WO2019147089A1 - Pharmaceutical composition for preventing or treating cancer comprising, as active ingredient, calcium channel inhibitor or pharmaceutically acceptable salt thereof - Google Patents

Pharmaceutical composition for preventing or treating cancer comprising, as active ingredient, calcium channel inhibitor or pharmaceutically acceptable salt thereof Download PDF

Info

Publication number
WO2019147089A1
WO2019147089A1 PCT/KR2019/001143 KR2019001143W WO2019147089A1 WO 2019147089 A1 WO2019147089 A1 WO 2019147089A1 KR 2019001143 W KR2019001143 W KR 2019001143W WO 2019147089 A1 WO2019147089 A1 WO 2019147089A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
calcium channel
pharmaceutical composition
cells
channel inhibitor
Prior art date
Application number
PCT/KR2019/001143
Other languages
French (fr)
Korean (ko)
Inventor
민상현
김준우
이희진
유지훈
최동규
송영우
최재헌
김재호
Original Assignee
재단법인 대구경북첨단의료산업진흥재단
부산대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 재단법인 대구경북첨단의료산업진흥재단, 부산대학교 산학협력단 filed Critical 재단법인 대구경북첨단의료산업진흥재단
Publication of WO2019147089A1 publication Critical patent/WO2019147089A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Stem cells are immature cells that can undergo self-renewal and can differentiate into the cells of each organ and achieve organogenesis.
  • Adult stem cells are distinguishable from embryonic stem cells in that they are identical to embryonic stem cells in that they can regenerate and differentiate, but differentiation is mainly made up of specific endothelial cells.
  • a representative example of adult stem cells is hematopoietic stem cells.
  • Cancer stem cells like stem cells, can regenerate and differentiate through asymmetric division, but unlike normal stem cells, they are cells that produce tumors with impaired ability to regulate division.
  • signals such as Notch, Sonic hedgehog (SHH), Wnt, ⁇ -catenin, phosphatase and tensin homolog [HONG7] (PTEN), transforming growth factor
  • SHH Sonic hedgehog
  • Wnt Wnt
  • ⁇ -catenin phosphatase and tensin homolog [HONG7]
  • PTEN tensin homolog
  • the pathway was found to be deformed in malignant tumors.
  • the expression of PTEN, Hedgehog signaling pathway, nestin, epidermal growth factor (EGF) -receptor, and proliferative potential vasculature, immature expression pattern, and high motility, progeny diversity, , telomerase activity, and Wnt signaling pathway activity.
  • Patent Document 1 Korean Patent Laid-open Publication No. 1020160094861 discloses a pharmaceutical composition for inhibiting the growth of cancer stem cells comprising an aldehyde inhibitor and an acetylated compound,
  • Non-Patent Document 1 J Korean Assoc Oral Maxillofac Surg 2011; 37: 97-108 discloses the theory of cancer stem cell in oral squamous cell carcinoma and the latest knowledge.
  • Another object of the present invention is to provide a health functional food composition for preventing or ameliorating cancer.
  • a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a calcium channel inhibitor or a pharmaceutically acceptable salt thereof.
  • a combined preparation for preventing or treating cancer comprising a calcium channel inhibitor or a pharmaceutically acceptable salt thereof and an anticancer agent.
  • a health functional food composition for preventing or ameliorating a cancer containing, as an active ingredient, a calcium channel inhibitor or a pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition for preventing or treating cancer comprising the calcium channel blocker or a pharmaceutically acceptable salt thereof as an active ingredient, A method of preventing or treating cancer is provided.
  • a pharmaceutical composition for preventing or treating cancer comprising the calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient in the prevention or treatment of cancer.
  • the use of functional food compositions is provided.
  • the pharmaceutical composition comprising the calcium channel inhibitor of the present invention is excellent in the effect of inhibiting growth and proliferation of cancer stem cells even when used alone and exhibits a more excellent pharmacological effect when it is administered in combination with a conventional anticancer drug , Cancer recurrence, metastasis, and progression of cancer can be inhibited. Therefore, the composition can be usefully used as a pharmaceutical composition or a combined preparation for the prevention or treatment of cancer.
  • Figure 1 shows a dose-response curve of the viability of A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated.
  • Figure 1 a Manidipine
  • Figure 1 b Rashedipine
  • Figure 1 c Benidipine
  • Figure 1 d shows a dose-response curve of the viability of A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated.
  • Figure 1 a Manidipine
  • Figure 1 b Rashedipine
  • Figure 1 c Benidipine
  • Figure 1 d shows a dose-response curve of the viability of A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated.
  • Figure 1 a Manidipine
  • Figure 1 b Rashedipine
  • Figure 1 c Benidipine
  • Figure 1 d Benidipine
  • Figure 2 shows the dose-response curve of the viability of A2780 cells (ovarian cancer cells) when the example pharmaceutical composition was treated.
  • Figure 2a Manidipine
  • Figure 2b Rashedipine
  • Figure 2c Lomerizine HCl
  • Figure 2d Benidipine HCl
  • FIG. 3 is an image showing the result of analyzing the sphere formation of A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of the Example was treated.
  • Figure 3a Manidipine
  • Figure 3b Rashedipine
  • Figure 3c Lomerizine HCl
  • Figure 3d Benidipine HCl
  • FIG. 4 is a graph showing the results of measurement of the proliferation of A2780-SP cells by analyzing the sphere formation of A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of Example was treated.
  • Figure 4a Manidipine
  • Figure 4b Rashedipine
  • Figure 4c Romerizine HCl
  • Figure 4d Benidipine HCl
  • FIG. 5 is an image showing changes in the expression of markers related to cancer stem cell function in A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of Example is treated.
  • Figure 6 is a graph quantifying the expression changes of markers related to cancer stem cell function in A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated.
  • Figure 6a OCT 3/4
  • Figure 6b NANOG
  • Figure 6c SOX2
  • Figure 6d ALDHl
  • Figure 6e CD133
  • FIG. 7 is an image showing changes in the activity of proteins involved in cancer stem cell survival and proliferation in A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of the Example was treated.
  • FIG. 8 is a graph quantifying the change in the activity of a protein involved in cancer stem cell survival and proliferation in A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated.
  • Figure 8a pAKT
  • Figure 8b pERK
  • Figure 8c p-p38
  • FIG. 9 is a graph showing cell cycle changes of A2780-SP cells when the example pharmaceutical composition is treated.
  • Figure 9a DMSO
  • Figure 9b Manidipine
  • Figure 9c Rashedipine
  • Figure 9d Romerizin HCl
  • Figure 9e Benidipine HCl
  • FIG. 10 is a graph showing the cell death of A2780-SP cells when the pharmaceutical composition of Example is treated.
  • Fig. 11 shows quantitative RT-PCR quantification of the expression levels of calcium channel subunits (Cacna1d, Cacna1f, Cacna1h) mRNA in A2780-SP cells and A2780 cells.
  • 11a Cacna1d
  • Fig. 11b Cacna1f
  • Fig. 11c Cacna1h
  • 12 shows the results of knockdown of the calcium channel-related genes (si-Cacna1d, si-Cacna1f, si-Cacna1h) in A2780-SP cells and the mRNA expression level of each calcium channel subunit (Cacna1d, Cacna1f, Cacna1h) Was quantitated by quantitative RT-PCR to confirm knockdown.
  • 12a Cacna1d
  • Fig. 12b Cacna1f
  • Fig. 12c Cacna1h
  • FIG. 13 shows the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f and si-Cacna1h) in A2780-SP cells and then to identify markers (OCT3 / 4, NANOG, SOX2, ALDH1 , CD133) was quantified by quantitative RT-PCR.
  • Figure 13a OCT 3/4
  • Figure 13b NANOG
  • Figure 13c SOX2
  • Figure 13d ALDHl
  • Figure 13e CD133
  • FIG. 14 is a graph showing the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f and si-Cacna1h) in A2780-SP cells, and stem cell-associated markers and the expression of proteins involved in the survival and proliferation of cancer stem cells.
  • FIG. 15 is a graph showing the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f, si-Cacna1h) in A2780-SP cells, a stem cell-associated marker, and a stem cell-associated marker and the expression of a protein associated with survival and proliferation of cancer stem cells.
  • Figure 15a OCT 3/4
  • Figure 15b pERK
  • Figure 15c p-p38
  • Figure 16 is a graph showing that the amplitude of calcium current is reduced when the pharmaceutical composition of Example is treated with A2780-SP cells.
  • FIG. 17 is a graph showing the results of measuring the cell survival rate of A2780-SP cells when the pharmaceutical composition of Example and cisplatin were used in combination.
  • FIG. Figure 17a Manidipine
  • Figure 17b Rashedipine
  • Figure 17c Romerizin HCl
  • Figure 17d Benidipine HCl
  • FIG. 18 is a graph showing the results of analysis of cell proliferation of A2780-SP cells when the pharmaceutical composition of Example and cisplatin were used in combination.
  • Figure 18a Manidipine
  • Figure 18b Rashedipine
  • Figure 18c Romerizin HCl
  • Figure 18d Benidipine HCl
  • FIG. 19 is a graph showing the results of tumorigenesis (A2780-AD) in the case of the ovarian cancer cell tumor animal model (A2780-AD) and the ovarian cancer stem cell tumor animal model (A2780-SP), alone or in combination with paclitaxel Inhibitory effect and evaluating the results.
  • 19A A2780-AD
  • FIG. 19B A2780-SP
  • An aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a calcium channel inhibitor or a pharmaceutically acceptable salt thereof.
  • the calcium channel inhibitor may be any one selected from the following formulas (A) to (D).
  • the cancer may be selected from the group consisting of benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial lesion, ependymoma, brain tumor, laryngeal cancer, pancreatic cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, Cancer of the breast, cancer of the gallbladder, cancer of the gallbladder, cancer of the bile duct, pancreatic cancer, small bowel cancer, colon cancer, anal cancer, bladder cancer, breast cancer, Kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female gonadal cancer and skin cancer.
  • the cancer may be an ovarian cancer.
  • the pharmaceutical composition may inhibit expression of at least one selected from the group consisting of proteins consisting of OCT3 / 4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 and p38.
  • the pharmaceutical composition can inhibit the proliferation of cancer stem cells.
  • the anticancer effect of the pharmaceutical composition was measured.
  • the pharmaceutical composition reduced the viability of cancer cells and cancer stem cells, suppressed proliferation, inhibited the formation and proliferation of cancer stem cells
  • the anticancer effect was excellent because the cancer stem cell-related marker and the effect of suppressing the expression of the protein related to the proliferation were excellent and the cell death of the cancer stem cell was increased (Experimental Examples 2 to 5 and FIGS. 10).
  • the pharmaceutical composition according to one aspect of the present invention can be usefully used as a pharmaceutical composition for preventing or treating cancer.
  • the calcium channel inhibitor may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful.
  • Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid and the like, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, And organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like are obtained from non-toxic organic acids such as dicarboxylic acids, Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,
  • the acid addition salt can be prepared by a conventional method.
  • the calcium channel inhibitor is dissolved in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., and a precipitate formed by adding an organic acid or an inorganic acid is filtered, dried Or by distilling the solvent and excess acid under reduced pressure, followed by drying and crystallization in an organic solvent.
  • 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 an 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.
  • the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).
  • the calcium channel inhibitor may be used in the form of a pharmaceutically acceptable salt thereof, as well as a solvate, an optical isomer, a hydrate, etc., which may be prepared therefrom.
  • the calcium channel blocker or a pharmaceutically acceptable salt thereof may be administered in various forms of oral and parenteral administration at the time of clinical administration.
  • 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 one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like.
  • lubricants such as magnesium stearate, talc, and the like are also used.
  • Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like.
  • excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, and emulsions.
  • the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • the pharmaceutical composition comprising the calcium channel blocker or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and the parenteral administration may be a subcutaneous injection, an intravenous injection, an intramuscular injection, And.
  • the calcium channel blocker or pharmaceutically acceptable salt thereof is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, which is then prepared into an ampoule or vial unit dosage form .
  • the compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.
  • formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and troches, , Dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants (such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols).
  • the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain binders such as starch, agar, alginic acid or sodium salts thereof Release or boiling mixture and / or absorbent, colorant, flavor, and sweetening agent.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain binders such as starch, agar, alginic acid or sodium salts thereof Release or boiling mixture and / or absorbent, colorant, flavor, and sweetening agent.
  • Another aspect of the present invention provides a combined preparation for prevention or treatment of cancer comprising a calcium channel inhibitor or a pharmaceutically acceptable salt thereof and an anticancer agent.
  • the calcium channel inhibitor may be any one selected from the following formulas (A) to (D).
  • the cancer may be selected from the group consisting of benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial lesion, ependymoma, brain tumor, laryngeal cancer, pancreatic cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, Cancer of the breast, cancer of the gallbladder, cancer of the gallbladder, cancer of the bile duct, pancreatic cancer, small bowel cancer, colon cancer, anal cancer, bladder cancer, breast cancer, Kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female gonadal cancer and skin cancer.
  • the cancer may be an ovarian cancer.
  • the combined preparation may inhibit expression of at least one selected from the group consisting of OCT3 / 4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 and p38.
  • the combined preparation can inhibit the proliferation of cancer stem cells.
  • anticancer agent is a generic term for known drugs used in conventional cancer therapy that act on various metabolic pathways of cancer cells and exhibit cytotoxicity or cytostatic effects on cancer cells. Metabolic antagonists, botanical alkaloids, topoisomerase inhibitors, alkylating agents, anticancer antibiotics, hormones and other medicaments.
  • the anticancer agent is selected from the group consisting of doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, (Including but not limited to: teniposide, bisantrene, homoharringtonine, Gleevec (STI-571), cisplatin, 5-fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, and nitrosourea.
  • doxorubicin doxorubicin
  • paclitaxel paclitaxel
  • vincristine daunorubicin
  • vinblastine actinomycin-D
  • docetaxel etoposide
  • etoposide including but not limited to: teniposide, bisantrene, homoharringtonine, Gleevec
  • the anticancer agent may be cisplatin or paclitaxel.
  • the pharmaceutical composition containing the calcium channel blocker exhibits excellent cell proliferation inhibitory effect even when it is administered alone.
  • the cell proliferation of stem cells is further suppressed (see Experimental Example 7 and Figs. 17 and 18).
  • the calcium channel inhibitor of the present invention or a pharmaceutically acceptable salt thereof exhibits excellent anticancer effects even when used alone, and can exhibit higher synergistic effects when used in combination with existing anticancer drugs.
  • the combined preparation according to one aspect of the present invention can be usefully used for the prevention or treatment of cancer by the combined use with the conventional anticancer agent.
  • Another aspect of the present invention provides a combination therapy for prevention or treatment of cancer, which is used by administering a combination of a calcium channel inhibitor, a pharmaceutically acceptable salt thereof and an anticancer agent.
  • the combined administration can be carried out simultaneously, separately or sequentially.
  • Another aspect of the present invention provides a health functional food composition for preventing or ameliorating cancer, which comprises a calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the calcium channel inhibitor may be any one selected from the following formulas (A) to (D).
  • the cancer may be selected from the group consisting of benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial lesion, ependymoma, brain tumor, laryngeal cancer, pancreatic cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, Cancer of the breast, cancer of the gallbladder, cancer of the gallbladder, cancer of the bile duct, pancreatic cancer, small bowel cancer, colon cancer, anal cancer, bladder cancer, breast cancer, Kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female gonadal cancer and skin cancer.
  • the cancer may be an ovarian cancer.
  • health functional food refers to a food prepared by adding the calcium channel inhibitor to food materials such as beverage, tea, spices, gum, confectionery, etc., encapsulation, powdering, suspension, etc., But it has the advantage that there are no side effects that can occur when a drug is used for a long period of time using a food as a raw material.
  • the health functional food of the present invention thus obtained can be ingested on a daily basis, so that a high skin whitening effect can be expected, which is very useful.
  • the calcium channel blocker or pharmaceutically acceptable salt thereof may be added directly to the food or may be used together with other food or food ingredients and may be suitably used according to conventional methods.
  • the amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the compound in the health functional food may be 0.1 to 90 parts by weight based on the total weight of the food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.
  • the health functional beverage composition according to one aspect of the present invention has no particular limitation on other ingredients except that the calcium channel inhibitor is contained as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates As an additional component.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • Natural flavors 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 g of the composition of the present invention.
  • the calcium channel inhibitor of the present invention or a pharmaceutically acceptable salt thereof may be in the form of a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), manufacturing flavors and natural flavors, Etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like.
  • the calcium channel inhibitor of the present invention or a pharmaceutically acceptable salt thereof may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.
  • a method for preventing or treating cancer comprising the calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, And a method for preventing or treating cancer.
  • Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer comprising the calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient in the prevention or treatment of cancer.
  • a food composition comprising the calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient in the prevention or treatment of cancer.
  • compositions of Examples 1 to 3 were prepared using the following three test substances as calcium channel inhibitors.
  • A2780-SP cells (ovarian cancer stem cells) were plated in Ultra-Low Attachment round bottom 96-well corning at a viable cell density of 500 cells per well and cultured in Cancer stem cell medium.
  • Cancer stem cell culture medium consists of neurobasal medium supplemented with bFGF 20 ng / ml, EGF 10 ng / ml, Amphotericin B 2.5 ng / ml, HEPES, Glutamax and B27. The cells were centrifuged lightly and cultured.
  • apigenin-treated control group was treated with 0% and DMSO-treated control group was treated with 100%. As a result, the size and viability of spherical cells were reduced to less than 10% of DMSO treatment.
  • Normal fibroblast cells BJ6 and NIH-3T3, were plated at a viable cell density of 3000 cells per well on a 96-well cell culture plate to determine if these compounds are generally cytotoxic stem cell culture medium. After culturing for 24 hours, the selected compounds were treated with a single concentration of 10 nM and cultured for 72 hours. Cell-titer glo (Promega, WC, USA) was then treated for luciferase activity to compare viability. Luciferase activity was detected using a Tecan plate reader (Biocompare, USA). DMSO-treated control cells were divided into 100% and 0% cells, respectively.
  • Example 1 Benidipine hydrochloride 5 0 2 (Example 2) Lacidipine (Lacipil, Motens) 5 0 3 (Example 3) Manidipine (Manyper) 2 0 4 (Example 4) Lomerizine HCl 2 0 5 Cilnidipine 18 14 6 Ranolazine dihydrochloride 86 87 7 Isradipine (Dynacirc) 48 20 8 Amlodipine (Norvasc) 21 4 9 Flunarizine 2HCl 12 0 10 Cleviprex (Clevidipine) 96 64 11 Nitrendipine 95 89 12 Tetracaine hydrochloride (Pontocaine) 46 17 13 Nilvadipine (ARC029) 28 32 14 Azelnidipine 98 0 15 Nicardipine HCl 29 57 16 Nimodipine (Nimotop) 78 64 17 Nisoldipine (Waters) 78 52 18 Nife
  • a total of 20 kinds of calcium channel inhibitors were screened. However, only four kinds of compounds of the present invention show an area value of 5 or less, and at the same time, the ATP measurement value is zero.
  • A2780-SP cells were plated in Ultra-Low Attachment round-bottomed 96-well corning at a viable cell density of 500 cells per well and cultured in cancer stem cell medium.
  • Cancer stem cell culture medium consists of neurobasal medium supplemented with bFGF 20 ng / ml, EGF 10 ng / ml, Amphotericin B 2.5 ng / ml, HEPES, Glutamax and B27.
  • the cells were treated with 30 ⁇ M, 10 ⁇ M, 2 ⁇ M, 0.4 ⁇ M and 0.08 ⁇ M of the compounds of Examples 1 to 4, respectively, and cultured for 8 days. The medium containing the compound was added once again on the fourth day.
  • Sphere cell viability was assessed by Cell-titer Glo (Promega, WC, USA) and luciferase activity was detected using a Tecan plate reader (Biocompare, USA).
  • A2780 ovarian cancer cells were plated in 96-well plates at a density of 3000 viable cells per well and cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin Respectively. On the day following the incubation, the cells were treated with 100 ⁇ M, 30 ⁇ M, 10 ⁇ M, 3 ⁇ M and 0.3 ⁇ M of the compounds of Examples 1 to 4, respectively, and cultured for 3 days. Cell viability was assessed by Cell-titer Glo (Promega, WC, USA) and luciferase activity was detected using a Tecan plate reader (Biocompare, USA).
  • A2780-SP cells were plated in Ultra-Low Attachment round-bottomed 96-well corning at a viable cell density of 500 cells per well and cultured in cancer stem cell medium. On the day following the incubation, the cells were treated with 10 ⁇ M, 2 ⁇ M and 0.4 ⁇ M of the compounds of Examples 1 to 4, respectively, and cultured for 8 days at 37 ° C., 5% CO 2 and 95% humidity. Sphere size was analyzed using Image J software (NIH image).
  • A2780-SP cells were plated on Ultra-Low Attachment flat bottom 96-well corning at a viable cell density of 6000 cells per well and grown in cancer stem cell medium. The following day, the cells were treated with 10 ⁇ M of the compounds of Examples 1 to 4 and incubated for 60 hours at 37 ° C., 5% CO 2 and 95% humidity. Sphere forming confluence was analyzed using Incucyte (BioTek, VT, USA).
  • A2780-SP cells (ovarian cancer stem cells) were plated in Ultra-Low Attachment 6-well corning at 4 x 10 viable cell densities per well and cultured in cancer stem cell medium. The next day, the medium was replaced with Neuro Basal Medium (NBM), incubated for 16 hours in starvation, and treated with 10 ⁇ M of the compounds of Examples 1 to 4 for 1 hour. The medium was exchanged with a cancer stem cell medium containing the compound, and the cells were further cultured for 30 minutes before harvesting and dissolving in RiPA buffer. The extracted proteins were separated by SDS-PAGE gel and transferred to PVDF membranes for Western Blot analysis. After blocking with 5% skim milk, the membranes were incubated overnight with primary antibodies in blocking buffer at 4 ° C and then incubated with HRP-conjugated secondary antibody ( HRP-conjugated secondary antibodies).
  • HRP-conjugated secondary antibodies HRP-conjugated secondary antibodies
  • anti-phospho-AKT Ser473 (clone 193H12, Cell Signaling), anti-AKT (rabbit polyclonal, Cell Signaling), anti-phospho-ERK (Thr202 / Tyr204) monoclonal, Cell Signaling), anti-phospho-p38 (rabbit monoclonal, Cell Signaling), anti-p38 (rabbit monoclonal, Cell Signaling), anti-OCT3 / 4 (mouse monoclonal, Santa Cruz)
  • Anti-CD133 rabbit monoclonal, Abcam
  • anti-GAPDH mimouse monoclonal, Santa Cruz
  • anti-ALDH1 mimethyl-1
  • anti-SOX2 anti-SOX2 (rabbit monoclonal, Cell signaling).
  • the secondary antibodies used were as follows:
  • A2780-SP cells (ovarian cancer stem cells) were plated in Ultra-Low Attachment 6-well corning at a viable cell density of 1x10 6 cells per well and cultured in cancer stem cell medium. The following day, A2780-SP cells were treated with 10 ⁇ M of the compounds of Examples 1 to 4, cultured at 37 ° C, 5% CO 2, 95% humidity for 24 hours, and then harvested with a centrifuge. After washing once with PBS, the cells were resuspended in 0.3 ml of PBS. 0.7 ml of cold ethanol was slowly added dropwise to fix the cells, and the cells were incubated on ice for 1 hour.
  • Quantitative Real-time polymerase chain reaction Quantitative Real-time polymerase chain reaction
  • Trizol RNA extraction kit (Invitrogen) was used to extract the total RNA from the samples according to the manufacturer's instructions. 2 [mu] g of RNA was reverse transcribed with cDNA using the GoScriptTM cDNA synthesis system (GoScriptTM cDNA synthesis system, Promega). The synthesized cDNA was applied to Quantitative RT-PCR using FastStart SYBR green Master (Roche) and Bio-Rad S1000 Thermal cycler with indicated primers. GAPDH was used as a reference gene and the results were expressed relative to the control group using the ddCt method.
  • the composition of the external solution was 143 mM NaCl, 5.6 mM KCl, 10 mM CaCl 2 , 2 mM MgCl 2 , 10 mM HEPES, 5 mM glucose, 1 nM tetrodotoxin and 10 mM tetraethylammonium, The pH was adjusted to 7.4 using.
  • the composition of the internal pipette solution for the whole cell patch was 140 mM CsCl, 2 mM MgCl 2 , 3 mM Mg-ATP, 5 mM HEPES, 1.1 mM EGTA and pH was adjusted to 7.2 using CsOH .
  • the Ca 2+ current was measured using the Axopatch 700B, DigiData 1440A, pClamp 10.4 in a voltage clamp mode and after holding the basal membrane potential at -70 mV, starting at -70 mV and +10 mV The amount of current appearing while increasing the voltage was measured. After measuring the normal current, 10 ⁇ M of Manidipine of Example 3 was treated for 5 minutes, and the change in the amount of current was measured by applying the same voltage change. The access resistance (Ra) value of the whole cell patch clamp was 10 ⁇ 20 M ⁇ .
  • ovarian cancer stem cell line A2780-SP cells and ovarian cancer cell line A2780-AD cells were inoculated subcutaneously in nude mice 1x10 5 . At this time, ovarian cancer cells were inoculated on the left side of the nude mouse, and 8 ovarian cancer stem cells were inoculated on the right side. After the inoculation, the size of the tumor formation was measured from the 14th day.
  • control group (3 pBS treatment), Manidipine treatment group (3 animals -1 mpk treatment) Treatment group (3 animals -5mpk treatment), Manidipine and anti-cancer drug (Pacritaxel) treatment group were divided into two groups. Thereafter, drug administration and tumor size were measured at intervals of 3 to 4 days. After that, the animals were euthanized on the 31st day, and the weight and size of the tumors were finally measured.
  • cell viability analysis was carried out using the pharmaceutical compositions of Examples 1 to 4, 1 and 2, respectively.
  • the GI 50 values of the respective compounds were measured for each cell, and are shown in Table 3 below.
  • the specific experimental method is the same as the 1. cell viability assay of the above experimental protocol.
  • Figure 1 shows a dose-response curve of the viability of A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated. , And the dose-response curve of the viability of A2780 cells (ovarian cancer cells).
  • Examples 1 to 4 were found to reduce the viability of A2780-SP cells, which are ovarian cancer cells in a dose-dependent manner.
  • the pharmaceutical compositions of Examples 1 to 4 were found to reduce the viability of A2780 cells, which are ovarian cancer cells, in a dose-dependent manner.
  • the pharmaceutical compositions of Examples 1 to 4 showed a GI50 value of 15 ⁇ M or less, thereby confirming that the cell growth inhibitory effect was excellent.
  • the lower the value of stem cells the more excellent selective growth inhibition effect is obtained in stem cells.
  • the pharmaceutical composition comprising the calcium channel inhibitor of the present invention shows the inhibitory effect on the growth of cancer stem cells, and thus it has anti-cancer effect.
  • the cell spheres of cancer stem cells were analyzed using the pharmaceutical compositions of Examples 1 to 4, 3.
  • the specific experimental method is the same as the 2. spherical analysis of the above experimental protocol.
  • FIG. 3 is an image showing the result of analyzing the sphere formation of A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of the Example was treated.
  • compositions of Examples 1 to 4 reduce the sphere formation of A2780-SP cells, which are ovarian cancer cells in a dose-dependent manner.
  • the sphere is a characteristic form of cancer stem cells, which means that the growth of the sphere is the growth of cancer stem cells. Therefore, the pharmaceutical composition containing the calcium channel inhibitor according to the present invention inhibits the formation of the stem cells of cancer stem cells, so that it has anti-cancer effect.
  • the cell spheres of cancer stem cells were analyzed using the pharmaceutical compositions of Examples 1 to 4, 4.
  • the specific experimental method is the same as that of Experimental Protocol.
  • FIG. 4 is a graph showing the results of measurement of the proliferation of A2780-SP cells by analyzing the sphere formation of A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of Example was treated.
  • the pharmaceutical compositions of Examples 1 to 4 inhibited sphere fusion, and showed a fusion rate of 1/3 or less as compared with the control group (DMSO treatment group) after 60 hours, Proliferation inhibitory effect can be shown.
  • the pharmaceutical composition comprising the calcium channel inhibitor according to the present invention inhibits the proliferation of cancer stem cells, and thus has anticancer effects.
  • FIG. 5 is an image showing changes in the expression of markers related to cancer stem cell function in A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of Example is treated.
  • Figure 6 is a graph quantifying the expression changes of markers related to cancer stem cell function in A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated.
  • Example 1 bistyreine
  • Example 3 manidipine
  • Example 4 lomeligin
  • Example 2 reduces the expression of NANOG, SOX2, ALDH1, and CD133.
  • the pharmaceutical composition of the present invention can reduce the expression of the stem cells related to the stem cells of cancer stem cells which cause tolerance to existing anticancer drugs, and thus can be useful for cancer treatment.
  • FIG. 7 is an image showing changes in the activity of proteins involved in cancer stem cell survival and proliferation in A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of the Example was treated.
  • A2780-SP cells ovarian cancer stem cells
  • Example 1 bis(benidipine), Example 3 (manidipine), and Example 4 (lomeligin) was treated, the cancer stem cell survival and proliferation- The activity of AKT, ERK, and p38 was decreased.
  • Example 2 decreases the activity of ERK, p38.
  • the pharmaceutical composition of the present invention reduces the stem cell function of cancer stem cells, reduces the expression of proteins involved in cancer stem cell survival and proliferation, inhibits recurrence, metastasis, and progression of cancer, It can be useful for treatment.
  • Figure 9 is a graph showing the cell cycle changes of A2780-SP cells when the pharmaceutical composition of the example was treated.
  • Figure 10 is a graph showing the cell death of A2780-SP cells when the pharmaceutical composition of Example was treated. to be.
  • compositions of Examples 1 to 4 increase cell death of cancer stem cells.
  • the cell cycle of the cells can be analyzed by flow cytometry analysis.
  • the cell cycle of each cell is analyzed by flow cytometry, The number is expressed as a percentage.
  • the pharmaceutical composition of the present invention increases the apoptosis of cancer stem cells, and thus can be useful for the treatment of cancer.
  • Fig. 11 shows quantitative RT-PCR quantification of the expression levels of calcium channel subunits (Cacna1d, Cacna1f, Cacna1h) mRNA in A2780-SP cells and A2780 cells.
  • FIG. 13 shows the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f and si-Cacna1h) in A2780-SP cells and then to identify markers (OCT3 / 4, NANOG, SOX2, ALDH1 , CD133) was quantified by quantitative RT-PCR.
  • knockdown of a gene related to calcium channel in cancer stem cells revealed that the expression amount of mRNA of calcium channel subunit was remarkably decreased.
  • knockdown of genes related to calcium channels in cancer stem cells results in a decrease in mRNA expression of markers associated with cancer stem cells.
  • FIG. 14 is a graph showing the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f and si-Cacna1h) in A2780-SP cells, and stem cell-associated markers and the expression of proteins involved in the survival and proliferation of cancer stem cells.
  • FIG. 15 is a graph showing the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f, si-Cacna1h) in A2780-SP cells, a stem cell-associated marker, and a stem cell-associated marker and the expression of a protein associated with survival and proliferation of cancer stem cells.
  • Figure 16 is a graph showing that the amplitude of calcium current is reduced when the pharmaceutical composition of Example is treated with A2780-SP cells.
  • Example 3 which is a pharmaceutical composition containing the calcium channel inhibitor of the present invention, showed a larger amplitude of calcium current in ovarian cancer cell (A2780-SP) than ovarian cancer cell (A2780) -SP, the amplitude of the calcium current is remarkably suppressed.
  • the four pharmaceutical compositions of the present invention inhibit the cancer stem cell stem stem, which is a major factor in resistance to existing drugs through the inhibition of the calcium channel overexpressed in cancer stem cells, Suggesting that apoptosis may be induced.
  • cancer can be effectively used for cancer treatment by inhibiting recurrence, metastasis and progression of cancer.
  • FIG. 17 is a graph showing the results of measuring the cell survival rate of A2780-SP cells when the pharmaceutical composition of Example and cisplatin were used in combination.
  • FIG. 17 is a graph showing the results of measuring the cell survival rate of A2780-SP cells when the pharmaceutical composition of Example and cisplatin were used in combination.
  • Example 18 is a graph showing the results of analysis of cell proliferation of A2780-SP cells when the pharmaceutical composition of Example and cisplatin were used in combination.
  • the pharmaceutical composition containing the calcium channel inhibitor of the present invention when administered singly, it exhibits an excellent effect of reducing cell viability. However, when administered in combination with an anticancer agent, the cell viability of cancer stem cells is further reduced .
  • the pharmaceutical composition containing the calcium channel inhibitor of the present invention not only exhibits excellent anticancer effects even when used alone, but also can exhibit synergistic effects even higher when used in combination with existing anticancer drugs.
  • the cell viability was analyzed using the pharmaceutical compositions of Examples 1 to 4.
  • the results are shown in the following Table 5 Respectively.
  • the specific experimental method is the same as the 1. cell viability analysis of the above experimental protocol, and the normal cells were normal fibroblasts BJ6 and NIH-3T3.
  • the pharmaceutical compositions of Examples 1 to 4 exhibited cell viability of 80% or more, so that the pharmaceutical composition of the present invention had no effect on normal cells , And specifically shows cytotoxicity only to cancer cells, particularly cancer stem cells.
  • the pharmaceutical composition comprising the calcium channel inhibitor of the present invention does not affect normal cells, but exhibits an inhibitory effect on the growth of cancer stem cells.
  • the anticancer effect with less side effects can be exhibited.
  • the pharmaceutical composition containing the calcium channel inhibitor of the present invention is excellent in the effect of inhibiting the growth and proliferation of cancer stem cells even when used alone, As a result, it is possible to inhibit recurrence, metastasis and progression of cancer. Therefore, it can be used as a pharmaceutical composition or a combined preparation for cancer prevention or treatment.
  • FIG. 19 is a graph showing the results of tumorigenesis (A2780-AD) in the case of the ovarian cancer cell tumor animal model (A2780-AD) and the ovarian cancer stem cell tumor animal model (A2780-SP), alone or in combination with paclitaxel Inhibitory effect and evaluating the results.
  • the pharmaceutical composition containing the calcium channel inhibitor of the present invention can inhibit recurrence, metastasis and progression of ovarian cancer, and thus can be usefully used as a pharmaceutical composition for the prevention or treatment of ovarian cancer.
  • tablets were prepared by tableting according to a conventional method for producing tablets.
  • the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.
  • an injectable preparation was prepared by incorporating the aforementioned components in the amounts indicated.
  • the ointment was prepared by incorporating the above ingredients in the prescribed amounts according to the usual preparation method of ointment.
  • Vitamin A acetate 70 mg
  • Vitamin E 1.0mg
  • Vitamin B6 0.5mg
  • Vitamin B12 0.2mg
  • composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed , Granules may be prepared and used in the manufacture of a health functional food composition according to a conventional method.
  • the above components were mixed in accordance with a conventional health functional beverage 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 container, which was sealed and sterilized, Were used to prepare beverage compositions.
  • composition ratio is relatively mixed with the ingredient suitable for the favorite drink, it is also possible to arbitrarily modify the blending ratio according to the regional or national preference such as the demand class, demand country, use purpose, and the like.
  • the pharmaceutical composition containing the calcium channel inhibitor of the present invention can be usefully used as a pharmaceutical composition or a combined preparation for the prevention or treatment of cancer.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Disclosed is a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, a calcium channel inhibitor or a pharmaceutically acceptable salt thereof. The pharmaceutical composition not only exhibits an excellent effect of inhibiting growth and proliferation of cancer stem cells even when used alone but also exhibits a more excellent pharmacological effect when administering the pharmaceutical composition together with conventional anticancer drugs. Since it is possible to inhibit the recurrence, metastasis, and progression of cancer, the pharmaceutical composition can usefully be used as a pharmaceutical composition or a combined formulation for the prevention or treatment of cancer.

