KR20140015798A - Effective anticancer drugs against gefitinib-resistant human lung cancer cells comprising the daphnane diterpenoid compound - Google Patents

Abstract

The present invention relates to an anticancer drug useful for drug resistance cancer cells and more specifically, to a composition which provides excellent destructive effect against cancer cells showing resistance to conventional anticancer drugs containing a compound separated from a Daphne genkwa extract. Especially, the composition of the present invention enables a user to overcome the anticancer drug resistance in cells which is resistant to a Gefitinib anticancer drug, and an epithelial growth factor receptor (EGFR) inhibitor. The composition has an anticancer effect against regular cancer cells and cells with the anticancer drug resistance, and is capable of being used as a pharmaceutically useful anticancer drug. [Reference numerals] (AA) Yuanhuadine (nM); (BB,FF) Control group; (CC,GG) 24 hours; (DD,HH) 48 hours; (EE) Gefitinib (nM)

Description

Effective anticancer drugs against gefitinib-resistant human lung cancer cells inhibit the epidermal growth factor receptor (EWFR) inhibitor of daphnan diterpenoids daphnane diterpenoid compound}

The present invention relates to an anticancer agent useful for gefitinib resistant human lung cancer cells of daphnane diterpenoid-based substance isolated from the original extract.

1 Hardie WD et al (2008) Am J Physiol Lung Cell Mol Physiol 294: L1217-25.

Imaizumi M et al (1997) Lung Cancer 17: 69-84; Wu YY et al (2011) Oncogene 30: 3682-3693.

Suzuki S et al (2005) Cancer 103: 1265-73.

Cappuzzo F et al (2005) J Natl Cancer Inst 97: 643-55.

Zhang W et al (2005) Clin Cancer Res 11: 600-5.

6 Shepherd FA et al (1994) Semin Oncol 21: 7-18.

7 Kawamoto et al (1983) Proc. Natl. Acad. Sci. USA 80: 1337-1341.

William Pao et al (2005) J Clin Oncol 23: 2556-2568.

Herbst R et al (2004) Nat Rev Cancer 4: 956-65.

Miller VA et al (2004) J Clin Oncol 22: 1103-1109.

11 Kwak EL (2005) Proc Natl Acad Sci USA 102: 7665-70.

12 Kobayashi S et al (2005) N Engl J Med 352: 786-92.

[Ref. 13] Jung Sup and Shin Min-kyo, Antidrug Dictionary, Younglimsa, pp740-741, 1998.

Kai et al (2004) Yakugaku Zasshi 124: 349-354.

15 Yuanyuandine, Hong JY et al (2011) J Nat Prod 74: 2102-2108.

16 Zheng WF (2007) Int Immunopharmacol 7: 128-34.

He W et al (2002) Mini Rev Med Chem 2: 185-200.

18, Zhang S et al (2006) Bioorg Med Chem 14: 3888-95.

Lee SK et al (2008) Chem Biol Interact 115: 215-28.

Chung HJ et al (2011) Food Chem Toxicol 49: 2942.

Hong JY et al (2011) J Nat Prod 74: 2102-8.

Jurek A et al (2009) J Biol Chem 284: 4626-34.

23. Franklin WA (2002) Semin Oncol 29: 3-14.

24, Hong et al (2011) J Nat Prod 74: 2102-2108.

Suzuki S (2005) Cancer 103: 1265-73.

Jean Y et al (2008) Eur J Pharmacol 578: 323-31.

The present invention relates to a composition that provides an excellent killing effect on cancer cells that exhibit resistance to gefitinib anticancer agents, which are epidermal growth factor receptor (EGFR) inhibitors.

As the incidence and mortality rate of cancer continues to increase worldwide, the importance of developing effective anticancer drugs is also increasing. The market for anticancer drugs is the third largest in the world, along with cardiovascular and neurological diseases, with around 10 million cancer cases worldwide in 2000 and an estimated 15 million by 2020. . The market size is estimated to be 35 trillion won in 2003 and 60 trillion won in 2010. The findings testify to the fact that research for the development of anticancer drugs is being actively conducted worldwide.

According to data from the National Statistical Office, lung cancer is the number 1 cancer mortality disease in Korea, and it is the fastest growing rate compared to other cancers in the past 10 years, and the lung cancer incidence and mortality rate continues to increase. It is estimated to increase by more than twice. Treatment for lung cancer can be classified into surgery, radiation therapy, chemotherapy, etc. In most cases, additional chemotherapy drugs are used to obtain higher therapeutic effects.In advanced lung cancer, chemotherapy or radiation therapy is the main treatment. to be. In the last decade, the quality of life and survival have increased significantly due to the combination of platinum compounds and new anticancer drugs such as vinorelbine, gemcitabine, and taxanes. Nevertheless, the 1-year survival rate for advanced non-small cell lung cancer is only 35%, and the 2-year survival rate is only 15-20%. Lung cancer has reached the therapeutic limit. Recently, molecular targeted therapy, a new concept of therapy aimed at reducing specific biological mechanisms and increasing the therapeutic effect and reducing side effects, has been developed. Many drugs are also in clinical trials for the treatment of lung cancer. Gefitinib among them [gefitinib; Some drugs, such as Iressa, are the first to be approved by the US Food and Drug Administration as a molecular target therapy for non-small cell lung cancer, and have already been applied in clinical practice to help patients. However, since cancer develops in a very complicated route, these molecular target therapies that selectively inhibit only specific targets have the problem of resistance by long-term administration and have the disadvantage of showing selective therapeutic effects depending on the mutation status of specific genes. There is an urgent need for the development of anticancer drugs that can overcome these problems.

Until recently, with the development of various anticancer drugs, rapid progress has been made in the treatment of cancer by chemotherapy. However, the treatment of cancer by chemotherapy often fails because the cancer cells become resistant to drugs gradually from the beginning or during the treatment. Therefore, drug development that can overcome the resistance to anticancer drugs must be followed.

Currently developed target therapeutics target: (1) receptor tyrosine kinase (TK) that accepts growth signals from outside the cell: epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TK inhibitor), epithelium Cell growth factor receptor (EGFR) monoclonal antibodies, (2) non-receptor tyrosine kinases (Ras, raf, protein kinase C) in the cell membrane, and (3) MAPKs that signal to the nucleus in the cytoplasm kinase (MEK), mTOR and the like, (4) retinoic receptor (5) and angiogenesis related targets.

Cancer cells produce several types of peptide hormones, which stimulate the receptors on the cell wall and contribute to the normal growth and development of cancer. Among the factors related to growth, there are six known factors so far: epidermal growth factor (EGF), transforming growth factor [α] (TGF-α), and amphiregulin. (AR)], betacellulin (BTC), heparin-binding epithelial growth factor [heparin-binding-EGF (HB-EGF)], and heregulin (HRG). Among them, epidermal growth factor (EGF) is related to cancer cell proliferation and cancer growth factor system, whereas tumor growth factor (TGF-α), a cell-active substance of acute immune response, is used in patients with lung cancer. It has been shown to be shortened for survival and to be involved in cell degradation (Hardie WD et al (2008) Am J Physiol Lung Cell Mol Physiol 294: L1217-25; Imaizumi M et al (1997) Lung Cancer 17:69 Wu YY et al (2011) Oncogene 30: 3682-3693).

