WO2015199454A1

WO2015199454A1 - Receptor tyrosine kinase inhibitor-resistant anticancer drug composition comprising 3,4,5-trihydroxybenzoic acid, derivative thereof or salt thereof as active ingredient

Info

Publication number: WO2015199454A1
Application number: PCT/KR2015/006464
Authority: WO
Inventors: 정양식; 김기우; 모상현
Original assignee: 연세대학교 원주산학협력단
Priority date: 2014-06-25
Filing date: 2015-06-25
Publication date: 2015-12-30
Also published as: KR101751371B1; KR20160008105A

Abstract

The present invention relates to a composition for preventing or treating a cancer resistant to a target anticancer drug, the composition including, as an active ingredient, a 3,4,5-trihydroxybenzoic acid (gallic acid) compound or a derivative thereof which specifically inhibits cancer cells that exhibit resistance to an anticancer drug targeting receptor tyrosine kinase. Since the 3,4,5-trihydroxybenzoic acid (gallic acid) compound or a derivative thereof specifically kills cancer cells that exhibit resistance to an anticancer drug targeting receptor tyrosine kinase, cancer diseases that are resistant to a receptor tyrosine kinase inhibitor such as gefitinib can be prevented or treated.

Description

Receptor tyrosine kinase inhibitor resistant anticancer composition comprising 3,4,5-trihydroxybenzoic acid, derivatives thereof or salts thereof as an active ingredient

The present invention provides a 3,4,5-trihydroxybenzoic acid (Trihydroxybenzoic acid, gallic acid) compound or a derivative thereof that specifically inhibits cancer cells that are resistant to an anticancer agent targeting a receptor tyrosine kinase. It relates to a composition for preventing or treating cancer diseases resistant to a target anticancer agent comprising as an active ingredient.

According to the recent announcement of the Central Cancer Registry, cancer incidence in Korea has increased by 3.5% every year for the past 10 years. In 2010, more than 200,000 people were newly diagnosed with cancer. If a Korean male lives up to the age of 77, the probability of getting cancer is nearly two in five, and one in three if a female lives up to 84. Cancer is becoming an increasingly common disease. In 2008, there were 12.7 million new cancer patients, with 5.6 million in developed countries and 7.1 million in developing countries. It is also expected that 201 million new cancer cases will occur by 2030, with 13 million cancer deaths. As the cancer incidence continues to increase not only in Korea but also in the world, the importance of developing effective anticancer drugs is also increasing. The global market for cancer drugs exceeded 70 trillion won in 2010 and reached 84 trillion won in 2013. The results of this continuous market growth support the active research around the world for the development of anticancer drugs.

Existing anticancer drugs used drugs that have strong toxicity that can induce cytotoxicity to block the cell cycle of each stage, but this toxicity has caused side effects such as nausea, vomiting and hair loss by attacking normal cells. However, in recent years, in order to overcome the side effects of the existing cytotoxic anticancer drugs, researches on target anticancer drugs targeting only cancer cells have been in the spotlight. The target anticancer agent is to prevent cancer cells by administering a substance that reacts by targeting the factors that are related to the abnormal process of cell mutation by noting that the cancer cells are generated by the genetic mutation process of normal cells. . Since targeted anticancer drugs have little effect on normal cells, they are attracting attention as next-generation anticancer drugs with relatively low side effects. Accordingly, investments and researches for preoccupying the target anticancer drug market globally are proceeding competitively.

Recently developed tyrosine kinase inhibitors of epidermal growth factor receptors (EGFR-TKIs) have attracted attention as novel therapeutics for non-small cell lung cancer as molecular target therapeutics (Shepherd FA et al (1994) Semin Oncol 21: 7-18).

Epidermal growth factor receptor (EGFR) is the first known growth factor receptor among receptor tyrosine kinases and is a 170 kilodalton (kDa) membrane-binding protein expressed on the surface of epithelial cells. Epidermal growth factor receptors are members of the growth factor receptor family of protein tyrosine kinases, a class of cell cycle regulatory molecules (W. J. Gullick et al., 1986, Cancer Res., 46: 285-292). Epithelial growth factor receptors are activated when their ligand, epidermal growth factor (EGF), binds to the extracellular domain, resulting in autophosphorylation of the intracellular tyrosine kinase domain of the receptor (S. Cohen et al., 1980). , J. Biol. Chem, 255: 4834-4842; AB Schreiber et al., 1983, J. Biol. Chem., 258: 846-853) signaling pathways are activated, resulting in cell proliferation.

Epithelial growth factor receptors are protein products of the growth promoting oncogene erbB or ErbB1, members of the ERBB family of primary cancer genes and are believed to play a central role in the development and progression of many human cancers. In fact, the expression of receptors that are overexpressed or continuously activated in mutant form have been observed in glioblastma, as well as in breast cancer, bladder cancer, lung cancer, head cancer, cervical cancer and gastric cancer.

Cancer is caused by uncontrolled cell proliferation as a result of genetic mutations that affect the regulation of growth factor receptors or induce overexpression of receptors and / or ligands.