Description

칼슘 채널 억제제 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 암의 예방 또는 치료용 약학적 조성물A pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a calcium channel inhibitor or a pharmaceutically acceptable salt thereof
칼슘 채널 억제제 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 암의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.
줄기세포는 자가재생(self-renewal)을 할 수 있고, 각 기관의 세포로 분화(differentiation)하여 기관형성(organogenesis)을 이룰 수 있는 미성숙(immature) 세포를 말한다. 줄기세포는 크게 두 종류로 분류할 수 있다. 배아줄기세포(embryonic stem cell)는 신체의 어느 기관으로도 분화할 수 있고, 주머니배(blastocyst)에서 기원한다. 연구목적의 줄기세포는 수정 후 6일경에 세포 수가 대략 200개인 주머니배의 내세포괴(inner cell mass)에서 채취한다. 다른 종류의 줄기세포는 성인줄기세포(adult stem cell)이다. 성인줄기세포는 자가재생과 분화를 할 수 있다는 점에서는 배아줄기세포와 동일하지만 분화가 주로 특정 기관 내 세포로 이루어진다는 점에서 배아줄기세포와 구분된다. 성인줄기세포의 대표적인 예로 조혈줄기세포(hematopoietic stem cell)을 들 수 있다.Stem cells are immature cells that can undergo self-renewal and can differentiate into the cells of each organ and achieve organogenesis. There are two types of stem cells. Embryonic stem cells can differentiate into any organ of the body and originate from the blastocyst. The stem cells for the study are harvested from the inner cell mass of approximately 200 cell counts at 6 days after fertilization. Other types of stem cells are adult stem cells. Adult stem cells are distinguishable from embryonic stem cells in that they are identical to embryonic stem cells in that they can regenerate and differentiate, but differentiation is mainly made up of specific endothelial cells. A representative example of adult stem cells is hematopoietic stem cells.
암줄기세포(cancer stem cell)는 줄기세포와 같이 비대칭적 분열을 통해 자가재생과 분화를 할 수 있으나, 정상 줄기세포와 달리 분열조절능력에 장애가 생겨 종양을 생성하는 세포이다. 최근 연구를 통해 정상 줄기세포에서 발견되는 Notch, Sonic hedgehog (SHH), Wnt, β-catenin, phosphatase and tensin homolog [HONG7](PTEN), transforming growth factor (TGF)-β, Bmi-1 등의 신호경로가 악성종양에서 변형되어 있음이 밝혀졌다. 암줄기세포는 정상줄기세포와 높은 운동성, 자손(progeny)의 다양성, 강한 증식 잠재력, 혈관계(vasculature), 미성숙한 발현양상, nestin, epidermal growth factor (EGF)-receptor, PTEN의 발현, Hedgehog 신호경로 활성도, telomerase 활성도, Wnt 신호경로 활성도 등에서 공통된 특징을 보인다.Cancer stem cells, like stem cells, can regenerate and differentiate through asymmetric division, but unlike normal stem cells, they are cells that produce tumors with impaired ability to regulate division. Recent studies have shown that signals such as Notch, Sonic hedgehog (SHH), Wnt, β-catenin, phosphatase and tensin homolog [HONG7] (PTEN), transforming growth factor The pathway was found to be deformed in malignant tumors. The expression of PTEN, Hedgehog signaling pathway, nestin, epidermal growth factor (EGF) -receptor, and proliferative potential, vasculature, immature expression pattern, and high motility, progeny diversity, , telomerase activity, and Wnt signaling pathway activity.
상기와 같은 이유로 암줄기세포를 타겟으로 한 항암제 개발이 활발히 이루어지고 있다.For the above reasons, anti-cancer agents targeting cancer stem cells have been actively developed.
특허문헌 1(대한민국 공개특허 1020160094861)은 알데히드 억제제 및 비구아나이드 계열 화합물을 포함하는 암 줄기세포의 성장 억제용 약학적 조성물에 대하여 개시한 바 있고,Patent Document 1 (Korean Patent Laid-open Publication No. 1020160094861) discloses a pharmaceutical composition for inhibiting the growth of cancer stem cells comprising an aldehyde inhibitor and an acetylated compound,
비특허문헌 1(J Korean Assoc Oral Maxillofac Surg 2011;37:97-108)은 구강 편평세포암종에서의 암줄기세포 이론과 최신 지견에 대하여 개시한 바 있다.Non-Patent Document 1 (J Korean Assoc Oral Maxillofac Surg 2011; 37: 97-108) discloses the theory of cancer stem cell in oral squamous cell carcinoma and the latest knowledge.
본 발명의 일 목적은 암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer.
본 발명의 다른 목적은 암의 예방 또는 치료용 병용 제제를 제공하는 것이다.It is another object of the present invention to provide a combined preparation for the prevention or treatment of cancer.
본 발명의 다른 목적은 암의 예방 또는 개선용 건강기능식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a health functional food composition for preventing or ameliorating cancer.
상기 목적을 달성하기 위하여,In order to achieve the above object,
본 발명의 일 측면에 따라, 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 치료용 약학적 조성물이 제공된다.According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a calcium channel inhibitor or a pharmaceutically acceptable salt thereof.
또한, 본 발명의 다른 측면에 따라, 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염 및 항암제를 포함하는 암의 예방 또는 치료용 병용 제제가 제공된다.Further, according to another aspect of the present invention, there is provided a combined preparation for preventing or treating cancer, comprising a calcium channel inhibitor or a pharmaceutically acceptable salt thereof and an anticancer agent.
또한, 본 발명의 다른 측면에 따라, 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 개선용 건강기능식품 조성물이 제공된다.According to another aspect of the present invention, there is provided a health functional food composition for preventing or ameliorating a cancer containing, as an active ingredient, a calcium channel inhibitor or a pharmaceutically acceptable salt thereof.
또한, 본 발명의 다른 측면에 따라, 상기 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 치료용 약학적 조성물 병용 제제 또는 건강기능식품 조성물을 필요한 대상에게 투여하는 단계를 포함하는 암의 예방 또는 치료 방법이 제공된다.According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer comprising the calcium channel blocker or a pharmaceutically acceptable salt thereof as an active ingredient, A method of preventing or treating cancer is provided.
또한, 본 발명의 다른 측면에 따라, 암의 예방 또는 치료에 있어서의, 상기 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 치료용 약학적 조성물 병용 제제 또는 건강기능식품 조성물의 용도가 제공된다.According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer comprising the calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient in the prevention or treatment of cancer. The use of functional food compositions is provided.
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물은 단독으로 사용하여도 암 줄기세포의 성장 및 증식을 저해하는 효과가 우수할 뿐만 아니라, 종래 항암제와 병용 투여할 경우, 보다 우수한 약리 효과를 나타내는 바, 암의 재발, 전이, 진행을 억제시킬 수 있으므로, 암의 예방 또는 치료용 약학적 조성물 또는 병용 제제로 유용하게 사용될 수 있다.The pharmaceutical composition comprising the calcium channel inhibitor of the present invention is excellent in the effect of inhibiting growth and proliferation of cancer stem cells even when used alone and exhibits a more excellent pharmacological effect when it is administered in combination with a conventional anticancer drug , Cancer recurrence, metastasis, and progression of cancer can be inhibited. Therefore, the composition can be usefully used as a pharmaceutical composition or a combined preparation for the prevention or treatment of cancer.
도 1은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell) 생존능의 용량-반응(Dose-response) 곡선을 나타낸 것이다. 도 1a: 마니디핀, 도 1b: 라시디핀, 도 1c: 베니디핀, 도 1d: 로메리진Figure 1 shows a dose-response curve of the viability of A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated. Figure 1 a: Manidipine, Figure 1 b: Rashedipine, Figure 1 c: Benidipine, Figure 1 d:
도 2는 실시예 약학적 조성물을 처리하였을 때, A2780 세포(ovarian cancer cell) 생존능의 용량-반응(Dose-response) 곡선을 나타낸 것이다. 도 2a: 마니디핀, 도 2b: 라시디핀, 도 2c: 로메리진 HCl, 도 2d: 베니디핀 HClFigure 2 shows the dose-response curve of the viability of A2780 cells (ovarian cancer cells) when the example pharmaceutical composition was treated. Figure 2a: Manidipine, Figure 2b: Rashedipine, Figure 2c: Lomerizine HCl, Figure 2d: Benidipine HCl
도 3은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)의 구체 형성을 분석한 결과를 나타낸 이미지이다. 도 3a: 마니디핀, 도 3b: 라시디핀, 도 3c: 로메리진 HCl, 도 3d: 베니디핀 HClFIG. 3 is an image showing the result of analyzing the sphere formation of A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of the Example was treated. Figure 3a: Manidipine, Figure 3b: Rashedipine, Figure 3c: Lomerizine HCl, Figure 3d: Benidipine HCl
도 4는 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)의 구체 형성을 분석하여 A2780-SP 세포의 증식을 측정한 결과를 나타낸 그래프이다. 도 4a: 마니디핀, 도 4b: 라시디핀, 도 4c: 로메리진 HCl, 도 4d: 베니디핀 HClFIG. 4 is a graph showing the results of measurement of the proliferation of A2780-SP cells by analyzing the sphere formation of A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of Example was treated. Figure 4a: Manidipine, Figure 4b: Rashedipine, Figure 4c: Romerizine HCl, Figure 4d: Benidipine HCl
도 5는 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포능(stemness)과 관련된 마커들의 발현 변화를 관찰한 이미지이다.FIG. 5 is an image showing changes in the expression of markers related to cancer stem cell function in A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of Example is treated.
도 6은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포능(stemness)과 관련된 마커들의 발현 변화를 정량화한 그래프이다. 도 6a: OCT 3/4, 도 6b: NANOG, 도 6c: SOX2, 도 6d: ALDH1, 도 6e: CD133Figure 6 is a graph quantifying the expression changes of markers related to cancer stem cell function in A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated. Figure 6a: OCT 3/4, Figure 6b: NANOG, Figure 6c: SOX2, Figure 6d: ALDHl, Figure 6e: CD133
도 7은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포 생존 및 증식과 관련된 단백질의 활성 변화를 관찰한 이미지이다.FIG. 7 is an image showing changes in the activity of proteins involved in cancer stem cell survival and proliferation in A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of the Example was treated.
도 8은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포 생존 및 증식과 관련된 단백질의 활성 변화를 정량화한 그래프이다. 도 8a: pAKT, 도 8b: pERK, 도 8c: p-p388 is a graph quantifying the change in the activity of a protein involved in cancer stem cell survival and proliferation in A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated. Figure 8a: pAKT, Figure 8b: pERK, Figure 8c: p-p38
도 9는 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포의 세포 주기 변화를 나타낸 그래프이다. 도 9a: DMSO, 도 9b: 마니디핀, 도 9c: 라시디핀, 도 9d: 로메리진 HCl, 도 9e: 베니디핀 HCl9 is a graph showing cell cycle changes of A2780-SP cells when the example pharmaceutical composition is treated. Figure 9a: DMSO, Figure 9b: Manidipine, Figure 9c: Rashedipine, Figure 9d: Romerizin HCl, Figure 9e: Benidipine HCl
도 10은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포의 세포 사멸을 측정한 그래프이다.FIG. 10 is a graph showing the cell death of A2780-SP cells when the pharmaceutical composition of Example is treated. FIG.
도 11은 A2780-SP 세포 및 A2780세포에서 칼슘 채널 서브유닛(Cacna1d, Cacna1f, Cacna1h) mRNA의 발현 수준을 Quantitative RT-PCR로 정량화 하여 나타낸 것이다. 도 11a: Cacna1d, 도 11b: Cacna1f, 도 11c: Cacna1hFig. 11 shows quantitative RT-PCR quantification of the expression levels of calcium channel subunits (Cacna1d, Cacna1f, Cacna1h) mRNA in A2780-SP cells and A2780 cells. 11a: Cacna1d, Fig. 11b: Cacna1f, Fig. 11c: Cacna1h
도 12는 A2780-SP 세포에서 칼슘 채널과 관련된 유전자(si-Cacna1d, si-Cacna1f, si-Cacna1h)를 넉다운(knockdown)시킨 후, 각 칼슘 채널 서브유닛(Cacna1d, Cacna1f, Cacna1h)의 mRNA발현 수준을 Quantitative RT-PCR로 정량화 하여 넉다운을 확인한 것을 나타낸 것이다. 도 12a: Cacna1d, 도 12b: Cacna1f, 도 12c: Cacna1h12 shows the results of knockdown of the calcium channel-related genes (si-Cacna1d, si-Cacna1f, si-Cacna1h) in A2780-SP cells and the mRNA expression level of each calcium channel subunit (Cacna1d, Cacna1f, Cacna1h) Was quantitated by quantitative RT-PCR to confirm knockdown. 12a: Cacna1d, Fig. 12b: Cacna1f, Fig. 12c: Cacna1h
도 13은 A2780-SP 세포에서 칼슘 채널과 관련된 유전자(si-Cacna1d, si-Cacna1f, si-Cacna1h)를 넉다운(knockdown)시킨 후, 암 줄기세포와 관련된 마커(OCT3/4, NANOG, SOX2, ALDH1, CD133)의 mRNA 발현 수준을 Quantitative RT-PCR로 정량화한 것을 나타낸 것이다. 도 13a: OCT 3/4, 도 13b: NANOG, 도 13c: SOX2, 도 13d: ALDH1, 도 13e: CD133FIG. 13 shows the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f and si-Cacna1h) in A2780-SP cells and then to identify markers (OCT3 / 4, NANOG, SOX2, ALDH1 , CD133) was quantified by quantitative RT-PCR. Figure 13a: OCT 3/4, Figure 13b: NANOG, Figure 13c: SOX2, Figure 13d: ALDHl, Figure 13e: CD133
도 14는 A2780-SP 세포에서 칼슘 채널과 관련된 유전자(si-Cacna1d, si-Cacna1f, si-Cacna1h)를 넉다운(knockdown)시킨 후, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포능(stemness) 관련 마커와 암 줄기세포의 생존 및 증식과 관련된 단백질의 발현 변화를 관찰한 이미지이다.14 is a graph showing the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f and si-Cacna1h) in A2780-SP cells, and stem cell-associated markers and the expression of proteins involved in the survival and proliferation of cancer stem cells.
도 15는 A2780-SP 세포에서 칼슘 채널과 관련된 유전자(si-Cacna1d, si-Cacna1f, si-Cacna1h)를 넉다운(knockdown)시킨 후, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포능(stemness) 관련 마커와 암 줄기세포의 생존 및 증식과 관련된 단백질의 발현 변화를 정량화한 그래프이다. 도 15a: OCT 3/4, 도 15b: pERK, 도 15c: p-p3815 is a graph showing the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f, si-Cacna1h) in A2780-SP cells, a stem cell-associated marker, and a stem cell-associated marker and the expression of a protein associated with survival and proliferation of cancer stem cells. Figure 15a: OCT 3/4, Figure 15b: pERK, Figure 15c: p-p38
도 16은 A2780-SP 세포에 실시예 약학적 조성물을 처리하였을 때, 칼슘 전류 진폭이 줄어듦을 나타낸 그래프이다.Figure 16 is a graph showing that the amplitude of calcium current is reduced when the pharmaceutical composition of Example is treated with A2780-SP cells.
도 17은 실시예 약학적 조성물과 시스플라틴을 병용 처리하였을 때, A2780-SP 세포의 세포 생존율을 측정하여 그 결과를 나타낸 그래프이다. 도 17a: 마니디핀, 도 17b: 라시디핀, 도 17c: 로메리진 HCl, 도 17d: 베니디핀 HClFIG. 17 is a graph showing the results of measuring the cell survival rate of A2780-SP cells when the pharmaceutical composition of Example and cisplatin were used in combination. FIG. Figure 17a: Manidipine, Figure 17b: Rashedipine, Figure 17c: Romerizin HCl, Figure 17d: Benidipine HCl
도 18은 실시예 약학적 조성물과 시스플라틴을 병용 처리하였을 때, A2780-SP 세포의 세포 증식을 분석하여 그 결과를 나타낸 그래프이다. 도 18a: 마니디핀, 도 18b: 라시디핀, 도 18c: 로메리진 HCl, 도 18d: 베니디핀 HCl18 is a graph showing the results of analysis of cell proliferation of A2780-SP cells when the pharmaceutical composition of Example and cisplatin were used in combination. Figure 18a: Manidipine, Figure 18b: Rashedipine, Figure 18c: Romerizin HCl, Figure 18d: Benidipine HCl
도 19는 난소암 세포 종양 동물 모델(A2780-AD) 및 난소암 암 줄기세포 종양 동물모델(A2780-SP)에서, 실시예 약학적 조성물 단독처리 및 파클리탁셀(Paclitaxel)을 병용 처리하였을 때의 종양 형성 억제 효과를 평가하여 그 결과를 나타낸 그래프이다. 도 19a: A2780-AD, 도 19b: A2780-SPFIG. 19 is a graph showing the results of tumorigenesis (A2780-AD) in the case of the ovarian cancer cell tumor animal model (A2780-AD) and the ovarian cancer stem cell tumor animal model (A2780-SP), alone or in combination with paclitaxel Inhibitory effect and evaluating the results. 19A: A2780-AD, FIG. 19B: A2780-SP
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 측면은 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.An aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a calcium channel inhibitor or a pharmaceutically acceptable salt thereof.
상기 칼슘 채널 억제제는 하기 화학식 A 내지 화학식 D로부터 선택되는 어느 하나일 수 있다.The calcium channel inhibitor may be any one selected from the following formulas (A) to (D).
[화학식 A(베니디핀, Benidipine)][Chemical formula A (Benidipine)]
Figure PCTKR2019001143-appb-I000001
;
Figure PCTKR2019001143-appb-I000001
;
[화학식 B(라시디핀, Lacidipine)][Chemical formula (B) (Lacidipine)]
Figure PCTKR2019001143-appb-I000002
;
Figure PCTKR2019001143-appb-I000002
;
[화학식 C(마니디핀, Manidipine)][Chemical Formula C (Manidipine)]
Figure PCTKR2019001143-appb-I000003
;
Figure PCTKR2019001143-appb-I000003
;
[화학식 D(로메리진, Lomerizine)][Chemical Formula D (Lomerizine)]
Figure PCTKR2019001143-appb-I000004
.
Figure PCTKR2019001143-appb-I000004
.
상기 암은 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 구강암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 음경암, 전립선암, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성외부생식기암 및 피부암으로 이루어지는 군으로부터 선택되는 1종 이상일 수 있다.The cancer may be selected from the group consisting of benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial lesion, ependymoma, brain tumor, laryngeal cancer, pancreatic cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, Cancer of the breast, cancer of the gallbladder, cancer of the gallbladder, cancer of the bile duct, pancreatic cancer, small bowel cancer, colon cancer, anal cancer, bladder cancer, breast cancer, Kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female gonadal cancer and skin cancer.
또한, 상기 암은 난소암일 수 있다.Further, the cancer may be an ovarian cancer.
상기 약학적 조성물은 OCT3/4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 및 p38로 이루어지는 단백질 군으로부터 선택되는 하나 이상의 발현을 억제할 수 있다.The pharmaceutical composition may inhibit expression of at least one selected from the group consisting of proteins consisting of OCT3 / 4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 and p38.
상기 약학적 조성물은 암 줄기세포의 증식을 억제할 수 있다.The pharmaceutical composition can inhibit the proliferation of cancer stem cells.
다양한 in vitro 실험을 통하여 상기 약학적 조성물의 항암 효과를 측정한 결과, 상기 약학적 조성물은 암 세포 및 암 줄기세포의 생존능을 감소시키고, 증식을 억제하며, 암 줄기세포의 구체 형성 및 증식을 억제하고, 암 줄기세포와 관련된 마커 및 증식과 관련된 단백질의 발현을 억제하는 효과가 우수하고, 암 줄기세포의 세포 사멸을 증가시키므로, 항암 효과가 우수함을 확인하였다(실험예 2 내지 5 및 도 1 내지 10 참조).Through various in vitro experiments, the anticancer effect of the pharmaceutical composition was measured. As a result, the pharmaceutical composition reduced the viability of cancer cells and cancer stem cells, suppressed proliferation, inhibited the formation and proliferation of cancer stem cells And it was found that the anticancer effect was excellent because the cancer stem cell-related marker and the effect of suppressing the expression of the protein related to the proliferation were excellent and the cell death of the cancer stem cell was increased (Experimental Examples 2 to 5 and FIGS. 10).
따라서, 본 발명의 일 측면에 따른 약학적 조성물은 암의 예방 또는 치료용 약학적 조성물로 유용하게 사용될 수 있다.Accordingly, the pharmaceutical composition according to one aspect of the present invention can be usefully used as a pharmaceutical composition for preventing or treating cancer.