Epidermal growth factor receptor (EGFR) is the first known growth factor receptor among receptor TKs (80-100% of head and neck cancer, 25-77% of colorectal cancer, 30--30 of pancreatic cancer) It is known to be expressed in 50%, 40-80% of lung cancer, and the like. Epidermal growth factor receptor (EGFR) abnormalities, including protein overexpression, gene amplification or abnormal activation of epidermal growth factor receptor (EGFR), have been observed in several epithelial carcinomas including lung carcinoma (Suzuki S et al (2005) Cancer 103 Epithelial growth factor observed in cancer precursor bronchial epithelium, especially in cancer progenitor bronchial epithelium: 1265-73; Cappuzzo F et al (2005) J Natl Cancer Inst 97: 643-55; Zhang W et al (2005) Clin Cancer Res 11: 600-5) Receptors (EGFR) are known to play an important role in the development of lung carcinoma. In lung cancer tissues, it has been shown that signaling actually occurs through tyrosine phosphorylation of intracellular proteins (Imaizumi M (1997) Lung Cancer 17: 69-84). An increase in epidermal growth factor receptor (EGFR) expression was observed in abnormal lesions of bronchial epithelium and in epithelial growth factor receptor (EGFR) mutant glandular carcinoma. Mutations in the (EGFR) tyrosine kinase region (TK domain) have been observed, suggesting that epidermal growth factor receptor (EGFR) signaling plays an important role in the early pathogenesis of lung cancer.

Conventional anticancer chemotherapy is based on cytotoxic drugs that block each step of the cell cycle, but recently developed epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are molecular target therapeutics for non-small cell lung cancer. It is attracting attention as a new therapeutic agent (Shepherd FA et al (1994) Semin Oncol 21: 7-18). After Kawamoto et al. Hypothesized that inhibiting epidermal growth factor receptor (EGFR) in 1984 could be the target of new chemotherapy, two therapeutic strategies to inhibit epithelial growth factor receptor (EGFR) have been developed. The signaling pathway of epidermal growth factor receptor (EGFR) can be blocked in two different ways (Kawamoto et al (1983) Proc. Natl. Acad. Sci. USA 80: 1337-1341). First, it is a monoclonal antibody that inhibits binding of a ligand to a receptor, and second, a small molecule that inhibits autophosphorylation of tyrosine kinase in the intracellular domain. )to be.

Gefitinib is the first commercially available oral epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that has been shown to inhibit the growth of several solid cancers, including lung cancer, colorectal cancer, and breast cancer, in preclinical studies. William Pao et al (2005) J Clin Oncol 23: 2556-2568; Herbst R et al (2004) Nat Rev Cancer 4: 956-65; Miller VA et al (2004) J Clin Oncol 22: 1103-1109. Zephytinib is a small synthetic anilinoquinazoline that has minimal activity against other tyrosine kinases (TKs) and selectively inhibits epidermal growth factor receptor-tyrosine kinase (EGFR-TK). do. It also inhibits Mitogen-activated protein kinase (MAPK) and subsequent c-fos, a nuclear transcription factor. Basically, the drug inhibits growth, but at high doses it also induces cell death. Gefitinib has improved the problems of serious toxicity and side effects such as bone marrow suppression, nephrotoxicity, nausea, vomiting, hair loss, etc. of the existing cytotoxic anticancer drugs, as well as oral treatment. However, gefitinib, an epidermal growth factor receptor (EGFR) target inhibitor, has been developed as a chemotherapeutic agent for non-small cell lung cancer.However, the expression of T790M of the epidermal growth factor receptor (EGFR) or c-Met, a protooncogene, has been developed. It has a limit of obtaining drug resistance.

The epithelial growth factor receptor inhibitor (EGFR-TKI) used in the clinic is a small molecule reversible inhibitor, after which studies have shown that the use of irreversible inhibitors overcomes T790M resistance (Kwak EL (2005) Proc Natl Acad Sci USA 102 : 7665-70) Many types of irreversible inhibitors have been developed and are currently in clinical trials. These new drugs not only have irreversible properties, but also inhibit two or many kinase inhibitors that inhibit other receptors such as HER2 and have shown good results in preclinical studies. Efficacy in treating epidermal growth factor receptor inhibitors is sometimes quite dramatic in patients, resulting in long-term survivors that were nearly impossible in the past, and recovering enough to allow a normal patient to live in a respiratory failure due to cancer. In some cases. However, after taking medicine for about a year, most patients become resistant to the drug and make the disease worse again. Therefore, it is emerging as an important research task to identify the mechanism of tolerance and to come up with a way to overcome it.

It was reported for the first time in 2005 that drug resistance occurs when an additional T790M mutation occurs in exon 20 of a gene containing the tyrosine kinase domain of epidermal growth factor receptor (EGFR). The mechanism of methionine has been described as a method of methionine disrupting the hydrogen bonding of drugs in the ATP-binding pocket (Kobayashi S et al (2005) N Engl J Med 352: 786-92).

Many of the drugs currently in use are known to be substances or derivatives derived from natural products. Many anti-cancer chemotherapeutic agents currently used in clinical trials have active substances isolated from natural medicinal plants as the leading substances, and the target sample of the present invention is Wonhwa ( Daphne). genkwa SIEB. et ZUCC) is the bud of the red bean tree. The leaves are picked before blooming and dried. The main ingredients are diterpenoids and stimulating oil phases such as genkwadaphnin, apigenin, cytosterol, benzoic acid, yuanhuadine and yuanhuacine. Substances, etc. (Information and Shin Min-kyo, Antibiotics, Yeonglimsa, pp740-741, 1998). Wonhwa is a nerve hypersensitivity, heart is not stable, heart beats, surprised, and forgetful symptoms are effective. In addition, there is a clinical report that asthma, sea water, skin swelling, food indigestion is effective, and a significant improvement in schizophrenia, manic depression, neurological disorders. It is also used for diuresis, edema, nephritis (Kai et al (2004) Yakugaku Zasshi 124: 349-354).

Yuanhuadine (Hong et al (2011) J Nat Prod 74: 2102-8), Yuanhuacine, a substance that exhibits a growth inhibition effect on human lung cancer cells more specifically from the original revealed by the present inventors ) And daphnodorins isolated from diterpenoids and roots, such as yuanhualine and yuanhuahine, identified as novel compounds by the present inventors. It has been shown to be an effective anticancer substance (Zheng WF (2007) Int Immunopharmacol 7: 128-34).

Therefore, the inventors of the present invention confirmed that the novel compounds of the present invention isolated from the original patent not only show excellent anticancer activity but also have high specific selectivity for lung cancer and have an effect of inhibiting cancer cell growth compared to normal cells. Previous studies were conducted. In addition, human lung cancer cell line H292 resistant to gefitinib, a representative anticancer agent of epidermal growth factor receptor tyrosine kinase (EGFR TK), was applied in vitro and in vivo experimental models similar to those of actual clinical trials. The present invention has been completed by proceeding with the contents of the growth inhibitory effect of the target material against (H292-Gef) and the mechanism of action of overcoming resistance.

However, anywhere in the literature, any of the compositions for the treatment and prevention of existing anticancer drug resistant cancer diseases comprising yuanhuadine or yuanhuacine or a pharmaceutically acceptable salt thereof as an active ingredient isolated from the extracts of Wonhwa. There is no disclosure or suggestion.

Accordingly, the present inventors have found that yuanhuadine or yuanhuacine isolated from the original extract developed by the present inventors is gefitinib, an epidermal growth factor receptor (EGFR) inhibitor. The present invention was completed by confirming that the cells exhibiting resistance to anticancer drugs and those of normal cancer cells exhibited strong anticancer efficacy.

In order to solve the above object, the present invention provides a target comprising a daphnane diterpenoid compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof isolated from the original extract. Provided are pharmaceutical compositions for the treatment and prevention of anticancer drug resistant cancer diseases.

(I)

Where

R is a hydrogen atom, a C1-C3 lower alkyl group, a C1-C3 lower alkoxy group, or a phenyl group substituted with R ', wherein R' is a hydrogen atom, a C1-C3 lower alkyl group, or a C1-C3 lower alkoxy group.