In particular, epidermal growth factor receptors have been shown to play an important role in cell differentiation, enhanced cell motility, protein secretion, angiogenesis, invasion, metastasis and resistance of cancer cells to chemotherapeutic agents and radiation (MJ Oh et al. , 2000, Clin. Cancer Res., 6: 4760-4763).

Along with the hypothesis that inhibiting epidermal growth factor receptors may be a target of new chemotherapy, the therapeutic methods include monoclonal antibodies that inhibit receptor binding of ligands and small molecules that inhibit the autophosphorylation of tyrosine kinases of intracellular domains. There was a presentation of the compound (Kawamoto et al (1983) Proc. Natl. Acad. Sci. USA 80: 1337-1341).

Thus, epidermal growth factor receptors have been the target of choice for the development of several different cancer therapies. EGFR tyrosine kinase inhibitors (EGFR-TKIs) are one of these therapies because activation of the epidermal growth factor receptor pathway requires reversible phosphorylation of tyrosine residues. In other words, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) block the activity of receptors on the cell surface that are responsible for triggering and / or maintaining the signaling pathways of cells that cause tumor cells to grow and differentiate. Specifically, these inhibitors block the receptor's activity by blocking the adenosine triphosphate (ATP) binding domain of the epidermal growth factor receptor kinase termed HER-1, preventing ATP from binding anymore. . Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) are known as quinazolines pyridopyrimidines, and pyrrolopyrimidines compounds.

Recently, various inhibitors on epidermal growth factor receptors have been identified, and two new drug-approved quinazolines compounds after clinical development have been identified: Compound ZD1839 developed by Gefitinib (AstraZeneca UK Ltd.); Commercially available under the trademark IRESSA) and Erlotinib (compound OSI774 developed by Genentech, Inc. and OSI Pharmaceuticals, Inc .; available under the trade name TARCEVA), both of which gave encouraging clinical results ( Presentations at ASCO 2003 and WCLC, Aug. 2003).

In particular, gefitinib is the first oral epithelial growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), the first commercially available oral drug, which has been shown to inhibit the growth of various solid cancers such as 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 exhibits minimal inhibitory activity on other tyrosine kinases (TKs) and selectively modulates epidermal growth factor receptor tyrosine kinase (EGFR-TK). Suppress It also inhibits Mitogen-activated protein kinase (MAPK) and subsequently 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, and hair loss of existing cytotoxic anticancer agents, as well as oral treatment.

However, Gefitinib, an epidermal growth factor receptor (EGFR) target inhibitor, is limited to obtaining drug resistance by expression of T790M mutation of the epidermal growth factor receptor (EGFR) or c-Met, a protocogene. It has Indeed, zephytinib has been shown to be highly effective with a response rate of about 15% in patients, but after one year of use, it is important to come up with a method to overcome the cancer that is resistant to almost all treated patients. It is emerging as a task.

3,4,5-trihydroxybenzoic acid is called gallic acid, gallate and is found in various plant extracts. Gallic acid has been shown to have an anticancer effect of inhibiting cancer cell growth or inducing apoptosis such as lung cancer, gastric cancer and uterine cancer (Ohno Y et. Al (1999) Anticancer Drugs 10 (9): 845-51; Ho et al. (2010) Food and Chemical Toxicology 48 (8-9): 4834-4842; Zhao et al. (2013) Onco Letters 6: 1749-1755).

However, the anti-cancer effect of specifically killing cancer cells resistant to epidermal growth factor receptor inhibitors such as zephytinib and / or erlotinib is not known.

U.S. Patent No. 8,334,000 discloses that gallic acid and various derivatives of gallic acid contained in extracts of pomegranate and other plants inhibit angiogenesis and inhibit the growth of malignant tumors. There is no mention of therapeutic effects against cancers that are resistant to tyrosine kinase inhibitors.

In addition, Korean Laid-Open Patent Publication No. 2014-0015798 discloses a daphhnane diterpenoid-based substance as an anticancer agent that kills cancer cells resistant to gefitinib anticancer agents, but the substance exhibits resistance. As it kills not only cancer cells but also cancer cells that do not exhibit resistance, there is no specificity. Therefore, it is considered that a long verification time is required before actual use as a drug overcomer of Zephytinib.

Meanwhile, Korean Laid-Open Patent Publication No. 2007-0107693 (WO2006 / 084058) is an irreversible epidermal growth factor receptor inhibitor for gefitinib or erlotinib-resistant cancer, and EKB-569, HKI-272 or HKI- 357, but this is based on the second mutation T790M in the kinase domain of resistant cancer cases, which does not result in secondary mutations in all resistant cancers, and because the compounds are not naturally occurring components, they are relatively toxic. Since there is a possibility of appearing, it is considered that a long verification time is required before it can be used as a drug overcoming agent of zephytinib.

Therefore, it specifically acts on cancer cells that are resistant to epidermal growth factor receptor inhibitors such as zephytinib and / or erlotinib, thereby selectively killing the carcinoma that is resistant to the therapeutic effect of commercially available drugs. Naturally derived compounds are required that can be maximized while minimizing side effects.

