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WO2019013607A1 - Compositions for inhibiting oct4 and use thereof - Google Patents

Compositions for inhibiting oct4 and use thereof Download PDF

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Publication number
WO2019013607A1
WO2019013607A1 PCT/KR2018/008027 KR2018008027W WO2019013607A1 WO 2019013607 A1 WO2019013607 A1 WO 2019013607A1 KR 2018008027 W KR2018008027 W KR 2018008027W WO 2019013607 A1 WO2019013607 A1 WO 2019013607A1
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Prior art keywords
cell
compound
cells
oct4
composition
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PCT/KR2018/008027
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French (fr)
Korean (ko)
Inventor
한동초
권병목
김장환
강용국
김석호
김영미
박정선
심현아
정지예
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한국생명공학연구원
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Publication of WO2019013607A1 publication Critical patent/WO2019013607A1/en

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    • 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
    • A61K31/12—Ketones
    • 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

Definitions

  • the present invention relates to a composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) and its use.
  • a composition for inhibiting the formation of a teratoma or a teratocarcinoma of a cell therapeutic agent for removing undifferentiated cells using the composition A pharmaceutical composition for preventing or treating testicular cancer; A method for removing undifferentiated cells; Methods of inhibiting teratoma or teratocarcinoma formation; A method for producing a cell therapeutic agent; To a method of preventing or treating testicular cancer.
  • OCT4 Optamer-Binding Transcription Factor 4
  • OCT4 and SOX2 transcription factors are very important factors for the induction of stem cells in adult cells.
  • Myc gene plays a role in improving the efficiency of stem cell production.
  • OCT4 is important for maintaining the pluripotency of induced pluripotent stem cells.
  • FACS Fluorescent Activated Cells Sorter
  • Magnetic cell sorting has been used to label cells using antibodies against markers present in the cell membrane to avoid damage to therapeutic cells.
  • the Magnetic Separation Method has the advantage of eliminating the risk of cell damage to the laser used when using a flow cell separator.
  • both FACS and MACS methods can not completely exclude the possibility that pluripotent pluripotent pluripotent pluripotent stem cells are included in the therapeutic cell population. If there is a technical limitation in selecting only cells that have differentiated into 100% purity as therapeutic cells, serious safety problems may arise. Particularly, there is a possibility that undifferentiated cells expressing OCT4 are mixed in the differentiated cells derived from embryonic stem cells. Therefore, in the development of a cell therapy agent, there is a risk of tumor formation due to undifferentiated pluripotent stem cells called " teratoma " Problems are constantly emerging (Stem Cells. 2009, 27 (5): 1050-1056.).
  • ECC Embryonic carcinoma cells
  • ESC embryonic stem cells
  • the present inventors have completed the present invention by confirming that the capturing derivative compounds can specifically and selectively remove OCT4 expressing cells by regulating the expression and activity of the stem cell transcription factor OCT4.
  • One object of the present invention is to provide a composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) comprising a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.
  • OCT4 Optamer-Binding Transcription Factor 4
  • Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or for removing undifferentiated cells comprising said composition; Or a composition for inhibiting the formation of a teratoma or a teratocarcinoma of a cell therapy agent.
  • Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or a pharmaceutical composition for preventing or treating testicular cancer, which comprises the composition.
  • Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or treating the composition with a cell therapeutic agent; Or a method of inhibiting teratoma or teratocarcinoma formation.
  • Another object of the present invention is to provide a compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof; Or a method for producing a cell therapeutic agent comprising the step of treating the composition with a cell therapeutic agent.
  • Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or a method for the prophylaxis or treatment of testicular cancer, comprising the step of administering the composition.
  • the compound of the present invention inhibits the expression or activity of OCT4 protein, thereby killing the undifferentiated cells remaining in the cell treatment agent, inhibiting the generation of teratoma or teratocarcinoma, and preventing or treating testicular cancer And can be usefully utilized as a pharmaceutical composition.
  • FIG. 1 is a Western blot analysis image showing whether or not the expression of OCT4 is inhibited in NCCIT testicular cancer cells treated with 2 ⁇ M, 5 ⁇ M and 10 ⁇ M of the compounds K53, K55, K56, K58 and K59, respectively.
  • OCT4 expression quantification was performed using LAS-2000 image analysis instrument.
  • FIG. 2 is a Western blot analysis image of an OCT4 protein expressed by treating compound K53 with NCCIT testicular cancer cells at different concentrations, using an OCT4-specific antibody.
  • FIG. 2 is a Western blot analysis image of an OCT4 protein expressed by treating compound K53 with NCCIT testicular cancer cells at different concentrations, using an OCT4-specific antibody.
  • FIG. 3 is a graph showing changes in mRNA and protein levels of genes NANOG and USP44, which are induced by OCT4 expression when compound K53 is treated to NCCIT testicular cancer cells at different concentrations.
  • FIG. 4 is a graph showing the inhibition of OCT4 expression and the degree of cleavage of PARP, a cell death marker, by treating NCCIT testicular cancer cells with 20 ⁇ M concentration of compound K53 at different times.
  • 5A is a graph showing a change in body weight of nude mouse regression model by intraperitoneal administration of compound K53 (30 mg / kg).
  • FIG. 5B is a graph showing the tumor size change of the nude mouse regression model by the intraperitoneal administration of the compound K53 according to the administration period.
  • 5c is an image of tumor of nude mouse regression model by intraperitoneal administration of compound K53.
  • 6A is a graph showing the number of cells induced when compound K53 was treated with two testicular cancer cell lines (NCCIT, Tera-1) and two normal cell lines (HFF, MCF10A).
  • FIG. 6B is a Western blot analysis image showing the degree of expression of OCT4 in two testicular cancer cell lines (NCCIT, Tera-1) and two normal cell lines (HFF, MCF10A) using an OCT4-specific antibody.
  • FIG. 7 shows the results of affinity chromatography of K53-Biotin (KRIBB53-Biotin) synthesized by the specific binding of OCT4 protein to the K53 compound.
  • FIG. 8 is an image of western blot analysis in which OCT4 expression of human induced pluripotent stem cells (iPSC) and neural stem cells (NSC) differentiated therefrom is analyzed using an OCT4 specific antibody.
  • iPSC human induced pluripotent stem cells
  • NSC neural stem cells
  • Induced pluripotent stem cells express OCT4, but the expression of OCT4 was inhibited in the neural stem cells differentiated therefrom.
  • FIG. 9A is a micrograph showing the cell morphology observed by treating human induced pluripotent stem cells (iPSC) and neural stem cells (NSC) with compounds K53, K55, K56, K58 or K59 at a concentration of 5 ⁇ M for 24 hours.
  • iPSC human induced pluripotent stem cells
  • NSC neural stem cells
  • FIG. 9B is a graph showing survival cell numbers of human induced pluripotent stem cells (iPSC) and neural stem cells (NSC) treated with the compound K53, K55, K56, K58 or K59 at a concentration of 5 ⁇ M for 24 hours.
  • iPSC human induced pluripotent stem cells
  • NSC neural stem cells
  • 10 is a graph showing the number of surviving cells after treating NCCIT testicular cancer cells with the compounds K53, K55, K56, K58 and K59 at a concentration of 10 ⁇ M and 30 ⁇ M for 48 hours, respectively.
  • composition for inhibiting OCT4 comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof:
  • R 1, R 2, R 3, R 4, R 5, R 1 ', R 2', R 3 ', R 4' and R 5 ' are each independently selected from the group consisting of H, hydroxy, C 1-4 alkoxy and halogen.
  • R1 is H, or hydroxy
  • R2 is H, or Ci- 4 alkoxy
  • R3 is H, hydroxy, or C 1-4 alkoxy
  • R4 is H, C 1-4 alkoxy, or halogen
  • R5 is H
  • R1 ' is H, hydroxy or C 1-4 alkoxy
  • R2 ' is H, or halogen
  • R3 ' is H, or C 1-4 alkoxy
  • R4 ' is H
  • R5 ' may be a H, hydroxy, or C 1-4 alkoxy, but is not limited thereto.
  • R1, R2, R3, R4, R5, R1 ', R2', R3 ', R4' and R5 ' are each independently selected from the group consisting of H, hydroxy, methoxy and bromo But are not limited thereto.
  • R1 is H, or hydroxy
  • R2 is H, or methoxy
  • R3 is H, hydroxy, or methoxy
  • R4 is H, methoxy, or bromo
  • R5 is H
  • R1 ' is H, hydroxy, or methoxy
  • R2 ' is H, or bromo
  • R3 ' is H, or methoxy
  • R4 ' is H
  • the compounds represented by Formula 1 according to the present invention include all pharmaceutically acceptable salts thereof as well as possible solvates and hydrates thereof which may be prepared therefrom and include all possible stereoisomers.
  • the solvates, hydrates and stereoisomers of the compound represented by the formula (1) can be prepared from the compound represented by the formula (1) using conventional methods.
  • the compound represented by the formula (1) according to the present invention may be prepared in a crystalline form or in an amorphous form, and may be optionally hydrated or solvated when prepared in crystalline form.
  • compounds containing various amounts of water as well as stoichiometric hydrates of the compound represented by the formula (1) may be included.
  • Solvates of the compounds of formula (I) according to the present invention include both stoichiometric solvates and non-stoichiometric solvates.
  • the method for obtaining the compound represented by Formula 1 according to the present invention is not particularly limited and may be chemically synthesized by a method known in the art, or a commercially available substance may be used.
  • the compound represented by the above-mentioned compound 1 or a pharmaceutically acceptable salt thereof may be used as an inhibitor of the expression or activity of OCT4 protein. Through such physiological activity, it is possible to remove undifferentiated cells, inhibit the formation of teratoma or teratocarcinoma, Can be usefully used as agents for the prophylaxis and treatment of testicular cancer.
  • the term "pharmaceutically acceptable salt” means a salt commonly used in the medical industry.
  • the salt include inorganic ion salts such as calcium, potassium, sodium and magnesium, hydrochloric acid, nitric acid, phosphoric acid,
  • inorganic acid salts prepared from acid, iodic acid, perchloric acid, tartaric acid and sulfuric acid and the like, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, acetic acid, trifluoroacetic acid, , Acetic acid, carbonic acid, vanillic acid, lactic acid, glycolic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid , Hydroiodic acid, etc., sulfonic acid salts such
  • OCT4 'Octamer-Binding Transcription Factor 4'
  • POU5F1 POU domain, class 5, transcription factor 1
  • the OCT4 is known to be an important factor for stem cell induction and maintenance of pluripotency of induced pluripotent stem cells.
  • " OCT4 inhibition " means decreasing or inhibiting the expression and / or activity of OCT4. Since OCT4 inhibition inhibits cell growth and induces apoptosis, it can prevent or treat cell proliferative diseases and can selectively kill undifferentiated cells. As a result, compounds inhibiting OCT4 can be used as a therapeutic agent for cell therapy, Can be used as the compound. In addition, the compound inhibiting OCT4 can inhibit the generation of teratoma or teratocarcinoma due to the administration of a cell therapy agent, effectively kills cancer cells expressing OCT4, and can be used as an excellent chemotherapeutic agent.
  • the compound represented by Formula 1 may be any compound selected from the following Formulas 2 to 6, but is not limited thereto.
  • the compound of Formula 2 is a compound having the formula C 18 H 18 O 6 , and the IUPAC designation is 2 ', 4-dihydroxy-3,4', 6'-4-dihydroxy- 3,4 ', 6'-trimethoxychalcone).
  • the compound of Formula 2 is a compound of Formula 1 wherein R3 and R5 'are hydroxy, R4, R1', and R3 'are methoxy and the remainder is hydrogen.
  • the compound was designated KRIBB53 (K53).
  • the compound of Formula 3 is a compound having the formula C 18 H 14 O 5 , and the IUPAC name is 2'-hydroxy-3,4,5-trimethoxychalcone ).
  • the compound of Formula 3 is reacted with KRIBB55 (K55 (R), R < 3 >, R & ).
  • the compound of formula 4 is a compound having the formula C 17 H 18 BrO 5 , the IUPAC designation is 5-bromo-2,2'-dihydroxy-4 ', 6'-dimethyl- 2'-dihydroxy-4 ', 6'-dimethoxychalcone).
  • the compound of Formula 4 is the compound of Formula 1 wherein R1 and R1 'are hydroxy, R3' and R5 'are methoxy, R4 is bromo and the remainder is hydrogen.
  • the compound of formula 4 was designated KRIBB56 (K56).
  • the compound of formula (5) is a compound of formula (1) wherein R 2 to R 4 are methoxy, R 2 'is bromo, R 5' is hydroxy and the remainder is hydrogen.
  • KRIBB58 K58
  • the compound of Formula 6 is a compound having the formula C 15 H 12 O 2 , and the IUPAC name corresponds to a 2-hydroxychalcone.
  • the compound of formula (6) is a compound of formula (1) wherein R1 is substituted by hydroxy and the remainder by hydrogen.
  • the compound of formula (6) is designated as KRIBB59 (K59).
  • the inhibition of OCT4 may be, but is not limited to, in vitro, in vivo, or ex vivo.
  • the compounds of the above formulas 2 to 6 of the present invention were treated with NCCIT cells (testicular cancer cell line) to inhibit the activity of OCT4 protein (Table 1) and inhibit the expression of OCT4 in a concentration- (FIGS. 1 and 2), and it was confirmed that the composition could be usefully used as a composition for inhibiting OCT4.
  • NCCIT cells testicular cancer cell line
  • the present invention relates to a compound of the above formula (1), a compound of any one of the above formulas (2) to (6), or a pharmaceutically acceptable salt thereof; Or a composition comprising them.
  • the present invention also provides a composition for removing undifferentiated cells.
  • the composition may be a composition for removing undifferentiated cells of a cell therapy agent, but is not limited thereto.
  • the present invention also relates to the aforementioned compound of formula 1, a compound of any one of the above formulas 2 to 6, or a pharmaceutically acceptable salt thereof; Or a composition comprising them, for inhibiting the formation of a teratoma or teratocarcinoma of a cell therapy agent.
  • cell therapy products refers to living cells used for cell therapy, and more specifically refers to living cells used for autologous, allogenic, xenogenic, ) Means a drug that contains cells that have been proliferated and selected in vitro.
  • the types of the cell therapy agents can be divided into somatic cell therapy agents and stem cell treatment agents depending on the degree of differentiation.
  • the stem cell treatment agents can be divided into embryonic stem cell treatment agents, induced pluripotent stem cell treatment agents and adult stem cell therapy agents, It does not.
  • the cell therapeutic agent may include, but is not limited to, undifferentiated cells.
  • the term " undifferentiated cell " as used herein refers to a cell that does not yet undergo differentiation and has room for differentiation, and includes embryonic stem cells, induced pluripotent stem cells, tumor cells, and cancer cells. More specifically, the undifferentiated cell may be an undifferentiated cell expressing OCT4.
  • 'teratoma' refers to a teratoma composed of various cells and tissues such as skin cells, muscle cells, nerve cells, etc., and a problem that teratoma may be generated due to undifferentiated cells contained in a cell therapy agent have.
  • 'teratocarcinoma means an embryonal carcinoma in which embryonal carcinoma and teratoma coexist, and also called teratocarcinoma.
  • the removal of the undifferentiated cells, inhibition of teratoma or teratocarcinoma formation may be accomplished in vitro, in vivo, or ex vivo, but is not limited thereto.
  • the compound represented by the formula (1) or the compound represented by any one of the formulas (2) to (6) of the present invention can inhibit the expression or activity of the OCT4 protein to remove undifferentiated cells contained in the cell therapeutic agent, which can inhibit teratoma or teratocarcinoma.
  • An embodiment of the present invention relates to a method for treating a cell treatment agent before administration to a subject and / or treating a composition comprising the same and / or a composition comprising the compound of the present invention To remove undifferentiated cells of the cell therapy agent or to inhibit the formation of the teratoma or teratocarcinoma.
  • the compound of the present invention specifically inhibits OCT4-expressing cells, and thus can be used not only for the removal of undifferentiated cells of cell therapy agents, but also for suppressing the formation of teratoma or teratocarcinoma of cell therapy agents .
  • Yet another aspect of the present invention relates to a method for preventing or treating cancer, comprising the step of administering to a patient a composition comprising the compound of formula 1, the compound of any one of formulas 2 to 6, or a pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition is provided.
  • cancer examples include, but are not limited to, liver cancer, colon cancer, breast cancer, stomach cancer, lung cancer, pancreatic cancer, thyroid cancer, uterine cancer, prostate cancer or testicular cancer.
  • &quot refers to any act that inhibits or delays the onset of cancer, specifically testicular cancer, by administration of a composition comprising a compound of the invention or a pharmaceutically acceptable salt thereof.
  • " treatment " as used herein refers to any action in which administration of a composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof improves or alleviates the symptoms of the disease.
  • the compound represented by the formula (1) or the compound represented by any one of the formulas (2) to (6) of the present invention specifically inhibits the cancer cell expressing OCT4 by inhibiting the expression or activity of the OCT4 protein, .
  • the pharmaceutical composition of the present invention may further include, but is not limited to, an anticancer agent (for example, an anticancer agent for testicular cancer and the like).
  • an anticancer agent for example, an anticancer agent for testicular cancer and the like.
  • the anticancer agent may be selected depending on the purpose of prevention or treatment.
  • the compound of the present invention is treated with a testicular cancer cell line to confirm the cancer cell apoptosis inducing effect of the compound of the present invention.
  • a testicular cancer cell line to confirm the cancer cell apoptosis inducing effect of the compound of the present invention.
  • the "pharmaceutical composition” may further comprise suitable carriers, excipients or diluents conventionally used in the production of pharmaceutical compositions.
  • the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .
  • the carrier, excipient and diluent which may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.
  • Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like.
  • excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have.
  • excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc.
  • water and liquid paraffin which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • suspending agent examples include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.
  • suppository base examples include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
  • the amount of the compound or pharmaceutically acceptable salt thereof contained in the pharmaceutical composition according to an embodiment of the present invention is not particularly limited, but is preferably 0.0001 to 50% by weight, more preferably 0.01 to 50% by weight, 5% by weight.
  • the pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount in the present invention means a therapeutically effective amount for treating or preventing a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention
  • the effective dose level refers to the amount of the effective amount of the composition of the present invention, and the effective dose level is determined depending on the severity of the disease, the activity of the drug, the age, body weight, health, sex, The duration of the treatment, the factors including the drugs used in combination or concurrently with the composition of the present invention used, and other factors well known in the medical field.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.