As a preferable compound group of the said General formula (I) compound, a compound group whose R substituent is a hydrogen atom, a methyl group, an ethyl group, or a phenyl group is a compound group whose R substituent is a methyl group, an ethyl group, or a phenyl group.

Most preferred compounds of the general formula (I) include at least one compound selected from the group consisting of yuanhuadine or yuanhuacine.

In addition, the present invention provides a target anticancer drug-resistant cancer disease comprising a daphnane diterpenoid compound represented by the general formula (I) isolated from the original extract or a pharmaceutically acceptable salt thereof as an active ingredient. Provide health functional foods for prevention and improvement.

The compounds of the present invention are characterized by exhibiting cytotoxicity in cancer cells that are resistant to normal cancer cells and epidermal growth factor receptor (EGFR) target anticancer agents.

The compound of the present invention is characterized by exhibiting apoptosis-inducing effect similar to or better than that of normal cancer cells in drug resistant cancer cells.

The compounds of the present invention may be prepared by pharmaceutically acceptable salts and solvates according to methods conventional in the art.

As salts, acid addition salts formed by pharmaceutically acceptable free acids are useful. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, lactic acid, glycollic acid, gluconic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the above compounds include salts of acidic or basic groups which may be present in the compound, unless otherwise indicated. For example, pharmaceutically acceptable salts include the sodium, calcium, and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate rate, methane sulfonate (mesylate) and p - toluene sulfonate (tosylate) and a salt, the salt manufacturing method or manufacturing process known in the art ≪ / RTI >

Target anticancer agents as defined herein are gefitinib, temozolomide, bevacizumab, erlotinib, capecitabine, imatinib or other possible anticancer agents. And more preferably Gefitinib.

Cancer diseases as defined herein include gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, Anal rectus cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer Renal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma or pituitary adenoma, preferably lung cancer, or non-small cell lung cancer, more preferably Gefitinib, temozolomide, bevacizumab elo It characterized in that it comprises a lung cancer, or non-small cell lung cancer with a nip (erlotinib), capecitabine (capecitabine), double sheet tinip (imatinib), or any other resistant to available anticancer drugs.

In particular, the cancer cell line resistant to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) anti-cancer agent gefitinib (gefitinib) of the present invention is characterized in that it is selected as a lung cancer cell line.

Therefore, in the present invention, a daphnane diterpenoid compound represented by the general formula (I) isolated from the original extract or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable excipient or Provided is a pharmaceutical composition for treating and preventing a target anticancer drug resistant cancer disease containing a carrier.

The compound may be used as an anticancer adjuvant for drug-resistant cancer cells by showing cytotoxicity in normal cancer cells and drug-resistant cancer cells, and particularly in drug-resistant cancer cells, by inducing similar or better apoptosis-inducing effects.

Accordingly, the present invention is directed to a target anticancer drug-resistant cancer disease containing a daphnane diterpenoid compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient isolated from the original extract. Provided anticancer adjuvant for treatment and prevention.

In another aspect, the present invention provides a daphnane diterpenoid compound represented by the general formula (I) isolated from the original extract or a combination containing a pharmaceutically acceptable salt thereof and an existing anticancer agent as an active ingredient. do.

Existing anticancer agents as defined herein include metabolism inhibitors (antimetabolites) such as folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine) and pyrimidine derivatives (5-fluorouracil, cytarabine); An alkylating agent such as a chlorambucil, a cyclophosphamide, an ethylene imine compound, an alkyl sulfonate compound, a carnustine compound or a dacarbazine compound, ); Antitumor agents such as actinomycin D, doxorubicin, bleomycin, mitomycin, botanical alkaloids such as vincristine, vvenilastin, and mitotic inhibitors such as taxoid, a mitotic inhibitor including a taxane ring antimitotic drugs); Hormones such as corticosteroids or progesterone; Existing anticancer agents such as platinum-containing compounds such as cisplatin.

Daphnane diterpenoid compounds represented by the general formula (I) isolated from the original extract of the present invention are gefitinib, an epidermal growth factor receptor (EGFR) inhibitor. It was confirmed that the cells exhibiting resistance to anticancer drugs and normal cancer cells also exhibited strong anticancer efficacy in cells displaying anticancer drug resistance, which is useful for the treatment and prevention of target anticancer drug resistant cancer diseases.

The composition of the present invention contains 0.01 to 99% by weight of the compound, based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

The compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The present invention also provides a method for treating cancer disease, comprising administering to a subject exhibiting drug resistance to the target anticancer agent at an effective concentration that inhibits drug resistance.

In addition, the present invention provides a method for treating cancer disease, comprising administering an effective concentration of the anticancer adjuvant and one or more anticancer agents that inhibit drug resistance to a subject exhibiting drug resistance to a target anticancer agent.

The anticancer adjuvant of the present invention may further contain one or more active ingredients exhibiting the same or similar functions. The pharmaceutically acceptable carrier may be used by mixing one or more of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and these components.

In addition, the present invention provides a method for treating cancer disease, comprising administering an effective concentration of the anticancer adjuvant and one or more anticancer agents that inhibit drug resistance to a subject exhibiting drug resistance to a target anticancer agent.

The method of administering the compound of the present invention or the anticancer adjuvant and the anticancer agent is not particularly limited, but may be parenterally administered (for example, intravenous, subcutaneous, intraperitoneal or topical) or oral administration according to a desired method. Parenteral administration is preferred, and administration by intravenous injection is more preferred.

In addition, the dosage of the compound of the present invention or the anticancer adjuvant and the anticancer agent to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and is based on an adult patient having a weight of 70 kg. When used, it is generally 0.1 to 1,000 mg / day, preferably 1 to 500 mg / day, and the daily dose of the compound or anticancer and anticancer agent, or 1/2, 1/3 or 1 / its thereof, per unit dosage form. A dose of 4 may be contained, and may be dividedly administered 1 to 6 times a day at regular intervals according to the judgment of a doctor or a pharmacist.

The composition containing the compound according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The preferred dosage of the compound of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound is preferably administered at 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, including, for example, oral and rectal, or intravenous.

In another aspect, the present invention is to prevent the target anticancer drug-resistant cancer disease comprising a daphnane diterpenoid-based compound represented by the general formula (I) or pharmaceutically acceptable salt thereof isolated from the original extract And it provides a dietary supplement for improvement.

Health functional food containing the compound of the present invention can be used in various ways, such as drugs, foods and drinks for the prevention and improvement of the target disease. Examples of foods to which the compound of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used in powder, granule, tablet, have.

Accordingly, the present invention also provides a daphnane diterpenoid compound represented by the general formula (I) or a pharmaceutically acceptable compound thereof, which is isolated from the original extract having a prophylactic and improving effect of a target anticancer drug resistant cancer disease. It provides a food or food additive containing a salt as an active ingredient.

Food forms to which the compounds of the present invention can be added include various foods of candy, beverages, gums, tea, vitamin complexes, or foods that are health supplement foods.

The compounds of the present invention may be added to food or beverages for the purpose of preventing and ameliorating the desired disease. At this time, the amount of the compound in the food or beverage may generally be from 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is preferably 0.02 to 10 g based on 100 ml, Can be added in a proportion of 0.3 to 1 g.

The health beverage composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient, such as ordinary beverages, in addition to containing a mixture of the above-mentioned compounds as essential ingredients in the indicated ratios, have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia compounds (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Daphnane diterpenoid compounds represented by the following general formula (I) isolated from the original extract of the present invention are gefitinib, which is an epithelial growth factor receptor (EGFR) inhibitor. ) It was confirmed that the cells exhibiting strong anticancer efficacy in cancer cells resistant to cancer and normal cancer cells were useful for the treatment and prevention of existing cancer drug resistant cancer diseases.

