were performed in a cancer model. Epidermal growth factor receptor (EGFR) mutant (L858R / T790M) *C. elegans* (… C. elegans The cancer model differs from the wild-type, forming... Figure 6 The red arrow in A indicates the abnormal multivulva phenotype.

[0117] Regarding this, such as Figure 6 As shown in B, it can be confirmed that treatment with narcissin (NCS) on the epidermal growth factor receptor (EGFR) mutant (L858R / T790M) Caenorhabditis elegans (C. elegans) C. elegans In the case of a cancer model, the formation of the multivulva phenotype was significantly suppressed. In particular, as... Figure 6As shown in C, it can be confirmed that the existing first-generation epidermal growth factor receptor (EGFR) kinase inhibitor, namely gefitinib, does not have the effect of inhibiting the formation of the multivulva phenotype, but the narcissin (NCS) of the present invention has a significant effect of inhibiting the formation of the multivulva phenotype.

[0118] This confirms that the narcissin (NCS) of the present invention has an anticancer effect on epidermal growth factor receptor (EGFR) mutant (L858R / T790M) subjects.

[0119] The results confirm that the narcissin (NCS) of the present invention can significantly reduce the activity or expression of epidermal growth factor receptor (EGFR) by promoting the degradation of EGFR in EGFR-mutant cancers or tyrosine kinase inhibitor (TKI)-resistant cancers, thereby significantly inhibiting or suppressing the growth of cancer cells. Therefore, it can be effectively applied to the prevention and treatment of cancer.

[0120] From the foregoing description, those skilled in the art should understand that the present invention can be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood in all respects as exemplary rather than limiting. The scope of the present invention should not be limited to the description, but should be interpreted as including all modifications or variations that can be derived from the scope and meaning of the claims and their equivalents.

Claims

1. A pharmaceutical composition for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancer comprising narcissine or a pharmaceutically acceptable salt thereof.

2. The composition according to claim 1, The cancer in question is an epidermal growth factor receptor (EGFR) mutant cancer.

3. The composition according to claim 2, The solid tumor is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, colon cancer, pancreatic cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, prostate cancer, skin cancer, liver cancer, thyroid cancer, gallbladder cancer, bile duct cancer, esophageal cancer, bladder cancer, and oral cancer.

4. The composition according to claim 2, The solid tumor in question is lung cancer.

5. The composition according to claim 1, The epidermal growth factor receptor (EGFR) mutations include one or more mutations selected from the group consisting of the L858R mutation and the T790M mutation.

6. The composition according to claim 1, The composition inhibits or reduces the activity or expression of epidermal growth factor receptor (EGFR).

7. The composition according to claim 1, The composition promotes the degradation of epidermal growth factor receptor (EGFR) via the proteasome degradation pathway or the lysosome degradation pathway.

8. A pharmaceutical composition for the prevention or treatment of tyrosine kinase inhibitor (TKI) resistant cancers comprising narcissin or a pharmaceutically acceptable salt thereof.

9. A food composition for the prevention or improvement of epidermal growth factor receptor (EGFR) mutant cancer containing narcissin or a food science-acceptable salt thereof.

10. A food composition for the prevention or improvement of tyrosine kinase inhibitor (TKI) resistant cancers containing narcissin or a food-grade acceptable salt thereof.

11. An anticancer adjuvant comprising narcissin or a pharmaceutically acceptable salt thereof.

12. Use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancers.

13. Use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for the prevention or treatment of cancer resistant to tyrosine kinase inhibitors.

14. Use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancer.

15. Use of a composition comprising narcissin or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of tyrosine kinase inhibitor-resistant cancer.

16. A method for the prevention or treatment of epidermal growth factor receptor (EGFR) mutant cancer, comprising the step of administering to a subject a composition containing narcissin or a pharmaceutically acceptable salt thereof.

17. A method for the prevention or treatment of tyrosine kinase inhibitor-resistant cancer, comprising the step of administering to a subject a composition containing narcissin or a pharmaceutically acceptable salt thereof.

Images