칼슘 채널 억제제는 약학적으로 허용가능한 염의 형태로 사용할 수 있으며, 염으로는 약학적으로 허용가능한 유리산(free acid)에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요드화수소산, 아질산, 아인산 등과 같은 무기산류, 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸디오에이트, 방향족 산류, 지방족 및 방향족 설폰산류 등과 같은 무독성 유기산, 아세테이트, 안식향산, 구연산, 젖산, 말레인산, 글루콘산, 메탄설폰산, 4-톨루엔설폰산, 주석산, 푸마르산 등과 같은 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염의 종류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트, 니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 석시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, β-하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트, 만델레이트 등을 포함한다.The calcium channel inhibitor may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid and the like, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, And organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like are obtained from non-toxic organic acids such as dicarboxylic acids, Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, But are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, halides, halides, halides, halides, halides, halides, But are not limited to, lactose, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene Sulfonates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, and the like, as well as sulfonates such as benzyl sulfonate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, -Sulfonate, naphthalene-2-sulfonate, mandelate, and the like.
상기 산 부가염은 통상의 방법으로 제조할 수 있으며, 예를 들면 칼슘 채널 억제제를 메탄올, 에탄올, 아세톤, 메틸렌클로라이드, 아세토니트릴 등과 같은 유기용매에 녹이고 유기산 또는 무기산을 가하여 생성된 침전물을 여과, 건조시켜 제조하거나, 용매와 과량의 산을 감압 증류한 후 건조시켜 유기용매 하에서 결정화시켜서 제조할 수 있다. The acid addition salt can be prepared by a conventional method. For example, the calcium channel inhibitor is dissolved in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., and a precipitate formed by adding an organic acid or an inorganic acid is filtered, dried Or by distilling the solvent and excess acid under reduced pressure, followed by drying and crystallization in an organic solvent.
또한, 염기를 사용하여 약학적으로 허용가능한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조시켜 얻는다. 이때, 금속염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다. 또한, 이에 대응하는 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염(예, 질산은)과 반응시켜 얻는다.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 an 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 addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).
나아가, 상기 칼슘 채널 억제제는 이의 약학적으로 허용가능한 염뿐만 아니라, 이로부터 제조될 수 있는 용매화물, 광학 이성질체, 수화물 등의 형태로 사용될 수 있다.Further, the calcium channel inhibitor may be used in the form of a pharmaceutically acceptable salt thereof, as well as a solvate, an optical isomer, a hydrate, etc., which may be prepared therefrom.
상기 칼슘 채널 억제제 또는 이의 약학적으로 허용가능한 염은 임상 투여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등 이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다.The calcium channel blocker or a pharmaceutically acceptable salt thereof may be administered in various forms of oral and parenteral administration at the time of clinical administration. 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 one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, and emulsions. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
상기 칼슘 채널 억제제 또는 이의 약학적으로 허용가능한 염을 유효 성분으로 하는 약학적 조성물은 비경구 투여할 수 있으며, 비경구 투여는 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사를 주입하는 방법에 의한다. The pharmaceutical composition comprising the calcium channel blocker or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and the parenteral administration may be a subcutaneous injection, an intravenous injection, an intramuscular injection, And.
이때, 비경구 투여용 제형으로 제제화하기 위하여 상기 칼슘 채널 억제제 또는 이의 약학적으로 허용가능한 염을 안정제 또는 완충제와 함께 물에 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알 단위 투여형으로 제조할 수 있다. 상기 조성물은 멸균되고/되거나 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 및/또는 완충제 등의 보조제, 및 기타 치료적으로 유용한 물질을 함유할 수 있으며, 통상적인 방법인 혼합, 과립화 또는 코팅 방법에 따라 제제화할 수 있다.In order to formulate the composition for parenteral administration, the calcium channel blocker or pharmaceutically acceptable salt thereof is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, which is then prepared into an ampoule or vial unit dosage form . The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.
경구 투여용 제형으로는 예를 들면 정제, 환제, 경/연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제, 엘릭시르제, 트로키제 등이 있는데, 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)를 함유하고 있다. 정제는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및/또는 폴리비닐피롤리딘 등과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염 등과 같은 붕해제 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제, 및 감미제를 함유할 수 있다.Examples of formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and troches, , Dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants (such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols). The tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain binders such as starch, agar, alginic acid or sodium salts thereof Release or boiling mixture and / or absorbent, colorant, flavor, and sweetening agent.
또한, 본 발명의 다른 측면은 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염 및 항암제를 포함하는 암의 예방 또는 치료용 병용 제제를 제공한다.Another aspect of the present invention provides a combined preparation for prevention or treatment of cancer comprising a calcium channel inhibitor or a pharmaceutically acceptable salt thereof and an anticancer agent.
상기 칼슘 채널 억제제는 하기 화학식 A 내지 화학식 D로부터 선택되는 어느 하나일 수 있다.The calcium channel inhibitor may be any one selected from the following formulas (A) to (D).
[화학식 A(베니디핀, Benidipine)][Chemical formula A (Benidipine)]
Figure PCTKR2019001143-appb-I000005
;
Figure PCTKR2019001143-appb-I000005
;
[화학식 B(라시디핀, Lacidipine)][Chemical formula (B) (Lacidipine)]
Figure PCTKR2019001143-appb-I000006
;
Figure PCTKR2019001143-appb-I000006
;
[화학식 C(마니디핀, Manidipine)][Chemical Formula C (Manidipine)]
Figure PCTKR2019001143-appb-I000007
;
Figure PCTKR2019001143-appb-I000007
;
[화학식 D(로메리진, Lomerizine)][Chemical Formula D (Lomerizine)]
Figure PCTKR2019001143-appb-I000008
.
Figure PCTKR2019001143-appb-I000008
.
상기 암은 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 구강암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 음경암, 전립선암, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성외부생식기암 및 피부암으로 이루어지는 군으로부터 선택되는 1종 이상일 수 있다.The cancer may be selected from the group consisting of benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial lesion, ependymoma, brain tumor, laryngeal cancer, pancreatic cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, Cancer of the breast, cancer of the gallbladder, cancer of the gallbladder, cancer of the bile duct, pancreatic cancer, small bowel cancer, colon cancer, anal cancer, bladder cancer, breast cancer, Kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female gonadal cancer and skin cancer.
또한, 상기 암은 난소암일 수 있다.Further, the cancer may be an ovarian cancer.
상기 병용 제제는 OCT3/4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 및 p38로 이루어지는 단백질 군으로부터 선택되는 하나 이상의 발현을 억제할 수 있다.The combined preparation may inhibit expression of at least one selected from the group consisting of OCT3 / 4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 and p38.
상기 병용 제제는 암 줄기세포의 증식을 억제할 수 있다.The combined preparation can inhibit the proliferation of cancer stem cells.
용어 "항암제"란 암세포의 각종 대사경로에 작용하여 암세포에 대하여 세포독성(cytotoxicity)이나 성장억제효과(cytostatic effects)를 나타내는 기존의 암 치료에 사용되는 공지의 약제를 총칭하는 것이며, 지금까지 개발된 대사길항제, 식물성 알칼로이드, 토포이소머라제 저해제(topoisomerase inhibitor), 알킬화제, 항암성 항생물질, 호르몬제 및 기타 약제를 모두 포함하는 것이다. The term "anticancer agent" is a generic term for known drugs used in conventional cancer therapy that act on various metabolic pathways of cancer cells and exhibit cytotoxicity or cytostatic effects on cancer cells. Metabolic antagonists, botanical alkaloids, topoisomerase inhibitors, alkylating agents, anticancer antibiotics, hormones and other medicaments.
본 발명의 일 측면에서, 항암제는 독소루비신, 파클리탁셀, 빈크리스틴, 다우노루비신(daunorubicin), 빈블라스틴(vinblastine), 액티노마이신-D(actinomycin-D), 도세탁셀, 에토포사이드(etoposide), 테니포사이드(teniposide), 비산트렌 (bisantrene), 호모해링토닌(homoharringtonine), 글리벡(Gleevec; STI-571), 시스플라틴, 5-플로오로우라실, 아드리아마이신, 메토트렉세이트, 부설판(busulfan), 클로람부실(chlorambucil), 시클로포스파미드(cyclophosphamide), 멜팔란 (melphalan), 니트로겐 무스타드(nitrogen mustard) 및 니트로소우레아 (nitrosourea)로 이루어진 군으로부터 선택된 1종 이상일 수 있다.In one aspect of the invention, the anticancer agent is selected from the group consisting of doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, (Including but not limited to: teniposide, bisantrene, homoharringtonine, Gleevec (STI-571), cisplatin, 5-fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, and nitrosourea. The term " chlorambucil "
상기 항암제는 시스플라틴 또는 파클리탁셀일 수 있다.The anticancer agent may be cisplatin or paclitaxel.
상기 병용 제제를 항암제와 함께 투여하였을 때의 항암 효과를 측정한 결과, 상기 칼슘 채널 억제제를 포함하는 약학적 조성물을 단독으로 투여하였을 때도 우수한 세포 증식 억제 효과를 나타내나, 항암제와 병용투여 하였을 때 암 줄기세포의 세포 증식이 보다 더 억제되는 것을 알 수 있다(실험예 7 및 도 17 및 18 참조).As a result of measuring the anticancer effect when the combined preparation is administered together with the anticancer agent, the pharmaceutical composition containing the calcium channel blocker exhibits excellent cell proliferation inhibitory effect even when it is administered alone. However, when administered in combination with an anticancer agent, It can be seen that the cell proliferation of stem cells is further suppressed (see Experimental Example 7 and Figs. 17 and 18).
따라서, 본 발명의 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염은 단독으로 사용하여도 우수한 항암 효과를 나타낼 뿐만 아니라, 기존 항암제와 병용으로 사용하여 보다 더 높은 시너지 효과를 나타낼 수 있다.Therefore, the calcium channel inhibitor of the present invention or a pharmaceutically acceptable salt thereof exhibits excellent anticancer effects even when used alone, and can exhibit higher synergistic effects when used in combination with existing anticancer drugs.
따라서, 본 발명의 일 측면에 따른 병용 제제는 종래 항암제와 병용 투여함으로써 암의 예방 또는 치료에 유용하게 사용될 수 있다.Therefore, the combined preparation according to one aspect of the present invention can be usefully used for the prevention or treatment of cancer by the combined use with the conventional anticancer agent.
또한, 본 발명의 다른 측면은 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염 및 항암제를 병용투여하여 사용하는 암의 예방 또는 치료를 위한 병용 요법을 제공한다.Another aspect of the present invention provides a combination therapy for prevention or treatment of cancer, which is used by administering a combination of a calcium channel inhibitor, a pharmaceutically acceptable salt thereof and an anticancer agent.
이때, 상기 병용 투여는 동시에, 별도로 또는 순차적으로 병용할 수 있다.At this time, the combined administration can be carried out simultaneously, separately or sequentially.
또한, 본 발명의 다른 측면은 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 개선용 건강기능식품 조성물을 제공한다.Another aspect of the present invention provides a health functional food composition for preventing or ameliorating cancer, which comprises a calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 칼슘 채널 억제제는 하기 화학식 A 내지 화학식 D로부터 선택되는 어느 하나일 수 있다.The calcium channel inhibitor may be any one selected from the following formulas (A) to (D).
[화학식 A(베니디핀, Benidipine)][Chemical formula A (Benidipine)]
Figure PCTKR2019001143-appb-I000009
;
Figure PCTKR2019001143-appb-I000009
;
[화학식 B(라시디핀, Lacidipine)][Chemical formula (B) (Lacidipine)]
Figure PCTKR2019001143-appb-I000010
;
Figure PCTKR2019001143-appb-I000010
;
[화학식 C(마니디핀, Manidipine)][Chemical Formula C (Manidipine)]
Figure PCTKR2019001143-appb-I000011
;
Figure PCTKR2019001143-appb-I000011
;
[화학식 D(로메리진, Lomerizine)][Chemical Formula D (Lomerizine)]
Figure PCTKR2019001143-appb-I000012
.
Figure PCTKR2019001143-appb-I000012
.
상기 암은 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 구강암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 음경암, 전립선암, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성외부생식기암 및 피부암으로 이루어지는 군으로부터 선택되는 1종 이상일 수 있다.The cancer may be selected from the group consisting of benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial lesion, ependymoma, brain tumor, laryngeal cancer, pancreatic cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, Cancer of the breast, cancer of the gallbladder, cancer of the gallbladder, cancer of the bile duct, pancreatic cancer, small bowel cancer, colon cancer, anal cancer, bladder cancer, breast cancer, Kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female gonadal cancer and skin cancer.
또한, 상기 암은 난소암일 수 있다.Further, the cancer may be an ovarian cancer.
본 명세서에서 '건강기능식품'이란, 상기 칼슘 채널 억제제를 음료, 차류, 향신료, 껌, 과자류 등의 식품소재에 첨가하거나, 캡슐화, 분말화, 현탁액 등으로 제조한 식품으로, 이를 섭취할 경우 건강상 특정한 효과를 가져오는 것을 의미하나, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있다. 이와 같이 하여 얻어지는 본 발명의 건강기능식품은, 일상적으로 섭취하는 것이 가능하기 때문에 높은 피부 미백 효과를 기대할 수 있어 매우 유용하다.The term "health functional food" as used herein refers to a food prepared by adding the calcium channel inhibitor to food materials such as beverage, tea, spices, gum, confectionery, etc., encapsulation, powdering, suspension, etc., But it has the advantage that there are no side effects that can occur when a drug is used for a long period of time using a food as a raw material. The health functional food of the present invention thus obtained can be ingested on a daily basis, so that a high skin whitening effect can be expected, which is very useful.
칼슘 채널 억제제 또는 이의 약학적으로 허용가능한 염은 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 건강기능식품 중의 상기 화합물의 양은 전체 식품 중량의 0.1 내지 90 중량부로 가할 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The calcium channel blocker or pharmaceutically acceptable salt thereof may be added directly to the food or may be used together with other food or food ingredients and may be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the compound in the health functional food may be 0.1 to 90 parts by weight based on the total weight of the food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.
또한, 본 발명의 일 측면에 따른 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 칼슘 채널 억제제를 함유하는 것 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 제조 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 g당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.In addition, the health functional beverage composition according to one aspect of the present invention has no particular limitation on other ingredients except that the calcium channel inhibitor is contained as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates As an additional component. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and prepared 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 g of the composition of the present invention.
나아가, 상기 외에 본 발명의 칼슘 채널 억제제 또는 이의 약학적으로 허용가능한 염은 여러 가지 영양제, 비타민, 광물(전해질), 제조 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 칼슘 채널 억제제 또는 이의 약학적으로 허용가능한 염은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. In addition to the above, the calcium channel inhibitor of the present invention or a pharmaceutically acceptable salt thereof may be in the form of a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), manufacturing flavors and natural flavors, Etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. In addition, the calcium channel inhibitor of the present invention or a pharmaceutically acceptable salt thereof may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.
또한, 본 발명의 다른 측면은, 상기 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 치료용 약학적 조성물 병용 제제 또는 건강기능식품 조성물을 필요한 대상에게 투여하는 단계를 포함하는 암의 예방 또는 치료 방법을 제공한다.According to another aspect of the present invention, there is also provided a method for preventing or treating cancer comprising the calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient, And a method for preventing or treating cancer.
또한, 본 발명의 다른 측면은, 암의 예방 또는 치료에 있어서의, 상기 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 치료용 약학적 조성물 병용 제제 또는 건강기능식품 조성물의 용도를 제공한다.Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer comprising the calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient in the prevention or treatment of cancer. To provide the use of a food composition.
이하, 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 한정되는 것은 아니다.However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.
<실시예> 칼슘 채널 억제제를 포함하는 약학적 조성물EXAMPLES Pharmaceutical Compositions Containing Calcium Channel Inhibitors
칼슘 채널 억제제로서 하기 3종의 시험물질을 사용하여 실시예 1 내지 3의 약학적 조성물이 제조되었다.The pharmaceutical compositions of Examples 1 to 3 were prepared using the following three test substances as calcium channel inhibitors.
사용된 시험물질의 물질명 화학구조 및 구입처를 하기 표 1에 나타내었다.The chemical name of the test substance used and the place of purchase are shown in Table 1 below.
실시예Example 물질명Material name 화학구조Chemical structure 구입처Where to buy
1One 베니디핀(Benidipine)Benidipine
Figure PCTKR2019001143-appb-I000013
Figure PCTKR2019001143-appb-I000013
 		TocrisTocris
22 라시디핀(Lacidipine)Lacidipine
Figure PCTKR2019001143-appb-I000014
Figure PCTKR2019001143-appb-I000014
 		SigmaSigma
33 마니디핀(Manidipine)Manidipine
Figure PCTKR2019001143-appb-I000015
Figure PCTKR2019001143-appb-I000015
 		SelleckchemSelleckchem
44 로메리진(Lomerizine)Lomerizine
Figure PCTKR2019001143-appb-I000016
Figure PCTKR2019001143-appb-I000016
 		SelleckchemSelleckchem
<실험예 1> 칼슘 채널 억제제에 대한 약리 활성 스크리닝모든 칼슘 채널 억제제가 항암 효과가 있는 것은 아니라는 것을 증명하기 위하여, 종래 알려진 칼슘 채널 억제제에 대하여 하기와 같은 약리 활성 스크리닝을 수행하였다. 그 결과를 표 2에 나타내었다. &Lt; Experimental Example 1 > Pharmacological activity screening for calcium channel blockers To demonstrate that not all calcium channel blockers have anticancer activity, pharmacologically active screening was performed on the known calcium channel blockers as follows. The results are shown in Table 2.
구체적으로, 본 발명의 실시예 1 내지 4의 칼슘 채널 억제제를 포함하여 총 20종의 칼슘 채널 억제제에 대하여 스크리닝을 수행하였으며, 하기 표 2에서 area는 암 줄기 세포의 특징인 구체(sphere)형성을 저해하는지를 나타낸 값이며, area는 sphere 크기를 측정하여 나타낸 것이다. area의 값이 작을수록 암 줄기세포의 증식을 저해하는 것이다. 또한, ATP는 일반적인 화합물의 독성을 확인하는 것으로, 세포 내 ATP의 양을 측정하여 나타낸 것으로, ATP 값이 작을수록 세포 독성이 낮은 것이다.Specifically, 20 kinds of calcium channel inhibitors including calcium channel inhibitors of Examples 1 to 4 of the present invention were screened. In the following Table 2, area was defined as the sphere formation characteristic of cancer stem cells And the area is measured by measuring the sphere size. The smaller the area, the more proliferation of cancer stem cells is inhibited. In addition, ATP is a measure of the toxicity of a general compound, which is measured by measuring the amount of intracellular ATP. The smaller the ATP value, the lower the cytotoxicity.