1 shows micrographs of the growth inhibitory effect of gefitinib resistant lung cancer cells, which are epidermal growth factor receptor (EGFR) inhibitors of yuanhuadine and gefitinib of the present invention;
Figure 2 shows gefitinib resistance, which is an epithelial growth factor receptor (EGFR) inhibitor upon 24 hours (A) and 48 hours (B) treatment of yuanhuadine and gefitinib of the present invention. A cell cycle analysis of lung cancer cells;
FIG. 3 shows gefitinib resistance, which is an epidermal growth factor receptor (EGFR) inhibitor upon 24 and 48 hours treatment of yuanhuadine (A) and gefitinib (B) of the present invention. FIG. Graph showing cell cycle analysis of lung cancer cells;
4 is a diagram showing the effect on the expression of proteins by yuanhuadine and gefitinib in lung cancer cell lines and gefitinib resistant lung cancer cell lines;
FIG. 5 shows the efficacy of yuanhuadine in an in vivo tumor xenograft model of gefitinib resistant lung cancer cell line (H292-Gef); FIG.
FIG. 6 is a diagram showing the efficacy of yuanhuacine in an in vivo tumor xenograft model of a lung cancer cell line (H1993) having the characteristics of overexpressing epidermal growth factor receptor (EGFR) and c-Met;
FIG. 7 shows uanhuadine for Ki-67 in an ex vivo test of tissues obtained from an in vivo tumor xenograft model of lung cancer cell line (H292) and gefitinib resistant lung cancer cell line (H292-Gef). (yuanhuadine) shows the effect on the antiproliferative effect;
FIG. 8 shows huahua for epithelial growth factor receptor (EGFR) in tissue ex vivo tests from in vivo tumor xenograft models of lung cancer cell line (H292) and gefitinib resistant lung cancer cell line (H292-Gef). Shows the effect on the antiproliferative effect of Dean;
9 Inhibits PCNA and epidermal growth factor receptor (EGFR) protein of yuanhuadine from in vivo tumor xenograft models of lung cancer cell line (H292) and gefitinib resistant lung cancer cell line (H292-Gef) It is a figure which shows the effect on expression.

Hereinafter, the present invention will be described in detail with reference to the following Reference Examples and Experimental Examples.

However, the following Reference Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Reference Examples and Experimental Examples.

Example  1. From Original Extract Yuanhuadin ( yuanhuadine ) And Yuanhuacin ( yuanhuacine ) Separation of compounds

1-1. Preparation of Wonhwa Extract

10 kg of dried Wonhwa (Herbal Medicine) was extracted three times in a water bath three times using an ultrasonic extractor device with chloroform: acetone: methanol (1: 1: 1). The extract was filtered and concentrated under reduced pressure to obtain 386 g of the original extract.

1-2. Isolation of Compounds from Native Extracts

The original extract of 1-1 was dispersed in methanol: chloroform (2: 8), filtered and concentrated under reduced pressure to obtain 216 g of methanol and chloroform extract of the native extract, from which a daphnane type diterpenoids were obtained. ) Were isolated, identified and used in the present invention. WH obtained in Example 1-1 was subjected to silica gel column chromatography using chloroform, methylene chloride (1: 1 (v / v)) and methanol: chloroform (1: 1 (v / v)) solvent. To give three fractions. Among them, the second fraction having the highest cell growth inhibitory effect on human lung cancer cells (A549) was taken, and four fractions were obtained by ODS flash chromatography with 50-100% methanol. The third fraction having the highest activity was taken and six fractions were obtained by silica column chromatography using acetone: methylene chloride solvent gradient (5: 100 → 30: 100 (v: v)). The third fraction of this was 93% methanol using high performance liquid chromatography (ODS HPLC) to obtain yuanhuadine (340 mg) and yuanhuacine compound (202 mg) having physical properties as follows. Each was isolated and compared with the physical properties described in the literature to confirm the structure (He W et al (2002) Mini Rev Med Chem 2: 185-200; Zhang S et al (2006) Bioorg Med Chem 14: 3888-95). .

Yuanhuadin  ( yuanhuadine )

: White amorphous;

[?] _D ²⁰ +9.4 (c 0.09, CHCl ₃ );

UV (CHCl ₃ ) λ _max 245 (ε = 16853 M ⁻¹ cm ⁻¹ );

IR (film): 3464, 1702, 937 cm ^-1 ;

HRESIMS: m / z 587.2741 [M + H] ⁺ , 609.3289 [M + Na] ⁺ (C ₃₂ H ₄₂ O ₁₀ calcd 586.6795);

¹ H-NMR (CDCl ₃ , 300 MHz): δ 7.47 (1H, s, H-1), 6.61 (1H, dd, 15.6, 10.8, H-3 ′), 6.00 (1H, dd, 15.0, 10.8, H-4 '), 5.81 (1H, m, H-5'), 5.60 (1H, d, 15.6, H-2 '), 4.95 and 4.92 (2H, s, H-16), 4.91 (1H, s , H-12), 4.74 (1H, s, H-14), 4.20 (1H, s, H-5), 3.88 (1H, d, -14.8, H-20) and 3.69 (1H, d, -14.8 , H-20), 3.80 (1H, s, H-10), 3.48 (1H, d, overlapped, H-7), 3.47 (1H, s, H-8), 2.35 (1H, q, 7.2, H -11), 2.04 (2H, m, H-6 '), 1.93 (3H, s, H-2''), 1.78 (3H, s, H-17), 1.70 (3H, s, H-19) , 1.34 (2H, m, H-7 '), 1.26 (2H, m, H-9'), 1.24 (2H, m, H-8 '), 1.22 (3H, d, 7.2, H-18), 0.83 (3H, t, 3.0, H-10 ');

¹³ C-NMR (CDCl ₃ , 75 MHz): δ 209.1 (C-3), 169.8 (C-1``), 160.0 (C-1), 143.1 (C-15), 139.2 (C-5 '), 136.8 (C-2), 134.9 (C-3 '), 128.6 (C-4'), 122.5 (C-2 '), 117.0 (C-1'), 113.3 (C-16), 83.6 (C- 13), 80.4 (C-14), 78.2 (C-9), 78.3 (C-12), 72.7 (C-6), 70.7 (C-7), 65.1 (C-20), 64.0 (C-5 ), 61.3 (C-4), 47.4 (C-10), 43.9 (C-11), 35.2 (C-8), 32.6 (C-6 '), 31.3 (C-8'), 28.7 (C- 7 '), 22.5 (C-9'), 21.1 (C-2 ''), 18.7 (C-17), 18.2 (C-18), 14.0 (C-10 '), 9.9 (C-19).

Yuanhuacin  ( yuanhuacine )

: White amorphous;

[?] _D ²⁰ +9.3 (c 0.09, CHCl ₃ );

UV (CHCl ₃ ) λ _max 246 (ε = 20833 M- ¹ cm ^-1 );

IR (film): 3472, 1713, 937, 758 cm ⁻¹ ;

HRESIMS: m / z 649.2916 [M + H] ⁺ , 671.7529 [M + Na] ⁺ (C ₃₇ H ₄₄ O ₁₀ calcd 648.7504);

¹ H-NMR (CDCl ₃ , 300 MHz): δ 7.87 (2H, d, 7.2, H-3`` and 7 ''), 7.52 (1H, s, H-5 ''), 7.49 (1H, s , H-1), 7.38 (2H, t, 7.8, H-4 '' and H-6 ''), 6.68 (1H, d, 15. 6, 10. 5, H-3 '), 6.01 (1H , dd, 10. 5, 15, 0, H-4 '), 5.85 (1H, m, H-5'), 5.66 (1H, d, 15. 6, H-2 '), 5.19 (1H, s , H-12), 5.00 (1H, s, H-16), 4.96 (1H, s, H-16), 4.89 (1H, d, 2. 4, H-14), 4.13 (1H, s, H -5), 3.82 (1H, d, -12.4, H-20), 3.78 (1H, s, H-10), 3.74 (1H, d, -12.5, H-20), 3.59 (1H, d , 2.4, H-8), 3.57 (1H, s, H-5), 2.55 (1H, q, 7.2, H-11), 2.08 (2H, m, H-6 '), 1.84 (3H, s, H-17), 1.70 (3H, s, H-19), 1.37 (2H, m, H-7 '), 1.35 (3H, d, 7.2, H-18), 1.27 (2H, m, H-9 '), 1.26 (2H, m, H-8'), 0.86 (3H, t, 6. 6, H-10 '):

