KR20210019330A - Anti-cancer composition for pancreatic cancer using Lapathoside A - Google Patents

Abstract

The present invention discloses an anticancer composition for pancreatic cancer using Lapathoside A, which exhibits cytotoxicity to a pancreatic cancer cell line PANC-1 cell and inhibits the activation of AKT and FAK (phosphorylation of AKT and FAK). The anticancer composition of the present invention can be commercialized as a drug or food for purposes of treating, preventing, or inhibiting metastasis of pancreatic cancer.

Description

Anti-cancer composition for pancreatic cancer using Lapathoside A}

The present invention relates to an anti-cancer composition for pancreatic cancer using Lapathoside A.

Pancreatic cancer is very difficult to detect early, and in most cases it is detected at the end of the cancer, so the mortality rate is very high. Pancreatic cancer is an organ that is surrounded by several organs. It is difficult to operate and has a high recurrence rate. The survival rate for reoperation is only 25%, and 90% of the deaths have metastasized to other tissues well enough that pancreatic cancer has metastasized to other areas. It has been reported that the prognosis is very poor. There is also a report that only 5% of all patients with pancreatic cancer have an average survival rate of 5 years after diagnosis.

As a treatment method, the survival rate is improved by chemotherapy and radiation therapy rather than resection, but there is no significant change in the long-term survival rate.

Akt, called protein kinase B, is a serine/threonin kinase that is known to be activated through the upstream kinase PI3K pathway. It is a very important regulator that regulates various physiological activities such as cell proliferation and differentiation and angiogenesis. Works. It has been reported that the PI3K/Akt signaling pathway is overactivated in several carcinomas, and when a signal is transmitted to PI3K by stimulating factors such as insulin, Akt moves from the cytoplasm to the cell membrane, and serine 473 residue is phosphorylated to become an activated form ( Adv. Cancer Res. 94, 29-86, 2005). Through this activity, Akt activates sub-molecules such as GSK3β, FOXOs and mTOR to promote cell proliferation and differentiation, and by inactivating proapoptotic factors such as BAD and FKHR (procaspase-9, forkhead) transcription factors, etc. (Biomed. Pharmacother. 64. 600-604, 2010; Apoptosis 9, 667-676, 2004)

FAK (focal adhesion kinase) is a 125 kDa non-receptor protein tyrosine kinase (nRTK) that is known to be involved in cell survival, proliferation, metastasis, and invasion in normal and cancer cells. FAK is overexpressed in a variety of carcinomas including pancreatic cancer (Oncotarget. 2015, 6:4757?4772; BioMed Res. Int. 2015, 2015:690-690; Cell Biol. 2003, 160:753-767; J Clin Med. 2019) , 8(1):38; CANCER BIOLOGY & THERAPY 2018, 19(4):316-327), particularly in pancreatic cancer, inhibition of FAK expression has been reported to inhibit cancer metastasis and invasion (Anticancer Res. 2005;25(Anticancer Res. 2005;25( 3B):2017?2025). FAK is activated by phosphorylation by Src at the Y397 (tyrosine-397) position (Mol Cell Biol. 1995;15:954-63; J Cell Sci. 2003;116:1409-16), currently developing a therapeutic agent for pancreatic cancer. It has been proposed as a target of (CANCER BIOLOGY & THERAPY 2018, 19(4):316-327).

The present invention discloses that rapatoside A exhibits cytotoxicity to PANC-1 cells, a pancreatic cancer cell line, and inhibits the activation of AKT and FAK.

The present invention discloses an anticancer composition for pancreatic cancer using Rapatoside A.

Other or specific objects of the present invention will be presented below.

The present invention is completed by confirming that rapatoside A exhibits cytotoxicity to the pancreatic cancer cell line PANC-1 cell and inhibits the activation of AKT and FAK (phosphorylation of AKT and FAK), as confirmed in the examples below. It was done.

Considering the above, the anticancer composition for pancreatic cancer according to the present invention is characterized by comprising as an active ingredient rapatoside A, a pharmaceutically acceptable salt thereof, a solvate thereof, a hydrate thereof, or an isomer thereof.