구체적인 실험 방법은 하기와 같다.Specific experimental methods are as follows.
A2780-SP 세포(ovarian cancer stem cell)를 Ultra-Low Attachment 둥근 바닥96-웰 플레이트 (corning)에 각 웰당 500개의 생존가능한 세포밀도로 도말하고 암줄기세포(Cancer stem cell) 배지에서 배양하였다. 암 줄기세포 배지는 bFGF 20ng/ml, EGF 10ng/ml, Amphotericin B 2.5ng/ml, HEPES, Glutamax 및 B27이 보충된 신경세포 배양용 배지(neurobasal medium)로 구성된다. 가볍게 centrifuge하여 세포가 가운데 모이게 한 후 배양하였다. 배양 다음날, 세포에 20종의 칼슘 채널 억제제를 포함한 FDA 승인 화합물 1018개를 각각 10νM 단일 농도로 처리하고, 각 플레이트마다 양성 대조군으로 Apigenin 40 νM, 음성 대조군으로 DMSO 0.1% 처리하였다. 처리 4일째 화합물이 함유된 배지를 다시 한번 추가하였다. 처리 8일 후 구형세포(sphere cell)의 크기를 확인하기 위해 현미경을 이용하여 사진을 찍어 Image J software (NIH image)를 사용하여 분석하였다. 또한 사진 찍은 후 구형세포(sphere cell) 생존능(viability)을 비교하기 위해 Cell-titer Glo (Promega, WC, USA)에 의해 평가하였으며, 루시퍼라제 활성(luciferase activity)은 Tecan plate reader (Biocompare, USA)를 사용하여 검출하였다. 구형세포의 구체 형성능 분석과 생존능 분석을 위해 Apigenin을 처리한 대조군을 0%, DMSO를 처리한 대조군을 100%로 두어 분석하였다. 분석 결과 구형세포의 크기와 생존능 동시에 DMSO처리 대비 10% 이하로 감소시킨 화합물을 선별하었다.A2780-SP cells (ovarian cancer stem cells) were plated in Ultra-Low Attachment round bottom 96-well corning at a viable cell density of 500 cells per well and cultured in Cancer stem cell medium. Cancer stem cell culture medium consists of neurobasal medium supplemented with bFGF 20 ng / ml, EGF 10 ng / ml, Amphotericin B 2.5 ng / ml, HEPES, Glutamax and B27. The cells were centrifuged lightly and cultured. On the day following incubation, 1018 FDA-approved compounds containing 20 calcium channel blockers were treated with a single concentration of 10νM each, and each plate was treated with Apigenin 40 νM as a positive control and 0.1% DMSO as a negative control. On the fourth day of treatment, the medium containing the compound was again added. After 8 days of treatment, pictures were taken using a microscope to confirm the size of sphere cells and analyzed using Image J software (NIH image). The luciferase activity was measured using a Tecan plate reader (Biocompare, USA). Cell viability was assessed by Cell-titer Glo (Promega, WC, USA) . &Lt; / RTI &gt; For the spermatogenesis and viability analysis of spherical cells, apigenin-treated control group was treated with 0% and DMSO-treated control group was treated with 100%. As a result, the size and viability of spherical cells were reduced to less than 10% of DMSO treatment.
이 화합물들이 일반적으로 세포독성을 보이는지 확인하기 위해 정상섬유아세포(normal fibroblast cell)인 BJ6와 NIH-3T3를 96-웰 일반 세포 배양 플레이트에 각 웰당 3000개의 생존가능한 세포밀도로 도말하고 암줄기세포(Cancer stem cell) 배지에서 배양하였다. 24시간 배양 후 선별된 화합물들을 10νM 단일 농도로 처리하고 72시간 배양 후, 생존능을 비교하기 위해 Cell-titer Glo (Promega, WC, USA)를 처리하고, 루시퍼라제 활성(luciferase activity)을 분석하였다. 루시퍼라제 활성(luciferase activity)은 Tecan plate reader (Biocompare, USA)를 사용하여 검출하였으며, DMSO를 처리한 대조군을 100%, 세포를 도말하지 않은 군을 0%로 두어 분석하였다. Normal fibroblast cells, BJ6 and NIH-3T3, were plated at a viable cell density of 3000 cells per well on a 96-well cell culture plate to determine if these compounds are generally cytotoxic stem cell culture medium. After culturing for 24 hours, the selected compounds were treated with a single concentration of 10 nM and cultured for 72 hours. Cell-titer glo (Promega, WC, USA) was then treated for luciferase activity to compare viability. Luciferase activity was detected using a Tecan plate reader (Biocompare, USA). DMSO-treated control cells were divided into 100% and 0% cells, respectively.
번호number 칼슘 채널 억제제Calcium channel blocker areaarea ATPATP
1(실시예1)1 (Example 1) Benidipine hydrochloride Benidipine hydrochloride 55 00
2(실시예2)2 (Example 2) Lacidipine (Lacipil, Motens)Lacidipine (Lacipil, Motens) 55 00
3(실시예3)3 (Example 3) Manidipine (Manyper)Manidipine (Manyper) 22 00
4(실시예4)4 (Example 4) Lomerizine HClLomerizine HCl 22 00
55 CilnidipineCilnidipine 1818 1414
66 Ranolazine dihydrochlorideRanolazine dihydrochloride 8686 8787
77 Isradipine (Dynacirc)Isradipine (Dynacirc) 4848 2020
88 Amlodipine (Norvasc)Amlodipine (Norvasc) 2121 44
99 Flunarizine 2HCl Flunarizine 2HCl 1212 00
1010 Cleviprex (Clevidipine)Cleviprex (Clevidipine) 9696 6464
1111 NitrendipineNitrendipine 9595 8989
1212 Tetracaine hydrochloride (Pontocaine)Tetracaine hydrochloride (Pontocaine) 4646 1717
1313 Nilvadipine (ARC029)Nilvadipine (ARC029) 2828 3232
1414 AzelnidipineAzelnidipine 9898 00
1515 Nicardipine HClNicardipine HCl 2929 5757
1616 Nimodipine (Nimotop)Nimodipine (Nimotop) 7878 6464
1717 Nisoldipine (Sular)Nisoldipine (Waters) 7878 5252
1818 Nifedipine (Adalat)Nifedipine (Adalat) 8383 8484
1919 Diltiazem HCl (Tiazac)Diltiazem HCl (Tiazac) 5353 7373
2020 Felodipine (Plendil)Felodipine (Plendil) 3030 6565
상기 표 2에 나타난 바와 같이,As shown in Table 2,
총 20종의 칼슘 채널 억제제에 대하여 스크리닝을 수행하였으나, 본 발명 실시예의 4종류 화합물만이 5 이하의 area값을 나타내며, 동시에 ATP 측정값이 0임을 알 수 있다. A total of 20 kinds of calcium channel inhibitors were screened. However, only four kinds of compounds of the present invention show an area value of 5 or less, and at the same time, the ATP measurement value is zero.
즉, 상기 결과는 칼슘 채널 억제제라고 하여 모두 항암 효과를 나타내는 것은 아니며, 본 발명의 4종 화합물이 특이적으로 우수한 항암 효과를 나타내는 것을 입증한다.That is, the above results do not show all of the anticancer effects as calcium channel blockers, and demonstrate that the quaternary compounds of the present invention have a particularly excellent anticancer effect.
본 명세서의 실험예 2 내지 9는 하기 실험 프로토콜을 사용하여 수행하였으며, 구체적인 실험방법은 하기와 같다. Experimental Examples 2 to 9 of the present specification were carried out using the following experimental protocols, and specific experimental methods are as follows.
<실험 프로토콜(protocol)><Experimental Protocol>
1. 세포 생존능 분석(cell viability assay)1. Cell viability assay
A2780-SP 세포(ovarian cancer stem cell)를 Ultra-Low Attachment 둥근 바닥 96- 웰 플레이트 (corning)에 각 웰당 500개의 생존가능한 세포밀도로 도말하고 암 줄기세포(Cancer stem cell) 배지에서 배양하였다. 암 줄기세포 배지는 bFGF 20ng/ml, EGF 10ng/ml, Amphotericin B 2.5ng/ml, HEPES, Glutamax 및 B27가 보충된 신경세포 배양용 배지(neurobasal medium)로 구성된다. 배양 다음날, 세포에 실시예 1 내지 4의 화합물을 각각 30μM, 10μM, 2μM, 0.4μM 및 0.08μM로 처리하고 8일간 배양하였다. 화합물이 함유된 배지를 4일째에 다시 한번 추가하였다. 구형 세포(sphere cell) 생존능(viability)은 Cell-titer Glo (Promega, WC, USA)에 의해 평가하였으며, 루시퍼라제 활성(luciferase activity)은 Tecan plate reader (Biocompare, USA)를 사용하여 검출하였다. A2780 세포(ovarian cancer cell)를 96 웰 플레이트에 각 웰 당 3000개의 생존 가능한 세포의 밀도로 도말하고, 10 % FBS, 1 % 페니실린 / 스트렙토마이신(penicillin/streptomycin)을 보충한 RPMI-1640 배지에서 배양 하였다. 배양 다음날, 세포에 실시예 1 내지 4의 화합물을 각각 100μM, 30μM, 10μM, 3μM 및 0.3μM 로 처리하고 3일간 배양하였다. 세포 생존능은 Cell-titer Glo (Promega, WC, USA)에 의해 평가하였으며, 루시퍼라제 활성은 Tecan plate reader (Biocompare, USA)를 사용하여 검출하였다.A2780-SP cells were plated in Ultra-Low Attachment round-bottomed 96-well corning at a viable cell density of 500 cells per well and cultured in cancer stem cell medium. Cancer stem cell culture medium consists of neurobasal medium supplemented with bFGF 20 ng / ml, EGF 10 ng / ml, Amphotericin B 2.5 ng / ml, HEPES, Glutamax and B27. On the day following the incubation, the cells were treated with 30 μM, 10 μM, 2 μM, 0.4 μM and 0.08 μM of the compounds of Examples 1 to 4, respectively, and cultured for 8 days. The medium containing the compound was added once again on the fourth day. Sphere cell viability was assessed by Cell-titer Glo (Promega, WC, USA) and luciferase activity was detected using a Tecan plate reader (Biocompare, USA). A2780 ovarian cancer cells were plated in 96-well plates at a density of 3000 viable cells per well and cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin Respectively. On the day following the incubation, the cells were treated with 100 μM, 30 μM, 10 μM, 3 μM and 0.3 μM of the compounds of Examples 1 to 4, respectively, and cultured for 3 days. Cell viability was assessed by Cell-titer Glo (Promega, WC, USA) and luciferase activity was detected using a Tecan plate reader (Biocompare, USA).
2. 구체 형성 분석(Sphere formation assay)2. Sphere formation assay
A2780-SP 세포를 Ultra-Low Attachment 둥근 바닥 96- 웰 플레이트 (corning)에 각 웰당 500개의 생존가능한 세포밀도로 도말하고 암 줄기세포(Cancer stem cell) 배지에서 배양하였다. 배양 다음날, 세포에 실시예 1 내지 4의 화합물을 각각 10μM, 2μM, 0.4μM 로 처리하고 8일간 37℃, 5% CO2,95%습도 조건에서 배양하였다. 구체 크기는 Image J software (NIH image)를 사용하여 분석하였다.A2780-SP cells were plated in Ultra-Low Attachment round-bottomed 96-well corning at a viable cell density of 500 cells per well and cultured in cancer stem cell medium. On the day following the incubation, the cells were treated with 10 μM, 2 μM and 0.4 μM of the compounds of Examples 1 to 4, respectively, and cultured for 8 days at 37 ° C., 5% CO 2 and 95% humidity. Sphere size was analyzed using Image J software (NIH image).
3. 구체 증식 분석(Sphere proliferation assay)3. Sphere proliferation assay
A2780-SP 세포(ovarian cancer stem cell)를 Ultra-Low Attachment 평평한 바닥 96- 웰 플레이트(corning)에 각 웰 당 6000개의 생존가능한 세포밀도로 도말하고 암 줄기세포(Cancer stem cell) 배지에서 성장시켰다. 다음날, 세포에 실시예 1 내지 4의 화합물을 10μM로 처리하고 60시간동안 37℃, 5% CO2,95%습도 조건에서 배양하였다. 구체 형성 융합(sphere forming confluence)은 Incucyte (BioTek, VT, USA)를 사용하여 분석하였다.A2780-SP cells were plated on Ultra-Low Attachment flat bottom 96-well corning at a viable cell density of 6000 cells per well and grown in cancer stem cell medium. The following day, the cells were treated with 10 μM of the compounds of Examples 1 to 4 and incubated for 60 hours at 37 ° C., 5% CO 2 and 95% humidity. Sphere forming confluence was analyzed using Incucyte (BioTek, VT, USA).
4. 웨스턴 블랏(Western blotting)4. Western blotting
A2780-SP 세포(ovarian cancer stem cell)를 Ultra-Low Attachment 6- 웰 플레이트(corning)에 각 웰 당 4x105개의 생존가능한 세포밀도로 도말하고 암 줄기세포(Cancer stem cell) 배지에서 배양시켰다. 다음날, 배지를 신경세포 배양용 배지(Neuro Basal Medium, NBM)로 교체하고, 기아 상태(in starvation)에서 16 시간 동안 배양 한 다음 실시예 1 내지 4의 화합물을 10μM로 1시간 동안 처리 하였다. 배지를 화합물을 함유하는 암 줄기 세포 배지로 교환하고, 세포를 수확하고 RiPA 완충액에 용해시키기 전에 30 분 동안 더 배양하였다. 추출된 단백질을 SDS-PAGE 겔로 분리하고 Western Blot 분석을 위해 PVDF 멤브레인(membranes)으로 옮겼다. 5% 스킴밀크(skim milk)로 블로킹(blocking)한 후, 멤브레인을 4 ℃에서 블로킹 완충액에서 일차 항체(primary antibodies)로 밤샘 배양한 다음, 상온에서 2 시간 동안 HRP-컨쥬게이티드 2차 항체(HRP-conjugated secondary antibodies)로 배양 하였다. A2780-SP cells (ovarian cancer stem cells) were plated in Ultra-Low Attachment 6-well corning at 4 x 10 viable cell densities per well and cultured in cancer stem cell medium. The next day, the medium was replaced with Neuro Basal Medium (NBM), incubated for 16 hours in starvation, and treated with 10 μM of the compounds of Examples 1 to 4 for 1 hour. The medium was exchanged with a cancer stem cell medium containing the compound, and the cells were further cultured for 30 minutes before harvesting and dissolving in RiPA buffer. The extracted proteins were separated by SDS-PAGE gel and transferred to PVDF membranes for Western Blot analysis. After blocking with 5% skim milk, the membranes were incubated overnight with primary antibodies in blocking buffer at 4 ° C and then incubated with HRP-conjugated secondary antibody ( HRP-conjugated secondary antibodies).
사용된 일차 항체는 다음과 같다:The primary antibodies used were:
anti-phospho-AKT (Ser473)(clone 193H12, Cell Signaling), anti-AKT (rabbit polyclonal, Cell Signaling), anti-phospho-ERK (Thr202/Tyr204) (clone 20G11, Cell Signaling), anti-ERK (rabbit monoclonal, Cell Signaling), anti-phospho-p38 (rabbit monoclonal, Cell Signaling), anti-p38 (rabbit monoclonal, Cell Signaling), anti-OCT3/4 (mouse monoclonal, Santa Cruz), anti-NANOG (rabbit monoclonal, Cell Signaling), anti-SOX2 (rabbit monoclonal, Cell signaling), anti-ALDH1 (mouse monoclonal, BD), anti-CD133 (rabbit monoclonal, Abcam), anti-GAPDH (mouse monoclonal, Santa Cruz).anti-phospho-AKT (Ser473) (clone 193H12, Cell Signaling), anti-AKT (rabbit polyclonal, Cell Signaling), anti-phospho-ERK (Thr202 / Tyr204) monoclonal, Cell Signaling), anti-phospho-p38 (rabbit monoclonal, Cell Signaling), anti-p38 (rabbit monoclonal, Cell Signaling), anti-OCT3 / 4 (mouse monoclonal, Santa Cruz) Anti-CD133 (rabbit monoclonal, Abcam), anti-GAPDH (mouse monoclonal, Santa Cruz), anti-ALDH1 (mouse monoclonal, Cell signaling), anti-SOX2 (rabbit monoclonal, Cell signaling).
사용된 2차 항체는 다음과 같다:The secondary antibodies used were as follows:
Goat anti-mouse IgG-HRP (Bioss), Goat anti-rabbit IgG-HRP (Bioss). Goat anti-mouse IgG-HRP (Bioss), Goat anti-rabbit IgG-HRP (Bioss).
시그널(Signals)은 강화된 화학발광(chemiluminescence) HRP 기판 (Bio-Rad)으로 현상되었고 LAS-3000 mini (Fuji film)를 사용하여 검출시켰다. 상기 시그널은 Image J software (NIH Image)를 사용하여 정량화시켰다.Signals were developed with an enhanced chemiluminescence HRP substrate (Bio-Rad) and detected using a LAS-3000 mini (Fuji film). The signal was quantified using Image J software (NIH Image).
5. PI 염색(PI staining)5. PI staining (PI staining)
A2780-SP 세포(ovarian cancer stem cell)를 Ultra-Low Attachment 6- 웰 플레이트(corning)에 각 웰 당 1x106개의 생존가능한 세포밀도로 도말하고 암 줄기세포(Cancer stem cell) 배지에서 배양시켰다. 다음날, A2780-SP 세포에 실시예 1 내지 4의 화합물을 10μM로 처리하고, 37℃, 5% CO2,95%습도 조건에서 24시간 동안 배양시킨 후, 원심분리기(centrifuge)로 수확하였다. PBS로 한번 세척한 후, 세포를 PBS 0.3 ml에 재현탁시켰다. 세포를 고정시키기 위하여 차가운 에탄올 0.7 ml를 천천히 적가첨가하고, 세포를 1시간동안 얼음위에서 배양시켰다. 고정된 세포를 차가운 PBS로 한번 헹구고, 0.1 ml PBS에 재현탁시킨 후, 2μl의 10mg/ml RNase A을 첨가하였다. 37℃에서 1시간 배양시킨 후, 상기 세포에 5μl의 PI 용액(BD science)을 보충하고, 부드럽게 와류시킨(vortexed) 후, 어두운 곳에서 상온에서 15분간 더 배양하였다. 세포에 차가운 PBS 400 μl를 첨가한 후, 1시간 이내에 유동세포계수법(flow cytometry)을 수행하였다.A2780-SP cells (ovarian cancer stem cells) were plated in Ultra-Low Attachment 6-well corning at a viable cell density of 1x10 6 cells per well and cultured in cancer stem cell medium. The following day, A2780-SP cells were treated with 10 μM of the compounds of Examples 1 to 4, cultured at 37 ° C, 5% CO 2, 95% humidity for 24 hours, and then harvested with a centrifuge. After washing once with PBS, the cells were resuspended in 0.3 ml of PBS. 0.7 ml of cold ethanol was slowly added dropwise to fix the cells, and the cells were incubated on ice for 1 hour. Fixed cells were rinsed once with cold PBS, resuspended in 0.1 ml PBS, and then 2 μl of 10 mg / ml RNase A was added. After incubation at 37 ° C for 1 hour, the cells were supplemented with 5 μl of PI solution (BD science), gently vortexed, and further cultured in the dark at room temperature for 15 minutes. After adding 400 μl of cold PBS to the cells, flow cytometry was performed within 1 hour.
6. 실시간 중합효소연쇄반응(Quantitative Real-time polymerase chain reaction, Quantitative RT-PCR)6. Quantitative Real-time polymerase chain reaction (Quantitative RT-PCR)
Trizol RNA 추출 키트 (Trizol RNA extraction kit, Invitrogen)를 사용하여 제조업체의 지침에 따라 시료의 총 RNA를 추출했다. 2μg의 RNA를 GoScriptTM cDNA 합성 시스템 (GoScriptTM cDNA synthesis system,Promega)을 사용하여 cDNA로 역전사시켰다. 합성된 cDNA를 표시된 프라이머(indicated primers)가 있는 FastStart SYBR green Master (Roche) 및 Bio-Rad S1000 Thermal cycler를 사용하여 Quantitative RT-PCR에 적용했다. GAPDH를 표준 유전자(reference gene)로 사용하였으며, 결과는 ddCt method를 사용하여 대조군에 대한 상대적인 발현으로 나타내었다.Trizol RNA extraction kit (Invitrogen) was used to extract the total RNA from the samples according to the manufacturer's instructions. 2 [mu] g of RNA was reverse transcribed with cDNA using the GoScriptTM cDNA synthesis system (GoScriptTM cDNA synthesis system, Promega). The synthesized cDNA was applied to Quantitative RT-PCR using FastStart SYBR green Master (Roche) and Bio-Rad S1000 Thermal cycler with indicated primers. GAPDH was used as a reference gene and the results were expressed relative to the control group using the ddCt method.
7. 병용 투여 효과 실험(Combination effect test)7. Combination effect test
상기 실시예의 마니디핀 4μM, 라시디핀 8μM, 로메리진 HCl 10μM 또는 베니시핀 HCl 10μM을 각각 8μM의 시스플라틴(cisplatin)와 함께 A2780-SP 난소 암 줄기 세포에 동시 처리(co-treated)하였다. 상기 구체 형성 분석 및 구체 증식 분석법을 사용하여 실시예 화합물과 시스플라틴의 병용 효과를 확인 하였다.Treated with co-treatment with 8 μM of cisplatin to A2780-SP ovarian cancer stem cells, respectively, with 4 μM of manidipine, 8 μM of laminidin, 10 μM of romeridin HCl or 10 μM of benicipine HCl, respectively. The combined effect of the compound of Example and cisplatin was confirmed by the above-described spherical shape analysis and sperm propagation assay.
8. 전세포 패치 클램프 레코딩 실험(whole cell patch clamp recording)8. Whole cell patch clamp recording.
커버글라스에 미리 준비된 세포(난소암세포(A2780) 및 난소암줄기세포(A2780-SP))를 기록용 챔버(chamber)로 옮기고 외부 용액(external solution)을 계속해서 순환시켰다. 외부 용액의 조성은 143 mM NaCl, 5.6 mM KCl,10mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 5mM 글루코스(glucose), 1nM 테트로도톡신(tetrodotoxin), 10 mM 테트라에틸암모늄(tetraethylammonium)이며, NaOH를 사용하여 pH를 7.4로 보정하였다. 전세포 패치(Whole cell patch)를 위한 내부 피펫 용액(internal pipette solution)의 조성은 140 mM CsCl, 2mM MgCl2, 3 mM Mg-ATP, 5mM HEPES, 1.1 mM EGTA이며, CsOH를 사용하여 pH를 7.2로 보정하였다. (Ovarian cancer cells (A2780) and ovarian cancer stem cells (A2780-SP)) in a cover glass were transferred to a recording chamber and the external solution was continuously circulated. The composition of the external solution was 143 mM NaCl, 5.6 mM KCl, 10 mM CaCl 2 , 2 mM MgCl 2 , 10 mM HEPES, 5 mM glucose, 1 nM tetrodotoxin and 10 mM tetraethylammonium, The pH was adjusted to 7.4 using. The composition of the internal pipette solution for the whole cell patch was 140 mM CsCl, 2 mM MgCl 2 , 3 mM Mg-ATP, 5 mM HEPES, 1.1 mM EGTA and pH was adjusted to 7.2 using CsOH .
Ca2+ 전류는 전압 클램프 모드(voltage clamp mode)에서 Axopatch 700B, DigiData 1440A, pClamp10.4를 이용하여 측정하였고, -70 mV에서 기본 막전위를 홀딩 후, -70 mV를 시작으로 +10 mV 씩 막 전압을 증가시키면서 나타나는 전류의 양을 측정하였다. 앞의 정상 전류(normal current) 측정한 후 실시예 3의 마니디핀(Manidipine) 10 μM을 5분간 처리하고, 동일한 전압의 변화를 가하여 이에 의해 나타나는 전류 양의 변화를 측정하였다. 전세포 패치 클램프의 저항(access Resistance) (Ra) 값은 10~20 MΩ으로 사용하였다.The Ca 2+ current was measured using the Axopatch 700B, DigiData 1440A, pClamp 10.4 in a voltage clamp mode and after holding the basal membrane potential at -70 mV, starting at -70 mV and +10 mV The amount of current appearing while increasing the voltage was measured. After measuring the normal current, 10 μM of Manidipine of Example 3 was treated for 5 minutes, and the change in the amount of current was measured by applying the same voltage change. The access resistance (Ra) value of the whole cell patch clamp was 10 ~ 20 MΩ.
9. 동물 실험9. Animal experiment