¹³ C-NMR (CDCl ₃ , 75 MHz): δ 209.2 (C-3), 165.5 (C-1``), 160.1 (C-1), 143.1 (C-15), 139.4 (C-5 ') , 136.9 (C-2), 135.1 (C-3 '), 133.3 (C-5''), 129.8 (C-2''), 129.5 (C-3''andC-7''), 128.7 (C-4 ', C-4 "and H-6''), 122.5 (C-2'), 117.1 (C-1 '), 113.7 (C-16), 83.9 (C-13), 80.5 ( C-14), 79.1 (C-12), 78.3 (C-9), 72.5 (C-6), 71.1 (C-7), 64.8 (C-20), 64.0 (C-5), 61.1 (C -4), 47.5 (C-10), 44.1 (C-11), 35.8 (C-8), 32.7 (C-6 '), 31.4 (C-8'), 28.8 (C-7 '), 22.6 (C-9 '), 18.8 (C-17), 18.4 (C-18), 14.1 (C-10'), 9.9 (C-19).

Experimental Example  1. Measurement of Growth Inhibitory Effect on Human Lung Cancer Cells and Drug-Resistant Cell Lines in Vitro

In order to confirm the growth inhibitory effect on lung cancer cells and drug-resistant cell lines in vitro of the sample obtained in the above example, the experiment was performed as follows by applying the method described in the literature (Lee SK et al (2008) Chem Biol Interact 115: 215-28).

Human lung cancer cell lines H292 and A549 were distributed by the Korea Cell Line Bank (KCLB, Seoul, Korea).

Gefitinib-resistant human lung cancer cell line H292 (H292-Gef) was used after obtaining resistance by continuously treating 1 μM of gefitinib to ge29ini. A549, H292, and H292-Gef cells were heat inactivated by 10% fetal bovine serum (Fetal Bovine Serum, FBS, 100 units / mL penicillin, 100 μg / mL streptomycin and 250 ng / mL vesicles). Using Roswell Park Memorial Institute (RPMI) medium (640, RPMI 1640) containing atherin b (amphotericin B) at 1 week at 37 ° C, 5% CO ₂ One or two passages were used, in particular, the gefitinib-resistant lung cancer cell line (H292-Gef) was treated with 1 μM of gefitinib, one day after passage, to maintain resistance. After dissolving from nitrogen, it was used for the experiment after passing three times or more.

The effect on the cell growth of the compounds obtained in Example 1 was measured by the sulforhodamine B (SRB) method (Lee et al (1998) Chemico-Biol Interact 115: 215-228).

Human non-small cell lung cancer and gefitinib resistant lung cancer cell lines used in the present invention were passaged in RPMI / MEME / DMEM medium containing 10% FBS, 1% PSF and the like. In each well of a 96-well plate, 10 μl of sample dissolved in 10% DMSO and 190 μl of the cell suspension (5 × 10 ⁴ cells / ml) were added and incubated for 3 days. 190 μl of the cell suspension was added to at least 16 wells and incubated for 30 minutes to be used as a zero-day control before the experiment. The cultured cells were fixed with 10% TCA (trichloroacetic acid), stained with SRB solution, dissolved in 10 mM Tris-base, and then absorbed at 515 nm. In the case of incubation in 10% DMSO as a control cell viability according to each test material treatment was measured using the following equation (1).

When the control group was not treated with the sample at 100%, the value of the sample treatment group was expressed as a percentage of the control group, and each test substance treatment was calculated as the average value ± SEM of the double or triple test. IC ₅₀ value is the concentration of test substance for 50% survival. Table 1 shows the effects of yuanhuadine, a native phenotype of daphnane diterpenoids, on non-small cell lung cancer and gefitinib-resistant lung cancer cell lines.

As shown in Table 1, the IC ₅₀ for the A549 cell line of Juanhuadine, which was found to be 10,000-fold higher in selectivity for lung cancer cells, was 12 nM, and in the H292 cell line, it was 1 pM or less and zefitinib. For H292-Gef cell lines that are resistant to (gefitinib), IC ₅₀ was maintained at 1 pM or less, as was the case for non-resistant H292 cell lines, to overcome drug resistance by yuanhuadine.

Cell line IC ₅₀ (μM) A549 12ⅹ10 ^-3 H292 <10 ^-6 (1 pM) H292-Gef <10 ^-6 (1 pM)

Experimental Example 2 Observation of Cell Morphology in Vitro

In order to confirm the effect of the sample obtained in the above example on the cell morphology of in vitro lung cancer cells and drug-resistant cell lines, experiments were carried out as follows by applying the method described in the literature.

Human lung cancer cells H292 and gefitinib resistant cell lines (H292-Gef) were suspended in 10% FBS-RPMI at 1 × 10 ⁴ cells / ml. 10 ml of cell suspension was attached to a 10 cm cell culture dish. The yuanhuadine (5-100 nM concentration) of Example 1 was added and then incubated at 37 ° C., 5% CO ₂ for a certain time. First, in order to observe the change in the shape of the cells, the cells were incubated for a certain time, then the culture medium was removed, washed twice with phosphate buffered saline (PBS), and then observed under the microscope at 100 times magnification. It was. The yuanhuadine (0.1-100 nM concentration) of Example 1 was added, and then incubated at 37 ° C. and 5% CO ₂ for a predetermined time. The results are shown in FIG. 1 (see FIG. 1).

As a result, cells treated with 0.1, 1, 10, and 100 nM of huahuadine in lung cancer cell line H292 decreased in a concentration-dependent manner compared to control cells. In addition, when gefitinib-resistant cell line H292-Gef was treated with gefitinib at concentrations of 1, 10, 100, and 1,000 nM, the number of cells was increased, and the shape of cells was also compared with the control group. When treated with 0.1, 1, 10, and 100 nM yuanhuadine, as shown in FIG. 1, the number of cells was reduced in the same concentration-dependent manner as in H292 cells. A change in shape was observed and a form of cell death was also observed.

Experimental Example 3. Measurement of Cell Cycle in Drug-Resistant Cancer Cells

In order to confirm the effect on the cell cycle of drug-resistant cancer cells of the sample obtained in the above example, the experiment was performed as follows (Chung HJ et al (2011) Food Chem Toxicol 49: 2942).

H292 cells, which are lung cancer cell lines or gefitinib-resistant lung cancer cell lines (H292-Gef), were diluted to 1 로 10 ⁵ per 100 mm culture dish with medium containing 10% FBS at 37 ° C, 5% CO ₂ After 24 hours of incubation in the conditions, washed twice with phosphate buffered saline (PBS). Add 10 ml of each medium containing 10% FBS diluted sample by concentration, incubate for a certain period of time, and collect the unattached cells and attached cells in the cell medium twice with phosphate buffered saline (PBS). After washing, 500 μl of 100% methanol, cooled with ice, was added to fix the cells at 4 ° C. for 16 hours. Washed twice with phosphate buffered saline (PBS), shaded in propidium iodide buffer (PI buffer) containing 50 μg / ml RNase A (R5500, Sigma, St Louis, MO) for 30 minutes, and allowed to stand at room temperature. The cell cycle was analyzed by using a polystyrene round-bottom tube and a facsimile flow cytometer (BD FACSCalibur, FACScaliburflowcytometer, BD Biosciences).