[Preceding technical literature]

[Patent Documents]

US Patent No. 8,334,000

Korean Laid-Open Patent Publication No. 2014-0015798

Korean Patent Publication No. 2007-0107693 (International Publication No. WO 2006/084058)

[Non-Patent Documents]

Shepherd FA et al., 1994, Semin Oncol., 21: 7-18

W. J. Gullick et al., 1986, Cancer Res., 46: 285-292

S. Cohen et al., 1980, J. Biol. Chem., 255: 4834-4842; A. B. Schreiber et al., 1983, J. Biol. Chem., 258: 846-853

M. J. Oh et al., 2000, Clin. Cancer Res., 6: 4760-4763

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

Ohno Y et. al, 1999, Anticancer Drugs, 10 (9): 845-51

Ho et al., 2010, Food and Chemical Toxicology, 48 (8-9): 4834-4842 Zhao et al., 2013, Oncology Letters 6: 1749-1755

C. Locatelli et al., 2012, Euro. J. of Medi. Chem., 60: 233-239

It is an object of the present invention to provide a composition capable of inducing intracellular toxicity or apoptosis of cancer cells that exhibit resistance to anticancer agents targeting receptor tyrosine kinase.

In order to achieve the above object, the present invention prevents cancer disease resistant to a target anticancer agent comprising a 3,4,5-trihydroxybenzoic acid compound, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient. Or a composition that can be treated, an anticancer adjuvant, a functional health food that can prevent or improve resistant cancer disease, a food that can prevent and improve cancer, including the active ingredient, and an existing anticancer agent in addition to the active ingredient. It provides a combination that can prevent or treat a target anticancer drug-resistant cancer disease.

The composition according to the present invention specifically kills cancer cells that exhibit resistance to an anticancer agent targeting receptor tyrosine kinase, thereby receptor tyrosine kinase inhibitor resistant cancer diseases such as gefitinib. Can be prevented or treated.

1 shows the changes in cell growth responses of HCC827 cell lines that are not resistant to gefitinib and resistant HCC827GR (gefitinib resistnace) cell lines according to the concentration-specific treatment of gefitinib. It is a graph showing the result of the measurement.

Figure 2a shows the cell growth response of HCC827 cell line without resistance to gefitinib and resistant GCC827GR (Gefitinib Resistnace) cell line following treatment with 3,4,5-trihydroxybenzoic acid (gallic acid) It is a graph showing the result of the change measured by the MTT method.

Figure 2b is a 3,4,5-trihydroxybenzoic acid (Trihydroxybenzoic acid, gallic acid), gefitinib (gefitinib), 4-hydroxy-3-methoxybenzoic acid (4-hydroxy-3-methoxybenzoic acid, vanillic acid ), And colony formation assay results of resistant HCC827 cells and resistant HCC827GR (Gefitinib Resistnace) cells following treatment with vehicle and Veh. Indicates.

Figure 3a shows the sensitivity to gefitinib and 3,4,5-trihydroxybenzoic acid (gallic acid), which are represented by the cell lines H358, H1650, H1666, H1734, H1975, HCC827, and H3255. Is a graph measured by the MTT assay.

3B shows gefitinib for each treatment of 3,4,5-trihydroxybenzoic acid, gallic acid, gefitinib, and excipients (vehicle, Veh). Colony formation assay results of resistant H358 and H1975 cell lines are shown.

The present invention is a prevention of cancer disease resistant to a target anticancer agent comprising a 3,4,5-trihydroxybenzoic acid compound represented by the following formula (1), a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient Or to a therapeutic composition.

[Formula 1]

.

The present invention is a 3,4,5-trihydroxybenzoic acid compound represented by the following formula (1) found in various plant extracts Zephytinib is an epithelial growth factor receptor (EGFR) inhibitor (gefitinib) By confirming the specific response and killing of cancer cells resistant to anticancer drugs, 3,4,5-trihydroxybenzoic acid compound was found to be useful for the treatment or prevention of the target anticancer drug resistant cancer disease. .

[Formula 1]

.

In one embodiment according to the invention, the compound, derivative thereof or pharmaceutically acceptable salt thereof may exhibit cytotoxicity in cancer cells that are resistant to receptor tyrosine kinase target anticancer agents.

In one embodiment according to the invention, the compound, derivative thereof or pharmaceutically acceptable salt thereof may exhibit cytotoxicity in cancer cells resistant to epidermal growth factor receptor (EGFR) target anticancer agents.

In one embodiment according to the present invention, the compound, derivative thereof or pharmaceutically acceptable salt thereof may exhibit a higher apoptosis inducing effect in drug resistant cancer cells than normal cancer cells.

In one embodiment according to the present invention, the target anticancer agent is Gefitinib, temozolomide, bevacizumab, erlotinib, capecitabine and imatinib ( imatinib) may be selected from, but is not limited thereto.

In one embodiment according to the present invention, the cancer disease is breast cancer, lung cancer, non-small cell lung cancer, stomach cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin cancer, malignant melanoma, uterine cancer, ovarian cancer, colon cancer , Small bowel 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, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocyte lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS; central nervous system) may be selected from the group consisting of tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma.