  • the dosage of the pharmaceutical composition of the present invention can be administered, for example, such that the composition containing the compound is administered to an individual at 10 to 1,000 mg / kg, more specifically, 10 to 600 mg / kg for a day,
  • the dosage of the composition of the present invention is not particularly limited, but may be administered once a day or divided into several doses.
  • the present invention provides a pharmaceutical composition comprising the compound of Formula 1, the compound of any one of Formulas 2 to 6, or a pharmaceutically acceptable salt thereof; Or a composition comprising the same, to a cell therapy agent; Or a method of inhibiting teratoma or teratocarcinoma formation.
  • the compounds or compositions of the present invention may be administered to a cell therapy agent in vitro, in vivo, or ex vivo.
  • An embodiment of the present invention relates to a method for treating a cell treatment agent before administration to a subject and / or treating a composition comprising the same and / or a composition comprising the compound of the present invention To remove undifferentiated cells of the cell therapy agent or to inhibit the formation of the teratoma or teratocarcinoma.
  • a pharmaceutical preparation for preventing or treating teratoma or teratocarcinoma comprising a compound of Formula 1, a compound of Formula 2 to 6, Or a pharmaceutically acceptable salt thereof.
  • the removal of the undifferentiated cells, inhibition of teratoma or teratocarcinoma formation may be accomplished in vitro, in vivo, or ex vivo, but is not limited thereto.
  • the compound of the present invention specifically inhibits OCT4-expressing cells, and thus can be used not only for the removal of undifferentiated cells of cell therapy agents, but also for suppressing the formation of teratoma or teratocarcinoma of cell therapy agents .
  • the present invention provides a pharmaceutical composition comprising the compound of Formula 1, the compound of any one of Formulas 2 to 6, or a pharmaceutically acceptable salt thereof; Or a composition comprising the same, to a cell treatment agent.
  • the production method may be a method for preparing a cell therapy agent in which undifferentiated cells are removed, or the formation of a teratoma or a teratocarcinoma is inhibited.
  • the compound of the present invention or the composition for inhibiting OCT4 can kill undifferentiated cells remaining in a cell therapeutic agent through inhibition of the expression or activity of OCT4 protein and inhibit the generation of teratoma or teratocarcinoma, Can be produced.
  • the present invention provides a pharmaceutical composition comprising the compound of Formula 1, the compound of any one of Formulas 2 to 6, or a pharmaceutically acceptable salt thereof; Or a composition comprising the same, to a subject.
  • the present invention provides a method of preventing or treating testicular cancer, comprising the step of administering the composition to a subject.
  • the compound of formula (1), the compound of any one of the above formulas (2) to (6), the pharmaceutically acceptable salt and the cancer are as described above.
  • the term "individual" may refer to any animal, including a human, who has, or is likely to develop, cancer, specifically, testicular cancer.
  • the animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,
  • the preventive or therapeutic method of the present invention may specifically include the step of administering the composition in a pharmaceutically effective amount to a subject who has developed or is at risk of developing cancer, specifically testicular cancer.
  • the compound of the present invention is treated with a testicular cancer cell line to confirm the cancer cell apoptosis inducing effect of the compound of the present invention.
  • a testicular cancer cell line to confirm the cancer cell apoptosis inducing effect of the compound of the present invention.
  • Example 1 The compounds K53, K55, K56, K58 and K59 OCT4 Identification of the activity inhibition effect
  • the OCT4-binding ORE (OCT4 Response Element) was prepared by the PCR method and inserted into the pTA-Luc plasmid (manufactured by Clontech) at the Korea Research Institute of Bioscience and Biotechnology to produce Firefly luciferase, [PORE-TA-Luc], which is a plasmid in which the expression of [pORE-TA-Luc] is increased.
  • the [pRL-TK] vector a plasmid that constantly expresses Renilla luciferase, was purchased from Promega.
  • [pORE-TA-Luc] and [pRL-TK] plasmids were simultaneously infected with a testicular cancer cell line (NCCIT, purchased from ATCC) using X-tremeGENE (Roche, USA).
  • Plasmid-introduced testicular cancer cells were removed from the cell culture dish using 0.05% trypsin-EDTA and 20,000 cells were inoculated in each well of a 96-well assay plate.
  • K56, K58, and K59 were cultured in DMSO (Sigma Chemical Co.) in a medium containing 10% FBS for 18 hours in a 37 ° C incubator containing 5% 1000 ⁇ M was added at a concentration of 10 ⁇ M and cultured in a 37 ° C. incubator for 24 hours.
  • the luminescence intensity due to the decomposition of the substrate was measured using a GloMax TM 96 Microplate Luminometer (Promega), and the measured value was calculated from the following formula (1)
  • the measured firefly luciferase activity which proportionally reflects the activity, is characterized by the nonspecific cytotoxicity and transfection that occurs in each experimental group using the activity of Renilla luciferase expressed by a constantly expressing promoter, The deviation of the efficiency was corrected).
  • the sample RLU represents [pORE-TA-Luc] + [pRL-TK] + [compound 1]
  • [PORE-TA-Luc] is a firefly luciferase expression plasmid having a promoter regulated by OCT4, and [pRL-TK] means a plasmid always constantly expressing Renilla luciferase .
  • NCCIT Compound concentration 10 ⁇ M Compound name Activity (%) (Luc activity) K53 76 K55 52 K56 99 K58 26 K59 99
  • the compounds K53, K55, K56, K58, and K59 inhibited OCT4 gene expression.
  • NCCIT cells were inoculated in a 6-well plate at 2.5 ⁇ 10 5 cells. After 18 hours, the compounds K53, K55, K56, K58 and K59 were treated with 2 ⁇ M, 5 ⁇ M and 10 ⁇ M, respectively, for 24 hours.
  • the treated cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the concentration of OCT4 and GAPDH protein in the recovered cell lysate was measured using Bradford reagent (Bio-Rad protein assay, USA).
  • NCCIT cells were inoculated in a 6-well plate at 2.5 ⁇ 10 5 cells. After 18 hours, the compound K53 was treated at a concentration of 3 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M and 30 ⁇ M for 12 hours.
  • the treated cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g, and the supernatant was collected. The concentration of OCT4 and GAPDH protein in the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
  • NANOG which is induced by OCT4 transcription factors, plays an important role in stem cell maintenance. Therefore, the change in mRNA expression level of NANOG and USP44 induced by OCT4 when compound K53 was treated was measured by the following experimental method, and the inhibitory effect of compound K53 on NANOG and USP44 mRNA expression was confirmed.
  • the 6-well plate was inoculated with 2.5 x 10 < 5 > cells of NCCIT cells. After 18 hours, the compound K53 was treated at a concentration of 3 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M and 30 ⁇ M for 12 hours.
  • the treated cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
  • PCR real - time reverse transcriptase chain reaction
  • PARP Poly (ADP-Ribose) polymerase
  • a 60 mm cell culture dish was inoculated with 3 ⁇ 10 5 NCCIT cells, and the compound K53 was treated at a concentration of 20 ⁇ M and cultured for the indicated time.
  • the treated cells were dissolved in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate) .
  • the cell lysate was centrifuged at 15,000 g to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
  • the cleaved PARP form is observed in proportion to the treatment time of the compound K53. Therefore, it can be seen that the compound K53 induces cell death of NCCIT expressing OCT4, exhibits excellent anticancer effect, and can effectively kill undifferentiated cells or teratomas.
  • DMA N, N-dimethylacetamide
  • Cremophore 80% distilled water / v / v
  • compound K53 was intraperitoneally administered at a daily dose of 30 mg / kg for 50 days.
  • the results of the last day (day 51) showed no weight loss in the compound K53 (30 mg / kg) group as compared with the solvent control group. Therefore, it was confirmed that the compound K53 is a safe substance which does not cause side effects in vivo.
  • the tumor size change by daily repeated intraperitoneal administration of compound K53 in the initiation model was observed for 51 days to confirm the anticancer effect.
  • the tumor volume was measured to be 387.8 ⁇ 66.2 mm 3 in the solvent-treated group and 90.6 ⁇ 26.2 mm 3 in the case of the compound K53, which was about 1/4 of the volume of the control group.
  • Tumor growth inhibition rate (%) [1- (Test group of the t / vehicle control group t)] x 100
  • the tumor size extracted from the compound K53-treated group was remarkably small, and the compound K53 showed excellent anti-cancer effect, undifferentiated cell inhibitory effect and teratoma inhibitory effect.
  • testicular cancer cells NCCIT and Tera-1 were analyzed.
  • NCCIT and Tera-1 human testicular cancer cells were administered to each well of a 96-well plate and the cells were cultured in a medium containing 10% FBS in a 37 ° C incubator containing 5% carbon dioxide. After 24 hours, the medium was replaced with a medium containing the control (0.1% DMSO) or the compound K53 at 10 ⁇ M and 30 ⁇ M (the compound dissolved in DMSO was diluted with the medium) and then cultured for 72 hours. The number of cells was measured using a hematocytometer after mixing 1: 1 of the triplan blue and cells, and the results are shown in FIG. 6A.
  • the compound K53 specifically suppresses only cancer cells and teratomas without affecting normal cells.
  • NCCIT, Tera-1, HFF and MCF10A cells were inoculated in a 60 mm cell culture dish at 3 ⁇ 10 5 cells and cultured.
  • Cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate).
  • the cell lysate was centrifuged at 15,000 g to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
  • KRIBB53-biotin was synthesized by dissolving N-biotinylcaproic acid, EDC and DMAP in a DMF solvent and reacting K53 (KRIBB53) compound. The KRIBB53-biotin was then purified using HPLC.
  • the NCCIT lysate was prepared using homogenizer buffer (60 mM ⁇ -glycerophosphate, 25 mM MOPS (pH 7.2), 15 mM EGTA, 1 mM DTT, 1 mM phenyl phosphate, 100 ⁇ M benzamidine). After the KRIBB53-biotin was treated with 20 ⁇ M, the K53 compound was treated at 0, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M and 500 ⁇ M concentrations in the solution. Then, NeutrAvidin-Agarose resin was added.
  • the cell lysates not bound to KRIBB53-biotin were diluted with bead buffer (50 mM Tris (pH 7.4), 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Nonidet P-40, 10 ⁇ g / ml of leupeptin, aprotinin, and soybean trypsin inhibitor, and 100 ⁇ M benzamidine), followed by treatment with SDS-sample buffer to recover binding proteins.
  • bead buffer 50 mM Tris (pH 7.4), 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Nonidet P-40, 10 ⁇ g / ml of leupeptin, aprotinin, and soybean trypsin inhibitor, and 100 ⁇ M benzamidine
  • the recovered proteins were separated by using 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on a PVDF membrane (Millipore, USA), TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl , 0.1% tween 20) containing 5% skim milk.
  • the cells were incubated with OCT4 antibody (Cell signaling, USA) for 2 hours, reacted with HRP-conjugated secondary antibody (Jackson Immunolab, USA) for 1 hour and detected with chemiluminescence POD reagent (Roche, Germany). The results are shown in Fig.
  • the pellet was loosened in RIPA (sigma # R0278-50ML) buffer and placed on ice for 1 hour to allow the protein to dissolve. After centrifugation at 16000g at 4 ° C for 20 minutes using a centrifuge, proteins were quantified by Bradford (Biorad # 500-0006) only after obtaining supernatant. 20 ⁇ g of which was loaded onto polyacrylamide gel (Biorad # 456-1083) and transferred to PVDF membrane (Biorad # 162-0177). After blocking for 1 hour at room temperature using 5% low fat dry milk (Biorad # 170-6404), OCT3 / 4 antibody (Santa Cruz # sc9081) was treated at a ratio of 1: 4000. The antibody reaction was carried out at 4 ° C for 16 hours.
  • RIPA sigma # R0278-50ML
  • anti-rabbit-HRP antibody (Santa Cruz # sc2004) was treated at a ratio of 1: 10000 for 1 hour at room temperature.
  • Beta-actin antibody (Sigma # A5441) for internal control was treated at a 1: 5000 ratio.
  • OCT4 size: 45 kDa expression was observed only in induced pluripotent stem cells at the protein level and not in neural stem cells.
  • the compound K53 inhibiting OCT4 affects undifferentiated cells such as inducible pluripotent stem cells or teratoma cells, but has no effect on neural stem cells.
  • Example 10 Compounds of the compounds K53, K55, K56, K58 and K59 Induced pluripotent stem cells Specific growth inhibitory effect
  • the cell counting was carried out as follows to confirm the growth inhibitory effect on the induced pluripotent stem cells.
  • Treffan blue and cells were mixed 1: 1, then Countess TM AutomatedCellCounter (Invitrogen) was used and the number of viable cells was compared under each condition treated with the drug. Data were analyzed using T - test to compare the conditions of drug treatment with DMSO.
  • the compounds K53, K55, K56, K58, and K59 exhibit a specific killing effect on cancer cells, OCT4 expressing cancer cells, undifferentiated cells, or teratoma cells, and thus exhibit excellent anticancer effects and inhibit undifferentiated cells or teratoma cell formation As shown in FIG.
  • Example 11 Compounds of the compounds K53, K55, K56, K58 and K59 OCT4 Specific growth inhibition effect on expression testicular cancer cells
  • testicular cancer cell line NCCIT In order to confirm whether compounds K53, K55, K56, K58, and K59 specifically inhibited the growth of OCT4 expressing testicular cancer cell line NCCIT, the proliferation of testicular cancer cells NCCIT was analyzed.
  • 2x10 5 NCCIT human testicular cancer cells were administered to each well of a 6-well plate and the cells were cultured in a medium containing 10% FBS in a 37 ° C incubator containing 5% carbon dioxide. After 24 hours, the medium was replaced with a control (0.1% DMSO) or a medium containing the compounds K53, K55, K56, K58 and K59 at 10 ⁇ M or 30 ⁇ M (the compound dissolved in DMSO was diluted with the medium) and then cultured for 48 hours. The number of cells was measured using a hematocytometer after mixing 1: 1 of the trypan blue and the cells, and the results are shown in FIG.
  • the compounds K53, K55, K56, K58, and K59 inhibited proliferation of NCCIT cells expressing OCT4, and showed inhibitory effects on the death of undifferentiated cells, formation of teratoma and teratocarcinoma And can also have the preventive and therapeutic effects of testicular cancer.

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Abstract

The present invention relates to a composition for inhibiting octamer-binding transcription factor 4 (OCT4) and a use thereof. Specifically, the present invention relates to a composition for removing undifferentiated cells or inhibiting formation of a teratoma or a teratocarcinoma of a cell therapy agent by using the composition for inhibiting OCT4; a pharmaceutical composition for preventing or treating testicular cancer; a method for removing undifferentiated cells; a method for inhibiting formation of teratoma or teratocarcinoma; a method for preparing a cellular therapeutic agent; and a method for preventing or treating testicular cancer.

Description

OCT4 저해용 조성물 및 이의 용도Compositions for inhibiting OCT4 and uses thereof
본 발명은 OCT4 (Octamer-Binding Transcription Factor 4) 저해용 조성물 및 이의 용도에 관한 것이다. 구체적으로 상기 조성물을 이용한, 미분화 세포 (undifferentiated cell) 제거용 또는 세포 치료제의 테라토마 (teratoma) 또는 테라토칼시노마 (teratocarcinoma) 형성 억제용 조성물; 고환암의 예방 또는 치료용 약학 조성물; 미분화 세포 제거 방법; 테라토마 또는 테라토칼시노마 형성 억제 방법; 세포치료제의 제조방법; 고환암의 예방 또는 치료방법에 관한 것이다.The present invention relates to a composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) and its use. A composition for inhibiting the formation of a teratoma or a teratocarcinoma of a cell therapeutic agent for removing undifferentiated cells using the composition; A pharmaceutical composition for preventing or treating testicular cancer; A method for removing undifferentiated cells; Methods of inhibiting teratoma or teratocarcinoma formation; A method for producing a cell therapeutic agent; To a method of preventing or treating testicular cancer.
인간 유도만능줄기세포는, 인체를 구성하는 내배엽, 중배엽, 및 외배엽으로 분화될 수 있는 전분화능(Pluripotency) 특성을 가지며, 자가재생(self-renewal)을 지속하는 세포로서, 재생의학(regenerative medicine)에 필요한 치료용 세포로 분화될 수 있다. 즉 유도만능줄기세포는 손상된 조직을 치료할 수 있는 신경세포, 간세포, 혈액세포, 등으로의 분화될 수 있다. 유도만능줄기세포는 2006년에 야마나카 박사가 4개의 전사인자(Oct4, Sox2, c-Myc, Klf4 transcription factors)를 성체세포에 도입하여 제조하였으며 (Cell. 126 (4): 663-76), 이로부터 유도만능줄기세포 제조를 위한 다양한 방법이 제안되었다. 일반적으로 OCT4 및 SOX2 전사인자는 성체세포의 줄기성 유도에 매우 중요한 인자이며, Myc 유전자는 줄기세포 제조의 효율을 향상시키는 역할을 한다. 특히 OCT4의 발현은 유도만능줄기세포의 전분화능 유지에 중요하다. Human induced pluripotent stem cells are pluripotency cells that can be differentiated into endoderm, mesoderm, and ectoderm that constitute the human body. They are cells that sustain self-renewal. They are regenerative medicine, Lt; RTI ID = 0.0 > cells. ≪ / RTI > In other words, induced pluripotent stem cells can differentiate into neurons, hepatocytes, blood cells, etc. that can treat damaged tissue. Induced pluripotent stem cells were produced by Yamanaka in 2006 by introducing four transcription factors (Oct4, Sox2, c-Myc and Klf4 transcription factors) into adult cells (Cell. 126 (4): 663-76) Various methods for the production of inducible pluripotent stem cells have been proposed. In general, OCT4 and SOX2 transcription factors are very important factors for the induction of stem cells in adult cells. Myc gene plays a role in improving the efficiency of stem cell production. In particular, the expression of OCT4 is important for maintaining the pluripotency of induced pluripotent stem cells.
OCT4를 발현하는 인간 유도만능줄기세포로부터 분화된 세포를 얻기 위해서는, 인간 유도만능줄기세포를 특정 치료용 세포로 분화를 유도하고, 분화된 세포를 특정하는 세포막에 존재하는 표지인자(surface makers)의 항체를 이용하여 유세포 분리기(Fluorescent Activated Cells Sorter : FACS)를 사용하여 표지인자를 발현하는 분화된 세포만을 얻는다. 다만, 유세포 분리기를 이용할 경우 사용되는 레이저에 의해 치료용 세포의 손상 위험이 있다. In order to obtain cells differentiated from human induced pluripotent stem cells expressing OCT4, it is necessary to induce the differentiation of human induced pluripotent stem cells into specific therapeutic cells, and to remove the surface makers present in the cell membrane that specify the differentiated cells Using antibodies, Fluorescent Activated Cells Sorter (FACS) is used to obtain only the differentiated cells expressing the marker. However, there is a risk of damaging the therapeutic cell by the laser used when the flow cytometer separator is used.