난소암 및 난소암 암 줄기세포 종양모델에서 Manidipine의 저해 영향을 확인하기 위하여, 난소암 암 줄기세포주 A2780-SP 세포와 난소암 세포주 A2780-AD 세포를 각각 1x10^5개씩 누드 마우스에 피하에 접종하였다. 이 때 누드 마우스의 왼쪽에는 난소암 세포를, 오른쪽에는 난소암 암 줄기세포를 각각 8마리씩 접종하였다. 접종 이 후, 14일차부터 종양 형성 크기를 측정하기 시작하였으며, 동일 시점부터 약물의 영향을 확인하기 위하여 대조군 그룹 (3마리-PBS treatment)과 Manidipine 처리 그룹 (3마리-1mpk treatment), 항암제-Pacritaxel 처리 그룹 (3마리-5mpk treatment), Manidipine과 항암제(Pacritaxel) 병용 처리 그룹으로 나누어 약물을 투여하였다. 이 후, 3일에서 4일 간격으로 약물 투여 및 종양의 크기를 측정하였다. 이 후, 31일차에 안락사 시켰고, 최종적으로 종양의 무게 및 크기를 측정하였다. In order to examine the inhibitory effect of manidipine on ovarian cancer and ovarian cancer stem cell tumor model, ovarian cancer stem cell line A2780-SP cells and ovarian cancer cell line A2780-AD cells were inoculated subcutaneously in nude mice 1x10 5 . At this time, ovarian cancer cells were inoculated on the left side of the nude mouse, and 8 ovarian cancer stem cells were inoculated on the right side. After the inoculation, the size of the tumor formation was measured from the 14th day. In order to confirm the effect of the drug from the same time point, the control group (3 pBS treatment), Manidipine treatment group (3 animals -1 mpk treatment) Treatment group (3 animals -5mpk treatment), Manidipine and anti-cancer drug (Pacritaxel) treatment group were divided into two groups. Thereafter, drug administration and tumor size were measured at intervals of 3 to 4 days. After that, the animals were euthanized on the 31st day, and the weight and size of the tumors were finally measured.
10. 통계(Statistics)10. Statistics
각 데이터는 3회 이상의 독립적인 실험의 평균±표준 편차(mean ± standard deviation, SD)로 표시하였다. 통계적으로 유의한 차이는 GraphPad Prism 5 (CA, USA)를 사용하여 1-way ANOVA를 사용하여 결정되었다. 0.05 이하의 p 값은 통계적으로 유의하다고 간주되었다.Each data was expressed as mean ± standard deviation (SD) of three or more independent experiments. Statistically significant differences were determined using a 1-way ANOVA using GraphPad Prism 5 (CA, USA). P values less than 0.05 were considered statistically significant.
<실험예 2> 암 세포 및 암 줄기세포의 생존능 분석<Experimental Example 2> Survival analysis of cancer cells and cancer stem cells
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물 처리에 따른 암 세포 및 암 줄기세포의 생존능을 분석하기 위하여, 실시예 1 내지 4 약학적 조성물을 사용하여 세포 생존능 분석을 수행하였으며, 그 결과를 도 1 및 2에 나타내었다. 또한, 세포별로 각 화합물의 GI50값을 측정하여 하기 표 3에 나타내었다. 구체적인 실험방법은 상기 실험 프로토콜의 1. 세포 생존능 분석과 같다.In order to analyze the viability of cancer cells and cancer stem cells according to the pharmaceutical composition treatment containing the calcium channel inhibitor of the present invention, cell viability analysis was carried out using the pharmaceutical compositions of Examples 1 to 4, 1 and 2, respectively. The GI 50 values of the respective compounds were measured for each cell, and are shown in Table 3 below. The specific experimental method is the same as the 1. cell viability assay of the above experimental protocol.
실시예Example GI50(μM)A2780-SPGI 50 (μM) A2780-SP GI50(μM)A2780GI 50 ([mu] M) A2780
1One 5.3455.345 10.6910.69
22 2.8442.844 11.3611.36
33 1.8921.892 9.6669.666
44 5.9495.949 11.4011.40
도 1은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell) 생존능의 용량-반응(Dose-response) 곡선을 나타낸 것이다.도 2는 실시예 약학적 조성물을 처리하였을 때, A2780 세포(ovarian cancer cell) 생존능의 용량-반응(Dose-response) 곡선을 나타낸 것이다.Figure 1 shows a dose-response curve of the viability of A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated. , And the dose-response curve of the viability of A2780 cells (ovarian cancer cells).
도 1에 나타난 바와 같이, 실시예 1 내지 4의 약학적 조성물은 투여량 의존적(dose-dependent manner)으로 난소암줄기세포인 A2780-SP 세포의 생존능을 감소시키는 것을 확인하였다. As shown in Figure 1, the pharmaceutical compositions of Examples 1 to 4 were found to reduce the viability of A2780-SP cells, which are ovarian cancer cells in a dose-dependent manner.
도 2에 나타난 바와 같이, 실시예 1 내지 4의 약학적 조성물은 투여량 의존적(dose-dependent manner)으로 난소암세포인 A2780 세포의 생존능을 감소시키는 것을 확인하였다. As shown in FIG. 2, the pharmaceutical compositions of Examples 1 to 4 were found to reduce the viability of A2780 cells, which are ovarian cancer cells, in a dose-dependent manner.
또한, 도 1의 결과 및 도 2의 결과를 비교하였을 때, 실시예 1 내지 4의 약학적 조성물은 난소암줄기세포에 대하여 보다 우수한 세포 성장 저해 효과를 나타내는 것을 알 수 있다.In addition, when the results of FIG. 1 and FIG. 2 are compared, it can be seen that the pharmaceutical compositions of Examples 1 to 4 exhibit better cell growth inhibitory effect on ovarian cancer stem cells.
상기 표 3에 나타난 바와 같이, As shown in Table 3,
난소암 줄기세포 A2780-SP 및 난소암세포 A2780 세포에서 실시예 1 내지 4의 약학적 조성물은 15 μM 이하의 GI50값을 나타냄으로써, 세포 성장 저해효과가 우수함을 확인하였다. 한편, 줄기세포의 경우가 더 낮은 값을 나타냄으로써, 줄기세포에서 선택적으로 우수한 성장 저해 효과를 나타냄을 알 수 있다.In the ovarian cancer stem cell A2780-SP and the ovarian cancer cell A2780 cells, the pharmaceutical compositions of Examples 1 to 4 showed a GI50 value of 15 μM or less, thereby confirming that the cell growth inhibitory effect was excellent. On the other hand, the lower the value of stem cells, the more excellent selective growth inhibition effect is obtained in stem cells.
따라서, 본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물은 암 줄기세포의 성장 저해 효과를 나타내는 바, 항암 효과가 있음을 알 수 있다.Therefore, the pharmaceutical composition comprising the calcium channel inhibitor of the present invention shows the inhibitory effect on the growth of cancer stem cells, and thus it has anti-cancer effect.
<실험예 3> 암 줄기세포의 성장 억제 평가<Experimental Example 3> Evaluation of growth inhibition of cancer stem cells
3-1. 구체 형성 분석3-1. Sphere formation analysis
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물 처리에 따른 암 줄기세포의 성장 정도를 분석하기 위하여, 실시예 1 내지 4 약학적 조성물을 사용하여 암줄기세포의 세포 구체 형성을 분석하였으며, 실험 결과를 도 3에 나타내었다. 구체적인 실험방법은 상기 상기 실험 프로토콜의 2. 구체 형성 분석과 같다.In order to analyze the degree of growth of cancer stem cells according to the pharmaceutical composition treatment containing the calcium channel inhibitor of the present invention, the cell spheres of cancer stem cells were analyzed using the pharmaceutical compositions of Examples 1 to 4, 3. The specific experimental method is the same as the 2. spherical analysis of the above experimental protocol.
도 3은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)의 구체 형성을 분석한 결과를 나타낸 이미지이다.FIG. 3 is an image showing the result of analyzing the sphere formation of A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of the Example was treated.
도 3에 나타난 바와 같이, 실시예 1 내지 4의 약학적 조성물은 투여량 의존적(dose-dependent manner)으로 난소암줄기세포인 A2780-SP 세포의 구체 형성을 감소시키는 것을 알 수 있다. As shown in FIG. 3, it can be seen that the pharmaceutical compositions of Examples 1 to 4 reduce the sphere formation of A2780-SP cells, which are ovarian cancer cells in a dose-dependent manner.
구체(sphere)는 암 줄기세포의 특징적인 형태로서, 구체의 성장은 암 줄기세포가 성장함을 의미한다. 따라서, 본 발명에 따른 칼슘 채널 억제제를 포함하는 약학적 조성물은 암 줄기 세포의 구체 형성을 억제하므로, 항암 효과가 있음을 알 수 있다.The sphere is a characteristic form of cancer stem cells, which means that the growth of the sphere is the growth of cancer stem cells. Therefore, the pharmaceutical composition containing the calcium channel inhibitor according to the present invention inhibits the formation of the stem cells of cancer stem cells, so that it has anti-cancer effect.
3-2. 구체 증식 분석3-2. Sphere multiplication analysis
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물 처리에 따른 암 줄기세포의 성장 정도를 분석하기 위하여, 실시예 1 내지 4 약학적 조성물을 사용하여 암줄기세포의 세포 구체 중식을 분석하였으며, 그 결과를 도 4에 나타내었다. 구체적인 실험방법은 상기 실험 프로토콜의 3. 구체 증식 분석과 같다.In order to analyze the degree of growth of cancer stem cells according to the pharmaceutical composition treatment containing the calcium channel inhibitor of the present invention, the cell spheres of cancer stem cells were analyzed using the pharmaceutical compositions of Examples 1 to 4, 4. The specific experimental method is the same as that of Experimental Protocol.
도 4는 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)의 구체 형성을 분석하여 A2780-SP 세포의 증식을 측정한 결과를 나타낸 그래프이다.FIG. 4 is a graph showing the results of measurement of the proliferation of A2780-SP cells by analyzing the sphere formation of A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of Example was treated.
도 4에 나타난 바와 같이, 실시예 1 내지 4의 약학적 조성물은 구체 형성 융합을 억제하였으며, 60시간 후에는 대조군(DMSO처리군)과 비교하여 1/3 이하의 융합률을 나타내어 우수한 암 줄기세포 증식 억제효과를 나타냄을 알 수 있다.As shown in FIG. 4, the pharmaceutical compositions of Examples 1 to 4 inhibited sphere fusion, and showed a fusion rate of 1/3 or less as compared with the control group (DMSO treatment group) after 60 hours, Proliferation inhibitory effect can be shown.
따라서, 본 발명에 따른 칼슘 채널 억제제를 포함하는 약학적 조성물은 암 줄기 세포의 구체 증식을 억제하므로, 항암 효과가 있음을 알 수 있다.Therefore, the pharmaceutical composition comprising the calcium channel inhibitor according to the present invention inhibits the proliferation of cancer stem cells, and thus has anticancer effects.
<실험예 4> 암 줄기세포능(stemness), 생존 및 성장 관련 인자의 발현 분석<Experimental Example 4> Expression analysis of cancer stem cell function, survival and growth factors
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물 처리에 따른 암 줄기세포능(stemness)과 관련된 마커 및 암 줄기세포의 생존 및 증식과 관련된 단백질의 발현 변화를 통해 항암 효과를 확인하기 위하여, 웨스턴블랏을 통해 각종 인자의 발현 정도를 분석하였으며, 그 결과를 도 5-8에 나타내었다. 구체적인 실험방법은 상기 실험 프로토콜의 4. 웨스턴 블랏과 같다.In order to confirm the anticancer effect through the expression of the markers related to cancer stem cells by the pharmaceutical composition treatment containing the calcium channel inhibitor of the present invention and the expression of proteins associated with survival and proliferation of cancer stem cells, . The results are shown in FIGS. 5-8. The specific experimental method is the same as the 4. Western blot of the above experimental protocol.
도 5는 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포능(stemness)과 관련된 마커들의 발현 변화를 관찰한 이미지이다.FIG. 5 is an image showing changes in the expression of markers related to cancer stem cell function in A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of Example is treated.
도 6은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포능(stemness)과 관련된 마커들의 발현 변화를 정량화한 그래프이다.Figure 6 is a graph quantifying the expression changes of markers related to cancer stem cell function in A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated.
도 5 및 6에 나타난 바와 같이, 실시예 1(베니디핀), 실시예 3(마니디핀), 실시예 4(로메리진)의 약학적 조성물을 처리하였을 때, 암 줄기세포능(stemness)과 관련된 마커인 OCT3/4, NANOG, SOX2, ALDH1, CD133의 발현이 감소하는 것을 알 수 있다.As shown in FIGS. 5 and 6, when the pharmaceutical composition of Example 1 (benidipine), Example 3 (manidipine), and Example 4 (lomeligin) were treated, they were associated with cancer stemness The expression of the markers OCT3 / 4, NANOG, SOX2, ALDH1 and CD133 is decreased.
또한, 실시예 2(라시디핀)의 약학적 조성물은 NANOG, SOX2, ALDH1, CD133의 발현을 감소시키는 것을 알 수 있다.In addition, it can be seen that the pharmaceutical composition of Example 2 (lacidipine) reduces the expression of NANOG, SOX2, ALDH1, and CD133.
따라서, 본 발명의 약학적 조성물은 기존 항암제에 대한 내성을 유발하는 암 줄기세포의 줄기세포능(stemness)과 관련된 마커의 발현을 감소시켜, 암 치료에 유용하게 사용할 수 있다.Accordingly, the pharmaceutical composition of the present invention can reduce the expression of the stem cells related to the stem cells of cancer stem cells which cause tolerance to existing anticancer drugs, and thus can be useful for cancer treatment.
도 7은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포 생존 및 증식과 관련된 단백질의 활성 변화를 관찰한 이미지이다.FIG. 7 is an image showing changes in the activity of proteins involved in cancer stem cell survival and proliferation in A2780-SP cells (ovarian cancer stem cells) when the pharmaceutical composition of the Example was treated.
도 8은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포 생존 및 증식과 관련된 단백질의 활성 변화를 정량화한 그래프이다.8 is a graph quantifying the change in the activity of a protein involved in cancer stem cell survival and proliferation in A2780-SP cells (ovarian cancer stem cells) when the example pharmaceutical composition is treated.
도 7 및 8에 나타난 바와 같이, 실시예 1(베니디핀), 실시예 3(마니디핀), 실시예 4(로메리진)의 약학적 조성물을 처리하였을 때, 암 줄기세포 생존 및 증식과 관련된 단백질인 AKT, ERK, p38의 활성이 감소하는 것을 알 수 있다.As shown in FIGS. 7 and 8, when the pharmaceutical composition of Example 1 (benidipine), Example 3 (manidipine), and Example 4 (lomeligin) was treated, the cancer stem cell survival and proliferation- The activity of AKT, ERK, and p38 was decreased.
또한, 실시예 2(라시디핀)의 약학적 조성물은 ERK, p38의 활성을 감소시키는 것을 알 수 있다.In addition, it can be seen that the pharmaceutical composition of Example 2 (lacidipine) decreases the activity of ERK, p38.
따라서, 본 발명의 약학적 조성물은 암 줄기세포의 줄기세포능(stemness)을 감소시키는 것에 더해 암 줄기세포 생존 및 증식과 관련된 단백질의 발현을 감소시켜, 암의 재발, 전이, 진행을 억제함으로써 암 치료에 유용하게 사용할 수 있다.Accordingly, the pharmaceutical composition of the present invention reduces the stem cell function of cancer stem cells, reduces the expression of proteins involved in cancer stem cell survival and proliferation, inhibits recurrence, metastasis, and progression of cancer, It can be useful for treatment.
<실험예 5> 암 줄기세포의 세포 주기 및 세포사멸 분석<Experimental Example 5> Cell cycle and cell death of cancer stem cells
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물 처리에 따른 암 줄기세포의 세포 주기 및 세포사멸을 분석하여 항암 효과를 확인하기 위하여, PI 염색 실험을 수행하였으며, 그 결과를 도 9 및 10에 나타내었다. 또한, PI 염색 결과, 세포 주기별, 각 세포 주기에 포함되는 세포 수를 표 4에 정리하였다. 구체적인 실험방법은 상기 실험 프로토콜의 5. PI 염색과 같다.In order to confirm the anti-cancer effect by analyzing the cell cycle and apoptosis of cancer stem cells according to the pharmaceutical composition treatment containing the calcium channel inhibitor of the present invention, a PI staining experiment was performed. The results are shown in FIGS. 9 and 10 . As a result of PI staining, the number of cells contained in each cell cycle is shown in Table 4. The specific experimental method is the same as the 5. PI staining of the above experimental protocol.
DMSODMSO 실시예 1Example 1 실시예 2Example 2 실시예 3Example 3 실시예4Example 4
사멸 (%)(Apoptotic)Apoptotic (%) 5.25.2 7.17.1 14.514.5 11.911.9 6.26.2
4.34.3 7.87.8 15.015.0 10.510.5 6.46.4
G0-G1 기(%)(G0-G1 phase)G0-G1 phase (%) (G0-G1 phase) 50.750.7 44.244.2 42.142.1 45.945.9 43.843.8
48.548.5 43.943.9 42.642.6 45.145.1 45.645.6
S 기(%)(S phase)S phase (%) (S phase) 6.36.3 7.87.8 8.28.2 7.17.1 9.59.5
7.17.1 12.312.3 8.68.6 7.37.3 9.99.9
G2-M 기(%)(G2-M phase)G2-M group (%) (G2-M phase) 23.323.3 29.829.8 17.717.7 22.122.1 25.025.0
24.924.9 28.728.7 18.618.6 22.722.7 20.720.7
도 9는 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포의 세포 주기 변화를 나타낸 그래프이다.도 10은 실시예 약학적 조성물을 처리하였을 때, A2780-SP 세포의 세포 사멸을 측정한 그래프이다.Figure 9 is a graph showing the cell cycle changes of A2780-SP cells when the pharmaceutical composition of the example was treated. Figure 10 is a graph showing the cell death of A2780-SP cells when the pharmaceutical composition of Example was treated. to be.
도 9에 나타난 바와 같이, 실시예 1 내지 4의 약학적 조성물은 세포 주기에는 영향을 미치지 않음을 알 수 있다.As shown in Fig. 9, it can be seen that the pharmaceutical compositions of Examples 1 to 4 do not affect the cell cycle.
도 10에 나타난 바와 같이, 실시예 1 내지 4의 약학적 조성물은 암 줄기세포의 세포 사멸을 증가시킴을 알 수 있다.As shown in FIG. 10, it can be seen that the pharmaceutical compositions of Examples 1 to 4 increase cell death of cancer stem cells.
한편, PI 염색 후, 유세포 분석(Flow cytometry)을 이용한 분석으로 세포의 세포 주기를 분석할 수 있는데, 상기 표 4는 유세포 분석을 통해 각 세포의 세포 주기를 분석하고, 각 주기에 해당하는 세포의 수를 백분율로 나타낸 것이다.Meanwhile, after PI staining, the cell cycle of the cells can be analyzed by flow cytometry analysis. The cell cycle of each cell is analyzed by flow cytometry, The number is expressed as a percentage.
상기 표 4에 나타난 바와 같이,As shown in Table 4,
Sub G1 기(Sub G1 phase)라고도 불리는 사멸된 세포 수(Apoptotic cell number)가 DMSO 대조군에 비해 실시예 1 내지 4를 처리했을 때 증가하는 것을 알 수 있다. 나머지 G0-G1, S, G2-M 기의 세포 수의 변화에는 유의성이 없음을 알 수 있으며, 이를 통하여 실시예 1 내지 4의 약학적 조성물은 세포주기에는 영향을 미치지 않는 것을 확인하였다. It can be seen that the number of apoptotic cells, also called the Sub G1 phase, increases with the treatment of Examples 1-4 compared to the DMSO control. It was found that there was no significant difference in the cell numbers of the remaining G0-G1, S, and G2-M groups. Thus, it was confirmed that the pharmaceutical compositions of Examples 1 to 4 did not affect the cell cycle.
상기 결과로부터, 실시예 1 내지 4의 약학적 조성물은 암 줄기세포의 세포 주기를 멈추게 하여 증식을 억제하기 보다는 암 줄기세포의 세포 사멸(apoptosis)을 유도하여 증식을 억제하고 이를 통해 항암효과를 나타냄을 알 수 있다.The above results show that the pharmaceutical compositions of Examples 1 to 4 inhibit proliferation and induce anti-cancer effects by inducing apoptosis of cancer stem cells rather than inhibiting proliferation by stopping the cell cycle of cancer stem cells .
따라서, 본 발명의 약학적 조성물은 암 줄기세포의 세포 사멸(apoptosis)을 증가시키므로, 암 치료에 유용하게 사용할 수 있다.Therefore, the pharmaceutical composition of the present invention increases the apoptosis of cancer stem cells, and thus can be useful for the treatment of cancer.
<실험예 6> 암 줄기세포와 칼슘 채널 활성의 상관관계 분석<Experimental Example 6> Correlation Analysis of Calcium Channel Activity with Cancer Stem Cells
6-1. 칼슘 채널 서브유닛 mRNA 발현 분석6-1. Calcium channel subunit mRNA expression analysis
암 줄기세포와 칼슘 채널 활성과의 상관 관계를 분석하기 위하여, 암 줄기세포에서의 칼슘 재널 관련 서브유닛의 mRNA의 발현 정도를 분석하였으며, 그 결과를 도 11-13에 나타내었다. 구체적인 실험방법은 상기 실험 프토토콜의 6. 실시간 중합효소연쇄반응과 같다.In order to analyze the correlation between cancer stem cell and calcium channel activity, the expression level of the mRNA of the calcium-related subunit in cancer stem cells was analyzed, and the results are shown in Figs. The specific experimental method is the same as the 6. Real-time polymerase chain reaction of the above experimental protocol.
도 11은 A2780-SP 세포 및 A2780세포에서 칼슘 채널 서브유닛(Cacna1d, Cacna1f, Cacna1h) mRNA의 발현 수준을 Quantitative RT-PCR로 정량화 하여 나타낸 것이다.Fig. 11 shows quantitative RT-PCR quantification of the expression levels of calcium channel subunits (Cacna1d, Cacna1f, Cacna1h) mRNA in A2780-SP cells and A2780 cells.
도 12는 A2780-SP 세포에서 칼슘 채널과 관련된 유전자(si-Cacna1d, si-Cacna1f, si-Cacna1h)를 넉다운(knockdown)시킨 후, 각 칼슘 채널 서브유닛(Cacna1d, Cacna1f, Cacna1h)의 mRNA발현 수준을 Quantitative RT-PCR로 정량화 하여 넉다운을 확인한 것을 나타낸 것이다.12 shows the results of knockdown of the calcium channel-related genes (si-Cacna1d, si-Cacna1f, si-Cacna1h) in A2780-SP cells and the mRNA expression level of each calcium channel subunit (Cacna1d, Cacna1f, Cacna1h) Was quantitated by quantitative RT-PCR to confirm knockdown.