In one embodiment according to the invention, the composition may be used alone or in combination with other anticancer agents.

In one embodiment according to the present invention, the compounds of the present invention or derivatives thereof can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art.

In one embodiment according to the invention, acid addition salts formed with pharmaceutically acceptable free acids are useful as said salts. Acid addition salts may be prepared by conventional methods, such as dissolving the compound in an excess of aqueous acid solution and precipitating the salt with a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile, and equimolar amounts of the compound and Acids or alcohols in water (such as glycol monomethyl ether) may be heated and then the mixture may be evaporated to dryness or the precipitated salts may be suction filtered.

In one embodiment according to the present invention, the free acid may be an organic acid and an inorganic acid, and the inorganic acid may be hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like, and the organic acid may be methanesulfonic acid, p-toluenesulfonic acid, Acetic acid, trifluoroacetic acid, citric acid, maleic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid ( glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. Can be used, but is not limited thereto.

In one embodiment according to the invention, bases can be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salts, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

In one embodiment according to the invention, pharmaceutically acceptable salts of the compounds of the invention or derivatives thereof may include salts of acidic or basic groups which may be present in the compounds, 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, dihydrogen phosphate, acetate, Succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, which are prepared by methods or processes for preparing salts known in the art. Can be.

The present invention is for the prevention or treatment of a target anticancer drug-resistant cancer disease comprising a 3,4,5-trihydroxybenzoic acid compound, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient represented by the formula (1) It relates to anticancer supplements.

[Formula 1]

.

In one embodiment according to the invention, the compound, derivative thereof or pharmaceutically acceptable salt thereof may exhibit cytotoxicity in cancer cells resistant to receptor tyrosine kinase target anticancer agents.

In one embodiment according to the present invention, the anticancer adjuvant of the present invention may further contain one or more active ingredients exhibiting the same or similar functions, wherein the pharmaceutically acceptable carrier is saline, sterile water, Ringer's solution, buffer Saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components can be mixed and used.

The present invention provides a 3,4,5-trihydroxybenzoic acid compound represented by the following formula (1), a derivative thereof or a pharmaceutically acceptable salt thereof, and the prevention of a target anticancer drug-resistant cancer disease comprising an anticancer agent as an active ingredient Or to therapeutic combinations.

[Formula 1]

.

In one embodiment according to the present invention, the anticancer agent includes: antimetabolites such as folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine), pyrimidine derivatives (5-fluorouracil, cytarabine); Alkylating agents such as nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine) ); Mitosis inhibitors such as actinomycin D, anti-cancer drugs such as doxorubicin, bleomycin, mitomycin, phytoalkaloids such as vincristine, vinblastine, and mitosis inhibitors including taxane rings antimitotic drugs); Or hormones such as corticosteroids or progesterone; It may be a platinum-containing compound such as cisplatin, but is not limited thereto.

The present invention includes administering an effective amount of the composition, anticancer adjuvant, or combination to a patient in need of treatment with gefitinib or erlotinib resistant cancer.

In one embodiment according to the invention, the treatment may include, but is not limited to, treatment in combination with treatment with other receptor tyrosine kinases such as zephytinib or erlotinib, chemotherapy, radiotherapy, and the like. It doesn't happen.

In one embodiment according to the present invention, an effective amount of the composition, anticancer adjuvant, or combination can be administered to mammals, including humans, by various routes.

In one embodiment according to the present invention, the effective amount may vary depending on the age, weight, sex, health condition, drug form, dosage form, disease severity, tumor mass, drug used concurrently, etc. of the individual, and at least for the individual A statistically significant fraction can be determined by considering the amount that produces a useful effect.

In one embodiment according to the invention, the effective dosage is generally from 0.001 mg / kg to 10 g / kg per day, even more preferably from 0.01 mg / kg to 1 g / kg, once a day The dosage may be divided into several times as needed, but the dosage does not limit the scope of the present invention in any aspect.

In one embodiment according to the present invention, in addition to the active ingredient of the present invention, it may be prepared by including one or more pharmaceutically acceptable carriers.

In one embodiment according to the invention, the pharmaceutically acceptable carrier comprises saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes and one or more of these components It can be used, and other conventional additives such as antioxidants, buffers, bacteriostatics can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

In one embodiment according to the invention, the formulation may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. which are commonly used, but are not limited thereto.

In one embodiment according to the invention, the dosage form is not particularly limited but may be parenteral or oral, as desired.

In one embodiment according to the invention, the parenteral administration is intravenous (iv), intramuscular (im), subcutaneous (sc), intradermal (id), intraperitoneal (ip), intradural (it), pleural Intraluminal, intrauterine, intrarectal, intravaginal, topical, intratumoral administration, and the like, and may be administered parenterally by injection or by gradual infusion over time and delivered by peristaltic means.