치료용 세포의 손상을 회피하기 위하여 세포막에 존재하는 표지인자에 대한 항체를 이용하여 세포를 표지 시킨 후 자성을 이용한 분리법(Magnetic Cell sorting: MACS)이 사용되고 있다. 자성을 이용한 분리법(MACS)은 유세포 분리기를 이용할 경우 사용되는 레이저에 대한 세포의 손상 위험을 제거할 수 있다는 장점을 가지고 있다. Magnetic cell sorting (MACS) has been used to label cells using antibodies against markers present in the cell membrane to avoid damage to therapeutic cells. The Magnetic Separation Method (MACS) has the advantage of eliminating the risk of cell damage to the laser used when using a flow cell separator.
그러나 FACS 또는 MACS 방법 모두 치료용 세포군에 미분화된 유도만능줄기세포가 소수로 포함될 가능성을 완벽하게 배제할 수 없다. 만약 치료용 세포로서 100%의 순도로 분화된 세포만을 선별하는 것에 기술적인 한계가 있다면 안전성에 심각한 문제가 발생할 수 있다. 특히, 배아줄기세포에서 유래한 분화세포에 OCT4를 발현하는 미분화 세포가 혼재되어 있을 가능성이 존재하여, 세포치료제 개발에 있어서 '테라토마 (teratoma)'라는 미분화 만능 줄기세포에서 유래한 종양형성 위험에 대한 문제점이 끊임없이 대두되고 있다 (Stem Cells. 2009, 27 (5): 1050-1056.). However, both FACS and MACS methods can not completely exclude the possibility that pluripotent pluripotent pluripotent stem cells are included in the therapeutic cell population. If there is a technical limitation in selecting only cells that have differentiated into 100% purity as therapeutic cells, serious safety problems may arise. Particularly, there is a possibility that undifferentiated cells expressing OCT4 are mixed in the differentiated cells derived from embryonic stem cells. Therefore, in the development of a cell therapy agent, there is a risk of tumor formation due to undifferentiated pluripotent stem cells called " teratoma " Problems are constantly emerging (Stem Cells. 2009, 27 (5): 1050-1056.).
그동안 많은 이종간 연구에서는 마우스의 줄기세포를 랫의 뇌에 접종하여 세포치료제 관련 연구를 보고하였으며, 이 경우에 테라토마 형성은 억제되었다. 그러나 동종간 연구에서는 마우스 유래 배아줄기세포의 경우, 단지 500개의 줄기세포를 마우스의 뇌조직에 접종하였을 때 모든 마우스에서 종양이 형성되었다 (J. CEREBRAL BLOOD FLOW MET, 2003, 23:780). 따라서 분화된 세포에는 영향이 없이, 테라토마의 위험성이 내재된 OCT4를 발현하는 미분화 세포를 선택적으로 제거하는 기술의 개발이 절실히 요구되고 있다.Many interspecies studies have reported on studies of cell therapy by inoculating mouse stem cells into the brain of rats, in which case teratoma formation was inhibited. However, in allogeneic studies, in mouse embryonic stem cells, tumors were formed in all mice when only 500 stem cells were inoculated into mouse brain tissue (J. CEREBRAL BLOOD FLOW MET, 2003, 23: 780). Therefore, there is a desperate need to develop a technique for selectively removing undifferentiated cells expressing OCT4 in which there is a risk of teratoma without affecting the differentiated cells.
한편, 종양 발병과 배아의 발생과정은 여러 특성들이 유사하다. 배아종양세포(ECC; embryonic carcinoma cells)은 기형암종 (teratocarcinoma)의 줄기세포를 말하며, 이것은 배아의 내세포집단(inner cell mass) 유래 배아줄기세포(ESC; embryonic stem cells)의 종양에 해당한다. 흥미로운 점은 이들 ECC 및 ESC가 표현형 유연성(plasticity)을 갖고 있어 특정 환경에서 분화를 한다는 점이다. 이제까지 ECC 및 ESC의 자기 재생(self-renewal) 및 다능성 (pluripotency)에 관여하는 중요한 전사인자들이 보고되었다 (Jung et al. 2010, PLoS ONE 5, e10709; Young, 2011, Cell, 144, 940-954.). On the other hand, the onset of the tumor and the development of the embryo have similar characteristics. Embryonic carcinoma cells (ECC) refers to stem cells of teratocarcinoma, which are tumors of embryonic stem cells (ESC) from the inner cell mass of the embryo. Interestingly, these ECCs and ESCs have phenotypic plasticity and thus differentiate in certain circumstances. So far, important transcription factors involved in self-renewal and pluripotency of ECC and ESC have been reported (Jung et al. 2010, PLoS ONE 5, e10709; Young, 2011, Cell, 144, 940- 954.).
본 발명자들은 촬콘 유도체 화합물들이 줄기성 전사인자 OCT4의 발현 및 활성을 조절하여, OCT4를 발현하는 세포를 특이적/선택적으로 제거할 수 있음을 확인함으로써 본 발명을 완성하였다.The present inventors have completed the present invention by confirming that the capturing derivative compounds can specifically and selectively remove OCT4 expressing cells by regulating the expression and activity of the stem cell transcription factor OCT4.
본 발명의 하나의 목적은, 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염을 포함하는, OCT4(Octamer-Binding Transcription Factor 4) 저해용 조성물을 제공하는 것이다.One object of the present invention is to provide a composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) comprising a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.
본 발명의 또 다른 하나의 목적은, 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염; 또는 상기 조성물을 포함하는, 미분화 세포 (undifferentiated cell) 제거용; 또는 세포 치료제의 테라토마 (teratoma) 또는 테라토칼시노마 (teratocarcinoma) 형성 억제용 조성물을 제공하는 것이다.Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or for removing undifferentiated cells comprising said composition; Or a composition for inhibiting the formation of a teratoma or a teratocarcinoma of a cell therapy agent.
본 발명의 또 다른 하나의 목적은, 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염; 또는 상기 조성물을 포함하는, 고환암의 예방 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or a pharmaceutical composition for preventing or treating testicular cancer, which comprises the composition.
본 발명의 또 다른 하나의 목적은, 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염; 또는 상기 조성물을 세포 치료제에 처리하는 단계를 포함하는 미분화 세포 제거; 또는 테라토마 또는 테라토칼시노마 형성 억제 방법을 제공하는 것이다.Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or treating the composition with a cell therapeutic agent; Or a method of inhibiting teratoma or teratocarcinoma formation.
본 발명의 또 다른 하나의 목적은, 상기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염; 또는 상기 조성물을 세포 치료제에 처리하는 단계를 포함하는 세포 치료제의 제조 방법을 제공하는 것이다.Another object of the present invention is to provide a compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof; Or a method for producing a cell therapeutic agent comprising the step of treating the composition with a cell therapeutic agent.
본 발명의 또 다른 하나의 목적은, 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염; 또는 상기 조성물을 투여하는 단계를 포함하는, 고환암의 예방 또는 치료방법을 제공하는 것이다.Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or a method for the prophylaxis or treatment of testicular cancer, comprising the step of administering the composition.
본 발명의 화합물은 OCT4 단백질의 발현 또는 활성을 저해하여, 이에 따라 세포 치료제에 잔존하는 미분화 세포를 사멸시킬 수 있고, 테라토마 또는 테라토칼시노마의 발생을 억제할 수 있으며, 고환암의 예방 또는 치료용 약학 조성물로서 유용하게 활용될 수 있다. The compound of the present invention inhibits the expression or activity of OCT4 protein, thereby killing the undifferentiated cells remaining in the cell treatment agent, inhibiting the generation of teratoma or teratocarcinoma, and preventing or treating testicular cancer And can be usefully utilized as a pharmaceutical composition.
도 1은 화합물 K53, K55, K56, K58, K59를 각각 2 μM, 5 μM 및 10 μM 처리한 NCCIT 고환암세포에서 OCT4의 발현이 저해되는지 여부를 확인한 웨스턴 블랏 분석 이미지이다. OCT4 발현 정량은 LAS-2000 이미지 분석기기를 사용하였다. 1 is a Western blot analysis image showing whether or not the expression of OCT4 is inhibited in NCCIT testicular cancer cells treated with 2 μM, 5 μM and 10 μM of the compounds K53, K55, K56, K58 and K59, respectively. OCT4 expression quantification was performed using LAS-2000 image analysis instrument.
도 2는 화합물 K53을 농도 별로 NCCIT 고환암세포에 처리하여 발현되는 OCT4 단백질을 OCT4 특이적 항체를 사용하여 분석한 웨스턴 블랏 분석이미지이다.FIG. 2 is a Western blot analysis image of an OCT4 protein expressed by treating compound K53 with NCCIT testicular cancer cells at different concentrations, using an OCT4-specific antibody. FIG.
도 3은 화합물 K53을 농도 별로 NCCIT 고환암세포에 처리할 경우 나타나는, OCT4에 의해 발현이 유도되는 유전자 NANOG 및 USP44의 mRNA 및 단백질 양의 변화를 분석한 도이다.FIG. 3 is a graph showing changes in mRNA and protein levels of genes NANOG and USP44, which are induced by OCT4 expression when compound K53 is treated to NCCIT testicular cancer cells at different concentrations.
도 4는 화합물 K53을 20 μM 농도로 시간 별로 NCCIT 고환암세포에 처리하여, OCT4 발현 저해 및 세포사멸 마커인 PARP의 절단 정도를 확인한 도이다. FIG. 4 is a graph showing the inhibition of OCT4 expression and the degree of cleavage of PARP, a cell death marker, by treating NCCIT testicular cancer cells with 20 μM concentration of compound K53 at different times.
도 5a은 화합물 K53 (30 mg/kg)의 복강투여에 의한 nude mouse regression model의 체중변화를 나타낸 도이다.5A is a graph showing a change in body weight of nude mouse regression model by intraperitoneal administration of compound K53 (30 mg / kg).
도 5b는 화합물 K53의 복강투여에 의한 nude mouse regression model의 종양크기 변화를 투여 기간에 따라 나타낸 도이다.FIG. 5B is a graph showing the tumor size change of the nude mouse regression model by the intraperitoneal administration of the compound K53 according to the administration period.
도 5c은 화합물 K53의 복강투여에 의한 nude mouse regression model의 종양의 이미지를 나타낸 도이다.5c is an image of tumor of nude mouse regression model by intraperitoneal administration of compound K53.
도 6a은 화합물 K53을 2종의 고환암 세포주(NCCIT, Tera-1) 및 2종의 정상 세포주(HFF, MCF10A)에 대한 처리하였을 때 유도되는 세포의 수를 나타낸 도이다.6A is a graph showing the number of cells induced when compound K53 was treated with two testicular cancer cell lines (NCCIT, Tera-1) and two normal cell lines (HFF, MCF10A).
도 6b은 2종의 고환암 세포주(NCCIT, Tera-1) 및 2종의 정상 세포주(HFF, MCF10A)에서 OCT4의 발현 정도를 OCT4 특이적 항체를 사용하여 분석한 웨스턴 블랏 분석 이미지이다.FIG. 6B is a Western blot analysis image showing the degree of expression of OCT4 in two testicular cancer cell lines (NCCIT, Tera-1) and two normal cell lines (HFF, MCF10A) using an OCT4-specific antibody.
도 7은 K53 화합물에 OCT4 단백질이 특이적으로 결합한다는 사실을, K53-Biotin (KRIBB53-Biotin) 화합물을 합성하여 친화 크로마토그래피 (affinity chromatography) 방법으로 증명한 결과이다.FIG. 7 shows the results of affinity chromatography of K53-Biotin (KRIBB53-Biotin) synthesized by the specific binding of OCT4 protein to the K53 compound.
도 8은 인간 유도만능줄기세포(iPSC) 및 이로부터 분화시킨 신경줄기세포(NSC)의 OCT4 발현여부를, OCT4 특이적 항체를 사용하여 분석한 웨스턴 블랏 분석이미지이다. 유도만능줄기세포는 OCT4를 발현하지만 이로부터 분화된 신경줄기세포의 경우 OCT4의 발현이 억제되었다.8 is an image of western blot analysis in which OCT4 expression of human induced pluripotent stem cells (iPSC) and neural stem cells (NSC) differentiated therefrom is analyzed using an OCT4 specific antibody. Induced pluripotent stem cells express OCT4, but the expression of OCT4 was inhibited in the neural stem cells differentiated therefrom.
도 9a은 인간 유도만능줄기세포(iPSC) 및 신경줄기세포(NSC)에 화합물 K53, K55, K56, K58 또는 K59를 5 μM 농도로 24시간 처리하여 세포 형태를 관찰한 현미경 사진이다.FIG. 9A is a micrograph showing the cell morphology observed by treating human induced pluripotent stem cells (iPSC) and neural stem cells (NSC) with compounds K53, K55, K56, K58 or K59 at a concentration of 5 μM for 24 hours.
도 9b는 인간 유도만능줄기세포(iPSC) 및 신경줄기세포(NSC)에 화합물 K53, K55, K56, K58 또는 K59를 5 μM 농도로 24시간 처리하여 생존 세포 수를 나타낸 그래프이다.FIG. 9B is a graph showing survival cell numbers of human induced pluripotent stem cells (iPSC) and neural stem cells (NSC) treated with the compound K53, K55, K56, K58 or K59 at a concentration of 5 μM for 24 hours.
도 10은 NCCIT 고환암세포에 화합물 K53, K55, K56, K58, K59를 각각 10 μM 및 30 μM 농도로 48시간 처리한 후 생존 세포의 수를 나타낸 그래프이다.10 is a graph showing the number of surviving cells after treating NCCIT testicular cancer cells with the compounds K53, K55, K56, K58 and K59 at a concentration of 10 μM and 30 μM for 48 hours, respectively.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.
상기 목적을 달성하기 위한 하나의 양태로서, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염을 포함하는, OCT4 (Octamer-Binding Transcription Factor 4) 저해용 조성물을 제공한다:According to one aspect of the present invention, there is provided a composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof:
[화학식 1][Chemical Formula 1]
Figure PCTKR2018008027-appb-I000001
Figure PCTKR2018008027-appb-I000001
상기 화학식 1에서In Formula 1,
R1, R2, R3, R4, R5, R1', R2', R3', R4' 및 R5'는 각각 독립적으로 H, 하이드록시, C1-4 알콕시, 및 할로겐으로 이루어진 군에서 선택됨. R 1, R 2, R 3, R 4, R 5, R 1 ', R 2', R 3 ', R 4' and R 5 'are each independently selected from the group consisting of H, hydroxy, C 1-4 alkoxy and halogen.
또한, 본 발명의 일 실시 양태에서, Further, in one embodiment of the present invention,
R1은 H, 또는 하이드록시이고; R1 is H, or hydroxy;
R2는 H, 또는 C1-4 알콕시이고; R2 is H, or Ci- 4 alkoxy;
R3는 H, 하이드록시, 또는 C1-4 알콕시이고; R3 is H, hydroxy, or C 1-4 alkoxy;
R4는 H, C1-4 알콕시, 또는 할로겐이고; R4 is H, C 1-4 alkoxy, or halogen;
R5는 H이고; R5 is H;
R1'는 H, 하이드록시 또는 C1-4 알콕시이고; R1 'is H, hydroxy or C 1-4 alkoxy;
R2'는 H, 또는 할로겐이고;R2 ' is H, or halogen;
R3'는 H, 또는 C1-4 알콕시이고; R3 'is H, or C 1-4 alkoxy;
R4'는 H이고;R4 ' is H;
R5'는 H, 하이드록시, 또는 C1-4 알콕시일 수 있으나, 이에 제한되지 않는다.R5 'may be a H, hydroxy, or C 1-4 alkoxy, but is not limited thereto.
또한, 본 발명의 일 실시 양태에서, 상기 R1, R2, R3, R4, R5, R1', R2', R3', R4' 및 R5'는 각각 독립적으로 H, 하이드록시, 메톡시, 및 브로모로 이루어진 군에서 선택될 수 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, R1, R2, R3, R4, R5, R1 ', R2', R3 ', R4' and R5 'are each independently selected from the group consisting of H, hydroxy, methoxy and bromo But are not limited thereto.
또한, 본 발명의 일 실시 양태에서, Further, in one embodiment of the present invention,
R1은 H, 또는 하이드록시이고; R1 is H, or hydroxy;
R2는 H, 또는 메톡시이고; R2 is H, or methoxy;
R3는 H, 하이드록시, 또는 메톡시이고; R3 is H, hydroxy, or methoxy;
R4는 H, 메톡시, 또는 브로모이고; R4 is H, methoxy, or bromo;
R5는 H; R5 is H;
R1'는 H, 하이드록시, 또는 메톡시이고; R1 'is H, hydroxy, or methoxy;
R2'는 H, 또는 브로모이고;R2 ' is H, or bromo;
R3'는 H, 또는 메톡시이고; R3 ' is H, or methoxy;
R4'는 H이고;R4 ' is H;
R5'는 H, 하이드록시, 또는 메톡시일 수 있으나, 이에 제한되지 않는다.R5 'can be H, hydroxy, or methoxy, but is not limited thereto.
본 발명에 따른 상기 화학식 1로 표시되는 화합물은, 이의 약학적으로 허용 가능한 염뿐만 아니라 이로부터 제조될 수 있는 가능한 용매화물 및 수화물을 모두 포함하고, 가능한 모든 입체이성체도 포함한다. 상기 화학식 1로 표시되는 화합물의 용매화물, 수화물 및 입체이성체는 통상적인 방법들을 사용하여 화학식 1로 표시되는 화합물로부터 제조할 수 있다.The compounds represented by Formula 1 according to the present invention include all pharmaceutically acceptable salts thereof as well as possible solvates and hydrates thereof which may be prepared therefrom and include all possible stereoisomers. The solvates, hydrates and stereoisomers of the compound represented by the formula (1) can be prepared from the compound represented by the formula (1) using conventional methods.