In the following examples, the present inventors used Rapatoside A by separating and identifying it from Yangjeol buckwheat, one of the varieties of buckwheat, but Rapatoside A is a known substance reported to exist in Polygonum lapathifolium, etc.(Tropical Journal of Pharmaceutical Research 2014; 13 (10): 1749-1759) The chemical formula is as follows.

<Formula 1>

In the present specification, "anti-cancer against pancreatic cancer" is meant to include inhibition of proliferation, induction of death, and inhibition of metastasis to other organs.

In addition, in the present specification, the term "active ingredient" refers to an ingredient capable of exhibiting a desired activity alone or with a carrier that is not itself active.

Rapatoside A, an active ingredient in the present invention, may be used in the form of a pharmaceutically acceptable salt, and as such a salt, it may be used as an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, butin-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, me Toxoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol Rate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate, but are not limited thereto. The acid addition salt in the present invention is a conventional method, for example, by dissolving rapatoside A in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., and then adding an organic or inorganic acid to filter the precipitate. , Dried, or dried after distilling a solvent and an excess of acid under reduced pressure, or crystallized under an organic solvent.

In addition, rapatoside A, an active ingredient in the present invention, can make a pharmaceutically acceptable metal salt using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

In addition, rapatoside A, which is an active ingredient in the present invention, may be used not only in its pharmaceutically acceptable salts, but also in the form of possible solvates, hydrates, isomers, etc. that can be prepared therefrom.

In the composition of the present invention, the active ingredient may be included in an arbitrary amount (effective amount) according to the specific formulation, as long as it can exhibit anticancer activity, and the usual effective amount is 0.0001% by weight to 20.0 based on the total weight of the composition. It will be determined within the range of weight percent. Herein, "effective amount" refers to the intended medical and pharmacological effects such as anticancer effects on pancreatic cancer when the composition of the present invention is administered to a mammal, preferably a human, to which the composition of the present invention is administered during the administration period according to the recommendations of a medical professional It refers to the amount of the active ingredient contained in the composition of the present invention. Such effective amounts can be determined empirically within the range of ordinary skill in the art.

The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the meaning of "pharmaceutically acceptable" means that the application (prescription) does not have toxicity beyond adaptable without inhibiting the activity of the active ingredient.

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier It can be prepared in such a formulation. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable Yu, etc. are mentioned. In the case of formulation activity, if necessary, it may be formulated including diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

When the pharmaceutical composition of the present invention is prepared in a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterilization for injection Water, isotonic solutions such as 5% dextrose can be used. When formulated into a transdermal dosage form, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, liniment, air roll, and the like. In the case of nasal inhalants, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide can be used to form an aerosol spray.When formulated as a suppository, the base is Withepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like.

Regarding the formulation of a pharmaceutical composition, it is known in the art, and specifically, Remington's Pharmaceutical Sciences (19th ed., 1995), etc. may be referred to. These documents are considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. May be in the /kg range. Administration can be made once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any aspect.

The composition of the present invention can be grasped as a food composition in a specific aspect.

The food composition of the present invention can be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, ionized beverages, processed oils such as milk, yogurt, gums, rice cakes, Korean sweets, bread, sweets, noodles, etc. Foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, and health functional food preparations such as bars can be prepared. In addition, the food composition of the present invention can be classified as a product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food according to the Korean "Health Functional Food Act", or confectionery, beans, tea, beverages according to each food type according to the food code of the Korean Food Sanitation Act (notified by the Ministry of Food and Drug Safety, food standards and standards), It may be a special purpose food.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to food and mixed or infiltrated in manufacturing, processing, or preserving food. Since they are consumed daily and for a long time with food, their safety must be ensured. In the Food Additive Code (“Food Sanitation Act” in Korea) governing the manufacture and distribution of food, food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (KFDA notice "Food Additive Standards and Specifications), food additives are classified into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients. These food additives are sweeteners and flavoring agents in terms of function. , Preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart a suitable sweetness to foods, and both natural and synthetic ones can be used in the food composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to improve taste or flavor, and both natural and synthetic can be used. Preferably, it is the case of using a natural one. In the case of using natural ingredients, the purpose of nutrient enhancement can be combined in addition to flavor. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, green leaves, large leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, you can use those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, synthetic flavoring agents may be used. As the synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, and the like may be used.