In one embodiment according to the present invention, the preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

In one embodiment according to the invention, the solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like, which solid preparations comprise at least one excipient in one or more compounds of the invention For example, it is prepared by mixing starch, calcium carbonate, sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, or syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

In one embodiment according to the invention, the formulation for oral administration may be provided with an enhancer. Orally acceptable absorption enhancers include surfactants such as sodium lauryl sulfate, palmitoylcarnitine, laureth-9, phosphatidylcholine, cyclodextrin, and derivatives thereof; Bile salts such as sodium deoxycholate, sodium taurocholate, sodium glycocholate, and sodium fucidate; Chelating agents such as EDTA, citric acid and salicylate; And fatty acids (eg, oleic acid, lauric acid, acylcarnitines, mono and diglycerides). Other oral absorption enhancers include benzalkonium chloride, benzetonium chloride, CHAPS (3- (3-colamidopropyl) -dimethylammonio-1-propanesulfonate), Big-CHAPS (N, N-bis ( 3-D-glucoamidopropyl) -collamide), chlorobutanol, octoxynol-9, benzyl alcohol, phenols, cresols, and alkyl alcohols.

In one embodiment according to the present invention, the active ingredient of the present invention may be administered in liposomes or microspheres (or microparticles).

In one embodiment according to the present invention, the active ingredient of the present invention can be inserted into a microsphere or a composite of microspheres and implanted so as to be released slowly over days to months.

In one embodiment according to the invention, the microspheres are known to those skilled in the art formed of polymers or proteins and can be tailored to pass directly into the bloodstream through the gastrointestinal tract.

In one embodiment according to the present invention, the active ingredient of the present invention may be formulated into the liposomes or microparticles (microspheres) to be intravenously administered to a size suitable for lodge on the capillaries.

In one embodiment according to the invention, when the liposomes or microparticles reach the capillary phase around the ischemic tissue, the active ingredient can be administered locally to the site where they are most effective.

In one embodiment according to the present invention, liposomes are generally less than about 200 nm and may be multilayer vesicles to target ischemic tissue.

In one embodiment according to the present invention, the microparticles may be those made from biodegradable polymers such as polyglycolide, polylactide and their copolymers, and those skilled in the art will appreciate the desired rate of drug release and the desired administration. Suitable carrier systems can be readily determined depending on various factors such as amount.

In one embodiment according to the invention, the formulation comprising the active ingredient of the present invention can be administered directly inside the blood vessel through a catheter, wherein the active ingredient can be included in a biodegradable polymer hydrogel.

In one embodiment according to the present invention, the polymer hydrogel may be delivered inside the tissue lumen and the formulation comprising the active ingredient of the present invention may be released over time as the polymer is degraded.

In one embodiment according to the present invention, the polymer hydrogel may have microparticles or liposomes in which the active ingredient of the present invention is dispersed, which may provide another mechanism for controlled release of the active ingredient of the present invention. Can be.

In one embodiment according to the present invention, the formulation comprising the active ingredient of the present invention for administration may be prepared by methods known in the art of pharmacy and suitably provided in unit dosage form wherein the method is effective in the present invention. Contacting the component with a carrier that constitutes one or more accessory ingredients.

In one embodiment according to the invention, the preparation is brought into uniform and intimate contact with the active ingredient of the invention with a liquid carrier or a finely divided solid carrier, and then, if necessary, shaping the product into the desired unit dosage form. Can be prepared.

In one embodiment according to the invention, the formulation comprising the active ingredient comprises one or more optional auxiliary ingredients used in the pharmaceutical art, for example diluents, buffers, flavors, binders, surface active agents, thickeners, lubricants, suspensions It may further include an agent, a preservative (antioxidant, etc.).

In one embodiment according to the present invention, the active ingredient of the present invention is used as a snuff or aerosol or inhaler solution or as an inhalation ultrafine powder for administration to a respiratory tract, alone or with an inert carrier such as lactose. It can be provided in combination, in which case it is suitable that the particles of the active ingredient have a diameter of less than 50 microns (microns), preferably less than 10 microns, even more preferably 2 to 5 microns.

In one embodiment according to the present invention, the composition for nasal administration preferably has a weak pH, specifically pH of about 3 to 5 more specifically 3.5 to 3.9 more specifically 3.7 and the pH is It can be adjusted by adding a suitable acid such as hydrochloric acid.

The present invention is for preventing or improving a target anticancer drug-resistant cancer disease comprising a 3,4,5-trihydroxybenzoic acid compound, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient represented by the formula (1) It is about health food.

[Formula 1]

.

The health functional food can be used in a variety of drugs, foods, beverages, and the like and can be commercialized in the form of tablets, capsules, pills, powders, granules, liquid drinks, concentrates and the like.

In the case of the health functional food in the form of granules, tablets or capsules, usually 0.0001 to less than 100% by weight, more specifically 0.01 to 80% by weight, even more specifically 0.1 to 50% by weight, even more specifically 1 to 20% by weight The active ingredient of the present invention may be added in the range of, but is not limited thereto.

The present invention relates to a food for preventing and improving cancer comprising 3,4,5-trihydroxybenzoic acid, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient represented by the following formula (1). .