또한, 본 발명에 따른 상기 화학식 1로 표시되는 화합물 결정 형태 또는 비결정 형태로 제조될 수 있으며, 결정 형태로 제조될 경우 임의로 수화되거나 용매화될 수 있다. 본 발명에서는 상기 화학식 1로 표시되는 화합물의 화학양론적 수화물뿐만 아니라 다양한 양의 물을 함유하는 화합물이 포함될 수 있다. 본 발명에 따른 상기 화학식 1로 표시되는 화합물의 용매화물은 화학양론적 용매화물 및 비화학양론적 용매화물 모두를 포함한다.The compound represented by the formula (1) according to the present invention may be prepared in a crystalline form or in an amorphous form, and may be optionally hydrated or solvated when prepared in crystalline form. In the present invention, compounds containing various amounts of water as well as stoichiometric hydrates of the compound represented by the formula (1) may be included. Solvates of the compounds of formula (I) according to the present invention include both stoichiometric solvates and non-stoichiometric solvates.
본 발명에 따른 상기 화학식 1로 표시되는 화합물의 획득 방법은 특별히 한정되지 않으며, 당업계에 공지된 방법으로 화학적으로 합성하거나, 시판되는 물질을 사용할 수 있다.The method for obtaining the compound represented by Formula 1 according to the present invention is not particularly limited and may be chemically synthesized by a method known in the art, or a commercially available substance may be used.
상기 화합물 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염은 OCT4 단백질의 발현 또는 활성의 억제제로 활용될 수 있으며, 이러한 생리활성을 통하여 미분화 세포의 제거, 테라토마 또는 테라토칼시노마의 형성 억제, 고환암의 예방 및 치료용 제제로서 유용하게 사용될 수 있다.The compound represented by the above-mentioned compound 1 or a pharmaceutically acceptable salt thereof may be used as an inhibitor of the expression or activity of OCT4 protein. Through such physiological activity, it is possible to remove undifferentiated cells, inhibit the formation of teratoma or teratocarcinoma, Can be usefully used as agents for the prophylaxis and treatment of testicular cancer.
상기 화학식 1로 표시되는 화합물의 OCT4에 대한 특이적 저해 효과뿐만 아니라, 미분화 세포의 제거 효과, 테라토마 또는 테라토칼시노마에 대한 특이적인 형성 억제 효과, 고환암의 예방 및 치료 효과는 본 발명 이전에는 밝혀진 바 없는 새로운 효과이다. In addition to the specific inhibitory effect on the OCT4 of the compound represented by the above formula (1), the effect of removing undifferentiated cells, the specific inhibitory effect on the teratoma or teratocarcinoma, and the preventive and therapeutic effect of testicular cancer, It is a new effect without bar.
본 발명에서, '약제학적으로 허용 가능한 염'은 의약업계에서 통상적으로 사용되는 염을 의미하며, 예를 들어 칼슘, 칼륨, 나트륨 및 마그네슘 등으로 제조된 무기이온염, 염산, 질산, 인산, 브롬산, 요오드산, 과염소산, 주석산 및 황산 등으로 제조된 무기산염, 메탄설폰산, 에탄설폰산, 벤젠설폰산, p-톨루엔술폰산, 나프탈렌설폰산, 아세트산, 트리플루오로아세트산, 시트르산, 말레인산, 숙신산, 옥살산, 벤조산, 타르타르산, 푸마르산, 만데르산, 프로피온산, 구연산, 젖산, 글리콜산 , 글루콘산, 갈락투론산, 글루탐산, 글루타르산, 글루쿠론산, 아스파르트산, 아스코르브산, 카본산, 바닐릭산, 하이드로 아이오딕산 등으로 제조된 유기산염, 메탄설폰산, 에탄설폰산, 벤젠설폰산, p-톨루엔설폰산 및 나트탈렌설폰산 등으로 제조된 설폰산염, 글리신, 아르기닌, 라이신 등으로 제조된 아미노산염 및 트리메틸아민, 트리에틸아민, 암모니아, 피리딘, 피콜린 등으로 제조된 아민염 등이 있으나, 열거된 이들 염에 의해 본 발명에서 의미하는 염의 종류가 한정되는 것은 아니다.In the present invention, the term "pharmaceutically acceptable salt" means a salt commonly used in the medical industry. Examples of the salt include inorganic ion salts such as calcium, potassium, sodium and magnesium, hydrochloric acid, nitric acid, phosphoric acid, There may be mentioned inorganic acid salts prepared from acid, iodic acid, perchloric acid, tartaric acid and sulfuric acid and the like, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, acetic acid, trifluoroacetic acid, , Acetic acid, carbonic acid, vanillic acid, lactic acid, glycolic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid , Hydroiodic acid, etc., sulfonic acid salts such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and nattalenesulfonic acid, glycine, N, lysine and the like, and amine salts prepared with trimethylamine, triethylamine, ammonia, pyridine, picoline and the like. However, the types of salts defined in the present invention are limited by the listed salts no.
본 발명에서 'OCT4 (Octamer-Binding Transcription Factor 4)'는 POU5F1 (POU domain, class 5, transcription factor 1)로도 불리는 전사인자로서, 인간에서는 POU5F1 유전자에 의해 암호화 되는 단백질이다. 상기 OCT4는 성체세포의 줄기성 유도, 유도만능줄기세포의 다능성 (pluripotency) 유지에 중요한 인자로 알려져 있다.In the present invention, 'Octamer-Binding Transcription Factor 4' (OCT4) It is a transcription factor also called POU5F1 (POU domain, class 5, transcription factor 1), which is a protein encoded by the POU5F1 gene in humans. The OCT4 is known to be an important factor for stem cell induction and maintenance of pluripotency of induced pluripotent stem cells.
본 발명에서 “OCT4 저해”란 OCT4의 발현 및/또는 활성을 감소 또는 억제하는 것을 의미한다. OCT4 저해를 통하여 세포성장이 억제되고 세포 사멸이 유도되므로 세포 증식성 질환을 예방 또는 치료할 수 있으며, 미분화 세포를 선택적으로 사멸시킬 수 있는바, OCT4를 저해하는 화합물을 세포 치료 시 안전성(safety) 증진 화합물로 사용할 수 있다. 또한, OCT4를 저해하는 화합물은 세포 치료제 투여로 인한 테라토마 또는 테라토칼시노마의 발생을 억제할 수 있으며, OCT4를 발현하는 암 세포를 효과적으로 사멸시켜, 우수한 항암 치료제로 사용될 수 있다.In the present invention, " OCT4 inhibition " means decreasing or inhibiting the expression and / or activity of OCT4. Since OCT4 inhibition inhibits cell growth and induces apoptosis, it can prevent or treat cell proliferative diseases and can selectively kill undifferentiated cells. As a result, compounds inhibiting OCT4 can be used as a therapeutic agent for cell therapy, Can be used as the compound. In addition, the compound inhibiting OCT4 can inhibit the generation of teratoma or teratocarcinoma due to the administration of a cell therapy agent, effectively kills cancer cells expressing OCT4, and can be used as an excellent chemotherapeutic agent.
구체적으로, 상기 화학식 1로 표시되는 화합물은 하기 화학식 2 내지 6으로 이루어진 군에서 선택된 어느 하나의 화합물일 수 있으나, 이에 제한되지 않는다.Specifically, the compound represented by Formula 1 may be any compound selected from the following Formulas 2 to 6, but is not limited thereto.
[화학식 2](2)
Figure PCTKR2018008027-appb-I000002
Figure PCTKR2018008027-appb-I000002
상기 화학식 2의 화합물은 화학식 C18H18O6를 갖는 화합물로, IUPAC 명칭은 2',4-디하이드록시-3,4',6'-트리메톡시촬콘(2',4-dihydroxy-3,4',6'-trimthoxychalcone)에 해당한다. The compound of Formula 2 is a compound having the formula C 18 H 18 O 6 , and the IUPAC designation is 2 ', 4-dihydroxy-3,4', 6'-4-dihydroxy- 3,4 ', 6'-trimethoxychalcone).
상기 화학식 2의 화합물은 상기 화학식 1의 화합물에서 R3 및 R5'이 하이드록시로, R4, R1', 및 R3'이 메톡시로, 나머지가 수소로 치환된 화합물로서, 본 발명에서는 상기 화학식 2의 화합물을 KRIBB53 (K53)으로 명명하였다.The compound of Formula 2 is a compound of Formula 1 wherein R3 and R5 'are hydroxy, R4, R1', and R3 'are methoxy and the remainder is hydrogen. In the present invention, The compound was designated KRIBB53 (K53).
[화학식 3](3)
Figure PCTKR2018008027-appb-I000003
Figure PCTKR2018008027-appb-I000003
상기 화학식 3의 화합물은 화학식 C18H14O5을 갖는 화합물로, IUPAC 명칭은 2'-하이드록시-3,4,5-트리메톡시촬콘 (2'-hydroxy-3,4,5-trimethoxychalcone)에 해당한다. The compound of Formula 3 is a compound having the formula C 18 H 14 O 5 , and the IUPAC name is 2'-hydroxy-3,4,5-trimethoxychalcone ).
상기 화학식 3의 화합물은 상기 화학식 1의 화합물에서 R2, R3, R4가 메톡시로, R1'이 하이드록시로, 나머지가 수소로 치환된 화합물로서, 본 발명에서는 상기 화학식 3의 화합물을 KRIBB55 (K55)로 명명하였다.In the present invention, the compound of Formula 3 is reacted with KRIBB55 (K55 (R), R < 3 >, R & ).
[화학식 4][Chemical Formula 4]
Figure PCTKR2018008027-appb-I000004
Figure PCTKR2018008027-appb-I000004
상기 화학식 4의 화합물은 화학식 C17H18BrO5를 갖는 화합물로, IUPAC 명칭은 5-브로모-2,2'-디하이드록시-4',6'-디메틸촬콘(5-bromo-2,2'-dihydroxy-4',6'-dimethoxychalcone)에 해당한다. The compound of formula 4 is a compound having the formula C 17 H 18 BrO 5 , the IUPAC designation is 5-bromo-2,2'-dihydroxy-4 ', 6'-dimethyl- 2'-dihydroxy-4 ', 6'-dimethoxychalcone).
상기 화학식 4의 화합물은 상기 화학식 1의 화합물에서 R1 및 R1'이 하이드록시로, R3' 및 R5'이 메톡시로, R4가 브로모기로, 나머지가 수소로 치환된 화합물로서, 본 발명에서는 상기 화학식 4의 화합물을 KRIBB56 (K56)으로 명명하였다.The compound of Formula 4 is the compound of Formula 1 wherein R1 and R1 'are hydroxy, R3' and R5 'are methoxy, R4 is bromo and the remainder is hydrogen. In the present invention, The compound of formula 4 was designated KRIBB56 (K56).
[화학식 5][Chemical Formula 5]
Figure PCTKR2018008027-appb-I000005
Figure PCTKR2018008027-appb-I000005
상기 화학식 5의 화합물은 화학식 C18H17BrO5를 갖는 화합물로, IUPAC 명칭은 5'-브로모-2'-하이드록시-3,4,5-트리메틸촬콘(5'-bromo-2'-hydroxy-3,4,5-trimethoxychalcone)에 해당한다. The compound of formula 5 with a compound having the formula C 18 H 17 BrO 5, IUPAC designation 5'-bromo-2'-hydroxy -3,4,5-trimethyl chwalkon (5'-bromo-2'- hydroxy-3,4,5-trimethoxychalcone).
상기 화학식 5의 화합물은 상기 화학식 1의 화합물에서 R2 내지 R4가 메톡시로, R2'이 브로모로, R5'가 하이드록시로, 나머지가 수소로 치환된 화합물로서, 본 발명에서는 상기 화학식 5의 화합물을 KRIBB58 (K58)으로 명명하였다.The compound of formula (5) is a compound of formula (1) wherein R 2 to R 4 are methoxy, R 2 'is bromo, R 5' is hydroxy and the remainder is hydrogen. In the present invention, Was named KRIBB58 (K58).
[화학식 6][Chemical Formula 6]
Figure PCTKR2018008027-appb-I000006
Figure PCTKR2018008027-appb-I000006
상기 화학식 6의 화합물은 화학식 C15H12O2를 갖는 화합물로, IUPAC 명칭은 2-하이드록시촬콘 (2-hydroxychalcone)에 해당한다. The compound of Formula 6 is a compound having the formula C 15 H 12 O 2 , and the IUPAC name corresponds to a 2-hydroxychalcone.
상기 화학식 6의 화합물은 상기 화학식 1의 화합물에서 R1이 하이드록시로, 나머지가 수소로 치환된 화합물로서, 본 발명에서는 상기 화학식 6의 화합물을 KRIBB59 (K59)으로 명명하였다.The compound of formula (6) is a compound of formula (1) wherein R1 is substituted by hydroxy and the remainder by hydrogen. In the present invention, the compound of formula (6) is designated as KRIBB59 (K59).
상기 OCT4의 저해는 시험관 내 (in vitro), 생체 내 (in vivo), 또는 생체 외 (ex vivo)에서 이루어질 수 있으나, 이에 제한되지 않는다.The inhibition of OCT4 may be, but is not limited to, in vitro, in vivo, or ex vivo.
본 발명의 구체적인 일 실시예에서는, 본 발명의 상기 화학식 2 내지 6의 화합물을 NCCIT 세포 (고환암 세포주)에 처리한 결과 OCT4 단백질의 활성을 억제하고 (표 1), OCT4의 발현을 농도 의존적으로 억제함을 확인하여 (도 1 및 2), OCT4 저해용 조성물로서 유용하게 활용될 수 있음을 확인하였다.In one specific example of the present invention, the compounds of the above formulas 2 to 6 of the present invention were treated with NCCIT cells (testicular cancer cell line) to inhibit the activity of OCT4 protein (Table 1) and inhibit the expression of OCT4 in a concentration- (FIGS. 1 and 2), and it was confirmed that the composition could be usefully used as a composition for inhibiting OCT4.
본 발명은 상기 화학식 1의 화합물, 상기 화학식 2 내지 6 중 어느 하나의 화합물, 또는 이의 약제학적으로 허용 가능한 염; 또는 이들을 포함하는 조성물을 포함하는, 미분화 세포 (undifferentiated cell) 제거용 조성물을 제공한다. 구체적으로 상기 조성물은 세포 치료제의 미분화 세포 제거용 조성물일 수 있으나, 이에 제한되지 않는다.The present invention relates to a compound of the above formula (1), a compound of any one of the above formulas (2) to (6), or a pharmaceutically acceptable salt thereof; Or a composition comprising them. The present invention also provides a composition for removing undifferentiated cells. Specifically, the composition may be a composition for removing undifferentiated cells of a cell therapy agent, but is not limited thereto.
또한, 본 발명은 상기 화학식 1의 화합물, 상기 화학식 2 내지 6 중 어느 하나의 화합물, 또는 이의 약제학적으로 허용 가능한 염; 또는 이들을 포함하는 조성물을 포함하는, 세포 치료제의 테라토마 (teratoma) 또는 테라토칼시노마 (teratocarcinoma) 형성 억제용 조성물을 제공한다. The present invention also relates to the aforementioned compound of formula 1, a compound of any one of the above formulas 2 to 6, or a pharmaceutically acceptable salt thereof; Or a composition comprising them, for inhibiting the formation of a teratoma or teratocarcinoma of a cell therapy agent.
본 발명에서 '세포 치료제(cell therapy products)'란 세포 치료에 사용하는 살아있는 세포를 말하며, 보다 구체적으로 세포와 조직의 기능을 복원하기 위하여 살아 있는 자가(autologous), 동종(allogenic), 이종(xenogenic) 세포를 체외에서 증식, 선별한 세포를 포함하는 의약품을 의미한다. 상기 세포치료제의 종류로서, 분화 정도에 따라 체세포 치료제와, 줄기세포 치료제로 나눌 수 있으며, 상기 줄기세포 치료제는 배아줄기세포 치료제, 유도만능줄기세포 치료제, 성체줄기세포 치료제로 나눌 수 있으나, 이에 제한되지 않는다. 또한, 상기 세포 치료제는 미분화 세포를 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, "cell therapy products" refers to living cells used for cell therapy, and more specifically refers to living cells used for autologous, allogenic, xenogenic, ) Means a drug that contains cells that have been proliferated and selected in vitro. The types of the cell therapy agents can be divided into somatic cell therapy agents and stem cell treatment agents depending on the degree of differentiation. The stem cell treatment agents can be divided into embryonic stem cell treatment agents, induced pluripotent stem cell treatment agents and adult stem cell therapy agents, It does not. In addition, the cell therapeutic agent may include, but is not limited to, undifferentiated cells.
본 발명에서 '미분화 세포 (undifferentiated cell)'란 아직 분화가 끝나지 않고, 분화될 여지가 있는 세포를 의미하며, 배아줄기세포, 유도만능줄기세포, 종양 세포, 암 세포 등을 모두 포함한다. 보다 구체적으로, 상기 미분화 세포는 OCT4를 발현하는 미분화 세포일 수 있다.The term " undifferentiated cell " as used herein refers to a cell that does not yet undergo differentiation and has room for differentiation, and includes embryonic stem cells, induced pluripotent stem cells, tumor cells, and cancer cells. More specifically, the undifferentiated cell may be an undifferentiated cell expressing OCT4.
본 발명에서 '테라토마 (teratoma)'란, 피부세포, 근육세포, 신경세포 등 다양한 세포와 조직으로 이루어진 기형종을 의미하는 것으로, 세포 치료제에 포함된 미분화 세포로 인해 테라토마가 발생될 수 있는 문제가 있다. In the present invention, 'teratoma' refers to a teratoma composed of various cells and tissues such as skin cells, muscle cells, nerve cells, etc., and a problem that teratoma may be generated due to undifferentiated cells contained in a cell therapy agent have.
본 발명에서 '테라토칼시노마 (teratocarcinoma)'란 태생기암(embryonal carcinoma)와 테라토마가 혼재된 배아세포 종양을 의미하며, 기형암종으로도 명명된다.In the present invention, 'teratocarcinoma' means an embryonal carcinoma in which embryonal carcinoma and teratoma coexist, and also called teratocarcinoma.
상기 미분화 세포의 제거, 테라토마 또는 테라토칼시노마 형성의 억제는 시험관 내 (in vitro), 생체 내 (in vivo), 또는 생체 외 (ex vivo)에서 이루어질 수 있으나, 이에 제한되지 않는다.The removal of the undifferentiated cells, inhibition of teratoma or teratocarcinoma formation may be accomplished in vitro, in vivo, or ex vivo, but is not limited thereto.