As a result of this experiment, as shown in FIG. 2 (A), zefitinib [gefitinib, ZD 1839, Iressa; G0 / G1 phase increased from 49.9% to 73.9% (100 nM) when treated with yuanhuadine for 24 hours in AstraZeneca Pharmaceuticals LP, Wilmington, DE] resistant lung cancer cell line (H292-Gef) S phase decreased from 22.7% to 2.7% (100 nM). Such G0 / G1 phase cell cycle arrest was also observed when cultured for 48 hours after yuanhuadine treatment as shown in FIG. 2 (B). The G0 / G1 phase increased from 50.9% to 68.3% (100 nM) and the S phase decreased from 23.1% to 5.9% (100 nM) compared to the control group. In the case of gefitinib treatment, there was no significant change in the G0 / G1 and S phase ratios compared to the control group when cultured for 24 hours and 48 hours after treatment (FIG. 3).

Experimental Example 4. Measurement of effect on apoptosis signaling system in lung cancer cell line and drug resistant lung cancer cell line

Effects on the apoptosis signaling system in the lung cancer cell line and drug-resistant lung cancer cell line of the sample obtained in the above example In order to confirm the effect, an experiment was performed as follows by applying the method described in the literature. (Hong JY et al (2011) J Nat Prod 74: 2102-8)

Several studies have shown that Akt / mTOR signaling pathway is activated by growth factor receptor tyrosine kinase, such as epidermal growth factor receptor (EGFR). This signaling system, consisting of Phosphatidylinositol 3-kinase (PI3K), Akt, and the mammalian target of rapamycin (mTOR), plays an important role in regulating cell proliferation, growth, and differentiation, and in many cancers, including lung cancer. Abnormalities in the delivery system have been found. When activated, extracellular signal regulated kinase (ERK) induces cyclin D1 activation in the nucleus, altering cell cycle from G1 to S phase, leading to DNA synthesis, which is also associated with growth and development of cancer, including cell proliferation, survival and differentiation. Known. Jurek A et al (2009) J Biol Chem 284: 4626-34

Src phosphorylates Src or releases a protein that inhibits Src activity, resulting in phosphorylation of Src, which is known to inhibit apoptosis and promote angiogenesis and angiogenesis. . Thus, inhibition of EGFR, PI3K / Akt / mTOR, ERK and Src signaling systems may be one target for treating cancer (Franklin WA (2002) Semin Oncol 29: 3-14).

To examine the effects on various signaling systems, lung cancer cell lines (H292) were plated with RPMI medium containing 10% FBS at a concentration of 1 ⅹ 10 ⁵ in a 100 mm culture dish and treated at 37 ° C and 5% CO ₂ . After 24 hours of incubation, the cells were washed twice with phosphate buffered saline (PBS). Gefitinib-resistant lung cancer cell line (H292-Gef) was diluted to 1.5 ⅹ 10 ⁵ per 100 mm culture dish in the same medium, incubated for 24 hours under the same conditions, and washed twice with phosphate buffered saline (PBS). It was. 10 ml of RPMI medium containing 10% FBS in which the samples were previously diluted by concentration was added thereto, and then cultured for a predetermined time. Collect the non-attached cells and attached cells in the cell medium, wash them twice with PBS, and then boil 2 ⅹ sample loading buffer (250 mM Tris-HCl, pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol blue , 2% β-mercaptoethanol, 50 mM sodium fluoride and 5 mM sodium orthovanadate) were added to disrupt the cells and placed at 100 ° C for 5 minutes. After cooling, the product was stored at 20 ° C and immediately dissolved before use at 37 ° C for protein quantitation and electrophoresis. 80 μg of protein was electrophoresed at 138 V for 2 hours using 5-12% SDS-polyacrylamide gel (# 456-1036, Bio-rad, Hercules, CA). The isolated protein was transferred to PVDF membrane (membrane, ISEQ15150, Millipore, Bedford, Mass.) For 1 hour at 100 V, washed twice with PBST and blocked with buffer (5% non-fat dry milk in PBS 0.1% Tween-20). (PBST)) and incubated in shaker for 1 hour at room temperature. After washing three times for 5 minutes with PBST, the single antibody was diluted with 3% non-fat dry milk with PBST and reacted with a membrane for 18 hours at 4 ° C. After washing the membrane (Membrane, ISEQ 1S150, Millipore, Bedford, MA) three times with PBST for 5 minutes, HRP (horseradish peroxidase) -bound secondary antibody was washed with PBST at a ratio of 1: 1,000 to 1: 2,000 with 3% skim milk powder. Diluted and reacted with the membrane (membrane) at room temperature for 3 to 4 hours. After washing 3 times with PBST for 5 minutes and treated with a chemiluminescent reagent (LabFrontier, Suwon, Korea) was confirmed the degree of protein expression in LAS-3000 (Fuji Film Corp., Japan).

Specifically, lung cancer cell line (H292) and gefitinib-resistant lung cancer cell line (H292-Gef) were treated with yuanhuadine 1, 10 nM and gefitinib 10, 100 nM, and cultured for 24 hours. Post-protein expression analysis confirmed the inhibition of signaling system in yuanhuadine lung cancer cell line (H292) and gefitinib-resistant lung cancer cell line (H292-Gef), as shown in Figure 4 lung cancer P-EGFR (Tyr 1068), p-Akt (Ser 473), p-mTOR (Ser 2448), p-ERK 1 / in gefitinib resistant cell line (H292-Gef) compared to cell line (H292) 2, p-Src (Tyr 416) protein was found to be overexpressed. In addition, activation of EGFR, Akt, and ERK 1/2 were inhibited in both lung cancer cell lines (H292) and gefitinib-resistant lung cancer cell lines (H292-Gef) when yuanhuadine 1, 10 nM was treated for 24 hours. It became. Subregulators, signal transducer and activator of transcription 3 (STAT3), and mTOR and Src activation were also significantly inhibited in a concentration dependent manner. Total EGFR protein expression was significantly inhibited concentration-dependently by yuanhuadine treatment, and total Akt, STAT3, ERK 1/2, Src protein expression did not change. Taken together, yuanhuadine inhibits Akt / mTOR, EGFR, ERK and Src signaling, thereby inhibiting lung cancer cell line (H292) cells as well as gefitinib-resistant lung cancer cell line (H292-Gef). It is judged to suppress the proliferation of.

Experimental Example  5. Zetitnib ( Gefitinib ) Resistant human lung cancer cell line ( H292 - Gef ) In vivo  In tumor xenograft models Yuanhuadin ( yuanhuadine ) Validation

In order to confirm the efficacy in a tumor xenograft model in vivo using Gefitinib-resistant human lung cancer cell line (H292-Gef) of the sample obtained in the above example, the experiment was performed as follows by applying the method described in the literature. (Hong et al (2011) J Nat Prod 74: 2102-2108).

Yuanhuadine based on in vitro growth inhibitory activity of yuanhuadine against gefitinib-resistant human lung cancer cell line (H292-Gef) constructed to be resistant to Zefitinib (Gefitinib) 1 μM ) Tumor growth inhibition efficacy was identified in a gefitinib resistant human lung cancer cell (H292-Gef) tumor xenograft model. Gefitinib at a dose of 0.5, 1.0 mg / kg when yuanhuadine was injected subcutaneously with H292-Gef at a concentration of 1 × ¹⁰ 7/200 μL and the tumor size reached 310 mm ³ after 20 days of injection. Was administered orally for 22 days at 10, 50 mg / kg concentration. Tumor size and body weight were measured at intervals of 3-5 days for 22 days. Tumor size after tumor transplantation was measured using mouse calipers to check drug toxicity at intervals of 3-5 days for 21 days. Tumor volume was calculated by Equation 2 below.