[Formula 1]

.

The food is beverages, meat, chocolate, sweets, pizza. Ramen noodles, noodles, gum, ice cream, vitamin complexes, alcoholic beverages, dairy products, juices, pills, granules, tea bags and sticks, etc., may include all foods in a conventional sense.

In general, the food composition of the present invention may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 10 g, preferably 0.3 to 1 g based on 100 ml.

In one embodiment according to the present invention, the health beverage composition of the present invention contains the active ingredient mixture of the present invention as an essential ingredient in the indicated proportions, and there are no special limitations on the liquid components and various flavoring agents such as ordinary beverages. Or natural carbohydrate or the like as an additional component. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and conventional sugars such as dextrin, cyclodextrin and xylitol, sorbitol And sugar alcohols such as erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia compounds (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, more specifically about 5 to 12 g per 100 ml of the beverage composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples, Experimental Examples, and Preparation Examples. However, this is to help the understanding of the present invention, not intended to limit the scope of the present invention to this.

[실험예]Experimental Example

< 3,4,5-트리하이드록시벤조산(Trihydroxybenzoic acid) 화합물 ><3,4,5-trihydroxybenzoic acid compound>

The 3,4,5-trihydroxybenzoic acid compound used in the experimental example was purchased from Sigma-Aldrich and Alfa Aesar.

구입처Where to buy	제품번호(Catalog #)Serial number (Catalog #)
씨그마알드리치(Sigma-Aldrich)Sigma-Aldrich	G7384G7384
알파아에스타(Alfa Aesar)Alfa Aesar	B24887B24887

실험예 1: 제피티닙(gefitinib) 내성 세포주Experimental Example 1: Gefitinib Resistant Cell Line

In order to measure the cell growth response to gefitinib, HCC827 and HCC827GR # 1 and HCC827GR # 2 cell lines resistant to gefitinib were inoculated into 96-well plates, respectively, and zepitinib was 0.1, 0.3 after 24 hours of inoculation. , 5, and then treated with concentrations of 1, 3, and 5 uM, and 5 days later, cell viability was measured by an MTT assay to confirm cell growth responses. As a result, as shown in Figure 1, the resistance to gefitinib showed the highest pattern in the order of HCC827 <HCC827GR # 1 <HCC827GR # 2.

실험예2: HCC827 세포주들의 3,4,5-트리하이드록시벤조산(Trihydroxybenzoic acid)에 대한 세포성장 반응Experimental Example 2 Cell Growth Response of HCC827 Cell Lines to 3,4,5-Trihydroxybenzoic Acid

In order to measure cell growth response to 3,4,5-trihydroxybenzoic acid, HCC827GR # 1 and HCC827GR # 2 cell lines resistant to HCC827 and zefitinib were inoculated into 96-well plates, respectively. 24 hours after inoculation, 3,4,5-trihydroxybenzoic acid was treated at concentrations of 0, 5, 10, 25, 35, and 50 uM, respectively, and 5 days later, cell viability was determined by an MTT assay. ) To determine the growth response of the cells. As a result, as shown in Figure 2a, the most resistant to gefitinib (gefitinib) HCC827GR # 2 cell line was the most cell growth by treatment with 3,4,5-trihydroxybenzoic acid (gallic acid) It was confirmed that it is sensitively inhibited.

In addition, 6-well plates of HCC827GR # 1 and HCC827GR # 2 cells resistant to HCC827 and gefitinib were used to measure cell growth response to 3,4,5-trihydroxybenzoic acid. Inoculated into 0.3 uM zefitinib, 25 uM 3,4,5-trihydroxybenzoic acid, and 25 uM 4-hydroxy-3-methoxybenzoic acid, 10 days after each treatment with vanillic acid), cell viability was measured by colony formation assay through methylene blue staining to confirm cell growth response.

As a result, as shown in Figure 2b, in the colony formation assay (colony formation assay) cell growth inhibition and clonogenecity according to 3,4,5-trihydroxybenzoic acid treatment and the degree of resistance to gefitinib Proportion was confirmed. Moreover, no anticancer effect could be observed with 4-hydroxy-3-methoxybenzoic acid, a similar polyphenol. This result confirms that the anticancer effect of 3,4,5-trihydroxybenzoic acid is specific for zephytinib resistant strains compared to other polyphenols.

실험예 3: 다양한 폐암 세포주들로부터 3,4,5-트리하이드록시벤조산(Trihydroxybenzoic acid) 항암효과 검증Experimental Example 3: Verification of anticancer effect of 3,4,5-trihydroxybenzoic acid from various lung cancer cell lines

Table 2 below shows the IC ₅₀ values for gefitinib in a panel of various lung cancer cell lines with K-ras mutations and epidermal growth factor receptor (EGFR) mutation characteristics. Properties and IC ₅₀ values for gefitinib)

H358, H1975, H1650, HCC827, H1666, H3255 and Gefitinib-resistant HCC827GR # 1, HCC827GR # 2 cell lines listed in Table 2, respectively, were inoculated into 6-well plates and 0.3 uM zephyti after 24 hours of inoculation. Nip, 35 uM 3,4,5-trihydroxybenzoic acid, and 25 uM 4-hydroxy-3-methoxybenzoic acid (vanillic acid), respectively, after 5 days of treatment, empty Cell viability was measured by MTT assay to confirm the growth response of the cells.