본 발명의 화학식 1로 표시되는 화합물, 또는 화학식 2 내지 6 중 어느 하나의 화합물은 OCT4 단백질의 발현 또는 활성을 저해시킴으로써, 세포 치료제에 포함된 미분화 세포를 제거하거나, 세포 치료제의 투여로 인해 발생될 수 있는 테라토마 또는 테라토칼시노마를 억제할 수 있다.The compound represented by the formula (1) or the compound represented by any one of the formulas (2) to (6) of the present invention can inhibit the expression or activity of the OCT4 protein to remove undifferentiated cells contained in the cell therapeutic agent, Which can inhibit teratoma or teratocarcinoma.
본 발명의 일 실시 양태는 개체에 투여하기 전 세포 치료제에 본 발명의 화합물 또는 이를 포함하는 조성물을 처리하고/하거나, 세포 치료제가 투여되거나 또는 투여되기 전의 개체에 본 발명의 화합물 또는 이를 포함하는 조성물을 투여하여, 세포 치료제의 미분화 세포를 제거하거나, 테라토마 또는 테라토칼시노마의 형성을 억제할 수 있다.An embodiment of the present invention relates to a method for treating a cell treatment agent before administration to a subject and / or treating a composition comprising the same and / or a composition comprising the compound of the present invention To remove undifferentiated cells of the cell therapy agent or to inhibit the formation of the teratoma or teratocarcinoma.
본 발명의 구체적인 일 실시예에서는, OCT4의 단백질 수준의 발현이 유도만능줄기세포와 같은 미분화 세포에서만 관찰되며, 신경줄기세포에서는 관찰되지 않음을 확인하였으며 (도 8), 본 발명의 상기 화합물이 OCT4를 발현하는 미분화 세포인 유도만능줄기세포만을 선별적으로 억제함을 확인하였다.In a specific example of the present invention, it was confirmed that the expression of OCT4 protein level was observed only in undifferentiated cells such as inducible pluripotent stem cells and not observed in neural stem cells (FIG. 8) But also selectively induced pluripotent stem cells.
따라서, 본 발명의 상기 화합물은 OCT4를 발현하는 세포만을 특이적으로 억제하는바, 세포 치료제의 미분화 세포를 제거하는데 활용될 수 있을 뿐만 아니라, 세포 치료제의 테라토마 또는 테라토칼시노마 형성을 억제하는데 활용될 수 있다.Therefore, the compound of the present invention specifically inhibits OCT4-expressing cells, and thus can be used not only for the removal of undifferentiated cells of cell therapy agents, but also for suppressing the formation of teratoma or teratocarcinoma of cell therapy agents .
또한, 본 발명의 또 다른 하나의 양태는, 상기 화학식 1의 화합물, 상기 화학식 2 내지 6 중 어느 하나의 화합물, 또는 이의 약제학적으로 허용 가능한 염을 포함하는 조성물을 포함하는, 암의 예방 또는 치료용 약학 조성물을 제공한다.Yet another aspect of the present invention relates to a method for preventing or treating cancer, comprising the step of administering to a patient a composition comprising the compound of formula 1, the compound of any one of formulas 2 to 6, or a pharmaceutically acceptable salt thereof. A pharmaceutical composition is provided.
상기 암의 구체적인 예시로서, 간암, 대장암, 유방암, 위암, 폐암, 췌장암, 갑상선암, 자궁암, 전립선암, 또는 고환암 등이 있으며, 보다 구체적으로 고환암일 수 있으나, 이에 제한되지 않는다.Specific examples of the cancer include, but are not limited to, liver cancer, colon cancer, breast cancer, stomach cancer, lung cancer, pancreatic cancer, thyroid cancer, uterine cancer, prostate cancer or testicular cancer.
본 발명에서 사용된 용어 “예방”은, 본 발명의 화합물 또는 이의 약제학적으로 허용 가능한 염을 포함하는 조성물의 투여로 암, 구체적으로 고환암의 발병을 억제 또는 지연시키는 모든 행위를 말한다.The term " prophylactic, " as used herein, refers to any act that inhibits or delays the onset of cancer, specifically testicular cancer, by administration of a composition comprising a compound of the invention or a pharmaceutically acceptable salt thereof.
본 발명에서 사용된 용어 “치료”는, 본 발명의 화합물 또는 이의 약제학적으로 허용 가능한 염을 포함하는 조성물의 투여로 상기 질병의 증상이 호전되거나 이롭게 변경되는 모든 행위를 말한다.The term " treatment " as used herein refers to any action in which administration of a composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof improves or alleviates the symptoms of the disease.
본 발명의 화학식 1로 표시되는 화합물, 또는 화학식 2 내지 6 중 어느 하나의 화합물은 OCT4 단백질의 발현 또는 활성을 저해시킴으로써, OCT4를 발현하는 암세포만을 특이적으로 억제하여, 항암 제제로 유용하게 활용될 수 있다.The compound represented by the formula (1) or the compound represented by any one of the formulas (2) to (6) of the present invention specifically inhibits the cancer cell expressing OCT4 by inhibiting the expression or activity of the OCT4 protein, .
본 발명의 약학 조성물은 추가로 항암제(예들 들어, 고환암의 항암제 등)를 포함할 수 있으나, 이에 제한되지 않는다. 상기 항암제는 예방 또는 치료 목적에 따라 선택될 수 있다.The pharmaceutical composition of the present invention may further include, but is not limited to, an anticancer agent (for example, an anticancer agent for testicular cancer and the like). The anticancer agent may be selected depending on the purpose of prevention or treatment.
본 발명의 구체적인 일 실시예에서는, 본 발명의 상기 화합물의 암세포 세포사멸 유도 효과를 확인하기 위해 고환암 세포주에 본 발명의 상기 화합물을 처리한 결과, 생체 내에서 부작용을 유발하지 않으면서도 (도 5a) 종양의 부피 및 크기를 감소시키고 (도 5b 및 5c), 정상세포에는 영향을 미치지 않으면서도 암 세포만을 특이적으로 억제하여 (도 6a), 암의 예방 및 치료에 유용하게 사용될 수 있음을 확인하였다.In a specific embodiment of the present invention, the compound of the present invention is treated with a testicular cancer cell line to confirm the cancer cell apoptosis inducing effect of the compound of the present invention. As a result, (FIGS. 5B and 5C), it was confirmed that cancer cells can be specifically inhibited (FIG. 6A) without affecting normal cells, and thus can be useful for prevention and treatment of cancer .
본 발명에서 “약학 조성물”은, 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 구체적으로, 상기 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. In the present invention, the "pharmaceutical composition" may further comprise suitable carriers, excipients or diluents conventionally used in the production of pharmaceutical compositions. Specifically, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .
본 발명에서, 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. In the present invention, the carrier, excipient and diluent which may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 상엽 추출물과 이의 분획물들에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용된다. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는 데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 일 실시예에 따른 약학 조성물에 포함된 상기 화합물 또는 이의 약제학적으로 허용 가능한 염의 함량은 특별히 이에 제한되지 않으나, 최종 조성물 총 중량을 기준으로 0.0001 내지 50 중량%, 보다 바람직하게는 0.01 내지 5 중량%의 함량으로 포함할 수 있다. The amount of the compound or pharmaceutically acceptable salt thereof contained in the pharmaceutical composition according to an embodiment of the present invention is not particularly limited, but is preferably 0.0001 to 50% by weight, more preferably 0.01 to 50% by weight, 5% by weight.
상기 본 발명의 약학 조성물은 약학적으로 유효한 양으로 투여될 수 있는데, 본 발명에서 "약제학적으로 유효한 양"이란, 의학적 치료 또는 예방에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료 또는 예방하기에 충분한 양을 의미하며, 유효 용량 수준은 질환의 중증도, 약물의 활성, 개체의 연령, 체중, 건강, 성별, 개체의 약물에 대한 민감도, 사용된 본 발명의 조성물의 투여 시간, 투여 경로 및 배출 비율 치료기간, 사용된 본 발명의 조성물과 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하다.The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term " pharmaceutically effective amount " in the present invention means a therapeutically effective amount for treating or preventing a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention And the effective dose level refers to the amount of the effective amount of the composition of the present invention, and the effective dose level is determined depending on the severity of the disease, the activity of the drug, the age, body weight, health, sex, The duration of the treatment, the factors including the drugs used in combination or concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.
본 발명의 약학 조성물의 투여량은 예를 들어, 상기 화합물을 포함하는 조성물을 개체에 하루 동안 10 내지 1,000 mg/kg, 구체적으로는 10 내지 600 mg/kg으로 투여되도록 투여될 수 있고, 본 발명의 조성물의 투여빈도는 특별히 이에 제한되지 않으나, 1일 1회 투여하거나 또는 용량을 분할하여 수회 투여할 수 있다.The dosage of the pharmaceutical composition of the present invention can be administered, for example, such that the composition containing the compound is administered to an individual at 10 to 1,000 mg / kg, more specifically, 10 to 600 mg / kg for a day, The dosage of the composition of the present invention is not particularly limited, but may be administered once a day or divided into several doses.
본 발명의 또 다른 하나의 양태로서, 본 발명은 상기 화학식 1의 화합물, 상기 화학식 2 내지 6 중 어느 하나의 화합물, 또는 이의 약제학적으로 허용 가능한 염; 또는 이들을 포함하는 조성물을 세포 치료제에 처리하는 단계를 포함하는, 미분화 세포 제거; 또는 테라토마 또는 테라토칼시노마 형성 억제 방법을 제공한다.In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the compound of Formula 1, the compound of any one of Formulas 2 to 6, or a pharmaceutically acceptable salt thereof; Or a composition comprising the same, to a cell therapy agent; Or a method of inhibiting teratoma or teratocarcinoma formation.
상기 '화학식 1의 화합물', '상기 화학식 2 내지 6 중 어느 하나의 화합물', '약제학적으로 허용 가능한 염', '세포 치료제', '미분화 세포', '테라토마', 및 '테라토칼시노마'에 대해서는 상기 설명한 바와 같다.The term "compound of formula 1", "compound of any one of the above formulas 2 to 6", "pharmaceutically acceptable salt", "cell therapeutic agent", "undifferentiated cell" "teratoma" 'Have been described above.
본 발명의 화합물 또는 조성물은 세포 치료제에 시험관 내 (in vitro), 생체 내 (in vivo), 또는 생체 외 (ex vivo)에서 처리될 수 있다.The compounds or compositions of the present invention may be administered to a cell therapy agent in vitro, in vivo, or ex vivo.
본 발명의 일 실시 양태는 개체에 투여하기 전 세포 치료제에 본 발명의 화합물 또는 이를 포함하는 조성물을 처리하고/하거나, 세포 치료제가 투여되거나 또는 투여되기 전의 개체에 본 발명의 화합물 또는 이를 포함하는 조성물을 투여하여, 세포 치료제의 미분화 세포를 제거하거나, 테라토마 또는 테라토칼시노마의 형성을 억제할 수 있다.An embodiment of the present invention relates to a method for treating a cell treatment agent before administration to a subject and / or treating a composition comprising the same and / or a composition comprising the compound of the present invention To remove undifferentiated cells of the cell therapy agent or to inhibit the formation of the teratoma or teratocarcinoma.
또한, 본 발명의 또 다른 하나의 양태로서, 본 발명은 테라토마 또는 테라토칼시노마의 예방 또는 치료를 위한 약제학적 제제를 제조하는데 상기 화학식 1의 화합물, 상기 화학식 2 내지 6 중 어느 하나의 화합물, 또는 이의 약제학적으로 허용 가능한 염을 사용하는 용도를 제공한다.According to another aspect of the present invention, there is provided a pharmaceutical preparation for preventing or treating teratoma or teratocarcinoma, comprising a compound of Formula 1, a compound of Formula 2 to 6, Or a pharmaceutically acceptable salt thereof.
상기 '화학식 1의 화합물', '상기 화학식 2 내지 6 중 어느 하나의 화합물', '약제학적으로 허용 가능한 염', '세포 치료제', '미분화 세포', '테라토마', 및 '테라토칼시노마'에 대해서는 상기 설명한 바와 같다.The term "compound of formula 1", "compound of any one of the above formulas 2 to 6", "pharmaceutically acceptable salt", "cell therapeutic agent", "undifferentiated cell" "teratoma" 'Have been described above.
상기 미분화 세포의 제거, 테라토마 또는 테라토칼시노마 형성의 억제는 시험관 내 (in vitro), 생체 내 (in vivo), 또는 생체 외 (ex vivo)에서 이루어질 수 있으나, 이에 제한되지 않는다.The removal of the undifferentiated cells, inhibition of teratoma or teratocarcinoma formation may be accomplished in vitro, in vivo, or ex vivo, but is not limited thereto.
본 발명의 구체적인 일 실시예에서는, OCT4의 단백질 수준의 발현이 유도만능줄기세포와 같은 미분화 세포에서만 관찰되며, 신경줄기세포에서는 관찰되지 않음을 확인하였으며 (도 8), 본 발명의 상기 화합물이 OCT4를 발현하는 미분화 세포인 유도만능줄기세포만을 선별적으로 억제함을 확인하였다.In a specific example of the present invention, it was confirmed that the expression of OCT4 protein level was observed only in undifferentiated cells such as inducible pluripotent stem cells and not observed in neural stem cells (FIG. 8) But also selectively induced pluripotent stem cells.
따라서, 본 발명의 상기 화합물은 OCT4를 발현하는 세포만을 특이적으로 억제하는바, 세포 치료제의 미분화 세포를 제거하는데 활용될 수 있을 뿐만 아니라, 세포 치료제의 테라토마 또는 테라토칼시노마 형성을 억제하는데 활용될 수 있다.Therefore, the compound of the present invention specifically inhibits OCT4-expressing cells, and thus can be used not only for the removal of undifferentiated cells of cell therapy agents, but also for suppressing the formation of teratoma or teratocarcinoma of cell therapy agents .
본 발명의 또 다른 하나의 양태로서, 본 발명은 상기 화학식 1의 화합물, 상기 화학식 2 내지 6 중 어느 하나의 화합물, 또는 이의 약제학적으로 허용 가능한 염; 또는 이들을 포함하는 조성물을 세포 치료제에 처리하는 단계를 포함하는, 세포 치료제의 제조 방법을 제공한다. 구체적으로 상기 제조 방법은 미분화 세포가 제거되거나, 또는 테라토마 또는 테라토칼시노마의 형성이 억제된 세포 치료제의 제조 방법일 수 있다.In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the compound of Formula 1, the compound of any one of Formulas 2 to 6, or a pharmaceutically acceptable salt thereof; Or a composition comprising the same, to a cell treatment agent. Specifically, the production method may be a method for preparing a cell therapy agent in which undifferentiated cells are removed, or the formation of a teratoma or a teratocarcinoma is inhibited.
상기 '화학식 1의 화합물', '상기 화학식 2 내지 6 중 어느 하나의 화합물', '약제학적으로 허용 가능한 염', '세포 치료제', '미분화 세포', '테라토마', 및 '테라토칼시노마'에 대해서는 상기 설명한 바와 같다.The term "compound of formula 1", "compound of any one of the above formulas 2 to 6", "pharmaceutically acceptable salt", "cell therapeutic agent", "undifferentiated cell" "teratoma" 'Have been described above.
본 발명의 화합물 또는 OCT4 저해용 조성물은 OCT4 단백질의 발현 또는 활성 저해를 통해 세포 치료제에 잔존하는 미분화 세포를 사멸시킬 수 있어, 테라토마 또는 테라토칼시노마의 발생을 억제할 수 있는 바, 안전한 세포 치료제를 제조할 수 있다.The compound of the present invention or the composition for inhibiting OCT4 can kill undifferentiated cells remaining in a cell therapeutic agent through inhibition of the expression or activity of OCT4 protein and inhibit the generation of teratoma or teratocarcinoma, Can be produced.
본 발명의 또 다른 하나의 양태로서, 본 발명은 상기 화학식 1의 화합물, 상기 화학식 2 내지 6 중 어느 하나의 화합물, 또는 이의 약제학적으로 허용 가능한 염; 또는 이들을 포함하는 조성물을 개체에 투여하는 단계를 포함하는, 암의 예방 또는 치료방법을 제공한다.In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the compound of Formula 1, the compound of any one of Formulas 2 to 6, or a pharmaceutically acceptable salt thereof; Or a composition comprising the same, to a subject.
보다 구체적으로, 본 발명은 상기 조성물을 개체에 투여하는 단계를 포함하는, 고환암의 예방 또는 치료방법을 제공한다. More specifically, the present invention provides a method of preventing or treating testicular cancer, comprising the step of administering the composition to a subject.
상기 '화학식 1의 화합물', '상기 화학식 2 내지 6 중 어느 하나의 화합물', '약제학적으로 허용 가능한 염', '암'에 대해서는 상기 설명한 바와 같다.The compound of formula (1), the compound of any one of the above formulas (2) to (6), the pharmaceutically acceptable salt and the cancer are as described above.
본 발명에서 사용되는 용어, "개체"란, 암, 구체적으로 고환암이 발병되었거나 발병할 가능성이 있는 인간을 포함한 모든 동물을 의미할 수 있다. 상기 동물은 인간뿐만 아니라 이와 유사한 증상의 치료를 필요로 하는 소, 말, 양, 돼지, 염소, 낙타, 영양, 개, 고양이 등의 포유동물일 수 있으나, 이에 제한되지는 않는다.As used herein, the term " individual " may refer to any animal, including a human, who has, or is likely to develop, cancer, specifically, testicular cancer. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,
본 발명의 상기 예방 또는 치료 방법은 구체적으로, 암, 구체적으로 고환암이 발병하였거나 발병할 위험이 있는 개체에 상기 조성물을 약학적으로 유효한 양으로 투여하는 단계를 포함할 수 있다.The preventive or therapeutic method of the present invention may specifically include the step of administering the composition in a pharmaceutically effective amount to a subject who has developed or is at risk of developing cancer, specifically testicular cancer.
본 발명의 구체적인 일 실시예에서는, 본 발명의 상기 화합물의 암세포 세포사멸 유도 효과를 확인하기 위해 고환암 세포주에 본 발명의 상기 화합물을 처리한 결과, 생체 내에서 부작용을 유발하지 않으면서도 (도 5a) 종양의 부피 및 크기를 감소시키고 (도 5b 및 5c), 정상세포에는 영향을 미치지 않으면서도 암 세포만을 특이적으로 억제하여 (도 6a), 암의 예방 및 치료에 유용하게 사용될 수 있음을 확인하였다.In a specific embodiment of the present invention, the compound of the present invention is treated with a testicular cancer cell line to confirm the cancer cell apoptosis inducing effect of the compound of the present invention. As a result, (FIGS. 5B and 5C), it was confirmed that cancer cells can be specifically inhibited (FIG. 6A) without affecting normal cells, and thus can be useful for prevention and treatment of cancer .