As a result, in the group treated with gefitinib, tumor growth inhibition rate was lower than that in yuanhuadine group, and yuanhuadine was compared with zefitinib, regardless of the drug concentration increase. Inhibition of tumor growth was concentration dependent even at significantly low concentrations (FIG. 5). After 22 days of drug administration, tumor tissues were detached and size and weight were measured. Tumor growth inhibition rate was determined based on the control group not treated with active substance, and yuanhuadine 0.5 mg / kg was 60.4%, With 1.0 mg / kg of 68.8% inhibition rate, yuanhuadine inhibited tumor growth in a concentration-dependent manner, and gefitinib showed tumor inhibition with 24.7% for 10mg / kg and 33.3% for 50mg / kg. Growth was inhibited (Table 2). Weight change and other side effects were not observed by oral administration of yuanhuadine.

Tumor Volume Inhibition Rate in Xenograft Models of Gefitinib-resistant Human Lung Cancer Cell Lines density
(mg / kg) Yuanhuadine Zefitinib 0.5 One 10 50 Inhibition rate (%) 60.4 68.6 24.7 33.3

Experimental Example  6. Genetic variation or expression is different Lung cancer cell line  ( H1993 ) In vivo  In tumor xenograft models Yuanhuacin  ( yuanhuacine ) Validation

In order to confirm the efficacy of the tumor xenograft model in vivo using a lung cancer cell line (H1993) whose genetic variation or expression of the sample obtained in the above example was different, an experiment was performed as follows (Hong et. al (2011) J Nat Prod 74: 2102-2108).

Based on the growth inhibitory activity of yuanhuacine in vitro human lung cancer cells (H1993), tumor growth inhibition effect of yuanhuacine was confirmed in H1993 xenograft nude mouse animal model.

Human lung cancer cell H1993 is a mutated c-Met cell and has an overexpression of epidermal growth factor receptor (EGFR) and c-Met. When human lung cancer cells H1993 were injected subcutaneously at a concentration of 1 × ¹⁰ 7/200 μl cells, and when the tumor size reached 90 mm ³ after 22 days of injection, oral huahuacine (yuanhuacine) at a concentration of 0.5, 1.0 mg / kg daily for 21 days Administered. Changes in tumor size and body weight were measured at intervals of 4 to 6 days for 21 days. Yuanhuacine inhibited tumor growth in a concentration-dependent manner (FIG. 6). Tumor tissue was removed and size and weight measured. Tumor growth inhibition rate was obtained based on the control group that did not receive the active substance, and the concentration of yuanhuacine was increased at 33.4% when 0.5 mg / kg yuanhuacine was administered and 38.8% when 1.0 mg / kg was administered. Dependently inhibited tumor growth (Table 3). Weight change and other side effects were not observed by oral administration of the substance.

Tumor Volume Inhibition Rate in Xenograft Model of Human Lung Cancer Cell Line (H1993) density
(mg / kg) Yuanhuacine Zefitinib 0.5 One 10 Inhibition rate (%) 33.4 38.8 35.9

Experimental Example 7. Validation of yuanhuadine on the expression of Ki-67 and epidermal growth factor receptor (EGFR), a cell proliferation factor through immunohistochemical staining from tumor tissue

In order to confirm the effectiveness of Ki-67, which is a cell proliferation factor, and epidermal growth factor receptor (EGFR) expression through immunohistochemical staining from tumor tissues of the samples obtained in the above examples, the method described in the literature was applied as follows. Experiments were performed (Suzuki S (2005) Cancer 103: 1265-73)

7-1. Ki -67 effect on expression

It is believed that the tumor growth inhibitory effect of a substance derived from Won is related to inhibition of cell proliferation. Cell proliferation is associated with a nuclear protein called Ki-67, which accumulates during the G1 and mitosis stages, reaching peak levels during cell division. Since the amount of nuclear protein decreases to a minimum immediately after cell division, it is widely known as a factor for labeling proliferating cells. Based on this, immunohistochemical staining (IHC) experimental technique was used to investigate the morphological effect of yuanhuadine Ki-67 expression.

The experimental method was fixed in formalin and embedded in paraffin, and the paraffin blocks were cut into 4-6 thicknesses and attached to glass slides. Tumor-attached glass slides were soaked in xylene, 100% ethanol and 70% ethanol in order to remove paraffin and placed in primary distilled water to hydrate. Tissue slides were put in boiled citric acid buffer (PH 6.0) for antigen recovery and boiled in a microwave for 10 minutes. After cooling for 10 minutes at room temperature, 3% H ₂ O ₂ reagent was dropped on the tissue and allowed to stand at room temperature for 15 minutes to block the endogenous peroxidase in the tissues that were zoned and reacted with DAB. . After washing with primary distilled water, and washed in a chamber containing primary distilled water for 5 minutes, and then again washed in a chamber containing PBS for 5 minutes. Thereafter, 150 μl of the primary antibody previously diluted with PBS was dropped onto the tissue and left overnight at 4 ° C. Thereafter, the primary antibody was washed with PBS for 10 minutes, and 150 μl of the biotin-labeled secondary antibody was left in the refrigerator for 1 hour, reacted at room temperature for 1 hour, and then washed with PBS. 150 μl of Streptavidin-HRP (# K0679, LSAB + System-HRP Kit, Dako, Japan) was dropped into the tissue and then refrigerated for 1 hour, and then placed at room temperature for 1 hour and washed with PBS for 20 minutes. 1 drop of DAB chromogen (# K0679, LSAB + System-HRP Kit, Dako, Japan) and 1 ml of DAB were mixed immediately before treatment, treated with tissue and observed under a microscope while the tissue was properly developed. . After 7 minutes of washing with primary distilled water, a control dyeing reaction was performed with hematoxylin (MHS32-1L, Sigma, St Louis, MO) for 5 minutes at room temperature, and washed three times with primary distilled water for 5 minutes. Subsequently, it was dehydrated by soaking in 70% EtOH for 1 minute, 5 minutes in 100% EtOH, and 5 minutes in xylene. After dropping the mount solution on the tissue and cover with the cover, the mounting solution was hardened properly and the slide was turned over and observed under a microscope.

As a result, when the H292 xenograft tumor tissue was stained with Ki-67 antibody, Ki-67 positive cells were stained brown in the nucleus, and the degree of staining was higher in the yuanhuadine-treated group than in the control group. It was confirmed that the number of cells stained with (Fig. 7A). Similar trend was observed in gefitinib, a positive control. In the H292-Gef xenograft tumor tissues, the number of Ki-67 positive stained cells in the yuanhuadine-treated group decreased compared to the control group, but the gefitinib-treated group was not significantly different from the control group. 7B). Therefore, Yuanhuadine has tumor growth inhibitory effects on not only lung cancer cell line (H292) cells but also gefitinib-resistant lung cancer cell line (H292-Gef) in in vivo animal experiments. Tumor growth inhibitory effects in animal experiments were found to be related to the inhibition of Ki-67, a cell proliferation effector.

7-2. Epidermal growth factor receptor ( EGFR ) Influence on expression

The immunohistochemical staining technique was used to investigate the effect of yuanhuadine on epithelial growth factor receptor (EGFR) inhibition. When H292 xenograft tumor tissue, a human lung cancer cell line, was stained with an epidermal growth factor receptor (EGFR) antibody, the cells of the epidermal growth factor receptor (EGFR) positive stained the cell membrane and the cytoplasmic part in brown. It was confirmed that the number of cells stained with epidermal growth factor receptor (EGFR) positive in the yuanhuadine administration group (Fig. 8). Similar trend was observed in gefitinib, a positive control. In addition, in H292-Gef xenograft tumor tissues, the number of cells stained with EGFR positive was decreased in the yuanhuadine-administered group compared to the control group, but the gefitinib-treated group was significantly different from the control group. Seemed to be absent. Thus, yuanhuadine has an inhibitory effect on epidermal growth factor receptor (EGFR) expression not only in lung cancer cell line (H292) cells but also in gefitinib resistant lung cancer cell line (H292-Gef). Could confirm.