As a result, as shown in Figure 3a, the treatment of various lung cancer cell lines with 3,4,5-trihydroxybenzoic acid (Trihydroxybenzoic acid, gallic acid) to increase the sensitivity of gefitinib and 3,4,5-trihydroxybenzoic acid The results showed that the sensitivity of zephytinib and 3,4,5-trihydroxybenzoic acid was inversely proportional to almost all different lung cancer cell lines.

In addition, H358 and epithelium with K-ras mutations to measure the growth response to 3,4,5-trihydroxybenzoic acid, two cell lines with different mutations but commonly resistant to zephytinib. H1975 cell lines with cell growth factor receptor (EGFR) mutations were each inoculated into 6-well plates and 0.3 uM zefitinib, 25 uM 3,4,5-trihydroxybenzoic acid, and 25 uM 4- after 24 h of inoculation Cell viability after colony formation assay by methylene blue staining after 10 days of treatment with 4-hydroxy-3-methoxybenzoic acid and vanillic acid respectively (cell viability) was measured to confirm the growth response of the cells.

As a result, as shown in FIG. 3B, both H358 and H1975 cell lines disappeared by clonogenecity by 3,4,5-trihydroxybenzoic acid treatment. Therefore, it was confirmed that even if lung cancer cell lines caused by different mutations had gefitinib resistance, specific anticancer effects by 3,4,5-trihydroxybenzoic acid were observed.

실험예 4. 동물실험 (종양 성장 억제 효과)Experimental Example 4. Animal experiment (tumor growth inhibitory effect)

A xenograft experiment was performed to determine the inhibitory effect of 3,4,5-trihydroxybenzoic acid on tumor growth. The experimental method is as follows:

5 × 10 ⁶ H1650 lung cancer cells were injected subcutaneously into the flanks of nude mice. Nude mice transplanted with H1650 were randomly divided into two groups, and then 4 rats and 6 rats were assigned to the control group. Control mice received intraperitoneal DMSO for 32 days from the tumor volume of 20 mm ³ , and experimental mice received 200 mg / kg dose of 3,4,5-trihydroxybenzoic acid every two days. Tumor volume was calculated as (x width x length x height).

As a result, the experimental group treated with 3,4,5-trihydroxybenzoic acid significantly inhibited tumor growth compared to the control group treated with DMSO. Figure 4a graphically shows the tumor growth inhibitory effect of H1650 transplanted mice. (p = 0.0211) When the 3,4,5-trihydroxybenzoic acid according to the present invention is administered, it can be seen that the tumor size is significantly reduced.

[제조예][Production example]

제조예 1. 산제의 제조Preparation Example 1 Preparation of Powder

3,4,5-trihydroxybenzoic acid (gallic acid) --- 200 mg

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

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

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

제조예 2. 정제의 제조Preparation Example 2 Preparation of Tablet

3,4,5-trihydroxybenzoic acid (gallic acid) --- 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 a conventional method for preparing tablets.

제조예 3. 캅셀제의 제조Preparation Example 3 Preparation of Capsule

3,4,5-trihydroxybenzoic acid (gallic acid) --- 200 mg

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

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

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

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

제조예 4. 주사제의 제조Preparation Example 4 Preparation of Injection

3,4,5-trihydroxybenzoic acid (gallic acid) --- 200 mg

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

Sterile Distilled Water for Injection ---------------------------------------------- -1594 mg

Na ₂ HPO ₄ , 12H ₂ O ------------------------------------------ ---------- 26 mg

According to the conventional method for preparing an injection, it is prepared in the above-described content of ingredients per ampoules (2 mL).

제조예 5. 액제의 제조Preparation Example 5 Preparation of Liquid

3,4,5-trihydroxybenzoic acid (gallic acid) --- 200 mg

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

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

Purified water ------------------------------------------------- ----------- Suitable

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to 100 ml by adding purified water and filling into a brown bottle. The solution is prepared by sterilization.

제조예 6. 건강 식품의 제조Preparation Example 6 Preparation of Health Food

3,4,5-trihydroxybenzoic acid (gallic acid) --- 1000 mg

Vitamin Mixture ------------------------------------------------ ------ Suitable

Vitamin A Acetate ----------------------------------------------- -70 g

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 g

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

Mineral Mixtures ------ Suitable

Ferrous Sulfate ---------------------------------------------- ------------ 1.75 mg

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 mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

제조예 7. 건강 음료의 제조Preparation Example 7 Preparation of Healthy Drink

3,4,5-trihydroxybenzoic acid (gallic acid) --- 1000 mg

Citric Acid ------------------------------------------------- ----------- 1000 mg

oligosaccharide ------------------------------------------------- --------- 100 g

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

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

By adding purified water --------------------------------------------- Total 900 mL

After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

The pharmaceutical composition according to the present invention specifically kills cancer cells that exhibit resistance to an anticancer agent targeting receptor tyrosine kinase, thereby receptor tyrosine kinase inhibitor resistant cancer such as gefitinib. It is useful industrially to prevent or treat a disease.