도 13은 A2780-SP 세포에서 칼슘 채널과 관련된 유전자(si-Cacna1d, si-Cacna1f, si-Cacna1h)를 넉다운(knockdown)시킨 후, 암 줄기세포와 관련된 마커(OCT3/4, NANOG, SOX2, ALDH1, CD133)의 mRNA 발현 수준을 Quantitative RT-PCR로 정량화한 것을 나타낸 것이다.FIG. 13 shows the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f and si-Cacna1h) in A2780-SP cells and then to identify markers (OCT3 / 4, NANOG, SOX2, ALDH1 , CD133) was quantified by quantitative RT-PCR.
도 11에 나타난 바와 같이, 암 세포에서보다 암 줄기세포에서 특이적으로 칼슘 채널 서브유닛의 mRNA발현량이 현저하게 높은 것을 확인할 수 있다.As shown in FIG. 11, it can be confirmed that the expression level of calcium channel subunit mRNA specifically in cancer stem cells is significantly higher than in cancer cells.
도 12에 나타난 바와 같이, 암 줄기세포에서 칼슘 채널과 관련된 유전자를 넉다운 시킨 결과, 칼슘 채널 서브유닛의 mRNA발현량이 현저하게 감소한 것을 확인할 수 있다.As shown in FIG. 12, knockdown of a gene related to calcium channel in cancer stem cells revealed that the expression amount of mRNA of calcium channel subunit was remarkably decreased.
도 13에 나타난 바와 같이, 암 줄기세포에서 칼슘 채널과 관련된 유전자를 넉다운 시킨 결과, 암 줄기세포와 관련된 마커들의 mRNA 발현 또한 감소하는 것을 알 수 있다.As shown in FIG. 13, knockdown of genes related to calcium channels in cancer stem cells results in a decrease in mRNA expression of markers associated with cancer stem cells.
상기 결과는 암 줄기세포에 특이적으로 존재하는 특정 유형의 칼슘채널의 존재가 암 줄기세포의 특성을 유지하는데 중요한 역할을 하고 있으며, 이를 억제함으로써 줄기세포능(stemness)을 억제할 수 있음을 보여준다.The above results show that the presence of a specific type of calcium channel specifically present in cancer stem cells plays an important role in maintaining the characteristics of cancer stem cells and can inhibit stemness by inhibiting them .
6-2. 암 줄기 세포 관련 인자의 단백질 발현 분석6-2. Protein expression analysis of cancer stem cell-related factors
암 줄기세포와 칼슘 채널 활성과의 상관 관계를 분석하기 위하여, 암 줄기세포에서의 칼슘 재널 관련 서브유닛의 유전자를 넉다운 시킨 후, 암 줄기 세포능(stemness)과 관련된 마커(OCT 3/4) 와 암 줄기세포의 생존 및 증식과 관련된 단백질 (p-ERK, ERK, p-p38, p38) 발현 변화를 분석하였으며, 그 결과를 도 14 및 15에 나타내었다. 구체적인 실험방법은 상기 실험 프토토콜의 4. 웨스턴 블랏과 같다.In order to analyze the correlation between cancer stem cell and calcium channel activity, knockdown of the gene of the calcium-related subunit in the cancer stem cell was performed, and the marker (OCT 3/4) related to cancer stem cell function The expression of proteins (p-ERK, ERK, p-p38, p38) involved in the survival and proliferation of cancer stem cells was analyzed and the results are shown in Figs. The specific experimental method is the same as the 4. Western blot of the experimental protocol.
도 14는 A2780-SP 세포에서 칼슘 채널과 관련된 유전자(si-Cacna1d, si-Cacna1f, si-Cacna1h)를 넉다운(knockdown)시킨 후, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포능(stemness) 관련 마커와 암 줄기세포의 생존 및 증식과 관련된 단백질의 발현 변화를 관찰한 이미지이다.14 is a graph showing the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f and si-Cacna1h) in A2780-SP cells, and stem cell-associated markers and the expression of proteins involved in the survival and proliferation of cancer stem cells.
도 15는 A2780-SP 세포에서 칼슘 채널과 관련된 유전자(si-Cacna1d, si-Cacna1f, si-Cacna1h)를 넉다운(knockdown)시킨 후, A2780-SP 세포(ovarian cancer stem cell)에서의 암 줄기세포능(stemness) 관련 마커와 암 줄기세포의 생존 및 증식과 관련된 단백질의 발현 변화를 정량화한 그래프이다.15 is a graph showing the results of knockdown of calcium channel-related genes (si-Cacna1d, si-Cacna1f, si-Cacna1h) in A2780-SP cells, a stem cell-associated marker, and a stem cell-associated marker and the expression of a protein associated with survival and proliferation of cancer stem cells.
도 14 및 15에 나타난 바와 같이, 암 줄기세포에서 칼슘 채널 관련 유전자를 넉다운 한 결과, 6-1의 mRNA 분석과 유사하게 단백질 수준에서도 암 줄기세포능(stemness) 관련 마커인 OCT3/4의 발현을 감소시키는 것을 확인하였다. As shown in FIGS. 14 and 15, knockdown of the calcium channel-related gene in cancer stem cells revealed that expression of OCT3 / 4, which is a stem cell-related marker, at the protein level similar to that of 6-1 mRNA, Respectively.
또한, 암 줄기세포에서 칼슘 채널 관련 유전자를 넉다운 한 결과, 세포의 생존 및 증식과 관련된 단백질들인 ERK, P-38의 활성이 감소하는 것을 단백질 수준에서 확인하였다. 이러한 결과는 실험예 4의 칼슘채널 억제제의 결과와 그 궤를 같이 하는 것으로 볼 수 있다. In addition, knockdown of calcium channel-related genes in cancer stem cells revealed that the activity of ERK and P-38, which are related to cell survival and proliferation, is decreased at the protein level. These results are consistent with the results of the calcium channel inhibitor of Experimental Example 4.
6-3. 전세포 패치 클램프 레코딩 실험(whole cell patch clamp recording)6-3. Whole cell patch clamp recording.
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물의 난소암줄기세포(A2780-SP)에서의 칼슘 채널 억제 효과를 평가하기 위하여, 다음과 같은 실험을 수행하였으며, 그 결과를 그 결과를 도 16에 나타내었다. 구체적인 실험방법은 상기 실험 프로토콜의 8. 전세포 패치 클램프 레코딩 실험과 같다.In order to evaluate the calcium channel inhibitory effect in the ovarian cancer stem cell (A2780-SP) of the pharmaceutical composition comprising the calcium channel inhibitor of the present invention, the following experiment was conducted and the results are shown in FIG. 16 . The specific experimental method is the same as the 8. whole-cell patch clamp recording experiment of the above experimental protocol.
도 16은 A2780-SP 세포에 실시예 약학적 조성물을 처리하였을 때, 칼슘 전류 진폭이 줄어듦을 나타낸 그래프이다.Figure 16 is a graph showing that the amplitude of calcium current is reduced when the pharmaceutical composition of Example is treated with A2780-SP cells.
도 16에 나타난 바와 같이,As shown in Fig. 16,
난소암세포(A2780) 보다 난소암줄기세포(A2780-SP)에서 칼슘 전류(calcium current)의 진폭이 더 크게 나타나며, 본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물인 실시예 3(마니디핀)을 A2780-SP에 처리하였을 때, 칼슘 전류의 진폭히 현저하게 억제되는 것을 알 수 있다.Example 3 (manidipine), which is a pharmaceutical composition containing the calcium channel inhibitor of the present invention, showed a larger amplitude of calcium current in ovarian cancer cell (A2780-SP) than ovarian cancer cell (A2780) -SP, the amplitude of the calcium current is remarkably suppressed.
이는 본 발명의 4종의 약학적 조성물들이 암 줄기세포에 과발현되어 있는 칼슘 채널의 억제를 통해 기존 약물에 내성을 보이는 주요 요인인 암 줄기세포의 암 줄기세포능(stemness)을 억제하고, 세포의 사멸(apoptosis)을 유도할 수 있음을 시사한다. It is believed that the four pharmaceutical compositions of the present invention inhibit the cancer stem cell stem stem, which is a major factor in resistance to existing drugs through the inhibition of the calcium channel overexpressed in cancer stem cells, Suggesting that apoptosis may be induced.
이를 통해 암의 재발, 전이 및 진행을 억제함으로써 암 치료에 유용하게 사용될 수 있음을 알 수 있다.Thus, it can be shown that cancer can be effectively used for cancer treatment by inhibiting recurrence, metastasis and progression of cancer.
<실험예 7> 항암제와의 병용 투여 효과 실험&Lt; Experimental Example 7 >
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물과 종래 사용중인 항암제 시스플라틴(cisplatin)을 병용 투여하였을 때의 효과를 알아보기 위하여 실험을 수행하였으며, 그 결과를 도 17 및 18에 나타내었다. 구체적인 실험방법은 상기 실험 프로토콜의 7. 병용 투여 효과 실험과 같다.Experiments were conducted to examine the effects of the pharmaceutical composition comprising the calcium channel inhibitor of the present invention and the anticancer agent cisplatin, which are conventionally used, in combination. The results are shown in FIGS. 17 and 18. The specific experimental method is the same as the seventh concurrent administration experiment of the experimental protocol.
도 17은 실시예 약학적 조성물과 시스플라틴을 병용 처리하였을 때, A2780-SP 세포의 세포 생존율을 측정하여 그 결과를 나타낸 그래프이다.FIG. 17 is a graph showing the results of measuring the cell survival rate of A2780-SP cells when the pharmaceutical composition of Example and cisplatin were used in combination. FIG.
도 18은 실시예 약학적 조성물과 시스플라틴을 병용 처리하였을 때, A2780-SP 세포의 세포 증식을 분석하여 그 결과를 나타낸 그래프이다.18 is a graph showing the results of analysis of cell proliferation of A2780-SP cells when the pharmaceutical composition of Example and cisplatin were used in combination.
도 17에 나타난 바와 같이, 본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물을 단독으로 투여하였을 때도 우수한 세포 생존능 감소 효과를 나타내나, 항암제와 병용투여 하였을 때 암 줄기세포의 세포 생존능이 보다 더 감소하는 것을 알 수 있다.As shown in FIG. 17, when the pharmaceutical composition containing the calcium channel inhibitor of the present invention is administered singly, it exhibits an excellent effect of reducing cell viability. However, when administered in combination with an anticancer agent, the cell viability of cancer stem cells is further reduced .
도 18에 나타난 바와 같이, 본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물을 단독으로 투여하였을 때도 우수한 세포 증식 억제 효과를 나타내나, 항암제와 병용투여 하였을 때 암 줄기세포의 세포 증식이 보다 더 억제되는 것을 알 수 있다.As shown in Fig. 18, when the pharmaceutical composition containing the calcium channel inhibitor of the present invention is administered alone, it exhibits an excellent cell proliferation inhibitory effect. However, when the combination is administered with an anticancer agent, cell proliferation of cancer stem cells is further inhibited .
따라서, 본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물은 단독으로 사용하여도 우수한 항암 효과를 나타낼 뿐만 아니라, 기존 항암제와 병용으로 사용하여 보다 더 높은 시너지 효과를 나타낼 수 있다.Therefore, the pharmaceutical composition containing the calcium channel inhibitor of the present invention not only exhibits excellent anticancer effects even when used alone, but also can exhibit synergistic effects even higher when used in combination with existing anticancer drugs.
<실험예 8> 정상세포에서의 세포 생존능 평가<Experimental Example 8> Evaluation of cell viability in normal cells
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물 처리에 따른 정상세포에서의 생존능을 분석하기 위하여, 실시예 1 내지 4 약학적 조성물을 사용하여 세포 생존능 분석을 수행하였으며, 그 결과를 하기 표 5에 나타내었다. 구체적인 실험방법은 상기 실험 프로토콜의 1. 세포 생존능 분석과 같으며, 정상세포는 정상섬유아세포인 BJ6 및 NIH-3T3를 사용하였다.In order to analyze the viability of normal cells according to the pharmaceutical composition containing the calcium channel inhibitor of the present invention, the cell viability was analyzed using the pharmaceutical compositions of Examples 1 to 4. The results are shown in the following Table 5 Respectively. The specific experimental method is the same as the 1. cell viability analysis of the above experimental protocol, and the normal cells were normal fibroblasts BJ6 and NIH-3T3.
실시예Example 세포 생존능(%), BJ6Cell viability (%), BJ6 세포 생존능(%), NIH-3T3Cell viability (%), NIH-3T3
1(베니디핀)1 (Benidipine) 8585 9191
2(라시디핀)2 (Ladydipin) 8787 119119
3(마니디핀)3 (manidipine) 8181 9696
4(로메리진)4 (Romerizin) 8888 120120
상기 표 5에 나타난 바와 같이, 정상세포 BJ6 및 NIH-3T3 세포에서, 실시예 1 내지 4의 약학적 조성물은 80% 이상의 세포 생존능을 나타냄으로써, 본 발명의 약학적 조성물은 정상세포에는 영향을 미치지 않고, 암 세포, 특히, 암 줄기 세포에만 특이적으로 세포 독성을 나타내는 것을 알 수 있다.As shown in Table 5, in the normal cells BJ6 and NIH-3T3 cells, the pharmaceutical compositions of Examples 1 to 4 exhibited cell viability of 80% or more, so that the pharmaceutical composition of the present invention had no effect on normal cells , And specifically shows cytotoxicity only to cancer cells, particularly cancer stem cells.
따라서, 본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물은 정상세포에는 영향을 미치지 않고, 암 줄기세포의 성장 저해 효과를 나타내는 바, 부작용 이 적은 항암 효과를 나타낼 수 있음을 알 수 있다.Therefore, the pharmaceutical composition comprising the calcium channel inhibitor of the present invention does not affect normal cells, but exhibits an inhibitory effect on the growth of cancer stem cells. Thus, it can be seen that the anticancer effect with less side effects can be exhibited.
상기 결과를 통하여 본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물은 단독으로 사용하여도 암 줄기세포의 성장 및 증식을 저해하는 효과가 우수할 뿐만 아니라, 종래 항암제와 병용 투여할 경우, 보다 우수한 약리 효과를 나타내는 바, 암의 재발, 전이, 진행을 억제시킬 수 있으므로, 암의 예방 또는 치료용 약학적 조성물 또는 병용 제제로 유용하게 사용될 수 있음을 알 수 있다.The above results show that the pharmaceutical composition containing the calcium channel inhibitor of the present invention is excellent in the effect of inhibiting the growth and proliferation of cancer stem cells even when used alone, As a result, it is possible to inhibit recurrence, metastasis and progression of cancer. Therefore, it can be used as a pharmaceutical composition or a combined preparation for cancer prevention or treatment.
<실험예 9> 동물 모델 실험Experimental Example 9 Animal Model Experiment
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물의 난소암 치료 효과를 in vivo 수준에서 확인하기 위하여, 동물 모델 실험을 수행하였으며, 그 결과를 도 19에 나타내었다. 구체적인 실험방법은 상기 실험 프로토콜의 9. 동물 실험과 같다.In order to confirm the ovarian cancer therapeutic effect of the pharmaceutical composition comprising the calcium channel blocker of the present invention at the in vivo level, an animal model experiment was conducted and the results are shown in FIG. The specific experimental method is the same as that of the animal experiment of the above experimental protocol.
도 19는 난소암 세포 종양 동물 모델(A2780-AD) 및 난소암 암 줄기세포 종양 동물모델(A2780-SP)에서, 실시예 약학적 조성물 단독처리 및 파클리탁셀(Paclitaxel)을 병용 처리하였을 때의 종양 형성 억제 효과를 평가하여 그 결과를 나타낸 그래프이다.FIG. 19 is a graph showing the results of tumorigenesis (A2780-AD) in the case of the ovarian cancer cell tumor animal model (A2780-AD) and the ovarian cancer stem cell tumor animal model (A2780-SP), alone or in combination with paclitaxel Inhibitory effect and evaluating the results.
도 19 에 나타난 바와 같이,19,
난소암 암 줄기세포에서 특이적으로 종양의 형성을 저해하며, Paclitaxel과 병용투여시 더 좋은 효능을 보임을 알 수 있다.It inhibits the formation of tumors specifically in ovarian cancer stem cells and shows better efficacy when combined with paclitaxel.
따라서, 본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물은 난소암의 재발, 전이, 진행을 억제시킬 수 있으므로, 난소암의 예방 또는 치료용 약학적 조성물로 유용하게 사용될 수 있다.Therefore, the pharmaceutical composition containing the calcium channel inhibitor of the present invention can inhibit recurrence, metastasis and progression of ovarian cancer, and thus can be usefully used as a pharmaceutical composition for the prevention or treatment of ovarian cancer.
<제제예 1> 산제의 제조&Lt; Formulation Example 1 > Preparation of powders
칼슘 채널 억제제 2gCalcium Channel Inhibitor 2g
유당 1gLactose 1g
상기의 성분을 혼합하고 기밀포에 충진하여 산제를 제조하였다.The above components were mixed and packed in airtight bags to prepare powders.
<제제예 2> 정제의 제조&Lt; Formulation Example 2 > Preparation of tablet
칼슘 채널 억제제 100 ㎎100 mg calcium channel blocker
옥수수전분 100 ㎎ Corn starch 100 mg
유당 100 ㎎ Lactose 100 mg
스테아린산 마그네슘 2 ㎎2 mg of magnesium stearate
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.
<제제예 3> 캡슐제의 제조&Lt; Formulation Example 3 > Preparation of capsules
칼슘 채널 억제제 100 ㎎100 mg calcium channel blocker
옥수수전분 100 ㎎ Corn starch 100 mg
유당 100 ㎎ Lactose 100 mg
스테아린산 마그네슘 2 ㎎2 mg of magnesium stearate
상기의 성분을 혼합한 후, 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.
<제제예 4> 주사제의 제조&Lt; Formulation Example 4 > Preparation of injection
칼슘 채널 억제제 100 ㎎100 mg calcium channel blocker
만니톨 180 ㎎180 mg mannitol
Na2HPO4ㆍ2H2O 26 ㎎Na 2 HPO 4 .2H 2 O 26 mg
증류수 2974 ㎎2974 mg of distilled water
통상적인 주사제의 제조방법에 따라, 상기 성분들을 제시된 함량으로 함유시켜 주사제를 제조하였다.According to the conventional method for preparing an injectable preparation, an injectable preparation was prepared by incorporating the aforementioned components in the amounts indicated.
<제제예 5> 연고제의 제조&Lt; Formulation Example 5 > Preparation of ointment preparation
칼슘 채널 억제제 5 gCalcium Channel Inhibitor 5 g
세틸팔미테이트 20 gCetyl palmitate 20 g
세탄올 40 g40 g of cetanol
스테아릴알콜 40 g40 g of stearyl alcohol
미리스탄이소프로필 80 g80 g of myristanisopropyl
폴리솔베이트 60 gPolysorbate 60 g
파라옥시안식향산 프로필 1 g1 g of p-hydroxybenzoic acid propyl
파라옥시안식향산 메틸 1 g1 g of p-hydroxybenzoic acid methyl
인산 및 정제수 적당량Amount of phosphoric acid and purified water
통상적인 연고제의 제조방법에 따라, 상기 성분들을 제시된 함량으로 함유시켜 연고제를 제조하였다.The ointment was prepared by incorporating the above ingredients in the prescribed amounts according to the usual preparation method of ointment.
<제제예 6> 건강기능식품의 제조&Lt; Formulation Example 6 > Preparation of health functional foods
칼슘 채널 억제제 500ngCalcium channel inhibitor 500ng
비타민 혼합물 적량Vitamin mixture quantity
비타민 A 아세테이트 70mg Vitamin A acetate 70 mg
비타민 E 1.0mgVitamin E 1.0mg
비타민 0.13mg0.13mg of vitamin
비타민 B2 0.15mg0.15 mg of vitamin B2
비타민 B6 0.5mgVitamin B6 0.5mg
비타민 B12 0.2mgVitamin B12 0.2mg
비타민 C 10mgVitamin C 10mg
비오틴 10mgBiotin 10mg
니코틴산아미드 1.7mgNicotinic acid amide 1.7 mg
엽산 50mgFolic acid 50mg
판토텐산 칼슘 0.5mgCalcium pantothenate 0.5mg
무기질 혼합물 적량Mineral mixture quantity
황산제1철 1.75mg1.75 mg ferrous sulfate
산화아연 0.82mg0.82 mg of zinc oxide
탄산마그네슘 25.3mgMagnesium carbonate 25.3 mg
제1인산칼륨 15mg15 mg of potassium phosphate monobasic
제2인산칼슘 55mgCalcium phosphate diphosphate 55 mg
구연산칼륨 90mgPotassium citrate 90mg
탄산칼슘 100mg Calcium carbonate 100 mg
염화마그네슘 24.8mg24.8 mg of magnesium chloride
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강기능식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강기능식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강기능식품 조성물 제조에 사용할 수 있다.Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed , Granules may be prepared and used in the manufacture of a health functional food composition according to a conventional method.
<제제예 7> 건강기능음료의 제조Formulation Example 7 Preparation of Health Functional Drink
칼슘 채널 억제제 500ngCalcium channel inhibitor 500ng
구연산 1000mgCitric acid 1000mg
올리고당 100gOligosaccharide 100 g
매실농축액 2gPlum concentrate 2g
타우린 1gTaurine 1g
정제수를 가하여 전체 900mlPurified water was added to the entire 900 ml
통상의 건강 기능 음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간 동안 85℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 건강 기능 음료 조성물 제조에 사용하였다.The above components were mixed in accordance with a conventional health functional beverage 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 container, which was sealed and sterilized, Were used to prepare beverage compositions.
상기 조성비는 비교적 기호 음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만 수요계층이나, 수요국가, 사용용도 등 지역적, 민족적 기호 도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.Although the composition ratio is relatively mixed with the ingredient suitable for the favorite drink, it is also possible to arbitrarily modify the blending ratio according to the regional or national preference such as the demand class, demand country, use purpose, and the like.
본 발명의 칼슘 채널 억제제를 포함하는 약학적 조성물은 암의 예방 또는 치료용 약학적 조성물 또는 병용 제제로 유용하게 사용될 수 있다.The pharmaceutical composition containing the calcium channel inhibitor of the present invention can be usefully used as a pharmaceutical composition or a combined preparation for the prevention or treatment of cancer.