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
<< 실시예Example 1> 화합물 K53, K55, K56, K58 및 K59의  1 > The compounds K53, K55, K56, K58 and K59 OCT4OCT4 활성 억제효과 확인 Identification of the activity inhibition effect
화합물 K53, K55, K56, K58, 및 K59의 OCT4 활성 억제효과를 확인하기 위해 다음과 같은 이중 루시퍼라아제법을 수행하였다. To confirm the inhibitory effect of the compounds K53, K55, K56, K58, and K59 on OCT4 activity, the following dual luciferase method was performed.
한국생명공학연구원에서 OCT4가 결합하는 염기서열(ORE; OCT4 Response Element)을 PCR 방법으로 제조하여 pTA-Luc 플라스미드(제조사: Clontech)에 삽입하여, OCT4의 활성에 비례하여 반딧불 루시퍼라제(Firefly luciferase)의 발현이 증가하는 플라스미드인 [pORE-TA-Luc]를 제조하였다. OCT4의 활성과 무관하게, 레닐라 루시퍼라제(Renilla luciferase)를 항상 일정하게 발현하는 플라스미드인 [pRL-TK] 벡터를 Promega 사에서 구입하여 사용하였다. [pORE-TA-Luc]와 [pRL-TK] 플라스미드를 X-tremeGENE(Roche, USA)를 이용하여 고환암 세포주(NCCIT, 구입처: ATCC에서 구매)에 동시에 감염시켰다. The OCT4-binding ORE (OCT4 Response Element) was prepared by the PCR method and inserted into the pTA-Luc plasmid (manufactured by Clontech) at the Korea Research Institute of Bioscience and Biotechnology to produce Firefly luciferase, [PORE-TA-Luc], which is a plasmid in which the expression of [pORE-TA-Luc] is increased. Regardless of the activity of OCT4, the [pRL-TK] vector, a plasmid that constantly expresses Renilla luciferase, was purchased from Promega. [pORE-TA-Luc] and [pRL-TK] plasmids were simultaneously infected with a testicular cancer cell line (NCCIT, purchased from ATCC) using X-tremeGENE (Roche, USA).
0.05% 트립신-EDTA(trypsin-EDTA)를 이용하여 플라스미드들이 도입된 고환암 세포를 세포배양 접시로부터 떼어내고, 96-웰 검정 플레이트의 각각의 웰(well)에 20,000개의 세포를 접종하였다. 10% FBS가 포함된 배지에서 5% 이산화탄소를 포함하는 37℃ 배양기에 18시간 동안 배양한 후, 대조군[1% DMSO(Dimethylsulfoxide)]과, 화합물 K53, K55, K56, K58, 및 K59를 DMSO에 1000 μM로 녹인 것을 10μM의 농도로 첨가하고 24시간 동안 37℃배양기에서 배양하였다. 반딧불 루시퍼라제(Firefly luciferase)와 레닐라 루시퍼라제(Renilla luciferase)에 특이적인 별도의 기질 Beetle luciferin(구입처:Promega)과 coelenterazine(구입처:Promega)을 순차적으로 25 ㎕씩 첨가한 후 측정하였다. Plasmid-introduced testicular cancer cells were removed from the cell culture dish using 0.05% trypsin-EDTA and 20,000 cells were inoculated in each well of a 96-well assay plate. K56, K58, and K59 were cultured in DMSO (Sigma Chemical Co.) in a medium containing 10% FBS for 18 hours in a 37 ° C incubator containing 5% 1000 μM was added at a concentration of 10 μM and cultured in a 37 ° C. incubator for 24 hours. A separate substrate, Beetle luciferin (Promega) and coelenterazine (Promega), specific for Firefly luciferase and Renilla luciferase, were added to each well in an amount of 25 μl.
기질의 분해에 의한 발광 강도는 GloMaxTM 96 Microplate Luminometer (Promega)를 이용하여 측정하였으며, 그 측정값을 하기 수학식 1을 통해 활성저해도(%)를 계산하여 하기 표 1에 나타내었다 (OCT4의 활성을 비례적으로 반영하는 측정된 반딧불 루시퍼라제 활성은, 항상 일정하게 발현하는 프로모터에 의해 발현되는 레닐라 루시퍼라제 활성값을 사용하여 각각의 실험군에서 발생하는 비특이적인 세포독성 및 형질주입(transfection) 효율의 편차를 보정하였다). The luminescence intensity due to the decomposition of the substrate was measured using a GloMax TM 96 Microplate Luminometer (Promega), and the measured value was calculated from the following formula (1) The measured firefly luciferase activity, which proportionally reflects the activity, is characterized by the nonspecific cytotoxicity and transfection that occurs in each experimental group using the activity of Renilla luciferase expressed by a constantly expressing promoter, The deviation of the efficiency was corrected).
[수학식 1][Equation 1]
Figure PCTKR2018008027-appb-I000007
Figure PCTKR2018008027-appb-I000007
상기 식에서, 대조구 RLU는 [pORE-TA-Luc] + [pRL-TK] + [DMSO]를 의미하며, 시료구 RLU는 [pORE-TA-Luc] + [pRL-TK] + [화합물 1]을 의미하며, [pORE-TA-Luc]는 OCT4에 의해 조절되는 프로모터를 갖고 있는 반딧불 루시퍼레이즈 발현 플라스미드이고, [pRL-TK]는 레닐라 루시퍼라제(Renilla luciferase)를 항상 일정하게 발현하는 플라스미드을 의미한다.[PORE-TA-Luc] + [pRL-TK] + [DMSO], and the sample RLU represents [pORE-TA-Luc] + [pRL-TK] + [compound 1] , [PORE-TA-Luc] is a firefly luciferase expression plasmid having a promoter regulated by OCT4, and [pRL-TK] means a plasmid always constantly expressing Renilla luciferase .
NCCITNCCIT
화합물 농도: 10 μMCompound concentration: 10 μM
화합물명Compound name 활성저해도(%)(Luc activity)Activity (%) (Luc activity)
K53K53 7676
K55K55 5252
K56 K56 9999
K58K58 2626
K59 K59 9999
그 결과, 표 1에서 볼 수 있듯이, 화합물 K53, K55, K56 및 K59는 루시퍼라제의 활성을 50% 이상 저해하여, OCT4 활성을 억제하는 효과를 나타냄을 확인하였다. 특히, K56, K59는 거의 100%에 가까운 활성 저해도를 나타내어, OCT4 활성 저해 효과가 더욱 우수함을 알 수 있다.As a result, as shown in Table 1, it was confirmed that the compounds K53, K55, K56, and K59 inhibited the activity of luciferase by 50% or more, thereby inhibiting OCT4 activity. In particular, K56 and K59 exhibited almost 100% active inhibition, indicating that the OCT4 activity inhibiting effect is more excellent.
<< 실시예Example 2> 화합물 K53, K55, K56, K58 및 K59의  2> Compounds of the compounds K53, K55, K56, K58 and K59 OCT4OCT4 발현 억제효과 확인 Confirming expression suppression effect
OCT4 활성 억제효과에 더하여, 화합물 K53, K55, K56, K58, 및 K59의 OCT4 유전자 발현 억제효과를 확인하고자 하였다.In addition to the OCT4 activity inhibitory effect, the compounds K53, K55, K56, K58, and K59 inhibited OCT4 gene expression.
먼저, 6-웰 플레이트에 NCCIT 세포를 2.5×105개로 접종하였다. 18시간 후 화합물 K53, K55, K56, K58 및 K59를 각각 2μM, 5μM, 10μM 농도로 처리하여 24시간 동안 배양하였다. 화합물이 처리된 세포를 RIPA buffer (50mM Tris-HCl, pH7.4, 150mM NaCl, 5mM EDTA, 1mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate)로 용해시켰다. 세포 용해액을 15,000g로 원심 분리하여 상층 세포액을 회수하였으며, 브래드포드 시약 (Bradford reagent) (Bio-Rad protein assay, USA)을 이용하여 회수한 세포액의 OCT4 및 GAPDH 단백질 농도를 측정하였다.First, NCCIT cells were inoculated in a 6-well plate at 2.5 × 10 5 cells. After 18 hours, the compounds K53, K55, K56, K58 and K59 were treated with 2 μM, 5 μM and 10 μM, respectively, for 24 hours. The treated cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the concentration of OCT4 and GAPDH protein in the recovered cell lysate was measured using Bradford reagent (Bio-Rad protein assay, USA).
또한 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis)를 이용하여 15 μg의 세포 용해액을 단백질 분리한 후, PVDF 막 (Millipore, USA)으로 단백질들을 전기 이동하고 TBST (50mM Tris-HCl, pH7.6, 150mM NaCl, 0.1% tween 20)에 녹인 5% skim milk를 이용하여 블로킹(blocking) 하였다. OCT4 항체(Cell signaling, USA), GAPDH 항체(Santacruz, USA)로 2시간 동안 반응시키고 HRP가 conjugation된 2차 항체 (Jackson Immunolab, USA)와 30분 동안 반응 시킨 뒤, 화학발광 (chemiluminescence) POD 시약 (Roche, Germany)을 사용하여 OCT4 및 GAPDH 발현량을 검출하였다. After 15 μg of the cell lysate was separated by 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on a PVDF membrane (Millipore, USA) and TBST (50 mM Tris-HCl, 6, 150 mM NaCl, 0.1% tween 20). The cells were reacted with OCT4 antibody (Cell signaling, USA) and GAPDH antibody (Santacruz, USA) for 2 hours and reacted with HRP-conjugated secondary antibody (Jackson Immunolab, USA) for 30 minutes. Then, chemiluminescence POD reagent (Roche, Germany) were used to detect OCT4 and GAPDH expression levels.
그 결과, 도 1에서 알 수 있듯이, 화합물 K53, K55, K56, K58 및 K59는 농도 의존적으로 OCT4의 발현을 억제하는 활성을 나타냄을 확인하였다.As a result, as shown in FIG. 1, it was confirmed that the compounds K53, K55, K56, K58 and K59 showed an activity of inhibiting the expression of OCT4 in a concentration-dependent manner.
<< 실시예Example 3> 화합물 K53의 농도 비례적인  3> Concentration of Compound K53 OCT4OCT4 발현 억제효과 확인 Confirming expression suppression effect
다음으로, 화합물 K53이 농도 의존적으로 OCT4의 발현을 억제하는지 여부를 구체적으로 확인하고자 하였다. Next, it was tried to specifically confirm whether compound K53 inhibits OCT4 expression in a concentration-dependent manner.
먼저, 6-웰 플레이트에 NCCIT 세포를 2.5×105개로 접종하였다. 18시간 후 화합물 K53을 3 μM, 5 μM, 10 μM, 20 μM, 30 μM 농도로 처리하여 12시간 동안 배양하였다. 화합물이 처리된 세포를 RIPA buffer (50mM Tris-HCl, pH7.4, 150mM NaCl, 5mM EDTA, 1mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate)으로 용해시켰다. 세포 용해액을 15,000g로 원심 분리하여 상층 세포액을 회수하였으며, Bradford reagent (Bio-Rad protein assay, USA)를 이용하여 회수한 세포액의 OCT4 및 GAPDH 단백질 농도를 측정하였다.First, NCCIT cells were inoculated in a 6-well plate at 2.5 × 10 5 cells. After 18 hours, the compound K53 was treated at a concentration of 3 μM, 5 μM, 10 μM, 20 μM and 30 μM for 12 hours. The treated cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g, and the supernatant was collected. The concentration of OCT4 and GAPDH protein in the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
또한, 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis)를 이용하여 15 μg의 세포 용해액을 단백질 분리한 후, PVDF 막 (Millipore, USA)으로 단백질들을 전기 이동하고 TBST (50mM Tris-HCl, pH7.6, 150mM NaCl, 0.1% tween 20)에 녹인 5% skim milk를 이용하여 블로킹 하였다. OCT4 항체(Cell signaling, USA), GAPDH 항체(Santacruz, USA)로 2시간 동안 반응시키고 HRP가 conjugation된 2차 항체 (Jackson Immunolab, USA)와 30분 동안 반응 시킨 뒤, chemiluminescence POD 시약 (Roche, Germany)을 사용하여 OCT4 및 GAPDH 발현량을 검출하였다. After 15 μg of the cell lysate was separated by 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on PVDF membrane (Millipore, USA) and TBST (50 mM Tris- 6, 150 mM NaCl, 0.1% tween 20). After incubation with OCT4 antibody (Cell signaling, USA) and GAPDH antibody (Santacruz, USA) for 2 hours and reaction with HRP conjugated secondary antibody (Jackson Immunolab, USA) for 30 minutes, chemiluminescence POD reagent ) Were used to detect OCT4 and GAPDH expression levels.
그 결과, 도 2에서 알 수 있듯이, 화합물 K53는 농도 의존적으로 OCT4의 발현을 억제할 수 있음을 확인하였다.As a result, as shown in FIG. 2, it was confirmed that the compound K53 was able to inhibit the expression of OCT4 in a concentration-dependent manner.
<< 실시예Example 4> 화합물 K53의,  4> of compound K53, OCT4OCT4 다운스트림Downstream 유전자인  Gene NANOGNANOG 및  And USP44의USP44 발현 억제효과 확인 Confirming expression suppression effect
OCT4 전사인자에 의해서 발현이 유도되는 NANOG는 세포의 줄기성 유지에 중요한 역할을 한다. 따라서, 화합물 K53을 처리하였을 때 OCT4에 의해 유도되는 NANOG 및 USP44의 mRNA 발현량 변화를 다음과 같은 실험방법을 통해 측정하여, 화합물 K53의 NANOG 및 USP44의 mRNA 발현 억제효과를 확인하였다. NANOG, which is induced by OCT4 transcription factors, plays an important role in stem cell maintenance. Therefore, the change in mRNA expression level of NANOG and USP44 induced by OCT4 when compound K53 was treated was measured by the following experimental method, and the inhibitory effect of compound K53 on NANOG and USP44 mRNA expression was confirmed.
6-웰 플레이트에 NCCIT 세포를 2.5×105개로 접종하였다. 18시간 후 화합물 K53을 3 μM, 5 μM, 10 μM, 20 μM, 30 μM 농도로 처리하여 12시간 동안 배양하였다. 화합물이 처리된 세포를 RIPA buffer (50mM Tris-HCl, pH7.4, 150mM NaCl, 5mM EDTA, 1mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate)으로 용해시켰다. 세포 용해액을 15,000g로 원심 분리하여 상층 세포액을 회수하였으며, Bradford reagent (Bio-Rad protein assay, USA)를 이용하여 회수한 세포액의 단백질 농도를 측정하였다.The 6-well plate was inoculated with 2.5 x 10 &lt; 5 &gt; cells of NCCIT cells. After 18 hours, the compound K53 was treated at a concentration of 3 μM, 5 μM, 10 μM, 20 μM and 30 μM for 12 hours. The treated cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
또한, 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis)를 이용하여 15 μg의 세포 용해액을 단백질 분리한 후, PVDF 막 (Millipore, USA)으로 단백질들을 전기 이동하고 TBST (50mM Tris-HCl, pH7.6, 150mM NaCl, 0.1% tween 20)에 녹인 5% skim milk를 이용하여 블로킹 하였다. NANOG 항체(R&D systems, USA), USP44 항체 (Santacruz, USA), GAPDH 항체(Santacruz, USA)로 2시간 동안 반응시키고 HRP가 conjugation된 2차 항체 (Jackson Immunolab, USA)와 30분 동안 반응 시킨 뒤, chemiluminescence POD 시약 (Roche, Germany)을 사용하여 검출하였다. After 15 μg of the cell lysate was separated by 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on PVDF membrane (Millipore, USA) and TBST (50 mM Tris- 6, 150 mM NaCl, 0.1% tween 20). (Jackson Immunolab, USA) for 2 min. The reaction was then incubated with NANOG antibody (R & D systems, USA), USP44 antibody (Santacruz, USA) and GAPDH antibody (Santacruz, , and chemiluminescence POD reagent (Roche, Germany).
RNA는 TRIzol(Invitrogen)을 통해 수집하였으며, 얻어진 RNA에서 2㎍ 정도를 취하여 RevertAid first strand cDNA synthesis kit (Fermentas)을 이용하여 RNA에 상보적인 DNA를 합성하였다. 이 상보적인 DNA를 주형으로 하여 첨가한 IQ™ SYBR green supermix (BioRad)의 형광 발색 정도로 NANOG의 mRNA 발현량을 실시간 역전사 효소 연쇄 중합 반응을 통해 확인하였다. RNA was collected from TRIzol (Invitrogen). Approximately 2 μg of the obtained RNA was collected and the complementary DNA was synthesized using RevertAid first strand cDNA synthesis kit (Fermentas). The amount of mRNA expression of NANOG was confirmed by real - time reverse transcriptase chain reaction (PCR) using the fluorescence intensity of IQ ™ SYBR green supermix (BioRad) supplemented with this complementary DNA template.
도 3에서 알 수 있듯이, 화합물 K53을 12시간 처리하였을 때 K53 화합물의 농도가 증가함에 따라 NANOG의 mRNA 발현량이 감소하는 것을 확인하였다. 또한, 화합물 K53의 처리 농도가 증가함에 따라 USP44의 mRNA 발현량 또한 감소하는 것을 확인하였다.As can be seen from FIG. 3, when the compound K53 was treated for 12 hours, the amount of NANOG mRNA expression was decreased as the concentration of the K53 compound was increased. In addition, it was confirmed that the amount of mRNA expression of USP44 was also decreased as the treatment concentration of compound K53 was increased.
<< 실시예Example 5> 화합물 K53의 암세포 세포사멸 유도효과 확인 5> Confirmation of induction of cancer cell death by compound K53
poly (ADP-Ribose) polymerase[이하, PARP]은 세포 사멸 유도를 확인 할 수 있는 마커로, 외부 자극에 대해 유전자의 안정성을 유지할 수 있게 해주는 대표적인 세포 내의 수리 시스템이다. 세포 사멸이 일어나게 되면 PARP가 잘리게 되고 이러한 PARP의 절단은 세포사멸의 대표적인 신호로 여겨진다.Poly (ADP-Ribose) polymerase (hereinafter referred to as PARP) is a marker that can induce apoptosis and is a typical intracellular repair system that enables the maintenance of gene stability against external stimuli. When cell death occurs, PARP is cleaved and cleavage of such PARP is considered to be a typical signal of apoptosis.