Experimental Example 8. Validation of Yuanhuadine on the expression of proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR) by analyzing protein expression from tumor tissue

In order to confirm the effectiveness of proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR) expression through analysis of danprotein expression from tumor tissues of the sample obtained in the above example, the method described in the literature was applied as follows. Experiments were performed (Jean Y et al (2008) Eur J Pharmacol 578: 323-31).

PCNA is a kind of nuclear protein that shows cell proliferation ability. It is known to be the best in the cell cycle S phase and plays an important role in causing cell proliferation. Epidermal growth factor receptor (EGFR) is overexpressed in a variety of solid cancers, including non-small cell lung cancer, and proliferates cells during overexpression, inhibits apoptosis, and promotes invasion, metastasis and angiogenesis, resulting in tumor growth and progression. Will be promoted.

Lung cancer cell line (H292) and gefitinib resistance to determine whether tumor growth inhibition efficacy in xenograft experiments in vivo is due to PCNA inhibition or epidermal growth factor receptor (EGFR) inhibition of yuanhuadine. The expression level of PCNA and epidermal growth factor receptor (EGFR) protein in xenograft tumor tissue of lung cancer cell line (H292-Gef) was analyzed by western blot analysis. After oral administration of the drug yuanhuadine 1 mg / kg and gefitinib 50 mg / kg for 21 days, tumor tissues of nude mice were excised and protein was isolated and analyzed for protein expression. As a result, the yuanhuadine 1 mg / kg treated group showed PCNA and epithelial growth factor in xenograft tumor tissues of lung cancer cell line (H292) and gefitinib resistant lung cancer cell line (H292-Gef) in vivo. It was confirmed that the receptor (EGFR) protein expression is inhibited (FIG. 9).

Hereinafter, formulation examples of the composition containing the compound of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

Yuanhuadine ------------------------------------------ 200 mg

Lactose ------------------------------------------------ 100 mg

Talc ------------------------------------------------- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation Example 2. Preparation of tablets

Yuanhuacin ------------------------------------------ 200 mg

Corn Starch ------------------------------------------ 100 mg

Lactose ------------------------------------------------ 100 mg

Magnesium Stearate ----------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation Example 3. Preparation of capsules

Yuanhuacin ------------------------------------------ 200 mg

Crystalline Cellulose ------------------------------------- 3 mg

Lactose ------------------------------------------- 14.8 mg

Magnesium Stearate ------------------------------- 0.2 mg

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example  4. Preparation of injections

Yuanhuadine ------------------------------------------ 200 mg

Mannitol ---------------------------------------------- 180 mg

Sterile sterile distilled water for injection --------------------------------- 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O ----------------------------------------- 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

Yuanhuadine ------------------------------------------ 200 mg

Isomerized sugar ---------------------------------------------- 10 g

Mannitol ------------------------------------------------- 5 g

Purified Water ------------------------------------------------ Appropriate

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

Yuanhuacin ------------------------------------------ 1000 mg

Vitamin mixture -------------------------------------------

Vitamin A Acetate ----------------------------------- 70 μg

Vitamin E --------------------------------------------- 1.0 mg

Vitamin B1 ------------------------------------------- 0.13 mg

Vitamin B2 - 0.15 mg

Vitamin B6 -------------------------------------------- 0.5 mg

Vitamin B12 ------------------------------------------- 0.2 g

Vitamin C ---------------------------------------------- 10 mg

Biotin ------------------------------------------------ 10 G

Nicotinamide --------------------------------------- 1.7 mg

Folic acid ------------------------------------------------- - 50 [mu] g

Calcium Pantothenate ---------------------------------------- 0.5mg

Inorganic mixture --------------------------------------------------------------------------

Ferrous sulfate 1.75 mg &lt; RTI ID = 0.0 &gt;

Zinc oxide - 0.82 mg

Magnesium carbonate ---------------------------------------- 25.3 mg

Potassium monophosphate ------------------------------------------ 15 mg

Dibasic calcium phosphate ------------------------------------------- 55 mg

Potassium Citrate -------------------------------------------- 90 mg

Calcium Carbonate --------------------------------------------- 100 mg

Magnesium chloride ---------------------------------------- 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

Yuanhuadine ------------------------------------------ 1000 mg

Citric acid ---------------------------------------------- 1000 mg

Oligosaccharides ---------------------------------------------- 100 g

Plum concentrate ---------------------------------------------- 2 g

Taurine ------------------------------------------------- - 1 g

Add purified water --------------------------------- 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, &Lt; / RTI &gt;

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (13)

A pharmaceutical composition for treating and preventing a target anticancer drug-resistant cancer disease comprising a daphnane diterpenoid compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient:

(I)
In this formula,
R is a hydrogen atom, a C1-C3 lower alkyl group, a C1-C3 lower alkoxy group, or a phenyl group substituted with R ', wherein R' is a hydrogen atom, a C1-C3 lower alkyl group, or a C1-C3 lower alkoxy group. The method of claim 1,
A pharmaceutical composition wherein the R substituent of the compound of formula (I) is a hydrogen atom, a methyl group, an ethyl group, or a phenyl group. 3. The method of claim 2,
The general formula (I) compound is a pharmaceutical composition is one or more compounds selected from the group consisting of yuanhuadine and or yuanhuacine. The pharmaceutical composition of claim 1, wherein the compound exhibits cytotoxicity in cancer cells resistant to normal cancer cells and epidermal growth factor receptor (EGFR) target anticancer agents. The pharmaceutical composition of claim 1, wherein the compound exhibits apoptosis inducing effect similar to or better than that of normal cancer cells in drug resistant cancer cells. The method of claim 1, wherein the target anticancer agent is gefitinib, temozolomide, bevacizumab, erlotinib, capecitabine, or imatinib. Pharmaceutical composition characterized in that. According to claim 1, wherein the cancer disease gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine cancer, ovarian cancer, colon cancer, small intestine cancer , Rectal cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer Thyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor Pharmaceutical composition selected from primary CNS lymphoma, spinal cord tumor, brain stem glioma or pituitary adenoma. Treatment of a target anticancer drug-resistant cancer disease containing as an active ingredient a daphnane diterpenoid compound represented by the general formula (I) of claim 1 or an pharmaceutically acceptable salt thereof Anticancer supplements for prevention. A daphnane diterpenoid compound represented by the general formula (I) of claim 1 isolated from the original extract, or a combination containing a pharmaceutically acceptable salt thereof and an existing anticancer agent as an active ingredient. 10. The method of claim 9, wherein the existing anticancer agents include folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine), pyrimidine derivatives (5-fluorouracil, cytarabine); Nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine); Actinomycin D, doxorubicin, bleomycin, mitomycin, vincristine, vinvillastin, taxoid; Corticosteroids, progesterone; Or cisplatin. Prevention of a target anticancer drug-resistant cancer disease comprising as an active ingredient a daphnane diterpenoid compound represented by the general formula (I) of claim 1 isolated from the original extract or a pharmaceutically acceptable salt thereof; and Health functional food for improvement. 12. The dietary supplement of claim 11, wherein the dietary supplement is in the form of a powder, granule, tablet, capsule or beverage. A daphnane diterpenoid compound represented by the general formula (I) of claim 1 isolated from the original extract having the effect of preventing and improving the target anticancer drug resistant cancer disease or a pharmaceutically acceptable salt thereof is effective. Food or food additives contained as ingredients.

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