As a preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, pectin Etc. may be used, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like can be used. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to the purpose of enhancing taste.

As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include a physiologically active substance or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutritional properties and ensuring stability as a food additive.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, etc., and minerals include calcium preparations such as calcium citrate, magnesium stearate. Magnesium preparations, such as magnesium preparations, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

In the food composition of the present invention, the food additive as described above may be included in an appropriate amount to achieve the purpose of addition according to the product type.

With respect to other food additives that may be included in the food composition of the present invention, reference may be made to the food code or food additive code according to the Food Sanitation Act.

As described above, according to the present invention, it is possible to provide an anticancer composition for pancreatic cancer using rapatoside A.

The anticancer composition of the present invention may be commercialized as a drug or food for use in the treatment, prevention, and metastasis inhibition of pancreatic cancer.

FIG. 1 is a result showing that rapatoside A has cytotoxicity in PANC-1 cells, a pancreatic cancer cell line (A), and Western blot results for AKT, FAK and their active forms (B).

Hereinafter, the present invention will be described with reference to examples. However, the scope of the present invention is not limited to these examples.

<Example> Anticancer activity test of rapatoside A against pancreatic cancer

1. Experimental method

1.1 Cytotoxicity test on PANC-1 cell, a pancreatic cancer cell line

Cell viability for PANC-1 cells, a pancreatic cancer cell line, was measured using the WST-1 assay kit. Cells were seeded at 1.25×10 ⁴ /well in each 24 well plate, and Lapathoside A dissolved in DMSO after 24 hours was treated by concentration. DMSO was treated as a control group.

After 72 hours, for cell viability assay, it was exchanged with a medium to which 10% WST-1 reagent was added, and then reacted in a 37°C, 5% CO ₂ incubator for about 30 minutes. After the reaction was completed, the absorbance was measured at 450 nm using a Multiskan GO spectrophotometer (Thermo Scientific).

1.2 Western Blot

For Western blot samples, PANC-1 cells were seeded in a 6 well plate, and DMSO was treated in the control group 24 hours later, and lapathoside A was treated by concentration in the experimental group.

M-PER™ Mammalian Protein Extraction Reagent, the final concentration of 2mM sodium vanadate, 30mM sodium pyrophosphate, 100mM sodium fluoride, 0.1M PMSF, 1X Protein inhibitor was dissolved in a lysis buffer made to the cells and scraped off.

Quantify the amount of protein in each sample by the Bradford assay method, mix it with sample buffer, and heat for 10 minutes. Proteins were separated by SDS-PAGE on 12% SDS polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking this membrane in 1X TBST solution containing 5% skim milk, primary antibodies (Akt, FAK, caspase-3, GAPDH, etc.) were added and reacted overnight at 4°C. Secondary antibody was reacted at room temperature for 1 hour using Donkey anti-Mouse IgG Antibody or Donkey anti-Rabbit IgG Antibody, and then the expressed protein was measured using BS ECL plus kit (Biosesang).

2. Experiment result

2.1 Cytotoxicity test results

The results of cytotoxicity experiments on pancreatic cancer cell lines are shown in Fig. 1(A).

Referring to FIG. 1(A), rapatoside A showed cytotoxicity to pancreatic cancer cell lines in a concentration-dependent manner.

2.2 Western Blot Results

Western blot results are shown in Fig. 1(B).

Referring to Figure 1(B), it is shown that rapatoside A inhibits the activation of ATK and FAK.

Claims (4)

An anticancer composition for pancreatic cancer comprising rapatoside A as an active ingredient.
The method of claim 1,
The anti-cancer composition, characterized in that for inhibiting the proliferation of pancreatic cancer cells, inducing death or inhibiting metastasis to other organs.
The method according to claim 1 or 2,
The composition is a composition, characterized in that the food composition.
The method according to claim 1 or 2,
The composition is a pharmaceutical composition, characterized in that the composition.

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