따라서 PARP를 활용하여, 화합물 K53의 세포사멸 유도 활성과, 테라토마 억제 효과를 구체적으로 확인하고자 하였다. Therefore, we intend to confirm the cell apoptosis inducing activity and the teratoma inhibitory effect of compound K53 using PARP.
구체적으로, 60mm 세포배양접시에 NCCIT 세포를 3×105개로 접종한 후, 화합물 K53을 20 μM 농도로 처리하여 표시된 시간 동안 배양하였다. 화합물 K53에 의한 PARP의 절단을 분석하기 위해 처리된 세포를 RIPA buffer (50mM Tris-HCl, pH7.4, 150mM NaCl, 5mM EDTA, 1mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate)로 용해시켰다. 세포 용해액을 15,000g로 원심 분리하여 상층 세포액을 회수하였으며, Bradford reagent (Bio-Rad protein assay, USA)를 이용하여 회수한 세포액의 단백질 농도를 측정하였다.Specifically, a 60 mm cell culture dish was inoculated with 3 × 10 5 NCCIT cells, and the compound K53 was treated at a concentration of 20 μM and cultured for the indicated time. To analyze the cleavage of PARP by compound K53, the treated cells were dissolved in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate) . The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
또한 7.5% SDS-PAGE (SDS-polyacrylamide gel electrophoresis)를 이용하여 30 μg의 세포 용해액을 단백질 분리한 후, PVDF 막 (Millipore, USA)으로 단백질들을 전기 이동하고 TBST (50mM Tris-HCl, pH7.6, 150mM NaCl, 0.1% tween 20)에 녹인 5% skim milk를 이용하여 블로킹 하였다. PARP 항체(Cell signaling, USA), GAPDH 항체(Santacruz, USA)로 2시간 동안 반응시키고 HRP가 conjugation된 2차 항체 (Jackson Immunolab, USA)와 1시간 동안 반응 시킨 뒤, chemiluminescence POD 시약 (Roche, Germany)을 사용하여 검출하였다. 그 결과는 도 4에 도시하였다. After 30 μg of the cell lysate was separated by 7.5% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on a PVDF membrane (Millipore, USA) and TBST (50 mM Tris-HCl, 6, 150 mM NaCl, 0.1% tween 20). The cells were incubated with PARP antibody (Cell signaling, USA), GAPDH antibody (Santacruz, USA) for 2 hours and reacted with secondary antibody conjugated with HRP (Jackson Immunolab, USA) for 1 hour and then chemiluminescence POD reagent ). &Lt; / RTI &gt; The results are shown in Fig.
도 4에서 알 수 있듯이, 화합물 K53의 처리시간에 비례하여 절단된 PARP 형태가 관찰되는 것을 볼 수 있다. 따라서, 화합물 K53은 OCT4를 발현하는 NCCIT의 세포사멸이 유도하여 우수한 항암 효과를 나타내며, 나아가, 미분화 세포 또는 테라토마를 효과적으로 사멸시킬 수 있음을 알 수 있다.As can be seen from FIG. 4, it can be seen that the cleaved PARP form is observed in proportion to the treatment time of the compound K53. Therefore, it can be seen that the compound K53 induces cell death of NCCIT expressing OCT4, exhibits excellent anticancer effect, and can effectively kill undifferentiated cells or teratomas.
<< 실시예Example 6> Nude mouse initiation model에서 화합물 K53의 복강투여에 의한 부작용 및 항암 효과 확인  6> Nude mouse initiation model confirmed the side effects and anti-cancer effects of peritoneal administration of compound K53
NCCIT 5× 106 cells을 Matrigel Matrix (BD biosciences) 100 μl에 혼합하여 NOD.CB17-PrkdcscidIl2rgtm1Wjl/SzJmice (6주령)에 이식 하였다. 접종 1일 후에 마우스를 무작위적으로 6마리씩 2개의 그룹(n=6)으로 나누어, 여기에 본 발명의 화합물 K53을 10% DMA(N,N-dimethylacetamide), 10% Cremophore 및 80% 증류수(v/v/v)로 구성된 용액에 녹인 후, 50일 동안 화합물 K53을 일일 투여용량 30 mg/kg으로 하여 매일 복강 투여하였다. A mixture of 5 × 10 6 cells in NCCIT Matrigel Matrix (BD biosciences) 100 μl were transferred to NOD.CB17-Prkdc scid Il2rg tm1Wjl / SzJmice (6 weeks old). One day after the inoculation, the mice were randomly divided into two groups (n = 6) in groups of 6, and the compound K53 of the present invention was added to 10% DMA (N, N-dimethylacetamide), 10% Cremophore and 80% distilled water / v / v), and compound K53 was intraperitoneally administered at a daily dose of 30 mg / kg for 50 days.
시험 기간 동안 특이한 증상은 관찰되지 않았으며, 마우스 체중 변화 결과, 종양의 부피변화 결과 및 최종일의 종양 조직 적출사진은 각각 도 5a, 도 5b 및 도 5c과 같다.No specific symptoms were observed during the test period, and results of mouse weight change, tumor volume change, and tumor histology of the last day were as shown in Figs. 5A, 5B and 5C, respectively.
6-1) 체중변화 관찰6-1) Observation of weight change
도 5a에 나타낸 바와 같이, 최종일(day 51)의 결과를 보면 용매 대조군과 비교하여, 화합물 K53(30 mg/kg) 투여군에서 체중 감소는 없었다. 따라서, 화합물 K53은 생체 내에서 부작용을 유발하지 않는 안전한 물질임을 확인하였다.As shown in FIG. 5A, the results of the last day (day 51) showed no weight loss in the compound K53 (30 mg / kg) group as compared with the solvent control group. Therefore, it was confirmed that the compound K53 is a safe substance which does not cause side effects in vivo.
6-2) 종양 부피변화 관찰6-2) Observation of tumor volume change
도 5b에 나타난 바와 같이, Initiation model에서 화합물 K53의 매일 반복 복강 투여에 의한 종양크기 변화를 51일 동안 관찰하여 항암효과를 확인하였다. 최종일에 종양조직의 부피를 측정한 결과, 용매 투여군의 경우 387.8± 66.2mm3, 화합물 K53의 경우에는 90.6± 26.2mm3으로, 대조군에 비해 약 1/4 크기 정도로 감소하였음을 확인하였다. As shown in FIG. 5B, the tumor size change by daily repeated intraperitoneal administration of compound K53 in the initiation model was observed for 51 days to confirm the anticancer effect. On the last day, the tumor volume was measured to be 387.8 ± 66.2 mm 3 in the solvent-treated group and 90.6 ± 26.2 mm 3 in the case of the compound K53, which was about 1/4 of the volume of the control group.
또한, 도 5c의 결과를 하기 수학식 2를 통해 종양성장억제율(%)을 계산한 결과, 77% (p<0.001)의 우수한 억제율을 나타냄을 확인하였다. In addition, the results of FIG. 5c were calculated to show an excellent inhibition rate of 77% (p < 0.001) as a result of calculating tumor growth inhibition rate (%) through the following equation (2).
[수학식 2]&Quot; (2) &quot;
종양성장억제율(%)= [1-(시험군의 t/용매대조군의 t)] x 100Tumor growth inhibition rate (%) = [1- (Test group of the t / vehicle control group t)] x 100
{상기식에서, t= Vt - Vo , Vt(Measurement of the tumor volume)이다}(Where t = Vt - Vo , Vt is measurement of the tumor volume)
6-3) 종양 크기의 육안 확인6-3) Visual confirmation of tumor size
또한, 실험 최종일(day 51)에 용매 투여군(vehicle control) 및 화합물 K53 (30mg/kg)투여군의 조직을 적출하여 찍은 사진인 도 5c와 같이 나타내었다. 5c, which is a photograph showing the tissue of the vehicle control group and the compound K53 (30 mg / kg) administration group on the last day of the experiment (day 51).
그 결과, 화합물 K53 투여군에서 적출한 종양 크기가 현저히 작아, 화합물 K53이 우수한 항암효과, 미분화 세포 억제효과 및 테라토마 억제효과를 나타냄을 확인하였다.As a result, it was confirmed that the tumor size extracted from the compound K53-treated group was remarkably small, and the compound K53 showed excellent anti-cancer effect, undifferentiated cell inhibitory effect and teratoma inhibitory effect.
<< 실시예Example 7> 화합물 K53의,  7> of compound K53, OCT4OCT4 발현  Expression 고환암Testicular cancer 세포주 특이적 성장 억제 효과 확인  Identification of cell-specific growth inhibition
화합물 K53이 고환암 세포주를 선별적으로 성장 억제하는지 여부를 확인하기 위하여 고환암세포 NCCIT 및 Tera-1의 증식을 분석하였다. In order to confirm whether compound K53 selectively inhibited testicular cancer cell lines, the proliferation of testicular cancer cells NCCIT and Tera-1 was analyzed.
구체적으로, NCCIT 및 Tera-1 인간 고환암 세포 7,000개를 96-웰 플레이트의 각 웰에 투여하고, 5% 이산화탄소를 포함한 37℃ 배양기에서 10% FBS가 포함된 배지하에 세포를 배양시켰다. 24시간 후에 대조군(0.1 % DMSO) 또는 화합물 K53을 10μM 및 30μM로 포함(DMSO에 녹인 화합물을 배지로 희석)한 배지로 교환한 후, 72시간 동안 배양시켰다. 트리판블루와 세포를 1:1로 섞은 뒤 헤마토사이토미터(hematocytometer)를 사용하여 세포 수를 측정하였으며, 그 결과를 도 6a에 나타내었다. Specifically, 7,000 NCCIT and Tera-1 human testicular cancer cells were administered to each well of a 96-well plate and the cells were cultured in a medium containing 10% FBS in a 37 ° C incubator containing 5% carbon dioxide. After 24 hours, the medium was replaced with a medium containing the control (0.1% DMSO) or the compound K53 at 10 μM and 30 μM (the compound dissolved in DMSO was diluted with the medium) and then cultured for 72 hours. The number of cells was measured using a hematocytometer after mixing 1: 1 of the triplan blue and cells, and the results are shown in FIG. 6A.
그 결과 도 6a에서 알 수 있듯이, K53 화합물을 30 μM 처리하였을 때 NCCIT 및 Tera-1의 증식이 각각 88.69% 및 70.4% 저해되었다. 반면에 K53을 30 μM 처리한 정상세포주 HFF 및 MCF10A는 증식이 억제되지 않았다. As a result, as shown in FIG. 6 (a), when the K53 compound was treated at 30 μM, the proliferation of NCCIT and Tera-1 was inhibited by 88.69% and 70.4%, respectively. On the other hand, normal cell lines HFF and MCF10A treated with 30 μM of K53 did not inhibit proliferation.
따라서, 화합물 K53은 정상 세포에는 영향을 미치지 않으면서도, 암 세포, 테라토마만을 특이적으로 억제함을 알 수 있다.Therefore, it can be seen that the compound K53 specifically suppresses only cancer cells and teratomas without affecting normal cells.
한편, 화합물 K53의 세포사멸 유도 활성이 OCT4 활성 억제에 기인함을 을 확인하기 위하여 NCCIT, Tera-1, HFF 및 MCF10A 세포주에서의 OCT4 발현 정도를 분석하였다. On the other hand, in order to confirm that the induction activity of the compound K53 is due to the inhibition of OCT4 activity, the expression level of OCT4 in the NCCIT, Tera-1, HFF and MCF10A cell lines was analyzed.
구체적으로, 60mm 세포배양접시에 NCCIT, Tera-1, HFF 및 MCF10A 세포를 3×105개로 접종한 후 배양하였다. 세포를 RIPA buffer (50mM Tris-HCl, pH7.4, 150mM NaCl, 5mM EDTA, 1mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate)으로 용해시켰다. 세포 용해액을 15,000g로 원심 분리하여 상층 세포액을 회수하였으며, Bradford reagent (Bio-Rad protein assay, USA)를 이용하여 회수한 세포액의 단백질 농도를 측정하였다.Specifically, NCCIT, Tera-1, HFF and MCF10A cells were inoculated in a 60 mm cell culture dish at 3 × 10 5 cells and cultured. Cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
또한 7.5% SDS-PAGE (SDS-polyacrylamide gel electrophoresis)를 이용하여 30 μg의 세포 용해액을 단백질 분리한 후, PVDF 막 (Millipore, USA)으로 단백질들을 전기 이동하고 TBST (50mM Tris-HCl, pH7.6, 150mM NaCl, 0.1% tween 20)에 녹인 5% skim milk를 이용하여 blocking 하였다. OCT4 항체(Cell signaling, USA) 및 Actin 항체(Santa Cruz Biotechnology, USA)로 2시간 동안 반응시키고 HRP가 컨쥬게이션된 2차 항체 (Jackson Immunolab, USA)와 1시간 동안 반응 시킨 뒤, chemiluminescence POD 시약 (Roche, Germany)을 사용하여 검출하였다. 그 결과는 도 6b에 도시하였다. After 30 μg of the cell lysate was separated by 7.5% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on a PVDF membrane (Millipore, USA) and TBST (50 mM Tris-HCl, 6, 150 mM NaCl, 0.1% tween 20). The cells were reacted with OCT4 antibody (Cell signaling, USA) and Actin antibody (Santa Cruz Biotechnology, USA) for 2 hours. HRP conjugated secondary antibody (Jackson Immunolab, USA) was reacted for 1 hour and chemiluminescence POD reagent Roche, Germany). The results are shown in FIG. 6B.
그 결과 도 6b에서 알 수 있듯이, 화합물 K53은 OCT4를 발현하는 고환암 세포만을 특이적으로 저해하며, 암세포가 아닌 일반 세포에는 영향을 미치지 않음을 확인하였다.As a result, as shown in FIG. 6B, it was confirmed that the compound K53 specifically inhibited OCT4-expressing testicular cancer cells and did not affect normal cells other than cancer cells.
<< 실시예Example 8>  8> OCT4의OCT4 분자 표적에 대한 화합물 K53의 결합 확인 Confirmation of binding of compound K53 to the molecular target
OCT4 억제를 통한 K53의 활성에 대해 보다 구체적으로 분석하기 위하여, OCT4에 대한 K53의 결합 여부를 확인하고자 하였다.In order to analyze more specifically the activity of K53 through OCT4 inhibition, we examined whether K53 binds to OCT4.
먼저, DMF 용매에 N-biotinylcaproic acid, EDC 및 DMAP을 녹인 후 K53 (KRIBB53) 화합물을 반응시켜 KRIBB53-biotin을 합성하였다. 이후 HPLC를 사용하여 KRIBB53-biotin을 정제하였다. First, KRIBB53-biotin was synthesized by dissolving N-biotinylcaproic acid, EDC and DMAP in a DMF solvent and reacting K53 (KRIBB53) compound. The KRIBB53-biotin was then purified using HPLC.
NCCIT 용해액을 호모겐나이저 버퍼(60mM β-glycerophosphate, 25mM MOPS (pH7.2), 15mM EGTA, 1mM DTT, 1mM phenyl phosphate, 100μM benzamidine)를 사용하여 제조하였다. 용해액에 KRIBB53-biotin을 20 μM 처리한 후 K53 화합물을 0, 50 μM, 100 μM, 200 μM, 500 μM 농도로 처리하였다. 그 후에 NeutrAvidin-Agarose resin을 첨가하였다. KRIBB53-biotin에 결합하지 않은 세포 용해액은 비드 버퍼( 50mM Tris (pH 7.4), 5mM NaF, 250mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Nonidet P-40, 10μg/ml of leupeptin, aprotinin, and soybean trypsin inhibitor, and 100 μM benzamidine)로 세척하여 제거한 후 SDS-샘플 버퍼를 처리하여 결합단백질들을 회수하였다. 회수된 단백질들은 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis)를 이용하여 단백질 분리한 후, PVDF 막 (Millipore, USA)으로 단백질들을 전기 이동하고 TBST (50mM Tris-HCl, pH7.6, 150mM NaCl, 0.1% tween 20)에 녹인 5% skim milk를 이용하여 블로킹 하였다. OCT4 항체(Cell signaling, USA)로 2시간 동안 반응시키고 HRP가 conjugation된 2차 항체 (Jackson Immunolab, USA)와 1시간 동안 반응 시킨 뒤, chemiluminescence POD 시약 (Roche, Germany)을 사용하여 검출하였다. 그 결과는 도 7에 도시하였다.The NCCIT lysate was prepared using homogenizer buffer (60 mM β-glycerophosphate, 25 mM MOPS (pH 7.2), 15 mM EGTA, 1 mM DTT, 1 mM phenyl phosphate, 100 μM benzamidine). After the KRIBB53-biotin was treated with 20 μM, the K53 compound was treated at 0, 50 μM, 100 μM, 200 μM and 500 μM concentrations in the solution. Then, NeutrAvidin-Agarose resin was added. The cell lysates not bound to KRIBB53-biotin were diluted with bead buffer (50 mM Tris (pH 7.4), 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Nonidet P-40, 10 μg / ml of leupeptin, aprotinin, and soybean trypsin inhibitor, and 100 μM benzamidine), followed by treatment with SDS-sample buffer to recover binding proteins. The recovered proteins were separated by using 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on a PVDF membrane (Millipore, USA), TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl , 0.1% tween 20) containing 5% skim milk. The cells were incubated with OCT4 antibody (Cell signaling, USA) for 2 hours, reacted with HRP-conjugated secondary antibody (Jackson Immunolab, USA) for 1 hour and detected with chemiluminescence POD reagent (Roche, Germany). The results are shown in Fig.
그 결과 도 7에서 알 수 있듯이, OCT4가 KRIBB53-biotin에 결합하며, K53 (KRIBB53)을 처리하면 결합이 경쟁적으로 감소하는 것으로 나타났다. 따라서, K53은 OCT4에 직접적으로 결합하는 활성을 가짐을 알 수 있다.As a result, as shown in FIG. 7, the binding of OCT4 to KRIBB53-biotin and the treatment of K53 (KRIBB53) showed a competitive decrease. Thus, it can be seen that K53 has an activity of directly binding to OCT4.
<< 실시예Example 9>  9> 유도만능줄기세포Induced pluripotent stem cells 및  And 신경줄기세포에서의In neural stem cells OCT4OCT4 발현 여부 분석 Expression analysis
미분화 유도만능 줄기세포 및 신경 줄기세포에서 OCT4 발현여부를 확인하기 위하여 웨스턴 블롯 (western blot)을 수행하였다. Western blotting was performed to confirm the expression of OCT4 in undifferentiated pluripotent stem cells and neural stem cells.