Claims

A composition for preventing or treating cancer diseases resistant to a target anticancer agent comprising 3,4,5-trihydroxybenzoic acid compound represented by Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. :

[Formula 1]

.
The method of claim 1,

The compound, a derivative thereof, or a pharmaceutically acceptable salt thereof has a composition for preventing or treating cancer diseases, characterized in that it exhibits cytotoxicity in cancer cells resistant to receptor tyrosine kinase target anticancer agents.
The method of claim 1,

The compound, a derivative thereof or a pharmaceutically acceptable salt thereof has a composition for preventing or treating cancer diseases, characterized in that it shows cytotoxicity in cancer cells resistant to epidermal growth factor receptor (EGFR) target anticancer agent.
The method of claim 1,

The compound, a derivative thereof, or a pharmaceutically acceptable salt thereof has a composition for preventing or treating cancer diseases, characterized in that the drug-resistant cancer cells exhibit a higher apoptosis-inducing effect than normal cancer cells.
The method of claim 1,

The target anticancer agent is selected from the group consisting of gefitinib, temozolomide, bevacizumab, erlotinib, capecitabine and imatinib. Composition for the prevention or treatment of cancer diseases
The method of claim 1,

The cancer diseases include breast cancer, lung cancer, non-small cell lung cancer, gastric cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin cancer, malignant melanoma, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer and 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, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, Urethral Cancer, Penile Cancer, Prostate Cancer, Chronic Leukemia, Acute Leukemia, Lymphocyte Lymphoma, Bladder Cancer, Kidney Cancer, Urinary Tract Cancer, Kidney Cell Carcinoma, Kidney Pelvic Carcinoma, Central Nervous System (CNS) Tumor, Primary Central Neurologic lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma composition for the prevention or treatment of cancer diseases, characterized in that selected from the group consisting of.
The method of claim 1,

The composition is a composition for the prevention or treatment of cancer diseases, characterized in that it is used alone or in combination with other anticancer agents.
Anticancer adjuvant for the prevention or treatment of a target anticancer drug-resistant cancer disease comprising a 3,4,5-trihydroxybenzoic acid compound represented by Formula 1, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.
The method of claim 8,

The compound, a derivative thereof or a pharmaceutically acceptable salt thereof is an anticancer adjuvant for preventing or treating cancer disease, characterized in that it exhibits cytotoxicity in cancer cells resistant to receptor tyrosine kinase target anticancer agents.
The method of claim 8,

The compound, a derivative thereof or a pharmaceutically acceptable salt thereof is an anticancer adjuvant for preventing or treating cancer disease, characterized in that it exhibits cytotoxicity in cancer cells resistant to epidermal growth factor receptor (EGFR) target anticancer agent.
The method of claim 8,

The compound, a derivative thereof or a pharmaceutically acceptable salt thereof is an anticancer adjuvant for preventing or treating cancer diseases, characterized in that the drug-resistant cancer cells exhibit a higher apoptosis-inducing effect than normal cancer cells.
The method of claim 8,

The target anticancer agent is selected from the group consisting of gefitinib, temozolomide, bevacizumab, erlotinib, capecitabine and imatinib. An anticancer adjuvant for preventing or treating cancer diseases.
The method of claim 8,

The cancer diseases include breast cancer, lung cancer, non-small cell lung cancer, gastric cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin cancer, malignant melanoma, uterine cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer and 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, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, Urethral Cancer, Penile Cancer, Prostate Cancer, Chronic Leukemia, Acute Leukemia, Lymphocyte Lymphoma, Bladder Cancer, Kidney Cancer, Urinary Tract Cancer, Kidney Cell Carcinoma, Kidney Pelvic Carcinoma, Central Nervous System (CNS) Tumor, Primary Central An anticancer adjuvant for the prevention or treatment of cancer diseases, characterized in that selected from the group consisting of neurological lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma.
A 3,4,5-trihydroxybenzoic acid compound represented by Formula 1, a derivative thereof, or a pharmaceutically acceptable salt thereof; And

Combination for the prophylaxis or treatment of a target anticancer drug resistant cancer disease comprising an anticancer agent as an active ingredient:

[Formula 1]

.
The method of claim 14,

The anticancer agent is any one or more selected from the group consisting of metabolic antagonists, alkylating agents, anticancer anticancer agents, plant alkaloids, mitosis inhibitors, hormones, and platinum-containing compounds, a combination for the prevention or treatment of target anticancer drug resistant cancer diseases water.
A dietary supplement for the prevention or improvement of a target anticancer drug-resistant cancer disease comprising 3,4,5-trihydroxybenzoic acid compound, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.
Food for preventing or ameliorating cancer comprising 3,4,5-trihydroxybenzoic acid compound, a derivative thereof or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.