Claims (13)

  1. 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prophylaxis or treatment of cancer comprising as an active ingredient a calcium channel inhibitor or a pharmaceutically acceptable salt thereof.
  2. 제1항에 있어서,The method according to claim 1,
    상기 칼슘 채널 억제제는 하기 화학식 A 내지 화학식 D로부터 선택되는 어느 하나인 것을 특징으로 하는 약학적 조성물:Wherein the calcium channel inhibitor is any one selected from the following Formulas A to D:
    [화학식 A](A)
    Figure PCTKR2019001143-appb-I000017
    ;
    Figure PCTKR2019001143-appb-I000017
    ;
    [화학식 B][Chemical Formula B]
    Figure PCTKR2019001143-appb-I000018
    ;
    Figure PCTKR2019001143-appb-I000018
    ;
    [화학식 C]&Lt; RTI ID = 0.0 &
    Figure PCTKR2019001143-appb-I000019
    ; 및
    Figure PCTKR2019001143-appb-I000019
    ; And
    [화학식 D][Chemical Formula D]
    Figure PCTKR2019001143-appb-I000020
    .
    Figure PCTKR2019001143-appb-I000020
    .
  3. 제1항에 있어서,The method according to claim 1,
    상기 암은 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 구강암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 음경암, 전립선암, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성외부생식기암 및 피부암으로 이루어지는 군으로부터 선택되는 1종 이상인 것을 특징으로 하는 약학적 조성물.The cancer may be selected from the group consisting of benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial lesion, ependymoma, brain tumor, laryngeal cancer, pancreatic cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, Cancer of the breast, cancer of the gallbladder, cancer of the gallbladder, cancer of the bile duct, pancreatic cancer, small bowel cancer, colon cancer, anal cancer, bladder cancer, breast cancer, Wherein the composition is at least one selected from the group consisting of renal cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female germline cancer and skin cancer.
  4. 제1항에 있어서,The method according to claim 1,
    상기 약학적 조성물은 CT3/4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 및 p38로 이루어지는 단백질 군으로부터 선택되는 하나 이상의 발현을 억제하는 것을 특징으로 하는 약학적 조성물.Wherein said pharmaceutical composition inhibits the expression of at least one selected from the group consisting of CT3 / 4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 and p38 Gt;
  5. 제1항에 있어서,The method according to claim 1,
    상기 약학적 조성물은 암 줄기세포의 증식을 억제하는 것을 특징으로 하는 약학적 조성물.Wherein said pharmaceutical composition inhibits the proliferation of cancer stem cells.
  6. 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염 및 항암제를 포함하는 암의 예방 또는 치료용 병용 제제.A prophylactic or therapeutic agent for cancer, comprising a calcium channel inhibitor or a pharmaceutically acceptable salt thereof and an anticancer agent.
  7. 제6항에 있어서,The method according to claim 6,
    상기 칼슘 채널 억제제는 하기 화학식 A 내지 화학식 C로부터 선택되는 어느 하나인 것을 특징으로 하는 병용 제제:Wherein the calcium channel inhibitor is any one selected from the following Formulas A to C:
    [화학식 A](A)
    Figure PCTKR2019001143-appb-I000021
    ;
    Figure PCTKR2019001143-appb-I000021
    ;
    [화학식 B][Chemical Formula B]
    Figure PCTKR2019001143-appb-I000022
    ;
    Figure PCTKR2019001143-appb-I000022
    ;
    [화학식 C]&Lt; RTI ID = 0.0 &
    Figure PCTKR2019001143-appb-I000023
    ; 및
    Figure PCTKR2019001143-appb-I000023
    ; And
    [화학식 D][Chemical Formula D]
    Figure PCTKR2019001143-appb-I000024
    .
    Figure PCTKR2019001143-appb-I000024
    .
  8. 제6항에 있어서,The method according to claim 6,
    상기 암은 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 두개내인종, 상의세포종, 뇌간종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 구강암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 항문암, 방광암, 신장암, 음경암, 전립선암, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성외부생식기암 및 피부암으로 이루어지는 군으로부터 선택되는 1종 이상인 것을 특징으로 하는 병용 제제.The cancer may be selected from the group consisting of benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial lesion, ependymoma, brain tumor, laryngeal cancer, pancreatic cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, Cancer of the breast, cancer of the gallbladder, cancer of the gallbladder, cancer of the bile duct, pancreatic cancer, small bowel cancer, colon cancer, anal cancer, bladder cancer, breast cancer, Wherein the composition is at least one selected from the group consisting of renal cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female germline cancer and skin cancer.
  9. 제6항에 있어서,The method according to claim 6,
    상기 병용 제제는 CT3/4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 및 p38로 이루어지는 단백질 군으로부터 선택되는 하나 이상의 발현을 억제하는 것을 특징으로 하는 병용 제제.Wherein the combined preparation inhibits expression of at least one selected from the group consisting of CT3 / 4, NANOG, SOX2, ALDH1, CD133, pAKT, AKT, p-ERK, ERK, p-p38 and p38. .
  10. 제6항에 있어서,The method according to claim 6,
    상기 병용 제제는 암 줄기세포의 증식을 억제하는 것을 특징으로 하는 병용 제제.Wherein said combined preparation inhibits proliferation of cancer stem cells.
  11. 제6항에 있어서, The method according to claim 6,
    상기 항암제는 독소루비신, 파클리탁셀, 빈크리스틴, 다우노루비신(daunorubicin), 빈블라스틴(vinblastine), 액티노마이신-D(actinomycin-D), 도세탁셀, 에토포사이드(etoposide), 테니포사이드(teniposide), 비산트렌 (bisantrene), 호모해링토닌(homoharringtonine), 글리벡(Gleevec; STI-571), 시스플라틴, 5-플로오로우라실, 아드리아마이신, 메토트렉세이트, 부설판(busulfan), 클로람부실(chlorambucil), 시클로포스파미드(cyclophosphamide), 멜팔란 (melphalan), 니트로겐 무스타드(nitrogen mustard) 및 니트로소우레아 (nitrosourea)로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는 병용 제제.The anticancer agent may be selected from the group consisting of doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, teniposide, But are not limited to, bisantrene, homoharringtonine, Gleevec (STI-571), cisplatin, 5-fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil, Wherein the pharmaceutical preparation is at least one selected from the group consisting of cyclophosphamide, melphalan, nitrogen mustard, and nitrosoourea.
  12. 칼슘 채널 억제제 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 암의 예방 또는 개선용 건강기능식품 조성물.A health functional food composition for preventing or ameliorating a cancer containing an calcium channel inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.
  13. 제12항에 있어서,13. The method of claim 12,
    상기 칼슘 채널 억제제는 하기 화학식 A 내지 화학식 C로부터 선택되는 어느 하나인 것을 특징으로 하는 건강기능식품 조성물:Wherein the calcium channel inhibitor is any one selected from the following chemical formulas A to C:
    [화학식 A](A)
    Figure PCTKR2019001143-appb-I000025
    ;
    Figure PCTKR2019001143-appb-I000025
    ;
    [화학식 B][Chemical Formula B]
    Figure PCTKR2019001143-appb-I000026
    ;
    Figure PCTKR2019001143-appb-I000026
    ;
    [화학식 C]&Lt; RTI ID = 0.0 &
    Figure PCTKR2019001143-appb-I000027
    ; 및
    Figure PCTKR2019001143-appb-I000027
    ; And
    [화학식 D][Chemical Formula D]
    Figure PCTKR2019001143-appb-I000028
    .
    Figure PCTKR2019001143-appb-I000028
    .
PCT/KR2019/001143 2018-01-26 2019-01-28 Pharmaceutical composition for preventing or treating cancer comprising, as active ingredient, calcium channel inhibitor or pharmaceutically acceptable salt thereof WO2019147089A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20180010034 2018-01-26
KR10-2018-0010034 2018-01-26