ATCC에서 구매한 사람유래 섬유아세포 (CRL2097)로부터 리프로그래밍을 통해 만들어진 유도만능줄기세포 CRL2097 iPSC와 분화시킨 신경줄기세포 CRL2097 NSC를 이용하였다. 신경줄기세포로의 분화는 Dual-smad inhibition을 바탕으로 한 Jun Takahashi의 논문을 참고로 하였다. (Doi, Daisuke, et al. "Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation." Stem cell reports 2.3 (2014): 337-350.)Derived neural stem cell CRL2097 NSC differentiated from induced pluripotent stem cell CRL2097 iPSC produced by reprogramming from human-derived fibroblast (CRL2097) purchased from ATCC was used. The differentiation into neural stem cells was based on Jun Takahashi's paper based on dual-smad inhibition. (Doi, Daisuke, et al., "Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation." Stem cell reports 2.3 (2014): 337-350.)
RIPA (sigma #R0278-50ML) 버퍼에 펠릿을 풀어주고, ice에서 1시간 동안 두어 단백질이 용해되도록 하였다. 원심분리기를 이용하여 16000g, 4°C 조건에서 20분 동안 원심분리 후, 상층액만을 얻어 브래드포드 (Biorad #500-0006) 시험법을 통해 단백질을 정량하였다. 그 중 20 μg을 폴리아크릴아미드 겔 (Biorad #456-1083)에 로딩하였고, PVDF membrane (Biorad #162-0177)으로 트랜스퍼하였다. 5% low fat dry milk (Biorad #170-6404)를 이용하여 실온에서 1시간 동안 블로킹을 한 뒤, OCT3/4 항체 (Santa cruz #sc9081)를 1:4000 비율로 처리하였다. 항체 반응은 4°C 조건에서 16시간 동안 진행하였다. 그 후, Anti-rabbit-HRP 항체 (Santa cruz #sc2004)를 1:10000 비율로 실온에서 1시간 동안 처리하였다. 내부 대조군을 위한 Beta-actin 항체 (Sigma #A5441)는 1:5000 비율로 처리하였다. The pellet was loosened in RIPA (sigma # R0278-50ML) buffer and placed on ice for 1 hour to allow the protein to dissolve. After centrifugation at 16000g at 4 ° C for 20 minutes using a centrifuge, proteins were quantified by Bradford (Biorad # 500-0006) only after obtaining supernatant. 20 μg of which was loaded onto polyacrylamide gel (Biorad # 456-1083) and transferred to PVDF membrane (Biorad # 162-0177). After blocking for 1 hour at room temperature using 5% low fat dry milk (Biorad # 170-6404), OCT3 / 4 antibody (Santa Cruz # sc9081) was treated at a ratio of 1: 4000. The antibody reaction was carried out at 4 ° C for 16 hours. Then, anti-rabbit-HRP antibody (Santa Cruz # sc2004) was treated at a ratio of 1: 10000 for 1 hour at room temperature. Beta-actin antibody (Sigma # A5441) for internal control was treated at a 1: 5000 ratio.
그 결과, 도 8에서 알 수 있듯이, OCT4 (size : 45 kDa) 발현은 단백질 수준에서 유도만능줄기세포에서만 관찰할 수 있었으며, 신경줄기세포에서는 관찰되지 않았다. As a result, as shown in FIG. 8, OCT4 (size: 45 kDa) expression was observed only in induced pluripotent stem cells at the protein level and not in neural stem cells.
따라서 OCT4 저해하는 상기 화합물 K53 등은 유도만능줄기세포와 같은 미분화 세포, 또는 테라토마 세포에는 영향을 주지만, 신경줄기세포에는 아무런 영향을 미치지 않음을 알 수 있다.Therefore, it can be seen that the compound K53 inhibiting OCT4 affects undifferentiated cells such as inducible pluripotent stem cells or teratoma cells, but has no effect on neural stem cells.
<< 실시예Example 10> 화합물 K53, K55, K56, K58 및 K59의  10> Compounds of the compounds K53, K55, K56, K58 and K59 유도만능줄기세포에Induced pluripotent stem cells 대한 특이적 성장 억제 효과 Specific growth inhibitory effect
K53, K55, K56, K58 및 K59 화합물을 처리할 경우, 유도만능줄기세포에 대한 성장 억제 효과를 확인하기 위하여, 다음과 같이 세포 계수를 진행하였다.When the K53, K55, K56, K58, and K59 compounds were treated, the cell counting was carried out as follows to confirm the growth inhibitory effect on the induced pluripotent stem cells.
TeSR-E8 (stemcell #05940) 배지를 이용하여 배양 중인 유도만능줄기세포 CRL2097 iPSC를 Accutase (Millipore #SCR005)를 이용하여 단일세포로 분리한 뒤, 5X104/well(12)로 접종(seed)하였다. 2일 뒤, 세포가 안정적으로 성장하는 시기에 약물을 처리하였다. 신경줄기세포의 경우, 5X105/well(24)로 seed 한 뒤 11일간 분화시킨 세포에 약물을 처리하였다. 유의성을 확인하고자 각 조건은 n=3으로 반복실험을 진행하였다. DMSO (대조군), 화합물 K53 또는 K59를 각각 5 μM씩 24시간 동안 처리한 뒤, 세포 계수를 진행하였다. 트리판블루와 세포를 1:1로 섞은 뒤, CountessTMAutomatedCellCounter(Invitrogen)을 이용하였고, 약물을 처리한 각 조건에서 살아있는 세포 수를 비교하였다. DMSO와 약물 처리시 조건을 비교하기 위해 T 검정을 이용하여 데이터 분석을 진행하였다.Induced pluripotent stem cell CRL2097 iPSC cultured using TeSR-E8 (stemcell # 05940) medium was separated into single cells using Accutase (Millipore # SCR005) and seeded at 5 × 10 4 / well (12) . Two days later, the drug was treated at a time when cells were stably growing. In the case of neural stem cells, the cells were seeded at 5 × 10 5 / well (24) and treated for 11 days. To confirm the significance, each condition was repeated with n = 3. DMSO (control), compound K53 or K59 was treated with 5 [mu] M each for 24 hours, and then cell counting was carried out. Treffan blue and cells were mixed 1: 1, then Countess ™ AutomatedCellCounter (Invitrogen) was used and the number of viable cells was compared under each condition treated with the drug. Data were analyzed using T - test to compare the conditions of drug treatment with DMSO.
그 결과, 도 9a 및 도 9b에서 알 수 있듯이, OCT4를 발현하는 유도만능줄기세포 CRL2097 iPSC의 경우, K53, K55, K56, K58과 K59를 처리한 조건에서 세포 사멸이 일어나, 살아있는 세포의 수가 감소하였다. 반면, OCT4를 발현하지 않는 신경줄기세포 CRL2097 NSC의 경우, K53, K55, K56, K58과 K59를 처리하여도 대조군과 유의성 있는 차이를 관찰할 수 없었으며, 세포수가 거의 감소하지 않았다. As a result, as shown in FIGS. 9A and 9B, in the case of induced pluripotent stem cell CRL2097 iPSC expressing OCT4, apoptosis was observed under conditions of treatment with K53, K55, K56, K58 and K59, Respectively. On the other hand, in the case of neural stem cell CRL2097 NSC not expressing OCT4, no significant difference was observed between K53, K55, K56, K58 and K59, and the number of cells was not decreased.
따라서, 화합물 K53, K55, K56, K58과 K59는 OCT4를 발현하는 암 세포, 미분화 세포, 또는 테라토마 세포에 대한 특이적인 사멸 효과를 나타내므로, 우수한 항암 효과를 나타내며, 미분화 세포 또는 테라토마 세포 형성 억제 효과를 가짐을 알 수 있다.Therefore, the compounds K53, K55, K56, K58, and K59 exhibit a specific killing effect on cancer cells, OCT4 expressing cancer cells, undifferentiated cells, or teratoma cells, and thus exhibit excellent anticancer effects and inhibit undifferentiated cells or teratoma cell formation As shown in FIG.
<< 실시예Example 11> 화합물 K53, K55, K56, K58 및 K59의  11> Compounds of the compounds K53, K55, K56, K58 and K59 OCT4OCT4 발현 고환암세포에 대한 특이적 성장 억제 효과 Specific growth inhibition effect on expression testicular cancer cells
화합물 K53, K55, K56, K58, 및 K59가 OCT4를 특이적으로 저해함으로써 OCT4를 발현하는 고환암 세포주 NCCIT의 성장을 억제하는지 여부를 확인하기 위하여 고환암세포 NCCIT의 증식을 분석하였다. In order to confirm whether compounds K53, K55, K56, K58, and K59 specifically inhibited the growth of OCT4 expressing testicular cancer cell line NCCIT, the proliferation of testicular cancer cells NCCIT was analyzed.
구체적으로, NCCIT 인간 고환암 세포 2x105개를 6-웰 플레이트의 각 웰에 투여하고, 5% 이산화탄소를 포함한 37℃ 배양기에서 10% FBS가 포함된 배지하에 세포를 배양시켰다. 24시간 후에 대조군(0.1 % DMSO) 또는 화합물 K53, K55, K56, K58 및 K59을 10μM 또는 30μM로 포함(DMSO에 녹인 화합물을 배지로 희석)한 배지로 교환한 후, 48시간 동안 배양시켰다. 트리판블루와 세포를 1:1로 섞은 뒤 헤마토사이토미터(hematocytometer)를 사용하여 세포 수를 측정하였으며, 그 결과를 도 10에 나타내었다. Specifically, 2x10 5 NCCIT human testicular cancer cells were administered to each well of a 6-well plate and the cells were cultured in a medium containing 10% FBS in a 37 ° C incubator containing 5% carbon dioxide. After 24 hours, the medium was replaced with a control (0.1% DMSO) or a medium containing the compounds K53, K55, K56, K58 and K59 at 10 μM or 30 μM (the compound dissolved in DMSO was diluted with the medium) and then cultured for 48 hours. The number of cells was measured using a hematocytometer after mixing 1: 1 of the trypan blue and the cells, and the results are shown in FIG.
그 결과 도 10에서 알 수 있듯이, K53, K55, K56, K58 및 K59 화합물을 10 μM 처리하였을 때 NCCIT의 증식이 각각 27, 42, 87, 31, 59% 저해되었다. 또한 K53, K55, K56, K58 및 K59 화합물을 30 μM 처리하였을때 NCCIT의 증식이 각각 59, 97, 100, 96, 99% 저해되었다. As shown in FIG. 10, when 10 μM of K53, K55, K56, K58 and K59 compounds were treated, the proliferation of NCCIT was inhibited by 27, 42, 87, 31 and 59%, respectively. When 30 μM of K53, K55, K56, K58 and K59 compounds were treated, the proliferation of NCCIT was inhibited by 59, 97, 100, 96 and 99%, respectively.
따라서, 상기 화합물 K53, K55, K56, K58 및 K59 화합물은 OCT4를 발현하는 세포인 NCCIT 세포의 증식을 억제함을 확인하여, 미분화 세포의 사멸, 테라토마 및 테라토칼시노마 형성의 억제 효과를 나타낼 수 있으며, 또한 고환암의 예방 및 치료 효과를 가질 수 있음을 알 수 있다.Therefore, the compounds K53, K55, K56, K58, and K59 inhibited proliferation of NCCIT cells expressing OCT4, and showed inhibitory effects on the death of undifferentiated cells, formation of teratoma and teratocarcinoma And can also have the preventive and therapeutic effects of testicular cancer.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (14)

  1. 하기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용 가능한 염을 포함하는, OCT4(Octamer-Binding Transcription Factor 4) 저해용 조성물:A composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
    [화학식 1][Chemical Formula 1]
    Figure PCTKR2018008027-appb-I000008
    Figure PCTKR2018008027-appb-I000008
    상기 화학식 1에서In Formula 1,
    R1, R2, R3, R4, R5, R1', R2', R3', R4'및 R5'는 각각 독립적으로 H, 하이드록시, C1-4 알콕시, 및 할로겐으로 이루어진 군에서 선택되는 것임.Wherein each of R1, R2, R3, R4, R5, R1 ', R2', R3 ', R4' and R5 'is independently selected from the group consisting of H, hydroxy, C 1-4 alkoxy and halogen.
  2. 제1항에 있어서, The method according to claim 1,
    R1은 H, 또는 하이드록시이고; R1 is H, or hydroxy;
    R2는 H, 또는 C1-4 알콕시이고; R2 is H, or Ci- 4 alkoxy;
    R3는 H, 하이드록시, 또는 C1-4 알콕시이고; R3 is H, hydroxy, or C 1-4 alkoxy;
    R4는 H, C1-4 알콕시, 또는 할로겐이고; R4 is H, C 1-4 alkoxy, or halogen;
    R5는 H이고; R5 is H;
    R1'는 H, 하이드록시 또는 C1-4 알콕시이고; R1 'is H, hydroxy or C 1-4 alkoxy;
    R2'는 H, 또는 할로겐이고;R2 ' is H, or halogen;
    R3'는 H, 또는 C1-4 알콕시이고; R3 'is H, or C 1-4 alkoxy;
    R4'는 H이고;R4 ' is H;
    R5'는 H, 하이드록시, 또는 C1-4 알콕시임.R5 'is H, hydroxy, or C 1-4 alkoxy.
  3. 제1항에 있어서,The method according to claim 1,
    R1, R2, R3, R4, R5, R1', R2', R3', R4'및 R5'는 각각 독립적으로 H, 하이드록시, 메톡시, 및 브로모로 이루어진 군에서 선택되는 것임.R2, R3, R4, R5, R1 ', R2', R3 ', R4' and R5 'are each independently selected from the group consisting of H, hydroxy, methoxy and bromo.
  4. 제1항에 있어서, The method according to claim 1,
    R1은 H, 또는 하이드록시이고; R1 is H, or hydroxy;
    R2는 H, 또는 메톡시이고; R2 is H, or methoxy;
    R3는 H, 하이드록시, 또는 메톡시이고; R3 is H, hydroxy, or methoxy;
    R4는 H, 메톡시, 또는 브로모이고; R4 is H, methoxy, or bromo;
    R5는 H; R5 is H;
    R1'는 H, 하이드록시, 또는 메톡시이고; R1 'is H, hydroxy, or methoxy;
    R2'는 H, 또는 브로모이고;R2 ' is H, or bromo;
    R3'는 H, 또는 메톡시이고; R3 ' is H, or methoxy;
    R4'는 H이고;R4 ' is H;
    R5'는 H, 하이드록시, 또는 메톡시임.R5 'is H, hydroxy, or methoxy.
  5. 제1항에 있어서, 상기 화합물은 하기 화학식 2 내지 6로 이루어진 군에서 선택된 어느 하나의 화합물인 것인, 조성물:The composition according to claim 1, wherein the compound is any one selected from the group consisting of the following formulas (2) to (6):
    [화학식 2](2)
    Figure PCTKR2018008027-appb-I000009
    Figure PCTKR2018008027-appb-I000009
    [화학식 3](3)
    Figure PCTKR2018008027-appb-I000010
    Figure PCTKR2018008027-appb-I000010
    [화학식 4][Chemical Formula 4]
    Figure PCTKR2018008027-appb-I000011
    Figure PCTKR2018008027-appb-I000011
    [화학식 5][Chemical Formula 5]
    Figure PCTKR2018008027-appb-I000012
    Figure PCTKR2018008027-appb-I000012
    [화학식 6][Chemical Formula 6]
    Figure PCTKR2018008027-appb-I000013
    .
    Figure PCTKR2018008027-appb-I000013
    .
  6. 제1항 내지 제5항 중 어느 한 항의 조성물을 포함하는, 미분화 세포 (undifferentiated cell) 제거용; 또는 세포 치료제의 테라토마 (teratoma) 또는 테라토칼시노마 (teratocarcinoma) 형성 억제용 조성물.A composition for removing undifferentiated cells, comprising the composition of any one of claims 1 to 5; Or a composition for inhibiting the formation of a teratoma or a teratocarcinoma of a cell therapy agent.
  7. 제6항에 있어서, 상기 미분화 세포는 OCT4 (Octamer-Binding Transcription Factor 4)를 발현하는 것인, 조성물.7. The composition of claim 6, wherein the undifferentiated cell expresses OCT4 (Octamer-Binding Transcription Factor 4).
  8. 제6항에 있어서, 상기 미분화 세포 제거; 또는 테라토마 또는 테라토칼시노마 형성 억제는 시험관 내 (in vitro), 생체 내 (in vivo), 또는 생체 외 (ex vivo)에서 이루어지는 것인, 조성물.7. The method of claim 6, wherein the undifferentiated cell is removed; Or teratoma or teratocarcinoma formation inhibition is achieved in vitro, in vivo, or ex vivo.
  9. 제6항 내지 제8항 중 어느 한 항의 조성물을 세포 치료제에 처리하는 단계를 포함하는, 미분화 세포 제거; 또는 테라토마 또는 테라토칼시노마 형성 억제 방법.9. A method for the treatment of cancer, comprising: treating the composition of any one of claims 6 to 8 to a cell therapy agent; Or inhibiting the formation of a teratoma or teratocarcinoma.
  10. 제6항 내지 제8항 중 어느 한 항의 조성물을 세포 치료제에 처리하는 단계를 포함하는, 미분화 세포가 제거되거나, 또는 테라토마 또는 테라토칼시노마의 형성이 억제된 세포 치료제의 제조 방법.9. A method for producing a cell therapy agent, wherein the undifferentiated cell is removed, or the formation of a teratoma or a teratocarcinoma is inhibited, comprising the step of treating the composition of any one of claims 6 to 8 to a cell treatment agent.
  11. 제9항 또는 제10항에 있어서, 상기 세포 치료제는 체세포 치료제, 줄기세포 치료제 또는 이들의 조합인, 방법.11. The method according to claim 9 or 10, wherein the cell therapeutic agent is a somatic cell therapeutic agent, a stem cell therapeutic agent, or a combination thereof.
  12. 제1항 내지 제5항 중 어느 한 항의 조성물을 포함하는, 고환암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for the prophylaxis or treatment of testicular cancer, comprising the composition of any one of claims 1 to 5.
  13. 제12항에 있어서, 상기 약학 조성물은 항암제를 추가로 포함하는, 약학 조성물.13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition further comprises an anti-cancer agent.
  14. 제12항 또는 제13항의 조성물을 개체에 투여하는 단계를 포함하는, 고환암의 예방 또는 치료방법.13. A method for the prophylaxis or treatment of testicular cancer, comprising the step of administering the composition of claim 12 or 13 to a subject.
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