Publications (1)

Publication Number Publication Date
WO2019147089A1 true WO2019147089A1 (en) 2019-08-01

Family

ID=67395547

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2019/001143 WO2019147089A1 (en) 2018-01-26 2019-01-28 Pharmaceutical composition for preventing or treating cancer comprising, as active ingredient, calcium channel inhibitor or pharmaceutically acceptable salt thereof

Country Status (2)

Country Link
KR (1) KR102217010B1 (en)
WO (1) WO2019147089A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110742890A (en) * 2019-10-24 2020-02-04 暨南大学 Application of lomerizine in preparation of anti-colon cancer drug

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102528732B1 (en) * 2020-11-25 2023-05-03 재단법인 대구경북첨단의료산업진흥재단 Pharmaceutical composition for preventing or treating cancer comprising poziotinib and calcium channel blocker

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007008499A1 (en) * 2005-07-06 2007-01-18 Kanisa Pharmaceuticals, Inc. Treatment of cancer patients exhibiting activation of the p-glycoprotein efflux pump mechanism
WO2015157471A1 (en) * 2014-04-08 2015-10-15 The Methodist Hospital Inos-inhibitory compositions and their use as breast cancer therapeutics
KR20160124012A (en) * 2015-04-16 2016-10-26 서울대학교산학협력단 Method for inhibiting lung carcinogenesis of both smokers and non-smokers by using antihypertensive drugs
WO2019008516A2 (en) * 2017-07-03 2019-01-10 Menri Group Ltd. Treatment of cancer with dihydropyridines

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4906646A (en) * 1983-03-31 1990-03-06 Board Of Governors Of Wayne State University Method and composition for the treatment of tumors by administering a platinum coordination compound and a calcium channel blocker compound of the dihydropyridine class
JP2016506248A (en) * 2013-01-10 2016-03-03 ティーエーユー・セラピューティクス・エルエルシー T-type calcium channel inhibitors for the treatment of cancer
KR101765575B1 (en) 2015-02-02 2017-08-07 연세대학교 산학협력단 Pharmaceutical composition for inhibiting a growth of cancer stem cells comprising aldehyde inhibitor and biguanide compounds
WO2016167605A2 (en) * 2015-04-16 2016-10-20 서울대학교 산학협력단 Method for inhibiting lung cancer of smokers and non-smokers using hypertension medicine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007008499A1 (en) * 2005-07-06 2007-01-18 Kanisa Pharmaceuticals, Inc. Treatment of cancer patients exhibiting activation of the p-glycoprotein efflux pump mechanism
WO2015157471A1 (en) * 2014-04-08 2015-10-15 The Methodist Hospital Inos-inhibitory compositions and their use as breast cancer therapeutics
KR20160124012A (en) * 2015-04-16 2016-10-26 서울대학교산학협력단 Method for inhibiting lung carcinogenesis of both smokers and non-smokers by using antihypertensive drugs
WO2019008516A2 (en) * 2017-07-03 2019-01-10 Menri Group Ltd. Treatment of cancer with dihydropyridines

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHIRAKI, N. ET AL.: "Increase in doxorubicin cytotoxicity by inhibition of P-glycoprotein activity with lomerizine", BIOLOGICAL AND PHARMACEUTICAL BULLETIN, vol. 24, no. 5, 2001, pages 555 - 557, XP055628797 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110742890A (en) * 2019-10-24 2020-02-04 暨南大学 Application of lomerizine in preparation of anti-colon cancer drug

Also Published As

Publication number Publication date
KR20190091222A (en) 2019-08-05
KR102217010B1 (en) 2021-02-18

Similar Documents

Publication Publication Date Title
WO2020171606A1 (en) Novel heterotricyclic derivative compound and use of same
WO2021246797A1 (en) Pharmaceutical composition for preventing or treating cancer containing antiviral agent and antidepressant as active ingredients
WO2021162451A1 (en) Pharmaceutical composition for preventing or treating cancer, containing bile acids or derivatives thereof, biguanide-based compounds, and two or more kinds of antiviral agents as active ingredients
WO2018004213A1 (en) Novel compound having smo inhibitory activity and composition comprising same as active ingredient for preventing or treating cancer
WO2022035115A1 (en) Composition for prevention and treatment of skeletal muscle-related diseases containing alnus japonica extract or compound isolated therefrom and use thereof
WO2016108446A1 (en) Pharmaceutical composition for treating cancer, containing lactate metal salt
WO2019147089A1 (en) Pharmaceutical composition for preventing or treating cancer comprising, as active ingredient, calcium channel inhibitor or pharmaceutically acceptable salt thereof
WO2009125923A2 (en) Novel indol carboxylic acid bispyridyl carboxamide derivatives, pharmaceutically acceptable salt thereof, preparation method and composition containing the same as an active ingredient
WO2018012907A1 (en) Novel quinazolinone derivatives inhibiting pi3k and pharmaceutical composition containing same
WO2021230600A1 (en) Anticancer composition comprising metabolic anticancer drug
WO2018155947A1 (en) Pharmaceutical composition comprising compound capable of penetrating blood-brain barrier as effective ingredient for preventing or treating brain cancer
WO2013183920A1 (en) Pharmaceutical composition containing verbenone derivative for treating or preventing neurodegenerative disease
WO2019168237A1 (en) Novel compound and composition for preventing, ameliorating, or treating fibrosis or nonalcoholic steatohepatitis comprising same as active ingredient
WO2014030972A1 (en) Anticancer composition
WO2015037778A1 (en) Composition containing lignan compound as active ingredient for preventing or treating cancer
WO2014007447A1 (en) Composition for preventing or treating diseases caused by angiogenesis, containing hydroxychalcone compound as active ingredient
WO2015026172A1 (en) Indole amide compound as inhibitor of necrosis
WO2020096416A1 (en) Compound inhibiting yap-tead binding, and pharmaceutical composition for preventing or treating cancer, comprising compound as active ingredient
WO2018004263A1 (en) Optically active pyranochromenyl phenol derivative and pharmaceutical composition comprising same
WO2021096112A1 (en) Pyrrolopyrimidine, pyrrolopyridine and indazole compound derivative, and therapeutic pharmaceutical composition containing same
WO2019194583A1 (en) 3-phenyl-2,8-dihydropyrano[2,3-f]chromene derivative and pharmaceutical composition comprising same
WO2021230710A1 (en) Novel ido/tdo inhibitor, anticancer use thereof, and anticancer combination therapy thereof
WO2023080765A1 (en) Novel oxadiazole derivative and use thereof
WO2018101762A1 (en) Pharmaceutical composition for preventing or treating ischemic acute kidney injury, containing tricyclic derivative or pharmaceutically acceptable salt thereof
WO2012060482A1 (en) Cdk-inhibiting pyrrolopyrimidinone carboxamide derivative or pharmaceutically acceptable salt thereof, and pharmaceutical composition containing same as active ingredient for preventing or treating liver cell cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19743403

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19743403

Country of ref document: EP

Kind code of ref document: A1